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Thyroid_Cancer

thyroid cancer THCAprognosis and construct a polygene risk prediction model for prognosis predictionand improvementMethods The HTSeqCounts data of THCA were accessed from TCGA databaseincluding cancer samples and normal tissue samples œedgeR package wasutilized to perform differential analysis and weighted gene coexpression networkanalysis WGCNA was applied to screen the differential coexpression genes associatedwith THCA tissue types Univariant Cox regression analysis was further used for theselection of survivalrelated genes Then LASSO regression model was constructed toanalyze the genes and an optimal prognostic model was developed as well as evaluatedby KaplanMeier and ROC curvesResults Three thousand two hundred seven differentially expressed genes DEGs wereobtained by differential analysis and coexpression genes COR P were gained after WGCNA analysis In addition eight genes significantly related to THCAsurvival were screened by univariant Cox regression analysis and an optimal prognostic3gene risk prediction model was constructed after genes were analyzed by the LASSOregression model Based on this model patients were grouped into the highrisk groupand lowrisk group KaplanMeier curve showed that patients in the lowrisk group hadmuch better survival than those in the highrisk group Moreover great accuracy of the3gene model was revealed by ROC curve and the remarkable correlation between themodel and patients™ prognosis was verified using the multivariant Cox regression analysisConclusion The prognostic 3gene model composed by GHR GPR125 and ATP2C2three genes can be used as an independent prognostic factor and has better predictionfor the survival of THCA patientsKeywords THCA WGCNA prognostic 3gene risk prediction model prediction prognosisINTRODUCTIONThyroid cancer THCA derived from parafollicular cells or thyroid follicular cells is the mostcommon endocrine malignancy accounting for about of all kinds of human cancers Papillary PTC follicular anaplastic and medullary thyroid carcinomas are the four subtypes ofTHCA among which papillary and follicular carcinomas are common and have better prognosisEdited byChristoph ReinersUniversity HospitalW¼rzburg GermanyReviewed byTrevor Edmund AngellUniversity of Southern CaliforniaUnited StatesRoberto VitaUniversity of Messina ItalyCorrespondenceHaixing Fanghaixing01231163comSpecialty sectionThis was submitted toThyroid Endocrinologya section of the journalFrontiers in EndocrinologyReceived November Accepted June Published August CitationZhao H Zhang S Shao S and Fang H Identification of a Prognostic3Gene Risk Prediction Model forThyroid CancerFront Endocrinol 103389fendo202000510Frontiers in Endocrinology wwwfrontiersinAugust Volume 0cZhao et alA Prognostic 3Gene Model for Thyroid Cancer while anaplastic carcinoma is rare to be seen with extremelypoor prognosis Therefore it™s very important to find eï¬ectiveapproaches for the improvement of the overall THCA prognosisAt present the conventional prognostic model of THCA inclinical practice is constructed according to predictive factorslike age tumor size and lymph nodule metastasis Withthe development of highthroughput sequencing technologymRNA expression profiles of specific cancers are easy to obtainwhich helps us better find more robust prognostic signals For instance microarraybased gene expression analysis enablesus to identify the important genes during tumor progressionand helps to define and diagnose prognostic characteristics In this way many THCA prognostic biomarkers have beenverified However these markers are almost single genes and havenot been widely accepted Polygenic combination has beenreported to possess better predictive ability for cancer prognosisthan single genes Therefore recent studies have involvedin the identification of the biomarkers for THCA prognosis However restricted by research methods novel biologicalalgorithm needs to be explored to construct more accuratediagnostic or prognosis modelsIn the present study a large number of mRNA expressionprofiles of THCA patients were accessed from TCGA databaseand modules associated with THCA were identified by WGCNAA 3gene risk prediction model was constructed using Cox andLASSO regression models which could help us better predictTHCA prognosisMATERIALS AND METHODSData ResourceExpression profiles of THCA mRNA and corresponding clinicaldata were accessed from TCGA database cancergenomenihgovsamples and normaltissue samples The study was in line with the guidelinesreleased by TCGA httpcancergenomenihgovpublicationspublicationguidelinesincluding cancerIdentification and Confirmation ofTHCAAssociated GenesœedgeR packagebioconductorpackagesreleasebiochtmledgeRhtml was used to perform diï¬erential analysisbetween cancer tissues and normal tissues Genes met thecriteria logFC and P were considered to havesignificant diï¬erencesModule Selection With WGCNAThe mechanism of WGCNA is the research for coexpressionmodules and the exploration of the correlation between the genenetwork and the phenotypes which is motivated by the analysesof scalefree clustering and dynamic tree cut on expressionprofiles In the present study modules that were most relatedto THCA tissue types in the coexpression network constructedby WGCNA package cranrprojectwebpackagesWGCNAindexhtml were selected and genes meeting P and COR were extracted for further studyConstruction of the Prognostic RiskPrediction ModelTHCA prognosisassociated genes werescreened usingunivariant Cox regression analysis Then a prognostic modelwas constructed using the least absolute shrinkage and selectionoperator LASSO According to this model risk score of eachsample was calculated and patients were divided into thehighrisk group and lowrisk group with the median risk scoreas the threshold KaplanMeier was used to evaluate the survivalof the two groups The ROC curve was drawn for the evaluationof the prognosis performance of the model and the area underthe curve AUC was calculated Furthermore multivariantCox regression analysis was performed to assess the correlationbetween the risk score and patients™ prognosis KaplanMeierand ROC curves of each gene in this model were plotted to makea comparison with those curves of the modelStatistical AnalysisUnivariant and multivariant Cox regression analyses wereboth performed in TCGA dataset œglmnet package of theR software wwwrproject was used for LASSOstatistic algorithm IBM SPSS statistical software IBMCorp Armonk NY USA was applied for statistical analysis P was considered statistically significantRESULTSIdentification of THCAAssociatedModulesAs shown in A a total of DEGs were identifiedlogFC P WGCNA was used to screen THCArelated modules and appropriate adjacency matrix weightparameter power was selected to ensure the scalefreedistribution of the coexpression network as possible In therange of ‰ ‰ log k and log Pk were calculated for linearmodels™ construction respectively is the squared value of thecoefficient R As shown in B the soft threshold poweris higher with the elevated R2 suggesting that the network closelyapproaches to scalefree distribution In the present study R2 for the first time was selected to ensure the realizationof scalefree distribution as possible and make the values on thecurve approach to the minimum threshold When themean connectivity of RNA in the network was Cwhich was consistent with the smallworld network in the scalefree one Then cluster dendrogram was constructed Dand dynamic tree cut was performed deep split Modulesobtained were merged with the minimum size of and modules were eventually developedThe correlation and significance between the modulecharacteristics and sample phenotypes were calculated Amongthe modules genes in blue brown pink and turquoisemodules were verified to be most associated with THCAprognosis E THCA tissue typeassociated geneswere obtained from the four modules taking the P andCOR as the threshold FFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cZhao et alA Prognostic 3Gene Model for Thyroid CancerFIGURE Identification of the THCA tissue typeassociated RNA functional modules A Volcano plot of DEGs B Analysis of scaleindependence index for varioussoft threshold powers Horizontal axis is the soft threshold power and vertical axis is the scalefree topology fitting indices R2 The red line refers to the standardcorresponding to the R2 of C Analysis of the mean connectivity under different soft threshold powers D Cluster dendrogram of all DEGs clustered based on adissimilarity measure E Distribution of average gene significance and errors in the modules associated with the progression of THCA F Venn diagram of the genesin the four modules for coexpression genes selectionFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cZhao et alA Prognostic 3Gene Model for Thyroid CancerConstruction of a Prognostic 3Gene RiskPrediction Model for THCAUnivariant Cox regression was performed for analysis ofthe coexpression genes suggesting that eight genes weresignificantly correlated with survival as shown in Table LASSO regression model was constructed to analyze thegenes and an optimal prognostic risk prediction modelwasScore — GHR —GPR125 — Atp2c2 Risk prediction wasperformed according to this model and patients were rangedFigure 2A RiskeventuallydevelopedTABLE Basic information of the eight prognostic genesidHRHR95LHR95HPvaluebased on the risk scores Figure 2B The median risk score wasused as the critical value to group the patients into the highriskgroup n and lowrisk group n As shown inthe KaplanMeier curve in Figure 2C patients in the highriskgroup had worse overall survival OS than those in the lowriskgroup ROC curve was plotted to predict the 3year survival andthe results showed in Figure 2D revealed that AUC of the 3genemodel was which indicated the good performance of therisk score in survival prediction Multivariant Cox proportionalhazards regression analysis was then performed combined withclinical factors and the correlation between the risk score andprognosis of patients was verified Figure 2E From the heatmaps of the expression profiles of these three genes Figure 2Fthe expression levels of GHR GPR125 and Atp2c2 were found tobe positively correlated with the risk score and all of them wereregarded as highrisk genesAtp2c2GPR125GHRCLMNCYTH3PLA2R1RYR2C8orf88Evaluation of the 3Gene Risk PredictionModelKaplanMeier curves of the three genes were drawn using thelog rank test As shown in Figures 3A“C THCA patients withlow expression of GHR GPR125 and Atp2c2 had longer survivaltime indicting that these three genes were highrisk genes whichwas in agreement with the results predicted by univariant Coxregression analysis Furthermore ROC curves Figures 3D“FFIGURE Construction of a 3gene risk prediction model A LASSO regression model B 3gene based distribution of risk scores C Survival analysis of realhub genes in the TCGATHCA dataset D ROC curve of real hub genes in the TCGATHCA dataset E The correlation between the risk score and patients™prognosis F Heatmap of the genes expression profilesFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cZhao et alA Prognostic 3Gene Model for Thyroid CancerFIGURE The evaluation of the 3gene risk prediction model A“C Survival analyses of GHR GPR125 and Atp2c2 in the TCGATHCA dataset D“F ROCanalyses of GHR GPR125 and Atp2c2 in the TCGATHCA datasetrevealed that the AUC of GHR GPR125 and Atp2c2 was and respectively all of which were smaller than thatof the 3gene risk prediction model Findings above demonstratethat risk score is a good indicator for prognosis and the 3genemodel has a higher accuracyDISCUSSIONWith the development of the microarray and RNA sequencingtechnologies new era of large data on biology is coming It hasbeen reported that microarraybased gene expression analysiscould achieve characterization in human cancers identificationof the important genes during tumorigenesis and the definitionas well as the diagnosis of prognostic features However therole of genes as prognosis factors has been few investigated In the present study a large amount of RNAseq profiles andclinical prognosis data of THCA patients were accessed fromTCGA database and coexpression gene modules were screenedusing WGCNA Studies have shown that gene modules are muchreliable in cancer prognosis than biomarkers While there arefew studies on the crosstalk among the modules and someimportant modules might be ignored Therefore in ourstudy gene coexpression network was constructed via WGCNAand was used to identify THCA tissue typeassociated genemodules including blue brown pink and turquoise Twentythree common genes were obtained from the four modulesand an optimal prognostic 3gene risk prediction model wasthen constructed by univariant Cox and LASSO regressionanalyses Along with the LASSO model all independent variablescan be processed simultaneously verifying the more accurateperformance than the stepwise regression model GHRGPR125 and Atp2C2 were the three genes in this model GHR isa kind of proteincoding gene coding transmembrane receptorsof the growth hormone In prior studies GHR has been verifiedto be a oncogene in some cancers such as breast cancer pancreatic ductal carcinoma and melanoma but therole in THCA prognosis is firstly reported GPR125 a 57KDafactor for transmembrane signal transduction is considered toplay a key role in cell adhesion and signal transduction It™sreported that GPR125 is upregulated in human cerebral cancertissues and promotes cell adhesion as well as the formationof myelosarcoma In our study GHR and GPR125 wereverified as highrisk genes in THCA which was consistent withthe previous studies Moreover we found that these two genescould be used as independent risk predictive factors but theaccuracy was lower than that of the 3gene risk prediction modelwhich was further verified by ROC and KaplanMeier curvesAs the expression profiles of THCA and clinical informationare just from one dataset of TCGA the samples for analyzingthe prognostic 3gene model are limited In addition the modelconstructed in this study might be not available when it comesto other databases and it™s necessary to improve the model withFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cZhao et alA Prognostic 3Gene Model for Thyroid Cancermore datasets In a word a 3gene model is constructed to be anindependent predictor in this study which provides novel viewand approach for the prognosis of THCA patientsDATA AVAILABILITY STATEMENTAll datasets generated for this study are included in thesupplementary materialAUTHOR CONTRIBUTIONSHZ contributed to the study design and gave the finalapproval of the version to be submitted SZ conducted theliterature search and performed data analysis and draftedSS acquired the data and revised the HF wrote the All authors contributed to the and approved thesubmitted versionREFERENCES Hedayati M Zarif Yeganeh M Sheikholeslami S Afsari F Diversityof mutations in the RET protooncogene and its oncogenic mechanismin medullary thyroid cancer Crit Rev Clin Lab Sci “ Carling T Udelsman R Thyroid cancer Annu Rev Med “ 101146annurevmed061512105739 Dralle H Machens A Basa J Fatourechi V Franceschi S Hay IDet al Follicular cellderived thyroid cancer Nat Rev Dis Primers 101038nrdp201577 SmallridgeRCCoplandand emergingAnaplasticJAtherapies Clin Oncolthyroidcarcinoma “pathogenesis 101016jclon201003013 Shaha AR Implications of prognostic factors and risk groups in themanagement of diï¬erentiated thyroid cancer Laryngoscope “ Zhao QJ Zhang J Xu L Liu FF Identification of a fivelong noncoding RNA signature to improve the prognosis prediction for patientswith hepatocellular carcinoma World J Gastroenterol “ 103748wjgv24i303426et Hebrant A Dom G Dewaele M Andry G Tr©sallet C LeteurtreEthyroidcarcinoma molecular anatomy of a killing switch PLoS ONE 7e37807 101371journalpone0037807al mRNA expression in papillaryand anaplastic Brennan K Holsinger C Dosiou C Sunwoo JB Akatsu H Haile R et alDevelopment of prognostic signatures for intermediaterisk papillary thyroidcancer BMC Cancer 101186s1288501627716 ZuoS Dai G Ren XIdentification ofa6genepredicting prognosis 101186s1293501807247for colorectal cancer Cancer CellsignatureInt Cui ZJ Zhou XH Zhang HY DNA methylation module networkcancer Genestyping ofbased prognosisand molecular 103390genes10080571 Gu JX Zhang X Miao RC Xiang XH Fu YN Zhang JY et alrecurrencefreein hepatocellular carcinoma World J GastroenterolSixlongsurvival“ 103748wjgv25i2220noncodingsignaturepredictsRNA Arumugam A Subramani R Nandy SB Terreros D Dwivedi AK Saltzstein Eet al Silencing growth hormone receptor inhibits estrogen receptor negativebreast cancer through ATPbinding cassette subfamily G member Exp MolMed 101038s1227601801978 Subramani R LopezValdez R Salcido A Boopalan T Arumugam A NandyS et al Growth hormone receptor inhibition decreases the growth andmetastasis of pancreatic ductal adenocarcinoma Exp Mol Med 46e117 101038emm201461 Proudfoot NJ Gil A Whitelaw E Studies on messenger RNA ™ endformation in globin genes a transcriptional interference model for globin geneswitching Prog Clin Biol Res “ Wu Y Chen W Gong L Ke C Wang H Cai Y Elevated Gprotein receptor GPR125 expression predicts good outcomes in colorectal cancer andinhibits Wntbetacatenin signaling pathway Med Sci Monit “ 1012659MSM910105 Pickering C Hgglund M SzmydyngerChodobska J Marques F Palha JAWaller L et al The adhesion GPCR GPR125 is specifically expressed in thechoroid plexus and is upregulated following brain injury BMC Neurosci Fu JF Yen TH Chen Y Huang YJ Hsu CL Liang DC et alInvolvement of Gpr125 in the myeloid sarcoma formation inducedby cooperating MLLAF10OMLZ and oncogenic KRAS in a mousebone marrow transplantation model “ 101002ijc28195J CancerInt Li X Dai D Wang H Wu B Wang R Identification of prognostic signaturesassociated with longterm overall survival of thyroid cancer patients basedon a competing endogenous RNA network Genomics “ 101016jygeno201907005 Li J Yu X Liu Q Ou S Li K Kong Y et al Screening of importantadenocarcinomalncRNAsbased on integrated bioinformatics analysis Mol Med Rep“ 103892mmr201910061associated with the prognosis oflung Tavares C Melo M CameselleTeijeiro JM Soares P Sobrinhothyroid cancer 174R117“ 101530EJESimoes M Endocrine tumours genetic predictors ofoutcome EurJ EndocrinolConflict of Interest The authors declare that the research was conducted in theabsence of any commercial or financial relationships that could be construed as apotential conflict of interestCopyright Zhao Zhang Shao and Fang This is an openaccess distributed under the terms of the Creative Commons Attribution License CC BYThe use distribution or reproduction in other forums is permitted provided theoriginal authors and the copyright owners are credited and that the originalpublication in this journal is cited in accordance with accepted academic practiceNo use distribution or reproduction is permitted which does not comply with thesetermsFrontiers in Endocrinology wwwfrontiersinAugust Volume 0c'

cancer200

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: thyroid cancer THCAprognosis and construct a polygene risk prediction model for prognosis predictionand improvementMethods The HTSeqCounts data of THCA were accessed from TCGA databaseincluding cancer samples and normal tissue samples œedgeR package wasutilized to perform differential analysis and weighted gene coexpression networkanalysis WGCNA was applied to screen the differential coexpression genes associatedwith THCA tissue types Univariant Cox regression analysis was further used for theselection of survivalrelated genes Then LASSO regression model was constructed toanalyze the genes and an optimal prognostic model was developed as well as evaluatedby KaplanMeier and ROC curvesResults Three thousand two hundred seven differentially expressed genes DEGs wereobtained by differential analysis and coexpression genes COR P were gained after WGCNA analysis In addition eight genes significantly related to THCAsurvival were screened by univariant Cox regression analysis and an optimal prognostic3gene risk prediction model was constructed after genes were analyzed by the LASSOregression model Based on this model patients were grouped into the highrisk groupand lowrisk group KaplanMeier curve showed that patients in the lowrisk group hadmuch better survival than those in the highrisk group Moreover great accuracy of the3gene model was revealed by ROC curve and the remarkable correlation between themodel and patients™ prognosis was verified using the multivariant Cox regression analysisConclusion The prognostic 3gene model composed by GHR GPR125 and ATP2C2three genes can be used as an independent prognostic factor and has better predictionfor the survival of THCA patientsKeywords THCA WGCNA prognostic 3gene risk prediction model prediction prognosisINTRODUCTIONThyroid cancer THCA derived from parafollicular cells or thyroid follicular cells is the mostcommon endocrine malignancy accounting for about of all kinds of human cancers Papillary PTC follicular anaplastic and medullary thyroid carcinomas are the four subtypes ofTHCA among which papillary and follicular carcinomas are common and have better prognosisEdited byChristoph ReinersUniversity HospitalW¼rzburg GermanyReviewed byTrevor Edmund AngellUniversity of Southern CaliforniaUnited StatesRoberto VitaUniversity of Messina ItalyCorrespondenceHaixing Fanghaixing01231163comSpecialty sectionThis was submitted toThyroid Endocrinologya section of the journalFrontiers in EndocrinologyReceived November Accepted June Published August CitationZhao H Zhang S Shao S and Fang H Identification of a Prognostic3Gene Risk Prediction Model forThyroid CancerFront Endocrinol 103389fendo202000510Frontiers in Endocrinology wwwfrontiersinAugust Volume 0cZhao et alA Prognostic 3Gene Model for Thyroid Cancer while anaplastic carcinoma is rare to be seen with extremelypoor prognosis Therefore it™s very important to find eï¬ectiveapproaches for the improvement of the overall THCA prognosisAt present the conventional prognostic model of THCA inclinical practice is constructed according to predictive factorslike age tumor size and lymph nodule metastasis Withthe development of highthroughput sequencing technologymRNA expression profiles of specific cancers are easy to obtainwhich helps us better find more robust prognostic signals For instance microarraybased gene expression analysis enablesus to identify the important genes during tumor progressionand helps to define and diagnose prognostic characteristics In this way many THCA prognostic biomarkers have beenverified However these markers are almost single genes and havenot been widely accepted Polygenic combination has beenreported to possess better predictive ability for cancer prognosisthan single genes Therefore recent studies have involvedin the identification of the biomarkers for THCA prognosis However restricted by research methods novel biologicalalgorithm needs to be explored to construct more accuratediagnostic or prognosis modelsIn the present study a large number of mRNA expressionprofiles of THCA patients were accessed from TCGA databaseand modules associated with THCA were identified by WGCNAA 3gene risk prediction model was constructed using Cox andLASSO regression models which could help us better predictTHCA prognosisMATERIALS AND METHODSData ResourceExpression profiles of THCA mRNA and corresponding clinicaldata were accessed from TCGA database cancergenomenihgovsamples and normaltissue samples The study was in line with the guidelinesreleased by TCGA httpcancergenomenihgovpublicationspublicationguidelinesincluding cancerIdentification and Confirmation ofTHCAAssociated GenesœedgeR packagebioconductorpackagesreleasebiochtmledgeRhtml was used to perform diï¬erential analysisbetween cancer tissues and normal tissues Genes met thecriteria logFC and P were considered to havesignificant diï¬erencesModule Selection With WGCNAThe mechanism of WGCNA is the research for coexpressionmodules and the exploration of the correlation between the genenetwork and the phenotypes which is motivated by the analysesof scalefree clustering and dynamic tree cut on expressionprofiles In the present study modules that were most relatedto THCA tissue types in the coexpression network constructedby WGCNA package cranrprojectwebpackagesWGCNAindexhtml were selected and genes meeting P and COR were extracted for further studyConstruction of the Prognostic RiskPrediction ModelTHCA prognosisassociated genes werescreened usingunivariant Cox regression analysis Then a prognostic modelwas constructed using the least absolute shrinkage and selectionoperator LASSO According to this model risk score of eachsample was calculated and patients were divided into thehighrisk group and lowrisk group with the median risk scoreas the threshold KaplanMeier was used to evaluate the survivalof the two groups The ROC curve was drawn for the evaluationof the prognosis performance of the model and the area underthe curve AUC was calculated Furthermore multivariantCox regression analysis was performed to assess the correlationbetween the risk score and patients™ prognosis KaplanMeierand ROC curves of each gene in this model were plotted to makea comparison with those curves of the modelStatistical AnalysisUnivariant and multivariant Cox regression analyses wereboth performed in TCGA dataset œglmnet package of theR software wwwrproject was used for LASSOstatistic algorithm IBM SPSS statistical software IBMCorp Armonk NY USA was applied for statistical analysis P was considered statistically significantRESULTSIdentification of THCAAssociatedModulesAs shown in A a total of DEGs were identifiedlogFC P WGCNA was used to screen THCArelated modules and appropriate adjacency matrix weightparameter power was selected to ensure the scalefreedistribution of the coexpression network as possible In therange of ‰ ‰ log k and log Pk were calculated for linearmodels™ construction respectively is the squared value of thecoefficient R As shown in B the soft threshold poweris higher with the elevated R2 suggesting that the network closelyapproaches to scalefree distribution In the present study R2 for the first time was selected to ensure the realizationof scalefree distribution as possible and make the values on thecurve approach to the minimum threshold When themean connectivity of RNA in the network was Cwhich was consistent with the smallworld network in the scalefree one Then cluster dendrogram was constructed Dand dynamic tree cut was performed deep split Modulesobtained were merged with the minimum size of and modules were eventually developedThe correlation and significance between the modulecharacteristics and sample phenotypes were calculated Amongthe modules genes in blue brown pink and turquoisemodules were verified to be most associated with THCAprognosis E THCA tissue typeassociated geneswere obtained from the four modules taking the P andCOR as the threshold FFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cZhao et alA Prognostic 3Gene Model for Thyroid CancerFIGURE Identification of the THCA tissue typeassociated RNA functional modules A Volcano plot of DEGs B Analysis of scaleindependence index for varioussoft threshold powers Horizontal axis is the soft threshold power and vertical axis is the scalefree topology fitting indices R2 The red line refers to the standardcorresponding to the R2 of C Analysis of the mean connectivity under different soft threshold powers D Cluster dendrogram of all DEGs clustered based on adissimilarity measure E Distribution of average gene significance and errors in the modules associated with the progression of THCA F Venn diagram of the genesin the four modules for coexpression genes selectionFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cZhao et alA Prognostic 3Gene Model for Thyroid CancerConstruction of a Prognostic 3Gene RiskPrediction Model for THCAUnivariant Cox regression was performed for analysis ofthe coexpression genes suggesting that eight genes weresignificantly correlated with survival as shown in Table LASSO regression model was constructed to analyze thegenes and an optimal prognostic risk prediction modelwasScore — GHR —GPR125 — Atp2c2 Risk prediction wasperformed according to this model and patients were rangedFigure 2A RiskeventuallydevelopedTABLE Basic information of the eight prognostic genesidHRHR95LHR95HPvaluebased on the risk scores Figure 2B The median risk score wasused as the critical value to group the patients into the highriskgroup n and lowrisk group n As shown inthe KaplanMeier curve in Figure 2C patients in the highriskgroup had worse overall survival OS than those in the lowriskgroup ROC curve was plotted to predict the 3year survival andthe results showed in Figure 2D revealed that AUC of the 3genemodel was which indicated the good performance of therisk score in survival prediction Multivariant Cox proportionalhazards regression analysis was then performed combined withclinical factors and the correlation between the risk score andprognosis of patients was verified Figure 2E From the heatmaps of the expression profiles of these three genes Figure 2Fthe expression levels of GHR GPR125 and Atp2c2 were found tobe positively correlated with the risk score and all of them wereregarded as highrisk genesAtp2c2GPR125GHRCLMNCYTH3PLA2R1RYR2C8orf88Evaluation of the 3Gene Risk PredictionModelKaplanMeier curves of the three genes were drawn using thelog rank test As shown in Figures 3A“C THCA patients withlow expression of GHR GPR125 and Atp2c2 had longer survivaltime indicting that these three genes were highrisk genes whichwas in agreement with the results predicted by univariant Coxregression analysis Furthermore ROC curves Figures 3D“FFIGURE Construction of a 3gene risk prediction model A LASSO regression model B 3gene based distribution of risk scores C Survival analysis of realhub genes in the TCGATHCA dataset D ROC curve of real hub genes in the TCGATHCA dataset E The correlation between the risk score and patients™prognosis F Heatmap of the genes expression profilesFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cZhao et alA Prognostic 3Gene Model for Thyroid CancerFIGURE The evaluation of the 3gene risk prediction model A“C Survival analyses of GHR GPR125 and Atp2c2 in the TCGATHCA dataset D“F ROCanalyses of GHR GPR125 and Atp2c2 in the TCGATHCA datasetrevealed that the AUC of GHR GPR125 and Atp2c2 was and respectively all of which were smaller than thatof the 3gene risk prediction model Findings above demonstratethat risk score is a good indicator for prognosis and the 3genemodel has a higher accuracyDISCUSSIONWith the development of the microarray and RNA sequencingtechnologies new era of large data on biology is coming It hasbeen reported that microarraybased gene expression analysiscould achieve characterization in human cancers identificationof the important genes during tumorigenesis and the definitionas well as the diagnosis of prognostic features However therole of genes as prognosis factors has been few investigated In the present study a large amount of RNAseq profiles andclinical prognosis data of THCA patients were accessed fromTCGA database and coexpression gene modules were screenedusing WGCNA Studies have shown that gene modules are muchreliable in cancer prognosis than biomarkers While there arefew studies on the crosstalk among the modules and someimportant modules might be ignored Therefore in ourstudy gene coexpression network was constructed via WGCNAand was used to identify THCA tissue typeassociated genemodules including blue brown pink and turquoise Twentythree common genes were obtained from the four modulesand an optimal prognostic 3gene risk prediction model wasthen constructed by univariant Cox and LASSO regressionanalyses Along with the LASSO model all independent variablescan be processed simultaneously verifying the more accurateperformance than the stepwise regression model GHRGPR125 and Atp2C2 were the three genes in this model GHR isa kind of proteincoding gene coding transmembrane receptorsof the growth hormone In prior studies GHR has been verifiedto be a oncogene in some cancers such as breast cancer pancreatic ductal carcinoma and melanoma but therole in THCA prognosis is firstly reported GPR125 a 57KDafactor for transmembrane signal transduction is considered toplay a key role in cell adhesion and signal transduction It™sreported that GPR125 is upregulated in human cerebral cancertissues and promotes cell adhesion as well as the formationof myelosarcoma In our study GHR and GPR125 wereverified as highrisk genes in THCA which was consistent withthe previous studies Moreover we found that these two genescould be used as independent risk predictive factors but theaccuracy was lower than that of the 3gene risk prediction modelwhich was further verified by ROC and KaplanMeier curvesAs the expression profiles of THCA and clinical informationare just from one dataset of TCGA the samples for analyzingthe prognostic 3gene model are limited In addition the modelconstructed in this study might be not available when it comesto other databases and it™s necessary to improve the model withFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cZhao et alA Prognostic 3Gene Model for Thyroid Cancermore datasets In a word a 3gene model is constructed to be anindependent predictor in this study which provides novel viewand approach for the prognosis of THCA patientsDATA AVAILABILITY STATEMENTAll datasets generated for this study are included in thesupplementary materialAUTHOR CONTRIBUTIONSHZ contributed to the study design and gave the finalapproval of the version to be submitted SZ conducted theliterature search and performed data analysis and draftedSS acquired the data and revised the HF wrote the All authors contributed to the and approved thesubmitted versionREFERENCES Hedayati M Zarif Yeganeh M Sheikholeslami S Afsari F Diversityof mutations in the RET protooncogene and its oncogenic mechanismin medullary thyroid cancer Crit Rev Clin Lab Sci “ Carling T Udelsman R Thyroid cancer Annu Rev Med “ 101146annurevmed061512105739 Dralle H Machens A Basa J Fatourechi V Franceschi S Hay IDet al Follicular cellderived thyroid cancer Nat Rev Dis Primers 101038nrdp201577 SmallridgeRCCoplandand emergingAnaplasticJAtherapies Clin Oncolthyroidcarcinoma “pathogenesis 101016jclon201003013 Shaha AR Implications of prognostic factors and risk groups in themanagement of diï¬erentiated thyroid cancer Laryngoscope “ Zhao QJ Zhang J Xu L Liu FF Identification of a fivelong noncoding RNA signature to improve the prognosis prediction for patientswith hepatocellular carcinoma World J Gastroenterol “ 103748wjgv24i303426et Hebrant A Dom G Dewaele M Andry G Tr©sallet C LeteurtreEthyroidcarcinoma molecular anatomy of a killing switch PLoS ONE 7e37807 101371journalpone0037807al mRNA expression in papillaryand anaplastic Brennan K Holsinger C Dosiou C Sunwoo JB Akatsu H Haile R et alDevelopment of prognostic signatures for intermediaterisk papillary thyroidcancer BMC Cancer 101186s1288501627716 ZuoS Dai G Ren XIdentification ofa6genepredicting prognosis 101186s1293501807247for colorectal cancer Cancer CellsignatureInt Cui ZJ Zhou XH Zhang HY DNA methylation module networkcancer Genestyping ofbased prognosisand molecular 103390genes10080571 Gu JX Zhang X Miao RC Xiang XH Fu YN Zhang JY et alrecurrencefreein hepatocellular carcinoma World J GastroenterolSixlongsurvival“ 103748wjgv25i2220noncodingsignaturepredictsRNA Arumugam A Subramani R Nandy SB Terreros D Dwivedi AK Saltzstein Eet al Silencing growth hormone receptor inhibits estrogen receptor negativebreast cancer through ATPbinding cassette subfamily G member Exp MolMed 101038s1227601801978 Subramani R LopezValdez R Salcido A Boopalan T Arumugam A NandyS et al Growth hormone receptor inhibition decreases the growth andmetastasis of pancreatic ductal adenocarcinoma Exp Mol Med 46e117 101038emm201461 Proudfoot NJ Gil A Whitelaw E Studies on messenger RNA ™ endformation in globin genes a transcriptional interference model for globin geneswitching Prog Clin Biol Res “ Wu Y Chen W Gong L Ke C Wang H Cai Y Elevated Gprotein receptor GPR125 expression predicts good outcomes in colorectal cancer andinhibits Wntbetacatenin signaling pathway Med Sci Monit “ 1012659MSM910105 Pickering C Hgglund M SzmydyngerChodobska J Marques F Palha JAWaller L et al The adhesion GPCR GPR125 is specifically expressed in thechoroid plexus and is upregulated following brain injury BMC Neurosci Fu JF Yen TH Chen Y Huang YJ Hsu CL Liang DC et alInvolvement of Gpr125 in the myeloid sarcoma formation inducedby cooperating MLLAF10OMLZ and oncogenic KRAS in a mousebone marrow transplantation model “ 101002ijc28195J CancerInt Li X Dai D Wang H Wu B Wang R Identification of prognostic signaturesassociated with longterm overall survival of thyroid cancer patients basedon a competing endogenous RNA network Genomics “ 101016jygeno201907005 Li J Yu X Liu Q Ou S Li K Kong Y et al Screening of importantadenocarcinomalncRNAsbased on integrated bioinformatics analysis Mol Med Rep“ 103892mmr201910061associated with the prognosis oflung Tavares C Melo M CameselleTeijeiro JM Soares P Sobrinhothyroid cancer 174R117“ 101530EJESimoes M Endocrine tumours genetic predictors ofoutcome EurJ EndocrinolConflict of Interest The authors declare that the research was conducted in theabsence of any commercial or financial relationships that could be construed as apotential conflict of interestCopyright Zhao Zhang Shao and Fang This is an openaccess distributed under the terms of the Creative Commons Attribution License CC BYThe use distribution or reproduction in other forums is permitted provided theoriginal authors and the copyright owners are credited and that the originalpublication in this journal is cited in accordance with accepted academic practiceNo use distribution or reproduction is permitted which does not comply with thesetermsFrontiers in Endocrinology wwwfrontiersinAugust Volume 0c' Answer:

Thyroid_Cancer

Primary squamous cell carcinoma ofis anextremely rare aggressive malignancy with a poor prognosis However almost noreportthus far has investigated the microvasculature of ThyPSCC imaged usingcontrastenhanced ultrasoundthe thyroid ThyPSCCCase Report A 59yearold male patient presented to our hospital with progressivelyworsening hoarse voice symptoms for days and was diagnosed with left unilateralvocal fold palsy Ultrasonography revealed a solitary marked hypoechoic thyroid nodulewith an unclear boundary in the inferior part of the left lobe Color Doppler flow imagingshowed a poor blood flow signalinside this nodule Contrastenhanced ultrasoundimages showed a persistent low peak enhancement of the nodule from its periphery to itscenter The timeintensity curve displayed a washin time of s a time to peak of s apeak signal intensity of and a washout time of s for the thyroid tumor Finallyleft hemithyroidectomy of the thyroid tumor was performed and histopathologic andimmunohistochemical evaluations confirmed the diagnosis of ThyPSCC Postoperativelythe patient received a combination therapy of chemotherapy radiotherapy and targetedtherapy but the patient died months after surgeryPrimary squamous cell carcinoma ofConclusionthe thyroid is a rare butaggressive malignancy of the thyroid Herein we reported a case of ThyPSCC and itsultrasonography and pathologic findingsKeywords thyroid cancer thyroid nodules TNs thyroid ultrasound US primary squamous cell carcinomacontrast enhanced ultrasound CEUSINTRODUCTIONPrimary squamous cell carcinoma of the thyroid ThyPSCC is a rare thyroid malignancy withhigh aggressiveness and poor prognosis comprising ˆ¼“ of all primary thyroid carcinomas“ Owing to the rapidly progressing and highly invasive nature of the malignancy patients withThyPSCC often present at an advanced stage and are difficult to diagnose in the early stage becauseof its rare incidence and lack of typical imaging findings Thyroid ultrasonography and fineneedle aspiration biopsy FNAB are the diagnostic tools ofchoice for evaluating patients with suspected thyroid nodules Contrastenhanced ultrasoundCEUS as a relatively novel US technique is used to investigate the microvasculature of thyroidnodules and improve the diagnostic accuracy of thyroid nodules accompanied by the use of ThyroidEdited byChristoph ReinersUniversity HospitalW¼rzburg GermanyReviewed byPasqualino MalandrinoUniversity of Catania ItalyDaniela PasqualiUniversity of Campania LuigiVanvitelli ItalyCorrespondenceChengcheng NiuniuchengchengcsueducnSpecialty sectionThis was submitted toCancer Endocrinologya section of the journalFrontiers in EndocrinologyReceived February Accepted June Published August CitationChen S Peng Q Zhang Q and Niu C ContrastEnhanced Ultrasoundof Primary Squamous Cell Carcinomaof the Thyroid A Case ReportFront Endocrinol 103389fendo202000512Frontiers in Endocrinology wwwfrontiersinAugust Volume 0cChen et alThyroid Primary Squamous Cell CarcinomaImaging Reporting and Data Systems for ultrasonographicfeatures “ However very few published studies havereported the use of ultrasonography for ThyPSCC To ourknowledge this is the first case describing the CEUS findingsof ThyPSCCreached its peak [time to peak TTP] at s with a peakintensity of Then the nodule slowly declined until allthe microbubbles washed out at s Figures 1CD Based onits malignant conventional ultrasound features and the poormicrovasculature revealed by CEUS we inferred that the nodulewas a malignant tumorCASE REPORTA 59yearold male patient presented to our hospital withprogressively worsening hoarse voice symptoms for daysand was diagnosed with left unilateral vocal fold palsy Ahighresolution ultrasound instrument Siemens Acuson S3000Mountain View CA USA equipped with a to 9MHz linearprobe was used Thyroid ultrasonography revealed a solitary — — 26cm3 marked hypoechoic thyroid nodule with anunclear boundary in the inferior part of the left lobe AThis nodule exhibited many malignant ultrasound featuressuch as solid components hypoechogenicity and microlobulatedmargins Color Doppler flow imaging CDFI showed poorblood flow signals in the nodule B Contrastenhancedultrasound was performed with a bolus intravenous injectionof mL of SonoVue Bracco Milan Italy followed by mLof saline Contrast pulse sequencing technology was used andthe timeintensity curves TICs of the nodule were calculatedThe nodule began to be slowly enhanced from the peripheryto the center at s washin time and the enhancementAfterneckthepositronultrasonographyemissiontomography“computed tomography was carried for evaluatingthesituation of distant metastases Positron emissiontomography“computed tomography showed a mass withincreased glucose metabolism in the inferior part of the leftthyroid lobe A which indicated it as a malignantmass whereas there was no evidence of lymph nodes metastasisand distant metastases Then ultrasonographyguided FNABwas performed for the left thyroid mass immediately Cytologicexamination by fineneedle aspiration FNA revealed sheets oftumor cells with giant deepstained nuclei Bethesda categoryV B Finally a left hemithyroidectomy of the thyroidtumor was undertaken The lower edge of the tumor reachedthe upper mediastinum and the depth of the tumor invadedthe esophagus and trachea which could not be completelyremoved According to the eighth edition of the AmericanJoint Committee on CancerTumor Lymph Node MetastasisTNM staging system the patient was in TNM stage IIIT4a N0 M0 Histopathological examination of hematoxylinand eosin staining showed that a carcinoma in the inferiorFIGURE Ultrasonography images of primary squamous cell carcinoma of the thyroid A Longitudinal grayscale sonography revealed a solid marked hypoechoicthyroid nodule in the inferior part of the left lobe B Color Doppler flow imaging showed a poor blood flow signal inside this nodule C Contrastenhanced ultrasoundimage showed a persistent low peak enhancement of the nodule at s D Timeintensity curves displayed the washin time of s TTP of s peak signalintensity of and washout time of s for the thyroid tumorFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cChen et alThyroid Primary Squamous Cell CarcinomaFIGURE A A positron emission tomography“computed tomography scan showed increased 18Ffluorodeoxyglucose metabolism in the left neck mass BPreoperative fineneedle aspiration cytology of the mass demonstrated a few sheets of malignantlooking tumor cells with giant deep stained nuclei hematoxylin andeosin magnification — FIGURE Hematoxylin and eosin staining of primary squamous cell carcinoma of the thyroid A magnification — B magnification — C magnification — D magnification — part of the thyroid lobe A had no obvious palisadearrangementintercellular bridges or keratinization with acancer pearl Figures 3B“D Immunohistochemically tumorcells were positive for cytokeratin CK19 Acytokeratin and CK56 B epithelial membraneantigen EMA C p40 D p63 Aand Ki67 B and negative for thyroglobulinTG C and thyroid transcription factor TTF1D In view of these findings the tumor was diagnosedas poorly diï¬erentiated ThyPSCC Postoperatively the patientreceived two cycles of chemotherapy with docetaxelcisplatinintensitymodulated radiotherapy and nimotuzumabtargetedtherapy However the patient died months after surgeryDISCUSSIONPrimary squamous cell carcinoma of the thyroid is a thyroidmalignancy with extremely rare incidence and the clinicaldiagnosis and treatment guidelines for this disease have noconsensus The biological behavior of ThyPSCC is aggressiveFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cChen et alThyroid Primary Squamous Cell CarcinomaFIGURE Immunohistochemical staining of primary squamous cell carcinoma of the thyroid magnification — Immunohistochemical staining for A CK19 BCK56 C EMA D p40 all of which were deeply stained positiveFIGURE Immunohistochemical staining of primary squamous cell carcinoma of the thyroid magnification — Immunohistochemical staining for A p63 B Ki C TG D TTF1 and p63 was deeply stain positive Ki67 proliferation index was TG and TTF1 did not stain negativeFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cChen et alThyroid Primary Squamous Cell Carcinomaand the prognosis is poor with a median overall survival of “ months which depends on the diï¬erent tumor grades Yang et al using the Surveillance Epidemiology and End ResultsProgram database reported that poorly diï¬erentiated tumrade occupied the highest percentages of all graded tumors andthe median survival was months which is similar to the survivaltime in our case Highfrequency ultrasound as the basic imaging modality inthe diagnosis of thyroid nodules has found gradually increasingdiï¬erentiated thyroid cancers over recent years Theultrasonography imaging findings of ThyPSCC have seldombeen published Regarding the ultrasonography findings Chenet al reported that ThyPSCC presented as a thyroid masswith eggshell calcification peripheral soft tissue with a blurredmargin and minimal vascular signals on CDFI sonographyIn the case of Jang et al ThyPSCC presented as a largewelldefined lobulated heterogeneously hypoechoic mass withdiï¬use microcalcifications on ultrasonography Kondo et al reported that a welldiï¬erentiated ThyPSCC showed acystic hypoechoic mass with a smooth margin and rapidlygrew with margin change blurring in year In our case thispoorly diï¬erentiated ThyPSCC presented as a solitary markedhypoechoic thyroid mass with an irregular margin and unclearboundary with a normal thyroid The irregular margin andunclear boundary with normal thyroid corresponded to tumorinvasion with adjacent tissue infiltration which is consistentwith the findings during the operation that tumor invasion withthe esophagus cannot be completely removed Poor blood flowsignals on CDFI sonography and persistent hypoenhancement onCEUS of the mass are consistent with squamous cell carcinomawhich has no obvious vascularity on pathologic examinationMany studies have investigated the application of CEUS toimprove the diagnostic accuracy of thyroid nodules despiteits usage in ThyPSCC being scarce Zhang et al foundthat highcircularequal enhancement indicated benign thyroidnodules and low enhancement indicated malignant thyroidnodules Ma et al investigated whether incomplete noring or heterogeneous enhancement later washin time andlow peak intensity on CEUS were independent risk factorsin predicting malignantthyroid nodules Deng et al detected that papillary thyroid carcinomas PTCs exhibited lowenhancement a lower peak signal intensity and a lower areaunder the curve AUC than peripheral thyroid parenchyma onCEUS In our study the TICs of CEUS for ThyPSCCshowed a washin time of s a TTP of s a peak signalintensity as low as and a washout time of s Thisis similar to the results of PTCs with a slow washin time alower peak signal intensity and a lower AUC as in previousreports To our knowledge no reports on CEUS imagingfindings of ThyPSCC have appeared in the Englishlanguageliterature According to Jang et al ThyPSCC showed a largeheterogeneously enhancing thyroid mass with a large centralnonenhancing portion on enhanced CT which correspondedwell with the squamous cell carcinoma portion with a necroticportion in pathologic staining Because of the rapid growth ofsquamous tumor cells relatively few interstitial blood vessels intumors were related to the low peak signal intensity and low AUCon CEUSisusefulstainingWith increasing malignancy in squamous cell carcinoma thetypical squamous cell carcinoma findings of intercellular bridgesand keratinized cancer pearl can decrease or disappearImmunohistochemicalin diagnosingprimary thyroid cancer In this case positivity for CK56and EMA and negativity for TTF1 and TG expressionpredicted squamous cell carcinoma derivation and excludedthe possibility ofthese common tumors Furtherpositivity for p63 and Ki67 expression as poor prognosticmarkers was associated with its poorly diï¬erentiated tumrade CONCLUSIONPrimary squamous cell carcinoma of the thyroid is an extremelyrare tumor and very few studies describe its ultrasonographicimaging findings It is difficult to establish a clinical guidelinefor diagnosis Our case presents the CEUS features of ThyPSCCindicating that the TICs of ThyPSCC are similar to the enhancingparameters of PTCs with a slow washin time a lower peak signalintensity and a lower AUCDATA AVAILABILITY STATEMENTThe datasets generated for this study are available on request tothe corresponding authorETHICS STATEMENTThe studies involving human participants were reviewed andapproved by the Ethics Committee of Second Xiangya HospitalCentral South University China The patientsparticipantsprovided their written informed consentto participate inthis study Written informed consent was obtained from theindividuals for the publication of any potentially identifiableimages or data included in this AUTHOR CONTRIBUTIONSAll authors listed have made a substantial direct and intellectualcontribution to the work and approved it for publicationFUNDINGof ChinaThis project was funded by the National Natural ScienceFoundationProvincialNatural Science Foundation of China 2018JJ2575 andHunan Provincial Health Commission Research FoundationProject B2019166 HunanFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cChen et alREFERENCES Yang S Li C Shi X Ma B Xu W Jiang H et al Primary squamous cellcarcinoma in the thyroid gland a populationbased analysis using the SEERdatabase World J Surg “ 101007s00268019049062 Limberg J Ullmann TM Stefanova D Finnerty BM Beninato T Fahey TJet al Prognostic characteristics of primary squamous cell carcinoma of thethyroid a national cancer database analysis World J Surg “ 101007s00268019050985Thyroid Primary Squamous Cell Carcinomacontrastenhanced ultrasound Ultrasound Med Biol “ 1016jultrasmedbio201810020 Casella C Ministrini S Galani A Mastriale F Cappelli C Portolani NThe new TNM staging system for thyroid cancer and the risk of diseasedownstaging Front Endocrinol 103389fendo201800541 Zhang Y Zhou P Tian SM Zhao YF Li JL Li L Usefulness of combineduse of contrastenhanced ultrasound and TIRADS classification for thediï¬erentiation of benign from malignant lesions of thyroid nodules EurRadiol “ 101007s003300164508y Koyama S Fujiwara K Nosaka K Fukuhara T Morisaki T MiyakeN et al Immunohistochemical features of primary pure squamous cellcarcinoma in the thyroid an autopsy case Case Rep Oncol “ Zhang YZ Xu T Gong HY Li CY Ye XH Lin HJ et al Application ofhighresolution ultrasound realtime elastography and contrastenhancedultrasound in diï¬erentiating solid thyroid nodules Medicine95e5329 101097MD00000000000053290000579220161108000016 Wang SS Ye DX Wang B Xie C The expressions of keratins andP63 in primary squamous cell carcinoma ofthe thyroid gland anapplication of raman spectroscopy Onco Targets Ther “ 102147OTTS229436 Chen CY Tseng HS Lee CH Chan PW Primary squamous cellcarcinoma of the thyroid gland with eggshell calcification sonographicand computed tomographic findings J Ultrasound Med “ 107863jum201029111667 Yasumatsu R Sato M Uchi R Nakano T Hashimoto K Kogo R et al Thetreatment and outcome analysis of primary squamous cell carcinoma of thethyroid Auris Nasus Larynx “ 101016janl201707009 Kao NH Tan CS H Koh AJ The utility of immunohistochemistry indiï¬erentiating metastatic primary squamous cell carcinoma of the thyroidfrom a primary lung squamous cell carcinoma Case Rep Endocrinol “ Jang JY Kwon KW Kim SW Youn I Primary squamouscarcinoma ofandtomographic“ 1014366usg13022cellrecurrence ultrasonographicthyroid gland with localUltrasonographycomputedfindings Raggio B Barrcarcinomacell 1031486toj180002J Ghandour Z Friedlander P Primary squamousof“thyroid OchsnertheJ Kondo T Matsuyoshi A Matsuyoshi H Goto R Ono K Honda Y et alA case of primary thyroid squamous cell cancer transformation frombenign tumour associated with chronic thyroiditis BMJ Case Rep 2009bcr1020081137 101136bcr1020081137 Ma JJ Ding H Xu BH Xu C Song LJ Huang BJ et al Diagnosticand contrastmalignant101089thy201performances ofenhancedultrasonographythyroid nodules Thyroid“ color dopplergrayscalepredictingfindingsvariousin Deng J Zhou P Tian SM Zhang L Liofefficacydiagnosticofradiationdiï¬erentiating9e90674 101371journalpone0090674PONED1330329imagingthyroidandnodulescontrastenhancedimpulseforcesolidfocalJL Qian Y ComparisonacousticinuseultrasoundcombinedPLoS ONEtheir Haugen BR Alexander EK Bible KC Doherty GM Mandel SJ Nikiforov YEet al American thyroid association management guidelines for adultpatients with thyroid nodules and diï¬erentiated thyroid cancer the Americanthyroid association guidelines task force on thyroid nodules and diï¬erentiatedthyroid cancer Thyroid “ 101089thy20150020 Tessler FN Middleton WD Grant EG Hoang JK Berland LL Teefey SAet al ACR thyroid imaging reporting and data system TIRADS whitepaper of the ACR TIRADS committee J Am Coll Radiol “ 101016jjacr201701046 Kwak JY Han KH Yoon JH Moon HJ Son EJ Park SH et al Thyroidimaging reporting and data system for US features of nodules a step inestablishing better stratification of cancer risk Radiology “ 101148radiol11110206radiol11110206 Peng Q Niu C Zhang Q Zhang M Chen S Mummified thyroid nodulesconventional and contrastenhanced ultrasound features J Ultrasound Med “ 101002jum14712 Peng Q Niu C Zhang M Chen S Sonographic characteristics ofpapillary thyroid carcinoma with coexistent hashimoto™sthyroiditisconventional ultrasound acoustic radiation force impulse imaging and Struller F Senne M Falch C Kirschniak A Konigsrainer A Mullerthe thyroid case report andS Primary squamous cell carcinoma ofsystematic review of the literature Int J Surg Case Rep “ 101016jijscr201706011 Wang W Ouyang Q Meng C Jing L Li X Treatment optimization andprognostic considerations for primary squamous cell carcinoma of thethyroid Gland Surg “ 1021037gs20191107Conflict of Interest The authors declare that the research was conducted in theabsence of any commercial or financial relationships that could be construed as apotential conflict of interestCopyright Chen Peng Zhang and Niu This is an openaccess distributed under the terms of the Creative Commons Attribution License CC BYThe use distribution or reproduction in other forums is permitted provided theoriginal authors and the copyright owners are credited and that the originalpublication in this journal is cited in accordance with accepted academic practiceNo use distribution or reproduction is permitted which does not comply with thesetermsFrontiers in Endocrinology wwwfrontiersinAugust Volume 0c'

cancer201

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: Primary squamous cell carcinoma ofis anextremely rare aggressive malignancy with a poor prognosis However almost noreportthus far has investigated the microvasculature of ThyPSCC imaged usingcontrastenhanced ultrasoundthe thyroid ThyPSCCCase Report A 59yearold male patient presented to our hospital with progressivelyworsening hoarse voice symptoms for days and was diagnosed with left unilateralvocal fold palsy Ultrasonography revealed a solitary marked hypoechoic thyroid nodulewith an unclear boundary in the inferior part of the left lobe Color Doppler flow imagingshowed a poor blood flow signalinside this nodule Contrastenhanced ultrasoundimages showed a persistent low peak enhancement of the nodule from its periphery to itscenter The timeintensity curve displayed a washin time of s a time to peak of s apeak signal intensity of and a washout time of s for the thyroid tumor Finallyleft hemithyroidectomy of the thyroid tumor was performed and histopathologic andimmunohistochemical evaluations confirmed the diagnosis of ThyPSCC Postoperativelythe patient received a combination therapy of chemotherapy radiotherapy and targetedtherapy but the patient died months after surgeryPrimary squamous cell carcinoma ofConclusionthe thyroid is a rare butaggressive malignancy of the thyroid Herein we reported a case of ThyPSCC and itsultrasonography and pathologic findingsKeywords thyroid cancer thyroid nodules TNs thyroid ultrasound US primary squamous cell carcinomacontrast enhanced ultrasound CEUSINTRODUCTIONPrimary squamous cell carcinoma of the thyroid ThyPSCC is a rare thyroid malignancy withhigh aggressiveness and poor prognosis comprising ˆ¼“ of all primary thyroid carcinomas“ Owing to the rapidly progressing and highly invasive nature of the malignancy patients withThyPSCC often present at an advanced stage and are difficult to diagnose in the early stage becauseof its rare incidence and lack of typical imaging findings Thyroid ultrasonography and fineneedle aspiration biopsy FNAB are the diagnostic tools ofchoice for evaluating patients with suspected thyroid nodules Contrastenhanced ultrasoundCEUS as a relatively novel US technique is used to investigate the microvasculature of thyroidnodules and improve the diagnostic accuracy of thyroid nodules accompanied by the use of ThyroidEdited byChristoph ReinersUniversity HospitalW¼rzburg GermanyReviewed byPasqualino MalandrinoUniversity of Catania ItalyDaniela PasqualiUniversity of Campania LuigiVanvitelli ItalyCorrespondenceChengcheng NiuniuchengchengcsueducnSpecialty sectionThis was submitted toCancer Endocrinologya section of the journalFrontiers in EndocrinologyReceived February Accepted June Published August CitationChen S Peng Q Zhang Q and Niu C ContrastEnhanced Ultrasoundof Primary Squamous Cell Carcinomaof the Thyroid A Case ReportFront Endocrinol 103389fendo202000512Frontiers in Endocrinology wwwfrontiersinAugust Volume 0cChen et alThyroid Primary Squamous Cell CarcinomaImaging Reporting and Data Systems for ultrasonographicfeatures “ However very few published studies havereported the use of ultrasonography for ThyPSCC To ourknowledge this is the first case describing the CEUS findingsof ThyPSCCreached its peak [time to peak TTP] at s with a peakintensity of Then the nodule slowly declined until allthe microbubbles washed out at s Figures 1CD Based onits malignant conventional ultrasound features and the poormicrovasculature revealed by CEUS we inferred that the nodulewas a malignant tumorCASE REPORTA 59yearold male patient presented to our hospital withprogressively worsening hoarse voice symptoms for daysand was diagnosed with left unilateral vocal fold palsy Ahighresolution ultrasound instrument Siemens Acuson S3000Mountain View CA USA equipped with a to 9MHz linearprobe was used Thyroid ultrasonography revealed a solitary — — 26cm3 marked hypoechoic thyroid nodule with anunclear boundary in the inferior part of the left lobe AThis nodule exhibited many malignant ultrasound featuressuch as solid components hypoechogenicity and microlobulatedmargins Color Doppler flow imaging CDFI showed poorblood flow signals in the nodule B Contrastenhancedultrasound was performed with a bolus intravenous injectionof mL of SonoVue Bracco Milan Italy followed by mLof saline Contrast pulse sequencing technology was used andthe timeintensity curves TICs of the nodule were calculatedThe nodule began to be slowly enhanced from the peripheryto the center at s washin time and the enhancementAfterneckthepositronultrasonographyemissiontomography“computed tomography was carried for evaluatingthesituation of distant metastases Positron emissiontomography“computed tomography showed a mass withincreased glucose metabolism in the inferior part of the leftthyroid lobe A which indicated it as a malignantmass whereas there was no evidence of lymph nodes metastasisand distant metastases Then ultrasonographyguided FNABwas performed for the left thyroid mass immediately Cytologicexamination by fineneedle aspiration FNA revealed sheets oftumor cells with giant deepstained nuclei Bethesda categoryV B Finally a left hemithyroidectomy of the thyroidtumor was undertaken The lower edge of the tumor reachedthe upper mediastinum and the depth of the tumor invadedthe esophagus and trachea which could not be completelyremoved According to the eighth edition of the AmericanJoint Committee on CancerTumor Lymph Node MetastasisTNM staging system the patient was in TNM stage IIIT4a N0 M0 Histopathological examination of hematoxylinand eosin staining showed that a carcinoma in the inferiorFIGURE Ultrasonography images of primary squamous cell carcinoma of the thyroid A Longitudinal grayscale sonography revealed a solid marked hypoechoicthyroid nodule in the inferior part of the left lobe B Color Doppler flow imaging showed a poor blood flow signal inside this nodule C Contrastenhanced ultrasoundimage showed a persistent low peak enhancement of the nodule at s D Timeintensity curves displayed the washin time of s TTP of s peak signalintensity of and washout time of s for the thyroid tumorFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cChen et alThyroid Primary Squamous Cell CarcinomaFIGURE A A positron emission tomography“computed tomography scan showed increased 18Ffluorodeoxyglucose metabolism in the left neck mass BPreoperative fineneedle aspiration cytology of the mass demonstrated a few sheets of malignantlooking tumor cells with giant deep stained nuclei hematoxylin andeosin magnification — FIGURE Hematoxylin and eosin staining of primary squamous cell carcinoma of the thyroid A magnification — B magnification — C magnification — D magnification — part of the thyroid lobe A had no obvious palisadearrangementintercellular bridges or keratinization with acancer pearl Figures 3B“D Immunohistochemically tumorcells were positive for cytokeratin CK19 Acytokeratin and CK56 B epithelial membraneantigen EMA C p40 D p63 Aand Ki67 B and negative for thyroglobulinTG C and thyroid transcription factor TTF1D In view of these findings the tumor was diagnosedas poorly diï¬erentiated ThyPSCC Postoperatively the patientreceived two cycles of chemotherapy with docetaxelcisplatinintensitymodulated radiotherapy and nimotuzumabtargetedtherapy However the patient died months after surgeryDISCUSSIONPrimary squamous cell carcinoma of the thyroid is a thyroidmalignancy with extremely rare incidence and the clinicaldiagnosis and treatment guidelines for this disease have noconsensus The biological behavior of ThyPSCC is aggressiveFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cChen et alThyroid Primary Squamous Cell CarcinomaFIGURE Immunohistochemical staining of primary squamous cell carcinoma of the thyroid magnification — Immunohistochemical staining for A CK19 BCK56 C EMA D p40 all of which were deeply stained positiveFIGURE Immunohistochemical staining of primary squamous cell carcinoma of the thyroid magnification — Immunohistochemical staining for A p63 B Ki C TG D TTF1 and p63 was deeply stain positive Ki67 proliferation index was TG and TTF1 did not stain negativeFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cChen et alThyroid Primary Squamous Cell Carcinomaand the prognosis is poor with a median overall survival of “ months which depends on the diï¬erent tumor grades Yang et al using the Surveillance Epidemiology and End ResultsProgram database reported that poorly diï¬erentiated tumrade occupied the highest percentages of all graded tumors andthe median survival was months which is similar to the survivaltime in our case Highfrequency ultrasound as the basic imaging modality inthe diagnosis of thyroid nodules has found gradually increasingdiï¬erentiated thyroid cancers over recent years Theultrasonography imaging findings of ThyPSCC have seldombeen published Regarding the ultrasonography findings Chenet al reported that ThyPSCC presented as a thyroid masswith eggshell calcification peripheral soft tissue with a blurredmargin and minimal vascular signals on CDFI sonographyIn the case of Jang et al ThyPSCC presented as a largewelldefined lobulated heterogeneously hypoechoic mass withdiï¬use microcalcifications on ultrasonography Kondo et al reported that a welldiï¬erentiated ThyPSCC showed acystic hypoechoic mass with a smooth margin and rapidlygrew with margin change blurring in year In our case thispoorly diï¬erentiated ThyPSCC presented as a solitary markedhypoechoic thyroid mass with an irregular margin and unclearboundary with a normal thyroid The irregular margin andunclear boundary with normal thyroid corresponded to tumorinvasion with adjacent tissue infiltration which is consistentwith the findings during the operation that tumor invasion withthe esophagus cannot be completely removed Poor blood flowsignals on CDFI sonography and persistent hypoenhancement onCEUS of the mass are consistent with squamous cell carcinomawhich has no obvious vascularity on pathologic examinationMany studies have investigated the application of CEUS toimprove the diagnostic accuracy of thyroid nodules despiteits usage in ThyPSCC being scarce Zhang et al foundthat highcircularequal enhancement indicated benign thyroidnodules and low enhancement indicated malignant thyroidnodules Ma et al investigated whether incomplete noring or heterogeneous enhancement later washin time andlow peak intensity on CEUS were independent risk factorsin predicting malignantthyroid nodules Deng et al detected that papillary thyroid carcinomas PTCs exhibited lowenhancement a lower peak signal intensity and a lower areaunder the curve AUC than peripheral thyroid parenchyma onCEUS In our study the TICs of CEUS for ThyPSCCshowed a washin time of s a TTP of s a peak signalintensity as low as and a washout time of s Thisis similar to the results of PTCs with a slow washin time alower peak signal intensity and a lower AUC as in previousreports To our knowledge no reports on CEUS imagingfindings of ThyPSCC have appeared in the Englishlanguageliterature According to Jang et al ThyPSCC showed a largeheterogeneously enhancing thyroid mass with a large centralnonenhancing portion on enhanced CT which correspondedwell with the squamous cell carcinoma portion with a necroticportion in pathologic staining Because of the rapid growth ofsquamous tumor cells relatively few interstitial blood vessels intumors were related to the low peak signal intensity and low AUCon CEUSisusefulstainingWith increasing malignancy in squamous cell carcinoma thetypical squamous cell carcinoma findings of intercellular bridgesand keratinized cancer pearl can decrease or disappearImmunohistochemicalin diagnosingprimary thyroid cancer In this case positivity for CK56and EMA and negativity for TTF1 and TG expressionpredicted squamous cell carcinoma derivation and excludedthe possibility ofthese common tumors Furtherpositivity for p63 and Ki67 expression as poor prognosticmarkers was associated with its poorly diï¬erentiated tumrade CONCLUSIONPrimary squamous cell carcinoma of the thyroid is an extremelyrare tumor and very few studies describe its ultrasonographicimaging findings It is difficult to establish a clinical guidelinefor diagnosis Our case presents the CEUS features of ThyPSCCindicating that the TICs of ThyPSCC are similar to the enhancingparameters of PTCs with a slow washin time a lower peak signalintensity and a lower AUCDATA AVAILABILITY STATEMENTThe datasets generated for this study are available on request tothe corresponding authorETHICS STATEMENTThe studies involving human participants were reviewed andapproved by the Ethics Committee of Second Xiangya HospitalCentral South University China The patientsparticipantsprovided their written informed consentto participate inthis study Written informed consent was obtained from theindividuals for the publication of any potentially identifiableimages or data included in this AUTHOR CONTRIBUTIONSAll authors listed have made a substantial direct and intellectualcontribution to the work and approved it for publicationFUNDINGof ChinaThis project was funded by the National Natural ScienceFoundationProvincialNatural Science Foundation of China 2018JJ2575 andHunan Provincial Health Commission Research FoundationProject B2019166 HunanFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cChen et alREFERENCES Yang S Li C Shi X Ma B Xu W Jiang H et al Primary squamous cellcarcinoma in the thyroid gland a populationbased analysis using the SEERdatabase World J Surg “ 101007s00268019049062 Limberg J Ullmann TM Stefanova D Finnerty BM Beninato T Fahey TJet al Prognostic characteristics of primary squamous cell carcinoma of thethyroid a national cancer database analysis World J Surg “ 101007s00268019050985Thyroid Primary Squamous Cell Carcinomacontrastenhanced ultrasound Ultrasound Med Biol “ 1016jultrasmedbio201810020 Casella C Ministrini S Galani A Mastriale F Cappelli C Portolani NThe new TNM staging system for thyroid cancer and the risk of diseasedownstaging Front Endocrinol 103389fendo201800541 Zhang Y Zhou P Tian SM Zhao YF Li JL Li L Usefulness of combineduse of contrastenhanced ultrasound and TIRADS classification for thediï¬erentiation of benign from malignant lesions of thyroid nodules EurRadiol “ 101007s003300164508y Koyama S Fujiwara K Nosaka K Fukuhara T Morisaki T MiyakeN et al Immunohistochemical features of primary pure squamous cellcarcinoma in the thyroid an autopsy case Case Rep Oncol “ Zhang YZ Xu T Gong HY Li CY Ye XH Lin HJ et al Application ofhighresolution ultrasound realtime elastography and contrastenhancedultrasound in diï¬erentiating solid thyroid nodules Medicine95e5329 101097MD00000000000053290000579220161108000016 Wang SS Ye DX Wang B Xie C The expressions of keratins andP63 in primary squamous cell carcinoma ofthe thyroid gland anapplication of raman spectroscopy Onco Targets Ther “ 102147OTTS229436 Chen CY Tseng HS Lee CH Chan PW Primary squamous cellcarcinoma of the thyroid gland with eggshell calcification sonographicand computed tomographic findings J Ultrasound Med “ 107863jum201029111667 Yasumatsu R Sato M Uchi R Nakano T Hashimoto K Kogo R et al Thetreatment and outcome analysis of primary squamous cell carcinoma of thethyroid Auris Nasus Larynx “ 101016janl201707009 Kao NH Tan CS H Koh AJ The utility of immunohistochemistry indiï¬erentiating metastatic primary squamous cell carcinoma of the thyroidfrom a primary lung squamous cell carcinoma Case Rep Endocrinol “ Jang JY Kwon KW Kim SW Youn I Primary squamouscarcinoma ofandtomographic“ 1014366usg13022cellrecurrence ultrasonographicthyroid gland with localUltrasonographycomputedfindings Raggio B Barrcarcinomacell 1031486toj180002J Ghandour Z Friedlander P Primary squamousof“thyroid OchsnertheJ Kondo T Matsuyoshi A Matsuyoshi H Goto R Ono K Honda Y et alA case of primary thyroid squamous cell cancer transformation frombenign tumour associated with chronic thyroiditis BMJ Case Rep 2009bcr1020081137 101136bcr1020081137 Ma JJ Ding H Xu BH Xu C Song LJ Huang BJ et al Diagnosticand contrastmalignant101089thy201performances ofenhancedultrasonographythyroid nodules Thyroid“ color dopplergrayscalepredictingfindingsvariousin Deng J Zhou P Tian SM Zhang L Liofefficacydiagnosticofradiationdiï¬erentiating9e90674 101371journalpone0090674PONED1330329imagingthyroidandnodulescontrastenhancedimpulseforcesolidfocalJL Qian Y ComparisonacousticinuseultrasoundcombinedPLoS ONEtheir Haugen BR Alexander EK Bible KC Doherty GM Mandel SJ Nikiforov YEet al American thyroid association management guidelines for adultpatients with thyroid nodules and diï¬erentiated thyroid cancer the Americanthyroid association guidelines task force on thyroid nodules and diï¬erentiatedthyroid cancer Thyroid “ 101089thy20150020 Tessler FN Middleton WD Grant EG Hoang JK Berland LL Teefey SAet al ACR thyroid imaging reporting and data system TIRADS whitepaper of the ACR TIRADS committee J Am Coll Radiol “ 101016jjacr201701046 Kwak JY Han KH Yoon JH Moon HJ Son EJ Park SH et al Thyroidimaging reporting and data system for US features of nodules a step inestablishing better stratification of cancer risk Radiology “ 101148radiol11110206radiol11110206 Peng Q Niu C Zhang Q Zhang M Chen S Mummified thyroid nodulesconventional and contrastenhanced ultrasound features J Ultrasound Med “ 101002jum14712 Peng Q Niu C Zhang M Chen S Sonographic characteristics ofpapillary thyroid carcinoma with coexistent hashimoto™sthyroiditisconventional ultrasound acoustic radiation force impulse imaging and Struller F Senne M Falch C Kirschniak A Konigsrainer A Mullerthe thyroid case report andS Primary squamous cell carcinoma ofsystematic review of the literature Int J Surg Case Rep “ 101016jijscr201706011 Wang W Ouyang Q Meng C Jing L Li X Treatment optimization andprognostic considerations for primary squamous cell carcinoma of thethyroid Gland Surg “ 1021037gs20191107Conflict of Interest The authors declare that the research was conducted in theabsence of any commercial or financial relationships that could be construed as apotential conflict of interestCopyright Chen Peng Zhang and Niu This is an openaccess distributed under the terms of the Creative Commons Attribution License CC BYThe use distribution or reproduction in other forums is permitted provided theoriginal authors and the copyright owners are credited and that the originalpublication in this journal is cited in accordance with accepted academic practiceNo use distribution or reproduction is permitted which does not comply with thesetermsFrontiers in Endocrinology wwwfrontiersinAugust Volume 0c' Answer:

Thyroid_Cancer

Neurosteroids Biosynthesisand Physiological FunctionsShogo Haraguchi and Kazuyoshi Tsutsui Department of Biochemistry Showa University School of Medicine Tokyo Japan Graduate School of Integrated Sciencesfor Life Hiroshima University Hiroshima JapanSimilar to the adrenal glands gonads and placenta vertebrate brains also producevarious steroids which are known as œneurosteroids Neurosteroids are mainlysynthesized in the hippocampus hypothalamus and cerebellum however it has recentlybeen discovered that in birds the pineal gland a photosensitive region in the brainproduces more neurosteroids than other brain regions A series of experiments usingmolecular and biochemical techniques have found that the pineal gland producesvarious neurosteroids including sex steroids de novo from cholesterol For instanceallopregnanolone and 7αhydroxypregnenolone are actively produced in the pinealgland unlike in other brain regions Pineal 7αhydroxypregnenolone an upregulator oflocomotion enhances locomotor activity in response to light stimuli in birds Additionallypineal allopregnanolone acts on Purkinje cells in the cerebellum and prevents neuronalapoptosis within the developing cerebellum in juvenile birds Furthermore exposure tolight during nighttime hours can cause loss of diurnal variations of pineal allopregnanolonesynthesis during early posthatch life eventually leading to cerebellar Purkinje cell deathin juvenile birds In light of these new findings this review summarizes the biosynthesisand physiological functions of pineal neurosteroids Given that the circadian rhythms ofindividuals in modern societies are constantly interrupted by artificial light exposure thesefindings in birds which are excellent model diurnal animals may have direct implicationsfor addressing problems regarding the mental health and brain development of humansKeywords allopregnanolone 7αhydroxypregnenolone neurosteroid pineal gland cerebellum lightINTRODUCTIONSimilar to the gonads and placenta vertebrate brains actively also produce various steroidhormones These steroid hormones produced in the brain are named œneurosteroids Theproduction of neurosteroids was demonstrated firstly in mammals and then in other vertebrates“ Thus neurosteroid production appears to be a universal feature of the brain in vertebratesIt is known that neurosteroids are produced in glial cells and neurons of the centraland peripheral nervous systems However we have demonstrated thatthe pinealgland produces neurosteroids from cholesterol in birds during early posthatch period “Notably allopregnanolone also known as 3α5αtetrahydroprogesterone 3α5αTHP and 7αhydroxypregnenolone are the two major neurosteroids produced in the pineal gland Of thesetwo pineal allopregnanolone prevents the death of developing Purkinje cells and pineal 7αhydroxypregnenolone functions as an upregulator of locomotion regulating locomotor activity inresponse to light stimuli in birds Edited byVance L TrudeauUniversity of Ottawa CanadaReviewed byVincent M CassoneUniversity of Kentucky United StatesMaria Claudia Gonzalez DeniselleCONICET Instituto de Biolog­a yMedicina ExperimentalIBYME ArgentinaCorrespondenceShogo HaraguchishogoharaguchigmailcomSpecialty sectionThis was submitted toNeuroendocrine Sciencea section of the journalFrontiers in EndocrinologyReceived April Accepted July Published August CitationHaraguchi S and Tsutsui K Pineal Neurosteroids Biosynthesisand Physiological FunctionsFront Endocrinol 103389fendo202000549Frontiers in Endocrinology wwwfrontiersinAugust Volume 0cHaraguchi and TsutsuiPineal NeurosteroidsBIOSYNTHESIS OF PINEALNEUROSTEROIDSThe pineal glands of vertebrates respond to light stimuli andfulfillimportant functions in the anization of circadianrhythms The secretion of melatonin a major hormone producedby the pineal gland shows a clear daily rhythm with its peakconcentration occurring at night However it was notknown whether the pineal gland produces neurosteroids untilrecently We have recently demonstrated that the pineal gland isa newly found neurosteroidogenic an producing a variety ofneurosteroids from cholesterol αhydroxypregnenolone and allopregnanolone are activelyreleased Taken together these findings indicate that the pineal gland injuvenile birds produces various neurosteroids from cholesterolAccordingly this is the first demonstration of neurosteroidsynthesis in the pineal gland in a vertebratePHYSIOLOGICAL FUNCTION OF PINEAL7αHYDROXYPREGNENOLONE INLIGHTDEPENDENT LOCOMOTIONthatgene3hydroxysteroidPregnenolone is an anabolic intermediate of most endogenoussteroid hormones and is produced from cholesterol throughthe mitochondrial cholesterol side chain cleavage enzymecytochrome P450scc P450scc encoded by the Cyp11a geneWe have demonstrated by transcriptionpolymerase chainreaction RTPCR thatthe pineal gland in juvenile birdsexpresses P450scc mRNA Figure The proteinproduct of this mRNA is localized in the cells that form thefollicular structures in the pineal glands of birds We havedemonstrated by highperformance liquid chromatography3HHPLC with radioactive flow detector analysischolesterolis converted to radioactive pregnenolone whenincubated with pineal gland extract from juvenile birds This observation has confirmed the presence offunctionalP450scc in the pineal gland Figure which has also beendetected by gas chromatographymass spectrometry GCMS Subsequent RTPCR“based assessment has revealed thatkey steroidogenic enzymes cytochrome P450 7αhydroxylaseP4507α encoded by the Cyp7b gene 3αhydroxysteroiddehydrogenase 01 014isomerase 3αHSD encoded by thedehydrogenase 01 01Hsd3aisomerase 3HSD encoded by the Hsd3b gene 5αreductaseencoded by the Srd5a gene 5reductase encoded by theSrd5b gene cytochrome P450 17αhydroxylasec1720lyaseP45017αlyase encoded by the Cyp17 gene 17hydroxysteroiddehydrogenase 17HSD encoded by the Hsd17b gene andcytochrome P450 aromatase P450arom encoded by the Cyp19gene are expressed in the pineal gland of birds Figure We further demonstrated that steroid hormones are indeedpresent in the pineal gland Incubation of 3Hpregnenolonewith pineal glands from posthatch birds generates 7α andor7hydroxypregnenolone by the action of P4507α foundin the pineal glands Figure In addition to theseneurosteroid isomers progesterone allopregnanolone 3α 5αTHP andor epipregnanolone 3 5THP androstenedionetestosteroneandestradiol17 are also produced Figure These exvivo observations have confirmed that the pineal glands injuvenile birds have the biosynthetic machinery for majorsteroid hormones which have also been verified to be producedas neurosteroidsFigure Although HPLCanalysis has failed to resolve the isomers of these hormonesalloepipregnanolonesuch as 7αhydroxypregnenoloneand 5αdihydrotestosterone several sets ofisomers havebeen successfully isolated by GCMS analysis Especially5dihydrotestosteronein vivo andor5αanalysis hasactivation oftranscriptomicslightinduced transcriptionalfor studiesThe chick pineal gland is used as a modeltheon the lightdependent phaseshifting mechanism ofcircadian clock To search for genes involved in thisa diï¬erential GeneChip analysis has beenmechanismidentifiedperformed Thisthethefullset of genes in the pineal gland involved in cholesterolbiosynthesis When the pineal gland was exposed tolightit produced cholesterol and 7αhydroxypregnenoloneex vivo Interestingly this lightinduced production of 7αhydroxypregnenolone occurred only when the gland wasexposed to light at early night but not atlate night orduring the daytime During early night time the circadianclock is sensitive to light which causes phasedelay of theclock Thusthe lightsensitive pineal production of7αhydroxypregnenolone appearsto be regulated by thecircadian clockactivateslocomotorIn vertebratesan intracerebroventricularinjection of7αhydroxypregnenoloneactivities“ Thusthe intracerebroventricular injection of 7αhydroxypregnenolone was administered in a dosedependentmanner at early night in chicks After the injection chickswere placed individually for locomotor activity measurementin an open field apparatus for min Spontaneous locomotoractivities of chicks were stimulated by the intracerebroventricularinjection of 7αhydroxypregnenolone in a dosedependentmanner Furthermore when chicks are exposed to lightduring early nightreachthe daytime level These results suggest that pineal 7αhydroxypregnenolone reaches the target sites within the brain byvolume transmission upon light exposure at early nightlocomotor activitiestimetheirPHYSIOLOGICAL FUNCTION OF PINEALALLOPREGNANOLONE IN PURKINJECELL SURVIVAL DURING DEVELOPMENT7αHydroxypregnenolone and allopregnanolone are activelyreleased during early posthatch period compared with adulthood Therefore 7αhydroxypregnenolone and allopregnanolonemay play key roles in birds during early posthatch periodIn vertebrates pinealectomy decreases cell number in thedeveloping brain These findings suggest that these majorneurosteroids secreted from the pineal gland are involved in thedevelopment of brain cellsFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cHaraguchi and TsutsuiPineal NeurosteroidsFIGURE Biosynthetic pathways of pineal neurosteroids Allopregnanolone and 7αhydroxypregnenolone are the major neurosteroids produced in the pineal glandof birds P450scc cytochrome P450 sidechain cleavage enzyme P4507α cytochrome P450 7αhydroxylase 3HSD 3hydroxysteroiddehydrogenase 01 014isomerase 3αHSD 3αhydroxysteroid dehydrogenase 01 014isomerase 5αreductase 5reductase P45017αlyase cytochrome P45017αhydroxylasec1720lyase 17HSD 17hydroxysteroid dehydrogenase and P450arom cytochrome P450 aromataseFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cHaraguchi and TsutsuiPineal Neurosteroidstheincreasessupplementation oftheIn chicks pinealectomy decreases the concentration ofallopregnanolone and the number of cerebellar Purkinjeallopregnanolonecells whereasto pinealectomized birdsconcentration ofallopregnanolone and recovers the number of Purkinje cells Thus pineal allopregnanolone is considered to be an essentialfactor for the normal development of cerebellar Purkinje cellsIt thus appears that pineal allopregnanolone functions as anessential factor for Purkinje cells during posthatch periodIn addition pinealectomy in juvenile birds increases theexpression of active caspase3 in Purkinje cells whereasallopregnanolone supplementation decreases the expressionof active caspase3 during posthatch period Thus theneuroprotective action of pineal allopregnanolone on cerebellarPurkinje cells is exerted by suppressing the activation of caspase3Figure Allopregnanolone acts mainly as a ligand ofthe γaminobutyric acid type A GABAA receptor and may alsoact as an agonist of the membrane progesterone receptors αmPRα as well as the mPR and mPRγ “ Thereforeeither mPR siRNA orisoallopregnanolone an antagonistof allopregnanolone was delivered into the cerebellum ofposthatched chicks It was found that the silencing of mPRαincreases the number of Purkinje cells that express active caspase in the cerebellum of chicks Furthermore to uncoverthe mechanism of neuroprotective action of allopregnanoloneFIGURE A schematic model of the effect of pineal allopregnanolone on Purkinje cell survival immediately after hatching under a h lightdark cycle or with hlight exposure during the dark period lightatnight condition Left panel The normal cerebellar development under a h lightdark cycle during the first weekafter hatching Pineal allopregnanolone induces the expression of pituitary adenylate cyclaseactivating polypeptide PACAP a neuroprotective factor through themembrane progestin receptor α mPRα receptor binding mechanism in Purkinje cells Subsequently PACAP inhibits the activation of caspase3 that facilitates theapoptosis of cerebellar Purkinje cells Right panel The abnormal cerebellar development under the lightatnight condition during the first week after hatching Thelightatnight condition disrupts the diurnal rhythm in pineal allopregnanolone synthesis Decreased pineal allopregnanolone synthesis leads to decreased expressionof PACAP in Purkinje cells Consequently the active caspase3 level increases inducing the apoptosis of Purkinje cells in the cerebellumFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cHaraguchi and TsutsuiPineal Neurosteroidsin cerebellar Purkinje cells allopregnanolone action on theexpression of neuroprotectiveneurotoxic factors “ hasbeen investigated Pinealectomy decreases the mRNA levels ofpituitary adenylate cyclaseactivating polypeptide PACAP aneuroprotective factor in the cerebellum of juvenile birds It has been found that a daily injection of allopregnanolone inpinealectomized juvenile birds upregulates PACAP relative tothe levels in control birds These findings show that PACAPmediates the neuroprotective action of pineal allopregnanolonethrough mPRα receptor binding during cerebellar developmentFigure LIGHTATNIGHT AFFECTS THEDEVELOPMENT OF CEREBELLUMTHROUGH A MECHANISM MEDIATED BYPINEAL ALLOPREGNANOLONE ACTIONIt is known that environmental stimuli aï¬ect the developmentof animals including humans In vertebrate brain development anatural lightdark cycle promotes better brain development thanconstant conditions such as constant light or constant darkness“ Howeverthe molecular mechanisms that controlhow environmental light conditions aï¬ect brain developmentremain unclear The pineal gland is a photosensitive anTo investigate whether light conditions are involved in thesynthesis of allopregnanolone in the pineal gland the birdshave been incubated under either a h lightdark LDcycle or LD cycle with h light exposure during the darkperiod lightatnight Consequently it has been found that theallopregnanolone concentration and synthesis during the darkperiod are higher in the pineal glands of LD birds than in thoseof lightatnight birds Figure Furthermore the numberof cerebellar Purkinje cells is decreased by the lightatnightcondition Figure It is therefore considered that pinealallopregnanolone is a critical metabolite that aï¬ects cerebellardevelopment in vertebrates depending on the environmentallight conditionsREFERENCES Baulieu EE Neurosteroids of the nervous system by the nervous system forthe nervous system Rec Prog Hormone Res “ Tsutsui K Ukena K Takase M Kohchi C Lea RW Neurosteroidbiosynthesis in vertebrate brains Comp Biochem Physiol C “ 101016S0742841399000651 Compagnone NA Mellon SH Neurosteroids biosynthesis and functionthese novel neuromodulators Front Neuroendocrinol “of 101006frne19990188CONCLUSIONSthedatarecentreview summarizedThison pinealneurosteroids Studies have indicated that the pineal glandproduces neurosteroids from cholesterol in birds Pineal 7αhydroxypregnenolone regulates locomotion in response to lightstimuli in birds Pineal allopregnanolone prevents the death ofdeveloping Purkinje cells by suppressing neuronal apoptosisduring development In addition circadian disruption by lightexposure during nighttime leads to cell death of developingPurkinje cellsthrough pineal allopregnanolonedependentmechanisms in juvenile birds These observations suggest thatnighttime artificial light exposure in modern societies may alsoperturb the development of the human brainAlmost all animals have circadian rhythms However modernlife conditions chronically disrupt circadian rhythm throughartificial light exposure The disruption of circadian rhythm isassociated with a decline in mental and physical health “The most potent circadian rhythm disruption is inappropriatelytimed bright light exposure eg lightatnight To investigatethe eï¬ects of chronic circadian disruption in modern societieson mental and physical health which is efficiently modeled bythe lightatnight condition presented here many studies havebeen conducted on mice However it is important for us tobear in mind that laboratory mice are mainly nocturnal animalswhereas humans are diurnal Thus birds are excellent animalmodels to uncover the eï¬ect of lightatnight on diurnal animalsincluding humansAUTHOR CONTRIBUTIONSSH and KT wrote the manuscript All authors contributed to the and approved the submitted versionFUNDINGThis work wasJSPS GrantsinAid forScientific Research KAKENHI Grant Numbers JP15K18571and JP19K09033supported bysterol regulatory elementbinding protein Xboxbinding protein andheat shock factor pathways Proc Natl Acad Sci USA “ 101073pnas1015959108 Haraguchi S Hara S Ubuka T Mita M Tsutsui K Possible role of pinealallopregnanolone in Purkinje cell survival Proc Natl Acad Sci USA “ 101073pnas1210804109al Lightatnight Haraguchi S Kamata M Tokita T Tashiro KI Sato M Nozaki Mthroughaï¬ects brain developmenteLifeetallopregnanolonedependentpineal8e45306 107554eLife45306037mechanismsexposure Tsutsui K Matsunaga M Miyabara H Ukena K Neurosteroid biosynthesis in Reiter RJ Pineal melatonin cell biology of its synthesis and of its physiologicalthe quail brain J Exp Zool 305A733“ 101002jeza302interactions Endocr Rev “ 101210edrv122151 DoRego JL Seong JY Burel D Leprince J LuuThe V Tsutsui Ket al Neurosteroid biosynthesis enzymatic pathways and neuroendocrineregulation by neurotransmitters and neuropeptides Front Neuroendocrinol “ 101016jyfrne200905006 Hatori M Hirota T Iitsuka M Kurabayashi N Haraguchi S KokameK et al Lightdependent and circadian clockregulated activation of Fukada Y Okano T Circadian clock system in the pineal gland Mol Neurobiol “ 101385MN251019 Matsunaga M Ukena K Baulieu EE Tsutsui K 7αHydroxypregnenoloneacts as a neuronal activator to stimulate locomotor activity of breeding newtsby means of the dopaminergic system Proc Natl Acad Sci USA “ 101073pnas0407176101Frontiers in Endocrinology wwwfrontiersinAugust Volume 0cHaraguchi and TsutsuiPineal Neurosteroids Tsutsui KInoue K Miyabara H Suzuki S Ogura Y Haraguchi7αHydroxypregnenolone mediates melatonin action underlying“Sdiurnal 101523JNEUROSCI3562072008locomotorNeuroscirhythmsJ Haraguchi S Koyama T Hasunuma I Vaudry H Tsutsui K Prolactin increasesthe synthesis of 7αhydroxypregnenolone a key factor for induction oflocomotor activity in breeding male newts Endocrinology “ 101210en20091229 Haraguchi S Koyama T Hasunuma I Okuyama S Ubuka T Kikuyama S et alAcute stress increases the synthesis of 7αhydroxypregnenolone a new keyneurosteroid stimulating locomotor activity through corticosterone action innewts Endocrinology “ 101210en20111422 Haraguchi S Yamamoto Y Suzuki Y Hyung Chang J Koyama T Sato Met al 7αHydroxypregnenolone a key neuronal modulator of locomotionstimulates upstream migration by means of the dopaminergic system insalmon Sci Rep 101038srep12546 Fillenz M Volume transmission in the brain Novel mechanisms for neuronaltransmission In Fuxe K Agnati LF editors Advances in Neurosciences Vol New York NY Raven Press p “ Kilic E Hermann DM Isenmann S B¤hr M Eï¬ects of pinealectomy andmelatonin on the retrograde degeneration of retinal ganglion cells in a novelmodel of intraorbital optic nerve transection in mice J Pineal Res “ 101034j1600079x20021823x Tun§ AT Turgut M Aslan H Sahin B Yurtseven ME KaplantheS Neonatalcerebellum of“ 101016jbrainres200510011pinealectomythechick a stereologicalstudy Brain ResPurkinjeinduceslosscellin Pang Y Dong J Thomas P Characterization neurosteroid binding and braindistribution of human membrane progesterone receptors δ and ε mPRδand mPRε and mPRδ involvement in neurosteroid inhibition of apoptosisEndocrinology “ 101210en20121772 Schumacher M Mattern C Ghoumari A Oudinet JP Liere P LabombardaF et al Revisiting the roles of progesterone and allopregnanolone in thenervous system resurgence of the progesterone receptors Prog Neurobiol “ 101016jpneurobio201309004 Belelli D Lambert JJ Neurosteroids endogenous regulators of the GABAAreceptor Nat Rev Neurosci “ 101038nrn1703 Bernal J Thyroid hormone receptors in brain development and function NatClin Pract Endocrinol Metabol “ 101038ncpendmet0424 FalluelMorel A Vaudry D Aubert N Galas L Benard M Basille M et alPituitary adenylate cyclaseactivating polypeptide prevents the eï¬ects ofceramides on migration neurite outgrowth and cytoskeleton remodelingProc Natl Acad Sci USA “ 101073pnas04096 Koibuchi N Chin WW ThyroidEndocrinoldevelopment 101016S1043276000002381TrendshormoneMetabactionandbrain“ Vaudry D FalluelMorel A Leuillet S Vaudry H Gonzalez BJ Regulators ofcerebellar granule cell development act through specific signaling pathwaysScience “ 101126science1085260 Sasahara K Shikimi H Haraguchi S Sakamoto H Honda S Harada N et alMode of action and functional significance of estrogeninducing dendriticgrowth Spinogenesis and synaptogenesis in the developing Purkinje cell JNeurosci “ 101523JNEUROSCI0710072007 Bakkum BW Benevento LA Cohen RS Eï¬ects of lightdark and darkrearing on synaptic morphology in the superior colliculus and visualcortex ofJ Neurosci Res “ 101002jnr490280107the postnatal and adult rat Brooks E Waters E Farrington L Canal MM Diï¬erential hypothalamictyrosine hydroxylase distribution and activation by light in adult mice rearedunder diï¬erent light conditions during the suckling period Brain Struct Funct “ 101007s0042901103189 DulcisDSpitzerNCIlluminationcontrolsdopamineof“ 101038nature07569neuronsregulatingbehaviourdiï¬erentiationNature Li Y Komuro Y Fahrion JK Hu T Ohno N Fenner KB et al Lightstimuli control neuronal migration by altering ofinsulinlike growthfactor IGF1 signaling Proc Natl Acad Sci USA “ 101073pnas1111326109 Ohta H Mitchell AC McMahon DG ConstantPediatrthe“ 10120301pdr00002331141840366developing mousebiologicalclocklight disruptsRes Kantermann T Roenneberg TIsfactor or a health risk predictor Chronobiol lightatnightInthealthriska “ Wu J Dauchy RT Tirrell PC Wu SS Lynch DT Jitawatanarat P et alLight at night activates IGF1RPDK1 signaling and accelerates tumrowth in human breast cancer xenografts Cancer Res “ 10115800085472CAN103837 Smarr BL Grant AD Perez L Zucker I Kriegsfeld LJ Maternal andearlylife circadian disruption have longlasting negative consequenceson oï¬spring development and adult behavior in mice Sci Rep 101038s41598017034064Conflict of Interest The authors declare that the research was conducted in theabsence of any commercial or financial relationships that could be construed as apotential conflict of interestCopyright Haraguchi and Tsutsui This is an openaccess distributedunder the terms of the Creative Commons Attribution License CC BY The usedistribution or reproduction in other forums is permitted provided the originalauthors and the copyright owners are credited and that the original publicationin this journal is cited in accordance with accepted academic practice No usedistribution or reproduction is permitted which does not comply with these termsFrontiers in Endocrinology wwwfrontiersinAugust Volume 0c'

cancer202

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: Neurosteroids Biosynthesisand Physiological FunctionsShogo Haraguchi and Kazuyoshi Tsutsui Department of Biochemistry Showa University School of Medicine Tokyo Japan Graduate School of Integrated Sciencesfor Life Hiroshima University Hiroshima JapanSimilar to the adrenal glands gonads and placenta vertebrate brains also producevarious steroids which are known as œneurosteroids Neurosteroids are mainlysynthesized in the hippocampus hypothalamus and cerebellum however it has recentlybeen discovered that in birds the pineal gland a photosensitive region in the brainproduces more neurosteroids than other brain regions A series of experiments usingmolecular and biochemical techniques have found that the pineal gland producesvarious neurosteroids including sex steroids de novo from cholesterol For instanceallopregnanolone and 7αhydroxypregnenolone are actively produced in the pinealgland unlike in other brain regions Pineal 7αhydroxypregnenolone an upregulator oflocomotion enhances locomotor activity in response to light stimuli in birds Additionallypineal allopregnanolone acts on Purkinje cells in the cerebellum and prevents neuronalapoptosis within the developing cerebellum in juvenile birds Furthermore exposure tolight during nighttime hours can cause loss of diurnal variations of pineal allopregnanolonesynthesis during early posthatch life eventually leading to cerebellar Purkinje cell deathin juvenile birds In light of these new findings this review summarizes the biosynthesisand physiological functions of pineal neurosteroids Given that the circadian rhythms ofindividuals in modern societies are constantly interrupted by artificial light exposure thesefindings in birds which are excellent model diurnal animals may have direct implicationsfor addressing problems regarding the mental health and brain development of humansKeywords allopregnanolone 7αhydroxypregnenolone neurosteroid pineal gland cerebellum lightINTRODUCTIONSimilar to the gonads and placenta vertebrate brains actively also produce various steroidhormones These steroid hormones produced in the brain are named œneurosteroids Theproduction of neurosteroids was demonstrated firstly in mammals and then in other vertebrates“ Thus neurosteroid production appears to be a universal feature of the brain in vertebratesIt is known that neurosteroids are produced in glial cells and neurons of the centraland peripheral nervous systems However we have demonstrated thatthe pinealgland produces neurosteroids from cholesterol in birds during early posthatch period “Notably allopregnanolone also known as 3α5αtetrahydroprogesterone 3α5αTHP and 7αhydroxypregnenolone are the two major neurosteroids produced in the pineal gland Of thesetwo pineal allopregnanolone prevents the death of developing Purkinje cells and pineal 7αhydroxypregnenolone functions as an upregulator of locomotion regulating locomotor activity inresponse to light stimuli in birds Edited byVance L TrudeauUniversity of Ottawa CanadaReviewed byVincent M CassoneUniversity of Kentucky United StatesMaria Claudia Gonzalez DeniselleCONICET Instituto de Biolog­a yMedicina ExperimentalIBYME ArgentinaCorrespondenceShogo HaraguchishogoharaguchigmailcomSpecialty sectionThis was submitted toNeuroendocrine Sciencea section of the journalFrontiers in EndocrinologyReceived April Accepted July Published August CitationHaraguchi S and Tsutsui K Pineal Neurosteroids Biosynthesisand Physiological FunctionsFront Endocrinol 103389fendo202000549Frontiers in Endocrinology wwwfrontiersinAugust Volume 0cHaraguchi and TsutsuiPineal NeurosteroidsBIOSYNTHESIS OF PINEALNEUROSTEROIDSThe pineal glands of vertebrates respond to light stimuli andfulfillimportant functions in the anization of circadianrhythms The secretion of melatonin a major hormone producedby the pineal gland shows a clear daily rhythm with its peakconcentration occurring at night However it was notknown whether the pineal gland produces neurosteroids untilrecently We have recently demonstrated that the pineal gland isa newly found neurosteroidogenic an producing a variety ofneurosteroids from cholesterol αhydroxypregnenolone and allopregnanolone are activelyreleased Taken together these findings indicate that the pineal gland injuvenile birds produces various neurosteroids from cholesterolAccordingly this is the first demonstration of neurosteroidsynthesis in the pineal gland in a vertebratePHYSIOLOGICAL FUNCTION OF PINEAL7αHYDROXYPREGNENOLONE INLIGHTDEPENDENT LOCOMOTIONthatgene3hydroxysteroidPregnenolone is an anabolic intermediate of most endogenoussteroid hormones and is produced from cholesterol throughthe mitochondrial cholesterol side chain cleavage enzymecytochrome P450scc P450scc encoded by the Cyp11a geneWe have demonstrated by transcriptionpolymerase chainreaction RTPCR thatthe pineal gland in juvenile birdsexpresses P450scc mRNA Figure The proteinproduct of this mRNA is localized in the cells that form thefollicular structures in the pineal glands of birds We havedemonstrated by highperformance liquid chromatography3HHPLC with radioactive flow detector analysischolesterolis converted to radioactive pregnenolone whenincubated with pineal gland extract from juvenile birds This observation has confirmed the presence offunctionalP450scc in the pineal gland Figure which has also beendetected by gas chromatographymass spectrometry GCMS Subsequent RTPCR“based assessment has revealed thatkey steroidogenic enzymes cytochrome P450 7αhydroxylaseP4507α encoded by the Cyp7b gene 3αhydroxysteroiddehydrogenase 01 014isomerase 3αHSD encoded by thedehydrogenase 01 01Hsd3aisomerase 3HSD encoded by the Hsd3b gene 5αreductaseencoded by the Srd5a gene 5reductase encoded by theSrd5b gene cytochrome P450 17αhydroxylasec1720lyaseP45017αlyase encoded by the Cyp17 gene 17hydroxysteroiddehydrogenase 17HSD encoded by the Hsd17b gene andcytochrome P450 aromatase P450arom encoded by the Cyp19gene are expressed in the pineal gland of birds Figure We further demonstrated that steroid hormones are indeedpresent in the pineal gland Incubation of 3Hpregnenolonewith pineal glands from posthatch birds generates 7α andor7hydroxypregnenolone by the action of P4507α foundin the pineal glands Figure In addition to theseneurosteroid isomers progesterone allopregnanolone 3α 5αTHP andor epipregnanolone 3 5THP androstenedionetestosteroneandestradiol17 are also produced Figure These exvivo observations have confirmed that the pineal glands injuvenile birds have the biosynthetic machinery for majorsteroid hormones which have also been verified to be producedas neurosteroidsFigure Although HPLCanalysis has failed to resolve the isomers of these hormonesalloepipregnanolonesuch as 7αhydroxypregnenoloneand 5αdihydrotestosterone several sets ofisomers havebeen successfully isolated by GCMS analysis Especially5dihydrotestosteronein vivo andor5αanalysis hasactivation oftranscriptomicslightinduced transcriptionalfor studiesThe chick pineal gland is used as a modeltheon the lightdependent phaseshifting mechanism ofcircadian clock To search for genes involved in thisa diï¬erential GeneChip analysis has beenmechanismidentifiedperformed Thisthethefullset of genes in the pineal gland involved in cholesterolbiosynthesis When the pineal gland was exposed tolightit produced cholesterol and 7αhydroxypregnenoloneex vivo Interestingly this lightinduced production of 7αhydroxypregnenolone occurred only when the gland wasexposed to light at early night but not atlate night orduring the daytime During early night time the circadianclock is sensitive to light which causes phasedelay of theclock Thusthe lightsensitive pineal production of7αhydroxypregnenolone appearsto be regulated by thecircadian clockactivateslocomotorIn vertebratesan intracerebroventricularinjection of7αhydroxypregnenoloneactivities“ Thusthe intracerebroventricular injection of 7αhydroxypregnenolone was administered in a dosedependentmanner at early night in chicks After the injection chickswere placed individually for locomotor activity measurementin an open field apparatus for min Spontaneous locomotoractivities of chicks were stimulated by the intracerebroventricularinjection of 7αhydroxypregnenolone in a dosedependentmanner Furthermore when chicks are exposed to lightduring early nightreachthe daytime level These results suggest that pineal 7αhydroxypregnenolone reaches the target sites within the brain byvolume transmission upon light exposure at early nightlocomotor activitiestimetheirPHYSIOLOGICAL FUNCTION OF PINEALALLOPREGNANOLONE IN PURKINJECELL SURVIVAL DURING DEVELOPMENT7αHydroxypregnenolone and allopregnanolone are activelyreleased during early posthatch period compared with adulthood Therefore 7αhydroxypregnenolone and allopregnanolonemay play key roles in birds during early posthatch periodIn vertebrates pinealectomy decreases cell number in thedeveloping brain These findings suggest that these majorneurosteroids secreted from the pineal gland are involved in thedevelopment of brain cellsFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cHaraguchi and TsutsuiPineal NeurosteroidsFIGURE Biosynthetic pathways of pineal neurosteroids Allopregnanolone and 7αhydroxypregnenolone are the major neurosteroids produced in the pineal glandof birds P450scc cytochrome P450 sidechain cleavage enzyme P4507α cytochrome P450 7αhydroxylase 3HSD 3hydroxysteroiddehydrogenase 01 014isomerase 3αHSD 3αhydroxysteroid dehydrogenase 01 014isomerase 5αreductase 5reductase P45017αlyase cytochrome P45017αhydroxylasec1720lyase 17HSD 17hydroxysteroid dehydrogenase and P450arom cytochrome P450 aromataseFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cHaraguchi and TsutsuiPineal Neurosteroidstheincreasessupplementation oftheIn chicks pinealectomy decreases the concentration ofallopregnanolone and the number of cerebellar Purkinjeallopregnanolonecells whereasto pinealectomized birdsconcentration ofallopregnanolone and recovers the number of Purkinje cells Thus pineal allopregnanolone is considered to be an essentialfactor for the normal development of cerebellar Purkinje cellsIt thus appears that pineal allopregnanolone functions as anessential factor for Purkinje cells during posthatch periodIn addition pinealectomy in juvenile birds increases theexpression of active caspase3 in Purkinje cells whereasallopregnanolone supplementation decreases the expressionof active caspase3 during posthatch period Thus theneuroprotective action of pineal allopregnanolone on cerebellarPurkinje cells is exerted by suppressing the activation of caspase3Figure Allopregnanolone acts mainly as a ligand ofthe γaminobutyric acid type A GABAA receptor and may alsoact as an agonist of the membrane progesterone receptors αmPRα as well as the mPR and mPRγ “ Thereforeeither mPR siRNA orisoallopregnanolone an antagonistof allopregnanolone was delivered into the cerebellum ofposthatched chicks It was found that the silencing of mPRαincreases the number of Purkinje cells that express active caspase in the cerebellum of chicks Furthermore to uncoverthe mechanism of neuroprotective action of allopregnanoloneFIGURE A schematic model of the effect of pineal allopregnanolone on Purkinje cell survival immediately after hatching under a h lightdark cycle or with hlight exposure during the dark period lightatnight condition Left panel The normal cerebellar development under a h lightdark cycle during the first weekafter hatching Pineal allopregnanolone induces the expression of pituitary adenylate cyclaseactivating polypeptide PACAP a neuroprotective factor through themembrane progestin receptor α mPRα receptor binding mechanism in Purkinje cells Subsequently PACAP inhibits the activation of caspase3 that facilitates theapoptosis of cerebellar Purkinje cells Right panel The abnormal cerebellar development under the lightatnight condition during the first week after hatching Thelightatnight condition disrupts the diurnal rhythm in pineal allopregnanolone synthesis Decreased pineal allopregnanolone synthesis leads to decreased expressionof PACAP in Purkinje cells Consequently the active caspase3 level increases inducing the apoptosis of Purkinje cells in the cerebellumFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cHaraguchi and TsutsuiPineal Neurosteroidsin cerebellar Purkinje cells allopregnanolone action on theexpression of neuroprotectiveneurotoxic factors “ hasbeen investigated Pinealectomy decreases the mRNA levels ofpituitary adenylate cyclaseactivating polypeptide PACAP aneuroprotective factor in the cerebellum of juvenile birds It has been found that a daily injection of allopregnanolone inpinealectomized juvenile birds upregulates PACAP relative tothe levels in control birds These findings show that PACAPmediates the neuroprotective action of pineal allopregnanolonethrough mPRα receptor binding during cerebellar developmentFigure LIGHTATNIGHT AFFECTS THEDEVELOPMENT OF CEREBELLUMTHROUGH A MECHANISM MEDIATED BYPINEAL ALLOPREGNANOLONE ACTIONIt is known that environmental stimuli aï¬ect the developmentof animals including humans In vertebrate brain development anatural lightdark cycle promotes better brain development thanconstant conditions such as constant light or constant darkness“ Howeverthe molecular mechanisms that controlhow environmental light conditions aï¬ect brain developmentremain unclear The pineal gland is a photosensitive anTo investigate whether light conditions are involved in thesynthesis of allopregnanolone in the pineal gland the birdshave been incubated under either a h lightdark LDcycle or LD cycle with h light exposure during the darkperiod lightatnight Consequently it has been found that theallopregnanolone concentration and synthesis during the darkperiod are higher in the pineal glands of LD birds than in thoseof lightatnight birds Figure Furthermore the numberof cerebellar Purkinje cells is decreased by the lightatnightcondition Figure It is therefore considered that pinealallopregnanolone is a critical metabolite that aï¬ects cerebellardevelopment in vertebrates depending on the environmentallight conditionsREFERENCES Baulieu EE Neurosteroids of the nervous system by the nervous system forthe nervous system Rec Prog Hormone Res “ Tsutsui K Ukena K Takase M Kohchi C Lea RW Neurosteroidbiosynthesis in vertebrate brains Comp Biochem Physiol C “ 101016S0742841399000651 Compagnone NA Mellon SH Neurosteroids biosynthesis and functionthese novel neuromodulators Front Neuroendocrinol “of 101006frne19990188CONCLUSIONSthedatarecentreview summarizedThison pinealneurosteroids Studies have indicated that the pineal glandproduces neurosteroids from cholesterol in birds Pineal 7αhydroxypregnenolone regulates locomotion in response to lightstimuli in birds Pineal allopregnanolone prevents the death ofdeveloping Purkinje cells by suppressing neuronal apoptosisduring development In addition circadian disruption by lightexposure during nighttime leads to cell death of developingPurkinje cellsthrough pineal allopregnanolonedependentmechanisms in juvenile birds These observations suggest thatnighttime artificial light exposure in modern societies may alsoperturb the development of the human brainAlmost all animals have circadian rhythms However modernlife conditions chronically disrupt circadian rhythm throughartificial light exposure The disruption of circadian rhythm isassociated with a decline in mental and physical health “The most potent circadian rhythm disruption is inappropriatelytimed bright light exposure eg lightatnight To investigatethe eï¬ects of chronic circadian disruption in modern societieson mental and physical health which is efficiently modeled bythe lightatnight condition presented here many studies havebeen conducted on mice However it is important for us tobear in mind that laboratory mice are mainly nocturnal animalswhereas humans are diurnal Thus birds are excellent animalmodels to uncover the eï¬ect of lightatnight on diurnal animalsincluding humansAUTHOR CONTRIBUTIONSSH and KT wrote the manuscript All authors contributed to the and approved the submitted versionFUNDINGThis work wasJSPS GrantsinAid forScientific Research KAKENHI Grant Numbers JP15K18571and JP19K09033supported bysterol regulatory elementbinding protein Xboxbinding protein andheat shock factor pathways Proc Natl Acad Sci USA “ 101073pnas1015959108 Haraguchi S Hara S Ubuka T Mita M Tsutsui K Possible role of pinealallopregnanolone in Purkinje cell survival Proc Natl Acad Sci USA “ 101073pnas1210804109al Lightatnight Haraguchi S Kamata M Tokita T Tashiro KI Sato M Nozaki Mthroughaï¬ects brain developmenteLifeetallopregnanolonedependentpineal8e45306 107554eLife45306037mechanismsexposure Tsutsui K Matsunaga M Miyabara H Ukena K Neurosteroid biosynthesis in Reiter RJ Pineal melatonin cell biology of its synthesis and of its physiologicalthe quail brain J Exp Zool 305A733“ 101002jeza302interactions Endocr Rev “ 101210edrv122151 DoRego JL Seong JY Burel D Leprince J LuuThe V Tsutsui Ket al Neurosteroid biosynthesis enzymatic pathways and neuroendocrineregulation by neurotransmitters and neuropeptides Front Neuroendocrinol “ 101016jyfrne200905006 Hatori M Hirota T Iitsuka M Kurabayashi N Haraguchi S KokameK et al Lightdependent and circadian clockregulated activation of Fukada Y Okano T Circadian clock system in the pineal gland Mol Neurobiol “ 101385MN251019 Matsunaga M Ukena K Baulieu EE Tsutsui K 7αHydroxypregnenoloneacts as a neuronal activator to stimulate locomotor activity of breeding newtsby means of the dopaminergic system Proc Natl Acad Sci USA “ 101073pnas0407176101Frontiers in Endocrinology wwwfrontiersinAugust Volume 0cHaraguchi and TsutsuiPineal Neurosteroids Tsutsui KInoue K Miyabara H Suzuki S Ogura Y Haraguchi7αHydroxypregnenolone mediates melatonin action underlying“Sdiurnal 101523JNEUROSCI3562072008locomotorNeuroscirhythmsJ Haraguchi S Koyama T Hasunuma I Vaudry H Tsutsui K Prolactin increasesthe synthesis of 7αhydroxypregnenolone a key factor for induction oflocomotor activity in breeding male newts Endocrinology “ 101210en20091229 Haraguchi S Koyama T Hasunuma I Okuyama S Ubuka T Kikuyama S et alAcute stress increases the synthesis of 7αhydroxypregnenolone a new keyneurosteroid stimulating locomotor activity through corticosterone action innewts Endocrinology “ 101210en20111422 Haraguchi S Yamamoto Y Suzuki Y Hyung Chang J Koyama T Sato Met al 7αHydroxypregnenolone a key neuronal modulator of locomotionstimulates upstream migration by means of the dopaminergic system insalmon Sci Rep 101038srep12546 Fillenz M Volume transmission in the brain Novel mechanisms for neuronaltransmission In Fuxe K Agnati LF editors Advances in Neurosciences Vol New York NY Raven Press p “ Kilic E Hermann DM Isenmann S B¤hr M Eï¬ects of pinealectomy andmelatonin on the retrograde degeneration of retinal ganglion cells in a novelmodel of intraorbital optic nerve transection in mice J Pineal Res “ 101034j1600079x20021823x Tun§ AT Turgut M Aslan H Sahin B Yurtseven ME KaplantheS Neonatalcerebellum of“ 101016jbrainres200510011pinealectomythechick a stereologicalstudy Brain ResPurkinjeinduceslosscellin Pang Y Dong J Thomas P Characterization neurosteroid binding and braindistribution of human membrane progesterone receptors δ and ε mPRδand mPRε and mPRδ involvement in neurosteroid inhibition of apoptosisEndocrinology “ 101210en20121772 Schumacher M Mattern C Ghoumari A Oudinet JP Liere P LabombardaF et al Revisiting the roles of progesterone and allopregnanolone in thenervous system resurgence of the progesterone receptors Prog Neurobiol “ 101016jpneurobio201309004 Belelli D Lambert JJ Neurosteroids endogenous regulators of the GABAAreceptor Nat Rev Neurosci “ 101038nrn1703 Bernal J Thyroid hormone receptors in brain development and function NatClin Pract Endocrinol Metabol “ 101038ncpendmet0424 FalluelMorel A Vaudry D Aubert N Galas L Benard M Basille M et alPituitary adenylate cyclaseactivating polypeptide prevents the eï¬ects ofceramides on migration neurite outgrowth and cytoskeleton remodelingProc Natl Acad Sci USA “ 101073pnas04096 Koibuchi N Chin WW ThyroidEndocrinoldevelopment 101016S1043276000002381TrendshormoneMetabactionandbrain“ Vaudry D FalluelMorel A Leuillet S Vaudry H Gonzalez BJ Regulators ofcerebellar granule cell development act through specific signaling pathwaysScience “ 101126science1085260 Sasahara K Shikimi H Haraguchi S Sakamoto H Honda S Harada N et alMode of action and functional significance of estrogeninducing dendriticgrowth Spinogenesis and synaptogenesis in the developing Purkinje cell JNeurosci “ 101523JNEUROSCI0710072007 Bakkum BW Benevento LA Cohen RS Eï¬ects of lightdark and darkrearing on synaptic morphology in the superior colliculus and visualcortex ofJ Neurosci Res “ 101002jnr490280107the postnatal and adult rat Brooks E Waters E Farrington L Canal MM Diï¬erential hypothalamictyrosine hydroxylase distribution and activation by light in adult mice rearedunder diï¬erent light conditions during the suckling period Brain Struct Funct “ 101007s0042901103189 DulcisDSpitzerNCIlluminationcontrolsdopamineof“ 101038nature07569neuronsregulatingbehaviourdiï¬erentiationNature Li Y Komuro Y Fahrion JK Hu T Ohno N Fenner KB et al Lightstimuli control neuronal migration by altering ofinsulinlike growthfactor IGF1 signaling Proc Natl Acad Sci USA “ 101073pnas1111326109 Ohta H Mitchell AC McMahon DG ConstantPediatrthe“ 10120301pdr00002331141840366developing mousebiologicalclocklight disruptsRes Kantermann T Roenneberg TIsfactor or a health risk predictor Chronobiol lightatnightInthealthriska “ Wu J Dauchy RT Tirrell PC Wu SS Lynch DT Jitawatanarat P et alLight at night activates IGF1RPDK1 signaling and accelerates tumrowth in human breast cancer xenografts Cancer Res “ 10115800085472CAN103837 Smarr BL Grant AD Perez L Zucker I Kriegsfeld LJ Maternal andearlylife circadian disruption have longlasting negative consequenceson oï¬spring development and adult behavior in mice Sci Rep 101038s41598017034064Conflict of Interest The authors declare that the research was conducted in theabsence of any commercial or financial relationships that could be construed as apotential conflict of interestCopyright Haraguchi and Tsutsui This is an openaccess distributedunder the terms of the Creative Commons Attribution License CC BY The usedistribution or reproduction in other forums is permitted provided the originalauthors and the copyright owners are credited and that the original publicationin this journal is cited in accordance with accepted academic practice No usedistribution or reproduction is permitted which does not comply with these termsFrontiers in Endocrinology wwwfrontiersinAugust Volume 0c' Answer:

Thyroid_Cancer

Genomics Score Based onGenomeWide Network Analysis forPrediction of Survival in GastricCancer A Novel PrognosticSignatureZepang Sun  Hao Chen  Zhen Han  Weicai Huang Yanfeng Hu Mingli Zhao Tian LinJiang Yu Hao Liu Yuming Jiang and Guoxin LiDepartment of General Surgery Nanfang Hospital Southern Medical University Guangzhou ChinaGCPurpose Gastric canceris a product of multiple genetic abnormalitiesincluding genetic and epigenetic modifications This study aimed to integrate variousbiomolecules such as miRNAs mRNA and DNA methylation into a genomewidenetwork and develop a nomogram for predicting the overall survival OS of GCMaterials and Methods A total of GC cases as a training cohort with a random of examples included as a validation cohort were screened from The Cancer GenomeAtlas database A genomewide network was constructed based on a combination ofunivariate Cox regression and least absolute shrinkage and selection operator analysesand a nomogram was established to predict and 5year OS in the trainingcohort The nomogram was then assessed in terms of calibration discriminationand clinical usefulness in the validation cohort Afterward in order to confirm thesuperiority of the whole gene network model and further reduce the biomarkers for theimprovement of clinical usefulness we also constructed eight other models accordingto the different combinations of miRNAs mRNA and DNA methylation sites and madecorresponding comparisons Finally Gene Ontology GO and Kyoto Encyclopedia ofGenes and Genomes KEGG analyses were also performed to describe the function ofthis genomewide networkResults A multivariate analysis revealed a novel prognostic factor a genomics scoreGS comprising seven miRNAs eight mRNA and DNA methylation sites In thevalidation cohort comparing to patients with low GS highGS patients HR P were significantly associated with increased allcause mortality Furthermoreafter stratification of the TNM stage I II III and IV there were significant differencesrevealed in the survival rates between the highGS and lowGS groups as wellP The and 5year Cindex of whole genomicsbased nomogram were and respectively The other models have comparable or relativelypoor comprehensive performance while they had fewer biomarkers Besides thatDAVID further revealed multiple molecules and pathways related to the genomewidenetwork such as cytomembranes cell cycle and adipocytokine signalingEdited byXin Maizie ZhouStanford University United StatesReviewed byHailin TangSun Yatsen University Cancer CenterSYSUCC ChinaJie TianInstitute of Automation CAS ChinaCorrespondenceYuming Jiangjiangymbest163comGuoxin Ligzliguoxin163com These authors have contributedequally to this workSpecialty sectionThis was submitted toComputational Genomicsa section of the journalFrontiers in GeneticsReceived April Accepted July Published August CitationSun Z Chen H Han Z Huang WHu Y Zhao M Lin T Yu J Liu HJiang Y and Li G GenomicsScore Based on GenomeWideNetwork Analysis for Predictionof Survival in Gastric Cancer A NovelPrognostic SignatureFront Genet 103389fgene202000835Frontiers in Genetics wwwfrontiersinAugust Volume 0cSun et alGenomics ScoreConclusion We successfully developed a GS based on genomewide network whichmay represent a novel prognostic factor for GC A combination of GS and TNMstaging provides additional precision in stratifying patients with different OS prognosesconstituting a more comprehensive subtyping system This could potentially play animportant role in future clinical practiceKeywords gastric cancer genomewide network miRNA mRNA DNA methylation nomogramBACKGROUNDGastric cancer GC is one of the most common malignanthuman tumors and the third leading cause of cancerrelatedmortalities worldwide Reports estimate that nearly one millionnew cases and deaths occur each year across the worldTorre Despite the rapid research advancement GCrelated impacts on human life remain high around the globeAccording to the global cancer burden data hundreds of billionsof dollars in economic losses are incurred each year due to GCAt the same time stomach cancer has been reported to cause million disabilityadjusted life years with of these resultingfrom years of life lost and from years lived with disabilityGlobal Burden of Disease Cancer Collaboration Despite major breakthroughs in GC prevention diagnosisand treatment therapies reported over the past decade prognosisremains a challenge at diï¬erent TNM stages Jiang 2018aSun 2019ab Notably patients with similar clinicalfeatures and at the same tumor stage who receive uniformtreatment have exhibited varying clinical outcomes Bang Jiang 2018b Such evidence indicates the existingchallenges to traditional TNM staging Serra possiblydue to a lack of molecular tools for eï¬ectively predicting theprognosis and the therapeutic eï¬ect of GC patients Thereforemore rigorous and reliable systems that accurately reflect theheterogeneity of diï¬erent patients and guide the development oftreatment approaches are urgently needed Duarte Serra Tumors are a product of multiple genetic mutations includinggenetic gene expression and epigenetic DNA methylation andhistone modification modifications as well as deregulationsof tumorsuppressor genes and protooncogenes Anna Choi In addition changes in a set of geneticmaterials have been closely associated with cancer outcomesAnna Choi Therefore to eï¬ectivelypredict the prognosis of tumors such as GC a single biomarkeris insufficient necessitating the need for a gene networkA variety of mRNAs have been associated with GC prognosisCamargo with microRNAsmiRNAs alsoimplicated in tumor prediction in the recent years Li Ueda Camargo These smallnoncoding RNAs comprising nucleotides primarily functionto regulate protein translation by inhibiting the expressionoffrom geneticsepigenetics is currently receiving considerable research attentionDNA methylation isthe most common epigenetic eventassociated with cancer development and progression Anna Consequently numerous studies have implicated DNAtarget messenger RNAs mRNAs Apartmethylation in the diagnosis and the prognosis of various tumorsincluding GC Camargo Choi Althoughthese studies have revealed several biomarkers that have proved tobe prognostic predictors in GC only a handful have been adoptedin clinical therapies or are used to build predictive models forthe disease Anna Duarte Camargo Choi Serra Previous studies have identified and recommended numerousbiomarkers for GC However since malignant tumors ofteninvolve multiple layers and diï¬erent levels of genetic changesincluding the genome transcriptome and proteome or evenepigenetic content selecting reasonable candidate factors fromtens of thousands of biomarkers and comprehensively analyzingthem as an independent feature is imperative to eï¬ectivelydevelop a suitable prognostic target Therefore genetic networkscontaining a panel of abnormal factors from diï¬erent regulatorylevels represent the best chance for achieving prognostic valueThe whole genomewide network analysis is reported inseveral other cancers such as colorectal cancer breast cancer andlung cancer Hou Zhang and it showsgreat value in diï¬erentiating the prognosis of these patientsTherefore it is feasible and advantageous to apply genomewidenetwork analysis to GCIn the current study we performed a series of sophisticatedstatistical analyses and identified genetic molecules that werehighly correlated with the prognosis of GC Specifically wescreened The Cancer Genome Atlas TCGA a genome projectwith types of cancer including gene expression and DNAmethylation as well as other biological information Furthermorewe extended these independent prognostic factors to theœomics concept Then a genomewide network was constructedInterestingly the genomics score GS obtained herein couldsupplement TNM staging and enhance the prognostic value ofdiï¬erent patients Moreover we developed multiple prognosticmodels then validated and compared them to ascertain theirstrengths and weaknesses Finally we performed pathwayenrichment and gene oncology annotation analyses to elucidatethe function of this gene networkMATERIALS AND METHODSData Acquisition and PreprocessingLevel data were downloaded from the TCGA databaseusing TCGAAssembler Module Ain January whichwas then pretreated with Module B The dataset comprised ofclinical variables from patients including age sex stageFrontiers in Genetics wwwfrontiersinAugust Volume 0cSun et alGenomics Scoreprimary site grade treatment and survival as well as associatedgenomewide data In addition the expression levels of miRNAs mRNA and DNA methylation sitesIllumina methylation were obtained from and patients respectively Afterward an intersection with a totalof samples was eventually retained Furthermore patientswith missing active followup data were excluded from theanalysis leaving patients in the final cohort Figure Moreover genomewide level data whose expression levelsfor miRNAs mRNA and DNA methylation sites were missingin more than of all samples were excluded from the finalanalysis Finally GC patients with miRNAs mRNA and DNA methylation sites were chosen forfurther analysisGenomeWide Network AnalysisGene expression and DNA methylation data were normalizedusing R package before subsequent processing Univariate andleast absolute shrinkage and selection operator LASSO Coxregression models were combined and used to identify themost useful prognostic factors in miRNAs mRNA and DNAmethylation sites associated with survival Firstly univariate Coxregression was performed on each candidate miRNA mRNA andDNA methylation site to elucidate its role in patient survivalthen signatures with P value less than were retained forsubsequent analysis Thereafter the LASSO Cox regression modelwas applied to select and shrink the data SupplementaryFigure S3 Tibshirani Finally a GS based on a genomewide network comprising seven miRNAs eight mRNAs and DNA methylation sites was constructed for predicting survivalA summary of the whole screening process is displayed inSupplementary Figure S1Development and Comparison ofIndividualized Prediction ModelsThe TCGA cohort with cases was used as the trainingset with a random cases from the total cohort includedas a validation group The random number is Firstlywe developed the original GS based on biomarkers sevenmiRNAs eight mRNAs and DNA methylation sites Thenconsidering the complexity of the original GS and difficultclinical applicationin order to obtain a more concise andFIGURE A Venn diagram displays the patients™ screening processFrontiers in Genetics wwwfrontiersinAugust Volume 0cSun et alGenomics Scoreeï¬ective GS we also constructed eight other models accordingto the diï¬erent combinations of miRNAs mRNA and DNAmethylation and made corresponding comparisons Finally atotal of nine GS models based on the genomewide networkfrom LASSO were adopted to screen for the most appropriatemarkers These included the following models genomics sevenmiRNAs eight mRNA and DNA methylation sites miRNAsseven miRNAs mRNA eight mRNA methylation DNAmethylation sites miRNAs methylation seven miRNAsand DNA methylation sites miRNAs mRNA sevenmiRNAs and eight mRNA mRNA methylation eightmRNA and DNA methylation sites Coxmodel twomiRNAs six mRNA and nine DNA methylation sites andCoxmodel one miRNA one mRNA and seven DNAmethylation sites Among them markers from Coxmodel were separately detected from miRNAs mRNA or DNAmethylation sites using multivariate Cox regression analysis afterLASSO Supplementary Tables S2“S4 On the other handmarkers from Coxmodel depended on signatures from amultivariate Cox regression analysis combining the genomewide network and the clinical characteristics SupplementaryTable S5 Thereafter we constructed several nomograms byincorporating significant P GS variables and otherclinical features following multivariate Cox regression Iasonos and a clinical nomogram was built as a blank controlThe equations used for calculating the GS of these models arelisted in Supplementary Table S6To calculate the discrimination and the stability of diï¬erentCox regression models we applied Cstatistics and calibrationAdditionally we performed an analysis oftimedependentreceiver operator characteristics ROC based on the and 5year survival endpoints to assess the prognostic accuracyof the diï¬erent nomograms Furthermore we evaluated thepotential net benefit of diï¬erent predictive models using decisioncurve analysis DCA DCA compares the clinical usefulness ofdiï¬erent indicators by calculating the potential net benefit of eachdecision strategy at each threshold probability Thus DCA wasa significant novel approach for comparing the old and the newmodels Vickers and Elkin Screening for Potential miRNA TargetGenesWe predicted the potential target genes of the seven miRNAsfrom LASSO by screening the miRTarBase miRDB andTargetScan databases Common genes from each miRNA acrossthe three databases were then used for subsequent studies Morethan of the miRNAs showed negative regulation to targetgenes Consequently the expression data from TCGA were usedto perform a batch of correlation analysis of each miRNAwith corresponding target genes and the three genes with thelargest absolute negative correlation were retained as the mostlikely targets Additionally at least three potential target genesfrom miRTarBase which is coexpressed with miRNAs wereconsidered as equally important and were subjected to Cytoscapeversion for identification of miRNA“target genes coexpression network analysis Supplementary Figure S2Functional Enrichment AnalysisThe potential target genes that were negatively correlated withmiRNAs in TCGA as well as the coding sequences for mRNAand DNA methylation sites were used for functional enrichmentanalysis using the Kyoto Encyclopedia of Genes and GenomesKEGG pathway and Gene Ontology GO using DAVID Supplementary Figure S2 Functional enrichment analysisindicates why the gene network produces images on the survivalof GC from a molecular mechanism Visualization was then doneusing the œggplot2 package implemented in RStatistical AnalysisThe patients were divided into lowrisk and highrisk groupsby the median GS as the cutoï¬ point Survival estimates wereobtained according to the Kaplan“Meier method and comparedusing the logrank test Variables that reached significance withP were entered into the multivariable analyses usingthe Cox proportional hazards model with an entry stepwiseapproach to identify covariates associated with increased allcause mortality and then hazard ratio with confidenceintervals CIs of each variable was achieved All the statisticalsignificance values were set as twosided P LASSOCox regression was performed through the œglmnet packageTimedependent ROC analysis at diï¬erent followup times wasimplemented using the œtimeROC package of R project in orderto further expound the performance of diï¬erent GS modelsand DCA was used to compare their clinical use by œrmdapackage Finally nomogram based on the Cox regression modelwas constructed using the œrms package Cindex and calibrationto calculate the discrimination and the stability of these modelswere performed using cstatistics and Bootstrap sample Harrell™sconcordance index Cindex indicated a better prognostic modelif its value was closer to and the calibration diagram showedthat the better the prediction if the closer the correction line wasto the diagonal All statistical methods are applied to both thetraining group and the validation group Statistical analyses wereperformed using SPSS statistical software version and Rsoftware version RESULTSPatient CharacteristicsAmong the GC patients analyzed in this study were male whereas were female The average ageof the entire study population was ± years In termsof pathological stage cases were identified as stageI were stage II were stage III and were at stage IV With regards to treatment patients received surgery cases of R0 surgery R1 and nineR2 whereas were subjected to fluorouracilbasedchemotherapy The genomic nomogram classified samplesinto low GS GS ‰¤ and into high GS GS groups based on the median cutoï¬ Figure A detaileddescription of tumor location pathology grade and Laurenclassification is outlined in Supplementary Table S1 while aheat map of the genomic scores layered by clinicopathologicalFrontiers in Genetics wwwfrontiersinAugust Volume 0cSun et alGenomics ScoreFIGURE Distribution of patient cases and density based on genomics score GS in the total The Cancer Genome Atlas cohort AB Scatter plots of genomicsscores regarding the classification of low and high GS CD and the bold line represents the medianfactors is illustrated in Supplementary Figure S4 The medianmean CI survival time for OS was “days in the total cohort “ days in the highGS group and mean “ days in the lowGSgroup Supplementary Figure S5 Toward the last followupa total of deaths and censoring were recorded Theestimated cumulative and 5year OS in the total cohortwere and respectively although these ratesincrease to and respectivelyin the lowGS group Conversely the and 5year OS decreased to and respectively in the highGS group Thebaseline information of the validation cohort is also listed inSupplementary Table S1 and Supplementary Figure S6Survival AnalysisWe identified a basic genomewide network comprisingseven miRNAs eight mRNAs and DNA methylationsites as the prognostic factor for OSfrom hundreds ofthousands of univariate Cox regression and LASSO analysesThis network was then classified as other models in thetraining and the validation groups Among the featuresidentified poor prognosis was significantly associated witha high expression of seven miRNAs hsamir100 hsamir hsamir136 hsamir193b hsamir22 hsamir653and hsamir6808 six mRNAs NRP18829 RNF144A9781ZNF227570 DUSP11843 CPNE8144402 MAGED19500and LOC9145091450 and seven DNA methylation sitesFrontiers in Genetics wwwfrontiersinAugust Volume 0cSun et alGenomics Scorecg22813794cg26014401cg25361506cg07020967 cg08859156 cg12485556 cg15861578 cg15861578cg25161386 and cg22740006 Conversely poor prognosis wasstrongly associated with a low expression of SOX148403 and DNA methylation sites including cg02223323 cg00481239cg14791193 cg15486740 cg20100408 cg20350671 cg22395807cg24361571andcg26856948 Table Univariate analysis performed onclinical characteristics revealed a significant association betweenage pathological stage TNM and surgery with OS Table On the other hand results from multivariable Cox regressionshowed that age pathological stage and GS were significantlyassociated with allcause mortality in GC Table and Figure Furthermore stratification of the pathological stage I II IIIand IV revealed significant diï¬erences in survival rates betweenthe highGS and the lowGS groups Figure A similarresult was found when the data were stratified by demographicvariables sex and age clinical characteristics primary sitegrade and Lauren classification as well as treatments surgeryand chemotherapy Supplementary Figures S7 S8 Onthe other hand categorizing GS into high or low groupsusing the median value across diï¬erent modelsindicatedthe genomics nomogram had the highest HR valuethatInterestingly HR was almost equal to miRNAs methylationmRNA methylation Coxmodel and Coxmodel nomograms which contained fewer gene features Moreoverthe HR value showed a marked decrease in miRNAs mRNAmethylation and miRNAs mRNA nomograms which includedthe least characteristics Table Nomograms Based on GenomeWideNetworkA genomics nomogram was first constructed based on thegenomewide network comprising gene features Figure To obtain a more concise and eï¬ective nomogram we also builta Coxmodel gene features and Coxmodel nine genefeatures nomograms Supplementary Figures S9 S10 Nexta clinical nomogram based on stage and age was built as acontrol Supplementary Figure S11 Thereafter we performedinternal and external validation to evaluate the feasibility of allnomograms using a threegrouped random bootstrap samplingFigure and Supplementary Figures S9“S11 We observedgood predictive performance in the first three nomograms butnot in the simple clinical modelValidation of the Nomograms Using ROCand DCATo ensure a good comparison across diï¬erent GS nomogramswe performed a timedependent ROC at and years offollowup as well as DCA In the validation group genomicsnomogram revealed the best comprehensive performance with and 5year area under the curve AUC values of and respectively Table and Coxmodel miRNAs methylation and mRNA methylation nomogramshad a comparable performance with and 5year AUCvalues of “ “ and “ respectivelybut it had fewer biomarkers Table Although the Coxmodel nomograms had the least biomarkers including miRNA mRNAand DNA methylation sites it had a relatively poor performancewith and 5year AUC values of and respectively Besides that the miRNA mRNA methylationmiRNAs methylation and miRNAs mRNA nomogramsrecorded and 5year AUC values of “ “ and “ respectively Finally we found thatcompared to miRNA and and mRNAnomogram and methylation nomogramhad higher and 5year AUC values of and Nevertheless all of them showed better performance thanthe clinical nomogram which recorded and 5year AUCvalues of and respectively Figures 6AB andSupplementary Figure S12 The Cindex based on diï¬erentnomograms exhibited a similar eï¬ect Supplementary Table S8Additionally DCA showed that the genomics Coxmodel mRNA methylation and methylation nomograms had asignificant net benefit compared to other GS models and theclinical nomogram Figures 6CDPotential miRNA Target GenesA total of hsamir22 hsamir100 hsamir136 hsamir193b hsamir653 hsamir1304 and hsamir6808 potential target genes were identified from themiRTarBase miRDB and TargetScan databases SupplementaryFigure S14 We then performed a correlation analysis betweeneach target gene and miRNAs and finally generated a miRNA“potential target gene plot Supplementary Figure S15A as wellas a miRNA“target gene coexpression network SupplementaryFigure S15B using CytoscapeFunctional Analysis of GenomeWideNetworkWe imported the potentialtarget genes mRNA andDNA methylation sitecoding sequences identified above intoDAVID for KEGG and GO analyses and identified biologicalprocesses molecular functions as well as cellular componentsFigures 7A“C Their corresponding KEGG pathways were alsoplotted Figure 7DDISCUSSIONGC can be divided into two types or four main categoriesaccording to the Lauren and World Health anizationWHO classifications Lauren Nagtegaal although neither of these classifications is based on molecularmarkers In the last decade however three novel molecularbased classification systems have been suggested for GC TheSingaporeDuke Group was the first to describe a classificationwith two intrinsic genomic subtypes GINT and GDIF whichhad diï¬erent gene expression Tan Serra The subtypes have diï¬erent levels of resistance to variouschemotherapy drugs and show limited prognostic value LaterTCGA used molecular evaluation to propose a new classificationwith four subtypes EBV MSI GS and CIN The identification ofthese subtypes has provided a roadmap for patient stratificationFrontiers in Genetics wwwfrontiersinAugust Volume 0cSun et alGenomics ScoreTABLE Univariable and multivariable analyses of the genomics score and the clinicopathological characteristics for overall survival in the training group and thevalidation groupVariablesTraining group n Validation group n Univariable analysisMultivariable analysisUnivariable analysisMultivariable analysisHR CIP valueHR CIP valueHR CIP valueHR CIP valueAge at diagnosis tears‰¥Pathological stageIIIIIIIVSurgeryR0R1R2UnknownGenomics scoreaLowHighT stagingT1T2T3T4N stagingN0N1N2N3M stagingM0M1SexFemaleMalePrimary siteCardiaFundusbodyAntrumUnknownPathology gradeI“IIIII“IVUnknownLauren classificationIntestinal typeDiffused typeUnknownChemotherapyYesNo“NANA““NA“NA“““ NA““ “““““““NANANA“““ NA““ “““““““NANANA“““ “NANANANA““““““NANANA““““““““““NANANANANA““““““NANANA“ “““““““““NANANANANAaBased on biomarkers seven miRNAs eight mRNAs and DNA methylation sitesFrontiers in Genetics wwwfrontiersinAugust Volume 0cSun et alGenomics ScoreTABLE miRNAs mRNA and DNA methylation whose expression levels showed a significant association with overall survival in least absolute shrinkage andselection operatorMolecular probeID reference geneCoefficientmiRNAsmRNAcg02223323cg00481239cg07020967cg08859156cg12485556cg14791193cg15861578cg15486740cg20100408cg20350671cg22395807cg24361571cg25361506cg25622155cg25161386cg22740006cg22813794cg26014401cg26856948hsamir100hsamir1304hsamir136hsamir193bhsamir22hsamir653hsamir6808NRP18829RNF144A9781ZNF227570SOX148403DUSP11843CPNE8144402MAGED19500LOC9145091450MAP7D2SHC4EID1TMEM117RPS4XPREPC1orf144ZC3H10ACOT13TTRAPHLADPB1IL1RAPL1ATXN10MIR3652UnconfirmedUnconfirmedNUFIP2PCLRFN4STYXL1MDH2UnconfirmedGOLGA3ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’HR CI““““““““““““““““““““““““““““““““““SEz valuep valueˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’as well as targeted therapeutic trials Cancer Genome AtlasResearch However initial data on disease outcomes fromthis cohort did not show diï¬erences in survival among thefour groups A series of positive studies on prognosis basedon TCGA classification was also reported Sohn In addition the Asian Cancer Research group divided GCinto four subtypes MSI EMT MSSTP53 and MSS TP53based on gene expression data and found significantly diï¬erentsurvival outcomes across them Cristescu Serra Despite the significant milestones of these studiesthey are all mainly based on the analysis of gene expressionmRNA Besides that a study proposed a fivemiRNAmodel while it had a Cindex of only Zhang In the current study we included methylation data andperformed functional enrichment analysis making our workstronger The aforementioned classifications are also complicatedand need further optimization to increase clinical applicabilityFurthermore they focused on typing and finding new targetswhereas our study reports on prognostic analysisSome of the biomarkers we identified herein including hsamir22 hsamir100 hsamir136 hsamir193b hsamir1304NRP1 DUSP1 and MAP7D2 cg02223323 have previouslybeen reported in GC Grandclement and B Chen Mu Zuo Zheng Chen Kurata and Lin Liu KT Song Teng Liu Pan Wang Others such as CPNE8MAGED1 RNF144A SOX14 ACOT13 cg15486740 EID1cg00481239 RPS4X cg08859156 and TTRAP cg15486740have been identified in various tumors other than GC Kamio Zeng Zhou Kuang Stanisavljevic Liu X Tosic Nagasawa Yang The remainingbiomarkers including hsamir653 hsamir6808 LOC91450Frontiers in Genetics wwwfrontiersinAugust Volume 0cSun et alGenomics ScoreFIGURE Kaplan“Meier curve of overall survival in all patients then stratified by genomics score GS pathological stage and age Survival analysis in the lowand highGS groups was further divided based on stages stages I“IVcg20100408 LRFN4cg14791193 GOLGA3ZNF22 C1orf144cg26856948HLADPB1cg22740006 MDH2cg22813794 MIR3652 cg24361571 NUFIP2 cg25161386PREPcg22813794 TMEM117cg07020967 ZC3H10 ZC3H10 IL1RAPL1 cg20350671 PCcg22740006 SHC4 cg00481239 and ATXN10 cg22395807have not been previously reportedcg12485556STYXL1Currently focus has been directed on identifying prognosticmiRNAs for GC Particularly one miRNA can regulate multipletargets while multiple miRNAs can regulate a single mRNATherefore a single miRNA may play an opposite role incancer progression by regulating diï¬erent target genes Forexample Mir22 and Mir100 were found to be tumorsuppressors in various cancers including GC Chen Zuo Similarly a high expression of Mir136 wasfound to promote proliferation and invasion in GC cell linesby inhibiting PTEN expression Chen while acontrasting result was reported when HOXC10 was targetedZheng Similarly Mir193b reportedly inducedGC proliferation or apoptosis by mediating diï¬erent mRNAexpressions Mu Song whereas a highMir1304 expression in GC was reported as a negative predictorfor prognosis of lung and thyroid cancers Kurata and Lin Liu Pan However the function of Mir653and Mir6808 has not been previously reported In the currentstudy we found an association between a high expression of allmiRNAs and poor survival Diï¬erent outcomes may be observedin our study relative to previous reports owing to the hugeFrontiers in Genetics wwwfrontiersinAugust Volume 0cSun et alGenomics ScoreTABLE Comparison of different genomics score models based on the median value for overall survival in the training group and the validation groupVariablesTraining group n Validation group n Hazard ratio CIP valueHazard ratio CIP valueGenomics nomogramAgePathological stageGenomics scoreaClinical nomogramAgePathological stagemiRNAs nomogramAgePathological stageGenomics scorebmRNA nomogramAgePathological stageGenomics scorecMethylation nomogramAgePathological stageGenomics scoredmiRNAs methylation nomogramAgePathological stageGenomics scoreemiRNAs mRNA nomogramAgePathological stageGenomics scorefmRNA methylation nomogramAgePathological stageGenomics scoregCoxmodel nomogramAgePathological stageGenomics scorehCoxmodel nomogramAgePathological stageGenomics scorei““““““““““““““““““““““““““““““““““““““““““““““““““““““““““aBased on biomarkers seven miRNAs eight mRNA and DNA methylation sites bBased on seven miRNAs cBased on eight mRNA dBased on DNA methylationsites eBased on seven miRNAs DNA methylation sites fBased on seven miRNAs eight mRNA gBased on eight mRNA DNA methylation sites hBased ontwo miRNAs six mRNA nine DNA methylation sites iBased on one miRNAs one mRNA seven DNA methylation sitesnumber of corresponding miRNA target genes herein and lackof evidence on their role in GC developmentMessenger RNAs have been reported to play an essentialrole in GC cancer For example high NRP1 expression andhypermethylation were associated with poor GC prognosisWang whereas another study indicated thatit could be an antitumor target Grandclement and B In addition high DUSP1 expression levels were foundto promote progression drug resistance and poor prognosisof GC Teng On the other hand SOX14showed opposite prognostic values in cervical cancer andleukemia with antitumor and carcinogenic eï¬ects respectivelyStanisavljevic Tosic Studies have alsoimplicated CPNE8 MAGED1 and RNF144A in ovarian andbreast cancers Zeng Nagasawa Yang However LOC91450 and ZNF22 have not beenreported in cancerAccumulating evidence indicates that DNA methylation playsa significant role in cancer progression However only a handfulof studies have described the relationship between levels ofFrontiers in Genetics wwwfrontiersinAugust Volume 0cSun et alGenomics ScoreFIGURE Genomics nomogram to predict the probability of and 5year overall survival OS in the training cohort A and the validation cohort B todetermine how many points for each variable to the probability of OS locate the variable on its axis draw a line straight upward to the point axis repeat this processfor each variable sum up the points achieved for each of the risk factors locate the final sum on the total point axis and draw a line straight down to find thepatient™s probability of OSsinglesite methylation and GC prognosis Particularly highexpressions of MAP7D2 ACOT13 EID1 RPS4X and TTRAPhave been associated with poor prognosis in gastric lung andpancreatic cancers as well as hepatic carcinoma respectivelywhile a high TTRAP expression reportedly inhibits the growthof osteosarcoma Kamio Zhou Kuang Liu KT Liu X Notably therelationship between methylation levels and corresponding geneexpression profiles is unknown necessitating further researchFurthermore the remaining DNA methylation sites and theircorresponding genes have not been reported Lastly no studyhas described the prognostic significance using a genomewide networkLast but not l

cancer203

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: Genomics Score Based onGenomeWide Network Analysis forPrediction of Survival in GastricCancer A Novel PrognosticSignatureZepang Sun  Hao Chen  Zhen Han  Weicai Huang Yanfeng Hu Mingli Zhao Tian LinJiang Yu Hao Liu Yuming Jiang and Guoxin LiDepartment of General Surgery Nanfang Hospital Southern Medical University Guangzhou ChinaGCPurpose Gastric canceris a product of multiple genetic abnormalitiesincluding genetic and epigenetic modifications This study aimed to integrate variousbiomolecules such as miRNAs mRNA and DNA methylation into a genomewidenetwork and develop a nomogram for predicting the overall survival OS of GCMaterials and Methods A total of GC cases as a training cohort with a random of examples included as a validation cohort were screened from The Cancer GenomeAtlas database A genomewide network was constructed based on a combination ofunivariate Cox regression and least absolute shrinkage and selection operator analysesand a nomogram was established to predict and 5year OS in the trainingcohort The nomogram was then assessed in terms of calibration discriminationand clinical usefulness in the validation cohort Afterward in order to confirm thesuperiority of the whole gene network model and further reduce the biomarkers for theimprovement of clinical usefulness we also constructed eight other models accordingto the different combinations of miRNAs mRNA and DNA methylation sites and madecorresponding comparisons Finally Gene Ontology GO and Kyoto Encyclopedia ofGenes and Genomes KEGG analyses were also performed to describe the function ofthis genomewide networkResults A multivariate analysis revealed a novel prognostic factor a genomics scoreGS comprising seven miRNAs eight mRNA and DNA methylation sites In thevalidation cohort comparing to patients with low GS highGS patients HR P were significantly associated with increased allcause mortality Furthermoreafter stratification of the TNM stage I II III and IV there were significant differencesrevealed in the survival rates between the highGS and lowGS groups as wellP The and 5year Cindex of whole genomicsbased nomogram were and respectively The other models have comparable or relativelypoor comprehensive performance while they had fewer biomarkers Besides thatDAVID further revealed multiple molecules and pathways related to the genomewidenetwork such as cytomembranes cell cycle and adipocytokine signalingEdited byXin Maizie ZhouStanford University United StatesReviewed byHailin TangSun Yatsen University Cancer CenterSYSUCC ChinaJie TianInstitute of Automation CAS ChinaCorrespondenceYuming Jiangjiangymbest163comGuoxin Ligzliguoxin163com These authors have contributedequally to this workSpecialty sectionThis was submitted toComputational Genomicsa section of the journalFrontiers in GeneticsReceived April Accepted July Published August CitationSun Z Chen H Han Z Huang WHu Y Zhao M Lin T Yu J Liu HJiang Y and Li G GenomicsScore Based on GenomeWideNetwork Analysis for Predictionof Survival in Gastric Cancer A NovelPrognostic SignatureFront Genet 103389fgene202000835Frontiers in Genetics wwwfrontiersinAugust Volume 0cSun et alGenomics ScoreConclusion We successfully developed a GS based on genomewide network whichmay represent a novel prognostic factor for GC A combination of GS and TNMstaging provides additional precision in stratifying patients with different OS prognosesconstituting a more comprehensive subtyping system This could potentially play animportant role in future clinical practiceKeywords gastric cancer genomewide network miRNA mRNA DNA methylation nomogramBACKGROUNDGastric cancer GC is one of the most common malignanthuman tumors and the third leading cause of cancerrelatedmortalities worldwide Reports estimate that nearly one millionnew cases and deaths occur each year across the worldTorre Despite the rapid research advancement GCrelated impacts on human life remain high around the globeAccording to the global cancer burden data hundreds of billionsof dollars in economic losses are incurred each year due to GCAt the same time stomach cancer has been reported to cause million disabilityadjusted life years with of these resultingfrom years of life lost and from years lived with disabilityGlobal Burden of Disease Cancer Collaboration Despite major breakthroughs in GC prevention diagnosisand treatment therapies reported over the past decade prognosisremains a challenge at diï¬erent TNM stages Jiang 2018aSun 2019ab Notably patients with similar clinicalfeatures and at the same tumor stage who receive uniformtreatment have exhibited varying clinical outcomes Bang Jiang 2018b Such evidence indicates the existingchallenges to traditional TNM staging Serra possiblydue to a lack of molecular tools for eï¬ectively predicting theprognosis and the therapeutic eï¬ect of GC patients Thereforemore rigorous and reliable systems that accurately reflect theheterogeneity of diï¬erent patients and guide the development oftreatment approaches are urgently needed Duarte Serra Tumors are a product of multiple genetic mutations includinggenetic gene expression and epigenetic DNA methylation andhistone modification modifications as well as deregulationsof tumorsuppressor genes and protooncogenes Anna Choi In addition changes in a set of geneticmaterials have been closely associated with cancer outcomesAnna Choi Therefore to eï¬ectivelypredict the prognosis of tumors such as GC a single biomarkeris insufficient necessitating the need for a gene networkA variety of mRNAs have been associated with GC prognosisCamargo with microRNAsmiRNAs alsoimplicated in tumor prediction in the recent years Li Ueda Camargo These smallnoncoding RNAs comprising nucleotides primarily functionto regulate protein translation by inhibiting the expressionoffrom geneticsepigenetics is currently receiving considerable research attentionDNA methylation isthe most common epigenetic eventassociated with cancer development and progression Anna Consequently numerous studies have implicated DNAtarget messenger RNAs mRNAs Apartmethylation in the diagnosis and the prognosis of various tumorsincluding GC Camargo Choi Althoughthese studies have revealed several biomarkers that have proved tobe prognostic predictors in GC only a handful have been adoptedin clinical therapies or are used to build predictive models forthe disease Anna Duarte Camargo Choi Serra Previous studies have identified and recommended numerousbiomarkers for GC However since malignant tumors ofteninvolve multiple layers and diï¬erent levels of genetic changesincluding the genome transcriptome and proteome or evenepigenetic content selecting reasonable candidate factors fromtens of thousands of biomarkers and comprehensively analyzingthem as an independent feature is imperative to eï¬ectivelydevelop a suitable prognostic target Therefore genetic networkscontaining a panel of abnormal factors from diï¬erent regulatorylevels represent the best chance for achieving prognostic valueThe whole genomewide network analysis is reported inseveral other cancers such as colorectal cancer breast cancer andlung cancer Hou Zhang and it showsgreat value in diï¬erentiating the prognosis of these patientsTherefore it is feasible and advantageous to apply genomewidenetwork analysis to GCIn the current study we performed a series of sophisticatedstatistical analyses and identified genetic molecules that werehighly correlated with the prognosis of GC Specifically wescreened The Cancer Genome Atlas TCGA a genome projectwith types of cancer including gene expression and DNAmethylation as well as other biological information Furthermorewe extended these independent prognostic factors to theœomics concept Then a genomewide network was constructedInterestingly the genomics score GS obtained herein couldsupplement TNM staging and enhance the prognostic value ofdiï¬erent patients Moreover we developed multiple prognosticmodels then validated and compared them to ascertain theirstrengths and weaknesses Finally we performed pathwayenrichment and gene oncology annotation analyses to elucidatethe function of this gene networkMATERIALS AND METHODSData Acquisition and PreprocessingLevel data were downloaded from the TCGA databaseusing TCGAAssembler Module Ain January whichwas then pretreated with Module B The dataset comprised ofclinical variables from patients including age sex stageFrontiers in Genetics wwwfrontiersinAugust Volume 0cSun et alGenomics Scoreprimary site grade treatment and survival as well as associatedgenomewide data In addition the expression levels of miRNAs mRNA and DNA methylation sitesIllumina methylation were obtained from and patients respectively Afterward an intersection with a totalof samples was eventually retained Furthermore patientswith missing active followup data were excluded from theanalysis leaving patients in the final cohort Figure Moreover genomewide level data whose expression levelsfor miRNAs mRNA and DNA methylation sites were missingin more than of all samples were excluded from the finalanalysis Finally GC patients with miRNAs mRNA and DNA methylation sites were chosen forfurther analysisGenomeWide Network AnalysisGene expression and DNA methylation data were normalizedusing R package before subsequent processing Univariate andleast absolute shrinkage and selection operator LASSO Coxregression models were combined and used to identify themost useful prognostic factors in miRNAs mRNA and DNAmethylation sites associated with survival Firstly univariate Coxregression was performed on each candidate miRNA mRNA andDNA methylation site to elucidate its role in patient survivalthen signatures with P value less than were retained forsubsequent analysis Thereafter the LASSO Cox regression modelwas applied to select and shrink the data SupplementaryFigure S3 Tibshirani Finally a GS based on a genomewide network comprising seven miRNAs eight mRNAs and DNA methylation sites was constructed for predicting survivalA summary of the whole screening process is displayed inSupplementary Figure S1Development and Comparison ofIndividualized Prediction ModelsThe TCGA cohort with cases was used as the trainingset with a random cases from the total cohort includedas a validation group The random number is Firstlywe developed the original GS based on biomarkers sevenmiRNAs eight mRNAs and DNA methylation sites Thenconsidering the complexity of the original GS and difficultclinical applicationin order to obtain a more concise andFIGURE A Venn diagram displays the patients™ screening processFrontiers in Genetics wwwfrontiersinAugust Volume 0cSun et alGenomics Scoreeï¬ective GS we also constructed eight other models accordingto the diï¬erent combinations of miRNAs mRNA and DNAmethylation and made corresponding comparisons Finally atotal of nine GS models based on the genomewide networkfrom LASSO were adopted to screen for the most appropriatemarkers These included the following models genomics sevenmiRNAs eight mRNA and DNA methylation sites miRNAsseven miRNAs mRNA eight mRNA methylation DNAmethylation sites miRNAs methylation seven miRNAsand DNA methylation sites miRNAs mRNA sevenmiRNAs and eight mRNA mRNA methylation eightmRNA and DNA methylation sites Coxmodel twomiRNAs six mRNA and nine DNA methylation sites andCoxmodel one miRNA one mRNA and seven DNAmethylation sites Among them markers from Coxmodel were separately detected from miRNAs mRNA or DNAmethylation sites using multivariate Cox regression analysis afterLASSO Supplementary Tables S2“S4 On the other handmarkers from Coxmodel depended on signatures from amultivariate Cox regression analysis combining the genomewide network and the clinical characteristics SupplementaryTable S5 Thereafter we constructed several nomograms byincorporating significant P GS variables and otherclinical features following multivariate Cox regression Iasonos and a clinical nomogram was built as a blank controlThe equations used for calculating the GS of these models arelisted in Supplementary Table S6To calculate the discrimination and the stability of diï¬erentCox regression models we applied Cstatistics and calibrationAdditionally we performed an analysis oftimedependentreceiver operator characteristics ROC based on the and 5year survival endpoints to assess the prognostic accuracyof the diï¬erent nomograms Furthermore we evaluated thepotential net benefit of diï¬erent predictive models using decisioncurve analysis DCA DCA compares the clinical usefulness ofdiï¬erent indicators by calculating the potential net benefit of eachdecision strategy at each threshold probability Thus DCA wasa significant novel approach for comparing the old and the newmodels Vickers and Elkin Screening for Potential miRNA TargetGenesWe predicted the potential target genes of the seven miRNAsfrom LASSO by screening the miRTarBase miRDB andTargetScan databases Common genes from each miRNA acrossthe three databases were then used for subsequent studies Morethan of the miRNAs showed negative regulation to targetgenes Consequently the expression data from TCGA were usedto perform a batch of correlation analysis of each miRNAwith corresponding target genes and the three genes with thelargest absolute negative correlation were retained as the mostlikely targets Additionally at least three potential target genesfrom miRTarBase which is coexpressed with miRNAs wereconsidered as equally important and were subjected to Cytoscapeversion for identification of miRNA“target genes coexpression network analysis Supplementary Figure S2Functional Enrichment AnalysisThe potential target genes that were negatively correlated withmiRNAs in TCGA as well as the coding sequences for mRNAand DNA methylation sites were used for functional enrichmentanalysis using the Kyoto Encyclopedia of Genes and GenomesKEGG pathway and Gene Ontology GO using DAVID Supplementary Figure S2 Functional enrichment analysisindicates why the gene network produces images on the survivalof GC from a molecular mechanism Visualization was then doneusing the œggplot2 package implemented in RStatistical AnalysisThe patients were divided into lowrisk and highrisk groupsby the median GS as the cutoï¬ point Survival estimates wereobtained according to the Kaplan“Meier method and comparedusing the logrank test Variables that reached significance withP were entered into the multivariable analyses usingthe Cox proportional hazards model with an entry stepwiseapproach to identify covariates associated with increased allcause mortality and then hazard ratio with confidenceintervals CIs of each variable was achieved All the statisticalsignificance values were set as twosided P LASSOCox regression was performed through the œglmnet packageTimedependent ROC analysis at diï¬erent followup times wasimplemented using the œtimeROC package of R project in orderto further expound the performance of diï¬erent GS modelsand DCA was used to compare their clinical use by œrmdapackage Finally nomogram based on the Cox regression modelwas constructed using the œrms package Cindex and calibrationto calculate the discrimination and the stability of these modelswere performed using cstatistics and Bootstrap sample Harrell™sconcordance index Cindex indicated a better prognostic modelif its value was closer to and the calibration diagram showedthat the better the prediction if the closer the correction line wasto the diagonal All statistical methods are applied to both thetraining group and the validation group Statistical analyses wereperformed using SPSS statistical software version and Rsoftware version RESULTSPatient CharacteristicsAmong the GC patients analyzed in this study were male whereas were female The average ageof the entire study population was ± years In termsof pathological stage cases were identified as stageI were stage II were stage III and were at stage IV With regards to treatment patients received surgery cases of R0 surgery R1 and nineR2 whereas were subjected to fluorouracilbasedchemotherapy The genomic nomogram classified samplesinto low GS GS ‰¤ and into high GS GS groups based on the median cutoï¬ Figure A detaileddescription of tumor location pathology grade and Laurenclassification is outlined in Supplementary Table S1 while aheat map of the genomic scores layered by clinicopathologicalFrontiers in Genetics wwwfrontiersinAugust Volume 0cSun et alGenomics ScoreFIGURE Distribution of patient cases and density based on genomics score GS in the total The Cancer Genome Atlas cohort AB Scatter plots of genomicsscores regarding the classification of low and high GS CD and the bold line represents the medianfactors is illustrated in Supplementary Figure S4 The medianmean CI survival time for OS was “days in the total cohort “ days in the highGS group and mean “ days in the lowGSgroup Supplementary Figure S5 Toward the last followupa total of deaths and censoring were recorded Theestimated cumulative and 5year OS in the total cohortwere and respectively although these ratesincrease to and respectivelyin the lowGS group Conversely the and 5year OS decreased to and respectively in the highGS group Thebaseline information of the validation cohort is also listed inSupplementary Table S1 and Supplementary Figure S6Survival AnalysisWe identified a basic genomewide network comprisingseven miRNAs eight mRNAs and DNA methylationsites as the prognostic factor for OSfrom hundreds ofthousands of univariate Cox regression and LASSO analysesThis network was then classified as other models in thetraining and the validation groups Among the featuresidentified poor prognosis was significantly associated witha high expression of seven miRNAs hsamir100 hsamir hsamir136 hsamir193b hsamir22 hsamir653and hsamir6808 six mRNAs NRP18829 RNF144A9781ZNF227570 DUSP11843 CPNE8144402 MAGED19500and LOC9145091450 and seven DNA methylation sitesFrontiers in Genetics wwwfrontiersinAugust Volume 0cSun et alGenomics Scorecg22813794cg26014401cg25361506cg07020967 cg08859156 cg12485556 cg15861578 cg15861578cg25161386 and cg22740006 Conversely poor prognosis wasstrongly associated with a low expression of SOX148403 and DNA methylation sites including cg02223323 cg00481239cg14791193 cg15486740 cg20100408 cg20350671 cg22395807cg24361571andcg26856948 Table Univariate analysis performed onclinical characteristics revealed a significant association betweenage pathological stage TNM and surgery with OS Table On the other hand results from multivariable Cox regressionshowed that age pathological stage and GS were significantlyassociated with allcause mortality in GC Table and Figure Furthermore stratification of the pathological stage I II IIIand IV revealed significant diï¬erences in survival rates betweenthe highGS and the lowGS groups Figure A similarresult was found when the data were stratified by demographicvariables sex and age clinical characteristics primary sitegrade and Lauren classification as well as treatments surgeryand chemotherapy Supplementary Figures S7 S8 Onthe other hand categorizing GS into high or low groupsusing the median value across diï¬erent modelsindicatedthe genomics nomogram had the highest HR valuethatInterestingly HR was almost equal to miRNAs methylationmRNA methylation Coxmodel and Coxmodel nomograms which contained fewer gene features Moreoverthe HR value showed a marked decrease in miRNAs mRNAmethylation and miRNAs mRNA nomograms which includedthe least characteristics Table Nomograms Based on GenomeWideNetworkA genomics nomogram was first constructed based on thegenomewide network comprising gene features Figure To obtain a more concise and eï¬ective nomogram we also builta Coxmodel gene features and Coxmodel nine genefeatures nomograms Supplementary Figures S9 S10 Nexta clinical nomogram based on stage and age was built as acontrol Supplementary Figure S11 Thereafter we performedinternal and external validation to evaluate the feasibility of allnomograms using a threegrouped random bootstrap samplingFigure and Supplementary Figures S9“S11 We observedgood predictive performance in the first three nomograms butnot in the simple clinical modelValidation of the Nomograms Using ROCand DCATo ensure a good comparison across diï¬erent GS nomogramswe performed a timedependent ROC at and years offollowup as well as DCA In the validation group genomicsnomogram revealed the best comprehensive performance with and 5year area under the curve AUC values of and respectively Table and Coxmodel miRNAs methylation and mRNA methylation nomogramshad a comparable performance with and 5year AUCvalues of “ “ and “ respectivelybut it had fewer biomarkers Table Although the Coxmodel nomograms had the least biomarkers including miRNA mRNAand DNA methylation sites it had a relatively poor performancewith and 5year AUC values of and respectively Besides that the miRNA mRNA methylationmiRNAs methylation and miRNAs mRNA nomogramsrecorded and 5year AUC values of “ “ and “ respectively Finally we found thatcompared to miRNA and and mRNAnomogram and methylation nomogramhad higher and 5year AUC values of and Nevertheless all of them showed better performance thanthe clinical nomogram which recorded and 5year AUCvalues of and respectively Figures 6AB andSupplementary Figure S12 The Cindex based on diï¬erentnomograms exhibited a similar eï¬ect Supplementary Table S8Additionally DCA showed that the genomics Coxmodel mRNA methylation and methylation nomograms had asignificant net benefit compared to other GS models and theclinical nomogram Figures 6CDPotential miRNA Target GenesA total of hsamir22 hsamir100 hsamir136 hsamir193b hsamir653 hsamir1304 and hsamir6808 potential target genes were identified from themiRTarBase miRDB and TargetScan databases SupplementaryFigure S14 We then performed a correlation analysis betweeneach target gene and miRNAs and finally generated a miRNA“potential target gene plot Supplementary Figure S15A as wellas a miRNA“target gene coexpression network SupplementaryFigure S15B using CytoscapeFunctional Analysis of GenomeWideNetworkWe imported the potentialtarget genes mRNA andDNA methylation sitecoding sequences identified above intoDAVID for KEGG and GO analyses and identified biologicalprocesses molecular functions as well as cellular componentsFigures 7A“C Their corresponding KEGG pathways were alsoplotted Figure 7DDISCUSSIONGC can be divided into two types or four main categoriesaccording to the Lauren and World Health anizationWHO classifications Lauren Nagtegaal although neither of these classifications is based on molecularmarkers In the last decade however three novel molecularbased classification systems have been suggested for GC TheSingaporeDuke Group was the first to describe a classificationwith two intrinsic genomic subtypes GINT and GDIF whichhad diï¬erent gene expression Tan Serra The subtypes have diï¬erent levels of resistance to variouschemotherapy drugs and show limited prognostic value LaterTCGA used molecular evaluation to propose a new classificationwith four subtypes EBV MSI GS and CIN The identification ofthese subtypes has provided a roadmap for patient stratificationFrontiers in Genetics wwwfrontiersinAugust Volume 0cSun et alGenomics ScoreTABLE Univariable and multivariable analyses of the genomics score and the clinicopathological characteristics for overall survival in the training group and thevalidation groupVariablesTraining group n Validation group n Univariable analysisMultivariable analysisUnivariable analysisMultivariable analysisHR CIP valueHR CIP valueHR CIP valueHR CIP valueAge at diagnosis tears‰¥Pathological stageIIIIIIIVSurgeryR0R1R2UnknownGenomics scoreaLowHighT stagingT1T2T3T4N stagingN0N1N2N3M stagingM0M1SexFemaleMalePrimary siteCardiaFundusbodyAntrumUnknownPathology gradeI“IIIII“IVUnknownLauren classificationIntestinal typeDiffused typeUnknownChemotherapyYesNo“NANA““NA“NA“““ NA““ “““““““NANANA“““ NA““ “““““““NANANA“““ “NANANANA““““““NANANA““““““““““NANANANANA““““““NANANA“ “““““““““NANANANANAaBased on biomarkers seven miRNAs eight mRNAs and DNA methylation sitesFrontiers in Genetics wwwfrontiersinAugust Volume 0cSun et alGenomics ScoreTABLE miRNAs mRNA and DNA methylation whose expression levels showed a significant association with overall survival in least absolute shrinkage andselection operatorMolecular probeID reference geneCoefficientmiRNAsmRNAcg02223323cg00481239cg07020967cg08859156cg12485556cg14791193cg15861578cg15486740cg20100408cg20350671cg22395807cg24361571cg25361506cg25622155cg25161386cg22740006cg22813794cg26014401cg26856948hsamir100hsamir1304hsamir136hsamir193bhsamir22hsamir653hsamir6808NRP18829RNF144A9781ZNF227570SOX148403DUSP11843CPNE8144402MAGED19500LOC9145091450MAP7D2SHC4EID1TMEM117RPS4XPREPC1orf144ZC3H10ACOT13TTRAPHLADPB1IL1RAPL1ATXN10MIR3652UnconfirmedUnconfirmedNUFIP2PCLRFN4STYXL1MDH2UnconfirmedGOLGA3ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’HR CI““““““““““““““““““““““““““““““““““SEz valuep valueˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’as well as targeted therapeutic trials Cancer Genome AtlasResearch However initial data on disease outcomes fromthis cohort did not show diï¬erences in survival among thefour groups A series of positive studies on prognosis basedon TCGA classification was also reported Sohn In addition the Asian Cancer Research group divided GCinto four subtypes MSI EMT MSSTP53 and MSS TP53based on gene expression data and found significantly diï¬erentsurvival outcomes across them Cristescu Serra Despite the significant milestones of these studiesthey are all mainly based on the analysis of gene expressionmRNA Besides that a study proposed a fivemiRNAmodel while it had a Cindex of only Zhang In the current study we included methylation data andperformed functional enrichment analysis making our workstronger The aforementioned classifications are also complicatedand need further optimization to increase clinical applicabilityFurthermore they focused on typing and finding new targetswhereas our study reports on prognostic analysisSome of the biomarkers we identified herein including hsamir22 hsamir100 hsamir136 hsamir193b hsamir1304NRP1 DUSP1 and MAP7D2 cg02223323 have previouslybeen reported in GC Grandclement and B Chen Mu Zuo Zheng Chen Kurata and Lin Liu KT Song Teng Liu Pan Wang Others such as CPNE8MAGED1 RNF144A SOX14 ACOT13 cg15486740 EID1cg00481239 RPS4X cg08859156 and TTRAP cg15486740have been identified in various tumors other than GC Kamio Zeng Zhou Kuang Stanisavljevic Liu X Tosic Nagasawa Yang The remainingbiomarkers including hsamir653 hsamir6808 LOC91450Frontiers in Genetics wwwfrontiersinAugust Volume 0cSun et alGenomics ScoreFIGURE Kaplan“Meier curve of overall survival in all patients then stratified by genomics score GS pathological stage and age Survival analysis in the lowand highGS groups was further divided based on stages stages I“IVcg20100408 LRFN4cg14791193 GOLGA3ZNF22 C1orf144cg26856948HLADPB1cg22740006 MDH2cg22813794 MIR3652 cg24361571 NUFIP2 cg25161386PREPcg22813794 TMEM117cg07020967 ZC3H10 ZC3H10 IL1RAPL1 cg20350671 PCcg22740006 SHC4 cg00481239 and ATXN10 cg22395807have not been previously reportedcg12485556STYXL1Currently focus has been directed on identifying prognosticmiRNAs for GC Particularly one miRNA can regulate multipletargets while multiple miRNAs can regulate a single mRNATherefore a single miRNA may play an opposite role incancer progression by regulating diï¬erent target genes Forexample Mir22 and Mir100 were found to be tumorsuppressors in various cancers including GC Chen Zuo Similarly a high expression of Mir136 wasfound to promote proliferation and invasion in GC cell linesby inhibiting PTEN expression Chen while acontrasting result was reported when HOXC10 was targetedZheng Similarly Mir193b reportedly inducedGC proliferation or apoptosis by mediating diï¬erent mRNAexpressions Mu Song whereas a highMir1304 expression in GC was reported as a negative predictorfor prognosis of lung and thyroid cancers Kurata and Lin Liu Pan However the function of Mir653and Mir6808 has not been previously reported In the currentstudy we found an association between a high expression of allmiRNAs and poor survival Diï¬erent outcomes may be observedin our study relative to previous reports owing to the hugeFrontiers in Genetics wwwfrontiersinAugust Volume 0cSun et alGenomics ScoreTABLE Comparison of different genomics score models based on the median value for overall survival in the training group and the validation groupVariablesTraining group n Validation group n Hazard ratio CIP valueHazard ratio CIP valueGenomics nomogramAgePathological stageGenomics scoreaClinical nomogramAgePathological stagemiRNAs nomogramAgePathological stageGenomics scorebmRNA nomogramAgePathological stageGenomics scorecMethylation nomogramAgePathological stageGenomics scoredmiRNAs methylation nomogramAgePathological stageGenomics scoreemiRNAs mRNA nomogramAgePathological stageGenomics scorefmRNA methylation nomogramAgePathological stageGenomics scoregCoxmodel nomogramAgePathological stageGenomics scorehCoxmodel nomogramAgePathological stageGenomics scorei““““““““““““““““““““““““““““““““““““““““““““““““““““““““““aBased on biomarkers seven miRNAs eight mRNA and DNA methylation sites bBased on seven miRNAs cBased on eight mRNA dBased on DNA methylationsites eBased on seven miRNAs DNA methylation sites fBased on seven miRNAs eight mRNA gBased on eight mRNA DNA methylation sites hBased ontwo miRNAs six mRNA nine DNA methylation sites iBased on one miRNAs one mRNA seven DNA methylation sitesnumber of corresponding miRNA target genes herein and lackof evidence on their role in GC developmentMessenger RNAs have been reported to play an essentialrole in GC cancer For example high NRP1 expression andhypermethylation were associated with poor GC prognosisWang whereas another study indicated thatit could be an antitumor target Grandclement and B In addition high DUSP1 expression levels were foundto promote progression drug resistance and poor prognosisof GC Teng On the other hand SOX14showed opposite prognostic values in cervical cancer andleukemia with antitumor and carcinogenic eï¬ects respectivelyStanisavljevic Tosic Studies have alsoimplicated CPNE8 MAGED1 and RNF144A in ovarian andbreast cancers Zeng Nagasawa Yang However LOC91450 and ZNF22 have not beenreported in cancerAccumulating evidence indicates that DNA methylation playsa significant role in cancer progression However only a handfulof studies have described the relationship between levels ofFrontiers in Genetics wwwfrontiersinAugust Volume 0cSun et alGenomics ScoreFIGURE Genomics nomogram to predict the probability of and 5year overall survival OS in the training cohort A and the validation cohort B todetermine how many points for each variable to the probability of OS locate the variable on its axis draw a line straight upward to the point axis repeat this processfor each variable sum up the points achieved for each of the risk factors locate the final sum on the total point axis and draw a line straight down to find thepatient™s probability of OSsinglesite methylation and GC prognosis Particularly highexpressions of MAP7D2 ACOT13 EID1 RPS4X and TTRAPhave been associated with poor prognosis in gastric lung andpancreatic cancers as well as hepatic carcinoma respectivelywhile a high TTRAP expression reportedly inhibits the growthof osteosarcoma Kamio Zhou Kuang Liu KT Liu X Notably therelationship between methylation levels and corresponding geneexpression profiles is unknown necessitating further researchFurthermore the remaining DNA methylation sites and theircorresponding genes have not been reported Lastly no studyhas described the prognostic significance using a genomewide networkLast but not l Answer:

Thyroid_Cancer

predominant male sex hormones drive the development andmaintenance of male characteristics by binding to androgen receptor AR As androgensare systemically distributed throughout the whole anism they affect many tissues andcell types in addition to those in male sexual ans It is now clear that the immunesystem is a target of androgen action In the lungs many immune cells express ARs andare responsive to androgens In this review we describe the effects of androgens and ARson lung myeloid immune cells”monocytes and macrophages”as they relate to healthand disease In particular we highlight the effect of androgens on lung diseases such asasthma chronic obstructive pulmonary disease and lung fibrosis We also discuss thetherapeutic use of androgens and how circulating androgens correlate with lung diseaseIn addition to human studies we also discuss how mouse models have helped to uncoverthe effect of androgens on monocytes and macrophages in lung disease Although therole of estrogen and other female hormones has been broadly analyzed in the literaturewe focus on the new perspectives of androgens as modulators of the immune systemthat target myeloid cells during lung ‚ammationEdited byFlavia BazzoniUniversity of Verona School ofMedicine and Surgery ItalyReviewed byPaola ParronchiUniversity of Florence ItalyTim WillingerKarolinska Institutet SwedenSandra O GollnickUniversity at Buffalo United StatesCorrespondenceNicola HellernhellerjhmieduKeywords androgen androgen receptor monocyte macrophage asthma lung sex difference sex hormoneSpecialty sectionThis was submitted toCytokines and Soluble Mediators inImmunitya section of the journalFrontiers in ImmunologyReceived March Accepted June Published August CitationBecerraDiaz M Song M and Heller N Androgen and AndrogenReceptors as Regulators of Monocyteand Macrophage Biology in theHealthy and Diseased LungFront Immunol 103389fimmu202001698INTRODUCTIONThe immune system is essential for maintaining homeostasis within tissues and ans andprotecting them against threats such as harmful pathogens or cancerous transformation Itcomprises both innate and adaptive components The innate immune system is made up of theinnate lymphoid innate lymphoid cells [ILCs] natural killer cells [NKs] and lymphoid tissueinducers [LTi] and innate myeloid subsets The innate immune system consists of a networkof immune cells and molecules that provide rapid firstline defense against pathogens In contrastthe adaptive immune response made up of B and T lymphocytes takes days or even weeks tobecome established Innate immune cells express pattern recognition receptors that recognizeunique and conserved pathogenassociated molecular patterns such as lipopolysaccharide LPSviral ssRNA and fungal glucan B and T cells have evolved to recognize a finer repertoireof self and nonselfantigens that facilitate pathogenspecific actions immunologic memorygeneration and host immune homeostasis regulation To accomplish this the adaptiveimmune response involves a tightly regulated interplay between T and B lymphocytes andFrontiers in Immunology wwwfrontiersinAugust Volume 0cBecerraDiaz et alAndrogenAR in Lung MonocyteMacrophage Biologyantigenpresenting cells of the myeloid lineage such as dendriticcells DCs monocytes and macrophages Myeloid cells arisefrom the bone marrow The type and magnitude of the immuneresponse is ‚uenced by biological sex and age and thereforediï¬ers between males and females Sex diï¬erences in the functionof the immune system arise from both genetic chromosomalsex diï¬erences and diï¬erences mediated by the action of maleand female sex hormones Because the concentration of sexhormones changes over the lifespan and throughout the courseof the menstrual cycle in women the function of the immunesystem also changes during diï¬erent stages of life Innate myeloidimmune cells like other cell types express sex hormone receptorsand are responsive to sex hormones Sex hormones are synthesized from cholesterol through adefined enzymatic cascade predominately in the gonads and theadrenal glands Sex hormones are also produced in othertissues including the brain placenta mammary glands liver andadipose tissue “ In addition to driving sexual developmentof egg and sperm production sex hormones are responsiblefor the development of male and female secondary sexualcharacteristics like breast development and growth of facial hairthat occur during puberty Androgens include testosteronedihydrotestosterone DHT androstenedione androstenedioland dehydroepiandrosterone DHEA with DHT being the mostpotent The concentration of androgens in circulation isabout sevenfold higher in adult men than in adult women Estradiol and progesterone are the predominantfemale sex hormones synthesized by the ovaries andadrenal glands Both male and female sex hormones are boundto the plasma proteins albumin and sex hormone bindingglobulin SHBG and only a small percentage exists as freehormone “ Thus the bioavailability of sex hormones isregulated by their biosynthesis and also the amount of albuminand SHBGImportantly sex hormones mediate not only anatomicdiï¬erences between women and men but also direct sexdiï¬erences in immune responses leading to diï¬erent risks forimmunologic diseases Overall women have a greaterrisk for autoimmune diseases such as systemic sclerosis andsystemic lupus erythematosus whereas men are morelikely to die of infectious and parasitic diseases Moreovermen have a greater risk of nonreproductive cancers “Both gender and sex are important mediators of these andother health and disease diï¬erences observed between men andwomen While gender refers to the array of socially constructedroles attitudes personality traits and behaviors sex representsa biological characteristic of an individual includingthe hormonal milieu and chromosome complement Ingeneral estrogens are considered to have pro‚ammatoryproperties and androgens are thought to have anti‚ammatoryproperties In the United States and worldwide relevant evidence highlights important epidemiologic sexdiï¬erences in incidence susceptibility and severity of a numberof diseases that aï¬ect the respiratory tract In this reviewwe will focus on how male sex hormones the androgensmodulate the response of myeloid cells in the lung and howthis modulation impacts the outcome of diï¬erent diseases ofthe lungSEX DIFFERENCES IN HUMAN LUNG ANDLUNG DISEASESsex mediates diï¬erencesBiologicalin the incidence andpathophysiology of lung diseases These diï¬erences arise fromsex diï¬erences in the structure and function of the lung itselfand also in the immune cells that populate the lung and arerecruited to it during ‚ammation Before birth the female lunghas several structural advantages over the male lung Surfactantis produced earlier and although the female lung is smaller ithas more alveoli per unit area Neonatal females have higherexpiratory flow rates than do male neonates when corrected forsize Thus male sex is a major risk factor for the developmentof respiratory distress syndrome bronchopulmonary dysplasia inneonates “ and asthma in childhood In addition to the contribution of structural diï¬erences ofthe lung between the sexes sex diï¬erences in lung function andlung diseases are also dependent on the action of sex hormonesWe have summarized some broad concepts that define howtestosterone and estrogen aï¬ectlung macrophage functionand how this may contribute to the outcome of particularlung diseases in Figure As testosterone rises after pubertythe immunosuppressive eï¬ects of this hormone on protectiveimmune responses to infectious diseases in males can worsenpulmonary disease This would be exemplified by tuberculosisor ‚uenza Some of these eï¬ects are a result of androgeneï¬ects on critical ‚ammatory macrophage functions althoughthe eï¬ects on the adaptive immune system also have a significantcontribution to the overall outcome Thus testosterone appearsto play a key immunoregulatory role in lung macrophagesTestosterone™s immunoregulatory properties also appear to bedependent on the amount of cellular expression of AR andon the concentration of the hormone Low concentrations oftestosterone have been noted in patients with asthma COPD andtuberculosis Low testosterone may also be linked to insufficientcontrol of tissuedamaging ‚ammatory responses seen inCOPD and pulmonary fibrosis Estrogen tends to promotewound healing responses in macrophages Dysregulation ofwound healing responses and overactive tissue remodelingmacrophages in the lung could be broadly used to describe theTh2 response in allergic asthma which is worse in womenCancer could also be considered an aberrant wound healingresponse driven by M2like tumor associated macrophages Wehave highlighted here how sex hormones contribute to changesin lung macrophage function that contribute to lung diseaseHowever it should be pointed out that not every sex diï¬erencein lung disease is due to direct eï¬ects on macrophages but on thebroader coordinated immune response as a wholeAsthmaBefore puberty the structural diï¬erences in the lung as wellas gender diï¬erences likely account for the higher incidence ofFrontiers in Immunology wwwfrontiersinAugust Volume 0cBecerraDiaz et alAndrogenAR in Lung MonocyteMacrophage BiologyFIGURE Sex differences in lung diseases discussed in this Review and how they may be connected to the effects of androgens and estrogens on ‚ammatorymacrophages in the lungasthma in boys than in girls With the onset of puberty male andfemale sex hormones and their eï¬ects on the structural cells ofthe lung and on the immune system contribute to the incidenceof asthma The incidence and severity of asthma aregreater in adult women than in adult men and greaterin female than in male mice Female sex hormones suchas estrogen appear to worsen asthma although a straightforwardcorrelation between amount of female sex hormone and asthmasymptoms has not been concluded Androgens have multipleimmunoregulatory and bronchodilatory functions and maycontribute to or be biomarkers for better lung function inmen Accordingly serum testosterone is low in men withmoderate to severe asthma “ In one study each ngdLincrease in serum testosterone correlated with a CI P decrease in the likelihood of having asthma On the other hand high concentrations of testosterone andcyclic AMP in sputum of asthmatic women during the lutealphase of the menstrual cycle were thought to play a role inpremenstrual exacerbations The idea that sex hormonesmay be a causal factor in asthma was significantly strengthenedby a recent study of adults that quantified serum sexhormones and asthma outcomes That study showed thatlow testosterone in both women and men was associated with anincreased incidence of asthma The other interesting finding wasthat higher testosterone was protective against asthma in obesewomen Obesity is a risk factor for asthma “ Thereforehow high body mass index BMI and circulating sex hormonestogether aï¬ect asthma requires further investigationAnother androgen dehydroepiandrosterone DHEA alsoknown as androstenolone is an endogenous steroid hormoneand one of the most abundant circulating steroids in humansIt is a precursor for the synthesis of both testosterone andestrogen DHEA is sulfated at the C3 position into DHEAS by the action ofthe sulfotransferase enzymes SULT2A1and SULT1E1 in the adrenal glands The amount of DHEAS in the circulation is ˆ¼“ times those of DHEADHEA became of interest to the asthma field because womenwith severe asthma had very low concentrations of DHEAS and DHEAS concentration correlated with lung function Interestingly DHEAS is suppressed by oral or inhaledglucocorticoids the mainstay therapy for asthma HumanDHEA peaks at around age and then follows an agedependentdecline until they reach prepubertal concentrations Reducedsecretion of DHEA with age has been related to a numberof ageassociated conditions Replacement of DHEA has beenconsidered as a possible therapeutic that could activate protectiveresponses in an aging immune system DHEA is known todownregulate Th2‚ammatory cytokines while upregulatingIL2 synthesis in concanavalin Astimulated peripheralblood mononuclear cells from adult males with atopic dermatitis Thus it was hypothesized that it would be a usefultreatment for atopic diseases including asthma and the results ofthe clinical trials for DHEA in asthma patients show promiseThe results are discussed in a later section titled œEï¬ects ofandrogen exposure on monocytes macrophages in humans withlung diseaseCOPDSex diï¬erences also have been reported in chronic obstructivepulmonary disease COPD a heterogeneous chronic andprogressive respiratory disorder that includes chronic bronchitisand emphysema Chronic exposure of the airways to insultssuch as cigarette smoke leads to epithelial cell injury destructionof pulmonary capillary vasculature acceleration of epithelial cellsenescence and airway remodeling The loss of lung complianceultimately leads to COPD COPD was previously thoughtto aï¬ect mostly elderly men primarily because of the higherprevalence of smoking in men However as smoking ratesincreased in women the number of COPD cases in womenexceeded that of men These diï¬erences are not only basedon gender as women develop more severe COPD with earlyonset disease years and have greater susceptibility toCOPD with lower tobacco exposure Moreover increasingage in female smokers leads to a faster annual decline inFrontiers in Immunology wwwfrontiersinAugust Volume 0cBecerraDiaz et alAndrogenAR in Lung MonocyteMacrophage Biologyforced expiratory volume in the first second when compared tothat of male smokers even when they smoke fewer cigarettes Similarly pulmonary fibrosis is another lung disease thatmanifests sex diï¬erences with men being more aï¬ectedthan women It is characterized by destruction of thepulmonary parenchyma and deposition of extracellular matrixwith alterations in phenotype of both fibroblasts and alveolarepithelial cells InfluenzaThe lungs are also the target of respiratory viruses such as‚uenza A œï¬‚u respiratory syncytial virus and coronavirusessuch as severe acute respiratory syndrome and the MiddleEast respiratory syndrome The viruses infectthe airwayepithelial cells and cause damage to the epithelial barrierby themselves or as a result ofthe immune response tothe viralinfection Sex diï¬erences have been noted in theimmune response to ‚uenza A virus and to the ‚uenzavaccine In general women have a more robust protectiveimmune response to ‚uenza virus and vaccine than do menAlthough this elevated response is helpful in clearing viruswomen of reproductive age also experience higher mortalityand hospitalizations “ possibly from collateral tissuedamage to the lungs The vigorous immune response in womenalso means that women experience more adverse events aftervaccination Indeed a systems biology approach identifiedthat high testosterone was correlated with a blunted responseto the flu vaccine in men As testosterone wanes in elderlymen mortality increases Since the male immune responseto the virus is also less robustthe incidence of seasonalflu is generally higher in men than in women in developedcountries according to the World Health anization It is not yet known how fluctuations in sex hormones acrossthe menstrual cycle and lifespan aï¬ect the immune system™sresponse to the ‚uenza virus in humans Mouse studieshave revealed that estrogen is protective at high but notlow concentrations On the other hand testosteronereplacement in gonadectomized or aged male mice enhancedsurvival rates Despite these findings in mouse modelsstudies examining the eï¬ect of sex hormones on cellular andmolecular mechanisms in human immune cells during ‚uenzainfection are lackingTuberculosisLike ‚uenza infection tuberculosis TB a lung disease causedby Mycobacterium tuberculosis exhibits notable sex diï¬erencesin the number of cases worldwide with men being almosttwice as frequently aï¬ected than women Both sexand gender diï¬erences impact the incidence of TB AlthoughTB aï¬ects less women than men in adulthood womenin their economically active years “ years old have ahigher TB incidence compared to women in other age groups This indicates that factors associated with gender such asexposure to the bacteria are important in this disease Howeverbecause male predominance does not occur in children thissuggests that biological factors such as male sex hormones alsoplay a significant role This is supported by a study ofmedically castrated men who experienced a significantly smallerproportion of death from TB compared to in intactmen Understanding how androgens lead to the greatersusceptibility of men to TB is critical as TB is still one ofthe leading fatal infectious diseases worldwide and may alsomay favor the development of other diseases such as lungcancer Lung CancerLung cancer is a very complex disease that depends on anumber of variants such as sex gender race and socioeconomicstatus The development of lung cancer is also related toenvironmental factors such as pollution due to industrializationand urbanization An additional genderassociated riskfactor significantly linked to developing lung cancer is cigarettesmoking Historically more men develop lung cancer andsuï¬er lung cancerassociated deaths compared to women However the incidence of lung cancer has changed notably inboth women and men In men lung cancer incidence startedto increase in the 1920s and started to decrease in the early1990s while in women the mortality rates and incidence beganto rise in the 1960s Changes in smoking habits in the lastseveral decades with a rise in the number of women who smokecorrelate with an increase in the incidence of lung cancer in thisdemographic group Smoking is definitely a key factor inthe development of lung cancer however recent studies showa higher incidence of lung cancer in young women comparedto young men even when the prevalence of cigarettesmoking among young women has approached but not exceededthat among men This suggests that the higher incidenceof lung cancer in women is not explained simply by genderdiï¬erences in smoking habits a deeper analysis of diï¬erencesmediated by sex such as greater sensitivity to tobacco smoke inwomen is warranted Furthermore men and women develop diï¬erent specifictypes of lung cancer Malignant mesothelioma is more commonin men while women develop more adenocarcinoma particularly nonsmall cell lung cancer NSCLC Womenhave a superior survival rate for lung cancer compared tomen Tumorassociated macrophages are critical in tumorprogression yet how androgens ‚uence macrophage behaviorin lung cancer and in responses to treatment must be addressedmore deeply to develop better therapies and increase survivalrates in menTHE MYELOID IMMUNE SYSTEM IN LUNGHEALTH AND DISEASEAlveolar MacrophagesThe lungs are a primary interface with the external environmentThe delicate structures needed for gas exchange make themsusceptible to damage from invading pathogens and toxicmolecules Some insults to the lung can lead to the developmentof chronic conditions such as allergic asthma As a protectivemechanism alveolar macrophages clearspace ofinfectious toxic or allergenic ps to maintain homeostasisin the alveoli Thus alveolar macrophages have a dualthe airFrontiers in Immunology wwwfrontiersinAugust Volume 0cBecerraDiaz et alAndrogenAR in Lung MonocyteMacrophage Biologyfunction as ‚ammatory cells phagocytosing and killinginhaled bacteria or viruses and also as controllers ofthe‚ammatory immune response minimizing alveolar damageResident alveolar macrophages are seeded embryonically fromyolk sac and fetalliver monocytes “ In asthma andother lung diseases recruited alveolar macrophages derived fromblood monocytes can turn into pathogenic cells worseningthe condition Mouse alveolar macrophages arecharacterized by high surface expression of Siglec F and produceTGF TGF both supports AM development and theirmaintenance of immune homeostasis by induction of Tregs andsuppression of B and T cell proliferation Another importantfunction of AM is the clearance of surfactant AM from male andfemale mice respond diï¬erently to surfactant protein A SPA SPA acts as an opsonin and is important in clearanceof pathogens Sex diï¬erences in AM responses to surfactantcould aï¬ect bacterial clearance and regulate the production ofpro‚ammatory mediators The molecular mechanisms thatmediate these diï¬erences and how sex hormones change thisimportant AM function is an open questionIn the human lung there appears to be more diversity inthe subtypes of lung macrophages compared to mice The maindeterminant of the frequency of subtypes of macrophages inhumans appears to be their anatomicallocation within thelung AM are the predominantimmune cells in the lungairways bronchi and bronchioalveolar space Flow cytometricpanels have employed HLADR CD163 CD169 and CD206to diï¬erentiate between AM IM and monocytes Human AMwere identified as large highly autofluorescent CD14 CD16cells that also express CD206 CD169 and MARCO There appear to be two populations of AM distinguished byeither high or low expression of CD163 More recent approachesto characterize the macrophage populationsin the lunginvolve singlecell transcriptomic analysis Althoughmacrophages show a large variation in the transcriptionalphenotype expression of MARCO CCL18 APOC1 APOEPPARG and MRC1 was found in macrophages from healthydonors while CHI3L1 MARCKS IL1RN PLA2G7MMP9 and SPP1 were highly expressed in macrophages frompulmonary fibrosis patients Thus a second contributor todiversity is likely the activation state of the cells There are nodata that describe sex diï¬erences in human AM responses andthe eï¬ect of sex hormones on these cells From our mouse andhuman MDM studies we would predict that androgens augmentthe immune homeostatic functions of these cells in the malelung Further work is still needed to standardize characterizationof the diï¬erent subpopulations of human lung macrophagepopulations and their role in maintaining healthy lung functionand in diseaseIMsInterstitial MacrophagesInterstitial macrophagesanother macrophagepopulation found in the lung They are a minor populationof monocytederived macrophages which comprise“ of lung macrophages and are localized in the lungparenchyma IMs contribute to maintaining homeostasisthrough the spontaneous release of IL10 a cytokine thataredampens ‚ammation IMs can prevent the developmentof aberranttype allergic responses triggered by inhaledallergens and have been related to reduction of asthma Diï¬erent subpopulations of IMs have been foundin the lung however their characterization has not arrived at aconsensus due to difficulties in their identification and isolationIn the mouse lung diï¬erent subpopulations of IMs have beendescribed based on the expression of surface markers One reportdescribed three diï¬erent subpopulations of IMs based on thediï¬erential expression of pro‚ammatory cytokines chemokineligands MHCII CD11c CD206 and Lyve1 other groupidentified two subpopulations based on similar markers butincluding CX3CR1 Moreover IMs subpopulations canbe also described based on the diï¬erent anatomic locationsthese cells populate inside the mouse lung parenchyma Further work is needed to better characterize and define thediï¬erent IM populations as the diï¬erent subtypes may havediï¬erent functions during the ‚ammatory process Smallerin size than their AM counterparts human IMs express moreof the monocytic marker CD14 than AM perhaps suggestingtheir monocytic origin and have lower expression of CD169than human AM The responses of IM to androgen will dependon their expression of AR which has not been measured Thiswill be a challenge due to difficulties in clearly identifying thispopulation and its subpopulations from the monocytic AMand other myeloid populations in the lungMonocytesMonocytes are produced in the bone marrow along with anumber of other myeloid cells Myeloid cells originate fromcommon pluripotent hematopoietic stem cells and representthe major subset of white cells in circulation These cellscomprise basophils neutrophils eosinophils DCs monocytesand macrophages among others Monocytes are releasedinto circulationthen blood monocytes are recruited into‚amed tissue and can mature into macrophages or dendriticcells There are two main subsets of mouse monocytesœclassical or Ly6Chigh monocytes that originate directly fromLy6C precursors and œnonclassical or Ly6Clow monocytesthat derive from Ly6Chigh monocytes The origin ofLy6C low monocytes was demonstrated by Sunderkotteret al by tracking the maturation of DiIlabeled Ly6Chighmonocytes into DiIlabeled Ly6Clow monocytes Thisprocess depends on the transcription factor Nr4a1 whichregulates the development and survival of Ly6Clow monocytes These two monocyte subsets mirror the human CD14classical and CD16 nonclassical monocyte populationsrespectively Ly6Chigh monocytes highly express thechemokine receptor CCchemokine receptor CCR2 whereasLy6Clow monocytes highly express CX3CR1 ImportantlyCCR2 expression is required for Ly6C monocyte egress fromthe bone marrow into the circulation and entry into non‚amed and ‚amed tissues “ from the blood As monocytes migrate into tissue they mature into macrophagesdeveloping unique tissuedependent morphology and functions They lose expression of Ly6C and gain expression ofMHC class II becoming more efficient antigenpresenting cellsFrontiers in Immunology wwwfrontiersinAugust Volume 0cBecerraDiaz et alAndrogenAR in Lung MonocyteMacrophage Biology Some authors have proposed the concept of œtissuemonocytes which are monocytes that can enter nonlymphoidans without obligatory diï¬erentiation into macrophagesTherefore monocytes are much more than simply precursorsfor macrophagesIn human lungs monocytes which can be both beneficialand pathogenic in a variety of pulmonary diseases arepresent at steady state Multiplecolor cytometric analysison cells obtained from diï¬erent anatomical locations of the lungof healthy subjects nonsmokers with normal lung function andabsence of disease or infection revealed that while intermediatemonocytes CD14CD16 are more frequent in the airwaysclassical monocytes CD14CD16ˆ’ are more frequent in blood Moreover the diï¬erent monocyte subsets produced TNFα to diï¬erent degrees upon stimulation with TLR ligands and Thus the anatomic location where samples are obtainedshould be considered and reported when working with humanbronchoscopies as this may alter the type and abundance ofmonocytes and macrophages found Accurate identification ofmonocytes in the lung compartments in humans has been achallenge because monocytic œcontamination from the bloodvessels Overcoming this challenge Desch et alperformed a flow cytometric phenotyping study and identifiedtwo additional lung monocyte populations by analyzing lungsobtained from donors who died of nonpulmonary causes CD14 CD206ˆ’ CD1cˆ’ CD1aˆ’ intravascular monocyteswere similar to CD14 blood monocytes and CD14 CD206CD1cˆ’ CD1aˆ’ monocytes were described as tissue œmonocytesThese studies highlightthe beginningof understanding the complexity of lung monocyte subtypesand their functions depending on the ‚ammatory state ofthe lungthat we are just atOther myeloid populationslike DCs occupy the lungparenchyma at steady state and their relative numbers changeduring ‚ammation We refer readers to previous excellentreviews in this journal that cover the importance of DCs inimmune responses in the lung and how they are aï¬ectedby sex diï¬erences Therefore we will not discuss DCs here “Macrophage ActivationPolarization is a very important eï¬ector characteristic observedin monocytes and macrophages Polarization refers to the changein phenotype and function of monocytes and macrophagesas they are exposed to diï¬erent‚ammatory milieus orfactors in the tissue microenvironment To understand theeï¬ects of the diï¬ering ‚ammatory or tissue environments onmonocytemacrophage phenotype and function researchershave used cytokines and other factors in vitro to mimic diï¬erent‚ammatoryand tissue microenvironments Monocytesand macrophages stimulated with interferonγ LPS TNFαinterleukin IL12colonystimulating factor promote a pro‚ammatory macrophagephenotype denoted as M1 polarization The activation state wasalso known as œclassical activation M1polarized macrophagesmediate immunity to intracellular infections such as viruses andand granulocytemacrophagebacteria and they are generally considered tumoricidal “ M1 macrophages accomplish these functions by inducingproduction of nitric oxide reactive nitrogen intermediatesreactive oxygen species and hydrogen peroxide “ Incontrast activation of macrophages with IL4 or IL13 as inextracellular parasitic infections and allergic reactionsleadsto M2 polarization or œalternative activation of macrophages M2 macrophages produce ‚ammatory mediatorsand chemokines such as chitinaselike proteins IL13 CCL17 CCL18 CCL22 and CCL24 which activateTh2 cells and promote eosinophil ltration into the lungs In allergic asthma a Th2‚ammatory response to inhaledallergens drives lung macrophages toward an M2 phenotypeIncreased number and percent of M2 macrophages havebeen correlated with asthma severity and a decline in lungfunction in humans and mouse models “ SimilarlyM2 macrophages are the predominant subset seen in pulmonaryfibrosis and are responsible for fibrogenesis During COPDthe number of macrophages in airwayslung parenchymabronchoalveolar lavage fluid and sputum increases This increase may occur as a result of enhanced monocyterecruitment from circulation in response to chemokines suchas CCL2 and CXCchemokine ligand1 which are increased inthe sputum and bronchoalveolar lavage fluid of patients withCOPD Unlike in allergic asthma and pulmonary fibrosismacrophages in COPD are polarized toward an M1 profile In addition to aï¬ecting men and women diï¬erently anothercommonality of COPD is that macrophages both in the alveolarspace and in lung tissue present an altered activation phenotypeDiï¬erent concentrations of cytokines TNFα IL1 IL6 IL IL12 and chemokines CCL2 CCL5 CCL7 CCL13 CCL22IL8 CXCL9 and CXCL10 are found comparing smokers tohealthy subjects “ Thus the external provoking stimulusuniquely shapes macrophage phenotype and functionWhile the M1M2 designations are useful for in vitro studieswith stimulation with defined cytokines the in vivo phenotypeof macrophages exists on a spectrum somewhere in betweenthese two welldefined opposing phenotypes or does not fitthe paradigm at all For example M1 and M2 markers canexist simultaneously within the same cell in some cases “ The key factors dictating the macrophage phenotypeor activation state are the stage ofthe immune responseand the soluble factors and interactions in a particular tissuemicroenvironment For example the lung environment is richin GMCSF TGF and PPARγ and is critical for developmentof mature AMs after birth in both mice “and humans “ Furthermoreinteractions betweenCD200 on type II alveolar epithelial cells and CD200R on thesurface of the AM deliver regulatory signals to the AM toprevent pro‚ammatory signaling and macrophage activation Thus macrophage nomenclature has evolved as ourunderstanding of the phenotypes and functions of diï¬erenttypes of tissue resident macrophages recruited monocytes andmonocytederived macrophages advances Indepth studies ofthe eï¬ects of androgens and other sex hormones on tissuemacrophage plasticity and phenotype have yet to be carried outFrontiers in Immunology wwwfrontiersinAugust Volume 0cBecerraDiaz et alAndrogenAR in Lung MonocyteMacrophage BiologyMECHANISMS OF ANDROGEN SEXSTEROID ACTIONEFFECTS OF ANDROGEN EXPOSURE ONMONOCYTES MACROPHAGES IN VITROBecause androgens are lipophilic steroid hormones they caneasily diï¬use across cell membranes withoutthe need forreceptormediated import Androgens in circulation arefound mostly bound to sex hormonebinding globulin andalbumin Free unbound steroid sex hormones can signalthrough two diï¬erent mechanisms the classical ARlocate

cancer204

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: predominant male sex hormones drive the development andmaintenance of male characteristics by binding to androgen receptor AR As androgensare systemically distributed throughout the whole anism they affect many tissues andcell types in addition to those in male sexual ans It is now clear that the immunesystem is a target of androgen action In the lungs many immune cells express ARs andare responsive to androgens In this review we describe the effects of androgens and ARson lung myeloid immune cells”monocytes and macrophages”as they relate to healthand disease In particular we highlight the effect of androgens on lung diseases such asasthma chronic obstructive pulmonary disease and lung fibrosis We also discuss thetherapeutic use of androgens and how circulating androgens correlate with lung diseaseIn addition to human studies we also discuss how mouse models have helped to uncoverthe effect of androgens on monocytes and macrophages in lung disease Although therole of estrogen and other female hormones has been broadly analyzed in the literaturewe focus on the new perspectives of androgens as modulators of the immune systemthat target myeloid cells during lung ‚ammationEdited byFlavia BazzoniUniversity of Verona School ofMedicine and Surgery ItalyReviewed byPaola ParronchiUniversity of Florence ItalyTim WillingerKarolinska Institutet SwedenSandra O GollnickUniversity at Buffalo United StatesCorrespondenceNicola HellernhellerjhmieduKeywords androgen androgen receptor monocyte macrophage asthma lung sex difference sex hormoneSpecialty sectionThis was submitted toCytokines and Soluble Mediators inImmunitya section of the journalFrontiers in ImmunologyReceived March Accepted June Published August CitationBecerraDiaz M Song M and Heller N Androgen and AndrogenReceptors as Regulators of Monocyteand Macrophage Biology in theHealthy and Diseased LungFront Immunol 103389fimmu202001698INTRODUCTIONThe immune system is essential for maintaining homeostasis within tissues and ans andprotecting them against threats such as harmful pathogens or cancerous transformation Itcomprises both innate and adaptive components The innate immune system is made up of theinnate lymphoid innate lymphoid cells [ILCs] natural killer cells [NKs] and lymphoid tissueinducers [LTi] and innate myeloid subsets The innate immune system consists of a networkof immune cells and molecules that provide rapid firstline defense against pathogens In contrastthe adaptive immune response made up of B and T lymphocytes takes days or even weeks tobecome established Innate immune cells express pattern recognition receptors that recognizeunique and conserved pathogenassociated molecular patterns such as lipopolysaccharide LPSviral ssRNA and fungal glucan B and T cells have evolved to recognize a finer repertoireof self and nonselfantigens that facilitate pathogenspecific actions immunologic memorygeneration and host immune homeostasis regulation To accomplish this the adaptiveimmune response involves a tightly regulated interplay between T and B lymphocytes andFrontiers in Immunology wwwfrontiersinAugust Volume 0cBecerraDiaz et alAndrogenAR in Lung MonocyteMacrophage Biologyantigenpresenting cells of the myeloid lineage such as dendriticcells DCs monocytes and macrophages Myeloid cells arisefrom the bone marrow The type and magnitude of the immuneresponse is ‚uenced by biological sex and age and thereforediï¬ers between males and females Sex diï¬erences in the functionof the immune system arise from both genetic chromosomalsex diï¬erences and diï¬erences mediated by the action of maleand female sex hormones Because the concentration of sexhormones changes over the lifespan and throughout the courseof the menstrual cycle in women the function of the immunesystem also changes during diï¬erent stages of life Innate myeloidimmune cells like other cell types express sex hormone receptorsand are responsive to sex hormones Sex hormones are synthesized from cholesterol through adefined enzymatic cascade predominately in the gonads and theadrenal glands Sex hormones are also produced in othertissues including the brain placenta mammary glands liver andadipose tissue “ In addition to driving sexual developmentof egg and sperm production sex hormones are responsiblefor the development of male and female secondary sexualcharacteristics like breast development and growth of facial hairthat occur during puberty Androgens include testosteronedihydrotestosterone DHT androstenedione androstenedioland dehydroepiandrosterone DHEA with DHT being the mostpotent The concentration of androgens in circulation isabout sevenfold higher in adult men than in adult women Estradiol and progesterone are the predominantfemale sex hormones synthesized by the ovaries andadrenal glands Both male and female sex hormones are boundto the plasma proteins albumin and sex hormone bindingglobulin SHBG and only a small percentage exists as freehormone “ Thus the bioavailability of sex hormones isregulated by their biosynthesis and also the amount of albuminand SHBGImportantly sex hormones mediate not only anatomicdiï¬erences between women and men but also direct sexdiï¬erences in immune responses leading to diï¬erent risks forimmunologic diseases Overall women have a greaterrisk for autoimmune diseases such as systemic sclerosis andsystemic lupus erythematosus whereas men are morelikely to die of infectious and parasitic diseases Moreovermen have a greater risk of nonreproductive cancers “Both gender and sex are important mediators of these andother health and disease diï¬erences observed between men andwomen While gender refers to the array of socially constructedroles attitudes personality traits and behaviors sex representsa biological characteristic of an individual includingthe hormonal milieu and chromosome complement Ingeneral estrogens are considered to have pro‚ammatoryproperties and androgens are thought to have anti‚ammatoryproperties In the United States and worldwide relevant evidence highlights important epidemiologic sexdiï¬erences in incidence susceptibility and severity of a numberof diseases that aï¬ect the respiratory tract In this reviewwe will focus on how male sex hormones the androgensmodulate the response of myeloid cells in the lung and howthis modulation impacts the outcome of diï¬erent diseases ofthe lungSEX DIFFERENCES IN HUMAN LUNG ANDLUNG DISEASESsex mediates diï¬erencesBiologicalin the incidence andpathophysiology of lung diseases These diï¬erences arise fromsex diï¬erences in the structure and function of the lung itselfand also in the immune cells that populate the lung and arerecruited to it during ‚ammation Before birth the female lunghas several structural advantages over the male lung Surfactantis produced earlier and although the female lung is smaller ithas more alveoli per unit area Neonatal females have higherexpiratory flow rates than do male neonates when corrected forsize Thus male sex is a major risk factor for the developmentof respiratory distress syndrome bronchopulmonary dysplasia inneonates “ and asthma in childhood In addition to the contribution of structural diï¬erences ofthe lung between the sexes sex diï¬erences in lung function andlung diseases are also dependent on the action of sex hormonesWe have summarized some broad concepts that define howtestosterone and estrogen aï¬ectlung macrophage functionand how this may contribute to the outcome of particularlung diseases in Figure As testosterone rises after pubertythe immunosuppressive eï¬ects of this hormone on protectiveimmune responses to infectious diseases in males can worsenpulmonary disease This would be exemplified by tuberculosisor ‚uenza Some of these eï¬ects are a result of androgeneï¬ects on critical ‚ammatory macrophage functions althoughthe eï¬ects on the adaptive immune system also have a significantcontribution to the overall outcome Thus testosterone appearsto play a key immunoregulatory role in lung macrophagesTestosterone™s immunoregulatory properties also appear to bedependent on the amount of cellular expression of AR andon the concentration of the hormone Low concentrations oftestosterone have been noted in patients with asthma COPD andtuberculosis Low testosterone may also be linked to insufficientcontrol of tissuedamaging ‚ammatory responses seen inCOPD and pulmonary fibrosis Estrogen tends to promotewound healing responses in macrophages Dysregulation ofwound healing responses and overactive tissue remodelingmacrophages in the lung could be broadly used to describe theTh2 response in allergic asthma which is worse in womenCancer could also be considered an aberrant wound healingresponse driven by M2like tumor associated macrophages Wehave highlighted here how sex hormones contribute to changesin lung macrophage function that contribute to lung diseaseHowever it should be pointed out that not every sex diï¬erencein lung disease is due to direct eï¬ects on macrophages but on thebroader coordinated immune response as a wholeAsthmaBefore puberty the structural diï¬erences in the lung as wellas gender diï¬erences likely account for the higher incidence ofFrontiers in Immunology wwwfrontiersinAugust Volume 0cBecerraDiaz et alAndrogenAR in Lung MonocyteMacrophage BiologyFIGURE Sex differences in lung diseases discussed in this Review and how they may be connected to the effects of androgens and estrogens on ‚ammatorymacrophages in the lungasthma in boys than in girls With the onset of puberty male andfemale sex hormones and their eï¬ects on the structural cells ofthe lung and on the immune system contribute to the incidenceof asthma The incidence and severity of asthma aregreater in adult women than in adult men and greaterin female than in male mice Female sex hormones suchas estrogen appear to worsen asthma although a straightforwardcorrelation between amount of female sex hormone and asthmasymptoms has not been concluded Androgens have multipleimmunoregulatory and bronchodilatory functions and maycontribute to or be biomarkers for better lung function inmen Accordingly serum testosterone is low in men withmoderate to severe asthma “ In one study each ngdLincrease in serum testosterone correlated with a CI P decrease in the likelihood of having asthma On the other hand high concentrations of testosterone andcyclic AMP in sputum of asthmatic women during the lutealphase of the menstrual cycle were thought to play a role inpremenstrual exacerbations The idea that sex hormonesmay be a causal factor in asthma was significantly strengthenedby a recent study of adults that quantified serum sexhormones and asthma outcomes That study showed thatlow testosterone in both women and men was associated with anincreased incidence of asthma The other interesting finding wasthat higher testosterone was protective against asthma in obesewomen Obesity is a risk factor for asthma “ Thereforehow high body mass index BMI and circulating sex hormonestogether aï¬ect asthma requires further investigationAnother androgen dehydroepiandrosterone DHEA alsoknown as androstenolone is an endogenous steroid hormoneand one of the most abundant circulating steroids in humansIt is a precursor for the synthesis of both testosterone andestrogen DHEA is sulfated at the C3 position into DHEAS by the action ofthe sulfotransferase enzymes SULT2A1and SULT1E1 in the adrenal glands The amount of DHEAS in the circulation is ˆ¼“ times those of DHEADHEA became of interest to the asthma field because womenwith severe asthma had very low concentrations of DHEAS and DHEAS concentration correlated with lung function Interestingly DHEAS is suppressed by oral or inhaledglucocorticoids the mainstay therapy for asthma HumanDHEA peaks at around age and then follows an agedependentdecline until they reach prepubertal concentrations Reducedsecretion of DHEA with age has been related to a numberof ageassociated conditions Replacement of DHEA has beenconsidered as a possible therapeutic that could activate protectiveresponses in an aging immune system DHEA is known todownregulate Th2‚ammatory cytokines while upregulatingIL2 synthesis in concanavalin Astimulated peripheralblood mononuclear cells from adult males with atopic dermatitis Thus it was hypothesized that it would be a usefultreatment for atopic diseases including asthma and the results ofthe clinical trials for DHEA in asthma patients show promiseThe results are discussed in a later section titled œEï¬ects ofandrogen exposure on monocytes macrophages in humans withlung diseaseCOPDSex diï¬erences also have been reported in chronic obstructivepulmonary disease COPD a heterogeneous chronic andprogressive respiratory disorder that includes chronic bronchitisand emphysema Chronic exposure of the airways to insultssuch as cigarette smoke leads to epithelial cell injury destructionof pulmonary capillary vasculature acceleration of epithelial cellsenescence and airway remodeling The loss of lung complianceultimately leads to COPD COPD was previously thoughtto aï¬ect mostly elderly men primarily because of the higherprevalence of smoking in men However as smoking ratesincreased in women the number of COPD cases in womenexceeded that of men These diï¬erences are not only basedon gender as women develop more severe COPD with earlyonset disease years and have greater susceptibility toCOPD with lower tobacco exposure Moreover increasingage in female smokers leads to a faster annual decline inFrontiers in Immunology wwwfrontiersinAugust Volume 0cBecerraDiaz et alAndrogenAR in Lung MonocyteMacrophage Biologyforced expiratory volume in the first second when compared tothat of male smokers even when they smoke fewer cigarettes Similarly pulmonary fibrosis is another lung disease thatmanifests sex diï¬erences with men being more aï¬ectedthan women It is characterized by destruction of thepulmonary parenchyma and deposition of extracellular matrixwith alterations in phenotype of both fibroblasts and alveolarepithelial cells InfluenzaThe lungs are also the target of respiratory viruses such as‚uenza A œï¬‚u respiratory syncytial virus and coronavirusessuch as severe acute respiratory syndrome and the MiddleEast respiratory syndrome The viruses infectthe airwayepithelial cells and cause damage to the epithelial barrierby themselves or as a result ofthe immune response tothe viralinfection Sex diï¬erences have been noted in theimmune response to ‚uenza A virus and to the ‚uenzavaccine In general women have a more robust protectiveimmune response to ‚uenza virus and vaccine than do menAlthough this elevated response is helpful in clearing viruswomen of reproductive age also experience higher mortalityand hospitalizations “ possibly from collateral tissuedamage to the lungs The vigorous immune response in womenalso means that women experience more adverse events aftervaccination Indeed a systems biology approach identifiedthat high testosterone was correlated with a blunted responseto the flu vaccine in men As testosterone wanes in elderlymen mortality increases Since the male immune responseto the virus is also less robustthe incidence of seasonalflu is generally higher in men than in women in developedcountries according to the World Health anization It is not yet known how fluctuations in sex hormones acrossthe menstrual cycle and lifespan aï¬ect the immune system™sresponse to the ‚uenza virus in humans Mouse studieshave revealed that estrogen is protective at high but notlow concentrations On the other hand testosteronereplacement in gonadectomized or aged male mice enhancedsurvival rates Despite these findings in mouse modelsstudies examining the eï¬ect of sex hormones on cellular andmolecular mechanisms in human immune cells during ‚uenzainfection are lackingTuberculosisLike ‚uenza infection tuberculosis TB a lung disease causedby Mycobacterium tuberculosis exhibits notable sex diï¬erencesin the number of cases worldwide with men being almosttwice as frequently aï¬ected than women Both sexand gender diï¬erences impact the incidence of TB AlthoughTB aï¬ects less women than men in adulthood womenin their economically active years “ years old have ahigher TB incidence compared to women in other age groups This indicates that factors associated with gender such asexposure to the bacteria are important in this disease Howeverbecause male predominance does not occur in children thissuggests that biological factors such as male sex hormones alsoplay a significant role This is supported by a study ofmedically castrated men who experienced a significantly smallerproportion of death from TB compared to in intactmen Understanding how androgens lead to the greatersusceptibility of men to TB is critical as TB is still one ofthe leading fatal infectious diseases worldwide and may alsomay favor the development of other diseases such as lungcancer Lung CancerLung cancer is a very complex disease that depends on anumber of variants such as sex gender race and socioeconomicstatus The development of lung cancer is also related toenvironmental factors such as pollution due to industrializationand urbanization An additional genderassociated riskfactor significantly linked to developing lung cancer is cigarettesmoking Historically more men develop lung cancer andsuï¬er lung cancerassociated deaths compared to women However the incidence of lung cancer has changed notably inboth women and men In men lung cancer incidence startedto increase in the 1920s and started to decrease in the early1990s while in women the mortality rates and incidence beganto rise in the 1960s Changes in smoking habits in the lastseveral decades with a rise in the number of women who smokecorrelate with an increase in the incidence of lung cancer in thisdemographic group Smoking is definitely a key factor inthe development of lung cancer however recent studies showa higher incidence of lung cancer in young women comparedto young men even when the prevalence of cigarettesmoking among young women has approached but not exceededthat among men This suggests that the higher incidenceof lung cancer in women is not explained simply by genderdiï¬erences in smoking habits a deeper analysis of diï¬erencesmediated by sex such as greater sensitivity to tobacco smoke inwomen is warranted Furthermore men and women develop diï¬erent specifictypes of lung cancer Malignant mesothelioma is more commonin men while women develop more adenocarcinoma particularly nonsmall cell lung cancer NSCLC Womenhave a superior survival rate for lung cancer compared tomen Tumorassociated macrophages are critical in tumorprogression yet how androgens ‚uence macrophage behaviorin lung cancer and in responses to treatment must be addressedmore deeply to develop better therapies and increase survivalrates in menTHE MYELOID IMMUNE SYSTEM IN LUNGHEALTH AND DISEASEAlveolar MacrophagesThe lungs are a primary interface with the external environmentThe delicate structures needed for gas exchange make themsusceptible to damage from invading pathogens and toxicmolecules Some insults to the lung can lead to the developmentof chronic conditions such as allergic asthma As a protectivemechanism alveolar macrophages clearspace ofinfectious toxic or allergenic ps to maintain homeostasisin the alveoli Thus alveolar macrophages have a dualthe airFrontiers in Immunology wwwfrontiersinAugust Volume 0cBecerraDiaz et alAndrogenAR in Lung MonocyteMacrophage Biologyfunction as ‚ammatory cells phagocytosing and killinginhaled bacteria or viruses and also as controllers ofthe‚ammatory immune response minimizing alveolar damageResident alveolar macrophages are seeded embryonically fromyolk sac and fetalliver monocytes “ In asthma andother lung diseases recruited alveolar macrophages derived fromblood monocytes can turn into pathogenic cells worseningthe condition Mouse alveolar macrophages arecharacterized by high surface expression of Siglec F and produceTGF TGF both supports AM development and theirmaintenance of immune homeostasis by induction of Tregs andsuppression of B and T cell proliferation Another importantfunction of AM is the clearance of surfactant AM from male andfemale mice respond diï¬erently to surfactant protein A SPA SPA acts as an opsonin and is important in clearanceof pathogens Sex diï¬erences in AM responses to surfactantcould aï¬ect bacterial clearance and regulate the production ofpro‚ammatory mediators The molecular mechanisms thatmediate these diï¬erences and how sex hormones change thisimportant AM function is an open questionIn the human lung there appears to be more diversity inthe subtypes of lung macrophages compared to mice The maindeterminant of the frequency of subtypes of macrophages inhumans appears to be their anatomicallocation within thelung AM are the predominantimmune cells in the lungairways bronchi and bronchioalveolar space Flow cytometricpanels have employed HLADR CD163 CD169 and CD206to diï¬erentiate between AM IM and monocytes Human AMwere identified as large highly autofluorescent CD14 CD16cells that also express CD206 CD169 and MARCO There appear to be two populations of AM distinguished byeither high or low expression of CD163 More recent approachesto characterize the macrophage populationsin the lunginvolve singlecell transcriptomic analysis Althoughmacrophages show a large variation in the transcriptionalphenotype expression of MARCO CCL18 APOC1 APOEPPARG and MRC1 was found in macrophages from healthydonors while CHI3L1 MARCKS IL1RN PLA2G7MMP9 and SPP1 were highly expressed in macrophages frompulmonary fibrosis patients Thus a second contributor todiversity is likely the activation state of the cells There are nodata that describe sex diï¬erences in human AM responses andthe eï¬ect of sex hormones on these cells From our mouse andhuman MDM studies we would predict that androgens augmentthe immune homeostatic functions of these cells in the malelung Further work is still needed to standardize characterizationof the diï¬erent subpopulations of human lung macrophagepopulations and their role in maintaining healthy lung functionand in diseaseIMsInterstitial MacrophagesInterstitial macrophagesanother macrophagepopulation found in the lung They are a minor populationof monocytederived macrophages which comprise“ of lung macrophages and are localized in the lungparenchyma IMs contribute to maintaining homeostasisthrough the spontaneous release of IL10 a cytokine thataredampens ‚ammation IMs can prevent the developmentof aberranttype allergic responses triggered by inhaledallergens and have been related to reduction of asthma Diï¬erent subpopulations of IMs have been foundin the lung however their characterization has not arrived at aconsensus due to difficulties in their identification and isolationIn the mouse lung diï¬erent subpopulations of IMs have beendescribed based on the expression of surface markers One reportdescribed three diï¬erent subpopulations of IMs based on thediï¬erential expression of pro‚ammatory cytokines chemokineligands MHCII CD11c CD206 and Lyve1 other groupidentified two subpopulations based on similar markers butincluding CX3CR1 Moreover IMs subpopulations canbe also described based on the diï¬erent anatomic locationsthese cells populate inside the mouse lung parenchyma Further work is needed to better characterize and define thediï¬erent IM populations as the diï¬erent subtypes may havediï¬erent functions during the ‚ammatory process Smallerin size than their AM counterparts human IMs express moreof the monocytic marker CD14 than AM perhaps suggestingtheir monocytic origin and have lower expression of CD169than human AM The responses of IM to androgen will dependon their expression of AR which has not been measured Thiswill be a challenge due to difficulties in clearly identifying thispopulation and its subpopulations from the monocytic AMand other myeloid populations in the lungMonocytesMonocytes are produced in the bone marrow along with anumber of other myeloid cells Myeloid cells originate fromcommon pluripotent hematopoietic stem cells and representthe major subset of white cells in circulation These cellscomprise basophils neutrophils eosinophils DCs monocytesand macrophages among others Monocytes are releasedinto circulationthen blood monocytes are recruited into‚amed tissue and can mature into macrophages or dendriticcells There are two main subsets of mouse monocytesœclassical or Ly6Chigh monocytes that originate directly fromLy6C precursors and œnonclassical or Ly6Clow monocytesthat derive from Ly6Chigh monocytes The origin ofLy6C low monocytes was demonstrated by Sunderkotteret al by tracking the maturation of DiIlabeled Ly6Chighmonocytes into DiIlabeled Ly6Clow monocytes Thisprocess depends on the transcription factor Nr4a1 whichregulates the development and survival of Ly6Clow monocytes These two monocyte subsets mirror the human CD14classical and CD16 nonclassical monocyte populationsrespectively Ly6Chigh monocytes highly express thechemokine receptor CCchemokine receptor CCR2 whereasLy6Clow monocytes highly express CX3CR1 ImportantlyCCR2 expression is required for Ly6C monocyte egress fromthe bone marrow into the circulation and entry into non‚amed and ‚amed tissues “ from the blood As monocytes migrate into tissue they mature into macrophagesdeveloping unique tissuedependent morphology and functions They lose expression of Ly6C and gain expression ofMHC class II becoming more efficient antigenpresenting cellsFrontiers in Immunology wwwfrontiersinAugust Volume 0cBecerraDiaz et alAndrogenAR in Lung MonocyteMacrophage Biology Some authors have proposed the concept of œtissuemonocytes which are monocytes that can enter nonlymphoidans without obligatory diï¬erentiation into macrophagesTherefore monocytes are much more than simply precursorsfor macrophagesIn human lungs monocytes which can be both beneficialand pathogenic in a variety of pulmonary diseases arepresent at steady state Multiplecolor cytometric analysison cells obtained from diï¬erent anatomical locations of the lungof healthy subjects nonsmokers with normal lung function andabsence of disease or infection revealed that while intermediatemonocytes CD14CD16 are more frequent in the airwaysclassical monocytes CD14CD16ˆ’ are more frequent in blood Moreover the diï¬erent monocyte subsets produced TNFα to diï¬erent degrees upon stimulation with TLR ligands and Thus the anatomic location where samples are obtainedshould be considered and reported when working with humanbronchoscopies as this may alter the type and abundance ofmonocytes and macrophages found Accurate identification ofmonocytes in the lung compartments in humans has been achallenge because monocytic œcontamination from the bloodvessels Overcoming this challenge Desch et alperformed a flow cytometric phenotyping study and identifiedtwo additional lung monocyte populations by analyzing lungsobtained from donors who died of nonpulmonary causes CD14 CD206ˆ’ CD1cˆ’ CD1aˆ’ intravascular monocyteswere similar to CD14 blood monocytes and CD14 CD206CD1cˆ’ CD1aˆ’ monocytes were described as tissue œmonocytesThese studies highlightthe beginningof understanding the complexity of lung monocyte subtypesand their functions depending on the ‚ammatory state ofthe lungthat we are just atOther myeloid populationslike DCs occupy the lungparenchyma at steady state and their relative numbers changeduring ‚ammation We refer readers to previous excellentreviews in this journal that cover the importance of DCs inimmune responses in the lung and how they are aï¬ectedby sex diï¬erences Therefore we will not discuss DCs here “Macrophage ActivationPolarization is a very important eï¬ector characteristic observedin monocytes and macrophages Polarization refers to the changein phenotype and function of monocytes and macrophagesas they are exposed to diï¬erent‚ammatory milieus orfactors in the tissue microenvironment To understand theeï¬ects of the diï¬ering ‚ammatory or tissue environments onmonocytemacrophage phenotype and function researchershave used cytokines and other factors in vitro to mimic diï¬erent‚ammatoryand tissue microenvironments Monocytesand macrophages stimulated with interferonγ LPS TNFαinterleukin IL12colonystimulating factor promote a pro‚ammatory macrophagephenotype denoted as M1 polarization The activation state wasalso known as œclassical activation M1polarized macrophagesmediate immunity to intracellular infections such as viruses andand granulocytemacrophagebacteria and they are generally considered tumoricidal “ M1 macrophages accomplish these functions by inducingproduction of nitric oxide reactive nitrogen intermediatesreactive oxygen species and hydrogen peroxide “ Incontrast activation of macrophages with IL4 or IL13 as inextracellular parasitic infections and allergic reactionsleadsto M2 polarization or œalternative activation of macrophages M2 macrophages produce ‚ammatory mediatorsand chemokines such as chitinaselike proteins IL13 CCL17 CCL18 CCL22 and CCL24 which activateTh2 cells and promote eosinophil ltration into the lungs In allergic asthma a Th2‚ammatory response to inhaledallergens drives lung macrophages toward an M2 phenotypeIncreased number and percent of M2 macrophages havebeen correlated with asthma severity and a decline in lungfunction in humans and mouse models “ SimilarlyM2 macrophages are the predominant subset seen in pulmonaryfibrosis and are responsible for fibrogenesis During COPDthe number of macrophages in airwayslung parenchymabronchoalveolar lavage fluid and sputum increases This increase may occur as a result of enhanced monocyterecruitment from circulation in response to chemokines suchas CCL2 and CXCchemokine ligand1 which are increased inthe sputum and bronchoalveolar lavage fluid of patients withCOPD Unlike in allergic asthma and pulmonary fibrosismacrophages in COPD are polarized toward an M1 profile In addition to aï¬ecting men and women diï¬erently anothercommonality of COPD is that macrophages both in the alveolarspace and in lung tissue present an altered activation phenotypeDiï¬erent concentrations of cytokines TNFα IL1 IL6 IL IL12 and chemokines CCL2 CCL5 CCL7 CCL13 CCL22IL8 CXCL9 and CXCL10 are found comparing smokers tohealthy subjects “ Thus the external provoking stimulusuniquely shapes macrophage phenotype and functionWhile the M1M2 designations are useful for in vitro studieswith stimulation with defined cytokines the in vivo phenotypeof macrophages exists on a spectrum somewhere in betweenthese two welldefined opposing phenotypes or does not fitthe paradigm at all For example M1 and M2 markers canexist simultaneously within the same cell in some cases “ The key factors dictating the macrophage phenotypeor activation state are the stage ofthe immune responseand the soluble factors and interactions in a particular tissuemicroenvironment For example the lung environment is richin GMCSF TGF and PPARγ and is critical for developmentof mature AMs after birth in both mice “and humans “ Furthermoreinteractions betweenCD200 on type II alveolar epithelial cells and CD200R on thesurface of the AM deliver regulatory signals to the AM toprevent pro‚ammatory signaling and macrophage activation Thus macrophage nomenclature has evolved as ourunderstanding of the phenotypes and functions of diï¬erenttypes of tissue resident macrophages recruited monocytes andmonocytederived macrophages advances Indepth studies ofthe eï¬ects of androgens and other sex hormones on tissuemacrophage plasticity and phenotype have yet to be carried outFrontiers in Immunology wwwfrontiersinAugust Volume 0cBecerraDiaz et alAndrogenAR in Lung MonocyteMacrophage BiologyMECHANISMS OF ANDROGEN SEXSTEROID ACTIONEFFECTS OF ANDROGEN EXPOSURE ONMONOCYTES MACROPHAGES IN VITROBecause androgens are lipophilic steroid hormones they caneasily diï¬use across cell membranes withoutthe need forreceptormediated import Androgens in circulation arefound mostly bound to sex hormonebinding globulin andalbumin Free unbound steroid sex hormones can signalthrough two diï¬erent mechanisms the classical ARlocate Answer:

Thyroid_Cancer

progression of breast cancerare greatly affected by the immune environment Neutrophils are the most abundantleucocytes in circulation and act as the spearhead in ‚ammationincluding inbreast cancer Circulating neutrophils are closely related to the prognosis of breastcancer patients and tumorltrating neutrophils have varied functions at differentstages of breast cancer such as antitumor or tumorpromoting neutrophils which aretermed N1 and N2 neutrophils respectively In this review we will discuss the utilityof circulating neutrophils for predicting prognosis and therapeutic efficacy and theunderlying mechanisms of their chemotaxis the dynamic regulation of their antitumoror protumor functions and their different spatial distributions in tumor microenvironmentFinally we also discuss the possibility of targeting neutrophils as a therapeutic strategyin breast cancerKeywords breast cancer immunotherapy neutrophils neutrophiltolymphocyte ratio tumor microenvironmentSpecialty sectionThis was submitted toCancer Immunity and ImmunotherapyINTRODUCTIONa section of the journalFrontiers in ImmunologyReceived May Accepted July Published August CitationZhang W Shen Y Huang H Pan SJiang J Chen W Zhang T Zhang Cand Ni C A Rosetta Stone forBreast Cancer Prognostic Value andDynamic Regulation of Neutrophil inTumor MicroenvironmentFront Immunol 103389fimmu202001779Breast cancer BC is the most common malignancy in women worldwide Although BCis classified as a malignant disease with low immunogenicity recent evidence has revealeda promising outcome of therapies with blocking immune checkpoints in both early andadvanced stages “ The efficacy of immunotherapy is closely related to the tumor immunemicroenvironment especially to ltrating immune cells To date macrophages and Tcells are the most wellstudied immune cells in BC whereas increasing evidence has indicatedthat neutrophils are also key in the oncogenesis and metastasis of BC in addition circulatingneutrophils have been reported to have great prognostic prediction value Neutrophils are themost abundant leucocytes in blood and usually act as the first line of host defense against pathogens However due to their short life span an average of “ h in blood it is difficult to employthis subset of cells for experiments which has resulted in a poor understanding of their role in solidtumors In addition some contradictory results reported in vitro studies or animal experimentshave suggested a dual eï¬ect of neutrophils in tumor developmentFrontiers in Immunology wwwfrontiersinAugust Volume 0cZhang et alNeutrophils in Breast CancerNeutrophils can present both antitumorigenic œN1 andprotumorigenic œN2 phenotypesin various cancers orspecific circumstances The term neutrophil in several studiesalso includes both mature neutrophils and myeloidderivedsuppressor cells MDSCs MDSCs are described as a subsetof neutrophils with immunosuppressive functions that expressCD11b and Gr1 and can be divided into monocyticM CD11bLy6C MDSCs and GPMN CD11bLy6GMDSCs and GPMN MDSCs usually share a commonset of markers and similar morphologicalfeatures withneutrophils To avoid confusion we mainly focus on the biologicalfunction of mature neutrophils and related therapeutic strategiesfor targeting them in BC We provide a comprehensive reviewofthe prognostic value of circulating neutrophils and themechanisms of how tumorassociated neutrophils TANs exertantitumor or tumorpromoting functions in BC and in theend we also discuss the potential of targeting neutrophils as atherapeutic strategy in cancerPROGNOSTIC VALUE OF THENEUTROPHILTOLYMPHOCYTE RATIONLRTumors can be thought of as wounds that will not heal andare characterized by chronic ‚ammation Neutrophils are themost rapidly responding immune cells to ‚ammation andmany studies have found that the NLR is closely related to theprognosis and treatment response in patients bearing BC A recent metaanalysis of studies including patientswith both early and advanced BC revealed that patients witha higher NLR before treatment had poorer diseasefree survivalDFS than those with a lower NLR before treatment but theNLR was not related to overall survival OS the subgroupanalysis found that the NLR was associated with prognosisonly in earlystage patients but not in patients with metastasisAbbreviations BC Breast cancer MDSCs Myeloidderived suppressor cellsTANs Tumorassociated neutrophils NLR Neutrophiltolymphocyte ratio DFSDiseasefree survival OS Overall survival ALC Absolute lymphocyte count NCTNeoadjuvant chemotherapy pCR Pathological complete response PLR Platelettolymphocyte ratio TAMs Tumorassociated macrophages CTCs Circulatingtumor cells NETs Neutrophil extracellular traps MPO MyeloperoxidaseGCSF Granulocyte colonystimulating factor ECs Endothelial cells PMNsPolymorphonuclear neutrophils ICAM1 Intercellular adhesion molecule MMP9 Matrix metalloproteinases9 ROS Reactive oxygen species HMGB1Highmobility group box TLR4 Tolllike receptor TNBC Triplenegativebreast cancer MES Macrophageenriched subtype NES Neutrophilenrichedsubtype H2O2 Hydrogen peroxide TNFα Tumor necrosis factorα HOCIHypochlorous acid TRPM2Transientcation channelsubfamily M member ADCC Antibodydependent cellular cytotoxicityNK Natural killer NE Neutrophil elastase NRP1 Neuropilin1 IRS1 Insulinreceptorsubstrate1 PI3K Phosphatidylinositol 3kinase VEGF Vascularendothelial growth factor TIMP1 Tissue inhibitor of matrix metalloproteaseTGF Transforming growth factor 27HC 27hydroxycholesterol PAD4Peptidyl arginine deiminase TINs Tumorltrating neutrophils CRTConventionalradiotherapy MRT Microbeam radiation therapy DAMPsDamageassociated molecular patterns ICB Immune checkpoint blockadeLDNs Lowdensity neutrophils HDNs Highdensity neutrophils NAMPTNicotinamideadeninedinucleotide GTX granulocyte transfusiontransferase NAD Nicotinamidereceptor potentialphosphoribosyl Since similar metaanalyses were not based on individualpatient data which may cause significant bias we reviewed andcompared the individual reports and found some issues worthdiscussing here Widmann first reported the correlationbetween the NLR and BC prognosis in patients and itwas found that a higher NLR ‰¥ before treatment was anadverse factor for both short and longterm mortality Themajority of retrospective studies thereafter have drawn similars “ and the NLR was found to be consistentamong diï¬erent BC subtypes at baseline However aprospective substudy of GEICAM9906 which comprised patients did not find any prognostic value of the NLR afteradjustment for clinicopathological factors in addition a highNLR was independently associated with worse DFS in only highrisk patients the hormone receptornegativeHER2 populationand in patients with ‰¥ lymph node metastases Anotherstudy with early BC patients also found that the NLR beforesurgery was not associated with DFS indicating that thepresurgery NLR may be valuable only in patients with a hightumor burdenseveralIn addition to the above studiesstudies alsoexplored the prognostic value of the NLR posttreatment or withcontinuous assessment A retrospective study comparing theabsolute lymphocyte count ALC and the NLR eight consecutivetimes before and after chemotherapy found that patients whodied had lower ALC and higher NLR values than patients whoremained alive throughout the treatment course additionallyamong the patients who died a steady increase in the NLRover the baseline measurement was observed at subsequenttime points Another retrospective study included BCpatients with DFS values of more than years and it interestinglyfound that NLR sampled during followup rather than beforeany treatment was an independent prognostic factor for laterecurrence However there is still no compelling explanationfor the abovementioned inconsistent results In addition sincelymphocytes are critical in cancer immune surveillance andneutrophils have been reported to play a protumor role in moststudies low lymphocytes and high neutrophils in circulationmay also suggest immunosuppression status and studiesfocused on the relationship between neoadjuvant chemotherapyNCT and the NLR might support the above hypothesis Acomprehensive review of the existing reports shows that moststudies have found that a low NLR indicates a higher NCTresponse and pathological complete response pCR rate “in addition the NLR has showed predictive value not only inall molecular types of BC but also in both operable and locallyadvanced BC Interestingly although Suppan et aldid not find a significant correlation between the initial NLR andprognosis the same cohort revealed a low NLR as a significantparameter for predicting chemotherapy response p A low NLR was also reported to be associated with a higherresponse rate to primary endocrine therapy for locally advancedor metastatic BC Although increasing evidence suggests a close associationbetween the NLR and prognosis in BC several issues remain thatmake clinical application difficult One of the most importantreasons is the lack of a consensus cutoï¬ value As we listhere Table the cutoï¬ values for the NLR in the publishedFrontiers in Immunology wwwfrontiersinAugust Volume 0cZhang et alNeutrophils in Breast CancerTABLE Characteristics of the studies related to neutrophiltolymphocyte ratioReferencesCountry Study periodCancer typeMedianage ysNo patientslowhigh NLRTreatmentFollowupSignificance of NLRNR ysHigh NLR indicates lowersurvival rate p Surgery NCT moSurgery ysNR moUnivariate analysis indicateshigh NLR related to lower RFSp and OS p High NLR indicates lower5year OSMultivariate analysis indicateshigh pretreatment NLR iscorrelated with poor DFS p and OS p Adjuvanttranstuzumab moHigh pretreatment NLRindicates shorter DFSNoh Korea“Koh Korea“Yao China“Luminal ABHER2enrichedTNBCERPRpositiveHER2enrichedLuminal ABERPRpositiveHER2enrichedTNBCPistelli Italy“TNBCUlas Turkey“HER2enrichedJia China“ER PRpositiveHER2enrichedTNBCBozkurt Turkey“TNBCAsano Japan“TNBCn NLR n ‰¤ NLR ‰¥ n n NLR ‰¤ n NLR n n NLR NLR n NLR ‰¤ n NLR n n NLR n NLR n n NLR n NLR ‰¤ n n NLR ‰¤ n NLR n n NLR n NLR n NCT surgery mo moSurgeryadjuvantchemotherapyandradiotherapyNCT ysMultivariate analysis indicateslow NLR is related to superiorDFS p and p Multivariate analysis indicateshigh pretreatment NLR iscorrelated with poor DFS p and OS p Univariate analysis indicateslow NLR is related to favorableprognosis in TNBC patientswho achieved pCR p hazard ratio High pretreatment NLRindicates poor allcausemortality with a multivariableHR of CI“High NLR upon recurrenceindicates shorter OSrecurrence rates p Low NLR indicates higher OSp Rimando USA“Nonmetastatic BC n NLR ‰¤ n NLR n Radiotherapychemotherapy moIwase Japan“TNBCn NRL n NLR ChemotherapyNRNCT surgery moHernandez et alSpain“Miyagawa Japan“Ferroni Italy“Luminal ABERPRpositiveHER2enrichedTNBCLocally Advanced orMetastatic BCLuminal ABHER2enrichedTNBCn NLR n NLR ‰¥ n n NLR ‰¤ n NLR n Qiu China“NonmetastaticTNBCn NLR n NLR ‰¥ n EribulinNRLow NLR indicates better PFSp NCTchemotherapyendocrinetherapytrastuzumabregimensSurgery NCTchemotherapy moHigh pretreatment NLRindicates worse DFS HR and OS HR moLow NLR indicates higher OSp and DFS p ContinuedFrontiers in Immunology wwwfrontiersinAugust Volume 0cZhang et alTABLE ContinuedNeutrophils in Breast CancerReferencesCountry Study periodCancer typeMedianage ysNo patientslowhigh NLRTreatmentFollowupSignificance of NLRIimori Japan“Luminal ABHER2enrichedTNBCMando Argentina “Early stage BCLee Korea“TNBCXuan China“TNBCFujmoto Japan“With high counts oflymphocytesImamura Japan“HER2enrichedn NLR n NLR ‰¥ n n NRL n NLR ‰¤ n NLR n n NLR n NLR ‰¥ 293n n NLR n NLR n n NLR n NLR ‰¥ n Endocrinetherapy moSurgery moNCTNRSurgeryNRNRSurgeryadjuvantchemotherapiesendocrinetherapiesTrastuzumabemtansineNRLow NLR indicates aprolongation of PFS p and OS p High NLR indicates lower DFSp Low NLR indicates superiorOS p and DFS p Low NLR indicates longer DFSp Low NLR indicates better RFSp Low NLR at baseline indicatesbetter PFS p andOS p NLR Neutrophiltolymphocyte ratio ER Estrogen receptor PR Progesterone receptor HER2 Human epidermal growth factor receptor Mo Months Ys Years DFS Diseasefreesurvival OS Overall survival PFS Progressionfree survival RFS Relapse free survival pCR Pathological complete response TNBC Triplenegative breast cancer NCT Neoadjuvantchemotherapy NR Not recordedstudies were between and In addition based on individualstudies the sensitivity of the NLR fluctuates greatly “and the specificity is much lower “ Therefore some researchers have tried to determine a betteralternative parameter In addition to the NLR the platelettolymphocyte PLR ratio has also been investigated and comparedwith the NLR in BC A single central retrospective study with hormone receptornegative nonmetastatic BC patients reportedthat both elevated NLR and PLR were associated with poor OShowever the multivariate analysis revealed that only the NLR p but not the PLR p was a significant indicatorfor both DFS and OS Additionally since the absolutelymphocyte count has also been reported as a prognostic factorthe predictive values of the PLR and NLR were evaluated afteradjusting for the total lymphocyte count The results showed thatthe PLR was no longer a significant predictor for 5year mortalityand the NLR remained a significant predictor irrespective ofthe lymphocyte count Furthermore it was revealed thatthe combination of the NLR and PLR could further improvethe predictive value Two retrospective studies found that thehighest rate of pCR was in the group of patients withan NLRlowPLRlow profile and the lowest rate was inthe group with an NLRhighPLRhigh profile in additionwhen the cutoï¬ values for the NLR and PLR were applied thespecificity of predicting a pCR increased from to Howeverthe causal relationship between the NLR andpoor prognosis in malignant disease has yet to be illuminatedAccording to an assessment with paired peripheral bloodand pancreatic cancer specimens Takakura found thata high NLR was associated with increased tumorassociatedTAMsanditseemsdecreased Thereforetumorassociatedmacrophageslymphocytes but was not significantly related to CD66bltrating neutrophilsthat anincrease in neutrophils in peripheral blood is not necessarilyrelated to the number of TANs Several basic studies havesuggested a unique mechanism ofthe protumor functionof circulating neutrophils protecting circulating tumor cellsCTCs Circulating neutrophils can cluster around tumor cellsand induce tumor cell aggregation aiding tumor cell survivalby hiding them from immune surveillance Neutrophilextracellular traps NETs are webs of decondensed chromatbers conjugated together with histones myeloperoxidaseMPO elastase and other cytoplasmic proteins Recentstudies also found that neutrophils could form many NETs bothin circulation and in tumor lesions and could coordinate withplatelets to capture CTCs and facilitate cancer metastasis In addition neutropenia is very common in cancer patientsundergoing chemotherapy and supportive treatment withgranulocyte colonystimulating factor GCSF can inducea neutrophilic response as a consequence neutrophils areprimed toward a proNETotic phenotype and may suppress thecytotoxic activity of T cells as well as impair immune surveillance On the other hand lymphocytes have the propensityto mount an adaptive antitumor response in malignant disease and decreased lymphocyte numbers are considered to berelated to an insufficient immunologic reaction which mayincrease the risk of tumor relapse or metastasis Clearlya general association between prognosis and the NLR exists inBC but large prospective studies and rigorous research are stillrequired to determine its clinical significanceFrontiers in Immunology wwwfrontiersinAugust Volume 0cZhang et alNeutrophils in Breast CancerMECHANISM OF NEUTROPHILCHEMOTAXIS TO THE TUMORMICROENVIRONMENTNeutrophils are considered the main immune cells that provideprotection against invading pathogens which can be induced bytrauma infection and malignant disease The recruitmentof neutrophils is greatly dependent on certain chemokinesincluding interleukin IL8 also known as CXCL8 CXCL and CXCL2 IL8 is a pro‚ammatory cytokineand acknowledged as the most important chemoattractant forneutrophils in the tumor microenvironment IL8 mainlycomes from endothelial cells ECs and monocytes in the tumormicroenvironment upon certain stimulation such as physicalinjury hypoxia chemotherapy or radiotherapy and other celltypes including fibroblasts and keratinocytes can secrete IL8 aswell In addition to its chemotactic eï¬ect it was revealedthat IL8 could provoke neutrophils to release NETs to assistcancer cell migration By livecell fluorescence microscopyGupta confirmed that activated ECs could induceNETosis characterized by typical extracellular DNA latticeswhen cocultured with polymorphonuclear neutrophils PMNsand activated ECs In addition activated ECs produceother ‚ammatory cytokines such as Pselectin Eselectinand intercellular adhesion molecule ICAM1 to facilitateneutrophil adhesion to ECs and migration Furthermoretumorpromoting neutrophils in BC cells are also characterizedby high expression of matrix metalloproteinases9 MMP9 which was found to cleave CXCL5 potentiating itsaction in neutrophil recruitment as a positive feedback functionin tumors IL17 was also found to control neutrophilrecruitmentin lung metastasis of BC in a mouse modelCD3CD4 and γδ T cells were the major sources of IL17 and it was interesting to find that the absence of γδT cells or neutrophils markedly reduced pulmonary and lymphnode metastases without ‚uencing primary tumor progressionwhich suggested a collaborative relationship between γδ T cellsand neutrophils in promoting BC lung metastasis Howeverin an orthotopic hepatocellular carcinoma model Sofia et alreported that TANs exert an overt antitumor role by suppressingγδ T17 cells via reactive oxygen species ROS contraryto the phenomenon that within the 4T1derived BC modelCD11bLy6G neutrophils that ltrate and surround livermetastases were found to be tumor promoting Thesecontroversial results suggest both promoting and suppressiveroles of TANs in diï¬erent circumstancesHighmobility group box HMGB1 usually acts as adamageassociated molecular pattern that is released by dyingcells or stressed cells to initiate ‚ammation and was laterfound to be an important chemoattractant for neutrophils Epithelial cellderived HMGB1 was found to recruit neutrophilsto the necrotic site through its receptor RAGE Enrichmentof platelets has been reported in the microenvironment ofmultiple cancers including BC and ltrating plateletscould be activated by the large amounts of adenosine phosphatereleased by necrotic cells as a result of chemotherapy Activated plateletderived HMGB1 known as the major mediatorof injuryinduced thrombosis in vivo can also stimulateNETosis through Tolllike receptor TLR4 and RAGE onneutrophils and as a positive feedback mechanism releasedNETs strongly induce a prothrombotic state and activate platelets Meanwhile tumor cellderived exosomal HMGB1 wasalso found to activate neutrophils through the TLR4NFκB pathway which promotes its survival by increasing theautophagic response and polarizing TANs to a protumor type It is noteworthy that various reports imply the coreposition of the NFκB pathway in the activation and recruitmentof neutrophils In addition to HMGB1 tumor cellsincluding BC cells have been reported to secrete other peptidessuch as a2 isoform VATPase a2V to activate the NFκBpathway in neutrophils thereby promoting their recruitment andinhibiting their apoptosis Additionally breast involutionafter weaning is characterized by acute ‚ammation and anincrease in estrogen It was found that estrogen could inducethe mammary ltration of neutrophils and upregulate theexpression of protumor cytokineschemokines such as COX2and MMPs in mammary ltrating neutrophils similarIn additionto lymphocytes and macrophagesneutrophils are more likely to localize in tumors of triplenegativebreast cancer TNBC than to tumors of other BC subtypes Recently Zhang identified neutrophils and macrophages asthe most frequent ltrating immune cells in various BC murinemodels and BC could be classified into a macrophageenrichedsubtype MES and a neutrophilenriched subtype NES It wasinteresting to find that there were only a few neutrophils inthe MES but a large number of macrophages in the NES This mutual repelling phenomenon in the MES and NES mayresult in spatial segregation within the same tumor The authorsspeculated that a possible mechanism could be the factors derivedfrom macrophages that inhibit the IL8dependent chemotaxis ofneutrophils ANTITUMOR FUNCTION OF TANs IN BCThe polarization of neutrophils can be diï¬erentially regulatedin the tumor microenvironment In a mouse model Fridlender found that TANs from the early tumor stage were liketumorkilling cells which produce high levels of hydrogenperoxide H2O2 tumor necrosis factor TNFα and NOand that TANs are more likely to obtain a protumorigenicphenotype with tumor progression Although few studieshave directly compared the phenotype and function of TANsbetween early and latestage tumors there are still some clues tosupport this hypothesis A phenotypical and functional analysisof TANs in earlystage lung cancer found an activated phenotypeCD62lowCD54high that was able to stimulate T cell proliferationand IFNγ release which suggested a pro‚ammatory ratherthan immunosuppressive state of TANs in earlystage lungcancer MPO is an enzyme characteristic of mature œN1type neutrophils which are able to convert H2O2 to cytotoxichypochlorous acid HOCI Recently a retrospectivestudy of BC cases revealed that MPOpositive neutrophilsFrontiers in Immunology wwwfrontiersinAugust Volume 0cZhang et alNeutrophils in Breast Cancerin additiondefined as ‰¥ cellstissue punch were found in of evaluablecases while the luminal ERPR and Her2 Her2enriched andtriplenegative types had positive rates of and respectivelyltrationby MPOpositive neutrophils was a significant independentfavorable indicator for both OS and DFS Notably almost all ofthe patients included in this study had earlystage disease T1 N01 and the data suggested that MPOpositiveneutrophils were much more abundant in BC cases with low Tand N stages than in advanced cases in univariate analysesIn addition a direct tumor killing function of neutrophils hasalso been reported One of the classical factors working againsttumor cells is ROS Recent research in mouse BC models revealedthat ROSmediated cell lysis was dependent on Ca2 channelsand mediated by transient receptor potential cation channelsubfamily M member TRPM2 expression on tumor cells Although TCGA analysis revealed a high expression ofTRPM2 in BC cells httpgepia2cancerpkucnindex activeNOX1 catalase and SOD were also increased in the membraneof cancer cells forming a complex mechanism by which tumorcell apoptosis induced by ROS is prevented In additiontumor cells are characterized by enhanced metabolic activity andhigh levels of intracellular ROS which indicates that directcytotoxic eï¬ects of neutrophilproduced ROS are not sufficientIn addition to the direct cytotoxic eï¬ect TANs containing ROShave been found to strongly suppress IL17producing γδ Tcells which are critical for shaping the immune suppressivemicroenvironment in various solid tumors “ and have alsobeen reported to promote BC cell extravasation and metastasis In addition neutrophils could also express Fc receptors andexert antibodydependent cellular cytotoxicity ADCC eï¬ectssimilar to those of T cells and macrophagesleading to atrogocytosis eï¬ect to destroy cancer cells However somestudies have indicated that neutrophils are more likely to bedistributed at the periphery of tumors at the initiation stage which may make controlling tumor growth with thesecellcell contactdependent mechanisms ineï¬ectivePROTUMOR EFFECTS OF TANsMore studies suggest that neutrophils facilitate tumor promotionand metastasis in BC than antitumor eï¬ect Overexpression ofthe chemokines CCL2 and CCL17 is a recognized feature ofN2 neutrophils Richmond found that exogenousCCL2 enhances the killing eï¬ect of neutrophils against BCcells in vitro while this antitumor activity was not observedin vivo Instead intranasal delivery of CCL2 to BALBc micemarkedly enhanced lung metastasis of BC cells and increasedthe recruitment of CD4 T cells and CD8 central memory Tcells CCL17 secretion from TANs was found to support tumrowth by recruiting CD4 Treg cells and macrophages In addition to recruiting immunesuppressive cells TANs werereported to promote the accumulation of BC cells in the lungand directly inhibit natural killer NK cellmediated clearanceof tumor cells Human NK cells can be divided intoCD56dim antitumor and CD56bright protumor subsets andCD56bright NK cells are enriched in the tumor microenvironmentand draining lymph nodes Early reports revealed thatROS and arginase1 from neutrophils impair the maturation andcytotoxic function of NK cells but CD56brightCD16ˆ’ NKcell are resistant to neutrophilderived ROS perhaps due to theirhigh antioxidative capacity Meanwhile NK cells couldbe recruited by TANs via CCL2 and CCL5 which may explainthe preferential accumulation of CD56bright NK cells in tumormicroenvironments with high ROS levels Extracellular arginine is crucial to signal local CD8 cellsand increase their CD3ζ expression which is key for T cellsto survey antigens presented on MHC class I molecules andit was also found to be necessary for T cell activation andsurvival Tumor cellderived IL8 could lead to TANdegranulation resulting in arginase1 release and conversion ofextracellular arginine to ornithine and urea thereby dampeningthe survival and cytotoxic eï¬ect of CD8 T cells Neutrophil elastase NE is also released by TANs and canbe endocytosed by tumor cells via neuropilin1 NRP1 thisresults in the crosspresentation of PR1 which is an NEderivedHLAA2restricted peptide that may be an immunotherapeutictarget Besides upon endocytosis NE is to bind insulinreceptor substrate1 IRS1 which removes the inhibitory eï¬ectof IRS1 on phosphatidylinositol 3kinase PI3K to enhance theproliferation of cancer cells Recentleukocytesreports highlighted thethe important characteristics of malignantespeciallyneutrophils preferentially uptake tumor derived extracellularvesicles or named exosomes Hypercoagulability is oneoftumors andhas been reported associated with NETs Breast cancer cell4T1derived exosomes induced NETs formation in neutrophilsbesides tumorderived exosomes also interacted with NETsto significantly accelerate venous thrombosis in vivo Furthermore several reports also indicated the cancer derivedexosomes prolonged lifespan of neutrophils and also polarizedneutrophils toward protumor type increasingevidence hasIn addition to direct modulation ofthe protumormicroenvironmentfound thatneutrophils promote tumor cell migration and the formation ofa metastatic niche Tumor angiogenesis is regardedas a prerequisite for tumor metastasis and TANs have beenrecognized as an important source of vascular endothelialgrowth factor VEGF upon specific stimulation in the tumormicroenvironment Neutrophils were also found to beone of the main sources of MMP9 and the link betweenMMP9 and VEGF has been reported previously The absenceof MMP9 has been reported to have a similar function as theinhibition of VEGF signaling indicating that MMP9 serves asan angiogenic switch during tumorigenesis by inducing VEGFrelease from the matrix “ In addition Gabriele et alalso found that MMP9 was expressed by a small number ofcells in close proximity to the vasculature such as ltrating‚ammatory cells rather than tumor cells In additionseveral serine proteases are also produced by TANs such asNE cathepsin G and proteinase3 which have been reported toactivate MMP2 to promote tumor invasion and proliferation In addition although neutrophils were reportedFrontiers in Immunology wwwfrontiersinAugust Volume 0cZhang et alNeutrophils in Breast Cancerto produce little tissue inhibitor of matrix metalloproteaseTIMP1 Wang observed that BC cells with CD90positiveexpression could induce the TIMP1 secretion by TANs and asa reciprocal eï¬ect TIMP1 induced EMT and metastasis in BC Other neutrophilderived cytokines such as IL1 IL6and IL17α have been reported to initiate EMT of cancer cells byactivating JAK2STAT3 and ERK signaling Inadditiontheprimaryto modulatingtumormicroenvironment neutrophils can also assist the formationof the cancer premetastatic niche in distant ans CTCsare precursors for metastatic lesion formationintravascularNETs were found to protect CTCs from attack by circulatingimmune cells and dysregulated NETs were found to induce‚ammatory vascular injury EC shrinkage and tissue damage “ Moreover in vitro and in vivo experiments foundthat activated neutrophils promote the adherence of CTCs toECs and facilitate their lung and liver metastasis RecentlyAceto provided strong evidence that neutrophils escortCTCs in BC to assist metastasis With detection of cellsurface markers and Wright Giemsa staining they identified thatmost CTCassociated white blood cells were N2like neutrophilsIn addition singlecell RNA sequencing revealed higher Ki67expression in disseminated tumor cells from CTC neutrophilclusters than in standalone CTCs In the same study TNFαoncostatin M IL1 and IL6 were frequently expressed byCTCassociated neutrophils and matched by the receptors oncorresponding CTCs on the other hand CTCs from the CTCneutrophil clusters expressed high gene levels encoding GCSFtransforming growth factor TGF3 and IL15 which havebeen reported to activate neutrophils “ illuminating amechanism of neutrophilCTC cluster formationIn addition to escorting CTCs in circulation several studieshave found that neutrophil accumulation is a prerequisitefor cancer metastasis For both orthotopic transplantationand spontaneous BC models neutrophils were suggestedto accumulate in the distant an before cancer cellsltration Obesity and elevated cholesterol arerisk factors for BC development and poor prognosis Interestingly 27hydroxycholesterol 27HC increased thenumber of polymorphonuclearneutrophils and γδ T cells atdistal metastatic sites and neutrophils were required for themetastatic eï¬ects of 27HC Egeblad developeda confocal intravital lung imaging system and found that NETswere formed early in the lung and continued to form forthe next few days after tail vein injection of BC cells Inaddition based on immunofluorescence staining of humanprimary BC and matched metastatic lung lesions they foundthat the abundance of NETs was highest in TNBC but NETswere absent or very rare in luminal BC samples which mayexplain the higher metastatic ability of TNBCs than luminalBCs In ovarian cancer an ‚ux of neutrophils in the omentumwas also observed before metastasis and blockade of NETformation with peptidyl arginine deiminase PAD4 an enzymethat is essentia

cancer205

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: progression of breast cancerare greatly affected by the immune environment Neutrophils are the most abundantleucocytes in circulation and act as the spearhead in ‚ammationincluding inbreast cancer Circulating neutrophils are closely related to the prognosis of breastcancer patients and tumorltrating neutrophils have varied functions at differentstages of breast cancer such as antitumor or tumorpromoting neutrophils which aretermed N1 and N2 neutrophils respectively In this review we will discuss the utilityof circulating neutrophils for predicting prognosis and therapeutic efficacy and theunderlying mechanisms of their chemotaxis the dynamic regulation of their antitumoror protumor functions and their different spatial distributions in tumor microenvironmentFinally we also discuss the possibility of targeting neutrophils as a therapeutic strategyin breast cancerKeywords breast cancer immunotherapy neutrophils neutrophiltolymphocyte ratio tumor microenvironmentSpecialty sectionThis was submitted toCancer Immunity and ImmunotherapyINTRODUCTIONa section of the journalFrontiers in ImmunologyReceived May Accepted July Published August CitationZhang W Shen Y Huang H Pan SJiang J Chen W Zhang T Zhang Cand Ni C A Rosetta Stone forBreast Cancer Prognostic Value andDynamic Regulation of Neutrophil inTumor MicroenvironmentFront Immunol 103389fimmu202001779Breast cancer BC is the most common malignancy in women worldwide Although BCis classified as a malignant disease with low immunogenicity recent evidence has revealeda promising outcome of therapies with blocking immune checkpoints in both early andadvanced stages “ The efficacy of immunotherapy is closely related to the tumor immunemicroenvironment especially to ltrating immune cells To date macrophages and Tcells are the most wellstudied immune cells in BC whereas increasing evidence has indicatedthat neutrophils are also key in the oncogenesis and metastasis of BC in addition circulatingneutrophils have been reported to have great prognostic prediction value Neutrophils are themost abundant leucocytes in blood and usually act as the first line of host defense against pathogens However due to their short life span an average of “ h in blood it is difficult to employthis subset of cells for experiments which has resulted in a poor understanding of their role in solidtumors In addition some contradictory results reported in vitro studies or animal experimentshave suggested a dual eï¬ect of neutrophils in tumor developmentFrontiers in Immunology wwwfrontiersinAugust Volume 0cZhang et alNeutrophils in Breast CancerNeutrophils can present both antitumorigenic œN1 andprotumorigenic œN2 phenotypesin various cancers orspecific circumstances The term neutrophil in several studiesalso includes both mature neutrophils and myeloidderivedsuppressor cells MDSCs MDSCs are described as a subsetof neutrophils with immunosuppressive functions that expressCD11b and Gr1 and can be divided into monocyticM CD11bLy6C MDSCs and GPMN CD11bLy6GMDSCs and GPMN MDSCs usually share a commonset of markers and similar morphologicalfeatures withneutrophils To avoid confusion we mainly focus on the biologicalfunction of mature neutrophils and related therapeutic strategiesfor targeting them in BC We provide a comprehensive reviewofthe prognostic value of circulating neutrophils and themechanisms of how tumorassociated neutrophils TANs exertantitumor or tumorpromoting functions in BC and in theend we also discuss the potential of targeting neutrophils as atherapeutic strategy in cancerPROGNOSTIC VALUE OF THENEUTROPHILTOLYMPHOCYTE RATIONLRTumors can be thought of as wounds that will not heal andare characterized by chronic ‚ammation Neutrophils are themost rapidly responding immune cells to ‚ammation andmany studies have found that the NLR is closely related to theprognosis and treatment response in patients bearing BC A recent metaanalysis of studies including patientswith both early and advanced BC revealed that patients witha higher NLR before treatment had poorer diseasefree survivalDFS than those with a lower NLR before treatment but theNLR was not related to overall survival OS the subgroupanalysis found that the NLR was associated with prognosisonly in earlystage patients but not in patients with metastasisAbbreviations BC Breast cancer MDSCs Myeloidderived suppressor cellsTANs Tumorassociated neutrophils NLR Neutrophiltolymphocyte ratio DFSDiseasefree survival OS Overall survival ALC Absolute lymphocyte count NCTNeoadjuvant chemotherapy pCR Pathological complete response PLR Platelettolymphocyte ratio TAMs Tumorassociated macrophages CTCs Circulatingtumor cells NETs Neutrophil extracellular traps MPO MyeloperoxidaseGCSF Granulocyte colonystimulating factor ECs Endothelial cells PMNsPolymorphonuclear neutrophils ICAM1 Intercellular adhesion molecule MMP9 Matrix metalloproteinases9 ROS Reactive oxygen species HMGB1Highmobility group box TLR4 Tolllike receptor TNBC Triplenegativebreast cancer MES Macrophageenriched subtype NES Neutrophilenrichedsubtype H2O2 Hydrogen peroxide TNFα Tumor necrosis factorα HOCIHypochlorous acid TRPM2Transientcation channelsubfamily M member ADCC Antibodydependent cellular cytotoxicityNK Natural killer NE Neutrophil elastase NRP1 Neuropilin1 IRS1 Insulinreceptorsubstrate1 PI3K Phosphatidylinositol 3kinase VEGF Vascularendothelial growth factor TIMP1 Tissue inhibitor of matrix metalloproteaseTGF Transforming growth factor 27HC 27hydroxycholesterol PAD4Peptidyl arginine deiminase TINs Tumorltrating neutrophils CRTConventionalradiotherapy MRT Microbeam radiation therapy DAMPsDamageassociated molecular patterns ICB Immune checkpoint blockadeLDNs Lowdensity neutrophils HDNs Highdensity neutrophils NAMPTNicotinamideadeninedinucleotide GTX granulocyte transfusiontransferase NAD Nicotinamidereceptor potentialphosphoribosyl Since similar metaanalyses were not based on individualpatient data which may cause significant bias we reviewed andcompared the individual reports and found some issues worthdiscussing here Widmann first reported the correlationbetween the NLR and BC prognosis in patients and itwas found that a higher NLR ‰¥ before treatment was anadverse factor for both short and longterm mortality Themajority of retrospective studies thereafter have drawn similars “ and the NLR was found to be consistentamong diï¬erent BC subtypes at baseline However aprospective substudy of GEICAM9906 which comprised patients did not find any prognostic value of the NLR afteradjustment for clinicopathological factors in addition a highNLR was independently associated with worse DFS in only highrisk patients the hormone receptornegativeHER2 populationand in patients with ‰¥ lymph node metastases Anotherstudy with early BC patients also found that the NLR beforesurgery was not associated with DFS indicating that thepresurgery NLR may be valuable only in patients with a hightumor burdenseveralIn addition to the above studiesstudies alsoexplored the prognostic value of the NLR posttreatment or withcontinuous assessment A retrospective study comparing theabsolute lymphocyte count ALC and the NLR eight consecutivetimes before and after chemotherapy found that patients whodied had lower ALC and higher NLR values than patients whoremained alive throughout the treatment course additionallyamong the patients who died a steady increase in the NLRover the baseline measurement was observed at subsequenttime points Another retrospective study included BCpatients with DFS values of more than years and it interestinglyfound that NLR sampled during followup rather than beforeany treatment was an independent prognostic factor for laterecurrence However there is still no compelling explanationfor the abovementioned inconsistent results In addition sincelymphocytes are critical in cancer immune surveillance andneutrophils have been reported to play a protumor role in moststudies low lymphocytes and high neutrophils in circulationmay also suggest immunosuppression status and studiesfocused on the relationship between neoadjuvant chemotherapyNCT and the NLR might support the above hypothesis Acomprehensive review of the existing reports shows that moststudies have found that a low NLR indicates a higher NCTresponse and pathological complete response pCR rate “in addition the NLR has showed predictive value not only inall molecular types of BC but also in both operable and locallyadvanced BC Interestingly although Suppan et aldid not find a significant correlation between the initial NLR andprognosis the same cohort revealed a low NLR as a significantparameter for predicting chemotherapy response p A low NLR was also reported to be associated with a higherresponse rate to primary endocrine therapy for locally advancedor metastatic BC Although increasing evidence suggests a close associationbetween the NLR and prognosis in BC several issues remain thatmake clinical application difficult One of the most importantreasons is the lack of a consensus cutoï¬ value As we listhere Table the cutoï¬ values for the NLR in the publishedFrontiers in Immunology wwwfrontiersinAugust Volume 0cZhang et alNeutrophils in Breast CancerTABLE Characteristics of the studies related to neutrophiltolymphocyte ratioReferencesCountry Study periodCancer typeMedianage ysNo patientslowhigh NLRTreatmentFollowupSignificance of NLRNR ysHigh NLR indicates lowersurvival rate p Surgery NCT moSurgery ysNR moUnivariate analysis indicateshigh NLR related to lower RFSp and OS p High NLR indicates lower5year OSMultivariate analysis indicateshigh pretreatment NLR iscorrelated with poor DFS p and OS p Adjuvanttranstuzumab moHigh pretreatment NLRindicates shorter DFSNoh Korea“Koh Korea“Yao China“Luminal ABHER2enrichedTNBCERPRpositiveHER2enrichedLuminal ABERPRpositiveHER2enrichedTNBCPistelli Italy“TNBCUlas Turkey“HER2enrichedJia China“ER PRpositiveHER2enrichedTNBCBozkurt Turkey“TNBCAsano Japan“TNBCn NLR n ‰¤ NLR ‰¥ n n NLR ‰¤ n NLR n n NLR NLR n NLR ‰¤ n NLR n n NLR n NLR n n NLR n NLR ‰¤ n n NLR ‰¤ n NLR n n NLR n NLR n NCT surgery mo moSurgeryadjuvantchemotherapyandradiotherapyNCT ysMultivariate analysis indicateslow NLR is related to superiorDFS p and p Multivariate analysis indicateshigh pretreatment NLR iscorrelated with poor DFS p and OS p Univariate analysis indicateslow NLR is related to favorableprognosis in TNBC patientswho achieved pCR p hazard ratio High pretreatment NLRindicates poor allcausemortality with a multivariableHR of CI“High NLR upon recurrenceindicates shorter OSrecurrence rates p Low NLR indicates higher OSp Rimando USA“Nonmetastatic BC n NLR ‰¤ n NLR n Radiotherapychemotherapy moIwase Japan“TNBCn NRL n NLR ChemotherapyNRNCT surgery moHernandez et alSpain“Miyagawa Japan“Ferroni Italy“Luminal ABERPRpositiveHER2enrichedTNBCLocally Advanced orMetastatic BCLuminal ABHER2enrichedTNBCn NLR n NLR ‰¥ n n NLR ‰¤ n NLR n Qiu China“NonmetastaticTNBCn NLR n NLR ‰¥ n EribulinNRLow NLR indicates better PFSp NCTchemotherapyendocrinetherapytrastuzumabregimensSurgery NCTchemotherapy moHigh pretreatment NLRindicates worse DFS HR and OS HR moLow NLR indicates higher OSp and DFS p ContinuedFrontiers in Immunology wwwfrontiersinAugust Volume 0cZhang et alTABLE ContinuedNeutrophils in Breast CancerReferencesCountry Study periodCancer typeMedianage ysNo patientslowhigh NLRTreatmentFollowupSignificance of NLRIimori Japan“Luminal ABHER2enrichedTNBCMando Argentina “Early stage BCLee Korea“TNBCXuan China“TNBCFujmoto Japan“With high counts oflymphocytesImamura Japan“HER2enrichedn NLR n NLR ‰¥ n n NRL n NLR ‰¤ n NLR n n NLR n NLR ‰¥ 293n n NLR n NLR n n NLR n NLR ‰¥ n Endocrinetherapy moSurgery moNCTNRSurgeryNRNRSurgeryadjuvantchemotherapiesendocrinetherapiesTrastuzumabemtansineNRLow NLR indicates aprolongation of PFS p and OS p High NLR indicates lower DFSp Low NLR indicates superiorOS p and DFS p Low NLR indicates longer DFSp Low NLR indicates better RFSp Low NLR at baseline indicatesbetter PFS p andOS p NLR Neutrophiltolymphocyte ratio ER Estrogen receptor PR Progesterone receptor HER2 Human epidermal growth factor receptor Mo Months Ys Years DFS Diseasefreesurvival OS Overall survival PFS Progressionfree survival RFS Relapse free survival pCR Pathological complete response TNBC Triplenegative breast cancer NCT Neoadjuvantchemotherapy NR Not recordedstudies were between and In addition based on individualstudies the sensitivity of the NLR fluctuates greatly “and the specificity is much lower “ Therefore some researchers have tried to determine a betteralternative parameter In addition to the NLR the platelettolymphocyte PLR ratio has also been investigated and comparedwith the NLR in BC A single central retrospective study with hormone receptornegative nonmetastatic BC patients reportedthat both elevated NLR and PLR were associated with poor OShowever the multivariate analysis revealed that only the NLR p but not the PLR p was a significant indicatorfor both DFS and OS Additionally since the absolutelymphocyte count has also been reported as a prognostic factorthe predictive values of the PLR and NLR were evaluated afteradjusting for the total lymphocyte count The results showed thatthe PLR was no longer a significant predictor for 5year mortalityand the NLR remained a significant predictor irrespective ofthe lymphocyte count Furthermore it was revealed thatthe combination of the NLR and PLR could further improvethe predictive value Two retrospective studies found that thehighest rate of pCR was in the group of patients withan NLRlowPLRlow profile and the lowest rate was inthe group with an NLRhighPLRhigh profile in additionwhen the cutoï¬ values for the NLR and PLR were applied thespecificity of predicting a pCR increased from to Howeverthe causal relationship between the NLR andpoor prognosis in malignant disease has yet to be illuminatedAccording to an assessment with paired peripheral bloodand pancreatic cancer specimens Takakura found thata high NLR was associated with increased tumorassociatedTAMsanditseemsdecreased Thereforetumorassociatedmacrophageslymphocytes but was not significantly related to CD66bltrating neutrophilsthat anincrease in neutrophils in peripheral blood is not necessarilyrelated to the number of TANs Several basic studies havesuggested a unique mechanism ofthe protumor functionof circulating neutrophils protecting circulating tumor cellsCTCs Circulating neutrophils can cluster around tumor cellsand induce tumor cell aggregation aiding tumor cell survivalby hiding them from immune surveillance Neutrophilextracellular traps NETs are webs of decondensed chromatbers conjugated together with histones myeloperoxidaseMPO elastase and other cytoplasmic proteins Recentstudies also found that neutrophils could form many NETs bothin circulation and in tumor lesions and could coordinate withplatelets to capture CTCs and facilitate cancer metastasis In addition neutropenia is very common in cancer patientsundergoing chemotherapy and supportive treatment withgranulocyte colonystimulating factor GCSF can inducea neutrophilic response as a consequence neutrophils areprimed toward a proNETotic phenotype and may suppress thecytotoxic activity of T cells as well as impair immune surveillance On the other hand lymphocytes have the propensityto mount an adaptive antitumor response in malignant disease and decreased lymphocyte numbers are considered to berelated to an insufficient immunologic reaction which mayincrease the risk of tumor relapse or metastasis Clearlya general association between prognosis and the NLR exists inBC but large prospective studies and rigorous research are stillrequired to determine its clinical significanceFrontiers in Immunology wwwfrontiersinAugust Volume 0cZhang et alNeutrophils in Breast CancerMECHANISM OF NEUTROPHILCHEMOTAXIS TO THE TUMORMICROENVIRONMENTNeutrophils are considered the main immune cells that provideprotection against invading pathogens which can be induced bytrauma infection and malignant disease The recruitmentof neutrophils is greatly dependent on certain chemokinesincluding interleukin IL8 also known as CXCL8 CXCL and CXCL2 IL8 is a pro‚ammatory cytokineand acknowledged as the most important chemoattractant forneutrophils in the tumor microenvironment IL8 mainlycomes from endothelial cells ECs and monocytes in the tumormicroenvironment upon certain stimulation such as physicalinjury hypoxia chemotherapy or radiotherapy and other celltypes including fibroblasts and keratinocytes can secrete IL8 aswell In addition to its chemotactic eï¬ect it was revealedthat IL8 could provoke neutrophils to release NETs to assistcancer cell migration By livecell fluorescence microscopyGupta confirmed that activated ECs could induceNETosis characterized by typical extracellular DNA latticeswhen cocultured with polymorphonuclear neutrophils PMNsand activated ECs In addition activated ECs produceother ‚ammatory cytokines such as Pselectin Eselectinand intercellular adhesion molecule ICAM1 to facilitateneutrophil adhesion to ECs and migration Furthermoretumorpromoting neutrophils in BC cells are also characterizedby high expression of matrix metalloproteinases9 MMP9 which was found to cleave CXCL5 potentiating itsaction in neutrophil recruitment as a positive feedback functionin tumors IL17 was also found to control neutrophilrecruitmentin lung metastasis of BC in a mouse modelCD3CD4 and γδ T cells were the major sources of IL17 and it was interesting to find that the absence of γδT cells or neutrophils markedly reduced pulmonary and lymphnode metastases without ‚uencing primary tumor progressionwhich suggested a collaborative relationship between γδ T cellsand neutrophils in promoting BC lung metastasis Howeverin an orthotopic hepatocellular carcinoma model Sofia et alreported that TANs exert an overt antitumor role by suppressingγδ T17 cells via reactive oxygen species ROS contraryto the phenomenon that within the 4T1derived BC modelCD11bLy6G neutrophils that ltrate and surround livermetastases were found to be tumor promoting Thesecontroversial results suggest both promoting and suppressiveroles of TANs in diï¬erent circumstancesHighmobility group box HMGB1 usually acts as adamageassociated molecular pattern that is released by dyingcells or stressed cells to initiate ‚ammation and was laterfound to be an important chemoattractant for neutrophils Epithelial cellderived HMGB1 was found to recruit neutrophilsto the necrotic site through its receptor RAGE Enrichmentof platelets has been reported in the microenvironment ofmultiple cancers including BC and ltrating plateletscould be activated by the large amounts of adenosine phosphatereleased by necrotic cells as a result of chemotherapy Activated plateletderived HMGB1 known as the major mediatorof injuryinduced thrombosis in vivo can also stimulateNETosis through Tolllike receptor TLR4 and RAGE onneutrophils and as a positive feedback mechanism releasedNETs strongly induce a prothrombotic state and activate platelets Meanwhile tumor cellderived exosomal HMGB1 wasalso found to activate neutrophils through the TLR4NFκB pathway which promotes its survival by increasing theautophagic response and polarizing TANs to a protumor type It is noteworthy that various reports imply the coreposition of the NFκB pathway in the activation and recruitmentof neutrophils In addition to HMGB1 tumor cellsincluding BC cells have been reported to secrete other peptidessuch as a2 isoform VATPase a2V to activate the NFκBpathway in neutrophils thereby promoting their recruitment andinhibiting their apoptosis Additionally breast involutionafter weaning is characterized by acute ‚ammation and anincrease in estrogen It was found that estrogen could inducethe mammary ltration of neutrophils and upregulate theexpression of protumor cytokineschemokines such as COX2and MMPs in mammary ltrating neutrophils similarIn additionto lymphocytes and macrophagesneutrophils are more likely to localize in tumors of triplenegativebreast cancer TNBC than to tumors of other BC subtypes Recently Zhang identified neutrophils and macrophages asthe most frequent ltrating immune cells in various BC murinemodels and BC could be classified into a macrophageenrichedsubtype MES and a neutrophilenriched subtype NES It wasinteresting to find that there were only a few neutrophils inthe MES but a large number of macrophages in the NES This mutual repelling phenomenon in the MES and NES mayresult in spatial segregation within the same tumor The authorsspeculated that a possible mechanism could be the factors derivedfrom macrophages that inhibit the IL8dependent chemotaxis ofneutrophils ANTITUMOR FUNCTION OF TANs IN BCThe polarization of neutrophils can be diï¬erentially regulatedin the tumor microenvironment In a mouse model Fridlender found that TANs from the early tumor stage were liketumorkilling cells which produce high levels of hydrogenperoxide H2O2 tumor necrosis factor TNFα and NOand that TANs are more likely to obtain a protumorigenicphenotype with tumor progression Although few studieshave directly compared the phenotype and function of TANsbetween early and latestage tumors there are still some clues tosupport this hypothesis A phenotypical and functional analysisof TANs in earlystage lung cancer found an activated phenotypeCD62lowCD54high that was able to stimulate T cell proliferationand IFNγ release which suggested a pro‚ammatory ratherthan immunosuppressive state of TANs in earlystage lungcancer MPO is an enzyme characteristic of mature œN1type neutrophils which are able to convert H2O2 to cytotoxichypochlorous acid HOCI Recently a retrospectivestudy of BC cases revealed that MPOpositive neutrophilsFrontiers in Immunology wwwfrontiersinAugust Volume 0cZhang et alNeutrophils in Breast Cancerin additiondefined as ‰¥ cellstissue punch were found in of evaluablecases while the luminal ERPR and Her2 Her2enriched andtriplenegative types had positive rates of and respectivelyltrationby MPOpositive neutrophils was a significant independentfavorable indicator for both OS and DFS Notably almost all ofthe patients included in this study had earlystage disease T1 N01 and the data suggested that MPOpositiveneutrophils were much more abundant in BC cases with low Tand N stages than in advanced cases in univariate analysesIn addition a direct tumor killing function of neutrophils hasalso been reported One of the classical factors working againsttumor cells is ROS Recent research in mouse BC models revealedthat ROSmediated cell lysis was dependent on Ca2 channelsand mediated by transient receptor potential cation channelsubfamily M member TRPM2 expression on tumor cells Although TCGA analysis revealed a high expression ofTRPM2 in BC cells httpgepia2cancerpkucnindex activeNOX1 catalase and SOD were also increased in the membraneof cancer cells forming a complex mechanism by which tumorcell apoptosis induced by ROS is prevented In additiontumor cells are characterized by enhanced metabolic activity andhigh levels of intracellular ROS which indicates that directcytotoxic eï¬ects of neutrophilproduced ROS are not sufficientIn addition to the direct cytotoxic eï¬ect TANs containing ROShave been found to strongly suppress IL17producing γδ Tcells which are critical for shaping the immune suppressivemicroenvironment in various solid tumors “ and have alsobeen reported to promote BC cell extravasation and metastasis In addition neutrophils could also express Fc receptors andexert antibodydependent cellular cytotoxicity ADCC eï¬ectssimilar to those of T cells and macrophagesleading to atrogocytosis eï¬ect to destroy cancer cells However somestudies have indicated that neutrophils are more likely to bedistributed at the periphery of tumors at the initiation stage which may make controlling tumor growth with thesecellcell contactdependent mechanisms ineï¬ectivePROTUMOR EFFECTS OF TANsMore studies suggest that neutrophils facilitate tumor promotionand metastasis in BC than antitumor eï¬ect Overexpression ofthe chemokines CCL2 and CCL17 is a recognized feature ofN2 neutrophils Richmond found that exogenousCCL2 enhances the killing eï¬ect of neutrophils against BCcells in vitro while this antitumor activity was not observedin vivo Instead intranasal delivery of CCL2 to BALBc micemarkedly enhanced lung metastasis of BC cells and increasedthe recruitment of CD4 T cells and CD8 central memory Tcells CCL17 secretion from TANs was found to support tumrowth by recruiting CD4 Treg cells and macrophages In addition to recruiting immunesuppressive cells TANs werereported to promote the accumulation of BC cells in the lungand directly inhibit natural killer NK cellmediated clearanceof tumor cells Human NK cells can be divided intoCD56dim antitumor and CD56bright protumor subsets andCD56bright NK cells are enriched in the tumor microenvironmentand draining lymph nodes Early reports revealed thatROS and arginase1 from neutrophils impair the maturation andcytotoxic function of NK cells but CD56brightCD16ˆ’ NKcell are resistant to neutrophilderived ROS perhaps due to theirhigh antioxidative capacity Meanwhile NK cells couldbe recruited by TANs via CCL2 and CCL5 which may explainthe preferential accumulation of CD56bright NK cells in tumormicroenvironments with high ROS levels Extracellular arginine is crucial to signal local CD8 cellsand increase their CD3ζ expression which is key for T cellsto survey antigens presented on MHC class I molecules andit was also found to be necessary for T cell activation andsurvival Tumor cellderived IL8 could lead to TANdegranulation resulting in arginase1 release and conversion ofextracellular arginine to ornithine and urea thereby dampeningthe survival and cytotoxic eï¬ect of CD8 T cells Neutrophil elastase NE is also released by TANs and canbe endocytosed by tumor cells via neuropilin1 NRP1 thisresults in the crosspresentation of PR1 which is an NEderivedHLAA2restricted peptide that may be an immunotherapeutictarget Besides upon endocytosis NE is to bind insulinreceptor substrate1 IRS1 which removes the inhibitory eï¬ectof IRS1 on phosphatidylinositol 3kinase PI3K to enhance theproliferation of cancer cells Recentleukocytesreports highlighted thethe important characteristics of malignantespeciallyneutrophils preferentially uptake tumor derived extracellularvesicles or named exosomes Hypercoagulability is oneoftumors andhas been reported associated with NETs Breast cancer cell4T1derived exosomes induced NETs formation in neutrophilsbesides tumorderived exosomes also interacted with NETsto significantly accelerate venous thrombosis in vivo Furthermore several reports also indicated the cancer derivedexosomes prolonged lifespan of neutrophils and also polarizedneutrophils toward protumor type increasingevidence hasIn addition to direct modulation ofthe protumormicroenvironmentfound thatneutrophils promote tumor cell migration and the formation ofa metastatic niche Tumor angiogenesis is regardedas a prerequisite for tumor metastasis and TANs have beenrecognized as an important source of vascular endothelialgrowth factor VEGF upon specific stimulation in the tumormicroenvironment Neutrophils were also found to beone of the main sources of MMP9 and the link betweenMMP9 and VEGF has been reported previously The absenceof MMP9 has been reported to have a similar function as theinhibition of VEGF signaling indicating that MMP9 serves asan angiogenic switch during tumorigenesis by inducing VEGFrelease from the matrix “ In addition Gabriele et alalso found that MMP9 was expressed by a small number ofcells in close proximity to the vasculature such as ltrating‚ammatory cells rather than tumor cells In additionseveral serine proteases are also produced by TANs such asNE cathepsin G and proteinase3 which have been reported toactivate MMP2 to promote tumor invasion and proliferation In addition although neutrophils were reportedFrontiers in Immunology wwwfrontiersinAugust Volume 0cZhang et alNeutrophils in Breast Cancerto produce little tissue inhibitor of matrix metalloproteaseTIMP1 Wang observed that BC cells with CD90positiveexpression could induce the TIMP1 secretion by TANs and asa reciprocal eï¬ect TIMP1 induced EMT and metastasis in BC Other neutrophilderived cytokines such as IL1 IL6and IL17α have been reported to initiate EMT of cancer cells byactivating JAK2STAT3 and ERK signaling Inadditiontheprimaryto modulatingtumormicroenvironment neutrophils can also assist the formationof the cancer premetastatic niche in distant ans CTCsare precursors for metastatic lesion formationintravascularNETs were found to protect CTCs from attack by circulatingimmune cells and dysregulated NETs were found to induce‚ammatory vascular injury EC shrinkage and tissue damage “ Moreover in vitro and in vivo experiments foundthat activated neutrophils promote the adherence of CTCs toECs and facilitate their lung and liver metastasis RecentlyAceto provided strong evidence that neutrophils escortCTCs in BC to assist metastasis With detection of cellsurface markers and Wright Giemsa staining they identified thatmost CTCassociated white blood cells were N2like neutrophilsIn addition singlecell RNA sequencing revealed higher Ki67expression in disseminated tumor cells from CTC neutrophilclusters than in standalone CTCs In the same study TNFαoncostatin M IL1 and IL6 were frequently expressed byCTCassociated neutrophils and matched by the receptors oncorresponding CTCs on the other hand CTCs from the CTCneutrophil clusters expressed high gene levels encoding GCSFtransforming growth factor TGF3 and IL15 which havebeen reported to activate neutrophils “ illuminating amechanism of neutrophilCTC cluster formationIn addition to escorting CTCs in circulation several studieshave found that neutrophil accumulation is a prerequisitefor cancer metastasis For both orthotopic transplantationand spontaneous BC models neutrophils were suggestedto accumulate in the distant an before cancer cellsltration Obesity and elevated cholesterol arerisk factors for BC development and poor prognosis Interestingly 27hydroxycholesterol 27HC increased thenumber of polymorphonuclearneutrophils and γδ T cells atdistal metastatic sites and neutrophils were required for themetastatic eï¬ects of 27HC Egeblad developeda confocal intravital lung imaging system and found that NETswere formed early in the lung and continued to form forthe next few days after tail vein injection of BC cells Inaddition based on immunofluorescence staining of humanprimary BC and matched metastatic lung lesions they foundthat the abundance of NETs was highest in TNBC but NETswere absent or very rare in luminal BC samples which mayexplain the higher metastatic ability of TNBCs than luminalBCs In ovarian cancer an ‚ux of neutrophils in the omentumwas also observed before metastasis and blockade of NETformation with peptidyl arginine deiminase PAD4 an enzymethat is essentia Answer:

Thyroid_Cancer

Lasting and SexDependent Impactof Maternal Immune Activation onMolecular Pathways of the AmygdalaMarissa R Keever1 Pan Zhang2 Courtni R Bolt1 Adrienne M Antonson1Haley E Rymut1 Megan P Caputo1 Alexandra K Houser1 Alvaro G Hernandez3Bruce R Southey1 Laurie A Rund1 Rodney W Johnson14 andSandra L RodriguezZas12456 Department of Animal Sciences University of Illinois at UrbanaChampaign Urbana IL United States Illinois InformaticsInstitute University of Illinois at UrbanaChampaign Urbana IL United States Highthroughput Sequencingand Genotyping Unit Roy J Carver Biotechnology Center University of Illinois at UrbanaChampaign Urbana ILUnited States Neuroscience Program University of Illinois at UrbanaChampaign Urbana IL United States Departmentof Statistics University of Illinois at UrbanaChampaign Urbana IL United States Carl R Woese Institute for GenomicBiology University of Illinois at UrbanaChampaign Urbana IL United StatesThe prolonged and sexdependent impact of maternal immune activation MIA duringgestation on the molecular pathways of the amygdala a brain region that ‚uencessocial emotional and other behaviors is only partially understood To address thisgap we investigated the effects of viralelicited MIA during gestation on the amygdalatranscriptome of pigs a species of high molecular and developmental homology tohumans Gene expression levels were measured using RNASeq on the amygdalafor 3weekold female and male offspring from MIA and control groups Amongthe genes that exhibited significant MIA effect a prevalence of differentiallyexpressed genes annotated to the neuroactive ligand“receptor pathway glutamatergicfunctions neuropeptide systems and cilium morphogenesis were uncovered Genesin these categories included corticotropinreleasing hormone receptor glutamatemetabotropic receptor glycoprotein hormones alpha polypeptide parathyroidhormone receptor vasointestinal peptide receptor neurotensin proenkephalinand gastrinreleasing peptide These categories and genes have been associatedwith the MIArelated human neurodevelopmental disorders including schizophreniaand autism spectrum disorders Gene network reconstruction highlighted differentialvulnerability to MIA effects between sexes Our results advance the understandingnecessary for the development of multifactorial therapies targeting immune modulationand neurochemical dysfunction that can ameliorate the effects of MIA on offspringbehavior later in lifeKeywords immune activation pigs RNAseq neuropeptides glutamatergic pathway GABAergic pathwayINTRODUCTIONThe maternal immune response triggered by pathogens and other environmental stressors duringgestation can also elicit an indirect response by the fetal immune cells Kroismayr Odorizzi and Feeney Prins Viral infection during gestation for exampleactivates a cytokinerelated signaling cascade and molecules from this process can cross theEdited byNo¨lia Fern ndezCastilloCentre for Biomedical NetworkResearch CIBER SpainReviewed bySilvia PellegriniUniversity of Pisa ItalyTewarit SarachanaChulalongkorn University ThailandCorrespondenceSandra L RodriguezZasrodrgzzsillinoiseduSpecialty sectionThis was submitted toNeurogenomicsa section of the journalFrontiers in NeuroscienceReceived May Accepted July Published August CitationKeever MR Zhang P Bolt CRAntonson AM Rymut HE Caputo MPHouser AK Hernandez AGSouthey BR Rund LA Johnson RWand RodriguezZas SL Lastingand SexDependent Impactof Maternal Immune Activation onMolecular Pathways of the AmygdalaFront Neurosci 103389fnins202000774Frontiers in Neuroscience wwwfrontiersinAugust Volume 0cKeever et alGestational Activation of the Amygdalaplacenta and reach the fetal brain The resulting maternalimmune activation MIA can impactfetal developmentalprocesses and exert longterm postnatal eï¬ects in the oï¬springRutherford The relationship between MIAand neurodevelopmental disordersincluding schizophreniaspectrum disorders SSD and autism spectrum disorders ASDand neurodegenerative disorders such as Alzheimer™s diseaseAD in oï¬spring has been established Knuesel Canetta Mattei These diseases sharesome behavior symptoms comorbidities such as eating disordersand genetic and environmental ie MIA agents Canitanoand Pallagrosi The previous neurological disorders havebeen associated with abnormal structure and dysregulationof the amygdala Schumann FernandezIrigoyen and share genes and molecular mechanismsincluding histocompatibility complex MHC genes Andersand Kinney glutamatergic and GABAergicassociatedgenes Bourgeron Marin Li andmitochondrial activity processes Pieczenik and Neustadt Sragovich socialinteractionThe fetal amygdala is susceptible to ‚ammatory signals andthe plasticity of this brain structure to MIA can lead to alterationsof the developmental trajectory These disruptions may havelonglasting and maladaptive consequences for the oï¬springdue to the significant role that the amygdala plays in manyneurological pathways Located in the forebrain the amygdala‚uencescognition neuroendocrinebehavior learning memory emotion and autonomic systemsThe amygdala also modulates the response of these processesto stressors including pathogenic infections and those resultingfrom management practices such as weaning Tian The amygdala experiences high uptake of gonadalhormones and is anatomically connected to other sexuallydimorphic nuclei Therefore this brain region is involved inregulation of several dimorphic functions such as aggressionsexual behavior gonadotropin secretion and integration ofolfactory information Hines Evidence supports thediï¬erential activation of the amygdala to stimuli between malesand females Killgore and YurgelunTodd includingdiï¬erences in the sexual responses and emotional memoryHamann and diï¬erential vulnerability to insult Baird Due to the interconnected and multiregulatorynature of this brain structureinsults to the amygdala canimpact the individual™s social locomotor and feeding behaviorPetrovich and Gallagher growth and reproductivephysiology health status and immunological response tosecondary stressorsRecent studies lend support to the link between MIA andaltered amygdala function Carlezon In miceMIA elicited by polyinosinicpolycytidylic acid [PolyIC]increased the synaptic strength of glutamatergic projectionsfrom the prefrontal cortex to the amygdala Li In field tests mice exposed to MIA spentless timein the center and traveled a higher distanceindicativeof a higher anxiety behavior incidence than the controlcounterparts These findings suggest that the change in thebalance between excitation glutamatergic and inhibitiontherefore aï¬ecting brain circuitsspike output offeedforward GABAergic modified theamygdala neuronsthatcould regulate behavior in SSD and ASD A candidate genestudy of the eï¬ects of social stress during gestation reportedthatthe expression of a corticotropinreleasing hormonereceptor in the amygdala of 10weekold pigs was higherin females than in males Rutherford Thisstudy concluded that prenatal stress substantially increasedanxietyrelated behaviorstheimpact of maternal stressors during gestation on specificamygdala molecular profiles and associated neurological orbehavioral disorders in the oï¬spring later in life highlightthe complexity of the molecular mechanisms underlying thepathophysiology of MIAin female pigs Studies ofetalvirusadvantages ofrodents when consideringstudying a pig modelto pigsResearch on the lasting eï¬ects of MIA in pigs complementsAntonson et althe insights oï¬ered by rodent modelsstem Theratherfrom the greater homology of humansan physiologythan toin particular brain growth andsize development anddevelopment processesMurphy A pigmodel that has oï¬ered insights into MIA employs porcinereproductivePRRSVtothein the brain andmicroglia ie macrophagelike cellsisin neonatal pigsAntonson associated with behavioralelicit MIA Thisand respiratorysyndromechallengeactivatesimmunechangesThe study of MIA elicited by PRRSV allows for thecharacterization of the impact of a live viral pathogen thatselfreplicates in the host evoking extended activation ofimmune pathways PRRSV challenge during gestation is a wellcharacterized replicable and eï¬ective method for inducingMIA in pigs Antonson In additionPRRSV outbreaks impose a major economic burden to thelivestock industry PRRSV is an enveloped singlestranded RNAvirus thatinfects alveolar macrophages causing interstitialpneumonia and increased serum levels ofthe cytokinesinterleukin betainterleukin and tumor necrosis factoralpha Antonson The persistent repercussionsof MIA on the molecular pathways ofthe pig amygdalaare yet to be investigated Moreover the potentially distinctvulnerability to the prolonged eï¬ects of MIA between sexesremains unknownThe overarching goal of the present study is to advancethe understanding of the impact of MIA on the molecularmechanisms ofthe amygdala Three supporting objectivesare explored a characterization of prolonged transcriptomechanges elicited by viral MIA in pigs a species that hashigh neurodevelopmental homology with humansandfood production valueidentification of molecularpathwaysthat present diï¬erential vulnerability to MIAbetween sexes and c understanding the eï¬ect of MIA onmolecular interactions assisted by gene network inferenceThe findings from these complementary analyses supportthe use of multipleto amelioratethe potential detrimental eï¬ect of MIA on the oï¬springphysiology and behaviortherapeutictargetsbFrontiers in Neuroscience wwwfrontiersinAugust Volume 0cKeever et alGestational Activation of the AmygdalaMATERIALS AND METHODSAnimal ExperimentsAll experimental procedures used published protocols Antonson The animal studies were approved by theIllinois Institutional Animal Care and Use Committee IACUCat the University of Illinois and are in compliance with the USDAAnimal Welfare Act and the NIH Public Health Service Policy onthe Humane Care and Use of AnimalsCamborough gilts born and raised at the University of Illinoisat UrbanaChampaign herd were inseminated at days ofage using PIC boar sperm Antonson All gilts were PRRSV negative and were moved at gestationday GD into diseasecontainment chambers maintainedat —¦C and a h lightdark cycle with lights on at AM The gilts were fed daily kg of a gestational diet andhad ad libitum water access One week after acclimation fourgilts were intranasally inoculated with live PRRSV strain P129BV School of Veterinary Medicine at Purdue University WestLafayette IN United States using mL of — median tissueculture infectious dose TCID50 diluted in sterile Dulbecco™smodified Eagle medium DMEM mL total volume Thefour gilts in the Control group were intranasally inoculatedwith an equal volume of sterile DMEM PRRSV inoculationcorresponded to the last third of gestation in pigs and humansduring initiation of rapid fetal brain growth Antonson PRRSV and Control groups were housed in separatecontainment chambersThe rectal temperatures and diet consumption of the giltswere recorded daily until farrowing Antonson The PRRSVinoculated gilts were oï¬ered the maximumfed daily and feed refusal was measured The Control giltswere fed the same amount consumed by the PRRSVinoculatedgilts on the previous day The daily body temperature andfeed intake levels were compared using a mixedeï¬ects modelanalyzed with PROC MIXED SAS Institute Inc Cary NCUnited States The model included the eï¬ects of gilt treatmentand replicate while accommodating for heterogeneity of variancebetween MIA groupsFarrowing was induced with an intramuscular injection of mg of Lutalyse dinoprost tromethamine Pfizer New YorkNY United States on GD in consideration that the averagegestation length is approximately days Antonson Gilts farrowed in individual farrowing crates ofstandard dimensions — m After farrowing thegilts were fed twice a day up to kg of a nutritionallycomplete diet for the lactating period and water remainedavailable ad libitum Pigs received intramuscular injections ofiron dextran mgpig Butler Schein Animal Health DublinOH United States and Excede for Swine mgpig ZoetisParsippany NJ United States to control for respiratory diseasesThe pigs remained with their mothers until PD The bodyweight of pigs was measured daily and analyzed using the mixedeï¬ects model in SAS PROC MIXED SAS Institute Inc CaryNC United States The model included the eï¬ect of MIA andthe random eï¬ect of gilt accommodating for heteroscedasticitybetween pig treatment and sex groups The impact of MIA wasstudied at PD because this is a common age to wean pigsThe study of transcriptome profiles from older pigs could beconfounded with changes in diet and environment associatedwith weaning while profiles from younger pigs would hinder theassessment of the prolonged eï¬ects of MIARNA Extraction and SequencingA balanced experimental design was studiedincluding pigs evenly distributed between maternal PPRSV activatedMPA group of pigs and Control gilts CON group of pigseach group encompassing males and females denoted Maand Fe respectively At PD pigs were removed fromthe farrowing crate and anesthetized intramuscularly using atelazolketaminexylazine drug cocktail mg of tiletamine mg of zolazepam reconstituted with mL ketamine gL and mL xylazine gL Fort Dodge AnimalHealth Fort Dodge IA United States at a dose of mLkgbody weight following protocols Antonson Following anesthetization pigs were euthanized using anintracardiac injection of sodium pentobarbital mgkg bodyweight Fata Plus Vortech Pharmaceuticals Dearborn MIUnited States Pig brains were extracted the amygdalae wererecognized using the stereotaxic atlas of the pig brain Felix dissected out flash frozen on dry ice and stored at ˆ’—¦Cfollowing published protocols Antonson RNA wasisolated using EZNA isolation kit following the manufacturer™sinstructions Omega Biotek Norcross GA United States TheRNA integrity numbers of the samples were above indicatinglow RNA degradation The RNASeq libraries were preparedwith TruSeq Stranded mRNAseq Sample Prep kit Illumina IncSan Diego CA United States The libraries were quantitatedby qPCR and sequenced on one lane on a NovaSeq for cycles from each end of the fragments using NovaSeqS4 reagent kit FASTQ files were generated and demultiplexedwith the bcl2fastq v220 conversion software Pairedend reads nt long were obtained and the FASTQ files are availablein the National Center for Biotechnology Information GeneExpression Omnibus GEO database experiment accessionnumber GSE149695RNA Sequence Mapping and DifferentialExpression AnalysisThe average Phred quality score of the reads assessed usingFastQC Andrews was across all read positions andtherefore no reads were trimmed The pairedend reads fromthe individual samples were aligned to the Sus scrofa genomeversion Sscrofa Pruitt using kallisto v0430Bray with default settings The normalized trimmedmean of Mvalues gene expression values were described usinga generalized linear model encompassing the eï¬ects of the MIAgroup MPA or CON levels sex Fe or Ma levels and MIAbysex interaction and analyzed using edgeR version in the R v environment Robinson Genessupported by transcripts per million TPM by each MIA“sex combination were analyzed to ensure adequate representationacross comparisonsFrontiers in Neuroscience wwwfrontiersinAugust Volume 0cKeever et alGestational Activation of the AmygdalaOrthogonal pairwise contrasts between MIA and sex groupswere evaluated in addition to testing for the eï¬ects of MIAbysex interaction and main eï¬ects of MIA and sex Thefour groups compared in the contrasts identified by treatmentfollowed by the sex levels are MPA_Fe MPA_Ma CON_Fe andCON_Ma The Pvalues were adjusted for multiple testing usingthe Benjamini“Hochberg false discovery rate FDR approachBenjamini and Hochberg categoriesamongtheMFand KEGG pathways The GeneFunctional Enrichment and NetworkInferenceapproaches were used to identifyTwo complementaryoverrepresented functionalgenesexhibiting diï¬erential expression across MIA and sex groupsCaetanoAnoll©s GonzalezPena et al2016ab Functional categories investigated included GeneBPs GO molecularOntology GO biological processesfunctionsSetEnrichment Analysisapproach implemented inthesoftware package GSEAP Subramanian was used to identify category overrepresentationwith gene over and underexpressed while considering allgenes analyzed The normalized enrichmentscore NESofin the Molecular Signature DatabaseMSigDB was calculated using the maximum deviation ofthe cumulative sum based on the signed and standardizedfold change The statistical significance ofthe enrichmentwas assessed using the FDRadjusted Pvalue computed from permutationscategoriesGSEAtheThe overrepresentation of functional categories was alsoevaluated among genes that exhibited a significant MIAbysex interaction or main eï¬ect using the Database forAnnotation Visualization and Integrated Discovery DAVID Huang The enrichment of Direct GOcategories in the DAVID database was assessed The Susscrofa genome was used as the background for enrichmenttesting and enrichmentis reported using the ExpressionAnalysis Systematic Explorer EASE score that was computedusing a onetailed jackknifed Fisher hypergeometric exacttest Functional categories were clustered based on geneannotation and the statisticalissummarized as the geometric mean of the log10 EASE scoresofthe categories Delfino Serao Delfino and RodriguezZas significance of clustersWeighted Gene Coexpression NetworkAnalysis and Gene Network VisualizationAn approach complementary to the identification of diï¬erentiallyexpressed genes was used to uncover coexpression networksusing Weighted Gene Coexpression Network AnalysisWGCNA version Langfelder and Horvath The input data were voomtransformed read count valuesgenerated using the limma package version Ritchie in R version Genes were filtered to removethose with low expression levels or no variation across samplesper developer recommendations The number of genes usedfor network analysis was genes Considering potentialfor interaction patterns a sexdependent softthresholdingpower was used to call for network topology analysis Thelowest power values that support a scalefree topology powerused were for the CON_MaMPA_Ma contrast and for the MPA_FeMPA_Ma contrast The Pearson correlationcoefficient ofthe normalized expression values was usedto identify modules of connected genes The minimummodule size was set to with the deepSplit set to and themergeCutHeight set to Module profiles were identifiedusing the correlation between the eigengene of each moduleand pig group Enrichment of functional categories among thegenes in each module profile was explored with DAVID using theSus scrofa genome as background and testing included an FDRmultiple test adjustmentFurther understanding of the impact of the MIAbysexinteraction was gained through the reconstruction of genenetworks using the BisoGenet package Martin inthe Cytoscape platform Shannon Information fromgene and protein interactions annotated in databases includingBIOGRID HPRD DIP BIND INTACT and MINT was usedto visualize relationships between genes Salwinski Alfarano Mishra Stark Kerrien Licata Networks highlightingdiï¬erences in gene levels associated with MIA within sex iethe contrasts MPA_MaCON_Ma and MPA_FeCON_Fe werecompared The network framework includes genes that exhibiteda significant MIAbysex interaction eï¬ect FDRadjusted P and are annotated to enriched functional categoriesThe framework genes were identified by full nodes with sizereflecting the diï¬erential expression level between the MPAand CON groups The network edges depict known molecularrelationships curated in the BisoGenet databases The frameworkgenes were connected through correlated genes listed in theBisoGenet database of molecular interactions that did not reachsignificant MIAbysex interaction eï¬ect The comparison ofthese networks oï¬ered insights into the simultaneous eï¬ect ofMIA across interacting genes and enabled the detection of sharedand distinct coregulation patterns between MPA and CONpigs across sexesRESULTSMaternal Immune Activation andSequencing MetricsThe diï¬erences between MPA and CON giltsin rectaltemperatures and daily diet consumption indicated the activationof the maternal immune system in response to PRRSV Thediï¬erence in body temperature between CON and MPA giltson GD was —¦C standard error —¦C P Thediï¬erence in feed refusal between CON and MPA gilts on GD was g standard error g P A significantincrease in rectal temperatures and decrease in feed intakeP was observed within h of inoculation and returnedto baseline levels within days for body temperature and within days for feed intake At days of age CON pigs were kgFrontiers in Neuroscience wwwfrontiersinAugust Volume 0cKeever et alGestational Activation of the Amygdalaheavier than MPA pigs standard error P whileno significant sex or interaction eï¬ects were detectedThe sequencing of the RNA samples produced billionsequenced reads and million pairedend reads per sampleThe number of reads was consistent across MIA and sexgroups coefficient of variation and the eï¬ects ofMIA sex and MIAbysex interaction were tested on genes that surpassed the minimum number of reads per MIA“sex combinationTranscriptome Changes Associated WithMaternal Immune Activation That AreSexDependentOverall genes exhibited a significant FDRadjusted P MIAbysex interaction eï¬ect and among these genes hada significant eï¬ect at FDRadjusted P The profile ofthese genes indicated that the eï¬ect of MIA diï¬ered betweenfemales and males Fortysix genes that presented a MIAbysex interaction eï¬ect are listed in Table together with theirexpression pattern and Pvalue The majority of the genes inTable including neurotensin NTS displayed a reversal inthe expression level between CON and MPA groups across sexesie opposite Log2[fold change] sign across sexes An extendedlist including genes that exhibited a MIAbysex interactioneï¬ect at FDRadjusted P is provided in SupplementaryFile S1 Table AAnother frequent pattern among the genes that displayed aMIAbysex interaction eï¬ect was characterized by a consistentexpression profile between CON and MPA across sexes albeitthe magnitude diï¬ered between sexes Table For exampleglycoprotein hormones alpha polypeptide CGA was overexpressed in CON relative to MPA but the diï¬erential washigher in males than in females Other genes presentingthis pattern included guanylatebinding protein GBP1transthyretin TTR aldehyde dehydrogenase family memberA2 ALDH1A2 hemoglobin subunit beta HBB and basichelixloophelix family member e22 BHLHE22GRPNotable is the significant MIAbysex interaction eï¬ecton genes associated with neuropeptides and hormones andgenes that participate in glutamatergic processes Genes underexpressed in MPA relative to CON males while presentingthe opposite pattern in females Table included NTS theneuropeptide gene proenkephalin PENK the neuropeptidegene gastrinreleasing peptidethe neuropeptiderelated gene vasoactive intestinal peptide receptor VIPR2corticotropin releasing hormone receptor CRHR2 neuronderived neurotrophic factor NDNF reelin RELN glutamatemetabotropic receptor GRM4 solute carrier family member SLC17A6 calcium voltagegated channel auxiliarysubunit alpha delta CACNA2D3 EFhand domain familymember D1 EFHD1 glutathione peroxidase GPX3parathyroid hormone receptor PTH1R thyroid hormoneresponsive THRSP and CGA The CGA gene codes for thealpha subunit protein of the hormones chorionic gonadotropinCG luteinizing hormone LH folliclestimulating hormoneFSH and thyroidstimulating hormone TSHFunctional and Network Analysis ofGenes That Exhibit SexDependentAssociations With Maternal ImmuneActivationThe genes expressing significant MIAbysex interaction eï¬ectswere analyzed for functional enrichment Table presentsthe clusters of most enriched and informative categoriesfrom the DAVID analysis and the complete list of categoriesis in Supplementary File S1 Table B The categories inTable encompass genes presenting the mostfrequentinteraction profile characterized by underexpression in CONfemales relative to males but overexpression in MPA femalesrelative to males These genes include KEGG Autoimmunethyroid diseaseCluster and BP brain developmentGO0007420 Cluster Enrichment results from GSEA complemented the findingsfrom DAVID Highly enriched informative categories amonggenes that have a MIAbysex interaction eï¬ect are presentedin Table and the extended list of categories is presented inSupplementary File S1 Table C The categories in Table in Table including ion homeostasissupport pathwaysTable and regulation of voltagegated calcium channelactivity processesenrichmentinteraction pathwayofthe neuroactiveand the hormoneactivity processesinclude genes such as CGA and VIPR2 that were identifiedin Table ligand receptorand neuropeptideTable Notablythethe diï¬erentialNetwork visualization furthered the understanding ofthe impact of MIA on the relationships among genes thatexhibited a significant MIAbysex interaction eï¬ect Thenetworks in Figures depictthe relationships betweengenes in the enriched neuroactive ligand receptor pathwaythat highlightexpression between CONand MPA in males and females ie CON_MaMPA_Maand CON_FeMPA_Ferespectively Red andrectangular nodesblueframework genes andthe known associations between genesedgesrepresentbased on curated databases of molecularinteractionsRed and blue nodes denote over or underexpressionof the gene in CON relative to MPA and the size is anthe diï¬erential expressioninverse logarithmic function ofPvalue Thediï¬erentialexpression pattern and connectivity among genes highlightsthe discrepancyelicited by MIAbetween the sexesin network modulescontrastsrepresentsimultaneousstudytheofTranscriptome Changes Associated WithMaternal Immune ActivationOverall genes exhibited diï¬erential FDRadjusted P expression between MPA and CON pigs irrespective of sexTable lists notable highly diï¬erentially expressed genesis in Supplementary File S1 Tableand the complete listD The majority of these genes were overexpressed in MPArelative to CON pigs Among the genes overexpressed in MPAcompared to CON pigs were islet amyloid polypeptide IAPPFrontiers in Neuroscience wwwfrontiersinAugust Volume 0cKeever et alGestational Activation of the AmygdalaTABLE Genes exhibiting significant FDRadjusted Pvalue maternal immune activationbysex interaction effectGene symbolPvalueaCON FeCON Ma MPA FeMPA MaCON FeMPA FeCON MaMPA MaCON FeMPA MaCON MaMPA FeRGS16CGAPOMCGPX3RELNVIPR2ANKRD34CGBP1GRM4CCDC136SLC17A6BTBD11TTRCACNA2D3CRHR2NDNFCXCL12USP43CCDC17KCNIP4CAMK2N2ALDH1A2GRPPENKSYT12PTH1RHBBESYT1EFHD1BHLHE22ZFP37SLC2A2THRSPNR4A3LOC396781C1QTNF1RAB27ANTSGVIN1SSTR1CCDC9BCCDC33CCDC162PPTHSYNPO2LCHGB5E115E115E115E115E115E115E115E115E1153E0911E0844E0850E0848E0728E0628E0662E0664E0671E0672E0696E0614E0515E0516E0529E0537E0567E0585E0596E0510E0412E0415E0431E0433E0444E0445E0456E0479E0479E0486E0488E0414E0314E0314E0315E0318E03ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’aLog2[fold change] between two maternal immune activationsex groups MPA PRRSVinduced maternal immune activation CON control Fe females Ma malesankyrin repeat domain ANKRD24interferoninducedtransmembrane protein IFITM1 and IFITM3 cathepsinC CTSC mitogenactivated protein kinase kinase MAP2K7heparan sulfateglucosamine 3sulfotransferase HS3ST5secreted phosphoprotein SPP1 immunoglobulin heavy chainIGHG and transforming acidic coiledcoilcontaining protein TACC1 Among the genes underexpressed in MPA relative toCON pigs are insulinlike growth factor IGF2 cellular retinoicacidbinding protein CRABP2 and aldehyde dehydrogenase family member A1 ALDH1A1Frontiers in Neuroscience wwwfrontiersinAugust Volume 0cKeever et alGestational Activation of the AmygdalaFunctional Analysis of Genes AssociatedWith Maternal Immune ActivationTable presents the top significant clusters of informativeenriched categoriesfrom the DAVID analysis of genesTABLE Most enriched DAVID clusters and supporting functional categoriesenrichment score ES among the genes presenting significant maternalimmune activationbysex interaction effectaCategory Category identifier and namePvaluebFDRPvalueCluster KEGGBPKEGGKEGGKEGGCluster BPBPBPBPBPCluster BPCluster BPBPES ssc05320Autoimmune thyroid diseaseGO000250ˆ¼Antigen processing andpresentation of peptide orpolysaccharide antigen via MHC class IIssc04514Celladhesion moleculesCAMsssc05323Rheumatoid arthritisssc05164Influenza AES GO0051050ˆ¼Positive regulation oftransportGO0051049ˆ¼Regulation of transportGO0050801ˆ¼Ion homeostasisGO0048878ˆ¼Chemical homeostasisGO0030001ˆ¼Metal ion transportES GO0048871ˆ¼Multicellular anismalhomeostasisES GO0061564ˆ¼Axon developmentGO0007420ˆ¼Brain development290E06190E03450E04340E01230E03320E02480E03200E02560E02170E01440E03350E01450E03130E02280E02450E02320E01360E01480E01570E01200E04370E01640E05130E03170E01310E01aBP biological process KEGG KEGG pathway bFalse discovery rate adjustedPvalueTABLE Enriched informative categories NES using GSEA among thegenes based on the overall maternal immune activationbysex interactionaCategory Category identifier and namebNES PvalueKEGGKEGGMFBPBPBPKEGGBPˆ’ 10E10ˆ’ 10E10ˆ’ 10E10ˆ’ 10E10ssc04080Neuroactive ligand receptorinteractionssc04912GnRH signaling pathwayGO0005179ˆ¼Hormone activityGO0006970ˆ¼Response to osmoticstressGO0019221ˆ¼Cytokine mediatedsignaling pathwayGO1901385ˆ¼Regulation of voltagegated calcium channel activityssc04020Calcium signaling pathway ˆ’ 12E01GO0085029ˆ¼Extracellular matrixˆ’ 18E01assemblyˆ’ 91E02ˆ’ 12E01cFDRPvalue83E0299E0222E0135E0154E0154E0154E0155E01aMF molecularfunction KEGG KEGG pathway BP biological processbNormalized enrichment score negative values indicate genes underexpressionin CON females relative to males but overexpression in MPA females relative tomales cFalse discovery rate adjusted Pvaluediï¬erentially expressed between MPA and CON groupsacross sexes the extended list of categories is presented inSupplementary File S1 Table E Some categories identifiedby the DAVID analysis are consistent with the categoriesdetected at more significant levels among the genes presentingan MIAbysex interaction eï¬ect Table and include theBP angiogenesisand KEGG autoimmunethyroid disease and Epstein“Barr virus infection pathwaysTable Also enriched Supplementary File S1 Table Ewere the BP homeostatic GO0042592 MF ion bindingGO0043167 and BP anatomical structure formation inmorphogenesis GO0048646GO0001525The GSEA enrichment results within the gene expressionpatterns of CON relative to MPA groups complemented thefindings from DAVID The most informative enriched categoriesare presented in Table and the extended list of categories ispresented in Supplementary File S1 Table F Enriched clustersof genes overexpressed in CON relative to MPA detected byGSEA were the BP enrichment of microtubule bundle formationGO0001578 and cilium morphogenesis GO0060271Transcriptome Differences BetweenSexes Independent of Maternal ImmuneActivationOverall genes were diï¬erentially expressed between malesand females FDRadjusted P These genes exhibiteda consistent diï¬erential expression between sexes irrespectiveof the MIA group The complete list of genes diï¬erentiallyexpressed between sexes at FDRadjusted P is available inSupplementary File S1 Table G and the majority were overexpressed in males relative to females Among the previousgenes excluding those that presented MIAbysex interactioneï¬ect a selection of informative genes is listed in Table Genesoverexpressed in males relative to females included eukaryotictranslation initiation factor 1A Ylinked EIF1AYleptinreceptor LEPR luteinizing hormone beta polypeptide LHBLIM homeobox LHX9 luteinizing hormone beta polypeptideLHB and immunoglobulin family member IGSF1Informative categories among the DAVID clusters ofenriched categoriesthe genes diï¬erentially expressedbetween sexes are listed in Table a complete list i

cancer206

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: Lasting and SexDependent Impactof Maternal Immune Activation onMolecular Pathways of the AmygdalaMarissa R Keever1 Pan Zhang2 Courtni R Bolt1 Adrienne M Antonson1Haley E Rymut1 Megan P Caputo1 Alexandra K Houser1 Alvaro G Hernandez3Bruce R Southey1 Laurie A Rund1 Rodney W Johnson14 andSandra L RodriguezZas12456 Department of Animal Sciences University of Illinois at UrbanaChampaign Urbana IL United States Illinois InformaticsInstitute University of Illinois at UrbanaChampaign Urbana IL United States Highthroughput Sequencingand Genotyping Unit Roy J Carver Biotechnology Center University of Illinois at UrbanaChampaign Urbana ILUnited States Neuroscience Program University of Illinois at UrbanaChampaign Urbana IL United States Departmentof Statistics University of Illinois at UrbanaChampaign Urbana IL United States Carl R Woese Institute for GenomicBiology University of Illinois at UrbanaChampaign Urbana IL United StatesThe prolonged and sexdependent impact of maternal immune activation MIA duringgestation on the molecular pathways of the amygdala a brain region that ‚uencessocial emotional and other behaviors is only partially understood To address thisgap we investigated the effects of viralelicited MIA during gestation on the amygdalatranscriptome of pigs a species of high molecular and developmental homology tohumans Gene expression levels were measured using RNASeq on the amygdalafor 3weekold female and male offspring from MIA and control groups Amongthe genes that exhibited significant MIA effect a prevalence of differentiallyexpressed genes annotated to the neuroactive ligand“receptor pathway glutamatergicfunctions neuropeptide systems and cilium morphogenesis were uncovered Genesin these categories included corticotropinreleasing hormone receptor glutamatemetabotropic receptor glycoprotein hormones alpha polypeptide parathyroidhormone receptor vasointestinal peptide receptor neurotensin proenkephalinand gastrinreleasing peptide These categories and genes have been associatedwith the MIArelated human neurodevelopmental disorders including schizophreniaand autism spectrum disorders Gene network reconstruction highlighted differentialvulnerability to MIA effects between sexes Our results advance the understandingnecessary for the development of multifactorial therapies targeting immune modulationand neurochemical dysfunction that can ameliorate the effects of MIA on offspringbehavior later in lifeKeywords immune activation pigs RNAseq neuropeptides glutamatergic pathway GABAergic pathwayINTRODUCTIONThe maternal immune response triggered by pathogens and other environmental stressors duringgestation can also elicit an indirect response by the fetal immune cells Kroismayr Odorizzi and Feeney Prins Viral infection during gestation for exampleactivates a cytokinerelated signaling cascade and molecules from this process can cross theEdited byNo¨lia Fern ndezCastilloCentre for Biomedical NetworkResearch CIBER SpainReviewed bySilvia PellegriniUniversity of Pisa ItalyTewarit SarachanaChulalongkorn University ThailandCorrespondenceSandra L RodriguezZasrodrgzzsillinoiseduSpecialty sectionThis was submitted toNeurogenomicsa section of the journalFrontiers in NeuroscienceReceived May Accepted July Published August CitationKeever MR Zhang P Bolt CRAntonson AM Rymut HE Caputo MPHouser AK Hernandez AGSouthey BR Rund LA Johnson RWand RodriguezZas SL Lastingand SexDependent Impactof Maternal Immune Activation onMolecular Pathways of the AmygdalaFront Neurosci 103389fnins202000774Frontiers in Neuroscience wwwfrontiersinAugust Volume 0cKeever et alGestational Activation of the Amygdalaplacenta and reach the fetal brain The resulting maternalimmune activation MIA can impactfetal developmentalprocesses and exert longterm postnatal eï¬ects in the oï¬springRutherford The relationship between MIAand neurodevelopmental disordersincluding schizophreniaspectrum disorders SSD and autism spectrum disorders ASDand neurodegenerative disorders such as Alzheimer™s diseaseAD in oï¬spring has been established Knuesel Canetta Mattei These diseases sharesome behavior symptoms comorbidities such as eating disordersand genetic and environmental ie MIA agents Canitanoand Pallagrosi The previous neurological disorders havebeen associated with abnormal structure and dysregulationof the amygdala Schumann FernandezIrigoyen and share genes and molecular mechanismsincluding histocompatibility complex MHC genes Andersand Kinney glutamatergic and GABAergicassociatedgenes Bourgeron Marin Li andmitochondrial activity processes Pieczenik and Neustadt Sragovich socialinteractionThe fetal amygdala is susceptible to ‚ammatory signals andthe plasticity of this brain structure to MIA can lead to alterationsof the developmental trajectory These disruptions may havelonglasting and maladaptive consequences for the oï¬springdue to the significant role that the amygdala plays in manyneurological pathways Located in the forebrain the amygdala‚uencescognition neuroendocrinebehavior learning memory emotion and autonomic systemsThe amygdala also modulates the response of these processesto stressors including pathogenic infections and those resultingfrom management practices such as weaning Tian The amygdala experiences high uptake of gonadalhormones and is anatomically connected to other sexuallydimorphic nuclei Therefore this brain region is involved inregulation of several dimorphic functions such as aggressionsexual behavior gonadotropin secretion and integration ofolfactory information Hines Evidence supports thediï¬erential activation of the amygdala to stimuli between malesand females Killgore and YurgelunTodd includingdiï¬erences in the sexual responses and emotional memoryHamann and diï¬erential vulnerability to insult Baird Due to the interconnected and multiregulatorynature of this brain structureinsults to the amygdala canimpact the individual™s social locomotor and feeding behaviorPetrovich and Gallagher growth and reproductivephysiology health status and immunological response tosecondary stressorsRecent studies lend support to the link between MIA andaltered amygdala function Carlezon In miceMIA elicited by polyinosinicpolycytidylic acid [PolyIC]increased the synaptic strength of glutamatergic projectionsfrom the prefrontal cortex to the amygdala Li In field tests mice exposed to MIA spentless timein the center and traveled a higher distanceindicativeof a higher anxiety behavior incidence than the controlcounterparts These findings suggest that the change in thebalance between excitation glutamatergic and inhibitiontherefore aï¬ecting brain circuitsspike output offeedforward GABAergic modified theamygdala neuronsthatcould regulate behavior in SSD and ASD A candidate genestudy of the eï¬ects of social stress during gestation reportedthatthe expression of a corticotropinreleasing hormonereceptor in the amygdala of 10weekold pigs was higherin females than in males Rutherford Thisstudy concluded that prenatal stress substantially increasedanxietyrelated behaviorstheimpact of maternal stressors during gestation on specificamygdala molecular profiles and associated neurological orbehavioral disorders in the oï¬spring later in life highlightthe complexity of the molecular mechanisms underlying thepathophysiology of MIAin female pigs Studies ofetalvirusadvantages ofrodents when consideringstudying a pig modelto pigsResearch on the lasting eï¬ects of MIA in pigs complementsAntonson et althe insights oï¬ered by rodent modelsstem Theratherfrom the greater homology of humansan physiologythan toin particular brain growth andsize development anddevelopment processesMurphy A pigmodel that has oï¬ered insights into MIA employs porcinereproductivePRRSVtothein the brain andmicroglia ie macrophagelike cellsisin neonatal pigsAntonson associated with behavioralelicit MIA Thisand respiratorysyndromechallengeactivatesimmunechangesThe study of MIA elicited by PRRSV allows for thecharacterization of the impact of a live viral pathogen thatselfreplicates in the host evoking extended activation ofimmune pathways PRRSV challenge during gestation is a wellcharacterized replicable and eï¬ective method for inducingMIA in pigs Antonson In additionPRRSV outbreaks impose a major economic burden to thelivestock industry PRRSV is an enveloped singlestranded RNAvirus thatinfects alveolar macrophages causing interstitialpneumonia and increased serum levels ofthe cytokinesinterleukin betainterleukin and tumor necrosis factoralpha Antonson The persistent repercussionsof MIA on the molecular pathways ofthe pig amygdalaare yet to be investigated Moreover the potentially distinctvulnerability to the prolonged eï¬ects of MIA between sexesremains unknownThe overarching goal of the present study is to advancethe understanding of the impact of MIA on the molecularmechanisms ofthe amygdala Three supporting objectivesare explored a characterization of prolonged transcriptomechanges elicited by viral MIA in pigs a species that hashigh neurodevelopmental homology with humansandfood production valueidentification of molecularpathwaysthat present diï¬erential vulnerability to MIAbetween sexes and c understanding the eï¬ect of MIA onmolecular interactions assisted by gene network inferenceThe findings from these complementary analyses supportthe use of multipleto amelioratethe potential detrimental eï¬ect of MIA on the oï¬springphysiology and behaviortherapeutictargetsbFrontiers in Neuroscience wwwfrontiersinAugust Volume 0cKeever et alGestational Activation of the AmygdalaMATERIALS AND METHODSAnimal ExperimentsAll experimental procedures used published protocols Antonson The animal studies were approved by theIllinois Institutional Animal Care and Use Committee IACUCat the University of Illinois and are in compliance with the USDAAnimal Welfare Act and the NIH Public Health Service Policy onthe Humane Care and Use of AnimalsCamborough gilts born and raised at the University of Illinoisat UrbanaChampaign herd were inseminated at days ofage using PIC boar sperm Antonson All gilts were PRRSV negative and were moved at gestationday GD into diseasecontainment chambers maintainedat —¦C and a h lightdark cycle with lights on at AM The gilts were fed daily kg of a gestational diet andhad ad libitum water access One week after acclimation fourgilts were intranasally inoculated with live PRRSV strain P129BV School of Veterinary Medicine at Purdue University WestLafayette IN United States using mL of — median tissueculture infectious dose TCID50 diluted in sterile Dulbecco™smodified Eagle medium DMEM mL total volume Thefour gilts in the Control group were intranasally inoculatedwith an equal volume of sterile DMEM PRRSV inoculationcorresponded to the last third of gestation in pigs and humansduring initiation of rapid fetal brain growth Antonson PRRSV and Control groups were housed in separatecontainment chambersThe rectal temperatures and diet consumption of the giltswere recorded daily until farrowing Antonson The PRRSVinoculated gilts were oï¬ered the maximumfed daily and feed refusal was measured The Control giltswere fed the same amount consumed by the PRRSVinoculatedgilts on the previous day The daily body temperature andfeed intake levels were compared using a mixedeï¬ects modelanalyzed with PROC MIXED SAS Institute Inc Cary NCUnited States The model included the eï¬ects of gilt treatmentand replicate while accommodating for heterogeneity of variancebetween MIA groupsFarrowing was induced with an intramuscular injection of mg of Lutalyse dinoprost tromethamine Pfizer New YorkNY United States on GD in consideration that the averagegestation length is approximately days Antonson Gilts farrowed in individual farrowing crates ofstandard dimensions — m After farrowing thegilts were fed twice a day up to kg of a nutritionallycomplete diet for the lactating period and water remainedavailable ad libitum Pigs received intramuscular injections ofiron dextran mgpig Butler Schein Animal Health DublinOH United States and Excede for Swine mgpig ZoetisParsippany NJ United States to control for respiratory diseasesThe pigs remained with their mothers until PD The bodyweight of pigs was measured daily and analyzed using the mixedeï¬ects model in SAS PROC MIXED SAS Institute Inc CaryNC United States The model included the eï¬ect of MIA andthe random eï¬ect of gilt accommodating for heteroscedasticitybetween pig treatment and sex groups The impact of MIA wasstudied at PD because this is a common age to wean pigsThe study of transcriptome profiles from older pigs could beconfounded with changes in diet and environment associatedwith weaning while profiles from younger pigs would hinder theassessment of the prolonged eï¬ects of MIARNA Extraction and SequencingA balanced experimental design was studiedincluding pigs evenly distributed between maternal PPRSV activatedMPA group of pigs and Control gilts CON group of pigseach group encompassing males and females denoted Maand Fe respectively At PD pigs were removed fromthe farrowing crate and anesthetized intramuscularly using atelazolketaminexylazine drug cocktail mg of tiletamine mg of zolazepam reconstituted with mL ketamine gL and mL xylazine gL Fort Dodge AnimalHealth Fort Dodge IA United States at a dose of mLkgbody weight following protocols Antonson Following anesthetization pigs were euthanized using anintracardiac injection of sodium pentobarbital mgkg bodyweight Fata Plus Vortech Pharmaceuticals Dearborn MIUnited States Pig brains were extracted the amygdalae wererecognized using the stereotaxic atlas of the pig brain Felix dissected out flash frozen on dry ice and stored at ˆ’—¦Cfollowing published protocols Antonson RNA wasisolated using EZNA isolation kit following the manufacturer™sinstructions Omega Biotek Norcross GA United States TheRNA integrity numbers of the samples were above indicatinglow RNA degradation The RNASeq libraries were preparedwith TruSeq Stranded mRNAseq Sample Prep kit Illumina IncSan Diego CA United States The libraries were quantitatedby qPCR and sequenced on one lane on a NovaSeq for cycles from each end of the fragments using NovaSeqS4 reagent kit FASTQ files were generated and demultiplexedwith the bcl2fastq v220 conversion software Pairedend reads nt long were obtained and the FASTQ files are availablein the National Center for Biotechnology Information GeneExpression Omnibus GEO database experiment accessionnumber GSE149695RNA Sequence Mapping and DifferentialExpression AnalysisThe average Phred quality score of the reads assessed usingFastQC Andrews was across all read positions andtherefore no reads were trimmed The pairedend reads fromthe individual samples were aligned to the Sus scrofa genomeversion Sscrofa Pruitt using kallisto v0430Bray with default settings The normalized trimmedmean of Mvalues gene expression values were described usinga generalized linear model encompassing the eï¬ects of the MIAgroup MPA or CON levels sex Fe or Ma levels and MIAbysex interaction and analyzed using edgeR version in the R v environment Robinson Genessupported by transcripts per million TPM by each MIA“sex combination were analyzed to ensure adequate representationacross comparisonsFrontiers in Neuroscience wwwfrontiersinAugust Volume 0cKeever et alGestational Activation of the AmygdalaOrthogonal pairwise contrasts between MIA and sex groupswere evaluated in addition to testing for the eï¬ects of MIAbysex interaction and main eï¬ects of MIA and sex Thefour groups compared in the contrasts identified by treatmentfollowed by the sex levels are MPA_Fe MPA_Ma CON_Fe andCON_Ma The Pvalues were adjusted for multiple testing usingthe Benjamini“Hochberg false discovery rate FDR approachBenjamini and Hochberg categoriesamongtheMFand KEGG pathways The GeneFunctional Enrichment and NetworkInferenceapproaches were used to identifyTwo complementaryoverrepresented functionalgenesexhibiting diï¬erential expression across MIA and sex groupsCaetanoAnoll©s GonzalezPena et al2016ab Functional categories investigated included GeneBPs GO molecularOntology GO biological processesfunctionsSetEnrichment Analysisapproach implemented inthesoftware package GSEAP Subramanian was used to identify category overrepresentationwith gene over and underexpressed while considering allgenes analyzed The normalized enrichmentscore NESofin the Molecular Signature DatabaseMSigDB was calculated using the maximum deviation ofthe cumulative sum based on the signed and standardizedfold change The statistical significance ofthe enrichmentwas assessed using the FDRadjusted Pvalue computed from permutationscategoriesGSEAtheThe overrepresentation of functional categories was alsoevaluated among genes that exhibited a significant MIAbysex interaction or main eï¬ect using the Database forAnnotation Visualization and Integrated Discovery DAVID Huang The enrichment of Direct GOcategories in the DAVID database was assessed The Susscrofa genome was used as the background for enrichmenttesting and enrichmentis reported using the ExpressionAnalysis Systematic Explorer EASE score that was computedusing a onetailed jackknifed Fisher hypergeometric exacttest Functional categories were clustered based on geneannotation and the statisticalissummarized as the geometric mean of the log10 EASE scoresofthe categories Delfino Serao Delfino and RodriguezZas significance of clustersWeighted Gene Coexpression NetworkAnalysis and Gene Network VisualizationAn approach complementary to the identification of diï¬erentiallyexpressed genes was used to uncover coexpression networksusing Weighted Gene Coexpression Network AnalysisWGCNA version Langfelder and Horvath The input data were voomtransformed read count valuesgenerated using the limma package version Ritchie in R version Genes were filtered to removethose with low expression levels or no variation across samplesper developer recommendations The number of genes usedfor network analysis was genes Considering potentialfor interaction patterns a sexdependent softthresholdingpower was used to call for network topology analysis Thelowest power values that support a scalefree topology powerused were for the CON_MaMPA_Ma contrast and for the MPA_FeMPA_Ma contrast The Pearson correlationcoefficient ofthe normalized expression values was usedto identify modules of connected genes The minimummodule size was set to with the deepSplit set to and themergeCutHeight set to Module profiles were identifiedusing the correlation between the eigengene of each moduleand pig group Enrichment of functional categories among thegenes in each module profile was explored with DAVID using theSus scrofa genome as background and testing included an FDRmultiple test adjustmentFurther understanding of the impact of the MIAbysexinteraction was gained through the reconstruction of genenetworks using the BisoGenet package Martin inthe Cytoscape platform Shannon Information fromgene and protein interactions annotated in databases includingBIOGRID HPRD DIP BIND INTACT and MINT was usedto visualize relationships between genes Salwinski Alfarano Mishra Stark Kerrien Licata Networks highlightingdiï¬erences in gene levels associated with MIA within sex iethe contrasts MPA_MaCON_Ma and MPA_FeCON_Fe werecompared The network framework includes genes that exhibiteda significant MIAbysex interaction eï¬ect FDRadjusted P and are annotated to enriched functional categoriesThe framework genes were identified by full nodes with sizereflecting the diï¬erential expression level between the MPAand CON groups The network edges depict known molecularrelationships curated in the BisoGenet databases The frameworkgenes were connected through correlated genes listed in theBisoGenet database of molecular interactions that did not reachsignificant MIAbysex interaction eï¬ect The comparison ofthese networks oï¬ered insights into the simultaneous eï¬ect ofMIA across interacting genes and enabled the detection of sharedand distinct coregulation patterns between MPA and CONpigs across sexesRESULTSMaternal Immune Activation andSequencing MetricsThe diï¬erences between MPA and CON giltsin rectaltemperatures and daily diet consumption indicated the activationof the maternal immune system in response to PRRSV Thediï¬erence in body temperature between CON and MPA giltson GD was —¦C standard error —¦C P Thediï¬erence in feed refusal between CON and MPA gilts on GD was g standard error g P A significantincrease in rectal temperatures and decrease in feed intakeP was observed within h of inoculation and returnedto baseline levels within days for body temperature and within days for feed intake At days of age CON pigs were kgFrontiers in Neuroscience wwwfrontiersinAugust Volume 0cKeever et alGestational Activation of the Amygdalaheavier than MPA pigs standard error P whileno significant sex or interaction eï¬ects were detectedThe sequencing of the RNA samples produced billionsequenced reads and million pairedend reads per sampleThe number of reads was consistent across MIA and sexgroups coefficient of variation and the eï¬ects ofMIA sex and MIAbysex interaction were tested on genes that surpassed the minimum number of reads per MIA“sex combinationTranscriptome Changes Associated WithMaternal Immune Activation That AreSexDependentOverall genes exhibited a significant FDRadjusted P MIAbysex interaction eï¬ect and among these genes hada significant eï¬ect at FDRadjusted P The profile ofthese genes indicated that the eï¬ect of MIA diï¬ered betweenfemales and males Fortysix genes that presented a MIAbysex interaction eï¬ect are listed in Table together with theirexpression pattern and Pvalue The majority of the genes inTable including neurotensin NTS displayed a reversal inthe expression level between CON and MPA groups across sexesie opposite Log2[fold change] sign across sexes An extendedlist including genes that exhibited a MIAbysex interactioneï¬ect at FDRadjusted P is provided in SupplementaryFile S1 Table AAnother frequent pattern among the genes that displayed aMIAbysex interaction eï¬ect was characterized by a consistentexpression profile between CON and MPA across sexes albeitthe magnitude diï¬ered between sexes Table For exampleglycoprotein hormones alpha polypeptide CGA was overexpressed in CON relative to MPA but the diï¬erential washigher in males than in females Other genes presentingthis pattern included guanylatebinding protein GBP1transthyretin TTR aldehyde dehydrogenase family memberA2 ALDH1A2 hemoglobin subunit beta HBB and basichelixloophelix family member e22 BHLHE22GRPNotable is the significant MIAbysex interaction eï¬ecton genes associated with neuropeptides and hormones andgenes that participate in glutamatergic processes Genes underexpressed in MPA relative to CON males while presentingthe opposite pattern in females Table included NTS theneuropeptide gene proenkephalin PENK the neuropeptidegene gastrinreleasing peptidethe neuropeptiderelated gene vasoactive intestinal peptide receptor VIPR2corticotropin releasing hormone receptor CRHR2 neuronderived neurotrophic factor NDNF reelin RELN glutamatemetabotropic receptor GRM4 solute carrier family member SLC17A6 calcium voltagegated channel auxiliarysubunit alpha delta CACNA2D3 EFhand domain familymember D1 EFHD1 glutathione peroxidase GPX3parathyroid hormone receptor PTH1R thyroid hormoneresponsive THRSP and CGA The CGA gene codes for thealpha subunit protein of the hormones chorionic gonadotropinCG luteinizing hormone LH folliclestimulating hormoneFSH and thyroidstimulating hormone TSHFunctional and Network Analysis ofGenes That Exhibit SexDependentAssociations With Maternal ImmuneActivationThe genes expressing significant MIAbysex interaction eï¬ectswere analyzed for functional enrichment Table presentsthe clusters of most enriched and informative categoriesfrom the DAVID analysis and the complete list of categoriesis in Supplementary File S1 Table B The categories inTable encompass genes presenting the mostfrequentinteraction profile characterized by underexpression in CONfemales relative to males but overexpression in MPA femalesrelative to males These genes include KEGG Autoimmunethyroid diseaseCluster and BP brain developmentGO0007420 Cluster Enrichment results from GSEA complemented the findingsfrom DAVID Highly enriched informative categories amonggenes that have a MIAbysex interaction eï¬ect are presentedin Table and the extended list of categories is presented inSupplementary File S1 Table C The categories in Table in Table including ion homeostasissupport pathwaysTable and regulation of voltagegated calcium channelactivity processesenrichmentinteraction pathwayofthe neuroactiveand the hormoneactivity processesinclude genes such as CGA and VIPR2 that were identifiedin Table ligand receptorand neuropeptideTable Notablythethe diï¬erentialNetwork visualization furthered the understanding ofthe impact of MIA on the relationships among genes thatexhibited a significant MIAbysex interaction eï¬ect Thenetworks in Figures depictthe relationships betweengenes in the enriched neuroactive ligand receptor pathwaythat highlightexpression between CONand MPA in males and females ie CON_MaMPA_Maand CON_FeMPA_Ferespectively Red andrectangular nodesblueframework genes andthe known associations between genesedgesrepresentbased on curated databases of molecularinteractionsRed and blue nodes denote over or underexpressionof the gene in CON relative to MPA and the size is anthe diï¬erential expressioninverse logarithmic function ofPvalue Thediï¬erentialexpression pattern and connectivity among genes highlightsthe discrepancyelicited by MIAbetween the sexesin network modulescontrastsrepresentsimultaneousstudytheofTranscriptome Changes Associated WithMaternal Immune ActivationOverall genes exhibited diï¬erential FDRadjusted P expression between MPA and CON pigs irrespective of sexTable lists notable highly diï¬erentially expressed genesis in Supplementary File S1 Tableand the complete listD The majority of these genes were overexpressed in MPArelative to CON pigs Among the genes overexpressed in MPAcompared to CON pigs were islet amyloid polypeptide IAPPFrontiers in Neuroscience wwwfrontiersinAugust Volume 0cKeever et alGestational Activation of the AmygdalaTABLE Genes exhibiting significant FDRadjusted Pvalue maternal immune activationbysex interaction effectGene symbolPvalueaCON FeCON Ma MPA FeMPA MaCON FeMPA FeCON MaMPA MaCON FeMPA MaCON MaMPA FeRGS16CGAPOMCGPX3RELNVIPR2ANKRD34CGBP1GRM4CCDC136SLC17A6BTBD11TTRCACNA2D3CRHR2NDNFCXCL12USP43CCDC17KCNIP4CAMK2N2ALDH1A2GRPPENKSYT12PTH1RHBBESYT1EFHD1BHLHE22ZFP37SLC2A2THRSPNR4A3LOC396781C1QTNF1RAB27ANTSGVIN1SSTR1CCDC9BCCDC33CCDC162PPTHSYNPO2LCHGB5E115E115E115E115E115E115E115E115E1153E0911E0844E0850E0848E0728E0628E0662E0664E0671E0672E0696E0614E0515E0516E0529E0537E0567E0585E0596E0510E0412E0415E0431E0433E0444E0445E0456E0479E0479E0486E0488E0414E0314E0314E0315E0318E03ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’aLog2[fold change] between two maternal immune activationsex groups MPA PRRSVinduced maternal immune activation CON control Fe females Ma malesankyrin repeat domain ANKRD24interferoninducedtransmembrane protein IFITM1 and IFITM3 cathepsinC CTSC mitogenactivated protein kinase kinase MAP2K7heparan sulfateglucosamine 3sulfotransferase HS3ST5secreted phosphoprotein SPP1 immunoglobulin heavy chainIGHG and transforming acidic coiledcoilcontaining protein TACC1 Among the genes underexpressed in MPA relative toCON pigs are insulinlike growth factor IGF2 cellular retinoicacidbinding protein CRABP2 and aldehyde dehydrogenase family member A1 ALDH1A1Frontiers in Neuroscience wwwfrontiersinAugust Volume 0cKeever et alGestational Activation of the AmygdalaFunctional Analysis of Genes AssociatedWith Maternal Immune ActivationTable presents the top significant clusters of informativeenriched categoriesfrom the DAVID analysis of genesTABLE Most enriched DAVID clusters and supporting functional categoriesenrichment score ES among the genes presenting significant maternalimmune activationbysex interaction effectaCategory Category identifier and namePvaluebFDRPvalueCluster KEGGBPKEGGKEGGKEGGCluster BPBPBPBPBPCluster BPCluster BPBPES ssc05320Autoimmune thyroid diseaseGO000250ˆ¼Antigen processing andpresentation of peptide orpolysaccharide antigen via MHC class IIssc04514Celladhesion moleculesCAMsssc05323Rheumatoid arthritisssc05164Influenza AES GO0051050ˆ¼Positive regulation oftransportGO0051049ˆ¼Regulation of transportGO0050801ˆ¼Ion homeostasisGO0048878ˆ¼Chemical homeostasisGO0030001ˆ¼Metal ion transportES GO0048871ˆ¼Multicellular anismalhomeostasisES GO0061564ˆ¼Axon developmentGO0007420ˆ¼Brain development290E06190E03450E04340E01230E03320E02480E03200E02560E02170E01440E03350E01450E03130E02280E02450E02320E01360E01480E01570E01200E04370E01640E05130E03170E01310E01aBP biological process KEGG KEGG pathway bFalse discovery rate adjustedPvalueTABLE Enriched informative categories NES using GSEA among thegenes based on the overall maternal immune activationbysex interactionaCategory Category identifier and namebNES PvalueKEGGKEGGMFBPBPBPKEGGBPˆ’ 10E10ˆ’ 10E10ˆ’ 10E10ˆ’ 10E10ssc04080Neuroactive ligand receptorinteractionssc04912GnRH signaling pathwayGO0005179ˆ¼Hormone activityGO0006970ˆ¼Response to osmoticstressGO0019221ˆ¼Cytokine mediatedsignaling pathwayGO1901385ˆ¼Regulation of voltagegated calcium channel activityssc04020Calcium signaling pathway ˆ’ 12E01GO0085029ˆ¼Extracellular matrixˆ’ 18E01assemblyˆ’ 91E02ˆ’ 12E01cFDRPvalue83E0299E0222E0135E0154E0154E0154E0155E01aMF molecularfunction KEGG KEGG pathway BP biological processbNormalized enrichment score negative values indicate genes underexpressionin CON females relative to males but overexpression in MPA females relative tomales cFalse discovery rate adjusted Pvaluediï¬erentially expressed between MPA and CON groupsacross sexes the extended list of categories is presented inSupplementary File S1 Table E Some categories identifiedby the DAVID analysis are consistent with the categoriesdetected at more significant levels among the genes presentingan MIAbysex interaction eï¬ect Table and include theBP angiogenesisand KEGG autoimmunethyroid disease and Epstein“Barr virus infection pathwaysTable Also enriched Supplementary File S1 Table Ewere the BP homeostatic GO0042592 MF ion bindingGO0043167 and BP anatomical structure formation inmorphogenesis GO0048646GO0001525The GSEA enrichment results within the gene expressionpatterns of CON relative to MPA groups complemented thefindings from DAVID The most informative enriched categoriesare presented in Table and the extended list of categories ispresented in Supplementary File S1 Table F Enriched clustersof genes overexpressed in CON relative to MPA detected byGSEA were the BP enrichment of microtubule bundle formationGO0001578 and cilium morphogenesis GO0060271Transcriptome Differences BetweenSexes Independent of Maternal ImmuneActivationOverall genes were diï¬erentially expressed between malesand females FDRadjusted P These genes exhibiteda consistent diï¬erential expression between sexes irrespectiveof the MIA group The complete list of genes diï¬erentiallyexpressed between sexes at FDRadjusted P is available inSupplementary File S1 Table G and the majority were overexpressed in males relative to females Among the previousgenes excluding those that presented MIAbysex interactioneï¬ect a selection of informative genes is listed in Table Genesoverexpressed in males relative to females included eukaryotictranslation initiation factor 1A Ylinked EIF1AYleptinreceptor LEPR luteinizing hormone beta polypeptide LHBLIM homeobox LHX9 luteinizing hormone beta polypeptideLHB and immunoglobulin family member IGSF1Informative categories among the DAVID clusters ofenriched categoriesthe genes diï¬erentially expressedbetween sexes are listed in Table a complete list i Answer:

Thyroid_Cancer

Deoxyshikonin Inhibits Viability andGlycolysis by Suppressing theAktmTOR Pathway in Acute MyeloidLeukemia CellsHuijuan Wu   Hongmian Zhao   and Li Chen Telemedicine and Connected Health Center Huaihe Hospital of Henan University Kaifeng China Department ofHematology Huaihe Hospital of Henan University Kaifeng ChinaDeoxyshikonin was reported to exhibit an antitumor effect in colorectal cancer Howeverno studies are available to illustrate the effect of deoxyshikonin on acute myeloid leukemiaAML The effects of deoxyshikonin on viability apoptosis caspase37 activity andcytochrome Cyt C expression were evaluated by Cell Counting Kit8 assay flowcytometry analysis caspase37 activity assay and western blot analysis respectivelyGlucose consumption and lactate production were measured to determine the glycolysislevel The expression of pyruvate kinase M2 PKM2 was detected by quantitativerealtime polymerase chain reaction and western blot analysis The results showed thatdeoxyshikonin inhibited cell viability and increased the apoptotic rate the caspase37activity and the Cyt C protein level in AML cells in a dosedependent manner Additionallydeoxyshikonin concentrationdependently decreased glucose consumptionlactateproduction and PKM2 expression in AML cells Deoxyshikonin inactivated the proteinkinase B Aktmammalian target of the rapamycin mTOR pathway The activation ofthe AktmTOR pathway reversed the effects of deoxyshikonin on viability apoptosisand glycolysis in AML cells In deoxyshikonin dampened the viability and theglycolysis of AML cells by suppressing PKM2 via inactivation of the AktmTOR signalingEdited byCyrus KhandanpourUniversity Hospital M¼nster GermanyReviewed byPeng YangShanxi University ChinaHaili WuShanxi University ChinaHongyu ZhouKunming Medical University ChinaCorrespondenceHuijuan Wuhjwu297163com These authors share first authorshipKeywords deoxyshikonin glycolysis PKM2 the AktmTOR signaling acute myeloid leukemiaSpecialty sectionThis was submitted toHematologic Malignanciesa section of the journalFrontiers in OncologyReceived March Accepted June Published August CitationWu H Zhao H and Chen L Deoxyshikonin Inhibits Viability andGlycolysis by Suppressing theAktmTOR Pathway in Acute MyeloidLeukemia CellsFront Oncol 103389fonc202001253INTRODUCTIONAcute myeloid leukemia AML the most prevalent form of acute leukemia in adults is anaggressive malignancy derived from hemopoietic progenitor cells and with poor survival rate andfrequent relapse posing a threat to the health and life of aï¬ected patients AML is characterizedby rapid growth impaired apoptosis and abnormal clonal accumulation of hematopoietic stemcells in the bone marrow due to various genetic and epigenetic changes eventually leading to bonemarrow failure There were an estimated new cases diagnosed with AML and mortalities due to AML according to the statistics in Presently the efficacy of the standardchemotherapy for AML patients remains suboptimal owing to drug resistance and high clinicalrelapse rate In this regard searching for novel antileukemia drugs is urgently required toeï¬ectively improve the outcome of patients with AMLShikonin 58dihydroxy2[1S1hydroxy4methylpent3en1yl]naphthalene14 dionea naturally occurring naphthoquinone extracted from the oriental traditional medical herbFrontiers in Oncology wwwfrontiersinAugust Volume 0cWu et alDeoxyshikonin Inhibits AML Cellsand suppressed glycolysis in AML cells Mechanistically the antiAML eï¬ect of deoxyshikonin was mediated via repressing PKM2by inactivation of the AktmTOR pathwayMATERIALS AND METHODSCell Cultivation and TreatmentAML cell lines including THP1 and HL60 were purchasedfrom American Tissue Culture Collection ATCC ManassasVA USA and routinely cultured in Roswell Park MemorialInstitute1640 medium HyClone South Logan UT USAconjugated with heatinactivated fetal bovine serum ExCellBio Shanghai China and penicillinstreptomycin SigmaAldrich St Louis MO USA The cells were fostered at —¦Cin a drippy environment flushed with air plus CO2Deoxyshikonin purity Tauto Biotech Shanghai Chinawas dissolved in dimethyl sulfoxide SigmaAldrich St LouisMO USA as a reserve solution and diluted into diï¬erentconcentrations and µgml The THP1and HL60 cells were exposed to diverse doses of deoxyshikoninfor h The Aktoverexpressing plasmid pcDNAAkt andthe empty vectorpcDNA control were obtained fromRibobio Guangzhou China Transfection was performed usingLipofectamine reagent Invitrogen Carlsbad CA USACell Counting Kit8 AssayCell Counting Kit8 CCK8 Beyotime Shanghai China wastaken to evaluate the viability of AML cells THP1 and HL60cells were seeded into 96well plates at — cellswell anddealt with various concentrations of deoxyshikonin and µgml for h or incubated with µgmldeoxyshikonin in the presence or the absence of µM 740YP Tocris Bioscience Shanghai China an activator of theAktmTOR signaling pathway After treatment for h µl ofCCK8 solution was added to each well followed by incubationfor another h The optical density of each well was recorded at awavelength of nm using a microplate reader Thermo FisherScientific Waltham MA USAFlow Cytometry Analysis for ApoptosisAfter the treatments as aforementioned annexin V FITCand propidium iodide kitKeyGen Nanjing China wasimplemented to analyze the apoptosis of THP1 and HL60 cellsCell apoptotic rate was detected by means of a flow cytometerFACScan BD Biosciences San Diego CA USAtheastreatmentsCaspase37 Activity AssayFollowingaforementioned ApoONEHomogeneous CaspaseGlo Assay kit Promega MadisonWI USA was adopted to measure the caspase37 activityof THP1 and HL60 cells referring to the manufacturer™sdescription Finally M2000 Infinite Pro instrument TecanTrading AG Maennedorf Switzerland was used to determinethe luminescenceFIGURE Chemical structure of deoxyshikoninLithospermum erythrorhizon Sieb et Zucc has been extensivelyused for the treatment of many diseases including burnssore throats HIV1 infection and macular eruption Currently clinical and pharmacological propertiesstudieshave demonstrated that shikonin and its derivatives exhibitvarious biological activities such as immune regulation andantithrombotic anti‚ammatory antioxidative and antiglycolytic activities An increasing number of researchesreveal that shikonin derivatives have garnered much researchinterest due to its limited toxicity and stronger antitumoractivities in miscellaneous cancers Interestingly a previousinvestigation proved that deoxyshikonin its chemical structureis shown in Figure a derivative of shikonin exhibited an antitumor eï¬ect in colorectal cancer However no studies areavailable to illustrate the eï¬ect of deoxyshikonin on AMLAerobic glycolysis also known as the Warburg eï¬ect is wellrecognized as a metabolic pathway in the rapidly proliferatingcancer cells for the regeneration of energy and the biosynthesisof macromolecules even in the presence of sufficient oxygen Aerobic glycolysis has been extensively accepted as an importantcharacteristic of tumor cells including AML eventually resultingin increased glucose consumption and lactate production Pyruvate kinase PK is a ratelimiting glycolytic enzymePyruvate kinase M1 PKM1 is expressed in normal diï¬erentiatedtissues whereas pyruvate kinase M2 PKM2 is expressedin cancer cellsleading to increased glycolysis As weall know the protein kinase B Aktmammalian target ofrapamycin mTOR pathway widely existing in cells is one ofthe most important survival signaling pathways that participatein the regulation of diverse physiological processes such as cellgrowth apoptosis and metabolism It has been reportedthat the AktmTOR pathway is a positive regulator of PKM2expression Previous studies suggested that shikonininhibited glycolysis by suppression of PKM2 expression Therefore we hypothesized that deoxyshikonin inhibitedviability and glycolysis suppressing pyruvate kinase M2 via theAktmTOR pathway in acute myeloid leukemia cellsIn the present study we assessed the eï¬ects and the underlyingmechanisms of deoxyshikonin on viability apoptosis glycolysisand PKM2 expression in AML cells These results revealed thatdeoxyshikonin treatment inhibited viability induced apoptosisFrontiers in Oncology wwwfrontiersinAugust Volume 0cWu et alDeoxyshikonin Inhibits AML CellsDetermination of Glucose Consumptionand Lactate ProductionAfter the treatments as aforementioned the culture medium ofTHP1 and HL60 cells was collected for the measurement ofglucose and lactate levels using a glucose uptake colorimetricassay kit SigmaAldrich and a lactic acid assay kit JianchengBioengineering Institute Nanjing China respectivelyPKM2 Activity AssayPKM2 activity was detected by the lactate dehydrogenasecoupled assay as described earlier Five microliters of wholecelllysate was utilized in the assay The absorbance at awavelength of nm was measured using a microplate readerThermo Fisher Scientificicecold radioimmunoprecipitation assayWestern Blot AnalysisThe treated THP1 and HL60 cells were collected and rinsedwith phosphatebuï¬ered salinefollowed by the additionoflysis buï¬erBeyotime containing mM phenylmethylsulfonyl fluorideSigmaAldrich A total of µg of protein samples wasfractionated on sodium dodecyl sulfatepolyacrylamide gelelectrophoresis prior to electrotransfer onto polyvinylidenedifluoride membranes Millipore Bedford MA USA Afterblocking with defatted milkTrisbased saline with Tween atroom temperature for h the membranes were immunoblottedovernight at —¦C with corresponding primary antibodiesagainst cytochrome C Cyt C Cell Signaling TechnologyInc Danvers MA USA phosphorylated Akt pAkt Ser473Abcam Cambridge MA USA AktAbcam glycogensynthase kinase3β GSK3β Abcam phosphorylated GSK3βpGSK3β Ser9 Abcam mTOR Abcam phosphorylatedmTOR pmTOR Ser2448 Abcam p70 ribosomal S6 kinasep70S6K Cell Signaling TechnologyInc phosphorylatedp70S6K pp70S6K Thr389 Cell Signaling Technology Inceukaryotic translation initiation factor 4Ebinding protein 4EBP1 Cell Signaling Technology Inc phosphorylated4EBP1 p4EBP1 Thr70 Cell Signaling TechnologyIncPKM2 Abcam and βactin Abcam and then incubated withhorseradish peroxidaseconjugated secondary antibodies CellSignaling Technology Inc for h at room temperature Lastlyan enhanced chemiluminescence kit Amersham PharmaciaPiscataway NJ USA was implemented to examine the antigen“antibody complexes βActin was used as a loading control Theprotein bands were visualized by VersaDoc imaging systemBioRad Hercules CA USAQuantitative RealTime PCRRNAiso Plus TaKaRa Dalian China was used to extracttotal RNA from treated THP1 and HL60 cells and theconcentration of extracted RNA was measured using a NanoDrop spectrophotometer Thermo Fisher Scientific Reversetranscription was carried out using the PrimeScript RT ReagentKit Takara Dalian China SYBR Green Taq Mix TaKaRawas then implemented to detect PKM2 mRNA expressionon the StepOnePlus qPCR system Thermo Fisher Scientificwith βactin as an endogenous control The thermocyclingconditions were displayed as follows —¦C for s followedby cycles of —¦C for s —¦C for s and —¦C for sThe primers were as follows PKM2 ²GCTG CCAT CTACCACT TGC3² forward and ²CCAG ACTT GGTG AGGACGAT T3² reverse GAPDH ²ATGT CGTG GAGT CTACTGGC3² forward and ²TGAC CTTG CCCA CAGC CTTG² reverse The ˆ’\x01\x01Ct method was taken to quantify theexpression level of PKM2 mRNAStatisticsAll data are shown as mean ± standard deviation of threeindependent experiments Statistical assays were determinedusing SPSS software IBM Corp Armonk NY USA withStudent™s ttest or oneway ANOVA followed by Dunnett™s test P were regarded as statistically significantRESULTSDeoxyshikonin Inhibited the Viability ofAML CellsTo clarify the antitumor activity of deoxyshikonin in AMLcells CCK8 was taken to evaluate cell viability after THP and HL60 cells were exposed to a series of deoxyshikoninconcentrations and µgml for h Asshown in Figures 2AB cell viability was significantly declinedin a concentrationdependent manner in THP1 and HL60 cellsin response to deoxyshikoninDeoxyshikonin Enhanced the Apoptosis ofAML CellsThe eï¬ect of deoxyshikonin on the apoptotic consequences ofAML cells was investigated by annexin VFITC apoptosis assayAs a result deoxyshikonin treatment led to a concentrationdependent increase of apoptotic rate in THP1 Figure 3Aand HL60 cells Figure 3B In line with the results of theflow cytometry analysis an elevation of caspase37 activityin deoxyshikonintreated THP1 Figure 3C and HL60 cellsFigure 3D was observed The mitochondrial protein Cyt Cis known as the initiating factor of mitochondrial apoptosispathway The expression of apoptotic marker Cyt C in THP1and HL60 cells was further detected by western blot analysisThe results demonstrated that deoxyshikonin concentrationdependently increased Cyt C protein level in THP1 Figure 3Eand HL60 cells Figure 3F relative to the control group Theseresults suggested that deoxyshikonin facilitated the apoptosis ofAML cellsDeoxyshikonin Suppressed Glycolysis inAML CellsTo further characterize the eï¬ect of deoxyshikonin on glycolysisin AML cells we measured glucose consumption and lactateproduction in AML cells These results demonstrated thatdeoxyshikonin exposure decreased glucoseconsumptionFigures 4AB and lactate production Figures 4CD in THP1Frontiers in Oncology wwwfrontiersinAugust Volume 0cWu et alDeoxyshikonin Inhibits AML CellsFIGURE Deoxyshikonin dosedependently inhibited the viability of acute myeloid leukemia cells THP1 A and HL60 B cells were administrated with variousdoses of deoxyshikonin and µgml for h and cell viability was then assessed by CCK8 assay P compared to the control groupand HL60 cells in a dosedependent manner We concluded thatdeoxyshikonin suppressed glycolysis in AML cellsenzymeratelimitingan importantDeoxyshikonin Decreased the ExpressionLevel of PKM2 in AML CellsA previous study reported that shikonin suppressed tumoraerobic glycolysis through suppressing the activity of PKM2in regulatingcellular glycolysis Accordingly we supposed whetherdeoxyshikonin had an inhibitory eï¬ect on PKM2 expression Asexpected the quantitative realtime polymerase chain reactionqRTPCR results showed that the mRNA levels of PKM2were suppressed following the addition of deoxyshikonin in adosedependent manner in THP1 Figure 5A and HL60 cellsFigure 5B The western blot results showed that deoxyshikonintreatment inhibited the protein levels of PKM2 in a dosedependent manner in THP1 Figure 5C and HL60 cellsFigure 5D We also found that deoxyshikonin suppressedPKM2 activity in THP1 Figure 5E and HL60 cells Figure 5FThese results suggested that deoxyshikonin decreased theexpression level of PKM2 in AML cellsDeoxyshikonin Inactivated the AktmTORPathway in AML CellsThe aberrant AktmTOR signaling has been demonstrated tobe associated with the tumorigenesis of miscellaneous cancersincluding AML We further determined the ‚uence ofdeoxyshikonin on the AktmTOR pathway in AML cells Asdemonstrated by western blot analysis deoxyshikonin treatmentrestricted the phosphorylation of Akt GSK3β mTOR p70S6Kand 4EBP1 in a concentrationdependent manner but causedno noticeable change on the total protein levels of Akt GSK3β mTOR p70S6K and 4EBP1 in THP1 and HL60 cellsFigures 6A“C indicating that deoxyshikonin inactivated theAktmTOR pathway in AML cellsActivation of the AktmTOR PathwayReversed the Effects of Deoxyshikonin onViability and Apoptosis of AML CellsTo figure out whether the AktmTOR pathway was involved inmediating the antitumor eï¬ects of deoxyshikonin on AML cellsTHP1 and HL60 cells were treated with µgml deoxyshikoninin the presence or the absence of 740YP for h The 740YP treatment alone resulted in a notable enhancement of pAkt and pmTOR expressions but produced little alternationon Akt and mTOR protein levels in THP1 Figure 7A andHL60 cells Figure 7B suggesting the activation of AktmTORsignaling by 740YP The CCK8 assay presented thatthedeoxyshikonin treatmentinduced viability reduction in THP Figure 7C and HL60 cells Figure 7D was eï¬ectivelyameliorated following the addition of 740YP The increaseof apoptotic rate in deoxyshikonintreated THP1 Figure 7Eand HL60 cells Figure 7F was significantly abolished aftercotreatment with deoxyshikonin and 740YP Moreover caspase activity was enhanced in THP1 Figure 7G and HL60 cellsFigure 7H in response to deoxyshikonin which was attenuatedfollowing the addition of 740YP Furthermore the protein levelof Cyt C in the deoxyshikonin 740YP cotreatment groupin THP1 Figure 7I and HL60 cells Figure 7J was reducedwhen compared with that of the deoxyshikonin treatment groupCollectively these results suggested that the activation of theAktmTOR pathway reversed the eï¬ects of deoxyshikonin on theviability and the apoptosis of AML cellsActivation of the AktmTOR PathwayReversed the Effects of Deoxyshikonin onGlycolysis and PKM2 Expression in AMLCellsThe 740YP treatment overturned the reduction of glucoseconsumption Figure 8A and lactate production Figure 8Bmediated by deoxyshikonin in THP1 and HL60 cells ThedeoxyshikoninsuppressedPKM2 mRNA expression in THP1 and HL60 cells whichsignificantlytreatmentaloneFrontiers in Oncology wwwfrontiersinAugust Volume 0cWu et alDeoxyshikonin Inhibits AML CellsFIGURE Deoxyshikonin concentrationdependently promoted the apoptosis of acute myeloid leukemia cells THP1 and HL60 cells were treated with increasingdoses of deoxyshikonin and µgml followed by detection of the apoptotic rate caspase37 activity and Cyt C protein level by annexin VFITC apoptosisassay AB caspase37 activity assay CD and western blot analysis EF respectively P compared to the control groupFrontiers in Oncology wwwfrontiersinAugust Volume 0cWu et alDeoxyshikonin Inhibits AML CellsFIGURE Deoxyshikonin concentrationdependently suppressed glycolysis in acute myeloid leukemia cells After THP1 and HL60 cells were administrated withincreasing doses of deoxyshikonin and µgml for h glucose consumption AB and lactate production CD were then measured P compared to control the groupwas restored by the combined treatment of deoxyshikoninand 740YP Figure 8C Moreover 740YP resisted thedeoxyshikonininduced decrease of PKM2 protein levelFigures 8DE and PKM2 activity Figure 8F in THP1 andHL60 cells These results suggested that the activation of theAktmTOR pathway reversed the eï¬ects of deoxyshikonin onglycolysis and PKM2 expression in AML cells To confirmthe abovementioned results THP1 and HL60 cells weretransfected with Aktoverexpressing plasmid pcDNAAkt toactivate the AktmTOR pathway As shown in Figures 9ABthe ratios of pAktAkt and pmTORmTOR were increased h after transfection in THP1 and HL60 cells The CCK8assay showed that deoxyshikonin treatmentcaused viabilityreduction in THP1 and HL60 cells was attenuated aftertransfection with pcDNAAkt Figure 9C The increase ofapoptotic rate in deoxyshikonintreated THP1 and HL60 cellswas significantly abolished after transfection with pcDNAAktimpairedthe deoxyshikonin treatmentcaused decrease of glucoseconsumption Figure 9E and lactate production Figure 9Fin THP1 and HL60 cells The deoxyshikonin treatmentinhibited the expression of PKM2 mRNA Figure 9G andprotein Figures 9HI and the activity of PKM2 Figure 9Jwhereas these eï¬ects were attenuated after transfection withpcDNAAkt These results confirmed that the activation ofFigure 9D Transfection with pcDNAAktthe AktmTOR pathway reversed the eï¬ects of deoxyshikoninon viability apoptosis glycolysis and PKM2 expression inAML cellsDISCUSSIONIn spite of significant improvements in therapeutic interventionsof AML the prognosis of patients suï¬ering from AML remainsunfavorable and the 5year survival rate of AML patients islower than accompanied by a high mortality rate Forthe high mortality there is a great need to identify eï¬ectivealternative therapeutic agents specifically targeting AML It iscommonly reckoned that natural products have the potentialto induce apoptosis in cancer cells including AML and maytherefore be essential sources for anticancer drugs becauseof their extensive biological activities and limited side eï¬ects Shikonin and its derivatives the predominant typeof naphthoquinone derivatives extracted from the root ofLithospermum erythrorhizon Sieb et Zucc have been welldocumented to possess a wide range of pharmacologicalactivities including antitumor activity by suppression of cellproliferation For instance shikonin potently depressed theviability and the metastasis of triplenegative breast cancercells by reversing the epithelialtomesenchymal transition viaFrontiers in Oncology wwwfrontiersinAugust Volume 0cWu et alDeoxyshikonin Inhibits AML CellsFIGURE Deoxyshikonin decreased the expression level and the activity of PKM2 in acute myeloid leukemia cells THP1 and HL60 cells were exposed toincreasing doses of deoxyshikonin and µgml for h A“D The mRNA and protein levels of PKM2 were determined by qRTPCR and western blotrespectively EF PKM2 activity was detected by the lactate dehydrogenasecoupled assay P compared to the control groupFrontiers in Oncology wwwfrontiersinAugust Volume 0cWu et alDeoxyshikonin Inhibits AML CellsFIGURE Deoxyshikonin inactivated the AktmTOR pathway in AML cells A“C THP1 and HL60 cells were treated with different doses of deoxyshikonin and µgml for h and the protein levels of pAkt Akt pGSK3β GSK3β pmTOR mTOR pp70S6K p70S6K p4EBP1 and 4EBP1 were measured bywestern blot analysis P compared to the control groupglycogen synthase kinase 3βregulated repression of βcateninsignaling Additionally βdimethylacrylshikonin suppressedcell viability and induced mitochondriadependent apoptosisin human lung adenocarcinoma cells via the activation of thep38 signaling pathway Acetylshikonin another shikoninderivative significantly inhibited the anchorageindependentgrowth of pancreatic cancer cells by suppressing the nuclearfactorkappa B signaling pathway More importantly itwas previously demonstrated that deoxyshikonin exhibited antiproliferative and proapoptotic activities in colorectal cancercells through the phosphoinositide 3kinase PI3KAktmTORpathway Nevertheless whether deoxyshikonin showed anantitumor activity in AML remained far from being addressedTo our knowledgetime to demonstratethat deoxyshikonin dampened the viability of AML cellsin a dosedependent manner Meanwhile we found thatexposure to deoxyshikonin led to a concentrationdependentthis is the firstincrease of the apoptotic rate caspase37 activity and CytC protein levelin AML cells The increase of caspase activity is an important indicator of apoptosis These resultssuggested that deoxyshikonin exerted an antitumor activityin AML cells The eï¬ects of deoxyshikonin on the viabilityand the apoptosis of normal bone marrow stromal HS5cells were also evaluated in this study The results showedthat deoxyshikonin at µgml did not aï¬ect HS5 cellviability and apoptosis Supplementary Figure suggestingthat deoxyshikonin was selectively toxic to cancer cells but notto normal cellsIt has been proven that disruption of aerobic glycolysisrestricts cancer carcinogenesis suggesting that elevated aerobicglycolysis facilitates tumor development and oncogenesis Our study provided evidence that deoxyshikonin inhibitedglycolysis in AML cells as demonstrated by decreased glucoseconsumption and lactate production Moreover we found thatFrontiers in Oncology wwwfrontiersinAugust Volume 0cWu et alDeoxyshikonin Inhibits AML CellsFIGURE Activation of the AktmTOR pathway reversed the effects of deoxyshikonin on the viability and the apoptosis of acute myeloid leukemia cells AB Theprotein levels of pAkt Akt pmTOR and mTOR in the THP1 and HL60 cells treated with 740YP for h were detected by western blot analysis THP1 and HL60cells were treated with µgml deoxyshikonin in the presence or the absence of µM 740YP for h followed by assessment of cell viability CD apoptosisEF caspase37 activity GH and Cyt C protein level IJ by CCK8 assay flow cytometry analysis caspase37 activity assay and western blot analysisrespectively P compared to the control group P compared to the deoxyshikonin treatment groupdeoxyshikonin inhibited the expression of PKM2 in AML cellsPKM2 a critical ratelimiting enzyme of aerobic glycolysis isproposed to play a crucial role in glycolysis process and cancerprogression The robust expression of PKM2 has beenobserved in various human cancers which facilitates cancercell proliferation and growth These findings togetherrevealed that deoxyshikonin inhibited glycolysis in AML cells bysuppressing PKM2It has been demonstrated that the AktmTOR signalingnetwork is constitutively activated and associated with thedevelopment of several types of cancers including AML Overactivation of the AktmTOR signaling is involvedin the elevated aerobic glycolysis of cancer cellstherebycontributing to cancer cell survival and growth Thusthe AktmTOR pathway may be regarded as a promisingtherapeutic target for cancer treatment To elucidate themolecular mechanism underlying the antitumor eï¬ects ofdeoxyshikonin we detected the ‚uence of deoxyshikoninon the AktmTOR signaling in AML cells It was shownthat deoxyshikonin impeded the activation of the AktmTORsignaling in AML cells In the restoration assay activation ofthe AktmTOR signaling by 740YP or pcDNAAkt plasmidabolished the antitumor eï¬ect of deoxyshikonin in AML cellsTaken togetherthese results suggested that deoxyshikoninFrontiers in Oncology wwwfrontiersinAugust Volume 0cWu et alDeoxyshikonin Inhibits AML CellsFIGURE Activation of the AktmTOR pathway reversed the effects of deoxyshikonin on glycolysis and PKM2 expression in acute myeloid leukemia cells THP1and HL60 cells were exposed to µgml deoxyshikonin or together with µM 740YP for h AB Glucose consumption and lactate production in thesupernatants of treated THP1 and HL60 cells were measured C The mRNA level of PKM2 in treated THP1 and HL60 cells was estimated by quantitative realtimepolymerase chain reaction DE The protein level of PKM2 in the treated THP1 and HL60 cells was estimated by western blot F PKM2 activity was detected bythe lactate dehydrogenasecoupled assay P compared to the control group P compared to the deoxyshikonin treatment groupFrontiers in Oncology wwwfrontiersinAugust Volume 0cWu et alDeoxyshikonin Inhibits AML CellsFIGURE Transfection with pcDNAAkt reversed the effects of deoxyshikonin on viability apoptosis glycolysis and PKM2 expression in acute myeloid leukemiacells AB The Aktoverexpressing plasmid pcDNAAkt and empty vector pcDNA control were transfected into THP1 and HL60 cells After h the protein levelsof pAkt Akt pmTOR and mTOR were detected by western blot analysis Transfected THP1 and HL60 cells were exposed to µgml deoxyshikonin for hfollowed by assessment of cell viability C apoptosis D glucose consumption E and lactate production F as well as PKM2 mRNA G protein HI and activityJ P compared to the pcDNA group P compared to the pcDNA deoxyshikonin groupFrontiers in Oncology wwwfrontiersinAugust Volume 0cWu et alDeoxyshikonin Inhibits AML Cellsdampened the viability and the glycolysis of AML cells bysuppressing PKM2 via inactivation of the AktmTOR signalingThe main defect ofthis study is that experiments wereonly performed on two AML cell lines THP1 and HL60and no primary AML cells were tested The heterogeneityof AML is therefore not taken into account Future studiesshould explore the role of deoxyshikonin using primaryAML cellsCONCLUSIONTo sum up our study provided the first evidence thatdeoxyshikonin exerted antitumor and antiglycolytic activitiesin AML cells by suppressing PKM2 via inactivation of theAktmTOR signaling Our study provided novel insights intothe antitumor and antiglycolytic activities of deoxyshikonin inAML Deoxyshikonin may be a promising anticancer candidateagent in AML cellsREFERENCES Kavanagh S Murphy T Law A Yehudai D Ho JM Chan S et al Emergingtherapies for acute myeloid leukemia translating biology into the clinic JCIInsight 101172jciinsight95679 Papaemmanuil E Gerstung M Bullinger L Gaidzik VI Paschka PRoberts ND et al Genomic classification and prognosis in acute myeloidleukemia N EnglJ Med “ 101056NEJMoa1 Siegel RL Miller KD Jemal A Cancer statistics CA Cancer J Clin “ 103322caac21442 Krug U Berdel WE Gale RP Haferlach C Schnittger S MullerTidow C etal Increasing intensity of therapies assigned at diagnosis does not improvesurvival of adults with acute myeloid leukemia Leukemia “ 101038leu201625 Gao H Liu L Qu ZY Wei FX Wang SQ Chen G et al Antiadenovirus activities ofshikonin a component of Chinese herbalmedicine in vitro Biol Pharm Bull “ 101248bpb Li W Zhang C Ren A Li T Jin R Li G et al Shikonin suppressesskin carcinogenesis via inhibiting cell proliferation PLoS ONE 10e0126459 101371journalpone0126459 Andujar I Rios JL Giner RM Recio MC Pharmacological properties ofshikonin a review of literature since Planta Med “ 101055s00331350934 Li W Liu J Zhao Y PKM2 inhibitor shikonin suppresses TPAinducedmitochondrial malfunction and proliferation of skin epidermal JB6 cells MolCarcinog “ 101002mc21988 Boulos JC Rahama M Hegazy MF Eï¬erth T Shikonin derivatives “for cancer prevention and therapy Cancer Lett 101016jcanlet201904033 Zhu Y Zhong Y Long X Zhu Z Zhou Y Ye H et al Deoxyshikoninisolated from Arnebia euchroma inhibits colorectal cancer by downregulating the PI3KAktmTOR pathway PharmBiol “ Liberti MV Locasale JW The Warburg eï¬ect how does it benefit cancer cellsDATA AVAILABILITY STATEMENTAll datasets generated for this study are included in theSupplementary MaterialAUTHOR CONTRIBUTIONSHW conducted the experiments and participated in theconception and the design of the study HZ conducted theexperiments and performed the analysis LC contributed toanalyzing the data and drafting the manuscript All authorscontributed to the and approved the submitted versionSUPPLEMENTARY MATERIALThe Supplementary Materialonline202001253fullsupplementarymaterialfor this can be foundhttpswwwfrontiersins103389foncat Xu D Liang J Lin J Yu C PKM2 a potential regulator of rheumatoid arthritisvia glycolytic and nonglycolytic pathways Front Immunol 103389fimmu201902919 Gong T Cui L Wang H Wang H Han N Knockdown of KLF5 suppresseshypoxiainduced resistance to cisplatin in NSCLC cells by regulating HIF1αdependent glycolysis through inactivation of the PI3KAktmTOR pathway JTransl Med 101186s1296701815432 Nemazanyy I Espeillac C Pende M Panasyuk G Role of PI3K mTOR andAkt2 signalling in hepatic tumorigenesis via the control of PKM2 expressionBiochem Soc Trans “ 101042BST20130034 Zheng YL Li L Jia YX Zhang BZ Li JC Zhu YH et al LINC01554mediated glucose metabolism reprogramming suppresses tumorigenicityin hepatocellular carcinoma via downregulating PKM2 expression andinhibiting AktmTOR signaling pathway Theranostics “ 107150thno28992 Zhao X Zhu Y Hu J Jiang L Li L Jia S et al Shikonin inhibits tumor growthin mice by suppressing pyruvate kinase M2mediated aerobic glycolysis SciRep 101038s4159801831615y Tang J Ren YG Zhao J Long F Chen J Jiang Z Shikonin enhancessensitization of gefitinib against wildtype EGFR nonsmall cell lung cancervia inhibition PKM2stat3cyclinD1 signal pathway Life Sci “ 101016jlfs201805012 Popescu NC Cheng SY Chromosomal localization of the gene for a humancytosolic thyroid hormone binding protein homologous to the subunitof pyruvate kinase subtype M2 Somat Cell Mol Genet “ 101007BF01233100 Martelli AMF McCubreyEvangelisti C ChiariniJA Thephosphatidylinositol 3kinaseAktmTOR signaling network as a therapeutictarget in acute myelogenous leukemia patients Oncotarget “ 1018632oncotarget114 Yang D Zhang X Zhang X Xu Y The progress and current status ofimmunotherapy in acute myeloid leukemia Ann Hematol “ 101007s002770173148x Wang X Feng Y Chinese medicines induce cell death the molecular andcellular mechanisms for cancer therapy Biomed Res Int Trends Biochem Sci “ 101016jtibs201512001 Wang ZY Chen Z Acute promyelocytic leukemia from highly fatal to highly Hanahan D Weinberg RA Hallmarks of cancer the next generation Cellcurable Blood “ 101182blood200707102798 “ 101016jcell201102013 Herst PM Howma

cancer207

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: Deoxyshikonin Inhibits Viability andGlycolysis by Suppressing theAktmTOR Pathway in Acute MyeloidLeukemia CellsHuijuan Wu   Hongmian Zhao   and Li Chen Telemedicine and Connected Health Center Huaihe Hospital of Henan University Kaifeng China Department ofHematology Huaihe Hospital of Henan University Kaifeng ChinaDeoxyshikonin was reported to exhibit an antitumor effect in colorectal cancer Howeverno studies are available to illustrate the effect of deoxyshikonin on acute myeloid leukemiaAML The effects of deoxyshikonin on viability apoptosis caspase37 activity andcytochrome Cyt C expression were evaluated by Cell Counting Kit8 assay flowcytometry analysis caspase37 activity assay and western blot analysis respectivelyGlucose consumption and lactate production were measured to determine the glycolysislevel The expression of pyruvate kinase M2 PKM2 was detected by quantitativerealtime polymerase chain reaction and western blot analysis The results showed thatdeoxyshikonin inhibited cell viability and increased the apoptotic rate the caspase37activity and the Cyt C protein level in AML cells in a dosedependent manner Additionallydeoxyshikonin concentrationdependently decreased glucose consumptionlactateproduction and PKM2 expression in AML cells Deoxyshikonin inactivated the proteinkinase B Aktmammalian target of the rapamycin mTOR pathway The activation ofthe AktmTOR pathway reversed the effects of deoxyshikonin on viability apoptosisand glycolysis in AML cells In deoxyshikonin dampened the viability and theglycolysis of AML cells by suppressing PKM2 via inactivation of the AktmTOR signalingEdited byCyrus KhandanpourUniversity Hospital M¼nster GermanyReviewed byPeng YangShanxi University ChinaHaili WuShanxi University ChinaHongyu ZhouKunming Medical University ChinaCorrespondenceHuijuan Wuhjwu297163com These authors share first authorshipKeywords deoxyshikonin glycolysis PKM2 the AktmTOR signaling acute myeloid leukemiaSpecialty sectionThis was submitted toHematologic Malignanciesa section of the journalFrontiers in OncologyReceived March Accepted June Published August CitationWu H Zhao H and Chen L Deoxyshikonin Inhibits Viability andGlycolysis by Suppressing theAktmTOR Pathway in Acute MyeloidLeukemia CellsFront Oncol 103389fonc202001253INTRODUCTIONAcute myeloid leukemia AML the most prevalent form of acute leukemia in adults is anaggressive malignancy derived from hemopoietic progenitor cells and with poor survival rate andfrequent relapse posing a threat to the health and life of aï¬ected patients AML is characterizedby rapid growth impaired apoptosis and abnormal clonal accumulation of hematopoietic stemcells in the bone marrow due to various genetic and epigenetic changes eventually leading to bonemarrow failure There were an estimated new cases diagnosed with AML and mortalities due to AML according to the statistics in Presently the efficacy of the standardchemotherapy for AML patients remains suboptimal owing to drug resistance and high clinicalrelapse rate In this regard searching for novel antileukemia drugs is urgently required toeï¬ectively improve the outcome of patients with AMLShikonin 58dihydroxy2[1S1hydroxy4methylpent3en1yl]naphthalene14 dionea naturally occurring naphthoquinone extracted from the oriental traditional medical herbFrontiers in Oncology wwwfrontiersinAugust Volume 0cWu et alDeoxyshikonin Inhibits AML Cellsand suppressed glycolysis in AML cells Mechanistically the antiAML eï¬ect of deoxyshikonin was mediated via repressing PKM2by inactivation of the AktmTOR pathwayMATERIALS AND METHODSCell Cultivation and TreatmentAML cell lines including THP1 and HL60 were purchasedfrom American Tissue Culture Collection ATCC ManassasVA USA and routinely cultured in Roswell Park MemorialInstitute1640 medium HyClone South Logan UT USAconjugated with heatinactivated fetal bovine serum ExCellBio Shanghai China and penicillinstreptomycin SigmaAldrich St Louis MO USA The cells were fostered at —¦Cin a drippy environment flushed with air plus CO2Deoxyshikonin purity Tauto Biotech Shanghai Chinawas dissolved in dimethyl sulfoxide SigmaAldrich St LouisMO USA as a reserve solution and diluted into diï¬erentconcentrations and µgml The THP1and HL60 cells were exposed to diverse doses of deoxyshikoninfor h The Aktoverexpressing plasmid pcDNAAkt andthe empty vectorpcDNA control were obtained fromRibobio Guangzhou China Transfection was performed usingLipofectamine reagent Invitrogen Carlsbad CA USACell Counting Kit8 AssayCell Counting Kit8 CCK8 Beyotime Shanghai China wastaken to evaluate the viability of AML cells THP1 and HL60cells were seeded into 96well plates at — cellswell anddealt with various concentrations of deoxyshikonin and µgml for h or incubated with µgmldeoxyshikonin in the presence or the absence of µM 740YP Tocris Bioscience Shanghai China an activator of theAktmTOR signaling pathway After treatment for h µl ofCCK8 solution was added to each well followed by incubationfor another h The optical density of each well was recorded at awavelength of nm using a microplate reader Thermo FisherScientific Waltham MA USAFlow Cytometry Analysis for ApoptosisAfter the treatments as aforementioned annexin V FITCand propidium iodide kitKeyGen Nanjing China wasimplemented to analyze the apoptosis of THP1 and HL60 cellsCell apoptotic rate was detected by means of a flow cytometerFACScan BD Biosciences San Diego CA USAtheastreatmentsCaspase37 Activity AssayFollowingaforementioned ApoONEHomogeneous CaspaseGlo Assay kit Promega MadisonWI USA was adopted to measure the caspase37 activityof THP1 and HL60 cells referring to the manufacturer™sdescription Finally M2000 Infinite Pro instrument TecanTrading AG Maennedorf Switzerland was used to determinethe luminescenceFIGURE Chemical structure of deoxyshikoninLithospermum erythrorhizon Sieb et Zucc has been extensivelyused for the treatment of many diseases including burnssore throats HIV1 infection and macular eruption Currently clinical and pharmacological propertiesstudieshave demonstrated that shikonin and its derivatives exhibitvarious biological activities such as immune regulation andantithrombotic anti‚ammatory antioxidative and antiglycolytic activities An increasing number of researchesreveal that shikonin derivatives have garnered much researchinterest due to its limited toxicity and stronger antitumoractivities in miscellaneous cancers Interestingly a previousinvestigation proved that deoxyshikonin its chemical structureis shown in Figure a derivative of shikonin exhibited an antitumor eï¬ect in colorectal cancer However no studies areavailable to illustrate the eï¬ect of deoxyshikonin on AMLAerobic glycolysis also known as the Warburg eï¬ect is wellrecognized as a metabolic pathway in the rapidly proliferatingcancer cells for the regeneration of energy and the biosynthesisof macromolecules even in the presence of sufficient oxygen Aerobic glycolysis has been extensively accepted as an importantcharacteristic of tumor cells including AML eventually resultingin increased glucose consumption and lactate production Pyruvate kinase PK is a ratelimiting glycolytic enzymePyruvate kinase M1 PKM1 is expressed in normal diï¬erentiatedtissues whereas pyruvate kinase M2 PKM2 is expressedin cancer cellsleading to increased glycolysis As weall know the protein kinase B Aktmammalian target ofrapamycin mTOR pathway widely existing in cells is one ofthe most important survival signaling pathways that participatein the regulation of diverse physiological processes such as cellgrowth apoptosis and metabolism It has been reportedthat the AktmTOR pathway is a positive regulator of PKM2expression Previous studies suggested that shikonininhibited glycolysis by suppression of PKM2 expression Therefore we hypothesized that deoxyshikonin inhibitedviability and glycolysis suppressing pyruvate kinase M2 via theAktmTOR pathway in acute myeloid leukemia cellsIn the present study we assessed the eï¬ects and the underlyingmechanisms of deoxyshikonin on viability apoptosis glycolysisand PKM2 expression in AML cells These results revealed thatdeoxyshikonin treatment inhibited viability induced apoptosisFrontiers in Oncology wwwfrontiersinAugust Volume 0cWu et alDeoxyshikonin Inhibits AML CellsDetermination of Glucose Consumptionand Lactate ProductionAfter the treatments as aforementioned the culture medium ofTHP1 and HL60 cells was collected for the measurement ofglucose and lactate levels using a glucose uptake colorimetricassay kit SigmaAldrich and a lactic acid assay kit JianchengBioengineering Institute Nanjing China respectivelyPKM2 Activity AssayPKM2 activity was detected by the lactate dehydrogenasecoupled assay as described earlier Five microliters of wholecelllysate was utilized in the assay The absorbance at awavelength of nm was measured using a microplate readerThermo Fisher Scientificicecold radioimmunoprecipitation assayWestern Blot AnalysisThe treated THP1 and HL60 cells were collected and rinsedwith phosphatebuï¬ered salinefollowed by the additionoflysis buï¬erBeyotime containing mM phenylmethylsulfonyl fluorideSigmaAldrich A total of µg of protein samples wasfractionated on sodium dodecyl sulfatepolyacrylamide gelelectrophoresis prior to electrotransfer onto polyvinylidenedifluoride membranes Millipore Bedford MA USA Afterblocking with defatted milkTrisbased saline with Tween atroom temperature for h the membranes were immunoblottedovernight at —¦C with corresponding primary antibodiesagainst cytochrome C Cyt C Cell Signaling TechnologyInc Danvers MA USA phosphorylated Akt pAkt Ser473Abcam Cambridge MA USA AktAbcam glycogensynthase kinase3β GSK3β Abcam phosphorylated GSK3βpGSK3β Ser9 Abcam mTOR Abcam phosphorylatedmTOR pmTOR Ser2448 Abcam p70 ribosomal S6 kinasep70S6K Cell Signaling TechnologyInc phosphorylatedp70S6K pp70S6K Thr389 Cell Signaling Technology Inceukaryotic translation initiation factor 4Ebinding protein 4EBP1 Cell Signaling Technology Inc phosphorylated4EBP1 p4EBP1 Thr70 Cell Signaling TechnologyIncPKM2 Abcam and βactin Abcam and then incubated withhorseradish peroxidaseconjugated secondary antibodies CellSignaling Technology Inc for h at room temperature Lastlyan enhanced chemiluminescence kit Amersham PharmaciaPiscataway NJ USA was implemented to examine the antigen“antibody complexes βActin was used as a loading control Theprotein bands were visualized by VersaDoc imaging systemBioRad Hercules CA USAQuantitative RealTime PCRRNAiso Plus TaKaRa Dalian China was used to extracttotal RNA from treated THP1 and HL60 cells and theconcentration of extracted RNA was measured using a NanoDrop spectrophotometer Thermo Fisher Scientific Reversetranscription was carried out using the PrimeScript RT ReagentKit Takara Dalian China SYBR Green Taq Mix TaKaRawas then implemented to detect PKM2 mRNA expressionon the StepOnePlus qPCR system Thermo Fisher Scientificwith βactin as an endogenous control The thermocyclingconditions were displayed as follows —¦C for s followedby cycles of —¦C for s —¦C for s and —¦C for sThe primers were as follows PKM2 ²GCTG CCAT CTACCACT TGC3² forward and ²CCAG ACTT GGTG AGGACGAT T3² reverse GAPDH ²ATGT CGTG GAGT CTACTGGC3² forward and ²TGAC CTTG CCCA CAGC CTTG² reverse The ˆ’\x01\x01Ct method was taken to quantify theexpression level of PKM2 mRNAStatisticsAll data are shown as mean ± standard deviation of threeindependent experiments Statistical assays were determinedusing SPSS software IBM Corp Armonk NY USA withStudent™s ttest or oneway ANOVA followed by Dunnett™s test P were regarded as statistically significantRESULTSDeoxyshikonin Inhibited the Viability ofAML CellsTo clarify the antitumor activity of deoxyshikonin in AMLcells CCK8 was taken to evaluate cell viability after THP and HL60 cells were exposed to a series of deoxyshikoninconcentrations and µgml for h Asshown in Figures 2AB cell viability was significantly declinedin a concentrationdependent manner in THP1 and HL60 cellsin response to deoxyshikoninDeoxyshikonin Enhanced the Apoptosis ofAML CellsThe eï¬ect of deoxyshikonin on the apoptotic consequences ofAML cells was investigated by annexin VFITC apoptosis assayAs a result deoxyshikonin treatment led to a concentrationdependent increase of apoptotic rate in THP1 Figure 3Aand HL60 cells Figure 3B In line with the results of theflow cytometry analysis an elevation of caspase37 activityin deoxyshikonintreated THP1 Figure 3C and HL60 cellsFigure 3D was observed The mitochondrial protein Cyt Cis known as the initiating factor of mitochondrial apoptosispathway The expression of apoptotic marker Cyt C in THP1and HL60 cells was further detected by western blot analysisThe results demonstrated that deoxyshikonin concentrationdependently increased Cyt C protein level in THP1 Figure 3Eand HL60 cells Figure 3F relative to the control group Theseresults suggested that deoxyshikonin facilitated the apoptosis ofAML cellsDeoxyshikonin Suppressed Glycolysis inAML CellsTo further characterize the eï¬ect of deoxyshikonin on glycolysisin AML cells we measured glucose consumption and lactateproduction in AML cells These results demonstrated thatdeoxyshikonin exposure decreased glucoseconsumptionFigures 4AB and lactate production Figures 4CD in THP1Frontiers in Oncology wwwfrontiersinAugust Volume 0cWu et alDeoxyshikonin Inhibits AML CellsFIGURE Deoxyshikonin dosedependently inhibited the viability of acute myeloid leukemia cells THP1 A and HL60 B cells were administrated with variousdoses of deoxyshikonin and µgml for h and cell viability was then assessed by CCK8 assay P compared to the control groupand HL60 cells in a dosedependent manner We concluded thatdeoxyshikonin suppressed glycolysis in AML cellsenzymeratelimitingan importantDeoxyshikonin Decreased the ExpressionLevel of PKM2 in AML CellsA previous study reported that shikonin suppressed tumoraerobic glycolysis through suppressing the activity of PKM2in regulatingcellular glycolysis Accordingly we supposed whetherdeoxyshikonin had an inhibitory eï¬ect on PKM2 expression Asexpected the quantitative realtime polymerase chain reactionqRTPCR results showed that the mRNA levels of PKM2were suppressed following the addition of deoxyshikonin in adosedependent manner in THP1 Figure 5A and HL60 cellsFigure 5B The western blot results showed that deoxyshikonintreatment inhibited the protein levels of PKM2 in a dosedependent manner in THP1 Figure 5C and HL60 cellsFigure 5D We also found that deoxyshikonin suppressedPKM2 activity in THP1 Figure 5E and HL60 cells Figure 5FThese results suggested that deoxyshikonin decreased theexpression level of PKM2 in AML cellsDeoxyshikonin Inactivated the AktmTORPathway in AML CellsThe aberrant AktmTOR signaling has been demonstrated tobe associated with the tumorigenesis of miscellaneous cancersincluding AML We further determined the ‚uence ofdeoxyshikonin on the AktmTOR pathway in AML cells Asdemonstrated by western blot analysis deoxyshikonin treatmentrestricted the phosphorylation of Akt GSK3β mTOR p70S6Kand 4EBP1 in a concentrationdependent manner but causedno noticeable change on the total protein levels of Akt GSK3β mTOR p70S6K and 4EBP1 in THP1 and HL60 cellsFigures 6A“C indicating that deoxyshikonin inactivated theAktmTOR pathway in AML cellsActivation of the AktmTOR PathwayReversed the Effects of Deoxyshikonin onViability and Apoptosis of AML CellsTo figure out whether the AktmTOR pathway was involved inmediating the antitumor eï¬ects of deoxyshikonin on AML cellsTHP1 and HL60 cells were treated with µgml deoxyshikoninin the presence or the absence of 740YP for h The 740YP treatment alone resulted in a notable enhancement of pAkt and pmTOR expressions but produced little alternationon Akt and mTOR protein levels in THP1 Figure 7A andHL60 cells Figure 7B suggesting the activation of AktmTORsignaling by 740YP The CCK8 assay presented thatthedeoxyshikonin treatmentinduced viability reduction in THP Figure 7C and HL60 cells Figure 7D was eï¬ectivelyameliorated following the addition of 740YP The increaseof apoptotic rate in deoxyshikonintreated THP1 Figure 7Eand HL60 cells Figure 7F was significantly abolished aftercotreatment with deoxyshikonin and 740YP Moreover caspase activity was enhanced in THP1 Figure 7G and HL60 cellsFigure 7H in response to deoxyshikonin which was attenuatedfollowing the addition of 740YP Furthermore the protein levelof Cyt C in the deoxyshikonin 740YP cotreatment groupin THP1 Figure 7I and HL60 cells Figure 7J was reducedwhen compared with that of the deoxyshikonin treatment groupCollectively these results suggested that the activation of theAktmTOR pathway reversed the eï¬ects of deoxyshikonin on theviability and the apoptosis of AML cellsActivation of the AktmTOR PathwayReversed the Effects of Deoxyshikonin onGlycolysis and PKM2 Expression in AMLCellsThe 740YP treatment overturned the reduction of glucoseconsumption Figure 8A and lactate production Figure 8Bmediated by deoxyshikonin in THP1 and HL60 cells ThedeoxyshikoninsuppressedPKM2 mRNA expression in THP1 and HL60 cells whichsignificantlytreatmentaloneFrontiers in Oncology wwwfrontiersinAugust Volume 0cWu et alDeoxyshikonin Inhibits AML CellsFIGURE Deoxyshikonin concentrationdependently promoted the apoptosis of acute myeloid leukemia cells THP1 and HL60 cells were treated with increasingdoses of deoxyshikonin and µgml followed by detection of the apoptotic rate caspase37 activity and Cyt C protein level by annexin VFITC apoptosisassay AB caspase37 activity assay CD and western blot analysis EF respectively P compared to the control groupFrontiers in Oncology wwwfrontiersinAugust Volume 0cWu et alDeoxyshikonin Inhibits AML CellsFIGURE Deoxyshikonin concentrationdependently suppressed glycolysis in acute myeloid leukemia cells After THP1 and HL60 cells were administrated withincreasing doses of deoxyshikonin and µgml for h glucose consumption AB and lactate production CD were then measured P compared to control the groupwas restored by the combined treatment of deoxyshikoninand 740YP Figure 8C Moreover 740YP resisted thedeoxyshikonininduced decrease of PKM2 protein levelFigures 8DE and PKM2 activity Figure 8F in THP1 andHL60 cells These results suggested that the activation of theAktmTOR pathway reversed the eï¬ects of deoxyshikonin onglycolysis and PKM2 expression in AML cells To confirmthe abovementioned results THP1 and HL60 cells weretransfected with Aktoverexpressing plasmid pcDNAAkt toactivate the AktmTOR pathway As shown in Figures 9ABthe ratios of pAktAkt and pmTORmTOR were increased h after transfection in THP1 and HL60 cells The CCK8assay showed that deoxyshikonin treatmentcaused viabilityreduction in THP1 and HL60 cells was attenuated aftertransfection with pcDNAAkt Figure 9C The increase ofapoptotic rate in deoxyshikonintreated THP1 and HL60 cellswas significantly abolished after transfection with pcDNAAktimpairedthe deoxyshikonin treatmentcaused decrease of glucoseconsumption Figure 9E and lactate production Figure 9Fin THP1 and HL60 cells The deoxyshikonin treatmentinhibited the expression of PKM2 mRNA Figure 9G andprotein Figures 9HI and the activity of PKM2 Figure 9Jwhereas these eï¬ects were attenuated after transfection withpcDNAAkt These results confirmed that the activation ofFigure 9D Transfection with pcDNAAktthe AktmTOR pathway reversed the eï¬ects of deoxyshikoninon viability apoptosis glycolysis and PKM2 expression inAML cellsDISCUSSIONIn spite of significant improvements in therapeutic interventionsof AML the prognosis of patients suï¬ering from AML remainsunfavorable and the 5year survival rate of AML patients islower than accompanied by a high mortality rate Forthe high mortality there is a great need to identify eï¬ectivealternative therapeutic agents specifically targeting AML It iscommonly reckoned that natural products have the potentialto induce apoptosis in cancer cells including AML and maytherefore be essential sources for anticancer drugs becauseof their extensive biological activities and limited side eï¬ects Shikonin and its derivatives the predominant typeof naphthoquinone derivatives extracted from the root ofLithospermum erythrorhizon Sieb et Zucc have been welldocumented to possess a wide range of pharmacologicalactivities including antitumor activity by suppression of cellproliferation For instance shikonin potently depressed theviability and the metastasis of triplenegative breast cancercells by reversing the epithelialtomesenchymal transition viaFrontiers in Oncology wwwfrontiersinAugust Volume 0cWu et alDeoxyshikonin Inhibits AML CellsFIGURE Deoxyshikonin decreased the expression level and the activity of PKM2 in acute myeloid leukemia cells THP1 and HL60 cells were exposed toincreasing doses of deoxyshikonin and µgml for h A“D The mRNA and protein levels of PKM2 were determined by qRTPCR and western blotrespectively EF PKM2 activity was detected by the lactate dehydrogenasecoupled assay P compared to the control groupFrontiers in Oncology wwwfrontiersinAugust Volume 0cWu et alDeoxyshikonin Inhibits AML CellsFIGURE Deoxyshikonin inactivated the AktmTOR pathway in AML cells A“C THP1 and HL60 cells were treated with different doses of deoxyshikonin and µgml for h and the protein levels of pAkt Akt pGSK3β GSK3β pmTOR mTOR pp70S6K p70S6K p4EBP1 and 4EBP1 were measured bywestern blot analysis P compared to the control groupglycogen synthase kinase 3βregulated repression of βcateninsignaling Additionally βdimethylacrylshikonin suppressedcell viability and induced mitochondriadependent apoptosisin human lung adenocarcinoma cells via the activation of thep38 signaling pathway Acetylshikonin another shikoninderivative significantly inhibited the anchorageindependentgrowth of pancreatic cancer cells by suppressing the nuclearfactorkappa B signaling pathway More importantly itwas previously demonstrated that deoxyshikonin exhibited antiproliferative and proapoptotic activities in colorectal cancercells through the phosphoinositide 3kinase PI3KAktmTORpathway Nevertheless whether deoxyshikonin showed anantitumor activity in AML remained far from being addressedTo our knowledgetime to demonstratethat deoxyshikonin dampened the viability of AML cellsin a dosedependent manner Meanwhile we found thatexposure to deoxyshikonin led to a concentrationdependentthis is the firstincrease of the apoptotic rate caspase37 activity and CytC protein levelin AML cells The increase of caspase activity is an important indicator of apoptosis These resultssuggested that deoxyshikonin exerted an antitumor activityin AML cells The eï¬ects of deoxyshikonin on the viabilityand the apoptosis of normal bone marrow stromal HS5cells were also evaluated in this study The results showedthat deoxyshikonin at µgml did not aï¬ect HS5 cellviability and apoptosis Supplementary Figure suggestingthat deoxyshikonin was selectively toxic to cancer cells but notto normal cellsIt has been proven that disruption of aerobic glycolysisrestricts cancer carcinogenesis suggesting that elevated aerobicglycolysis facilitates tumor development and oncogenesis Our study provided evidence that deoxyshikonin inhibitedglycolysis in AML cells as demonstrated by decreased glucoseconsumption and lactate production Moreover we found thatFrontiers in Oncology wwwfrontiersinAugust Volume 0cWu et alDeoxyshikonin Inhibits AML CellsFIGURE Activation of the AktmTOR pathway reversed the effects of deoxyshikonin on the viability and the apoptosis of acute myeloid leukemia cells AB Theprotein levels of pAkt Akt pmTOR and mTOR in the THP1 and HL60 cells treated with 740YP for h were detected by western blot analysis THP1 and HL60cells were treated with µgml deoxyshikonin in the presence or the absence of µM 740YP for h followed by assessment of cell viability CD apoptosisEF caspase37 activity GH and Cyt C protein level IJ by CCK8 assay flow cytometry analysis caspase37 activity assay and western blot analysisrespectively P compared to the control group P compared to the deoxyshikonin treatment groupdeoxyshikonin inhibited the expression of PKM2 in AML cellsPKM2 a critical ratelimiting enzyme of aerobic glycolysis isproposed to play a crucial role in glycolysis process and cancerprogression The robust expression of PKM2 has beenobserved in various human cancers which facilitates cancercell proliferation and growth These findings togetherrevealed that deoxyshikonin inhibited glycolysis in AML cells bysuppressing PKM2It has been demonstrated that the AktmTOR signalingnetwork is constitutively activated and associated with thedevelopment of several types of cancers including AML Overactivation of the AktmTOR signaling is involvedin the elevated aerobic glycolysis of cancer cellstherebycontributing to cancer cell survival and growth Thusthe AktmTOR pathway may be regarded as a promisingtherapeutic target for cancer treatment To elucidate themolecular mechanism underlying the antitumor eï¬ects ofdeoxyshikonin we detected the ‚uence of deoxyshikoninon the AktmTOR signaling in AML cells It was shownthat deoxyshikonin impeded the activation of the AktmTORsignaling in AML cells In the restoration assay activation ofthe AktmTOR signaling by 740YP or pcDNAAkt plasmidabolished the antitumor eï¬ect of deoxyshikonin in AML cellsTaken togetherthese results suggested that deoxyshikoninFrontiers in Oncology wwwfrontiersinAugust Volume 0cWu et alDeoxyshikonin Inhibits AML CellsFIGURE Activation of the AktmTOR pathway reversed the effects of deoxyshikonin on glycolysis and PKM2 expression in acute myeloid leukemia cells THP1and HL60 cells were exposed to µgml deoxyshikonin or together with µM 740YP for h AB Glucose consumption and lactate production in thesupernatants of treated THP1 and HL60 cells were measured C The mRNA level of PKM2 in treated THP1 and HL60 cells was estimated by quantitative realtimepolymerase chain reaction DE The protein level of PKM2 in the treated THP1 and HL60 cells was estimated by western blot F PKM2 activity was detected bythe lactate dehydrogenasecoupled assay P compared to the control group P compared to the deoxyshikonin treatment groupFrontiers in Oncology wwwfrontiersinAugust Volume 0cWu et alDeoxyshikonin Inhibits AML CellsFIGURE Transfection with pcDNAAkt reversed the effects of deoxyshikonin on viability apoptosis glycolysis and PKM2 expression in acute myeloid leukemiacells AB The Aktoverexpressing plasmid pcDNAAkt and empty vector pcDNA control were transfected into THP1 and HL60 cells After h the protein levelsof pAkt Akt pmTOR and mTOR were detected by western blot analysis Transfected THP1 and HL60 cells were exposed to µgml deoxyshikonin for hfollowed by assessment of cell viability C apoptosis D glucose consumption E and lactate production F as well as PKM2 mRNA G protein HI and activityJ P compared to the pcDNA group P compared to the pcDNA deoxyshikonin groupFrontiers in Oncology wwwfrontiersinAugust Volume 0cWu et alDeoxyshikonin Inhibits AML Cellsdampened the viability and the glycolysis of AML cells bysuppressing PKM2 via inactivation of the AktmTOR signalingThe main defect ofthis study is that experiments wereonly performed on two AML cell lines THP1 and HL60and no primary AML cells were tested The heterogeneityof AML is therefore not taken into account Future studiesshould explore the role of deoxyshikonin using primaryAML cellsCONCLUSIONTo sum up our study provided the first evidence thatdeoxyshikonin exerted antitumor and antiglycolytic activitiesin AML cells by suppressing PKM2 via inactivation of theAktmTOR signaling Our study provided novel insights intothe antitumor and antiglycolytic activities of deoxyshikonin inAML Deoxyshikonin may be a promising anticancer candidateagent in AML cellsREFERENCES Kavanagh S Murphy T Law A Yehudai D Ho JM Chan S et al Emergingtherapies for acute myeloid leukemia translating biology into the clinic JCIInsight 101172jciinsight95679 Papaemmanuil E Gerstung M Bullinger L Gaidzik VI Paschka PRoberts ND et al Genomic classification and prognosis in acute myeloidleukemia N EnglJ Med “ 101056NEJMoa1 Siegel RL Miller KD Jemal A Cancer statistics CA Cancer J Clin “ 103322caac21442 Krug U Berdel WE Gale RP Haferlach C Schnittger S MullerTidow C etal Increasing intensity of therapies assigned at diagnosis does not improvesurvival of adults with acute myeloid leukemia Leukemia “ 101038leu201625 Gao H Liu L Qu ZY Wei FX Wang SQ Chen G et al Antiadenovirus activities ofshikonin a component of Chinese herbalmedicine in vitro Biol Pharm Bull “ 101248bpb Li W Zhang C Ren A Li T Jin R Li G et al Shikonin suppressesskin carcinogenesis via inhibiting cell proliferation PLoS ONE 10e0126459 101371journalpone0126459 Andujar I Rios JL Giner RM Recio MC Pharmacological properties ofshikonin a review of literature since Planta Med “ 101055s00331350934 Li W Liu J Zhao Y PKM2 inhibitor shikonin suppresses TPAinducedmitochondrial malfunction and proliferation of skin epidermal JB6 cells MolCarcinog “ 101002mc21988 Boulos JC Rahama M Hegazy MF Eï¬erth T Shikonin derivatives “for cancer prevention and therapy Cancer Lett 101016jcanlet201904033 Zhu Y Zhong Y Long X Zhu Z Zhou Y Ye H et al Deoxyshikoninisolated from Arnebia euchroma inhibits colorectal cancer by downregulating the PI3KAktmTOR pathway PharmBiol “ Liberti MV Locasale JW The Warburg eï¬ect how does it benefit cancer cellsDATA AVAILABILITY STATEMENTAll datasets generated for this study are included in theSupplementary MaterialAUTHOR CONTRIBUTIONSHW conducted the experiments and participated in theconception and the design of the study HZ conducted theexperiments and performed the analysis LC contributed toanalyzing the data and drafting the manuscript All authorscontributed to the and approved the submitted versionSUPPLEMENTARY MATERIALThe Supplementary Materialonline202001253fullsupplementarymaterialfor this can be foundhttpswwwfrontiersins103389foncat Xu D Liang J Lin J Yu C PKM2 a potential regulator of rheumatoid arthritisvia glycolytic and nonglycolytic pathways Front Immunol 103389fimmu201902919 Gong T Cui L Wang H Wang H Han N Knockdown of KLF5 suppresseshypoxiainduced resistance to cisplatin in NSCLC cells by regulating HIF1αdependent glycolysis through inactivation of the PI3KAktmTOR pathway JTransl Med 101186s1296701815432 Nemazanyy I Espeillac C Pende M Panasyuk G Role of PI3K mTOR andAkt2 signalling in hepatic tumorigenesis via the control of PKM2 expressionBiochem Soc Trans “ 101042BST20130034 Zheng YL Li L Jia YX Zhang BZ Li JC Zhu YH et al LINC01554mediated glucose metabolism reprogramming suppresses tumorigenicityin hepatocellular carcinoma via downregulating PKM2 expression andinhibiting AktmTOR signaling pathway Theranostics “ 107150thno28992 Zhao X Zhu Y Hu J Jiang L Li L Jia S et al Shikonin inhibits tumor growthin mice by suppressing pyruvate kinase M2mediated aerobic glycolysis SciRep 101038s4159801831615y Tang J Ren YG Zhao J Long F Chen J Jiang Z Shikonin enhancessensitization of gefitinib against wildtype EGFR nonsmall cell lung cancervia inhibition PKM2stat3cyclinD1 signal pathway Life Sci “ 101016jlfs201805012 Popescu NC Cheng SY Chromosomal localization of the gene for a humancytosolic thyroid hormone binding protein homologous to the subunitof pyruvate kinase subtype M2 Somat Cell Mol Genet “ 101007BF01233100 Martelli AMF McCubreyEvangelisti C ChiariniJA Thephosphatidylinositol 3kinaseAktmTOR signaling network as a therapeutictarget in acute myelogenous leukemia patients Oncotarget “ 1018632oncotarget114 Yang D Zhang X Zhang X Xu Y The progress and current status ofimmunotherapy in acute myeloid leukemia Ann Hematol “ 101007s002770173148x Wang X Feng Y Chinese medicines induce cell death the molecular andcellular mechanisms for cancer therapy Biomed Res Int Trends Biochem Sci “ 101016jtibs201512001 Wang ZY Chen Z Acute promyelocytic leukemia from highly fatal to highly Hanahan D Weinberg RA Hallmarks of cancer the next generation Cellcurable Blood “ 101182blood200707102798 “ 101016jcell201102013 Herst PM Howma Answer:

Thyroid_Cancer

RANK are expressed in different cell types and tissues throughout thebody They were originally described for their essential roles in bone remodeling and theimmune system but have subsequently been shown to provide essential signals fromregulating mammary gland homeostasis during pregnancy to modulating tumorigenesisThe success of RANKLRANK research serves as a paragon for translational researchfrom the laboratory to the bedside The case in point has been the development ofDenosumab a RANKLblocking monoclonal antibody which has already helped millionsof patients suffering from postmenopausal osteoporosis and skeletal related events incancer Here we will provide an overview of the pathway from its origins to its clinicalrelevance in disease with a special focus on emerging evidence demonstrating thetherapeutic value of targeting the RANKLRANKOPG axis not only in breast cancer butalso as an addition to the cancer immunotherapy arsenalKeywords osteoimmunology RANKLRANKOPG malignant tumor targeted therapy DenosumabEdited byLinda ConnellyCalifornia University of Science andMedicine United StatesReviewed byDhivya R SudhanINTRODUCTIONUniversity of Texas SouthwesternMedical Center United StatesSophia HL GeeUniversity of Miami United StatesCorrespondenceJosef M PenningerjosefpenningerubccaSpecialty sectionThis was submitted toWomen™s Cancera section of the journalFrontiers in OncologyReceived February Accepted June Published August CitationMing J Cronin SJF and Penninger JM Targeting theRANKLRANKOPG Axis for CancerTherapy Front Oncol 103389fonc202001283In the œseed and soil theory was first proposed by Stephen Paget for tumor metastasesto distant ans When tumor cells œseeds leave their primary site of origin and spreador metastasize the microenvironment œsoil of the target an is usually favorable for tumorcell anchoring and expansion of metastatic cells Bone is not only the site for primary bonetumors such as giant cell tumors and osteosarcoma but is also one of the most common distantmetastatic sites for solid tumors such as multiple myeloma MM breast cancer prostate cancerand nonsmall cell lung cancer NSCLC suggesting that the bone environment can serve asœsoil for tumor development and might also serve as a œseed for further metastatic spread Recentresearch on the bone microenvironment and its involvement in cancer biology has focused on thefield of osteoimmunology which includes the crosstalk between bone stromal cells osteoblastsand osteoclasts and immune cells Identifying key players regulating bone homeostasis couldpave the way for potential therapeutic cancer targets in particular to break the vicious circle ofmetastasis to the bonesThe receptor activator of the nuclear factor kappaB ligand RANKL also known as TNFSF11together with its receptor RANK TNFRSF11A the decoy receptor osteoprotegerin OPGTNFRSF11B and the recently identified receptor Leucinerich repeatcontaining Gproteincoupled receptor LGR4 has been shown to play critical bottleneck functions not only inregulating bone metabolism but also in immunity and tumorigenesis In this review we will brieflyintroduce the key functions of the RANKLRANKOPG axis in maintaining bone homeostasisand regulating immunity Furthermore we will discuss the role of this pathway from primaryFrontiers in Oncology wwwfrontiersinAugust Volume 0cMing et alRANKLRANK in Cancertumorigenesis to cancer metastasis with particular attention tobreast cancer and the hormonal control of this pathway We willalso discuss recent data pointing to the RANKLRANK axis as anovel therapeutic target in BRCAmutated breast cancers and asa novel promising cancer immunotherapy agentRANKLRANKOPG AND BONEHOMEOSTASISBone provides strength and structure protects vital ans storesminerals such as calcium and is essential in the productionof hematopoietic cells Bone homeostasis is maintained by thebalance between mainly two types of cells osteoblasts derivedfrom mesenchymal cells which build bone and osteoclastsderived from bone marrow hematopoietic precursor cells whichresorb bone Figure Osteoblasts act as both mechanicalsensors together with osteocytes and coordinators for the boneremodeling process which is controlled by local growth factorsand systemic factors for example calcitonin or sex hormonessuch as estrogen The pathological imbalance between boneformation and resorption leads to the development of local orsystemic bone diseases such as osteopetrosis and osteoporosis The interaction and communication between osteoclasts andosteoblasts is intricately regulated in feedback loops to maintainbone homeostasis and this constant remodeling process of thebone matrix is critical for healthy bone strength and efficienthematopoiesis RANK TNFRSF11A OFE ODFR TRANCER ODARCD265 and RANKL TNFSF11 TRANCE ODF andOPGL “ are a receptorligand pair of the TNF receptorsuperfamily discovered at the end of the last millennium and wereidentified as key regulators of osteoclast development and bonemetabolism Figure Factors that can induce boneresorption such as the sex hormone progesterone vitamin D3PTHrP IL1 IL11 IL17 or TNFα “ act on osteoblaststo induce RANKL expression which then binds to its receptorRANK on the surface of osteoclast progenitor cells inducing preosteoclast diï¬erentiation into multinucleated fullyfunctionalosteoclasts RANKL also plays an important role in the continuedsurvival and function of osteoclasts “ Figure RANKLis produced as a membranebound protein which can alsobe shed as a soluble trimeric protein SheddaseresistantRANKL mice have been generated in which soluble RANKLis undetectable in the circulation bone mass or boneFIGURE Role of RANKLRANKOPG axis on bone homeostasis and immune system RANKL is secreted by osteoblasts and osteocytes when stimulated byparathyroid hormone PTH vitamin D andor prostaglandin PGE2 RANKL binds to RANK on the membrane of osteoclast progenitors preosteoclasts whichresults in bone resorption by mature osteoclasts Osteoprotegerin OPG binds to RANKL thus inhibiting RANK signaling and bone resorption RANKRANKL alsoplays a role in immune cell regulation and the crosstalk between both systems termed osteoimmunology T cells can also express RANKL which can both act onpreosteoclasts but can also act on dendritic cells DCs to promote their survival and to prolong T“DC interactions DCs can exhibit modulating effects onRANKmediated osteoclastogenesis through the secretion of OPG HSC hematopoietic stem cell MSC mesenchymal stem cellFrontiers in Oncology wwwfrontiersinAugust Volume 0cMing et alRANKLRANK in Cancerstructure was not aï¬ected during development in these mice butadult mice displayed reduced osteoclast numbers and increasedcancellous bone mass Importantly the bone loss caused byestrogen deficiency was unaï¬ected by the lack of soluble RANKLThus these data show that it is the membranebound formof RANKL which is largely responsible for the physiologicalfunctions of RANKL although the soluble form can contributeto bone remodeling in adult mice Osteoprotegerin OPG TNFRSF11B acts as a decoy receptorfor RANKL and is induced by estrogen IL4 or transforminggrowth factor beta TGF OPG competitively binds toRANKL thereby interfering with RANKL“RANK interactionsand blocking bone resorption “ The relative levels ofOPG and RANKL are precisely controlled to ensure healthybone During pathological conditions such as menopauserelatedosteoporosis decreased estrogen levels result in decreased OPGand subsequently increased RANKL resulting in enhancedosteoclast activation and bone loss Recently leucinerichrepeat G proteincoupled receptor LGR4 was identifiedas an additional receptor for RANKL Similar to RANKLGR4 is expressed on osteoclasts but unlike RANK LGR4 is anegative regulator for osteoclast diï¬erentiation Therefore bothOPG and LGR4 are endogenous inhibitors of RANKLRANKsignaling A recent study has shown that RANKL reversesignaling from osteoclasts to osteoblasts couples bone resorptionto bone formation processes This is achieved through thesecretion of small extracellular vesicles from osteoclasts thatcontain RANK The authors showed that these RANK vesiclesbind membranebound RANKL on the osteoblasts and therebypromote bone formation by triggering RANKL reverse signalingvia activation of Runtrelated transcription factor Runx2Targeting RANKL reverse signaling represents a novel strategyto avoid the reduced bone production associated with inhibitionof osteoclastogenesis As RANKL is an important regulator of bone loss in bonemetastases associated with cancers such as multiple myelomaand in postmenopausal osteoporosis a specific fully human IgG2monoclonal RANKL antibody mAb has been developed whichneutralizes the activity of RANKL which has been designated asDenosumab The efficacy of Denosumab has been confirmed inmultiple clinical trials and Denosumab therapy is now approvedand widely used for the treatment of various boneassociateddiseases “RANKLRANKOPG IN THE IMMUNESYSTEMApart from bone homeostasis the RANKLRANKOPG axis isalso involved in various physiological immune processes RANKwas originally discovered on dendritic cells DCs and RANKLmediates the survival of DCs The interaction betweenactivated T cellderived RANKL and RANK expressed on DCsincreases the antigenpresenting capabilities of the latter thusaugmenting the number and cell cycle of antigenspecific Tcells as well as enhancing the immune response of memory Tcells Interestingly phenotyping of rankl and rankdeficient micerevealed a complete absence of peripheral lymph nodes but intactspleen and Peyer™s plaque structures “ Subsequent studieshave found that during embryogenesis RANKL is expressedby hematopoietic lymphoid tissue inducing LTi cells andmesenchymallymphoid tissue anizer LTo cells “RANKL has been demonstrated to stimulate lymphotoxin LTexpression and regulate LTi cell accumulation FurthermoreRANKL also triggers the proliferation of adult lymph nodestroma indicating that RANKL may directly activate LTo cells“ In the thymus the RANKLRANK pathway is criticalfor CD80 AIRE medullary thymic epithelial cell mTECmaturation involved in central immune tolerance RANKdeficient mice display mild autoimmunity at an advancedage RANKLRANK activation in lymphatic endothelialcells LECs is important for the tissueresident macrophagesnamely sinusoidal macrophage maturation not only duringembryogenesis but also after inflammationinduced loss of thesecells Moreover group innate lymphoid cells ILC3s inthe intestine use RANKLRANK interactions to control theirown abundance and intestinal homeostasis Genetic ablation ofRANKL specifically in IL3C cells leads to an increased number ofthese cells with enhanced levels of proinflammatory cytokinessuch as interleukin17A IL17A and IL22 during intestinalinfection Human patients carry RANK mutations and mice lackingRANKL or RANK exhibit a defect in B cell developmentresulting in a significant reduction in B cell numbers however these eï¬ects might be indirect because in themousetissuespecific deletion of Rank in B cells showedno diï¬erence in function nor development of B cells andblocking RANKRANKL with Denosumab does not apparentlyaï¬ect B cell physiology in osteoporosis patients In addition reports using Bcellspecific rankldeficient micehave shown that B cellderived RANKL increases osteoclastnumbers and bone loss brought on by estrogen deficiency Overexpression of RANKL in keratinocytes results in functionalalterations of epidermal dendritic cells and systemic increases inregulatory CD4CD25 T cells Tregs numbers Thereforeenvironmental stimuli can rewire the local and systemic immunesystems via RANKL The RANKLRANK system is alsoinvolved in M microfoldcell development a specific antigensampling cellular subtype found in the intestine as mesenchymalcells produce RANKL that can directly interact with intestinalepithelial cells to regulate M cell diï¬erentiation “Inhibition of mesenchymal RANKL impairs M celldependentantigen sampling and B celldendritic cell interaction in thesubepithelial dome SED resulting in decreased IgA productionand microbial diversity In addition B cells are absentin cryptopatches CPs and isolated lymphoid follicle ILFsformation was abrogated in rankl null mice Whether B cells or T cells are essential for bone loss isstill controversial Ovariectomy has been shown to enhance Tcelldependent TNFalpha production in a bone loss mousemodel because of the enhanced macrophage colonystimulatingfactor MCSF and RANKL In contrast anotherstudy suggested T cells are not involved in ovariectomyinducedFrontiers in Oncology wwwfrontiersinAugust Volume 0cMing et alRANKLRANK in Cancertrabecular bone loss Nevertheless it has been reportedin postmenopausal women that increased T cell activity andincreased RANKL production by T cells are associated withosteoporosis Furthermore studies in conditionalknockout mice to specifically eliminate RANKL in B cells or Tcells have shown that RANKL produced by B cells but not T cellsleads to bone loss by the induction of osteoclastogenesis Thelack of mature B cells does not prevent bone loss suggestingthat RANKL is derived from immature B cells Moreover it hasbeen reported that deletion of rankl in T cells does not change thenumber of T cells but results in impaired mature B cell numbersin the bone marrow suggesting that RANKL might promote Bcell maturation via paracrine signaling RANKLRANKLOPG IN MAMMARYGLAND PHYSIOLOGY AND BREASTCANCERBreast cancer is the most prevalent female malignancy Studies based on large populations have shown that womenwho receive estrogen plus progesterone hormone replacementtherapy called combined HRT are more vulnerable to breastcancer compared to women who receive estrogen only “furthermore progesterone levels have been demonstrated to bean independent risk factor for increased breast cancer incidence In rankl knockout mice our group was the first to reportthat during pregnancy RANKL deficiency results in a totalblock in the development oflobuloalveolar milksecretingstructures Whereas estrogen triggers the expansion ofthe mammary epithelium in puberty progesterone drives theproliferation of mammary epithelial cells in the estrous cycleand in pregnancy induces the growth and diï¬erentiation of themammary epithelium into ultimately milksecreting acini Figure Mechanistically progesterone induces progesteronereceptor PRpositive mammary epithelial cells to expressRANKL resulting in the proliferation of neighboring RANKmammary epithelial progenitor cells in an autocrine and alsoparacrine fashion “ Moreover RANKL can induce theproliferation of RANKpositive ductal epithelial cells through theFIGURE RANKRANKL pathway in mammary gland physiology and breast cancer A RANK is constitutively expressed on the membrane of luminal and basalepithelial cells including mammary stem cells MaSCs Stimulation with progesterone induces RANKL expression and secretion in progesterone receptor PRpositiveluminal epithelial cells RANKL binds in an autocrine fashion to RANK on luminal epithelial cells which stimulates further RANKL expression and in a paracrine fashionto RANK on basal epithelial cells resulting in enhanced RANK expression on basal mammary epithelial cells and the activation of the IKKαNFκB“cyclin D1 signalingaxis to induce a variety of physiological responses necessary for mammary gland development B Heterozygous BRCA1 mutationcarrying women canspontaneously lose the remaining wildtype BRCA1 gene from somatic mutation or epigenetic silencing Subsequently loss of BRCA1 protein can result in increasedgenomic instability DNA damage and genetic mutations eg TP53 Progesterone as well as synthetic progestins upregulate RANKL expression in PR luminalbreast epithelial cells which stimulates RANKmediated cell proliferation of adjacent progenitor cells as discussed in A Altogether the genotoxic stress and amplifiedproliferation cues culminate in uncontrolled proliferation and the development of breast cancerFrontiers in Oncology wwwfrontiersinAugust Volume 0cMing et alRANKLRANK in Cancerinduction of Rspondin Therefore RANK and RANKL linksex hormones to mammary progenitor cell proliferation duringthe estrous cycle and in pregnancy Figure Clinically an increase in serum progesterone and RANKLlevels is associated with an increase in breast cancer risk inpostmenopausal women Higher concentrations of solubleRANKL are positively correlated with an increased risk ofestrogen receptorpositive but not estrogen receptornegativebreast cancer indicating that the RANKRANKLOPG axis maybe involved in the tumorigenesis of ER breast cancer Indeed in a hormoneinduced spontaneous mouse breast cancermodel RANKL is critical for the development of sex hormonedriven breast cancer Deletion of RANK and Ikkα akey downstream regulator of the RANK signaling pathway inmammary epithelial cells also significantly delayed progestinMPA and DNAmutation DMBAinduced mammary tumorformation further indicating that the RANKRANKL pathwaydrives breast cancer Furthermore the selective inhibitionof RANKL by RANKFc not only attenuated breast tumorprogression in a hormone and carcinogendriven mouse breastcancer model but also decreased the progression of breast cancerin a transgenic spontaneous tumor model BRCA1 and BRCA2 mutations are the most prevalent geneticdrivers for hereditary breast cancer in humans Interestinglywomen with germline BRCA12 mutations usually exhibithigher progesterone and estrogen levels during the gestationalphase ofthe estrous cycle compared to women withoutthese mutations Inversely decreased serum OPG levelsare associated with increased breast cancer incidence Moreover high levels of RANK expression were observed inbreast cancer samples from premalignant lesions and patientswith BRCA1 mutations SNP data analysis from theCooperative Tumor GeneEnvironmental Research iCOGSincluding approximately BRCA1 and BRCA2mutation carriers identified SNPs which were significantlyassociated with breast cancer risk at the TNFRSF11A locusencoding RANK Altogether these human data stronglysupport the idea that the RANKLRANKOPG axis is intimatelyinvolved in the tumorigenesis of BRCA12 mutationdrivenbreast cancerSubsequent animal studies provided direct evidence thatRANKL and RANK are critically involved in the oncogenesis ofBRCA1 mutationdriven hereditary breast cancer Figure Genetically engineered mice carrying Brca1 and Tp53 mutationsshowed hyperproliferation and malignancy in their mammaryglands at months of age the inactivation of the RANKLRANKpathway in these mice largely prevented the occurrence ofmalignanttumors and resulted in significantly prolongedsurvival Additionally the pharmacological blockade of RANKLusing RANKFc completely abolished the development ofprecancerousin the Brca1Tp53 doublemutatedbreast cancer model Amplification of RANKexpressingmammary duct progenitor cells can be found in the nontumorbreast tissue of BRCA1 mutant carriers and these cells havesimilar molecular characteristics as basallike breast cancercells RANKL inhibition also significantly suppressedthe proliferation oftumor anoids derived from BRCA1mutant human breast biopsy specimens and RANKLRANKlesionspathway blockade strongly reduced tumorigenesis in patientderived xenograft PDX breasttumor mouse model Thus independent work among diï¬erent laboratories usingdiï¬erent mouse models as well as studies using humanbreast epithelial progenitor assays hasled to the sameconclusion RANKLRANK aï¬ect mammaryprogenitorcells and are critically involved in the BRCA1mutation drivenmammary tumorigenesisTherefore we and others have proposed that the monoclonalantibody Denosumab which specifically inhibits RANKRANKLinteractions could potentially be used for the prophylactictreatment of breast cancer in BRCA12 carriers Indeed weposit that healthy women with BRCA1 mutation will benefit notexcluding an eï¬ect on other TNBCs In a pilot clinical studytermed BRCAD the proliferation marker Ki67 was significantlydownregulated in the breast biopsy of BRCA1 mutation carrierswho received shortterm treatment with Denosumab suggestingthat RANKL inhibition may be a feasible method for the chemoprevention of breast cancer in women with BRCA1 mutationsThis study requires additional patient data which is currentlyongoing Another clinical study DBEYOND which aimedto investigate whether neoadjuvant RANKL inhibition therapycan reduce tumor proliferation in premenopausal early breastcancer patients found no significant change in Ki67positivetumor cells in the breast cancer tissues treated with Denosumabbut the density of tumorinfiltrating lymphocytes TILs wasincreased in the stroma and tumor tissues upon Denosumabtreatment In addition to the now experimentally well validated role ofRANKLRANKOPG in the sex hormone and BRCA1 mutationdriven mammary cancer tumorigenesis it has also been reportedthat this pathway can induce epithelialmesenchymal transitionEMT in breast cancer cells as well as in prostate andendometrial cancers “ suggesting that RANKLRANKsupports tumorigenesis in various epithelial cancers Moreoverour group has recently reported on the role of RANKL andRANK in lung cancer We demonstrated that the inactivation ofrank in lung epithelial cells disrupts mitochondrial bioenergeticsand significantly reduceslung cancer development bothculminating in increased survival This genetic modeling inthe mouse supports findings in human clinical trials in whichRANK inhibition with the monoclonal antibody Denosumabresulted in prolonged survival especially in patients withnonsmall celllung cancer NSCLC adenocarcinomas andsquamous tumors Notably this Denosumabdependent survivaladvantage occurred in lung cancer patients irrespective of visceralmetastasis hinting that the underlying eï¬ects of RANKLRANKblockade in addition to those targeting the bone are involved Epidemiological reports have also uncovered genderdiï¬erences particularly in lung cancer with respect to etiologyprogression and treatment response believed to be due tosexrelated hormonal factors “ though the underlyingmolecular mechanisms are poorly understood We have recentlyshown in our experimental lung cancer model that by ablatingthe sex hormones in female mice we could eï¬ectively eliminatethe survival advantages brought about by loss of rank in the lungtumors Furthermore synthetic progesterone MPAdependentenhanced lung cancer initiation required RANK expressionFrontiers in Oncology wwwfrontiersinAugust Volume 0cMing et alRANKLRANK in CancerTogether these data suggest that the sex hormone regulation ofRANKLRANK could also explain the gender diï¬erences seen inhuman lung cancerRANKRANKL AS REGULATORS OFMETASTASISStudies have now shown that the RANKLRANKOPG axis playsa role in the progression of malignant tumors by promotingtumor cell migration stimulating tumor neovascularizationand promoting distant metastasis of tumor cells Disseminated tumor cells are responsible for the earlymetastasis of tumors which frequently can be detected inthe bone marrow of patients with malignant tumors Thisœmicrometastases niche forms a favorable microenvironmentfor the development of metastatic spread protecting cancer cellsfrom various antitumor treatments and modulating anticancerimmune responses thereby allowing the tumor cells to escapeimmune surveillance The tumor microenvironment is acomplex milieu composed of distinct factors such as cytokinesextracellular matrix components and various cell types suchas fibroblasts endothelial cells and immune cells all ofwhich participate in cancer development progression andmetastasis In bone tissue the tumor microenvironmentincludes immune and tumor cells as well as osteoblasts andosteoclasts all of which participate in a œvicious cycle thataccelerates osteolysis and cancer cell proliferation through inpart the RANKRANKLOPG axis For instance cancercells can increase the expression of RANKL in osteoclastsby secreting parathyroid hormonerelated peptide PTHrP Tumor cells can also directly express RANKLand secrete cytokines such as interleukin IL1α TNFα macrophage colonystimulating factor MCSF orprostaglandin E2 PGE2 all of which promote osteoclastdiï¬erentiation and survival resulting in local osteolysis whichsupports metastatic growth “ Subsequently growthfactors released by the bone matrix such as insulinlike growthfactors IGFs fibroblast growth factor FGFs plateletderivedgrowth factor PDGF or bone morphogenetic proteins BMPspromote cancer cell proliferation “ In addition tocytotoxic drugs and endocrine disruptive drugstherapiestargeting the RANKRANKLOPG axis exhibit direct andorindirect antitumor eï¬ects by blocking the vicious cycle betweenbone and cancer cells “In a murine model of melanoma metastasis it was foundthat for malignant tumors with RANK expression RANKLproduced by osteoblasts and bone marrow stromal cells couldact as a chemical attractant and promote the migration andmetastasis of malignant tumors to these sites Similareï¬ects were also found in malignant tumors such as breastcancer “ prostate cancer “ and lung cancer The activation of phospholipase C PLC proteinkinase C PKC ERK and phosphatidylinositol3OH kinasePI3K pathways were involved in RANKinduced tumor cellmigration “ RANK engagement by RANKL inducestrimerization of the RANK receptor which then stimulates therecruitment and activation of the adapter protein TRAF6 viaTRAF6binding sites in the Cterminus of RANK™s cytoplasmictail TRAF6 in turn complexes with many other downstreamadapters and kinases to activate the aforementioned pathwaysMoreoverthe RANKLRANK pathway was also shown topromote the formation of new blood vessels and regulate thetumor microenvironment at the primary tumor site to promotethe migration of tumor cells into the bloodstream and formetastasis to distant ans “In breast cancer RANKL is also produced by Foxp3expressing Tregs and tumorassociated macrophages TAMsthat can aï¬ect tumor growth tumor cell dissemination andmetastasis RANKL expression on tumorinfiltratingregulatory T cells may also be involved in cancer metastasis TAMs are either M1 or M2 macrophages with M1 being antitumor and M2 TAMs promoting tumorigenesis ImportantlyM2 macrophages express RANK and are attracted by RANKLproduced by the tumor microenvironment The RANKLRANKpathway in M2 macrophages can regulate the production ofchemokines and promote the proliferation of Treg lymphocyteswhich supports the immunosuppressive milieu within the tumormicroenvironment Recently it has been reported that estrogenrelatedreceptoralpha ERRα an important factor of cancer cell invasivenesspromotes breast cancer cell dissemination from primarymammary tumors to the bone Intriguingly RANK hasbeen shown to be a target for ERRα Furthermore the metaexpression analysis of breast cancer patients has uncovereda positive association between metastases and ERRαRANKexpression as well as a positive correlation between ERRαand BRCA1 mutation carriers revealing a novel pathwaywhereby ERRα in primary breast cancer could promote earlydissemination of cancer cells to bone Moreover it wasrecently shown that RANKL serum levels are significantlyincreased in breast cancer patients who developed bonemetastases p and patients within the highest quartileof RANKL had a significantly increased risk of developing bonemetastases compared to those in the lowest HR 95CI“ p This study further suggests a role ofRANKL in breast cancer metastasis TARGETING RANKLRANK IN HUMANCANCERIn light of the diï¬erent roles of the RANKLRANK pathwayin bone metabolism and immune system functions therapytargeting this axis may not only control primary tumordevelopment such as in the case of breast cancer and reducebone metastasis which has been demonstrated in clinical trials but also exert a direct antitumor eï¬ect via regulatinglocal tumorassociated immune responses as observed in studiesusing the monoclonal RANKL antibody inhibitor Denosumab In randomized clinical trials Denosumab has shown rapideï¬ectiveness by directly impairing osteoclast activity andinducing osteoclast apoptosis Moreover Denosumabwassignificantly more eï¬ective in reducing urinary Nterminal peptides a biochemical marker for bone turnoverFrontiers in Oncology wwwfrontiersinAugust Volume 0cMing et alRANKLRANK in Cancerand more eï¬ective in delaying skeletalrelated events SREssuch as pathologic fractures spinal cord compression andhypercalcemia which greatly aï¬ect quality of life in patients withbreast cancer and castrationresistant prostate cancer CRPCbone metastases However the eï¬ect of Denosumab to delaySREs in patients with NSCLC and multiple myeloma MMpatients with bone metastases is comparable to bisphosphonatedrugs “ Moreover the benefit of Denosumab andbisphosphonates is not only restricted to osteolytic cancers suchas breast myeloma and NSCLC but also evident in osteoblasticcancers Recently it was demonstrated in osteoblastic cancerssuch as prostate cancer that Denosumab or bisphosphonate canaï¬ect the osteoclastosteoblast balance in the œvicious cycle ofbone destruction induced by metastasized cancer cells which highlights the potential rationale in treating osteoblasticcancer patients with Denosumab or bisphosphonatesIn a randomized phase III clinical trial comparing Denosumaband bisphosphonate zoledronic acid ZA in patients with solidtumors breast cancer prostate cancer multiple myeloma andbone metastases the results showed that Denosumab was similarto ZA in preventing or delaying the onset of primary SREs However in nonsmallcell lung carcinoma NSCLCn treatment with Denosumab showed a significantimprovement in overall survival In these patients nostatistically significant SRE delay was observed in Denosumabtreated patients suggesting that this survival advantage maybe independent of the bone system The result of arandomized phase III trial of multiple myeloma MM patientsn also demonstrated the eï¬ectivity of Denosumab toreduce the occurrence of primary SRE events moreover the useof Denosumab significantly improved progressionfree survivalPFS Whether this survival benefit is due to the decreasein the incidence of bone metastasis or whether Denosumab hasother antitumor eï¬ects requires further researchIn the randomized placebocontrolled phase III ABCSG18trial which enrolled postmenopausal female patients withearly hormone receptorpositive breast cancer the first clinicalfracture of the Denosumabtreated group was compared with theplacebo group and a significant protection of bone breaks wasdemonstrated hazard ratio [HR] · [ CI ·“·] p A median followup of months showeda significant improvement in the diseasefree survival DFS inthe Denosumabtreated group HR CI “Cox p These data suggest that adjuvant Denosumabcan significantly improve the DFS rate of HR postmenopausalbreast cancer patients However in another randomizedphase III clinical trial of breast cancer DCARE recent reportshave shown that adjuvant Denosumab does not reduce therisk of breast cancer recurrence or death in earlystage breastcancer patients receiving standard adjuvant therapy Theseinconsistencies which could be explained by diï¬erent cohortsfor patient stratifications eg more advanced early cases ofbreast cancer were included in the DCARE trials as comparedto the ABCSG18 study need to be further evaluated with largercohorts of patients and multiplecenter analysis Importantlya recent followup study of the ABCSG18 trial confirmed thethat blocking RANKL in an adjuvant breast cancer therapysetting not only markedly reduces the risk of breaking bones butalso significantly reduces the reoccurrence of the breast tumors It should be also noted that although there was nodiï¬erence in bonemetastasesfreesurvival in the DCARE trialDenosumab treatment significantly reduced the time to bonemetastasis at the site of first occurrence DENOSUMAB AS A NOVEL CANCERIMMUNOTHERAPYThe field of cancer immunotherapy has paved the way for a newparadigm to combat cancer by coaxing the body™s own immunesystem to seek out specifically target and destroy cancer cellsAmong the various approaches immunecheckpoint inhibitorsthattarget CTLA4 as

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You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: RANK are expressed in different cell types and tissues throughout thebody They were originally described for their essential roles in bone remodeling and theimmune system but have subsequently been shown to provide essential signals fromregulating mammary gland homeostasis during pregnancy to modulating tumorigenesisThe success of RANKLRANK research serves as a paragon for translational researchfrom the laboratory to the bedside The case in point has been the development ofDenosumab a RANKLblocking monoclonal antibody which has already helped millionsof patients suffering from postmenopausal osteoporosis and skeletal related events incancer Here we will provide an overview of the pathway from its origins to its clinicalrelevance in disease with a special focus on emerging evidence demonstrating thetherapeutic value of targeting the RANKLRANKOPG axis not only in breast cancer butalso as an addition to the cancer immunotherapy arsenalKeywords osteoimmunology RANKLRANKOPG malignant tumor targeted therapy DenosumabEdited byLinda ConnellyCalifornia University of Science andMedicine United StatesReviewed byDhivya R SudhanINTRODUCTIONUniversity of Texas SouthwesternMedical Center United StatesSophia HL GeeUniversity of Miami United StatesCorrespondenceJosef M PenningerjosefpenningerubccaSpecialty sectionThis was submitted toWomen™s Cancera section of the journalFrontiers in OncologyReceived February Accepted June Published August CitationMing J Cronin SJF and Penninger JM Targeting theRANKLRANKOPG Axis for CancerTherapy Front Oncol 103389fonc202001283In the œseed and soil theory was first proposed by Stephen Paget for tumor metastasesto distant ans When tumor cells œseeds leave their primary site of origin and spreador metastasize the microenvironment œsoil of the target an is usually favorable for tumorcell anchoring and expansion of metastatic cells Bone is not only the site for primary bonetumors such as giant cell tumors and osteosarcoma but is also one of the most common distantmetastatic sites for solid tumors such as multiple myeloma MM breast cancer prostate cancerand nonsmall cell lung cancer NSCLC suggesting that the bone environment can serve asœsoil for tumor development and might also serve as a œseed for further metastatic spread Recentresearch on the bone microenvironment and its involvement in cancer biology has focused on thefield of osteoimmunology which includes the crosstalk between bone stromal cells osteoblastsand osteoclasts and immune cells Identifying key players regulating bone homeostasis couldpave the way for potential therapeutic cancer targets in particular to break the vicious circle ofmetastasis to the bonesThe receptor activator of the nuclear factor kappaB ligand RANKL also known as TNFSF11together with its receptor RANK TNFRSF11A the decoy receptor osteoprotegerin OPGTNFRSF11B and the recently identified receptor Leucinerich repeatcontaining Gproteincoupled receptor LGR4 has been shown to play critical bottleneck functions not only inregulating bone metabolism but also in immunity and tumorigenesis In this review we will brieflyintroduce the key functions of the RANKLRANKOPG axis in maintaining bone homeostasisand regulating immunity Furthermore we will discuss the role of this pathway from primaryFrontiers in Oncology wwwfrontiersinAugust Volume 0cMing et alRANKLRANK in Cancertumorigenesis to cancer metastasis with particular attention tobreast cancer and the hormonal control of this pathway We willalso discuss recent data pointing to the RANKLRANK axis as anovel therapeutic target in BRCAmutated breast cancers and asa novel promising cancer immunotherapy agentRANKLRANKOPG AND BONEHOMEOSTASISBone provides strength and structure protects vital ans storesminerals such as calcium and is essential in the productionof hematopoietic cells Bone homeostasis is maintained by thebalance between mainly two types of cells osteoblasts derivedfrom mesenchymal cells which build bone and osteoclastsderived from bone marrow hematopoietic precursor cells whichresorb bone Figure Osteoblasts act as both mechanicalsensors together with osteocytes and coordinators for the boneremodeling process which is controlled by local growth factorsand systemic factors for example calcitonin or sex hormonessuch as estrogen The pathological imbalance between boneformation and resorption leads to the development of local orsystemic bone diseases such as osteopetrosis and osteoporosis The interaction and communication between osteoclasts andosteoblasts is intricately regulated in feedback loops to maintainbone homeostasis and this constant remodeling process of thebone matrix is critical for healthy bone strength and efficienthematopoiesis RANK TNFRSF11A OFE ODFR TRANCER ODARCD265 and RANKL TNFSF11 TRANCE ODF andOPGL “ are a receptorligand pair of the TNF receptorsuperfamily discovered at the end of the last millennium and wereidentified as key regulators of osteoclast development and bonemetabolism Figure Factors that can induce boneresorption such as the sex hormone progesterone vitamin D3PTHrP IL1 IL11 IL17 or TNFα “ act on osteoblaststo induce RANKL expression which then binds to its receptorRANK on the surface of osteoclast progenitor cells inducing preosteoclast diï¬erentiation into multinucleated fullyfunctionalosteoclasts RANKL also plays an important role in the continuedsurvival and function of osteoclasts “ Figure RANKLis produced as a membranebound protein which can alsobe shed as a soluble trimeric protein SheddaseresistantRANKL mice have been generated in which soluble RANKLis undetectable in the circulation bone mass or boneFIGURE Role of RANKLRANKOPG axis on bone homeostasis and immune system RANKL is secreted by osteoblasts and osteocytes when stimulated byparathyroid hormone PTH vitamin D andor prostaglandin PGE2 RANKL binds to RANK on the membrane of osteoclast progenitors preosteoclasts whichresults in bone resorption by mature osteoclasts Osteoprotegerin OPG binds to RANKL thus inhibiting RANK signaling and bone resorption RANKRANKL alsoplays a role in immune cell regulation and the crosstalk between both systems termed osteoimmunology T cells can also express RANKL which can both act onpreosteoclasts but can also act on dendritic cells DCs to promote their survival and to prolong T“DC interactions DCs can exhibit modulating effects onRANKmediated osteoclastogenesis through the secretion of OPG HSC hematopoietic stem cell MSC mesenchymal stem cellFrontiers in Oncology wwwfrontiersinAugust Volume 0cMing et alRANKLRANK in Cancerstructure was not aï¬ected during development in these mice butadult mice displayed reduced osteoclast numbers and increasedcancellous bone mass Importantly the bone loss caused byestrogen deficiency was unaï¬ected by the lack of soluble RANKLThus these data show that it is the membranebound formof RANKL which is largely responsible for the physiologicalfunctions of RANKL although the soluble form can contributeto bone remodeling in adult mice Osteoprotegerin OPG TNFRSF11B acts as a decoy receptorfor RANKL and is induced by estrogen IL4 or transforminggrowth factor beta TGF OPG competitively binds toRANKL thereby interfering with RANKL“RANK interactionsand blocking bone resorption “ The relative levels ofOPG and RANKL are precisely controlled to ensure healthybone During pathological conditions such as menopauserelatedosteoporosis decreased estrogen levels result in decreased OPGand subsequently increased RANKL resulting in enhancedosteoclast activation and bone loss Recently leucinerichrepeat G proteincoupled receptor LGR4 was identifiedas an additional receptor for RANKL Similar to RANKLGR4 is expressed on osteoclasts but unlike RANK LGR4 is anegative regulator for osteoclast diï¬erentiation Therefore bothOPG and LGR4 are endogenous inhibitors of RANKLRANKsignaling A recent study has shown that RANKL reversesignaling from osteoclasts to osteoblasts couples bone resorptionto bone formation processes This is achieved through thesecretion of small extracellular vesicles from osteoclasts thatcontain RANK The authors showed that these RANK vesiclesbind membranebound RANKL on the osteoblasts and therebypromote bone formation by triggering RANKL reverse signalingvia activation of Runtrelated transcription factor Runx2Targeting RANKL reverse signaling represents a novel strategyto avoid the reduced bone production associated with inhibitionof osteoclastogenesis As RANKL is an important regulator of bone loss in bonemetastases associated with cancers such as multiple myelomaand in postmenopausal osteoporosis a specific fully human IgG2monoclonal RANKL antibody mAb has been developed whichneutralizes the activity of RANKL which has been designated asDenosumab The efficacy of Denosumab has been confirmed inmultiple clinical trials and Denosumab therapy is now approvedand widely used for the treatment of various boneassociateddiseases “RANKLRANKOPG IN THE IMMUNESYSTEMApart from bone homeostasis the RANKLRANKOPG axis isalso involved in various physiological immune processes RANKwas originally discovered on dendritic cells DCs and RANKLmediates the survival of DCs The interaction betweenactivated T cellderived RANKL and RANK expressed on DCsincreases the antigenpresenting capabilities of the latter thusaugmenting the number and cell cycle of antigenspecific Tcells as well as enhancing the immune response of memory Tcells Interestingly phenotyping of rankl and rankdeficient micerevealed a complete absence of peripheral lymph nodes but intactspleen and Peyer™s plaque structures “ Subsequent studieshave found that during embryogenesis RANKL is expressedby hematopoietic lymphoid tissue inducing LTi cells andmesenchymallymphoid tissue anizer LTo cells “RANKL has been demonstrated to stimulate lymphotoxin LTexpression and regulate LTi cell accumulation FurthermoreRANKL also triggers the proliferation of adult lymph nodestroma indicating that RANKL may directly activate LTo cells“ In the thymus the RANKLRANK pathway is criticalfor CD80 AIRE medullary thymic epithelial cell mTECmaturation involved in central immune tolerance RANKdeficient mice display mild autoimmunity at an advancedage RANKLRANK activation in lymphatic endothelialcells LECs is important for the tissueresident macrophagesnamely sinusoidal macrophage maturation not only duringembryogenesis but also after inflammationinduced loss of thesecells Moreover group innate lymphoid cells ILC3s inthe intestine use RANKLRANK interactions to control theirown abundance and intestinal homeostasis Genetic ablation ofRANKL specifically in IL3C cells leads to an increased number ofthese cells with enhanced levels of proinflammatory cytokinessuch as interleukin17A IL17A and IL22 during intestinalinfection Human patients carry RANK mutations and mice lackingRANKL or RANK exhibit a defect in B cell developmentresulting in a significant reduction in B cell numbers however these eï¬ects might be indirect because in themousetissuespecific deletion of Rank in B cells showedno diï¬erence in function nor development of B cells andblocking RANKRANKL with Denosumab does not apparentlyaï¬ect B cell physiology in osteoporosis patients In addition reports using Bcellspecific rankldeficient micehave shown that B cellderived RANKL increases osteoclastnumbers and bone loss brought on by estrogen deficiency Overexpression of RANKL in keratinocytes results in functionalalterations of epidermal dendritic cells and systemic increases inregulatory CD4CD25 T cells Tregs numbers Thereforeenvironmental stimuli can rewire the local and systemic immunesystems via RANKL The RANKLRANK system is alsoinvolved in M microfoldcell development a specific antigensampling cellular subtype found in the intestine as mesenchymalcells produce RANKL that can directly interact with intestinalepithelial cells to regulate M cell diï¬erentiation “Inhibition of mesenchymal RANKL impairs M celldependentantigen sampling and B celldendritic cell interaction in thesubepithelial dome SED resulting in decreased IgA productionand microbial diversity In addition B cells are absentin cryptopatches CPs and isolated lymphoid follicle ILFsformation was abrogated in rankl null mice Whether B cells or T cells are essential for bone loss isstill controversial Ovariectomy has been shown to enhance Tcelldependent TNFalpha production in a bone loss mousemodel because of the enhanced macrophage colonystimulatingfactor MCSF and RANKL In contrast anotherstudy suggested T cells are not involved in ovariectomyinducedFrontiers in Oncology wwwfrontiersinAugust Volume 0cMing et alRANKLRANK in Cancertrabecular bone loss Nevertheless it has been reportedin postmenopausal women that increased T cell activity andincreased RANKL production by T cells are associated withosteoporosis Furthermore studies in conditionalknockout mice to specifically eliminate RANKL in B cells or Tcells have shown that RANKL produced by B cells but not T cellsleads to bone loss by the induction of osteoclastogenesis Thelack of mature B cells does not prevent bone loss suggestingthat RANKL is derived from immature B cells Moreover it hasbeen reported that deletion of rankl in T cells does not change thenumber of T cells but results in impaired mature B cell numbersin the bone marrow suggesting that RANKL might promote Bcell maturation via paracrine signaling RANKLRANKLOPG IN MAMMARYGLAND PHYSIOLOGY AND BREASTCANCERBreast cancer is the most prevalent female malignancy Studies based on large populations have shown that womenwho receive estrogen plus progesterone hormone replacementtherapy called combined HRT are more vulnerable to breastcancer compared to women who receive estrogen only “furthermore progesterone levels have been demonstrated to bean independent risk factor for increased breast cancer incidence In rankl knockout mice our group was the first to reportthat during pregnancy RANKL deficiency results in a totalblock in the development oflobuloalveolar milksecretingstructures Whereas estrogen triggers the expansion ofthe mammary epithelium in puberty progesterone drives theproliferation of mammary epithelial cells in the estrous cycleand in pregnancy induces the growth and diï¬erentiation of themammary epithelium into ultimately milksecreting acini Figure Mechanistically progesterone induces progesteronereceptor PRpositive mammary epithelial cells to expressRANKL resulting in the proliferation of neighboring RANKmammary epithelial progenitor cells in an autocrine and alsoparacrine fashion “ Moreover RANKL can induce theproliferation of RANKpositive ductal epithelial cells through theFIGURE RANKRANKL pathway in mammary gland physiology and breast cancer A RANK is constitutively expressed on the membrane of luminal and basalepithelial cells including mammary stem cells MaSCs Stimulation with progesterone induces RANKL expression and secretion in progesterone receptor PRpositiveluminal epithelial cells RANKL binds in an autocrine fashion to RANK on luminal epithelial cells which stimulates further RANKL expression and in a paracrine fashionto RANK on basal epithelial cells resulting in enhanced RANK expression on basal mammary epithelial cells and the activation of the IKKαNFκB“cyclin D1 signalingaxis to induce a variety of physiological responses necessary for mammary gland development B Heterozygous BRCA1 mutationcarrying women canspontaneously lose the remaining wildtype BRCA1 gene from somatic mutation or epigenetic silencing Subsequently loss of BRCA1 protein can result in increasedgenomic instability DNA damage and genetic mutations eg TP53 Progesterone as well as synthetic progestins upregulate RANKL expression in PR luminalbreast epithelial cells which stimulates RANKmediated cell proliferation of adjacent progenitor cells as discussed in A Altogether the genotoxic stress and amplifiedproliferation cues culminate in uncontrolled proliferation and the development of breast cancerFrontiers in Oncology wwwfrontiersinAugust Volume 0cMing et alRANKLRANK in Cancerinduction of Rspondin Therefore RANK and RANKL linksex hormones to mammary progenitor cell proliferation duringthe estrous cycle and in pregnancy Figure Clinically an increase in serum progesterone and RANKLlevels is associated with an increase in breast cancer risk inpostmenopausal women Higher concentrations of solubleRANKL are positively correlated with an increased risk ofestrogen receptorpositive but not estrogen receptornegativebreast cancer indicating that the RANKRANKLOPG axis maybe involved in the tumorigenesis of ER breast cancer Indeed in a hormoneinduced spontaneous mouse breast cancermodel RANKL is critical for the development of sex hormonedriven breast cancer Deletion of RANK and Ikkα akey downstream regulator of the RANK signaling pathway inmammary epithelial cells also significantly delayed progestinMPA and DNAmutation DMBAinduced mammary tumorformation further indicating that the RANKRANKL pathwaydrives breast cancer Furthermore the selective inhibitionof RANKL by RANKFc not only attenuated breast tumorprogression in a hormone and carcinogendriven mouse breastcancer model but also decreased the progression of breast cancerin a transgenic spontaneous tumor model BRCA1 and BRCA2 mutations are the most prevalent geneticdrivers for hereditary breast cancer in humans Interestinglywomen with germline BRCA12 mutations usually exhibithigher progesterone and estrogen levels during the gestationalphase ofthe estrous cycle compared to women withoutthese mutations Inversely decreased serum OPG levelsare associated with increased breast cancer incidence Moreover high levels of RANK expression were observed inbreast cancer samples from premalignant lesions and patientswith BRCA1 mutations SNP data analysis from theCooperative Tumor GeneEnvironmental Research iCOGSincluding approximately BRCA1 and BRCA2mutation carriers identified SNPs which were significantlyassociated with breast cancer risk at the TNFRSF11A locusencoding RANK Altogether these human data stronglysupport the idea that the RANKLRANKOPG axis is intimatelyinvolved in the tumorigenesis of BRCA12 mutationdrivenbreast cancerSubsequent animal studies provided direct evidence thatRANKL and RANK are critically involved in the oncogenesis ofBRCA1 mutationdriven hereditary breast cancer Figure Genetically engineered mice carrying Brca1 and Tp53 mutationsshowed hyperproliferation and malignancy in their mammaryglands at months of age the inactivation of the RANKLRANKpathway in these mice largely prevented the occurrence ofmalignanttumors and resulted in significantly prolongedsurvival Additionally the pharmacological blockade of RANKLusing RANKFc completely abolished the development ofprecancerousin the Brca1Tp53 doublemutatedbreast cancer model Amplification of RANKexpressingmammary duct progenitor cells can be found in the nontumorbreast tissue of BRCA1 mutant carriers and these cells havesimilar molecular characteristics as basallike breast cancercells RANKL inhibition also significantly suppressedthe proliferation oftumor anoids derived from BRCA1mutant human breast biopsy specimens and RANKLRANKlesionspathway blockade strongly reduced tumorigenesis in patientderived xenograft PDX breasttumor mouse model Thus independent work among diï¬erent laboratories usingdiï¬erent mouse models as well as studies using humanbreast epithelial progenitor assays hasled to the sameconclusion RANKLRANK aï¬ect mammaryprogenitorcells and are critically involved in the BRCA1mutation drivenmammary tumorigenesisTherefore we and others have proposed that the monoclonalantibody Denosumab which specifically inhibits RANKRANKLinteractions could potentially be used for the prophylactictreatment of breast cancer in BRCA12 carriers Indeed weposit that healthy women with BRCA1 mutation will benefit notexcluding an eï¬ect on other TNBCs In a pilot clinical studytermed BRCAD the proliferation marker Ki67 was significantlydownregulated in the breast biopsy of BRCA1 mutation carrierswho received shortterm treatment with Denosumab suggestingthat RANKL inhibition may be a feasible method for the chemoprevention of breast cancer in women with BRCA1 mutationsThis study requires additional patient data which is currentlyongoing Another clinical study DBEYOND which aimedto investigate whether neoadjuvant RANKL inhibition therapycan reduce tumor proliferation in premenopausal early breastcancer patients found no significant change in Ki67positivetumor cells in the breast cancer tissues treated with Denosumabbut the density of tumorinfiltrating lymphocytes TILs wasincreased in the stroma and tumor tissues upon Denosumabtreatment In addition to the now experimentally well validated role ofRANKLRANKOPG in the sex hormone and BRCA1 mutationdriven mammary cancer tumorigenesis it has also been reportedthat this pathway can induce epithelialmesenchymal transitionEMT in breast cancer cells as well as in prostate andendometrial cancers “ suggesting that RANKLRANKsupports tumorigenesis in various epithelial cancers Moreoverour group has recently reported on the role of RANKL andRANK in lung cancer We demonstrated that the inactivation ofrank in lung epithelial cells disrupts mitochondrial bioenergeticsand significantly reduceslung cancer development bothculminating in increased survival This genetic modeling inthe mouse supports findings in human clinical trials in whichRANK inhibition with the monoclonal antibody Denosumabresulted in prolonged survival especially in patients withnonsmall celllung cancer NSCLC adenocarcinomas andsquamous tumors Notably this Denosumabdependent survivaladvantage occurred in lung cancer patients irrespective of visceralmetastasis hinting that the underlying eï¬ects of RANKLRANKblockade in addition to those targeting the bone are involved Epidemiological reports have also uncovered genderdiï¬erences particularly in lung cancer with respect to etiologyprogression and treatment response believed to be due tosexrelated hormonal factors “ though the underlyingmolecular mechanisms are poorly understood We have recentlyshown in our experimental lung cancer model that by ablatingthe sex hormones in female mice we could eï¬ectively eliminatethe survival advantages brought about by loss of rank in the lungtumors Furthermore synthetic progesterone MPAdependentenhanced lung cancer initiation required RANK expressionFrontiers in Oncology wwwfrontiersinAugust Volume 0cMing et alRANKLRANK in CancerTogether these data suggest that the sex hormone regulation ofRANKLRANK could also explain the gender diï¬erences seen inhuman lung cancerRANKRANKL AS REGULATORS OFMETASTASISStudies have now shown that the RANKLRANKOPG axis playsa role in the progression of malignant tumors by promotingtumor cell migration stimulating tumor neovascularizationand promoting distant metastasis of tumor cells Disseminated tumor cells are responsible for the earlymetastasis of tumors which frequently can be detected inthe bone marrow of patients with malignant tumors Thisœmicrometastases niche forms a favorable microenvironmentfor the development of metastatic spread protecting cancer cellsfrom various antitumor treatments and modulating anticancerimmune responses thereby allowing the tumor cells to escapeimmune surveillance The tumor microenvironment is acomplex milieu composed of distinct factors such as cytokinesextracellular matrix components and various cell types suchas fibroblasts endothelial cells and immune cells all ofwhich participate in cancer development progression andmetastasis In bone tissue the tumor microenvironmentincludes immune and tumor cells as well as osteoblasts andosteoclasts all of which participate in a œvicious cycle thataccelerates osteolysis and cancer cell proliferation through inpart the RANKRANKLOPG axis For instance cancercells can increase the expression of RANKL in osteoclastsby secreting parathyroid hormonerelated peptide PTHrP Tumor cells can also directly express RANKLand secrete cytokines such as interleukin IL1α TNFα macrophage colonystimulating factor MCSF orprostaglandin E2 PGE2 all of which promote osteoclastdiï¬erentiation and survival resulting in local osteolysis whichsupports metastatic growth “ Subsequently growthfactors released by the bone matrix such as insulinlike growthfactors IGFs fibroblast growth factor FGFs plateletderivedgrowth factor PDGF or bone morphogenetic proteins BMPspromote cancer cell proliferation “ In addition tocytotoxic drugs and endocrine disruptive drugstherapiestargeting the RANKRANKLOPG axis exhibit direct andorindirect antitumor eï¬ects by blocking the vicious cycle betweenbone and cancer cells “In a murine model of melanoma metastasis it was foundthat for malignant tumors with RANK expression RANKLproduced by osteoblasts and bone marrow stromal cells couldact as a chemical attractant and promote the migration andmetastasis of malignant tumors to these sites Similareï¬ects were also found in malignant tumors such as breastcancer “ prostate cancer “ and lung cancer The activation of phospholipase C PLC proteinkinase C PKC ERK and phosphatidylinositol3OH kinasePI3K pathways were involved in RANKinduced tumor cellmigration “ RANK engagement by RANKL inducestrimerization of the RANK receptor which then stimulates therecruitment and activation of the adapter protein TRAF6 viaTRAF6binding sites in the Cterminus of RANK™s cytoplasmictail TRAF6 in turn complexes with many other downstreamadapters and kinases to activate the aforementioned pathwaysMoreoverthe RANKLRANK pathway was also shown topromote the formation of new blood vessels and regulate thetumor microenvironment at the primary tumor site to promotethe migration of tumor cells into the bloodstream and formetastasis to distant ans “In breast cancer RANKL is also produced by Foxp3expressing Tregs and tumorassociated macrophages TAMsthat can aï¬ect tumor growth tumor cell dissemination andmetastasis RANKL expression on tumorinfiltratingregulatory T cells may also be involved in cancer metastasis TAMs are either M1 or M2 macrophages with M1 being antitumor and M2 TAMs promoting tumorigenesis ImportantlyM2 macrophages express RANK and are attracted by RANKLproduced by the tumor microenvironment The RANKLRANKpathway in M2 macrophages can regulate the production ofchemokines and promote the proliferation of Treg lymphocyteswhich supports the immunosuppressive milieu within the tumormicroenvironment Recently it has been reported that estrogenrelatedreceptoralpha ERRα an important factor of cancer cell invasivenesspromotes breast cancer cell dissemination from primarymammary tumors to the bone Intriguingly RANK hasbeen shown to be a target for ERRα Furthermore the metaexpression analysis of breast cancer patients has uncovereda positive association between metastases and ERRαRANKexpression as well as a positive correlation between ERRαand BRCA1 mutation carriers revealing a novel pathwaywhereby ERRα in primary breast cancer could promote earlydissemination of cancer cells to bone Moreover it wasrecently shown that RANKL serum levels are significantlyincreased in breast cancer patients who developed bonemetastases p and patients within the highest quartileof RANKL had a significantly increased risk of developing bonemetastases compared to those in the lowest HR 95CI“ p This study further suggests a role ofRANKL in breast cancer metastasis TARGETING RANKLRANK IN HUMANCANCERIn light of the diï¬erent roles of the RANKLRANK pathwayin bone metabolism and immune system functions therapytargeting this axis may not only control primary tumordevelopment such as in the case of breast cancer and reducebone metastasis which has been demonstrated in clinical trials but also exert a direct antitumor eï¬ect via regulatinglocal tumorassociated immune responses as observed in studiesusing the monoclonal RANKL antibody inhibitor Denosumab In randomized clinical trials Denosumab has shown rapideï¬ectiveness by directly impairing osteoclast activity andinducing osteoclast apoptosis Moreover Denosumabwassignificantly more eï¬ective in reducing urinary Nterminal peptides a biochemical marker for bone turnoverFrontiers in Oncology wwwfrontiersinAugust Volume 0cMing et alRANKLRANK in Cancerand more eï¬ective in delaying skeletalrelated events SREssuch as pathologic fractures spinal cord compression andhypercalcemia which greatly aï¬ect quality of life in patients withbreast cancer and castrationresistant prostate cancer CRPCbone metastases However the eï¬ect of Denosumab to delaySREs in patients with NSCLC and multiple myeloma MMpatients with bone metastases is comparable to bisphosphonatedrugs “ Moreover the benefit of Denosumab andbisphosphonates is not only restricted to osteolytic cancers suchas breast myeloma and NSCLC but also evident in osteoblasticcancers Recently it was demonstrated in osteoblastic cancerssuch as prostate cancer that Denosumab or bisphosphonate canaï¬ect the osteoclastosteoblast balance in the œvicious cycle ofbone destruction induced by metastasized cancer cells which highlights the potential rationale in treating osteoblasticcancer patients with Denosumab or bisphosphonatesIn a randomized phase III clinical trial comparing Denosumaband bisphosphonate zoledronic acid ZA in patients with solidtumors breast cancer prostate cancer multiple myeloma andbone metastases the results showed that Denosumab was similarto ZA in preventing or delaying the onset of primary SREs However in nonsmallcell lung carcinoma NSCLCn treatment with Denosumab showed a significantimprovement in overall survival In these patients nostatistically significant SRE delay was observed in Denosumabtreated patients suggesting that this survival advantage maybe independent of the bone system The result of arandomized phase III trial of multiple myeloma MM patientsn also demonstrated the eï¬ectivity of Denosumab toreduce the occurrence of primary SRE events moreover the useof Denosumab significantly improved progressionfree survivalPFS Whether this survival benefit is due to the decreasein the incidence of bone metastasis or whether Denosumab hasother antitumor eï¬ects requires further researchIn the randomized placebocontrolled phase III ABCSG18trial which enrolled postmenopausal female patients withearly hormone receptorpositive breast cancer the first clinicalfracture of the Denosumabtreated group was compared with theplacebo group and a significant protection of bone breaks wasdemonstrated hazard ratio [HR] · [ CI ·“·] p A median followup of months showeda significant improvement in the diseasefree survival DFS inthe Denosumabtreated group HR CI “Cox p These data suggest that adjuvant Denosumabcan significantly improve the DFS rate of HR postmenopausalbreast cancer patients However in another randomizedphase III clinical trial of breast cancer DCARE recent reportshave shown that adjuvant Denosumab does not reduce therisk of breast cancer recurrence or death in earlystage breastcancer patients receiving standard adjuvant therapy Theseinconsistencies which could be explained by diï¬erent cohortsfor patient stratifications eg more advanced early cases ofbreast cancer were included in the DCARE trials as comparedto the ABCSG18 study need to be further evaluated with largercohorts of patients and multiplecenter analysis Importantlya recent followup study of the ABCSG18 trial confirmed thethat blocking RANKL in an adjuvant breast cancer therapysetting not only markedly reduces the risk of breaking bones butalso significantly reduces the reoccurrence of the breast tumors It should be also noted that although there was nodiï¬erence in bonemetastasesfreesurvival in the DCARE trialDenosumab treatment significantly reduced the time to bonemetastasis at the site of first occurrence DENOSUMAB AS A NOVEL CANCERIMMUNOTHERAPYThe field of cancer immunotherapy has paved the way for a newparadigm to combat cancer by coaxing the body™s own immunesystem to seek out specifically target and destroy cancer cellsAmong the various approaches immunecheckpoint inhibitorsthattarget CTLA4 as Answer:

Thyroid_Cancer

Hepatitis B virus HBV infection has been associated with the risk andprognosis of many malignancies Nevertheless the association between HBV and theprognosis of breast cancer is unclear The objectives of this study were to investigate theprognostic role of hepatitis B surface antigen HBsAg and to integrate HBsAg to establishnomograms for better prognostic prediction of very young patients with breast cancerMethods This analysis was performed retrospectively in a cohort of consecutivevery young ‰ at diagnosis patients who received curative resection for breastcancer The significance of HBsAg in the prognosis of these patients was investigatedUnivariate and multivariate analyses were used to identify independent variables fordiseasefree survival DFS and overall survival OS Nomograms were built based onthose identified variablesResults Overall of the patients were seropositive for HBsAgThe median followup was CI “ months forthe entirepopulation The HBsAgpositive cohort had significantly inferior DFS HR CI “ P and OS HR CI “ P as compared with the HBsAgnegative cohort The rates of 10year DFS andOS were and in the HBsAgpositive group and and in the HBsAgnegative group respectively In multivariable analysis HBsAg statuswas identified as an independent significant unfavorable prognostic factor for DFSP and OS P in very young patients with breast cancer Nomogramswere established and displayed good calibration and acceptable discrimination TheCindex values for DFS and OS were CI “ and CI “ respectively Based on the total prognostic scores TPSof the nomograms different prognosis groups were identified for DFS and OSEdited byImtiaz Ahmad SiddiquiUniversity of Colorado AnschutzMedical Campus United StatesReviewed byAbhinit NagarUMass Memorial Medical CenterUnited StatesNidhi JainBeckman Coulter IndiaCorrespondenceLu YangyanglusysucccnWeiDong Weiweiwdsysucccn These authors have contributedequally to this workSpecialty sectionThis was submitted toCancer Epidemiology and Preventiona section of the journalFrontiers in OncologyReceived March Accepted July Published August CitationLi N Zhong QQ Yang XRWang QC Zhang DT Zheng SYang L and Wei WD Prognostic Value of Hepatitis B VirusInfection in Very Young Patients WithCuratively Resected Breast CancerAnalyses From an Endemic Area inChina Front Oncol 103389fonc202001403Frontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast CancerConclusions HBsAg is an independent unfavorable prognostic factor for DFS andOS in very young patients with curatively resected breast cancer and nomogramsintegrating HBsAg provide individual survival prediction to benefit prognosis evaluationand individualized therapyKeywords HBV young breast cancer prognosis nomogram survivalINTRODUCTIONGlobally breast cancer is the most common cancer and theleading cause of cancer death for women accounting for of total cancer cases and of total cancer deaths Breast cancer has also been the top one malignancy in termsof incidence in Chinese women constituting of newlydiagnosed cases and of all deaths from breast cancer in theworld Although breast cancer occurs at a lower incidence inChinese women than in western women this disease occurs at ayounger age in China than in highincome countries and China™scontribution to global breast cancer rate is increasing rapidly The disparities between young and old breast cancer include ahigher mortality rate higher risk of recurrence poorer treatmentresponse and more aggressive phenotypes “ Thereforeunderstanding the etiology and identifying novel prognosticfactors are essential for early diagnosis prognosis evaluationearly intervention and personalized therapy in young patientswith breast cancerHepatitis B virus HBV infection is a serious public healthdilemma with ˆ¼ million chronic carriers worldwide China account for about a third of infectionassociated cancerglobally driven by high prevalence of HBV and H pylori infection Although China has made tremendous eï¬orts in controllingHBV over the past years and the prevalence of HBV ininfants and children has remarkably declined the hepatitisB surface antigen HBsAg prevalence is still high in Chineseadults ranging from to “ HBV is the leading causeof hepatocellular carcinoma and cholangiocarcinoma Inaddition there is also accumulating evidence that HBV infectionis associated with many extrahepatic malignancies includingnonHodgkin™s lymphoma pancreatic cancer gastriccancer nasopharyngeal carcinoma lung cancer esophageal cancer and ovarian cancer Thus it seemsreasonable that HBV is an important factor in the developmentof extrahepatic malignancies in endemic areasDespite the facts that HBsAg status is one of the routineexaminations in patients with operable breast cancer and severalstudies have showed that HBV is not associated with therisk of breast cancer the impact of HBV on theclinicopathological characteristics and prognosis of very youngpatients with breast cancer remains to be determined Giventhat both early breast cancer and HBV are endemic in China itis possible that HBV infection is associated with the prognosisof early breast cancer even though the precise mechanismsare yet to be determined It is crucial to address this issuesince HBV has been reported to be found in breast cancertissue We therefore performed this study to investigate theHBsAg prevalence in very young breast cancer and the impactof HBsAg on the survival of these patients and to establishnomograms to better predict prognosis for very young patientswith breast cancerMATERIALS AND METHODSPatient SelectionA retrospective review was conducted in a cohort of consecutive breast tumor women who were aged ‰ years oldand received curative resection for breast cancer at Sun Yatsen University Cancer Center between May and July This study was conducted according to the ethicalstandards of the Declaration of Helsinki Institutional ReviewBoard approval was obtained from the Medical Ethics Committeeof this cancer center All patients were restaged by the eighthinternational classification system for breast cancer Dueto the retrospective nature of this studyinformed consentwas waivedInformation was collected from electronic patient recordsand survival data were obtained from the followup registryofthis center The information collected included HBsAgstatus laterality type of breast surgery type of axillary surgeryhistological type tumor grade tumornodemetastasis TNMstage dates of surgeryrelapsedeath status of estrogen receptorER progesterone receptor PR human epidermal growthfactor receptor HER2 and Ki67 Breast cancers wereclassified as luminal Alike ER PR‰¥ HER2“ andKi6715luminal Blike ER andor PR HER2“HER2enriched ER“ PR“ HER2 or triplenegative ER“PR“ HER2“ subtypesPotentially eligible patients had to have curatively resectedbreast cancer without previous therapy other than neoadjuvanttherapy be aged years old or below and have definiteinformation of HBsAg The main exclusion criteria includedbenign tumor not having surgery having incomplete resectionprevious malignant disease hepatitis viral infections other thanHBV men patients and insufficient data of survival or HBsAgStatistical AnalysisThe main objectives of this study were to compare diseasefreesurvival DFS and overall survival OS between HBsAgpositivepatients and HBsAgnegative patients DFS was defined as theinterval from the date of being diagnosed to the date of diseaserecurrencemetastasis or death from any cause OS was defined asthe interval from the date of being diagnosed to the date of deathfrom any cause Median followup was estimated by KaplanMeier analysis with reversed meaning of status indicator Frontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast Cancerforindependent variablesDFS and OS were estimated by the Kaplan“Meier methodand diï¬erences were compared by the logrank test Univariateand multivariate analyses with a Cox proportional hazardsmodel were used to testforDFS and OS Covariates included laterality left vs righttype of surgery breastconserving surgery vs mastectomytype of axillary surgery sentinellymph node dissection vsaxillary lymph node dissection histological type ductal vsotherstumor grade grade III vs grade III T stageT34 vs T12 N stage N23 vs N1 HER2statustriplenegativeHER2enriched vs luminal All variables reaching asignificance of in univariate analyses were included inmultivariate analysispositive vs negative molecularsubtypesThe nomograms for predicting and 10year DFSand OS were formulated based on the results of multivariateanalysis by the œrms package of R The discriminationofthe nomogram models was estimated by the Harrell™sconcordance index Cindex The value of the Cindex rangesfrom to with implying a random chance and indicating a perfect prediction Calibration curves of thenomogram models for DFS OS were plotted to assess thepredictive value of the model In addition patients weredivided into three diï¬erent risk groups highintermediatelow according to total prognostic scores TPS The totalprognostic scores of patients were transformed into categoricalvariables based on cutoï¬ points which were determined by theminimum Pvalue from logrank × statistics with the Xtileprogram Pearson™s chisquare test was used to compare categoricaldata All Pvalues were twosided and P was consideredstatistically significant Statistical analyses were performed by theSPSS software SPSS Inc version Chicago IL USA and Rfor Windows version httpwwwrprojectData AvailabilityThe authenticity of this has been validated by uploadingthe key raw data onto the Research Data Deposit public platformwwwresearchdatacn with the approval RDD numberas RDDA2020001410 The data that support the findings ofthe study are available from the corresponding authors uponreasonable requestRESULTSPatient CharacteristicsA total of very young ‰ at diagnosis breasttumor patients were screened of whom were excludedbecause of benign tumor n not having surgeryn not having R0 resection n or unknownHBsAg status n With further exclusions forinsufficientfollowup data a total of patients withcurative resection for breast cancer and aged years orbelow were included in this study Figure The patients™FIGURE The process of patient selectionFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast CancerTABLE Patient characteristics by HBsAg statusCharacteristicsHBsAgpositiveN No HBsAgnegativeN No Pvalue FIGURE Number of recurrences by year of entire patientsLateralityLeftRightBilateralType of breast surgeryMastectomyBreastconserving surgeryType of axillary surgeryALNDSLNDHistological typeDuctalInvasive lobularOtherTumor gradeIIIIIIUnknownT StageT1T2T3T4N StageN0N1N2N3HER2 statusPositiveNegativeUnknownMolecular subtypesLuminal ALuminal BHER2enrichedTriple negativeUnknown HBV Hepatitis B Virus ALND axillary lymph node dissection SLND sentinelnode dissectionHER2 human epidermal growth factor receptor2lymphcharacteristics are shown in Table Overall ofthe patients were seropositive for HBsAg HBsAgpositive group and HBsAgnegative group were wellmatchedfor basic characteristics including laterality type of breast andaxillary surgery histological type tumor grade T stage Nstage and molecular subtypes About in patients receivedmastectomy and most patients received axillary lymph nodedissection ALNDAssociations Between the Status of HBsAgand SurvivalThe median followup was CI “ months forthe entire population By the time of analysis December instances of disease recurrence had occurred Thenumber of recurrences in each year of the followup is shown inFigure Of note although HBsAgpositive patients had a higherfrequency of extrahepatic metastasis vs P thanHBsAgnegative patients they had a comparable frequency ofliver metastasis vs P when compared withHBsAgnegative patientsDFS was significantly shorter among those who were HBsAgpositive than among those who were HBsAgnegative HR CI “ P The rates of 10yearDFS were in the HBsAgpositive group and inthe HBsAgnegative group respectively Figure 3A A totalof death events had occurred by the data cutoï¬ HBsAgpositive group had significantly inferior OS compared withHBsAgnegative group HR CI “ P with a 10year OS of and respectivelyFigure 3B The association of HBsAg status and survival ineach molecular subtype was further analyzed As expectedDFS and OS were significantly longer among those in theluminal A subgroup and the HER2enriched and triplenegativegroups had significantly shorter DFS and OS Figures 4ABNotably HBsAgpositive status was associated with shorterDFS P and OS P in the luminalB cohort HBsAgpositive status was also associated with aslightly shorter DFS in the triplenegative cohort and shorterOS in the HER2enriched cohort but statistical significancewas not reached Supplementary Figures There was nosignificant diï¬erence in DFS between HBsAgpositive patientsand HBsAgnegative patients in luminal A and HER2enrichedcohorts and in OS in luminal A and triplenegative cohortsSupplementary Figures In the univariate analysis type of breast surgery tumor gradeT stage N stage molecular subtype and HBsAg status wereFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast Cancerindividual patient Figures 6AB The model™s explanatorycovariables consisted of HBsAg status T stage N stage andmolecular subtype Patients with higher scores correspondedto inferior survival The scatter plots for the TPS of DFSand OS and percentage of patient number were presentedin Figures 6CD The Cindex values for DFS and OS were CI “ and CI “ respectively The calibration curves for the probabilityof DFS and OS at or year presented an optimalagreement between the prediction by nomogram and actualobservation Supplementary Figure Next we divided thepatients into the following groups based on the TPS ofthe nomogram modelfor DFS using the Xtile programlowrisk group TPS “ patients intermediateriskgroup TPS “ patients and highrisk group TPS patients The 10year DFS for lowrisk groupintermediaterisk group and highrisk group were and respectively Survival analyses for DFS demonstratedsignificant discrimination between these three groups P Figure 7A Same procedures were performed for OS in theentire population and patients were divided into the following groups based on the TPS of the nomogram model for OS withthe Xtile program lowrisk group TPS “ patientsintermediaterisk group TPS “ patients and highrisk group TPS patients OS diï¬erences were alsoobserved among three subgroups with a 10year OS of and for lowrisk intermediaterisk and highrisk groupsP Figure 7BDISCUSSIONIn this study we investigated the association of HBsAg statusand very young breast cancer and to our knowledge reportfor the firsttime that HBsAgpositive status is associatedwith inferior DFS and OS from a population with a highprevalence of both HBV infection and young breast canceridentified as a significant unfavorableHBsAg status wasprognostic predictor for DFS and OSindependent of anyother clinicopathological features of breast cancer includingT stage N stage and molecular subtype We also integratedHBsAg to build nomograms to better predict prognosis foryoung patients with breast cancer The results of our studydemonstrated that the prevalence of HBsAg in young patientswith breast cancer in southern China was which wasin accordance with the “ reported in the populationof this endemic area This result suggests that unlikecervical cancer young breast cancer is not correlatedwith an increased prevalence of HBV infection Indeed breastcancer patients with HBsAg did not demonstrate a diï¬erentpattern of characteristics It is noteworthy that in our studyHBsAg did not increase the rate of liver metastases for veryyoung patients with breast cancer This results are comparableto those reported in previous studies for esophageal cancerand colorectal cancer which suggested that HBVinfection is associated with decreased risk of liver metastasis inthese malignanciesFIGURE Kaplan“Meier curves for A diseasefree survival and B overallsurvival stratified by HBsAg status in very young patients with breast canceridentified as significant prognostic factors for DFS Figure 5AWhen those variables were further analyzed in the multivariateanalysis we found that T stage P N stage P molecular subtype P and HBsAg status P remained statistically significant indicating that theyare significant independent predictors for DFS Figure 5ABy the same methods for OSthe results showed that Tstage P N stage P molecular subtype and HBsAg status P were independentprognostic factors for OS Figure 5B HBsAgpositive status isan independent negative prognostic factor for survival in veryyoung breast cancerPrognostic Nomograms For Very YoungBreast Cancer PatientsTo better assess the DFS and OS of very young breastcancer patients prognostic nomograms for DFS and OS wereestablished respectively All the independent predictors of DFSand OS in the multivariate analysis were integrated into thenomogram models and and 10year survivals weregraphically computed according to the characteristics of anFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast CancerFIGURE Kaplan“Meier curves for A diseasefree survival and B overall survival stratified by molecular subtype in very young patients with breast cancerOur study shows that HBsAgpositive status is associatedwith inferior DFS and OS in young patients with curativelyresected breast cancer decreasing the 10year DFS and OS by and respectively The association between HBsAgpositive status and poor prognosis is in keeping with thatdemonstrated in nasopharyngeal carcinoma lung cancer and ovarian cancer However some studies regardingother cancers indicate that HBsAgpositive cancer patientshad a favorable survival as compared with HBsAgnegativepatients This discrepancy can be partly explainedby the diversity and heterogeneity of diï¬erent malignanciesThe genetic or biological mechanisms underlying the inferiorFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast CancerFIGURE Univariate and multivariate analysis for diseasefree survival A and overall survival B for very young patients with breast cancerprognosis remain to be elucidated butthis may largelyrelate to the presence of hepatitis B Xinteracting proteinHBXIP which has been welldocumented to function asan oncoprotein in breast cancer HBXIP can act as atransactivator by activating certain genes including cMyc E2F1STAT4 and Sp1 to play a crucial role in the progression ofbreast cancer HBXIP is associated with controlling cellapoptosis and promoting cell proliferation by mTOC1 activation HBXIP can also act as a modulation factor of cellularoxidative stress by competitively binding KEAP1 to enhancethe progression of breast cancer Previously studies showedthat HBV is not associated with risk of breast cancer These results combined with the data of our study suggestthat HBV is not a risk factor but a prognostic factor forbreast cancerAnother reason for this might relate to the HBV reactivationin HBsAgpositive patients with breast cancer who werereceiving chemotherapy HBV reactivation occurs frequentlyin breast cancer patients who are HBV carriers while receivingcytotoxic chemotherapy HBV reactivation can result inliver failure and interruption of the chemotherapy scheduleOther potential mechanisms underlyingassociationtheFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast CancerFIGURE AB Nomograms predicting and 10year A diseasefree survival and B overall survival for very young patients with breast cancer CD Thescatter plots of percentage of patient number and groups of C total prognostic score for DFS and D total prognostic score for OSbetween the HBsAg and patient survival include the immunesuppression Chronic HBV infection is characterized by thefailure to elicit an eï¬ective adaptive immune response andthe immune modulation of key innate immune response Chronic HBV infection can lead to immune anergy andimpair the function of the immune system which has longbeen deemed to protect the host against the developmentof nonviral cancerstogether couldpartially explain the positive association between HBsAgpositive status and the poor prognosis in young patients withbreast cancer These reasonsInthisstudy wecharacteristicscombined HBsAgstatus withclinicopathologicaleï¬ectiveprognostic nomogram models of DFS and OS for very youngpatients with breast cancer Both nomograms showed goodcalibration and acceptable discrimination These nomogrammodels can be used for prognosis evaluation at diagnosis forvery young patients with breast cancer and may benefit patientestablishtocounseling and personalized therapy for these patients Weadopted Xtile program to divide these patients into three riskgroups based on TPS from nomograms for DFS and OS Thesurvival curves for DFS and OS separated very well Thusspecial attention should be paid to and active surveillanceshould be conducted over patients with high risk group for DFSand OSThis study nevertheless has certain limitations that shouldbe noted First this study was retrospective in nature andwe cannot rule outthe impact of selection bias Secondthe sample size was relatively small and the sample sizesof the two cohorts were unequal as only patients wereHBsAgpositive The small sample size may be insufficientto allow us to perform subgroup analysis by each molecularsubtype Anotherthat Cantonese constitutemost of our study population The monotonicity ofthestudy population confines the universality of our resultsFurthermore the information was insufficient to perform otherlimitation isFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast CancerFIGURE Kaplan“Meier curves for A diseasefree survival and B overall survival stratified by risk groups based on total prognostic scores fromnomogram modelsanalysis such as that of hepatic function and HBV DNAcopy number Nevertheless the results of our study providewhat to our knowledge is the first evidence of the impactof HBsAg on the prognosis of very young patients withbreast cancerIn conclusion we have demonstrated in a retrospectivestudy that HBsAg is an independent unfavorable prognosticfactor for patients with very young breast cancer Furtherprospective studies involving varied ethnic populations arewarranted to confirm the prognostic value of HBsAg status invery young breast cancer and simultaneously other potentialclinicopathologic factors for breast cancer and HBV infection arerequired to be taken into account The mechanisms of the impactof HBV infection on the progression of breast cancer also need tobe elucidatedDATA AVAILABILITY STATEMENTThe datasets generated for this study are available on request tothe corresponding authorETHICS STATEMENTThe studiesinvolving human participants were reviewedand approved by the Medical Ethics Committee of SunYatsen University Cancer Center WritteninformedFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast Cancerconsentin accordance with the nationalinstitutional requirementsfor participation was not required for this studylegislation and theAUTHOR CONTRIBUTIONSNL QQZ LY and WDW designed the study NL QQZXRY QCW DTZ and SZ collected the data NL QQZ LYand WDW interpreted and analyzed the data All authors wereinvolved in writing the and approved the final versionREFERENCES Bray F Ferlay J Soerjomataram I Siegel RL Torre LA Jemal A Globalcancer statistics GLOBOCAN estimates of incidence and mortalityworldwide for cancers in countries CA Cancer J Clin “ 103322caac21492 Fan L StrasserWeippl K LiJJ St LouisJ Finkelstein DM Yu 15e279“KD et al Breast cancer 101016S1470204513705679in China Lancet Oncol Colleoni M Rotmensz N Robertson C Orlando L Viale G Renneet al 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lymphoma a systematic review and metaanalysisInternal Med J “ 101111j14455994200902060x Luo G Hao NB Hu CJ Yong X Lu MH Cheng BJ et al HBV infectionincreases the risk of pancreatic cancer a metaanalysis Cancer Causes Control “ 101007s1055201201442FUNDINGThis work was supported by the National Natural ScienceFoundation of China No SUPPLEMENTARY MATERIALThe Supplementary Materialonline202001403fullsupplementarymaterialfor this can be foundhttpswwwfrontiersins103389foncat Wei XL Qiu MZ Jin Y Huang YX Wang RY Chen WW et al Hepatitis Bvirus infection is associated with gastric cancer in China an endemic area ofboth diseases Br J Cancer “ 101038bjc2014406 Ye YF Xiang YQ Fang F Gao R Zhang LF Xie SHHepatitis B virusin southern China Cancer Epidemiol Biomarkers Prevent“ 10115810559965EPI150344et alinfection and risk of nasopharyngeal carcinoma Peng JW Liu DY Lin GN Xiao JJ Xia ZJ Hepatitis B virusinfection is associated with poor prognosis in patients with advancednon small cell“ 107314APJCP201516135285lung cancer Asian Pacific J Cancer Prevent Zou J Chen J Xie X Liu Z Cai X Liu Q et al Hepatitis B virus infectionis a prognostic biomarker for better survival in operable esophageal canceranalysis of patients from an endemic area in China Cancer EpidemiolBiomarkers Prevent “ 10115810559965EPI181095 Wong L Cheung TH Yim SF Lao TT Prevalence and impact of hepatitis Bvirus infection in ovarian cancer patients in an endemic areaA retrospectivecohort study J Viral Hepat “ 101111jvh13250 Song C Lv J Liu Y Chen JG Ge Z Zhu J et al Associations between hepatitisB virus infection and risk of all cancer types JAMA Netw Open 2e195718 101001jamanetworkopen20195718 Su FH Chang SN Chen PC Sung FC Su CT Yeh CC Associationrisk abetween chronic viral hepatitisinfection and breast cancernationwide populationbased casecontrol study BMC Cancer Qin B Zhao K Wei J Wang X Xu M Lang J et al Novel evidence indicatesthe presence and replication of hepatitis B virus in breast cancer tissue OncolRep “ 103892or20197393 Te HS Jensen DM Epidemiology of hepatitis B and C viruses a globaloverview Clinics Liver Dis “ vii 101016jcld200911009 Siu SS Cheung TH Chan PK Lin CK Lo KW Patients with malignant or premalignant cervical lesion have increased risk of becoming hepatitis B carrierJ Exp Clin Cancer Res “in patients with colorectal Zhao Y Lin J Peng J Deng Y Zhao R Sui Q et al Hepatitis B virus infectionpredicts better survivalliveronly metastasesundergoing liver resection J Cancer “ 107150jca24544 Liu X Li X Jiang N Lei Y Tang LL Chen L et al Prognostic value ofchronic hepatitis B virus infection in patients with nasopharyngeal carcinomaanalysis of patients from an endemic area in China Cancer “ 101002cncr28377 Zhao Y Li H Zhang Y Li L Fang R Li Y et al Oncoprotein HBXIPmodulates abnormal lipid metabolism and growth of breast cancer cells byactivating the LXRsSREBP1cFAS signaling cascade Cancer Res “ 10115800085472CAN151734 Zhang Y Zhao Y Li H Li Y Cai X Shen Y et al The nuclear import ofoncoprotein hepatitis B Xinteracting protein depends on interacting with cFos and phosphorylation of both proteins in breast cancer cells J Biol Chem “ 101074jbcM113458638 BarPeled L Schweitzer LD Zoncu R Sabatini DM Ragulator is a GEFfor the rag GTPases that signal amino acid levels to mTORC1 Cell “ 101016jcell201207032 Zhou XL Zhu CY Wu ZG Guo X Zou W The oncoproteinHBXIP competitively binds KEAP1 to activate NRF2 and enhanceFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast Cancercancerbreast“ 101038s4138801906985growthcelland metastasis Oncogene Liu Z Jiang L Liang G Song E Jiang W Zheng Y et al Hepatitis B virusreactivation in breast cancer patients undergoing chemotherapy a reviewand metaanalysis of prophylaxis management J Viral Hepat “ 101111jvh12672 Yuen MF Chen DS Dusheiko GM Janssen HLA Lau DTY LocarniniSA et al Hepatitis B virus infection Nat Rev Dis Primers 101038nrdp201835 Dunn GP Old LJ Schreiber RD Theimmunobiology ofimmunosurveillance“ 101016jimmuni200407017immunoeditingandImmunitycancer Giuliano AE Edge SB Hortobagyi GN Eighth edition ofthe AJCCcancer staging manual breast cancer Ann Surg Oncol “ 101245s1043401864866 Schemper MSmith TL A note on quantifyingstudies 101016019724569600075Xfailuretime Control ClinofTrialsfollowup in“ Vickers AJ Elkin EB Decision curve analysis a novel method for “evaluating prediction models Med Decision Making 1011770272989X06295361 Camp RL DolledFilhart M Rimm DL Xtile a new bioinformatics toolfor biomarker assessment and outcomebased cutpoint optimization ClinCancer Res “ 10115810780432CCR040713Conflict of Interest The authors declare that the research was conducted in theabsence of any commercial or financial relationships that could be construed as apotential conflict of interestCopyright Li Zhong Yang Wang Zhang Zheng Yang and Wei This is anopenaccess distributed under the terms of the Creative Commons AttributionLicense CC BY The use distribution or reproduction in other forums is permittedprovided the original authors and the copyright owners are credited and that theoriginal publication in this journal is cited in accordance with accepted academicpractice No use distribution or reproduction is permitted which does not complywith these termsFrontiers in Oncology wwwfrontiersinAugust Volume 0c'

cancer209

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: Hepatitis B virus HBV infection has been associated with the risk andprognosis of many malignancies Nevertheless the association between HBV and theprognosis of breast cancer is unclear The objectives of this study were to investigate theprognostic role of hepatitis B surface antigen HBsAg and to integrate HBsAg to establishnomograms for better prognostic prediction of very young patients with breast cancerMethods This analysis was performed retrospectively in a cohort of consecutivevery young ‰ at diagnosis patients who received curative resection for breastcancer The significance of HBsAg in the prognosis of these patients was investigatedUnivariate and multivariate analyses were used to identify independent variables fordiseasefree survival DFS and overall survival OS Nomograms were built based onthose identified variablesResults Overall of the patients were seropositive for HBsAgThe median followup was CI “ months forthe entirepopulation The HBsAgpositive cohort had significantly inferior DFS HR CI “ P and OS HR CI “ P as compared with the HBsAgnegative cohort The rates of 10year DFS andOS were and in the HBsAgpositive group and and in the HBsAgnegative group respectively In multivariable analysis HBsAg statuswas identified as an independent significant unfavorable prognostic factor for DFSP and OS P in very young patients with breast cancer Nomogramswere established and displayed good calibration and acceptable discrimination TheCindex values for DFS and OS were CI “ and CI “ respectively Based on the total prognostic scores TPSof the nomograms different prognosis groups were identified for DFS and OSEdited byImtiaz Ahmad SiddiquiUniversity of Colorado AnschutzMedical Campus United StatesReviewed byAbhinit NagarUMass Memorial Medical CenterUnited StatesNidhi JainBeckman Coulter IndiaCorrespondenceLu YangyanglusysucccnWeiDong Weiweiwdsysucccn These authors have contributedequally to this workSpecialty sectionThis was submitted toCancer Epidemiology and Preventiona section of the journalFrontiers in OncologyReceived March Accepted July Published August CitationLi N Zhong QQ Yang XRWang QC Zhang DT Zheng SYang L and Wei WD Prognostic Value of Hepatitis B VirusInfection in Very Young Patients WithCuratively Resected Breast CancerAnalyses From an Endemic Area inChina Front Oncol 103389fonc202001403Frontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast CancerConclusions HBsAg is an independent unfavorable prognostic factor for DFS andOS in very young patients with curatively resected breast cancer and nomogramsintegrating HBsAg provide individual survival prediction to benefit prognosis evaluationand individualized therapyKeywords HBV young breast cancer prognosis nomogram survivalINTRODUCTIONGlobally breast cancer is the most common cancer and theleading cause of cancer death for women accounting for of total cancer cases and of total cancer deaths Breast cancer has also been the top one malignancy in termsof incidence in Chinese women constituting of newlydiagnosed cases and of all deaths from breast cancer in theworld Although breast cancer occurs at a lower incidence inChinese women than in western women this disease occurs at ayounger age in China than in highincome countries and China™scontribution to global breast cancer rate is increasing rapidly The disparities between young and old breast cancer include ahigher mortality rate higher risk of recurrence poorer treatmentresponse and more aggressive phenotypes “ Thereforeunderstanding the etiology and identifying novel prognosticfactors are essential for early diagnosis prognosis evaluationearly intervention and personalized therapy in young patientswith breast cancerHepatitis B virus HBV infection is a serious public healthdilemma with ˆ¼ million chronic carriers worldwide China account for about a third of infectionassociated cancerglobally driven by high prevalence of HBV and H pylori infection Although China has made tremendous eï¬orts in controllingHBV over the past years and the prevalence of HBV ininfants and children has remarkably declined the hepatitisB surface antigen HBsAg prevalence is still high in Chineseadults ranging from to “ HBV is the leading causeof hepatocellular carcinoma and cholangiocarcinoma Inaddition there is also accumulating evidence that HBV infectionis associated with many extrahepatic malignancies includingnonHodgkin™s lymphoma pancreatic cancer gastriccancer nasopharyngeal carcinoma lung cancer esophageal cancer and ovarian cancer Thus it seemsreasonable that HBV is an important factor in the developmentof extrahepatic malignancies in endemic areasDespite the facts that HBsAg status is one of the routineexaminations in patients with operable breast cancer and severalstudies have showed that HBV is not associated with therisk of breast cancer the impact of HBV on theclinicopathological characteristics and prognosis of very youngpatients with breast cancer remains to be determined Giventhat both early breast cancer and HBV are endemic in China itis possible that HBV infection is associated with the prognosisof early breast cancer even though the precise mechanismsare yet to be determined It is crucial to address this issuesince HBV has been reported to be found in breast cancertissue We therefore performed this study to investigate theHBsAg prevalence in very young breast cancer and the impactof HBsAg on the survival of these patients and to establishnomograms to better predict prognosis for very young patientswith breast cancerMATERIALS AND METHODSPatient SelectionA retrospective review was conducted in a cohort of consecutive breast tumor women who were aged ‰ years oldand received curative resection for breast cancer at Sun Yatsen University Cancer Center between May and July This study was conducted according to the ethicalstandards of the Declaration of Helsinki Institutional ReviewBoard approval was obtained from the Medical Ethics Committeeof this cancer center All patients were restaged by the eighthinternational classification system for breast cancer Dueto the retrospective nature of this studyinformed consentwas waivedInformation was collected from electronic patient recordsand survival data were obtained from the followup registryofthis center The information collected included HBsAgstatus laterality type of breast surgery type of axillary surgeryhistological type tumor grade tumornodemetastasis TNMstage dates of surgeryrelapsedeath status of estrogen receptorER progesterone receptor PR human epidermal growthfactor receptor HER2 and Ki67 Breast cancers wereclassified as luminal Alike ER PR‰¥ HER2“ andKi6715luminal Blike ER andor PR HER2“HER2enriched ER“ PR“ HER2 or triplenegative ER“PR“ HER2“ subtypesPotentially eligible patients had to have curatively resectedbreast cancer without previous therapy other than neoadjuvanttherapy be aged years old or below and have definiteinformation of HBsAg The main exclusion criteria includedbenign tumor not having surgery having incomplete resectionprevious malignant disease hepatitis viral infections other thanHBV men patients and insufficient data of survival or HBsAgStatistical AnalysisThe main objectives of this study were to compare diseasefreesurvival DFS and overall survival OS between HBsAgpositivepatients and HBsAgnegative patients DFS was defined as theinterval from the date of being diagnosed to the date of diseaserecurrencemetastasis or death from any cause OS was defined asthe interval from the date of being diagnosed to the date of deathfrom any cause Median followup was estimated by KaplanMeier analysis with reversed meaning of status indicator Frontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast Cancerforindependent variablesDFS and OS were estimated by the Kaplan“Meier methodand diï¬erences were compared by the logrank test Univariateand multivariate analyses with a Cox proportional hazardsmodel were used to testforDFS and OS Covariates included laterality left vs righttype of surgery breastconserving surgery vs mastectomytype of axillary surgery sentinellymph node dissection vsaxillary lymph node dissection histological type ductal vsotherstumor grade grade III vs grade III T stageT34 vs T12 N stage N23 vs N1 HER2statustriplenegativeHER2enriched vs luminal All variables reaching asignificance of in univariate analyses were included inmultivariate analysispositive vs negative molecularsubtypesThe nomograms for predicting and 10year DFSand OS were formulated based on the results of multivariateanalysis by the œrms package of R The discriminationofthe nomogram models was estimated by the Harrell™sconcordance index Cindex The value of the Cindex rangesfrom to with implying a random chance and indicating a perfect prediction Calibration curves of thenomogram models for DFS OS were plotted to assess thepredictive value of the model In addition patients weredivided into three diï¬erent risk groups highintermediatelow according to total prognostic scores TPS The totalprognostic scores of patients were transformed into categoricalvariables based on cutoï¬ points which were determined by theminimum Pvalue from logrank × statistics with the Xtileprogram Pearson™s chisquare test was used to compare categoricaldata All Pvalues were twosided and P was consideredstatistically significant Statistical analyses were performed by theSPSS software SPSS Inc version Chicago IL USA and Rfor Windows version httpwwwrprojectData AvailabilityThe authenticity of this has been validated by uploadingthe key raw data onto the Research Data Deposit public platformwwwresearchdatacn with the approval RDD numberas RDDA2020001410 The data that support the findings ofthe study are available from the corresponding authors uponreasonable requestRESULTSPatient CharacteristicsA total of very young ‰ at diagnosis breasttumor patients were screened of whom were excludedbecause of benign tumor n not having surgeryn not having R0 resection n or unknownHBsAg status n With further exclusions forinsufficientfollowup data a total of patients withcurative resection for breast cancer and aged years orbelow were included in this study Figure The patients™FIGURE The process of patient selectionFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast CancerTABLE Patient characteristics by HBsAg statusCharacteristicsHBsAgpositiveN No HBsAgnegativeN No Pvalue FIGURE Number of recurrences by year of entire patientsLateralityLeftRightBilateralType of breast surgeryMastectomyBreastconserving surgeryType of axillary surgeryALNDSLNDHistological typeDuctalInvasive lobularOtherTumor gradeIIIIIIUnknownT StageT1T2T3T4N StageN0N1N2N3HER2 statusPositiveNegativeUnknownMolecular subtypesLuminal ALuminal BHER2enrichedTriple negativeUnknown HBV Hepatitis B Virus ALND axillary lymph node dissection SLND sentinelnode dissectionHER2 human epidermal growth factor receptor2lymphcharacteristics are shown in Table Overall ofthe patients were seropositive for HBsAg HBsAgpositive group and HBsAgnegative group were wellmatchedfor basic characteristics including laterality type of breast andaxillary surgery histological type tumor grade T stage Nstage and molecular subtypes About in patients receivedmastectomy and most patients received axillary lymph nodedissection ALNDAssociations Between the Status of HBsAgand SurvivalThe median followup was CI “ months forthe entire population By the time of analysis December instances of disease recurrence had occurred Thenumber of recurrences in each year of the followup is shown inFigure Of note although HBsAgpositive patients had a higherfrequency of extrahepatic metastasis vs P thanHBsAgnegative patients they had a comparable frequency ofliver metastasis vs P when compared withHBsAgnegative patientsDFS was significantly shorter among those who were HBsAgpositive than among those who were HBsAgnegative HR CI “ P The rates of 10yearDFS were in the HBsAgpositive group and inthe HBsAgnegative group respectively Figure 3A A totalof death events had occurred by the data cutoï¬ HBsAgpositive group had significantly inferior OS compared withHBsAgnegative group HR CI “ P with a 10year OS of and respectivelyFigure 3B The association of HBsAg status and survival ineach molecular subtype was further analyzed As expectedDFS and OS were significantly longer among those in theluminal A subgroup and the HER2enriched and triplenegativegroups had significantly shorter DFS and OS Figures 4ABNotably HBsAgpositive status was associated with shorterDFS P and OS P in the luminalB cohort HBsAgpositive status was also associated with aslightly shorter DFS in the triplenegative cohort and shorterOS in the HER2enriched cohort but statistical significancewas not reached Supplementary Figures There was nosignificant diï¬erence in DFS between HBsAgpositive patientsand HBsAgnegative patients in luminal A and HER2enrichedcohorts and in OS in luminal A and triplenegative cohortsSupplementary Figures In the univariate analysis type of breast surgery tumor gradeT stage N stage molecular subtype and HBsAg status wereFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast Cancerindividual patient Figures 6AB The model™s explanatorycovariables consisted of HBsAg status T stage N stage andmolecular subtype Patients with higher scores correspondedto inferior survival The scatter plots for the TPS of DFSand OS and percentage of patient number were presentedin Figures 6CD The Cindex values for DFS and OS were CI “ and CI “ respectively The calibration curves for the probabilityof DFS and OS at or year presented an optimalagreement between the prediction by nomogram and actualobservation Supplementary Figure Next we divided thepatients into the following groups based on the TPS ofthe nomogram modelfor DFS using the Xtile programlowrisk group TPS “ patients intermediateriskgroup TPS “ patients and highrisk group TPS patients The 10year DFS for lowrisk groupintermediaterisk group and highrisk group were and respectively Survival analyses for DFS demonstratedsignificant discrimination between these three groups P Figure 7A Same procedures were performed for OS in theentire population and patients were divided into the following groups based on the TPS of the nomogram model for OS withthe Xtile program lowrisk group TPS “ patientsintermediaterisk group TPS “ patients and highrisk group TPS patients OS diï¬erences were alsoobserved among three subgroups with a 10year OS of and for lowrisk intermediaterisk and highrisk groupsP Figure 7BDISCUSSIONIn this study we investigated the association of HBsAg statusand very young breast cancer and to our knowledge reportfor the firsttime that HBsAgpositive status is associatedwith inferior DFS and OS from a population with a highprevalence of both HBV infection and young breast canceridentified as a significant unfavorableHBsAg status wasprognostic predictor for DFS and OSindependent of anyother clinicopathological features of breast cancer includingT stage N stage and molecular subtype We also integratedHBsAg to build nomograms to better predict prognosis foryoung patients with breast cancer The results of our studydemonstrated that the prevalence of HBsAg in young patientswith breast cancer in southern China was which wasin accordance with the “ reported in the populationof this endemic area This result suggests that unlikecervical cancer young breast cancer is not correlatedwith an increased prevalence of HBV infection Indeed breastcancer patients with HBsAg did not demonstrate a diï¬erentpattern of characteristics It is noteworthy that in our studyHBsAg did not increase the rate of liver metastases for veryyoung patients with breast cancer This results are comparableto those reported in previous studies for esophageal cancerand colorectal cancer which suggested that HBVinfection is associated with decreased risk of liver metastasis inthese malignanciesFIGURE Kaplan“Meier curves for A diseasefree survival and B overallsurvival stratified by HBsAg status in very young patients with breast canceridentified as significant prognostic factors for DFS Figure 5AWhen those variables were further analyzed in the multivariateanalysis we found that T stage P N stage P molecular subtype P and HBsAg status P remained statistically significant indicating that theyare significant independent predictors for DFS Figure 5ABy the same methods for OSthe results showed that Tstage P N stage P molecular subtype and HBsAg status P were independentprognostic factors for OS Figure 5B HBsAgpositive status isan independent negative prognostic factor for survival in veryyoung breast cancerPrognostic Nomograms For Very YoungBreast Cancer PatientsTo better assess the DFS and OS of very young breastcancer patients prognostic nomograms for DFS and OS wereestablished respectively All the independent predictors of DFSand OS in the multivariate analysis were integrated into thenomogram models and and 10year survivals weregraphically computed according to the characteristics of anFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast CancerFIGURE Kaplan“Meier curves for A diseasefree survival and B overall survival stratified by molecular subtype in very young patients with breast cancerOur study shows that HBsAgpositive status is associatedwith inferior DFS and OS in young patients with curativelyresected breast cancer decreasing the 10year DFS and OS by and respectively The association between HBsAgpositive status and poor prognosis is in keeping with thatdemonstrated in nasopharyngeal carcinoma lung cancer and ovarian cancer However some studies regardingother cancers indicate that HBsAgpositive cancer patientshad a favorable survival as compared with HBsAgnegativepatients This discrepancy can be partly explainedby the diversity and heterogeneity of diï¬erent malignanciesThe genetic or biological mechanisms underlying the inferiorFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast CancerFIGURE Univariate and multivariate analysis for diseasefree survival A and overall survival B for very young patients with breast cancerprognosis remain to be elucidated butthis may largelyrelate to the presence of hepatitis B Xinteracting proteinHBXIP which has been welldocumented to function asan oncoprotein in breast cancer HBXIP can act as atransactivator by activating certain genes including cMyc E2F1STAT4 and Sp1 to play a crucial role in the progression ofbreast cancer HBXIP is associated with controlling cellapoptosis and promoting cell proliferation by mTOC1 activation HBXIP can also act as a modulation factor of cellularoxidative stress by competitively binding KEAP1 to enhancethe progression of breast cancer Previously studies showedthat HBV is not associated with risk of breast cancer These results combined with the data of our study suggestthat HBV is not a risk factor but a prognostic factor forbreast cancerAnother reason for this might relate to the HBV reactivationin HBsAgpositive patients with breast cancer who werereceiving chemotherapy HBV reactivation occurs frequentlyin breast cancer patients who are HBV carriers while receivingcytotoxic chemotherapy HBV reactivation can result inliver failure and interruption of the chemotherapy scheduleOther potential mechanisms underlyingassociationtheFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast CancerFIGURE AB Nomograms predicting and 10year A diseasefree survival and B overall survival for very young patients with breast cancer CD Thescatter plots of percentage of patient number and groups of C total prognostic score for DFS and D total prognostic score for OSbetween the HBsAg and patient survival include the immunesuppression Chronic HBV infection is characterized by thefailure to elicit an eï¬ective adaptive immune response andthe immune modulation of key innate immune response Chronic HBV infection can lead to immune anergy andimpair the function of the immune system which has longbeen deemed to protect the host against the developmentof nonviral cancerstogether couldpartially explain the positive association between HBsAgpositive status and the poor prognosis in young patients withbreast cancer These reasonsInthisstudy wecharacteristicscombined HBsAgstatus withclinicopathologicaleï¬ectiveprognostic nomogram models of DFS and OS for very youngpatients with breast cancer Both nomograms showed goodcalibration and acceptable discrimination These nomogrammodels can be used for prognosis evaluation at diagnosis forvery young patients with breast cancer and may benefit patientestablishtocounseling and personalized therapy for these patients Weadopted Xtile program to divide these patients into three riskgroups based on TPS from nomograms for DFS and OS Thesurvival curves for DFS and OS separated very well Thusspecial attention should be paid to and active surveillanceshould be conducted over patients with high risk group for DFSand OSThis study nevertheless has certain limitations that shouldbe noted First this study was retrospective in nature andwe cannot rule outthe impact of selection bias Secondthe sample size was relatively small and the sample sizesof the two cohorts were unequal as only patients wereHBsAgpositive The small sample size may be insufficientto allow us to perform subgroup analysis by each molecularsubtype Anotherthat Cantonese constitutemost of our study population The monotonicity ofthestudy population confines the universality of our resultsFurthermore the information was insufficient to perform otherlimitation isFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast CancerFIGURE Kaplan“Meier curves for A diseasefree survival and B overall survival stratified by risk groups based on total prognostic scores fromnomogram modelsanalysis such as that of hepatic function and HBV DNAcopy number Nevertheless the results of our study providewhat to our knowledge is the first evidence of the impactof HBsAg on the prognosis of very young patients withbreast cancerIn conclusion we have demonstrated in a retrospectivestudy that HBsAg is an independent unfavorable prognosticfactor for patients with very young breast cancer Furtherprospective studies involving varied ethnic populations arewarranted to confirm the prognostic value of HBsAg status invery young breast cancer and simultaneously other potentialclinicopathologic factors for breast cancer and HBV infection arerequired to be taken into account The mechanisms of the impactof HBV infection on the progression of breast cancer also need tobe elucidatedDATA AVAILABILITY STATEMENTThe datasets generated for this study are available on request tothe corresponding authorETHICS STATEMENTThe studiesinvolving human participants were reviewedand approved by the Medical Ethics Committee of SunYatsen University Cancer Center WritteninformedFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast Cancerconsentin accordance with the nationalinstitutional requirementsfor participation was not required for this studylegislation and theAUTHOR CONTRIBUTIONSNL QQZ LY and WDW designed the study NL QQZXRY QCW DTZ and SZ collected the data NL QQZ LYand WDW interpreted and analyzed the data All authors wereinvolved in writing the and approved the final versionREFERENCES Bray F Ferlay J Soerjomataram I Siegel RL Torre LA Jemal A Globalcancer statistics GLOBOCAN estimates of incidence and mortalityworldwide for cancers in countries CA Cancer J Clin “ 103322caac21492 Fan L StrasserWeippl K LiJJ St LouisJ Finkelstein DM Yu 15e279“KD et al Breast cancer 101016S1470204513705679in China Lancet Oncol Colleoni M Rotmensz N Robertson C Orlando L Viale G Renneet al Very young women years with operable breastGcancer“ 101093annoncmdf039at presentation Ann Oncolfeaturesof disease Anders CK Hsu DS Broadwater G Acharya CR Foekens JA Zhang Y et alYoung age at diagnosis correlates with worse prognosis and defines a subsetof breast cancers with shared patterns of gene expression J Clin Oncol “ 101200JCO2007142471 Narod SA Breast cancer in young women Nat Rev Clin Oncol “ 101038nrclinonc2012102 Trepo C Chan HL Lok A Hepatitis B virus infection Lancet “ 101016S0140673614602208 de Martel C Gees D Bray F Ferlay J Cliï¬ord GM Global burden of cancerattributable to infections in a worldwide incidence analysis LancetGlobal Health 8e180“ 101016S2214109X19304887 Lu FM Li T Liu S Zhuang H Epidemiology and prevention of hepatitisJ Viral Hepatitis 17Suppl “B virus infection in China 101111j13652893201001266x Liu J Zhang S Wang Q Shen H Zhang M Zhang Y et al Seroepidemiologyof hepatitis B virus infection in million men aged years in ruralChina a populationbased crosssectional study Lancet Infect Dis “ 101016S1473309915002182 Zhang Y Fang W Fan L Gao X Guo Y Huang W et al HepatitisB surface antigen prevalence among rural women of childbearingage in Hainan Province China a crosssectional study Virol J 1011861743422X1025 Cui Y Jia J Update on epidemiology of hepatitis B and C in China JGastroenterol Hepatol 28Suppl “ 101111jgh12220 Chan SL Wong VW Qin S Chan HL Infection and cancer the case ofHepatitis B J Clin Oncol “ 101200JCO2015615724 Fwu CW Chien YC You SL Nelson KE Kirk GD Kuo HS et al Hepatitis Bvirus infection and risk of intrahepatic cholangiocarcinoma and nonHodgkinlymphoma a cohort study of parous women in Taiwan Hepatology “ 101002hep24150 Kamiza AB Su FH Wang WC Sung FC Chang SN Yeh CC Chronichepatitis infection is associated with extrahepatic cancer developmenta nationwide populationbased study in Taiwan BMC Cancer 101186s1288501629185 Nath A Agarwal R Malhotra P Varma S Prevalence of hepatitis B virusinfection in nonHodgkin lymphoma a systematic review and metaanalysisInternal Med J “ 101111j14455994200902060x Luo G Hao NB Hu CJ Yong X Lu MH Cheng BJ et al HBV infectionincreases the risk of pancreatic cancer a metaanalysis Cancer Causes Control “ 101007s1055201201442FUNDINGThis work was supported by the National Natural ScienceFoundation of China No SUPPLEMENTARY MATERIALThe Supplementary Materialonline202001403fullsupplementarymaterialfor this can be foundhttpswwwfrontiersins103389foncat Wei XL Qiu MZ Jin Y Huang YX Wang RY Chen WW et al Hepatitis Bvirus infection is associated with gastric cancer in China an endemic area ofboth diseases Br J Cancer “ 101038bjc2014406 Ye YF Xiang YQ Fang F Gao R Zhang LF Xie SHHepatitis B virusin southern China Cancer Epidemiol Biomarkers Prevent“ 10115810559965EPI150344et alinfection and risk of nasopharyngeal carcinoma Peng JW Liu DY Lin GN Xiao JJ Xia ZJ Hepatitis B virusinfection is associated with poor prognosis in patients with advancednon small cell“ 107314APJCP201516135285lung cancer Asian Pacific J Cancer Prevent Zou J Chen J Xie X Liu Z Cai X Liu Q et al Hepatitis B virus infectionis a prognostic biomarker for better survival in operable esophageal canceranalysis of patients from an endemic area in China Cancer EpidemiolBiomarkers Prevent “ 10115810559965EPI181095 Wong L Cheung TH Yim SF Lao TT Prevalence and impact of hepatitis Bvirus infection in ovarian cancer patients in an endemic areaA retrospectivecohort study J Viral Hepat “ 101111jvh13250 Song C Lv J Liu Y Chen JG Ge Z Zhu J et al Associations between hepatitisB virus infection and risk of all cancer types JAMA Netw Open 2e195718 101001jamanetworkopen20195718 Su FH Chang SN Chen PC Sung FC Su CT Yeh CC Associationrisk abetween chronic viral hepatitisinfection and breast cancernationwide populationbased casecontrol study BMC Cancer Qin B Zhao K Wei J Wang X Xu M Lang J et al Novel evidence indicatesthe presence and replication of hepatitis B virus in breast cancer tissue OncolRep “ 103892or20197393 Te HS Jensen DM Epidemiology of hepatitis B and C viruses a globaloverview Clinics Liver Dis “ vii 101016jcld200911009 Siu SS Cheung TH Chan PK Lin CK Lo KW Patients with malignant or premalignant cervical lesion have increased risk of becoming hepatitis B carrierJ Exp Clin Cancer Res “in patients with colorectal Zhao Y Lin J Peng J Deng Y Zhao R Sui Q et al Hepatitis B virus infectionpredicts better survivalliveronly metastasesundergoing liver resection J Cancer “ 107150jca24544 Liu X Li X Jiang N Lei Y Tang LL Chen L et al Prognostic value ofchronic hepatitis B virus infection in patients with nasopharyngeal carcinomaanalysis of patients from an endemic area in China Cancer “ 101002cncr28377 Zhao Y Li H Zhang Y Li L Fang R Li Y et al Oncoprotein HBXIPmodulates abnormal lipid metabolism and growth of breast cancer cells byactivating the LXRsSREBP1cFAS signaling cascade Cancer Res “ 10115800085472CAN151734 Zhang Y Zhao Y Li H Li Y Cai X Shen Y et al The nuclear import ofoncoprotein hepatitis B Xinteracting protein depends on interacting with cFos and phosphorylation of both proteins in breast cancer cells J Biol Chem “ 101074jbcM113458638 BarPeled L Schweitzer LD Zoncu R Sabatini DM Ragulator is a GEFfor the rag GTPases that signal amino acid levels to mTORC1 Cell “ 101016jcell201207032 Zhou XL Zhu CY Wu ZG Guo X Zou W The oncoproteinHBXIP competitively binds KEAP1 to activate NRF2 and enhanceFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast Cancercancerbreast“ 101038s4138801906985growthcelland metastasis Oncogene Liu Z Jiang L Liang G Song E Jiang W Zheng Y et al Hepatitis B virusreactivation in breast cancer patients undergoing chemotherapy a reviewand metaanalysis of prophylaxis management J Viral Hepat “ 101111jvh12672 Yuen MF Chen DS Dusheiko GM Janssen HLA Lau DTY LocarniniSA et al Hepatitis B virus infection Nat Rev Dis Primers 101038nrdp201835 Dunn GP Old LJ Schreiber RD Theimmunobiology ofimmunosurveillance“ 101016jimmuni200407017immunoeditingandImmunitycancer Giuliano AE Edge SB Hortobagyi GN Eighth edition ofthe AJCCcancer staging manual breast cancer Ann Surg Oncol “ 101245s1043401864866 Schemper MSmith TL A note on quantifyingstudies 101016019724569600075Xfailuretime Control ClinofTrialsfollowup in“ Vickers AJ Elkin EB Decision curve analysis a novel method for “evaluating prediction models Med Decision Making 1011770272989X06295361 Camp RL DolledFilhart M Rimm DL Xtile a new bioinformatics toolfor biomarker assessment and outcomebased cutpoint optimization ClinCancer Res “ 10115810780432CCR040713Conflict of Interest The authors declare that the research was conducted in theabsence of any commercial or financial relationships that could be construed as apotential conflict of interestCopyright Li Zhong Yang Wang Zhang Zheng Yang and Wei This is anopenaccess distributed under the terms of the Creative Commons AttributionLicense CC BY The use distribution or reproduction in other forums is permittedprovided the original authors and the copyright owners are credited and that theoriginal publication in this journal is cited in accordance with accepted academicpractice No use distribution or reproduction is permitted which does not complywith these termsFrontiers in Oncology wwwfrontiersinAugust Volume 0c' Answer:

Thyroid_Cancer

Supraclavicular Recurrence inCompletely Resected ypN2NonSmall Cell Lung CancerImplications for PostoperativeRadiotherapyLiang Liu   Zhiqin Zheng   Juan Li Yuan Li and Jianjiao Ni Department of Radiation Oncology Fudan University Shanghai Cancer Center Shanghai China Department of OncologyShanghai Medical College Fudan University Shanghai China Department of Radiation Oncology Minhang BranchHospital Fudan University Shanghai Cancer Center Shanghai China Department of Pathology Fudan University ShanghaiCancer Center Shanghai Chinatarget volume CTVBackground The clinical value and delineation of clinicalof postoperative radiotherapy PORTin completely resected ypN2 nonsmall celllung cancer NSCLC remain controversial Investigations specifically focusing on thecumulative incidence and prognostic significance of initial disease recurrence at thesupraclavicular region SCR in this disease population are seldom reportedMethods Consecutive patients with curatively resected ypN2 NSCLC who receivedadjuvant chemotherapy from January to December at our cancer center wereretrospectively examined Disease recurrence at the surgical margin ipsilateral hilumandor mediastinum was defined as locoregional recurrence LRR Disease recurrencebeyond LRR and SCR was defined as distant metastasis DM Overall survival OS1 andOS2 were calculated from surgery and disease recurrence to death of any cause in theentire cohort and in patients with recurrent disease respectivelyResults Among the patients enrolled PORT without elective supraclavicularnodal irradiation ESRT was performed in patients and neoadjuvant chemotherapywas administered in patients With a median followup of months patientsdeveloped recurrent disease including SCRs among which were without DMand involved the ipsilateral supraclavicular region The and 5year cumulativeincidence of SCR were and respectively Chosen DM as a competingevent cN2 ypN2 not receiving lobectomy and negative expression of CK7 weresignificantly associated with SCR using the univariate competing risk analysis whileypN2 was identified as the only independent risk factor of SCR p PORTsignificantly reduced LRR p and prolonged OS1 p but didn™timpact SCR p Pattern of failure analyses indicated that the majority of LRRsdeveloped within the actuarial or virtual CTV of PORT and of the ipsilateralSCRs could be covered by the virtual CTV of proposed ESRT In terms of OS2patients who developed SCR but without DM had intermediate prognosis comparedwith those who had DM p and those who had only LRR p Edited byStephen V LiuGeetown University MedicalCenter United StatesReviewed byTim KruserNorthwestern Medicine United StatesHeloisa De Andrade CarvalhoUniversity of S£o Paulo BrazilCorrespondenceJianjiao Ninijianjiao8sinacom These authors have contributedequally to this workSpecialty sectionThis was submitted toThoracic Oncologya section of the journalFrontiers in OncologyReceived May Accepted July Published August CitationLiu L Zheng Z Li J Li Y and Ni J Supraclavicular Recurrence inCompletely Resected ypN2NonSmall Cell Lung CancerImplications for PostoperativeRadiotherapy Front Oncol 103389fonc202001414Frontiers in Oncology wwwfrontiersinAugust Volume 0cLiu et alSupraclavicular Recurrence in ypN2 NSCLCConclusions SCR is not uncommon and has important prognostic significance incompletely resected ypN2 NSCLC The clinical value of PORT and ESRT in suchpatients need to be further investigatedKeywords supraclavicular recurrence postoperative radiotherapy nonsmall cell lung cancer overall survivalclinical target volumeINTRODUCTIONStage III nonsmall cell lung cancer NSCLC is a heterogeneousdisease and surgical resection with or without neoadjuvanttherapy could be carried out in selected patients Aftercurative resection disease recurrence poses a considerablethreat and it has been demonstrated that platinumbasedadjuvant chemotherapy could significantly reduce postoperativerecurrence and improve 5year survival Howeveralthough numerous retrospective studies and several populationbased investigations “ have suggested a beneficial role ofpostoperative radiotherapy PORT in reducing locoregionalrecurrence LRR prolonging diseasefree survival DFS andeven improving overall survival OS among patients withcompletely resected ypN2 NSCLC the clinical valueof PORT is still controversial due to a lack of convincing datafrom large randomized clinical trials Moreover there is no definite agreement on the delineationof clinical target volume CTV during PORT for completelyresected ypN2 NSCLC and it varies between diï¬erentinstitutions and clinical trials The rationales of CTVdelineation are mostly based on the patterns of disease recurrencein surgical resected patients who don™t receive PORT and patternsof treatment failure in those who receive PORT In these studiescumulative incidence anatomic locations and risk factors of LRRwere extensively examined However the definitions of LRR arediï¬erent some of which include the initial disease recurrencedeveloped in the supraclavicular region SCR whileothers don™t Investigations specifically focused on SCR areseldom reported and elective supraclavicular nodal irradiationESRT is not routinely performedIn the current study we investigated the cumulative incidencerisk factor and prognostic significance of SCR in completelyresected ypN2 NSCLC Additionally our recent study findscrucial prognostic value of routine immunohistochemical IHCmarkers in completely resected NSCLC Hence besidescommon clinicpathological variables a list of routine IHCmarkers were examined when investigating the risk factorsof SCRMATERIALS AND METHODSPatientsLung cancer patients who received surgery at Fudan Universityfrom January toShanghai Cancer Center FUSCCreviewed PatientsDecemberwho underwentresection withpathologically confirmed N2 disease and received standardretrospectively werecompletesurgicaladjuvant chemotherapy were included in the study Patientsreceived PORT or not as well as neoadjuvant chemotherapyor not were both allowed to be included Exclusion criteriaincluded a second primary tumor compromised resectionpositive surgical margins neoadjuvant radiotherapy receivingno adjuvant chemotherapy death due to surgical complicationsand postoperative follow up monthsinvasion perineuralFor each patient common clinicpathological parameterswere gathered from the electronic medical records including agesex smoking history the Eastern Corporative Oncology GroupECOG performance score clinical TNM stage pathologicalTNM stage primary tumor size tumor diï¬erentiation tumorlymphovascular invasion visceralhistology tumor locationpleuralinvasion and type of surgeryPathologic TNM stage was in accordance with the eighth editionLung Cancer Stage Classification Tumor diï¬erentiationand tumor histology were determined on the basis of the World Health anization Classification of Tumors of the LungPleura Thymus and Heart Besides the expression statusof IHC markers ie HER2 TTF1 ERCC1 CK20 CK56CK7 P63 NapsinA Syn RRM1 EGFR and Ki67 were collectedThe IHC staining and evaluation were routinely performedin the Immunohistochemistry Diagnostic Laboratory of ourcancer center Our study followed The Declaration of HelsinkiThe institutional review board of FUSCC approved the studyInformed consent was waived by the institutional review boardbecause this was a retrospective studyTreatmentPretreatment evaluation generally included clinical assessmentblood test bronchoscopy contrastenhanced chest computedtomography CT scan ultrasonographic examination or CTthe abdomen brain magnetic resonance imagingscan ofMRI and bone scans Patients with mediastinallymphnode enlargement cm in the short axis on CT scan orpathologically proven to be malignant were defined as harboringclinical N2 cN2 disease Of note positron emission tomographyPETCT as well as invasive staging of the mediastinum wasstrongly recommended for patients with cN2 disease at ourcancer centerNeoadjuvanttherapy generally consisted of “ cyclesof platinumbased doublet regimen and surgicaltreatmentincluded lobectomy sublobectomy and pneumonectomy withsystematic multilevel mediastinallymph node dissection oradequate mediastinal sampling no N2 stations must includethe subcarinal station PORT was performed according toour institutional protocol using the intensitymodulatedradiation therapy technique employing a linear accelerator withFrontiers in Oncology wwwfrontiersinAugust Volume 0cLiu et alSupraclavicular Recurrence in ypN2 NSCLC6MV Xrays Briefly the CTV for left lung cancers included thebronchial stump and 2R 2L 4R 4L and 11L lymphnode stations while the CTV for right lung cancers included thebronchial stump and 2R 4R and 11R stations ESRT wasnot performed The total radiation dose prescibed to the planningtarget volume PTV was generally Gy administered daily at Gy per fraction days per weekFollow UpFollowups were at the discretion of the treating physicians andwere generally scheduled at regular intervals every monthsafter surgery in the first years every months for the next years and annually thereafter During followup blood testschest CT scans and CT scans or ultrasonographic examination ofabdominal and cervical regions were routinely performed whilebrain MRI and bone scans were not mandatory Telephone callswere also implemented when necessaryPostoperative recurrence was diagnosed considering allthe evidence provided by imaging scans and pathologicconfirmation Initial disease recurrence in the supraclavicularregion was defined as SCR and first relapse developed at thesurgical marginipsilateral hilum andor mediastinum wasconsidered LRR Initial disease recurrence beyond LRR and SCRwas categorized as distant metastasis DMPattern of Failure AnalysesFor patients with LRR the PTVs were restored for thosewho received PORT and virtual PTVs were created for thosewho didn™t receive PORT by independent radiation oncologistaccording to ourinstitutional protocol mentioned aboveMeanwhile for patients with SCR individual virtual PTVs werecreated for ipsilateral ESRT PTVsc by independent radiationoncologist according to the CT atlas proposed by Lynch et al Then we plotted the sites of LRRs andor SCRs and overlaidthem with restored or created PTVs Coverage of the LRRs andSCRs by the PTVs were investigatedStatistical AnalysesRecurrence free survival RFS was calculated from surgery toinitial disease recurrence Overall survival OS1 was calculatedfrom surgery to death of any cause in the entire cohort andOS2 was calculated from initial disease recurrence to deathof any cause in patients with recurrent disease Diï¬erences andbetween clinical parameters were compared using the χFisher™s exact tests The predictors of SCR were selected usingcompeting risk methodology and Stata version softwareStataCorp College Station TX USA The associations betweenclinicpathological parameters and OS were identified using theCox proportional hazard regression model The hazard ratioHR and the confidence interval CI were calculatedusing coefficients from the model Kaplan“Meier methodwas used to estimate survival and diï¬erences among groupswere investigated by the logrank test Statistical analysis wasperformed using SPSS SPSS Chicago IL USA Allassessment is considered to be significant when twosided pvalueis RESULTSPatients CharacteristicsA total of patients were finally enrolled and a flowchartfor patient selection was presented in Supplementary Figure Detailed baseline disease characteristics of the patientswere summarized in Table The majority of patients had ahistology of nonsquamous NSCLC and received lobectomyTABLE Disease characteristicsVariablesNumber of patients Age at diagnosis years‰¤SexFemaleMaleSmoking historyEver smokerNever smokerECOG performance scoreClinical N stagecN0“cN2Neoadjuvant chemotherapyYesNoSurgery typeSublobarLobectomyPneumonectomyPathological T stagepT0“pT3“Lymphovascular invasionAbsentPresentVisceral pleural invasionAbsentPresentTumor locationLeft lower lobeLeft upper lobeRight lower lobeRight middle lobeRight upper lobeHistologySquamous cell carcinomaNonsquamous nonsmall cell lung cancerECOG Eastern Corporative Oncology Group Frontiers in Oncology wwwfrontiersinAugust Volume 0cLiu et alSupraclavicular Recurrence in ypN2 NSCLCFIGURE Patterns of supraclavicular recurrence A Venn diagram demonstrating the distribution of initial postoperative recurrence B Pie chart demonstrating thedistribution of SCR SCR supraclavicular recurrence LRR locoregional recurrence DM distant metastasisFIGURE Cumulative incidence and dynamics of supraclavicular recurrence A Cumulative incidence of supraclavicular recurrence in the entire cohort andstratified by pathological status ypN2 vs pN2 B The dynamics of hazard ratio of supraclavicular recurrenceThe positive rate of HER2 TTF1 ERCC1 CK20 CK56 CK7P63 NapsinA Syn RRM1 and EGFR was and respectivelyAdditionally Ki67 ‰¥ was detected in of the patientsPretreatment PETCT was performed in patients andinvasive staging of the mediastinum was underwent in patients One hundred and sixtyfour patients were found tohave cN2 disease among whom patients receivedpretreatment PETCT and patients had invasivestaging of the mediastinum A total of patientsreceived neoadjuvant chemotherapyCumulative Incidence and Risk Factors ofSCRPost surgery patients received PORT and with a medianfollow up of range “ months patients developedrecurrent disease including SCRs Of note of the SCRswere pathologically confirmed and the rest were diagnosed byclinical assessments and radiographic findings The and year RFS were and in patients without PORTrespectively and were and in patients withPORT respectively Among the patients with SCR patients developed SCR without DM Figure 1A and patients developed SCR involving the ipsilateral supraclavicularregion Figure 1B Moreover among the patients with leftlung cancer who developed SCR seven were ipsilateral threebilateral and two contralateral Among the patients with rightlung cancer who developed SCR nine were ipsilateral threebilateral and three contralateralThe and 5year cumulative incidence of SCR were and respectively Figure 2A and the dynamicof hazard ratio of SCR was presented in Figure 2B ChosenDM as a competing event cN2 disease ypN2lobectomyand CK7 were identified as significant risk factors of SCRFrontiers in Oncology wwwfrontiersinAugust Volume 0cLiu et alSupraclavicular Recurrence in ypN2 NSCLCTABLE Competing risk analyses of clinicalpathological variables associatedwith supraclavicular recurrenceVariablesUnivariate Analyses Multivariate AnalysesHR 95CIpHR 95CIpAge vs ‰¤ “ Sex Male vs Female “ Smoking Never vs Ever “ ECOG vs cN2 vs “ “ “ pT stage T3“ vs T0“ “ pN1 vs “ “ Multiple levels of pN2 vs “ “ Histology SCC vs NonSCC “ Differentiation P vs WM “ LVI vs “VPI vs “PNI vs “ypN2 vs pN2 “ “ “ “ “ Tumor Location Left vs Right “ Tumor Lobe Upper vs Others “ TLN ‰¥ vs “ PLN ‰¥ vs “ LNR ‰¥ vs “ Surgery Others vs Lobectomy “ “ PORT vs “ERCC1 vs “Her2 vs “ “ “ “ Ki67 ‰¥ vs “ TTF1 vs “CK20 vs “CK7 vs “CK56 vs “P63 vs “ “ “ “ “ “ “ NapsinA vs “ “ Syn vs “RRM1 vs “EGFR vs “ “ “ “ HR hazard ratios CI confidence intervals SCC squamous cell carcinoma LVILymphovascular invasion VPI Visceral pleuralinvasion ECOGthe Eastern Corporative Oncology Group TLN total lymph node examined PLN positivelymph node LNR positive lymph node ratio PORT postoperative radiotherapy WMwellmoderate P poor Bold values indicates statistical significantinvasion PI perineuralusing the univariate competing risk analysis Table Sincethere was a significant association between cN2 disease and testreceiving neoadjuvant chemotherapy p χwe excluded cN2 disease and included the otherthreesignificant risk factors in the multivariate competing riskanalyses The result showed that only ypN2 were identifiedas an independent risk factor of SCR Table The and 5year cumulative incidence of SCR were and among ypN2 patients respectively and were and among pN2 patients respectivelyFigure 2APattern of Failure AnalysesIn the entire cohort of the patients who receivedPORT developed LRR while of the patientswho did not receive PORT developed LRR PORT significantlyreduced the risk of LRR Figure 3A Among the six patients whoreceived PORT and subsequently developed LRR five developedLRR only within the PTV and the rest one developed LRRboth within and outside the PTV Among the patients whodid not receive PORT and subsequently developed LRR developed LRR only within the proposed PTV three developedLRR both within and outside the proposed PTV and the restone developed LRR outside the proposed PTV That patienthad adenocarcinoma in the middle lobe of right lung withpathologically proven metastatic lymph node in the right hilumand station but developed recurrent disease at mediastinallymph node stations and On the other hand of the patients who receivedPORT developed SCR while of the patients whodid not receive PORT developed SCR in the entire cohort PORTwithout ESRT didn™t reduce the incidence of SCR Figure 3BFifteen of the ipsilateral SCRs could be covered by theproposed PTVsc and the ipsilateral parts of the six bilateral SCRscould all be covered by the proposed PTVscSurvival AnalysesBy the time of data cutoï¬ patients had died and the medianOS1 was 95CI “ months PORT was found tosignificant prolong OS1 in the entire cohort Figure 3C Agesex ECOG scorelymphovascular invasion total number ofpositive lymph node positive lymph node ratio PORT and Ki67were found to be significantly associated with OS1 in univariateCox analyses while age ECOG score PORT and Ki67 wereidentified to be independent indicators of OS1 in multivariateCox analyses Table Among the patients with recurrentdisease the median OS2 was 95CI “ monthsAge sex ECOG score and DM were revealed to be significantlyassociated with OS1 in univariate and multivariate Cox analysesTable In order to investigate the prognostic significance of SCRpatients with recurrent disease were further divided into threegroups Group A consisted of patients who had DM n Group B consisted of patients who did not have DM but have SCRn and Group C consisted of patients who only had LRR n In terms of OS2 patients in Group B had an intermediateprognosis when compared with patients in Group A and GroupC Figure 3DDISCUSSIONTo the best of our knowledge this is the first comprehensivestudy specifically focusing on SCR in completely resected ypN2NSCLC with a relatively large sample size in the era of modernradiation technique SCR was not uncommon and had imperativeprognostic significance indicating that treatment modalities ableto reduce the incidence of SCR may be beneficial AdditionallyPORT without ESRT significantly reduced LRR and prolongedOS but did not decrease SCR in our study suggesting that theFrontiers in Oncology wwwfrontiersinAugust Volume 0cLiu et alSupraclavicular Recurrence in ypN2 NSCLCFIGURE Prognostic significance of postoperative radiotherapy and supraclavicular recurrence The impact of postoperative radiotherapy PORT on locoregionalrecurrence LRR A supraclavicular recurrence SCR B overall survival OS1 C in the entire cohort Kaplan“Meier survival curve stratified by the diseaserecurrence patterns among patients with recurrent disease Dclinical value of ESRT may be reconsidered in selected patientswith high risks of SCRSCR is not uncommon in completely resected ypN2 NSCLCespecially among those with extra risk factors Although therewas limited historical data published that could be directlycompared the incidence of SCR in our study was reliable sincethe overall recurrence rate and the percentage of SCR amongpatients with recurrent disease were in accordance with previousfindings The cumulative incidence of postoperative recurrencein the PORT group and nonPORT group were generallycomparable with recent studies Furthermorestudies from our institution and others had reported asimilar percentage of SCR among patients with recurrent disease“ in the literature in our study Compared withtheir counterpart patients staged cN2 or ypN2 generally had amore advanced and aggressive disease and thus it was reasonablefor them to have a higher risk developing disease recurrenceincluding SCR “ Compared with those receivinglobectomy patients receiving pneumonectomy generally hada higher tumor burden and those receiving sublobectomycommonly had unfavorable prognostic factors such as morecomorbidities and poorer preoperative lung functions that madethem unsuitable for lobectomy Therefore patients whodidn™t receive lobectomy were also at a higher risk developingpostoperative recurrence which is generally consistent with arecent retrospective study using the SEER database Inaddition two recent studies found that positive expression ofCK7 were associated with more advanced disease and shorteroverall survival In our study distant metastasis waschosen as a competing event and negative expression of CK7was identified as a risk factor of SCR which need to befurther verifiedCompared with patients developing only LRR and thosedeveloping DM patients developing SCR but without DM hadintermediate OS2 highlighting the vital prognostic significanceof SCR in curatively resected ypN2 NSCLC The TNMstaging system is one ofindicators ofpatient™s prognosis in NSCLC among which patients havingsupraclavicular lymph node metastasis N3 generally haveintermediate prognosis when compared with those having distantmetastasis M1 and those harboring metastatic tumor lesionslimited to the ipsilateral hilar N1 or mediastinal N2 lymphthe most powerfulFrontiers in Oncology wwwfrontiersinAugust Volume 0cLiu et alSupraclavicular Recurrence in ypN2 NSCLCTABLE Cox analyses of clinicalpathological variables associated with overallsurvival OS1TABLE Cox analyses of clinicalpathological variables associated with OS2 inpatients with recurrent diseaseVariablesUnivariate Analyses Multivariate AnalysesVariablesUnivariate AnalysesMultivariate AnalysesHR 95CIpHR 95CIpHR 95CIpHR 95CIpAge vs ‰¤ “ “ Sex Male vs Female “ “ Smoking Never vs Ever “ ECOG vs cN2 vs “ “ “ “ pT stage T3“ vs T0“ “ pN1 vs “ “ Multiple levels of pN2 vs “ “ Histology SCC vs NonSCC “ Differentiation P vs WM “ LVI vs “VPI vs “PNI vs “ypN2 vs pN2 “ “ “ “ “ Tumor Location Left vs Right “ Tumor Lobe Upper vs Others “ TLN ‰¥ vs “ Age vs ‰¤ “ “Sex Male vs Female “ “Smoking Never vs Ever “ECOG vs cN2 vs “ “ “ “pT stage T3“ vs T0“ “pN1 vs “ “Multiple levels of pN2 vs “ “Histology SCC vs NonSCCDifferentiation P vs WM “ “LVI vs “VPI vs “PNI vs “ypN2 vs pN2 “ “ “ “Tumor Location Left vs Right “Tumor Lobe Upper vs Others “TLN ‰¥ vs “PLN ‰¥ vs “LNR ‰¥ vs “PLN ‰¥ vs “ “ Surgery Others vs Lobectomy “LNR ‰¥ vs “ “ Surgery Others vs Lobectomy “ PORT vs “ERCC1 vs “Her2 vs “ “ “ “ “ Ki67 ‰¥ vs “ “ TTF1 vs “CK20 vs “CK7 vs “CK56 vs “P63 vs “ “ “ “ “ “ NapsinA vs “ “ Syn vs “RRM1 vs “EGFR vs “ “ “ “ HR hazard ratios CI confidence intervals SCC squamous cell carcinoma LVILymphovascular invasion VPI Visceral pleuralinvasion ECOGthe Eastern Corporative Oncology Group TLN total lymph node examined PLN positivelymph node LNR positive lymph node ratio PORT postoperative radiotherapy WMwellmoderate P poor Bold values indicates statistical significantinvasion PI perineuralnodes Similarly SCR represented an unfavorable sign ofsubsequent disease metastasis to distant ans and thus wasreasonable to have worse prognosis when compared with thosewho had only LRR On the other hand when compared withthose who already had DM patients who had recurrent diseaselimited to the thoracic region ie LRR and SCR could beconsidered as harboring locoregional disease and may benefitfrom aggressive locoregional treatment as well as systematictherapies and thus may still have a chance of longterm survival In fact among the patients with SCR but without DMthe 3year survival rate exceeded in our study Figure 3DHowever due to the advancement of adjuvant chemotherapy andPORT vs “DM vs “ERCC1 vs “Her2 vs “ “ “ “ “ “Ki67 ‰¥ vs “TTF1 vs “CK20 vs “CK7 vs “CK56 vs “P63 vs “NapsinA vs “Syn vs “RRM1 vs “EGFR vs ““ “ “ “ “ “ “ “ “OS overall survival HR hazard ratios CI confidence intervals SCC squamouscell carcinoma LVI Lymphovascularinvasion PIperineural invasion ECOG the Eastern Corporative Oncology Group TLN total lymphnode examined PLN positive lymph node LNR positive lymph node ratio PORTpostoperative radiotherapy DM distant metastasis WM wellmoderate P poor Boldvalues indicates statistical significantinvasion VPI Visceral pleuralPORT the number of patients who developed localized recurrentdisease ie LRR and SCR was small patients in group Band patients in group C although a total of patients wereenrolled and followed up for a median of months Hencethe prognostic significance of SCR needed to be interpreted withcaution and future investigations with larger sample size andprospective design are warrantedThe clinical value of PORT in completely resected ypN2NSCLC was demonstrated again in our study but the delineationof CTV remain controversial In the current study PORTsignificantly reduced LRR and improved OS1 which havebeen demonstrated in various studies “ However since ESRT was not routinely performed in our cancerFrontiers in Oncology wwwfrontiersinAugust Volume 0cLiu et alSupraclavicular Recurrence in ypN2 NSCLCindicating thatinstitution PORT failed to reduce SCRthe majority of SCRs represented the outgrowth of subclinicaltumor lesions already in the supraclavicular region and werenot originated from the locoregional recurrent disease throughlymphatic metastasis In fact of the patientswith SCR had no LRR in the current study These dataindicated a potential role of ESRT in selected patients withhigh risks Actuallyfor locally advanced NSCLC receivingchemoradiotherapy there is no significant diï¬erence of patient™ssurvival between those with or without N3 disease highlighting that the treatment efficacy of chemoradiotherapyin locally advanced NSCLC was largely dependent on theintrinsic biology of the tumor and the prognosis of patients withor without macroscopic supraclavicular tumor lesions seemedsimilar PORT with adjuvant chemotherapy has been repeatedlyshown to significantly reduce LRR indicating the beneficial roleof adjuvant chemoradiotherapy in treating microscopic N1N2disease It is possible that adjuvant chemoradiotherapy ieadjuvant chemotherapy in combination with ESRT may alsoplay a role in reducing SCR and subsequently improve patient™ssurvival Furthermore nearly of the ipsilateral SCRs couldbe covered with the virtual CTV of ESRT in our study Howeverthere are also evidence against the use of ESRT for patients withcompletely resected NSCLC Elective irradiation of mediastinalcontralateral hilar and supraclavicular lymph nodes failed toimprove patient™s survivalin unresectable stage III NSCLCwithout clinical N3 disease And pattern of failure analyses ofa prospective trial of PORT without ESRT suggested that the useof limited CTV including only the involved lymph node stationsand those with a risk of invasion was associated withacceptable risk of geographic miss Taken together PORTwithout ESRT provided significant clinical benefit for patientswith completely resected ypN2 NSCLC and the clinical valueof ESRT in highly selected patients for example those withpersistent N2 ypN2 disease after neoadjuvant chemotherapyneed to be further investigatedOur study also has some limitations Firstly since ESRT is notroutinely performed in our cancer center we could not directlyexamine the clinical value and prognostic significance of ESRTSecondly as this was a retrospectively study treatment decisionsand followup strategies were at the discretion of the treatingphysicians Diï¬erent neoadjuvant and adjuvant chemotherapyregimens were used and the protocols of followup were notidentical Moreover since brain MRI and bone scans were notmandatory asymptomatic brain andor bone metastasis may beunderestimated Despite these limitations we believe our studyprovided valuable information about the cumulative incidenceand prognostic significance of SCR in completely resected ypN2NSCLC which may guide better design of adjuvant treatmentmodalities and individualized surveillance strategiesDATA AVAILABILITY STATEMENTThe raw data supporting the conclusions of this will bemade available by the authors without undue reservationETHICS STATEMENTThe studiesinvolving human participants were reviewedand approved by the institutional review board of FudanUniversity Shanghai Cancer Center Written informed consentfor participation was not required for this study in accordancewith the national legislation and the institutional requirementsAUTHOR CONTRIBUTIONSLL ZZ and JN conceptualization LL and ZZ methodologyvalidation and writing”original draft preparation LL ZZ andJL formal analysis and investigation LL ZZ and YL resourcesand data curation LL and JN writing”review and editing Allthe authors have approved the final manuscriptFUNDINGThis study was supported by the National Natural ScienceFoundation of China No to JN and grant provided bythe Shanghai Municipal Health Commission No 20194Y0501to JNSUPPLEMENTARY MATERIALThe Supplementary Materialonline202001414fullsupplementarymaterialfor this can be foundhttpswwwfrontiersins103389foncatSupplementary Figure Flowchart of patient enrollment NSCLC nonsmallcell lung cancerREFERENCES Eberhardt WE De Ruysscher D Weder W Le Pechoux C De Leyn PHoï¬mann H et al 2nd ESMO Consensus Conference in Lung Cancerlocally advanced stage III nonsmallcell lung cancer Ann Oncol “ 101093annoncmdv187 Tan WL Chua KLM Lin CC Lee VHF Tho LM Chan AW et alAsian thoracic oncology research group expert consensus statement onoptimal management of stage III NSCLC J Thorac Oncol “ 101016jjtho201910022 Kenmotsu H Yamamoto N Yamanaka T Yoshiya K Takahashi T Uenostudy of pemetrexed plus cisplatintoT et al Randomized phase IIIversus vinorelbine plus cisplatin for completely resected stage IIIIIA nonsquamous nonsmallcell“ 102139ssrn3460654lung cancerJ Clin Oncol Kato H Tsuboi M Kato Y Ikeda N Okunaka T Hamada C Postoperativeadjuvant therapy for completely resected earlystage nonsmall cell lungcancer Int J Clin Oncol “ 101007s101470050493x Herskovic A Mauer E Christos P Nagar H Role of postoperativeradiotherapy in pathologic stage IIIA N2 nonsmall cell lung cancer in aprospective nationwide oncology outcomes database J Thorac Oncol “ 101016jjtho201609135 Deng W Xu T Xu Y Wang Y Liu X Zhao Y et al Survival patterns for patientswith resected N2 nonsmall cell lung cancer and postoperative radiotherapya prognostic scoring model and heat map approach J Thorac Oncol “ 101016jjtho2018082021Frontiers in Oncology wwwfrontiersinAugust Volume 0cLiu et alSupraclavicular Recurrence in ypN2 NSCLC Feng W Fu XL Cai XW Yang HJ Wu KL Fan M et al Patternsof localregional failure in completely resected stage IIIAN2 nonsmallcellimplications for postoperative radiation therapyclinical target volume design Int J Radiat Oncol Biol Phys “ 101016jijrobp201312048lung cancer cases Dai H Hui Z Ji W Liang J Lu J Ou G et al Postoperative radiotherapy forresected pathological stage IIIAN2 nonsmall cell lung cancer a retrospectivestudy of cases from a single institution Oncologist “ 101634theoncologist20100343 Zou B Xu Y Li T Li W Tang B Zhou L et al A multicenter retrospectiveanalysis of survival outcome following postoperative chemoradiotherapy innonsmallcell lung cancer patients with N2 nodal disease Int J Radiat OncolBiol Phys “ 101016jijrobp200905044 Billiet C De Ruysscher D Peeters S Decaluwe H Vansteenkiste J Dooms Cet al Patterns of locoregional relapses in patients with contemporarily stagedstage IIIN2 NSCLC treated with induction c

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You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: Supraclavicular Recurrence inCompletely Resected ypN2NonSmall Cell Lung CancerImplications for PostoperativeRadiotherapyLiang Liu   Zhiqin Zheng   Juan Li Yuan Li and Jianjiao Ni Department of Radiation Oncology Fudan University Shanghai Cancer Center Shanghai China Department of OncologyShanghai Medical College Fudan University Shanghai China Department of Radiation Oncology Minhang BranchHospital Fudan University Shanghai Cancer Center Shanghai China Department of Pathology Fudan University ShanghaiCancer Center Shanghai Chinatarget volume CTVBackground The clinical value and delineation of clinicalof postoperative radiotherapy PORTin completely resected ypN2 nonsmall celllung cancer NSCLC remain controversial Investigations specifically focusing on thecumulative incidence and prognostic significance of initial disease recurrence at thesupraclavicular region SCR in this disease population are seldom reportedMethods Consecutive patients with curatively resected ypN2 NSCLC who receivedadjuvant chemotherapy from January to December at our cancer center wereretrospectively examined Disease recurrence at the surgical margin ipsilateral hilumandor mediastinum was defined as locoregional recurrence LRR Disease recurrencebeyond LRR and SCR was defined as distant metastasis DM Overall survival OS1 andOS2 were calculated from surgery and disease recurrence to death of any cause in theentire cohort and in patients with recurrent disease respectivelyResults Among the patients enrolled PORT without elective supraclavicularnodal irradiation ESRT was performed in patients and neoadjuvant chemotherapywas administered in patients With a median followup of months patientsdeveloped recurrent disease including SCRs among which were without DMand involved the ipsilateral supraclavicular region The and 5year cumulativeincidence of SCR were and respectively Chosen DM as a competingevent cN2 ypN2 not receiving lobectomy and negative expression of CK7 weresignificantly associated with SCR using the univariate competing risk analysis whileypN2 was identified as the only independent risk factor of SCR p PORTsignificantly reduced LRR p and prolonged OS1 p but didn™timpact SCR p Pattern of failure analyses indicated that the majority of LRRsdeveloped within the actuarial or virtual CTV of PORT and of the ipsilateralSCRs could be covered by the virtual CTV of proposed ESRT In terms of OS2patients who developed SCR but without DM had intermediate prognosis comparedwith those who had DM p and those who had only LRR p Edited byStephen V LiuGeetown University MedicalCenter United StatesReviewed byTim KruserNorthwestern Medicine United StatesHeloisa De Andrade CarvalhoUniversity of S£o Paulo BrazilCorrespondenceJianjiao Ninijianjiao8sinacom These authors have contributedequally to this workSpecialty sectionThis was submitted toThoracic Oncologya section of the journalFrontiers in OncologyReceived May Accepted July Published August CitationLiu L Zheng Z Li J Li Y and Ni J Supraclavicular Recurrence inCompletely Resected ypN2NonSmall Cell Lung CancerImplications for PostoperativeRadiotherapy Front Oncol 103389fonc202001414Frontiers in Oncology wwwfrontiersinAugust Volume 0cLiu et alSupraclavicular Recurrence in ypN2 NSCLCConclusions SCR is not uncommon and has important prognostic significance incompletely resected ypN2 NSCLC The clinical value of PORT and ESRT in suchpatients need to be further investigatedKeywords supraclavicular recurrence postoperative radiotherapy nonsmall cell lung cancer overall survivalclinical target volumeINTRODUCTIONStage III nonsmall cell lung cancer NSCLC is a heterogeneousdisease and surgical resection with or without neoadjuvanttherapy could be carried out in selected patients Aftercurative resection disease recurrence poses a considerablethreat and it has been demonstrated that platinumbasedadjuvant chemotherapy could significantly reduce postoperativerecurrence and improve 5year survival Howeveralthough numerous retrospective studies and several populationbased investigations “ have suggested a beneficial role ofpostoperative radiotherapy PORT in reducing locoregionalrecurrence LRR prolonging diseasefree survival DFS andeven improving overall survival OS among patients withcompletely resected ypN2 NSCLC the clinical valueof PORT is still controversial due to a lack of convincing datafrom large randomized clinical trials Moreover there is no definite agreement on the delineationof clinical target volume CTV during PORT for completelyresected ypN2 NSCLC and it varies between diï¬erentinstitutions and clinical trials The rationales of CTVdelineation are mostly based on the patterns of disease recurrencein surgical resected patients who don™t receive PORT and patternsof treatment failure in those who receive PORT In these studiescumulative incidence anatomic locations and risk factors of LRRwere extensively examined However the definitions of LRR arediï¬erent some of which include the initial disease recurrencedeveloped in the supraclavicular region SCR whileothers don™t Investigations specifically focused on SCR areseldom reported and elective supraclavicular nodal irradiationESRT is not routinely performedIn the current study we investigated the cumulative incidencerisk factor and prognostic significance of SCR in completelyresected ypN2 NSCLC Additionally our recent study findscrucial prognostic value of routine immunohistochemical IHCmarkers in completely resected NSCLC Hence besidescommon clinicpathological variables a list of routine IHCmarkers were examined when investigating the risk factorsof SCRMATERIALS AND METHODSPatientsLung cancer patients who received surgery at Fudan Universityfrom January toShanghai Cancer Center FUSCCreviewed PatientsDecemberwho underwentresection withpathologically confirmed N2 disease and received standardretrospectively werecompletesurgicaladjuvant chemotherapy were included in the study Patientsreceived PORT or not as well as neoadjuvant chemotherapyor not were both allowed to be included Exclusion criteriaincluded a second primary tumor compromised resectionpositive surgical margins neoadjuvant radiotherapy receivingno adjuvant chemotherapy death due to surgical complicationsand postoperative follow up monthsinvasion perineuralFor each patient common clinicpathological parameterswere gathered from the electronic medical records including agesex smoking history the Eastern Corporative Oncology GroupECOG performance score clinical TNM stage pathologicalTNM stage primary tumor size tumor diï¬erentiation tumorlymphovascular invasion visceralhistology tumor locationpleuralinvasion and type of surgeryPathologic TNM stage was in accordance with the eighth editionLung Cancer Stage Classification Tumor diï¬erentiationand tumor histology were determined on the basis of the World Health anization Classification of Tumors of the LungPleura Thymus and Heart Besides the expression statusof IHC markers ie HER2 TTF1 ERCC1 CK20 CK56CK7 P63 NapsinA Syn RRM1 EGFR and Ki67 were collectedThe IHC staining and evaluation were routinely performedin the Immunohistochemistry Diagnostic Laboratory of ourcancer center Our study followed The Declaration of HelsinkiThe institutional review board of FUSCC approved the studyInformed consent was waived by the institutional review boardbecause this was a retrospective studyTreatmentPretreatment evaluation generally included clinical assessmentblood test bronchoscopy contrastenhanced chest computedtomography CT scan ultrasonographic examination or CTthe abdomen brain magnetic resonance imagingscan ofMRI and bone scans Patients with mediastinallymphnode enlargement cm in the short axis on CT scan orpathologically proven to be malignant were defined as harboringclinical N2 cN2 disease Of note positron emission tomographyPETCT as well as invasive staging of the mediastinum wasstrongly recommended for patients with cN2 disease at ourcancer centerNeoadjuvanttherapy generally consisted of “ cyclesof platinumbased doublet regimen and surgicaltreatmentincluded lobectomy sublobectomy and pneumonectomy withsystematic multilevel mediastinallymph node dissection oradequate mediastinal sampling no N2 stations must includethe subcarinal station PORT was performed according toour institutional protocol using the intensitymodulatedradiation therapy technique employing a linear accelerator withFrontiers in Oncology wwwfrontiersinAugust Volume 0cLiu et alSupraclavicular Recurrence in ypN2 NSCLC6MV Xrays Briefly the CTV for left lung cancers included thebronchial stump and 2R 2L 4R 4L and 11L lymphnode stations while the CTV for right lung cancers included thebronchial stump and 2R 4R and 11R stations ESRT wasnot performed The total radiation dose prescibed to the planningtarget volume PTV was generally Gy administered daily at Gy per fraction days per weekFollow UpFollowups were at the discretion of the treating physicians andwere generally scheduled at regular intervals every monthsafter surgery in the first years every months for the next years and annually thereafter During followup blood testschest CT scans and CT scans or ultrasonographic examination ofabdominal and cervical regions were routinely performed whilebrain MRI and bone scans were not mandatory Telephone callswere also implemented when necessaryPostoperative recurrence was diagnosed considering allthe evidence provided by imaging scans and pathologicconfirmation Initial disease recurrence in the supraclavicularregion was defined as SCR and first relapse developed at thesurgical marginipsilateral hilum andor mediastinum wasconsidered LRR Initial disease recurrence beyond LRR and SCRwas categorized as distant metastasis DMPattern of Failure AnalysesFor patients with LRR the PTVs were restored for thosewho received PORT and virtual PTVs were created for thosewho didn™t receive PORT by independent radiation oncologistaccording to ourinstitutional protocol mentioned aboveMeanwhile for patients with SCR individual virtual PTVs werecreated for ipsilateral ESRT PTVsc by independent radiationoncologist according to the CT atlas proposed by Lynch et al Then we plotted the sites of LRRs andor SCRs and overlaidthem with restored or created PTVs Coverage of the LRRs andSCRs by the PTVs were investigatedStatistical AnalysesRecurrence free survival RFS was calculated from surgery toinitial disease recurrence Overall survival OS1 was calculatedfrom surgery to death of any cause in the entire cohort andOS2 was calculated from initial disease recurrence to deathof any cause in patients with recurrent disease Diï¬erences andbetween clinical parameters were compared using the χFisher™s exact tests The predictors of SCR were selected usingcompeting risk methodology and Stata version softwareStataCorp College Station TX USA The associations betweenclinicpathological parameters and OS were identified using theCox proportional hazard regression model The hazard ratioHR and the confidence interval CI were calculatedusing coefficients from the model Kaplan“Meier methodwas used to estimate survival and diï¬erences among groupswere investigated by the logrank test Statistical analysis wasperformed using SPSS SPSS Chicago IL USA Allassessment is considered to be significant when twosided pvalueis RESULTSPatients CharacteristicsA total of patients were finally enrolled and a flowchartfor patient selection was presented in Supplementary Figure Detailed baseline disease characteristics of the patientswere summarized in Table The majority of patients had ahistology of nonsquamous NSCLC and received lobectomyTABLE Disease characteristicsVariablesNumber of patients Age at diagnosis years‰¤SexFemaleMaleSmoking historyEver smokerNever smokerECOG performance scoreClinical N stagecN0“cN2Neoadjuvant chemotherapyYesNoSurgery typeSublobarLobectomyPneumonectomyPathological T stagepT0“pT3“Lymphovascular invasionAbsentPresentVisceral pleural invasionAbsentPresentTumor locationLeft lower lobeLeft upper lobeRight lower lobeRight middle lobeRight upper lobeHistologySquamous cell carcinomaNonsquamous nonsmall cell lung cancerECOG Eastern Corporative Oncology Group Frontiers in Oncology wwwfrontiersinAugust Volume 0cLiu et alSupraclavicular Recurrence in ypN2 NSCLCFIGURE Patterns of supraclavicular recurrence A Venn diagram demonstrating the distribution of initial postoperative recurrence B Pie chart demonstrating thedistribution of SCR SCR supraclavicular recurrence LRR locoregional recurrence DM distant metastasisFIGURE Cumulative incidence and dynamics of supraclavicular recurrence A Cumulative incidence of supraclavicular recurrence in the entire cohort andstratified by pathological status ypN2 vs pN2 B The dynamics of hazard ratio of supraclavicular recurrenceThe positive rate of HER2 TTF1 ERCC1 CK20 CK56 CK7P63 NapsinA Syn RRM1 and EGFR was and respectivelyAdditionally Ki67 ‰¥ was detected in of the patientsPretreatment PETCT was performed in patients andinvasive staging of the mediastinum was underwent in patients One hundred and sixtyfour patients were found tohave cN2 disease among whom patients receivedpretreatment PETCT and patients had invasivestaging of the mediastinum A total of patientsreceived neoadjuvant chemotherapyCumulative Incidence and Risk Factors ofSCRPost surgery patients received PORT and with a medianfollow up of range “ months patients developedrecurrent disease including SCRs Of note of the SCRswere pathologically confirmed and the rest were diagnosed byclinical assessments and radiographic findings The and year RFS were and in patients without PORTrespectively and were and in patients withPORT respectively Among the patients with SCR patients developed SCR without DM Figure 1A and patients developed SCR involving the ipsilateral supraclavicularregion Figure 1B Moreover among the patients with leftlung cancer who developed SCR seven were ipsilateral threebilateral and two contralateral Among the patients with rightlung cancer who developed SCR nine were ipsilateral threebilateral and three contralateralThe and 5year cumulative incidence of SCR were and respectively Figure 2A and the dynamicof hazard ratio of SCR was presented in Figure 2B ChosenDM as a competing event cN2 disease ypN2lobectomyand CK7 were identified as significant risk factors of SCRFrontiers in Oncology wwwfrontiersinAugust Volume 0cLiu et alSupraclavicular Recurrence in ypN2 NSCLCTABLE Competing risk analyses of clinicalpathological variables associatedwith supraclavicular recurrenceVariablesUnivariate Analyses Multivariate AnalysesHR 95CIpHR 95CIpAge vs ‰¤ “ Sex Male vs Female “ Smoking Never vs Ever “ ECOG vs cN2 vs “ “ “ pT stage T3“ vs T0“ “ pN1 vs “ “ Multiple levels of pN2 vs “ “ Histology SCC vs NonSCC “ Differentiation P vs WM “ LVI vs “VPI vs “PNI vs “ypN2 vs pN2 “ “ “ “ “ Tumor Location Left vs Right “ Tumor Lobe Upper vs Others “ TLN ‰¥ vs “ PLN ‰¥ vs “ LNR ‰¥ vs “ Surgery Others vs Lobectomy “ “ PORT vs “ERCC1 vs “Her2 vs “ “ “ “ Ki67 ‰¥ vs “ TTF1 vs “CK20 vs “CK7 vs “CK56 vs “P63 vs “ “ “ “ “ “ “ NapsinA vs “ “ Syn vs “RRM1 vs “EGFR vs “ “ “ “ HR hazard ratios CI confidence intervals SCC squamous cell carcinoma LVILymphovascular invasion VPI Visceral pleuralinvasion ECOGthe Eastern Corporative Oncology Group TLN total lymph node examined PLN positivelymph node LNR positive lymph node ratio PORT postoperative radiotherapy WMwellmoderate P poor Bold values indicates statistical significantinvasion PI perineuralusing the univariate competing risk analysis Table Sincethere was a significant association between cN2 disease and testreceiving neoadjuvant chemotherapy p χwe excluded cN2 disease and included the otherthreesignificant risk factors in the multivariate competing riskanalyses The result showed that only ypN2 were identifiedas an independent risk factor of SCR Table The and 5year cumulative incidence of SCR were and among ypN2 patients respectively and were and among pN2 patients respectivelyFigure 2APattern of Failure AnalysesIn the entire cohort of the patients who receivedPORT developed LRR while of the patientswho did not receive PORT developed LRR PORT significantlyreduced the risk of LRR Figure 3A Among the six patients whoreceived PORT and subsequently developed LRR five developedLRR only within the PTV and the rest one developed LRRboth within and outside the PTV Among the patients whodid not receive PORT and subsequently developed LRR developed LRR only within the proposed PTV three developedLRR both within and outside the proposed PTV and the restone developed LRR outside the proposed PTV That patienthad adenocarcinoma in the middle lobe of right lung withpathologically proven metastatic lymph node in the right hilumand station but developed recurrent disease at mediastinallymph node stations and On the other hand of the patients who receivedPORT developed SCR while of the patients whodid not receive PORT developed SCR in the entire cohort PORTwithout ESRT didn™t reduce the incidence of SCR Figure 3BFifteen of the ipsilateral SCRs could be covered by theproposed PTVsc and the ipsilateral parts of the six bilateral SCRscould all be covered by the proposed PTVscSurvival AnalysesBy the time of data cutoï¬ patients had died and the medianOS1 was 95CI “ months PORT was found tosignificant prolong OS1 in the entire cohort Figure 3C Agesex ECOG scorelymphovascular invasion total number ofpositive lymph node positive lymph node ratio PORT and Ki67were found to be significantly associated with OS1 in univariateCox analyses while age ECOG score PORT and Ki67 wereidentified to be independent indicators of OS1 in multivariateCox analyses Table Among the patients with recurrentdisease the median OS2 was 95CI “ monthsAge sex ECOG score and DM were revealed to be significantlyassociated with OS1 in univariate and multivariate Cox analysesTable In order to investigate the prognostic significance of SCRpatients with recurrent disease were further divided into threegroups Group A consisted of patients who had DM n Group B consisted of patients who did not have DM but have SCRn and Group C consisted of patients who only had LRR n In terms of OS2 patients in Group B had an intermediateprognosis when compared with patients in Group A and GroupC Figure 3DDISCUSSIONTo the best of our knowledge this is the first comprehensivestudy specifically focusing on SCR in completely resected ypN2NSCLC with a relatively large sample size in the era of modernradiation technique SCR was not uncommon and had imperativeprognostic significance indicating that treatment modalities ableto reduce the incidence of SCR may be beneficial AdditionallyPORT without ESRT significantly reduced LRR and prolongedOS but did not decrease SCR in our study suggesting that theFrontiers in Oncology wwwfrontiersinAugust Volume 0cLiu et alSupraclavicular Recurrence in ypN2 NSCLCFIGURE Prognostic significance of postoperative radiotherapy and supraclavicular recurrence The impact of postoperative radiotherapy PORT on locoregionalrecurrence LRR A supraclavicular recurrence SCR B overall survival OS1 C in the entire cohort Kaplan“Meier survival curve stratified by the diseaserecurrence patterns among patients with recurrent disease Dclinical value of ESRT may be reconsidered in selected patientswith high risks of SCRSCR is not uncommon in completely resected ypN2 NSCLCespecially among those with extra risk factors Although therewas limited historical data published that could be directlycompared the incidence of SCR in our study was reliable sincethe overall recurrence rate and the percentage of SCR amongpatients with recurrent disease were in accordance with previousfindings The cumulative incidence of postoperative recurrencein the PORT group and nonPORT group were generallycomparable with recent studies Furthermorestudies from our institution and others had reported asimilar percentage of SCR among patients with recurrent disease“ in the literature in our study Compared withtheir counterpart patients staged cN2 or ypN2 generally had amore advanced and aggressive disease and thus it was reasonablefor them to have a higher risk developing disease recurrenceincluding SCR “ Compared with those receivinglobectomy patients receiving pneumonectomy generally hada higher tumor burden and those receiving sublobectomycommonly had unfavorable prognostic factors such as morecomorbidities and poorer preoperative lung functions that madethem unsuitable for lobectomy Therefore patients whodidn™t receive lobectomy were also at a higher risk developingpostoperative recurrence which is generally consistent with arecent retrospective study using the SEER database Inaddition two recent studies found that positive expression ofCK7 were associated with more advanced disease and shorteroverall survival In our study distant metastasis waschosen as a competing event and negative expression of CK7was identified as a risk factor of SCR which need to befurther verifiedCompared with patients developing only LRR and thosedeveloping DM patients developing SCR but without DM hadintermediate OS2 highlighting the vital prognostic significanceof SCR in curatively resected ypN2 NSCLC The TNMstaging system is one ofindicators ofpatient™s prognosis in NSCLC among which patients havingsupraclavicular lymph node metastasis N3 generally haveintermediate prognosis when compared with those having distantmetastasis M1 and those harboring metastatic tumor lesionslimited to the ipsilateral hilar N1 or mediastinal N2 lymphthe most powerfulFrontiers in Oncology wwwfrontiersinAugust Volume 0cLiu et alSupraclavicular Recurrence in ypN2 NSCLCTABLE Cox analyses of clinicalpathological variables associated with overallsurvival OS1TABLE Cox analyses of clinicalpathological variables associated with OS2 inpatients with recurrent diseaseVariablesUnivariate Analyses Multivariate AnalysesVariablesUnivariate AnalysesMultivariate AnalysesHR 95CIpHR 95CIpHR 95CIpHR 95CIpAge vs ‰¤ “ “ Sex Male vs Female “ “ Smoking Never vs Ever “ ECOG vs cN2 vs “ “ “ “ pT stage T3“ vs T0“ “ pN1 vs “ “ Multiple levels of pN2 vs “ “ Histology SCC vs NonSCC “ Differentiation P vs WM “ LVI vs “VPI vs “PNI vs “ypN2 vs pN2 “ “ “ “ “ Tumor Location Left vs Right “ Tumor Lobe Upper vs Others “ TLN ‰¥ vs “ Age vs ‰¤ “ “Sex Male vs Female “ “Smoking Never vs Ever “ECOG vs cN2 vs “ “ “ “pT stage T3“ vs T0“ “pN1 vs “ “Multiple levels of pN2 vs “ “Histology SCC vs NonSCCDifferentiation P vs WM “ “LVI vs “VPI vs “PNI vs “ypN2 vs pN2 “ “ “ “Tumor Location Left vs Right “Tumor Lobe Upper vs Others “TLN ‰¥ vs “PLN ‰¥ vs “LNR ‰¥ vs “PLN ‰¥ vs “ “ Surgery Others vs Lobectomy “LNR ‰¥ vs “ “ Surgery Others vs Lobectomy “ PORT vs “ERCC1 vs “Her2 vs “ “ “ “ “ Ki67 ‰¥ vs “ “ TTF1 vs “CK20 vs “CK7 vs “CK56 vs “P63 vs “ “ “ “ “ “ NapsinA vs “ “ Syn vs “RRM1 vs “EGFR vs “ “ “ “ HR hazard ratios CI confidence intervals SCC squamous cell carcinoma LVILymphovascular invasion VPI Visceral pleuralinvasion ECOGthe Eastern Corporative Oncology Group TLN total lymph node examined PLN positivelymph node LNR positive lymph node ratio PORT postoperative radiotherapy WMwellmoderate P poor Bold values indicates statistical significantinvasion PI perineuralnodes Similarly SCR represented an unfavorable sign ofsubsequent disease metastasis to distant ans and thus wasreasonable to have worse prognosis when compared with thosewho had only LRR On the other hand when compared withthose who already had DM patients who had recurrent diseaselimited to the thoracic region ie LRR and SCR could beconsidered as harboring locoregional disease and may benefitfrom aggressive locoregional treatment as well as systematictherapies and thus may still have a chance of longterm survival In fact among the patients with SCR but without DMthe 3year survival rate exceeded in our study Figure 3DHowever due to the advancement of adjuvant chemotherapy andPORT vs “DM vs “ERCC1 vs “Her2 vs “ “ “ “ “ “Ki67 ‰¥ vs “TTF1 vs “CK20 vs “CK7 vs “CK56 vs “P63 vs “NapsinA vs “Syn vs “RRM1 vs “EGFR vs ““ “ “ “ “ “ “ “ “OS overall survival HR hazard ratios CI confidence intervals SCC squamouscell carcinoma LVI Lymphovascularinvasion PIperineural invasion ECOG the Eastern Corporative Oncology Group TLN total lymphnode examined PLN positive lymph node LNR positive lymph node ratio PORTpostoperative radiotherapy DM distant metastasis WM wellmoderate P poor Boldvalues indicates statistical significantinvasion VPI Visceral pleuralPORT the number of patients who developed localized recurrentdisease ie LRR and SCR was small patients in group Band patients in group C although a total of patients wereenrolled and followed up for a median of months Hencethe prognostic significance of SCR needed to be interpreted withcaution and future investigations with larger sample size andprospective design are warrantedThe clinical value of PORT in completely resected ypN2NSCLC was demonstrated again in our study but the delineationof CTV remain controversial In the current study PORTsignificantly reduced LRR and improved OS1 which havebeen demonstrated in various studies “ However since ESRT was not routinely performed in our cancerFrontiers in Oncology wwwfrontiersinAugust Volume 0cLiu et alSupraclavicular Recurrence in ypN2 NSCLCindicating thatinstitution PORT failed to reduce SCRthe majority of SCRs represented the outgrowth of subclinicaltumor lesions already in the supraclavicular region and werenot originated from the locoregional recurrent disease throughlymphatic metastasis In fact of the patientswith SCR had no LRR in the current study These dataindicated a potential role of ESRT in selected patients withhigh risks Actuallyfor locally advanced NSCLC receivingchemoradiotherapy there is no significant diï¬erence of patient™ssurvival between those with or without N3 disease highlighting that the treatment efficacy of chemoradiotherapyin locally advanced NSCLC was largely dependent on theintrinsic biology of the tumor and the prognosis of patients withor without macroscopic supraclavicular tumor lesions seemedsimilar PORT with adjuvant chemotherapy has been repeatedlyshown to significantly reduce LRR indicating the beneficial roleof adjuvant chemoradiotherapy in treating microscopic N1N2disease It is possible that adjuvant chemoradiotherapy ieadjuvant chemotherapy in combination with ESRT may alsoplay a role in reducing SCR and subsequently improve patient™ssurvival Furthermore nearly of the ipsilateral SCRs couldbe covered with the virtual CTV of ESRT in our study Howeverthere are also evidence against the use of ESRT for patients withcompletely resected NSCLC Elective irradiation of mediastinalcontralateral hilar and supraclavicular lymph nodes failed toimprove patient™s survivalin unresectable stage III NSCLCwithout clinical N3 disease And pattern of failure analyses ofa prospective trial of PORT without ESRT suggested that the useof limited CTV including only the involved lymph node stationsand those with a risk of invasion was associated withacceptable risk of geographic miss Taken together PORTwithout ESRT provided significant clinical benefit for patientswith completely resected ypN2 NSCLC and the clinical valueof ESRT in highly selected patients for example those withpersistent N2 ypN2 disease after neoadjuvant chemotherapyneed to be further investigatedOur study also has some limitations Firstly since ESRT is notroutinely performed in our cancer center we could not directlyexamine the clinical value and prognostic significance of ESRTSecondly as this was a retrospectively study treatment decisionsand followup strategies were at the discretion of the treatingphysicians Diï¬erent neoadjuvant and adjuvant chemotherapyregimens were used and the protocols of followup were notidentical Moreover since brain MRI and bone scans were notmandatory asymptomatic brain andor bone metastasis may beunderestimated Despite these limitations we believe our studyprovided valuable information about the cumulative incidenceand prognostic significance of SCR in completely resected ypN2NSCLC which may guide better design of adjuvant treatmentmodalities and individualized surveillance strategiesDATA AVAILABILITY STATEMENTThe raw data supporting the conclusions of this will bemade available by the authors without undue reservationETHICS STATEMENTThe studiesinvolving human participants were reviewedand approved by the institutional review board of FudanUniversity Shanghai Cancer Center Written informed consentfor participation was not required for this study in accordancewith the national legislation and the institutional requirementsAUTHOR CONTRIBUTIONSLL ZZ and JN conceptualization LL and ZZ methodologyvalidation and writing”original draft preparation LL ZZ andJL formal analysis and investigation LL ZZ and YL resourcesand data curation LL and JN writing”review and editing Allthe authors have approved the final manuscriptFUNDINGThis study was supported by the National Natural ScienceFoundation of China No to JN and grant provided bythe Shanghai Municipal Health Commission No 20194Y0501to JNSUPPLEMENTARY MATERIALThe Supplementary Materialonline202001414fullsupplementarymaterialfor this can be foundhttpswwwfrontiersins103389foncatSupplementary Figure Flowchart of patient enrollment NSCLC nonsmallcell lung cancerREFERENCES Eberhardt WE De Ruysscher D Weder W Le Pechoux C De Leyn PHoï¬mann H et al 2nd ESMO Consensus Conference in Lung Cancerlocally advanced stage III nonsmallcell lung cancer Ann Oncol “ 101093annoncmdv187 Tan WL Chua KLM Lin CC Lee VHF Tho LM Chan AW et alAsian thoracic oncology research group expert consensus statement onoptimal management of stage III NSCLC J Thorac Oncol “ 101016jjtho201910022 Kenmotsu H Yamamoto N Yamanaka T Yoshiya K Takahashi T Uenostudy of pemetrexed plus cisplatintoT et al Randomized phase IIIversus vinorelbine plus cisplatin for completely resected stage IIIIIA nonsquamous nonsmallcell“ 102139ssrn3460654lung cancerJ Clin Oncol Kato H Tsuboi M Kato Y Ikeda N Okunaka T Hamada C Postoperativeadjuvant therapy for completely resected earlystage nonsmall cell lungcancer Int J Clin Oncol “ 101007s101470050493x Herskovic A Mauer E Christos P Nagar H Role of postoperativeradiotherapy in pathologic stage IIIA N2 nonsmall cell lung cancer in aprospective nationwide oncology outcomes database J Thorac Oncol “ 101016jjtho201609135 Deng W Xu T Xu Y Wang Y Liu X Zhao Y et al Survival patterns for patientswith resected N2 nonsmall cell lung cancer and postoperative radiotherapya prognostic scoring model and heat map approach J Thorac Oncol “ 101016jjtho2018082021Frontiers in Oncology wwwfrontiersinAugust Volume 0cLiu et alSupraclavicular Recurrence in ypN2 NSCLC Feng W Fu XL Cai XW Yang HJ Wu KL Fan M et al Patternsof localregional failure in completely resected stage IIIAN2 nonsmallcellimplications for postoperative radiation therapyclinical target volume design Int J Radiat Oncol Biol Phys “ 101016jijrobp201312048lung cancer cases Dai H Hui Z Ji W Liang J Lu J Ou G et al Postoperative radiotherapy forresected pathological stage IIIAN2 nonsmall cell lung cancer a retrospectivestudy of cases from a single institution Oncologist “ 101634theoncologist20100343 Zou B Xu Y Li T Li W Tang B Zhou L et al A multicenter retrospectiveanalysis of survival outcome following postoperative chemoradiotherapy innonsmallcell lung cancer patients with N2 nodal disease Int J Radiat OncolBiol Phys “ 101016jijrobp200905044 Billiet C De Ruysscher D Peeters S Decaluwe H Vansteenkiste J Dooms Cet al Patterns of locoregional relapses in patients with contemporarily stagedstage IIIN2 NSCLC treated with induction c Answer:

Thyroid_Cancer

Computed Tomography Features andClinicopathological Characteristicsof Gastric Sarcomatoid CarcinomaYiyang Liu1 Pan Liang1 Kaixiang Feng2 Kuisheng Chen3 Songwei Yue1 Jiang Ji4Weiwei Li1 Xitong Zhao1 and Jianbo Gao1 Department of Radiology The First Affiliated Hospital of Zhengzhou University Zhengzhou China Departmentof Thyroid Surgery The First Affiliated Hospital of Zhengzhou University Zhengzhou China Department of PathologyThe First Affiliated Hospital of Zhengzhou University Zhengzhou China Department of Radiology General Hospital NingxiaMedical University Yinchuan ChinaPurpose Gastric sarcomatoid carcinoma GSC is a very rare malignant tumor Thepurpose of this study is to describe the clinical computed tomography CT andpathologic features of GSC to increase awareness of this entityEdited byZiwen LiuPeking Union Medical CollegeHospital CAMS ChinaMethods The CT features and clinical data of five patients with pathologicallydocumented GSC were retrospectively analyzed and compared with the correspondingdata of gastric adenocarcinoma and lymphomaReviewed bySavio Gee BarretoMedanta The Medicity IndiaHaruhiko SugimuraHamamatsu University Schoolof Medicine JapanXiuying XiaoShanghai Jiao Tong University ChinaCorrespondenceJianbo GaofccyisunshinegszzueducnSpecialty sectionThis was submitted toGastrointestinal Cancersa section of the journalFrontiers in OncologyReceived May Accepted July Published August CitationLiu Y Liang P Feng K Chen KYue S Ji J Li W Zhao X and Gao J Computed TomographyFeatures and ClinicopathologicalCharacteristics of GastricSarcomatoid CarcinomaFront Oncol 103389fonc202001611Results Among the patients were male and was female The median agewas years Of the cases of GSC were in the gastric fundus and cardia was in the gastric body and was in the gastric fundus The gastric wall had localthickening in cases and mass formation in case with stenosis and deformationof the adjacent gastric cavity The longaxis diameter of the lesions ranged from to cm mean cm and was cm in cases and cm in case The tumorshowed predominantly inhomogeneous density with radiodensity values ranging from to HU In addition ulcers with an irregular base and slightly raised borders wereobserved in of cases After an injection of contrast material heterogeneous n or homogeneous n enhancement was observed After contrast medium injectionobvious enhancement was seen in cases and moderate enhancement was seen in cases the peak tumor signal was observed in the portal phase Two of the patientsdemonstrated evidence of lymph node involvement and in one patient the boundarybetween the lesion and the left lobe of the liver was unclear with low attenuation in theright lobe of the liver with circular enhancement The remaining two patients showed noevidence of metastasisConclusion Although GSC is extremely rare it should be considered in the differentialdiagnosis of gastric adenocarcinoma and lymphoma CT findings combined withpatient age and sex can provide support for the diagnosis of GSC However the finaldiagnosis must be confirmed with histopathologyKeywords sarcomatoid carcinoma stomach gastric cancer tomography Xray computed diagnosisFrontiers in Oncology wwwfrontiersinAugust Volume 0cLiu et alINTRODUCTIONSarcomatoid carcinomas SCs are extremely rare aggressivemalignant tumors characterized by distinct cellular morphology The features of this tumor were first described in by Snover SCs can occur in a wide variety of sitesincluding the respiratory tract digestive tract genitourinary tractbreast and thyroid glands However these tumors are rarein the digestive tract especially in the stomach As of April there are only six cases of gastric sarcomatoid carcinomaGSC reported in the English medical literature These previousreports focused on the pathological and clinical manifestationsthem have not systematically described the radiologic appearanceof the tumor Due to the more invasive nature and poorerprognosis of GSC than pure gastric adenocarcinoma GACand gastric lymphoma GL it is clinically beneficial to narrowdown the diï¬erential diagnoses by understanding the computedtomography CT characteristics of GSC The present studyanalyzed our experience in diagnosing five patients with GSCin terms of the imaging findings and clinical features To thebest of our knowledge our study represents the largest seriesof GSCs to dateIn addition due to the rarity of GSC the diï¬erential diagnosisbetween GSC and other types of malignant gastric tumors hasnot received much attention so we also initially explored thediï¬erential diagnosis of GSC from GAC and GLMATERIALS AND METHODSThe protocol was approved by the Medical Ethics Committeeof Zhengzhou UniversityInformed consent was obtainedfrom all patientsPatient SelectionFrom August to January we searched the pathologyrecords and the Picture Archiving and Communication SystemsPACS of our hospital The search terms included stomachand sarcomatoid carcinomas A total of five patients wereidentified as having SC and were enrolled in the present study Weretrospectively reviewed all clinical data demographic featureslaboratory findings clinical interventions and the histologicfindings of the five biopsy or operation specimensCT EvaluationFive GSC patients underwent CT examinations The CTscans were acquired with a 64row multidetector deviceDiscoveryCT750HD GE Healthcare Waukesha WIUnited States Conventional axial scanning was performedbefore and after an intravenous iv injection of nonioniciohexol iopromide mgmL GE Medical Systems mLkgand mLs through a dualhead pump injector MedradWarrendale PA United States The imaging parameters wereas follows tube voltage kV tube current mA fieldof view FOV mm matrix — mm and sectionthickness mm Finally a 20mL saline flush was performedat a rate of mLsGastric Sarcomatoid CarcinomaContrastenhanced CT scans were acquired with scanningdelays of s arterial phase AP and s portal venous phasePP after the iv injection of the contrast agent started The CTdose index volume for the three phases was mSvImage AnalysisTwo experienced radiologists and years of abdominal CTexperience performed a retrospective analysis of the CT imagesAll analyses were performed with an AW47 workstation GEHealthcare and the radiologists were blinded to the clinicalinformation of the patients The evaluated parameters includedthe tumor location gastric cardia gastric fundus gastric bodygastric angle and gastric antrum longaxis diameter shapegrowth pattern serosa condition attenuation and enhancementcharacteristics such as the enhancement pattern and degreeof enhancement The enhancement pattern of the tumor wasclassified as homogeneous or heterogeneous based on the APimage The degree of enhancement of the tumor was based ondynamic CT imaging using HU attenuation where œobviousenhancement was defined as HU œmoderate enhancementas HU and œmildly enhancement as HUThe GSCs were staged with the Union for InternationalCancer Control UICC TNM staging standard All imagingfindings were compared with the postoperative pathologicalfindings The accuracy rate the number of CTs coincidentwith the pathological diagnosisthe number of actual pathologicaldiagnoses — Pathological EvaluationThree patients underwent gastrectomy and two underwentendoscopic biopsy The three gastrectomy specimens measured cm — cm — cm cm — cm — cmcm — cm — cm respectively in two of theseand tumors the mucosal surface of the excised specimen showedulcerative masses of approximately cm — cm — cmand cm — cm The remaining specimen was a soft massmeasuring cm — cm — cm For biopsy multiple sampleswere acquired and the diameter of each sample was cmAccording to the relevant literature the diagnostic criteria fSC were generally as follows the tumor originated fromthe stomach and the tumor consisted of both carcinomatousand sarcomatoid components and the sarcomatoid componentaccounted for more than of the tissue In addition if biopsywas performed the sarcomatoid component can be seen in everysample Furthermore sarcomatoid regions express epithelialmarkers such as CK or EMAThe specimens were fully stretched fixed and soaked in formaldehyde solution for h All biopsy specimenswere analyzed The specimens underwent routine dehydrationparaffin embedding sectioning into µm thick sectionsand hematoxylin eosin HE staining Immunohistochemicalstaining was performed using a Roche BenchMark XT automaticimmunohistochemical detector The antibodies used in thisstudy included AE1AE3 CKL CK818 epithelial membraneantigen EMA vimentin P40 P63 and antigen KI67 Ki67All antibodies listed above were purchased from DAKO DakoGlostrup DenmarkFrontiers in Oncology wwwfrontiersinAugust Volume 0cLiu et alGastric Sarcomatoid CarcinomaTABLE Comparison between GSC and GAC GLAge median age rangeMain symptomsEpigastric discomfortpainIntermittent vomitingAcute hematemesisBloody stoolDysphagiaLocationCardia and FundusBodyAntrumThe longaxis diameter median size rangeShapeFocal thickeningDiffuse thickeningMassSerosal surfacebare areaClearUnclearUlcersYesNoDensity characteristicsHeterogeneousHomogeneousEnhancement patterHeterogeneousHomogeneousLymph node involvementYesNoLiver involvementYesNoTherapyResectionChemotherapyResection and ChemotherapyNeoadjuvant chemotherapyRadiation therapyGSCGACGL “ years “ years “ years “ cm “ cm “ cm Comprehensive Comparative AnalysisEach patient with GSC was matched by age ± years year ofdiagnosis and sex to four patients with GAC GL patients witheach disease were retrieved from PACS Patients with GSC werecompared with those with GAC GL in terms of demographicclinical and CT characteristics Table RESULTSPatient CharacteristicsThe patients included four men and one woman ranging inage from to years with a median age of years Theclinical and CT features of these patients are summarized inTables All patients had nonspecific symptoms includingabdominal discomfort epigastric discomfort nausea or vomitingThe other presenting symptoms included hematemesis or weightloss Three patients underwent radical resectionin whichonly one patient was treated with adjuvant chemotherapyafter surgery And two patients chose to deny treatment Inaddition we also reviewed the upper gastrointestinal radiographyresults Figure The laboratory findings revealed that patient was positivefor tumor abnormal protein TAP and patient was positivefor carbohydrate antigen CA125 Before treatmenthemoglobin and erythrocyte count decreased in three patientsFrontiers in Oncology wwwfrontiersinAugust Volume 0cLiu et alGastric Sarcomatoid CarcinomaTABLE Clinical and pathological factors of the five GSC patientsCaseSexAge yearsComplaintLocationMaximumdiameterTumor markercmAnemiaTherapyMetastasisMMFMMSuddenhematemesisEpigastricdiscomfortIntermittentvomitingEpigastric painEpigastric painEpigastric painLesser curvatureRemnant stomachCardia andFundusCardia and FundusFundusCardia and FundusNormalTAP CA125 NormalNA““RRnNoneNoneRC“““œ yespresentpositive œ“ noabsentnegative F female M male age in years R Radical gastrectomy Rn Remnant gastrectomy C chemotherapyNA not availableTABLE Computed tomography features of the five GSC patientsCaseGross features of the tumorUlcersGrowth modeDensity characteristicsEnhancement patterFocal thickeningFocal thickeningMassFocal thickeningFocal thickening“IntracavityIntracavityIntracavityIntracavityIntracavityHeterogeneousHeterogeneousHomogeneousHeterogeneousHeterogeneousHeterogeneousHeterogeneousHomogeneousHeterogeneousHeterogeneousMarginUnclearUnclearUnclearClearUnclearœ yespresentpositive œ“ noabsentnegativeFIGURE Characteristics of Xray examinations of a 65yearold male patient with GSC AB Reveals that there is a huge niche with irregular shapes at the smallcurvature of the stomach the niche is located inside the outline of the stomach the niche is surrounded by transparent bands with different widths that is ringembankments with irregular outlines The surrounding mucosa is thickened interrupted and the local gastric cavity is narrowedpatients and and platelet count was elevated in fourpatients patients and Pathological FeaturesMicropathologically the gastric tumor cells showed infiltrativegrowth The cytological characteristics ofthe tumor cellsshowed obvious malignant characteristics Microscopicallythe spindle cell structure and the nucleus were obviouslyatypical pleomorphic and enlarged Mitotic figures were visibleFigures 2AB On immunohistochemical examination thetumor cells showed positive staining for AE1AE3 CKLCK818 EMA P40 vimentin The Ki67 index was higher than Figures 2C“I All five tumors were diagnosed as GSCIn addition the sarcomatoid component showed spindle cellsarcomatoid morphologyCT FindingsOf the cases of GSC were in the gastric fundus and cardiaFigure was in the gastric body and was in the gastricfundus of these tumors one was a recurrence in the remnantstomach The CT manifestations of this tumor included localthickening n mass formation n The longaxisFrontiers in Oncology wwwfrontiersinAugust Volume 0cLiu et alGastric Sarcomatoid CarcinomaFIGURE Histological and immunohistochemical features of GSC AB Hematoxylineosin HE staining showing tumor cells demonstrated spindleshapedstructures significant atypical nuclei pleomorphic nuclei and giant nuclei Mitotic figures visible Tumor cells showed infiltrative growth Cells were stained withhematoxylin and eosin stain magnification A — B — By immunohistochemistry the tumor cells were positive for AE1AE3 C CKL D CK818 E EMAF P40 G and vimentin H Moreover of them were positive for Ki67 I The final diagnosis was SC [magnification C“I —]diameter of the lesions ranged from to cm mean size cm In addition ulcers with an irregular base and slightlyraised borders were observed in of cases Among the threepatients who underwent surgery two lesions invaded the gastricserosa and the remaining lesion invaded the gastric bare areaAmong the two patients with biopsyproven GSC one patientexhibited tumor invasion of the gastric bare area The majorchanges in the CT imaging characteristics were an irregularouter layer of the gastric wall and obscuration of the perigastricfat Initially the CT findings were interpreted as GAC in fourcases and GL in The tumor showed predominantly inhomogeneous densityand the radiodensity values were “ HU in the noncontrastphase Heterogeneous enhancement was seen in four casesdue to necrotic or cystic areas and the other tumor revealedhomogeneous enhancement The radiodensity values on the APimages ranged from to HU and to HU in thevenous phase After contrast medium injection two tumorsshowed obvious enhancement and moderate enhancementwas seen in the other three tumors the peak tumor valuewas observed in the portal phase One of the three patientswho underwentlymphsurgery demonstrated evidence ofin one patientthe boundary betweennode involvementthe lesion and the left lobe of the liver was unclear andthe area with low attenuation was confirmed by pathologythe liver withas a metastatic lesion in the rightcircular enhancement The remaining patientshowed noevidence of metastasis Among the two patients with biopsyspecimens one patient was identified as having lymph nodemetastasis on CTlobe ofCT Staging Versus Pathological Stagingof GSCNone of the GSCs were staged as T1T2 by CT or pathologyThe accuracy of CT staging T3 and T4 GSC was and respectively The overall diagnostic accuracy of CTfor determining the T stage of GSC was None of the GSCs were staged as N2N3 by CT or pathologyThe accuracy of CT in staging N3 and N4 GSC was and respectively The overall diagnostic accuracy of CT fordetermining the N stage of GSC was The comparison of TN staging based on CT and pathology isshown in Table Frontiers in Oncology wwwfrontiersinAugust Volume 0cLiu et alGastric Sarcomatoid CarcinomaFIGURE Sarcomatoid carcinoma of the stomach in 62yearold women A Unenhanced CT image of stomach reveals an intraluminal mass of homogeneousattenuation with an irregular surface at the gastric fundus and cardiac region B“D Contrastenhanced CT image shows obvious homogeneous enhancement ofmass with the peak value of the tumor on the portal phase In perigastric lymph nodes an enlarged and significantly enhancement lymph node can be seenB Arterial phase of contrast enhancement image C Portal phase of contrast enhancement image D Portal phase of contrast enhancement coronal imageDISCUSSIONTABLE CT and pathological TN staging for comparisonSarcomatoid carcinoma is an extremely rare and complicatedmalignant tumor composed of malignant epithelial componentsand atypical spindle cells However the spindle cells of SCsappear to show evidence of epithelial diï¬erentiationforexample showing epithelial markers or epithelial ultrastructuralinstead of a specific line of mesenchymalcharacteristicsdiï¬erentiation Moreoverliteratureemphasizes that the sarcomatous components occupy ofthe elements involved In the present study our patients™tumor cells displayed atypical spindle shapes that expressed theepithelial phenotypethe currentsome ofSarcomatoid carcinomas can occur in almost any an wherecarcinoma can occur In the digestive system the incidencesof SCs in the esophagus and liver are relatively high but SCsare exceedingly rare in the stomach we could find only sixprevious reports in the English literature Table Between and patients with SC confirmed by pathologywere retrospectively analyzed with only five tumors occurringin the stomach The average age of the reported patientswas years range “ and that in our series was years range “ A previous study reported that the sexCaseNO NO NO NO NO CTT4aN0T3N0T3N1T3N0T4aN1NA not available T tumor N nodePathological stageT4aN1T3N0NANAT4aN0distribution of male to female GSC patients was and thecorresponding proportion in our patients was “ Ithas been noticed that SCs are more common in male patientsand sex is a probable risk factor GSC patients may present withepigastric pain or discomfort dysphagia nausea and vomitinghematemesis and emaciation Due to thickening of the gastricwall pain or discomfort in the upper abdomen is common Thesymptoms can last from a few days to several years withoutobvious specificityIn the present study of the cases of GSC were recognizedin the proximal stomach and the remaining tumor was founddistal to the stomach Four cases of GSC in the present study hadFrontiers in Oncology wwwfrontiersinAugust Volume 0cLiu et alGastric Sarcomatoid CarcinomaTABLE Clinical and imaging features of six previously reported cases of GCSCaseGenderAgeyearsLocationSize cmShapeUlcersEnhanceappearanceRecurrenceMetastasisTherapyPrognosis MMFFMMRemnant stomachGreater curvatureLesser curvatureGastroesophageal junctionRemnant stomachDistal stomachPolypoidPolypoidPolypoidUlceratedPolypoidMass“““NENENENENEHyperNE“““““NoneSurgerySurgerySurgeryEndoscopySurgeryNA Mo D Mo D Mo D Mo D Mo DThe patient succumbed to heart failure before the surgical treatment An autopsy was performed œ yespresentpositive œ“ noabsentnegative Hyper hyperdenseNE no evaluate Mo Month D Dieareregarded asa longaxis diameter less than cm and the remaining tumorhad the largest longaxis diameter among our patients cmThe location distribution and longaxis diameters of the GSCs inour patients were similar to those in previous reports “in the actual diagnosis processThe diagnosis of SC has always been difficult for cliniciansand pathologists especially the diï¬erential diagnosis fromcarcinosarcoma Carcinosarcomastrulybiphasic neoplasms composed of distinct malignant epithelialcarcinomatous and mesenchymal sarcomatous componentsThe sarcoma components show typical specialized diï¬erentiation Howeverthe termsœsarcomatoid carcinoma and œcarcinosarcoma have been usedinterchangeably in some cases Therefore the understanding ofthese tumors has been hampered Nevertheless we can try tofocus on whether there is a diï¬erence between these tumors froma new perspective The CT finding SC in the stomach have notbeen previously scientific reported or even detailed descriptionThere are only four simple descriptions ChunChao reported that a patient with a giant SC presented a mass witha cm diameter in the antrum and body of stomach whichinfiltrated the gastric serosa The hepatic flexure of the colon andgallbladder were also involved on CT Contrastenhanced CTimages showed obvious enhancement of the two lesions Sato reported a patient with SC of the remnant stomachand the radiographic examination showed an elevated lesionwith a large ulcer at the gastric cardiac lesser curvature thatmeasured cm in diameter The other two reports only describeda soft tissue mass or a large tumor in the dilated stomach On the other hand within in the upper gastrointestinal tractalthough there are fewer reports of carcinosarcoma localized inthe stomach this type of tumor is still more common than SC Gastric carcinosarcoma showed an elevated lesion or thickenedgastric walls in “ of all reviewed cases “ Tomoaki reported a 79yearold man with gastric carcinosarcomaand his veins showed severe invasion Enhanced abdominalCT showed irregular thickening and slight enhancement of thegastric wall on the side of the lesser curvature with suspiciousbulky lymph nodes Yoshiyuki reported a 70yearoldJapanese woman who presented with a soft tissue mass adjacentto the lesser curvature of the stomach that was lobulated andCT revealed an ulcer on the lesion The contrastenhanced CTimages showed heterogeneous enhancement of the mass Thefinal pathological diagnosis was gastric carcinosarcoma Inthe present study we found that GSC showed local thickeningof the gastric wall and mass formation often accompaniedby ulcers The site of the disease was mostly in the proximalpart of the stomach but these tumors can also occur in theremnant stomach The signal of the tumor was homogeneousor heterogeneous on plain CT scans After contrast mediuminjection of tumors demonstrated heterogeneousenhancement on AP images due to cystic areas or necrosis inthe lesions In this study the enhancement degree of all tumorsreached a peak in the PP after contrast enhancement For thesetumors the enhancement degree in the delayed phase wasnot significantly reduced The overall enhancement mode wasdelayed enhancement In addition CT showed that four patientshad invasion into the gastric serosal region or gastric bare areatwo patients had the characteristics of enlarged perigastric orretroperitoneal lymph nodes and uneven enhancement and onepatient had invasion into the adjacent liver tissue These findingsreflect the metastatic and highly invasive characteristics of GSCOverall CT and contrastenhanced CT can clearly show theprimary lesion infiltration range lymph node metastasis anddistant metastasis of GSCTomographic diagnosis of GSC has not been attemptedbecause of the rarity of this entity According to the findingsof our study GSC needs to be diï¬erentiated from GAC andGL on CT Adenocarcinoma is the most common pathologicaltype of gastric tumor and is mainly distributed in the antrumseldomly in the body and fundus of the stomach The incidenceof GAC is high in men and the median patient age is years The most common CT signs of GAC are localor extensive thickening of the gastric wall mass formationincluding fungoidestype polypoidtype masses rough orsmooth serous surfaces and continuous interruption of themucosal layer Tumors involving the mucosal surface can appearenhanced “ s after injecting a contrast agent The peakvalue for tumors invading the muscular layer usually appearsafter “ s and after the mucosal surface is strengthenedthe duration is longer Primary GL accounts for “ ofmalignant gastric tumors and is predominantly situated in thegastric antrum gastric body and gastric fundus The incidenceof GL is high among males with a median patient age of yearsThe clinical symptoms included epigastric pain bleeding earlysatiety and fatigue The most common CT manifestations ofGL are diï¬use thickening of the gastric wall or a homogeneousFrontiers in Oncology wwwfrontiersinAugust Volume 0cLiu et alGastric Sarcomatoid Carcinomatissue mass with slight attenuation or an appearancesoftsimilar to that of the normal gastric wall For GL becauseof hemorrhage necrosis submucosal edema or infarction thegastric wall may be heterogenous on CT GL originates froma submucosal process and gastric mucosa is commonly intactin the early stage but shows interruptions or ulceration in thelater stage After contrast medium injection most GL showedhomogeneous and slight enhancement in the delayed phase Lymphoma is considered when distant structures the mesenteryretroperitoneum or other parts of the abdomen have lymphnode metastasis The CT findings may only reflect features of GSC but cannotaccurately diagnose GSC let alone explore the origin of thesarcomatous portion Immunohistochemistry IHC also failedto conclusively establish the origin of GSC Rodrigues usedfluorescence in situ hybridization FISH to confirm that SC andadenocarcinoma have a common origin that is the epithelium while primary GL originated from gastric submucosallymphoid tissueThe main treatment for localized lymphomas is eradicationof Helicobacter pylori and surgical treatment whereas advanceddisease often requires radiation or chemotherapy alone Surgery is the only treatment option for patients with GACAdjuvant chemotherapy and chemoradiotherapy are also oftenused Targeted therapy isin the exploration stage However there are currently no specific National ComprehensiveCancer Network guidelines for the treatment of only GSCbecause the tumor is relatively rare although complete surgicalresection is the most important treatment method For examplewhile chemotherapy is considered in clinical practice whetherchemotherapy can be applied for GSC and the efficacy ofchemotherapy remain controversial Domblides firstevaluated the efficacy of immune checkpoint inhibitors ICIs forSC and found that lung SC patients exhibited high response ratesand prolonged overall survival OS with ICIs This studyprovides a new idea for the treatment of GSCBecause GL tends to be confined to the gastric wall forprolonged periods before tumor spread its prognosis is betterthan that of GAC Previous literature has found that SCin the parotid gland lung hypopharynx liver and pancreashave poor prognoses due to metastasis or recurrence with asurvival period of a few months “ Similarly GSCpatients also died or developed metastasis or recurrence withina few months or it was already in the advanced stage at thefirst diagnosis All these clinical manifestations suggest that GSChas a poorer prognosis than GAC and GL In additionGSC can metastasize through the blood and lymph nodesand the most common sites of metastasis are the local lymphnodes and liver This conclusion is consistent with ourresearch resultsCONCLUSIONThe incidence rate of GSC is extremely low so clinicians andradiologists are not familiar with the features of this tumorBased on systematic research of this rare tumor and comparisonswith common gastric cancers we found that GSC is morecommon in men who are approximately years old and isoften accompanied by ulcers The disease is mostly located in theproximal part of the stomach and can also occur in the remnantstomach with delayed enhancement on contrastenhanced CTimages These characteristics can provide a reference for furtherresearch on GSCs in the future However an accurate diagnosisof GSC depends on the combination of clinical imaging andhistopathological features Due to the aggressive nature and poorprognosis of the tumor rapid clinical intervention and detailedfollowup with CT are essentialDATA AVAILABILITY STATEMENTThe original contributions presented in the study are includedin the supplementary material further inquiries can bedirected to the corresponding authorETHICS STATEMENTThe studies involving human participants were reviewed andapproved by the Medical Ethical Committee of the ZhengzhouUniversity The patientsparticipants provided their writteninformed consent to participate in this study Written informedconsent was obtained from the individuals for the publication ofany potentially identifiable images or data included in this AUTHOR CONTRIBUTIONSYL manuscript preparationliterature research and dataanalysis PL literature research and data analysis KF manuscriptreview and data collection KC guidance of pathologicalknowledge SY guidance of imaging knowledge JJ imaging datacollection and analysis WL and XZ manuscript editing JGstudy conception and design manuscript review and guarantor ofintegrity of the entire study All authors have read and approvedthe final manuscriptFUNDINGThis work was supported by the National Natural and ScienceFund of China No REFERENCES Zhu CC Li MR Lin TL Zhao G Sarcomatoid carcinoma of the stomach acase report and literature review Oncol Lett “ ol20153460 Snover DC Levine GD RosaiJ Thymic carcinoma Five distinctivehistological variants Am J Surg Pathol “ Zhou DK Gao BQ Zhang W Qian XH Ying LX Wang WL Sarcomatoidcarcinoma of the pancreas a case report World J Clin Cases “ 1012998wjccv7i2236 Xie Y Xiang Y Zhang D Yao X Sheng J Yang Y Sarcomatoidthe “ 103892mmr2018and review ofthe pancreasreportcaseofcarcinomaliterature Mol Med RepaFrontiers in Oncology wwwfrontiersinAugust Volume 0cLiu et alGastric Sarcomatoid Carcinoma Sato A Oki E Kohso H Endo Y Uchida H Hiroshige S Sarcomatoidcarcinoma of the remnant stomach report of a case Surg Today “ 101007s0059501204027 Nakayama Y Murayama H Iwasaki H Iwanaga S Kikuchi M Ikeda S et alGastric carcinosarcoma sarcomatoid carcinoma with rhabdomyoblastic andosteoblastic diï¬erentiation Pathol Int “ 101111j144018271997tb04540x RobeyCaï¬erty SS Grignon DJ Ro JY Cleary KR Ayala AG OrdonezNG Sarcomatoid carcinoma of the stomach A report of three caseswith immunohistochemical and ultrastructural observations Cancer “ 101002109701421990040165730co2n Ruess DA Kayser C Neubauer J FichtnerFeigl S Hopt UT Wittel UACarcinosarcoma of the pancreas case report with comprehensive literaturereview Pancreas “ 101097mpa0000000000000904 Fujiie M Yamamoto M Taguchi K Iwanaga A Ohgaki K Egashira A et alGastric carcinosarcoma with rhabdomyosarcomatous diï¬erentiation a casereport and review Surg Case Rep 101186s407920160176z Tanimura H Furuta M Carcinosarcoma of the stomach Am J Surg “ 101016000296106790325x Kitamura S Study on carcinosarcoma of stomach Gan “ Kumagai K Kawai K Kusano H Matsuo K Irie J Tsuchiyama H A caseof socalled carcinosarcoma of the stomach Gan No Rinsho “ Bekki T Fujikuni N Tanabe K Yonehara S Amano H Noriyuki T Thegastric carcinosarcoma with severe venous invasion a case report Surg CaseRep 101186s4079201804218 Ikeda Y Kosugi S Nishikura K Ohashi M Kanda T Kobayashi T Gastriccarcinosarcoma presenting as a huge epigastric mass Gastric Cancer “ 101007s1012000604054 Cid³n EU Cuenca IJ Gastric adenocarcinoma is computed tomography CTuseful in preoperative staging Clin Med Oncol “ cmos2641 Hallinan JT Venkatesh SK Gastric carcinoma imaging diagnosis stagingand assessment of treatment response Cancer Imaging “ Gossios K Katsimbri P Tsianos E CT features of gastric lymphoma EurRadiol “ 101007s003300050069 Rodrigues DN Hazell S Miranda S Crespo M Fisher C de Bono JS Sarcomatoid carcinoma of the prostate ERG fluorescence insituhybridization confirms epithelial origin Histopathology “ 101111his12493 Levine MS Rubesin SE PantongragBrown L Buck JL Herlinger H NonHodgkin™s lymphoma of the gastrointestinal tract radiographic findings AJRAm J Roentgenol “ 102214ajr16818976941 Russo AE Strong VE Gastric cancer etiology and management in Asia and theWest Annu Rev Med “ 101146annurevmed081117 Domblides C Leroy K Monnet I Mazi¨res J Barlesi F Gounant V et alEfficacy of immune checkpoint inhibitors in lung sarcomatoid carcinoma JThor Oncol “ 101016jjtho202001014 Niu X Sarcomatoid carcinoma in the parotid gland a review of years ofexperience Laryngoscope “ 101002lary27474 Li S Jiang L He Q Wei W Wang Y Zhang X The prognostic significa

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You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: Computed Tomography Features andClinicopathological Characteristicsof Gastric Sarcomatoid CarcinomaYiyang Liu1 Pan Liang1 Kaixiang Feng2 Kuisheng Chen3 Songwei Yue1 Jiang Ji4Weiwei Li1 Xitong Zhao1 and Jianbo Gao1 Department of Radiology The First Affiliated Hospital of Zhengzhou University Zhengzhou China Departmentof Thyroid Surgery The First Affiliated Hospital of Zhengzhou University Zhengzhou China Department of PathologyThe First Affiliated Hospital of Zhengzhou University Zhengzhou China Department of Radiology General Hospital NingxiaMedical University Yinchuan ChinaPurpose Gastric sarcomatoid carcinoma GSC is a very rare malignant tumor Thepurpose of this study is to describe the clinical computed tomography CT andpathologic features of GSC to increase awareness of this entityEdited byZiwen LiuPeking Union Medical CollegeHospital CAMS ChinaMethods The CT features and clinical data of five patients with pathologicallydocumented GSC were retrospectively analyzed and compared with the correspondingdata of gastric adenocarcinoma and lymphomaReviewed bySavio Gee BarretoMedanta The Medicity IndiaHaruhiko SugimuraHamamatsu University Schoolof Medicine JapanXiuying XiaoShanghai Jiao Tong University ChinaCorrespondenceJianbo GaofccyisunshinegszzueducnSpecialty sectionThis was submitted toGastrointestinal Cancersa section of the journalFrontiers in OncologyReceived May Accepted July Published August CitationLiu Y Liang P Feng K Chen KYue S Ji J Li W Zhao X and Gao J Computed TomographyFeatures and ClinicopathologicalCharacteristics of GastricSarcomatoid CarcinomaFront Oncol 103389fonc202001611Results Among the patients were male and was female The median agewas years Of the cases of GSC were in the gastric fundus and cardia was in the gastric body and was in the gastric fundus The gastric wall had localthickening in cases and mass formation in case with stenosis and deformationof the adjacent gastric cavity The longaxis diameter of the lesions ranged from to cm mean cm and was cm in cases and cm in case The tumorshowed predominantly inhomogeneous density with radiodensity values ranging from to HU In addition ulcers with an irregular base and slightly raised borders wereobserved in of cases After an injection of contrast material heterogeneous n or homogeneous n enhancement was observed After contrast medium injectionobvious enhancement was seen in cases and moderate enhancement was seen in cases the peak tumor signal was observed in the portal phase Two of the patientsdemonstrated evidence of lymph node involvement and in one patient the boundarybetween the lesion and the left lobe of the liver was unclear with low attenuation in theright lobe of the liver with circular enhancement The remaining two patients showed noevidence of metastasisConclusion Although GSC is extremely rare it should be considered in the differentialdiagnosis of gastric adenocarcinoma and lymphoma CT findings combined withpatient age and sex can provide support for the diagnosis of GSC However the finaldiagnosis must be confirmed with histopathologyKeywords sarcomatoid carcinoma stomach gastric cancer tomography Xray computed diagnosisFrontiers in Oncology wwwfrontiersinAugust Volume 0cLiu et alINTRODUCTIONSarcomatoid carcinomas SCs are extremely rare aggressivemalignant tumors characterized by distinct cellular morphology The features of this tumor were first described in by Snover SCs can occur in a wide variety of sitesincluding the respiratory tract digestive tract genitourinary tractbreast and thyroid glands However these tumors are rarein the digestive tract especially in the stomach As of April there are only six cases of gastric sarcomatoid carcinomaGSC reported in the English medical literature These previousreports focused on the pathological and clinical manifestationsthem have not systematically described the radiologic appearanceof the tumor Due to the more invasive nature and poorerprognosis of GSC than pure gastric adenocarcinoma GACand gastric lymphoma GL it is clinically beneficial to narrowdown the diï¬erential diagnoses by understanding the computedtomography CT characteristics of GSC The present studyanalyzed our experience in diagnosing five patients with GSCin terms of the imaging findings and clinical features To thebest of our knowledge our study represents the largest seriesof GSCs to dateIn addition due to the rarity of GSC the diï¬erential diagnosisbetween GSC and other types of malignant gastric tumors hasnot received much attention so we also initially explored thediï¬erential diagnosis of GSC from GAC and GLMATERIALS AND METHODSThe protocol was approved by the Medical Ethics Committeeof Zhengzhou UniversityInformed consent was obtainedfrom all patientsPatient SelectionFrom August to January we searched the pathologyrecords and the Picture Archiving and Communication SystemsPACS of our hospital The search terms included stomachand sarcomatoid carcinomas A total of five patients wereidentified as having SC and were enrolled in the present study Weretrospectively reviewed all clinical data demographic featureslaboratory findings clinical interventions and the histologicfindings of the five biopsy or operation specimensCT EvaluationFive GSC patients underwent CT examinations The CTscans were acquired with a 64row multidetector deviceDiscoveryCT750HD GE Healthcare Waukesha WIUnited States Conventional axial scanning was performedbefore and after an intravenous iv injection of nonioniciohexol iopromide mgmL GE Medical Systems mLkgand mLs through a dualhead pump injector MedradWarrendale PA United States The imaging parameters wereas follows tube voltage kV tube current mA fieldof view FOV mm matrix — mm and sectionthickness mm Finally a 20mL saline flush was performedat a rate of mLsGastric Sarcomatoid CarcinomaContrastenhanced CT scans were acquired with scanningdelays of s arterial phase AP and s portal venous phasePP after the iv injection of the contrast agent started The CTdose index volume for the three phases was mSvImage AnalysisTwo experienced radiologists and years of abdominal CTexperience performed a retrospective analysis of the CT imagesAll analyses were performed with an AW47 workstation GEHealthcare and the radiologists were blinded to the clinicalinformation of the patients The evaluated parameters includedthe tumor location gastric cardia gastric fundus gastric bodygastric angle and gastric antrum longaxis diameter shapegrowth pattern serosa condition attenuation and enhancementcharacteristics such as the enhancement pattern and degreeof enhancement The enhancement pattern of the tumor wasclassified as homogeneous or heterogeneous based on the APimage The degree of enhancement of the tumor was based ondynamic CT imaging using HU attenuation where œobviousenhancement was defined as HU œmoderate enhancementas HU and œmildly enhancement as HUThe GSCs were staged with the Union for InternationalCancer Control UICC TNM staging standard All imagingfindings were compared with the postoperative pathologicalfindings The accuracy rate the number of CTs coincidentwith the pathological diagnosisthe number of actual pathologicaldiagnoses — Pathological EvaluationThree patients underwent gastrectomy and two underwentendoscopic biopsy The three gastrectomy specimens measured cm — cm — cm cm — cm — cmcm — cm — cm respectively in two of theseand tumors the mucosal surface of the excised specimen showedulcerative masses of approximately cm — cm — cmand cm — cm The remaining specimen was a soft massmeasuring cm — cm — cm For biopsy multiple sampleswere acquired and the diameter of each sample was cmAccording to the relevant literature the diagnostic criteria fSC were generally as follows the tumor originated fromthe stomach and the tumor consisted of both carcinomatousand sarcomatoid components and the sarcomatoid componentaccounted for more than of the tissue In addition if biopsywas performed the sarcomatoid component can be seen in everysample Furthermore sarcomatoid regions express epithelialmarkers such as CK or EMAThe specimens were fully stretched fixed and soaked in formaldehyde solution for h All biopsy specimenswere analyzed The specimens underwent routine dehydrationparaffin embedding sectioning into µm thick sectionsand hematoxylin eosin HE staining Immunohistochemicalstaining was performed using a Roche BenchMark XT automaticimmunohistochemical detector The antibodies used in thisstudy included AE1AE3 CKL CK818 epithelial membraneantigen EMA vimentin P40 P63 and antigen KI67 Ki67All antibodies listed above were purchased from DAKO DakoGlostrup DenmarkFrontiers in Oncology wwwfrontiersinAugust Volume 0cLiu et alGastric Sarcomatoid CarcinomaTABLE Comparison between GSC and GAC GLAge median age rangeMain symptomsEpigastric discomfortpainIntermittent vomitingAcute hematemesisBloody stoolDysphagiaLocationCardia and FundusBodyAntrumThe longaxis diameter median size rangeShapeFocal thickeningDiffuse thickeningMassSerosal surfacebare areaClearUnclearUlcersYesNoDensity characteristicsHeterogeneousHomogeneousEnhancement patterHeterogeneousHomogeneousLymph node involvementYesNoLiver involvementYesNoTherapyResectionChemotherapyResection and ChemotherapyNeoadjuvant chemotherapyRadiation therapyGSCGACGL “ years “ years “ years “ cm “ cm “ cm Comprehensive Comparative AnalysisEach patient with GSC was matched by age ± years year ofdiagnosis and sex to four patients with GAC GL patients witheach disease were retrieved from PACS Patients with GSC werecompared with those with GAC GL in terms of demographicclinical and CT characteristics Table RESULTSPatient CharacteristicsThe patients included four men and one woman ranging inage from to years with a median age of years Theclinical and CT features of these patients are summarized inTables All patients had nonspecific symptoms includingabdominal discomfort epigastric discomfort nausea or vomitingThe other presenting symptoms included hematemesis or weightloss Three patients underwent radical resectionin whichonly one patient was treated with adjuvant chemotherapyafter surgery And two patients chose to deny treatment Inaddition we also reviewed the upper gastrointestinal radiographyresults Figure The laboratory findings revealed that patient was positivefor tumor abnormal protein TAP and patient was positivefor carbohydrate antigen CA125 Before treatmenthemoglobin and erythrocyte count decreased in three patientsFrontiers in Oncology wwwfrontiersinAugust Volume 0cLiu et alGastric Sarcomatoid CarcinomaTABLE Clinical and pathological factors of the five GSC patientsCaseSexAge yearsComplaintLocationMaximumdiameterTumor markercmAnemiaTherapyMetastasisMMFMMSuddenhematemesisEpigastricdiscomfortIntermittentvomitingEpigastric painEpigastric painEpigastric painLesser curvatureRemnant stomachCardia andFundusCardia and FundusFundusCardia and FundusNormalTAP CA125 NormalNA““RRnNoneNoneRC“““œ yespresentpositive œ“ noabsentnegative F female M male age in years R Radical gastrectomy Rn Remnant gastrectomy C chemotherapyNA not availableTABLE Computed tomography features of the five GSC patientsCaseGross features of the tumorUlcersGrowth modeDensity characteristicsEnhancement patterFocal thickeningFocal thickeningMassFocal thickeningFocal thickening“IntracavityIntracavityIntracavityIntracavityIntracavityHeterogeneousHeterogeneousHomogeneousHeterogeneousHeterogeneousHeterogeneousHeterogeneousHomogeneousHeterogeneousHeterogeneousMarginUnclearUnclearUnclearClearUnclearœ yespresentpositive œ“ noabsentnegativeFIGURE Characteristics of Xray examinations of a 65yearold male patient with GSC AB Reveals that there is a huge niche with irregular shapes at the smallcurvature of the stomach the niche is located inside the outline of the stomach the niche is surrounded by transparent bands with different widths that is ringembankments with irregular outlines The surrounding mucosa is thickened interrupted and the local gastric cavity is narrowedpatients and and platelet count was elevated in fourpatients patients and Pathological FeaturesMicropathologically the gastric tumor cells showed infiltrativegrowth The cytological characteristics ofthe tumor cellsshowed obvious malignant characteristics Microscopicallythe spindle cell structure and the nucleus were obviouslyatypical pleomorphic and enlarged Mitotic figures were visibleFigures 2AB On immunohistochemical examination thetumor cells showed positive staining for AE1AE3 CKLCK818 EMA P40 vimentin The Ki67 index was higher than Figures 2C“I All five tumors were diagnosed as GSCIn addition the sarcomatoid component showed spindle cellsarcomatoid morphologyCT FindingsOf the cases of GSC were in the gastric fundus and cardiaFigure was in the gastric body and was in the gastricfundus of these tumors one was a recurrence in the remnantstomach The CT manifestations of this tumor included localthickening n mass formation n The longaxisFrontiers in Oncology wwwfrontiersinAugust Volume 0cLiu et alGastric Sarcomatoid CarcinomaFIGURE Histological and immunohistochemical features of GSC AB Hematoxylineosin HE staining showing tumor cells demonstrated spindleshapedstructures significant atypical nuclei pleomorphic nuclei and giant nuclei Mitotic figures visible Tumor cells showed infiltrative growth Cells were stained withhematoxylin and eosin stain magnification A — B — By immunohistochemistry the tumor cells were positive for AE1AE3 C CKL D CK818 E EMAF P40 G and vimentin H Moreover of them were positive for Ki67 I The final diagnosis was SC [magnification C“I —]diameter of the lesions ranged from to cm mean size cm In addition ulcers with an irregular base and slightlyraised borders were observed in of cases Among the threepatients who underwent surgery two lesions invaded the gastricserosa and the remaining lesion invaded the gastric bare areaAmong the two patients with biopsyproven GSC one patientexhibited tumor invasion of the gastric bare area The majorchanges in the CT imaging characteristics were an irregularouter layer of the gastric wall and obscuration of the perigastricfat Initially the CT findings were interpreted as GAC in fourcases and GL in The tumor showed predominantly inhomogeneous densityand the radiodensity values were “ HU in the noncontrastphase Heterogeneous enhancement was seen in four casesdue to necrotic or cystic areas and the other tumor revealedhomogeneous enhancement The radiodensity values on the APimages ranged from to HU and to HU in thevenous phase After contrast medium injection two tumorsshowed obvious enhancement and moderate enhancementwas seen in the other three tumors the peak tumor valuewas observed in the portal phase One of the three patientswho underwentlymphsurgery demonstrated evidence ofin one patientthe boundary betweennode involvementthe lesion and the left lobe of the liver was unclear andthe area with low attenuation was confirmed by pathologythe liver withas a metastatic lesion in the rightcircular enhancement The remaining patientshowed noevidence of metastasis Among the two patients with biopsyspecimens one patient was identified as having lymph nodemetastasis on CTlobe ofCT Staging Versus Pathological Stagingof GSCNone of the GSCs were staged as T1T2 by CT or pathologyThe accuracy of CT staging T3 and T4 GSC was and respectively The overall diagnostic accuracy of CTfor determining the T stage of GSC was None of the GSCs were staged as N2N3 by CT or pathologyThe accuracy of CT in staging N3 and N4 GSC was and respectively The overall diagnostic accuracy of CT fordetermining the N stage of GSC was The comparison of TN staging based on CT and pathology isshown in Table Frontiers in Oncology wwwfrontiersinAugust Volume 0cLiu et alGastric Sarcomatoid CarcinomaFIGURE Sarcomatoid carcinoma of the stomach in 62yearold women A Unenhanced CT image of stomach reveals an intraluminal mass of homogeneousattenuation with an irregular surface at the gastric fundus and cardiac region B“D Contrastenhanced CT image shows obvious homogeneous enhancement ofmass with the peak value of the tumor on the portal phase In perigastric lymph nodes an enlarged and significantly enhancement lymph node can be seenB Arterial phase of contrast enhancement image C Portal phase of contrast enhancement image D Portal phase of contrast enhancement coronal imageDISCUSSIONTABLE CT and pathological TN staging for comparisonSarcomatoid carcinoma is an extremely rare and complicatedmalignant tumor composed of malignant epithelial componentsand atypical spindle cells However the spindle cells of SCsappear to show evidence of epithelial diï¬erentiationforexample showing epithelial markers or epithelial ultrastructuralinstead of a specific line of mesenchymalcharacteristicsdiï¬erentiation Moreoverliteratureemphasizes that the sarcomatous components occupy ofthe elements involved In the present study our patients™tumor cells displayed atypical spindle shapes that expressed theepithelial phenotypethe currentsome ofSarcomatoid carcinomas can occur in almost any an wherecarcinoma can occur In the digestive system the incidencesof SCs in the esophagus and liver are relatively high but SCsare exceedingly rare in the stomach we could find only sixprevious reports in the English literature Table Between and patients with SC confirmed by pathologywere retrospectively analyzed with only five tumors occurringin the stomach The average age of the reported patientswas years range “ and that in our series was years range “ A previous study reported that the sexCaseNO NO NO NO NO CTT4aN0T3N0T3N1T3N0T4aN1NA not available T tumor N nodePathological stageT4aN1T3N0NANAT4aN0distribution of male to female GSC patients was and thecorresponding proportion in our patients was “ Ithas been noticed that SCs are more common in male patientsand sex is a probable risk factor GSC patients may present withepigastric pain or discomfort dysphagia nausea and vomitinghematemesis and emaciation Due to thickening of the gastricwall pain or discomfort in the upper abdomen is common Thesymptoms can last from a few days to several years withoutobvious specificityIn the present study of the cases of GSC were recognizedin the proximal stomach and the remaining tumor was founddistal to the stomach Four cases of GSC in the present study hadFrontiers in Oncology wwwfrontiersinAugust Volume 0cLiu et alGastric Sarcomatoid CarcinomaTABLE Clinical and imaging features of six previously reported cases of GCSCaseGenderAgeyearsLocationSize cmShapeUlcersEnhanceappearanceRecurrenceMetastasisTherapyPrognosis MMFFMMRemnant stomachGreater curvatureLesser curvatureGastroesophageal junctionRemnant stomachDistal stomachPolypoidPolypoidPolypoidUlceratedPolypoidMass“““NENENENENEHyperNE“““““NoneSurgerySurgerySurgeryEndoscopySurgeryNA Mo D Mo D Mo D Mo D Mo DThe patient succumbed to heart failure before the surgical treatment An autopsy was performed œ yespresentpositive œ“ noabsentnegative Hyper hyperdenseNE no evaluate Mo Month D Dieareregarded asa longaxis diameter less than cm and the remaining tumorhad the largest longaxis diameter among our patients cmThe location distribution and longaxis diameters of the GSCs inour patients were similar to those in previous reports “in the actual diagnosis processThe diagnosis of SC has always been difficult for cliniciansand pathologists especially the diï¬erential diagnosis fromcarcinosarcoma Carcinosarcomastrulybiphasic neoplasms composed of distinct malignant epithelialcarcinomatous and mesenchymal sarcomatous componentsThe sarcoma components show typical specialized diï¬erentiation Howeverthe termsœsarcomatoid carcinoma and œcarcinosarcoma have been usedinterchangeably in some cases Therefore the understanding ofthese tumors has been hampered Nevertheless we can try tofocus on whether there is a diï¬erence between these tumors froma new perspective The CT finding SC in the stomach have notbeen previously scientific reported or even detailed descriptionThere are only four simple descriptions ChunChao reported that a patient with a giant SC presented a mass witha cm diameter in the antrum and body of stomach whichinfiltrated the gastric serosa The hepatic flexure of the colon andgallbladder were also involved on CT Contrastenhanced CTimages showed obvious enhancement of the two lesions Sato reported a patient with SC of the remnant stomachand the radiographic examination showed an elevated lesionwith a large ulcer at the gastric cardiac lesser curvature thatmeasured cm in diameter The other two reports only describeda soft tissue mass or a large tumor in the dilated stomach On the other hand within in the upper gastrointestinal tractalthough there are fewer reports of carcinosarcoma localized inthe stomach this type of tumor is still more common than SC Gastric carcinosarcoma showed an elevated lesion or thickenedgastric walls in “ of all reviewed cases “ Tomoaki reported a 79yearold man with gastric carcinosarcomaand his veins showed severe invasion Enhanced abdominalCT showed irregular thickening and slight enhancement of thegastric wall on the side of the lesser curvature with suspiciousbulky lymph nodes Yoshiyuki reported a 70yearoldJapanese woman who presented with a soft tissue mass adjacentto the lesser curvature of the stomach that was lobulated andCT revealed an ulcer on the lesion The contrastenhanced CTimages showed heterogeneous enhancement of the mass Thefinal pathological diagnosis was gastric carcinosarcoma Inthe present study we found that GSC showed local thickeningof the gastric wall and mass formation often accompaniedby ulcers The site of the disease was mostly in the proximalpart of the stomach but these tumors can also occur in theremnant stomach The signal of the tumor was homogeneousor heterogeneous on plain CT scans After contrast mediuminjection of tumors demonstrated heterogeneousenhancement on AP images due to cystic areas or necrosis inthe lesions In this study the enhancement degree of all tumorsreached a peak in the PP after contrast enhancement For thesetumors the enhancement degree in the delayed phase wasnot significantly reduced The overall enhancement mode wasdelayed enhancement In addition CT showed that four patientshad invasion into the gastric serosal region or gastric bare areatwo patients had the characteristics of enlarged perigastric orretroperitoneal lymph nodes and uneven enhancement and onepatient had invasion into the adjacent liver tissue These findingsreflect the metastatic and highly invasive characteristics of GSCOverall CT and contrastenhanced CT can clearly show theprimary lesion infiltration range lymph node metastasis anddistant metastasis of GSCTomographic diagnosis of GSC has not been attemptedbecause of the rarity of this entity According to the findingsof our study GSC needs to be diï¬erentiated from GAC andGL on CT Adenocarcinoma is the most common pathologicaltype of gastric tumor and is mainly distributed in the antrumseldomly in the body and fundus of the stomach The incidenceof GAC is high in men and the median patient age is years The most common CT signs of GAC are localor extensive thickening of the gastric wall mass formationincluding fungoidestype polypoidtype masses rough orsmooth serous surfaces and continuous interruption of themucosal layer Tumors involving the mucosal surface can appearenhanced “ s after injecting a contrast agent The peakvalue for tumors invading the muscular layer usually appearsafter “ s and after the mucosal surface is strengthenedthe duration is longer Primary GL accounts for “ ofmalignant gastric tumors and is predominantly situated in thegastric antrum gastric body and gastric fundus The incidenceof GL is high among males with a median patient age of yearsThe clinical symptoms included epigastric pain bleeding earlysatiety and fatigue The most common CT manifestations ofGL are diï¬use thickening of the gastric wall or a homogeneousFrontiers in Oncology wwwfrontiersinAugust Volume 0cLiu et alGastric Sarcomatoid Carcinomatissue mass with slight attenuation or an appearancesoftsimilar to that of the normal gastric wall For GL becauseof hemorrhage necrosis submucosal edema or infarction thegastric wall may be heterogenous on CT GL originates froma submucosal process and gastric mucosa is commonly intactin the early stage but shows interruptions or ulceration in thelater stage After contrast medium injection most GL showedhomogeneous and slight enhancement in the delayed phase Lymphoma is considered when distant structures the mesenteryretroperitoneum or other parts of the abdomen have lymphnode metastasis The CT findings may only reflect features of GSC but cannotaccurately diagnose GSC let alone explore the origin of thesarcomatous portion Immunohistochemistry IHC also failedto conclusively establish the origin of GSC Rodrigues usedfluorescence in situ hybridization FISH to confirm that SC andadenocarcinoma have a common origin that is the epithelium while primary GL originated from gastric submucosallymphoid tissueThe main treatment for localized lymphomas is eradicationof Helicobacter pylori and surgical treatment whereas advanceddisease often requires radiation or chemotherapy alone Surgery is the only treatment option for patients with GACAdjuvant chemotherapy and chemoradiotherapy are also oftenused Targeted therapy isin the exploration stage However there are currently no specific National ComprehensiveCancer Network guidelines for the treatment of only GSCbecause the tumor is relatively rare although complete surgicalresection is the most important treatment method For examplewhile chemotherapy is considered in clinical practice whetherchemotherapy can be applied for GSC and the efficacy ofchemotherapy remain controversial Domblides firstevaluated the efficacy of immune checkpoint inhibitors ICIs forSC and found that lung SC patients exhibited high response ratesand prolonged overall survival OS with ICIs This studyprovides a new idea for the treatment of GSCBecause GL tends to be confined to the gastric wall forprolonged periods before tumor spread its prognosis is betterthan that of GAC Previous literature has found that SCin the parotid gland lung hypopharynx liver and pancreashave poor prognoses due to metastasis or recurrence with asurvival period of a few months “ Similarly GSCpatients also died or developed metastasis or recurrence withina few months or it was already in the advanced stage at thefirst diagnosis All these clinical manifestations suggest that GSChas a poorer prognosis than GAC and GL In additionGSC can metastasize through the blood and lymph nodesand the most common sites of metastasis are the local lymphnodes and liver This conclusion is consistent with ourresearch resultsCONCLUSIONThe incidence rate of GSC is extremely low so clinicians andradiologists are not familiar with the features of this tumorBased on systematic research of this rare tumor and comparisonswith common gastric cancers we found that GSC is morecommon in men who are approximately years old and isoften accompanied by ulcers The disease is mostly located in theproximal part of the stomach and can also occur in the remnantstomach with delayed enhancement on contrastenhanced CTimages These characteristics can provide a reference for furtherresearch on GSCs in the future However an accurate diagnosisof GSC depends on the combination of clinical imaging andhistopathological features Due to the aggressive nature and poorprognosis of the tumor rapid clinical intervention and detailedfollowup with CT are essentialDATA AVAILABILITY STATEMENTThe original contributions presented in the study are includedin the supplementary material further inquiries can bedirected to the corresponding authorETHICS STATEMENTThe studies involving human participants were reviewed andapproved by the Medical Ethical Committee of the ZhengzhouUniversity The patientsparticipants provided their writteninformed consent to participate in this study Written informedconsent was obtained from the individuals for the publication ofany potentially identifiable images or data included in this AUTHOR CONTRIBUTIONSYL manuscript preparationliterature research and dataanalysis PL literature research and data analysis KF manuscriptreview and data collection KC guidance of pathologicalknowledge SY guidance of imaging knowledge JJ imaging datacollection and analysis WL and XZ manuscript editing JGstudy conception and design manuscript review and guarantor ofintegrity of the entire study All authors have read and approvedthe final manuscriptFUNDINGThis work was supported by the National Natural and ScienceFund of China No REFERENCES Zhu CC Li MR Lin TL Zhao G Sarcomatoid carcinoma of the stomach acase report and literature review Oncol Lett “ ol20153460 Snover DC Levine GD RosaiJ Thymic carcinoma Five distinctivehistological variants Am J Surg Pathol “ Zhou DK Gao BQ Zhang W Qian XH Ying LX Wang WL Sarcomatoidcarcinoma of the pancreas a case report World J Clin Cases “ 1012998wjccv7i2236 Xie Y Xiang Y Zhang D Yao X Sheng J Yang Y Sarcomatoidthe “ 103892mmr2018and review ofthe pancreasreportcaseofcarcinomaliterature Mol Med RepaFrontiers in Oncology wwwfrontiersinAugust Volume 0cLiu et alGastric Sarcomatoid Carcinoma Sato A Oki E Kohso H Endo Y Uchida H Hiroshige S Sarcomatoidcarcinoma of the remnant stomach report of a case Surg Today “ 101007s0059501204027 Nakayama Y Murayama H Iwasaki H Iwanaga S Kikuchi M Ikeda S et alGastric carcinosarcoma sarcomatoid carcinoma with rhabdomyoblastic andosteoblastic diï¬erentiation Pathol Int “ 101111j144018271997tb04540x RobeyCaï¬erty SS Grignon DJ Ro JY Cleary KR Ayala AG OrdonezNG Sarcomatoid carcinoma of the stomach A report of three caseswith immunohistochemical and ultrastructural observations Cancer “ 101002109701421990040165730co2n Ruess DA Kayser C Neubauer J FichtnerFeigl S Hopt UT Wittel UACarcinosarcoma of the pancreas case report with comprehensive literaturereview Pancreas “ 101097mpa0000000000000904 Fujiie M Yamamoto M Taguchi K Iwanaga A Ohgaki K Egashira A et alGastric carcinosarcoma with rhabdomyosarcomatous diï¬erentiation a casereport and review Surg Case Rep 101186s407920160176z Tanimura H Furuta M Carcinosarcoma of the stomach Am J Surg “ 101016000296106790325x Kitamura S Study on carcinosarcoma of stomach Gan “ Kumagai K Kawai K Kusano H Matsuo K Irie J Tsuchiyama H A caseof socalled carcinosarcoma of the stomach Gan No Rinsho “ Bekki T Fujikuni N Tanabe K Yonehara S Amano H Noriyuki T Thegastric carcinosarcoma with severe venous invasion a case report Surg CaseRep 101186s4079201804218 Ikeda Y Kosugi S Nishikura K Ohashi M Kanda T Kobayashi T Gastriccarcinosarcoma presenting as a huge epigastric mass Gastric Cancer “ 101007s1012000604054 Cid³n EU Cuenca IJ Gastric adenocarcinoma is computed tomography CTuseful in preoperative staging Clin Med Oncol “ cmos2641 Hallinan JT Venkatesh SK Gastric carcinoma imaging diagnosis stagingand assessment of treatment response Cancer Imaging “ Gossios K Katsimbri P Tsianos E CT features of gastric lymphoma EurRadiol “ 101007s003300050069 Rodrigues DN Hazell S Miranda S Crespo M Fisher C de Bono JS Sarcomatoid carcinoma of the prostate ERG fluorescence insituhybridization confirms epithelial origin Histopathology “ 101111his12493 Levine MS Rubesin SE PantongragBrown L Buck JL Herlinger H NonHodgkin™s lymphoma of the gastrointestinal tract radiographic findings AJRAm J Roentgenol “ 102214ajr16818976941 Russo AE Strong VE Gastric cancer etiology and management in Asia and theWest Annu Rev Med “ 101146annurevmed081117 Domblides C Leroy K Monnet I Mazi¨res J Barlesi F Gounant V et alEfficacy of immune checkpoint inhibitors in lung sarcomatoid carcinoma JThor Oncol “ 101016jjtho202001014 Niu X Sarcomatoid carcinoma in the parotid gland a review of years ofexperience Laryngoscope “ 101002lary27474 Li S Jiang L He Q Wei W Wang Y Zhang X The prognostic significa Answer:

Thyroid_Cancer

ShortTerm Consequences ofPediatric Anticancer TreatmentRegarding Blood Pressure MotorPerformance Physical Activity andReintegration Into Sports StructuresTina Keiser Dominik Gaser Christiane Peters Renate OberhofferFritz Sabine Kesting   and Irene von Luettichau  Edited byKirsten K NessSt Jude Children™s Research Hospital Department of Sports Medicine and Exercise JustusLiebig University GieŸen GieŸen Germany Department of Sport andHealth Sciences Institute of Preventive Pediatrics Technical University of Munich Munich Germany Department ofPediatrics and Children™s Cancer Research Center Kinderklinik M¼nchen Schwabing TUM School of Medicine TechnicalUnited StatesReviewed bySeth E KarolSt Jude Children™s Research HospitalUnited StatesJacques GrillInstitut Gustave Roussy FranceCorrespondenceSabine KestingsabinekestingtumdeIrene von LuettichauIreneTeichertvonLuettichaumritumde These authors sharesenior authorshipSpecialty sectionThis was submitted toPediatric Oncologya section of the journalFrontiers in PediatricsReceived January Accepted July Published August CitationKeiser T Gaser D Peters COberhofferFritz R Kesting S and vonLuettichau I ShortTermConsequences of PediatricAnticancer Treatment RegardingBlood Pressure Motor PerformancePhysical Activity and ReintegrationInto Sports StructuresFront Pediatr 103389fped202000463University of Munich Munich GermanyBackground Cardiovascular diseases in childhood cancer survivors are knownlate sequelae following treatment Arterial stiffness pulse wave velocity PWV andcentral systolic blood pressure cSBP are potential predictors to assess the statusof cardiovascular health Frequent inpatient stays and reduced physical activity PAduring treatment can lead to noticeable impairments regarding motor skills and physicalperformance The present study examined parameters of cardiovascular health motorperformance and the status of integration into sports structures shortly after cessationof treatmentMethods A crosssectional monocentric study was conducted from April to June Participants “ yrs mixed cancer entities during maintenance therapy andfollowup care were recruited Peripheral and central systolicdiastolic blood pressurepSBP pDBP cSBP and PWV were assessed using the MobilOGraph® The MOONtest MOtor performance in pediatric ONcology was used to scale motor performancePA levels and status ofintegration into sports structures were assessed with aquestionnaire referring to the KiGGS study All measured data were compared topublished reference valuesResults Forty participants ± years female were recruited ± years posttreatment PSBP zscore ± p pDBP ± p and cSBP ‰¥ years ± p were significantly increasedcompared to reference values PWV was also elevated but not significantly Motorperformance was reduced in almost all motor abilities Thirtysix percent of the examinedgroup did not participate in physical education at school to the full extent Only reported hour of daily moderatetovigorous PA as recommended for children andadolescents by the World Health anization Half of the participants were active sportsclub members before treatment but one third did not resume their former membershipFrontiers in Pediatrics wwwfrontiersinAugust Volume 0cKeiser et alPhysical Consequences After Childhood CancerConclusionIncreased cardiovascular parameters and impaired motor performanceshortly after cessation of treatment physical inactivity and low rates of integration intoregular sports programs highlight the support needed Young cancer patients shouldreceive early support in coping with physical limitations preferably soon after diagnosisMotor deficits could be reduced by applying targeted interventions Furthermorea regular sports therapy program during in and outpatient care could increaseengagement in PA to possibly counteract risk factors and improve cardiovascular healthKeywords childhood cancer cardiovascular health motor performance physical activity sports reintegrationblood pressure arterial stiffnessINTRODUCTIONExtensive research and optimized treatment regimens resulted inan increase of the 5year survival rate to in the USA and of the 15year survival rate to for patients under theage of in Germany However childhood cancer is a raredisease It contributes only around to all malignant diseasesin developed countries Worldwide children underthe age of and adolescents aged between and are diagnosed with cancer every year As a consequence ofthe success in the treatment of childhood cancer the importanceof survival quality and prevention of late sequelae have receivedmore attention during the last yearsKnown negativelongterm consequences ofintensivetreatment for childhood and adolescent cancer patients ofteninclude adverse eï¬ects on the cardiovascular system Cardiovascular diseases are the most frequently reported causesof death in childhood cancer survivors following secondarytumors Arterial stiï¬ness pulse wave velocity PWV andcentral systolic blood pressure cSBP are potential predictorsfor cardiovascular diseases frequently investigated in medicalresearch to evaluate the status of a patient™s cardiovascularhealth PWV describes the velocity of the pressure wave in the aortawhich spreads from the left ventricle through the arterial vascularsystem during systole Noninvasive investigation of the PWVvia ultrasound or oscillometric methods provides informationon the elasticity ofthe vascular system and enables earlyrecognition of damages in the vessels Thus in order to detectpotential structural modifications in the vascular system andindicators for arterial stiï¬ness at an early stage this subclinicalparameter should be surveyed continually Previous data indicatea positive correlation of PWV with arterial vascular stiï¬ness Moreover elevated PWV reflecting subclinical vasculardamage was shown in pediatric patients after hematopoieticstem cell transplantation On the contrary another studyinvestigated elevated blood pressure levels but no statisticallyAbbreviations PWV Pulse Wave Velocity WHO World Health anizationcSBP central systolic blood pressure pSBP peripheral systolic blood pressurepDBP peripheral diastolic blood pressure BMI Body mass index MOONMotor performance in pediatric oncology KiGGS German Health Interviews andExamination Survey for Children and Adolescentssignificant variation for PWV in pediatric cancer survivorscompared to healthy children and adolescents In addition to the abovementioned late sequelae severalproblems already arise during treatment and often persistthroughout survivorship For instancelongterminpatient stays and reduced physical activity during treatmentcan lead to noticeably reduced physical performance ofsurvivors and reintegration into sportschildhood cancerstructures mightberehabilitationprocess throughoutfrequentaï¬ectedIn healthy populations reduced physical activity leadsto negative consequencesfor cardiovascular health Additionally the necessary use of anthracyclines in almost ofapplied therapy regimens in childhood cancer increases the riskof cardiovascular morbidity and mortality eightfold comparedto agematched patients not receiving anthracyclines indicatingthe importance of reducing such longterm consequences Due to a poor state of health and impaired immune functionsports options such as physical education at school engagementin sports clubs or recreational sports are no longer feasibleduring therapy Consequently reintegration after cessation oftreatment is associated with even more barriers due to diseaseand treatmentrelated impairments Circumstances ofanticancer treatment can lead to inactivity resulting in deficitsof fine and gross motor skills reduced muscle strength andpoor physical fitness following treatment Especiallymotor performance in pediatric bone tumor patients oftenremains reduced until at least years after cessation of treatment Impairments of physical performance have been shown topersist throughout survivorship which may complicate thesurvivors™ reintegration into both social and sports structures aswell as the development of a longterm active lifestyle According to a questionnairebased study childhood cancersurvivors™ reintegration rate into physical education at school isvery low especially after treatment for bone tumors The lackof comprehensive oï¬ers of physical activity promotion and motordevelopment might exacerbate motor impairments and problemsof reintegration into sports structures Von Korn et al examined motor performance usingthe Fitnessgram Rcid13 as well as peripheral blood pressure centralblood pressure and PWV using the MobilOGraph Rcid13 in childrenafter treatment for childhood cancer n aged ± years ± years postdiagnosis Their results show reducedFrontiers in Pediatrics wwwfrontiersinAugust Volume 0cKeiser et alPhysical Consequences After Childhood Cancermotor performance of childhood cancer survivors compared toreference values of healthy children However no correlationcould be drawn regarding cardiovascular parameters and motorperformance The present crosssectional study aimed atinvestigatingvarious parameters of cardiovascular health motor performanceand status of physical activity in children and adolescentsshortly after cessation of anticancer treatment or during ongoingoral maintenance therapy The collection of such data isof considerable importance for the early detection of healthimplications related to both disease and treatment Moreoverthe findings will help to support the development of preventivestrategies regarding the health of children and adolescents treatedfor cancer Appropriate strategies during primary and secondaryprevention and following cancer treatment tertiary preventionneed improvementsMATERIALS AND METHODSDesignThe crosssectional monocentric study was performed over aperiod of months April“June at our institution Theassessment of cardiovascular parameters using the MobilOGraph Rcid13 was followed by the MOON test MOtor performance inpediatric ONcology to evaluate motor performance Finally theparticipants completed a standardized questionnaire referring tothe KiGGS study German Health Interview and ExaminationSurvey for Children and Adolescents to collect data regardingtheir current level of physical activity and status of integrationinto sports structures The Ethics Committee of the School ofMedicine of the Technical University of Munich approved thestudy project number SSR Participation was voluntaryand informed written consent was signed by each participant aswell as by his or her legal guardian All data was collected encodedpseudonym and in accordance with privacy policy standardsParticipantsPrior to addressing the participants all eligible children andadolescents were screened using the electronic patient recordSAP Rcid13 ERP Patients were recruited during routine followupvisits The following inclusion criteria were applied childrenand adolescents during maintenance therapy and followupcare of a pediatric oncological disease and currently agedbetween and years No restriction was applied regardingthe period posttreatment Exclusion criteria were medicalcontraindications such as fever acute infection orthopedicrestrictions and mental retardation insufficient knowledgeof the German language and absence of written informedconsent The attending physician confirmed participation forall recruited children and adolescents Following these inclusioncriteria children and adolescents were initially found eligibleFigure Outcome MeasuresAnamnestic and Anthropometric DataAnamnestic and clinical data ie type of cancer treatmentregime end of therapy was obtained from the electronic patientrecord SAP Rcid13 ERP The nursing staï¬ assessed anthropometricdata height and weight during routine medical examinationseca electronic column scaleseca mechanicalmeasuring rod Body mass index BMI was calculated as aratio of body weight kg per square body height m2 By usingthe reference values of a healthy German cohort BMI wasconverted into percentiles and classified in underweight 10thpercentile normal weight 10thˆ’90th percentile and overweight90th percentile Cardiovascular ParametersPrior to the measurement the participants had to rest for atleast min in a supine position PWV central blood pressureand peripheral blood pressure were assessed using the MobilOGraph Rcid13 IEM GmbH Stolberg Germany and HMS ClientServer Version an oscillometric noninvasive methodMeasurements were performed on the left upper arm The cuï¬was inflated twice with a rest of s in between Cuï¬ sizewas chosen according to the circumference of the participant™sleft upper arm An ARCSolver Algorithm calculated the cSBPindirectly as well as the PWV from recorded brachial pulseRaw data was transformed into zscores and compared by usingzscores of a healthy reference cohort PSBP and pDBPwere compared to references from the national cohort of children and adolescents KiGGS study For the assessmentof PWV and cSBP values references from Elmenhorst et al of healthy children and young adults were used To evaluatethe results of the parameters cSBP and PWV the examinedparticipants were separated into two groups years and‰¥ years According to the age distribution of the referencevalues participants years were compared to heightmatchedreference values and participants ‰¥ years were compared toagematched referencesThe measurement using the MobilOGraph Rcid13 was previouslyused several times in pediatric patients “ and was alsosuccessfully applied in childhood cancer survivors Motor PerformanceTo quantify motor performancethe MOON test MOtorperformance in pediatric ONcology was applied Thetest assesses motor abilities coordination speed flexibility andstrength and consists of eight test items eyehand coordinationinserting pins static balance static stand upper extremitycoordination throwing at a targetspeed reaction testmuscular endurance sittostand flexibility stand and reachhand grip strength handheld dynamometry and muscularexplosive strength medicine ball shot The test lasts min onaverage Data of each item was compared to published age andsexmatched reference values of a healthy population within anage range of to years Data of participants older than years was compared to reference values of healthy 17yearoldssince reference values of healthy 18yearolds are not availableCalculation of a total score is not possible within this toolInstead each item was analyzed individually and the percentagedeviation to reference values was computedFrontiers in Pediatrics wwwfrontiersinAugust Volume 0cKeiser et alPhysical Consequences After Childhood CancerFIGURE Flow chart of recruitment Out of participants eligible could not be addressed due to several medical examinations in different departments of thehospital and resulting in missing time slots Longer periods posttreatment are associated with fewer appointments for followups and more medical examinationstake place in day This does not necessarily mean that the children who could not be included in the study due to missing time slots are medically more complexTen participants refused participation Thus the sample included participantsPhysical Activity and Reintegration Into SportsStructures After Acute TreatmentPhysical activity levels and status of integration into sportsstructures were assessed with a standardized selfreportingreferring to the KiGGS study Thequestionnairequestionnaire wassupplemented by several disease andtreatmentrelated aspects in accordance with the study ofKesting et al to investigate potential barriers regardingreintegration and participation in sports activities eg barrierswith respect to exemption from physical education at school ornonparticipation in sports clubs sports therapy oï¬ers duringtreatment The KiGGS study oï¬ers the reference values ofhealthy children and adolescents n for comparison ofour dataData AnalysisCardiovascular parameters were analyzed and compared to thehealthy reference population with the one sample ttest Motorperformance was analyzed using the Wilcoxon signedranktest in comparison to age and sexmatched reference valuesPearson correlation was applied to calculate possible associationsbetween motor performance and cardiovascular parametersBMI and the period posttreatment The MannWhitneyUtestwas performed to evaluate anthracyclinemediated eï¬ects oncardiovascular health as well as diï¬erences within subgroupsregarding diï¬erent entities and levels of physical activity motorperformance physical education at school and achievement ofphysical activity recommendationsExplorative twosided statistical tests were conducted andp ‰¤ was considered statistically significant No adjustmentfor multiple comparison was conducted Correlations coefficientρ were classified according to Cohen Descriptive statistics were calculated with Microsoft Excelversion for demographic characteristics and medicaldata GraphPad Prism version was used to perform allfurther statistical analyses Data analysis was performed inconsultation with the Institute of Medical Informatics Statisticsand Epidemiology of the Technical University of MunichRESULTSParticipantsOut of eligible children and adolescents who met the inclusioncriteria a total of participants female with variouscancer entities were recruited and examined Table Performed AssessmentsFor various reasons not alltests could be realized withall participants In participants cardiovascularparameters could not be evaluated due to missing time slotsFrontiers in Pediatrics wwwfrontiersinAugust Volume 0cKeiser et alPhysical Consequences After Childhood CancerTABLE Anthropometric and medical characteristics of the participants n CharacteristicsN Mean ± SD MedianRangeAge at diagnosis years ± Age at assessment years ± ““ years‰¥ years Period postdiagnosis years ± Period posttreatment years ± ““ year“ years yearsLeukemiaLymphomaBone tumorBrain tumorOther solid tumors Body mass index¢ kgm2 ± “UnderweightNormal weightOverweightChemotherapyAnthracycline applicationCumulative dose mgm2RadiotherapyChestdirected radiation Anthracycline chest radiation Surgical tumor resectionRelapse ± “FIGURE Cardiovascular parameters shown in zscores and compared topublished reference values pSBP peripheral systolic blood pressurepDBP peripheral diastolic blood pressure PWV pulse wave velocity cSBPcentral systolic blood pressure Significant values p ‰¤ Results are given in Mean ± SD M median RangeOther solid tumors alveolar rhabdomyosarcoma n carcinoid tumor of the appendixn nephroblastoma n focal nodular hyperplasia liver n mature cysticteratoma ovary n thoracic ganglioneuroma n papillary thyroid carcinoman neuroblastoma n ¢BMI was converted into percentiles and classified in underweight 10th percentilenormal weight 10thˆ’90th percentile and overweight 90th percentile Thirteen participants received a combination of anthracyclines mainly the combination ofDoxorubicin and Daunorubicinduring routine appointment and a lack of willingness to prolongthe outpatient visit Six of the participants could not performthe MOON test due to medical limitations current orthopedicrestrictions n examinationrelated limitations ie a draintube in the crook of the arm for followup MRT n andabandonment due to lack of time n The central venousdevice has already been explanted in all participants prior toour studyOf the remaining participants not everyone performedevery test item Two participants could not perform the testitem speed due to a drain tube in the crook of the armFour participants could not accomplish the test item muscularexplosive strength due to orthopedic restrictions as well asexaminationrelated limitations Three participants did notperform the item muscular endurance of the legs n hadcrutches n severe muscular deficit in the legs n lackof time Two participants could not perform the test item handgrip strength with both hands due to lack of time n andinfusion needle in the crook of the arm n The test itemupper extremity coordination was measured in n participantsbecause reference values are provided for children between and years onlyCardiovascular HealthIn participants all parameters were assessed Based on theunderlying reference values for cSBP and PWV of Elmenhorstet al the group was separated into participants aged years heightmatched reference values and ‰¥ years agematched reference values PSBP zscore ± p pDBP zscore ± p as well as cSBP values‰¥ years zscore ± p were significantlyincreased compared to reference values of healthy children andadolescents Figure PWV was elevated but not significantly years zscore ± p ‰¥ years zscore ± p Comparison of cardiovascular parameters of participantswho received anthracyclines during intense therapy with acumulative dose of ± mgm2 and subjects whodid not receive cardiotoxic agents did not show statisticallysignificant diï¬erencesMotor PerformanceThe participants™ n motor performance wasreduced in almost all motor abilities compared to the referencevalues of healthy children and adolescents Table Significantimpairments became obvious in the following dimensionsmuscular explosive strength p upper extremitycoordination p muscular endurance of the legs p Frontiers in Pediatrics wwwfrontiersinAugust Volume 0cKeiser et alPhysical Consequences After Childhood CancerTABLE Results of the MOONtest compared to reference values n Motor abilityTest itemEyehand coordinationStatic balanceφSpeedInserting pins timeStatic stand contactsReaction test timeUpper extremity coordinationθFlexibilityThrowing at a target pointsStand and reach cmMuscular explosive strengthMedicine ball shot meterMuscular endurance legsSittostand secHand grip strengthHandheld dynamometry kgRightLeftNMean ± SD of differenceMedian pvalueto reference values ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’All results were compared to the reference values of each single test item Speed could only be measured in participants muscular explosive strength in participants andhand grip strength in right and left participants due to lack of time orthopedic restrictions or drain tube in the crook of the armFor the test items static balance and flexibility the absolute differences were used as the measured values would have fluctuated around zero and would have givenoversized percentagesφ Static balance was assessed counting the contacts with a foot to the ground while balancing on a rail in this context a negative difference to reference values represents fewercontacts and therefore better resultsθ Although all participants completed this test item reference values are provided for children between and years onlyM median abs indicates absoluteBold numbers indicate significant values p ‰¤ TABLE Results of the MOONtest comparing participants treated for leukemialymphoma and participants treated for brain tumors compared to reference valuesLeukemiaLymphoma n Brain tumor n Motor abilityEyehand coordinationStatic balanceφSpeedFlexibilityMuscular explosive strengthMuscular endurance legsHand grip strength rightHand grip strength leftNMean ± SD Median ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’NMean ± SD Median pvalueˆ’ ± ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ All results were compared to the reference values of each single test item φ Static balance was assessed counting the contacts with a foot to the ground while balancing on a rail in this context a negative difference to reference values represents fewercontacts and therefore better resultsFor the test items static balance and flexibility the absolute differences were used as the measured values would have fluctuated around zero and would have givenoversized percentagesDue to insufficient number of participants the comparison regarding the test item upper extremity coordination was disregarded reference values only provided for children aged and yearsM median abs indicates absoluteBold numbers indicate significant values p ‰¤ and hand grip strength on the right hand p The performance of eyehand coordination speed flexibility andhand grip strength on the left hand were also reduced but notsignificantly In the test item static stand the study participantsperformed slightly better compared to the reference populationFor upper extremity coordination throwing at a target onlyreference values from children aged “ years are availableTherefore comparison of the collected data was only possiblewith the same age group n Data for olderparticipants was not collectedComparing motor performance of participants diagnosedwith leukemialymphoma n and participantsn diagnosed with brain tumorsrevealedsome diï¬erences Table Children and adolescents treatedfor brain tumors performed significantly worse in eyehandcoordination than participants treated for leukemialymphomap Moreover the performances in all other testedmotoric dimensions of participants diagnosed with braintumor were deteriorated compared to participants treated forleukemialymphomas but not significantlyTo determine influencing factors on motor performance thecorrelation between motor performance results and BMI as wellas the period posttreatment was performed With increasingBMI values of static balance deteriorated significantly ρ Frontiers in Pediatrics wwwfrontiersinAugust Volume 0cKeiser et alPhysical Consequences After Childhood CancerFIGURE Pearson correlation between static balance and BMI kgm2¢ leukemialymphoma —¦ bone tumor –³ brain tumor cid7 other solid tumorsHorizontal line indicates the reference values The absolute difference fromreference values is given number of contacts to the ground A negativedifference means fewer ground contacts and therefore better performanceThirty four participants performed the testp which corresponds to a moderate to high correlationFigure A negative diï¬erence to reference values means fewercontacts to the ground and therefore better performance in staticbalance Furthermore some nonsignificant correlations werefound Deteriorated eyehand coordination ρ ˆ’ p and flexibility ρ ˆ’ p were also associatedwith a higher BMI However superior values in upper extremitycoordination ρ p muscular explosive strengthρ p hand grip strength right ρ p left ρ p and muscle endurance ofthe legs ρ p were associated with increasedBMI The test item speed showed no association with BMIρ p A longer period posttreatment was significantly associatedwith decreased eyehand coordination ρ ˆ’ p corresponding to a moderate correlation Figure Especially participants treated for a brain tumor with a longerperiod posttreatment showed deteriorated values in eyehandcoordination Speed performance was deteriorated in participantswith longer posttreatment period ρ ˆ’ p Physical Activity and Reintegration IntoSports StructuresAccording to the selfreported questionnaire n did not participate in physical education at school to full extend n were not admitted to school sports activitiesand n were partly excluded Table Neitherof the two participants treated for bone tumor was takingpart in physical education at school n whereaschildren with other tumors participated to a notably higherrate Treatmentrelated muscular deficits n andosteonecrosis n were the most common reasons forparticipants not taking part in physical education at schoolFIGURE Pearson correlation between eyehand coordination and periodposttreatment months ¢ leukemialymphoma —¦ bone tumor –³ brain tumorcid7 other solid tumors Horizontal line indicates the reference values Thirtyfour participants performed the test itemTABLE Participation in physical education at school subdivided into entitiesn Participation Partial exemption Full exemptionN N N Entire group n LeukemiaLymphoma n Bone tumor n Brain tumor n Other solid tumors n Abs N number Four out of children were still attending kindergarten and could notanswer the question regarding participation in physical education at schoolOnly n reported moderatetovigorous physicalactivity for min daily as generally recommended by the WHOfor healthy children and adolescents Table This percentageis comparable to the achievements in the healthy referencepopulation n Every second participant questioned was an active memberin a sports club whereas n did not return to asports club following cancer treatment Almost onethird n has never been a sports club member Reasons fornot engaging in sports club activities of participants wereno interestfun n physical weakness n no time n anxiety n andphysicianbased prohibition due to clear medical reasons n Nearly all participants n were active inrecreational sportsFurther analyses pointed toward diï¬erences in physicalactivity and sports club participation especially betweenparticipants with brain tumors and leukemialymphomas Thenumber of participants in recreational sports was reported highin both groups leukemialymphoma and brain tumor In contrast a diï¬erence was found in sports club activitySixtysix percent of leukemialymphoma patients were membersFrontiers in Pediatrics wwwfrontiersinAugust Volume 0cKeiser et alPhysical Consequences After Childhood CancerTABLE Physical activity and engagement in sports club and recreationalsportsEntire groupReference populationn n Physical activity guidelinesWHO mindayDaily physical activityDaily walking distance kmDaily walking distance “ kmDaily walking distance kmSports club activity currentlyFormer membershipRecreational sports activityAbs N number Reference values derived from the national cohort of healthy childrenand adolescents in the KiGGS study German Health Interviews and Examination Surveyfor Children and Adolescents of a sports club whereas only of participants with a braintumor were active in a sports club On the other hand almosthalf of the children treated for brain tumor and of thechildren treated for leukemialymphoma were former membersConcerning possible correlations between motor abilities andphysical education at school participation in sports clubs orrecreational sports defined as activeinactive as well as meetingthe physical activity recommendations no significant associationscould be determined Likewise the comparison of motor abilitiesof participants receiving sports therapy during treatment didnot show any correlation Almost half of the participants n took part in a sports therapy programme duringtreatment which was mainly oï¬ered as care and varied greatlyin terms of training interventions without any standardizationDISCUSSIONtreatmentThe results of our study clearly present evidence for deterioratedcardiovascular function in children and adolescents shortlyafter cessation of cancerIncreased pSBP andincreased pDBP are risk factors for cardiovascular diseasesregarding guidelines for arterial hypertension Potentialcardiovascular consequences such as stroke sudden deathheart failure and peripheral artery disease due to elevatedblood pressure values are described in the aforementionedguidelines as well as in the literature Accordinglychildhood cancer survivors with elevated blood pressure are atrisk to experience such cardiovascular late eï¬ects Regarding10years survivors of childhood cancer a higher prevalenceof hypertension is assumed and cardiovascular diseaserelated deaths are eighttimes more likely in childhoodcancer survivors compared to the general population Our findings support previous study results which depictcomplications such as increased blood pressure prehypertensionand hypertension in children and adolescents treated for cancer This study aimed at investigating specific parameters thatcould serve as early predictors for potential damage to thecardiovascular system Recent evidence of cSBP as a suitableparameter to determine the elasticity of blood vessels suggeststhat cSBP is more closely related to cardiovascular events in thefuture than brachial blood pressure Increased cSBP inparticipants in our study may result from early changes in arterialwall stiï¬ness As a further parameter to detect early impairmentsin elasticity of the vascular system PWV was investigated Incontrast to prior studies no decisive change was observedin PWV While anthracycl

cancer212

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: ShortTerm Consequences ofPediatric Anticancer TreatmentRegarding Blood Pressure MotorPerformance Physical Activity andReintegration Into Sports StructuresTina Keiser Dominik Gaser Christiane Peters Renate OberhofferFritz Sabine Kesting   and Irene von Luettichau  Edited byKirsten K NessSt Jude Children™s Research Hospital Department of Sports Medicine and Exercise JustusLiebig University GieŸen GieŸen Germany Department of Sport andHealth Sciences Institute of Preventive Pediatrics Technical University of Munich Munich Germany Department ofPediatrics and Children™s Cancer Research Center Kinderklinik M¼nchen Schwabing TUM School of Medicine TechnicalUnited StatesReviewed bySeth E KarolSt Jude Children™s Research HospitalUnited StatesJacques GrillInstitut Gustave Roussy FranceCorrespondenceSabine KestingsabinekestingtumdeIrene von LuettichauIreneTeichertvonLuettichaumritumde These authors sharesenior authorshipSpecialty sectionThis was submitted toPediatric Oncologya section of the journalFrontiers in PediatricsReceived January Accepted July Published August CitationKeiser T Gaser D Peters COberhofferFritz R Kesting S and vonLuettichau I ShortTermConsequences of PediatricAnticancer Treatment RegardingBlood Pressure Motor PerformancePhysical Activity and ReintegrationInto Sports StructuresFront Pediatr 103389fped202000463University of Munich Munich GermanyBackground Cardiovascular diseases in childhood cancer survivors are knownlate sequelae following treatment Arterial stiffness pulse wave velocity PWV andcentral systolic blood pressure cSBP are potential predictors to assess the statusof cardiovascular health Frequent inpatient stays and reduced physical activity PAduring treatment can lead to noticeable impairments regarding motor skills and physicalperformance The present study examined parameters of cardiovascular health motorperformance and the status of integration into sports structures shortly after cessationof treatmentMethods A crosssectional monocentric study was conducted from April to June Participants “ yrs mixed cancer entities during maintenance therapy andfollowup care were recruited Peripheral and central systolicdiastolic blood pressurepSBP pDBP cSBP and PWV were assessed using the MobilOGraph® The MOONtest MOtor performance in pediatric ONcology was used to scale motor performancePA levels and status ofintegration into sports structures were assessed with aquestionnaire referring to the KiGGS study All measured data were compared topublished reference valuesResults Forty participants ± years female were recruited ± years posttreatment PSBP zscore ± p pDBP ± p and cSBP ‰¥ years ± p were significantly increasedcompared to reference values PWV was also elevated but not significantly Motorperformance was reduced in almost all motor abilities Thirtysix percent of the examinedgroup did not participate in physical education at school to the full extent Only reported hour of daily moderatetovigorous PA as recommended for children andadolescents by the World Health anization Half of the participants were active sportsclub members before treatment but one third did not resume their former membershipFrontiers in Pediatrics wwwfrontiersinAugust Volume 0cKeiser et alPhysical Consequences After Childhood CancerConclusionIncreased cardiovascular parameters and impaired motor performanceshortly after cessation of treatment physical inactivity and low rates of integration intoregular sports programs highlight the support needed Young cancer patients shouldreceive early support in coping with physical limitations preferably soon after diagnosisMotor deficits could be reduced by applying targeted interventions Furthermorea regular sports therapy program during in and outpatient care could increaseengagement in PA to possibly counteract risk factors and improve cardiovascular healthKeywords childhood cancer cardiovascular health motor performance physical activity sports reintegrationblood pressure arterial stiffnessINTRODUCTIONExtensive research and optimized treatment regimens resulted inan increase of the 5year survival rate to in the USA and of the 15year survival rate to for patients under theage of in Germany However childhood cancer is a raredisease It contributes only around to all malignant diseasesin developed countries Worldwide children underthe age of and adolescents aged between and are diagnosed with cancer every year As a consequence ofthe success in the treatment of childhood cancer the importanceof survival quality and prevention of late sequelae have receivedmore attention during the last yearsKnown negativelongterm consequences ofintensivetreatment for childhood and adolescent cancer patients ofteninclude adverse eï¬ects on the cardiovascular system Cardiovascular diseases are the most frequently reported causesof death in childhood cancer survivors following secondarytumors Arterial stiï¬ness pulse wave velocity PWV andcentral systolic blood pressure cSBP are potential predictorsfor cardiovascular diseases frequently investigated in medicalresearch to evaluate the status of a patient™s cardiovascularhealth PWV describes the velocity of the pressure wave in the aortawhich spreads from the left ventricle through the arterial vascularsystem during systole Noninvasive investigation of the PWVvia ultrasound or oscillometric methods provides informationon the elasticity ofthe vascular system and enables earlyrecognition of damages in the vessels Thus in order to detectpotential structural modifications in the vascular system andindicators for arterial stiï¬ness at an early stage this subclinicalparameter should be surveyed continually Previous data indicatea positive correlation of PWV with arterial vascular stiï¬ness Moreover elevated PWV reflecting subclinical vasculardamage was shown in pediatric patients after hematopoieticstem cell transplantation On the contrary another studyinvestigated elevated blood pressure levels but no statisticallyAbbreviations PWV Pulse Wave Velocity WHO World Health anizationcSBP central systolic blood pressure pSBP peripheral systolic blood pressurepDBP peripheral diastolic blood pressure BMI Body mass index MOONMotor performance in pediatric oncology KiGGS German Health Interviews andExamination Survey for Children and Adolescentssignificant variation for PWV in pediatric cancer survivorscompared to healthy children and adolescents In addition to the abovementioned late sequelae severalproblems already arise during treatment and often persistthroughout survivorship For instancelongterminpatient stays and reduced physical activity during treatmentcan lead to noticeably reduced physical performance ofsurvivors and reintegration into sportschildhood cancerstructures mightberehabilitationprocess throughoutfrequentaï¬ectedIn healthy populations reduced physical activity leadsto negative consequencesfor cardiovascular health Additionally the necessary use of anthracyclines in almost ofapplied therapy regimens in childhood cancer increases the riskof cardiovascular morbidity and mortality eightfold comparedto agematched patients not receiving anthracyclines indicatingthe importance of reducing such longterm consequences Due to a poor state of health and impaired immune functionsports options such as physical education at school engagementin sports clubs or recreational sports are no longer feasibleduring therapy Consequently reintegration after cessation oftreatment is associated with even more barriers due to diseaseand treatmentrelated impairments Circumstances ofanticancer treatment can lead to inactivity resulting in deficitsof fine and gross motor skills reduced muscle strength andpoor physical fitness following treatment Especiallymotor performance in pediatric bone tumor patients oftenremains reduced until at least years after cessation of treatment Impairments of physical performance have been shown topersist throughout survivorship which may complicate thesurvivors™ reintegration into both social and sports structures aswell as the development of a longterm active lifestyle According to a questionnairebased study childhood cancersurvivors™ reintegration rate into physical education at school isvery low especially after treatment for bone tumors The lackof comprehensive oï¬ers of physical activity promotion and motordevelopment might exacerbate motor impairments and problemsof reintegration into sports structures Von Korn et al examined motor performance usingthe Fitnessgram Rcid13 as well as peripheral blood pressure centralblood pressure and PWV using the MobilOGraph Rcid13 in childrenafter treatment for childhood cancer n aged ± years ± years postdiagnosis Their results show reducedFrontiers in Pediatrics wwwfrontiersinAugust Volume 0cKeiser et alPhysical Consequences After Childhood Cancermotor performance of childhood cancer survivors compared toreference values of healthy children However no correlationcould be drawn regarding cardiovascular parameters and motorperformance The present crosssectional study aimed atinvestigatingvarious parameters of cardiovascular health motor performanceand status of physical activity in children and adolescentsshortly after cessation of anticancer treatment or during ongoingoral maintenance therapy The collection of such data isof considerable importance for the early detection of healthimplications related to both disease and treatment Moreoverthe findings will help to support the development of preventivestrategies regarding the health of children and adolescents treatedfor cancer Appropriate strategies during primary and secondaryprevention and following cancer treatment tertiary preventionneed improvementsMATERIALS AND METHODSDesignThe crosssectional monocentric study was performed over aperiod of months April“June at our institution Theassessment of cardiovascular parameters using the MobilOGraph Rcid13 was followed by the MOON test MOtor performance inpediatric ONcology to evaluate motor performance Finally theparticipants completed a standardized questionnaire referring tothe KiGGS study German Health Interview and ExaminationSurvey for Children and Adolescents to collect data regardingtheir current level of physical activity and status of integrationinto sports structures The Ethics Committee of the School ofMedicine of the Technical University of Munich approved thestudy project number SSR Participation was voluntaryand informed written consent was signed by each participant aswell as by his or her legal guardian All data was collected encodedpseudonym and in accordance with privacy policy standardsParticipantsPrior to addressing the participants all eligible children andadolescents were screened using the electronic patient recordSAP Rcid13 ERP Patients were recruited during routine followupvisits The following inclusion criteria were applied childrenand adolescents during maintenance therapy and followupcare of a pediatric oncological disease and currently agedbetween and years No restriction was applied regardingthe period posttreatment Exclusion criteria were medicalcontraindications such as fever acute infection orthopedicrestrictions and mental retardation insufficient knowledgeof the German language and absence of written informedconsent The attending physician confirmed participation forall recruited children and adolescents Following these inclusioncriteria children and adolescents were initially found eligibleFigure Outcome MeasuresAnamnestic and Anthropometric DataAnamnestic and clinical data ie type of cancer treatmentregime end of therapy was obtained from the electronic patientrecord SAP Rcid13 ERP The nursing staï¬ assessed anthropometricdata height and weight during routine medical examinationseca electronic column scaleseca mechanicalmeasuring rod Body mass index BMI was calculated as aratio of body weight kg per square body height m2 By usingthe reference values of a healthy German cohort BMI wasconverted into percentiles and classified in underweight 10thpercentile normal weight 10thˆ’90th percentile and overweight90th percentile Cardiovascular ParametersPrior to the measurement the participants had to rest for atleast min in a supine position PWV central blood pressureand peripheral blood pressure were assessed using the MobilOGraph Rcid13 IEM GmbH Stolberg Germany and HMS ClientServer Version an oscillometric noninvasive methodMeasurements were performed on the left upper arm The cuï¬was inflated twice with a rest of s in between Cuï¬ sizewas chosen according to the circumference of the participant™sleft upper arm An ARCSolver Algorithm calculated the cSBPindirectly as well as the PWV from recorded brachial pulseRaw data was transformed into zscores and compared by usingzscores of a healthy reference cohort PSBP and pDBPwere compared to references from the national cohort of children and adolescents KiGGS study For the assessmentof PWV and cSBP values references from Elmenhorst et al of healthy children and young adults were used To evaluatethe results of the parameters cSBP and PWV the examinedparticipants were separated into two groups years and‰¥ years According to the age distribution of the referencevalues participants years were compared to heightmatchedreference values and participants ‰¥ years were compared toagematched referencesThe measurement using the MobilOGraph Rcid13 was previouslyused several times in pediatric patients “ and was alsosuccessfully applied in childhood cancer survivors Motor PerformanceTo quantify motor performancethe MOON test MOtorperformance in pediatric ONcology was applied Thetest assesses motor abilities coordination speed flexibility andstrength and consists of eight test items eyehand coordinationinserting pins static balance static stand upper extremitycoordination throwing at a targetspeed reaction testmuscular endurance sittostand flexibility stand and reachhand grip strength handheld dynamometry and muscularexplosive strength medicine ball shot The test lasts min onaverage Data of each item was compared to published age andsexmatched reference values of a healthy population within anage range of to years Data of participants older than years was compared to reference values of healthy 17yearoldssince reference values of healthy 18yearolds are not availableCalculation of a total score is not possible within this toolInstead each item was analyzed individually and the percentagedeviation to reference values was computedFrontiers in Pediatrics wwwfrontiersinAugust Volume 0cKeiser et alPhysical Consequences After Childhood CancerFIGURE Flow chart of recruitment Out of participants eligible could not be addressed due to several medical examinations in different departments of thehospital and resulting in missing time slots Longer periods posttreatment are associated with fewer appointments for followups and more medical examinationstake place in day This does not necessarily mean that the children who could not be included in the study due to missing time slots are medically more complexTen participants refused participation Thus the sample included participantsPhysical Activity and Reintegration Into SportsStructures After Acute TreatmentPhysical activity levels and status of integration into sportsstructures were assessed with a standardized selfreportingreferring to the KiGGS study Thequestionnairequestionnaire wassupplemented by several disease andtreatmentrelated aspects in accordance with the study ofKesting et al to investigate potential barriers regardingreintegration and participation in sports activities eg barrierswith respect to exemption from physical education at school ornonparticipation in sports clubs sports therapy oï¬ers duringtreatment The KiGGS study oï¬ers the reference values ofhealthy children and adolescents n for comparison ofour dataData AnalysisCardiovascular parameters were analyzed and compared to thehealthy reference population with the one sample ttest Motorperformance was analyzed using the Wilcoxon signedranktest in comparison to age and sexmatched reference valuesPearson correlation was applied to calculate possible associationsbetween motor performance and cardiovascular parametersBMI and the period posttreatment The MannWhitneyUtestwas performed to evaluate anthracyclinemediated eï¬ects oncardiovascular health as well as diï¬erences within subgroupsregarding diï¬erent entities and levels of physical activity motorperformance physical education at school and achievement ofphysical activity recommendationsExplorative twosided statistical tests were conducted andp ‰¤ was considered statistically significant No adjustmentfor multiple comparison was conducted Correlations coefficientρ were classified according to Cohen Descriptive statistics were calculated with Microsoft Excelversion for demographic characteristics and medicaldata GraphPad Prism version was used to perform allfurther statistical analyses Data analysis was performed inconsultation with the Institute of Medical Informatics Statisticsand Epidemiology of the Technical University of MunichRESULTSParticipantsOut of eligible children and adolescents who met the inclusioncriteria a total of participants female with variouscancer entities were recruited and examined Table Performed AssessmentsFor various reasons not alltests could be realized withall participants In participants cardiovascularparameters could not be evaluated due to missing time slotsFrontiers in Pediatrics wwwfrontiersinAugust Volume 0cKeiser et alPhysical Consequences After Childhood CancerTABLE Anthropometric and medical characteristics of the participants n CharacteristicsN Mean ± SD MedianRangeAge at diagnosis years ± Age at assessment years ± ““ years‰¥ years Period postdiagnosis years ± Period posttreatment years ± ““ year“ years yearsLeukemiaLymphomaBone tumorBrain tumorOther solid tumors Body mass index¢ kgm2 ± “UnderweightNormal weightOverweightChemotherapyAnthracycline applicationCumulative dose mgm2RadiotherapyChestdirected radiation Anthracycline chest radiation Surgical tumor resectionRelapse ± “FIGURE Cardiovascular parameters shown in zscores and compared topublished reference values pSBP peripheral systolic blood pressurepDBP peripheral diastolic blood pressure PWV pulse wave velocity cSBPcentral systolic blood pressure Significant values p ‰¤ Results are given in Mean ± SD M median RangeOther solid tumors alveolar rhabdomyosarcoma n carcinoid tumor of the appendixn nephroblastoma n focal nodular hyperplasia liver n mature cysticteratoma ovary n thoracic ganglioneuroma n papillary thyroid carcinoman neuroblastoma n ¢BMI was converted into percentiles and classified in underweight 10th percentilenormal weight 10thˆ’90th percentile and overweight 90th percentile Thirteen participants received a combination of anthracyclines mainly the combination ofDoxorubicin and Daunorubicinduring routine appointment and a lack of willingness to prolongthe outpatient visit Six of the participants could not performthe MOON test due to medical limitations current orthopedicrestrictions n examinationrelated limitations ie a draintube in the crook of the arm for followup MRT n andabandonment due to lack of time n The central venousdevice has already been explanted in all participants prior toour studyOf the remaining participants not everyone performedevery test item Two participants could not perform the testitem speed due to a drain tube in the crook of the armFour participants could not accomplish the test item muscularexplosive strength due to orthopedic restrictions as well asexaminationrelated limitations Three participants did notperform the item muscular endurance of the legs n hadcrutches n severe muscular deficit in the legs n lackof time Two participants could not perform the test item handgrip strength with both hands due to lack of time n andinfusion needle in the crook of the arm n The test itemupper extremity coordination was measured in n participantsbecause reference values are provided for children between and years onlyCardiovascular HealthIn participants all parameters were assessed Based on theunderlying reference values for cSBP and PWV of Elmenhorstet al the group was separated into participants aged years heightmatched reference values and ‰¥ years agematched reference values PSBP zscore ± p pDBP zscore ± p as well as cSBP values‰¥ years zscore ± p were significantlyincreased compared to reference values of healthy children andadolescents Figure PWV was elevated but not significantly years zscore ± p ‰¥ years zscore ± p Comparison of cardiovascular parameters of participantswho received anthracyclines during intense therapy with acumulative dose of ± mgm2 and subjects whodid not receive cardiotoxic agents did not show statisticallysignificant diï¬erencesMotor PerformanceThe participants™ n motor performance wasreduced in almost all motor abilities compared to the referencevalues of healthy children and adolescents Table Significantimpairments became obvious in the following dimensionsmuscular explosive strength p upper extremitycoordination p muscular endurance of the legs p Frontiers in Pediatrics wwwfrontiersinAugust Volume 0cKeiser et alPhysical Consequences After Childhood CancerTABLE Results of the MOONtest compared to reference values n Motor abilityTest itemEyehand coordinationStatic balanceφSpeedInserting pins timeStatic stand contactsReaction test timeUpper extremity coordinationθFlexibilityThrowing at a target pointsStand and reach cmMuscular explosive strengthMedicine ball shot meterMuscular endurance legsSittostand secHand grip strengthHandheld dynamometry kgRightLeftNMean ± SD of differenceMedian pvalueto reference values ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’All results were compared to the reference values of each single test item Speed could only be measured in participants muscular explosive strength in participants andhand grip strength in right and left participants due to lack of time orthopedic restrictions or drain tube in the crook of the armFor the test items static balance and flexibility the absolute differences were used as the measured values would have fluctuated around zero and would have givenoversized percentagesφ Static balance was assessed counting the contacts with a foot to the ground while balancing on a rail in this context a negative difference to reference values represents fewercontacts and therefore better resultsθ Although all participants completed this test item reference values are provided for children between and years onlyM median abs indicates absoluteBold numbers indicate significant values p ‰¤ TABLE Results of the MOONtest comparing participants treated for leukemialymphoma and participants treated for brain tumors compared to reference valuesLeukemiaLymphoma n Brain tumor n Motor abilityEyehand coordinationStatic balanceφSpeedFlexibilityMuscular explosive strengthMuscular endurance legsHand grip strength rightHand grip strength leftNMean ± SD Median ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’NMean ± SD Median pvalueˆ’ ± ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ All results were compared to the reference values of each single test item φ Static balance was assessed counting the contacts with a foot to the ground while balancing on a rail in this context a negative difference to reference values represents fewercontacts and therefore better resultsFor the test items static balance and flexibility the absolute differences were used as the measured values would have fluctuated around zero and would have givenoversized percentagesDue to insufficient number of participants the comparison regarding the test item upper extremity coordination was disregarded reference values only provided for children aged and yearsM median abs indicates absoluteBold numbers indicate significant values p ‰¤ and hand grip strength on the right hand p The performance of eyehand coordination speed flexibility andhand grip strength on the left hand were also reduced but notsignificantly In the test item static stand the study participantsperformed slightly better compared to the reference populationFor upper extremity coordination throwing at a target onlyreference values from children aged “ years are availableTherefore comparison of the collected data was only possiblewith the same age group n Data for olderparticipants was not collectedComparing motor performance of participants diagnosedwith leukemialymphoma n and participantsn diagnosed with brain tumorsrevealedsome diï¬erences Table Children and adolescents treatedfor brain tumors performed significantly worse in eyehandcoordination than participants treated for leukemialymphomap Moreover the performances in all other testedmotoric dimensions of participants diagnosed with braintumor were deteriorated compared to participants treated forleukemialymphomas but not significantlyTo determine influencing factors on motor performance thecorrelation between motor performance results and BMI as wellas the period posttreatment was performed With increasingBMI values of static balance deteriorated significantly ρ Frontiers in Pediatrics wwwfrontiersinAugust Volume 0cKeiser et alPhysical Consequences After Childhood CancerFIGURE Pearson correlation between static balance and BMI kgm2¢ leukemialymphoma —¦ bone tumor –³ brain tumor cid7 other solid tumorsHorizontal line indicates the reference values The absolute difference fromreference values is given number of contacts to the ground A negativedifference means fewer ground contacts and therefore better performanceThirty four participants performed the testp which corresponds to a moderate to high correlationFigure A negative diï¬erence to reference values means fewercontacts to the ground and therefore better performance in staticbalance Furthermore some nonsignificant correlations werefound Deteriorated eyehand coordination ρ ˆ’ p and flexibility ρ ˆ’ p were also associatedwith a higher BMI However superior values in upper extremitycoordination ρ p muscular explosive strengthρ p hand grip strength right ρ p left ρ p and muscle endurance ofthe legs ρ p were associated with increasedBMI The test item speed showed no association with BMIρ p A longer period posttreatment was significantly associatedwith decreased eyehand coordination ρ ˆ’ p corresponding to a moderate correlation Figure Especially participants treated for a brain tumor with a longerperiod posttreatment showed deteriorated values in eyehandcoordination Speed performance was deteriorated in participantswith longer posttreatment period ρ ˆ’ p Physical Activity and Reintegration IntoSports StructuresAccording to the selfreported questionnaire n did not participate in physical education at school to full extend n were not admitted to school sports activitiesand n were partly excluded Table Neitherof the two participants treated for bone tumor was takingpart in physical education at school n whereaschildren with other tumors participated to a notably higherrate Treatmentrelated muscular deficits n andosteonecrosis n were the most common reasons forparticipants not taking part in physical education at schoolFIGURE Pearson correlation between eyehand coordination and periodposttreatment months ¢ leukemialymphoma —¦ bone tumor –³ brain tumorcid7 other solid tumors Horizontal line indicates the reference values Thirtyfour participants performed the test itemTABLE Participation in physical education at school subdivided into entitiesn Participation Partial exemption Full exemptionN N N Entire group n LeukemiaLymphoma n Bone tumor n Brain tumor n Other solid tumors n Abs N number Four out of children were still attending kindergarten and could notanswer the question regarding participation in physical education at schoolOnly n reported moderatetovigorous physicalactivity for min daily as generally recommended by the WHOfor healthy children and adolescents Table This percentageis comparable to the achievements in the healthy referencepopulation n Every second participant questioned was an active memberin a sports club whereas n did not return to asports club following cancer treatment Almost onethird n has never been a sports club member Reasons fornot engaging in sports club activities of participants wereno interestfun n physical weakness n no time n anxiety n andphysicianbased prohibition due to clear medical reasons n Nearly all participants n were active inrecreational sportsFurther analyses pointed toward diï¬erences in physicalactivity and sports club participation especially betweenparticipants with brain tumors and leukemialymphomas Thenumber of participants in recreational sports was reported highin both groups leukemialymphoma and brain tumor In contrast a diï¬erence was found in sports club activitySixtysix percent of leukemialymphoma patients were membersFrontiers in Pediatrics wwwfrontiersinAugust Volume 0cKeiser et alPhysical Consequences After Childhood CancerTABLE Physical activity and engagement in sports club and recreationalsportsEntire groupReference populationn n Physical activity guidelinesWHO mindayDaily physical activityDaily walking distance kmDaily walking distance “ kmDaily walking distance kmSports club activity currentlyFormer membershipRecreational sports activityAbs N number Reference values derived from the national cohort of healthy childrenand adolescents in the KiGGS study German Health Interviews and Examination Surveyfor Children and Adolescents of a sports club whereas only of participants with a braintumor were active in a sports club On the other hand almosthalf of the children treated for brain tumor and of thechildren treated for leukemialymphoma were former membersConcerning possible correlations between motor abilities andphysical education at school participation in sports clubs orrecreational sports defined as activeinactive as well as meetingthe physical activity recommendations no significant associationscould be determined Likewise the comparison of motor abilitiesof participants receiving sports therapy during treatment didnot show any correlation Almost half of the participants n took part in a sports therapy programme duringtreatment which was mainly oï¬ered as care and varied greatlyin terms of training interventions without any standardizationDISCUSSIONtreatmentThe results of our study clearly present evidence for deterioratedcardiovascular function in children and adolescents shortlyafter cessation of cancerIncreased pSBP andincreased pDBP are risk factors for cardiovascular diseasesregarding guidelines for arterial hypertension Potentialcardiovascular consequences such as stroke sudden deathheart failure and peripheral artery disease due to elevatedblood pressure values are described in the aforementionedguidelines as well as in the literature Accordinglychildhood cancer survivors with elevated blood pressure are atrisk to experience such cardiovascular late eï¬ects Regarding10years survivors of childhood cancer a higher prevalenceof hypertension is assumed and cardiovascular diseaserelated deaths are eighttimes more likely in childhoodcancer survivors compared to the general population Our findings support previous study results which depictcomplications such as increased blood pressure prehypertensionand hypertension in children and adolescents treated for cancer This study aimed at investigating specific parameters thatcould serve as early predictors for potential damage to thecardiovascular system Recent evidence of cSBP as a suitableparameter to determine the elasticity of blood vessels suggeststhat cSBP is more closely related to cardiovascular events in thefuture than brachial blood pressure Increased cSBP inparticipants in our study may result from early changes in arterialwall stiï¬ness As a further parameter to detect early impairmentsin elasticity of the vascular system PWV was investigated Incontrast to prior studies no decisive change was observedin PWV While anthracycl Answer:

Thyroid_Cancer

Activation by NaturalPhytochemicals An OverviewConcetta Iside Marika Scafuro Angela Nebbioso  and Lucia Altucci Department of Precision Medicine University of Campania œLuigi Vanvitelli Naples ItalySirtuins are class III histone deacetylases whose enzymatic activity is dependent on NADas a cofactor Sirtuins are reported to modulate numerous activities by controlling geneexpression DNA repair metabolism oxidative stress response mitochondrial functionand biogenesis Deregulation of their expression andor action may lead to tissuespecificdegenerative events involved in the development of several human pathologies includingcancer neurodegeneration and cardiovascular disease The most studied member of thisclass of enzymes is sirtuin SIRT1 whose expression is associated with increasinginsulin sensitivity SIRT1 has been implicated in both tumorigenic and anticancerprocesses and is reported to regulate essential metabolic pathways suggesting thatits activation might be beneficial against disorders of the metabolism Via regulation of p53deacetylation and modulation of autophagy SIRT1 is implicated in cellular response tocaloric restriction and lifespan extension In recent years scientific interest focusing on theidentification of SIRT1 modulators has led to the discovery of novel small moleculestargeting SIRT1 activity This review will examine compounds of natural origin recentlyfound to upregulate SIRT1 activity such as polyphenolic products in fruits vegetablesand plants including resveratrol fisetin quercetin and curcumin We will also discuss thepotential therapeutic effects of these natural compounds in the prevention and treatmentof human disorders with particular emphasis on their metabolic impactKeywords sirtuin natural compounds oxidative stress human disorders polyphenolsEdited byCecilia BattistelliSapienza University of Rome ItalyReviewed byNarasimham L ParinandiThe Ohio State UniversityUnited StatesCarmen JeronimoPortuguese Oncology InstitutePortugalCorrespondenceAngela NebbiosoangelanebbiosounicampaniaitLucia Altucciluciaaltucciunicampaniait These authors share last authorshipINTRODUCTIONSpecialty sectionThis was submitted toTranslational Pharmacologya section of the journalFrontiers in PharmacologyReceived April Accepted July Published August CitationIside C Scafuro M Nebbioso A andAltucci L SIRT1 Activation byNatural Phytochemicals An OverviewFront Pharmacol 103389fphar202001225Epigenetic modifications are associated with genome stability gene transcription and metabolicregulation Acetylation is one of the most characterized histone modifications Histoneacetyltransferase HAT and histone deacetylase HDAC enzymes control the levels of histoneacetylation modulating gene expression Cavalli and Heard Sirtuins SIRT “ are enzymes classified as class III HDACs They exhibit different subcellularlocalizations SIRT1 SIRT6 and SIRT7 are nuclear although SIRT1 isoforms were also identified inAbbreviations SIRT1 Sirtuin HATs Histone acetyl transferases HDACs Histone deacetylases ROS Reactive oxygenspecies PPAR Receptor peroxisome proliferatoractivated receptor NRF Nuclear respiratory factor TFAM Mitochondrialtranscription factor A SOD Superoxide dismutase TNFa Tumor necrosis factor a IAP Apoptosis protein inhibitor Bcl2Bcell lymphoma2 family MnSOD Manganese superoxide dismutase RSV Resveratrol Que Quercetin oxLDL OxidizedLDL BBR Berberine Cur Curcumin COX Cytochrome c oxidase T2D Type II diabetes NAFLD Nonalcoholic fatty liverdisease CRM Caloric restriction mimeticFrontiers in Pharmacology wwwfrontiersinAugust Volume 0cIside et alNatural SIRT1 Activatorsthe cytoplasm SIRT2 is mainly cytosolic SIRT3 SIRT4 andSIRT5 are mitochondrial and can shuttle to the nucleus Changand Guarente The enzymatic activity of SIRTs is dependent on NAD as acofactor and plays an important role in controlling geneexpression DNA repair metabolism oxidative stress responsemitochondrial function and biogenesis Deregulation of theiractivity may lead to tissuespecific degenerative events thatunderlie several human pathologiesincluding cancerdiabetes and cardiovascular diseases Haigis and Sinclair O™Callaghan and Vassilopoulos Waldman The most studied member of this enzymatic class isSIRT1 SIRT1 regulates metabolic pathways cell survivalcellular senescence and ‚ammation and acts in thepathogenesis of chronic conditions such as diabetes as well aspulmonary neurodegenerative and cardiovascular diseasesIndeed SIRT1 has been reported to play a key role intumorigenesis as an oncogene or tumor suppressor dependingon the context specificity BiasonLauber It is able tocontrol these processes via deacetylation of lysine groups ofhistone and nonhistone proteins including known transcriptionfactors FOXO MyoD p53 PGC1a Kupis Chronic ‚ammation caused by oxidative damage increasesthe risk of many chronic disordersincluding heartcardiovascular and neurodegenerative diseases obesity insulinresistance and type diabetes T2D Geto Oxidative stress plays a key role in the pathogenesis of theseconditions The overproduction of reactive oxygen speciesROS including free radicals and reactive nitrogen speciesRNS can lead to damage of cellular components such aslipids proteins and DNA The imbalance between oxidantsand antioxidants can result in cellular dysfunction apoptosisand necrosis Liguori SIRT1 guards against oxidative stress by activating genetranscription of PGC1a via deacetylation and by regulatingtranscription of factors such as the nuclear receptor peroxisomeproliferatoractivated receptor PPAR nuclear respiratory factorNRF and mitochondrial transcription factor A TFAMinvolved in modulation of biogenesis and mitochondrialfunction Ren and metabolism of glucose and lipidsRodgers SIRT1 is also able to regulate the expressionof superoxide dismutase SOD and glutathione peroxidase Sun In addition since mitochondrial dysfunction leads tothe activation of apoptosis SIRT1 can directly regulate theapoptotic process by modulating acetylation of PGC1a Zhang SIRT1 also regulates ‚ammatory responseKauppinen By modulating the acetylation level ofNFkB p65 SIRT1 is able to control transcription of genes such asIL interleukin1 tumor necrosis factor a TNFa IL8 IL6and other ‚ammatory factors Rodgers Ren Yeung Through NFkB SIRT1 also regulatesthe expression of genes such as inhibitor of apoptosis proteinIAP and Bcell lymphoma2 Bcl2 and tumor necrosis factorreceptor TNFR Ren SIRT1 protects against oxidative stress via regulation ofFOXO protein acetylation which is involved in antioxidantprocesses apoptosis and cell proliferation Wong andWoodcock By activating FOXOMsSOD pathwaySIRT1 increases the expression of manganese superoxidedismutase MnSOD and catalase counteracting oxidativestress and promoting damage repair Gu SIRT1also increases the expression of MnSOD by deacetylating p53thus enhancing cellular antioxidant capacity Brunet Zhang Ren Over the past few years the evergrowing awareness that goodhealth goes hand in hand with a healthy and balanced diet hasencouraged people to eat more fruit and vegetables and to takesupplements to make up for any deficiency D™Angelo Bioactive compounds in the diet can act as antioxidantand anti‚ammatory agents thereby reducing the negativeeffects of oxidative stress and the incidence of chronicdiseases such as obesity diabetes and cardiovascular disordersWang Several moleculesincluding naturalphytochemical compounds can modulate SIRT1 activityMiceli Numerous studies have provided evidenceof the protective effects of natural polyphenolic substances suchas resveratrol quercetin curcumin and fisetin and ofnatural nonpolyphenolic substances such as berberineMcCubrey Natural polyphenols are the largestgroup of phytonutrients and are considered potential agents forthe prevention and treatment of stressrelated oxidative diseasesThey are found in many plants and foods such as fruitsvegetables tea cereals and wine and longterm intake isassociated with health benefits Mediterranean diets are in factlinked to a reduced risk of chronic diseases due to theconsumption of olive oil and red wine which contain highamounts of polyphenols Romagnolo and Selmin Most of the evidence supporting the beneficial effects ofphytochemical compounds comes from in vitro or animalstudies while human studies evaluating the longterm impact ofphytomolecules are particularly few or inconsistent Interventionalstudies are in fact limited by issues of bioavailability andmetabolism However in vitro studies aimed at identifyingcellular targets linked to the beneficial actions of phytonutrientrich foods at concentrations ranging from nM to µM challenge thetranslatability of data After ingestion these compounds are in factdetected as phase II metabolites and their blood level does notexceed concentrations in the nM range Substantial amounts of thecompounds and their metabolites are degraded in the colon byintestinal microbiota giving rise to small phenolic acids andaromatic catabolises which are absorbed by the circulatorysystem Del Rio Interesting studies showed thatthese natural polyphenol and nonpolyphenol substances couldaffect SIRT1 expressionactivity Table de Boer Themain mechanisms of action common to polyphenol and nonpolyphenol molecules that lead to antioxidant and anti‚ammatory effects via SIRT1 activation are reported in Figure Here we focus on the natural molecules resveratrolquercetin fisetin curcumin and berberine and elucidate theireffect on SIRT1 activation and their potential to treat andorprevent several human pathologies mainly associated withmetabolic disorders Figure Frontiers in Pharmacology wwwfrontiersinAugust Volume 0cIside et alNatural SIRT1 ActivatorsTABLE Classification of nutraceuticals based on their action and food sourceNaturalSIRT1activatorsEffectSourceReferencesResveratrol Positive effect on bloodlipid profile antioxidantDark grapesraisins peanutsQuercetinBerberineCurcuminFisetinAnticancer positiveeffect on blood lipidprofile antioxidant anti‚ammatoryAntioxidant anti‚ammatoryAnticancer antioxidantanti‚ammatoryAnticancercardiovascularpreventive anti‚ammatoryantioxidantFruits vegetablesnutsNatural componentof traditionalChinese herbCoptidis rhizomaActive componentin Curcuma longaApplespersimmonsgrapes onionskiwi kalestrawberriesD™Angelo Zordoky Hung Nabavi Nabavi Wu Hung Zendedel Kim Chen NATURAL COMPOUNDS ENHANCINGSIRT1 EXPRESSION AND ACTIVITYResveratrolResveratrol RSV a nonflavonoid polyphenol found in grapesand grape products such as red wine exerts an antioxidant actionwith reported cancer preventive properties KrisEtherton RSV also has anti‚ammatory anticancer andantineurodegenerative effects Piotrowska The roleof RSV as an immune response modulator was demonstrated inboth in vitro and in vivo studies where it reversed immunesenescence in older rats reduced ‚ammatory responses inrodents and improved immunological activity against cancercells Malaguarnera RSV was shown to be involved in theactivation of macrophages T cells and natural killer cells as wellas in the suppression processes of CD4 CD25 regulatory T cellsYang Svajger and Jeras All these effects aredue to its ability to remove ROSinhibit cyclooxygenaseCOX and trigger anti‚ammatory pathways via SIRT1activation Miceli Malaguarnera ActivatedSIRT1 interrupts TLR4NFkBSTAT axis reduces cytokineproduction by inactivated immune cells and inhibits pro‚ammatory factors derived from macrophagesmast cellssuch as plateletactivating factor and TNFa Capiralla RSVSIRT1 interaction modifies SIRT1 structure andpromotes binding activity with its substrates including p65RelA Yeung a component of the NFkB complexwhich regulates activation of leukocytes and ‚ammatorycytokines SIRT1 activated by RSV inhibits acetylation of RelAby reducing the expression of ‚ammatory factors such as TNFa IL1b IL6 metalloprotease MMP1 MMP3 and NFkBmediated Cox2 Malaguarnera AMP activatedprotein kinase AMPK is also a target of RSV as it controlsSIRT1 activity via regulation of cellular levels of NAD thusacting as an energy sensor Price Cyclicadenosine monophosphate cAMP levels activate proteinkinase A resulting in phosphorylation and activation of SIRT1FIGURE Basic mechanisms and effects of SIRT1 activation by polyphenol and nonpolyphenol moleculesFrontiers in Pharmacology wwwfrontiersinAugust Volume 0cIside et alNatural SIRT1 ActivatorsFIGURE Nutraceutical action on SIRT1 expression Natural substances have beneficial effects on human health by regulating SIRT1 action in different cellularprocesses wwwpubchemncbinlmnihgovWan Activated SIRT1 catalyzes the deacetylationand activation of PGC1a thereby promoting beneficial effectsin the metabolism Ren In different anisms S cerevisiae C elegans and Dmelanogaster expressing SIRT1 or its homologous genesRSV treatment is able to extend life span In mammaliansRSV administration can improve SIRT1dependent cellularprocesses such as axonal protection Araki fatmobilization Chaplin and inhibition of NFkBdependent transcription Yeung these effects areabolished in SIRT1 knockdown models Numerous studiesinvestigated the beneficial effects of RSV in cardiovasculardiseases including hypertension Theodotou cardiac ischemia Fourny and atherosclerosisChassot RSV has an effect on blood vesselsreduces ‚ammation and prevents thrombus formation andplatelet oxidation Zordoky It can also reducecardiac dysfunction oxidative stress fibrosis and apoptosis inthe heart Gupta Yamagata In addition RSVwas found to improve heart and kidney damage in rats Li et al2020a The protective effect of RSV is associated with anincrease in SIRT1 activity which deacetylates FOXO1 andactivates MnSOD downstream RSVinduced MnSOD alsoreduces oxidative stress Li 2020a A recent in vitrostudy showed that RSV reduces hypoxiainduced apoptosis inH9C2 cells through activation of SIRT1miR30d5pNFkB axisHan RSV treatment decreased cortical andhippocampal malondialdehyde levels while increasing SODactivity and SIRT1 expression in a diabetic rat model Ma RSV was shown to activate SIRT1 and improve endothelialfunction in obese mice via upregulation of PPARd expressionactivity in PPARd mutant mice Cheang It hadpreviously been observed that Akt activation together withPPARd is involved in vascularization of dbdb mice Tian RSV was subsequently reported to increasephosphorylation of Akt and transcriptional activity of PPARdin the aorta of wildtype mice thus supporting the hypothesis ofSIRT1PPARd interaction and to strongly decrease LPCinduced mitochondrial ROS in the aortic endothelium ofC57BL6 mice Cheang Taken together thesefindings highlight the beneficial effects of RSV against oxidativestress which is involved in major pathologies such as heart andmetabolic disorders Although RSV is beneficialin manycontexts its pleiotropic actions need to be better studied inorder to understand which of its described activities are directlydue to SIRT1 modulation and whether this effect is always directBecause of the pleiotropic actions of RSV clinical trials arecurrently testing its therapeutic potential in a wide range ofhuman diseases However of all the mechanisms described in invitro and in vivo studies only a few have been confirmed inhumans such as gene and protein regulation in blood or musclecells and Akt signaling pathways Ghanim Brasnyo Many clinical studies conducted in healthy patientsand volunteers using both high and low doses of RSV highlightits potential cardioprotective benefit through improvement ofendothelial function‚ammatory markers and glucosemetabolism Nevertheless the mechanisms of action are notyet well defined Despite clinical evidence of its effects thepoor bioavailability and rapid metabolism of RSV severelylimit the potential use of this molecule in the clinic Futurescientific research should focus on identifying actual metabolitesor mediators of these observed effectsTo date clinical trials have tested the efficacy safety andpharmacokinetics of RSV in the prevention and treatment of different pathological conditions wwwclinicaltrialsgovFrontiers in Pharmacology wwwfrontiersinAugust Volume 0cIside et alNatural SIRT1 ActivatorsRestricting the search to interventional phase studiescompleted and terminated clinical trials addressed theability of RSV to improve the pathological conditions ofpatients affected by several diseases Most of these studiestested RSVmediated effects in central nervous systemdisorders Friedreich ataxia Alzheimer™s disease Parkinson™sdisease metabolic disorders [T2Dinsulin resistancedyslipidemia hypercholesterolemia metabolic syndrome Xnonalcoholic fatty liver disease NAFLD] A phase clinicaltrial NCT01640197 tested the effects of chronic resveratrolsupplementation mg daily for days in healthy humansand found considerable improvements in cognitive performancecerebral blood flow subjective sleep mood health and bloodpressure A list of completed and terminated clinical trials inwhich RSV was tested for metabolic disorders is reported inTable Focusing on completed clinical trials with availableresults NCT02114892 evaluated the effect of RSV on metabolicsyndrome demonstrating that when administered three timesper day mgdie before meals RSV was able to treat andprotect from obesity and diabetes with beneficial effects onglucose and lipid metabolism blood pressure and bodyweight Another phase study NCT02095873 evaluated theeffects of a formulation composed of RSV and hesperetin inobese subjects and found that these molecules are dietaryinducers of glyxalase improving metabolic and vascularhealth of obese subjects Xue QuercetinThe flavonoid polyphenol quercetin Que ²²pentahydroxyflavone is a natural safe dietary supplementfound in a glycoside form in fruits vegetables and nuts whichhas antioxidant and anti‚ammatory properties Nabavi Wu In recent years the scientific community has focused on thepotential antiproliferative chemopreventive and anticarcinogenicactivities of Que as well as on its role as a modulator of geneexpression However Que was also found to have potentially toxiceffects including mutagenicity prooxidant activity mitochondrialtoxicity and inhibition of enzymes involved in hormonalmetabolism Li Due to its poor solubility short halflife and low bioavailability its medical use is limited Konrad andNieman In humans Que bioavailability is very low and absorption varies from to in subjects receiving mgdie Costa Que may reduce infection Li hepatic lipemicoxidative damage Cui Zhang et al2016b and antioxidant risk Xu In addition Que isknown to exert a modulating action on immunity Galleggiante As regards its mechanism of action in some cell linesQue was able to inhibit the production of TNF in macrophagesTang IL8 in A549 lung cells induced bylipopolysaccharide LPS Geraets and TNFa andIL1a mRNA levels in glial cells causing a decrease in neuronal celldeath induced by microglial activation Li MainlyTABLE Resveratrol in clinical trials for metabolic disordersStatusStudy TitleConditionsInterventionPhaseNCT NumberCompleted Effects of Resveratrol in Patients With TypeType Diabetes DiabetesTerminated Effect of Administration of Resveratrol onType Diabetes Mellitus mg to a maximum dose of g daily mg times dailyPhase NCT01677611Phase NCT02549924Glycemic Variability in Individuals With Type Diabetes MellitusCompleted Effect of Resveratrol on Agerelated InsulinResistance and Inflammation in HumansCompleted Regulation of Intestinal and HepaticLipoprotein Secretion by ResveratrolType Diabetes Mellitus InsulinResistanceDyslipidaemia Insulin ResistanceCompleted Effects of Dietary Antioxidants to PreventHypercholesterolemia HealthyCardiovascular DiseaseHealthy Aging Through Functional FoodCompletedwith resultsCompleted Effects of Resveratrol on Inflammation inType Diabetic PatientsCompletedwith resultsEffect of Resveratrol Administration onMetabolic Syndrome Insulin Sensitivity andInsulin SecretionCompleted Resveratrol for the Treatment of NonAlcoholic Fatty Liver Disease and InsulinResistance in Overweight AdolescentsGlucose Intolerance Aortic Stiffness VasodilationType Diabetes Mellitus Inflammation Insulin Resistance Other Disorders ofBone Density and StructureMetabolic Syndrome XNAFLD Type Diabetes MetabolicSyndromeCompleted A Study of Resveratrol as Treatment forFriedreich AtaxiaFriedreich AtaxiaCompleted Effect of Banaba Lagerstroemia Speciosaon Metabolic Syndrome Insulin Secretionand Insulin SensitivityMetabolic Syndrome X mg twice daily for daysPhase NCT01354977 mg for week followed by g for weekDietary Supplement red wine for monthDietary Supplementresveratrol for monthTransresveratrol mg hesperetin mg combination months mg daily then months mg daily mg times daily beforemeals with a total dose of mg daily mg twice daily for a total dailydose of mg for days g daily mg twice daily for weeks then g daily gtwice daily for weeksBanaba capsules mg times daily before meals for daysPhase NCT01451918Phase NCT02409537Phase NCT02095873Phase NCT02244879Phase NCT02114892Phase NCT02216552Phase NCT01339884Phase NCT02767869Frontiers in Pharmacology wwwfrontiersinAugust Volume 0cIside et alNatural SIRT1 Activatorsin immunity and ‚ammation Que acts on leukocytes and targetsmany intracellular signaling kinases and phosphatases as well asenzymes and membrane proteins Li Theimmunostimulating effect of Que is due to induction of theexpression of interferong IFNg derived from Th1 andinhibition of IL4 derived from Th2 in normal peripheral bloodmononuclear cells Nair In addition Que reduces T cellproliferation by blocking IL12induced tyrosine phosphorylationof JAK2 TYK2 STAT3 and STAT4 Muthian and Bright Nabavi In ‚ammation Que inhibits the enzymesCOX and lipoxygenase Lee and Min Savikin Inthe RAW cell line Que was also shown to counteract LPSinduced ‚ammation by phosphorylation of tyrosinephosphatidylinositol3kinase PI3Kp85 and complexformation of tolllike receptor TLR4MyD88PI3K Endale Oxidative stress occurs following an imbalance of the body™santioxidant defence mechanisms and excessive generation of freeradicals and is involved in various pathologies such as diabetesatherosclerosis hypertension neurodegenerative diseases‚ammation and cancer Oboh Que is apowerful ROS scavenger and its antioxidant action is due tothe presence of two pharmacophores within the molecularstructure which confer a favorable configuration for freeradical elimination Costa Generally Que reducesthe effects of free radicals by transferring the hydrogen atom andstabilizing the radicals a feature that has a structurefunctionrelationship Oboh Que can also act as both an antioxidant and prooxidant agentAt low concentrations “ µM Que displayed a protective effectagainst oxidative DNA damage in vitro in human lymphocytes Li At concentrations between µM and µM Que wasable to directly eliminate ROS in vitro Costa Howeverits effect in vivo is very likely not direct but due to its ability tomodulate the cell™s antioxidant defense mechanisms moderateoxidative stress can in fact increase the cell™s antioxidant defensesresulting in general cytoprotection Halliwell Recentresearch showed that oxidized LDL oxLDL induces oxidativestress LaraGuzman Oxidative injuries promote ROSgeneration in human endothelial cells and SIRT1 regulatesendothelial function Therefore enhancement of SIRT1 activityand SIRT1AMPK axis upregulation inhibits oxidative injuryinducing endothelial dysfunction Chen Shentu Que may reduce oxLDLinduced oxidative damageby upregulating SIRT1 and AMPK Hung thereforepotentially preventing oxLDLimpaired SIRT1 inhibition linked toendothelial dysfunction These findings indicate that SIRT1 canfunction as a regulator to improve AMPK activity under oxLDLstimulation Hung It was very recently shown that Que mgkg can reduceinsulin resistance and improve glucose metabolism by reducingsensitivity to T2Dinsulin resistance in obob mice via SIRT1activation Hu In this context another study showedthat in streptozotocininduced diabetic rats Que mgkginhibits oxidative damage by increasing SIRT1 expression anddecreasing levels of NFkB a SIRT1 substrate Iskender In recent years the scientific community has focused on therole of apoptosis in cardiovascular disease showing thatoxidative stress myocardial ischemia hypoxia and ischemiareperfusion injury may induce myocardial apoptosis Donniacuo Tang and colleagues evaluated the effects of Que inimproving myocardialischemiareperfusion injury MIRinduced cell apoptosis both in vitro and in vivo SIRT1 andPGC1a expression levels were decreased in rat MIR groups butwere significantly increased after treatment with Que Tang Furthermore activation of SIRT1PGC1a pathwayupregulated Bcl2 expression and downregulated Bax exertingantiapoptotic effects The authors hypothesized that Que mightimprove MIRinduced myocardial damage via regulation ofSIRT1PGC1a and Bcl2Bax pathways Tang Que is also reported to regulate ROS generation and mitigatemitochondrial dysfunction by promoting their biogenesisSpecifically in a study to develop a therapeutic strategy forosteoarthritis Que was shown to increase expression levels ofSIRT1 PGC1a NRF1 and NFR2 TFAM and phosphoAMPKa in osteoarthritis rats confirming the hypothesis that Quemight act via the AMPKSIRT1 signaling pathway Qiu Overall these findings suggest that Que may counteractcardiovascular disease and oxidative damageThe growing body of evidence supporting the beneficial effects ofQue has led to its clinical use as demonstrated by the number ofclinical trials studies on ClinicalTrialsgov A list of completedstudies using Que in different metabolic and ‚ammatoryconditions is reported in Table Specifically a phase clinicaltrial NCT01839344 measured the effect of Que on glucosetolerance and postprandial endothelial function in subjects withT2D compared to the effect of an alphaglusidase inhibitor acarboseThe administration of g of Que led to a decrease in postprandialblood glucose NCT01839344 Given its antioxidative and anti‚ammatory capacities this flavonoid was considered a goodcandidate for antioxidant therapy in mucositis NCT01732393hepatitis C NCT01438320 idiopathic pulmonary fibrosisNCT02874989 osteoporosis NCT00330096 uric acidmetabolism NCT01881919 cytokine release NCT01106170and chronic obstructive pulmonary disease NCT01708278 Inthe latter study Que supplementation was safely tolerated bypatients with mildtosevere chronic obstructive pulmonarydisease opening the way towards the potential use of Que as atherapeutic agent for this conditionHowever as for RSV and nutraceuticals in general the resultsof molecular studies on Que obtained from in vitro investigationsand animal models are often inconsistent with data from clinicaltrials Concentration factor dose and timing of administrationand bioavailability are the two main issues that require furtherclarification Additional studies are needed to identify theoptimal concentration of Que for it to exert a beneficial effectfor example on insulin sensitivityBerberineBerberine BBR is an isoquinoline alkaloid reported to haveanalgesic anticancer anti‚ammatory and myocardialprotective properties Cicero and Baggioni It was foundFrontiers in Pharmacology wwwfrontiersinAugust Volume 0cIside et alNatural SIRT1 ActivatorsTABLE Quercetin in clinical trials for metabolic and ‚ammatory disordersStatusStudy TitleConditionsInterventionPhaseNCT NumberCompleted Effect of Quercetin in Prevention andTreatment of Oral MucositisBeneficial Effects of Quercetin in ChronicObstructive Pulmonary Disease COPDCompletedwith resultsCompleted QTrial in Patients With Hepatitis CCompleted Effects of Quercetin on Blood Sugar andChemotherapy Induced OralMucositisChronic ObstructivePulmonary DiseaseChronic Hepatitis CDiabetes Mellitus Type mg daily for weeksPhase NCT01732393 to mg daily for weekPhase NCT01708278 days mg oral single dose of mgPhase Phase NCT01438320NCT01839344Blood Vessel Function in Type DiabetesCompleted Effect of Quercetin Supplements onCompletedHealthy Males a 4Week RandomizedCrossOver TrialTargeting ProInflammatory Cells inIdiopathic Pulmonary Fibrosis a HumanTrialCompleted Efficacy of Provex CV Supplement toReduce Inflammation Cytokines andBlood PressureHyperuricemia Gout KidneyCalculi DiabetesCardiovascular DiseaseIdiopathic Pulmonary FibrosisIPFBlood Pressure mg tablet for days with meal breakfastpreferredEarly PhaseNCT01881919 doses administered over consecutive days in consecutive weeks oral administration ofquercetin mg daily mg of Provex CV supplement by mouth perday for weeksPhase NCT02874989Phase NCT01106170Completed Effects of Hesperidin on Bone MineralOsteoporosis OsteopeniaPhase NCT00330096Density and Bone Metabolism ofPostmenopausal Womento exert protective antioxidative effects in different physiologicand pathologic conditions Huang Li 2020bHowever the mechanisms underlying these effects remainunclear BBR was described as a potential antitumor agent thatinduces cell cycle arrest in G0G1 phase increases Cipp21 andKipp27 protein expression decreases expression of cyclin D1D2 and DE and the cyclindependent kinases Cdk2 Cdk4 andCdk6 promoting apoptosis in HL60 human leukemia cellsLi BBR can also deregulate telomerase activityand promote mitochondriadependent apoptosis in HepG2human hepatocarcinoma cells through caspase and caspase activation PARP cleavage induction increased expression ofthe proapoptotic protein Bax through activation of FOXOtranscription factors and inhibition of Bcl2 and Bclx antiapoptotic protein expression Hwang BBR wasobserved to exert an apoptotic effect by inducing ROSproduction and increasing MAPK and JNK activity of p38 inSW620 human colon carcinoma cells and by increasing Ca andcytochrome C release in HSC3 squamous cells Song In addition BBR inhibits the proliferation of cancer cellsthrough an anti‚ammatory pathway In oral carcinoma celllines and in SCC4 cells BBR inhibits expression of COX2 andAP1 bond decreases prostaglandin E2 PGE2 production andsuppresses NFkB IKK ERK and JNK activities FurthermoreBBR can inhibit colon cancer cell growth by activating retinoid Xreceptor a RXRa which binds RXRa and promoting bcatenin degradation Ruan However some studieshighlighted the potential ability of BBR to prevent oxidativestressinduced senescence by activating AMPK and restoringNAD levels Song Initial research revealed a significant role of SIRT1 signalingin mediating the antioxidant effect of BBR in diabetes Pang and in lipid metabolism Hasanein Thelipidlowering activity mediated by cotreatment with BBR andRSV was investigated in mice exposed to a high fat diet Zhu In vivo data showed that BBR combined with RSVlowered total cholesterol triglyceride and LDL cholesterol levelsin mice These findings were also confirmed in vitro with 3T3L1adipocytes treated with BBR or RSV alone Specifically BBR andRSV cotreatment was able to reduce lipid accumulation morerobustly than single treatments BBR in combination with RSVdisplayed hypolipidemic effects likely mediated by SIRT1expression regulation Moreover BBR pretreatment seemed tocounteract SIRT1 downregulation Zhu The antioxidant and anti‚ammatory effects of BBR werealso investigated in heart Yu BBRmediated SIRT1activation reduced MIR injury by affecting oxidative damageand ‚ammation signaling Specifically BBR exerted anantioxidant effect by decreasing the generation of cardiacsuperoxide and gp91phox expression and by increasing SODlevels Yu A previous study had also shown thatSIRT1 activation promotes antioxidant molecule production anddecreases proapoptotic proteins through FOXO1 activationthus protecting against MIR lesions Hsu As well as activating SIRT1 BBR is also able to decreaseFOXO1 acetylation triggering antiapoptotic signaling pathwaysvia Bcl2 expression and Bax and caspase3 downregulation Yu A very recent report described the protective effect of BBRagainst doxorubicininduced cardiovascular damage Wu This effect is

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You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: Activation by NaturalPhytochemicals An OverviewConcetta Iside Marika Scafuro Angela Nebbioso  and Lucia Altucci Department of Precision Medicine University of Campania œLuigi Vanvitelli Naples ItalySirtuins are class III histone deacetylases whose enzymatic activity is dependent on NADas a cofactor Sirtuins are reported to modulate numerous activities by controlling geneexpression DNA repair metabolism oxidative stress response mitochondrial functionand biogenesis Deregulation of their expression andor action may lead to tissuespecificdegenerative events involved in the development of several human pathologies includingcancer neurodegeneration and cardiovascular disease The most studied member of thisclass of enzymes is sirtuin SIRT1 whose expression is associated with increasinginsulin sensitivity SIRT1 has been implicated in both tumorigenic and anticancerprocesses and is reported to regulate essential metabolic pathways suggesting thatits activation might be beneficial against disorders of the metabolism Via regulation of p53deacetylation and modulation of autophagy SIRT1 is implicated in cellular response tocaloric restriction and lifespan extension In recent years scientific interest focusing on theidentification of SIRT1 modulators has led to the discovery of novel small moleculestargeting SIRT1 activity This review will examine compounds of natural origin recentlyfound to upregulate SIRT1 activity such as polyphenolic products in fruits vegetablesand plants including resveratrol fisetin quercetin and curcumin We will also discuss thepotential therapeutic effects of these natural compounds in the prevention and treatmentof human disorders with particular emphasis on their metabolic impactKeywords sirtuin natural compounds oxidative stress human disorders polyphenolsEdited byCecilia BattistelliSapienza University of Rome ItalyReviewed byNarasimham L ParinandiThe Ohio State UniversityUnited StatesCarmen JeronimoPortuguese Oncology InstitutePortugalCorrespondenceAngela NebbiosoangelanebbiosounicampaniaitLucia Altucciluciaaltucciunicampaniait These authors share last authorshipINTRODUCTIONSpecialty sectionThis was submitted toTranslational Pharmacologya section of the journalFrontiers in PharmacologyReceived April Accepted July Published August CitationIside C Scafuro M Nebbioso A andAltucci L SIRT1 Activation byNatural Phytochemicals An OverviewFront Pharmacol 103389fphar202001225Epigenetic modifications are associated with genome stability gene transcription and metabolicregulation Acetylation is one of the most characterized histone modifications Histoneacetyltransferase HAT and histone deacetylase HDAC enzymes control the levels of histoneacetylation modulating gene expression Cavalli and Heard Sirtuins SIRT “ are enzymes classified as class III HDACs They exhibit different subcellularlocalizations SIRT1 SIRT6 and SIRT7 are nuclear although SIRT1 isoforms were also identified inAbbreviations SIRT1 Sirtuin HATs Histone acetyl transferases HDACs Histone deacetylases ROS Reactive oxygenspecies PPAR Receptor peroxisome proliferatoractivated receptor NRF Nuclear respiratory factor TFAM Mitochondrialtranscription factor A SOD Superoxide dismutase TNFa Tumor necrosis factor a IAP Apoptosis protein inhibitor Bcl2Bcell lymphoma2 family MnSOD Manganese superoxide dismutase RSV Resveratrol Que Quercetin oxLDL OxidizedLDL BBR Berberine Cur Curcumin COX Cytochrome c oxidase T2D Type II diabetes NAFLD Nonalcoholic fatty liverdisease CRM Caloric restriction mimeticFrontiers in Pharmacology wwwfrontiersinAugust Volume 0cIside et alNatural SIRT1 Activatorsthe cytoplasm SIRT2 is mainly cytosolic SIRT3 SIRT4 andSIRT5 are mitochondrial and can shuttle to the nucleus Changand Guarente The enzymatic activity of SIRTs is dependent on NAD as acofactor and plays an important role in controlling geneexpression DNA repair metabolism oxidative stress responsemitochondrial function and biogenesis Deregulation of theiractivity may lead to tissuespecific degenerative events thatunderlie several human pathologiesincluding cancerdiabetes and cardiovascular diseases Haigis and Sinclair O™Callaghan and Vassilopoulos Waldman The most studied member of this enzymatic class isSIRT1 SIRT1 regulates metabolic pathways cell survivalcellular senescence and ‚ammation and acts in thepathogenesis of chronic conditions such as diabetes as well aspulmonary neurodegenerative and cardiovascular diseasesIndeed SIRT1 has been reported to play a key role intumorigenesis as an oncogene or tumor suppressor dependingon the context specificity BiasonLauber It is able tocontrol these processes via deacetylation of lysine groups ofhistone and nonhistone proteins including known transcriptionfactors FOXO MyoD p53 PGC1a Kupis Chronic ‚ammation caused by oxidative damage increasesthe risk of many chronic disordersincluding heartcardiovascular and neurodegenerative diseases obesity insulinresistance and type diabetes T2D Geto Oxidative stress plays a key role in the pathogenesis of theseconditions The overproduction of reactive oxygen speciesROS including free radicals and reactive nitrogen speciesRNS can lead to damage of cellular components such aslipids proteins and DNA The imbalance between oxidantsand antioxidants can result in cellular dysfunction apoptosisand necrosis Liguori SIRT1 guards against oxidative stress by activating genetranscription of PGC1a via deacetylation and by regulatingtranscription of factors such as the nuclear receptor peroxisomeproliferatoractivated receptor PPAR nuclear respiratory factorNRF and mitochondrial transcription factor A TFAMinvolved in modulation of biogenesis and mitochondrialfunction Ren and metabolism of glucose and lipidsRodgers SIRT1 is also able to regulate the expressionof superoxide dismutase SOD and glutathione peroxidase Sun In addition since mitochondrial dysfunction leads tothe activation of apoptosis SIRT1 can directly regulate theapoptotic process by modulating acetylation of PGC1a Zhang SIRT1 also regulates ‚ammatory responseKauppinen By modulating the acetylation level ofNFkB p65 SIRT1 is able to control transcription of genes such asIL interleukin1 tumor necrosis factor a TNFa IL8 IL6and other ‚ammatory factors Rodgers Ren Yeung Through NFkB SIRT1 also regulatesthe expression of genes such as inhibitor of apoptosis proteinIAP and Bcell lymphoma2 Bcl2 and tumor necrosis factorreceptor TNFR Ren SIRT1 protects against oxidative stress via regulation ofFOXO protein acetylation which is involved in antioxidantprocesses apoptosis and cell proliferation Wong andWoodcock By activating FOXOMsSOD pathwaySIRT1 increases the expression of manganese superoxidedismutase MnSOD and catalase counteracting oxidativestress and promoting damage repair Gu SIRT1also increases the expression of MnSOD by deacetylating p53thus enhancing cellular antioxidant capacity Brunet Zhang Ren Over the past few years the evergrowing awareness that goodhealth goes hand in hand with a healthy and balanced diet hasencouraged people to eat more fruit and vegetables and to takesupplements to make up for any deficiency D™Angelo Bioactive compounds in the diet can act as antioxidantand anti‚ammatory agents thereby reducing the negativeeffects of oxidative stress and the incidence of chronicdiseases such as obesity diabetes and cardiovascular disordersWang Several moleculesincluding naturalphytochemical compounds can modulate SIRT1 activityMiceli Numerous studies have provided evidenceof the protective effects of natural polyphenolic substances suchas resveratrol quercetin curcumin and fisetin and ofnatural nonpolyphenolic substances such as berberineMcCubrey Natural polyphenols are the largestgroup of phytonutrients and are considered potential agents forthe prevention and treatment of stressrelated oxidative diseasesThey are found in many plants and foods such as fruitsvegetables tea cereals and wine and longterm intake isassociated with health benefits Mediterranean diets are in factlinked to a reduced risk of chronic diseases due to theconsumption of olive oil and red wine which contain highamounts of polyphenols Romagnolo and Selmin Most of the evidence supporting the beneficial effects ofphytochemical compounds comes from in vitro or animalstudies while human studies evaluating the longterm impact ofphytomolecules are particularly few or inconsistent Interventionalstudies are in fact limited by issues of bioavailability andmetabolism However in vitro studies aimed at identifyingcellular targets linked to the beneficial actions of phytonutrientrich foods at concentrations ranging from nM to µM challenge thetranslatability of data After ingestion these compounds are in factdetected as phase II metabolites and their blood level does notexceed concentrations in the nM range Substantial amounts of thecompounds and their metabolites are degraded in the colon byintestinal microbiota giving rise to small phenolic acids andaromatic catabolises which are absorbed by the circulatorysystem Del Rio Interesting studies showed thatthese natural polyphenol and nonpolyphenol substances couldaffect SIRT1 expressionactivity Table de Boer Themain mechanisms of action common to polyphenol and nonpolyphenol molecules that lead to antioxidant and anti‚ammatory effects via SIRT1 activation are reported in Figure Here we focus on the natural molecules resveratrolquercetin fisetin curcumin and berberine and elucidate theireffect on SIRT1 activation and their potential to treat andorprevent several human pathologies mainly associated withmetabolic disorders Figure Frontiers in Pharmacology wwwfrontiersinAugust Volume 0cIside et alNatural SIRT1 ActivatorsTABLE Classification of nutraceuticals based on their action and food sourceNaturalSIRT1activatorsEffectSourceReferencesResveratrol Positive effect on bloodlipid profile antioxidantDark grapesraisins peanutsQuercetinBerberineCurcuminFisetinAnticancer positiveeffect on blood lipidprofile antioxidant anti‚ammatoryAntioxidant anti‚ammatoryAnticancer antioxidantanti‚ammatoryAnticancercardiovascularpreventive anti‚ammatoryantioxidantFruits vegetablesnutsNatural componentof traditionalChinese herbCoptidis rhizomaActive componentin Curcuma longaApplespersimmonsgrapes onionskiwi kalestrawberriesD™Angelo Zordoky Hung Nabavi Nabavi Wu Hung Zendedel Kim Chen NATURAL COMPOUNDS ENHANCINGSIRT1 EXPRESSION AND ACTIVITYResveratrolResveratrol RSV a nonflavonoid polyphenol found in grapesand grape products such as red wine exerts an antioxidant actionwith reported cancer preventive properties KrisEtherton RSV also has anti‚ammatory anticancer andantineurodegenerative effects Piotrowska The roleof RSV as an immune response modulator was demonstrated inboth in vitro and in vivo studies where it reversed immunesenescence in older rats reduced ‚ammatory responses inrodents and improved immunological activity against cancercells Malaguarnera RSV was shown to be involved in theactivation of macrophages T cells and natural killer cells as wellas in the suppression processes of CD4 CD25 regulatory T cellsYang Svajger and Jeras All these effects aredue to its ability to remove ROSinhibit cyclooxygenaseCOX and trigger anti‚ammatory pathways via SIRT1activation Miceli Malaguarnera ActivatedSIRT1 interrupts TLR4NFkBSTAT axis reduces cytokineproduction by inactivated immune cells and inhibits pro‚ammatory factors derived from macrophagesmast cellssuch as plateletactivating factor and TNFa Capiralla RSVSIRT1 interaction modifies SIRT1 structure andpromotes binding activity with its substrates including p65RelA Yeung a component of the NFkB complexwhich regulates activation of leukocytes and ‚ammatorycytokines SIRT1 activated by RSV inhibits acetylation of RelAby reducing the expression of ‚ammatory factors such as TNFa IL1b IL6 metalloprotease MMP1 MMP3 and NFkBmediated Cox2 Malaguarnera AMP activatedprotein kinase AMPK is also a target of RSV as it controlsSIRT1 activity via regulation of cellular levels of NAD thusacting as an energy sensor Price Cyclicadenosine monophosphate cAMP levels activate proteinkinase A resulting in phosphorylation and activation of SIRT1FIGURE Basic mechanisms and effects of SIRT1 activation by polyphenol and nonpolyphenol moleculesFrontiers in Pharmacology wwwfrontiersinAugust Volume 0cIside et alNatural SIRT1 ActivatorsFIGURE Nutraceutical action on SIRT1 expression Natural substances have beneficial effects on human health by regulating SIRT1 action in different cellularprocesses wwwpubchemncbinlmnihgovWan Activated SIRT1 catalyzes the deacetylationand activation of PGC1a thereby promoting beneficial effectsin the metabolism Ren In different anisms S cerevisiae C elegans and Dmelanogaster expressing SIRT1 or its homologous genesRSV treatment is able to extend life span In mammaliansRSV administration can improve SIRT1dependent cellularprocesses such as axonal protection Araki fatmobilization Chaplin and inhibition of NFkBdependent transcription Yeung these effects areabolished in SIRT1 knockdown models Numerous studiesinvestigated the beneficial effects of RSV in cardiovasculardiseases including hypertension Theodotou cardiac ischemia Fourny and atherosclerosisChassot RSV has an effect on blood vesselsreduces ‚ammation and prevents thrombus formation andplatelet oxidation Zordoky It can also reducecardiac dysfunction oxidative stress fibrosis and apoptosis inthe heart Gupta Yamagata In addition RSVwas found to improve heart and kidney damage in rats Li et al2020a The protective effect of RSV is associated with anincrease in SIRT1 activity which deacetylates FOXO1 andactivates MnSOD downstream RSVinduced MnSOD alsoreduces oxidative stress Li 2020a A recent in vitrostudy showed that RSV reduces hypoxiainduced apoptosis inH9C2 cells through activation of SIRT1miR30d5pNFkB axisHan RSV treatment decreased cortical andhippocampal malondialdehyde levels while increasing SODactivity and SIRT1 expression in a diabetic rat model Ma RSV was shown to activate SIRT1 and improve endothelialfunction in obese mice via upregulation of PPARd expressionactivity in PPARd mutant mice Cheang It hadpreviously been observed that Akt activation together withPPARd is involved in vascularization of dbdb mice Tian RSV was subsequently reported to increasephosphorylation of Akt and transcriptional activity of PPARdin the aorta of wildtype mice thus supporting the hypothesis ofSIRT1PPARd interaction and to strongly decrease LPCinduced mitochondrial ROS in the aortic endothelium ofC57BL6 mice Cheang Taken together thesefindings highlight the beneficial effects of RSV against oxidativestress which is involved in major pathologies such as heart andmetabolic disorders Although RSV is beneficialin manycontexts its pleiotropic actions need to be better studied inorder to understand which of its described activities are directlydue to SIRT1 modulation and whether this effect is always directBecause of the pleiotropic actions of RSV clinical trials arecurrently testing its therapeutic potential in a wide range ofhuman diseases However of all the mechanisms described in invitro and in vivo studies only a few have been confirmed inhumans such as gene and protein regulation in blood or musclecells and Akt signaling pathways Ghanim Brasnyo Many clinical studies conducted in healthy patientsand volunteers using both high and low doses of RSV highlightits potential cardioprotective benefit through improvement ofendothelial function‚ammatory markers and glucosemetabolism Nevertheless the mechanisms of action are notyet well defined Despite clinical evidence of its effects thepoor bioavailability and rapid metabolism of RSV severelylimit the potential use of this molecule in the clinic Futurescientific research should focus on identifying actual metabolitesor mediators of these observed effectsTo date clinical trials have tested the efficacy safety andpharmacokinetics of RSV in the prevention and treatment of different pathological conditions wwwclinicaltrialsgovFrontiers in Pharmacology wwwfrontiersinAugust Volume 0cIside et alNatural SIRT1 ActivatorsRestricting the search to interventional phase studiescompleted and terminated clinical trials addressed theability of RSV to improve the pathological conditions ofpatients affected by several diseases Most of these studiestested RSVmediated effects in central nervous systemdisorders Friedreich ataxia Alzheimer™s disease Parkinson™sdisease metabolic disorders [T2Dinsulin resistancedyslipidemia hypercholesterolemia metabolic syndrome Xnonalcoholic fatty liver disease NAFLD] A phase clinicaltrial NCT01640197 tested the effects of chronic resveratrolsupplementation mg daily for days in healthy humansand found considerable improvements in cognitive performancecerebral blood flow subjective sleep mood health and bloodpressure A list of completed and terminated clinical trials inwhich RSV was tested for metabolic disorders is reported inTable Focusing on completed clinical trials with availableresults NCT02114892 evaluated the effect of RSV on metabolicsyndrome demonstrating that when administered three timesper day mgdie before meals RSV was able to treat andprotect from obesity and diabetes with beneficial effects onglucose and lipid metabolism blood pressure and bodyweight Another phase study NCT02095873 evaluated theeffects of a formulation composed of RSV and hesperetin inobese subjects and found that these molecules are dietaryinducers of glyxalase improving metabolic and vascularhealth of obese subjects Xue QuercetinThe flavonoid polyphenol quercetin Que ²²pentahydroxyflavone is a natural safe dietary supplementfound in a glycoside form in fruits vegetables and nuts whichhas antioxidant and anti‚ammatory properties Nabavi Wu In recent years the scientific community has focused on thepotential antiproliferative chemopreventive and anticarcinogenicactivities of Que as well as on its role as a modulator of geneexpression However Que was also found to have potentially toxiceffects including mutagenicity prooxidant activity mitochondrialtoxicity and inhibition of enzymes involved in hormonalmetabolism Li Due to its poor solubility short halflife and low bioavailability its medical use is limited Konrad andNieman In humans Que bioavailability is very low and absorption varies from to in subjects receiving mgdie Costa Que may reduce infection Li hepatic lipemicoxidative damage Cui Zhang et al2016b and antioxidant risk Xu In addition Que isknown to exert a modulating action on immunity Galleggiante As regards its mechanism of action in some cell linesQue was able to inhibit the production of TNF in macrophagesTang IL8 in A549 lung cells induced bylipopolysaccharide LPS Geraets and TNFa andIL1a mRNA levels in glial cells causing a decrease in neuronal celldeath induced by microglial activation Li MainlyTABLE Resveratrol in clinical trials for metabolic disordersStatusStudy TitleConditionsInterventionPhaseNCT NumberCompleted Effects of Resveratrol in Patients With TypeType Diabetes DiabetesTerminated Effect of Administration of Resveratrol onType Diabetes Mellitus mg to a maximum dose of g daily mg times dailyPhase NCT01677611Phase NCT02549924Glycemic Variability in Individuals With Type Diabetes MellitusCompleted Effect of Resveratrol on Agerelated InsulinResistance and Inflammation in HumansCompleted Regulation of Intestinal and HepaticLipoprotein Secretion by ResveratrolType Diabetes Mellitus InsulinResistanceDyslipidaemia Insulin ResistanceCompleted Effects of Dietary Antioxidants to PreventHypercholesterolemia HealthyCardiovascular DiseaseHealthy Aging Through Functional FoodCompletedwith resultsCompleted Effects of Resveratrol on Inflammation inType Diabetic PatientsCompletedwith resultsEffect of Resveratrol Administration onMetabolic Syndrome Insulin Sensitivity andInsulin SecretionCompleted Resveratrol for the Treatment of NonAlcoholic Fatty Liver Disease and InsulinResistance in Overweight AdolescentsGlucose Intolerance Aortic Stiffness VasodilationType Diabetes Mellitus Inflammation Insulin Resistance Other Disorders ofBone Density and StructureMetabolic Syndrome XNAFLD Type Diabetes MetabolicSyndromeCompleted A Study of Resveratrol as Treatment forFriedreich AtaxiaFriedreich AtaxiaCompleted Effect of Banaba Lagerstroemia Speciosaon Metabolic Syndrome Insulin Secretionand Insulin SensitivityMetabolic Syndrome X mg twice daily for daysPhase NCT01354977 mg for week followed by g for weekDietary Supplement red wine for monthDietary Supplementresveratrol for monthTransresveratrol mg hesperetin mg combination months mg daily then months mg daily mg times daily beforemeals with a total dose of mg daily mg twice daily for a total dailydose of mg for days g daily mg twice daily for weeks then g daily gtwice daily for weeksBanaba capsules mg times daily before meals for daysPhase NCT01451918Phase NCT02409537Phase NCT02095873Phase NCT02244879Phase NCT02114892Phase NCT02216552Phase NCT01339884Phase NCT02767869Frontiers in Pharmacology wwwfrontiersinAugust Volume 0cIside et alNatural SIRT1 Activatorsin immunity and ‚ammation Que acts on leukocytes and targetsmany intracellular signaling kinases and phosphatases as well asenzymes and membrane proteins Li Theimmunostimulating effect of Que is due to induction of theexpression of interferong IFNg derived from Th1 andinhibition of IL4 derived from Th2 in normal peripheral bloodmononuclear cells Nair In addition Que reduces T cellproliferation by blocking IL12induced tyrosine phosphorylationof JAK2 TYK2 STAT3 and STAT4 Muthian and Bright Nabavi In ‚ammation Que inhibits the enzymesCOX and lipoxygenase Lee and Min Savikin Inthe RAW cell line Que was also shown to counteract LPSinduced ‚ammation by phosphorylation of tyrosinephosphatidylinositol3kinase PI3Kp85 and complexformation of tolllike receptor TLR4MyD88PI3K Endale Oxidative stress occurs following an imbalance of the body™santioxidant defence mechanisms and excessive generation of freeradicals and is involved in various pathologies such as diabetesatherosclerosis hypertension neurodegenerative diseases‚ammation and cancer Oboh Que is apowerful ROS scavenger and its antioxidant action is due tothe presence of two pharmacophores within the molecularstructure which confer a favorable configuration for freeradical elimination Costa Generally Que reducesthe effects of free radicals by transferring the hydrogen atom andstabilizing the radicals a feature that has a structurefunctionrelationship Oboh Que can also act as both an antioxidant and prooxidant agentAt low concentrations “ µM Que displayed a protective effectagainst oxidative DNA damage in vitro in human lymphocytes Li At concentrations between µM and µM Que wasable to directly eliminate ROS in vitro Costa Howeverits effect in vivo is very likely not direct but due to its ability tomodulate the cell™s antioxidant defense mechanisms moderateoxidative stress can in fact increase the cell™s antioxidant defensesresulting in general cytoprotection Halliwell Recentresearch showed that oxidized LDL oxLDL induces oxidativestress LaraGuzman Oxidative injuries promote ROSgeneration in human endothelial cells and SIRT1 regulatesendothelial function Therefore enhancement of SIRT1 activityand SIRT1AMPK axis upregulation inhibits oxidative injuryinducing endothelial dysfunction Chen Shentu Que may reduce oxLDLinduced oxidative damageby upregulating SIRT1 and AMPK Hung thereforepotentially preventing oxLDLimpaired SIRT1 inhibition linked toendothelial dysfunction These findings indicate that SIRT1 canfunction as a regulator to improve AMPK activity under oxLDLstimulation Hung It was very recently shown that Que mgkg can reduceinsulin resistance and improve glucose metabolism by reducingsensitivity to T2Dinsulin resistance in obob mice via SIRT1activation Hu In this context another study showedthat in streptozotocininduced diabetic rats Que mgkginhibits oxidative damage by increasing SIRT1 expression anddecreasing levels of NFkB a SIRT1 substrate Iskender In recent years the scientific community has focused on therole of apoptosis in cardiovascular disease showing thatoxidative stress myocardial ischemia hypoxia and ischemiareperfusion injury may induce myocardial apoptosis Donniacuo Tang and colleagues evaluated the effects of Que inimproving myocardialischemiareperfusion injury MIRinduced cell apoptosis both in vitro and in vivo SIRT1 andPGC1a expression levels were decreased in rat MIR groups butwere significantly increased after treatment with Que Tang Furthermore activation of SIRT1PGC1a pathwayupregulated Bcl2 expression and downregulated Bax exertingantiapoptotic effects The authors hypothesized that Que mightimprove MIRinduced myocardial damage via regulation ofSIRT1PGC1a and Bcl2Bax pathways Tang Que is also reported to regulate ROS generation and mitigatemitochondrial dysfunction by promoting their biogenesisSpecifically in a study to develop a therapeutic strategy forosteoarthritis Que was shown to increase expression levels ofSIRT1 PGC1a NRF1 and NFR2 TFAM and phosphoAMPKa in osteoarthritis rats confirming the hypothesis that Quemight act via the AMPKSIRT1 signaling pathway Qiu Overall these findings suggest that Que may counteractcardiovascular disease and oxidative damageThe growing body of evidence supporting the beneficial effects ofQue has led to its clinical use as demonstrated by the number ofclinical trials studies on ClinicalTrialsgov A list of completedstudies using Que in different metabolic and ‚ammatoryconditions is reported in Table Specifically a phase clinicaltrial NCT01839344 measured the effect of Que on glucosetolerance and postprandial endothelial function in subjects withT2D compared to the effect of an alphaglusidase inhibitor acarboseThe administration of g of Que led to a decrease in postprandialblood glucose NCT01839344 Given its antioxidative and anti‚ammatory capacities this flavonoid was considered a goodcandidate for antioxidant therapy in mucositis NCT01732393hepatitis C NCT01438320 idiopathic pulmonary fibrosisNCT02874989 osteoporosis NCT00330096 uric acidmetabolism NCT01881919 cytokine release NCT01106170and chronic obstructive pulmonary disease NCT01708278 Inthe latter study Que supplementation was safely tolerated bypatients with mildtosevere chronic obstructive pulmonarydisease opening the way towards the potential use of Que as atherapeutic agent for this conditionHowever as for RSV and nutraceuticals in general the resultsof molecular studies on Que obtained from in vitro investigationsand animal models are often inconsistent with data from clinicaltrials Concentration factor dose and timing of administrationand bioavailability are the two main issues that require furtherclarification Additional studies are needed to identify theoptimal concentration of Que for it to exert a beneficial effectfor example on insulin sensitivityBerberineBerberine BBR is an isoquinoline alkaloid reported to haveanalgesic anticancer anti‚ammatory and myocardialprotective properties Cicero and Baggioni It was foundFrontiers in Pharmacology wwwfrontiersinAugust Volume 0cIside et alNatural SIRT1 ActivatorsTABLE Quercetin in clinical trials for metabolic and ‚ammatory disordersStatusStudy TitleConditionsInterventionPhaseNCT NumberCompleted Effect of Quercetin in Prevention andTreatment of Oral MucositisBeneficial Effects of Quercetin in ChronicObstructive Pulmonary Disease COPDCompletedwith resultsCompleted QTrial in Patients With Hepatitis CCompleted Effects of Quercetin on Blood Sugar andChemotherapy Induced OralMucositisChronic ObstructivePulmonary DiseaseChronic Hepatitis CDiabetes Mellitus Type mg daily for weeksPhase NCT01732393 to mg daily for weekPhase NCT01708278 days mg oral single dose of mgPhase Phase NCT01438320NCT01839344Blood Vessel Function in Type DiabetesCompleted Effect of Quercetin Supplements onCompletedHealthy Males a 4Week RandomizedCrossOver TrialTargeting ProInflammatory Cells inIdiopathic Pulmonary Fibrosis a HumanTrialCompleted Efficacy of Provex CV Supplement toReduce Inflammation Cytokines andBlood PressureHyperuricemia Gout KidneyCalculi DiabetesCardiovascular DiseaseIdiopathic Pulmonary FibrosisIPFBlood Pressure mg tablet for days with meal breakfastpreferredEarly PhaseNCT01881919 doses administered over consecutive days in consecutive weeks oral administration ofquercetin mg daily mg of Provex CV supplement by mouth perday for weeksPhase NCT02874989Phase NCT01106170Completed Effects of Hesperidin on Bone MineralOsteoporosis OsteopeniaPhase NCT00330096Density and Bone Metabolism ofPostmenopausal Womento exert protective antioxidative effects in different physiologicand pathologic conditions Huang Li 2020bHowever the mechanisms underlying these effects remainunclear BBR was described as a potential antitumor agent thatinduces cell cycle arrest in G0G1 phase increases Cipp21 andKipp27 protein expression decreases expression of cyclin D1D2 and DE and the cyclindependent kinases Cdk2 Cdk4 andCdk6 promoting apoptosis in HL60 human leukemia cellsLi BBR can also deregulate telomerase activityand promote mitochondriadependent apoptosis in HepG2human hepatocarcinoma cells through caspase and caspase activation PARP cleavage induction increased expression ofthe proapoptotic protein Bax through activation of FOXOtranscription factors and inhibition of Bcl2 and Bclx antiapoptotic protein expression Hwang BBR wasobserved to exert an apoptotic effect by inducing ROSproduction and increasing MAPK and JNK activity of p38 inSW620 human colon carcinoma cells and by increasing Ca andcytochrome C release in HSC3 squamous cells Song In addition BBR inhibits the proliferation of cancer cellsthrough an anti‚ammatory pathway In oral carcinoma celllines and in SCC4 cells BBR inhibits expression of COX2 andAP1 bond decreases prostaglandin E2 PGE2 production andsuppresses NFkB IKK ERK and JNK activities FurthermoreBBR can inhibit colon cancer cell growth by activating retinoid Xreceptor a RXRa which binds RXRa and promoting bcatenin degradation Ruan However some studieshighlighted the potential ability of BBR to prevent oxidativestressinduced senescence by activating AMPK and restoringNAD levels Song Initial research revealed a significant role of SIRT1 signalingin mediating the antioxidant effect of BBR in diabetes Pang and in lipid metabolism Hasanein Thelipidlowering activity mediated by cotreatment with BBR andRSV was investigated in mice exposed to a high fat diet Zhu In vivo data showed that BBR combined with RSVlowered total cholesterol triglyceride and LDL cholesterol levelsin mice These findings were also confirmed in vitro with 3T3L1adipocytes treated with BBR or RSV alone Specifically BBR andRSV cotreatment was able to reduce lipid accumulation morerobustly than single treatments BBR in combination with RSVdisplayed hypolipidemic effects likely mediated by SIRT1expression regulation Moreover BBR pretreatment seemed tocounteract SIRT1 downregulation Zhu The antioxidant and anti‚ammatory effects of BBR werealso investigated in heart Yu BBRmediated SIRT1activation reduced MIR injury by affecting oxidative damageand ‚ammation signaling Specifically BBR exerted anantioxidant effect by decreasing the generation of cardiacsuperoxide and gp91phox expression and by increasing SODlevels Yu A previous study had also shown thatSIRT1 activation promotes antioxidant molecule production anddecreases proapoptotic proteins through FOXO1 activationthus protecting against MIR lesions Hsu As well as activating SIRT1 BBR is also able to decreaseFOXO1 acetylation triggering antiapoptotic signaling pathwaysvia Bcl2 expression and Bax and caspase3 downregulation Yu A very recent report described the protective effect of BBRagainst doxorubicininduced cardiovascular damage Wu This effect is Answer:

Thyroid_Cancer

Deacetylases inSkeletal Muscle Physiology andSystemic Energy HomeostasisImplications for Metabolic Diseasesand TherapyHaili Tian1  Sujuan Liu2  Jun Ren3 Jason Kai Wei Lee456 Ru Wang1 and Peijie Chen1 School of Kinesiology Shanghai University of Sport Shanghai China Department of Anatomy and Histology Schoolof Basic Medical Sciences Tianjin Medical University Tianjin China Department of Cardiology Shanghai Instituteof Cardiovascular Diseases Zhongshan Hospital Fudan University Shanghai China Department of Physiology Yong LooLin School of Medicine National University of Singapore Singapore Singapore Global Asia Institute National Universityof Singapore Singapore Singapore N1 Institute for Health National University of Singapore Singapore SingaporeSkeletal muscle is the largest metabolic an in the human body and is able torapidly adapt to drastic changes during exercise Histone acetyltransferases HATs andhistone deacetylases HDACs which target histone and nonhistone proteins are twomajor enzyme families that control the biological process of histone acetylation anddeacetylation Balance between these two enzymes serves as an essential elementfor gene expression and metabolic and physiological function Genetic KOTG murinemodels reveal that HDACs possess pivotal roles in maintaining skeletal muscles™metabolic homeostasis regulating skeletal muscles motor adaptation and exercisecapacity HDACs may be involved in mitochondrial remodelinginsulin sensitivityregulationturn onoff of metabolic fuel switching and orchestrating physiologicalhomeostasis of skeletal muscles from the process of myogenesis Moreover manymyogenic factors and metabolic factors are modulated by HDACs HDACs areconsidered as therapeutic targets in clinicaltreatment of cancer‚ammation and neurological and metabolicrelated diseases This review will focuson physiological function of HDACs in skeletal muscles and provide new ideas for thetreatment of metabolic diseasesresearch forKeywords histone deacetylases exercise capacity skeletal muscle metabolism muscle physiologyINTRODUCTIONSkeletal muscle is the largest metabolic an in the human body consuming about of theentire body daily expenditure of energy Durnin It produces various secrete factors andparticipates in the interplay among multiple tissues and ans Pedersen and Febbraio Mizgier Muscular contraction is one of the main physiological functions of skeletalmuscles and plays a role in maintaining an and systemic metabolic homeostasis Guo Proper exercises help build up body defense to combat various diseases including obesitytype diabetes Alzheimer disease osteoarthritis and so on Luan These importantEdited byBrian James MorrisThe University of Sydney AustraliaReviewed bySean L McGeeDeakin University AustraliaViviana MoresiSapienza University of Rome ItalyCorrespondenceRu WangwangrutysuseducnPeijie Chenchenpeijiesuseducn These authors have contributedequally to this workSpecialty sectionThis was submitted toIntegrative Physiologya section of the journalFrontiers in PhysiologyReceived May Accepted July Published August CitationTian H Liu S Ren J Lee JKWWang R and Chen P Roleof Histone Deacetylases in SkeletalMuscle Physiology and SystemicEnergy Homeostasis Implicationsfor Metabolic Diseases and TherapyFront Physiol 103389fphys202000949Frontiers in Physiology wwwfrontiersinAugust Volume 0cTian et alRole of HDACs in Metabolism and Exercise Capacityfunctions require delicate regulations in muscle from thelevel of genome to signal transduction establishing muscleplasticity and responses to environmental stress AcetylCoAas a central metabolite of amino acidfatty acidglucose notonly serves as fuel for energy expenditure but also bridgesthe gap between environmental stress and anismal responseLempradl Liu This control occursthrough posttranslational modification on nucleosomes histonelysine residues and other signal transducing proteins whichin turn controls accessibility of DNA to regulatory factorsand protein activity Acetylation one of the most prevalentmodifications of proteinis believed to function as a keymodulator of chromatin structure and signal transduction andprovides an avenue to couple extracellular stimuli with genomeduring muscle metabolism process by regulating acetylation anddeacetylation Haberland histone deacetylasesHDACs as a family of proteindeacetylases have been demonstrated to moderate physiologicalhomeostasis and development by deacetylation Haberland Table In humans there are types ofHDACs which are classified into four categories based onhomologous proteins in yeast namely Class I Rpd3like proteinHDAC1 Class II Hda1like proteins are classifiedas Class IIa HDAC4 and Class IIb HDAC6 Class III Sir2plike nicotinamide adenine dinucleotideNADdependent SIRT1 and ClassIV HDAC11 containing homologous domain with bothRpd3 and Hda1 Seto and Yoshida HDACs modulategene expression and protein activity through deacetylatingproteins When histone lysine εamino acid in the nucleosomeis acetylated it can neutralize positive charge before looseningchromatin structuretranscriptionfactors to DNA and expression of downstream target genesBy contrast histone deacetylation compresseschromatinstructure thereby inhibiting transcriptional gene expressionShahbazian and Grunstein to promote binding ofBiochemical properties of HDACs have been comprehensivelyreviewed Shahbazian and Grunstein Howeverthephysiological functions of HDACs have yet to be well examined inskeletal muscles A series of researches shed light on the potentialof drugs targeting HDACs to improve muscle fitness and cardiacmuscle disease which needs further clarification of HDACsphysiological function to understand the mechanisms Bai Galmozzi Xie Zhang and Ren Gaur Ample work has revealed the role of HDACin muscle physiology such as skeletal muscles me olism and thusexercise capacity McGee and Hargreaves Table Class IHDACs can interact with myocyte enhancer factor MEF2MyoD regulating myogenesis and exercise capacity Mal Puri Ohkawa Gregoire HDAC3 participates in myocytes diï¬erentiation and is linked toskeletal muscles metabolic fuel switching Gregoire Hong Class II HDACs can be phosphorylated byHDAC kinases and are transduced from nucleus to cytoplasmin response to cellular stress mediating muscle fiber switch andaï¬ecting the pathway of insulin sensitivity such as Glut4 andAKT Haberland McGee and Hargreaves Class III HDACs target a crucial mitochondrial biogenesis factorperoxisome proliferatoractivated receptor gamma coactivator alpha PGC1α in skeletal muscles liver and fat tissue Whiteand Schenk Therefore we will further discuss howthese HDACs ‚uence respective downstream targets exercisecapacity and therapeutic eï¬ects in human diseasesCLASS I HDAC HDAC123Regulation of Myogenesis by Class IHDACsIn previous studies HDAC123 was shown to be associated withthe process of myogenesis or myocyte diï¬erentiation Mal Puri Ohkawa Gregoire HDAC1 tightly binds to MyoD and deacetylates specific sites toinhibit the expression of musclespecific genes such as MHC andMCK in myoblasts Mimicking muscle diï¬erentiation conditionby serum deprivation HDAC1 protein gradually decreasesduring myocyte diï¬erentiation and transfers to bind to the tumorsuppressor pRb accompanied by isolation of HDAC1MyoD andtranscriptional activation of musclespecific genes Puri A HDAC1 H141A mutant incapable of binding withMyoD loses its inhibitory property on musclespecific genes inmyoblast state Mal In the late stage of myocytediï¬erentiation activating factor gradually switches from MyoDto myogenin occurring with a decrease in the MEF2D inhibitoryregulator HDAC2 Simultaneous overexpression of myogeninand MEF2D can enhance the expression of the musclespecificgene MHC in the absence of MyoD Ohkawa MEF2D a key factor that controls myocyte diï¬erentiationcan only be eï¬ectively deacetylated by HDAC3 rather thanHDAC128 Gregoire In addition HDAC3 caninhibit autoacetylation of acetyltransferases p300 and p300CBPassociated factor PCAF Thus HDAC3 impedes MyoDMEF2PCAF to form a multicomplex disturbing MEF2dependentmyogenic transcription Gregoire Figure Redundant Roles of HDAC12 inMaintaining Sarcomere Homeostasis inMuscleLoss of function of HDAC12 inhibits autophagy flux in skeletalmuscles tissue accompanied by decreased LC3 III ratio andp62 accumulation under fasting condition Moresi Toxic autophagy intermediates accumulate in muscle fibersin which class I HDACs deficiency led to impaired exercisecapacity Moresi This oï¬ers convincing evidenceon the role of HDAC12 in stabilizing basic structure andfacilitating development in muscles Genetic models suggestthat Class I HDACs control the physiological homeostasis ofskeletal muscles A germline deletion shows that wholebodyknockout of either HDAC1 or HDAC2 leads to mortalityin mice prior to the perinatal period Montgomery However a tissuespecific single deletion of HDAC1or HDAC2 in myocardium does not cause any phenotypeMontgomery Double knockout HDAC12 in theFrontiers in Physiology wwwfrontiersinAugust Volume 0cTian et alRole of HDACs in Metabolism and Exercise CapacityTABLE The targets of HADCs in skeletal muscles and their physiological functionsClassMembersDown targetsGenetic KOTG phenotypeReferencesIHDAC1MyoD†“ PTEN†“ FoxOHDAC2HDAC3HDAC8HDAC4HDAC5HDAC9HDAC7HDAC6MyoD†“ MEF2D†“ NFκBp65†“MEF2D PCAF†“ Ampd3†“RCAN1 MEF2ACpt1b†“MEF2†“ Glut4†“ Myosin HeavyChain PGC1α and Hsc70“Pax7†“MEF2†“ Glut4†“ Baf60c†“Atg7 Beclin1 LC3†“Dach2Myog Gdf5†“MEF2†“Pax7 MFN1†“ Fam65bdysferlin and MAFbxHDAC10SIRT1“PGC1α STAT3†“IIAIIBIII“MnSOD Hexokinase II PDHsubunit E1αMalonyl CoA decarboxylase†““NFκBMstnSIRT2SIRT3SIRT4SIRT5SIRT6SIRT7Functionally redundant HDAC12 DKO mice causesmitochondrial abnormalities and sarcomeredegenerationPuri Ohkawa Beharry Cho Zhu Bin mKO mice Enhanced amino acid\\lipid metabolism andendurance exercise Causes IR under basal conditionGregoire Han Yuan Hong “Functionally redundant HDAC45 DKO HDAC59 DKOHDAC459 TKO increases type I fiber percentage andMcKinsey 2000a Choi Niu Luo oxidation metabolismYuan Meng Niu Macpherson Zhang Dressel “KO mice Causes mitochondrial oxidative damagemitofusion defect Protect against muscle wastingBalasubramanian Lee Ratti “mKO mice Mediates CR induced insulin sensitivity hasno effect on glucose homeostasis or exercise capacityTG mice no effect on glucose homeostasis or exercisecapacityKO mice Exacerbates obesity and IR in HFDKO mice Exacerbates obesity and IR in HFDKO mice Increased exercise tolerance and protectagainst HFDinduced obesity“Rodgers Schenk Boyle Jing Lantier Laurent KO mice Abnormal hypoglycemia TG mice Protectagainst HFD mKO mice Impaired insulin sensitivity lossKanfi Xiao Cui Samant of muscle mass““HDAC11IVActivation inhibition†“ unknown KO wholebody deletion TG wholebody overexpression DKO double knockout TKO triple knockout mKO musclespecificknockout CR caloric restriction HFD high fat diet IR insulin resistance““““heart leads to severe cardiomyopathy in mice indicating theobligatory role for HDAC12 in specific tissues Montgomery In skeletal muscle double knockout of HDAC12by myogeninCre causes mitochondrial abnormalities andsarcomere degeneration disrupting fundamental structural unitsof myofibers Moresi Therefore these findingsdemonstrate that HDAC12 redundantly maintains sarcomerehomeostasis in skeletal muscleHDAC3 Functions as a Fuel Switch inMuscle Energy Metabolismthe enzymatic core of nuclearHistone deacetylases isreceptor corepressorNCoR and silencing mediator ofretinoic acid and thyroid hormone receptors SMRT corepressor correspondingly NCoR and SMRT corepressoractivate HDAC3 through its SANT domain enzyme activityKaragianni and Wong Mice suï¬er from insulin resistanceafter specifically knocking out HDAC3 in skeletal musclesmKO while their endurance exercise abilities are enhancedHong It seems a selfcontradictory phenomenonbecause former studies pointed out that increased enduranceexercise capacity enhances insulin sensitivity The study showsthat insulin signaling cascades including pAKTAKT pIRS1S1101 and pGSKGSK have no change in HDAC3 mKO musclewhile glucose uptake and insulin sensitivity are impaired inglucose tolerance test GTT and insulin tolerance test ITTHong which is called the œdissociation eï¬ect byresearchers Lipid tends to be used as energy fuel in HDAC mKOmice which inhibits glucose absorption but does not ‚uenceinsulin signaling sensitivity Hong Further workfound that the HDAC3 knockout upregulates the expressionof AMP deaminase AMPD3 the first ratelimiting enzymein purine metabolism in skeletal muscles Fortuin Hong AMPD3 can deaminate AMP to form IMPfacilitating aspartic acid to transmit into fumarate and malate theintermediate metabolites of tricarboxylic acid cycle TCA cycleHong Research studies found that exercise inducedglucose labeled 13C6 expressed a lower glycolysis flux rate inmuscles of HDAC3 mKO but a higher expression in the TCAFrontiers in Physiology wwwfrontiersinAugust Volume 0cTian et alRole of HDACs in Metabolism and Exercise CapacityFIGURE Controls of myogenesis by Class I HDACs The inhibitory effects of HDACs on myogenic factors MEF2 and MyoD maintain a primary myoblast stateDuring myocyte differentiation and myogenic process the inhibition is lifted by other factors facilitating MEF2MyoD complex formation to promote myogenesis“Ac deacetylationcycle intermediates Hong Gong Thus ageneral increase in TCA cycle metabolites activates the oxidationin mitochondria In vitro radioactive aspartic acid isotope tracertest showed that inhibiting HDAC3 or overexpressing AMPD3can increase amino acid metabolism rate and enhance fatty acidmetabolism thereby downregulating glucose metabolism Hong Figure In liver knockout HDAC3 causes severeliver steatosis and can be rescued by wildtype or catalyticallyinactive mutants of HDAC3 indicating an enzymatic activityindependent function Sun However the musclefuel switching in HDAC3 mKO mice cannot be rescued by anenzymatic inactivity mutant HDAC3 Song Thisfinding demonstrates that HDAC3 controls the fuel utilizationin skeletal muscles and is dependent on its enzymatic activityThe relationship between exercise and glucose uptake is far morecomplex than one could expectCLASS II HDACs HDAC45796Phosphorylation and NucleusCytoplasmShuttle of Class II HDACsThe MEF2 family is a class of transcription factors that areclosely associated with myogenic basic helixloophelix domainsincluding MyoD Taylor and Hughes MEF2 may interactwith Class IIa histone deacetylase HDAC45 to inhibit thetranscription of MEF2dependent genes thereby impeding thediï¬erentiation from myoblast to myotube McKinsey et al2000a During exercise Ca2 flows out from sarcoplasmicreticulum and activate calciumcalmodulindependent proteinkinase CaMK which phosphorylates HDAC45 and mediatesHDAC45 shuttle from the nucleus to the cytoplasm thusreleasing the repressive eï¬ect of HDAC45 on MEF2dependentgenes McKinsey 2000b Yuan Further researchdemonstrates that the nucleoplasmic shuttle of HDAC4 relies on binding while the shuttle of HDAC5 depends on CaMKphosphorylation at serine and sites first and thenexporting from the nucleus by binding with McKinsey 2000b As expected HDAC7 in Class IIa also possessesthe ability to be exported from nucleus to cytoplasm Gao suggesting that there may be redundant functions of ClassIIa HDACs Except for Class IIa knocking out Class IIb HDAC6in embryonic stem cells ESCs can promote ESCs to diï¬erentiateinto myoblast cells and transplantation of ESCs with HDAC6knockdown can also help the regeneration of wound skeletalmuscles Lee type I fiberthe proportion ofRegulation of Class II HDACs onFiberType Switch MitochondriaRemodeling and Energy Stress inSkeletal MuscleEvidence showsinthatskeletal muscles has no change in animal knocked out aloneany member of Class IIa Potthoï¬ Howeverthe proportion of type I fiber is significantly increased inHDAC45 DKO HDAC59 DKO HDAC459 TKO miceas well as exercise capacity of skeletal muscles Potthoï¬ Altogether the finding indicated a functionalredundant mechanism in Class II HDACs family Previousstudy found that Class IIa HDACs are closely associated withthe MEF2 family and can repress their downstream targetgenes McKinsey 2000a Therefore similar to Class IIaHDACs DKO the proportion of type I fiber is decreased in theMEF2C and MEF2D knockout mouse models overexpressingMEF2C results in increasing type I fiber and exercise capacityHDAC45 not only regulates exercise capacity but also governsmotor adaptation Several studies had shown that exerciseFrontiers in Physiology wwwfrontiersinAugust Volume 0cTian et alRole of HDACs in Metabolism and Exercise CapacityFIGURE Class I HDACs regulates sarcomere structure and exercise capacity in mature muscle A schematic diagram shows Class I HDACs regulate exercisecapacity on different levels HDAC12 have redundant roles in modulating autophagy flux in muscle eliminating toxic factors in muscle HDAC3 serves as a fuelswitch and promotes muscle to use fatty acid to adapt endurance exercisepromotes phosphorylation of HDAC45 mediated by CaMK andadenosine monophosphateactivated protein kinase AMPKthen increasing a MEF2dependent transcription of Glut4 andmusclespecific genes which participates in the adaptationand plasticity of skeletal muscles McGee McGee and Hargreaves Besides exercise glucose can alsoactivate KATP channeldependent calcium signaling and CaMKwhich induce phosphorylationdependent nuclearcytoplasmtransportation of HDAC5 Meng The leavingHDAC5 releases its repressive eï¬ect on Baf60c and enhancinginsulinindependent AKT activation Meng Usingspecific inhibitor of class IIa HDAC activity myosin heavy chainPGC1α and heatshock cognate HSC70 are confirmed to beone of the substrates of HDAC4 in denervationinduced atrophymuscle in comparison with acetylated protein enrichment withuntreated group Luo Taken together Class IIaHDACs play critical roles in exercise capacity remodeling andnutrient sensing of skeletal muscles Figure For Class IIbit was revealed that glucose deprivationinduced mitochondrialfusion mediated by mitofusion1MFN1 is significantly disrupted in HDAC6 KO mice causingmitochondria degeneration which isreversed followingapplication of MFN1 acetylationresistant mutant Lee This study suggested that MFN1 deacetylation by HDAC6plays an important role in mitochondrial adaptive energyproduction and remodeling of skeletal musclesCLASS III HDACsSir2HOMOLOGSIRT1634NuclearLocated SIRT16Numerous studies have found that caloric restriction CRextends lifespan of mammals Caenorhabditis elegans and fruitflies while such eï¬ect is lost in SIR2 or NPT1 mutant C elegansstrains Imai Lin SIR2 encodes thesilencing protein Sir2p and NPT1 participates in the synthesis ofNAD NAD is an essential cofactor for Sir2pClass III HDACsto exert enzyme activity rather than Class I II and IV relayingon the zinc Imai Lin In mammalsSir2 has homologous proteins and these proteins are essentialto mitochondrial energy homeostasis antioxidant defense cellproliferation and DNA repair VargasOrtiz SIRT1 Mediates Energy Stress Adaptation in SkeletalMuscles reported that SIRT1 promotes theRodgers et altranscriptional activity of PGC1α by deacetylating the PGC1αat K13 site and mediates CRinduced gluconeogenesisrelatedgenes G6P PEPCK expression in liver Further research showedthat the NADNADH ratio and the enzymatic activity of Sir2 asensor of redox state were decreased in diï¬erentiating myocyteswhich relieved the inhibitory eï¬ect on MyoD and then promotedcell diï¬erentiation Resveratrol RSV a compound that maytarget SIRT1PGC1α can improve mitochondrial function andmetabolic homeostasis owning a potential to prolong lifespanRSV loses its activating eï¬ect on PGC1α in SIRT1 ˆ’ˆ’ MEFsLagouge Moreover RSVtreated mice at a dose of gkg increased their exercise capacity oxygen consumptionand improved insulin sensitivity against highfat induced obesityLagouge In addition RSV could enhance thedeacetylation of PGC1α in brown fat tissue BAT and skeletalmuscles which promotes mitochondrial production oxygenconsumption and thus improves metabolic syndrome Lagouge The function of SIRT1 in regulating skeletal musclesmetabolic homeostasis and exercise capacity has attracted greatattention from researchers Researchers further generated skeletalmusclespecific SIRT1mKO mice and found that mKO miceFrontiers in Physiology wwwfrontiersinAugust Volume 0cTian et alRole of HDACs in Metabolism and Exercise CapacityFIGURE Control of muscle exercise adaption and mitochondrial function by Class II HDACs A Schematic of the Class IIa HDACs nucleoplasm shuttle Understress condition CaMK and AMPK could be activated and phosphorylate Class IIa HDACs Phosphorylated HDACs then bind with and shuttle to cytoplasmrelieving inhibitory effects on MEF2 and Baf60c B Class IIb HDAC6 can deacetylate MFN1 and facilitates its mitofusion function P phosphorylationlost the eï¬ect of CRinduced increasing insulin sensitivity andwere unable to deacetylate and inactivate STAT3 resulting inupregulating the phosphatidylinositol3kinase PI3K inhibitoryregulator p55αp50α expression Schenk Althoughknockout or overexpression of SIRT1 in skeletal muscles hasno eï¬ect on endurance capacity or glucose homeostasis in miceGurd Philp White Svensson some studies have shown that AMPK regulatesenergy metabolism of skeletal muscles partially mediated bySIRT1 and SIRT1 is significantly increased after enduranceexercise Suwa Canto Overexpressionof Sirt1 in skeletal muscles by adenoassociated virus AAV1promotes the expression of oxidationrelated genes includingPpargc1a Tfam Cpt1b and Pdk4 while it has no eï¬ect on insulinsensitivity of body Vila But overexpressing Sirt1in liver by AAV8 can protect from fatty liver induced by highcarbohydrate food HCD Vila Taken togetherthe aforementioned studies suggest that SIRT1 possesses limitedregulation capacity of skeletal muscles motility and SIRT1may modulate mitochondrial homeostasis and mediate skeletalmuscles adaptation under certain physiological conditions suchas CR aging and regeneration Gomes Ryall Figure In addition there may be more beneficial eï¬ectsof SIRT1 on metabolism in the liver or fat tissue Lagouge Vila Stefanowicz SIRT6 Improves Muscle Fitness andExercise CapacitySIRT6 is another Sir2like deacetylase located in the nucleusWhole body deletion of SIRT6 causes mice death around weeks owning to hypoglycemia Xiao Neverthelessthe skeletal musclespecific knockout of SIRT6 causes insulinresistance and impairs glucose homeostasis of mKO mice Cui Because the activity of AMPK is reduced inSIRT6mKO mice the glycolipids absorption and utilizationof skeletal muscles are impaired and the exercise capacity ofthe mice was also attenuated Cui Furthermoreresearchers constructed the overexpressing SIRT6 Sirt6BACmice and found that their body weight and fat content werenormal but the Sirt6BAC mice could protect from HCDinducedhyperglycemia via increasing pAKTAKT induced by insulinstimulation and glucose absorption Anderson Vitroexperiments further demonstrated that the ability of glucoseuptake was enhanced mainly in skeletal muscles not in brainiWAT eWAT tissues of Sirt6BAC mice Anderson These results shows the potential eï¬ects of SIRT6 targeted inskeletal muscles on improving exercise performance and treatingmetabolic diseases Figure MitochondriaLocated SIRT34SIRT34 Maintains Mitochondria Function in SkeletalMusclesSIRT34 are mitochondrialocalized deacetylases and responsiblefor regulating the deacetylation of proteins in mitochondria andmaintaining mitochondrial homeostasis After feeding highfatdiet HFD accelerated obesity attenuated insulin sensitivityworsened fatty liver and increased acetylation of proteins inmitochondria were observed in SIRT3 KO mice Hirschey Multitissue proteomics and physiological examinationreveals that SIRT3 is responsible for mitochondrial acetylatedFrontiers in Physiology wwwfrontiersinAugust Volume 0cTian et alRole of HDACs in Metabolism and Exercise CapacityFIGURE Class III HDACs promote a stressinduced adaptation in muscle and regulate mitochondrial function Class III HDACs are NADdependentdeacetylases and may function as a redox sensor in muscle cell SIRT1 could activate PGC1alpha by directly deacetylasing K13 site and regulate insulin sensitivityby targeting STAT3 Mitochondrialocated Class III HDACs control mitochondrial function by deacetylating certain mitochondrial proteins modulating muscle fuelutilization and exercise capacityproteome regulation and metabolic fuel switching in brainheart kidneyliver and skeletal muscles DittenhaferReed Protein enrichment analysis discovers that itsregulatory proteins were mainly concentrated in lipid metabolismDittenhaferReed Knocking out SIRT3 leadsto reduction in the levels of deacetylation of manganesesuperoxide dismutase MnSOD mitochondrial complex II andpyruvate dehydrogenase PDH subunit E1α a downregulationof hexokinase II HK II binding with mitochondria resultingin impaired glucose and lipid metabolism of skeletal musclesLantier Interestingly SIRT3 liverspecific knockouthepˆ’ˆ’ and skeletal musclespecific knockoutskmˆ’ˆ’mice did not aï¬ect glucose homeostasis under chow or HFDconditions FernandezMarcos The above resultsindicate that SIRT3 has an important role in metabolic regulationbut in specific physiological processes such as redox stateexercise and aging and its regulatory mechanism is yet definedin skeletal muscles Kong Robin Williams A recent study found that SIRT4 knockoutcan resist HFDinduced obesity and increase endurance exercisein mice by repressing malonyl CoA decarboxylase which wasa key enzyme controlling fatty acid betaoxidation and wasreported to regulate muscle fuel switching between carbohydratesand fatty acids Koves Laurent Moreover knockdown of SIRT4 by adenoviral shRNA canincrease the mRNA and protein content of SIRT1 therebyenhancing the expression of fatty acid oxidation genes andmitochondrial oxidation capacity in hepatocytes and myotubesNasrin Table THERAPEUTIC TARGETS OF HDACsAND THEIR POTENTIAL FORMETABOLIC DISEASESA comparative analysis used HDAC paninhibitor SAHA aclass I HDAC selective inhibitor MS275 and a class IIHDAC selective inhibitor MC1568 to treat C2C12 separatelyIt was found that only MS275 could significantly stimulatemitochondria biogenesis and oxygen consumption Galmozzi In obese diabetic mice it was found that specificallyinhibiting Class I rather than Class II HDACs improvedGTT and insulin sensitivityincreased oxidative metabolismof skeletal muscles and adipose tissue and reduced bodyweight Galmozzi Similar to the eï¬ect of MS275knockdown HDAC3 in C2C12 could also increase Pgc1αGlut4 Tfam Idh3α transcription suggesting that HDAC3 andits target genes played an important role in the above eventsGalmozzi For Class II HDACs studies have found that scriptaid a ClassIIa HDAC inhibitor has similar eï¬ects to exercise Six weeksof scriptaid administration significantly improved enduranceperformance and significantly increased the wholebody energyexpenditure and the expression of lipid oxidation related genesPdk4 Cpt1b Pgc1α Pparδ etc of C57BL6 mice Gaur One possible explanation of above observation is thepotential target of scriptaid inhibition of titin a structure proteinof sarcomere deacetylation and downstream genes of Class IIaHDACs Gaur Huang Frontiers in Physiology wwwfrontiersinAugust Volume 0cTian et alRole of HDACs in Metabolism and Exercise CapacityIn mice treated with SPT1720 another activator of SIRT1overtly enhanced endurance exercise ability was noted Moreoverthese mice were protected from HFDinduced obesity and insulinresistance owing to an upregulation of the oxidative metabolismin skeletal muscles liver and BAT tissues Pacholec In order to further explore the principle of SIRT1 activation theresearchers purified SIRT1 in vitro and added SRT1720 and itsanalogs SRT2183 SRT1460 and RSV and they found that theenzymatic activity of SIRT1 was not enhanced suggesting thatthese drugs may indirectly activate SIRT1 Pacholec The enzymatic activity of SIRT1 largely depends on the contentof cofactor NAD This implied that the enhancement of SIRT1activity may be achieved through indirect upregulation of NADStudy has discovered that knocking out poly ADPribosepolymerase1 PARP1 which is a NAD consuming enzymecould increase NAD content and enhance the activity of SIRT1in BAT and skeletal muscles Bai PARP1 inhibitorscan upregulate the proteins of mitochondrial respiratory chaincomplexes in mice enhance the aerobic oxidation capacity ofmitochondria and improve mitochondrial defects in the primarymyotubes of obese humans Bai Pirinen et alResveratrol was initially reported as an SIRT1 activatorthat improves mitochondrial function and exercise capacity inmice and resists HFDinduced obesity Lagouge However subsequent studies have discovered that administeringthe same dose of RSV to rats or mice does not increasemitochondrial protein content Higashida Bycontrast overexpression of SIRT1 in the triceps muscle of ratsdecreases the mitochondrial protein content Higashida In a doubleblind human trial healthy and obesemen were supplied for 30day RSV in which the data showedthat RSV can improve systolic blood pressure and homeostasismodel assessment HOMA indexindicating glucose metabolismability simulating the eï¬ect of CR Timmers However some researchers found that the overexpression ofSIRT1 alone cannot mimic the CR eï¬ect in transgenic mice andthe transcriptomic changes in various tissues were quite diï¬erentor even opposite Boutant Such an opposite situationmay explain that the genetic model and compound stimulationare not completely consistent RSV as a potential metabolicsyndrome treatment drug still needs largescale populationsample verificationCONCLUSIONThe very first mammalian histone deacetylase HDAC1 wascloned and isolated by Taunton and sabout HDACs have been published in the last years CurrentlyREFERENCESAnderson J G Ramadori G Ioris R M Galie M Berglund E D CoateK C Enhanced insulin sensitivity in skeletal muscle and liver byphysiological overexpression of SIRT6 Mol Metab “ 101016jmolmet201509003we know at least HDAC proteins They are responsible foreradicating epigenetic modifications establishing an epigeneticoï¬ chromatin state and regulating heritable gene expressionYang and Seto Haberland In these processeseach HDACs may play a diï¬erent role Table What kind ofgeneprotein is the specific downstream target of these HDACsIs its enzyme activity related to intracellular localization andthe formation of multiprotein complex It is still one of thekey and difficult issues in this field Based on previous researchexperience some techniques may be used to further experimentsas follows HDACs interacting proteinscomplex coIP Proteinacetylation western Histone acetylation target geneChIP High through put proteomicsacetylome with specificHDACs inhibitor etctherapeuticare potentialHistone deacetylasestargetsand clinical drugs such as SAHA Vorinostat and FK228romidepsin have been used for antitumor treatment Bolden However they have two disadvantages greattoxic and side eï¬ects and difficulty in specific inhibition ofHDACs activity With the development of computer simulationtechnology and structural biology it is believed that more specificHDACs inhibitorsactivators can be constructed And researchesshould attach importance to HDACs regulatory factors likePARP1 when direct targeting on HDACs fails to show its eï¬ectsFurther development of their inhibitors with more specificityand trials for the treatment of metabolic diseases may have greatpotential as well

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You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: Deacetylases inSkeletal Muscle Physiology andSystemic Energy HomeostasisImplications for Metabolic Diseasesand TherapyHaili Tian1  Sujuan Liu2  Jun Ren3 Jason Kai Wei Lee456 Ru Wang1 and Peijie Chen1 School of Kinesiology Shanghai University of Sport Shanghai China Department of Anatomy and Histology Schoolof Basic Medical Sciences Tianjin Medical University Tianjin China Department of Cardiology Shanghai Instituteof Cardiovascular Diseases Zhongshan Hospital Fudan University Shanghai China Department of Physiology Yong LooLin School of Medicine National University of Singapore Singapore Singapore Global Asia Institute National Universityof Singapore Singapore Singapore N1 Institute for Health National University of Singapore Singapore SingaporeSkeletal muscle is the largest metabolic an in the human body and is able torapidly adapt to drastic changes during exercise Histone acetyltransferases HATs andhistone deacetylases HDACs which target histone and nonhistone proteins are twomajor enzyme families that control the biological process of histone acetylation anddeacetylation Balance between these two enzymes serves as an essential elementfor gene expression and metabolic and physiological function Genetic KOTG murinemodels reveal that HDACs possess pivotal roles in maintaining skeletal muscles™metabolic homeostasis regulating skeletal muscles motor adaptation and exercisecapacity HDACs may be involved in mitochondrial remodelinginsulin sensitivityregulationturn onoff of metabolic fuel switching and orchestrating physiologicalhomeostasis of skeletal muscles from the process of myogenesis Moreover manymyogenic factors and metabolic factors are modulated by HDACs HDACs areconsidered as therapeutic targets in clinicaltreatment of cancer‚ammation and neurological and metabolicrelated diseases This review will focuson physiological function of HDACs in skeletal muscles and provide new ideas for thetreatment of metabolic diseasesresearch forKeywords histone deacetylases exercise capacity skeletal muscle metabolism muscle physiologyINTRODUCTIONSkeletal muscle is the largest metabolic an in the human body consuming about of theentire body daily expenditure of energy Durnin It produces various secrete factors andparticipates in the interplay among multiple tissues and ans Pedersen and Febbraio Mizgier Muscular contraction is one of the main physiological functions of skeletalmuscles and plays a role in maintaining an and systemic metabolic homeostasis Guo Proper exercises help build up body defense to combat various diseases including obesitytype diabetes Alzheimer disease osteoarthritis and so on Luan These importantEdited byBrian James MorrisThe University of Sydney AustraliaReviewed bySean L McGeeDeakin University AustraliaViviana MoresiSapienza University of Rome ItalyCorrespondenceRu WangwangrutysuseducnPeijie Chenchenpeijiesuseducn These authors have contributedequally to this workSpecialty sectionThis was submitted toIntegrative Physiologya section of the journalFrontiers in PhysiologyReceived May Accepted July Published August CitationTian H Liu S Ren J Lee JKWWang R and Chen P Roleof Histone Deacetylases in SkeletalMuscle Physiology and SystemicEnergy Homeostasis Implicationsfor Metabolic Diseases and TherapyFront Physiol 103389fphys202000949Frontiers in Physiology wwwfrontiersinAugust Volume 0cTian et alRole of HDACs in Metabolism and Exercise Capacityfunctions require delicate regulations in muscle from thelevel of genome to signal transduction establishing muscleplasticity and responses to environmental stress AcetylCoAas a central metabolite of amino acidfatty acidglucose notonly serves as fuel for energy expenditure but also bridgesthe gap between environmental stress and anismal responseLempradl Liu This control occursthrough posttranslational modification on nucleosomes histonelysine residues and other signal transducing proteins whichin turn controls accessibility of DNA to regulatory factorsand protein activity Acetylation one of the most prevalentmodifications of proteinis believed to function as a keymodulator of chromatin structure and signal transduction andprovides an avenue to couple extracellular stimuli with genomeduring muscle metabolism process by regulating acetylation anddeacetylation Haberland histone deacetylasesHDACs as a family of proteindeacetylases have been demonstrated to moderate physiologicalhomeostasis and development by deacetylation Haberland Table In humans there are types ofHDACs which are classified into four categories based onhomologous proteins in yeast namely Class I Rpd3like proteinHDAC1 Class II Hda1like proteins are classifiedas Class IIa HDAC4 and Class IIb HDAC6 Class III Sir2plike nicotinamide adenine dinucleotideNADdependent SIRT1 and ClassIV HDAC11 containing homologous domain with bothRpd3 and Hda1 Seto and Yoshida HDACs modulategene expression and protein activity through deacetylatingproteins When histone lysine εamino acid in the nucleosomeis acetylated it can neutralize positive charge before looseningchromatin structuretranscriptionfactors to DNA and expression of downstream target genesBy contrast histone deacetylation compresseschromatinstructure thereby inhibiting transcriptional gene expressionShahbazian and Grunstein to promote binding ofBiochemical properties of HDACs have been comprehensivelyreviewed Shahbazian and Grunstein Howeverthephysiological functions of HDACs have yet to be well examined inskeletal muscles A series of researches shed light on the potentialof drugs targeting HDACs to improve muscle fitness and cardiacmuscle disease which needs further clarification of HDACsphysiological function to understand the mechanisms Bai Galmozzi Xie Zhang and Ren Gaur Ample work has revealed the role of HDACin muscle physiology such as skeletal muscles me olism and thusexercise capacity McGee and Hargreaves Table Class IHDACs can interact with myocyte enhancer factor MEF2MyoD regulating myogenesis and exercise capacity Mal Puri Ohkawa Gregoire HDAC3 participates in myocytes diï¬erentiation and is linked toskeletal muscles metabolic fuel switching Gregoire Hong Class II HDACs can be phosphorylated byHDAC kinases and are transduced from nucleus to cytoplasmin response to cellular stress mediating muscle fiber switch andaï¬ecting the pathway of insulin sensitivity such as Glut4 andAKT Haberland McGee and Hargreaves Class III HDACs target a crucial mitochondrial biogenesis factorperoxisome proliferatoractivated receptor gamma coactivator alpha PGC1α in skeletal muscles liver and fat tissue Whiteand Schenk Therefore we will further discuss howthese HDACs ‚uence respective downstream targets exercisecapacity and therapeutic eï¬ects in human diseasesCLASS I HDAC HDAC123Regulation of Myogenesis by Class IHDACsIn previous studies HDAC123 was shown to be associated withthe process of myogenesis or myocyte diï¬erentiation Mal Puri Ohkawa Gregoire HDAC1 tightly binds to MyoD and deacetylates specific sites toinhibit the expression of musclespecific genes such as MHC andMCK in myoblasts Mimicking muscle diï¬erentiation conditionby serum deprivation HDAC1 protein gradually decreasesduring myocyte diï¬erentiation and transfers to bind to the tumorsuppressor pRb accompanied by isolation of HDAC1MyoD andtranscriptional activation of musclespecific genes Puri A HDAC1 H141A mutant incapable of binding withMyoD loses its inhibitory property on musclespecific genes inmyoblast state Mal In the late stage of myocytediï¬erentiation activating factor gradually switches from MyoDto myogenin occurring with a decrease in the MEF2D inhibitoryregulator HDAC2 Simultaneous overexpression of myogeninand MEF2D can enhance the expression of the musclespecificgene MHC in the absence of MyoD Ohkawa MEF2D a key factor that controls myocyte diï¬erentiationcan only be eï¬ectively deacetylated by HDAC3 rather thanHDAC128 Gregoire In addition HDAC3 caninhibit autoacetylation of acetyltransferases p300 and p300CBPassociated factor PCAF Thus HDAC3 impedes MyoDMEF2PCAF to form a multicomplex disturbing MEF2dependentmyogenic transcription Gregoire Figure Redundant Roles of HDAC12 inMaintaining Sarcomere Homeostasis inMuscleLoss of function of HDAC12 inhibits autophagy flux in skeletalmuscles tissue accompanied by decreased LC3 III ratio andp62 accumulation under fasting condition Moresi Toxic autophagy intermediates accumulate in muscle fibersin which class I HDACs deficiency led to impaired exercisecapacity Moresi This oï¬ers convincing evidenceon the role of HDAC12 in stabilizing basic structure andfacilitating development in muscles Genetic models suggestthat Class I HDACs control the physiological homeostasis ofskeletal muscles A germline deletion shows that wholebodyknockout of either HDAC1 or HDAC2 leads to mortalityin mice prior to the perinatal period Montgomery However a tissuespecific single deletion of HDAC1or HDAC2 in myocardium does not cause any phenotypeMontgomery Double knockout HDAC12 in theFrontiers in Physiology wwwfrontiersinAugust Volume 0cTian et alRole of HDACs in Metabolism and Exercise CapacityTABLE The targets of HADCs in skeletal muscles and their physiological functionsClassMembersDown targetsGenetic KOTG phenotypeReferencesIHDAC1MyoD†“ PTEN†“ FoxOHDAC2HDAC3HDAC8HDAC4HDAC5HDAC9HDAC7HDAC6MyoD†“ MEF2D†“ NFκBp65†“MEF2D PCAF†“ Ampd3†“RCAN1 MEF2ACpt1b†“MEF2†“ Glut4†“ Myosin HeavyChain PGC1α and Hsc70“Pax7†“MEF2†“ Glut4†“ Baf60c†“Atg7 Beclin1 LC3†“Dach2Myog Gdf5†“MEF2†“Pax7 MFN1†“ Fam65bdysferlin and MAFbxHDAC10SIRT1“PGC1α STAT3†“IIAIIBIII“MnSOD Hexokinase II PDHsubunit E1αMalonyl CoA decarboxylase†““NFκBMstnSIRT2SIRT3SIRT4SIRT5SIRT6SIRT7Functionally redundant HDAC12 DKO mice causesmitochondrial abnormalities and sarcomeredegenerationPuri Ohkawa Beharry Cho Zhu Bin mKO mice Enhanced amino acid\\lipid metabolism andendurance exercise Causes IR under basal conditionGregoire Han Yuan Hong “Functionally redundant HDAC45 DKO HDAC59 DKOHDAC459 TKO increases type I fiber percentage andMcKinsey 2000a Choi Niu Luo oxidation metabolismYuan Meng Niu Macpherson Zhang Dressel “KO mice Causes mitochondrial oxidative damagemitofusion defect Protect against muscle wastingBalasubramanian Lee Ratti “mKO mice Mediates CR induced insulin sensitivity hasno effect on glucose homeostasis or exercise capacityTG mice no effect on glucose homeostasis or exercisecapacityKO mice Exacerbates obesity and IR in HFDKO mice Exacerbates obesity and IR in HFDKO mice Increased exercise tolerance and protectagainst HFDinduced obesity“Rodgers Schenk Boyle Jing Lantier Laurent KO mice Abnormal hypoglycemia TG mice Protectagainst HFD mKO mice Impaired insulin sensitivity lossKanfi Xiao Cui Samant of muscle mass““HDAC11IVActivation inhibition†“ unknown KO wholebody deletion TG wholebody overexpression DKO double knockout TKO triple knockout mKO musclespecificknockout CR caloric restriction HFD high fat diet IR insulin resistance““““heart leads to severe cardiomyopathy in mice indicating theobligatory role for HDAC12 in specific tissues Montgomery In skeletal muscle double knockout of HDAC12by myogeninCre causes mitochondrial abnormalities andsarcomere degeneration disrupting fundamental structural unitsof myofibers Moresi Therefore these findingsdemonstrate that HDAC12 redundantly maintains sarcomerehomeostasis in skeletal muscleHDAC3 Functions as a Fuel Switch inMuscle Energy Metabolismthe enzymatic core of nuclearHistone deacetylases isreceptor corepressorNCoR and silencing mediator ofretinoic acid and thyroid hormone receptors SMRT corepressor correspondingly NCoR and SMRT corepressoractivate HDAC3 through its SANT domain enzyme activityKaragianni and Wong Mice suï¬er from insulin resistanceafter specifically knocking out HDAC3 in skeletal musclesmKO while their endurance exercise abilities are enhancedHong It seems a selfcontradictory phenomenonbecause former studies pointed out that increased enduranceexercise capacity enhances insulin sensitivity The study showsthat insulin signaling cascades including pAKTAKT pIRS1S1101 and pGSKGSK have no change in HDAC3 mKO musclewhile glucose uptake and insulin sensitivity are impaired inglucose tolerance test GTT and insulin tolerance test ITTHong which is called the œdissociation eï¬ect byresearchers Lipid tends to be used as energy fuel in HDAC mKOmice which inhibits glucose absorption but does not ‚uenceinsulin signaling sensitivity Hong Further workfound that the HDAC3 knockout upregulates the expressionof AMP deaminase AMPD3 the first ratelimiting enzymein purine metabolism in skeletal muscles Fortuin Hong AMPD3 can deaminate AMP to form IMPfacilitating aspartic acid to transmit into fumarate and malate theintermediate metabolites of tricarboxylic acid cycle TCA cycleHong Research studies found that exercise inducedglucose labeled 13C6 expressed a lower glycolysis flux rate inmuscles of HDAC3 mKO but a higher expression in the TCAFrontiers in Physiology wwwfrontiersinAugust Volume 0cTian et alRole of HDACs in Metabolism and Exercise CapacityFIGURE Controls of myogenesis by Class I HDACs The inhibitory effects of HDACs on myogenic factors MEF2 and MyoD maintain a primary myoblast stateDuring myocyte differentiation and myogenic process the inhibition is lifted by other factors facilitating MEF2MyoD complex formation to promote myogenesis“Ac deacetylationcycle intermediates Hong Gong Thus ageneral increase in TCA cycle metabolites activates the oxidationin mitochondria In vitro radioactive aspartic acid isotope tracertest showed that inhibiting HDAC3 or overexpressing AMPD3can increase amino acid metabolism rate and enhance fatty acidmetabolism thereby downregulating glucose metabolism Hong Figure In liver knockout HDAC3 causes severeliver steatosis and can be rescued by wildtype or catalyticallyinactive mutants of HDAC3 indicating an enzymatic activityindependent function Sun However the musclefuel switching in HDAC3 mKO mice cannot be rescued by anenzymatic inactivity mutant HDAC3 Song Thisfinding demonstrates that HDAC3 controls the fuel utilizationin skeletal muscles and is dependent on its enzymatic activityThe relationship between exercise and glucose uptake is far morecomplex than one could expectCLASS II HDACs HDAC45796Phosphorylation and NucleusCytoplasmShuttle of Class II HDACsThe MEF2 family is a class of transcription factors that areclosely associated with myogenic basic helixloophelix domainsincluding MyoD Taylor and Hughes MEF2 may interactwith Class IIa histone deacetylase HDAC45 to inhibit thetranscription of MEF2dependent genes thereby impeding thediï¬erentiation from myoblast to myotube McKinsey et al2000a During exercise Ca2 flows out from sarcoplasmicreticulum and activate calciumcalmodulindependent proteinkinase CaMK which phosphorylates HDAC45 and mediatesHDAC45 shuttle from the nucleus to the cytoplasm thusreleasing the repressive eï¬ect of HDAC45 on MEF2dependentgenes McKinsey 2000b Yuan Further researchdemonstrates that the nucleoplasmic shuttle of HDAC4 relies on binding while the shuttle of HDAC5 depends on CaMKphosphorylation at serine and sites first and thenexporting from the nucleus by binding with McKinsey 2000b As expected HDAC7 in Class IIa also possessesthe ability to be exported from nucleus to cytoplasm Gao suggesting that there may be redundant functions of ClassIIa HDACs Except for Class IIa knocking out Class IIb HDAC6in embryonic stem cells ESCs can promote ESCs to diï¬erentiateinto myoblast cells and transplantation of ESCs with HDAC6knockdown can also help the regeneration of wound skeletalmuscles Lee type I fiberthe proportion ofRegulation of Class II HDACs onFiberType Switch MitochondriaRemodeling and Energy Stress inSkeletal MuscleEvidence showsinthatskeletal muscles has no change in animal knocked out aloneany member of Class IIa Potthoï¬ Howeverthe proportion of type I fiber is significantly increased inHDAC45 DKO HDAC59 DKO HDAC459 TKO miceas well as exercise capacity of skeletal muscles Potthoï¬ Altogether the finding indicated a functionalredundant mechanism in Class II HDACs family Previousstudy found that Class IIa HDACs are closely associated withthe MEF2 family and can repress their downstream targetgenes McKinsey 2000a Therefore similar to Class IIaHDACs DKO the proportion of type I fiber is decreased in theMEF2C and MEF2D knockout mouse models overexpressingMEF2C results in increasing type I fiber and exercise capacityHDAC45 not only regulates exercise capacity but also governsmotor adaptation Several studies had shown that exerciseFrontiers in Physiology wwwfrontiersinAugust Volume 0cTian et alRole of HDACs in Metabolism and Exercise CapacityFIGURE Class I HDACs regulates sarcomere structure and exercise capacity in mature muscle A schematic diagram shows Class I HDACs regulate exercisecapacity on different levels HDAC12 have redundant roles in modulating autophagy flux in muscle eliminating toxic factors in muscle HDAC3 serves as a fuelswitch and promotes muscle to use fatty acid to adapt endurance exercisepromotes phosphorylation of HDAC45 mediated by CaMK andadenosine monophosphateactivated protein kinase AMPKthen increasing a MEF2dependent transcription of Glut4 andmusclespecific genes which participates in the adaptationand plasticity of skeletal muscles McGee McGee and Hargreaves Besides exercise glucose can alsoactivate KATP channeldependent calcium signaling and CaMKwhich induce phosphorylationdependent nuclearcytoplasmtransportation of HDAC5 Meng The leavingHDAC5 releases its repressive eï¬ect on Baf60c and enhancinginsulinindependent AKT activation Meng Usingspecific inhibitor of class IIa HDAC activity myosin heavy chainPGC1α and heatshock cognate HSC70 are confirmed to beone of the substrates of HDAC4 in denervationinduced atrophymuscle in comparison with acetylated protein enrichment withuntreated group Luo Taken together Class IIaHDACs play critical roles in exercise capacity remodeling andnutrient sensing of skeletal muscles Figure For Class IIbit was revealed that glucose deprivationinduced mitochondrialfusion mediated by mitofusion1MFN1 is significantly disrupted in HDAC6 KO mice causingmitochondria degeneration which isreversed followingapplication of MFN1 acetylationresistant mutant Lee This study suggested that MFN1 deacetylation by HDAC6plays an important role in mitochondrial adaptive energyproduction and remodeling of skeletal musclesCLASS III HDACsSir2HOMOLOGSIRT1634NuclearLocated SIRT16Numerous studies have found that caloric restriction CRextends lifespan of mammals Caenorhabditis elegans and fruitflies while such eï¬ect is lost in SIR2 or NPT1 mutant C elegansstrains Imai Lin SIR2 encodes thesilencing protein Sir2p and NPT1 participates in the synthesis ofNAD NAD is an essential cofactor for Sir2pClass III HDACsto exert enzyme activity rather than Class I II and IV relayingon the zinc Imai Lin In mammalsSir2 has homologous proteins and these proteins are essentialto mitochondrial energy homeostasis antioxidant defense cellproliferation and DNA repair VargasOrtiz SIRT1 Mediates Energy Stress Adaptation in SkeletalMuscles reported that SIRT1 promotes theRodgers et altranscriptional activity of PGC1α by deacetylating the PGC1αat K13 site and mediates CRinduced gluconeogenesisrelatedgenes G6P PEPCK expression in liver Further research showedthat the NADNADH ratio and the enzymatic activity of Sir2 asensor of redox state were decreased in diï¬erentiating myocyteswhich relieved the inhibitory eï¬ect on MyoD and then promotedcell diï¬erentiation Resveratrol RSV a compound that maytarget SIRT1PGC1α can improve mitochondrial function andmetabolic homeostasis owning a potential to prolong lifespanRSV loses its activating eï¬ect on PGC1α in SIRT1 ˆ’ˆ’ MEFsLagouge Moreover RSVtreated mice at a dose of gkg increased their exercise capacity oxygen consumptionand improved insulin sensitivity against highfat induced obesityLagouge In addition RSV could enhance thedeacetylation of PGC1α in brown fat tissue BAT and skeletalmuscles which promotes mitochondrial production oxygenconsumption and thus improves metabolic syndrome Lagouge The function of SIRT1 in regulating skeletal musclesmetabolic homeostasis and exercise capacity has attracted greatattention from researchers Researchers further generated skeletalmusclespecific SIRT1mKO mice and found that mKO miceFrontiers in Physiology wwwfrontiersinAugust Volume 0cTian et alRole of HDACs in Metabolism and Exercise CapacityFIGURE Control of muscle exercise adaption and mitochondrial function by Class II HDACs A Schematic of the Class IIa HDACs nucleoplasm shuttle Understress condition CaMK and AMPK could be activated and phosphorylate Class IIa HDACs Phosphorylated HDACs then bind with and shuttle to cytoplasmrelieving inhibitory effects on MEF2 and Baf60c B Class IIb HDAC6 can deacetylate MFN1 and facilitates its mitofusion function P phosphorylationlost the eï¬ect of CRinduced increasing insulin sensitivity andwere unable to deacetylate and inactivate STAT3 resulting inupregulating the phosphatidylinositol3kinase PI3K inhibitoryregulator p55αp50α expression Schenk Althoughknockout or overexpression of SIRT1 in skeletal muscles hasno eï¬ect on endurance capacity or glucose homeostasis in miceGurd Philp White Svensson some studies have shown that AMPK regulatesenergy metabolism of skeletal muscles partially mediated bySIRT1 and SIRT1 is significantly increased after enduranceexercise Suwa Canto Overexpressionof Sirt1 in skeletal muscles by adenoassociated virus AAV1promotes the expression of oxidationrelated genes includingPpargc1a Tfam Cpt1b and Pdk4 while it has no eï¬ect on insulinsensitivity of body Vila But overexpressing Sirt1in liver by AAV8 can protect from fatty liver induced by highcarbohydrate food HCD Vila Taken togetherthe aforementioned studies suggest that SIRT1 possesses limitedregulation capacity of skeletal muscles motility and SIRT1may modulate mitochondrial homeostasis and mediate skeletalmuscles adaptation under certain physiological conditions suchas CR aging and regeneration Gomes Ryall Figure In addition there may be more beneficial eï¬ectsof SIRT1 on metabolism in the liver or fat tissue Lagouge Vila Stefanowicz SIRT6 Improves Muscle Fitness andExercise CapacitySIRT6 is another Sir2like deacetylase located in the nucleusWhole body deletion of SIRT6 causes mice death around weeks owning to hypoglycemia Xiao Neverthelessthe skeletal musclespecific knockout of SIRT6 causes insulinresistance and impairs glucose homeostasis of mKO mice Cui Because the activity of AMPK is reduced inSIRT6mKO mice the glycolipids absorption and utilizationof skeletal muscles are impaired and the exercise capacity ofthe mice was also attenuated Cui Furthermoreresearchers constructed the overexpressing SIRT6 Sirt6BACmice and found that their body weight and fat content werenormal but the Sirt6BAC mice could protect from HCDinducedhyperglycemia via increasing pAKTAKT induced by insulinstimulation and glucose absorption Anderson Vitroexperiments further demonstrated that the ability of glucoseuptake was enhanced mainly in skeletal muscles not in brainiWAT eWAT tissues of Sirt6BAC mice Anderson These results shows the potential eï¬ects of SIRT6 targeted inskeletal muscles on improving exercise performance and treatingmetabolic diseases Figure MitochondriaLocated SIRT34SIRT34 Maintains Mitochondria Function in SkeletalMusclesSIRT34 are mitochondrialocalized deacetylases and responsiblefor regulating the deacetylation of proteins in mitochondria andmaintaining mitochondrial homeostasis After feeding highfatdiet HFD accelerated obesity attenuated insulin sensitivityworsened fatty liver and increased acetylation of proteins inmitochondria were observed in SIRT3 KO mice Hirschey Multitissue proteomics and physiological examinationreveals that SIRT3 is responsible for mitochondrial acetylatedFrontiers in Physiology wwwfrontiersinAugust Volume 0cTian et alRole of HDACs in Metabolism and Exercise CapacityFIGURE Class III HDACs promote a stressinduced adaptation in muscle and regulate mitochondrial function Class III HDACs are NADdependentdeacetylases and may function as a redox sensor in muscle cell SIRT1 could activate PGC1alpha by directly deacetylasing K13 site and regulate insulin sensitivityby targeting STAT3 Mitochondrialocated Class III HDACs control mitochondrial function by deacetylating certain mitochondrial proteins modulating muscle fuelutilization and exercise capacityproteome regulation and metabolic fuel switching in brainheart kidneyliver and skeletal muscles DittenhaferReed Protein enrichment analysis discovers that itsregulatory proteins were mainly concentrated in lipid metabolismDittenhaferReed Knocking out SIRT3 leadsto reduction in the levels of deacetylation of manganesesuperoxide dismutase MnSOD mitochondrial complex II andpyruvate dehydrogenase PDH subunit E1α a downregulationof hexokinase II HK II binding with mitochondria resultingin impaired glucose and lipid metabolism of skeletal musclesLantier Interestingly SIRT3 liverspecific knockouthepˆ’ˆ’ and skeletal musclespecific knockoutskmˆ’ˆ’mice did not aï¬ect glucose homeostasis under chow or HFDconditions FernandezMarcos The above resultsindicate that SIRT3 has an important role in metabolic regulationbut in specific physiological processes such as redox stateexercise and aging and its regulatory mechanism is yet definedin skeletal muscles Kong Robin Williams A recent study found that SIRT4 knockoutcan resist HFDinduced obesity and increase endurance exercisein mice by repressing malonyl CoA decarboxylase which wasa key enzyme controlling fatty acid betaoxidation and wasreported to regulate muscle fuel switching between carbohydratesand fatty acids Koves Laurent Moreover knockdown of SIRT4 by adenoviral shRNA canincrease the mRNA and protein content of SIRT1 therebyenhancing the expression of fatty acid oxidation genes andmitochondrial oxidation capacity in hepatocytes and myotubesNasrin Table THERAPEUTIC TARGETS OF HDACsAND THEIR POTENTIAL FORMETABOLIC DISEASESA comparative analysis used HDAC paninhibitor SAHA aclass I HDAC selective inhibitor MS275 and a class IIHDAC selective inhibitor MC1568 to treat C2C12 separatelyIt was found that only MS275 could significantly stimulatemitochondria biogenesis and oxygen consumption Galmozzi In obese diabetic mice it was found that specificallyinhibiting Class I rather than Class II HDACs improvedGTT and insulin sensitivityincreased oxidative metabolismof skeletal muscles and adipose tissue and reduced bodyweight Galmozzi Similar to the eï¬ect of MS275knockdown HDAC3 in C2C12 could also increase Pgc1αGlut4 Tfam Idh3α transcription suggesting that HDAC3 andits target genes played an important role in the above eventsGalmozzi For Class II HDACs studies have found that scriptaid a ClassIIa HDAC inhibitor has similar eï¬ects to exercise Six weeksof scriptaid administration significantly improved enduranceperformance and significantly increased the wholebody energyexpenditure and the expression of lipid oxidation related genesPdk4 Cpt1b Pgc1α Pparδ etc of C57BL6 mice Gaur One possible explanation of above observation is thepotential target of scriptaid inhibition of titin a structure proteinof sarcomere deacetylation and downstream genes of Class IIaHDACs Gaur Huang Frontiers in Physiology wwwfrontiersinAugust Volume 0cTian et alRole of HDACs in Metabolism and Exercise CapacityIn mice treated with SPT1720 another activator of SIRT1overtly enhanced endurance exercise ability was noted Moreoverthese mice were protected from HFDinduced obesity and insulinresistance owing to an upregulation of the oxidative metabolismin skeletal muscles liver and BAT tissues Pacholec In order to further explore the principle of SIRT1 activation theresearchers purified SIRT1 in vitro and added SRT1720 and itsanalogs SRT2183 SRT1460 and RSV and they found that theenzymatic activity of SIRT1 was not enhanced suggesting thatthese drugs may indirectly activate SIRT1 Pacholec The enzymatic activity of SIRT1 largely depends on the contentof cofactor NAD This implied that the enhancement of SIRT1activity may be achieved through indirect upregulation of NADStudy has discovered that knocking out poly ADPribosepolymerase1 PARP1 which is a NAD consuming enzymecould increase NAD content and enhance the activity of SIRT1in BAT and skeletal muscles Bai PARP1 inhibitorscan upregulate the proteins of mitochondrial respiratory chaincomplexes in mice enhance the aerobic oxidation capacity ofmitochondria and improve mitochondrial defects in the primarymyotubes of obese humans Bai Pirinen et alResveratrol was initially reported as an SIRT1 activatorthat improves mitochondrial function and exercise capacity inmice and resists HFDinduced obesity Lagouge However subsequent studies have discovered that administeringthe same dose of RSV to rats or mice does not increasemitochondrial protein content Higashida Bycontrast overexpression of SIRT1 in the triceps muscle of ratsdecreases the mitochondrial protein content Higashida In a doubleblind human trial healthy and obesemen were supplied for 30day RSV in which the data showedthat RSV can improve systolic blood pressure and homeostasismodel assessment HOMA indexindicating glucose metabolismability simulating the eï¬ect of CR Timmers However some researchers found that the overexpression ofSIRT1 alone cannot mimic the CR eï¬ect in transgenic mice andthe transcriptomic changes in various tissues were quite diï¬erentor even opposite Boutant Such an opposite situationmay explain that the genetic model and compound stimulationare not completely consistent RSV as a potential metabolicsyndrome treatment drug still needs largescale populationsample verificationCONCLUSIONThe very first mammalian histone deacetylase HDAC1 wascloned and isolated by Taunton and sabout HDACs have been published in the last years CurrentlyREFERENCESAnderson J G Ramadori G Ioris R M Galie M Berglund E D CoateK C Enhanced insulin sensitivity in skeletal muscle and liver byphysiological overexpression of SIRT6 Mol Metab “ 101016jmolmet201509003we know at least HDAC proteins They are responsible foreradicating epigenetic modifications establishing an epigeneticoï¬ chromatin state and regulating heritable gene expressionYang and Seto Haberland In these processeseach HDACs may play a diï¬erent role Table What kind ofgeneprotein is the specific downstream target of these HDACsIs its enzyme activity related to intracellular localization andthe formation of multiprotein complex It is still one of thekey and difficult issues in this field Based on previous researchexperience some techniques may be used to further experimentsas follows HDACs interacting proteinscomplex coIP Proteinacetylation western Histone acetylation target geneChIP High through put proteomicsacetylome with specificHDACs inhibitor etctherapeuticare potentialHistone deacetylasestargetsand clinical drugs such as SAHA Vorinostat and FK228romidepsin have been used for antitumor treatment Bolden However they have two disadvantages greattoxic and side eï¬ects and difficulty in specific inhibition ofHDACs activity With the development of computer simulationtechnology and structural biology it is believed that more specificHDACs inhibitorsactivators can be constructed And researchesshould attach importance to HDACs regulatory factors likePARP1 when direct targeting on HDACs fails to show its eï¬ectsFurther development of their inhibitors with more specificityand trials for the treatment of metabolic diseases may have greatpotential as well Answer:

Thyroid_Cancer

CytoskeletonAssociated Role ofPDLIM5Xiaolan Huang Rongmei Qu Jun Ouyang Shizhen Zhong and Jingxing DaiGuangdong Provincial Key Laboratory of Medical Biomechanics Department of Anatomy School of Basic MedicalSciences Southern Medical University Guangzhou ChinaRegenerative medicine represented by stem cell technology has become one of thepillar medical technologies for human disease treatment Cytoskeleton plays importantroles in maintaining cell morphology bearing externalforces and maintaining theeffectiveness of cellinternal structure among which cytoskeleton related proteins areinvolved in and play an indispensable role in the changes of cytoskeleton PDLIM5 isa cytoskeletonrelated protein that like other cytoskeletal proteins acts as a bindingprotein PDZ and LIM domain PDLIM5 also known as ENH Enigma homolog isa cytoplasmic protein with a molecular mass of about KDa that consists of a PDZdomain at the Nterminus and three LIM domains at the Cterminus PDLIM5 bindsto the cytoskeleton and membrane proteins through its PDZ domain and interactswith various signaling molecules including protein kinases and transcription factorsthrough its LIM domain As a cytoskeletonrelated protein PDLIM5 plays an importantrole in regulating cell proliferation differentiation and cellfate decision in multipletissues and cell types In this review we briefly summarize the state of knowledge onthe PDLIM5 gene structural properties and molecular functional mechanisms of thePDLIM5 protein and its role in cells tissues and an systems and describe thepossible underlying molecular signaling pathways In the last part of this review wewill focus on discussing the limitations of existing research and the future prospects ofPDLIM5 research in turnKeywords PDZ and LIM domain microfilament actin cytoskeleton cytoskeletonassociated proteinINTRODUCTIONPDZ and LIM domain also known as ENH ENH1 L9 and LIM is a cytoskeletonrelated proteinthat was first discovered by Kuroda using yeast twohybrid technology with proteinkinase C PKC as the bait protein PDLIM5 which consists of a PDZ domain and one or moreLIM domains is a PDZLIM family member whose sequence is highly conserved across speciesThe proteins of the PDZLIM family EnigmaLMP1 ENH ZASPCipher RIL ALP and CLP36have been suggested to act as linkers to direct LIM binding proteins to the cytoskeleton Vallenius PDZLIM protein can act as a signal modulator to aï¬ect actin dynamics regulate cellstructure and control gene transcription to promote the assembly of protein complexes The PDZLIM protein family which function as protein“protein interaction modules act as scaï¬olds to bindto filamentous actinassociated proteins a range of cytoplasmic signaling molecules and nuclearEdited byMarina PajicGarvan Institute of Medical ResearchAustraliaReviewed byClment ThomasLuxembourg Institute of HealthLuxembourgHongqiang ChengZhejiang University ChinaCorrespondenceJun OuyangjouyangsmueducnShizhen ZhongzhszhsmueducnJingxing Daidaijxsmueducndaijx2013163comSpecialty sectionThis was submitted toIntegrative Physiologya section of the journalFrontiers in PhysiologyReceived May Accepted July Published August CitationHuang X Qu R Ouyang JZhong S and Dai J AnOverview of theCytoskeletonAssociated Roleof PDLIM5 Front Physiol 103389fphys202000975Frontiers in Physiology wwwfrontiersinAugust Volume 0cHuang et alAn Overview of PDLIM5proteins allowing this family to carry out diverse functionsduring development and adulthood Krcmery Several members of the PDZLIM proteins family play aregulatory role in the invasion and migration of cancer cellsDysfunction of the proteins of the PDZLIM family is knownto aï¬ect the maintenance of an function and weaken theinvasion ability and metastatic potential of cancer cells BagheriYarmand TanakaOkamoto Accordingto recent studies PDLIM5 may be involved in the progressionof multiple tumor types Eeckhoute Edlund Heiliger Li The important role ofPDLIM5 in various anizational systems have led to a deeperunderstanding of its physiological function This review aimsto summarize the state of knowledge and progress related toPDLIM5 from multidisciplinary perspectivesTHE PDLIM5 GENE AND ITSEXPRESSION AND THE STRUCTUREAND DISTRIBUTION OF PDLIM5The PDLIM5 GeneThe PDLIM5 gene is located on the human chromosome 4q223between markers W12900 and W13273 and spans exonsUeki Te Velthuis and Bagowski PDLIM5can be categorized as long isoforms with three LIM domainsand short isoforms without LIM domains according to thepresence or lack of three LIM domains and the long and shortisoforms each contain ˆ¼ subtypes Cheng Ito Mouse PDLIM5 isoform I mENH1 encodes afulllength 591aminoacid protein containing a PDZ domainand three LIM domains two smaller transcripts called mousePDLIM5 isoform and mENH2 and mENH3 encode a aminoacid protein and a 239aminoacid protein respectivelyBoth mouse PDLIM5 isoforms and lack three LIM domainsNiederlander Zheng In humans fourPDLIM5 splice isoforms have been identified one long isoformENH1 which contains three LIM domains at its Cterminaland is widely expressed in all tissues and three short isoformsENH24 which are mainly expressed in cardiac and skeletalmuscle Kuroda Ueki Analysis of humanPDLIM5 transcripts showed that three transcripts hENH12 were similar to those of mice while the fourth transcripthENH4 encodes a 215aminoacid protein lacking three LIMdomains Niederlander The Expression Structure andDistribution of PDLIM5is a memberThe PDLIM5 protein also known as ENHofthe Enigma family which consists of an NterminalPDZ domain and three Cterminal LIM domains The mainfunction of PDZ and LIM domains is to participate inproteinprotein interactions Table The PDZ domain oneofischaracterized by a highly conserved amino acid sequenceconsisting of six antiparallel strands and two αhelicesthe most common proteinprotein binding domainsTABLE Binding partners of PDLIM5 and their functionsProteinDomain FunctionsReferencesProtein kinaseA PKAαactininMyotilinLtype calciumchannelYAPProtein kinaseC PKCProtein kinaseD1 PKD1CREBID2PDZPDZPDZPDZPDZLIMLIMLIMLIMNtype Ca2channelsAMP activatedprotein kinaseKAE1Protein kinaseLin Sarcomere Zline proteinSarcomere Zline proteinCalcium channels inmyocytesTranscriptionalcoactivatorProtein kinaseProtein kinaseCAMP relatedtranscription factorsDifferentiation inhibitorNtype calcium channelsin nervous systemProtein kinaseNakagawa Ito Ito Maturana Elbediwy Kuroda Maturana Maturana Ito Lasorella and Iavarone Nakatani et alMaenoHikichi et alYan Liu Kidney anion exchanger Su Fanning and Anderson Sheng and Sala The PDZdomain provides a proteinbinding interface that facilitates theformation of multiprotein complexes with a variety of partnersincluding membraneassociated proteins cytoplasmic signalingproteins and cytoskeleton proteins Jelen Krcmery Zheng Ito The LIM domainis approximately amino acids long and is characterized byhighly conserved and spatially defined cysteine and histidineresidues that coordinate the binding of two zinc ions to formtwo zinc fingerlike structures LIM domains can exist in proteinsalone or in combination with other domains Dawid Bach Kadrmas and Beckerle Te Velthuisand Bagowski The LIM domains can combine highlydiverse partners ranging from signaling molecules and actincytoskeletal components to transcription factors and it alsosupport cellular functions Dawid In particularthe crosslinking with actin cytoskeleton such as LIM domainprotein can maintain the functional structure of cardiomyocytesby a mechanism involving its own binding and actin filamentcrosslinking which plays an important role in the developmentof heart disease Hoï¬mann In addition LIM domainproteins have also been reported to be involved in the invasionand metastasis of cancer as components and targets of thecytoskeleton Hoï¬mann exhibitThe PDLIM5 splicing isoformstissuespecificexpression the long isoforms are widely expressed in varioustissues while the short isoforms are only highly expressedin cardiac and skeletal muscle Yamazaki Thisdiï¬erential expression of PDLIM5 may be related to theirdiï¬erent roles in diï¬erent tissues and an systems Table Frontiers in Physiology wwwfrontiersinAugust Volume 0cHuang et alAn Overview of PDLIM5TABLE List of different disease involved in PDLIM5Disease and developmentReferencesRelated signalingpathwaysRASERKAMPKPKCMicroRNA17ˆ¼Papillary thyroid carcinomaProstate cancerWei Koutros Liu Li Gastric cancernonsmall cell carcinomaEdlund Cancer associated with steroid use Wang Heart developmentCardiomyocyte hypertrophyTGFSmadPulmonary hypertensionDilated CardiomyopathyBipolar disorderDepressive disorderSchizophreniaAlcohol dependence type diabetes and hypertensionNakagawa Yamazaki Bang Chen Cheng Cheng Zhao Liu Numata Owusu THE DIFFERENTIAL ROLES OF PDLIM5IN VARIOUS AN SYSTEMSThe Role of PDLIM5 in the NervousSystemPDZ and LIM domain is widely expressed in diï¬erent regionsof the brain such as the hippocampus thalamus hypothalamuscerebral cortex and amygdala MaenoHikichi Incentral neurons PDLIM5 is mainly localized in the membraneand cytoplasm where it regulates neuronal calcium signaling andcolocalizes with neurotransmitterprotruding vesicles Numata these observations indicate that PDLIM5 playsa role in brain development Some studies have shown thatthe expression of PDLIM5 is associated with multiple mentaldisorders such as bipolar disorder major depression andsusceptibility to schizophrenia Liu Zhao Herrick Wang In the nervous system PDLIM5 plays an important rolein the formation of nerve growth cones and promotes thediï¬erentiation of nerve cells Some studies have shownthat PDLIM5 expression is upregulated during neuraldiï¬erentiation and it has been shown that its ectopic expressionin neuroblastoma cells leads to the translocation of ID2 whichis one of the four members of the ID protein family called adiï¬erentiation inhibitor from the nucleus to the cytoplasmresulting in the inactivation of transcriptional and cellcyclepromoting functions ofthe latter Lasorella and Iavarone Furthermore PDLIM5 can form large complexes withPKC and Ntype Ca2 channels to promote the regulationof Ntype calcium channel activity MaenoHikichi Ren showed that PDLIM5 and PKCεcoexist in the nerve growth cone Through interaction withαactinin PDLIM5 may be involved in regulating microfilamentremodeling in neurons and the formation of the PDLIM5PKCεcomplex in the nerve growth cone which acts as a scaï¬old tomediate PKCε translocation to the membrane during PMAinduced growth cone collapse It is suggested that PDLIM5participates in a variety of functions of the nervous systemas well as in a signaling pathway involving the sequestrationof the transcription factor ID2 in the cytoplasm Howeverthe precise mechanisms by which PDLIM5 regulates thefunctions of the nervous system via ID2 blockade requiresfurther elucidationPDLIM5 in the Heart and Skeletal MusclePhysiological Roles of PDLIM5 in the HeartThe heart undergoes development and begins to function in theearly stages of embryonic development PDLIM5 is expressedat high levels in the skeletal muscle and myocardium and isconsidered to be a heart and skeletalmusclespecific scaï¬oldprotein to regulates mouse heart development Mu PDLIM5 is capable of binding to αactinin throughthe PDZ domain and colocalizing in the zdisk region ofcardiomyocytesindicating that this protein plays a role incardiac development Studies have shown that PDLIM5 mRNAsare mainly expressed in the heart and skeletal muscle of adultrats and that PDLIM5 acts as a scaï¬old protein to mediatethe transmission of PKC signals in cardiomyocytes playing animportant role in development of the muscle cell in an earlydevelopmental stage Nakagawa Zheng The PDZLIM protein family is highly expressed in the striatedmuscle PDLIM5 is similar to other PDZLIM members in thestriated muscle in which the PDZ domain binds to αactininwhile the LIM domain binds to several protein kinases C andprotein kinase D Kuroda Nakagawa For example PDLIM5 traditionally activates the PKC throughthe direct binding of its LIM domain Maturana and interacts to PKA Lin Transcription factorCREB which is one of the first transcription factors activated byneurohumoral factors stimulation is a transcription factor cAMPresponse element binding protein is a known target of the PKCand protein kinase D1 PKD1 pathways Thonberg Ozgen The interaction between PDLIM5 isoform and CREB is necessary for the phosphorylation of CREB at theaminoacid residue Ser133 which promotes the transcriptionalactivation and nuclear localization of CREB the phosphorylatedCREB enters the cardiomyocyte nucleus to play the role oftranscription factor and promote the growth of cardiomyocytesIto Moreover in neonatal rat cardiomyocytesPDLIM5 interacts with PKD1 through its LIM domain and formscomplexes with PKD1 and cardiac Ltype voltagegated calciumchannelα1C subunits to regulate the activity of Ltype voltagegated calcium channels Maturana Although theformation of protein complexes such as PDLIM5PKCPKD1is well understood the downstream molecular events remainto be elucidatedThese results suggest that the localization of PDLIM5 at somesubcellular sites and its ability to interact with multiple functionalproteins play an important role in cellular and physiologicalfunctions Furthermore the role of PDLIM5 in the heart wasFrontiers in Physiology wwwfrontiersinAugust Volume 0cHuang et alAn Overview of PDLIM5studied using a heartspecific PDLIM5knockout mouse modelIt was found that the ablation of PDLIM5 disrupted the stabilityof the PDLIM5CiphersCalsarcin complex formed in the zdiskregion thus interfering with the connection between adjacentsarcomeres and extracellular matrix These eï¬ects were found toresult in the loss of optimal force transmission and a significantdecrease in cardiac shortening fractionleading to dilatedcardiomyopathy Cheng Novel polymorphisms inthe PDLIM5 gene encoding the Zline protein have also beenshown to increase the risk of idiopathic dilated cardiomyopathyWang that underpinsPDZ and LIM domain is additionally involved in skeletalmuscle development Myogenesis is an important biologicalprocessskeletal muscle regeneration andpostnatal growth The silencing of PDLIM5 increases the nuclearaccumulation of diï¬erentiation inhibitor Id2 which inhibits theproliferation and diï¬erentiation of myoblasts Nakatani Qiu In addition the diï¬erentiation of andmorphological changes in skeletal muscle is regulated by a groupof transcription factors known as myogenic regulators PDLIM5isoform overexpression leads to the upregulation of MyoDand myogenin while PDLIM5 isoform knockout significantlydecreases the expression of these two proteins these findingsindicate that the main eï¬ect of PDLIM5 isoform on musclecells is to stimulate the transcription of MyoD andor myogeninencoding genes Ito Although the main role of PDLIM5 is as a specific scaï¬oldprotein which to bridge the connection between cytoskeletonand membrane proteins and promote the formation of proteincomplexes it is capable of generating numerous splicing isoformsENH24 that exert various eï¬ects on the development ofheart and skeletal muscle In vitro PDLIM5 isoform hasbeen shown to promote the expression of myogenic genes andmyotube formation while the short PDLIM5 isoform hasbeen found to abrogate myotubelike morphological changeswithout altering the expression of the myogenic transcriptionfactors MyoD and myogenin Ito Furthermorethe overexpression of PDLIM5 isoform prevented ventricularcardiomyocyte hypertrophy induced by vascular stress hormonesYamazaki Western blotting analysis of muscle tissueusing a nonisoformspecific antiPDLIM5 antibody showed thatPDLIM5 isoform is only expressed in skeletal muscle witha specific distribution of PDLIM5 members between skeletalmuscle and myocardium Niederlander These results suggest that PDLIM5 plays a key role in thedevelopment of the myocardium and skeletal muscle Howeverthe signal transduction mechanisms underlying the role ofPDLIM5 in heart and skeletal muscle remain to be furtherstudied For example the specific molecular mechanisms bywhich PDLIM5 regulates the development of myocardium andskeletal muscle through binding protein kinases are unknownFurthermore the mechanisms by which PDLIM5 sequestersnuclear protein Id2 in the cytoplasm remain to be elucidatedThe Role of PDLIM5 in Cardiovascular DiseasesPDZ and LIM domain is mainly distributed on the zline ofthe sarcomere of cardiomyocytes therefore the eï¬ect of PDLIM5on myocytes may be related to the contractile function of thesecells PDLIM5knockout mice exhibit dilated cardiomyopathywhich is characterized by thinning of the left ventricular wallenlargement of the left ventricular cavityimpaired cardiaccontraction and reduced ejection function Cheng PDLIM5 can regulates vascular remodeling which canas a new proatherosclerotic factor to be a therapeuticallytargeted Huang Cardiac remodeling which isindicative of progression in many cardiovascular diseases ischaracterized by cardiomyocyte hypertrophy and myocardialfibrosis which lead to heartfailure Swynghedauw Barry and Townsend microRNA miR21 derivedfrom cardiac fibroblasts exosomes is a strong paracrine RNAmolecule that induces cardiomyocyte hypertrophy Thum Recentlyit has been reported that PDLIM5 is thedirect target of miR17ˆ¼ Bang Chen By acting on its target gene PDLIM5 miR participates in the interaction between cardiac fibroblastsand cardiomyocytesthus inducing myocardial pathologicalhypertrophy Bang PDLIM5 also plays a role invascular smooth muscle AMPactivated protein kinase is anintracellular energy receptor of AMPK which is activatedunder hypoxia ischemia glucose loss and stress Steinberg andKemp AMPK is generally considered to be an energysensor kinase and requires AMP for its activation Carling Nakano reported that AMPK controls microtubuledynamics by phosphorylating cytoplasmic connexin170 CLIPthus regulating cell migration Nakano In addition AMPK is involved in the regulation of actincytoskeleton dynamics and plasma membrane reanizationBae Kondratowicz Studies haveshown that AMPK activation plays an important role inneovascularization and metastasis HoyerHansen and Jaattela In vascular smooth muscle cells AMPK phosphorylatesPDLIM5 at Ser177inhibiting the downstream Rac1Arp23signaling pathway to mediate cell migration Yan In pulmonary artery smooth muscle cells PASMCsSMCspecific knockout of PDLIM5 enhances hypoxiamediatedvascular remodeling while overexpression of PDLIM5 inhibitsthe TGFSmad signal pathway and prevents hypoxiainducedpulmonary hypertension elevation in vivo Cheng In addition PDLIM5 silencing induces the activity of TGF3 TR1 and TGF and increases the overall expressionlevel of Smad2 The suppression of PDLIM5 has beenfound to enhance the nuclear staining of Samd23 Chen indicating that PDLIM5 participates in thedevelopment of cardiovascular disease by negatively regulatingthe TGF3Smad23 signal pathway Additionally demonstratedthat PDLIM5 plays an important role in the cardiovascularsystem through miRmediated regulation of the phenotype ofpulmonary artery smooth muscle cells miR17ˆ¼ regulatesthe diï¬erentiation of PASMCs through its target PDLIM5indicating that the miR17ˆ¼92PDLIM5TGFSmad pathwayis essential for vascular remodeling during the developmentof pulmonary hypertension PDLIM5 therefore represents apromising therapeutic target for future cardiovascular drugdiscovery eï¬ortsFrontiers in Physiology wwwfrontiersinAugust Volume 0cHuang et alAn Overview of PDLIM5The Role of PDLIM5 in TumorAs an actin adaptor protein PDLIM5 is not only involved incytoskeletal tissue cellular processes and an development butis also considered to play roles in tumorigenesis and developmentEeckhoute Edlund Heiliger Li PDLIM5 is expressed in many cancer celllines In a study of neurologic tumor it was found that thetranscription factor ID2 binds to the PDLIM5 LIM domains andin these cancer cells high levels of PDLIM5 sequester ID2 in thecytoplasm preventing neuronal diï¬erentiation and promotingcell proliferation Lasorella and Iavarone PDZ and LIM domain is additionally upregulated inpapillary thyroid carcinoma PTC tissues elevated PDLIM5expression promotes the migration invasion and proliferation ofPTC cells by activating the RASERK pathway Wei PDLIM5 may therefore serve as a therapeutic target in a varietyof cancers It can promote the invasion and metastasis of cancercells Genotyping chip detection experiments have shown thatPDLIM5 is overexpressed in prostate cancer tissue Koutros and some studies have proved that the utility of serumand urine PDLIM5 levels as indicators for auxiliary diagnosisof prostate cancer with potential value in predicting the risk ofprogression in advanced prostate cancer PCA Ma PDLIM5 plays a key role in regulating the proliferation invasionand migration of malignant tumor cells by binding to AMPKand regulating its activation and degradation Liu PDLIM5 may therefore act as an oncogene in the developmentand progression of PCAIn view of the important role of PDLIM5 in cancer somestudies have indicated that it is involved in the growth of gastriccancer cells and suggested that PDLIM5 silencing through theuse of small interfering RNAs siRNA may be a potentialstrategy for the treatment of gastric cancer Li In addition Edlund found that the increased expressionof PDLIM5 is related to high tumor proliferation rates innonsmall cell carcinoma Edlund Steroids suchas corticosteroid medications play an important role in thedevelopment of cancer it has been reported that singlenucleotide polymorphisms SNPs in PDLIM5 interact withsteroids thus aï¬ecting the occurrence and development of cancerWang The above findings show that PDLIM5 plays an important rolein the occurrence and development of cancer and that PDLIM5represents a candidate oncogene in various cancersThe Role of PDLIM5 in Other DiseasesreportedIn addition to playing a role in the diseasesinvolved in the link between alcoholabove PDLIM5 isdependence and diabetes Owusu found thatPDLIM5 gene polymorphism is associated alcoholdependentAD type diabetes T2D and hypertension and Severalgenetic variants of the PDLIM5 gene can aï¬ect AD T2Dand hypertension indicating that PDLIM5 is a shared geneamong the three diseases Therefore elucidation oftheunderlying molecular mechanisms and identification of hithertoundiscovered molecular functions of PDLIM5 are expectedto enable the development of eï¬ective clinicalfor these diseasestherapiesIn addition PDLIM5 plays a role in the formation of cellextensions Being a scaï¬old protein PDLIM5 is involved inpromoting the activity of microfilamentassociated proteinsMicrofilamentassociated proteins play a central role in theprocess of cell extension Lanier Some studieshave found that PDLIM5 recruitment to cell extensions and isnecessary to form these extensions and that PDLIM5 knockoutreduces the assembly of actin filaments in cell extensionsYuda THE RELATIONSHIP BETWEEN PDLIM5AND INTEGRINS AND ITS POTENTIALROLE IN THE REGULATION OF STEMCELL DIFFERENTIATIONSome studies have shown that the PDZ domain of PDZLIMprotein binds to αactinin protein at the adhesion junctionwhich is the site of integrin localization Xia Pomies Vallenius Tamura The functional interaction with integrin indicates that PDZLIM protein can participate in the adhesion signal cascadewhich transmits extracellular signals via intracellular regulatorypathwaysthereby modifying the actin cytoskeleton Thesefindings show that the PDZLIM protein plays an overall role incell“cell and cell“matrix interaction and cell migration Krcmery The regulation of PDZLIM activity plays animportant role in preventing uncontrolled actin recombinationproliferation and cell migration For example PDLIM5 canalso bind to the integrin protein kinase ILK acting as ascaï¬old bridge between renal ion exchanger KAE1 and ILKproviding a bridge between KAE1 and potential microfilamentsSu It has been reported that PDLIM5 is recruited from thecytoplasm to the integrin adhesion and Factin stress fibersand responds to stress by directly binding to the key stressfiber component αactinin Microfilaments control the nuclearand cytoplasmic localization oftranscriptional coactivatorsYAP and TAZ to regulate gene expression and mediate thediï¬erentiation of MSCs Dupont Halder The eï¬ective domains of some proteins that are recruited intothe actin structure in a forcedependent manner through theLIM domain regulate actin signal transduction Smith The action of mechanical force on integrin results in therecruitment of PDLIM5 to activate the YAP pathway duringmechanical transduction Elbediwy PDLIM5 acomponents of the integrin adhesion complex mediates therelationship between integrin and the cytoskeleton Horton et al2016ab Proteomic analysis of integrinrelated complexes fromMSCs has demonstrated the formation of significant amountsof a vinculinpositive adhesion complex on a hard substratecoated with fibronectin FN in MSCs a subset of whichcolocalized with or closely to clusters of PDLIM1 or PDLIM5Ajeian Frontiers in Physiology wwwfrontiersinAugust Volume 0cHuang et alAn Overview of PDLIM5PDZ and LIM domain undergoes tensiondependentrelocalization in cells Both embryonic and induced pluripotentstem cells can diï¬erentiate into derivatives of the third germlayer as a result human pluripotent cells are important toolsin regenerative medicine Thomson Takahashiand Yamanaka Studies have reported that duringcardiogenesis embryonic stem cells exhibit dramatic changes inthe expression of metabolic enzymes and cytoskeleton proteinsKonze in particular Zdisk related proteins withPDZ and LIM domain proteins including PDLIM5 are upregulated during cardiogenesisFurthermorethe expression of NKX25 an importantmyocardial transcription factor results in the generation ofspecific PDLIM5 splicing variants during the early developmentof cardiomyocytes which in turn aï¬ectsthe myogenicdiï¬erentiation of myocardium Konze At presentthere are few studies on PDLIM5 in stem cells elucidation of itsmechanism in diï¬erent stem cell types is warranted to identify itsfunctions in these cellsPROBLEMS AND PERSPECTIVESIn this we briefly reviewed the known functionsof the PDLIM5 protein the progress in elucidation ofitsroles in various cellular and physiological processes and thesignaling pathways in which it participates As a member ofthe PDZLIM protein family PDLIM5 is involved in actinbinding αactinin binding protein kinase binding acts asa scaï¬old bridge between connective proteins and plays anindispensable role in various cellular processes However so farthe specific molecular mechanisms underlying the functions ofPDLIM5 remain unclearFuture research directions in investigation of PDLIM5 shouldseek to answer the following questionsFirst research on PDLIM5 has mainly focused on its roles intumor the nervous system and the cardiovascular system As amicrofilamentassociated protein does PDLIM5 play the samerole in other physiological systems or cell lines Is the PDLIM5gene expressed in multiple systemsREFERENCESAjeian J N Horton E R Astudillo P Byron A Askari J A and MillonFrmillon A Proteomic analysis of integrinassociated complexes frommesenchymal stem cells Proteomics Clin Appl “ 101002prcaBach I The LIM domain regulation by association Mech Dev “ 101016S0925477399003147Bae H B Zmijewski J W Deshane J S Tadie J M Chaplin D D andTakashima S AMPactivated protein kinase enhances the phagocyticability of macrophages and neutrophils FASEB J “ fj11190587BagheriYarmand R Mazumdar A Sahin A A and Kumar R LIMkinase increases tumor metastasis of human breast cancer cellsvia regulationof the urokinasetype plasminogen activator system Int J Cancer “ 101002ijc21650Bang C Batkai S Dangwal S Gupta S K Foinquinos A and HolzmannA Cardiac fibroblastderived microRNA passenger strandenrichedSecond although the role of PDLIM5 in diseases hasbeen studied eg its expression is upregulated during tumordevelopment the specific mechanisms by which it exerts theseeï¬ects are unknown and further elucidation of the underlyingmechanisms and other functions is warrantedThird PDLIM5 has four splicing isoforms which performdiï¬erent functions what are the mechanisms by which they playdiï¬erent roles Are these diï¬erences in their roles attributable tothe presence or absence of LIM domains Additionally what isthe mechanism by which they play diï¬erent rolesFourth in regard to strategies used for the inhibition ofPDLIM5 expression only viral transfection has been reportedto date no pharmaceutical compounds that inhibit PDLIM5expression have been identified Therefore additional research onPDLIM5 inhibitors is also criticalFinally although PDLIM5 plays a crucial role in binding actinand has attracted much attention as a connecting protein thereare few studies on its eï¬ects on the actin cytoskeleton or othercytoskeleton such as binding to cytoskeletonrelated proteinsbridging the connection with the cytoskeleton Does PDLIM5aï¬ect the shape and location of the cytoskeleton in the processof participating in the biological functions of cellTheeï¬ects of PDLIM5mediated modulation ofthecytoskeleton on cell diï¬erentiation proliferation and othercellular functions remain to be explored in detail in future studiesAUTHOR CONTRIBUTIONSAll authors participated in the conception and writing of themanuscript SZ JO and JD reviewed and suggested modificationsto the content JD designed the structure of the reviewFUNDINGThis work wassupported by the National Key RDProgram of China grant number 2017YFC1105000 andthe Sanming Project of Medicinein Shenzhen grantnumber SZSM201612019exosomes mediate cardiomyocyte hypertrophy J Clin Invest “ 101172JCI70577Barry S P and Townsend P A What causes a broken heart“molecularinsights into heart failure Int Rev Cell Mol Biol “ S1937644810840031Carling D Mayer F V Sanders M J and Gamblin S J AMPactivatedprotein kinase nature™s energy sensor Nat Chem Biol “ nchembio610Chen T HuangJ B DaiJ Zhou Q RajJ U and Zhou Gthe miR17ˆ¼ signaling that PAI1 is a novel component ofregulates pulmonary artery smooth muscle cell phenotypes Am J PhysiolLung Cell Mol Physiol L149“L161 101152ajplung00137Chen T Zhou G Zhou Q Tang H Ibe J C and Cheng H Loss of microRNA17 approximately in smooth muscle cells attenuatesexperimental pulmonary hypertension via induction of PDZ and LIM domain Am J Respir Crit Care Med “ 101164rccm2014050941OCFrontiers in Physiology wwwfrontiersinAugust Volume 0cHuang et alAn Overview of PDLIM5Cheng H Chen T Tor M Park D Zhou Q and Huang J B A highthroughput screening platform targeting PDLIM5 for pulmonary hypertensionJ Biomol Screen “ Cheng H Kimura K Peter A K Cui L Ouyang K and Shen T Lossof enigma homolog protein results in dilated cardiomyopathy Circ Res “ 101161CIRCRESAHA110218735Dawid I B Breen J J and Toyama R LIM domains multiple roles asadapters and functional modifiers in protein interactions Trends Genet “ 101016s0168952598014243Dupont S Morsut L Aragona M Enzo E Giulitti S and Cordenonsi M Role of YAPTAZ in mechanotransduction Nature “ 101038nature10137Edlund K Lindskog C Saito A Berglund A Pontn F and G¶ranssonKultima H CD99 is a novel prognostic stromal marker in nonsmallcell lung cancer Int J Cancer “ 101002ijc27518Eeckhoute J A celltypespecific transcriptional network required forestrogen regulation of cyclin D1 and cell cycle progression in breast cancerGene Dev “ 101101gad1446006Elbediwy A Vanyai H DiazdelaLoza M Frith D Snijders A P andThompson B J Enigma proteins regulate YAP mechanotransductionJ Cell Sci 131jcs221788 101242jcs221788Fanning A S and Anderson J M PDZ domains fundamental buildingblocks in the anization of protein compl

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You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: CytoskeletonAssociated Role ofPDLIM5Xiaolan Huang Rongmei Qu Jun Ouyang Shizhen Zhong and Jingxing DaiGuangdong Provincial Key Laboratory of Medical Biomechanics Department of Anatomy School of Basic MedicalSciences Southern Medical University Guangzhou ChinaRegenerative medicine represented by stem cell technology has become one of thepillar medical technologies for human disease treatment Cytoskeleton plays importantroles in maintaining cell morphology bearing externalforces and maintaining theeffectiveness of cellinternal structure among which cytoskeleton related proteins areinvolved in and play an indispensable role in the changes of cytoskeleton PDLIM5 isa cytoskeletonrelated protein that like other cytoskeletal proteins acts as a bindingprotein PDZ and LIM domain PDLIM5 also known as ENH Enigma homolog isa cytoplasmic protein with a molecular mass of about KDa that consists of a PDZdomain at the Nterminus and three LIM domains at the Cterminus PDLIM5 bindsto the cytoskeleton and membrane proteins through its PDZ domain and interactswith various signaling molecules including protein kinases and transcription factorsthrough its LIM domain As a cytoskeletonrelated protein PDLIM5 plays an importantrole in regulating cell proliferation differentiation and cellfate decision in multipletissues and cell types In this review we briefly summarize the state of knowledge onthe PDLIM5 gene structural properties and molecular functional mechanisms of thePDLIM5 protein and its role in cells tissues and an systems and describe thepossible underlying molecular signaling pathways In the last part of this review wewill focus on discussing the limitations of existing research and the future prospects ofPDLIM5 research in turnKeywords PDZ and LIM domain microfilament actin cytoskeleton cytoskeletonassociated proteinINTRODUCTIONPDZ and LIM domain also known as ENH ENH1 L9 and LIM is a cytoskeletonrelated proteinthat was first discovered by Kuroda using yeast twohybrid technology with proteinkinase C PKC as the bait protein PDLIM5 which consists of a PDZ domain and one or moreLIM domains is a PDZLIM family member whose sequence is highly conserved across speciesThe proteins of the PDZLIM family EnigmaLMP1 ENH ZASPCipher RIL ALP and CLP36have been suggested to act as linkers to direct LIM binding proteins to the cytoskeleton Vallenius PDZLIM protein can act as a signal modulator to aï¬ect actin dynamics regulate cellstructure and control gene transcription to promote the assembly of protein complexes The PDZLIM protein family which function as protein“protein interaction modules act as scaï¬olds to bindto filamentous actinassociated proteins a range of cytoplasmic signaling molecules and nuclearEdited byMarina PajicGarvan Institute of Medical ResearchAustraliaReviewed byClment ThomasLuxembourg Institute of HealthLuxembourgHongqiang ChengZhejiang University ChinaCorrespondenceJun OuyangjouyangsmueducnShizhen ZhongzhszhsmueducnJingxing Daidaijxsmueducndaijx2013163comSpecialty sectionThis was submitted toIntegrative Physiologya section of the journalFrontiers in PhysiologyReceived May Accepted July Published August CitationHuang X Qu R Ouyang JZhong S and Dai J AnOverview of theCytoskeletonAssociated Roleof PDLIM5 Front Physiol 103389fphys202000975Frontiers in Physiology wwwfrontiersinAugust Volume 0cHuang et alAn Overview of PDLIM5proteins allowing this family to carry out diverse functionsduring development and adulthood Krcmery Several members of the PDZLIM proteins family play aregulatory role in the invasion and migration of cancer cellsDysfunction of the proteins of the PDZLIM family is knownto aï¬ect the maintenance of an function and weaken theinvasion ability and metastatic potential of cancer cells BagheriYarmand TanakaOkamoto Accordingto recent studies PDLIM5 may be involved in the progressionof multiple tumor types Eeckhoute Edlund Heiliger Li The important role ofPDLIM5 in various anizational systems have led to a deeperunderstanding of its physiological function This review aimsto summarize the state of knowledge and progress related toPDLIM5 from multidisciplinary perspectivesTHE PDLIM5 GENE AND ITSEXPRESSION AND THE STRUCTUREAND DISTRIBUTION OF PDLIM5The PDLIM5 GeneThe PDLIM5 gene is located on the human chromosome 4q223between markers W12900 and W13273 and spans exonsUeki Te Velthuis and Bagowski PDLIM5can be categorized as long isoforms with three LIM domainsand short isoforms without LIM domains according to thepresence or lack of three LIM domains and the long and shortisoforms each contain ˆ¼ subtypes Cheng Ito Mouse PDLIM5 isoform I mENH1 encodes afulllength 591aminoacid protein containing a PDZ domainand three LIM domains two smaller transcripts called mousePDLIM5 isoform and mENH2 and mENH3 encode a aminoacid protein and a 239aminoacid protein respectivelyBoth mouse PDLIM5 isoforms and lack three LIM domainsNiederlander Zheng In humans fourPDLIM5 splice isoforms have been identified one long isoformENH1 which contains three LIM domains at its Cterminaland is widely expressed in all tissues and three short isoformsENH24 which are mainly expressed in cardiac and skeletalmuscle Kuroda Ueki Analysis of humanPDLIM5 transcripts showed that three transcripts hENH12 were similar to those of mice while the fourth transcripthENH4 encodes a 215aminoacid protein lacking three LIMdomains Niederlander The Expression Structure andDistribution of PDLIM5is a memberThe PDLIM5 protein also known as ENHofthe Enigma family which consists of an NterminalPDZ domain and three Cterminal LIM domains The mainfunction of PDZ and LIM domains is to participate inproteinprotein interactions Table The PDZ domain oneofischaracterized by a highly conserved amino acid sequenceconsisting of six antiparallel strands and two αhelicesthe most common proteinprotein binding domainsTABLE Binding partners of PDLIM5 and their functionsProteinDomain FunctionsReferencesProtein kinaseA PKAαactininMyotilinLtype calciumchannelYAPProtein kinaseC PKCProtein kinaseD1 PKD1CREBID2PDZPDZPDZPDZPDZLIMLIMLIMLIMNtype Ca2channelsAMP activatedprotein kinaseKAE1Protein kinaseLin Sarcomere Zline proteinSarcomere Zline proteinCalcium channels inmyocytesTranscriptionalcoactivatorProtein kinaseProtein kinaseCAMP relatedtranscription factorsDifferentiation inhibitorNtype calcium channelsin nervous systemProtein kinaseNakagawa Ito Ito Maturana Elbediwy Kuroda Maturana Maturana Ito Lasorella and Iavarone Nakatani et alMaenoHikichi et alYan Liu Kidney anion exchanger Su Fanning and Anderson Sheng and Sala The PDZdomain provides a proteinbinding interface that facilitates theformation of multiprotein complexes with a variety of partnersincluding membraneassociated proteins cytoplasmic signalingproteins and cytoskeleton proteins Jelen Krcmery Zheng Ito The LIM domainis approximately amino acids long and is characterized byhighly conserved and spatially defined cysteine and histidineresidues that coordinate the binding of two zinc ions to formtwo zinc fingerlike structures LIM domains can exist in proteinsalone or in combination with other domains Dawid Bach Kadrmas and Beckerle Te Velthuisand Bagowski The LIM domains can combine highlydiverse partners ranging from signaling molecules and actincytoskeletal components to transcription factors and it alsosupport cellular functions Dawid In particularthe crosslinking with actin cytoskeleton such as LIM domainprotein can maintain the functional structure of cardiomyocytesby a mechanism involving its own binding and actin filamentcrosslinking which plays an important role in the developmentof heart disease Hoï¬mann In addition LIM domainproteins have also been reported to be involved in the invasionand metastasis of cancer as components and targets of thecytoskeleton Hoï¬mann exhibitThe PDLIM5 splicing isoformstissuespecificexpression the long isoforms are widely expressed in varioustissues while the short isoforms are only highly expressedin cardiac and skeletal muscle Yamazaki Thisdiï¬erential expression of PDLIM5 may be related to theirdiï¬erent roles in diï¬erent tissues and an systems Table Frontiers in Physiology wwwfrontiersinAugust Volume 0cHuang et alAn Overview of PDLIM5TABLE List of different disease involved in PDLIM5Disease and developmentReferencesRelated signalingpathwaysRASERKAMPKPKCMicroRNA17ˆ¼Papillary thyroid carcinomaProstate cancerWei Koutros Liu Li Gastric cancernonsmall cell carcinomaEdlund Cancer associated with steroid use Wang Heart developmentCardiomyocyte hypertrophyTGFSmadPulmonary hypertensionDilated CardiomyopathyBipolar disorderDepressive disorderSchizophreniaAlcohol dependence type diabetes and hypertensionNakagawa Yamazaki Bang Chen Cheng Cheng Zhao Liu Numata Owusu THE DIFFERENTIAL ROLES OF PDLIM5IN VARIOUS AN SYSTEMSThe Role of PDLIM5 in the NervousSystemPDZ and LIM domain is widely expressed in diï¬erent regionsof the brain such as the hippocampus thalamus hypothalamuscerebral cortex and amygdala MaenoHikichi Incentral neurons PDLIM5 is mainly localized in the membraneand cytoplasm where it regulates neuronal calcium signaling andcolocalizes with neurotransmitterprotruding vesicles Numata these observations indicate that PDLIM5 playsa role in brain development Some studies have shown thatthe expression of PDLIM5 is associated with multiple mentaldisorders such as bipolar disorder major depression andsusceptibility to schizophrenia Liu Zhao Herrick Wang In the nervous system PDLIM5 plays an important rolein the formation of nerve growth cones and promotes thediï¬erentiation of nerve cells Some studies have shownthat PDLIM5 expression is upregulated during neuraldiï¬erentiation and it has been shown that its ectopic expressionin neuroblastoma cells leads to the translocation of ID2 whichis one of the four members of the ID protein family called adiï¬erentiation inhibitor from the nucleus to the cytoplasmresulting in the inactivation of transcriptional and cellcyclepromoting functions ofthe latter Lasorella and Iavarone Furthermore PDLIM5 can form large complexes withPKC and Ntype Ca2 channels to promote the regulationof Ntype calcium channel activity MaenoHikichi Ren showed that PDLIM5 and PKCεcoexist in the nerve growth cone Through interaction withαactinin PDLIM5 may be involved in regulating microfilamentremodeling in neurons and the formation of the PDLIM5PKCεcomplex in the nerve growth cone which acts as a scaï¬old tomediate PKCε translocation to the membrane during PMAinduced growth cone collapse It is suggested that PDLIM5participates in a variety of functions of the nervous systemas well as in a signaling pathway involving the sequestrationof the transcription factor ID2 in the cytoplasm Howeverthe precise mechanisms by which PDLIM5 regulates thefunctions of the nervous system via ID2 blockade requiresfurther elucidationPDLIM5 in the Heart and Skeletal MusclePhysiological Roles of PDLIM5 in the HeartThe heart undergoes development and begins to function in theearly stages of embryonic development PDLIM5 is expressedat high levels in the skeletal muscle and myocardium and isconsidered to be a heart and skeletalmusclespecific scaï¬oldprotein to regulates mouse heart development Mu PDLIM5 is capable of binding to αactinin throughthe PDZ domain and colocalizing in the zdisk region ofcardiomyocytesindicating that this protein plays a role incardiac development Studies have shown that PDLIM5 mRNAsare mainly expressed in the heart and skeletal muscle of adultrats and that PDLIM5 acts as a scaï¬old protein to mediatethe transmission of PKC signals in cardiomyocytes playing animportant role in development of the muscle cell in an earlydevelopmental stage Nakagawa Zheng The PDZLIM protein family is highly expressed in the striatedmuscle PDLIM5 is similar to other PDZLIM members in thestriated muscle in which the PDZ domain binds to αactininwhile the LIM domain binds to several protein kinases C andprotein kinase D Kuroda Nakagawa For example PDLIM5 traditionally activates the PKC throughthe direct binding of its LIM domain Maturana and interacts to PKA Lin Transcription factorCREB which is one of the first transcription factors activated byneurohumoral factors stimulation is a transcription factor cAMPresponse element binding protein is a known target of the PKCand protein kinase D1 PKD1 pathways Thonberg Ozgen The interaction between PDLIM5 isoform and CREB is necessary for the phosphorylation of CREB at theaminoacid residue Ser133 which promotes the transcriptionalactivation and nuclear localization of CREB the phosphorylatedCREB enters the cardiomyocyte nucleus to play the role oftranscription factor and promote the growth of cardiomyocytesIto Moreover in neonatal rat cardiomyocytesPDLIM5 interacts with PKD1 through its LIM domain and formscomplexes with PKD1 and cardiac Ltype voltagegated calciumchannelα1C subunits to regulate the activity of Ltype voltagegated calcium channels Maturana Although theformation of protein complexes such as PDLIM5PKCPKD1is well understood the downstream molecular events remainto be elucidatedThese results suggest that the localization of PDLIM5 at somesubcellular sites and its ability to interact with multiple functionalproteins play an important role in cellular and physiologicalfunctions Furthermore the role of PDLIM5 in the heart wasFrontiers in Physiology wwwfrontiersinAugust Volume 0cHuang et alAn Overview of PDLIM5studied using a heartspecific PDLIM5knockout mouse modelIt was found that the ablation of PDLIM5 disrupted the stabilityof the PDLIM5CiphersCalsarcin complex formed in the zdiskregion thus interfering with the connection between adjacentsarcomeres and extracellular matrix These eï¬ects were found toresult in the loss of optimal force transmission and a significantdecrease in cardiac shortening fractionleading to dilatedcardiomyopathy Cheng Novel polymorphisms inthe PDLIM5 gene encoding the Zline protein have also beenshown to increase the risk of idiopathic dilated cardiomyopathyWang that underpinsPDZ and LIM domain is additionally involved in skeletalmuscle development Myogenesis is an important biologicalprocessskeletal muscle regeneration andpostnatal growth The silencing of PDLIM5 increases the nuclearaccumulation of diï¬erentiation inhibitor Id2 which inhibits theproliferation and diï¬erentiation of myoblasts Nakatani Qiu In addition the diï¬erentiation of andmorphological changes in skeletal muscle is regulated by a groupof transcription factors known as myogenic regulators PDLIM5isoform overexpression leads to the upregulation of MyoDand myogenin while PDLIM5 isoform knockout significantlydecreases the expression of these two proteins these findingsindicate that the main eï¬ect of PDLIM5 isoform on musclecells is to stimulate the transcription of MyoD andor myogeninencoding genes Ito Although the main role of PDLIM5 is as a specific scaï¬oldprotein which to bridge the connection between cytoskeletonand membrane proteins and promote the formation of proteincomplexes it is capable of generating numerous splicing isoformsENH24 that exert various eï¬ects on the development ofheart and skeletal muscle In vitro PDLIM5 isoform hasbeen shown to promote the expression of myogenic genes andmyotube formation while the short PDLIM5 isoform hasbeen found to abrogate myotubelike morphological changeswithout altering the expression of the myogenic transcriptionfactors MyoD and myogenin Ito Furthermorethe overexpression of PDLIM5 isoform prevented ventricularcardiomyocyte hypertrophy induced by vascular stress hormonesYamazaki Western blotting analysis of muscle tissueusing a nonisoformspecific antiPDLIM5 antibody showed thatPDLIM5 isoform is only expressed in skeletal muscle witha specific distribution of PDLIM5 members between skeletalmuscle and myocardium Niederlander These results suggest that PDLIM5 plays a key role in thedevelopment of the myocardium and skeletal muscle Howeverthe signal transduction mechanisms underlying the role ofPDLIM5 in heart and skeletal muscle remain to be furtherstudied For example the specific molecular mechanisms bywhich PDLIM5 regulates the development of myocardium andskeletal muscle through binding protein kinases are unknownFurthermore the mechanisms by which PDLIM5 sequestersnuclear protein Id2 in the cytoplasm remain to be elucidatedThe Role of PDLIM5 in Cardiovascular DiseasesPDZ and LIM domain is mainly distributed on the zline ofthe sarcomere of cardiomyocytes therefore the eï¬ect of PDLIM5on myocytes may be related to the contractile function of thesecells PDLIM5knockout mice exhibit dilated cardiomyopathywhich is characterized by thinning of the left ventricular wallenlargement of the left ventricular cavityimpaired cardiaccontraction and reduced ejection function Cheng PDLIM5 can regulates vascular remodeling which canas a new proatherosclerotic factor to be a therapeuticallytargeted Huang Cardiac remodeling which isindicative of progression in many cardiovascular diseases ischaracterized by cardiomyocyte hypertrophy and myocardialfibrosis which lead to heartfailure Swynghedauw Barry and Townsend microRNA miR21 derivedfrom cardiac fibroblasts exosomes is a strong paracrine RNAmolecule that induces cardiomyocyte hypertrophy Thum Recentlyit has been reported that PDLIM5 is thedirect target of miR17ˆ¼ Bang Chen By acting on its target gene PDLIM5 miR participates in the interaction between cardiac fibroblastsand cardiomyocytesthus inducing myocardial pathologicalhypertrophy Bang PDLIM5 also plays a role invascular smooth muscle AMPactivated protein kinase is anintracellular energy receptor of AMPK which is activatedunder hypoxia ischemia glucose loss and stress Steinberg andKemp AMPK is generally considered to be an energysensor kinase and requires AMP for its activation Carling Nakano reported that AMPK controls microtubuledynamics by phosphorylating cytoplasmic connexin170 CLIPthus regulating cell migration Nakano In addition AMPK is involved in the regulation of actincytoskeleton dynamics and plasma membrane reanizationBae Kondratowicz Studies haveshown that AMPK activation plays an important role inneovascularization and metastasis HoyerHansen and Jaattela In vascular smooth muscle cells AMPK phosphorylatesPDLIM5 at Ser177inhibiting the downstream Rac1Arp23signaling pathway to mediate cell migration Yan In pulmonary artery smooth muscle cells PASMCsSMCspecific knockout of PDLIM5 enhances hypoxiamediatedvascular remodeling while overexpression of PDLIM5 inhibitsthe TGFSmad signal pathway and prevents hypoxiainducedpulmonary hypertension elevation in vivo Cheng In addition PDLIM5 silencing induces the activity of TGF3 TR1 and TGF and increases the overall expressionlevel of Smad2 The suppression of PDLIM5 has beenfound to enhance the nuclear staining of Samd23 Chen indicating that PDLIM5 participates in thedevelopment of cardiovascular disease by negatively regulatingthe TGF3Smad23 signal pathway Additionally demonstratedthat PDLIM5 plays an important role in the cardiovascularsystem through miRmediated regulation of the phenotype ofpulmonary artery smooth muscle cells miR17ˆ¼ regulatesthe diï¬erentiation of PASMCs through its target PDLIM5indicating that the miR17ˆ¼92PDLIM5TGFSmad pathwayis essential for vascular remodeling during the developmentof pulmonary hypertension PDLIM5 therefore represents apromising therapeutic target for future cardiovascular drugdiscovery eï¬ortsFrontiers in Physiology wwwfrontiersinAugust Volume 0cHuang et alAn Overview of PDLIM5The Role of PDLIM5 in TumorAs an actin adaptor protein PDLIM5 is not only involved incytoskeletal tissue cellular processes and an development butis also considered to play roles in tumorigenesis and developmentEeckhoute Edlund Heiliger Li PDLIM5 is expressed in many cancer celllines In a study of neurologic tumor it was found that thetranscription factor ID2 binds to the PDLIM5 LIM domains andin these cancer cells high levels of PDLIM5 sequester ID2 in thecytoplasm preventing neuronal diï¬erentiation and promotingcell proliferation Lasorella and Iavarone PDZ and LIM domain is additionally upregulated inpapillary thyroid carcinoma PTC tissues elevated PDLIM5expression promotes the migration invasion and proliferation ofPTC cells by activating the RASERK pathway Wei PDLIM5 may therefore serve as a therapeutic target in a varietyof cancers It can promote the invasion and metastasis of cancercells Genotyping chip detection experiments have shown thatPDLIM5 is overexpressed in prostate cancer tissue Koutros and some studies have proved that the utility of serumand urine PDLIM5 levels as indicators for auxiliary diagnosisof prostate cancer with potential value in predicting the risk ofprogression in advanced prostate cancer PCA Ma PDLIM5 plays a key role in regulating the proliferation invasionand migration of malignant tumor cells by binding to AMPKand regulating its activation and degradation Liu PDLIM5 may therefore act as an oncogene in the developmentand progression of PCAIn view of the important role of PDLIM5 in cancer somestudies have indicated that it is involved in the growth of gastriccancer cells and suggested that PDLIM5 silencing through theuse of small interfering RNAs siRNA may be a potentialstrategy for the treatment of gastric cancer Li In addition Edlund found that the increased expressionof PDLIM5 is related to high tumor proliferation rates innonsmall cell carcinoma Edlund Steroids suchas corticosteroid medications play an important role in thedevelopment of cancer it has been reported that singlenucleotide polymorphisms SNPs in PDLIM5 interact withsteroids thus aï¬ecting the occurrence and development of cancerWang The above findings show that PDLIM5 plays an important rolein the occurrence and development of cancer and that PDLIM5represents a candidate oncogene in various cancersThe Role of PDLIM5 in Other DiseasesreportedIn addition to playing a role in the diseasesinvolved in the link between alcoholabove PDLIM5 isdependence and diabetes Owusu found thatPDLIM5 gene polymorphism is associated alcoholdependentAD type diabetes T2D and hypertension and Severalgenetic variants of the PDLIM5 gene can aï¬ect AD T2Dand hypertension indicating that PDLIM5 is a shared geneamong the three diseases Therefore elucidation oftheunderlying molecular mechanisms and identification of hithertoundiscovered molecular functions of PDLIM5 are expectedto enable the development of eï¬ective clinicalfor these diseasestherapiesIn addition PDLIM5 plays a role in the formation of cellextensions Being a scaï¬old protein PDLIM5 is involved inpromoting the activity of microfilamentassociated proteinsMicrofilamentassociated proteins play a central role in theprocess of cell extension Lanier Some studieshave found that PDLIM5 recruitment to cell extensions and isnecessary to form these extensions and that PDLIM5 knockoutreduces the assembly of actin filaments in cell extensionsYuda THE RELATIONSHIP BETWEEN PDLIM5AND INTEGRINS AND ITS POTENTIALROLE IN THE REGULATION OF STEMCELL DIFFERENTIATIONSome studies have shown that the PDZ domain of PDZLIMprotein binds to αactinin protein at the adhesion junctionwhich is the site of integrin localization Xia Pomies Vallenius Tamura The functional interaction with integrin indicates that PDZLIM protein can participate in the adhesion signal cascadewhich transmits extracellular signals via intracellular regulatorypathwaysthereby modifying the actin cytoskeleton Thesefindings show that the PDZLIM protein plays an overall role incell“cell and cell“matrix interaction and cell migration Krcmery The regulation of PDZLIM activity plays animportant role in preventing uncontrolled actin recombinationproliferation and cell migration For example PDLIM5 canalso bind to the integrin protein kinase ILK acting as ascaï¬old bridge between renal ion exchanger KAE1 and ILKproviding a bridge between KAE1 and potential microfilamentsSu It has been reported that PDLIM5 is recruited from thecytoplasm to the integrin adhesion and Factin stress fibersand responds to stress by directly binding to the key stressfiber component αactinin Microfilaments control the nuclearand cytoplasmic localization oftranscriptional coactivatorsYAP and TAZ to regulate gene expression and mediate thediï¬erentiation of MSCs Dupont Halder The eï¬ective domains of some proteins that are recruited intothe actin structure in a forcedependent manner through theLIM domain regulate actin signal transduction Smith The action of mechanical force on integrin results in therecruitment of PDLIM5 to activate the YAP pathway duringmechanical transduction Elbediwy PDLIM5 acomponents of the integrin adhesion complex mediates therelationship between integrin and the cytoskeleton Horton et al2016ab Proteomic analysis of integrinrelated complexes fromMSCs has demonstrated the formation of significant amountsof a vinculinpositive adhesion complex on a hard substratecoated with fibronectin FN in MSCs a subset of whichcolocalized with or closely to clusters of PDLIM1 or PDLIM5Ajeian Frontiers in Physiology wwwfrontiersinAugust Volume 0cHuang et alAn Overview of PDLIM5PDZ and LIM domain undergoes tensiondependentrelocalization in cells Both embryonic and induced pluripotentstem cells can diï¬erentiate into derivatives of the third germlayer as a result human pluripotent cells are important toolsin regenerative medicine Thomson Takahashiand Yamanaka Studies have reported that duringcardiogenesis embryonic stem cells exhibit dramatic changes inthe expression of metabolic enzymes and cytoskeleton proteinsKonze in particular Zdisk related proteins withPDZ and LIM domain proteins including PDLIM5 are upregulated during cardiogenesisFurthermorethe expression of NKX25 an importantmyocardial transcription factor results in the generation ofspecific PDLIM5 splicing variants during the early developmentof cardiomyocytes which in turn aï¬ectsthe myogenicdiï¬erentiation of myocardium Konze At presentthere are few studies on PDLIM5 in stem cells elucidation of itsmechanism in diï¬erent stem cell types is warranted to identify itsfunctions in these cellsPROBLEMS AND PERSPECTIVESIn this we briefly reviewed the known functionsof the PDLIM5 protein the progress in elucidation ofitsroles in various cellular and physiological processes and thesignaling pathways in which it participates As a member ofthe PDZLIM protein family PDLIM5 is involved in actinbinding αactinin binding protein kinase binding acts asa scaï¬old bridge between connective proteins and plays anindispensable role in various cellular processes However so farthe specific molecular mechanisms underlying the functions ofPDLIM5 remain unclearFuture research directions in investigation of PDLIM5 shouldseek to answer the following questionsFirst research on PDLIM5 has mainly focused on its roles intumor the nervous system and the cardiovascular system As amicrofilamentassociated protein does PDLIM5 play the samerole in other physiological systems or cell lines Is the PDLIM5gene expressed in multiple systemsREFERENCESAjeian J N Horton E R Astudillo P Byron A Askari J A and MillonFrmillon A Proteomic analysis of integrinassociated complexes frommesenchymal stem cells Proteomics Clin Appl “ 101002prcaBach I The LIM domain regulation by association Mech Dev “ 101016S0925477399003147Bae H B Zmijewski J W Deshane J S Tadie J M Chaplin D D andTakashima S AMPactivated protein kinase enhances the phagocyticability of macrophages and neutrophils FASEB J “ fj11190587BagheriYarmand R Mazumdar A Sahin A A and Kumar R LIMkinase increases tumor metastasis of human breast cancer cellsvia regulationof the urokinasetype plasminogen activator system Int J Cancer “ 101002ijc21650Bang C Batkai S Dangwal S Gupta S K Foinquinos A and HolzmannA Cardiac fibroblastderived microRNA passenger strandenrichedSecond although the role of PDLIM5 in diseases hasbeen studied eg its expression is upregulated during tumordevelopment the specific mechanisms by which it exerts theseeï¬ects are unknown and further elucidation of the underlyingmechanisms and other functions is warrantedThird PDLIM5 has four splicing isoforms which performdiï¬erent functions what are the mechanisms by which they playdiï¬erent roles Are these diï¬erences in their roles attributable tothe presence or absence of LIM domains Additionally what isthe mechanism by which they play diï¬erent rolesFourth in regard to strategies used for the inhibition ofPDLIM5 expression only viral transfection has been reportedto date no pharmaceutical compounds that inhibit PDLIM5expression have been identified Therefore additional research onPDLIM5 inhibitors is also criticalFinally although PDLIM5 plays a crucial role in binding actinand has attracted much attention as a connecting protein thereare few studies on its eï¬ects on the actin cytoskeleton or othercytoskeleton such as binding to cytoskeletonrelated proteinsbridging the connection with the cytoskeleton Does PDLIM5aï¬ect the shape and location of the cytoskeleton in the processof participating in the biological functions of cellTheeï¬ects of PDLIM5mediated modulation ofthecytoskeleton on cell diï¬erentiation proliferation and othercellular functions remain to be explored in detail in future studiesAUTHOR CONTRIBUTIONSAll authors participated in the conception and writing of themanuscript SZ JO and JD reviewed and suggested modificationsto the content JD designed the structure of the reviewFUNDINGThis work wassupported by the National Key RDProgram of China grant number 2017YFC1105000 andthe Sanming Project of Medicinein Shenzhen grantnumber SZSM201612019exosomes mediate cardiomyocyte hypertrophy J Clin Invest “ 101172JCI70577Barry S P and Townsend P A What causes a broken heart“molecularinsights into heart failure Int Rev Cell Mol Biol “ S1937644810840031Carling D Mayer F V Sanders M J and Gamblin S J AMPactivatedprotein kinase nature™s energy sensor Nat Chem Biol “ nchembio610Chen T HuangJ B DaiJ Zhou Q RajJ U and Zhou Gthe miR17ˆ¼ signaling that PAI1 is a novel component ofregulates pulmonary artery smooth muscle cell phenotypes Am J PhysiolLung Cell Mol Physiol L149“L161 101152ajplung00137Chen T Zhou G Zhou Q Tang H Ibe J C and Cheng H Loss of microRNA17 approximately in smooth muscle cells attenuatesexperimental pulmonary hypertension via induction of PDZ and LIM domain Am J Respir Crit Care Med “ 101164rccm2014050941OCFrontiers in Physiology wwwfrontiersinAugust Volume 0cHuang et alAn Overview of PDLIM5Cheng H Chen T Tor M Park D Zhou Q and Huang J B A highthroughput screening platform targeting PDLIM5 for pulmonary hypertensionJ Biomol Screen “ Cheng H Kimura K Peter A K Cui L Ouyang K and Shen T Lossof enigma homolog protein results in dilated cardiomyopathy Circ Res “ 101161CIRCRESAHA110218735Dawid I B Breen J J and Toyama R LIM domains multiple roles asadapters and functional modifiers in protein interactions Trends Genet “ 101016s0168952598014243Dupont S Morsut L Aragona M Enzo E Giulitti S and Cordenonsi M Role of YAPTAZ in mechanotransduction Nature “ 101038nature10137Edlund K Lindskog C Saito A Berglund A Pontn F and G¶ranssonKultima H CD99 is a novel prognostic stromal marker in nonsmallcell lung cancer Int J Cancer “ 101002ijc27518Eeckhoute J A celltypespecific transcriptional network required forestrogen regulation of cyclin D1 and cell cycle progression in breast cancerGene Dev “ 101101gad1446006Elbediwy A Vanyai H DiazdelaLoza M Frith D Snijders A P andThompson B J Enigma proteins regulate YAP mechanotransductionJ Cell Sci 131jcs221788 101242jcs221788Fanning A S and Anderson J M PDZ domains fundamental buildingblocks in the anization of protein compl Answer:

Thyroid_Cancer

IFNBased Biotherapeutics toHarness the Host AgainstFootAndMouth DiseaseGisselle N Medina Teresa de los Santos and Fayna DiazSan Segundo Plum Island Animal Disease Center PIADC ARS USDA Orient Point NY United States Kansas State University Collegeof Veterinary Medicine Manhattan KS United StatesFootandmouth disease FMD is a highly contagious vesicular disease of clovenhoofedanimals that severely constrains international trade of livestock and animal productsCurrently disease control measures include broad surveillance enforcement of sanitarypolicy and use of an inactivated vaccine While use of these measures has contributedto eliminating footandmouth disease virus FMDV from a vast area of the worldthe disease remains endemic in three continents and outbreaks occasionally appearin previously declared FMDfree zones causing economic and social devastationAmong others a very fast rate of viral replication and the need for days to achievevaccineinduced protection are the main limitations in controlling the disease Newfastacting antiviral strategies targeted to boost the innate immunity of the host to blockviral replication are needed Here we review the knowledge on the multiple strategiesFMDV has evolved to block the host innate immunity with particularly focus on the pastand current research toward the development of interferon IFNbased biotherapeuticsin relevant livestock speciesKeywords footandmouth disease virus FMDVIFNλ IFNωinterferon IFN antivirals biotherapeutics IFNα IFNÎINTRODUCTIONThe Disease FootAndMouth DiseaseFootandmouth disease FMD is one the most serious livestock diseases that aï¬ects clovenhoofedanimals including cattle swine sheep and goats as well as numerous species of wild species The disease displays high morbidity but is usually not lethal except when it aï¬ects young animalsthat may develop myocarditis Infected animals secrete copious amounts of virus ps beforethe onset of the clinical phase of the disease Typical FMD clinical signs include fever and theappearance of vesicular lesions on the tongue mouth feet and teats Among ruminants thatrecovered from the disease a relatively large number become asymptomatic virus carriers although it is not clear what is the contribution of these carrier animals to disease transmissionin nature The World anization for Animal Health OIE lists FMD as a reportable diseaseand therefore by law participating nations are required to inform the anization about all FMDoutbreaks OIE member nations with reported cases of FMD are forbidden to engage in tradingof FMDsusceptible animals or their products Thus the presence of FMD in a country can havesevere economic consequencesDiï¬erent interventions to control an FMD outbreak include restriction of susceptible animalmovement slaughter of infectedcontact animals decontamination of infected and surroundingEdited byMariano PrezFilgueiraNational Agricultural TechnologyInstitute ArgentinaReviewed byMargarita S¡izSevero Ochoa Molecular BiologyCenter CSICUAM SpainKenneth James GenoveseAgricultural Research ServiceUnited States Department ofAgriculture United StatesCorrespondenceGisselle N MedinagissellemedinausdagovTeresa de los SantosteresadelossantosusdagovFayna DiazSan SegundofaynadiazsansegundousdagovSpecialty sectionThis was submitted toVeterinary Infectious Diseasesa section of the journalFrontiers in Veterinary ScienceReceived April Accepted June Published August CitationMedina GN de los Santos T andDiazSan Segundo F Use ofIFNBased Biotherapeutics to Harnessthe Host Against FootAndMouthDisease Front Vet Sci 103389fvets202000465Frontiers in Veterinary Science wwwfrontiersinAugust Volume 0cMedina et alIFN Against FMDVpremises and vaccination Vaccination is an option used mostlyin countries in which FMD is endemic but diseasefree nationsprefer to abstain from such practice In general FMDfreecountries that occasionally opted to vaccinate to better containthe outbreak did slaughter all vaccinated animals to regaincommerce rights faster as occurred in the outbreak inthe UK and the Netherlands The current approvedFMD vaccine consists of purified chemically inactivated virus[binary ethylenimine BEItreated] formulated with oilbased oraluminum adjuvants that induces serotypespecific protection inapproximately days and it is applied with a boosting protocolfor ensuring longterm protection While this vaccine hasbeen successfully used for many decades leading to diseaseeradication of a vast area of our planet challenges remainFMD is endemic in most of Africa and Asia and occasionallyepizootics appear in South America or in nations that havebeen diseasefree for many years as it happened in the UKthe Netherlands South Korea Taiwan and Japan Novelvaccine technologies have been developed but to this end noneof them has fully addressed the limitations of the commerciallyavailable vaccine or is currently approved for massive use Alternatives or additional therapeutics that could complementor in some instances substitute for vaccination protocols includethe use of antivirals and biotherapeutics that act quickly priorto induction of vaccineinduced immunity The development ofsuch molecules requires a thorough understanding of the biologyof the virus and its intricate interactions particularly with theinnate immune molecular and cellular mechanisms evolved bythe hostThe AgentFootandmouth disease virus FMDV is a member of theAphthovirus genus within the Picornaviridae family and it is theetiologic agent of FMD The virus contains a singlestrandedRNA of positive polarity Its genome of ˆ¼ nucleotidesconsists of a long reading frame ORF flanked by a ² anda ²untranslated region UTR The ORF encodes a polyproteinof about amino acids which is processed by virusencodedproteases Processing results in the generation of precursors andmature protein products including four structural [1A VP4 1BVP2 1C VP3 1D VP1] and ten nonstructural NS proteins[Lpro 2A 2B 2C 3A three distinct copies of 3B VPg 3Cproand 3Dpol] Due to high genetic variability FMDV is categorizedin seven distinct serotypes A Asia1 C O and Southern AfricanTerritories “ SAT “ and numerous subtypes or topotypesUpon infection the virus spreads very rapidly usually achieving morbidity Depending on the route of entry less than tissue culture infectious doses are required to infect andcause disease in animals In fact FMDV is one of thefastest replicating RNA viruses in nature taking as little as “ h to induce cytopathic eï¬ects in susceptible tissue culture cellsOne could envisage that during FMDV replication almost everycomponent of the virus must play a role in dampening interferingcellular responses to allow such rapid virus replicationInnate Immunity and Interferon ActivationEarly protection against viralfundamentallymediated by the action of interferons IFNs the pillar moleculesof the innate immune system “ Expression of IFN isinfection istriggered by the recognition of molecular signatures collectivelynamed pathogenassociated molecular patterns PAMPs viacellular receptors pattern recognition receptors PRRs that candistinguish œself from nonself  molecules Figure Bindingof PAMPs to PRRs triggers a series of signal transduction eventsand posttranslational modifications PTMs phosphorylationubiquitination ISGylation etc that ultimately activate latenttranscription factors to induce IFN transcription Subsequentlysecreted IFN proteins bind to specific receptors on the plasmamembrane to activate in an autocrine and paracrine mannerdiscrete and overlapping cellular signal transduction pathwaysDepending on the cell type and aï¬ected tissue over specificIFNstimulated genes ISGs may be induced many of whichdisplay antiviral activity to control the viral infection There are three families of IFNs based on the specific receptorusage types I II and III Table “ Type I IFNsie IFNα and IFN signal through a heterodimeric receptorcomplex formed by IFNAR1IFNAR2 type II IFN IFNÎsignals through the complex IFNÎR1IFNÎR2 and type IIIIFNs bind the receptor complex IL28RαIL10R Despitethe receptor diï¬erencesthe three IFN families transducesignals through the Janus kinase JAK“signal transducer andactivator of transcription STAT pathway and type I and typeIII IFNs induce redundant responses Figure Overall therapid production of IFN helps to limit viral replication whilemodulating other immune functionsFOOTANDMOUTH DISEASE VIRUSIMPAIRS INNATE IMMUNITY MOLECULARINTERACTIONSRecognition of FMDV RNA by the host cell results in theestablishment of a rapid antiviral state to limit and controlinfection This selective pressure has allowed FMDV to evolvemany strategiesto ensure enhanced virulence and rapidinfectivity In general RNA viruses can bypass the IFN responseby blocking i global cellular transcription and translationii IFN induction and iii IFN signaling Similarly to otherRNA viruses FMDV can also target IFNindependent antiviralresponses mostly associated with cellular metabolic functionsie autophagy apoptosis stress granule formation etc thathave been extensively described elsewhere In thissection we will summarize the current literature on studiesconducted in vitro that explain how FMDV counteracts the hostinnate immune response at the molecular level including RNAsensing activation of adaptoreï¬ector proteins and regulation ofsignaling pathways by specific PTMsBlock on Cellular Transcription andTranslationFMDV inhibition of cellular gene expression and proteinsynthesis during infection is mainly driven by the viralencoded proteases Leader Lpro and 3C FMDV Lpro isa papainlike protease PLP thatthetranslation initiation factor eIF4G including eIF4GI and eIF4GII to disable capdependent protein synthesis AlsoFMDV Lpro causes degradation of the transcription factorinduces cleavage ofFrontiers in Veterinary Science wwwfrontiersinAugust Volume 0cMedina et alIFN Against FMDVFIGURE Antiviral signaling pathways induced during viral infection Cellular detection of microbial molecules known as pathogenassociated molecular patternsPAMPs ie viral RNA is mediated by pattern recognition receptors PRRs including cytosolic RNA sensors ie RIGI MDA5 or LGP2 andor membraneboundTLRs PAMPPRR interaction activates signal transduction cascades black arrows that result in the production of IFN and inflammatory cytokines RIGI and MDA5contain two caspase recruitment domains CARD and an RNA helicase domain In the case of RIGI ubiquitination green circles is required for its effective activationActivated signals from either RIGI or MDA5 are transmitted downstream via the mitochondrial adaptor MAVS resulting in the formation of MAVS filaments At thisstage different PTMs such as ubiquitination or ISGylation black circles can regulate their functions Endosomal RNAs are detected by TLR3 or TLR78 which signalthrough adaptor proteins TRIF and MyD88 respectively MyD88 uses other adaptors IRAK14 to allow for interaction with TRAF proteins In addition to their role asadaptor proteins TRAFs also serve as E3 ubiquitin Ub ligases to regulate signaling TRAFmediated induction of polyUb is sensed by NEMO thus recruitingdownstream effector kinases such as TBK1 or IKK These proteins form different signaling complexes ie NEMOTBK1 and NEMOIKK leading to phosphorylationblue arrows of transcription factors IRF37 to a lesser extent IRF1 and IRF5 are also phosphorylated IRF phosphorylation triggers dimerization and translocationContinuedFrontiers in Veterinary Science wwwfrontiersinAugust Volume 0cMedina et alIFN Against FMDVFIGURE orange arrows to the nucleus where they bind mainly to IFN promotersenhancers Alongside with this pathway TRAF6E3 ligases can activate MAPK3and other kinases including ERK12 and JNK which phosphorylate the components of the AP1 heterodimer allowing for translocation to the nucleus and binding tothe IFN promoterenhancer to activate transcription Activated IKK also phosphorylates IκB releasing NFκB which then translocates to the nucleus and binds at theIFN promoter AP1 activating protein CARD caspase activation and recruitment domain DUB deubiquitinase ER endoplasmic reticulum IκB inhibitor of KBkinases IKK IκB kinase IL interleukin IRAK interleukin1 receptorassociated kinase IRF IFN regulatory factor LGP2 laboratory of genetics protein MAPKmitogenactivated protein kinase MAVS mitochondrial antiviral signaling protein MDA5 melanoma differentiationassociated gene MyD88 myeloid differentiationprimary response protein 88d NEMO NFκB essential modulator NFκB nuclear factorκB PKR protein kinase R PTM posttranslational modification RIGIretinoic acidinducible gene I TANK TRAF family memberassociated NFκB activator TBK TANK binding kinase TLR Tolllike receptor TRAF TNF receptorassociated factor TRIF TIRdomaincontaining adapterinducing interferonTABLE Use of IFNbased therapies against FMDVTypeReceptSignalSubtypeSpeciesMilestoneType IIFNAR1IFNAR2JAK1 TYK2IFNαPorcinebovine ¢ Recombinant bacterial expressed IFNα is a potent biotherapeutic againstIFNαPorcine¢ Ad5 delivered poIFNα protects swine against different serotypes of FMDVFMDV in vitro IFNIFNδIFNω7IFNαωIFNτType IIIFNÎ R1 IFNÎ R2JAK1 JAK2IFNÎType IIIIFNλR1IL10R2JAK2 TYK2IFNλ1IFNΓ¢ poIFNαprotection correlates with enhanced tissuespecific innate immune cellinfiltration in swine ¢ poIFNα protection correlates with upregulation of essential ISGs in vitro ¢ Ad5 delivered porcine poIFN protects swine against FMDV ¢ Bacterially expressed poIFNδ8 significantly inhibits FMDV replication in vitro ¢ E coli produced poIFNω7 protects cells against FMDV ¢ Bacterially expressed IFNαω added prior to infection resulted in a significantreduction in FMDV replication in vitro ¢ Ovine IFNτ has antiviral effect against FMDV in vitro ¢ Recombinant bovine IFNÎ reduced FMDV replication in BTY cell culture ¢ High dose of Ad5poIFNÎ protects swine against FMD ¢ Replication of FMDV in IBRS2 cells is inhibited by treatment with the purifiedrecombinant poIFNλ1 PorcinePorcinePorcinePorcineOvineBovinePorcinePorcineIFN CombosOtherIFNvaccinecombosIFNλ3Bovine¢ Inoculation with Ad5boIFNλ3 resulted in the induction of several ISGs in tissuesof the upper respiratory tract and protected cattle against challenge withFMDV PorcinePorcineIFNαIFN΢ Ad5poIFNλ3 protects swine against challenge with FMDV ¢ Use of a combination of Ad5poIFNÎ and Ad5poIFNα or Ad5poIFNαΠshowed an enhancement of the antiviral activity against FMDV in swinePoly ICPorcine¢ Double stranded ds RNA poly ICLC in combination with Ad5poIFNαprotected swine against FMDV siRNAPorcine¢ Combination of Ad5poIFNαΠwith Ad3siRNA targeting FMDV NS codingregions blocked replication of all serotypes of FMDV in vitro IRF73Porcine¢ Inoculation with Ad5IRF735D resulted in induction of IFNα and fully protectedmice and swine challenged with FMDV day after treatment IRESPorcine¢ Use of synthetic IRES in combination with adjuvanted typeO FMD improvedimmune response and protection against FMDV challenge IFNαPorcine¢ Use of a combination of Ad5poIFNα and Ad5A24 in swine resulted incomplete protection after challenge IFNαÎPorcine¢ Ad5poIFNαΠcoadministered with Ad5siRNA targeting NS regions of FMDVand a commercial inactivated FMD vaccine partially protected swine IFNλ3Bovine¢ Use of a combination of Ad5bovIFNÎ3 and AdtO1M in cattle resulted incomplete protection after aerosol challenge nuclear factor NFκB and results in blockage of specificdownstream signaling eï¬ectors Studies in porcine cellsdemonstrated that FMDV Lpro can promote its selfbindingto the transcription factor activitydependent neuroprotectiveprotein ADNP and negatively regulate the activity of the IFNα promoter In contrast chromatin changes that favor theupregulation of IFN and ISGs can inhibit FMDV replication Interestingly deletion or mutations in diï¬erent domainsof Lpro result in viral attenuation in vitro and in vivo “ Furthermore these studies have shown a strong type I IFNactivity upon infection with diï¬erent versions of FMDV Lpromutants Frontiers in Veterinary Science wwwfrontiersinAugust Volume 0cMedina et alIFN Against FMDVFIGURE Type I II and III interferon IFNmediated signaling All type I and type III IFN subtypes bind to respective receptors IFNAR1IFNAR2 and IFNLR1IL10R2These interactions trigger the phosphorylation of JAK1 and TYK2 kinases which in turn phosphorylate STAT1 and STAT2 JAK2 mediates type III IFNdependent STATphosphorylation Phosphorylated heterodimers of STAT1STAT2 bind to IRF9 forming the ISGF3G complex which then translocates to the nucleus and binds toIFNresponsive elements ISREs present in the promoters of over ISGs Type II IFN binds to the heterodimeric IFNÎR1IFNÎR2 receptor also inducingphosphorylation of JAK1JAK2 kinases In turn mostly STAT1 is phosphorylated Phosphorylated homodimers of STAT1 translocate to the nucleus and induce theexpression of genes controlled by gammaactivated sequence GASdependent promoter sequences IFNAR12 IFN alpha receptor12 IFNÎR12 IFNgammareceptor12 IFNALR1 IFNlambda receptor IL10R2 IL10 receptor ISGs IFNstimulated genes ISGF3G ISG factor gamma JAK12 Janus kinase STATsignal transducer and activator of transcriptionInterruption of cellular translation during infection can alsobe mediated by FMDV 3Cpro a chymotrypsinlike cysteineprotease that similarly to Lpro targets eIF4G and the capbindingcomplex eIF4A for cleavage although these events occur later inthe infection 3Cpro can also participate in the inhibitionof host“cell transcription by cleaving histone H3 upon FMDVinfection Block on Interferon InductionDuring infection the initial event that leads to the productionof IFN and proinflammatory cytokines is the recognition ofviral RNA Figure Sensing of FMDVRNA is mediated byMDA5 a protein that belongs to a family of helicasesknown as retinoic acidinducible geneI RIGIlike receptorsRLRs Recent studies have shown that the interaction betweenRLRs RIGI and LGP2 and the FMDV proteins Lpro 2Band 3A interferes with the induction of type I IFN “Indeed overexpression of either FMDV 2B or 3A resulted in thedownregulation of RIGI and MDA5 mRNA expression In contrast upregulation of LGP2 transcripts has been observedduring FMDV infection in porcine cells despite a detectablereduction of LGP2 protein levels presumably due to FMDVFrontiers in Veterinary Science wwwfrontiersinAugust Volume 0cMedina et alIFN Against FMDVLproinduced cleavage The apparent inconsistencybetween the levels of LGP2 mRNA and protein during FMDVinfection may be explained by LGP2™s ability to serve as a positiveand negative regulator of RIGI and MDA5 signaling presumablyaï¬ecting multiple steps of the IFN induction pathway Inaddition to RLRs nucleotidebinding oligomerization domainNODlike receptors NLRs NOD1 and NOD2 also participatein the recognition of RNA A study by Liu et al describedthe association of NOD2 with FMDV 2B 2C and 3Cpro to blockinnate immunity activation Protein kinase R PKR is anotherrecognized PRR that acts as an RNA sensor Binding ofRNA to PKR induces a conformational change that leads toautophosphorylation and activation The primary target ofactivated PKR is the eukaryotic initiation factor α subuniteIF2α whose phosphorylation results in the blockage of cellularprotein synthesis a relatively common process during viralinfection Although no direct interaction between FMDVRNA and PKR has been demonstrated it has been reportedthat PKR activity modulates FMDV infectivity In fact in tissueculture experiments depletion of endogenous levels of PKR usingsiRNA resulted in increased FMDV titers Furthermoreit has been recently shown that overexpression of autophagyrelated ATG5ATG12 proteins induces transcription of PKR andsubsequent reduction of FMDV replication These resultssuggest that PKR has a complex role as an RNA sensor but also asan antiviral agent during FMDV infectionIt has been demonstrated that FMDV also targets DExDHbox RNA helicases formally accepted as PRRs and modulatorsof the antiviral signaling pathway In vitro experimentsintending to analyze protein“protein interactions revealed theassociation between the RNA helicase DDX1 and FMDV 3D Interestingly these studies indicated that during FMDVinfection in porcine cells cleavage of DDX1 was detected whileoverexpression of DDX1 resulted in the upregulation of IFNand other ISG mRNAs which correlated with virus inhibition Other DExDHbox RNA helicases such as RNA helicaseH RHA are hijacked during FMDV infection and interact withFMDV ™UTR 2C and 3A to facilitate virus replication Signaling pathways downstream from RNA sensing involvethe activation of diï¬erent adaptor and eï¬ector proteins Oneof the pathways that lead to signal activation requires theformation of specific complexes such as NFκB essentialmodulator NEMO and the kinase IKK which bridges theactivation of NFκB and IFN regulatory factor IRF signalingpathways It has been demonstrated that FMDV 3Cpro interactswith NEMO and induces its cleavage resulting in impairedinnate immune signaling IRFmediated signals driven byIRF3 and IRF7 can also be targeted by FMDV proteinsSpecifically overexpression of Lpro in PK15 cells resulted inthe downregulation of IRF3 and IRF7 protein levels andinactivation of IFN and IFNλ1 promoter Other factors involved in the activation of IFN includeconventional PTMs such as phosphorylation and ubiquitinationwhich ensure eï¬ective regulation of these signaling pathways Also diï¬erent cellular deubiquitinases DUBs can reverseubiquitination to control the intensity of the immune signalingresponse Interestingly it has been shown that FMDV Lprocan remove ubiquitin Ub molecules from several proteinsrequired for IFN mRNA expression and those involved in theactivationrepression of the IFN loop This role became moreevident by the observation that during infection FMDV Lprocan cleave cellular substrates modified with the Ublike moleculeISG15 Furthermore mutation of Lpro thatimpairsdeISGylaseDUB function results in viral attenuation Inthis regard identification of FMDV targets for deubiquitinationand deISGylation may contribute to elucidate the role ofthose factors in counteracting the innate response and developnovel countermeasuresBlock on Interferon SignalingThe ligandmediated association of the specific IFN receptorspromotes a signaling cascade that results in the phosphorylationof the receptor by the action of JAKs These events result inthe generation of docking sites for downstream adaptor andeï¬ector proteins including signaltransducer and activatoroftranscription STAT proteins that associate with otherfactors and translocate to the nucleus inducing transcriptionof a plethora of ISGs described above and in Figure Although blockage of the JAK“STAT signaling pathway hasnot been reported during FMDV infection overexpressionof either FMDV 3Cpro or VP3 can inhibit this response Forinstance IFNtreated HeLa cells overexpressing FMDV 3Cprosuppressed IFNstimulated promoter activities and inducedproteasome and caspaseindependent protein degradationof karyopherin α1 KPNA1 the nuclear localization signalreceptor Thisinteraction inhibited the nuclear translocation of STAT1STAT2impeding maximal ISG promoter activity In another studyin HEK293T cells overexpression of VP3 followed by coimmunoprecipitation revealed the association between VP3 andJAK1 FMDV VP3 also inhibited virustriggered activation of theIFN promoter leading to the decrease in transcription of ISGspresumably due to lysosomalinduced degradation of JAK1 A yeast twohybrid screen identified FMDV 2C in complex withNmyc and STAT interactor Nmi a protein known to augmentimmune function dependent on STATmediated transcriptionInterestingly such interaction resulted in the recruitment ofNmi to vesicular compartments followed by the induction ofapoptosis in BHK21 cells tyrosinephosphorylated STAT1forEvidently FMDV proteins can also target crosstalk pathwaysinduced by JAKSTAT signaling and due to this versatilityunderstanding of these signaling events during FMDV infectionis challengingFOOTANDMOUTH DISEASE VIRUSIMPAIRS INTERFERONMEDIATEDCELLULAR INNATE IMMUNE RESPONSESSimilarly to what happens in vitro FMDV manipulates theearly innate immune response in vivo to ensure a windowof opportunity that favors viral replication and spread beforeFrontiers in Veterinary Science wwwfrontiersinAugust Volume 0cMedina et alIFN Against FMDVthe onset of eï¬ective adaptive immunity required for virusclearance During infection FMDV interacts with a range of hostcells including natural killer NK cells dendritic cells DCsmonocytesMφ and Îδ T cells All these cells play an importantrole in innate immune responses that trigger the productionof large quantities of IFN and other cytokines which serve asautocrine agents “Shortly after FMDV infection in swinethe number ofcirculating NK cells transiently decreases and the remaining NKcells show a dysfunctional lytic activity against target cells and areduction of IFNÎ production In parallel FMDV blocks theability of porcine DCs to mature into conventional DCs cDCs dampening their response against Tolllike receptor TLRligands Another subset of porcine DCs plasmacytoid DCspDCs also referred to as the major professional systemic IFNαproducers are also aï¬ected by FMDV During infectionpartial depletion of pDCs in the peripheral blood has beendetected and the remaining pDCs are less capable of producingIFNα in response to ex vivo stimulation by TLR ligands or virus Similar to pDCs FMDV infection reduces the productionof IFNα on Langerhans cells LCs a distinct subset oftissueresident DCs of the skin It has also been suggestedthat porcine Îδ T cells and Mφ can serve as targets for FMDVinfection in swine although the interplay betweenthese cells and FMDV remains unclearComparably to swine FMDV infection in cattle triggersseveral early events in the innate immune system althoughthe eï¬ects are not exactly the same For instance bovine NKcells originated from FMDVinfected cows have an elevatedcytotoxic function against bovine target cells in vitro In addition some subsets of cDCs are significantly decreasedduring the peak of viremia while the expression of majorhistocompatibility complex MHC class II molecules on allbovine cDCs is reduced and the processing of exogenous antigenis impaired Furthermore during FMDV infection thenumber of systemic mature bovine pDCs characterized bythe expression of CD4 and MHC class II is increasedpresumably to intensify a humoral response and T cell activationwhile levels ofimmature CD4 MHC class IIpDCs aredeclined Examination of bovine Îδ T cells revealed thatthese cells with the surface expression marker WC1 showa transient activated phenotype and increased expression ofIFNÎ FMDV also aï¬ects the innate immune response at the cytokinelevel in the natural host In vivo cytokine profile analysis duringthe clinical phase of disease shows a systemic decrease of proinflammatory cytokines [IL1 IL6 and tumor necrosis factorTNFα] and an increase of the antiinflammatory cytokine IL and IFNα Most likely these changesare related to the early T cell unresponsiveness and lymph iadescribed in swine and cattle during FMDV infection Interestingly a significant induction of inflammatoryand antiviral factors at the local level is detected in cattle in sitesof abundant viral amplification such as the nasaloropharynx orvesicular lesions “ A consistent upregulation of IFNα Î and λ mRNA in distinct microanatomical compartmentsof the nasopharyngeal mucosa concurrent with occurrence ofviremia has also been detected in cattle In contrast studiesin swine demonstrated that IFN expression in infected swineskin is inhibited These diï¬erences may be due to theanalysis of follicleassociated epithelium of the nasopharyngealmucosa in cattle vs skin in swine or to the specific samplingtechnique used in each experiment While in the cattle studylasercapture microscopy was used to focus only in areas of highFMDV replication in the swine study RNA was extracted froma piece of skin without discriminating between microanatomicalcompartments Evidently more studies are needed to elucidatethe intricate interactions between FMDV and the innate immunesystem of specific animal hostsEFFECTIVE USE OF INTERFERONAGAINST FOOTANDMOUTH DISEASEVIRUS IN VITROType I InterferonThe role of IFN in controlling FMDV replication was firstproposed in when Dinter and Philipson demonstrated thatcalf kidney cells exposed to FMDV could become persistentlyinfected and proposed this was a consequence of the inductionof an IFNlike inhibitor present in the supernatant of infectedcells Later studies also suggested that swine leukocytestreated with phytohemagglutinin produced an inhibitor ofFMDV replication with properties similar to IFN It wasnot until that new studies demonstrated that the abilityof FMDV to form plaques in cell culture correlated with thesuppression of type I IFN α protein expression Theseresults were further supported by detection of IFN protein andantiviral activity in the supernatants of primary porcine ovineand bovine kidney cells infected with an attenuated FMDVmutant leaderless as compared to the supernatants of cellsinfected with wildtype WT virus Later studies by the samegroup provided proof of concept on the use of recombinantbacterial expressed IFNα as a potent biotherapeutic againstFMDV This approach was further developed by deliveringrecombinant porcine IFNα using a replicationdefectivehuman Adenovirus vector Ad5poIFNα Infection ofIBRS2 cells with Ad5poIFNα resulted in secreted poIFNα IFN protein detected as early as h postinfection hpiand lasting for at least h Most important expressed IFNprotein displayed strong biological antiviral activity againstFMDV Followup studies by the same group showed that allFMDV serotypes are very sensitive to Ad5delivered poIFNαand sterile protection could be achieved in vivo highlightingthe potential of this approach for the development into abroad biotherapeutic strategy to control FMDV replicationIn the last years advancements is genomics have ledto the characterization of almost all type I IFN subtypes inthe porcine and bovine genome “ which are morenumerous than those identified in primates and mice This hasrevealed diï¬erent functional genes and pseudogenes with diverseexpression profiles and antiviralfunctions against diï¬erentviruses mostly in swine In fact a recent studyFrontiers in Veterinary Science wwwfrontiersinAugust Volume 0cMedina et alIFN Against FMDVdemonstrated that poIFNω7 known for its ability to inducethe highest levels of antiviral activity when compared to otherpoIFNω subtypes elicits an antiviral state against FMDV inIBRS2 cells treated with the recombinant form of poIFNω7produced in Escherichia coli Other subclasses of type IIFN known to be produced in swine and cattle include IFNalphaomega IFNαω also known as IFNµ and IFN delta IFNδ Significant reduction in FMDV replication has been observedupon treatment of porcine cells with bacterially expressed IFNαω or IFNδ8 prior to viral infection Recently another member of type I IFN family IFNτ whichis only produced in ruminants has been evaluated as an antiviralagainst FMDV IFNτ is a paracrine reproductive hormonesecreted constitutively by trophoblasts and endometrial cells toincrease the life span of the corpus luteum however productionis not induced upon viral infection While its secretionis restricted to ruminantsit has a broadspectrum activityagainst various crossspecies viruses Interestingly IFNτ has homology with the amino acids of IFNα which allows forbinding to type I IFN receptors The property of IFNτ thatmakes it an interesting therapeutic candidate for the treatmentof various viral diseases is its significantly lower toxicity ascompared to other type I IFNsType II InterferonIn contrast to type I IFN the type II IFN family is composedof only one member IFNÎ which exerts its actions througha specific receptor IFNGR1IFNGR2 IFNÎ is weakly resistantto heat and acid and it is able to activate leukocytes suchas macrophages and granulocytes also exerting regulatoryfunctions on T and B lymphocytes Indeed productionof IFNÎ is used as a tool to measure cellmediated immuneresponses against FMDV in vaccinated cattle “ andin swine Interestingly IFNÎ responses as measured b

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You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: IFNBased Biotherapeutics toHarness the Host AgainstFootAndMouth DiseaseGisselle N Medina Teresa de los Santos and Fayna DiazSan Segundo Plum Island Animal Disease Center PIADC ARS USDA Orient Point NY United States Kansas State University Collegeof Veterinary Medicine Manhattan KS United StatesFootandmouth disease FMD is a highly contagious vesicular disease of clovenhoofedanimals that severely constrains international trade of livestock and animal productsCurrently disease control measures include broad surveillance enforcement of sanitarypolicy and use of an inactivated vaccine While use of these measures has contributedto eliminating footandmouth disease virus FMDV from a vast area of the worldthe disease remains endemic in three continents and outbreaks occasionally appearin previously declared FMDfree zones causing economic and social devastationAmong others a very fast rate of viral replication and the need for days to achievevaccineinduced protection are the main limitations in controlling the disease Newfastacting antiviral strategies targeted to boost the innate immunity of the host to blockviral replication are needed Here we review the knowledge on the multiple strategiesFMDV has evolved to block the host innate immunity with particularly focus on the pastand current research toward the development of interferon IFNbased biotherapeuticsin relevant livestock speciesKeywords footandmouth disease virus FMDVIFNλ IFNωinterferon IFN antivirals biotherapeutics IFNα IFNÎINTRODUCTIONThe Disease FootAndMouth DiseaseFootandmouth disease FMD is one the most serious livestock diseases that aï¬ects clovenhoofedanimals including cattle swine sheep and goats as well as numerous species of wild species The disease displays high morbidity but is usually not lethal except when it aï¬ects young animalsthat may develop myocarditis Infected animals secrete copious amounts of virus ps beforethe onset of the clinical phase of the disease Typical FMD clinical signs include fever and theappearance of vesicular lesions on the tongue mouth feet and teats Among ruminants thatrecovered from the disease a relatively large number become asymptomatic virus carriers although it is not clear what is the contribution of these carrier animals to disease transmissionin nature The World anization for Animal Health OIE lists FMD as a reportable diseaseand therefore by law participating nations are required to inform the anization about all FMDoutbreaks OIE member nations with reported cases of FMD are forbidden to engage in tradingof FMDsusceptible animals or their products Thus the presence of FMD in a country can havesevere economic consequencesDiï¬erent interventions to control an FMD outbreak include restriction of susceptible animalmovement slaughter of infectedcontact animals decontamination of infected and surroundingEdited byMariano PrezFilgueiraNational Agricultural TechnologyInstitute ArgentinaReviewed byMargarita S¡izSevero Ochoa Molecular BiologyCenter CSICUAM SpainKenneth James GenoveseAgricultural Research ServiceUnited States Department ofAgriculture United StatesCorrespondenceGisselle N MedinagissellemedinausdagovTeresa de los SantosteresadelossantosusdagovFayna DiazSan SegundofaynadiazsansegundousdagovSpecialty sectionThis was submitted toVeterinary Infectious Diseasesa section of the journalFrontiers in Veterinary ScienceReceived April Accepted June Published August CitationMedina GN de los Santos T andDiazSan Segundo F Use ofIFNBased Biotherapeutics to Harnessthe Host Against FootAndMouthDisease Front Vet Sci 103389fvets202000465Frontiers in Veterinary Science wwwfrontiersinAugust Volume 0cMedina et alIFN Against FMDVpremises and vaccination Vaccination is an option used mostlyin countries in which FMD is endemic but diseasefree nationsprefer to abstain from such practice In general FMDfreecountries that occasionally opted to vaccinate to better containthe outbreak did slaughter all vaccinated animals to regaincommerce rights faster as occurred in the outbreak inthe UK and the Netherlands The current approvedFMD vaccine consists of purified chemically inactivated virus[binary ethylenimine BEItreated] formulated with oilbased oraluminum adjuvants that induces serotypespecific protection inapproximately days and it is applied with a boosting protocolfor ensuring longterm protection While this vaccine hasbeen successfully used for many decades leading to diseaseeradication of a vast area of our planet challenges remainFMD is endemic in most of Africa and Asia and occasionallyepizootics appear in South America or in nations that havebeen diseasefree for many years as it happened in the UKthe Netherlands South Korea Taiwan and Japan Novelvaccine technologies have been developed but to this end noneof them has fully addressed the limitations of the commerciallyavailable vaccine or is currently approved for massive use Alternatives or additional therapeutics that could complementor in some instances substitute for vaccination protocols includethe use of antivirals and biotherapeutics that act quickly priorto induction of vaccineinduced immunity The development ofsuch molecules requires a thorough understanding of the biologyof the virus and its intricate interactions particularly with theinnate immune molecular and cellular mechanisms evolved bythe hostThe AgentFootandmouth disease virus FMDV is a member of theAphthovirus genus within the Picornaviridae family and it is theetiologic agent of FMD The virus contains a singlestrandedRNA of positive polarity Its genome of ˆ¼ nucleotidesconsists of a long reading frame ORF flanked by a ² anda ²untranslated region UTR The ORF encodes a polyproteinof about amino acids which is processed by virusencodedproteases Processing results in the generation of precursors andmature protein products including four structural [1A VP4 1BVP2 1C VP3 1D VP1] and ten nonstructural NS proteins[Lpro 2A 2B 2C 3A three distinct copies of 3B VPg 3Cproand 3Dpol] Due to high genetic variability FMDV is categorizedin seven distinct serotypes A Asia1 C O and Southern AfricanTerritories “ SAT “ and numerous subtypes or topotypesUpon infection the virus spreads very rapidly usually achieving morbidity Depending on the route of entry less than tissue culture infectious doses are required to infect andcause disease in animals In fact FMDV is one of thefastest replicating RNA viruses in nature taking as little as “ h to induce cytopathic eï¬ects in susceptible tissue culture cellsOne could envisage that during FMDV replication almost everycomponent of the virus must play a role in dampening interferingcellular responses to allow such rapid virus replicationInnate Immunity and Interferon ActivationEarly protection against viralfundamentallymediated by the action of interferons IFNs the pillar moleculesof the innate immune system “ Expression of IFN isinfection istriggered by the recognition of molecular signatures collectivelynamed pathogenassociated molecular patterns PAMPs viacellular receptors pattern recognition receptors PRRs that candistinguish œself from nonself  molecules Figure Bindingof PAMPs to PRRs triggers a series of signal transduction eventsand posttranslational modifications PTMs phosphorylationubiquitination ISGylation etc that ultimately activate latenttranscription factors to induce IFN transcription Subsequentlysecreted IFN proteins bind to specific receptors on the plasmamembrane to activate in an autocrine and paracrine mannerdiscrete and overlapping cellular signal transduction pathwaysDepending on the cell type and aï¬ected tissue over specificIFNstimulated genes ISGs may be induced many of whichdisplay antiviral activity to control the viral infection There are three families of IFNs based on the specific receptorusage types I II and III Table “ Type I IFNsie IFNα and IFN signal through a heterodimeric receptorcomplex formed by IFNAR1IFNAR2 type II IFN IFNÎsignals through the complex IFNÎR1IFNÎR2 and type IIIIFNs bind the receptor complex IL28RαIL10R Despitethe receptor diï¬erencesthe three IFN families transducesignals through the Janus kinase JAK“signal transducer andactivator of transcription STAT pathway and type I and typeIII IFNs induce redundant responses Figure Overall therapid production of IFN helps to limit viral replication whilemodulating other immune functionsFOOTANDMOUTH DISEASE VIRUSIMPAIRS INNATE IMMUNITY MOLECULARINTERACTIONSRecognition of FMDV RNA by the host cell results in theestablishment of a rapid antiviral state to limit and controlinfection This selective pressure has allowed FMDV to evolvemany strategiesto ensure enhanced virulence and rapidinfectivity In general RNA viruses can bypass the IFN responseby blocking i global cellular transcription and translationii IFN induction and iii IFN signaling Similarly to otherRNA viruses FMDV can also target IFNindependent antiviralresponses mostly associated with cellular metabolic functionsie autophagy apoptosis stress granule formation etc thathave been extensively described elsewhere In thissection we will summarize the current literature on studiesconducted in vitro that explain how FMDV counteracts the hostinnate immune response at the molecular level including RNAsensing activation of adaptoreï¬ector proteins and regulation ofsignaling pathways by specific PTMsBlock on Cellular Transcription andTranslationFMDV inhibition of cellular gene expression and proteinsynthesis during infection is mainly driven by the viralencoded proteases Leader Lpro and 3C FMDV Lpro isa papainlike protease PLP thatthetranslation initiation factor eIF4G including eIF4GI and eIF4GII to disable capdependent protein synthesis AlsoFMDV Lpro causes degradation of the transcription factorinduces cleavage ofFrontiers in Veterinary Science wwwfrontiersinAugust Volume 0cMedina et alIFN Against FMDVFIGURE Antiviral signaling pathways induced during viral infection Cellular detection of microbial molecules known as pathogenassociated molecular patternsPAMPs ie viral RNA is mediated by pattern recognition receptors PRRs including cytosolic RNA sensors ie RIGI MDA5 or LGP2 andor membraneboundTLRs PAMPPRR interaction activates signal transduction cascades black arrows that result in the production of IFN and inflammatory cytokines RIGI and MDA5contain two caspase recruitment domains CARD and an RNA helicase domain In the case of RIGI ubiquitination green circles is required for its effective activationActivated signals from either RIGI or MDA5 are transmitted downstream via the mitochondrial adaptor MAVS resulting in the formation of MAVS filaments At thisstage different PTMs such as ubiquitination or ISGylation black circles can regulate their functions Endosomal RNAs are detected by TLR3 or TLR78 which signalthrough adaptor proteins TRIF and MyD88 respectively MyD88 uses other adaptors IRAK14 to allow for interaction with TRAF proteins In addition to their role asadaptor proteins TRAFs also serve as E3 ubiquitin Ub ligases to regulate signaling TRAFmediated induction of polyUb is sensed by NEMO thus recruitingdownstream effector kinases such as TBK1 or IKK These proteins form different signaling complexes ie NEMOTBK1 and NEMOIKK leading to phosphorylationblue arrows of transcription factors IRF37 to a lesser extent IRF1 and IRF5 are also phosphorylated IRF phosphorylation triggers dimerization and translocationContinuedFrontiers in Veterinary Science wwwfrontiersinAugust Volume 0cMedina et alIFN Against FMDVFIGURE orange arrows to the nucleus where they bind mainly to IFN promotersenhancers Alongside with this pathway TRAF6E3 ligases can activate MAPK3and other kinases including ERK12 and JNK which phosphorylate the components of the AP1 heterodimer allowing for translocation to the nucleus and binding tothe IFN promoterenhancer to activate transcription Activated IKK also phosphorylates IκB releasing NFκB which then translocates to the nucleus and binds at theIFN promoter AP1 activating protein CARD caspase activation and recruitment domain DUB deubiquitinase ER endoplasmic reticulum IκB inhibitor of KBkinases IKK IκB kinase IL interleukin IRAK interleukin1 receptorassociated kinase IRF IFN regulatory factor LGP2 laboratory of genetics protein MAPKmitogenactivated protein kinase MAVS mitochondrial antiviral signaling protein MDA5 melanoma differentiationassociated gene MyD88 myeloid differentiationprimary response protein 88d NEMO NFκB essential modulator NFκB nuclear factorκB PKR protein kinase R PTM posttranslational modification RIGIretinoic acidinducible gene I TANK TRAF family memberassociated NFκB activator TBK TANK binding kinase TLR Tolllike receptor TRAF TNF receptorassociated factor TRIF TIRdomaincontaining adapterinducing interferonTABLE Use of IFNbased therapies against FMDVTypeReceptSignalSubtypeSpeciesMilestoneType IIFNAR1IFNAR2JAK1 TYK2IFNαPorcinebovine ¢ Recombinant bacterial expressed IFNα is a potent biotherapeutic againstIFNαPorcine¢ Ad5 delivered poIFNα protects swine against different serotypes of FMDVFMDV in vitro IFNIFNδIFNω7IFNαωIFNτType IIIFNÎ R1 IFNÎ R2JAK1 JAK2IFNÎType IIIIFNλR1IL10R2JAK2 TYK2IFNλ1IFNΓ¢ poIFNαprotection correlates with enhanced tissuespecific innate immune cellinfiltration in swine ¢ poIFNα protection correlates with upregulation of essential ISGs in vitro ¢ Ad5 delivered porcine poIFN protects swine against FMDV ¢ Bacterially expressed poIFNδ8 significantly inhibits FMDV replication in vitro ¢ E coli produced poIFNω7 protects cells against FMDV ¢ Bacterially expressed IFNαω added prior to infection resulted in a significantreduction in FMDV replication in vitro ¢ Ovine IFNτ has antiviral effect against FMDV in vitro ¢ Recombinant bovine IFNÎ reduced FMDV replication in BTY cell culture ¢ High dose of Ad5poIFNÎ protects swine against FMD ¢ Replication of FMDV in IBRS2 cells is inhibited by treatment with the purifiedrecombinant poIFNλ1 PorcinePorcinePorcinePorcineOvineBovinePorcinePorcineIFN CombosOtherIFNvaccinecombosIFNλ3Bovine¢ Inoculation with Ad5boIFNλ3 resulted in the induction of several ISGs in tissuesof the upper respiratory tract and protected cattle against challenge withFMDV PorcinePorcineIFNαIFN΢ Ad5poIFNλ3 protects swine against challenge with FMDV ¢ Use of a combination of Ad5poIFNÎ and Ad5poIFNα or Ad5poIFNαΠshowed an enhancement of the antiviral activity against FMDV in swinePoly ICPorcine¢ Double stranded ds RNA poly ICLC in combination with Ad5poIFNαprotected swine against FMDV siRNAPorcine¢ Combination of Ad5poIFNαΠwith Ad3siRNA targeting FMDV NS codingregions blocked replication of all serotypes of FMDV in vitro IRF73Porcine¢ Inoculation with Ad5IRF735D resulted in induction of IFNα and fully protectedmice and swine challenged with FMDV day after treatment IRESPorcine¢ Use of synthetic IRES in combination with adjuvanted typeO FMD improvedimmune response and protection against FMDV challenge IFNαPorcine¢ Use of a combination of Ad5poIFNα and Ad5A24 in swine resulted incomplete protection after challenge IFNαÎPorcine¢ Ad5poIFNαΠcoadministered with Ad5siRNA targeting NS regions of FMDVand a commercial inactivated FMD vaccine partially protected swine IFNλ3Bovine¢ Use of a combination of Ad5bovIFNÎ3 and AdtO1M in cattle resulted incomplete protection after aerosol challenge nuclear factor NFκB and results in blockage of specificdownstream signaling eï¬ectors Studies in porcine cellsdemonstrated that FMDV Lpro can promote its selfbindingto the transcription factor activitydependent neuroprotectiveprotein ADNP and negatively regulate the activity of the IFNα promoter In contrast chromatin changes that favor theupregulation of IFN and ISGs can inhibit FMDV replication Interestingly deletion or mutations in diï¬erent domainsof Lpro result in viral attenuation in vitro and in vivo “ Furthermore these studies have shown a strong type I IFNactivity upon infection with diï¬erent versions of FMDV Lpromutants Frontiers in Veterinary Science wwwfrontiersinAugust Volume 0cMedina et alIFN Against FMDVFIGURE Type I II and III interferon IFNmediated signaling All type I and type III IFN subtypes bind to respective receptors IFNAR1IFNAR2 and IFNLR1IL10R2These interactions trigger the phosphorylation of JAK1 and TYK2 kinases which in turn phosphorylate STAT1 and STAT2 JAK2 mediates type III IFNdependent STATphosphorylation Phosphorylated heterodimers of STAT1STAT2 bind to IRF9 forming the ISGF3G complex which then translocates to the nucleus and binds toIFNresponsive elements ISREs present in the promoters of over ISGs Type II IFN binds to the heterodimeric IFNÎR1IFNÎR2 receptor also inducingphosphorylation of JAK1JAK2 kinases In turn mostly STAT1 is phosphorylated Phosphorylated homodimers of STAT1 translocate to the nucleus and induce theexpression of genes controlled by gammaactivated sequence GASdependent promoter sequences IFNAR12 IFN alpha receptor12 IFNÎR12 IFNgammareceptor12 IFNALR1 IFNlambda receptor IL10R2 IL10 receptor ISGs IFNstimulated genes ISGF3G ISG factor gamma JAK12 Janus kinase STATsignal transducer and activator of transcriptionInterruption of cellular translation during infection can alsobe mediated by FMDV 3Cpro a chymotrypsinlike cysteineprotease that similarly to Lpro targets eIF4G and the capbindingcomplex eIF4A for cleavage although these events occur later inthe infection 3Cpro can also participate in the inhibitionof host“cell transcription by cleaving histone H3 upon FMDVinfection Block on Interferon InductionDuring infection the initial event that leads to the productionof IFN and proinflammatory cytokines is the recognition ofviral RNA Figure Sensing of FMDVRNA is mediated byMDA5 a protein that belongs to a family of helicasesknown as retinoic acidinducible geneI RIGIlike receptorsRLRs Recent studies have shown that the interaction betweenRLRs RIGI and LGP2 and the FMDV proteins Lpro 2Band 3A interferes with the induction of type I IFN “Indeed overexpression of either FMDV 2B or 3A resulted in thedownregulation of RIGI and MDA5 mRNA expression In contrast upregulation of LGP2 transcripts has been observedduring FMDV infection in porcine cells despite a detectablereduction of LGP2 protein levels presumably due to FMDVFrontiers in Veterinary Science wwwfrontiersinAugust Volume 0cMedina et alIFN Against FMDVLproinduced cleavage The apparent inconsistencybetween the levels of LGP2 mRNA and protein during FMDVinfection may be explained by LGP2™s ability to serve as a positiveand negative regulator of RIGI and MDA5 signaling presumablyaï¬ecting multiple steps of the IFN induction pathway Inaddition to RLRs nucleotidebinding oligomerization domainNODlike receptors NLRs NOD1 and NOD2 also participatein the recognition of RNA A study by Liu et al describedthe association of NOD2 with FMDV 2B 2C and 3Cpro to blockinnate immunity activation Protein kinase R PKR is anotherrecognized PRR that acts as an RNA sensor Binding ofRNA to PKR induces a conformational change that leads toautophosphorylation and activation The primary target ofactivated PKR is the eukaryotic initiation factor α subuniteIF2α whose phosphorylation results in the blockage of cellularprotein synthesis a relatively common process during viralinfection Although no direct interaction between FMDVRNA and PKR has been demonstrated it has been reportedthat PKR activity modulates FMDV infectivity In fact in tissueculture experiments depletion of endogenous levels of PKR usingsiRNA resulted in increased FMDV titers Furthermoreit has been recently shown that overexpression of autophagyrelated ATG5ATG12 proteins induces transcription of PKR andsubsequent reduction of FMDV replication These resultssuggest that PKR has a complex role as an RNA sensor but also asan antiviral agent during FMDV infectionIt has been demonstrated that FMDV also targets DExDHbox RNA helicases formally accepted as PRRs and modulatorsof the antiviral signaling pathway In vitro experimentsintending to analyze protein“protein interactions revealed theassociation between the RNA helicase DDX1 and FMDV 3D Interestingly these studies indicated that during FMDVinfection in porcine cells cleavage of DDX1 was detected whileoverexpression of DDX1 resulted in the upregulation of IFNand other ISG mRNAs which correlated with virus inhibition Other DExDHbox RNA helicases such as RNA helicaseH RHA are hijacked during FMDV infection and interact withFMDV ™UTR 2C and 3A to facilitate virus replication Signaling pathways downstream from RNA sensing involvethe activation of diï¬erent adaptor and eï¬ector proteins Oneof the pathways that lead to signal activation requires theformation of specific complexes such as NFκB essentialmodulator NEMO and the kinase IKK which bridges theactivation of NFκB and IFN regulatory factor IRF signalingpathways It has been demonstrated that FMDV 3Cpro interactswith NEMO and induces its cleavage resulting in impairedinnate immune signaling IRFmediated signals driven byIRF3 and IRF7 can also be targeted by FMDV proteinsSpecifically overexpression of Lpro in PK15 cells resulted inthe downregulation of IRF3 and IRF7 protein levels andinactivation of IFN and IFNλ1 promoter Other factors involved in the activation of IFN includeconventional PTMs such as phosphorylation and ubiquitinationwhich ensure eï¬ective regulation of these signaling pathways Also diï¬erent cellular deubiquitinases DUBs can reverseubiquitination to control the intensity of the immune signalingresponse Interestingly it has been shown that FMDV Lprocan remove ubiquitin Ub molecules from several proteinsrequired for IFN mRNA expression and those involved in theactivationrepression of the IFN loop This role became moreevident by the observation that during infection FMDV Lprocan cleave cellular substrates modified with the Ublike moleculeISG15 Furthermore mutation of Lpro thatimpairsdeISGylaseDUB function results in viral attenuation Inthis regard identification of FMDV targets for deubiquitinationand deISGylation may contribute to elucidate the role ofthose factors in counteracting the innate response and developnovel countermeasuresBlock on Interferon SignalingThe ligandmediated association of the specific IFN receptorspromotes a signaling cascade that results in the phosphorylationof the receptor by the action of JAKs These events result inthe generation of docking sites for downstream adaptor andeï¬ector proteins including signaltransducer and activatoroftranscription STAT proteins that associate with otherfactors and translocate to the nucleus inducing transcriptionof a plethora of ISGs described above and in Figure Although blockage of the JAK“STAT signaling pathway hasnot been reported during FMDV infection overexpressionof either FMDV 3Cpro or VP3 can inhibit this response Forinstance IFNtreated HeLa cells overexpressing FMDV 3Cprosuppressed IFNstimulated promoter activities and inducedproteasome and caspaseindependent protein degradationof karyopherin α1 KPNA1 the nuclear localization signalreceptor Thisinteraction inhibited the nuclear translocation of STAT1STAT2impeding maximal ISG promoter activity In another studyin HEK293T cells overexpression of VP3 followed by coimmunoprecipitation revealed the association between VP3 andJAK1 FMDV VP3 also inhibited virustriggered activation of theIFN promoter leading to the decrease in transcription of ISGspresumably due to lysosomalinduced degradation of JAK1 A yeast twohybrid screen identified FMDV 2C in complex withNmyc and STAT interactor Nmi a protein known to augmentimmune function dependent on STATmediated transcriptionInterestingly such interaction resulted in the recruitment ofNmi to vesicular compartments followed by the induction ofapoptosis in BHK21 cells tyrosinephosphorylated STAT1forEvidently FMDV proteins can also target crosstalk pathwaysinduced by JAKSTAT signaling and due to this versatilityunderstanding of these signaling events during FMDV infectionis challengingFOOTANDMOUTH DISEASE VIRUSIMPAIRS INTERFERONMEDIATEDCELLULAR INNATE IMMUNE RESPONSESSimilarly to what happens in vitro FMDV manipulates theearly innate immune response in vivo to ensure a windowof opportunity that favors viral replication and spread beforeFrontiers in Veterinary Science wwwfrontiersinAugust Volume 0cMedina et alIFN Against FMDVthe onset of eï¬ective adaptive immunity required for virusclearance During infection FMDV interacts with a range of hostcells including natural killer NK cells dendritic cells DCsmonocytesMφ and Îδ T cells All these cells play an importantrole in innate immune responses that trigger the productionof large quantities of IFN and other cytokines which serve asautocrine agents “Shortly after FMDV infection in swinethe number ofcirculating NK cells transiently decreases and the remaining NKcells show a dysfunctional lytic activity against target cells and areduction of IFNÎ production In parallel FMDV blocks theability of porcine DCs to mature into conventional DCs cDCs dampening their response against Tolllike receptor TLRligands Another subset of porcine DCs plasmacytoid DCspDCs also referred to as the major professional systemic IFNαproducers are also aï¬ected by FMDV During infectionpartial depletion of pDCs in the peripheral blood has beendetected and the remaining pDCs are less capable of producingIFNα in response to ex vivo stimulation by TLR ligands or virus Similar to pDCs FMDV infection reduces the productionof IFNα on Langerhans cells LCs a distinct subset oftissueresident DCs of the skin It has also been suggestedthat porcine Îδ T cells and Mφ can serve as targets for FMDVinfection in swine although the interplay betweenthese cells and FMDV remains unclearComparably to swine FMDV infection in cattle triggersseveral early events in the innate immune system althoughthe eï¬ects are not exactly the same For instance bovine NKcells originated from FMDVinfected cows have an elevatedcytotoxic function against bovine target cells in vitro In addition some subsets of cDCs are significantly decreasedduring the peak of viremia while the expression of majorhistocompatibility complex MHC class II molecules on allbovine cDCs is reduced and the processing of exogenous antigenis impaired Furthermore during FMDV infection thenumber of systemic mature bovine pDCs characterized bythe expression of CD4 and MHC class II is increasedpresumably to intensify a humoral response and T cell activationwhile levels ofimmature CD4 MHC class IIpDCs aredeclined Examination of bovine Îδ T cells revealed thatthese cells with the surface expression marker WC1 showa transient activated phenotype and increased expression ofIFNÎ FMDV also aï¬ects the innate immune response at the cytokinelevel in the natural host In vivo cytokine profile analysis duringthe clinical phase of disease shows a systemic decrease of proinflammatory cytokines [IL1 IL6 and tumor necrosis factorTNFα] and an increase of the antiinflammatory cytokine IL and IFNα Most likely these changesare related to the early T cell unresponsiveness and lymph iadescribed in swine and cattle during FMDV infection Interestingly a significant induction of inflammatoryand antiviral factors at the local level is detected in cattle in sitesof abundant viral amplification such as the nasaloropharynx orvesicular lesions “ A consistent upregulation of IFNα Î and λ mRNA in distinct microanatomical compartmentsof the nasopharyngeal mucosa concurrent with occurrence ofviremia has also been detected in cattle In contrast studiesin swine demonstrated that IFN expression in infected swineskin is inhibited These diï¬erences may be due to theanalysis of follicleassociated epithelium of the nasopharyngealmucosa in cattle vs skin in swine or to the specific samplingtechnique used in each experiment While in the cattle studylasercapture microscopy was used to focus only in areas of highFMDV replication in the swine study RNA was extracted froma piece of skin without discriminating between microanatomicalcompartments Evidently more studies are needed to elucidatethe intricate interactions between FMDV and the innate immunesystem of specific animal hostsEFFECTIVE USE OF INTERFERONAGAINST FOOTANDMOUTH DISEASEVIRUS IN VITROType I InterferonThe role of IFN in controlling FMDV replication was firstproposed in when Dinter and Philipson demonstrated thatcalf kidney cells exposed to FMDV could become persistentlyinfected and proposed this was a consequence of the inductionof an IFNlike inhibitor present in the supernatant of infectedcells Later studies also suggested that swine leukocytestreated with phytohemagglutinin produced an inhibitor ofFMDV replication with properties similar to IFN It wasnot until that new studies demonstrated that the abilityof FMDV to form plaques in cell culture correlated with thesuppression of type I IFN α protein expression Theseresults were further supported by detection of IFN protein andantiviral activity in the supernatants of primary porcine ovineand bovine kidney cells infected with an attenuated FMDVmutant leaderless as compared to the supernatants of cellsinfected with wildtype WT virus Later studies by the samegroup provided proof of concept on the use of recombinantbacterial expressed IFNα as a potent biotherapeutic againstFMDV This approach was further developed by deliveringrecombinant porcine IFNα using a replicationdefectivehuman Adenovirus vector Ad5poIFNα Infection ofIBRS2 cells with Ad5poIFNα resulted in secreted poIFNα IFN protein detected as early as h postinfection hpiand lasting for at least h Most important expressed IFNprotein displayed strong biological antiviral activity againstFMDV Followup studies by the same group showed that allFMDV serotypes are very sensitive to Ad5delivered poIFNαand sterile protection could be achieved in vivo highlightingthe potential of this approach for the development into abroad biotherapeutic strategy to control FMDV replicationIn the last years advancements is genomics have ledto the characterization of almost all type I IFN subtypes inthe porcine and bovine genome “ which are morenumerous than those identified in primates and mice This hasrevealed diï¬erent functional genes and pseudogenes with diverseexpression profiles and antiviralfunctions against diï¬erentviruses mostly in swine In fact a recent studyFrontiers in Veterinary Science wwwfrontiersinAugust Volume 0cMedina et alIFN Against FMDVdemonstrated that poIFNω7 known for its ability to inducethe highest levels of antiviral activity when compared to otherpoIFNω subtypes elicits an antiviral state against FMDV inIBRS2 cells treated with the recombinant form of poIFNω7produced in Escherichia coli Other subclasses of type IIFN known to be produced in swine and cattle include IFNalphaomega IFNαω also known as IFNµ and IFN delta IFNδ Significant reduction in FMDV replication has been observedupon treatment of porcine cells with bacterially expressed IFNαω or IFNδ8 prior to viral infection Recently another member of type I IFN family IFNτ whichis only produced in ruminants has been evaluated as an antiviralagainst FMDV IFNτ is a paracrine reproductive hormonesecreted constitutively by trophoblasts and endometrial cells toincrease the life span of the corpus luteum however productionis not induced upon viral infection While its secretionis restricted to ruminantsit has a broadspectrum activityagainst various crossspecies viruses Interestingly IFNτ has homology with the amino acids of IFNα which allows forbinding to type I IFN receptors The property of IFNτ thatmakes it an interesting therapeutic candidate for the treatmentof various viral diseases is its significantly lower toxicity ascompared to other type I IFNsType II InterferonIn contrast to type I IFN the type II IFN family is composedof only one member IFNÎ which exerts its actions througha specific receptor IFNGR1IFNGR2 IFNÎ is weakly resistantto heat and acid and it is able to activate leukocytes suchas macrophages and granulocytes also exerting regulatoryfunctions on T and B lymphocytes Indeed productionof IFNÎ is used as a tool to measure cellmediated immuneresponses against FMDV in vaccinated cattle “ andin swine Interestingly IFNÎ responses as measured b Answer:

Thyroid_Cancer

"Gastroenterol Res Primary Localized Amyloidosis of the Intestine A Pathologist ViewpointSaeed Ali Alshehria c Mahmoud Rezk Abdelwahed HusseinbAbstractBackground Localized amyloidosis of the intestine is a rare entity which can clinically masquerade several conditions such as colitis polyps and malignant tumors This study aims to evaluate the clinicopathological features of this entityMethods To evaluate the clinicopathological features of this entity a comprehensive search of the literature to was done using the following keywords œamyloidosis and œsmall intestine or œduodenum or œileum or œjejunum or œcolon We identified studies about gastrointestinal amyloidosis Data were examined for studies about localized intestinal amyloidosis The clinicopathological features were describedResults The age at presentation ranged from to years The male to female ratio was The jejunum and sigmoid colon were the most commonly involved sites Abdominal pain and intestinal obstruction small intestine or rectal bleeding sigmoid region were the most common clinical presentations Colonoscopic findings included wall thickening mucosal ulcerations small intestine and tumorlike masses colonConclusions The clinical presentations of localized intestinal amyloidosis depend on the site of the deposition of the amyloid In most cases amyloid deposits consisted of light chain proteinKeywords Amyloidosis Amyloid Colon Small intestine Rectum Gastrointestinal tractIntroductionThe term œamyloid was initially coined to describe materials Manuscript submitted June accepted July Published online August aDepartment of Pathology Armed Forces Hospitals Southern Region King Fahd Hospital Saudi ArabiabDepartment of Pathology Faculty of Medicine Assuit University Hospitals Assuit EgyptcCorresponding Author Saeed Ali Alshehri Department of Pathology Chemistry Section Armed Forces Hospital Southern Region Khamis Mushyte Saudi Arabia Email syd46118gmailcom 1014740gr1303found in plants and has been thought to represent carbohydrate Greek œamylon starch In medical literature the term œAmyloidosis has been known for more than years It was named after the deposition of the hallmark protein called crosssheet amyloid fibrils which represents either misfolded or misassembled proteins The nomenclature of the amyloid protein uses two letters The first letter œA refers to amyloid whereas the second refers to the precursor proteins [] The amyloid proteins include several types such as immunoglobulin light chains AL in primary amyloidosis transthyretin TTR beta2microglobulin in hemodialysisassociated amyloidosis and serum amyloid A SAA protein in secondary AA amyloidosis [] There are some associations between AL amyloid deposition and several hematological conditions including myeloma lymphoplasmacytic disorders and plasma cell dyscrasia Moreover AL amyloidosis and myeloma may show similar genetic abnormalities such as 14q translocations and 13q deletion []Amyloidoses are degenerative conditions associated with significant suffering not only for patients but also for their families as well [] They are characterized by the deposition of extracellular protein fibrils in the ans such as the kidneys nerves gastrointestinal tract heart and skin Amyloidoses are associated with significant morbidities such as malabsorption renal insufficiency and hematological disorders or even lifethreatening events such as intestinal obstruction renal failure and fatal arrhythmia The clinicopathological features of amyloidosis are nonspecific They depend on the type of the precursor protein site and severity of the amyloid deposition The diagnosis of amyloidosis depends on the examination of the tissue biopsies from the lesions It is established by the detection of amyloid deposits with its characteristic birefringence under polarized light following staining with Congo red or thioflavinT or immunohistochemical stains Management of amyloidosis includes reduction of the supply of amyloid fibril precursor proteins stabilization of the precursor protein formation administration of antiplasma therapies general care systemic amyloidosis or surgical resection in localized amyloidosis [ ]Amyloidosis includes both primary and secondary types In the former also known as AL for amyloid light chain there is no evidence of coexisting disease and it has an immunoglobulin light chain as a major protein component In secondary amyloidosis also known as AA for amyloidassociated there is evidence of coexisting disease resulting in the production of inherently amyloidogenic proteins or overproduction of potentially amyloidogenic normal proteins and it has serum amyloidassociated protein SAA The deposition of amys The authors Journal compilation Gastroenterol Res and Elmer Press Inc„¢ wwwgastroresThis is distributed under the terms of the Creative Commons Attribution NonCommercial International License which permits unrestricted noncommercial use distribution and reproduction in any medium provided the original work is properly cited 0cLocalized Amyloidosis of the Intestine Gastroenterol Res loid may be either widespread systemic amyloidosis [ ] or restricted to a specific an localized amyloidosis In systemic amyloidosis the amyloidogenic proteins are synthesized at sites far away from the sites of amyloid deposition The amyloid protein is deposited at several sites and therefore the patients have disparate clinical manifestations such as arrhythmia neuropathies malabsorption and proteinuria due to cardiac neural intestinal and renal involvement respectively [] Currently the classification of amyloidosis is based on the type of precursor proteins []Localized amyloidosis refers to the infiltration of the tissues by amyloid where the precursor proteins are synthesized at or close to the site of deposition It includes both anspecific amyloidoses where the amyloid is produced by the an itself and nonan specific amyloidosis where the fibrillar protein is not specific to the affected an [] Localized amyloidosis of the intestine is a rare condition that involves the duodenum jejunum and ileum appendix or colorectum The clinicopathological features are nonspecific and therefore they can masquerade several conditions such as collagenous colitis ischemic colitis bowel infarction perforation polyps and malignant tumors [] The colonoscopic features include mucosal granularity friability erosions ulcerations petechial hemorrhage and polypoid lesions [] The radiological findings include ulcerations thickening of the mucosal folds narrowing of the colonic lumen effacement of haustrations and nonspecific mucosal nodularity []To date few cases of localized intestinal amyloidosis have been reported [ ] In Tada examined patients with small intestinal amyloidosis Clinical findings included malabsorption mass lesions diarrhea and hematochezia Endoscopy of the jejunum revealed polypoid lesions wall thickening erosions ulcerations and fine granular appearance The amyloid proteins included amyloid A protein AA light chain protein AL beta 2microglobulin and prealbumin Histologically amyloid deposits were seen in the submucosa and muscle layer [] Chen reported a case of localized amyloidosis of the transverse colon in a 52yearold male patient The clinical presentations included abdominal pain bloody stool and weight loss Colonoscopy revealed a marked narrowing of the transverse colon associated with the presence of multiple polypoid growths The clinical and radiological findings supported the initial impression of carcinoma and therefore the patient underwent right hemicolectomy with lymph node dissection [] Grossly there was an ulcerative mass with marked narrowing of the lumen Histologically amyloid deposits were seen in the lamina propria []To date our knowledge about the clinicopathological features of the localized intestinal amyloidosis is limited To gain insights into these issues we conducted a comprehensive literature search and metaanalysis of the relevant published literature Also we presented a rare case of primary isolated intestinal amyloidosisMaterials and MethodsThis is a viewpoints study not an original research investigation that did not include any interaction or intervention with human subjects or any access to any identifiable private information Therefore the study did not require institutional review board review [] This study was performed in accordance with the principles of the Declaration of Helsinki []Search strategy and selection criteriaThe authors adhered to Preferred Reporting Items for Systematic Reviews and MetaAnalyses PRISMA guidelines [] The literature was reviewed by searching the PubMed electronic database using the key term œamyloidosis and the following words œintestine or œsmall intestine or œduodenum or œileum or œjejunum or œappendix or œcolon or œsigmoid or œrectum andor œcecum to identify the eligible studies The search results were considered based on their titles abstracts and publication date Their full texts were examined to confirm their eligibility and then were included in the study A summary of the search strategy and selection criteria is shown in Figure [ ]Data extraction a methodological assessment and statistical analysisThe clinicopathological findings were extracted including the family name of the first author year of publication patient age gender symptoms endoscopic features histology special stains and presence or absence of systemic involvement The authors independently read and analyzed each study They defined the final diagnosis as œthe histopathological diagnosis to ensure homogeneous evaluation of the findings Statistical computations were performed using IBMSPSS IBMSPSS Inc Chicago IL USA Descriptive statistics mean standard error of mean and ranges were calculatedResultsFlow trialOur search yielded previous studies about gastrointestinal amyloidosis of which were excluded The remaining s have undergone œabstract review These studies covered a period of years to This relatively long period allowed proper evaluation of a good number of cases in the literature Twentyseven studies with the final diagnosis of localized intestinal amyloidosis were included in the present study In all cases the workup for monoclonal gammopathy and systemic amyloidosis serum protein electrophoresis and pathological examination of the tissue specimens was performed The diagnosis of localized amyloidosis was substantiated by the use of ancillary studies including radiological examination such as abdominal ultrasound barium enema []wholebody computed tomography [] magnetic resonance imaging positron emission tomography [] skeletal surveys electrocardiography and echocardiography [ ] s The authors Journal compilation Gastroenterol Res and Elmer Press Inc„¢ wwwgastrores 0cAlshehri Gastroenterol Res Figure Flow chart of literature search and study selection for cases of localized intestinal amyloidosis The inclusion criteria included patients who had amyloid deposition in the intestine without evidence of systemic involvement or an associated condition leading to secondary amyloidosis [] The exclusion criteria included patients with one or more of the followings Evidence of chronic diseases such as collagen vascular disease that may have led to secondary amyloidosis [] Additional an involvement by amyloidosis Intestinal symptoms suggestive of amyloidosis but without confirmatory diagnostic tissue biopsy Plasma cell dyscrasia myeloma lymphoplasmacytic disorders or other Bcell malignancies such as Waldenstrom™s macroglobulinemia [] Systemic AL type [] Laboratory findings revealing positive detection of monoclonal light chains in serum andor urine []The reported cases did not have any evidence of generalized metabolic complications or extraintestinal complications of accumulated amyloidosis [ ] A summary of the flow chart of the literature search and study selection criteria is shown in Figure [ ]Clinical presentations and treatment modalities in localized intestinal amyloidosisThe colorectum jejunum transverse colon ileum and duodenum were the most commonly affected sites The lesions were more common in males than in females The mean age at presentation was ± years small intestine and ± years colorectum [ ] In the small intestine the main symptoms were abdominal pain [ ] and intestinal obstruction [ ] followed by diarrhea and vomiting [ ] bleeding [ ] malabsorption [] and constipation [] In the colorectum the main symptoms were rectal bleeding [ ] followed by abdominal pain [ ] anemia [] intestinal obstruction [] and weight loss [] Chen described a case of localized amyloidosis of the transverse colon in a 52yearold male patient who presented with a periumbilical pain rectal bleeding and weight loss Colonoscopic examination revealed mucosal ulcerations and luminal narrowing by multiple polypoid lesions [] Treatment modalities included in these cases included drug therapy prednisolone azathioprine melphalan and colchicines [ ] intestinal resection partial jejunectomy endoscopic submucosal resection [ ] and colonic resection [ ] with close followup of the patients [ ] A summary of these results is presented in Tables [ ]s The authors Journal compilation Gastroenterol Res and Elmer Press Inc„¢ wwwgastrores 0cLocalized Amyloidosis of the Intestine Gastroenterol Res Table Data From Individual Studies of Primary Localized Amyloidosis of the Small IntestineCase Sex Age years SitesIleumClinical manifestationsColonoscopic findingsStudies[][][][][][][][][][][][][][]FMFMFMMMMMMFMiddle ageAgedAcute abdominal pain diarrhea and vomitingPseuntestinal obstructionMelena and anemiaJejunumJejunumSmall intestineIntestinal obstructionJejunum Chronic diarrhea and and ileummalabsorptionSmall intestine Acute abdomenDuodenum and jejunumSmall intestineAbdominal pain nausea and constipationSmall bowel obstructionSmall intestine Abdominal pains and hematemesisThickening of the wall of the ileum and ascending colonThickening of the mucosal folds and multiple polypoid protrusionsDiffuse thickening of the mucosa with friability nodularity erosions and polypoid protrusionsDiffuse thickening of the wall of the small intestineThickening of the jejunal and proximal ileal mucosal foldsPerforation of the wallMultiple shallow ulcers and several erosionsBand of amyloid and connective tissue surrounding the small intestineMucosal ulcerations with discrete nodulesSmall intestine Nonspecific digestive symptoms Multiple mucosal polypsJejunumSmall intestine Nonspecific digestive symptoms Thickening of the wall with rough and Nonspecific digestive symptoms Polypoid pseudotumoral mucosal formationsHeartburn and constipationpolypoid intestinal mucosal foldsF M MSmall intestine three casesDuodenumScreening for cancer stomachA localized depressed lesionEndoscopic and histological findings in localized intestinal amyloidosisIn the small intestine the most common endoscopic findings were thickening of the intestinal wall [ ] and polypoid mucosal protrusions [] followed by mucosal ulceration [ ] friability and nodularity of the mucosa [ ] and wall perforations [] In the colorectum polypoid masses with luminal narrowing were the most common endoscopic finding [ ] followed by mucosal ulcerations solitary or multiple ulcers [ ] or and thickening of the wall [ ] nodularity and friability of the mucosa [ ] and perforation [] In all cases deposition of pale eosinophilic amorphous amyloid material with the prominent cracking artifact in the mucosa submucosa muscle layer or walls of the blood vessels represented the main histological feature [ ] The further Congo red staining revealed the characteristic salmonpink color confirming the presence of amyloid deposits The deposits showed the characteristic applegreen birefringence under polarized light Immunohistochemical stains were also performed in some cases to substantiate the diagnosis Other histological features included mucosal ulceration amyloid angiopathy necrotizing angiitis [] foreign body giant cell reaction [] ischemic changes focal active colitis and cryptitis a personal observation The amyloid type was AL light chain protein in the majority of the cases of the small intestine [ ] and colorectum [ ] Other amyloid protein types included AAamyloid [] and high serum amyloid SAA in some cases [] Summary of clinicopathological findings in patients with localized intestinal amyloidosis is presented in Tables [ ]Case reportPrimary localized amyloidosis of the intestineA 55yearold female patient presented with anorexia diarrhea rectal bleeding and weight loss Colonoscopy revealed picture reminiscent of active pancolitis friable mucosa with mucosal erosions ulcerations and hemorrhage and six biopsies from the ileum cecum and colon ascending transverse descending colons and rectum were obtained On histology all biopsies showed deposition of pale eosinophilic amorphous material with the prominent cracking artifact characteristic of amyloid in the lamina propria and submucosa In some areas s The authors Journal compilation Gastroenterol Res and Elmer Press Inc„¢ wwwgastrores 0cAlshehri Gastroenterol Res Table Data From Individual Studies of Primary Localized Amyloidosis of the ColonCase SexSitesClinical presentationColonoscopic findingsStudiesAge yearsMMMMMFMMMFMFTransverse colonbleeding and weight lossIntestinal obstructionTransverse colon A periumbilical pain rectal Descending colon AnemiaSigmoid colonUlcerations and luminal narrowing by multiple polypoid lesionsElderlyA stenosing massMiddle age Transverse colon Nonspecific digestive symptoms Submucosal massUlcerative lesionsMucosal ulceration perforation and thickening of the bowel wallA submucosal tumorlike massMucosal ulcerA single friable rounded mucosal lesionA 15cm shallow depressed mucosal lesionAcute abdominal pain rectal bleeding and fecal peritonitisHematocheziaHemepositive stoolsRectal bleedingRoutine colonoscopyNonspecific digestive symptoms Several irregularlyshaped discrete ulcerationsSigmoid colonSigmoid colonSigmoid colonSigmoid colonSigmoidDescending colon Abdominal pain bloating flatulence and hematocheziaRectal bleedingHemorrhagic mucosa and amass lesionDiffuse nodular friable lesions ascending colon and several irregular large ulcers with nodularity descending colonThickening of the wall and mucosal ulcerationsAscending and descending colonsColon seven casesAscending colonF M Middle age ColonF M Rectal bleeding two casesHeartburn and constipationFAcute abdominal pain diarrhea and vomitingThickening of the wall of the ileum and ascending colon[][][][][][][][][][][][][][][]Table Clinical Colonoscopic and Histological Findings in the Primary Localized Amyloidosis of the IntestineAspectsAge mean ± SEMMale to female ratioSite of involvementClinical presentationsColonoscopic findingsHistological featuresSmall intestine duodenum jejunum and ilium ± years [ ]Jejunum Ileum Colorectum ± years [ ]The left colon Transverse colon Abdominal pain and intestinal obstruction [ ]Rectal bleeding [ ] followed by abdominal pain [ ] and anemia []Thickening of the wall [ ] polyps and polypoid mucosal protrusions [] ulcerations [ ] friability and nodularity of the mucosa [ ] and perforation []Mass lesions tumorlike lesion polypoid protrusions and polyps with narrowing were the most common [ ] Ulcerations with solitary or multiple ulcers [ ] thickening of the wall [ ] nodularity and friability of the mucosa [ ] and perforation []Amyloid deposits increased density of mixed inflammatory cells in the lamina propria light chain protein [ ]Amyloid deposits increased density of mixed inflammatory cells in the lamina propria foreign body giant cell reaction amyloid angiopathy necrotizing angiitis focal active colitis and immunoglobulin lightchain AL [ ]s The authors Journal compilation Gastroenterol Res and Elmer Press Inc„¢ wwwgastrores 0cLocalized Amyloidosis of the Intestine Gastroenterol Res Figure Isolated primary amyloidosis of the intestine case report a e Colonic mucosa with deposition of pale eosinophilic amorphous material star in the submucosa with abundant deposits of pink amorphous materials with some cracking artifacts characteristic of amyloid Focal surface mucosal ulceration is noted The background mucosa shows focal active inflammation including cryptitis arrow and there is patchy crypt distortion No granulomas are seen hematoxylin and eosin a × b × c d × and e × f g Congo red stain which reveals the characteristic salmonpink color confirming the presence of amyloid deposits Congo red f × and g × h i The deposits exhibit characteristic applegreen birefringence under polarized light Congo red under polarized light h × and i × the lamina propria was entirely replaced by these deposits The background mucosa showed focal active inflammation focal ulceration cryptitis occasional crypt abscess This focal chronic active inflammatory change may be secondary to erosionulceration associated with amyloid deposits [] Congo red stain confirmed the presence of amyloid deposits with applegreen birefringence under polarized light Fig Immunohistochemical analysis revealed that the amyloid deposits were positive for amyloid lambda but negative for amyloid A These findings were consistent with amyloid lightchain AL amyloidosis We performed several laboratory and radiological investigations to rule out the possibility of systemic involvement The hematological indices red blood cells RBCs count hemoglobin mean corpuscular volume MCV mean corpuscular hemoglobin MCH platelets and white blood cells count and the biochemical tests total protein albumin globulin alkaline phosphatase aspartate aminotransferase AST total bilirubin ferritin vitamin B12 folic acid and prostatespecific antigen PSA urea creatinine electrolytes and spot urine proteincreatinine ratio protein electrophoresis and immunoelectrophoresis of serum and urine were within normal limits Thyroid function tests were within normal limits Skeletal surveys electrocardiograms and radiological findings liver spleen and kidneys were unremarkable Bone marrow biopsy showed no abnormalities Therefore the diagnosis of localized primary intestinal AL amyloidosis involving terminal ileum cecum colon and rectum associated ulceration and inflammatoryregenerative changes was establishedThe study presents a representative retrospective case in which the archival formalinfixed paraffinembedded tissues from the consultation files of the authors were used [] All the materials paraffinembedded tissue block slides and pathology report were coded The information obtained was analyzed and reported in such a way that the identity of the case presented cannot absolutely be ascertained The patient consent was obtainedDiscussionLocalized amyloidosis of the intestine is of interest to the pathologists and gastroenterologists because some cases may s The authors Journal compilation Gastroenterol Res and Elmer Press Inc„¢ wwwgastrores 0cAlshehri Gastroenterol Res mimic tumors Here we present a comprehensive review of the literature about this entity In agreement with previous reports [ ] our analysis indicates that the primary localized intestinal amyloidosis is a rare condition with disparate and nonspecific clinicopathological and endoscopic features Also its outcome depends on the site of the deposition of the amyloid small intestine versus colorectum [ ] In Cowan examined the clinicopathological features of patients with amyloidosis Seventysix patients had gastrointestinal amyloidosis The age range of the patients was years The patients include cases with localized gastrointestinal amyloidosis and cases with gastrointestinal involvement as a part of systemic amyloidosis In the latter mutations of the transthyretin gene were found Gastrointestinal bleeding and weight loss were the most common presenting symptoms []Threlkeld and Nguyen reported as a case of isolated colonic amyloidosis in a 46yearold male patient who presented with hematochezia and abdominal pain Colonoscopic studies revealed the presence of a tumorlike mass in the descending colon and therefore partial colectomy was performed Grossly the mucosa was necrotic but no mass lesion was identified There were light chainspositive amyloid deposits in the muscle layers submucosa and submucosal blood vessels Laboratory investigations for inflammatory condition and plasma cell disorders were negative and therefore the diagnosis of isolated gastrointestinal amyloidosis was established The patient received colchicine with marked clinical improvement [] Similarly Diaz Del Arco reported a case of globular amyloid deposits involving the left colon in a 74yearold male patient presenting with anemia and colitis Histologically there were Congo red positive deposits in the lamina propria of the colon []The duodenum is an important site for the deposition of amyloid protein Patients with primary duodenal amyloidosis may present with epigastric pain nausea constipation steatorrhea protein loss malabsorption hemorrhage and vomiting Endoscopic findings include the thickening of the duodenal wall and the presence of polypoid lesions [ ] Deguchi described a case of an isolated duodenal and jejunal amyloidosis in a 47yearold male patient who presented with features of intestinal pseudoobstruction Endoscopy revealed thickening and polypoid lesions of the duodenal mucosal folds The further histological evaluation confirmed the presence of amyloid deposits in the duodenal and jejunal walls [] Similarly Choi reported a case of localized amyloidosis of the small intestine duodenum and jejunum in a 62yearold male patient who presented with abdominal pain nausea and constipation of several years duration Endoscopy revealed mucosal erosions ulceration and tumorlike masses Immunohistological analysis revealed the presence of lambda light chain protein therefore establishing the diagnosis of amyloidosis []In the primary localized amyloidosis the isolated deposition of amyloid proteins may be due to the local tissue damage with the synthesis and deposition of amyloid substances in the individual ans [] The synthesis of amyloid results from plasma cell reaction to the inflammatory antigens The otherwise benign polyclonal plasma cells produce monoclonal immunoglobulin light chains L that are deposited as AL amyloid [] It is still possible that the deposition of amyloid results from the inability of the body to clear light chains produced by plasma cells [] Amyloid deposits in the intestine are usually irreversible but can sometimes be reversible with chemotherapy treatment especially with melphalan and steroid therapy []In primary localized intestinal amyloidosis we found variations in the site of deposition of amyloid proteins There was the deposition of the amyloid proteins in the walls of the blood vessels mucosa submucosa muscularis mucosae muscularis propria and the neural elements [] The destructive effects of amyloid usually resulted from the local replacement of these important histological compartments For instance amyloid deposition in the wall of the blood vessels usually leads to ischemia bowel infarction [] or intestinal perforation [] The involvement of the mucosa by amyloid deposits usually produces mucosal ulcerations leading to malabsorption [] hematochezia and anemia [ ] The deposition of amyloid in the submucosa and the muscle layers usually leads to the formation of the mass lesions amyloidoma or amyloid tumor resulting in intestinal obstruction [] The involvement of the neural elements results in altered bowel motility and chronic ileus [] Hemorrhage melena and bleeding per rectum is reasoned to bowel ischemia hemorrhagic necrosis of the mucosa damage of the vessel walls and abnormal platelet aggregation []The variations in the gross findings of intestinal amyloidosis may be reasoned to the type and the site of deposition of the amyloid protein When the deposition of the amyloid protein occurs predominantly in the perivascular and interstitial spaces it usually produces diffuse thickening of the intestinal wall rather than polypoid or tumorlike lesions [ ] Elevations or localized tumorlike lesions may reflect the presence of nodular aggregates of plasma cells locally producing abundant amyloid proteins which usually originate from AL protein produced by local aggregates of plasma cells involving the mucosa submucosa or the muscle layer [ ] Fine granular lesions or mucosal erosion or ulcerations are commonly associated with the deposition of AA amyloidosis [ ]To conclude the data presented here provides an indepth characterization of the clinicopathological features of primary localized intestinal amyloidosis It indicates that this entity is rare and its presenting features are generally nonspecific and therefore the diagnosis is challenging and can be easily missed Knowledge of this type of amyloidosis is warranted to avoid aggressive management such as treatment with an alkylating agent A high index of suspicion should be maintained both on the part of the gastroenterologists and the histopathologistsAcknowledgmentsNone to declareFinancial DisclosureNone to declares The authors Journal compilation Gastroenterol Res and Elmer Press Inc„¢ wwwgastrores 0cLocalized Amyloidosis of the Intestine Gastroenterol Res Conflict of InterestNone to declareInformed ConsentInformed consent was obtainedAuthor ContributionsSaeed Ali Alshehri conceived and designed the analyses collected the data biochemical and clinicopathologic aspects of amyloidosis performed the analyses tools including the statistical data analysis and wrote the paper Mahmoud Rezk Abdelwahed Hussein supervised the data analysis and contributed to the conception of the idea of the manuscript data collection Histological aspects of amyloidosis writing up and revising of the manuscriptData AvailabilityThe authors declare that data supporting the findings of this study are available within the References Westermark P Benson MD Buxbaum JN Cohen AS Frangione B Ikeda S Masters CL Amyloid fibril protein nomenclature Amyloid Monge M Chauveau D Cordonnier C Noel LH Presne C Makdassi R Jaureguy M Localized amyloidosis of the genitourinary tract report of new cases and review of the literature Medicine Baltimore Mathis H [Rokitansky Virchow and Heschl on the problem of amyloidosis a historical study] Zentralbl Allg Pathol Sack GH Jr Serum amyloid A a review Mol Med Gillmore JD Wechalekar A Bird J Cavenagh J Hawkins S Kazmi M Lachmann HJ Guidelines on the diagnosis and investigation of AL amyloidosis Br J Haematol Cowan AJ Skinner M Seldin DC Berk JL Lichtenstein DR O'Hara CJ Doros G Amyloidosis of the gastrointestinal tract a 13year singlecenter referral experience Haematologica Ankarcrona M Winblad B Monteiro C Fearns C Powers ET Johansson J Westermark GT Current and future treatment of amyloid diseases J Intern Med Selkoe DJ Folding proteins in fatal ways Nature Otaka Y Goda F Nakazato Y Tsutsui T Systemic heavy and lightchain amyloidosis presenting nephrotic syndrome and congestive heart failure a case presentation and literature review Amyloid 201926sup19596 Picken MM The pathology of amyloidosis in classification a review Acta Haematol Trinh TD Jones B Fishman EK Amyloidosis of the colon presenting as ischemic colitis a case report and review of the literature Gastrointest Radiol Threlkeld C Nguyen TH Isolated amyloidosis of the colon J Am Osteopath Assoc GarciaGonzalez R Fernandez FA Garijo MF Fernando ValBernal J Amyloidosis of the rectum mimicking collagenous colitis Pathol Res Pract Tada S Iida M Yao T Kawakubo K Yao T Okada M Fujishima M Endoscopic features in amyloidosis of the small intestine clinical and morphologic differences between chemical types of amyloid protein Gastrointest Endosc

cancer217

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: "Gastroenterol Res Primary Localized Amyloidosis of the Intestine A Pathologist ViewpointSaeed Ali Alshehria c Mahmoud Rezk Abdelwahed HusseinbAbstractBackground Localized amyloidosis of the intestine is a rare entity which can clinically masquerade several conditions such as colitis polyps and malignant tumors This study aims to evaluate the clinicopathological features of this entityMethods To evaluate the clinicopathological features of this entity a comprehensive search of the literature to was done using the following keywords œamyloidosis and œsmall intestine or œduodenum or œileum or œjejunum or œcolon We identified studies about gastrointestinal amyloidosis Data were examined for studies about localized intestinal amyloidosis The clinicopathological features were describedResults The age at presentation ranged from to years The male to female ratio was The jejunum and sigmoid colon were the most commonly involved sites Abdominal pain and intestinal obstruction small intestine or rectal bleeding sigmoid region were the most common clinical presentations Colonoscopic findings included wall thickening mucosal ulcerations small intestine and tumorlike masses colonConclusions The clinical presentations of localized intestinal amyloidosis depend on the site of the deposition of the amyloid In most cases amyloid deposits consisted of light chain proteinKeywords Amyloidosis Amyloid Colon Small intestine Rectum Gastrointestinal tractIntroductionThe term œamyloid was initially coined to describe materials Manuscript submitted June accepted July Published online August aDepartment of Pathology Armed Forces Hospitals Southern Region King Fahd Hospital Saudi ArabiabDepartment of Pathology Faculty of Medicine Assuit University Hospitals Assuit EgyptcCorresponding Author Saeed Ali Alshehri Department of Pathology Chemistry Section Armed Forces Hospital Southern Region Khamis Mushyte Saudi Arabia Email syd46118gmailcom 1014740gr1303found in plants and has been thought to represent carbohydrate Greek œamylon starch In medical literature the term œAmyloidosis has been known for more than years It was named after the deposition of the hallmark protein called crosssheet amyloid fibrils which represents either misfolded or misassembled proteins The nomenclature of the amyloid protein uses two letters The first letter œA refers to amyloid whereas the second refers to the precursor proteins [] The amyloid proteins include several types such as immunoglobulin light chains AL in primary amyloidosis transthyretin TTR beta2microglobulin in hemodialysisassociated amyloidosis and serum amyloid A SAA protein in secondary AA amyloidosis [] There are some associations between AL amyloid deposition and several hematological conditions including myeloma lymphoplasmacytic disorders and plasma cell dyscrasia Moreover AL amyloidosis and myeloma may show similar genetic abnormalities such as 14q translocations and 13q deletion []Amyloidoses are degenerative conditions associated with significant suffering not only for patients but also for their families as well [] They are characterized by the deposition of extracellular protein fibrils in the ans such as the kidneys nerves gastrointestinal tract heart and skin Amyloidoses are associated with significant morbidities such as malabsorption renal insufficiency and hematological disorders or even lifethreatening events such as intestinal obstruction renal failure and fatal arrhythmia The clinicopathological features of amyloidosis are nonspecific They depend on the type of the precursor protein site and severity of the amyloid deposition The diagnosis of amyloidosis depends on the examination of the tissue biopsies from the lesions It is established by the detection of amyloid deposits with its characteristic birefringence under polarized light following staining with Congo red or thioflavinT or immunohistochemical stains Management of amyloidosis includes reduction of the supply of amyloid fibril precursor proteins stabilization of the precursor protein formation administration of antiplasma therapies general care systemic amyloidosis or surgical resection in localized amyloidosis [ ]Amyloidosis includes both primary and secondary types In the former also known as AL for amyloid light chain there is no evidence of coexisting disease and it has an immunoglobulin light chain as a major protein component In secondary amyloidosis also known as AA for amyloidassociated there is evidence of coexisting disease resulting in the production of inherently amyloidogenic proteins or overproduction of potentially amyloidogenic normal proteins and it has serum amyloidassociated protein SAA The deposition of amys The authors Journal compilation Gastroenterol Res and Elmer Press Inc„¢ wwwgastroresThis is distributed under the terms of the Creative Commons Attribution NonCommercial International License which permits unrestricted noncommercial use distribution and reproduction in any medium provided the original work is properly cited 0cLocalized Amyloidosis of the Intestine Gastroenterol Res loid may be either widespread systemic amyloidosis [ ] or restricted to a specific an localized amyloidosis In systemic amyloidosis the amyloidogenic proteins are synthesized at sites far away from the sites of amyloid deposition The amyloid protein is deposited at several sites and therefore the patients have disparate clinical manifestations such as arrhythmia neuropathies malabsorption and proteinuria due to cardiac neural intestinal and renal involvement respectively [] Currently the classification of amyloidosis is based on the type of precursor proteins []Localized amyloidosis refers to the infiltration of the tissues by amyloid where the precursor proteins are synthesized at or close to the site of deposition It includes both anspecific amyloidoses where the amyloid is produced by the an itself and nonan specific amyloidosis where the fibrillar protein is not specific to the affected an [] Localized amyloidosis of the intestine is a rare condition that involves the duodenum jejunum and ileum appendix or colorectum The clinicopathological features are nonspecific and therefore they can masquerade several conditions such as collagenous colitis ischemic colitis bowel infarction perforation polyps and malignant tumors [] The colonoscopic features include mucosal granularity friability erosions ulcerations petechial hemorrhage and polypoid lesions [] The radiological findings include ulcerations thickening of the mucosal folds narrowing of the colonic lumen effacement of haustrations and nonspecific mucosal nodularity []To date few cases of localized intestinal amyloidosis have been reported [ ] In Tada examined patients with small intestinal amyloidosis Clinical findings included malabsorption mass lesions diarrhea and hematochezia Endoscopy of the jejunum revealed polypoid lesions wall thickening erosions ulcerations and fine granular appearance The amyloid proteins included amyloid A protein AA light chain protein AL beta 2microglobulin and prealbumin Histologically amyloid deposits were seen in the submucosa and muscle layer [] Chen reported a case of localized amyloidosis of the transverse colon in a 52yearold male patient The clinical presentations included abdominal pain bloody stool and weight loss Colonoscopy revealed a marked narrowing of the transverse colon associated with the presence of multiple polypoid growths The clinical and radiological findings supported the initial impression of carcinoma and therefore the patient underwent right hemicolectomy with lymph node dissection [] Grossly there was an ulcerative mass with marked narrowing of the lumen Histologically amyloid deposits were seen in the lamina propria []To date our knowledge about the clinicopathological features of the localized intestinal amyloidosis is limited To gain insights into these issues we conducted a comprehensive literature search and metaanalysis of the relevant published literature Also we presented a rare case of primary isolated intestinal amyloidosisMaterials and MethodsThis is a viewpoints study not an original research investigation that did not include any interaction or intervention with human subjects or any access to any identifiable private information Therefore the study did not require institutional review board review [] This study was performed in accordance with the principles of the Declaration of Helsinki []Search strategy and selection criteriaThe authors adhered to Preferred Reporting Items for Systematic Reviews and MetaAnalyses PRISMA guidelines [] The literature was reviewed by searching the PubMed electronic database using the key term œamyloidosis and the following words œintestine or œsmall intestine or œduodenum or œileum or œjejunum or œappendix or œcolon or œsigmoid or œrectum andor œcecum to identify the eligible studies The search results were considered based on their titles abstracts and publication date Their full texts were examined to confirm their eligibility and then were included in the study A summary of the search strategy and selection criteria is shown in Figure [ ]Data extraction a methodological assessment and statistical analysisThe clinicopathological findings were extracted including the family name of the first author year of publication patient age gender symptoms endoscopic features histology special stains and presence or absence of systemic involvement The authors independently read and analyzed each study They defined the final diagnosis as œthe histopathological diagnosis to ensure homogeneous evaluation of the findings Statistical computations were performed using IBMSPSS IBMSPSS Inc Chicago IL USA Descriptive statistics mean standard error of mean and ranges were calculatedResultsFlow trialOur search yielded previous studies about gastrointestinal amyloidosis of which were excluded The remaining s have undergone œabstract review These studies covered a period of years to This relatively long period allowed proper evaluation of a good number of cases in the literature Twentyseven studies with the final diagnosis of localized intestinal amyloidosis were included in the present study In all cases the workup for monoclonal gammopathy and systemic amyloidosis serum protein electrophoresis and pathological examination of the tissue specimens was performed The diagnosis of localized amyloidosis was substantiated by the use of ancillary studies including radiological examination such as abdominal ultrasound barium enema []wholebody computed tomography [] magnetic resonance imaging positron emission tomography [] skeletal surveys electrocardiography and echocardiography [ ] s The authors Journal compilation Gastroenterol Res and Elmer Press Inc„¢ wwwgastrores 0cAlshehri Gastroenterol Res Figure Flow chart of literature search and study selection for cases of localized intestinal amyloidosis The inclusion criteria included patients who had amyloid deposition in the intestine without evidence of systemic involvement or an associated condition leading to secondary amyloidosis [] The exclusion criteria included patients with one or more of the followings Evidence of chronic diseases such as collagen vascular disease that may have led to secondary amyloidosis [] Additional an involvement by amyloidosis Intestinal symptoms suggestive of amyloidosis but without confirmatory diagnostic tissue biopsy Plasma cell dyscrasia myeloma lymphoplasmacytic disorders or other Bcell malignancies such as Waldenstrom™s macroglobulinemia [] Systemic AL type [] Laboratory findings revealing positive detection of monoclonal light chains in serum andor urine []The reported cases did not have any evidence of generalized metabolic complications or extraintestinal complications of accumulated amyloidosis [ ] A summary of the flow chart of the literature search and study selection criteria is shown in Figure [ ]Clinical presentations and treatment modalities in localized intestinal amyloidosisThe colorectum jejunum transverse colon ileum and duodenum were the most commonly affected sites The lesions were more common in males than in females The mean age at presentation was ± years small intestine and ± years colorectum [ ] In the small intestine the main symptoms were abdominal pain [ ] and intestinal obstruction [ ] followed by diarrhea and vomiting [ ] bleeding [ ] malabsorption [] and constipation [] In the colorectum the main symptoms were rectal bleeding [ ] followed by abdominal pain [ ] anemia [] intestinal obstruction [] and weight loss [] Chen described a case of localized amyloidosis of the transverse colon in a 52yearold male patient who presented with a periumbilical pain rectal bleeding and weight loss Colonoscopic examination revealed mucosal ulcerations and luminal narrowing by multiple polypoid lesions [] Treatment modalities included in these cases included drug therapy prednisolone azathioprine melphalan and colchicines [ ] intestinal resection partial jejunectomy endoscopic submucosal resection [ ] and colonic resection [ ] with close followup of the patients [ ] A summary of these results is presented in Tables [ ]s The authors Journal compilation Gastroenterol Res and Elmer Press Inc„¢ wwwgastrores 0cLocalized Amyloidosis of the Intestine Gastroenterol Res Table Data From Individual Studies of Primary Localized Amyloidosis of the Small IntestineCase Sex Age years SitesIleumClinical manifestationsColonoscopic findingsStudies[][][][][][][][][][][][][][]FMFMFMMMMMMFMiddle ageAgedAcute abdominal pain diarrhea and vomitingPseuntestinal obstructionMelena and anemiaJejunumJejunumSmall intestineIntestinal obstructionJejunum Chronic diarrhea and and ileummalabsorptionSmall intestine Acute abdomenDuodenum and jejunumSmall intestineAbdominal pain nausea and constipationSmall bowel obstructionSmall intestine Abdominal pains and hematemesisThickening of the wall of the ileum and ascending colonThickening of the mucosal folds and multiple polypoid protrusionsDiffuse thickening of the mucosa with friability nodularity erosions and polypoid protrusionsDiffuse thickening of the wall of the small intestineThickening of the jejunal and proximal ileal mucosal foldsPerforation of the wallMultiple shallow ulcers and several erosionsBand of amyloid and connective tissue surrounding the small intestineMucosal ulcerations with discrete nodulesSmall intestine Nonspecific digestive symptoms Multiple mucosal polypsJejunumSmall intestine Nonspecific digestive symptoms Thickening of the wall with rough and Nonspecific digestive symptoms Polypoid pseudotumoral mucosal formationsHeartburn and constipationpolypoid intestinal mucosal foldsF M MSmall intestine three casesDuodenumScreening for cancer stomachA localized depressed lesionEndoscopic and histological findings in localized intestinal amyloidosisIn the small intestine the most common endoscopic findings were thickening of the intestinal wall [ ] and polypoid mucosal protrusions [] followed by mucosal ulceration [ ] friability and nodularity of the mucosa [ ] and wall perforations [] In the colorectum polypoid masses with luminal narrowing were the most common endoscopic finding [ ] followed by mucosal ulcerations solitary or multiple ulcers [ ] or and thickening of the wall [ ] nodularity and friability of the mucosa [ ] and perforation [] In all cases deposition of pale eosinophilic amorphous amyloid material with the prominent cracking artifact in the mucosa submucosa muscle layer or walls of the blood vessels represented the main histological feature [ ] The further Congo red staining revealed the characteristic salmonpink color confirming the presence of amyloid deposits The deposits showed the characteristic applegreen birefringence under polarized light Immunohistochemical stains were also performed in some cases to substantiate the diagnosis Other histological features included mucosal ulceration amyloid angiopathy necrotizing angiitis [] foreign body giant cell reaction [] ischemic changes focal active colitis and cryptitis a personal observation The amyloid type was AL light chain protein in the majority of the cases of the small intestine [ ] and colorectum [ ] Other amyloid protein types included AAamyloid [] and high serum amyloid SAA in some cases [] Summary of clinicopathological findings in patients with localized intestinal amyloidosis is presented in Tables [ ]Case reportPrimary localized amyloidosis of the intestineA 55yearold female patient presented with anorexia diarrhea rectal bleeding and weight loss Colonoscopy revealed picture reminiscent of active pancolitis friable mucosa with mucosal erosions ulcerations and hemorrhage and six biopsies from the ileum cecum and colon ascending transverse descending colons and rectum were obtained On histology all biopsies showed deposition of pale eosinophilic amorphous material with the prominent cracking artifact characteristic of amyloid in the lamina propria and submucosa In some areas s The authors Journal compilation Gastroenterol Res and Elmer Press Inc„¢ wwwgastrores 0cAlshehri Gastroenterol Res Table Data From Individual Studies of Primary Localized Amyloidosis of the ColonCase SexSitesClinical presentationColonoscopic findingsStudiesAge yearsMMMMMFMMMFMFTransverse colonbleeding and weight lossIntestinal obstructionTransverse colon A periumbilical pain rectal Descending colon AnemiaSigmoid colonUlcerations and luminal narrowing by multiple polypoid lesionsElderlyA stenosing massMiddle age Transverse colon Nonspecific digestive symptoms Submucosal massUlcerative lesionsMucosal ulceration perforation and thickening of the bowel wallA submucosal tumorlike massMucosal ulcerA single friable rounded mucosal lesionA 15cm shallow depressed mucosal lesionAcute abdominal pain rectal bleeding and fecal peritonitisHematocheziaHemepositive stoolsRectal bleedingRoutine colonoscopyNonspecific digestive symptoms Several irregularlyshaped discrete ulcerationsSigmoid colonSigmoid colonSigmoid colonSigmoid colonSigmoidDescending colon Abdominal pain bloating flatulence and hematocheziaRectal bleedingHemorrhagic mucosa and amass lesionDiffuse nodular friable lesions ascending colon and several irregular large ulcers with nodularity descending colonThickening of the wall and mucosal ulcerationsAscending and descending colonsColon seven casesAscending colonF M Middle age ColonF M Rectal bleeding two casesHeartburn and constipationFAcute abdominal pain diarrhea and vomitingThickening of the wall of the ileum and ascending colon[][][][][][][][][][][][][][][]Table Clinical Colonoscopic and Histological Findings in the Primary Localized Amyloidosis of the IntestineAspectsAge mean ± SEMMale to female ratioSite of involvementClinical presentationsColonoscopic findingsHistological featuresSmall intestine duodenum jejunum and ilium ± years [ ]Jejunum Ileum Colorectum ± years [ ]The left colon Transverse colon Abdominal pain and intestinal obstruction [ ]Rectal bleeding [ ] followed by abdominal pain [ ] and anemia []Thickening of the wall [ ] polyps and polypoid mucosal protrusions [] ulcerations [ ] friability and nodularity of the mucosa [ ] and perforation []Mass lesions tumorlike lesion polypoid protrusions and polyps with narrowing were the most common [ ] Ulcerations with solitary or multiple ulcers [ ] thickening of the wall [ ] nodularity and friability of the mucosa [ ] and perforation []Amyloid deposits increased density of mixed inflammatory cells in the lamina propria light chain protein [ ]Amyloid deposits increased density of mixed inflammatory cells in the lamina propria foreign body giant cell reaction amyloid angiopathy necrotizing angiitis focal active colitis and immunoglobulin lightchain AL [ ]s The authors Journal compilation Gastroenterol Res and Elmer Press Inc„¢ wwwgastrores 0cLocalized Amyloidosis of the Intestine Gastroenterol Res Figure Isolated primary amyloidosis of the intestine case report a e Colonic mucosa with deposition of pale eosinophilic amorphous material star in the submucosa with abundant deposits of pink amorphous materials with some cracking artifacts characteristic of amyloid Focal surface mucosal ulceration is noted The background mucosa shows focal active inflammation including cryptitis arrow and there is patchy crypt distortion No granulomas are seen hematoxylin and eosin a × b × c d × and e × f g Congo red stain which reveals the characteristic salmonpink color confirming the presence of amyloid deposits Congo red f × and g × h i The deposits exhibit characteristic applegreen birefringence under polarized light Congo red under polarized light h × and i × the lamina propria was entirely replaced by these deposits The background mucosa showed focal active inflammation focal ulceration cryptitis occasional crypt abscess This focal chronic active inflammatory change may be secondary to erosionulceration associated with amyloid deposits [] Congo red stain confirmed the presence of amyloid deposits with applegreen birefringence under polarized light Fig Immunohistochemical analysis revealed that the amyloid deposits were positive for amyloid lambda but negative for amyloid A These findings were consistent with amyloid lightchain AL amyloidosis We performed several laboratory and radiological investigations to rule out the possibility of systemic involvement The hematological indices red blood cells RBCs count hemoglobin mean corpuscular volume MCV mean corpuscular hemoglobin MCH platelets and white blood cells count and the biochemical tests total protein albumin globulin alkaline phosphatase aspartate aminotransferase AST total bilirubin ferritin vitamin B12 folic acid and prostatespecific antigen PSA urea creatinine electrolytes and spot urine proteincreatinine ratio protein electrophoresis and immunoelectrophoresis of serum and urine were within normal limits Thyroid function tests were within normal limits Skeletal surveys electrocardiograms and radiological findings liver spleen and kidneys were unremarkable Bone marrow biopsy showed no abnormalities Therefore the diagnosis of localized primary intestinal AL amyloidosis involving terminal ileum cecum colon and rectum associated ulceration and inflammatoryregenerative changes was establishedThe study presents a representative retrospective case in which the archival formalinfixed paraffinembedded tissues from the consultation files of the authors were used [] All the materials paraffinembedded tissue block slides and pathology report were coded The information obtained was analyzed and reported in such a way that the identity of the case presented cannot absolutely be ascertained The patient consent was obtainedDiscussionLocalized amyloidosis of the intestine is of interest to the pathologists and gastroenterologists because some cases may s The authors Journal compilation Gastroenterol Res and Elmer Press Inc„¢ wwwgastrores 0cAlshehri Gastroenterol Res mimic tumors Here we present a comprehensive review of the literature about this entity In agreement with previous reports [ ] our analysis indicates that the primary localized intestinal amyloidosis is a rare condition with disparate and nonspecific clinicopathological and endoscopic features Also its outcome depends on the site of the deposition of the amyloid small intestine versus colorectum [ ] In Cowan examined the clinicopathological features of patients with amyloidosis Seventysix patients had gastrointestinal amyloidosis The age range of the patients was years The patients include cases with localized gastrointestinal amyloidosis and cases with gastrointestinal involvement as a part of systemic amyloidosis In the latter mutations of the transthyretin gene were found Gastrointestinal bleeding and weight loss were the most common presenting symptoms []Threlkeld and Nguyen reported as a case of isolated colonic amyloidosis in a 46yearold male patient who presented with hematochezia and abdominal pain Colonoscopic studies revealed the presence of a tumorlike mass in the descending colon and therefore partial colectomy was performed Grossly the mucosa was necrotic but no mass lesion was identified There were light chainspositive amyloid deposits in the muscle layers submucosa and submucosal blood vessels Laboratory investigations for inflammatory condition and plasma cell disorders were negative and therefore the diagnosis of isolated gastrointestinal amyloidosis was established The patient received colchicine with marked clinical improvement [] Similarly Diaz Del Arco reported a case of globular amyloid deposits involving the left colon in a 74yearold male patient presenting with anemia and colitis Histologically there were Congo red positive deposits in the lamina propria of the colon []The duodenum is an important site for the deposition of amyloid protein Patients with primary duodenal amyloidosis may present with epigastric pain nausea constipation steatorrhea protein loss malabsorption hemorrhage and vomiting Endoscopic findings include the thickening of the duodenal wall and the presence of polypoid lesions [ ] Deguchi described a case of an isolated duodenal and jejunal amyloidosis in a 47yearold male patient who presented with features of intestinal pseudoobstruction Endoscopy revealed thickening and polypoid lesions of the duodenal mucosal folds The further histological evaluation confirmed the presence of amyloid deposits in the duodenal and jejunal walls [] Similarly Choi reported a case of localized amyloidosis of the small intestine duodenum and jejunum in a 62yearold male patient who presented with abdominal pain nausea and constipation of several years duration Endoscopy revealed mucosal erosions ulceration and tumorlike masses Immunohistological analysis revealed the presence of lambda light chain protein therefore establishing the diagnosis of amyloidosis []In the primary localized amyloidosis the isolated deposition of amyloid proteins may be due to the local tissue damage with the synthesis and deposition of amyloid substances in the individual ans [] The synthesis of amyloid results from plasma cell reaction to the inflammatory antigens The otherwise benign polyclonal plasma cells produce monoclonal immunoglobulin light chains L that are deposited as AL amyloid [] It is still possible that the deposition of amyloid results from the inability of the body to clear light chains produced by plasma cells [] Amyloid deposits in the intestine are usually irreversible but can sometimes be reversible with chemotherapy treatment especially with melphalan and steroid therapy []In primary localized intestinal amyloidosis we found variations in the site of deposition of amyloid proteins There was the deposition of the amyloid proteins in the walls of the blood vessels mucosa submucosa muscularis mucosae muscularis propria and the neural elements [] The destructive effects of amyloid usually resulted from the local replacement of these important histological compartments For instance amyloid deposition in the wall of the blood vessels usually leads to ischemia bowel infarction [] or intestinal perforation [] The involvement of the mucosa by amyloid deposits usually produces mucosal ulcerations leading to malabsorption [] hematochezia and anemia [ ] The deposition of amyloid in the submucosa and the muscle layers usually leads to the formation of the mass lesions amyloidoma or amyloid tumor resulting in intestinal obstruction [] The involvement of the neural elements results in altered bowel motility and chronic ileus [] Hemorrhage melena and bleeding per rectum is reasoned to bowel ischemia hemorrhagic necrosis of the mucosa damage of the vessel walls and abnormal platelet aggregation []The variations in the gross findings of intestinal amyloidosis may be reasoned to the type and the site of deposition of the amyloid protein When the deposition of the amyloid protein occurs predominantly in the perivascular and interstitial spaces it usually produces diffuse thickening of the intestinal wall rather than polypoid or tumorlike lesions [ ] Elevations or localized tumorlike lesions may reflect the presence of nodular aggregates of plasma cells locally producing abundant amyloid proteins which usually originate from AL protein produced by local aggregates of plasma cells involving the mucosa submucosa or the muscle layer [ ] Fine granular lesions or mucosal erosion or ulcerations are commonly associated with the deposition of AA amyloidosis [ ]To conclude the data presented here provides an indepth characterization of the clinicopathological features of primary localized intestinal amyloidosis It indicates that this entity is rare and its presenting features are generally nonspecific and therefore the diagnosis is challenging and can be easily missed Knowledge of this type of amyloidosis is warranted to avoid aggressive management such as treatment with an alkylating agent A high index of suspicion should be maintained both on the part of the gastroenterologists and the histopathologistsAcknowledgmentsNone to declareFinancial DisclosureNone to declares The authors Journal compilation Gastroenterol Res and Elmer Press Inc„¢ wwwgastrores 0cLocalized Amyloidosis of the Intestine Gastroenterol Res Conflict of InterestNone to declareInformed ConsentInformed consent was obtainedAuthor ContributionsSaeed Ali Alshehri conceived and designed the analyses collected the data biochemical and clinicopathologic aspects of amyloidosis performed the analyses tools including the statistical data analysis and wrote the paper Mahmoud Rezk Abdelwahed Hussein supervised the data analysis and contributed to the conception of the idea of the manuscript data collection Histological aspects of amyloidosis writing up and revising of the manuscriptData AvailabilityThe authors declare that data supporting the findings of this study are available within the References Westermark P Benson MD Buxbaum JN Cohen AS Frangione B Ikeda S Masters CL Amyloid fibril protein nomenclature Amyloid Monge M Chauveau D Cordonnier C Noel LH Presne C Makdassi R Jaureguy M Localized amyloidosis of the genitourinary tract report of new cases and review of the literature Medicine Baltimore Mathis H [Rokitansky Virchow and Heschl on the problem of amyloidosis a historical study] Zentralbl Allg Pathol Sack GH Jr Serum amyloid A a review Mol Med Gillmore JD Wechalekar A Bird J Cavenagh J Hawkins S Kazmi M Lachmann HJ Guidelines on the diagnosis and investigation of AL amyloidosis Br J Haematol Cowan AJ Skinner M Seldin DC Berk JL Lichtenstein DR O'Hara CJ Doros G Amyloidosis of the gastrointestinal tract a 13year singlecenter referral experience Haematologica Ankarcrona M Winblad B Monteiro C Fearns C Powers ET Johansson J Westermark GT Current and future treatment of amyloid diseases J Intern Med Selkoe DJ Folding proteins in fatal ways Nature Otaka Y Goda F Nakazato Y Tsutsui T Systemic heavy and lightchain amyloidosis presenting nephrotic syndrome and congestive heart failure a case presentation and literature review Amyloid 201926sup19596 Picken MM The pathology of amyloidosis in classification a review Acta Haematol Trinh TD Jones B Fishman EK Amyloidosis of the colon presenting as ischemic colitis a case report and review of the literature Gastrointest Radiol Threlkeld C Nguyen TH Isolated amyloidosis of the colon J Am Osteopath Assoc GarciaGonzalez R Fernandez FA Garijo MF Fernando ValBernal J Amyloidosis of the rectum mimicking collagenous colitis Pathol Res Pract Tada S Iida M Yao T Kawakubo K Yao T Okada M Fujishima M Endoscopic features in amyloidosis of the small intestine clinical and morphologic differences between chemical types of amyloid protein Gastrointest Endosc Answer:

Thyroid_Cancer

"Long non‘coding RNA FOXD2‘AS1 regulates the tumorigenesis and progression of breast cancer via the S100 calcium binding protein A1Hippo signaling pathwayPEI HUANG1 and JINHUI XUE21Department of Pathology The First Affiliated Hospital of Zhengzhou University Zhengzhou Henan 2Department of Pathology Zhengzhou Central Hospital Affiliated to Zhengzhou University Zhengzhou Henan PR ChinaReceived November Accepted April 103892ijmm20204699Abstract Breast cancer is one of the most prevalent cancer types and is accompanied by a high incidence and mortality rate severely threatening women's health globally Long noncoding RNA forkhead box d2 adjacent apposite strand RNA lncRNA FOXD2‘AS1 has been identified to function as an oncogene in human cancers however it has rarely been investigated in breast cancer The aim of the present study was to investigate the role of FOXd2AS1 in breast cancer and to clarify the underlying mechanisms The expression of FOXD2‘AS1 in breast cancer cell lines was first quantified by reverse transcription‘quantitative PCR and the biological function of FOXD2‘AS1 was then determined Cellular proliferative ability was determined by Cell Counting kit‘ assay and wound healing and Transwell assays were conducted to assess the cell migratory and invasive ability corresponding protein expression levels were determined by western blot analysis In addition experimental animal models were established by the subcutaneous injection of MDA‘MB‘ cells into the right axillary lymph nodes of BALBc nude mice and the effects of FOXD2‘AS1 on tumor growth were observed The results indicated that FOXD2‘AS1 expression was upregulated in breast cancer cell lines and that FOXD2‘AS1 downregulation significantly inhibited the proliferation migration and invasiveness of MCF‘ and MDA‘MB‘ cells S100 calcium binding protein A1 S100A1 was also upregulated in breast cancer cell lines and was positively regulated by FOXD2‘AS1 Furthermore the inhibition of S100A1 and the overexpression of the serinethreonine‘protein kinase large tumor suppressor homolog LATS1 inhibited the FOXD2‘AS1‘induced Correspondence to Dr Pei Huang Department of Pathology The First Affiliated Hospital of Zhengzhou University East Jianshe Road Zhengzhou Henan PR ChinaE‘mail huangpei193163comKey words FOXD2‘AS1 S100 calcium binding protein A1 breast cancer large tumor suppressor homolog Hippocellular proliferation migration and invasiveness in breast cancer Experimental mouse models revealed that FOXD2‘AS1 downregulation significantly inhibited tumor growth and that the levels of phosphorylated p‘YAP and p‘LATS1 were upregulated by FOXD2‘AS1 knockdown indicating that the inhibition of FOXd2AS1 activated Hippoyesassociated protein signaling On the whole the findings of the present study suggest that the FOXD2‘AS1S100A1Hippo axis is involved in the tumorigenesis and progression of breast cancer In the future these may contribution to the identification of more effective breast cancer treatmentsIntroductionAs one of the most prevalent malignancies breast cancer is a primary cause of mortality among gynecological cancer cases and with increasing morbidity and mortality rates it poses a considerable threat to women's health worldwide In statistics from the American cancer Society estimated newly diagnosed cases and deaths from breast cancer in the United States The leading causes of the high death rate are distal metastasis and resistance to the existing treatments despite improvements in early diagnosis and systemic treatment the incidence of breast cancer and metastasisrelated mortality is steadily increasing Therefore there is an urgent need to elucidate the mechanisms responsible for the disordered cellular metastasis and to enhance our understanding of the tumorigenesis and development processes hence facilitating the identification of more efficient breast cancer treatmentsLong noncoding RNAs lncRNAs are a group of RNAs nucleotides in length which lack protein‘coding capacity Numerous studies have revealed that lncRNAs have versatile biological functions in pathological and physiological processes including tumorigenesis ‘ lncRNAs are considered to regulate the development of various types of cancer including breast cancer For instance LINC01089 is downregulated in breast cancer tissues and cell lines and LINC01089 overexpression increases tumor cell proliferation migration and invasiveness As an oncogene that regulates breast cancer cell proliferation and apoptosis hepatocellular 0cHUANG and XUE DOWNREGULATION OF FOXD2‘AS1 INHIBITS THE TUMORIGENESIS OF BREAST CANCERcarcinoma upregulated EZH2‘associated lncRNA is closely associated with the clinical progression of breast cancer These results indicate the indispensability of research on lncRNAs and breast cancerlncRNA forkhead box d2 adjacent apposite strand RNA FOXD2‘AS1 is a novel non‘coding RNA identified to be an oncogene in human cancers FOXD2‘AS1 has been shown to be upregulated in various types of cancer including glioma osteosarcoma and papillary thyroid cancer as well as breast cancer ‘ A previous study indicated that FOXD2‘AS1 participates in regulating the development of breast cancer via the miR‘‘5pPFN2 axis and that it may be a potential biomarker for the diagnosis and prognosis of breast cancer However to the best of our knowledge there are no additional data regarding the investigation of FOXd2AS1 in breast cancer and its effects and the underlying mechanisms on the regulation of breast cancer cell invasion and metastasis Thus the aim of the present study was to determine the role and potential mechanisms of action of FOXd2AS1 in breast cancer and to provide further support for its use in clinical diagnosis and treatmentMaterials and methodsDatasets The present study evaluated the expression level of FOXD2‘AS1 in breast cancer samples using The Cancer Genome Atlas TCGA dataset which was downloaded from the TCGA data portal tcga‘data‘nci‘nih‘govezxjtlueducn The TCGA data subset for breast cancer included normal samples and tumor samples The Mann‘Whitney test was used to determine statistically significant differences between normal and tumor samples P005 was considered to indicate a statistically significant differenceCell culture A human normal breast epithelial cell line MCF‘10A and human breast cancer cell lines MCF‘ MDA‘MB‘ MDA‘MB‘ and BT‘ were purchased from the American Type Culture Collection ATCC The cells were incubated in RPMI‘ medium supplemented with fetal bovine serum FBS in a humidified incubator at ˚C CO2 Transfection To overexpress FOXd2AS1 an overexpression vector pcdNA FOXd2AS1 and its corresponding negative control vector pcDNA‘NC were synthesized by Shanghai GenePharma Co Ltd Short hairpin shRNAs targeting FOXD2‘AS1 nM shRNA‘FOXD2‘AS1‘ and shRNAFOXd2AS12 and a negative scramble control shRNA shRNA also purchased from Shanghai GenePharma Co Ltd were used to knock down FOXD2‘AS1 expression In addition pcdNALATS1 shRNAS100A11 and shRNAS100A12 were obtained from Shanghai GenePharma Co Ltd to overexpress LATS1 or to knock down S100A1 respectively The shRNA sequences were as follows shRNA‘FOXD2‘AS1‘ targets GGA CTC CAC TCT TCG CTT A shRNA‘FOXD2‘AS1‘ targets GCT TCC AGG TAT GTG GGA A shRNA‘S100A1‘ targets GAT CCG GAG ACC CTC ATC AAC GTG TTC TTC CTG TCA GAA ACA CGT TGA TGA GGG TCT CCT TTT TG shRNA‘S100A1‘ targets GAT CCG TGG ACT TCC AGG AGT ATG TGC TTC CTG TCA GAC ACA TAC TCC TGG AAG TCC ACT TTT TG Cells were transfected with pcDNA FOXD2‘AS1 nM pcdNALATS1 nM pcdNANc nM shRNAFOXd2AS11 ngµl shRNAFOXd2AS12 ngµl shRNAS100A11 ngµl shRNAS100A12 ngµl shRNA ngµl or co transfected with pcdNA FOXd2AS1 and pcdNALATS1 or cotransfected with pcDNA FOXD2‘AS1 and shRNA‘S100A1 using Lipofectamine® transfection reagent Invitrogen Thermo Fisher Scientific Inc according to the manufacturer's instructions Lipofectamine reagent was first mixed with vectors to form a reagent‘vector complex followed by incubation with cells at ˚C for h The transfection efficacy was assessed by reverse transcription‘quantitative PCR RT‘qPCR after h of transfectionRNA extraction and RT‘qPCR Total RNA was extracted from all cell lines using TRIzol® reagent Takara Bio Inc according to the manufacturer's instructions Total RNA was then reverse transcribed into cDNA using the PrimeScript„¢ RT Master kit and the mRNA expression levels were quantified using the SYBR Premix Ex Taq„¢ kit Both Takara Bio Inc with a Fast Real‘Time PCR System Applied Biosystems Thermo Fisher Scientific Inc The sequences of specific primers used for RT‘qPCR were as follows FOXDA‘AS1 forward '‘TGG ACC TAG CTG CAG CTC CA‘' and reverse '‘AGT TGA AGG TGC ACA CAC TG‘' S100A1 forward '‘GAG TAT GTG GTG CTT GTG GC‘' and reverse '‘CTT GGA CCG CTA CTC TTG CG‘' large tumor suppressor homolog LATS1 forward '‘ACC GCT TCA AAT GTG ACT GTG ATG CCA C CT‘' and reverse '‘CTT CCT TGG GCA AGC TTG GCT GAT CCT CT‘' and GAPDH forward '‘GCG AGA TCG CAC TCA TCA TCT ‘' and reverse '‘TCA GTG GTG GAC CTG ACC ‘' The data were displayed as ‘ΔΔCq values with GAPDH as the constitutive marker The PCR conditions were as follows ˚C for min cycles of ˚C for sec and ˚C for sec followed by ˚C for minCell Counting Kit‘ CCK‘ assay cell proliferation was determined using the Cell Counting Kit‘ assay CCK‘ Dojindo Molecular Technologies Inc Briefly cells were seeded into a ‘well plate and incubated for h at ˚C Following culture for the indicated periods of time and h µl of the CCK‘ reagent were added to each well and the plate was incubated at ˚C for a further h The optical density values at nm were then measured using a microplate spectrophotometer Thermo Fisher Scientific IncWestern blot analysis The total protein was extracted from the cells using RIPA lysis buffer Beyotime Institute of Biotechnology and quantified using a BCA protein assay kit Thermo Fisher Scientific Inc The same amount of each protein sample µg was subjected to SDS‘PAGE the proteins were then transferred onto PVDF membranes EMd Millipore and was blocked in nonfat milk for h at room temperature The membranes were then incubated with primary antibodies against cyclinE1 cat no ab33911 Abcam cyclin‘dependent kinase‘ CDK2 cat no ab32147 Abcam p21 cat no ab109520 Abcam matrix metalloproteinases MMP2 cat no ab92536 Abcam MMP9 0cINTERNATIONAL JOURNAL OF MOLEcULAR MEdIcINE was added to the lower 24well chamber After h cells on the upper surface were removed and cells attached to the lower surface were stained with crystal violet Beijing Solarbio Science Technology Co Ltd at room temperature for min The cells were viewed under a light microscope magnification x100 CKX41 Olympus Corporation and the invasive ability of the cells was determined using ImageJ software version by counting the number of cells attached to the lower surfaceCell cycle analysis The cell cycle distribution was determined by flow cytometry After being subjected to the indicated treatments the cells were collected and fixed in ethanol at ‘ËšC overnight The cells were then washed twice with PBS and incubated in the dark with RNase A and PI staining solution Roche Diagnostics at ˚C for min Finally the cell samples were analyzed using a FACSCalibur flow cytometer and CellQuest software version both from BD BiosciencesIn vivo experiments The present study was approved by the First Affiliated Hospital of Zhengzhou University and the animal experiments were performed according to the National Institute of Health Guidelines for the Care and Use of Laboratory Animals A total of 5weekold male BALBc nude mice were purchased from the Experimental Animal Center of Shanghai Institute for Biological Sciences and housed in a standard environment ˚C humidity h lightdark cycle with free access to food and water Each mouse was subcutaneously injected at the right axillary lymph nodes with 10x107 MdAMB468 cells which were stably transfected with either the shRNA negative control shRNA or shRNA‘FOXD2‘AS1 The weights and tumor volumes tumor volume x length x width2 of the mice were monitored every days until the mice were sacrificed At days after the injection all the mice were sacrificed by cervical dislocation that caused a sharp section of the spinal cord followed by an instantaneous cardiac arrest After the cessation of the heartbeat and respiratory arrest of the mice was confirmed the tumors were excised photographed and stored for the further investigationStatistical analysis data are presented as the means ± standard deviation SD from ‰¥ independent experiments and each experiment was conducted in triplicate The data were analyzed using SPSS statistical software version SPSS Inc and the differences among groups were analyzed using one‘way analysis of variance followed by Tukey's post hoc test P005 was considered to indicate a statistically significant differenceResultsFOXD2‘AS1 expression is upregulated in breast cancer cells The TCGA database cancergovtcga was used to identify the association between FOXD2‘AS1 and breast cancer by evaluating the expression profiles of FOXD2‘AS1 in breast cancer tissues and normal tissues The results of TCGA analysis revealed a significantly higher FOXd2AS1 expression in breast cancer tissues than in normal tissues Fig 1A Human Figure FOXD2‘AS1 is upregulated in breast cancer cells A The Cancer Genome Atlas TCGA database cancergovtcga was used to identify the association of FOXD2‘AS1 with breast cancer by collecting the profiles of FOXD2‘AS1 in breast cancer tissues and normal tissues P0001 vs normal samples B mRNA level of FOXD2‘AS1 in human normal breast epithelial cell line MCF‘10A and human breast cancer cell lines MCF‘ MDA‘MB‘ MDA‘MB‘ and BT‘ was determined by RT‘qPCR P001 and P0001 vs MCF‘10A cells cat no ab38898 Abcam S100 calcium binding protein A1 S100A1 cat no Cell Signaling Technology Inc phosphorylated p‘yes‘associated protein YAP cat no Cell Signaling Technology Inc YAP cat no Cell Signaling Technology Inc serinethreonine‘protein kinase LATS1 cat no Cell Signaling Technology Inc p‘LATS1 cat no Cell Signaling Technology Inc mammalian STE20‘like protein kinase MST1 cat no Cell Signaling Technology Inc MST2 cat no Cell Signaling Technology Inc and GAPDH cat no ab8245 Abcam at ˚C overnight The membranes were washed with Tris‘buffered saline with Tween TBST and incubated with horseradish peroxidase HRP‘conjugated goat anti‘mouse IgG secondary antibody cat no sc‘ Santa Cruz Biotechnology Inc at room temperature for h The protein bands were visualized using an enhanced chemiluminescence kit Amersham Pharmacia Biotech and quantified using ImageJ software version National Institutes of HealthWound healing assay The cellular migration rate was determined using a wound healing assay The cells were seeded into a ‘well plate and cultured to confluence A wound was produced in each monolayer using a ‘µl pipette tip and the plate was washed times with PBS to remove detached cells The cells were then cultured in the fresh medium without FBS Following incubation for h the wound‘healing ability was assessed under a light microscope magnification x100 CKX41 Olympus Corporation and the widths of the wounds were measured at and hTranswell assay The cell invasive rate was determined with a Transwell assay cells 4x104well in serum‘free medium were placed in the upper chamber of each insert [which had been precoated with µl of Matrigel Bd biosciences at ˚C for h] and complete medium containing FBS 0cHUANG and XUE DOWNREGULATION OF FOXD2‘AS1 INHIBITS THE TUMORIGENESIS OF BREAST CANCERFigure Knockdown of FOXD2‘AS1 suppresses cell proliferation migration and invasion in breast cancer A and B Following transfection with shRNA‘FOXD2‘AS the mRNA level of FOXD2‘AS1 was measured by RT‘qPCR in MCF‘ and MDA‘MB‘ cells C and D CCK‘ assay was performed to determine cell proliferation following transfection E‘G Cell cycle distribution was determined and analyzed by FACS H and I The protein expression of cyclin E1 CDK2 and p21 was determined by western blot analysisnormal breast epithelial cell line MCF‘10A and human breast cancer cell lines McF7 MdAMB468 MdAMB453 and BT549 were also obtained to detect the mRNA levels of FOXD2‘AS1 The results revealed that FOXD2‘AS1 0cINTERNATIONAL JOURNAL OF MOLEcULAR MEdIcINE Figure Continued Knockdown of FOXD2‘AS1 suppresses cell proliferation migration and invasion in breast cancer J Wound healing assay was performed to detect the migration of both MCF‘ and MDA‘MB‘ cells K and L Relative migration rate of MCF‘ and MDA‘MB‘ cells was quantified respectively M Transwell assay was performed to detect the invasion of both McF7 and MdAMB468 cells N and O Relative cell invasive rate of McF7 and MDA‘MB‘ cells was quantified respectively P MMP‘ and MMP‘ protein expression in MCF‘ transfected with shRNA‘FOXD2‘AS1 was detected and quantified Q MMP‘ and MMP‘ protein expression in MDA‘MB‘ transfected with shRNA‘FOXD2‘AS1 was detected and quantified P005 P001 and P0001 vs shRNA‘NC CDK2 cyclin‘dependent kinase‘ MMP matrix metalloproteinase expression was markedly upregulated in all breast cancer cells particularly in the MCF‘ ER‘positive breast cancer cell line and MdAMB468 cells triplenegative breast cancer cell line Fig 1B which were used for further experiments even 0cHUANG and XUE DOWNREGULATION OF FOXD2‘AS1 INHIBITS THE TUMORIGENESIS OF BREAST CANCERFigure Knockdown of FOXD2‘AS1 regulates the HippoYAP signaling pathway A The protein expression of HippoYAP signaling pathway‘related genes p‘YAP YAP p‘LATS1 LATS1 MST1 and MST2 was determined by western blot analysis in MCF‘ cells B The protein expression of HippoYAP signaling pathway‘related genes p‘YAP YAP p‘LATS1 LATS1 MST1 and MST2 was determined by western blot analysis in MDA‘MB‘ cells P005 P001 and P0001 vs shRNA‘NC LATS1 large tumor suppressor homolog MST mammalian STE20‘like protein kinase YAP yes‘associated protein though there may be some variability in the results between the cell lines These findings indicate that FOXD2‘AS1 is upregulated in breast cancerFOXD2‘AS1 knockdown suppresses breast cancer cell proliferation migration and invasiveness To further elucidate the role of FOXd2AS1 in breast cancer FOXd2AS1 was knocked down in both the McF7 and MdAMB468 cells Due to a higher transfection efficacy shRNA‘FOXD2‘AS1‘ referred to as shRNA‘FOXD2‘AS1 was subsequently used for breast cancer cell experimentation Fig 2A and B The results of CCK‘ assay indicated that FOXD2‘AS1 knockdown significantly inhibited the proliferative ability of the MCF‘ and MdAMB468 cells Fig 2c and d The cell cycle was then analyzed by flow cytometry which revealed that FOXd2AS1 knockdown increased the percentage of cells in the G1 phase whereas it decreased that in the S phase for both the MCF‘ and MDA‘MB‘ cells Fig 2E‘G Furthermore FOXd2AS1 knockdown decreased the protein expression levels of cyclin E1 and CDK2 and increased the expression of 0cINTERNATIONAL JOURNAL OF MOLEcULAR MEdIcINE Figure S100A1 is upregulated in breast cancer cells A The mRNA level of S100A1 in MCF‘10A and human breast cancer cell lines MCF‘ MDA‘MB‘ MDA‘MB‘ and BT‘ was determined by RT‘qPCR P005 P001 and P0001 vs MCF‘10A cells B and C In shRNA‘FOXD2‘AS1‘transfected McF7 and MdAMB468 cells the protein expression of Sl00A1 was detected by western blot analysis P005 and P0001 vs shRNA‘NC D and E Following transfection with pcDNA‘FOXD2‘AS1 the mRNA level of FOXD2‘AS1 in MCF‘ and MDA‘MB‘ cells was detected by RT‘qPCR P001 and P0001 vs pcDNA‘NC MCF‘ and MDA‘MB‘ cells were transfected with shRNA‘S100A1 and the protein expression and mRNA level of S100A1 in F and G MCF‘ and H and I MDA‘MB‘ cells were determined by western blot analysis and RT‘qPCR respectively P001 and P0001 vs shRNA‘NC S100A1 S100 calcium binding protein A1 p21 Fig 2H and I These findings indicate that FOXD2‘AS1 knockdown suppresses cellular proliferation by regulating the cell cycle specifically by preventing G1 to S phase progression Moreover FOXD2‘AS1 knockdown significantly decreased the migration rate Fig 2JL and the invasiveness Fig 2MO of the McF7 and MdAMB468 cells The 0cHUANG and XUE DOWNREGULATION OF FOXD2‘AS1 INHIBITS THE TUMORIGENESIS OF BREAST CANCERFigure S100A1 mediates FOXD2‘AS1‘induced cell proliferation migration and invasion in breast cancer Following transfection with pcDNA‘FOXD2‘AS1 cells were transfected with shRNA‘S100A1 A and B CCK‘ assay was performed to determine the proliferation of the differently treated cells C Migratory ability of MCF‘ cells in the different treatment groups was determined by wound healing assay D Invasive ability of MCF‘ cells in the different treatment groups was determined by Transwell assay E Migratory ability of MDA‘MB‘ cells in the different treatment groups was determined by wound healing assay F Invasive ability of MDA‘MB‘ cells in the different treatment groups was determined by Transwell assay P0001 vs pcDNA‘NC P001 and P0001 vs FOXD2‘AS1 shRNA‘NC S100A1 S100 calcium binding protein A1 expression levels of MMP‘ and ‘ which are critical to the migration invasion and metastasis of breast cancer cells were both downregulated following FOXD2‘AS1 knockdown Fig 2P and Q indicating that FOXD2‘AS1 may enhance cellular migration and invasiveness by regulating MMP‘ and ‘FOXD2‘AS1 knockdown regulates the HippoYAP signaling pathway The HippoYAP signaling pathway is reportedly involved in the progression of breast cancer In the present study the levels of specific proteins involved in the YAPHippo signaling pathway were assessed in the MCF‘ and MdAMB468 cells following FOXd2AS1 knockdown Western blot analysis revealed that the levels of p‘YAP and p‘LATS1 were significantly upregulated while those of MST1 and were significantly downregulated by FOXD2‘AS1 knockdown Fig 3A and B Thus the results confirmed that FOXD2‘AS1 regulates the HippoYAP signaling pathway in breast cancer cellsS100A1 mediates FOXD2‘AS1‘induced breast cancer cell proliferation migration and invasiveness S100A1 is a 0cINTERNATIONAL JOURNAL OF MOLEcULAR MEdIcINE Figure Overexpression of LATS1 inhibits FOXD2‘AS‘induced cell proliferation migration and invasion A and B mRNA level of LATS1 in MCF‘ and MDA‘MB‘ cells was detected by RT‘qPCR in LATS1‘overexpressing cells P0001 vs overexpression‘NC oe‘NC Following transfection with pcDNA‘FOXD2‘AS1 cells were transfected with pcDNA‘LATS1 to overexpress LATS1 C and D CCK‘ assay was performed to determine the proliferation of the differently treated cells E Migratory ability of MCF‘ cells in the different treatment groups was determined by wound healing assay F Invasive ability of MCF‘ cells in the different treatment groups was determined by Transwell assay G Migratory ability of MDA‘MB‘ cells in the different treatment groups was determined by wound healing assay H Invasive ability of MDA‘MB‘ cells in the different treatment groups was determined by Transwell assay P0001 vs pcDNA‘NC P0001 vs FOXD2‘AS1 shRNA‘NC LATS1 large tumor suppressor homolog calcium‘binding protein of the S100 protein family which is not only upregulated in but is also involved in the progression of ovarian cancer In the present study the expression of S100A1 was evaluated in the breast cancer cell lines indicating that S100A1 was significantly upregulated in breast cancer cells particularly in the MCF‘ and MDA‘MB‘ cells compared with the McF10A cells Fig 4A In McF7 and MdAMB468 cells transfected with shRNAFOXd2AS1 it was found that the protein expression level of S100A1 was downregulated Fig 4B and C To further investigate the role of S100A1 in FOXD2‘AS1‘mediated cellular proliferation migration and invasiveness breast cancer cells were transfected with an expression vector pcdNAFOXd2AS1 Fig 4d and E and shRNAS100A1 to inhibit S100A1 protein and mRNA expression Fig 4FI As shown in Fig 5A and B the overexpression of FOXd2AS1 significantly promoted the proliferation of McF7 and MdAMB468 cells which was subsequently reversed by the downregulation of S100A1 0cHUANG and XUE DOWNREGULATION OF FOXD2‘AS1 INHIBITS THE TUMORIGENESIS OF BREAST CANCERFigure Knockdown of FOXD2‘AS1 suppresses tumor progression of breast cancer in vivo A Each mouse was injected with 10x107 MdAMB468 cells which were stably transfected with either shRNA negative control or shRNA‘FOXD2‘AS1 subcutaneously at the right axillary lymph node Following sacrifice the tumors were excised and photographed B and C During the experiment the mouse weight and tumor volume tumor volume x length x width2 was monitored every days until the mice were sacrificed D Protein expression levels of p‘YAP YAP p‘LATS1 and LATS1 in the extracted tumors were determined by western blot analysis and were then quantified P0001 vs shRNA‘NC LATS1 large tumor suppressor homolog YAP yes‘associated protein Wound healing and Transwell assays demonstrated that FOXD2‘AS1 overexpression significantly increased MCF‘ cell migration and invasiveness respectively which were also reversed by the downregulation of S100A1 Fig 5C and D A similar result was observed in the MDA‘MB‘ cells Fig 5E and F These results suggest that S100A1 regulates the FOXd2AS1mediated proliferation migration and invasiveness of breast cancer cellsOverexpression of LATS1 inhibits FOXD2‘AS‘induced cell proliferation migration and invasiveness Since the HippoYAP signaling pathway is involved in the FOXd2AS1mediated characteristics of breast cancer cells HippoYAP signaling was further investigated for its regulatory role in breast cancer cell proliferation migration and invasiveness For this purpose LATS1 was overexpressed in the McF7 and MdAMB468 cells Fig 6A and B and the results of CCK‘ assay revealed that LATS1 overexpression significantly inhibited FOXD2‘AS1‘induced cellular proliferation Fig 6C and D Furthermore the results of wound healing and Transwell assays revealed that LATS1 overexpression significantly inhibited the FOXD2‘AS1‘induced migration and invasiveness of both the McF7 Fig 6E and F and MDA‘MB‘ cells Fig 6G and HFOXD2‘AS1 knockdown suppresses breast cancer tumor progression in vivo From the aforementioned results the role of FOXd2AS1 in both the McF7 and MdAMB468 cells was confirmed To explore the role of FOXD2‘AS1 in breast cancer in vivo mice were injected with MdAMB468 cells which were stably transfected with either an shRNA negative control or shRNA‘FOXD2‘AS1 Following sacrifice 0cINTERNATIONAL JOURNAL OF MOLEcULAR MEdIcINE the tumors were excised and photographed Fig 7A Tumors in the shRNA‘FOXD2‘AS1 group were the smallest in size directly reflecting the suppressive effect of FOXD2‘AS1 knockdown on tumor growth During the experiment body weights and tumor volumes were recorded every days Body weight increased at a slower rate in the shRNAFOXd2AS1 group and the tumor volumes of this group also increased at a slower rate than those in the other groups Fig 7B and C Additionally western blot analysis of the extracted tumor tissues indicated a significant increase in p‘YAP and p‘LATS1 expression in the shRNA‘FOXD2‘AS1 group Fig 7D which was consistent with the in vitro results These findings thus suggest that FOXD2‘AS1 knockdown suppresses the progression of breast cancer and regulates the HippoYAP signaling pathway in vivoDiscussionBreast cancer is one of the most common types of human tumors particularly among females The high rates of metastasis and recurrence typically result in the deterioration and death of patients with breast cancer Increasing evidence suggests that lncRNAs function as oncogenic or antitumor genes in various tumor types cells types and the microenvironment and that they may be used as effective and specific biomarkers for clinical diagnosis and prognosis Due to its oncogenic properties lncRNA FOXd2AS1 has been investigated in several malignant tumors In the present study FOXD2‘AS1 expression was found to be upregulated in breast cancer cell lines thus it was knocked down in MCF‘ and MdAMB468 cell to investigate its role in breast cancer FOXd2AS1 knockdown inhibited the proliferation migration and invasiveness of McF7 and MdAMB468 cells and inhibited tumor growth in vivo Notably S100A1 expression was also found to be upregulated in breast cancer cells and further investigation revealed that S100A1 was inhibited following FOXd2AS1 knockdown indicating that the expression of FOXd2AS1 and S100A1 was positively associated in breast cancer cells Subsequent experiments revealed that the overexpression of FOXD2‘AS1 significantly accelerated tumorigenesis by promoting cellular proliferation migration and invasiveness However the effects of FOXd2AS1 were reversed by the downregulation of S100A1 These results suggest that both FOXD2‘AS1 and S100A1 knockdown exert antitumor effects on the progression of breast cancer and that FOXD2‘AS1 may exert its oncogenic functions by regulating S100A1S100A1 is a calcium‘binding protein belonging to the S100 protein family which exhibit a range of biological properties surrounding cellular proliferation metastasis immune evasion and angiogenesis and are also involved in tumorigenesis For example S100A4 enhances p53dependent apoptosis and facilitates more aggressive tumor progression S100A6 has been reported to be upregulated in human osteosarcoma colorectal carcinoma and hepatocellular carcinoma which was mostly associated with its suppressive properties towards cancer cell migration and tumor metastasis Therefore S100 proteins play an important role in the development and progression of tumors highlighting the necessity to further understand their roles and potential underlying mechanisms In the present study S100A1 expression was found to be upregulated in breast cancer cell lines FOXd2AS1 overexpression was shown to accelerate breast cancer progression by promoting cellular proliferation migration and invasiveness and functional experiments demonstrated that the knockdown of S100A1 reversed the effects induced by FOXD2‘AS1 Furthermore S100A1 knockdown suppressed breast cancer progression by inhibiting the proliferation migration and invasiveness of McF7 and MDA‘MB‘ cells In agreement with these findings S100A1 has been reported to be overexpressed in ovarian cancer and to be associated with lymph mode metastasis the overexpression of S100A1 was shown to enhance cellular proliferation and migration whilst its inhibition exerted an opposite effect on ovarian cancer cells Moreover high tumor expression levels of S100A1 have been shown to be positively associated with decreased relapse‘free survival time in an endometrioid subtype of ovarian and endometrial cancers It was thus hypothesized that S100A1 functions as an important regulator in breast cancer and may therefore be a promising therapeutic target for this as well as other types of gynecological cancerThe Hippo pathway is an important signaling pathway that regulates cellular proliferation and apoptosis the activation of which is triggered by the phosphorylation of the large tumor suppressor kinases LATS1 and LATS2 The Hippo pathway is very complex as a number of kinases relay upstream signals to LATS to regulate this pathway The STE20 protein kinases MST12 as the core components of the Hippo pathway are considered responsible for the phosphorylation and activation of LATS12 YAP a downstream effector of the Hippo pathway is highly activated in various types of cancer and targeting YAP may effectively suppress tumorigenesis Both dysregulated Hippo signaling and aberrant YAP activation contribute to cancer progression In the present study FOXD2‘AS1 knockdown significantly increased the phosphorylation of YAP and LATS1 indicating that the Hippo signaling pathway was activated by FOXD2‘AS1 downregulation Notably it was found that MST12 expression levels were downregulated by

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You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: "Long non‘coding RNA FOXD2‘AS1 regulates the tumorigenesis and progression of breast cancer via the S100 calcium binding protein A1Hippo signaling pathwayPEI HUANG1 and JINHUI XUE21Department of Pathology The First Affiliated Hospital of Zhengzhou University Zhengzhou Henan 2Department of Pathology Zhengzhou Central Hospital Affiliated to Zhengzhou University Zhengzhou Henan PR ChinaReceived November Accepted April 103892ijmm20204699Abstract Breast cancer is one of the most prevalent cancer types and is accompanied by a high incidence and mortality rate severely threatening women's health globally Long noncoding RNA forkhead box d2 adjacent apposite strand RNA lncRNA FOXD2‘AS1 has been identified to function as an oncogene in human cancers however it has rarely been investigated in breast cancer The aim of the present study was to investigate the role of FOXd2AS1 in breast cancer and to clarify the underlying mechanisms The expression of FOXD2‘AS1 in breast cancer cell lines was first quantified by reverse transcription‘quantitative PCR and the biological function of FOXD2‘AS1 was then determined Cellular proliferative ability was determined by Cell Counting kit‘ assay and wound healing and Transwell assays were conducted to assess the cell migratory and invasive ability corresponding protein expression levels were determined by western blot analysis In addition experimental animal models were established by the subcutaneous injection of MDA‘MB‘ cells into the right axillary lymph nodes of BALBc nude mice and the effects of FOXD2‘AS1 on tumor growth were observed The results indicated that FOXD2‘AS1 expression was upregulated in breast cancer cell lines and that FOXD2‘AS1 downregulation significantly inhibited the proliferation migration and invasiveness of MCF‘ and MDA‘MB‘ cells S100 calcium binding protein A1 S100A1 was also upregulated in breast cancer cell lines and was positively regulated by FOXD2‘AS1 Furthermore the inhibition of S100A1 and the overexpression of the serinethreonine‘protein kinase large tumor suppressor homolog LATS1 inhibited the FOXD2‘AS1‘induced Correspondence to Dr Pei Huang Department of Pathology The First Affiliated Hospital of Zhengzhou University East Jianshe Road Zhengzhou Henan PR ChinaE‘mail huangpei193163comKey words FOXD2‘AS1 S100 calcium binding protein A1 breast cancer large tumor suppressor homolog Hippocellular proliferation migration and invasiveness in breast cancer Experimental mouse models revealed that FOXD2‘AS1 downregulation significantly inhibited tumor growth and that the levels of phosphorylated p‘YAP and p‘LATS1 were upregulated by FOXD2‘AS1 knockdown indicating that the inhibition of FOXd2AS1 activated Hippoyesassociated protein signaling On the whole the findings of the present study suggest that the FOXD2‘AS1S100A1Hippo axis is involved in the tumorigenesis and progression of breast cancer In the future these may contribution to the identification of more effective breast cancer treatmentsIntroductionAs one of the most prevalent malignancies breast cancer is a primary cause of mortality among gynecological cancer cases and with increasing morbidity and mortality rates it poses a considerable threat to women's health worldwide In statistics from the American cancer Society estimated newly diagnosed cases and deaths from breast cancer in the United States The leading causes of the high death rate are distal metastasis and resistance to the existing treatments despite improvements in early diagnosis and systemic treatment the incidence of breast cancer and metastasisrelated mortality is steadily increasing Therefore there is an urgent need to elucidate the mechanisms responsible for the disordered cellular metastasis and to enhance our understanding of the tumorigenesis and development processes hence facilitating the identification of more efficient breast cancer treatmentsLong noncoding RNAs lncRNAs are a group of RNAs nucleotides in length which lack protein‘coding capacity Numerous studies have revealed that lncRNAs have versatile biological functions in pathological and physiological processes including tumorigenesis ‘ lncRNAs are considered to regulate the development of various types of cancer including breast cancer For instance LINC01089 is downregulated in breast cancer tissues and cell lines and LINC01089 overexpression increases tumor cell proliferation migration and invasiveness As an oncogene that regulates breast cancer cell proliferation and apoptosis hepatocellular 0cHUANG and XUE DOWNREGULATION OF FOXD2‘AS1 INHIBITS THE TUMORIGENESIS OF BREAST CANCERcarcinoma upregulated EZH2‘associated lncRNA is closely associated with the clinical progression of breast cancer These results indicate the indispensability of research on lncRNAs and breast cancerlncRNA forkhead box d2 adjacent apposite strand RNA FOXD2‘AS1 is a novel non‘coding RNA identified to be an oncogene in human cancers FOXD2‘AS1 has been shown to be upregulated in various types of cancer including glioma osteosarcoma and papillary thyroid cancer as well as breast cancer ‘ A previous study indicated that FOXD2‘AS1 participates in regulating the development of breast cancer via the miR‘‘5pPFN2 axis and that it may be a potential biomarker for the diagnosis and prognosis of breast cancer However to the best of our knowledge there are no additional data regarding the investigation of FOXd2AS1 in breast cancer and its effects and the underlying mechanisms on the regulation of breast cancer cell invasion and metastasis Thus the aim of the present study was to determine the role and potential mechanisms of action of FOXd2AS1 in breast cancer and to provide further support for its use in clinical diagnosis and treatmentMaterials and methodsDatasets The present study evaluated the expression level of FOXD2‘AS1 in breast cancer samples using The Cancer Genome Atlas TCGA dataset which was downloaded from the TCGA data portal tcga‘data‘nci‘nih‘govezxjtlueducn The TCGA data subset for breast cancer included normal samples and tumor samples The Mann‘Whitney test was used to determine statistically significant differences between normal and tumor samples P005 was considered to indicate a statistically significant differenceCell culture A human normal breast epithelial cell line MCF‘10A and human breast cancer cell lines MCF‘ MDA‘MB‘ MDA‘MB‘ and BT‘ were purchased from the American Type Culture Collection ATCC The cells were incubated in RPMI‘ medium supplemented with fetal bovine serum FBS in a humidified incubator at ˚C CO2 Transfection To overexpress FOXd2AS1 an overexpression vector pcdNA FOXd2AS1 and its corresponding negative control vector pcDNA‘NC were synthesized by Shanghai GenePharma Co Ltd Short hairpin shRNAs targeting FOXD2‘AS1 nM shRNA‘FOXD2‘AS1‘ and shRNAFOXd2AS12 and a negative scramble control shRNA shRNA also purchased from Shanghai GenePharma Co Ltd were used to knock down FOXD2‘AS1 expression In addition pcdNALATS1 shRNAS100A11 and shRNAS100A12 were obtained from Shanghai GenePharma Co Ltd to overexpress LATS1 or to knock down S100A1 respectively The shRNA sequences were as follows shRNA‘FOXD2‘AS1‘ targets GGA CTC CAC TCT TCG CTT A shRNA‘FOXD2‘AS1‘ targets GCT TCC AGG TAT GTG GGA A shRNA‘S100A1‘ targets GAT CCG GAG ACC CTC ATC AAC GTG TTC TTC CTG TCA GAA ACA CGT TGA TGA GGG TCT CCT TTT TG shRNA‘S100A1‘ targets GAT CCG TGG ACT TCC AGG AGT ATG TGC TTC CTG TCA GAC ACA TAC TCC TGG AAG TCC ACT TTT TG Cells were transfected with pcDNA FOXD2‘AS1 nM pcdNALATS1 nM pcdNANc nM shRNAFOXd2AS11 ngµl shRNAFOXd2AS12 ngµl shRNAS100A11 ngµl shRNAS100A12 ngµl shRNA ngµl or co transfected with pcdNA FOXd2AS1 and pcdNALATS1 or cotransfected with pcDNA FOXD2‘AS1 and shRNA‘S100A1 using Lipofectamine® transfection reagent Invitrogen Thermo Fisher Scientific Inc according to the manufacturer's instructions Lipofectamine reagent was first mixed with vectors to form a reagent‘vector complex followed by incubation with cells at ˚C for h The transfection efficacy was assessed by reverse transcription‘quantitative PCR RT‘qPCR after h of transfectionRNA extraction and RT‘qPCR Total RNA was extracted from all cell lines using TRIzol® reagent Takara Bio Inc according to the manufacturer's instructions Total RNA was then reverse transcribed into cDNA using the PrimeScript„¢ RT Master kit and the mRNA expression levels were quantified using the SYBR Premix Ex Taq„¢ kit Both Takara Bio Inc with a Fast Real‘Time PCR System Applied Biosystems Thermo Fisher Scientific Inc The sequences of specific primers used for RT‘qPCR were as follows FOXDA‘AS1 forward '‘TGG ACC TAG CTG CAG CTC CA‘' and reverse '‘AGT TGA AGG TGC ACA CAC TG‘' S100A1 forward '‘GAG TAT GTG GTG CTT GTG GC‘' and reverse '‘CTT GGA CCG CTA CTC TTG CG‘' large tumor suppressor homolog LATS1 forward '‘ACC GCT TCA AAT GTG ACT GTG ATG CCA C CT‘' and reverse '‘CTT CCT TGG GCA AGC TTG GCT GAT CCT CT‘' and GAPDH forward '‘GCG AGA TCG CAC TCA TCA TCT ‘' and reverse '‘TCA GTG GTG GAC CTG ACC ‘' The data were displayed as ‘ΔΔCq values with GAPDH as the constitutive marker The PCR conditions were as follows ˚C for min cycles of ˚C for sec and ˚C for sec followed by ˚C for minCell Counting Kit‘ CCK‘ assay cell proliferation was determined using the Cell Counting Kit‘ assay CCK‘ Dojindo Molecular Technologies Inc Briefly cells were seeded into a ‘well plate and incubated for h at ˚C Following culture for the indicated periods of time and h µl of the CCK‘ reagent were added to each well and the plate was incubated at ˚C for a further h The optical density values at nm were then measured using a microplate spectrophotometer Thermo Fisher Scientific IncWestern blot analysis The total protein was extracted from the cells using RIPA lysis buffer Beyotime Institute of Biotechnology and quantified using a BCA protein assay kit Thermo Fisher Scientific Inc The same amount of each protein sample µg was subjected to SDS‘PAGE the proteins were then transferred onto PVDF membranes EMd Millipore and was blocked in nonfat milk for h at room temperature The membranes were then incubated with primary antibodies against cyclinE1 cat no ab33911 Abcam cyclin‘dependent kinase‘ CDK2 cat no ab32147 Abcam p21 cat no ab109520 Abcam matrix metalloproteinases MMP2 cat no ab92536 Abcam MMP9 0cINTERNATIONAL JOURNAL OF MOLEcULAR MEdIcINE was added to the lower 24well chamber After h cells on the upper surface were removed and cells attached to the lower surface were stained with crystal violet Beijing Solarbio Science Technology Co Ltd at room temperature for min The cells were viewed under a light microscope magnification x100 CKX41 Olympus Corporation and the invasive ability of the cells was determined using ImageJ software version by counting the number of cells attached to the lower surfaceCell cycle analysis The cell cycle distribution was determined by flow cytometry After being subjected to the indicated treatments the cells were collected and fixed in ethanol at ‘ËšC overnight The cells were then washed twice with PBS and incubated in the dark with RNase A and PI staining solution Roche Diagnostics at ˚C for min Finally the cell samples were analyzed using a FACSCalibur flow cytometer and CellQuest software version both from BD BiosciencesIn vivo experiments The present study was approved by the First Affiliated Hospital of Zhengzhou University and the animal experiments were performed according to the National Institute of Health Guidelines for the Care and Use of Laboratory Animals A total of 5weekold male BALBc nude mice were purchased from the Experimental Animal Center of Shanghai Institute for Biological Sciences and housed in a standard environment ˚C humidity h lightdark cycle with free access to food and water Each mouse was subcutaneously injected at the right axillary lymph nodes with 10x107 MdAMB468 cells which were stably transfected with either the shRNA negative control shRNA or shRNA‘FOXD2‘AS1 The weights and tumor volumes tumor volume x length x width2 of the mice were monitored every days until the mice were sacrificed At days after the injection all the mice were sacrificed by cervical dislocation that caused a sharp section of the spinal cord followed by an instantaneous cardiac arrest After the cessation of the heartbeat and respiratory arrest of the mice was confirmed the tumors were excised photographed and stored for the further investigationStatistical analysis data are presented as the means ± standard deviation SD from ‰¥ independent experiments and each experiment was conducted in triplicate The data were analyzed using SPSS statistical software version SPSS Inc and the differences among groups were analyzed using one‘way analysis of variance followed by Tukey's post hoc test P005 was considered to indicate a statistically significant differenceResultsFOXD2‘AS1 expression is upregulated in breast cancer cells The TCGA database cancergovtcga was used to identify the association between FOXD2‘AS1 and breast cancer by evaluating the expression profiles of FOXD2‘AS1 in breast cancer tissues and normal tissues The results of TCGA analysis revealed a significantly higher FOXd2AS1 expression in breast cancer tissues than in normal tissues Fig 1A Human Figure FOXD2‘AS1 is upregulated in breast cancer cells A The Cancer Genome Atlas TCGA database cancergovtcga was used to identify the association of FOXD2‘AS1 with breast cancer by collecting the profiles of FOXD2‘AS1 in breast cancer tissues and normal tissues P0001 vs normal samples B mRNA level of FOXD2‘AS1 in human normal breast epithelial cell line MCF‘10A and human breast cancer cell lines MCF‘ MDA‘MB‘ MDA‘MB‘ and BT‘ was determined by RT‘qPCR P001 and P0001 vs MCF‘10A cells cat no ab38898 Abcam S100 calcium binding protein A1 S100A1 cat no Cell Signaling Technology Inc phosphorylated p‘yes‘associated protein YAP cat no Cell Signaling Technology Inc YAP cat no Cell Signaling Technology Inc serinethreonine‘protein kinase LATS1 cat no Cell Signaling Technology Inc p‘LATS1 cat no Cell Signaling Technology Inc mammalian STE20‘like protein kinase MST1 cat no Cell Signaling Technology Inc MST2 cat no Cell Signaling Technology Inc and GAPDH cat no ab8245 Abcam at ˚C overnight The membranes were washed with Tris‘buffered saline with Tween TBST and incubated with horseradish peroxidase HRP‘conjugated goat anti‘mouse IgG secondary antibody cat no sc‘ Santa Cruz Biotechnology Inc at room temperature for h The protein bands were visualized using an enhanced chemiluminescence kit Amersham Pharmacia Biotech and quantified using ImageJ software version National Institutes of HealthWound healing assay The cellular migration rate was determined using a wound healing assay The cells were seeded into a ‘well plate and cultured to confluence A wound was produced in each monolayer using a ‘µl pipette tip and the plate was washed times with PBS to remove detached cells The cells were then cultured in the fresh medium without FBS Following incubation for h the wound‘healing ability was assessed under a light microscope magnification x100 CKX41 Olympus Corporation and the widths of the wounds were measured at and hTranswell assay The cell invasive rate was determined with a Transwell assay cells 4x104well in serum‘free medium were placed in the upper chamber of each insert [which had been precoated with µl of Matrigel Bd biosciences at ˚C for h] and complete medium containing FBS 0cHUANG and XUE DOWNREGULATION OF FOXD2‘AS1 INHIBITS THE TUMORIGENESIS OF BREAST CANCERFigure Knockdown of FOXD2‘AS1 suppresses cell proliferation migration and invasion in breast cancer A and B Following transfection with shRNA‘FOXD2‘AS the mRNA level of FOXD2‘AS1 was measured by RT‘qPCR in MCF‘ and MDA‘MB‘ cells C and D CCK‘ assay was performed to determine cell proliferation following transfection E‘G Cell cycle distribution was determined and analyzed by FACS H and I The protein expression of cyclin E1 CDK2 and p21 was determined by western blot analysisnormal breast epithelial cell line MCF‘10A and human breast cancer cell lines McF7 MdAMB468 MdAMB453 and BT549 were also obtained to detect the mRNA levels of FOXD2‘AS1 The results revealed that FOXD2‘AS1 0cINTERNATIONAL JOURNAL OF MOLEcULAR MEdIcINE Figure Continued Knockdown of FOXD2‘AS1 suppresses cell proliferation migration and invasion in breast cancer J Wound healing assay was performed to detect the migration of both MCF‘ and MDA‘MB‘ cells K and L Relative migration rate of MCF‘ and MDA‘MB‘ cells was quantified respectively M Transwell assay was performed to detect the invasion of both McF7 and MdAMB468 cells N and O Relative cell invasive rate of McF7 and MDA‘MB‘ cells was quantified respectively P MMP‘ and MMP‘ protein expression in MCF‘ transfected with shRNA‘FOXD2‘AS1 was detected and quantified Q MMP‘ and MMP‘ protein expression in MDA‘MB‘ transfected with shRNA‘FOXD2‘AS1 was detected and quantified P005 P001 and P0001 vs shRNA‘NC CDK2 cyclin‘dependent kinase‘ MMP matrix metalloproteinase expression was markedly upregulated in all breast cancer cells particularly in the MCF‘ ER‘positive breast cancer cell line and MdAMB468 cells triplenegative breast cancer cell line Fig 1B which were used for further experiments even 0cHUANG and XUE DOWNREGULATION OF FOXD2‘AS1 INHIBITS THE TUMORIGENESIS OF BREAST CANCERFigure Knockdown of FOXD2‘AS1 regulates the HippoYAP signaling pathway A The protein expression of HippoYAP signaling pathway‘related genes p‘YAP YAP p‘LATS1 LATS1 MST1 and MST2 was determined by western blot analysis in MCF‘ cells B The protein expression of HippoYAP signaling pathway‘related genes p‘YAP YAP p‘LATS1 LATS1 MST1 and MST2 was determined by western blot analysis in MDA‘MB‘ cells P005 P001 and P0001 vs shRNA‘NC LATS1 large tumor suppressor homolog MST mammalian STE20‘like protein kinase YAP yes‘associated protein though there may be some variability in the results between the cell lines These findings indicate that FOXD2‘AS1 is upregulated in breast cancerFOXD2‘AS1 knockdown suppresses breast cancer cell proliferation migration and invasiveness To further elucidate the role of FOXd2AS1 in breast cancer FOXd2AS1 was knocked down in both the McF7 and MdAMB468 cells Due to a higher transfection efficacy shRNA‘FOXD2‘AS1‘ referred to as shRNA‘FOXD2‘AS1 was subsequently used for breast cancer cell experimentation Fig 2A and B The results of CCK‘ assay indicated that FOXD2‘AS1 knockdown significantly inhibited the proliferative ability of the MCF‘ and MdAMB468 cells Fig 2c and d The cell cycle was then analyzed by flow cytometry which revealed that FOXd2AS1 knockdown increased the percentage of cells in the G1 phase whereas it decreased that in the S phase for both the MCF‘ and MDA‘MB‘ cells Fig 2E‘G Furthermore FOXd2AS1 knockdown decreased the protein expression levels of cyclin E1 and CDK2 and increased the expression of 0cINTERNATIONAL JOURNAL OF MOLEcULAR MEdIcINE Figure S100A1 is upregulated in breast cancer cells A The mRNA level of S100A1 in MCF‘10A and human breast cancer cell lines MCF‘ MDA‘MB‘ MDA‘MB‘ and BT‘ was determined by RT‘qPCR P005 P001 and P0001 vs MCF‘10A cells B and C In shRNA‘FOXD2‘AS1‘transfected McF7 and MdAMB468 cells the protein expression of Sl00A1 was detected by western blot analysis P005 and P0001 vs shRNA‘NC D and E Following transfection with pcDNA‘FOXD2‘AS1 the mRNA level of FOXD2‘AS1 in MCF‘ and MDA‘MB‘ cells was detected by RT‘qPCR P001 and P0001 vs pcDNA‘NC MCF‘ and MDA‘MB‘ cells were transfected with shRNA‘S100A1 and the protein expression and mRNA level of S100A1 in F and G MCF‘ and H and I MDA‘MB‘ cells were determined by western blot analysis and RT‘qPCR respectively P001 and P0001 vs shRNA‘NC S100A1 S100 calcium binding protein A1 p21 Fig 2H and I These findings indicate that FOXD2‘AS1 knockdown suppresses cellular proliferation by regulating the cell cycle specifically by preventing G1 to S phase progression Moreover FOXD2‘AS1 knockdown significantly decreased the migration rate Fig 2JL and the invasiveness Fig 2MO of the McF7 and MdAMB468 cells The 0cHUANG and XUE DOWNREGULATION OF FOXD2‘AS1 INHIBITS THE TUMORIGENESIS OF BREAST CANCERFigure S100A1 mediates FOXD2‘AS1‘induced cell proliferation migration and invasion in breast cancer Following transfection with pcDNA‘FOXD2‘AS1 cells were transfected with shRNA‘S100A1 A and B CCK‘ assay was performed to determine the proliferation of the differently treated cells C Migratory ability of MCF‘ cells in the different treatment groups was determined by wound healing assay D Invasive ability of MCF‘ cells in the different treatment groups was determined by Transwell assay E Migratory ability of MDA‘MB‘ cells in the different treatment groups was determined by wound healing assay F Invasive ability of MDA‘MB‘ cells in the different treatment groups was determined by Transwell assay P0001 vs pcDNA‘NC P001 and P0001 vs FOXD2‘AS1 shRNA‘NC S100A1 S100 calcium binding protein A1 expression levels of MMP‘ and ‘ which are critical to the migration invasion and metastasis of breast cancer cells were both downregulated following FOXD2‘AS1 knockdown Fig 2P and Q indicating that FOXD2‘AS1 may enhance cellular migration and invasiveness by regulating MMP‘ and ‘FOXD2‘AS1 knockdown regulates the HippoYAP signaling pathway The HippoYAP signaling pathway is reportedly involved in the progression of breast cancer In the present study the levels of specific proteins involved in the YAPHippo signaling pathway were assessed in the MCF‘ and MdAMB468 cells following FOXd2AS1 knockdown Western blot analysis revealed that the levels of p‘YAP and p‘LATS1 were significantly upregulated while those of MST1 and were significantly downregulated by FOXD2‘AS1 knockdown Fig 3A and B Thus the results confirmed that FOXD2‘AS1 regulates the HippoYAP signaling pathway in breast cancer cellsS100A1 mediates FOXD2‘AS1‘induced breast cancer cell proliferation migration and invasiveness S100A1 is a 0cINTERNATIONAL JOURNAL OF MOLEcULAR MEdIcINE Figure Overexpression of LATS1 inhibits FOXD2‘AS‘induced cell proliferation migration and invasion A and B mRNA level of LATS1 in MCF‘ and MDA‘MB‘ cells was detected by RT‘qPCR in LATS1‘overexpressing cells P0001 vs overexpression‘NC oe‘NC Following transfection with pcDNA‘FOXD2‘AS1 cells were transfected with pcDNA‘LATS1 to overexpress LATS1 C and D CCK‘ assay was performed to determine the proliferation of the differently treated cells E Migratory ability of MCF‘ cells in the different treatment groups was determined by wound healing assay F Invasive ability of MCF‘ cells in the different treatment groups was determined by Transwell assay G Migratory ability of MDA‘MB‘ cells in the different treatment groups was determined by wound healing assay H Invasive ability of MDA‘MB‘ cells in the different treatment groups was determined by Transwell assay P0001 vs pcDNA‘NC P0001 vs FOXD2‘AS1 shRNA‘NC LATS1 large tumor suppressor homolog calcium‘binding protein of the S100 protein family which is not only upregulated in but is also involved in the progression of ovarian cancer In the present study the expression of S100A1 was evaluated in the breast cancer cell lines indicating that S100A1 was significantly upregulated in breast cancer cells particularly in the MCF‘ and MDA‘MB‘ cells compared with the McF10A cells Fig 4A In McF7 and MdAMB468 cells transfected with shRNAFOXd2AS1 it was found that the protein expression level of S100A1 was downregulated Fig 4B and C To further investigate the role of S100A1 in FOXD2‘AS1‘mediated cellular proliferation migration and invasiveness breast cancer cells were transfected with an expression vector pcdNAFOXd2AS1 Fig 4d and E and shRNAS100A1 to inhibit S100A1 protein and mRNA expression Fig 4FI As shown in Fig 5A and B the overexpression of FOXd2AS1 significantly promoted the proliferation of McF7 and MdAMB468 cells which was subsequently reversed by the downregulation of S100A1 0cHUANG and XUE DOWNREGULATION OF FOXD2‘AS1 INHIBITS THE TUMORIGENESIS OF BREAST CANCERFigure Knockdown of FOXD2‘AS1 suppresses tumor progression of breast cancer in vivo A Each mouse was injected with 10x107 MdAMB468 cells which were stably transfected with either shRNA negative control or shRNA‘FOXD2‘AS1 subcutaneously at the right axillary lymph node Following sacrifice the tumors were excised and photographed B and C During the experiment the mouse weight and tumor volume tumor volume x length x width2 was monitored every days until the mice were sacrificed D Protein expression levels of p‘YAP YAP p‘LATS1 and LATS1 in the extracted tumors were determined by western blot analysis and were then quantified P0001 vs shRNA‘NC LATS1 large tumor suppressor homolog YAP yes‘associated protein Wound healing and Transwell assays demonstrated that FOXD2‘AS1 overexpression significantly increased MCF‘ cell migration and invasiveness respectively which were also reversed by the downregulation of S100A1 Fig 5C and D A similar result was observed in the MDA‘MB‘ cells Fig 5E and F These results suggest that S100A1 regulates the FOXd2AS1mediated proliferation migration and invasiveness of breast cancer cellsOverexpression of LATS1 inhibits FOXD2‘AS‘induced cell proliferation migration and invasiveness Since the HippoYAP signaling pathway is involved in the FOXd2AS1mediated characteristics of breast cancer cells HippoYAP signaling was further investigated for its regulatory role in breast cancer cell proliferation migration and invasiveness For this purpose LATS1 was overexpressed in the McF7 and MdAMB468 cells Fig 6A and B and the results of CCK‘ assay revealed that LATS1 overexpression significantly inhibited FOXD2‘AS1‘induced cellular proliferation Fig 6C and D Furthermore the results of wound healing and Transwell assays revealed that LATS1 overexpression significantly inhibited the FOXD2‘AS1‘induced migration and invasiveness of both the McF7 Fig 6E and F and MDA‘MB‘ cells Fig 6G and HFOXD2‘AS1 knockdown suppresses breast cancer tumor progression in vivo From the aforementioned results the role of FOXd2AS1 in both the McF7 and MdAMB468 cells was confirmed To explore the role of FOXD2‘AS1 in breast cancer in vivo mice were injected with MdAMB468 cells which were stably transfected with either an shRNA negative control or shRNA‘FOXD2‘AS1 Following sacrifice 0cINTERNATIONAL JOURNAL OF MOLEcULAR MEdIcINE the tumors were excised and photographed Fig 7A Tumors in the shRNA‘FOXD2‘AS1 group were the smallest in size directly reflecting the suppressive effect of FOXD2‘AS1 knockdown on tumor growth During the experiment body weights and tumor volumes were recorded every days Body weight increased at a slower rate in the shRNAFOXd2AS1 group and the tumor volumes of this group also increased at a slower rate than those in the other groups Fig 7B and C Additionally western blot analysis of the extracted tumor tissues indicated a significant increase in p‘YAP and p‘LATS1 expression in the shRNA‘FOXD2‘AS1 group Fig 7D which was consistent with the in vitro results These findings thus suggest that FOXD2‘AS1 knockdown suppresses the progression of breast cancer and regulates the HippoYAP signaling pathway in vivoDiscussionBreast cancer is one of the most common types of human tumors particularly among females The high rates of metastasis and recurrence typically result in the deterioration and death of patients with breast cancer Increasing evidence suggests that lncRNAs function as oncogenic or antitumor genes in various tumor types cells types and the microenvironment and that they may be used as effective and specific biomarkers for clinical diagnosis and prognosis Due to its oncogenic properties lncRNA FOXd2AS1 has been investigated in several malignant tumors In the present study FOXD2‘AS1 expression was found to be upregulated in breast cancer cell lines thus it was knocked down in MCF‘ and MdAMB468 cell to investigate its role in breast cancer FOXd2AS1 knockdown inhibited the proliferation migration and invasiveness of McF7 and MdAMB468 cells and inhibited tumor growth in vivo Notably S100A1 expression was also found to be upregulated in breast cancer cells and further investigation revealed that S100A1 was inhibited following FOXd2AS1 knockdown indicating that the expression of FOXd2AS1 and S100A1 was positively associated in breast cancer cells Subsequent experiments revealed that the overexpression of FOXD2‘AS1 significantly accelerated tumorigenesis by promoting cellular proliferation migration and invasiveness However the effects of FOXd2AS1 were reversed by the downregulation of S100A1 These results suggest that both FOXD2‘AS1 and S100A1 knockdown exert antitumor effects on the progression of breast cancer and that FOXD2‘AS1 may exert its oncogenic functions by regulating S100A1S100A1 is a calcium‘binding protein belonging to the S100 protein family which exhibit a range of biological properties surrounding cellular proliferation metastasis immune evasion and angiogenesis and are also involved in tumorigenesis For example S100A4 enhances p53dependent apoptosis and facilitates more aggressive tumor progression S100A6 has been reported to be upregulated in human osteosarcoma colorectal carcinoma and hepatocellular carcinoma which was mostly associated with its suppressive properties towards cancer cell migration and tumor metastasis Therefore S100 proteins play an important role in the development and progression of tumors highlighting the necessity to further understand their roles and potential underlying mechanisms In the present study S100A1 expression was found to be upregulated in breast cancer cell lines FOXd2AS1 overexpression was shown to accelerate breast cancer progression by promoting cellular proliferation migration and invasiveness and functional experiments demonstrated that the knockdown of S100A1 reversed the effects induced by FOXD2‘AS1 Furthermore S100A1 knockdown suppressed breast cancer progression by inhibiting the proliferation migration and invasiveness of McF7 and MDA‘MB‘ cells In agreement with these findings S100A1 has been reported to be overexpressed in ovarian cancer and to be associated with lymph mode metastasis the overexpression of S100A1 was shown to enhance cellular proliferation and migration whilst its inhibition exerted an opposite effect on ovarian cancer cells Moreover high tumor expression levels of S100A1 have been shown to be positively associated with decreased relapse‘free survival time in an endometrioid subtype of ovarian and endometrial cancers It was thus hypothesized that S100A1 functions as an important regulator in breast cancer and may therefore be a promising therapeutic target for this as well as other types of gynecological cancerThe Hippo pathway is an important signaling pathway that regulates cellular proliferation and apoptosis the activation of which is triggered by the phosphorylation of the large tumor suppressor kinases LATS1 and LATS2 The Hippo pathway is very complex as a number of kinases relay upstream signals to LATS to regulate this pathway The STE20 protein kinases MST12 as the core components of the Hippo pathway are considered responsible for the phosphorylation and activation of LATS12 YAP a downstream effector of the Hippo pathway is highly activated in various types of cancer and targeting YAP may effectively suppress tumorigenesis Both dysregulated Hippo signaling and aberrant YAP activation contribute to cancer progression In the present study FOXD2‘AS1 knockdown significantly increased the phosphorylation of YAP and LATS1 indicating that the Hippo signaling pathway was activated by FOXD2‘AS1 downregulation Notably it was found that MST12 expression levels were downregulated by Answer:

Thyroid_Cancer

LncRNA SNHG15 predicts poor prognosis in uveal melanoma and its potential pathwaysXue Wu123 XiaoFeng Li123 Qian Wu4 RuiQi Ma123 Jiang Qian123 Rui Zhang123·Basic Research·1Department of Ophthalmology Eye ENT Hospital of Fudan University Shanghai China2NHC Key Laboratory of Myopia Fudan University Shanghai China 3Laboratory of Myopia Chinese Academy of Medical Sciences Shanghai China4Department of Pathology West China Hospital Sichuan University Chengdu Sichuan Province ChinaCofirst authors Xue Wu and XiaoFeng LiCorrespondence to Rui Zhang Department of Ophthalmology Eye ENT Hospital of Fudan University Fen Yang Road Shanghai China zhangrui936163comReceived Accepted Our research suggests that SNHG15 may play a vital role as a potential marker in UM that predicts poor prognosis Besides GSEA indicates the underlying signaling pathways enriched differentially in SNHG15 high expression phenotype KEYWORDS SNHG15 uveal melanoma the Cancer Genome Atlas pathology prognosis Gene Set Enrichment Analysis1018240ijo20200804Citation Wu X Li XF Wu Q Ma RQ Qian J Zhang R LncRNA SNHG15 predicts poor prognosis in uveal melanoma and its potential pathways Int J Ophthalmol Abstract— AIM To evaluate the role of long noncoding RNA lncRNA SNHG15 and its potential pathways in uveal melanoma UM METHODS The SNHG15 mRNA expression level and corresponding clinicopathological characteristics of patients with UM were obtained from the Cancer Genome Atlas TCGA database and further analyzed The SPSS statistical software package was used for statistical analyses To investigate the potential function of SNHG15 in UM we conducted indepth research on Gene Set Enrichment Analysis GSEA— RESULTS The univariate analysis revealed that the age tumor diameter pathological type extrascleral extension cancer status and high expression of SNHG15 were statistical risk factors for death from all causes The multivariate analysis suggested that the mRNA expression level of SNHG15 was an independent risk factor for death from all causes as was age and pathological type KaplanMeier survival analysis confirmed that UM patients with high SNHG15 expression might have a poor prognosis In addition SNHG15 was significantly differentially expressed in the different groups of tumor pathologic stage metastasis and living status Besides the logistic regression analysis indicated that high SNHG15 expression group in UM was significantly associated with cancer status pathologic stage metastasis and living status Moreover the GSEA indicated the potential pathways regulated by SNHG15 in UM INTRODUCTIONU veal melanoma UM the most common intraocular cancer in adult worldwide[] is a malignant tumor that originates in melanocytes of the choroid plexus ciliary body and iris of the eye At present despite definitive radiotherapy or removal of the primary lesion numerous patients eventually develop metastases and subsequently prognosis is significantly poor[] In addition UM tends to metastasize to liver through hematogenous pathway a distant site relative to their origins in the eye[] There is an incubation period between the enucleation of the primary tumor and the emergence of metastasis which can range from a few months to several decades[] Despite the advancement of UM management there are currently no effective therapy once the metastases occurred[] Therefore close followup and further research on the pathogenesis and novel makers exploration of UM are of great significance for accurate diagnosis appropriate therapy and prognosis prediction Long noncoding RNA lncRNA is a class of noncoding transcripts with a length of larger than nucleotides[] which has been involved widely in biological processes of different cancers including cell cycle apoptosis cell differentiation[] In the development of UM lncRNA is also reported to play a vital role in cell cycle cell proliferation apoptosis invasion and autophagy[] For example silencing of lncRNA PVT1 prevents the development of UM by impairing microRNA173pdependent MDM2 upregulation[] ZNNT1 can suppress Int J Ophthalmol Vol No Aug18 wwwijocnTel Email ijopress163com 0cLncRNA SNHG15 predicts prognosis in uveal melanomathe progression of UM by inducing the expression of crucial autophagy gene[] The lncRNA RHPN1AS1 facilitates the tumorigenesis of UM by influencing cell proliferation and migration[] However the study of vital lncRNAs in UM still remains to be exploredSNHG15 a novel lncRNA located on chromosome 7p13[] is identified to play a key role in many types of human tumors such as osteosarcoma[] papillary thyroid carcinoma[] pancreatic ductal adenocarcinoma[] colorectal carcinoma[] hepatocellular carcinoma[] prostate cancer[] and breast cancer[] To our knowledge the potential impact of SNHG15 on the tumorigenesis of UM seems unclear recently Thus the purpose of this study was to evaluate the pivotal role of SNHG15 in the progression of UM In addition the relationship between SNHG15 expression and clinicopathologic characteristics in UM was preliminarily demonstrated To explore the underlying mechanisms of the biological pathways involved in UM we conducted a research on Gene Set Enrichment Analysis GSEA MATERIALS AND METHODSEthical Approval The study protocol was approved by the Ethics Committee of the Eye ENT Hospital of Fudan University and all procedures were complied with the principles of the Declaration of Helsinki All datasets of our present study were downloaded from an database TCGA so there was no written informed consent from participantsRNASequencing Patient Data and Bioinformatics Analysis The RNASeq gene expression level and clinicopathological characteristics including cases were obtained from the official website of the Cancer Genome Atlas TCGA UM project portalgdccancergov Patients with UM were classified as two groups based on the median SNHG15 expression level cutoff value794 FPKM Finally patients with UM were retained and their clinicopathological characteristics were further analyzed including the detailed information of age gender tumor diameter thickness pathological type extrascleral extension cancer status pathological stage metastasis living status SNHG15 expressionGene Set Enrichment Analysis GSEA is a common bioanalysis used to interpret and analyze microarray and other similar data and to speculate related pathways that can significantly enrich regulatory genes[] Through TCGA UM project we obtained the RNASeq gene expression level of UM patients And the analysis was conducted using GSEA v30 software In this study according to the association with SNHG15 expression the ordered gene list was generated firstly by GSEA Subsequently GSEA was conducted to clarify statistically significant differences between the two groups with high and low SNHG15 expression A total of permutations were performed The SNHG15 expression level was identified as a phenotype label The related pathways statistically enriched in each phenotype were selected with the nominal P005 and an false discovery rate FDR Statistical Analysis The SPSS statistical software package SPSS Inc USA was used for statistical analyses Both the univariate and multivariate analyses using Cox analysis were performed to demonstrate independent prognostic biomarkers for UM patients The survival curve was generated by conducting KaplanMeier method To compare the significant differences in overall survival OS the logrank test was conducted The plot chart was performed to visualize the difference of SNHG15 expression level for diverse variables through Graphpad The relationship between the SNHG15 expression and clinicopathological characteristics were analyzed using logistic regression The median value of SNHG15 expression was selected as the cutoff value P005 was considered statistically significantRESULTSPatient Characteristics The records of primary UM with both RNASeq gene expression level and clinicopathological characteristics were obtained from TCGA database The mean age of UM patients was years old including males and females The mean value of tumor diameter and thickness were and mm respectively In our study cohort the pathological type of UM included epithelioid cell dominant type and spindle cell dominant type of tumors were epithelioid cell dominant and were spindle cell dominant There were cases without extrascleral extension and cases with extrascleral extension The cancer status included tumorfree cases and cases with tumor Pathologic stage II was found in cases and stage IIIIV in cases And of cases had metastases of cases had no metastases Of cases cases died of all causes Survival Outcomes and Multivariate Analysis Prognostic factors of UM were analyzed using univariate and multivariate Cox regression The univariate analysis suggested that high SNHG15 expression was a risk factor for death from all causes Other clinicopathologic variables related to poor prognosis included age tumor diameter pathological type extrascleral extension cancer status Table In a multivariate analysis SNHG15 was an independent risk factor for death from all causes as was age and pathological typeSNHG15 Expression Associated with Clinical Pathological Characteristics A total of UM cases with SNHG15 expression data and clinicopathologic characteristics were analyzed from TCGA KaplanMeier survival analysis 0cTable Prognostic parameters in UM were analyzed using univariate and multivariate Cox regressionDeath from all causesParametersnmeanUnivariate analysisPHR95CIAge yGenderFemaleMaleTumor diameter mmThickness mmPathological typeEpithelioid cell dominantSpindle cell dominantExtrascleral extensionNoYesCancer statusTumor freeWith tumorPathological stageIIIIIIVSNHG15HighLowUM Uveal melanomaMultivariate analysisPHR95CIdemonstrated that high SNHG15 expression group had a worse prognosis when compared to low SNHG15 expression group Figure 1A P005 As shown in Figure 1B1D SNHG15 was statistically differentially expressed in diverse groups of the tumor pathologic stage stage II vs IIIIV P00257 metastasis P00071 living status P00017 To clarify the clinicopathologic impact of SNHG15 we also used logistic regression and concluded that the SNHG15 expression based on median value of FPKM as a categorical variable was statistically related to clinicopathologic features Table High SNHG15 expression was significantly related to cancer status pathologic stage metastasis living status in UM all P005 Table These results demonstrated that UM with high SNHG15 expression were prone to progress to cancer status of survival with tumor a more advanced stage metastasis and poor living status when compared to the low SNHG15 expression group However there was no statistically significant difference in age gender tumor diameter thickness pathological type extrascleral invasionMain Enriched Pathways in UM Tissues with High SNHG15 Expression To explore the SNHG15related potential signaling pathways activated in UM GSEA was performed In the current study based on the association with SNHG15 expression the gene list was generated firstly Figure The SNHG15 expression was associated with clinical pathological characteristics A Patients with high SNHG15 expression had a shorter OS when compared with the low SNHG15 expression group P002 BD The expression of SNHG15 was statistically different in diverse groups of the tumor pathologic stage P00257 metastasis P00071 living status P00017 aP005 bP001by GSEA To clarify the statistically significant differences between high and low SNHG15 expression groups GSEA was Int J Ophthalmol Vol No Aug18 wwwijocnTel Email ijopress163com 0cLncRNA SNHG15 predicts prognosis in uveal melanomaTable Association between SNHG15 expression and clinicopathologic variables using logistic regressionParametersAge yGenderFemaleMaleTumor diameter mmThickness mmPathological typeEpithelialNonepithelialExtrascleral invasionNoYesCancer statusTumor freeWith tumorPathological stageIIIIIIVMetastasesNoYesLiving statusAliveDeadnmeanSNHG15 expressionLowHighPOR95CINESNominal PvalESTable Enriched pathways for differential SNHG15 expression in UMNameSpliceosomeCell cyclePyrimidine metabolismDNA replicationNucleotide excision repairRNA degradationHomologous recombinationMismatch repairUM Uveal melanoma ES Enrichment score NES Normal enrichment score FDR False discovery rateFDR Qvalconducted subsequently The results indicated that there were significant differences in spliceosome cell cycle pyrimidine metabolism DNA replication nucleotide excision repair RNA degradation homologous recombination and mismatch repair among patients with high SNHG15 expression phenotype Figure Table DISCUSSIONAccumulating evidences indicate that SNHG15 plays a dual role in the tumorigenesis and development of different tumors[] Previously SNHG15 has been demonstrated as a carcinogenic lncRNA which is usually upregulated in tumor tissues compared with normal tissues[] It exerts 0cFigure Enrichment plots from GSEA Spliceosome cell cycle pyrimidine metabolism DNA replication nucleotide excision repair RNA degradation homologous recombination and mismatch repair are enriched significantly in SNHG15 high expression phenotypean oncogenic effect via various epigenetic mechanisms[] For example it can suppress the expression of miR3383p and facilitate the proliferation of colorectal cancer cells[] It plays a carcinogenic role by affecting miR3383pFKBP1A axis in prostate cancer[] It can also enhance hepatocellular carcinoma progression by negative regulation of miR1413p[] However there are reports that SNHG15 has a tumor suppressive effect suggesting that low SNHG15 expression is related to poor prognosis in thyroid cancer and upregulating expression of SNHG15 can significantly suppress cell proliferation[] At present the impact of SNHG15 on UM is still unclear Therefore vital roles and potential biological mechanism of SNHG15 in UM needs to be elucidated In this study we revealed that high SNHG15 expression was related to clinicopathologic features in UM Through RNASeq gene expression level and clinicopathological characteristics obtained from the TCGA UM project we analyzed the relationship among SNHG15 expression clinicopathological features and prognosis of UM The univariate analysis demonstrated that SNHG15 expression level age tumor diameter pathological type extrascleral extension and cancer status were risk factors for death from all causes The multivariate analysis suggested that high SNHG15 expression along with age and pathological type was an independent risk factor for death from all causes Therefore the results demonstrated that high SNHG15 expression was an independent predictor of poor prognosis in UM through univariate and multivariate analysis KaplanMeier survival analysis also indicated that high SNHG15 expression group had a worse prognosis when compared to low SNHG15 expression group in UM In addition an analysis was conducted to further explore the relationship between SNHG15 and clinicopathological features The SNHG15 expression was statistically different in diverse groups of the tumor pathologic stage metastasis and living status Besides high SNHG15 expression based on median expression value of FPKM in UM was associated with cancer status of survival with tumor advanced pathologic stage metastasis and living status It demonstrated that high SNHG15 expression in UM was strongly related to poor prognosis The mechanisms of SNHG15 dysregulation in malignant tumors are quite complex and are far from being completely understood Previous studies have suggested that SNHG15 is involved in diverse pathological and physiological processes of many tumors through their abnormal expressions including cell proliferation invasion migration and autophagy[] To explore the biological mechanism of SNHG15 in UM GSEA was conducted It indicated that spliceosome cell cycle pyrimidine metabolism DNA replication nucleotide excision repair RNA degradation homologous recombination and mismatch repair were all enriched differentially in SNHG15 high expression phenotype Alternative splicing is essential for gene regulation and abnormal splicing plays a vital role in inactivating tumor suppressor genes or activating oncogenes[] SNHG15 may have an impact on the invasion Int J Ophthalmol Vol No Aug18 wwwijocnTel Email ijopress163com 0cLncRNA SNHG15 predicts prognosis in uveal melanomaand migration of UM cells by affecting spliceosomal related factors The abnormal cell proliferation of tumor is related to the lack of checkpoint control over the cell cycle which is the basis of genetic instability[] Evidence shows that the lack of homologous recombination may facilitate the disturbance of cell cycle the instability and accumulated mutations of genome during the progression and development[] Mismatch repair proteins have an significant role in DNA hypermethylation alteration and tumorigenesis[] SNHG15 is closely related to DNA replication and mismatch repair demonstrating that SNHG15 may promote the occurrence of UM by affecting DNA replication and DNA mismatch repair It indicated that SNHG15 may be identified as a novel marker of diagnosis therapeutic and prognosis prediction in UM However the related mechanism needs to be further elucidated This research also has some limitations The most important one is the limited number of patients and time of followup In addition some patient characteristics such as ciliary body involvement were not completely recorded in the database In fact ciliary body involvement plays a critical role in UM[]In conclusion this study aims to demonstrate the vital role of SNHG15 in UM and the potential relationship between SNHG15 expression and clinical parameters SNHG15 expression may be a valuable biomarker for poor survival in UM Moreover we have preliminarily explored the crucial pathway associated with SNHG15 in UM However further experimental validation is needed to be performed for clarifying the significant impact of SNHG15 And it is of great significance to further identify its independent prognostic value in a largescale standardized researches on UMACKNOWLEDGEMENTSFoundations Supported by the National Natural Science Foundation of China No81970835 No81800867 Conflicts of Interest Wu X None Li XF None Wu Q None Ma RQ None Qian J None Zhang R NoneREFERENCES van Raamsdonk CD Griewank KG Crosby MB Garrido MC Vemula S Wiesner T Obenauf AC Wackernagel W Green G Bouvier N Sozen MM Baimukanova G Roy R Heguy A Dolgalev I Khanin R Busam K Speicher MR O€™Brien J Bastian BC Mutations in GNA11 in uveal melanoma N Engl J Med Carvajal RD Sosman JA Quevedo JF Milhem MM Joshua AM Kudchadkar RR Linette GP Gajewski TF Lutzky J Lawson DH Lao CD Flynn PJ Albertini MR Sato T Lewis K Doyle A Ancell K Panageas KS Bluth M Hedvat C Erinjeri J Ambrosini G Marr B Abramson DH Dickson MA Wolchok JD Chapman PB Schwartz GK Effect of selumetinib vs chemotherapy on progressionfree survival in uveal melanoma a randomized clinical trial JAMA Shain AH Bagger MM Yu R Chang D Liu SS Vemula S Weier JF Wadt K Heegaard S Bastian BC Kiilgaard JF The genetic evolution of metastatic uveal melanoma Nat Genet Bagger M SmidtNielsen I Andersen MK Jensen PK Heegaard S Andersen KK Friis S Kiilgaard JF Longterm metastatic risk after biopsy of posterior uveal melanoma Ophthalmology Kujala E Mäkitie T Kivelä T Very longterm prognosis of patients with malignant uveal melanoma Invest Ophthalmol Vis Sci Chandran SS Somerville RPT Yang JC Sherry RM Klebanoff CA Goff SL Wunderlich JR Danforth DN Zlott D Paria BC Sabesan AC Srivastava AK Xi LQ Pham TH Raffeld M White DE Toomey MA Rosenberg SA Kammula US Treatment of metastatic uveal melanoma with adoptive transfer of tumourinfiltrating lymphocytes a singlecentre twostage singlearm phase study Lancet Oncol Mendell JT Targeting a long noncoding RNA in breast cancer N Engl J Med Lan Y Xiao XW He ZC Luo Y Wu CF Li L Song X Long noncoding RNA OCC1 suppresses cell growth through destabilizing HuR protein in colorectal cancer Nucleic Acids Res Cao CH Sun JY Zhang DY Guo XJ Xie LW Li X Wu DH Liu L The long intergenic noncoding RNA UFC1 a target of microRNA 34a interacts with the mRNA stabilizing protein HuR to increase levels of βcatenin in HCC cells Gastroenterology 20151482415426e18 Wang P Xue YQ Han YM Lin L Wu C Xu S Jiang ZP Xu JF Liu QY Cao XT The STAT3binding long noncoding RNA lncDC controls human dendritic cell differentiation Science Zheng XL Tang HW Zhao XF Sun YM Jiang YF Liu YH Long noncoding RNA FTH1P3 facilitates uveal melanoma cell growth and invasion through miR2245p PLoS One 20171211e0184746 Lu QK Zhao N Zha GP Wang HY Tong QH Xin SH LncRNA HOXA11AS exerts oncogenic functions by repressing p21 and miR in uveal melanoma DNA Cell Biol Lu LN Yu XY Zhang LL Ding X Pan H Wen XY Xu SQ Xing Y Fan JY Ge SF Zhang H Jia RB Fan XQ The long noncoding RNA RHPN1AS1 promotes uveal melanoma progression Int J Mol Sci Wu S Chen H Han N Zhang CX Yan HT Long noncoding RNA PVT1 silencing prevents the development of uveal melanoma by impairing MicroRNA173pdependent MDM2 upregulation Invest Ophthalmol Vis Sci Li P He J Yang Z Ge SF Zhang H Zhong Q Fan XQ ZNNT1 long noncoding RNA induces autophagy to inhibit tumorigenesis of uveal melanoma by regulating key autophagy gene expression Autophagy Dong YZ Meng XM Li GS Long noncoding RNA SNHG15 indicates poor prognosis of nonsmall cell lung cancer and promotes 0ccell proliferation and invasion Eur Rev Med Pharmacol Sci SNHG15 serves as an oncogene and predicts poor prognosis in epithelial ovarian cancer Onco Targets Ther Liu K Hou Y Liu YK Zheng J LncRNA SNHG15 contributes to proliferation invasion and autophagy in osteosarcoma cells by sponging miR141 J Biomed Sci Wu DM Wang S Wen X Han XR Wang YJ Shen M Fan SH Zhang ZF Shan Q Li MQ Hu B Lu J Chen GQ Zheng YL LncRNA SNHG15 acts as a ceRNA to regulate YAP1Hippo signaling pathway by sponging miR200a3p in papillary thyroid carcinoma Cell Death Dis Guo XB Yin HS Wang JY Evaluating the diagnostic and prognostic value of long noncoding RNA SNHG15 in pancreatic ductal adenocarcinoma Eur Rev Med Pharmacol Sci Sun XT Bai Y Yang C Hu SY Hou ZL Wang GX Long noncoding RNA SNHG15 enhances the development of colorectal carcinoma via functioning as a ceRNA through miR141SIRT1Wntβcatenin axis Artif Cells Nanomed Biotechnol Ye JF Tan LD Fu Y Xu HJ Wen LJ Deng Y Liu K LncRNA SNHG15 promotes hepatocellular carcinoma progression by sponging miR1413p J Cell Biochem Zhang JH Wei HW Yang HG Long noncoding RNA SNHG15 a potential prognostic biomarker for hepatocellular carcinoma Eur Rev Med Pharmacol Sci Zhang YL Zhang DH Lv J Wang S Zhang Q LncRNA SNHG15 Acts as an oncogene in prostate cancer by regulating miR3383pFKBP1A axis Gene Kong QL Qiu M Long noncoding RNA SNHG15 promotes human breast cancer proliferation migration and invasion by sponging miR2113p Biochem Biophys Res Commun Wang TQ Sun HB Bao Y En R Tian YJ Zhao W Jia LZ POM121 overexpression is related to a poor prognosis in colorectal cancer Expert Rev Mol Diagn Shuai Y Ma ZH Lu JW Feng JF LncRNA SNHG15 a new budding star in human cancers Cell Prolif 2020531e12716 Qu C Dai CM Guo YH Qin R Liu JB Long noncoding RNA Ma YW Xue YX Liu XB Qu CB Cai H Wang P Li ZQ Li Z Liu YH SNHG15 affects the growth of glioma microvascular endothelial cells by negatively regulating miR153 Oncol Rep Li M Bian ZH Jin GY Zhang J Yao SR Feng YY Wang X Yin Y Fei BJ You QJ Huang ZH LncRNASNHG15 enhances cell proliferation in colorectal cancer by inhibiting miR3383p Cancer Med Liu YC Li JL Li F Li M Shao Y Wu LP SNHG15 functions as a tumor suppressor in thyroid cancer J Cell Biochem Liu YC Li JL Li M Li F Shao Y Wu LP microRNA5105p promotes thyroid cancer cell proliferation migration and invasion through suppressing SNHG15 J Cell Biochem Li YW Guo HY Jin CJ Qiu CP Gao M Zhang L Liu ZJ Kong BH Spliceosomeassociated factor CTNNBL1 promotes proliferation and invasion in ovarian cancer Exp Cell Res Williams GH Stoeber K The cell cycle and cancer J Pathol Yu B Ding YM Liao XF Wang CH Wang B Chen XY Overexpression of PARPBP correlates with tumor progression and poor prognosis in hepatocellular carcinoma Dig Dis Sci Maiuri AR Peng M Podicheti R Sriramkumar S Kamplain CM Rusch DB DeStefano Shields CE Sears CL O€™Hagan HM Mismatch repair proteins initiate epigenetic alterations during inflammationdriven tumorigenesis Cancer Res Berry D Seider M Stinnett S Mruthyunjaya P Schefler AC Ocular Oncology Study Consortium Relationship of clinical features and baseline tumor size with gene expression profile status in uveal melanoma a Multiinstitutional study Retina Jiang ZM Yu FH Li M Upregulation of BCL2 kD proteininteracting protein BNIP3 is predictive of unfavorable prognosis in uveal melanoma Med Sci Monit Int J Ophthalmol Vol No Aug18 wwwijocnTel Email ijopress163com 0c'

cancer219

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: LncRNA SNHG15 predicts poor prognosis in uveal melanoma and its potential pathwaysXue Wu123 XiaoFeng Li123 Qian Wu4 RuiQi Ma123 Jiang Qian123 Rui Zhang123·Basic Research·1Department of Ophthalmology Eye ENT Hospital of Fudan University Shanghai China2NHC Key Laboratory of Myopia Fudan University Shanghai China 3Laboratory of Myopia Chinese Academy of Medical Sciences Shanghai China4Department of Pathology West China Hospital Sichuan University Chengdu Sichuan Province ChinaCofirst authors Xue Wu and XiaoFeng LiCorrespondence to Rui Zhang Department of Ophthalmology Eye ENT Hospital of Fudan University Fen Yang Road Shanghai China zhangrui936163comReceived Accepted Our research suggests that SNHG15 may play a vital role as a potential marker in UM that predicts poor prognosis Besides GSEA indicates the underlying signaling pathways enriched differentially in SNHG15 high expression phenotype KEYWORDS SNHG15 uveal melanoma the Cancer Genome Atlas pathology prognosis Gene Set Enrichment Analysis1018240ijo20200804Citation Wu X Li XF Wu Q Ma RQ Qian J Zhang R LncRNA SNHG15 predicts poor prognosis in uveal melanoma and its potential pathways Int J Ophthalmol Abstract— AIM To evaluate the role of long noncoding RNA lncRNA SNHG15 and its potential pathways in uveal melanoma UM METHODS The SNHG15 mRNA expression level and corresponding clinicopathological characteristics of patients with UM were obtained from the Cancer Genome Atlas TCGA database and further analyzed The SPSS statistical software package was used for statistical analyses To investigate the potential function of SNHG15 in UM we conducted indepth research on Gene Set Enrichment Analysis GSEA— RESULTS The univariate analysis revealed that the age tumor diameter pathological type extrascleral extension cancer status and high expression of SNHG15 were statistical risk factors for death from all causes The multivariate analysis suggested that the mRNA expression level of SNHG15 was an independent risk factor for death from all causes as was age and pathological type KaplanMeier survival analysis confirmed that UM patients with high SNHG15 expression might have a poor prognosis In addition SNHG15 was significantly differentially expressed in the different groups of tumor pathologic stage metastasis and living status Besides the logistic regression analysis indicated that high SNHG15 expression group in UM was significantly associated with cancer status pathologic stage metastasis and living status Moreover the GSEA indicated the potential pathways regulated by SNHG15 in UM INTRODUCTIONU veal melanoma UM the most common intraocular cancer in adult worldwide[] is a malignant tumor that originates in melanocytes of the choroid plexus ciliary body and iris of the eye At present despite definitive radiotherapy or removal of the primary lesion numerous patients eventually develop metastases and subsequently prognosis is significantly poor[] In addition UM tends to metastasize to liver through hematogenous pathway a distant site relative to their origins in the eye[] There is an incubation period between the enucleation of the primary tumor and the emergence of metastasis which can range from a few months to several decades[] Despite the advancement of UM management there are currently no effective therapy once the metastases occurred[] Therefore close followup and further research on the pathogenesis and novel makers exploration of UM are of great significance for accurate diagnosis appropriate therapy and prognosis prediction Long noncoding RNA lncRNA is a class of noncoding transcripts with a length of larger than nucleotides[] which has been involved widely in biological processes of different cancers including cell cycle apoptosis cell differentiation[] In the development of UM lncRNA is also reported to play a vital role in cell cycle cell proliferation apoptosis invasion and autophagy[] For example silencing of lncRNA PVT1 prevents the development of UM by impairing microRNA173pdependent MDM2 upregulation[] ZNNT1 can suppress Int J Ophthalmol Vol No Aug18 wwwijocnTel Email ijopress163com 0cLncRNA SNHG15 predicts prognosis in uveal melanomathe progression of UM by inducing the expression of crucial autophagy gene[] The lncRNA RHPN1AS1 facilitates the tumorigenesis of UM by influencing cell proliferation and migration[] However the study of vital lncRNAs in UM still remains to be exploredSNHG15 a novel lncRNA located on chromosome 7p13[] is identified to play a key role in many types of human tumors such as osteosarcoma[] papillary thyroid carcinoma[] pancreatic ductal adenocarcinoma[] colorectal carcinoma[] hepatocellular carcinoma[] prostate cancer[] and breast cancer[] To our knowledge the potential impact of SNHG15 on the tumorigenesis of UM seems unclear recently Thus the purpose of this study was to evaluate the pivotal role of SNHG15 in the progression of UM In addition the relationship between SNHG15 expression and clinicopathologic characteristics in UM was preliminarily demonstrated To explore the underlying mechanisms of the biological pathways involved in UM we conducted a research on Gene Set Enrichment Analysis GSEA MATERIALS AND METHODSEthical Approval The study protocol was approved by the Ethics Committee of the Eye ENT Hospital of Fudan University and all procedures were complied with the principles of the Declaration of Helsinki All datasets of our present study were downloaded from an database TCGA so there was no written informed consent from participantsRNASequencing Patient Data and Bioinformatics Analysis The RNASeq gene expression level and clinicopathological characteristics including cases were obtained from the official website of the Cancer Genome Atlas TCGA UM project portalgdccancergov Patients with UM were classified as two groups based on the median SNHG15 expression level cutoff value794 FPKM Finally patients with UM were retained and their clinicopathological characteristics were further analyzed including the detailed information of age gender tumor diameter thickness pathological type extrascleral extension cancer status pathological stage metastasis living status SNHG15 expressionGene Set Enrichment Analysis GSEA is a common bioanalysis used to interpret and analyze microarray and other similar data and to speculate related pathways that can significantly enrich regulatory genes[] Through TCGA UM project we obtained the RNASeq gene expression level of UM patients And the analysis was conducted using GSEA v30 software In this study according to the association with SNHG15 expression the ordered gene list was generated firstly by GSEA Subsequently GSEA was conducted to clarify statistically significant differences between the two groups with high and low SNHG15 expression A total of permutations were performed The SNHG15 expression level was identified as a phenotype label The related pathways statistically enriched in each phenotype were selected with the nominal P005 and an false discovery rate FDR Statistical Analysis The SPSS statistical software package SPSS Inc USA was used for statistical analyses Both the univariate and multivariate analyses using Cox analysis were performed to demonstrate independent prognostic biomarkers for UM patients The survival curve was generated by conducting KaplanMeier method To compare the significant differences in overall survival OS the logrank test was conducted The plot chart was performed to visualize the difference of SNHG15 expression level for diverse variables through Graphpad The relationship between the SNHG15 expression and clinicopathological characteristics were analyzed using logistic regression The median value of SNHG15 expression was selected as the cutoff value P005 was considered statistically significantRESULTSPatient Characteristics The records of primary UM with both RNASeq gene expression level and clinicopathological characteristics were obtained from TCGA database The mean age of UM patients was years old including males and females The mean value of tumor diameter and thickness were and mm respectively In our study cohort the pathological type of UM included epithelioid cell dominant type and spindle cell dominant type of tumors were epithelioid cell dominant and were spindle cell dominant There were cases without extrascleral extension and cases with extrascleral extension The cancer status included tumorfree cases and cases with tumor Pathologic stage II was found in cases and stage IIIIV in cases And of cases had metastases of cases had no metastases Of cases cases died of all causes Survival Outcomes and Multivariate Analysis Prognostic factors of UM were analyzed using univariate and multivariate Cox regression The univariate analysis suggested that high SNHG15 expression was a risk factor for death from all causes Other clinicopathologic variables related to poor prognosis included age tumor diameter pathological type extrascleral extension cancer status Table In a multivariate analysis SNHG15 was an independent risk factor for death from all causes as was age and pathological typeSNHG15 Expression Associated with Clinical Pathological Characteristics A total of UM cases with SNHG15 expression data and clinicopathologic characteristics were analyzed from TCGA KaplanMeier survival analysis 0cTable Prognostic parameters in UM were analyzed using univariate and multivariate Cox regressionDeath from all causesParametersnmeanUnivariate analysisPHR95CIAge yGenderFemaleMaleTumor diameter mmThickness mmPathological typeEpithelioid cell dominantSpindle cell dominantExtrascleral extensionNoYesCancer statusTumor freeWith tumorPathological stageIIIIIIVSNHG15HighLowUM Uveal melanomaMultivariate analysisPHR95CIdemonstrated that high SNHG15 expression group had a worse prognosis when compared to low SNHG15 expression group Figure 1A P005 As shown in Figure 1B1D SNHG15 was statistically differentially expressed in diverse groups of the tumor pathologic stage stage II vs IIIIV P00257 metastasis P00071 living status P00017 To clarify the clinicopathologic impact of SNHG15 we also used logistic regression and concluded that the SNHG15 expression based on median value of FPKM as a categorical variable was statistically related to clinicopathologic features Table High SNHG15 expression was significantly related to cancer status pathologic stage metastasis living status in UM all P005 Table These results demonstrated that UM with high SNHG15 expression were prone to progress to cancer status of survival with tumor a more advanced stage metastasis and poor living status when compared to the low SNHG15 expression group However there was no statistically significant difference in age gender tumor diameter thickness pathological type extrascleral invasionMain Enriched Pathways in UM Tissues with High SNHG15 Expression To explore the SNHG15related potential signaling pathways activated in UM GSEA was performed In the current study based on the association with SNHG15 expression the gene list was generated firstly Figure The SNHG15 expression was associated with clinical pathological characteristics A Patients with high SNHG15 expression had a shorter OS when compared with the low SNHG15 expression group P002 BD The expression of SNHG15 was statistically different in diverse groups of the tumor pathologic stage P00257 metastasis P00071 living status P00017 aP005 bP001by GSEA To clarify the statistically significant differences between high and low SNHG15 expression groups GSEA was Int J Ophthalmol Vol No Aug18 wwwijocnTel Email ijopress163com 0cLncRNA SNHG15 predicts prognosis in uveal melanomaTable Association between SNHG15 expression and clinicopathologic variables using logistic regressionParametersAge yGenderFemaleMaleTumor diameter mmThickness mmPathological typeEpithelialNonepithelialExtrascleral invasionNoYesCancer statusTumor freeWith tumorPathological stageIIIIIIVMetastasesNoYesLiving statusAliveDeadnmeanSNHG15 expressionLowHighPOR95CINESNominal PvalESTable Enriched pathways for differential SNHG15 expression in UMNameSpliceosomeCell cyclePyrimidine metabolismDNA replicationNucleotide excision repairRNA degradationHomologous recombinationMismatch repairUM Uveal melanoma ES Enrichment score NES Normal enrichment score FDR False discovery rateFDR Qvalconducted subsequently The results indicated that there were significant differences in spliceosome cell cycle pyrimidine metabolism DNA replication nucleotide excision repair RNA degradation homologous recombination and mismatch repair among patients with high SNHG15 expression phenotype Figure Table DISCUSSIONAccumulating evidences indicate that SNHG15 plays a dual role in the tumorigenesis and development of different tumors[] Previously SNHG15 has been demonstrated as a carcinogenic lncRNA which is usually upregulated in tumor tissues compared with normal tissues[] It exerts 0cFigure Enrichment plots from GSEA Spliceosome cell cycle pyrimidine metabolism DNA replication nucleotide excision repair RNA degradation homologous recombination and mismatch repair are enriched significantly in SNHG15 high expression phenotypean oncogenic effect via various epigenetic mechanisms[] For example it can suppress the expression of miR3383p and facilitate the proliferation of colorectal cancer cells[] It plays a carcinogenic role by affecting miR3383pFKBP1A axis in prostate cancer[] It can also enhance hepatocellular carcinoma progression by negative regulation of miR1413p[] However there are reports that SNHG15 has a tumor suppressive effect suggesting that low SNHG15 expression is related to poor prognosis in thyroid cancer and upregulating expression of SNHG15 can significantly suppress cell proliferation[] At present the impact of SNHG15 on UM is still unclear Therefore vital roles and potential biological mechanism of SNHG15 in UM needs to be elucidated In this study we revealed that high SNHG15 expression was related to clinicopathologic features in UM Through RNASeq gene expression level and clinicopathological characteristics obtained from the TCGA UM project we analyzed the relationship among SNHG15 expression clinicopathological features and prognosis of UM The univariate analysis demonstrated that SNHG15 expression level age tumor diameter pathological type extrascleral extension and cancer status were risk factors for death from all causes The multivariate analysis suggested that high SNHG15 expression along with age and pathological type was an independent risk factor for death from all causes Therefore the results demonstrated that high SNHG15 expression was an independent predictor of poor prognosis in UM through univariate and multivariate analysis KaplanMeier survival analysis also indicated that high SNHG15 expression group had a worse prognosis when compared to low SNHG15 expression group in UM In addition an analysis was conducted to further explore the relationship between SNHG15 and clinicopathological features The SNHG15 expression was statistically different in diverse groups of the tumor pathologic stage metastasis and living status Besides high SNHG15 expression based on median expression value of FPKM in UM was associated with cancer status of survival with tumor advanced pathologic stage metastasis and living status It demonstrated that high SNHG15 expression in UM was strongly related to poor prognosis The mechanisms of SNHG15 dysregulation in malignant tumors are quite complex and are far from being completely understood Previous studies have suggested that SNHG15 is involved in diverse pathological and physiological processes of many tumors through their abnormal expressions including cell proliferation invasion migration and autophagy[] To explore the biological mechanism of SNHG15 in UM GSEA was conducted It indicated that spliceosome cell cycle pyrimidine metabolism DNA replication nucleotide excision repair RNA degradation homologous recombination and mismatch repair were all enriched differentially in SNHG15 high expression phenotype Alternative splicing is essential for gene regulation and abnormal splicing plays a vital role in inactivating tumor suppressor genes or activating oncogenes[] SNHG15 may have an impact on the invasion Int J Ophthalmol Vol No Aug18 wwwijocnTel Email ijopress163com 0cLncRNA SNHG15 predicts prognosis in uveal melanomaand migration of UM cells by affecting spliceosomal related factors The abnormal cell proliferation of tumor is related to the lack of checkpoint control over the cell cycle which is the basis of genetic instability[] Evidence shows that the lack of homologous recombination may facilitate the disturbance of cell cycle the instability and accumulated mutations of genome during the progression and development[] Mismatch repair proteins have an significant role in DNA hypermethylation alteration and tumorigenesis[] SNHG15 is closely related to DNA replication and mismatch repair demonstrating that SNHG15 may promote the occurrence of UM by affecting DNA replication and DNA mismatch repair It indicated that SNHG15 may be identified as a novel marker of diagnosis therapeutic and prognosis prediction in UM However the related mechanism needs to be further elucidated This research also has some limitations The most important one is the limited number of patients and time of followup In addition some patient characteristics such as ciliary body involvement were not completely recorded in the database In fact ciliary body involvement plays a critical role in UM[]In conclusion this study aims to demonstrate the vital role of SNHG15 in UM and the potential relationship between SNHG15 expression and clinical parameters SNHG15 expression may be a valuable biomarker for poor survival in UM Moreover we have preliminarily explored the crucial pathway associated with SNHG15 in UM However further experimental validation is needed to be performed for clarifying the significant impact of SNHG15 And it is of great significance to further identify its independent prognostic value in a largescale standardized researches on UMACKNOWLEDGEMENTSFoundations Supported by the National Natural Science Foundation of China No81970835 No81800867 Conflicts of Interest Wu X None Li XF None Wu Q None Ma RQ None Qian J None Zhang R NoneREFERENCES van Raamsdonk CD Griewank KG Crosby MB Garrido MC Vemula S Wiesner T Obenauf AC Wackernagel W Green G Bouvier N Sozen MM Baimukanova G Roy R Heguy A Dolgalev I Khanin R Busam K Speicher MR O€™Brien J Bastian BC Mutations in GNA11 in uveal melanoma N Engl J Med Carvajal RD Sosman JA Quevedo JF Milhem MM Joshua AM Kudchadkar RR Linette GP Gajewski TF Lutzky J Lawson DH Lao CD Flynn PJ Albertini MR Sato T Lewis K Doyle A Ancell K Panageas KS Bluth M Hedvat C Erinjeri J Ambrosini G Marr B Abramson DH Dickson MA Wolchok JD Chapman PB Schwartz GK Effect of selumetinib vs chemotherapy on progressionfree survival in uveal melanoma a randomized clinical trial JAMA Shain AH Bagger MM Yu R Chang D Liu SS Vemula S Weier JF Wadt K Heegaard S Bastian BC Kiilgaard JF The genetic evolution of metastatic uveal melanoma Nat Genet Bagger M SmidtNielsen I Andersen MK Jensen PK Heegaard S Andersen KK Friis S Kiilgaard JF Longterm metastatic risk after biopsy of posterior uveal melanoma Ophthalmology Kujala E Mäkitie T Kivelä T Very longterm prognosis of patients with malignant uveal melanoma Invest Ophthalmol Vis Sci Chandran SS Somerville RPT Yang JC Sherry RM Klebanoff CA Goff SL Wunderlich JR Danforth DN Zlott D Paria BC Sabesan AC Srivastava AK Xi LQ Pham TH Raffeld M White DE Toomey MA Rosenberg SA Kammula US Treatment of metastatic uveal melanoma with adoptive transfer of tumourinfiltrating lymphocytes a singlecentre twostage singlearm phase study Lancet Oncol Mendell JT Targeting a long noncoding RNA in breast cancer N Engl J Med Lan Y Xiao XW He ZC Luo Y Wu CF Li L Song X Long noncoding RNA OCC1 suppresses cell growth through destabilizing HuR protein in colorectal cancer Nucleic Acids Res Cao CH Sun JY Zhang DY Guo XJ Xie LW Li X Wu DH Liu L The long intergenic noncoding RNA UFC1 a target of microRNA 34a interacts with the mRNA stabilizing protein HuR to increase levels of βcatenin in HCC cells Gastroenterology 20151482415426e18 Wang P Xue YQ Han YM Lin L Wu C Xu S Jiang ZP Xu JF Liu QY Cao XT The STAT3binding long noncoding RNA lncDC controls human dendritic cell differentiation Science Zheng XL Tang HW Zhao XF Sun YM Jiang YF Liu YH Long noncoding RNA FTH1P3 facilitates uveal melanoma cell growth and invasion through miR2245p PLoS One 20171211e0184746 Lu QK Zhao N Zha GP Wang HY Tong QH Xin SH LncRNA HOXA11AS exerts oncogenic functions by repressing p21 and miR in uveal melanoma DNA Cell Biol Lu LN Yu XY Zhang LL Ding X Pan H Wen XY Xu SQ Xing Y Fan JY Ge SF Zhang H Jia RB Fan XQ The long noncoding RNA RHPN1AS1 promotes uveal melanoma progression Int J Mol Sci Wu S Chen H Han N Zhang CX Yan HT Long noncoding RNA PVT1 silencing prevents the development of uveal melanoma by impairing MicroRNA173pdependent MDM2 upregulation Invest Ophthalmol Vis Sci Li P He J Yang Z Ge SF Zhang H Zhong Q Fan XQ ZNNT1 long noncoding RNA induces autophagy to inhibit tumorigenesis of uveal melanoma by regulating key autophagy gene expression Autophagy Dong YZ Meng XM Li GS Long noncoding RNA SNHG15 indicates poor prognosis of nonsmall cell lung cancer and promotes 0ccell proliferation and invasion Eur Rev Med Pharmacol Sci SNHG15 serves as an oncogene and predicts poor prognosis in epithelial ovarian cancer Onco Targets Ther Liu K Hou Y Liu YK Zheng J LncRNA SNHG15 contributes to proliferation invasion and autophagy in osteosarcoma cells by sponging miR141 J Biomed Sci Wu DM Wang S Wen X Han XR Wang YJ Shen M Fan SH Zhang ZF Shan Q Li MQ Hu B Lu J Chen GQ Zheng YL LncRNA SNHG15 acts as a ceRNA to regulate YAP1Hippo signaling pathway by sponging miR200a3p in papillary thyroid carcinoma Cell Death Dis Guo XB Yin HS Wang JY Evaluating the diagnostic and prognostic value of long noncoding RNA SNHG15 in pancreatic ductal adenocarcinoma Eur Rev Med Pharmacol Sci Sun XT Bai Y Yang C Hu SY Hou ZL Wang GX Long noncoding RNA SNHG15 enhances the development of colorectal carcinoma via functioning as a ceRNA through miR141SIRT1Wntβcatenin axis Artif Cells Nanomed Biotechnol Ye JF Tan LD Fu Y Xu HJ Wen LJ Deng Y Liu K LncRNA SNHG15 promotes hepatocellular carcinoma progression by sponging miR1413p J Cell Biochem Zhang JH Wei HW Yang HG Long noncoding RNA SNHG15 a potential prognostic biomarker for hepatocellular carcinoma Eur Rev Med Pharmacol Sci Zhang YL Zhang DH Lv J Wang S Zhang Q LncRNA SNHG15 Acts as an oncogene in prostate cancer by regulating miR3383pFKBP1A axis Gene Kong QL Qiu M Long noncoding RNA SNHG15 promotes human breast cancer proliferation migration and invasion by sponging miR2113p Biochem Biophys Res Commun Wang TQ Sun HB Bao Y En R Tian YJ Zhao W Jia LZ POM121 overexpression is related to a poor prognosis in colorectal cancer Expert Rev Mol Diagn Shuai Y Ma ZH Lu JW Feng JF LncRNA SNHG15 a new budding star in human cancers Cell Prolif 2020531e12716 Qu C Dai CM Guo YH Qin R Liu JB Long noncoding RNA Ma YW Xue YX Liu XB Qu CB Cai H Wang P Li ZQ Li Z Liu YH SNHG15 affects the growth of glioma microvascular endothelial cells by negatively regulating miR153 Oncol Rep Li M Bian ZH Jin GY Zhang J Yao SR Feng YY Wang X Yin Y Fei BJ You QJ Huang ZH LncRNASNHG15 enhances cell proliferation in colorectal cancer by inhibiting miR3383p Cancer Med Liu YC Li JL Li F Li M Shao Y Wu LP SNHG15 functions as a tumor suppressor in thyroid cancer J Cell Biochem Liu YC Li JL Li M Li F Shao Y Wu LP microRNA5105p promotes thyroid cancer cell proliferation migration and invasion through suppressing SNHG15 J Cell Biochem Li YW Guo HY Jin CJ Qiu CP Gao M Zhang L Liu ZJ Kong BH Spliceosomeassociated factor CTNNBL1 promotes proliferation and invasion in ovarian cancer Exp Cell Res Williams GH Stoeber K The cell cycle and cancer J Pathol Yu B Ding YM Liao XF Wang CH Wang B Chen XY Overexpression of PARPBP correlates with tumor progression and poor prognosis in hepatocellular carcinoma Dig Dis Sci Maiuri AR Peng M Podicheti R Sriramkumar S Kamplain CM Rusch DB DeStefano Shields CE Sears CL O€™Hagan HM Mismatch repair proteins initiate epigenetic alterations during inflammationdriven tumorigenesis Cancer Res Berry D Seider M Stinnett S Mruthyunjaya P Schefler AC Ocular Oncology Study Consortium Relationship of clinical features and baseline tumor size with gene expression profile status in uveal melanoma a Multiinstitutional study Retina Jiang ZM Yu FH Li M Upregulation of BCL2 kD proteininteracting protein BNIP3 is predictive of unfavorable prognosis in uveal melanoma Med Sci Monit Int J Ophthalmol Vol No Aug18 wwwijocnTel Email ijopress163com 0c' Answer:

Thyroid_Cancer

Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly citedAccumulating evidence has supported an increased risk of osteoporotic fracture in postmenopausal women and elderly mendiagnosed with diabetes mellitus However it is not uncommon for young and middleaged male patients diagnosed with type diabetes mellitus T2DM to suï¬er from oste ia or osteoporosis Few studies focused on this population group are availableThe aim of this study is to evaluate bone metabolic status and investigate the ‚uence of T2DM on bone metabolism in yearold men Anthropometric assessment and blood samples were obtained from patients with T2DM and nondiabeticvolunteers Serum parathyroid hormone PTH and bone turnover markers BTMs including serum procollagen type I Nterminal peptide PINP osteocalcin OC and crosslinked Ctelopeptide of type I collagen CTX were analysed Nosignificant diï¬erences were observed based on age body mass index systolic blood pressure serum calcium phosphoruscreatinine total protein and albumin levels when comparing T2DM and control groups Fasting blood glucose HbA1ctriglyceride TG total cholesterol and lowdensity lipoprotein cholesterol were significantly increased while highdensitylipoprotein cholesterol was significantly decreased in the T2DM group Compared with controls diabetic patients showed lowerserum PINP OC and PTH levels whereas serum CTX levels were similar between the two groups Moreover HbA1c levelswere positively correlated with PINP and inversely associated with PTH levels TG levels were negatively correlated with OC orCTX levels Furthermore multiple linear regression revealed a positive correlation between HbA1c and PINP levels Theseresults also revealed a negative association between HbA1c and PTH and between TG and OC levels even after adjusting forexpected confounder factors Collectively these findings indicated that young and middleaged male patients with T2DMshowed a lower turnover state resulting from bone formation inhibition Glucose and lipid metabolic disorders may aï¬ect boneformation through diï¬erent pathways IntroductionType diabetes mellitus T2DM is a common chronic metabolic disease caused by insufficient insulin secretion andoractivity leading to chronic hyperglycaemia Its high prevalence has resulted in a heavy burden on social financialand health care systems [] There is a large amount of evidence revealing an increased risk of fracture in diabeticpatients particularly hip fracture [ ] Recent metaanalyses indicated that hip fracture risk increases times in patients with T2DM [ ] In addition studies haddemonstrated that severe vertebral fracture in patients withT2DM was associated with increased allcause mortality [] Osteoporotic fracture has been increasingly recognizedas another complication of T2DM High morbidity and mortality make the two diseases be more serious global healthproblem The association between osteoporosis and T2DMshould be paid close attentionOsteoporosis is a skeletal chronic metabolic disease characterized by low bone mass and destroyed bone microarchitecture resulting in the high risk of fragility fracture []Therefore bone metabolism should be further studied inpatients with T2DM Bone metabolism is a dynamic cyclicalprocess where osteoblasts are involved in bone formationand osteoclasts are involved in bone resorption [] Metabolites known as bone turnovers markers BTMs are generated 0cJournal of Diabetes Researchfrom bone tissue and cells during the dynamic process andreflect bone metabolism during a relatively short period oftime [] and are thus better at predicting more recentchanges Specifically procollagen type I Nterminal peptidePINP is the degradation product during the formation oftype I collagen secreted by osteoblasts serum osteocalcinOC is released by osteoblasts during bone formation crosslinked Ctelopeptide of type I collagen CTX is abreakdown product during the degradation of mature typeI collagen secreted by osteoclasts [] Consequently PINPand OC are key markers of bone formation and CTX is akey marker for bone resorption The International Osteoporosis Foundation IOF recommends PINP and CTX as thereference markers for bone formation and bone resorptionrespectively due to their high sensitivity and specificity[] Recently these BTMs have been used to assess bonemetabolism evaluate the clinical efficacy of osteoporosistherapies and predict fracture risk [] Additionally BTMsare shown to be associated with energy metabolism []which is closely related to glucose metabolism Studying theeï¬ect of glucose metabolism disorders on BTMs is importantto evaluate bone metabolic status in T2DMMost research has focused on studying postmenopausalwomen and elderly men since these two groups of individualsare at a high risk for fractures especially those diagnosedwith T2DM Bone formation and bone mass are highest inthe third decade and then decrease with age [ ] However oste ia or osteoporosis in young and middleagedmale patients with T2DM is not uncommon in clinical practice Yet only a few studies focused on these populationgroups are available It is important to study how bonemetabolism disorders aï¬ect younger patients with T2DMTherefore young and middleaged male patients withT2DM were recruited as the subjects in the study presentedhere We aim to assess bone metabolism by determiningserum PINP OC CTX and parathyroid hormone PTHlevels and investigate the association among these markersand glucose metabolism The goal is to explore the ‚uenceof T2DM on bone metabolism which may allow for an accurate assessment of fracture risk and an earlier management ofbone metabolism disorders Materials and Methods Participants The study presented here is a crosssectional study conducted in men aged years oldPatients with T2DM who were admitted to the TianjinMetabolic Diseases Hospital from December to February were included in the T2DM group Nondiabeticmale volunteers from the physical examination centre wererecruited and included in the control group during thesame periodbloodfastingThe diagnosis of T2DM was based on the guidelinesprovided by the World Health anization [] includ°FBGž level ‰¥ mmolling mgdl or h blood glucose ‰¥ mmoll mgdlduring an oral glucose tolerance test OGTT Diabeticpatients were treated with oral antidiabetic agents or incombination with insulin Exclusion criteria included theglucosepresence of kidney disease eGFR mLmin173 m2hepatic disease ALT or AST ‰¥ times than the upperreference cancer rheumatic diseases rheumatic arthritisand rheumatoid arthritis other bone metabolic diseasesosteitis and osteomalacia hypercalcemia or other endocrine diseases Cushing™s syndrome primary hyperparathyroidism and thyroid dysfunction Participants takingmedications that may ‚uence bone metabolism were alsoexcluded These medications included glucocorticoids calcium vitamin D antiosteoporosis drugs steroids and thyroid hormonesThis study was conducted following the Declaration ofHelsinki and was approved by the Ethics Committee of the Tianjin Medical University Chu HsienI Memorial Hospital Each participant signed a written informedconsent form Clinical Measurements Anthropometric and biochemical assessments were performed in all participants Diabeticduration height weight body mass index BMI and bloodpressure data were collected BMI was calculated by the formula as weight in kg divided by height squared in m2All overnight fasting blood samples were obtained in themorning Serum samples were separated by centrifugationand stored at °C Blood calcium phosphorus total protein albumin alanine aminotransferase aspartate aminotransferase alkaline phosphatase ALP serum creatinineuric acid urea nitrogen haemoglobin A1c HbA1c FBGinsulin CpeptidetriglycerideTG highdensity lipoprotein cholesterol HDLc andlowdensity lipoprotein cholesterol LDLc were measuredusing standard methods Serum parathyroid hormonePTH and BTM levels including PINP OC and CTXwere measured using an IDSiSYS automated analyserRoche Germany The intraassay and interassay coefficients of variation CVs of BTMs were below and respectivelycholesterolTCtotal Statistical Analyses The statistical analyses were performed with SPSS SPSS Inc Chicago IL USA Normality testing was conducted in all continuous variablesVariables with normal distributions were described as mean± standard deviation and the diï¬erences were determinedusing Student™s ttest between the two groups Those withskewed distributions were expressed as median interquartilerange and diï¬erences between groups were assessed usingthe Mann“Whitney U test The Pearson or Spearman correlation analysis was used to determine the correlation betweenblood glucose or lipid and bone metabolism markers Multiple linear regression analyses were conducted to evaluate theassociation between HbA1c TG and BTMs P value was considered statistically significant ResultsA total of diabetic patients were included in the T2DMgroup The mean age of these patients was ± yearsand the mean diabetic duration was years ranging from to years The mean FBG was ± mmoll 0cJournal of Diabetes ResearchTable Comparison of characteristics between diabetic patientsand controlsVariablesPatients with T2DMn ± Nondiabeticcontrols n P ± ””””””” ± ± ± ± ” ± ± ± ± ± ± ± ± ± ± ± ± ± Age yDiabeticduration yHeight cmWeight kgBMI kgm2SBP mmHgDBP mmHgFBG mmollHbA1c INS mIUlCP ngmlTG mmollTC mmollHDLcmmollLDLcmmollCa mmollP mmollTP glALB glALT IUlAST IUlALP IUlCr μmollSUA μmollBUNmmolly years T2DM type diabetes mellitus BMI body mass index SBP systolicblood pressure DBP diastolic blood pressure FBG fasting blood glucoseHbA1c haemoglobin A1c INS fasting insulin CP fasting Cpeptide TGtotaltriglyceride TCcholesterol HDLc highdensity lipoproteinlowdensity lipoprotein cholesterol Ca calcium Pcholesterol LDLcalaninephosphorus TPtotalaminotransferase ASTalkalinephosphatase Cr serum creatinine SUA serum uric acid BUN blood ureanitrogen P value was considered significant ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± protein ALBaspartatealbumin ALTaminotransferase ALPand the mean HbA1c value was ± A total of nondiabetic volunteers were recruited in the control groupthat had a mean age of ± years and a mean FBG of ± mmollBaseline clinical characteristics of the two groups areshown in Table No significant diï¬erences were observedbetween the control or T2DM groups for age P height P weight P BMI P orsystolic blood pressure P There were also no significant diï¬erences between the two groups for serum calciumP phosphorus P creatinine P total protein P albumin P or ALPP As expected patients in the T2DM groupshowed significantly higher FBG levels P comparedwith the control group In addition significantly higher TGP TC P and LDLc P levelsand significantly lower HDLc P levels wereobserved in diabetic patients compared with controlsComparison of BTMs and PTH between diabetic patientsand controls is shown in Table There were significantdecreases in serum PINP P OC P andPTH P levels in patients with T2DM comparedwith controls In contrast serum CTX levels were similarbetween the two groups P Moreover univariate correlation analyses were performed to investigate the association between blood glucoseor lipid and bone metabolism markers The results revealedthat HbA1c was positively correlated with PINP rs P and inversely associated with PTH r ˆ’P There was a significant negative correlationbetween OC or CTX and TG rs ˆ’ P rs ˆ’ P levels Figure There was no significantassociation observed between PINP and TG or between OCand HbA1c levels Age was negatively correlated with PINPrs ˆ’ P OC rs ˆ’ P andPTH r P but not with CTX levels TheBTMs and PTH levels did not correlate with BMI bloodpressure calcium or phosphorous levelstheFurthermore multiple linear regression analyses wereperformed to examinecrosssectional associationbetween blood glucose or lipid and BTMs after adjustingfor expected confounder factors Serum PINP OC orPTH levels were used as dependent variables while HbA1cor TG levels were used as independent variables Thesefindings indicated that HbA1c was positively correlatedwith PINP P and inversely associatedwith PTH ˆ’ P after adjusting for ageBMI systolic blood pressure TG HDLc LDLc calciumand phosphorus Our results also showed a significantnegative correlation between TG and OC ˆ’ P after adjusting for age BMI systolic blood pressure HbA1c HDLc LDLc calcium and phosphorusTable All independent variables used in multiple linearanalyses are shown in Table S1 DiscussionMost previous studies investigating postmenopausal womenand elderly men have shown that the markers for bone formation andor resorption were reduced in patients withT2DM compared with nondiabetic individuals [] indicating a lower bone turnover state It is unclear whetheryoung and middleaged diabetic patients shared similarresults In this study we focused on young and middleaged male patients with T2DM Results demonstrated thatdiabetic patients had significantly lower serum PINP andOC levels compared with the control individuals In contrast serum CTX levels were not significantly diï¬erentbetween the two groups Results indicated that inhibition 0cJournal of Diabetes ResearchTable Comparison of BTMs and PTH between diabetic patients and controlsPatients with T2DM VariablesPINP ngmlOC ngmlCTX ngmlPTH pgmlT2DM type diabetes mellitus PINP procollagen type I Nterminal peptide OC osteocalcin CTX crosslinked Ctelopeptide of type I collagen PTHparathyroid hormone P value was considered significant ± Nondiabetic controls ± Plmgn PNIPlmgn COrs P HbA1c ars “P lmgp HTPlmgn XTC𝛽r “P HbA1c brs “P TG mmolldTG mmollcFigure Correlation between serum glucose or lipid levels and BTMs or PTH a Correlation between PINP and HbA1c b Correlationbetween PTH and HbA1c c Correlation between OC and TG d Correlation between CTX and TG HbA1c haemoglobin A1c TGtriglyceride PINP procollagen type I Nterminal peptide OC osteocalcin CTX crosslinked Ctelopeptide of type I collagen PTHparathyroid hormone r Pearson™s correlation coefficient rs Spearman™s correlation coefficient P value was considered significantTable Multiple linear regression analyses between serum glucose or lipid and bone metabolism markersIndependent variablesDependent variablePINPOCPTHHbA1c haemoglobin A1c TG triglyceride PINP procollagen type I Nterminal peptide OC osteocalcin PTH parathyroid hormone P value wasconsidered significantUnstandardized coefficients Standardized coefficients HbA1cTGHbA1cPof bone formation phase rather than resorption led to alower bone turnover state in young and middleaged malepatients with T2DM Moreover this study demonstratedthat HbA1c was an independent factor for PINP suggesting the ‚uence of glycaemic control on PINP in youngand middleaged male patients with T2DM Early glycaemic control may reduce the risk of fracture by delayingbone formation reduction 0cJournal of Diabetes ResearchReduced serum OC levels were previously reported inmale patients with T2DM [“] Bezerra dos SantosMagalhaes further demonstrated a weak negative correlation between FBG and OC levels [] Whereas serumPINP was not available in these studies A recent studyrevealed a decrease in serum PINP levels in patients withimpaired fasting glucose and diabetes [] which was in linewith our research Further analyses revealed that serum PINPlevels were significantly reduced in younger diabetic patients years old compared with the older patients ‰¥ yearsold but serum CTX was also significantly decreased []The controversial conclusions may be related to diï¬erencesin age race diabetic duration and glycaemic control Astudy by Kulkarni [] shared a similar relationshipbetween HbA1c and PINP levels Additionally a largescale crosssectional study performed in Germany indirectly supported this conjecture The authors revealed thatchances for metabolic syndrome or T2DM significantlydecreased with the higher serum PINP and CTX levelsin men aged years old [] However two largescale studies performed in China one involving men andwomen aged years old [] and the other includingmen aged years old [] indicated that serum OCwas negatively correlated with chances for T2DM evenafter adjusting age BMI waist circumference blood pressure FBG and TG As described by these studies theclose relationship between glucose and BTMs has beeninvestigated but needs further understandingIn addition compared with controls diabetic patientsshowed higher TG TC and LDLc and lower HDLc levelswhich may represent a high probability of lipid metabolismdisorders in patients with T2DM Further analyses investigating the correlation between lipid and BTMs revealed a significant negative correlation between serum TG and OClevels High TG levels may reduce serum OC levels andinhibit bone formation in young and middleaged malepatients with T2DM These observations were similar towhat was found in a recent male populationbased studywhere serum TG levels were also inversely correlated withOC levels [] Some research investigating male diabeticpatients showed no relationship between serum OC levelsand blood lipid metabolism [ ] These findings are contradictory to one another Diï¬erences in age race and metabolic status may account for these controversial resultsThe impact of blood glucose and lipid metabolism disorders on BTMs needs further studies to elucidate mechanismsIt is known that hyperglycaemia can lead to osmotic diuresiswhich causes renal calcium leakage and a negative calciumbalance Improved blood glucose control contributes to thereduction of urinary calcium levels [] The calciumsensing defect and secondary chronic hypomagnesaemiainduced by osmotic diuresis may be responsible for impairedPTH secretion [] The pathological regulation of PTH onBTMs in patients with T2DM is not clear In this studyserum PTH levels were decreased and were negatively associated with HbA1c levels in T2DM implying that diabeticpatients especially those with poor glycaemic control hadlower PTH levels These observations were in line with previous studies [ ] Relative hypoparathyroidism may disrupt bone metabolism in patients with T2DM Previousstudies demonstrated that low PTH levels directly inhibitedosteoblast activity and contribute to bone demineralizationIn the nondiabetic population PTH inhibited transcriptionalsuppression of sclerostin produced by osteocytes As a Wntantagonist sclerostin inhibited Wntcatenin signallingand osteoblast activity However the regulation of PTH onsclerostin may be impaired in diabetes [] As mentionedabove the negative relationship between blood glucose andbone metabolism is probably explainedOtherwise chronic ‚ammatory conditions and turbulence of adipokines increased the risk of osteoporosis inpatients with T2DM [] Advanced glycation endproductsAGEs were accumulated in diabetes and played a key rolein chronic ‚ammatory complications [] Previous studieshave shown that BTMs were suppressed by hyperinsulinemiaand the accumulation of AGEs [] AGEs promoted theproduction of both ‚ammatory cytokines and reactive oxygen species ROS in the body by activating ligands furthertriggering chronic ‚ammation and bone resorption []In vitro studies reported that AGE2 and AGE3 inhibitedthe maturation of human marrow mesenchymal stem cellsMSCs and their diï¬erentiation into cartilage and bone tissues resulting in decreased osteoblasts [] Moreover theformation and accumulation of AGEs inhibited synthesis ofosteocalcin and osteoblastic ossein matrix [] increasednonenzymatic crosslinked folding of the collagen fibres[] and disturbed osteoblast development A recent studyindicated that hyperglycaemia directly inhibited the diï¬erentiation of osteoblasts and then decreased bone formationenhanced osteoclast activity and increased bone absorptioneventually leading to a reduction of bone mass [ ] Glucose and insulin signalling involved receptor activation of thenuclear factor κB ligandosteoprotegerin RANKLOPGpathway [ ] Analyses revealed thatlower serumRANKL levels were associated with higher TG levels []This inverse relationship may explain the results generatedin this study Furthermore adiponectin a recently uncoveredadipocytokineis produced exclusivity in adipose tissueResearch shows that adiponectin stimulated osteoblast proliferation diï¬erentiation and mineralization [] Howeverserum adiponectin concentrations decreased in patients withT2DM [] The turbulence of adipocytokines may lead to animbalance of bone metabolismAntidiabetic agents may have diï¬erent eï¬ects on bonemetabolism Agents that may have an eï¬ect include thiazolidinediones TZDs sodiumglucoselinked transporter2SGLT2 inhibitors insulin and glucagonlike peptide1receptor agonists GLP1 RA A previous work shows thatrosiglitazone a type of TZDs promoted osteoblastosteocyteapoptosis and led to a negative balance in bone metabolism[] Analyses demonstrated a gender diï¬erence when itcame to the eï¬ects of TZDs on fracture in patients withT2DM and confirmed that TZDs only increased fracture riskin female patients and not male patients [] SGLT2 inhibitors improved blood glucose levels by promoting urinaryglucose excretion which may aï¬ect urinary calcium excretionand bone metabolism Canagliflozin treatment was associatedwith a higher fracture rate in patients with T2DM [] A 0cJournal of Diabetes Researchmetaanalysis indicated no relationship between three SGLT2inhibitors canagliflozin dapagliflozin and empagliflozin andfracture risk Clinical studies on adverse skeletal events ofSGLT2 inhibitors are still lacking Few studies have assessedthe association between insulin injection and BTMs Severalstudies reported an increased fracture risk in insulintreatedpatients with T2DM [] A high incidence of hypoglycaemicevents and falling [] may be the main reasons in olderadults Longterm disease and the presence of multiple diabetic complications may also disrupt bone metabolism Nosignificant diï¬erences were observed between diabetic patientsunder treatment with n or without n TZDswith n or without n SGLT2 inhibitors andwith n or without n insulin in this study Liraglutide and exenatide two GLP1 RAs may improve skeletalblood supply increase bone mineral density BMD andreduce the risk of osteoporosis and fracture in animal andhuman studies [ ] However the bone protective eï¬ectsbehind this require clinical studies There were no significantdiï¬erences observed between diabetic patients under treatment with n or without n GLP1 RAs in ourstudy In addition there were also no significant diï¬erencesbetween patients under treatment with n or withoutn dipeptidyl peptidase4 DPP4 inhibitors withn or without n insulin secretagogues withn or without n metformin and with n or without n alphaglucosidase inhibitors AGI in thepresent study Table S2 As a multiple metabolic diseasethe treatment of T2DM is complex and requires additionalclinical studies to evaluate the ‚uence of these therapies onbone metabolismOne advantage of this study is that it focused on male diabetic patients aged years old where BTMs varied withsmall changes and there was a restriction on gender and agebeing an ‚uence on the results With this it was easier toinvestigate the relationship between blood glucose lipidsand bone metabolism However this study still faces somelimitations First the crosssectional design prevents onefrom drawing a causal relationship and failed to explorechanges in BTMs after improving blood glucose and lipidmetabolism disorders Further prospective research mayoï¬er additional information about this Second the samplesize number between the two groups was unequal and thenumber of controls used was inadequate Besides this studywas a singlecentre study that only analysed patients with relatively severe diabetes Therefore the results presented heremay not be generalizable to all young and middleaged malepopulations diagnosed with T2DM Largescale and multicentre studies remained to verify these issues Third the‚uence of antidiabetic agents on bone metabolism remainscontradictory Consequently potential confounder factorsmay exist Fourth serum levels of bonespecific alkalinephosphatase BAP vitamin D or steroids all of which ‚uence bone metabolism were not determined in this studySerum ALP is mainly derived from liver isoform LAPand its specificity for bone metabolism is lacking [] BAPis a more bonespecific marker of bone formation while thecurrent immunoassays available for BAP still show up to crossreactivity toward LAP [] As recommended bythe IOF [] serum PINP was preferred for bone formationbecause of high specificity in our study Vitamin D promotesthe absorption of calcium and may aï¬ect bone metabolismHowever relatively limited data about the eï¬ect of vitaminD on BTMs are available A prospective partial interventionstudy in postmenopausal women with T2DM shows that25OHD was positively correlated with PINP especially inpatients taking alfacalcidol [] The MINOS study a prospective study of men aged years revealed thatserum 25OHD was not associated with BTMs in men under years of age n [] The relationship between vitamin D and BTMs still needs further research Fifth we didnot take BMD into consideration BMD altogether withBTMs may be helpful to evaluate bone metabolism BMDreflects mineral density of bone and is the cumulative resultof longterm bone metabolic activities This study mainlyfocused on the impact of T2DM on BTMs and evaluatedthe recent changes of bone metabolism Further studiesshould be conducted to investigate the longterm eï¬ect ofT2DM on BMD ConclusionsThis study demonstrated that young and middleaged malepatients with T2DM showed a lower turnover state resultingfrom bone formation inhibition HbA1c levels were positively correlated with PINP levels and inversely associatedwith PTH levels These findings also revealed a negative correlation between TG and OC levels even after adjusting forexpected confounder factors Glucose and lipid metabolismdisorders may aï¬ect bone formation through diï¬erent pathways The study presented here provides evidence of T2DM‚uencing bone metabolism in young and middleagedmen The improvement of blood glucose and lipids may bebeneficial to bone metabolism and reduce fracture risk inpatients with T2DMData AvailabilityThe data used to support the findings of this study are available from the corresponding author upon requestConflicts of InterestThe authors declare that there is no conflict of interestregarding the publication of this paperAcknowledgmentsThis work was supported by Scientific Research Funding ofTianjin Medical University Chu HsienI Memorial Hospitalgrant numbers 2018ZDKF07 We gratefully acknowledgethe participants in this studySupplementary MaterialsTable S1 multiple linear regression between serum glucoseor lipid levels and BTMs or PTH Table S2 the informationaboutthe patients with T2DMSupplementary Materialsthe medications of 0cJournal of Diabetes ResearchReferences[] K Ogurtsova J D da Rocha Fernandes Y Huang œIDFDiabetes Atlas Global estimates for the prevalence of diabetesfor and  Diabetes Research and Clinical Practicevol pp “ [] A V Schwartz D E Sellmeyer K E Ensrud œOlderwomen with diabetes have an increased risk of fracture a prospective study Journal of Clinical Endocrinology and Metabolism vol no pp “ [] L Forsen H E Meyer K Midthjell and T H Edna œDiabetesmellitus and the incidence of hip fracture results from theNordTr¸ndelag Health Survey Diabetologia vol no pp “ [] Y Fan F Wei Y Lang and Y Liu œDiabetes mellitus and riskof hip fractures a metaanalysis Osteoporosis Internationalvol no pp “ [] M Janghorbani R M Van Dam W C Willett and F B HuœSystematic review of type and type diabetes mellitus andrisk of fracture American Journal of Epidemiology vol no pp “ [] I KostoglouAthanassiou P Athanassiou A Gkountouvasand P Kaldrymides œVitamin D and glycemic control in diabetes mellitus type  Therapeutic Advances in Endocrinologyand Metabolism vol no pp “ [] H Miyake I Kanazawa and T Sugimoto œAssociation ofbone mineral density bone turnover markers and vertebralfractures with allcause mortality in type diabetes mellitusCalcified Tissue International vol no pp “ [] E S Siris R Adler J Bilezikian œThe clinical diagnosis ofosteoporosis a position statement from the National BoneHealth Alliance Working Group Osteoporosis Internationalvol no pp “ [] M B Greenblatt J N Tsai and M N Wein œBone turnovermarkers in the diagnosis and monitoring of metabolic bonedisease Clinical Chemistry vol no pp “ [] S Vasikaran for the IOFIFCC Bone Marker Standards Working Group R Eastell œMarkers of bone turnover for theprediction of fracture risk and monitoring of osteoporosistreatment a need for international reference standards Osteoporosis International vol no pp “ [] H W S Cabral B F G Andolphi B V C Ferreira œTheuse of biomarkers in clinical osteoporosis Revista da Associa§£o M©dica Brasileira vol no pp “ [] P Iglesias F Arrieta M Pi±era œSerum concentrationsof osteocalcin procollagen type Nterminal propeptide andbetaCrossLaps in obese subjects with varying degrees of glucose tolerance Clinical Endocrinology vol no pp “ [] J M Wishart A O Need M Horowitz H A Morris  andB E C Nordin œEï¬ect of age on bone density and bone turnover in men Clinical Endocrinology vol no pp “ [] P Szulc P Garnero F Munoz F Marchand and P D Delmas œCrosssectional evaluation of bone metabolism inmen Journal of Bone and Mineral Research vol no pp “ [] K G M M Alberti P Z Zimmet and WHO ConsultationœDefinition diagnosis and classification of diabetes mellitusand its complications Part diagnosis and classification ofdiabetes mellitus provisional report of a WHO consultationDiabetic Medicine vol no pp “ [] J StarupLinde and P Vestergaard œBiochemical bone turnover markers in diabetes mellitus a systematic review Bonevol pp “ [] L Achemlal S Tellal F Rkiouak œBone metabolism inmale patients with type diabetes Clinical Rheumatologyvol no pp “ [] S V Kulkarni S Meenatchi R Reeta R Ramesh A R Srinivasan and C Lenin œAssociation of glycemic status with boneturnover markers in type diabetes mellitus InternationalJournal of Applied Basic Medical Research vol no pp “ [] K B dos Santos Magalh£es M M Magalh£es E T DinizC S Lucena L Griz and F Bandeira œMetabolic syndromeand central fat distribution are related to lower serum osteocalcin concentrations Annals of Nutrition and Metabolismvol no pp “ [] K L HollowayKew L L F De Abreu M A Kotowicz M ASajjad and J A Pasco œBone turnover markers in men andwomen with impaired fasting glucose and diabetes CalcifiedTissue International vol no pp “ [] E Lerchbaum VSchwetz M Nauck H V¶lzkeH Wallaschofski and A Hannemann œLower bone turnovermarkersthepopulationbased study of health in Pomerania NutritionMetabolism and Cardiovascular Diseases vol no pp “ in metabolicsyndromediabetesand[] H Shu Y Pei K Chen and J Lu œSignificant inverse association between serum osteocalcin and incident type diabetesin a middleaged cohort DiabetesMetabolism Research andReviews vol no pp “ [] A Tan Y Gao X Yang œLow serum osteocalcin level is apotential marker for metabolic syndrome results from a Chinese male population survey Metabolism vol no pp “ [] X Y Ma F Q Chen H Hong X J Lv M Dong and Q YWang œThe relationship between serum osteocalcin concentration and glucose and lipid metabolism in patients with type diabetes mellitus the role of osteocalcin in energy metabolism Annals of Nutrition and Metabolism vol no pp “ [] Y Chen Q Zhao G Du and Y Xu œAssociation betweenserum osteocalcin and glucoselipid metabolism in ChineseHan and Uygur populations with type diabetes mellitus inXinjiang two crosssectional studies Lipids in Health andDisease vol no p [] N C Thalassinos P Hadjiyanni M Tzanela C Alevizaki a

cancer220

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly citedAccumulating evidence has supported an increased risk of osteoporotic fracture in postmenopausal women and elderly mendiagnosed with diabetes mellitus However it is not uncommon for young and middleaged male patients diagnosed with type diabetes mellitus T2DM to suï¬er from oste ia or osteoporosis Few studies focused on this population group are availableThe aim of this study is to evaluate bone metabolic status and investigate the ‚uence of T2DM on bone metabolism in yearold men Anthropometric assessment and blood samples were obtained from patients with T2DM and nondiabeticvolunteers Serum parathyroid hormone PTH and bone turnover markers BTMs including serum procollagen type I Nterminal peptide PINP osteocalcin OC and crosslinked Ctelopeptide of type I collagen CTX were analysed Nosignificant diï¬erences were observed based on age body mass index systolic blood pressure serum calcium phosphoruscreatinine total protein and albumin levels when comparing T2DM and control groups Fasting blood glucose HbA1ctriglyceride TG total cholesterol and lowdensity lipoprotein cholesterol were significantly increased while highdensitylipoprotein cholesterol was significantly decreased in the T2DM group Compared with controls diabetic patients showed lowerserum PINP OC and PTH levels whereas serum CTX levels were similar between the two groups Moreover HbA1c levelswere positively correlated with PINP and inversely associated with PTH levels TG levels were negatively correlated with OC orCTX levels Furthermore multiple linear regression revealed a positive correlation between HbA1c and PINP levels Theseresults also revealed a negative association between HbA1c and PTH and between TG and OC levels even after adjusting forexpected confounder factors Collectively these findings indicated that young and middleaged male patients with T2DMshowed a lower turnover state resulting from bone formation inhibition Glucose and lipid metabolic disorders may aï¬ect boneformation through diï¬erent pathways IntroductionType diabetes mellitus T2DM is a common chronic metabolic disease caused by insufficient insulin secretion andoractivity leading to chronic hyperglycaemia Its high prevalence has resulted in a heavy burden on social financialand health care systems [] There is a large amount of evidence revealing an increased risk of fracture in diabeticpatients particularly hip fracture [ ] Recent metaanalyses indicated that hip fracture risk increases times in patients with T2DM [ ] In addition studies haddemonstrated that severe vertebral fracture in patients withT2DM was associated with increased allcause mortality [] Osteoporotic fracture has been increasingly recognizedas another complication of T2DM High morbidity and mortality make the two diseases be more serious global healthproblem The association between osteoporosis and T2DMshould be paid close attentionOsteoporosis is a skeletal chronic metabolic disease characterized by low bone mass and destroyed bone microarchitecture resulting in the high risk of fragility fracture []Therefore bone metabolism should be further studied inpatients with T2DM Bone metabolism is a dynamic cyclicalprocess where osteoblasts are involved in bone formationand osteoclasts are involved in bone resorption [] Metabolites known as bone turnovers markers BTMs are generated 0cJournal of Diabetes Researchfrom bone tissue and cells during the dynamic process andreflect bone metabolism during a relatively short period oftime [] and are thus better at predicting more recentchanges Specifically procollagen type I Nterminal peptidePINP is the degradation product during the formation oftype I collagen secreted by osteoblasts serum osteocalcinOC is released by osteoblasts during bone formation crosslinked Ctelopeptide of type I collagen CTX is abreakdown product during the degradation of mature typeI collagen secreted by osteoclasts [] Consequently PINPand OC are key markers of bone formation and CTX is akey marker for bone resorption The International Osteoporosis Foundation IOF recommends PINP and CTX as thereference markers for bone formation and bone resorptionrespectively due to their high sensitivity and specificity[] Recently these BTMs have been used to assess bonemetabolism evaluate the clinical efficacy of osteoporosistherapies and predict fracture risk [] Additionally BTMsare shown to be associated with energy metabolism []which is closely related to glucose metabolism Studying theeï¬ect of glucose metabolism disorders on BTMs is importantto evaluate bone metabolic status in T2DMMost research has focused on studying postmenopausalwomen and elderly men since these two groups of individualsare at a high risk for fractures especially those diagnosedwith T2DM Bone formation and bone mass are highest inthe third decade and then decrease with age [ ] However oste ia or osteoporosis in young and middleagedmale patients with T2DM is not uncommon in clinical practice Yet only a few studies focused on these populationgroups are available It is important to study how bonemetabolism disorders aï¬ect younger patients with T2DMTherefore young and middleaged male patients withT2DM were recruited as the subjects in the study presentedhere We aim to assess bone metabolism by determiningserum PINP OC CTX and parathyroid hormone PTHlevels and investigate the association among these markersand glucose metabolism The goal is to explore the ‚uenceof T2DM on bone metabolism which may allow for an accurate assessment of fracture risk and an earlier management ofbone metabolism disorders Materials and Methods Participants The study presented here is a crosssectional study conducted in men aged years oldPatients with T2DM who were admitted to the TianjinMetabolic Diseases Hospital from December to February were included in the T2DM group Nondiabeticmale volunteers from the physical examination centre wererecruited and included in the control group during thesame periodbloodfastingThe diagnosis of T2DM was based on the guidelinesprovided by the World Health anization [] includ°FBGž level ‰¥ mmolling mgdl or h blood glucose ‰¥ mmoll mgdlduring an oral glucose tolerance test OGTT Diabeticpatients were treated with oral antidiabetic agents or incombination with insulin Exclusion criteria included theglucosepresence of kidney disease eGFR mLmin173 m2hepatic disease ALT or AST ‰¥ times than the upperreference cancer rheumatic diseases rheumatic arthritisand rheumatoid arthritis other bone metabolic diseasesosteitis and osteomalacia hypercalcemia or other endocrine diseases Cushing™s syndrome primary hyperparathyroidism and thyroid dysfunction Participants takingmedications that may ‚uence bone metabolism were alsoexcluded These medications included glucocorticoids calcium vitamin D antiosteoporosis drugs steroids and thyroid hormonesThis study was conducted following the Declaration ofHelsinki and was approved by the Ethics Committee of the Tianjin Medical University Chu HsienI Memorial Hospital Each participant signed a written informedconsent form Clinical Measurements Anthropometric and biochemical assessments were performed in all participants Diabeticduration height weight body mass index BMI and bloodpressure data were collected BMI was calculated by the formula as weight in kg divided by height squared in m2All overnight fasting blood samples were obtained in themorning Serum samples were separated by centrifugationand stored at °C Blood calcium phosphorus total protein albumin alanine aminotransferase aspartate aminotransferase alkaline phosphatase ALP serum creatinineuric acid urea nitrogen haemoglobin A1c HbA1c FBGinsulin CpeptidetriglycerideTG highdensity lipoprotein cholesterol HDLc andlowdensity lipoprotein cholesterol LDLc were measuredusing standard methods Serum parathyroid hormonePTH and BTM levels including PINP OC and CTXwere measured using an IDSiSYS automated analyserRoche Germany The intraassay and interassay coefficients of variation CVs of BTMs were below and respectivelycholesterolTCtotal Statistical Analyses The statistical analyses were performed with SPSS SPSS Inc Chicago IL USA Normality testing was conducted in all continuous variablesVariables with normal distributions were described as mean± standard deviation and the diï¬erences were determinedusing Student™s ttest between the two groups Those withskewed distributions were expressed as median interquartilerange and diï¬erences between groups were assessed usingthe Mann“Whitney U test The Pearson or Spearman correlation analysis was used to determine the correlation betweenblood glucose or lipid and bone metabolism markers Multiple linear regression analyses were conducted to evaluate theassociation between HbA1c TG and BTMs P value was considered statistically significant ResultsA total of diabetic patients were included in the T2DMgroup The mean age of these patients was ± yearsand the mean diabetic duration was years ranging from to years The mean FBG was ± mmoll 0cJournal of Diabetes ResearchTable Comparison of characteristics between diabetic patientsand controlsVariablesPatients with T2DMn ± Nondiabeticcontrols n P ± ””””””” ± ± ± ± ” ± ± ± ± ± ± ± ± ± ± ± ± ± Age yDiabeticduration yHeight cmWeight kgBMI kgm2SBP mmHgDBP mmHgFBG mmollHbA1c INS mIUlCP ngmlTG mmollTC mmollHDLcmmollLDLcmmollCa mmollP mmollTP glALB glALT IUlAST IUlALP IUlCr μmollSUA μmollBUNmmolly years T2DM type diabetes mellitus BMI body mass index SBP systolicblood pressure DBP diastolic blood pressure FBG fasting blood glucoseHbA1c haemoglobin A1c INS fasting insulin CP fasting Cpeptide TGtotaltriglyceride TCcholesterol HDLc highdensity lipoproteinlowdensity lipoprotein cholesterol Ca calcium Pcholesterol LDLcalaninephosphorus TPtotalaminotransferase ASTalkalinephosphatase Cr serum creatinine SUA serum uric acid BUN blood ureanitrogen P value was considered significant ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± protein ALBaspartatealbumin ALTaminotransferase ALPand the mean HbA1c value was ± A total of nondiabetic volunteers were recruited in the control groupthat had a mean age of ± years and a mean FBG of ± mmollBaseline clinical characteristics of the two groups areshown in Table No significant diï¬erences were observedbetween the control or T2DM groups for age P height P weight P BMI P orsystolic blood pressure P There were also no significant diï¬erences between the two groups for serum calciumP phosphorus P creatinine P total protein P albumin P or ALPP As expected patients in the T2DM groupshowed significantly higher FBG levels P comparedwith the control group In addition significantly higher TGP TC P and LDLc P levelsand significantly lower HDLc P levels wereobserved in diabetic patients compared with controlsComparison of BTMs and PTH between diabetic patientsand controls is shown in Table There were significantdecreases in serum PINP P OC P andPTH P levels in patients with T2DM comparedwith controls In contrast serum CTX levels were similarbetween the two groups P Moreover univariate correlation analyses were performed to investigate the association between blood glucoseor lipid and bone metabolism markers The results revealedthat HbA1c was positively correlated with PINP rs P and inversely associated with PTH r ˆ’P There was a significant negative correlationbetween OC or CTX and TG rs ˆ’ P rs ˆ’ P levels Figure There was no significantassociation observed between PINP and TG or between OCand HbA1c levels Age was negatively correlated with PINPrs ˆ’ P OC rs ˆ’ P andPTH r P but not with CTX levels TheBTMs and PTH levels did not correlate with BMI bloodpressure calcium or phosphorous levelstheFurthermore multiple linear regression analyses wereperformed to examinecrosssectional associationbetween blood glucose or lipid and BTMs after adjustingfor expected confounder factors Serum PINP OC orPTH levels were used as dependent variables while HbA1cor TG levels were used as independent variables Thesefindings indicated that HbA1c was positively correlatedwith PINP P and inversely associatedwith PTH ˆ’ P after adjusting for ageBMI systolic blood pressure TG HDLc LDLc calciumand phosphorus Our results also showed a significantnegative correlation between TG and OC ˆ’ P after adjusting for age BMI systolic blood pressure HbA1c HDLc LDLc calcium and phosphorusTable All independent variables used in multiple linearanalyses are shown in Table S1 DiscussionMost previous studies investigating postmenopausal womenand elderly men have shown that the markers for bone formation andor resorption were reduced in patients withT2DM compared with nondiabetic individuals [] indicating a lower bone turnover state It is unclear whetheryoung and middleaged diabetic patients shared similarresults In this study we focused on young and middleaged male patients with T2DM Results demonstrated thatdiabetic patients had significantly lower serum PINP andOC levels compared with the control individuals In contrast serum CTX levels were not significantly diï¬erentbetween the two groups Results indicated that inhibition 0cJournal of Diabetes ResearchTable Comparison of BTMs and PTH between diabetic patients and controlsPatients with T2DM VariablesPINP ngmlOC ngmlCTX ngmlPTH pgmlT2DM type diabetes mellitus PINP procollagen type I Nterminal peptide OC osteocalcin CTX crosslinked Ctelopeptide of type I collagen PTHparathyroid hormone P value was considered significant ± Nondiabetic controls ± Plmgn PNIPlmgn COrs P HbA1c ars “P lmgp HTPlmgn XTC𝛽r “P HbA1c brs “P TG mmolldTG mmollcFigure Correlation between serum glucose or lipid levels and BTMs or PTH a Correlation between PINP and HbA1c b Correlationbetween PTH and HbA1c c Correlation between OC and TG d Correlation between CTX and TG HbA1c haemoglobin A1c TGtriglyceride PINP procollagen type I Nterminal peptide OC osteocalcin CTX crosslinked Ctelopeptide of type I collagen PTHparathyroid hormone r Pearson™s correlation coefficient rs Spearman™s correlation coefficient P value was considered significantTable Multiple linear regression analyses between serum glucose or lipid and bone metabolism markersIndependent variablesDependent variablePINPOCPTHHbA1c haemoglobin A1c TG triglyceride PINP procollagen type I Nterminal peptide OC osteocalcin PTH parathyroid hormone P value wasconsidered significantUnstandardized coefficients Standardized coefficients HbA1cTGHbA1cPof bone formation phase rather than resorption led to alower bone turnover state in young and middleaged malepatients with T2DM Moreover this study demonstratedthat HbA1c was an independent factor for PINP suggesting the ‚uence of glycaemic control on PINP in youngand middleaged male patients with T2DM Early glycaemic control may reduce the risk of fracture by delayingbone formation reduction 0cJournal of Diabetes ResearchReduced serum OC levels were previously reported inmale patients with T2DM [“] Bezerra dos SantosMagalhaes further demonstrated a weak negative correlation between FBG and OC levels [] Whereas serumPINP was not available in these studies A recent studyrevealed a decrease in serum PINP levels in patients withimpaired fasting glucose and diabetes [] which was in linewith our research Further analyses revealed that serum PINPlevels were significantly reduced in younger diabetic patients years old compared with the older patients ‰¥ yearsold but serum CTX was also significantly decreased []The controversial conclusions may be related to diï¬erencesin age race diabetic duration and glycaemic control Astudy by Kulkarni [] shared a similar relationshipbetween HbA1c and PINP levels Additionally a largescale crosssectional study performed in Germany indirectly supported this conjecture The authors revealed thatchances for metabolic syndrome or T2DM significantlydecreased with the higher serum PINP and CTX levelsin men aged years old [] However two largescale studies performed in China one involving men andwomen aged years old [] and the other includingmen aged years old [] indicated that serum OCwas negatively correlated with chances for T2DM evenafter adjusting age BMI waist circumference blood pressure FBG and TG As described by these studies theclose relationship between glucose and BTMs has beeninvestigated but needs further understandingIn addition compared with controls diabetic patientsshowed higher TG TC and LDLc and lower HDLc levelswhich may represent a high probability of lipid metabolismdisorders in patients with T2DM Further analyses investigating the correlation between lipid and BTMs revealed a significant negative correlation between serum TG and OClevels High TG levels may reduce serum OC levels andinhibit bone formation in young and middleaged malepatients with T2DM These observations were similar towhat was found in a recent male populationbased studywhere serum TG levels were also inversely correlated withOC levels [] Some research investigating male diabeticpatients showed no relationship between serum OC levelsand blood lipid metabolism [ ] These findings are contradictory to one another Diï¬erences in age race and metabolic status may account for these controversial resultsThe impact of blood glucose and lipid metabolism disorders on BTMs needs further studies to elucidate mechanismsIt is known that hyperglycaemia can lead to osmotic diuresiswhich causes renal calcium leakage and a negative calciumbalance Improved blood glucose control contributes to thereduction of urinary calcium levels [] The calciumsensing defect and secondary chronic hypomagnesaemiainduced by osmotic diuresis may be responsible for impairedPTH secretion [] The pathological regulation of PTH onBTMs in patients with T2DM is not clear In this studyserum PTH levels were decreased and were negatively associated with HbA1c levels in T2DM implying that diabeticpatients especially those with poor glycaemic control hadlower PTH levels These observations were in line with previous studies [ ] Relative hypoparathyroidism may disrupt bone metabolism in patients with T2DM Previousstudies demonstrated that low PTH levels directly inhibitedosteoblast activity and contribute to bone demineralizationIn the nondiabetic population PTH inhibited transcriptionalsuppression of sclerostin produced by osteocytes As a Wntantagonist sclerostin inhibited Wntcatenin signallingand osteoblast activity However the regulation of PTH onsclerostin may be impaired in diabetes [] As mentionedabove the negative relationship between blood glucose andbone metabolism is probably explainedOtherwise chronic ‚ammatory conditions and turbulence of adipokines increased the risk of osteoporosis inpatients with T2DM [] Advanced glycation endproductsAGEs were accumulated in diabetes and played a key rolein chronic ‚ammatory complications [] Previous studieshave shown that BTMs were suppressed by hyperinsulinemiaand the accumulation of AGEs [] AGEs promoted theproduction of both ‚ammatory cytokines and reactive oxygen species ROS in the body by activating ligands furthertriggering chronic ‚ammation and bone resorption []In vitro studies reported that AGE2 and AGE3 inhibitedthe maturation of human marrow mesenchymal stem cellsMSCs and their diï¬erentiation into cartilage and bone tissues resulting in decreased osteoblasts [] Moreover theformation and accumulation of AGEs inhibited synthesis ofosteocalcin and osteoblastic ossein matrix [] increasednonenzymatic crosslinked folding of the collagen fibres[] and disturbed osteoblast development A recent studyindicated that hyperglycaemia directly inhibited the diï¬erentiation of osteoblasts and then decreased bone formationenhanced osteoclast activity and increased bone absorptioneventually leading to a reduction of bone mass [ ] Glucose and insulin signalling involved receptor activation of thenuclear factor κB ligandosteoprotegerin RANKLOPGpathway [ ] Analyses revealed thatlower serumRANKL levels were associated with higher TG levels []This inverse relationship may explain the results generatedin this study Furthermore adiponectin a recently uncoveredadipocytokineis produced exclusivity in adipose tissueResearch shows that adiponectin stimulated osteoblast proliferation diï¬erentiation and mineralization [] Howeverserum adiponectin concentrations decreased in patients withT2DM [] The turbulence of adipocytokines may lead to animbalance of bone metabolismAntidiabetic agents may have diï¬erent eï¬ects on bonemetabolism Agents that may have an eï¬ect include thiazolidinediones TZDs sodiumglucoselinked transporter2SGLT2 inhibitors insulin and glucagonlike peptide1receptor agonists GLP1 RA A previous work shows thatrosiglitazone a type of TZDs promoted osteoblastosteocyteapoptosis and led to a negative balance in bone metabolism[] Analyses demonstrated a gender diï¬erence when itcame to the eï¬ects of TZDs on fracture in patients withT2DM and confirmed that TZDs only increased fracture riskin female patients and not male patients [] SGLT2 inhibitors improved blood glucose levels by promoting urinaryglucose excretion which may aï¬ect urinary calcium excretionand bone metabolism Canagliflozin treatment was associatedwith a higher fracture rate in patients with T2DM [] A 0cJournal of Diabetes Researchmetaanalysis indicated no relationship between three SGLT2inhibitors canagliflozin dapagliflozin and empagliflozin andfracture risk Clinical studies on adverse skeletal events ofSGLT2 inhibitors are still lacking Few studies have assessedthe association between insulin injection and BTMs Severalstudies reported an increased fracture risk in insulintreatedpatients with T2DM [] A high incidence of hypoglycaemicevents and falling [] may be the main reasons in olderadults Longterm disease and the presence of multiple diabetic complications may also disrupt bone metabolism Nosignificant diï¬erences were observed between diabetic patientsunder treatment with n or without n TZDswith n or without n SGLT2 inhibitors andwith n or without n insulin in this study Liraglutide and exenatide two GLP1 RAs may improve skeletalblood supply increase bone mineral density BMD andreduce the risk of osteoporosis and fracture in animal andhuman studies [ ] However the bone protective eï¬ectsbehind this require clinical studies There were no significantdiï¬erences observed between diabetic patients under treatment with n or without n GLP1 RAs in ourstudy In addition there were also no significant diï¬erencesbetween patients under treatment with n or withoutn dipeptidyl peptidase4 DPP4 inhibitors withn or without n insulin secretagogues withn or without n metformin and with n or without n alphaglucosidase inhibitors AGI in thepresent study Table S2 As a multiple metabolic diseasethe treatment of T2DM is complex and requires additionalclinical studies to evaluate the ‚uence of these therapies onbone metabolismOne advantage of this study is that it focused on male diabetic patients aged years old where BTMs varied withsmall changes and there was a restriction on gender and agebeing an ‚uence on the results With this it was easier toinvestigate the relationship between blood glucose lipidsand bone metabolism However this study still faces somelimitations First the crosssectional design prevents onefrom drawing a causal relationship and failed to explorechanges in BTMs after improving blood glucose and lipidmetabolism disorders Further prospective research mayoï¬er additional information about this Second the samplesize number between the two groups was unequal and thenumber of controls used was inadequate Besides this studywas a singlecentre study that only analysed patients with relatively severe diabetes Therefore the results presented heremay not be generalizable to all young and middleaged malepopulations diagnosed with T2DM Largescale and multicentre studies remained to verify these issues Third the‚uence of antidiabetic agents on bone metabolism remainscontradictory Consequently potential confounder factorsmay exist Fourth serum levels of bonespecific alkalinephosphatase BAP vitamin D or steroids all of which ‚uence bone metabolism were not determined in this studySerum ALP is mainly derived from liver isoform LAPand its specificity for bone metabolism is lacking [] BAPis a more bonespecific marker of bone formation while thecurrent immunoassays available for BAP still show up to crossreactivity toward LAP [] As recommended bythe IOF [] serum PINP was preferred for bone formationbecause of high specificity in our study Vitamin D promotesthe absorption of calcium and may aï¬ect bone metabolismHowever relatively limited data about the eï¬ect of vitaminD on BTMs are available A prospective partial interventionstudy in postmenopausal women with T2DM shows that25OHD was positively correlated with PINP especially inpatients taking alfacalcidol [] The MINOS study a prospective study of men aged years revealed thatserum 25OHD was not associated with BTMs in men under years of age n [] The relationship between vitamin D and BTMs still needs further research Fifth we didnot take BMD into consideration BMD altogether withBTMs may be helpful to evaluate bone metabolism BMDreflects mineral density of bone and is the cumulative resultof longterm bone metabolic activities This study mainlyfocused on the impact of T2DM on BTMs and evaluatedthe recent changes of bone metabolism Further studiesshould be conducted to investigate the longterm eï¬ect ofT2DM on BMD ConclusionsThis study demonstrated that young and middleaged malepatients with T2DM showed a lower turnover state resultingfrom bone formation inhibition HbA1c levels were positively correlated with PINP levels and inversely associatedwith PTH levels These findings also revealed a negative correlation between TG and OC levels even after adjusting forexpected confounder factors Glucose and lipid metabolismdisorders may aï¬ect bone formation through diï¬erent pathways The study presented here provides evidence of T2DM‚uencing bone metabolism in young and middleagedmen The improvement of blood glucose and lipids may bebeneficial to bone metabolism and reduce fracture risk inpatients with T2DMData AvailabilityThe data used to support the findings of this study are available from the corresponding author upon requestConflicts of InterestThe authors declare that there is no conflict of interestregarding the publication of this paperAcknowledgmentsThis work was supported by Scientific Research Funding ofTianjin Medical University Chu HsienI Memorial Hospitalgrant numbers 2018ZDKF07 We gratefully acknowledgethe participants in this studySupplementary MaterialsTable S1 multiple linear regression between serum glucoseor lipid levels and BTMs or PTH Table S2 the informationaboutthe patients with T2DMSupplementary Materialsthe medications of 0cJournal of Diabetes ResearchReferences[] K Ogurtsova J D da Rocha Fernandes Y Huang œIDFDiabetes Atlas Global estimates for the prevalence of diabetesfor and  Diabetes Research and Clinical Practicevol pp “ [] A V Schwartz D E Sellmeyer K E Ensrud œOlderwomen with diabetes have an increased risk of fracture a prospective study Journal of Clinical Endocrinology and Metabolism vol no pp “ [] L Forsen H E Meyer K Midthjell and T H Edna œDiabetesmellitus and the incidence of hip fracture results from theNordTr¸ndelag Health Survey Diabetologia vol no pp “ [] Y Fan F Wei Y Lang and Y Liu œDiabetes mellitus and riskof hip fractures a metaanalysis Osteoporosis Internationalvol no pp “ [] M Janghorbani R M Van Dam W C Willett and F B HuœSystematic review of type and type diabetes mellitus andrisk of fracture American Journal of Epidemiology vol no pp “ [] I KostoglouAthanassiou P Athanassiou A Gkountouvasand P Kaldrymides œVitamin D and glycemic control in diabetes mellitus type  Therapeutic Advances in Endocrinologyand Metabolism vol no pp “ [] H Miyake I Kanazawa and T Sugimoto œAssociation ofbone mineral density bone turnover markers and vertebralfractures with allcause mortality in type diabetes mellitusCalcified Tissue International vol no pp “ [] E S Siris R Adler J Bilezikian œThe clinical diagnosis ofosteoporosis a position statement from the National BoneHealth Alliance Working Group Osteoporosis Internationalvol no pp “ [] M B Greenblatt J N Tsai and M N Wein œBone turnovermarkers in the diagnosis and monitoring of metabolic bonedisease Clinical Chemistry vol no pp “ [] S Vasikaran for the IOFIFCC Bone Marker Standards Working Group R Eastell œMarkers of bone turnover for theprediction of fracture risk and monitoring of osteoporosistreatment a need for international reference standards Osteoporosis International vol no pp “ [] H W S Cabral B F G Andolphi B V C Ferreira œTheuse of biomarkers in clinical osteoporosis Revista da Associa§£o M©dica Brasileira vol no pp “ [] P Iglesias F Arrieta M Pi±era œSerum concentrationsof osteocalcin procollagen type Nterminal propeptide andbetaCrossLaps in obese subjects with varying degrees of glucose tolerance Clinical Endocrinology vol no pp “ [] J M Wishart A O Need M Horowitz H A Morris  andB E C Nordin œEï¬ect of age on bone density and bone turnover in men Clinical Endocrinology vol no pp “ [] P Szulc P Garnero F Munoz F Marchand and P D Delmas œCrosssectional evaluation of bone metabolism inmen Journal of Bone and Mineral Research vol no pp “ [] K G M M Alberti P Z Zimmet and WHO ConsultationœDefinition diagnosis and classification of diabetes mellitusand its complications Part diagnosis and classification ofdiabetes mellitus provisional report of a WHO consultationDiabetic Medicine vol no pp “ [] J StarupLinde and P Vestergaard œBiochemical bone turnover markers in diabetes mellitus a systematic review Bonevol pp “ [] L Achemlal S Tellal F Rkiouak œBone metabolism inmale patients with type diabetes Clinical Rheumatologyvol no pp “ [] S V Kulkarni S Meenatchi R Reeta R Ramesh A R Srinivasan and C Lenin œAssociation of glycemic status with boneturnover markers in type diabetes mellitus InternationalJournal of Applied Basic Medical Research vol no pp “ [] K B dos Santos Magalh£es M M Magalh£es E T DinizC S Lucena L Griz and F Bandeira œMetabolic syndromeand central fat distribution are related to lower serum osteocalcin concentrations Annals of Nutrition and Metabolismvol no pp “ [] K L HollowayKew L L F De Abreu M A Kotowicz M ASajjad and J A Pasco œBone turnover markers in men andwomen with impaired fasting glucose and diabetes CalcifiedTissue International vol no pp “ [] E Lerchbaum VSchwetz M Nauck H V¶lzkeH Wallaschofski and A Hannemann œLower bone turnovermarkersthepopulationbased study of health in Pomerania NutritionMetabolism and Cardiovascular Diseases vol no pp “ in metabolicsyndromediabetesand[] H Shu Y Pei K Chen and J Lu œSignificant inverse association between serum osteocalcin and incident type diabetesin a middleaged cohort DiabetesMetabolism Research andReviews vol no pp “ [] A Tan Y Gao X Yang œLow serum osteocalcin level is apotential marker for metabolic syndrome results from a Chinese male population survey Metabolism vol no pp “ [] X Y Ma F Q Chen H Hong X J Lv M Dong and Q YWang œThe relationship between serum osteocalcin concentration and glucose and lipid metabolism in patients with type diabetes mellitus the role of osteocalcin in energy metabolism Annals of Nutrition and Metabolism vol no pp “ [] Y Chen Q Zhao G Du and Y Xu œAssociation betweenserum osteocalcin and glucoselipid metabolism in ChineseHan and Uygur populations with type diabetes mellitus inXinjiang two crosssectional studies Lipids in Health andDisease vol no p [] N C Thalassinos P Hadjiyanni M Tzanela C Alevizaki a Answer:

Thyroid_Cancer

properly citedIntroduction Endogenously produced antiganglioside antibodies could aï¬ect the evolution of cutaneous melanoma Epidemiologicaland experimental evidence suggest œchronic ‚ammation to be one of the hallmarks in skin cancers The aim of the study was tocharacterize the relation between antiganglioside antibodies and ‚ammation in cutaneous melanoma focusing on gangliosidesGM1 GM2 GM3 GD1a GD1b GT1b GQ1b Material and Method We performed an observational study that included subjects subdivided into three groups patients with metastatic melanoma cases patients with primary melanoma casesand healthy subjects subjects The assessment of antiganglioside antibodies IgG and IgM classes against GM1 GM2GM3 GD1a GD1b GT1b GQ1b was performed using immunoblot technique EUROLine kit Results The presence of IgGand IgM antiganglioside antibodies in primary melanoma was as follows antiGM1 and GM2 and GM3 and GD1a and GD1b and GT1b and GQ1b and In metastaticmelanoma the level of antiganglioside antibodies was significantly lower compared with primary melanoma p while inthe control group they were absent Antiganglioside antibodies antiGM1 and GD1a were positively correlated while antiGM3GD1b and GT1b were negatively associated with the ‚ammatory markers interleukin IL8 and C reactive protein CRPConclusions Tumour ganglioside antigens generate an immune response in patients with primary melanomas The host™s ability toelaborate an early antiganglioside response could be considered as a defence mechanism directed toward eliminating a dangersignal from the tumour microenvironment Antiganglioside antibodies associated with ‚ammation markers could be used asdiagnostic monitoring and treatment tools in patients with cutaneous melanoma IntroductionGangliosides are a group of bioactive glycolipids located onthe outer face of cell membranes These glycolipids play amajor role in cell proliferation diï¬erentiation migrationapoptosis signal transduction cell adhesion modulatinggrowth factor or hormone receptor antigen recognition protein trafficking viral transformation and oncogenesis [“]Atypical expression of some ganglioside antigens associatedwith certain tumours neuroblastomas melanomas gliomaslymphomas small cell lung cancer and prostate cancer andfurthermore could play an importantrole in cancer 0cJournal of Immunology Researchimmunotherapy [“] Gangliosides that are released inextracellular spaces could have dual action antitumor andprotumour eï¬ect [“] Data regarding the endogenousimmune response directed toward tumour gangliosides andthe significance of this response are limited A series of studiesperformed in in vivo experimental models and in vitro inmurine and human cancer cells have shown that monoclonalantiganglioside antibodies have antitumor potential Theseantibodies exert numerous antitumor eï¬ects through variousmechanisms An important mechanism is the translocationof gangliosides from the plasmatic membrane into theintracellular spaces so binding of antibodies to the surfaceof the tumor cells and complement activation that leads to celllysis mediated by complementdependent cytotoxicity andantibodymediated cellular cytotoxicity [ ] Antiganglioside antibodies modulate ceramide synthesis [ ]reception and transduction of the cytotoxic signal [] theyare involved in suppression or induction of cell death throughdiï¬erent pathways apoptosis necrosis oncogeneslike structural and functional changes of mitochondria accumulationof reactive oxygen species acetylation of gangliosides accumulation of sphingosine sphingamine ceramides [ ]Proteomic studies showed that antiganglioside antibodiescould induce changes like the disruption of signalling systemsP38MAPK PARP JNK123 METc ERK12 P13KAKTand FAK modulation of the level and function of transcription factors P53 SP1 MYCN and HSF1 regulating the balance between apoptosisinducing and apoptosissuppressingfactors cysteineaspartylproteases Bax Bcl2 [ “]These antibodies stimulate the cytotoxicity of chemotherapeutic drugs and small molecule inhibitors [ ] As a result antiganglioside antibodies could be used as diagnostic monitoringand treatment tools in cancer patients [ ]Ganglioside levels are increased in malignant melanocytes and represent an important topic of research [ ]Several researchers have emphasized the role of glycolipidsas markers of melanoma A study analysing the expressionof gangliosides in melanocyte lines and melanoma cell linesfound out an increased expression of GD3 synthase genesin melanoma cells but not in melanocytes The same resultswere obtained for GM2GD2 synthase [] It seems thatgangliosides induce cell proliferation and invasion throughp130Cas and paxillin in melanoma cells []Inflammatory mechanisms play an important role inmelanoma Multiple studies have shown that plasma levelsof C reactive protein CRP increase during tumor proliferation and several relations have been evaluated CRPsurvivalrelationship CRPresponse therapy CRP‚ammationNowadays CRP is considered a true marker for assessing‚ammation in melanoma as well as a marker for responseto treatment Prospective studies have provided consistentresults in the predictive value of CRP in neoplastic diseaseproving high sensitivity and specificity [] In addition inmelanoma elevated levels of CRP may reflect the amountand activity of circulating pro‚ammatory cytokines eginterleukin IL8 IL8 plays a crucial role in regulating cellfunction for host defence and for developing natural immunity [ ] Moreover IL8 is released by various cell typesincluding polymorphonuclear neutrophils PMNs monoSerum dilution Incubation with fixed on strip antigens degrees min with balanceWashing with universal tampon times min each with balanceIncubation with conjugated enzyme degrees min with balanceWashing with universal tampon times min each with balanceIncubation with fixed on strip antigens degrees min with balanceWashing with universal tampon times min each with balanceEUROLine scan evaluationFigure Antiganglioside detectioncytes T lymphocytes and endothelial cells upon exposureto ‚ammatory stimuli Melanoma cells have been reportedto express IL8 and this ‚uences their oncogenic properties[ ] IL8 follows the evolution of melanoma progression and regression under treatment reflecting the stage ofthe disease [“]Based on these accumulating data we have investigatedantiganglioside antibodies in correlation with other ‚ammatory markers IL8 CRP and the clinical evolution of the melanoma patients Clarifying these relations could significantlyimprove the prediction of clinical outcomes Furthermore itcan lead to the development of appropriate therapeutic strategies in patients with cutaneous melanoma Material and Method Patients We performed an observational prospectivestudy during years in Clinical Hospital for Infectious andTropical Diseases œVictor Babes”Dermatology Department Bucharest The study was approved by the Ethics Committee of the Hospital All participants agreed to be includedin research studies without prejudice of the diagnosis orpersonal image and signed the informed consent accordingto the Declaration of HelsinkiThe study included adult patients with cutaneous melanoma with no other pathologies and no treatment for theprimary disease Exclusion criteria were age under yearspregnancy alcohol use melanoma under treatmentWe performed an observational study that included subjects subdivided in three groups patients with metastaticmelanoma cases patients with primary melanoma cases and healthy subjects with matching sex and age subjects Patients were selected and examined accordingto ESMO Clinical Practice Guidelines for melanoma 0cJournal of Immunology ResearchTable Test strips coated with parallel lines of purified antigensAntigenGM1GM2GM3GD1aGD1bGT1bGanglioside typeSourceMonosialoganglioside GM1Monosialoganglioside GM2Monosialoganglioside GM3 Dog erythrocytesBovine brainBovine brainDisialoganglioside GD1aDisialoganglioside GD1bTrisialoganglioside GT1bBovine brainBovine brainBovine brainGQ1bTetrasialoganglioside GQ1bBovine brainStructureGal3GalNAc4[Neu5Ac3]Gal4GlcCerGalNAc4[Neu5Ac3]Gal4GlcCerNeu5Ac3Gal4GlcCerNeu5Ac3Gal3GalNAc4[Neu5Ac3]Gal4GlcCerGal3GalNAc4[Neu5Ac8Neu5Ac3]Gal4GlcCerNeu5Ac3Gal3GalNAc4[Neu5Ac8Neu5Ac3]Gal4GlcCerNeu5Ac8Neu5Ac3Gal3GalNAc4[Neu5Ac8Neu5Ac3]Gal4GlcCer Glcglucose Gal galactose GalNAc Nacetylgalactosaminediagnosis based on clinical histopathological immunohistochemical and imagistic data All the events related to theprogression of the disease were recorded relapse metastasisneurotoxicity hyper reactivation of the immune system upontreatment The group characteristics were similar for age andsex the primary melanoma group included women and men with a mean age of ± years the metastaticmelanoma group included women and men with a meanage of ± years and the control group included women and men with mean age of ± years Materials and Reagents In this work the assessment ofantiganglioside antibodies was made by the immunoblottechnique using EUROLine kits Figure This methodallows the evaluation of antibodies IgG and IgM classesagainst GM1 GM2 GM3 GD1a GD1b GT1b and GQ1bfrom serumplasma The kit contains strips marked withpurified antigens Table Theevaluation ofantigangliosideantibodies wasperformed using the EUROLine Scan software After readingthe signal intensity on the strips marked with ganglioside antigens the results were evaluated and the results are presented asoptical sensibility The assessment of IL8 was performed bythe ELISA method using Enzo Life Science reagents withTECAN analyser and the results are presented as pgdl CRPwas assessed by immunoturbidimetry using Human reagentsand HumaStar300 analyser the results are presented as mgdl Statistical Analysis All the results were analysed usingIBM SPSS Statistics We evaluated the normality of datadistribution using the KolmogorovSmirnov test The variationbetween groups was determined using the parametric teststtest when two groups were compared or ANOVA test whenmore groups were compared and nonparametric tests likeMannWhitney or Wilcoxon The correlation between groupswas evaluated using linear regression and Pearson coefficientp was considered with statistical significance ResultsAntiGM1 GM2 GM3 GD1a GD1b GT1b andGQ1b autoantibodies determined in primary metastaticmelanoma had diï¬erent serological profiles compared tothe control group The presence of IgG and IgM antiganglioside antibodies in primary melanoma was as followsantiGM1 and antiGM2 and antiGM3 and antiGD1a and antiGD1b and antiGT1b and antiGQ1b and Inmetastatic melanoma IgG and IgM antiganglioside antibodies had the following profile antiGM1 and antiGM2 and antiGM3 and antiGD1a and antiGD1b and antiGT1b and and antiGQ1b and In the control group antiganglioside autoantibodies were absentThe assessment for IgG antiGM1 antiGM2 antiGM3antiGD1a antiGD1b antiGT1a and antiGT1b showedextremely low signal intensity in all groups Figure Whencomparing the mean of signal intensity for IgG no statisticaldiï¬erences were observed between groups We obtained a statistically significant diï¬erence in IgM antiGM1 antiGM2antiGM3 antiGD1a antiGD1b antiGT1a and antiGT1b when comparing primary melanoma respectively metastatic melanoma to the control group and once more whencomparing primary versus metastatic melanoma Table To evaluate if the presence of IgM antibodies was associatedwith melanoma development we determined their relation to‚ammatory factors IL8 and CRP recommended by AJCCfor melanoma staging Table IL8 levels were statistically significantly increased in primary melanoma ± pgmland in metastatic melanoma ± pgml when compared with the control group ± pgml CRP levelswere found in primary ± ngml and metastaticmelanoma ± ngml significantly higher when compared with the control group ± ngml IL8 andCRP had no statistically significant variation when comparedto primary versus metastatic melanoma groups Positive correlations with statistical significance were determined betweenantiGM1 and CRP respectively IL8 between antiGD1aand CRP respectively IL8 Figure Negative significant correlations were observed between antiGM3 antiGT1b andCRP respectively IL8 Figure High levels of CRP and IL were associated with an increase in antiGM1 antiGD1aand a decrease in antiGM3 antiGM2 antibodies of IgM typeTable DiscussionsMelanoma the most aggressive skin tumour is a multifactorial cancer being the result of the interplay between geneticimmunological and environmental factors [“] Gangliosides due to their expression on tumor cells have beeninvolved in tumor biology and immunogenicity and hence 0cJournal of Immunology ResearchAntiGM1 IgM and IgG signalintensity in all groups AntiGM2 IgM and IgG signalintensity in all groups ControlPrimary melanomaMetastatic melanomaControlPrimary melanomaMetastatic melanomaAntiGM1 IgM Ž AntiGM1 IgGaAntiGM3 IgM and IgGintensity levels in all groups AntiGM2 IgM Ž AntiGM2 IgGbAntiGD1a IgM and IgGintensity signal in all groups ControlPrimary melanomaMetastatic melanomaControlPrimary melanomaMetastatic melanomaAntiGM3 IgM Ž AntiGM3 IgGcControlPrimary melanomaMetastatic melanomaAntiGD1b IgM and IgG signalintensity in all groupsControlPrimary melanomaMetastatic melanomaAntiGD1b IgM Ž AntiGD1b IgGAntiGD1a IgM Ž AntiGD1a IgGdAntiGT1b IgM and IgGintensity levels in all groups AntiGT1b IgM Ž AntiGT1b IgGefAntiGQ1b IgM and IgGintensity levels in all groupsControlPrimary melanomaMetastatic melanomaAntiGQ1b IgM Ž AntiGQ1b IgGgFigure Antiganglioside signal intensity in all groups ˆ—p have been considered as targets for cancer immunotherapy[“] The probability thatsome tumourassociatedganglioside determinants induce a human immune responsegenerated much interest in medical research [ “]If endogenously synthesized antiganglioside antibodies reactonly with human cancer cells these antibodies could play animportant role in the host™s protective immunity to thetumor There is little information about quantitative variations of serum antigangliosides their origin and progressionof melanoma Tumour ganglioside antigens generate a significantly increased immune response in patients with primarymelanoma versus metastatic melanoma In our study thehost™s ability to generate an early antiganglioside responseis supported by a significantly increased titter of IgMantibodies in patients with primary versus metastatic melanoma and the control groupfor antiGM1 antiGM2antiGM3 antiGD1a antiGD1b antiGT1b and antiGQ1b Figure The range of antiganglioside antibodiescould serve as an indicator of diï¬erentiation between patientswith primary melanoma and metastatic melanoma Based on 0cJournal of Immunology ResearchTable IgM antiganglioside signal intensity in all groupsAntibodies Classp significancePM vs MM PM vs control MM vs controlIgMAntiGM1IgMAntiGM2IgMAntiGM3AntiGD1aIgMAntiGD1b IgMAntiGT1bIgMAntiGQ1b IgMMM metastatic melanoma PM primary melanoma psignificancestatisticalTable IL8 and CRP in all studied groupsStudy groupPrimarymelanomaMetastaticmelanomaControlgroupCRPmgdl ± ± ± psignificance”IL8pgml ± ± ± psignificance”our findings we estimate that the levels of the antigangliosideantibodies could provide information regarding the clinicalstaging of melanomaIn addition the capacity of patients to develop an antiganglioside response in the early stage of development of melanoma could be understood as a mean of defence of the bodythrough eliminating a danger signal from the tumour microenvironment represented by the stimulation of glycosphingolipid synthesis [ ] Synthesis of antiganglioside antibodiescould confer a survival advantage in patients with primarymelanoma [] In metastatic melanoma patients we observeda reduction of antiganglioside antibody synthesis a result thatcould suggest the immunosuppressive eï¬ect exerted by theoverproduction of gangliosides associated with tumour metastasis andor due to the overall decreased immune responseIt has been shown in a previous study that in patients withuntreated primary melanoma there is a significant statisticalcorrelation between antiGM1 type IgM level and clinicalstage of the disease Breslow index Clark level tumour localization histologic type presenceabsence of ulceration [ ]In our study patients with cutaneous melanoma had detectable levels of antiGM1 in primary stages The presence of apositive significant relationship between IgM antiGM1 leveland IL8 and CRP in our study justified our statement regarding the involvement of these antibodies in tumour proliferation by stimulating ‚ammation [ “] The presentstudy is the first one that evaluated the relation betweenantiganglioside antibodies and IL8 and CRP based on theirrole in melanoma diagnosis progression and outcome [“] in metabolic disorders [ ]Previous studies in patients with prostate cancer [] orsarcoma [] have shown that antiGM1 antibodies had nodiagnostic or prognostic value in these pathologies In patientswith diï¬erentiated thyroid cancer antiGM1 type IgG andIgM were associated with carcinogenesis but the lack of correlation between antibody level and clinical status indicated thatantiGM1 had no diagnostic value in diï¬erentiated thyroidcancer []Another study performed by our group showed thatpatients with primary melanoma with a high level of IgMantiGM3 had a favourable prognosis compared withpatients displaying a low antibody titer [] In a study onpatients with primary untreated melanoma stages I andII lymph nodes clear of metastasis it was shown that theantiGM2 antibody titer for IgMtype was not diï¬erentiatedin correlation to the tumor thickness For antiGM3 it wasobtained a direct relationship between the serum titer andthe thickness of the tumor [] AntiGM2 and antiGM3antibodies have no diagnostic significance in thyroid cancerdue to the low prevalence of these antibodies [] In ourpresent study antiGM3 negative correlations with IL8and CRP are suitable with the hypothesis that patients withprimary melanoma with a high level of IgM antiGM3 havea favourable prognosisGD1a was thought to generate an immune response inpatients with earlystage melanoma [] In patients withT1T2 stage prostate cancer there were identified increasedIgM antiGD1a values compared to the T3T4 stage whichsustains the development of an early endogenous immuneresponse able to eliminate the danger signal from the tumormicroenvironment These data support the role of antiGD1ain the early diagnosis of localized prostate disease [] TheantiGD1a IgMtype titer was defined as a negative predictivefactor of survival in patients with soft tissue sarcoma [] andin patients with primary melanoma [] In patients™ serumdiagnosed with ovarian cancer an increased titer of IgMantiGD1 was found the authors pointing out that theseantibodies could represent immunological markers associated with ovarian cancer progression [] In cutaneousmelanoma IgM antiGD1a could be considered a markerassociated with melanoma progression based on the negativecorrelation with CRP and IL8 as shown in our studyThe antiGD1b immune response in patients with gastricneoplasm can be used as a prognostic marker [] On thecontrary the lack of correlation between the presence ofantiGD1b and the clinical status of patients with thyroidcancer has indicated that antigangliosides do not have diagnostic significance in this neoplasm []The antiGT1b titer can be an overall positive factor associated to global survival in sarcoma [] AntiGD1b GT1band GQ1b antibodies that are negatively correlated with IL8and CRP suggest that they could indirectly suppress tumorgrowth and angiogenesis AntiGD1b GT1b and GQ1bIgM type ‚uence the progression of melanoma [ ]soft tissue sarcomas [ ] Ehrlich subcutaneous solidtumors [ ] Ehrlich carcinomas accompanied by ascites[ ] and gastric cancer []Our study limitations could be considered the semiquantitative assessment method of antiganglioside antibodies as aquantitative determination technique could oï¬er more sensitive data ing the door for further studies One more 0cJournal of Immunology ResearchMGitnAMGitnAaDGitnA CRP CRP CRP MGitnAMGitnAaDGitnAIL8IL8IL8Figure AntiGM1 antiGM3 and antiGD1a in relation to CRP and IL8 in the primary melanoma groupimportant limitation of the study is that we evaluated thecorrelation between antiganglioside antibodies and ‚ammation markers in melanoma IL8 and CRP only after orbetween the surgical treatment of melanoma newer therapiesbeing also in place This could be the first study which otherresearchers and clinicians can use and analyze in order toevaluate the ‚uence of diï¬erent melanoma treatments onantiganglioside antibodies The pathogenic mechanismsinvolved in melanoma are complex [“] therefore theevaluation during the followup period of melanoma patientsat diï¬erent points is needed for a better antigangliosideprofile characterization 0cJournal of Immunology ResearchTable AntiGM1 GM2 GM3 GD1a GD1b GT1b andGQ1b relation with ‚ammatory markers in primary melanomagroupAntiGM1AntiGM2AntiGM3AntiGD1aAntiGD1bAntiGT1bAntiGQ1bCRPr p ‰¤ r ˆ’p r ˆ’p r p ‰¤ r ˆ’p ‰¤ r ˆ’p ‰¤ r ˆ’p IL8r p ‰¤ r ˆ’p r ˆ’p r p ‰¤ r ˆ’p ‰¤ r ˆ’p ‰¤ r ˆ’p ConclusionsAntiganglioside antibodies antiGM1 and GD1a werepositively correlated while antiGM3 GD1b and GT1bwere negatively associated with the ‚ammatory markersIL8 and CRP The host™s ability to elaborate an early antiganglioside response could be considered as a defence mechanism directed toward eliminating a danger signal from thetumour microenvironment Moreover our results suggest thattumour ganglioside antigens generate a significantly increasedimmune response in patients with primary versus metastaticmelanoma Antiganglioside antibodies associated with ‚ammation markers could be used as diagnostic monitoring andtreatment tools in patients with cutaneous melanomaData AvailabilityThe data used to support the findings of this study areincluded within the articleConflicts of InterestThe authors declare no conflicts of interestAuthors™ ContributionsAll authors have equally contributed to the writing and editing of the manuscriptAcknowledgmentsThis research and article processing charges were funded by agrant of the Romanian Ministry of Research and InnovationCCCDIUEFISCDI project number 61PCCDI2018 PNIIIP112PCCDI2017034References[] YH Xu S Barnes Y Sun and G A Grabowski œMultisystem disorders of glycosphingolipid and ganglioside metabolism Journal of Lipid Research vol no pp “ [] I Horwacik and H Rokita œTargeting of tumorassociatedgangliosides with antibodies aï¬ects signaling pathways andleads to cell death including apoptosis Apoptosis vol no pp “ [] J L Daniotti R D Lardone and A A Vilcaes œDysregulatedexpression of glycolipids in Tumor cells from negative modulator of Antitumor immunity to promising targets for developing therapeutic agents Frontiers in Oncology vol p [] I Nicolae A Caragheorgheopol S Schipor œGnagliosides and sex hormones in human melanoma Acta Endocrinologica vol no pp “ [] Y Ohmi M Kambe Y Ohkawa œDiï¬erential roles ofgangliosides in malignant properties of melanomas PLoSONE vol no article e0206881 [] M H Ravindranath S Muthugounder N Presser A D Santin R S Selvan and D L Morton œGanglioside GD1a present in ovarian cancer cells ascites and sera of patients elicitsendogenous IgM response Proceedings of the American Association for Cancer Research vol p [] V B Doronin T A Parkhomenko M Castellazzi et alœComparison of antibodies hydrolyzing myelin basic proteinfrom the cerebrospinal fluid and serum of patients with multiple sclerosis PLoS One vol no article e107807 [] C D Ene and I Nicolae œGangliosides and Antigangliosidesin Malignant Melanoma Melanoma Current Clinical Management and Future Therapeutics M Murph Ed MandiMurph Intech ISBN [] N AlvarezRueda S Leprieur B Clemenceau œBindingactivities and antitumor properties of a new mousehumanchimeric antibody specific for GD2 ganglioside antigen Clinical Cancer Research vol no pp 5613s“5620s [] K Bennaceur I Popa J A Chapman œDiï¬erent mechanisms are involved in apoptosis induced by melanoma ganglicellsosidesGlycobiology vol no pp “ on human monocytederived dendritic[] C D Nicolae and I Nicolae œAntibodies against GM1 gangliosides associated with metastatic melanoma Acta Dermatovenerologica Croatica vol no pp “ [] S GrouxDegroote Y Guerardel and P Delannoy œGangliosides structures biosynthesis analysis and roles in cancerChembiochem vol no pp “ [] C Tringali I Silvestri F Testa œMolecular subtyping ofmetastatic melanoma based on cell ganglioside metabolismprofiles BMC Cancer vol no p [] M Neagu C Constantin C Caruntu C Dumitru M Surceland S Zurac œInflammation a key process in skin tumorigenesis Oncology Letters vol no pp “ [] R Takeuchi M Kambe M Miyata œTNFαsignal andcAMPmediated signals oppositely regulate melanomaassociated ganglioside GD3 synthase gene in human melanocytes Scientific Reports vol no p [] A H Otake R de Freitas Saito A P M Duarte A F Ramosand R Chammas œGD3 gangliosideenriched extracellular vesiclesetstimulate melanocyte migration Biochimica 0cJournal of Immunology ResearchBiophysica Acta Molecular and Cell Biology of Lipidsvol no pp “ [] K Hamamura K Furukawa T Hayashi œGangliosideGD3 promotes cell growth and invasion through p130Casand paxillin in malignant melanoma cells Proceedings of theNational Academy of Sciences of the United States of Americavol no pp “ [] M Neagu C Constantin C Caruntu M Surcel D Boda andS Zurac œCytokine pattern for improving immunoscore inmelanoma patients European Journal ofImmunologyvol pp [] M Neagu C Constantin and C Longo œChemokines in themelanoma metastasis biomarkers portrait Journal of Immunoassay Immunochemistry vol no pp “ [] K A Timani B Gyorï¬y Y Liu K S Mohammad and J J HeœTip110SART3 regulates IL8 expression and predicts theclinical outcomes in melanoma Molecular Cancer vol no p [] S J Wigmore K C H Fearon J P Maingay P B S Lai andJ A Ross œInterleukin8 can mediate acutephase protein production by isolated human hepatocytes American Journal ofPhysiologyEndocrinology and Metabolism vol no pp E720“E726 [] M Bickel œThe role of interleukin8 in ‚ammation andJournal of Periodontologyregulationmechanisms ofvol no pp “ [] M Neagu C Constantin and S Zurac œImmune Parametersin The Prognosis and Therapy Monitoring of Cutaneous Melanoma Patients Experience Role and Limitations BioMedResearch International vol Article ID pages[] S R Georgescu M R Ioghen M I Sarbu œBiologicaltherapy in the treatment of melanoma Journal of Mind andMedical Sciences vol no pp “ [] A V Dumitru M Tampa S R Georgescu œImmunohistochemical mismatch in a case of rhabdomyoblastic metastaticmelanoma Romanian Journal of Morphology and Embryology vol no pp “ [] S N Pavri J Clune S Ariyan and D Narayan œMalignantMelanoma Plastic and Reconstructive Surgery vol no pp 330e“340e [] M Rastrelli S Tropea C R Rossi and M Alaibac œMelanoma epidemiology risk factors pathogenesis Diagnosis andClassification In Vivo vol no pp “ [] L LugovićMihić D Ćesić P Vuković G Novak BilićM Å itum and S Å poljar œMelanoma development currentknowledge on melanoma pathogenesis Acta Dermatovenerologica Croatica vol no pp “ [] M Costache A V Dumitru O M Pătraşcu œA challenging case of ocular melanoma Romanian Journal of Morphology and Embryology vol Suppl pp “ [] R Ancuceanu and M Neagu œImmune based therapy for melanoma Indian Journal of Medical Research vol no pp “ [] C A Perez M H Ravindranath D Soh A Gonzales W Yeand D L Morton œSerum antiganglioside IgM antibodies insoft tissue sarcoma clinical prognostic implications CancerJournal vol no pp “ [] B Mondal and S Sahal œInhibition of subcutaneous growth ofEhrlich ascites carcinoma EAC tumor by postimmunizationwith EACcell gangliosides and its antiidiotype antibody inrelation to tumor angiogenesis apoptosis cell cycle and ltration of CD4 CD8 lymphocytes NK cells suppressorcells and APCcells in tumor Indian Journal of ExperimentalBiology vol no pp “ [] S Sahal and S Mondal œSupression of Ehrlich subcutaneoussolid tumor growth by immunization with ganglioside GT1bof its origin its IgM antibody or antiidiotype antibody Journal of Experimental Clinical Cancer Research vol no p [] M M Konstandoulakis K N Syrigos M LeandrosA Charalabopoulos A Manouras and B C GolematisœAutoantibodies in the serum of patients with gastric cancertheir prognostic importance Hybridoma vol no pp “ [] M H Ravindranath S Muthugounder X Ye and D L Morton œInnate immune response to gangliosides of primary melanoma favors danger hypothesis Proceedings of the AmericanAssociation for Cancer Research vol p [] M H Ravindranath S Muthugounder and N Presser œGanglioside signatures of primary and nodal metastatic melanomacell lines from the same patient Melanoma Research vol no pp “ [] A Lewartowska T Pacuszka G Adler M Panasiewicz andW Wojciechowska œGanglioside reactive antibodies of IgGand IgM class in sera of patients with diï¬erentiated thyroidcancer Immunology Letters vol no pp “ [] I Nicolae C D Nicolae O A Coman M StefanescuL Coman and C Ardeleanu œSerum total gangliosides levelclinical prognostic implication Romanian Journal of Morphology and Embryology vol no pp “ [] C Nicolae and I Nicolae œHeterogeneity of gangliosides inmelanocytic tumors Acta Endocrinologica vol no pp “ [] S GrouxDegroote M RodríguezWalker J H Dewald J LDaniotti and P Delannoy œGangliosides in cancer cell signaling Progress in Molecular Biology and Translational Sciencevol pp “ [] I Nicolae C D E Nicolae and E Ceauşu œInvestigation onantigangliosides antibodies in asymptomatic HIV patientsBMC Infectious Diseases vol no S4 p [] G N Tzanakakis M Neagu A M Tsatsakis and D NikitovicœProteoglycans and immunobiology of cancer therapeuticimplications Frontiers in Immunology vol p [] Q Li M Sun M Yu œGangliosides profiling in serum ofbreast cancer patient GM3 as a potential diagnostic biomarker Glycoconjugate Journal vol no pp “[] C D Ene A E Anghel M Neagu and I Nicolae œ25OHvitamin D and interleukin8 emerging biomarkers in cutaneous melanoma development and progression Mediators ofInflammation vol Article ID pages [] N R Sproston and J J Ashworth œRole of Creactive proteinat sites of ‚ammation and infection Frontiers In Immunology vol p [] A E Anghel C D Ene M Neagu and I Nicolae œThe relationship between interleukin8 and Ki67 in cutaneous malignant melanoma HVM Bioflux vol no pp “[] C D E Nicolae and I Nicolae œInterleukin 8serumconcentration but not lactate dehydrogenase activity positively correlates to CD34 antigen in melanoma tumors Journal of 0cJournal of Immunology ResearchImmunoassay and Immunochemistry vol no pp “ [] A E Anghel C D Ene I Nicolae V A Budu C Constantinand M Neagu œInterleukin major player in cutaneous melanoma metastasic process Romanian Biotechnological Letters vol no pp “ [] M Neagu œMetabolic traits in cutaneous melanoma Frontiers in oncology vol p [] C D Ene A E Anghel M Neagu and I Nicolae œInterleukin and diabetic nephropathy Human and Veterinary Medicine vol no pp “ [] M Neagu C Constantin I D Popescu œInflammationand metabolism in cancer cell “ mitochondria key playerFrontiers in Oncology vol p [] C Nicolae I Nicolae and O Coman œGD1b GT1b

cancer221

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: properly citedIntroduction Endogenously produced antiganglioside antibodies could aï¬ect the evolution of cutaneous melanoma Epidemiologicaland experimental evidence suggest œchronic ‚ammation to be one of the hallmarks in skin cancers The aim of the study was tocharacterize the relation between antiganglioside antibodies and ‚ammation in cutaneous melanoma focusing on gangliosidesGM1 GM2 GM3 GD1a GD1b GT1b GQ1b Material and Method We performed an observational study that included subjects subdivided into three groups patients with metastatic melanoma cases patients with primary melanoma casesand healthy subjects subjects The assessment of antiganglioside antibodies IgG and IgM classes against GM1 GM2GM3 GD1a GD1b GT1b GQ1b was performed using immunoblot technique EUROLine kit Results The presence of IgGand IgM antiganglioside antibodies in primary melanoma was as follows antiGM1 and GM2 and GM3 and GD1a and GD1b and GT1b and GQ1b and In metastaticmelanoma the level of antiganglioside antibodies was significantly lower compared with primary melanoma p while inthe control group they were absent Antiganglioside antibodies antiGM1 and GD1a were positively correlated while antiGM3GD1b and GT1b were negatively associated with the ‚ammatory markers interleukin IL8 and C reactive protein CRPConclusions Tumour ganglioside antigens generate an immune response in patients with primary melanomas The host™s ability toelaborate an early antiganglioside response could be considered as a defence mechanism directed toward eliminating a dangersignal from the tumour microenvironment Antiganglioside antibodies associated with ‚ammation markers could be used asdiagnostic monitoring and treatment tools in patients with cutaneous melanoma IntroductionGangliosides are a group of bioactive glycolipids located onthe outer face of cell membranes These glycolipids play amajor role in cell proliferation diï¬erentiation migrationapoptosis signal transduction cell adhesion modulatinggrowth factor or hormone receptor antigen recognition protein trafficking viral transformation and oncogenesis [“]Atypical expression of some ganglioside antigens associatedwith certain tumours neuroblastomas melanomas gliomaslymphomas small cell lung cancer and prostate cancer andfurthermore could play an importantrole in cancer 0cJournal of Immunology Researchimmunotherapy [“] Gangliosides that are released inextracellular spaces could have dual action antitumor andprotumour eï¬ect [“] Data regarding the endogenousimmune response directed toward tumour gangliosides andthe significance of this response are limited A series of studiesperformed in in vivo experimental models and in vitro inmurine and human cancer cells have shown that monoclonalantiganglioside antibodies have antitumor potential Theseantibodies exert numerous antitumor eï¬ects through variousmechanisms An important mechanism is the translocationof gangliosides from the plasmatic membrane into theintracellular spaces so binding of antibodies to the surfaceof the tumor cells and complement activation that leads to celllysis mediated by complementdependent cytotoxicity andantibodymediated cellular cytotoxicity [ ] Antiganglioside antibodies modulate ceramide synthesis [ ]reception and transduction of the cytotoxic signal [] theyare involved in suppression or induction of cell death throughdiï¬erent pathways apoptosis necrosis oncogeneslike structural and functional changes of mitochondria accumulationof reactive oxygen species acetylation of gangliosides accumulation of sphingosine sphingamine ceramides [ ]Proteomic studies showed that antiganglioside antibodiescould induce changes like the disruption of signalling systemsP38MAPK PARP JNK123 METc ERK12 P13KAKTand FAK modulation of the level and function of transcription factors P53 SP1 MYCN and HSF1 regulating the balance between apoptosisinducing and apoptosissuppressingfactors cysteineaspartylproteases Bax Bcl2 [ “]These antibodies stimulate the cytotoxicity of chemotherapeutic drugs and small molecule inhibitors [ ] As a result antiganglioside antibodies could be used as diagnostic monitoringand treatment tools in cancer patients [ ]Ganglioside levels are increased in malignant melanocytes and represent an important topic of research [ ]Several researchers have emphasized the role of glycolipidsas markers of melanoma A study analysing the expressionof gangliosides in melanocyte lines and melanoma cell linesfound out an increased expression of GD3 synthase genesin melanoma cells but not in melanocytes The same resultswere obtained for GM2GD2 synthase [] It seems thatgangliosides induce cell proliferation and invasion throughp130Cas and paxillin in melanoma cells []Inflammatory mechanisms play an important role inmelanoma Multiple studies have shown that plasma levelsof C reactive protein CRP increase during tumor proliferation and several relations have been evaluated CRPsurvivalrelationship CRPresponse therapy CRP‚ammationNowadays CRP is considered a true marker for assessing‚ammation in melanoma as well as a marker for responseto treatment Prospective studies have provided consistentresults in the predictive value of CRP in neoplastic diseaseproving high sensitivity and specificity [] In addition inmelanoma elevated levels of CRP may reflect the amountand activity of circulating pro‚ammatory cytokines eginterleukin IL8 IL8 plays a crucial role in regulating cellfunction for host defence and for developing natural immunity [ ] Moreover IL8 is released by various cell typesincluding polymorphonuclear neutrophils PMNs monoSerum dilution Incubation with fixed on strip antigens degrees min with balanceWashing with universal tampon times min each with balanceIncubation with conjugated enzyme degrees min with balanceWashing with universal tampon times min each with balanceIncubation with fixed on strip antigens degrees min with balanceWashing with universal tampon times min each with balanceEUROLine scan evaluationFigure Antiganglioside detectioncytes T lymphocytes and endothelial cells upon exposureto ‚ammatory stimuli Melanoma cells have been reportedto express IL8 and this ‚uences their oncogenic properties[ ] IL8 follows the evolution of melanoma progression and regression under treatment reflecting the stage ofthe disease [“]Based on these accumulating data we have investigatedantiganglioside antibodies in correlation with other ‚ammatory markers IL8 CRP and the clinical evolution of the melanoma patients Clarifying these relations could significantlyimprove the prediction of clinical outcomes Furthermore itcan lead to the development of appropriate therapeutic strategies in patients with cutaneous melanoma Material and Method Patients We performed an observational prospectivestudy during years in Clinical Hospital for Infectious andTropical Diseases œVictor Babes”Dermatology Department Bucharest The study was approved by the Ethics Committee of the Hospital All participants agreed to be includedin research studies without prejudice of the diagnosis orpersonal image and signed the informed consent accordingto the Declaration of HelsinkiThe study included adult patients with cutaneous melanoma with no other pathologies and no treatment for theprimary disease Exclusion criteria were age under yearspregnancy alcohol use melanoma under treatmentWe performed an observational study that included subjects subdivided in three groups patients with metastaticmelanoma cases patients with primary melanoma cases and healthy subjects with matching sex and age subjects Patients were selected and examined accordingto ESMO Clinical Practice Guidelines for melanoma 0cJournal of Immunology ResearchTable Test strips coated with parallel lines of purified antigensAntigenGM1GM2GM3GD1aGD1bGT1bGanglioside typeSourceMonosialoganglioside GM1Monosialoganglioside GM2Monosialoganglioside GM3 Dog erythrocytesBovine brainBovine brainDisialoganglioside GD1aDisialoganglioside GD1bTrisialoganglioside GT1bBovine brainBovine brainBovine brainGQ1bTetrasialoganglioside GQ1bBovine brainStructureGal3GalNAc4[Neu5Ac3]Gal4GlcCerGalNAc4[Neu5Ac3]Gal4GlcCerNeu5Ac3Gal4GlcCerNeu5Ac3Gal3GalNAc4[Neu5Ac3]Gal4GlcCerGal3GalNAc4[Neu5Ac8Neu5Ac3]Gal4GlcCerNeu5Ac3Gal3GalNAc4[Neu5Ac8Neu5Ac3]Gal4GlcCerNeu5Ac8Neu5Ac3Gal3GalNAc4[Neu5Ac8Neu5Ac3]Gal4GlcCer Glcglucose Gal galactose GalNAc Nacetylgalactosaminediagnosis based on clinical histopathological immunohistochemical and imagistic data All the events related to theprogression of the disease were recorded relapse metastasisneurotoxicity hyper reactivation of the immune system upontreatment The group characteristics were similar for age andsex the primary melanoma group included women and men with a mean age of ± years the metastaticmelanoma group included women and men with a meanage of ± years and the control group included women and men with mean age of ± years Materials and Reagents In this work the assessment ofantiganglioside antibodies was made by the immunoblottechnique using EUROLine kits Figure This methodallows the evaluation of antibodies IgG and IgM classesagainst GM1 GM2 GM3 GD1a GD1b GT1b and GQ1bfrom serumplasma The kit contains strips marked withpurified antigens Table Theevaluation ofantigangliosideantibodies wasperformed using the EUROLine Scan software After readingthe signal intensity on the strips marked with ganglioside antigens the results were evaluated and the results are presented asoptical sensibility The assessment of IL8 was performed bythe ELISA method using Enzo Life Science reagents withTECAN analyser and the results are presented as pgdl CRPwas assessed by immunoturbidimetry using Human reagentsand HumaStar300 analyser the results are presented as mgdl Statistical Analysis All the results were analysed usingIBM SPSS Statistics We evaluated the normality of datadistribution using the KolmogorovSmirnov test The variationbetween groups was determined using the parametric teststtest when two groups were compared or ANOVA test whenmore groups were compared and nonparametric tests likeMannWhitney or Wilcoxon The correlation between groupswas evaluated using linear regression and Pearson coefficientp was considered with statistical significance ResultsAntiGM1 GM2 GM3 GD1a GD1b GT1b andGQ1b autoantibodies determined in primary metastaticmelanoma had diï¬erent serological profiles compared tothe control group The presence of IgG and IgM antiganglioside antibodies in primary melanoma was as followsantiGM1 and antiGM2 and antiGM3 and antiGD1a and antiGD1b and antiGT1b and antiGQ1b and Inmetastatic melanoma IgG and IgM antiganglioside antibodies had the following profile antiGM1 and antiGM2 and antiGM3 and antiGD1a and antiGD1b and antiGT1b and and antiGQ1b and In the control group antiganglioside autoantibodies were absentThe assessment for IgG antiGM1 antiGM2 antiGM3antiGD1a antiGD1b antiGT1a and antiGT1b showedextremely low signal intensity in all groups Figure Whencomparing the mean of signal intensity for IgG no statisticaldiï¬erences were observed between groups We obtained a statistically significant diï¬erence in IgM antiGM1 antiGM2antiGM3 antiGD1a antiGD1b antiGT1a and antiGT1b when comparing primary melanoma respectively metastatic melanoma to the control group and once more whencomparing primary versus metastatic melanoma Table To evaluate if the presence of IgM antibodies was associatedwith melanoma development we determined their relation to‚ammatory factors IL8 and CRP recommended by AJCCfor melanoma staging Table IL8 levels were statistically significantly increased in primary melanoma ± pgmland in metastatic melanoma ± pgml when compared with the control group ± pgml CRP levelswere found in primary ± ngml and metastaticmelanoma ± ngml significantly higher when compared with the control group ± ngml IL8 andCRP had no statistically significant variation when comparedto primary versus metastatic melanoma groups Positive correlations with statistical significance were determined betweenantiGM1 and CRP respectively IL8 between antiGD1aand CRP respectively IL8 Figure Negative significant correlations were observed between antiGM3 antiGT1b andCRP respectively IL8 Figure High levels of CRP and IL were associated with an increase in antiGM1 antiGD1aand a decrease in antiGM3 antiGM2 antibodies of IgM typeTable DiscussionsMelanoma the most aggressive skin tumour is a multifactorial cancer being the result of the interplay between geneticimmunological and environmental factors [“] Gangliosides due to their expression on tumor cells have beeninvolved in tumor biology and immunogenicity and hence 0cJournal of Immunology ResearchAntiGM1 IgM and IgG signalintensity in all groups AntiGM2 IgM and IgG signalintensity in all groups ControlPrimary melanomaMetastatic melanomaControlPrimary melanomaMetastatic melanomaAntiGM1 IgM Ž AntiGM1 IgGaAntiGM3 IgM and IgGintensity levels in all groups AntiGM2 IgM Ž AntiGM2 IgGbAntiGD1a IgM and IgGintensity signal in all groups ControlPrimary melanomaMetastatic melanomaControlPrimary melanomaMetastatic melanomaAntiGM3 IgM Ž AntiGM3 IgGcControlPrimary melanomaMetastatic melanomaAntiGD1b IgM and IgG signalintensity in all groupsControlPrimary melanomaMetastatic melanomaAntiGD1b IgM Ž AntiGD1b IgGAntiGD1a IgM Ž AntiGD1a IgGdAntiGT1b IgM and IgGintensity levels in all groups AntiGT1b IgM Ž AntiGT1b IgGefAntiGQ1b IgM and IgGintensity levels in all groupsControlPrimary melanomaMetastatic melanomaAntiGQ1b IgM Ž AntiGQ1b IgGgFigure Antiganglioside signal intensity in all groups ˆ—p have been considered as targets for cancer immunotherapy[“] The probability thatsome tumourassociatedganglioside determinants induce a human immune responsegenerated much interest in medical research [ “]If endogenously synthesized antiganglioside antibodies reactonly with human cancer cells these antibodies could play animportant role in the host™s protective immunity to thetumor There is little information about quantitative variations of serum antigangliosides their origin and progressionof melanoma Tumour ganglioside antigens generate a significantly increased immune response in patients with primarymelanoma versus metastatic melanoma In our study thehost™s ability to generate an early antiganglioside responseis supported by a significantly increased titter of IgMantibodies in patients with primary versus metastatic melanoma and the control groupfor antiGM1 antiGM2antiGM3 antiGD1a antiGD1b antiGT1b and antiGQ1b Figure The range of antiganglioside antibodiescould serve as an indicator of diï¬erentiation between patientswith primary melanoma and metastatic melanoma Based on 0cJournal of Immunology ResearchTable IgM antiganglioside signal intensity in all groupsAntibodies Classp significancePM vs MM PM vs control MM vs controlIgMAntiGM1IgMAntiGM2IgMAntiGM3AntiGD1aIgMAntiGD1b IgMAntiGT1bIgMAntiGQ1b IgMMM metastatic melanoma PM primary melanoma psignificancestatisticalTable IL8 and CRP in all studied groupsStudy groupPrimarymelanomaMetastaticmelanomaControlgroupCRPmgdl ± ± ± psignificance”IL8pgml ± ± ± psignificance”our findings we estimate that the levels of the antigangliosideantibodies could provide information regarding the clinicalstaging of melanomaIn addition the capacity of patients to develop an antiganglioside response in the early stage of development of melanoma could be understood as a mean of defence of the bodythrough eliminating a danger signal from the tumour microenvironment represented by the stimulation of glycosphingolipid synthesis [ ] Synthesis of antiganglioside antibodiescould confer a survival advantage in patients with primarymelanoma [] In metastatic melanoma patients we observeda reduction of antiganglioside antibody synthesis a result thatcould suggest the immunosuppressive eï¬ect exerted by theoverproduction of gangliosides associated with tumour metastasis andor due to the overall decreased immune responseIt has been shown in a previous study that in patients withuntreated primary melanoma there is a significant statisticalcorrelation between antiGM1 type IgM level and clinicalstage of the disease Breslow index Clark level tumour localization histologic type presenceabsence of ulceration [ ]In our study patients with cutaneous melanoma had detectable levels of antiGM1 in primary stages The presence of apositive significant relationship between IgM antiGM1 leveland IL8 and CRP in our study justified our statement regarding the involvement of these antibodies in tumour proliferation by stimulating ‚ammation [ “] The presentstudy is the first one that evaluated the relation betweenantiganglioside antibodies and IL8 and CRP based on theirrole in melanoma diagnosis progression and outcome [“] in metabolic disorders [ ]Previous studies in patients with prostate cancer [] orsarcoma [] have shown that antiGM1 antibodies had nodiagnostic or prognostic value in these pathologies In patientswith diï¬erentiated thyroid cancer antiGM1 type IgG andIgM were associated with carcinogenesis but the lack of correlation between antibody level and clinical status indicated thatantiGM1 had no diagnostic value in diï¬erentiated thyroidcancer []Another study performed by our group showed thatpatients with primary melanoma with a high level of IgMantiGM3 had a favourable prognosis compared withpatients displaying a low antibody titer [] In a study onpatients with primary untreated melanoma stages I andII lymph nodes clear of metastasis it was shown that theantiGM2 antibody titer for IgMtype was not diï¬erentiatedin correlation to the tumor thickness For antiGM3 it wasobtained a direct relationship between the serum titer andthe thickness of the tumor [] AntiGM2 and antiGM3antibodies have no diagnostic significance in thyroid cancerdue to the low prevalence of these antibodies [] In ourpresent study antiGM3 negative correlations with IL8and CRP are suitable with the hypothesis that patients withprimary melanoma with a high level of IgM antiGM3 havea favourable prognosisGD1a was thought to generate an immune response inpatients with earlystage melanoma [] In patients withT1T2 stage prostate cancer there were identified increasedIgM antiGD1a values compared to the T3T4 stage whichsustains the development of an early endogenous immuneresponse able to eliminate the danger signal from the tumormicroenvironment These data support the role of antiGD1ain the early diagnosis of localized prostate disease [] TheantiGD1a IgMtype titer was defined as a negative predictivefactor of survival in patients with soft tissue sarcoma [] andin patients with primary melanoma [] In patients™ serumdiagnosed with ovarian cancer an increased titer of IgMantiGD1 was found the authors pointing out that theseantibodies could represent immunological markers associated with ovarian cancer progression [] In cutaneousmelanoma IgM antiGD1a could be considered a markerassociated with melanoma progression based on the negativecorrelation with CRP and IL8 as shown in our studyThe antiGD1b immune response in patients with gastricneoplasm can be used as a prognostic marker [] On thecontrary the lack of correlation between the presence ofantiGD1b and the clinical status of patients with thyroidcancer has indicated that antigangliosides do not have diagnostic significance in this neoplasm []The antiGT1b titer can be an overall positive factor associated to global survival in sarcoma [] AntiGD1b GT1band GQ1b antibodies that are negatively correlated with IL8and CRP suggest that they could indirectly suppress tumorgrowth and angiogenesis AntiGD1b GT1b and GQ1bIgM type ‚uence the progression of melanoma [ ]soft tissue sarcomas [ ] Ehrlich subcutaneous solidtumors [ ] Ehrlich carcinomas accompanied by ascites[ ] and gastric cancer []Our study limitations could be considered the semiquantitative assessment method of antiganglioside antibodies as aquantitative determination technique could oï¬er more sensitive data ing the door for further studies One more 0cJournal of Immunology ResearchMGitnAMGitnAaDGitnA CRP CRP CRP MGitnAMGitnAaDGitnAIL8IL8IL8Figure AntiGM1 antiGM3 and antiGD1a in relation to CRP and IL8 in the primary melanoma groupimportant limitation of the study is that we evaluated thecorrelation between antiganglioside antibodies and ‚ammation markers in melanoma IL8 and CRP only after orbetween the surgical treatment of melanoma newer therapiesbeing also in place This could be the first study which otherresearchers and clinicians can use and analyze in order toevaluate the ‚uence of diï¬erent melanoma treatments onantiganglioside antibodies The pathogenic mechanismsinvolved in melanoma are complex [“] therefore theevaluation during the followup period of melanoma patientsat diï¬erent points is needed for a better antigangliosideprofile characterization 0cJournal of Immunology ResearchTable AntiGM1 GM2 GM3 GD1a GD1b GT1b andGQ1b relation with ‚ammatory markers in primary melanomagroupAntiGM1AntiGM2AntiGM3AntiGD1aAntiGD1bAntiGT1bAntiGQ1bCRPr p ‰¤ r ˆ’p r ˆ’p r p ‰¤ r ˆ’p ‰¤ r ˆ’p ‰¤ r ˆ’p IL8r p ‰¤ r ˆ’p r ˆ’p r p ‰¤ r ˆ’p ‰¤ r ˆ’p ‰¤ r ˆ’p ConclusionsAntiganglioside antibodies antiGM1 and GD1a werepositively correlated while antiGM3 GD1b and GT1bwere negatively associated with the ‚ammatory markersIL8 and CRP The host™s ability to elaborate an early antiganglioside response could be considered as a defence mechanism directed toward eliminating a danger signal from thetumour microenvironment Moreover our results suggest thattumour ganglioside antigens generate a significantly increasedimmune response in patients with primary versus metastaticmelanoma Antiganglioside antibodies associated with ‚ammation markers could be used as diagnostic monitoring andtreatment tools in patients with cutaneous melanomaData AvailabilityThe data used to support the findings of this study areincluded within the articleConflicts of InterestThe authors declare no conflicts of interestAuthors™ ContributionsAll authors have equally contributed to the writing and editing of the manuscriptAcknowledgmentsThis research and article processing charges were funded by agrant of the Romanian Ministry of Research and InnovationCCCDIUEFISCDI project number 61PCCDI2018 PNIIIP112PCCDI2017034References[] YH Xu S Barnes Y Sun and G A Grabowski œMultisystem disorders of glycosphingolipid and ganglioside metabolism Journal of Lipid Research vol no pp “ [] I Horwacik and H Rokita œTargeting of tumorassociatedgangliosides with antibodies aï¬ects signaling pathways andleads to cell death including apoptosis Apoptosis vol no pp “ [] J L Daniotti R D Lardone and A A Vilcaes œDysregulatedexpression of glycolipids in Tumor cells from negative modulator of Antitumor immunity to promising targets for developing therapeutic agents Frontiers in Oncology vol p [] I Nicolae A Caragheorgheopol S Schipor œGnagliosides and sex hormones in human melanoma Acta Endocrinologica vol no pp “ [] Y Ohmi M Kambe Y Ohkawa œDiï¬erential roles ofgangliosides in malignant properties of melanomas PLoSONE vol no article e0206881 [] M H Ravindranath S Muthugounder N Presser A D Santin R S Selvan and D L Morton œGanglioside GD1a present in ovarian cancer cells ascites and sera of patients elicitsendogenous IgM response Proceedings of the American Association for Cancer Research vol p [] V B Doronin T A Parkhomenko M Castellazzi et alœComparison of antibodies hydrolyzing myelin basic proteinfrom the cerebrospinal fluid and serum of patients with multiple sclerosis PLoS One vol no article e107807 [] C D Ene and I Nicolae œGangliosides and Antigangliosidesin Malignant Melanoma Melanoma Current Clinical Management and Future Therapeutics M Murph Ed MandiMurph Intech ISBN [] N AlvarezRueda S Leprieur B Clemenceau œBindingactivities and antitumor properties of a new mousehumanchimeric antibody specific for GD2 ganglioside antigen Clinical Cancer Research vol no pp 5613s“5620s [] K Bennaceur I Popa J A Chapman œDiï¬erent mechanisms are involved in apoptosis induced by melanoma ganglicellsosidesGlycobiology vol no pp “ on human monocytederived dendritic[] C D Nicolae and I Nicolae œAntibodies against GM1 gangliosides associated with metastatic melanoma Acta Dermatovenerologica Croatica vol no pp “ [] S GrouxDegroote Y Guerardel and P Delannoy œGangliosides structures biosynthesis analysis and roles in cancerChembiochem vol no pp “ [] C Tringali I Silvestri F Testa œMolecular subtyping ofmetastatic melanoma based on cell ganglioside metabolismprofiles BMC Cancer vol no p [] M Neagu C Constantin C Caruntu C Dumitru M Surceland S Zurac œInflammation a key process in skin tumorigenesis Oncology Letters vol no pp “ [] R Takeuchi M Kambe M Miyata œTNFαsignal andcAMPmediated signals oppositely regulate melanomaassociated ganglioside GD3 synthase gene in human melanocytes Scientific Reports vol no p [] A H Otake R de Freitas Saito A P M Duarte A F Ramosand R Chammas œGD3 gangliosideenriched extracellular vesiclesetstimulate melanocyte migration Biochimica 0cJournal of Immunology ResearchBiophysica Acta Molecular and Cell Biology of Lipidsvol no pp “ [] K Hamamura K Furukawa T Hayashi œGangliosideGD3 promotes cell growth and invasion through p130Casand paxillin in malignant melanoma cells Proceedings of theNational Academy of Sciences of the United States of Americavol no pp “ [] M Neagu C Constantin C Caruntu M Surcel D Boda andS Zurac œCytokine pattern for improving immunoscore inmelanoma patients European Journal ofImmunologyvol pp [] M Neagu C Constantin and C Longo œChemokines in themelanoma metastasis biomarkers portrait Journal of Immunoassay Immunochemistry vol no pp “ [] K A Timani B Gyorï¬y Y Liu K S Mohammad and J J HeœTip110SART3 regulates IL8 expression and predicts theclinical outcomes in melanoma Molecular Cancer vol no p [] S J Wigmore K C H Fearon J P Maingay P B S Lai andJ A Ross œInterleukin8 can mediate acutephase protein production by isolated human hepatocytes American Journal ofPhysiologyEndocrinology and Metabolism vol no pp E720“E726 [] M Bickel œThe role of interleukin8 in ‚ammation andJournal of Periodontologyregulationmechanisms ofvol no pp “ [] M Neagu C Constantin and S Zurac œImmune Parametersin The Prognosis and Therapy Monitoring of Cutaneous Melanoma Patients Experience Role and Limitations BioMedResearch International vol Article ID pages[] S R Georgescu M R Ioghen M I Sarbu œBiologicaltherapy in the treatment of melanoma Journal of Mind andMedical Sciences vol no pp “ [] A V Dumitru M Tampa S R Georgescu œImmunohistochemical mismatch in a case of rhabdomyoblastic metastaticmelanoma Romanian Journal of Morphology and Embryology vol no pp “ [] S N Pavri J Clune S Ariyan and D Narayan œMalignantMelanoma Plastic and Reconstructive Surgery vol no pp 330e“340e [] M Rastrelli S Tropea C R Rossi and M Alaibac œMelanoma epidemiology risk factors pathogenesis Diagnosis andClassification In Vivo vol no pp “ [] L LugovićMihić D Ćesić P Vuković G Novak BilićM Å itum and S Å poljar œMelanoma development currentknowledge on melanoma pathogenesis Acta Dermatovenerologica Croatica vol no pp “ [] M Costache A V Dumitru O M Pătraşcu œA challenging case of ocular melanoma Romanian Journal of Morphology and Embryology vol Suppl pp “ [] R Ancuceanu and M Neagu œImmune based therapy for melanoma Indian Journal of Medical Research vol no pp “ [] C A Perez M H Ravindranath D Soh A Gonzales W Yeand D L Morton œSerum antiganglioside IgM antibodies insoft tissue sarcoma clinical prognostic implications CancerJournal vol no pp “ [] B Mondal and S Sahal œInhibition of subcutaneous growth ofEhrlich ascites carcinoma EAC tumor by postimmunizationwith EACcell gangliosides and its antiidiotype antibody inrelation to tumor angiogenesis apoptosis cell cycle and ltration of CD4 CD8 lymphocytes NK cells suppressorcells and APCcells in tumor Indian Journal of ExperimentalBiology vol no pp “ [] S Sahal and S Mondal œSupression of Ehrlich subcutaneoussolid tumor growth by immunization with ganglioside GT1bof its origin its IgM antibody or antiidiotype antibody Journal of Experimental Clinical Cancer Research vol no p [] M M Konstandoulakis K N Syrigos M LeandrosA Charalabopoulos A Manouras and B C GolematisœAutoantibodies in the serum of patients with gastric cancertheir prognostic importance Hybridoma vol no pp “ [] M H Ravindranath S Muthugounder X Ye and D L Morton œInnate immune response to gangliosides of primary melanoma favors danger hypothesis Proceedings of the AmericanAssociation for Cancer Research vol p [] M H Ravindranath S Muthugounder and N Presser œGanglioside signatures of primary and nodal metastatic melanomacell lines from the same patient Melanoma Research vol no pp “ [] A Lewartowska T Pacuszka G Adler M Panasiewicz andW Wojciechowska œGanglioside reactive antibodies of IgGand IgM class in sera of patients with diï¬erentiated thyroidcancer Immunology Letters vol no pp “ [] I Nicolae C D Nicolae O A Coman M StefanescuL Coman and C Ardeleanu œSerum total gangliosides levelclinical prognostic implication Romanian Journal of Morphology and Embryology vol no pp “ [] C Nicolae and I Nicolae œHeterogeneity of gangliosides inmelanocytic tumors Acta Endocrinologica vol no pp “ [] S GrouxDegroote M RodríguezWalker J H Dewald J LDaniotti and P Delannoy œGangliosides in cancer cell signaling Progress in Molecular Biology and Translational Sciencevol pp “ [] I Nicolae C D E Nicolae and E Ceauşu œInvestigation onantigangliosides antibodies in asymptomatic HIV patientsBMC Infectious Diseases vol no S4 p [] G N Tzanakakis M Neagu A M Tsatsakis and D NikitovicœProteoglycans and immunobiology of cancer therapeuticimplications Frontiers in Immunology vol p [] Q Li M Sun M Yu œGangliosides profiling in serum ofbreast cancer patient GM3 as a potential diagnostic biomarker Glycoconjugate Journal vol no pp “[] C D Ene A E Anghel M Neagu and I Nicolae œ25OHvitamin D and interleukin8 emerging biomarkers in cutaneous melanoma development and progression Mediators ofInflammation vol Article ID pages [] N R Sproston and J J Ashworth œRole of Creactive proteinat sites of ‚ammation and infection Frontiers In Immunology vol p [] A E Anghel C D Ene M Neagu and I Nicolae œThe relationship between interleukin8 and Ki67 in cutaneous malignant melanoma HVM Bioflux vol no pp “[] C D E Nicolae and I Nicolae œInterleukin 8serumconcentration but not lactate dehydrogenase activity positively correlates to CD34 antigen in melanoma tumors Journal of 0cJournal of Immunology ResearchImmunoassay and Immunochemistry vol no pp “ [] A E Anghel C D Ene I Nicolae V A Budu C Constantinand M Neagu œInterleukin major player in cutaneous melanoma metastasic process Romanian Biotechnological Letters vol no pp “ [] M Neagu œMetabolic traits in cutaneous melanoma Frontiers in oncology vol p [] C D Ene A E Anghel M Neagu and I Nicolae œInterleukin and diabetic nephropathy Human and Veterinary Medicine vol no pp “ [] M Neagu C Constantin I D Popescu œInflammationand metabolism in cancer cell “ mitochondria key playerFrontiers in Oncology vol p [] C Nicolae I Nicolae and O Coman œGD1b GT1b Answer:

Thyroid_Cancer

"radiotherapy in combination with anti cytotoxic T lymphocyte associated antigen monoclonal antibody ipilimumab in patients with metastatic melanoma Nathalie Chaput Gras2 Emilie Lanoy3 Alicia Larive3 Celine Boutros Christine Mateus4 Emilie Routier4 Roger Sun5 Yun Gan Tao5 Christophe Massard6 Rastilav Bahleda6 Dominique Schwob3 Nathalie Ibrahim7 Rita Maria Khoury Abboud7 Caroline Caramella8 Andrea Lancia Lydie Cassard2 Severine Roy4 J C Soria10 Caroline Robert11 Eric Deutsch12 To cite Boutros a0C Chaput Gras a0N Lanoy a0E et a0al Dose escalation phase study of radiotherapy in combination with anti cytotoxic T lymphocyte associated antigen monoclonal antibody ipilimumab in patients with metastatic melanoma Journal for ImmunoTherapy of Cancer 20208e000627 101136jitc2020000627 –º Additional material is published online only To view please visit the journal online http dx jitc CR and ED contributed equallyAccepted June Authors or their employers Re use permitted under CC BY NC No commercial re use See rights and permissions Published by BMJFor numbered affiliations see end of Correspondence toDr Eric Deutsch eric deutsch gustaveroussy frBackground A synergy between radiotherapy and anti cytotoxic T lymphocyte associated antigen anti CTLA4 monoclonal antibody has been demonstrated preclinically The Mel Ipi Rx phase study aimed to determine the maximum tolerated dose MTD and safety profile of radiotherapy combined with ipilimumab in patients with metastatic melanomaPatients and methods A dose escalation design was used with and Gy dose of radiotherapy at week combined with mgkg ipilimumab every weeks for four doses Patients with evidence of clinical benefit at week were eligible for maintenance with ipilimumab mgkg every weeks starting at week until severe toxicity or disease progression The database lock occurred on April Tumor growth rate of irradiated lesions and non irradiated lesions were analyzed to assess the systemic immunologic antitumor response Blood immune monitoring was performed before and during treatment to determine if radiotherapy could modify ipilimumab pharmacodynamicsResults patients received ipilimumab between August and July Nine patients received the four doses of ipilimumab All patients received the combined radiotherapy Grade adverse events occurred in nine patients the most common being colitis and hepatitis No drug related death occurred Dose limiting toxicity occurred in two of six patients in the cohort receiving Gy The MTD was Gy Two patients had complete response three had partial response response and seven had stable disease giving an objective response rate of and a clinical benefit rate of at week The median duration of follow up was years Q145 Q368 The median overall survival CI was estimated at years “ The median progression free survival PFS CI was “ Radiotherapy combined with ipilimumab was associated with increased CD4 and CD8ICOS T cells Increased CD8 was significantly associated with PFSConclusion When combined with ipilimumab at mgkg the MTD of radiotherapy was Gy This combination of ipilimumab and radiotherapy appears to be associated with antitumor activity Increased CD8 was significantly associated with PFS Thus immune biomarkers may be useful for early response evaluationTrial registration number NCT01557114INTRODUCTIONImmunotherapy using immune checkpoint inhibition has revolutionized the management of patients with advanced stage melanoma and is an emerging approach for many other cancers1 The first immune checkpoint inhibitor that was developed was ipilimumab2 Ipilimumab targets the cytotoxic T lymphocyte associated antigen CTLA4 and significantly improves the overall survival in patients with metastatic melanoma2 However the objective response rate ORR and the disease control rates proportion of patients with a partial response PR or complete response CR or stable disease SD are relatively low and respectively3 As such increased interest has been drawn to enhance the induction of systemic immune responses of ipilimumab by combining it with radiotherapy4“ One of the rationale put forward to combine these therapies is that ipilimumab is able to deplete T regulatory cells Treg cells through antibody dependent cell cytotoxicity and consequently increases the CD8 T cell to Treg ratio whereas radiotherapy promotes the diversity of the T cell receptor TCR Boutros a0C et a0al J Immunother Cancer 20208e000627 101136jitc2020000627 0c access repertoire of intratumoral T cells7“ When combined ipilimumab promotes the expansion of T cells while radiation enhances the TCR repertoire of the expanded peripheral clones4 Ipilimumab at a dose of mgkg is approved in several countries for the treatment of unresectable or metastatic melanoma In a recent prospective phase study patients were treated with ipilimumab mgkg combined with concurrent or sequential stereotactic ablative radiotherapy11 Dose limiting toxicities DLTs and grade toxicities were reported in and of patients respectively PR and clinical benefit were reported in and of patients respectively However few clinical studies were conducted to assess the efficacy of ipilimumab mgkg combined with radiotherapy11“ although overall survival was significantly improved with the ipilimumab mgkg monotherapy compared with the mgkg dose2 Here we present the phase ˜Mel Ipi Rx™ study investigating the safety and efficacy of ipilimumab mgkg combined with radiotherapy in patients with metastatic melanoma Although the treatment landscape of patients with advanced melanoma has changed since this study was initiated the increased survival benefit of ipilimumab mgkg compared with mgkg suggests that the clinical utility of ipilimumab in refractory patients with high unmet medical need could warrant further assessment The primary objective of this study was to determine the maximum tolerated dose MTD DLT and the recommended phase dose RPTD of radiotherapy administered in combination with ipilimumab at a dose of mgkg in patients with metastatic melanoma The secondary objectives were to determine the adverse event AE profiles to describe the preliminary antitumor activity following escalating doses of radiation combined to ipilimumab using the immune related response criteria irRC and to evaluate the overall survival in patients treated with this combination The exploratory objectives were to evaluate the systemic immunologic antitumor response and factors influencing this responseMETHODSPatientsEligible patients had unresectable locally advanced or metastatic melanoma with at least one measurable metastasis accessible to radiotherapy Subcutaneous nodules and lymph nodes were considered as targets for irradiation Tumor lesions located within vital ans or gastrointestinal tract were not considered as target for radiotherapy All patients had measurable and non measurable disease evaluable according to irRC Patients were ‰¥ years of age were able to give written informed consent had Eastern Cooperative Oncology Group ECOG performance status of to and had normal renal and liver functions on blood tests Patients were excluded if they had one of the following criteria suspected or known central nervous system tumors including brain metastasis any other malignancy with a disease free for less than years an autoimmune disease a history of prior treatment with ipilimumab prior radiotherapy within the same body area or radiotherapy in fields containing flat bones volume If chemotherapy or immunotherapy were previously used a wash out period of weeks at least was required before the first administration of ipilimumabStudy designThis phase I dose escalation study evaluated the MTD of radiotherapy in combination with ipilimumab in patients with unresectable locally advanced or metastatic malignant melanoma Eligible patients received ipilimumab at mgkg at weeks and for a total of four doses at week intervals online supplementary figure S1 Patients without progressive disease PD who tolerated the treatment continued ipilimumab dosing in week intervals until progression or withdrawal of consent Radiotherapy was delivered on week on Monday Tuesday and Friday at the time of the second cycle of ipilimumab on measurable superficial lesions including subcutaneous nodules and lymph nodes Radiotherapy was delivered using MV photons or electrons with standard field encompassing Maximal field had to be at least × cm but not be more than × cm maximal dimensions on a lesion Millimetric margins were used to take into account microscopic spreading and patient movements clinical and planning target volumes of mm Dose escalation of ionizing radiation was planned in cohorts of three to six patients depending on the occurrence of DLTs during the first combination treatment cycle A hypofractionated radiation regimen higher doses per fraction was used Dose escalation was used with total doses of and Grays Gy administered in three fractions every Monday Wednesday and Friday and planned in cohorts of three to six patients depending on the occurrence of DLTs from week to week A minimum deadline of hours between two radiotherapy sessions had to be respected If necessary the radiotherapy could be put back to Tuesday Thursday Saturday Given the small size of the radiation field and the fact that irradiated lesions were superficial vital ans and gastrointestinal tract were not involved in the irradiated fieldToxicities were evaluated according to the Common Terminology Criteria for Adverse Events CTCAE version DLT observation period ranged from week to Any toxicity before the start of radiotherapy was not considered as a DLT but as a toxicity from ipilimumab alone The DLT was defined by the appearance of at least one of the following study combination related event within weeks grade vomiting diarrhea or gastrointestinal bleeding grade or non hematologic toxicity excluding grade nausea vomiting diarrhea and transient fever or grade or radiation dermatitis except if return to grade ‰¤ within weeks If none of the first three subjects in a given dosing cohort had experienced DLT dose escalation was realized If Boutros a0C et a0al J Immunother Cancer 20208e000627 101136jitc2020000627 0cone of the first three subjects in a given dosing cohort had experienced DLT an additional three subjects were enrolled to that dose level before further escalation was considered A dose escalation was realized if none of these additional three subjects had experienced DLT If two of the first three subjects in a given dosing cohort had experienced DLT dose escalation was stopped Dose escalation was continued until one third or more of the subjects at a particular dose level experienced DLT or up to the fourth cohort This was considered the maximum administered dose MAD The MTD was defined as the highest dose at which less than one third of the subjects experienced DLT The RPTD was defined as the MTD or the dose that was considered to give the optimal clinical andor immunological results Tumor assessments consisting of CT scans and assessment of skin lesions were performed at weeks and every weeks thereafter until the patient withdrew from the study or entered the follow up phase MRI or CT scan of the brain had to be realized when clinically indicated Evidence of PD was confirmed by a second assessment performed “ weeks later Definitions of lesions were based on irRC Measurable lesions were defined as lesions that could be accurately measured in two perpendicular diameters with at least one diameter ‰¥ mm and the other dimension ‰¥ mm It was possible to consider skin lesions if they were measurable All measurable lesions up to a maximum of lesions per an and lesions in total were identified as index lesions measured and recorded at screening and follow up The index lesions were representative of all involved ans Non index lesions corresponded to measurable lesions that were recorded and evaluated at the same assessment time points as the index lesions and that were irradiated according to the trial irradiation designBlood immune monitoringBlood samples were collected at baseline T0 at week W4 before second injection of ipilimumab and at week W6 after radiotherapy and before third injection of ipilimumab online supplementary figure S2 Phenotyping was performed on fresh whole blood and peripheral blood mononuclear cells PBMCs isolated by Ficoll density gradient were frozen for later analyses Data analysis from standard blood tests was realized to estimate the derived neutrophil to lymphocyte ratio dNLR at baseline W4 and W6 to see whether or not granulocytes could impact the prognosis of patients Whole blood or PBMCs were incubated with fluorochrome conjugated antibodies for min at room temperature or at °C respectively followed by min of lysis Versalyse Beckman Coulter Mervue Galway Ireland The following fluorochrome conjugated antibodies were used fluorescein isothiocyanate anti ICOS CD278 clone DX29 phycoerythrin PE conjugated anti CD25 clone B1499 allophycocyanin cyanine APC Cy7 conjugated CD45RA clone HI100 phycoerythrin cyanine PE Cy7 conjugated anti CD45RA clone access2H4 allophycocyanin Alexa Fluor AA700 conjugated anti CD3 clone UCHT1 Pacific Blue PB conjugated anti CD4 clone 13B82 and Krome orange conjugated anti CD8 clone B911 were obtained from Beckman Coulter Stained cells were acquired using a FACS Canto II cytometer BD Bioscience or a Gallios Cytometer Beckman Coulter and analyzed using Kaluza software Beckman Coulter Conventional T cells CD3CD4 and CD3CD8 were respectively defined by CCR7CD45RA for naïve T cells CCR7CD45RA for central memory T cells TCM CCR7CD45RA for effector T cells TEM and CCR7CD45RA for terminally differentiated T cells TEMRA Treg cells were defined as CD3CD127lowCD25Tumor growth rate analysisTo assess the systemic immunologic antitumor response tumor growth rate TGR of irradiated lesions and non irradiated lesions were analyzed Briefly TGR estimates the variation in tumor volume over time using each patient as his own control and has shown to be interesting to identify the specific therapeutic effect of a treatment regardless of the disease course of each patient16 TGR was expressed as a percentage increase in tumor volume during month in accordance with Hiniker et al11 using the following formula TGR100 [exp[3LogDtDt0t] ˆ’] with D0tumor size defined as the sum of the longest diameters of the lesions at baseline Dttumor size defined as the sum of the longest diameters of the lesions at the time t of evolution evaluation in monthsTGR100 [exp3LogDtDt0tˆ’]The TGR was computed for irradiated lesions TGRirr and non irradiated target lesions defined by the radiologist for the irRC evaluation TGRnon irr for two treatment periods when data were available the Reference TGR REF TGR assessed before the onset of the treatment using the baseline CT and a CT realized before the baseline prebaseline CT the Experimental TGR EXP TGR assessed between the onset of the treatment and the first evaluation The TGR was computed using the same target lesions at each evaluation time A positive value of the EXP TGR reflected a bigger lesion at the first evaluation than at baseline Therefore new lesions related to PD were not included in the TGR computation Difference between EXP TGR and REF TGR was used to assess the effect of the treatment ΔTGREXP TGRˆ’REF TGR a negative value reflecting a slowdown of the natural course of the disease ie a slower tumor growth or a tumor response between the two periodsStatistical analysisDemographic and baseline characteristics were summarized for all registered subjects using descriptive statistics Toxicity grades per subject were tabulated for AEs and on study laboratory measurements by using the NCI CTCAE version Analyses of efficacy endpoints were based on all subjects evaluable for efficacyBoutros a0C et a0al J Immunother Cancer 20208e000627 101136jitc2020000627 0c access The Kaplan Meier method was used to estimate overall survival and immune related progression free survival PFS Overall survival was defined as the time from treatment initiation to the date of death or date of last follow up in persons alive at last follow up Immune related progression was defined according to irRC An event was defined as progression or death whichever comes first PFS was defined as the time from treatment initiation to the date of occurrence of the event Follow up of patients who did not experience was censored at the date of last evaluation Duration of follow up was estimated using the Schemper and Smith method18 All statistical analyses were done using SAS software V94Evolution of innate immune cells was analyzed using GraphPAD PRISM software values at W4 and at W6 were described and compared with baseline by using Friedman test followed by Dunn's multiple comparisons testStudy oversightThe study was conducted in accordance with the protocol Good Clinical Practice guidelines and the provisions of the Declaration of Helsinki All patients provided written informed consentRESULTSPatient populationNineteen patients with advanced melanoma were treated at Gustave Roussy in the Mel Ipi Rx phase trial between August and July online supplementary table S1 The database lock occurred on April Nine patients received the four doses of ipilimumab and two patients received maintenance ipilimumab one and two cycles respectively All patients received the combined radiotherapy at week in three fractions Thirteen patients were enrolled at dose level Gy and six patients at dose level Gy Demographic data of patients are summarized in table Overall patients presented visceral metastases M1c had elevated level of lactate dehydrogenase LDH tumors were BRAF mutated and none had a history of brain metastases All patients had received systemic therapy previously Prior therapy included chemotherapy in patients BRAF inhibitors in patients radiotherapy in patient and surgery in patients respectively and all of them were immune checkpoint inhibitor naïve Irradiation was delivered on subcutaneous lesions in patients and patients in the cohorts treated with and Gy respectively and on lymph nodes in patients and patients in the cohorts treated with and Gy respectively online supplementary table S2 The average irradiated tumor volumes were relatively homogeneous with a median of mm IQR “SafetyAll patients presented at least one AE of any grade These AEs were felt to be probably related to ipilimumab The role of concurrent radiotherapy on these AEs was difficult Table Patient baseline characteristicsCharacteristics SexMaleFemaleAge yearsMedianRangeMutation BRAF statusNon mutantMutantUnknownLactate dehydrogenaseNormalElevatedM staging extent of metastasesM0M1aM1bM1cBrain metastasesNoYesSite of irradiated lesionsLymph nodesSubcutaneous nodulesVisceral ansPrevious treatments No prior systemic treatmentIpilimumabChemotherapyAnti BRAFRadiotherapySurgeryOverall populationN19 No “ The normal range for lactate dehydrogenase is UIL All patients were naïve to immune checkpoint inhibitorsto assess However no radiation induced necrosis or local symptoms were observed inside the radiation field AEs of all grades are summarized in table regardless of their attributionAsthenia was the most commonly reported AE of any grade It occurred in patients Among them nine patients were treated in the Gy cohort and five patients in the Gy cohort The median time to onset of asthenia was Q1 Q34“ weeks and the median duration was Q1 Q34“ weeks The other most common AEs of Boutros a0C et a0al J Immunother Cancer 20208e000627 101136jitc2020000627 0cTable Adverse events of all grade safety populationNo of patients with at least one adverse eventsFatigueDiarrheaDisease related painFeverPainNauseavomitingAnorexiaConstipationColitisPruritusWeight lossAnemiaEdema of limbsDyspneaSkin eruptionVitiligoHypereosinophiliaAlanineaspartate aminotransferase increasedCoughLymphedemaLevel dose N13 Level dose N6 TotalN19 any grade included diarrhea disease related pain fever nausea and vomitingThirteen patients discontinued the study owing to treatment related AEs nine patients in the cohort receiving Gy and four patients in the cohort receiving Gy Nine of these patients had AEs of grade Multiple AEs of grade occurred in some patients AEs of grade included colitis n2 hepatitis n2 asthenia n2 thyroid disorders n1 DRESS drug rash with eosinophilia and systemic symptoms syndrome n1 and nauseavomiting n1 Online supplementary table S3 presents the grade and grade events for each dose level for all events There were no treatment related deathsFour of the patients who discontinued the study did not have grade or AEs Indeed three patients had grade colitis associated with either contraindication to corticosteroids because of uncontrolled diabetes mellitus or complete remission after four cycles of ipilimumab leading to regular follow up without maintenance ipilimumab One patient had a grade asthenia after four cycles of ipilimumab leading to regular follow up without maintenance ipilimumab accessDose escalation and DLTsEighteen patients were evaluable for DLTs in this study patients in the cohort treated with Gy and patients in the cohort treated with Gy Among them four patients experienced DLTs All the DLTs occurred outside the radiation fieldIn the cohort treated with Gy of evaluable patients experienced DLTs after two cycles of ipilimumab combined with radiotherapy Indeed one of these patients presented grade hepatitis and the other one presented grade colitis with grade hypokalemia grade anorexia and grade thyroid disorders In both patients the three fractions of radiotherapy on Monday Wednesday and Friday were delivered on week DLTs led to permanent ipilimumab discontinuation in both patientsIn the cohort treated with Gy two of the six evaluable patients experienced a DLT after two and three cycles of ipilimumab respectively combined with radiotherapy One of these patients presented grade hepatitis and the other one presented grade colitis with unusually normal macroscopic colonoscopy but a total villous atrophy mimicking celiac disease on biopsies In both patients the radiotherapy fractions on Monday Wednesday and Friday were delivered on week DLTs led to permanent ipilimumab discontinuation in both patientsThe MTD of radiotherapy was thus Gy when combined with ipilimumab mgkg in the present design Consequently the RPTD of radiotherapy administered in combination with ipilimumab at mgkg in patients with metastatic melanoma was GyClinical outcomesAt the time of analysis the median duration of follow up was years Q145 Q368 The median overall survival CI was estimated at years “ The median PFS CI was “ Figure shows Kaplan Meier median overall survival and PFS curves with CI According to irRC the best response within the trial was CR for two patients both in the cohort receiving Gy PR for three patients two patients in the cohort receiving Gy and one patient in the cohort receiving Gy and SD for seven patients six patients in the cohort receiving Gy and one patient in the cohort receiving Gy giving an ORR of and a clinical benefit rate of at week online supplementary table S2 The initial melanoma staging of the patients who had CR was M1a for one patient multiple subcutaneous nodules and M1c multiple subcutaneous nodules and lymph nodes associated with an elevated LDH for the other patient The initial melanoma staging of the patients who had PR was M1a for one patient multiple subcutaneous nodules and M1c for two patients one of these patients had a subcutaneous nodule a lymph node and elevated LDH The other patient had multiple lymph nodes and bone metastasisBoutros a0C et a0al J Immunother Cancer 20208e000627 101136jitc2020000627 0c access Figure A Waterfall plot of patients according to the variation of tumor growth rate ΔTGR between reference before treatment and experimental period on treatment For each patient specific ΔTGR of irradiated and non irradiated lesions are represented ΔTGR on treatment lesions at the first evaluation are bigger than at baseline ΔTGR of non irradiated lesions was superior to the irradiated lesion B Changes of the sum of diameters of the target lesions irradiated and non irradiated respectively at months in compared with the baselineVariation of tumor growth rate across lesionsTGR variation for irradiated and non irradiated lesions before and after the treatment was evaluable for patients figure A total of non irradiated lesions with up to lesions for one patient were evaluated for the REF and EXP TGRnon irr median IQR “ non irradiated lesions by patient and irradiated lesions for the REF and EXP TGRirr one patient had two irradiated lesions Median reference and experimental period were IQR “ and IQR “ months respectively The sum of diameters of lesions at the three evaluation times prebaseline baseline first evaluation and corresponding TGR are presented in table The EXP TGR on treatment was not correlated with the REF TGR before treatment Spearman™s rho011 p073 Decrease of the TGR was ˆ’ of the tumor sizemonths IQR “ to “ for irradiated lesions and ˆ’ month IQR “ to “ for non irradiated lesions although the difference was not significantly different p082 Response Figure Kaplan Meier median overall survival A and progression free survival B curves with CINo pseudoprogression was observed among the patients treated in this study nor radiation induced necrosis or edema Of note three patients were not evaluable for response because they progressed and died before the radiologic assessment scheduled in this studyTreatments administered after the ˜Mel Ipi Rx™ study in patients with disease progression are summarized in online supplementary figure S3Boutros a0C et a0al J Immunother Cancer 20208e000627 101136jitc2020000627 0cTable Characteristics of lesions evaluated for tumor growth rate TGRPatients nLesions n sumLesions n median IQRSum of diameters prebaseline median IQRSum of diameters baseline median IQRSum of diameters at first evaluation median IQRREFperiod median IQREXPperiod median IQRREF TGR median IQREXP TGR median IQRTGRdiff median IQR accessWilcoxon p valueOverallIrradiated lesionsNon irradiated lesions to to to to to to to to to to to ˆ’ ˆ’ to to ˆ’ to ˆ’ ˆ’ to ˆ’ˆ’ ˆ’ to ˆ’ to to to ˆ’ ˆ’ to ˆ’ˆ’ ˆ’ to ˆ’REF TGR corresponds to TGR before the start of the treatment EXP TGR corresponds to TGR between the start of the treatment and the first evaluation at monthsof non irradiated lesions seemed more representative of patient outcomes vs irradiated lesion EXP TGRnon irr was significantly higher in patients with PD Although the number of patients was too low for statistical test p values are shown for information in table figure and online supplementary figure S4Immune analysesWe analyzed immune parameters in the blood that have been previously described to be changed during ipilimumab treatment to determine if radiotherapy could modify ipilimumab pharmacodynamics19 For CD4 T cell counts and as expected ipilimumab alone W4 could favor accumulation of TEM Treg and ICOSCD4 T cells Interestingly at W6 after ipilimumabradiotherapy only TEM and ICOSCD4 T cells remained significantly increased suggesting that combination favored accumulation of activated memory CD4 T cells rather than Treg cells For CD8 T cell counts no accumulation could be observed at W4 while augmentation of TCM and TEMRA could be depicted between W4 and W6 suggesting that adjunction of radiotherapy to ipilimumab was more prone to boost these CD8 T populations online supplementary figure S2 High fold change in CD8 from baseline to week was significantly correlated with PFS p00223 but not significantly correlated with overall survival p02355 online supplementary figure S5Innate immune cells and NLR absolute neutrophils count ANC divided by the number of lymphocytes or dNLR ANC divided by the number of white blood cellsˆ’ANC have been shown to have a prognostic role in patients treated with immunotherapy and even might represent a predictive biomarker of response We took advantage of standard blood tests to determine if Table Univariate analysis of tumor growth rate TGRAll lesions n37Progressive disease WilcoxonNop valueYesIrradiated lesion n13Progressive diseaseNoYesWilcoxon p valueNon irradiated lesions n24Progressive diseaseNoWilcoxon p valueYesSum of diameters prebaseline meanSum of diameters baseline meanSum of diameters at first evaluation meanREF TGR meanExp TGR meanΔTGR meanˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’TGR is evaluated in percentage per months ΔTGR EXP TGR “ REF TGR A negative value corresponds to a slowdown of the tumor growthBoutros a0C et a0al J Immunother Cancer 20208e000627 101136jitc2020000627 0c access neutrophils monocytes NLR or dNLR at baseline W4 and W6 could correlate with the prognosis in our study ANC or monocytes did not correlate with the prognosis of patients while absolute count of lymphocytes significantly increased at W6 compared with baseline in only patients with a clinical benefit CRPRSD Both NLR and dNLR were significantly lower at W6 only in patients with clinical benefit online supplementary figure S6 Note that we did not found an association with the dose of radiotherapy data not shownDISCUSSIONIn this dose escalation phase study four patients experienced DLTs All the DLTs occurred outside the radiation field Therefore it was difficult to assess the role of concurrent radiotherapy on the DLTs The MTD of radiotherapy combined with ipilimumab at mgkg was Gy The RPTD of radiotherapy administered in combination with ipilimumab at mgkg in patients with metastatic melanoma was Gy A hypofractionated radiation regimen higher doses per fraction was used in this study It is usually preferred for melanoma which displays low alphabeta ratio Our three fractions hypofractionated regimen is in line with the radiation standard in the metastatic setting Moreover it has been shown recently that radiation doses per session inferior to Gy combine more favorably with immunotherapy through interferon type induction21 Of note our study is the only one that combines radiotherapy and high dose of ipilimumab mgkgThe incidence of treatment related AEs was high with this combination in our study but numerically similar to the incidence reported previously with ipilimumab monotherapy at mgkg Grade or AEs occurred in of patients in our study whereas they occurred in “ of patients treated with ipilimumab monotherapy at mgkg2 The overall AE spectrum of the combination in this study was consistent with previous findings with the drug™s immune based mechanism of action The high rate of AEs might be partially attributed to the high dose of ipilimumab The dose of mgkg was chosen based on data from the randomized phase trial that compared various

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You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: "radiotherapy in combination with anti cytotoxic T lymphocyte associated antigen monoclonal antibody ipilimumab in patients with metastatic melanoma Nathalie Chaput Gras2 Emilie Lanoy3 Alicia Larive3 Celine Boutros Christine Mateus4 Emilie Routier4 Roger Sun5 Yun Gan Tao5 Christophe Massard6 Rastilav Bahleda6 Dominique Schwob3 Nathalie Ibrahim7 Rita Maria Khoury Abboud7 Caroline Caramella8 Andrea Lancia Lydie Cassard2 Severine Roy4 J C Soria10 Caroline Robert11 Eric Deutsch12 To cite Boutros a0C Chaput Gras a0N Lanoy a0E et a0al Dose escalation phase study of radiotherapy in combination with anti cytotoxic T lymphocyte associated antigen monoclonal antibody ipilimumab in patients with metastatic melanoma Journal for ImmunoTherapy of Cancer 20208e000627 101136jitc2020000627 –º Additional material is published online only To view please visit the journal online http dx jitc CR and ED contributed equallyAccepted June Authors or their employers Re use permitted under CC BY NC No commercial re use See rights and permissions Published by BMJFor numbered affiliations see end of Correspondence toDr Eric Deutsch eric deutsch gustaveroussy frBackground A synergy between radiotherapy and anti cytotoxic T lymphocyte associated antigen anti CTLA4 monoclonal antibody has been demonstrated preclinically The Mel Ipi Rx phase study aimed to determine the maximum tolerated dose MTD and safety profile of radiotherapy combined with ipilimumab in patients with metastatic melanomaPatients and methods A dose escalation design was used with and Gy dose of radiotherapy at week combined with mgkg ipilimumab every weeks for four doses Patients with evidence of clinical benefit at week were eligible for maintenance with ipilimumab mgkg every weeks starting at week until severe toxicity or disease progression The database lock occurred on April Tumor growth rate of irradiated lesions and non irradiated lesions were analyzed to assess the systemic immunologic antitumor response Blood immune monitoring was performed before and during treatment to determine if radiotherapy could modify ipilimumab pharmacodynamicsResults patients received ipilimumab between August and July Nine patients received the four doses of ipilimumab All patients received the combined radiotherapy Grade adverse events occurred in nine patients the most common being colitis and hepatitis No drug related death occurred Dose limiting toxicity occurred in two of six patients in the cohort receiving Gy The MTD was Gy Two patients had complete response three had partial response response and seven had stable disease giving an objective response rate of and a clinical benefit rate of at week The median duration of follow up was years Q145 Q368 The median overall survival CI was estimated at years “ The median progression free survival PFS CI was “ Radiotherapy combined with ipilimumab was associated with increased CD4 and CD8ICOS T cells Increased CD8 was significantly associated with PFSConclusion When combined with ipilimumab at mgkg the MTD of radiotherapy was Gy This combination of ipilimumab and radiotherapy appears to be associated with antitumor activity Increased CD8 was significantly associated with PFS Thus immune biomarkers may be useful for early response evaluationTrial registration number NCT01557114INTRODUCTIONImmunotherapy using immune checkpoint inhibition has revolutionized the management of patients with advanced stage melanoma and is an emerging approach for many other cancers1 The first immune checkpoint inhibitor that was developed was ipilimumab2 Ipilimumab targets the cytotoxic T lymphocyte associated antigen CTLA4 and significantly improves the overall survival in patients with metastatic melanoma2 However the objective response rate ORR and the disease control rates proportion of patients with a partial response PR or complete response CR or stable disease SD are relatively low and respectively3 As such increased interest has been drawn to enhance the induction of systemic immune responses of ipilimumab by combining it with radiotherapy4“ One of the rationale put forward to combine these therapies is that ipilimumab is able to deplete T regulatory cells Treg cells through antibody dependent cell cytotoxicity and consequently increases the CD8 T cell to Treg ratio whereas radiotherapy promotes the diversity of the T cell receptor TCR Boutros a0C et a0al J Immunother Cancer 20208e000627 101136jitc2020000627 0c access repertoire of intratumoral T cells7“ When combined ipilimumab promotes the expansion of T cells while radiation enhances the TCR repertoire of the expanded peripheral clones4 Ipilimumab at a dose of mgkg is approved in several countries for the treatment of unresectable or metastatic melanoma In a recent prospective phase study patients were treated with ipilimumab mgkg combined with concurrent or sequential stereotactic ablative radiotherapy11 Dose limiting toxicities DLTs and grade toxicities were reported in and of patients respectively PR and clinical benefit were reported in and of patients respectively However few clinical studies were conducted to assess the efficacy of ipilimumab mgkg combined with radiotherapy11“ although overall survival was significantly improved with the ipilimumab mgkg monotherapy compared with the mgkg dose2 Here we present the phase ˜Mel Ipi Rx™ study investigating the safety and efficacy of ipilimumab mgkg combined with radiotherapy in patients with metastatic melanoma Although the treatment landscape of patients with advanced melanoma has changed since this study was initiated the increased survival benefit of ipilimumab mgkg compared with mgkg suggests that the clinical utility of ipilimumab in refractory patients with high unmet medical need could warrant further assessment The primary objective of this study was to determine the maximum tolerated dose MTD DLT and the recommended phase dose RPTD of radiotherapy administered in combination with ipilimumab at a dose of mgkg in patients with metastatic melanoma The secondary objectives were to determine the adverse event AE profiles to describe the preliminary antitumor activity following escalating doses of radiation combined to ipilimumab using the immune related response criteria irRC and to evaluate the overall survival in patients treated with this combination The exploratory objectives were to evaluate the systemic immunologic antitumor response and factors influencing this responseMETHODSPatientsEligible patients had unresectable locally advanced or metastatic melanoma with at least one measurable metastasis accessible to radiotherapy Subcutaneous nodules and lymph nodes were considered as targets for irradiation Tumor lesions located within vital ans or gastrointestinal tract were not considered as target for radiotherapy All patients had measurable and non measurable disease evaluable according to irRC Patients were ‰¥ years of age were able to give written informed consent had Eastern Cooperative Oncology Group ECOG performance status of to and had normal renal and liver functions on blood tests Patients were excluded if they had one of the following criteria suspected or known central nervous system tumors including brain metastasis any other malignancy with a disease free for less than years an autoimmune disease a history of prior treatment with ipilimumab prior radiotherapy within the same body area or radiotherapy in fields containing flat bones volume If chemotherapy or immunotherapy were previously used a wash out period of weeks at least was required before the first administration of ipilimumabStudy designThis phase I dose escalation study evaluated the MTD of radiotherapy in combination with ipilimumab in patients with unresectable locally advanced or metastatic malignant melanoma Eligible patients received ipilimumab at mgkg at weeks and for a total of four doses at week intervals online supplementary figure S1 Patients without progressive disease PD who tolerated the treatment continued ipilimumab dosing in week intervals until progression or withdrawal of consent Radiotherapy was delivered on week on Monday Tuesday and Friday at the time of the second cycle of ipilimumab on measurable superficial lesions including subcutaneous nodules and lymph nodes Radiotherapy was delivered using MV photons or electrons with standard field encompassing Maximal field had to be at least × cm but not be more than × cm maximal dimensions on a lesion Millimetric margins were used to take into account microscopic spreading and patient movements clinical and planning target volumes of mm Dose escalation of ionizing radiation was planned in cohorts of three to six patients depending on the occurrence of DLTs during the first combination treatment cycle A hypofractionated radiation regimen higher doses per fraction was used Dose escalation was used with total doses of and Grays Gy administered in three fractions every Monday Wednesday and Friday and planned in cohorts of three to six patients depending on the occurrence of DLTs from week to week A minimum deadline of hours between two radiotherapy sessions had to be respected If necessary the radiotherapy could be put back to Tuesday Thursday Saturday Given the small size of the radiation field and the fact that irradiated lesions were superficial vital ans and gastrointestinal tract were not involved in the irradiated fieldToxicities were evaluated according to the Common Terminology Criteria for Adverse Events CTCAE version DLT observation period ranged from week to Any toxicity before the start of radiotherapy was not considered as a DLT but as a toxicity from ipilimumab alone The DLT was defined by the appearance of at least one of the following study combination related event within weeks grade vomiting diarrhea or gastrointestinal bleeding grade or non hematologic toxicity excluding grade nausea vomiting diarrhea and transient fever or grade or radiation dermatitis except if return to grade ‰¤ within weeks If none of the first three subjects in a given dosing cohort had experienced DLT dose escalation was realized If Boutros a0C et a0al J Immunother Cancer 20208e000627 101136jitc2020000627 0cone of the first three subjects in a given dosing cohort had experienced DLT an additional three subjects were enrolled to that dose level before further escalation was considered A dose escalation was realized if none of these additional three subjects had experienced DLT If two of the first three subjects in a given dosing cohort had experienced DLT dose escalation was stopped Dose escalation was continued until one third or more of the subjects at a particular dose level experienced DLT or up to the fourth cohort This was considered the maximum administered dose MAD The MTD was defined as the highest dose at which less than one third of the subjects experienced DLT The RPTD was defined as the MTD or the dose that was considered to give the optimal clinical andor immunological results Tumor assessments consisting of CT scans and assessment of skin lesions were performed at weeks and every weeks thereafter until the patient withdrew from the study or entered the follow up phase MRI or CT scan of the brain had to be realized when clinically indicated Evidence of PD was confirmed by a second assessment performed “ weeks later Definitions of lesions were based on irRC Measurable lesions were defined as lesions that could be accurately measured in two perpendicular diameters with at least one diameter ‰¥ mm and the other dimension ‰¥ mm It was possible to consider skin lesions if they were measurable All measurable lesions up to a maximum of lesions per an and lesions in total were identified as index lesions measured and recorded at screening and follow up The index lesions were representative of all involved ans Non index lesions corresponded to measurable lesions that were recorded and evaluated at the same assessment time points as the index lesions and that were irradiated according to the trial irradiation designBlood immune monitoringBlood samples were collected at baseline T0 at week W4 before second injection of ipilimumab and at week W6 after radiotherapy and before third injection of ipilimumab online supplementary figure S2 Phenotyping was performed on fresh whole blood and peripheral blood mononuclear cells PBMCs isolated by Ficoll density gradient were frozen for later analyses Data analysis from standard blood tests was realized to estimate the derived neutrophil to lymphocyte ratio dNLR at baseline W4 and W6 to see whether or not granulocytes could impact the prognosis of patients Whole blood or PBMCs were incubated with fluorochrome conjugated antibodies for min at room temperature or at °C respectively followed by min of lysis Versalyse Beckman Coulter Mervue Galway Ireland The following fluorochrome conjugated antibodies were used fluorescein isothiocyanate anti ICOS CD278 clone DX29 phycoerythrin PE conjugated anti CD25 clone B1499 allophycocyanin cyanine APC Cy7 conjugated CD45RA clone HI100 phycoerythrin cyanine PE Cy7 conjugated anti CD45RA clone access2H4 allophycocyanin Alexa Fluor AA700 conjugated anti CD3 clone UCHT1 Pacific Blue PB conjugated anti CD4 clone 13B82 and Krome orange conjugated anti CD8 clone B911 were obtained from Beckman Coulter Stained cells were acquired using a FACS Canto II cytometer BD Bioscience or a Gallios Cytometer Beckman Coulter and analyzed using Kaluza software Beckman Coulter Conventional T cells CD3CD4 and CD3CD8 were respectively defined by CCR7CD45RA for naïve T cells CCR7CD45RA for central memory T cells TCM CCR7CD45RA for effector T cells TEM and CCR7CD45RA for terminally differentiated T cells TEMRA Treg cells were defined as CD3CD127lowCD25Tumor growth rate analysisTo assess the systemic immunologic antitumor response tumor growth rate TGR of irradiated lesions and non irradiated lesions were analyzed Briefly TGR estimates the variation in tumor volume over time using each patient as his own control and has shown to be interesting to identify the specific therapeutic effect of a treatment regardless of the disease course of each patient16 TGR was expressed as a percentage increase in tumor volume during month in accordance with Hiniker et al11 using the following formula TGR100 [exp[3LogDtDt0t] ˆ’] with D0tumor size defined as the sum of the longest diameters of the lesions at baseline Dttumor size defined as the sum of the longest diameters of the lesions at the time t of evolution evaluation in monthsTGR100 [exp3LogDtDt0tˆ’]The TGR was computed for irradiated lesions TGRirr and non irradiated target lesions defined by the radiologist for the irRC evaluation TGRnon irr for two treatment periods when data were available the Reference TGR REF TGR assessed before the onset of the treatment using the baseline CT and a CT realized before the baseline prebaseline CT the Experimental TGR EXP TGR assessed between the onset of the treatment and the first evaluation The TGR was computed using the same target lesions at each evaluation time A positive value of the EXP TGR reflected a bigger lesion at the first evaluation than at baseline Therefore new lesions related to PD were not included in the TGR computation Difference between EXP TGR and REF TGR was used to assess the effect of the treatment ΔTGREXP TGRˆ’REF TGR a negative value reflecting a slowdown of the natural course of the disease ie a slower tumor growth or a tumor response between the two periodsStatistical analysisDemographic and baseline characteristics were summarized for all registered subjects using descriptive statistics Toxicity grades per subject were tabulated for AEs and on study laboratory measurements by using the NCI CTCAE version Analyses of efficacy endpoints were based on all subjects evaluable for efficacyBoutros a0C et a0al J Immunother Cancer 20208e000627 101136jitc2020000627 0c access The Kaplan Meier method was used to estimate overall survival and immune related progression free survival PFS Overall survival was defined as the time from treatment initiation to the date of death or date of last follow up in persons alive at last follow up Immune related progression was defined according to irRC An event was defined as progression or death whichever comes first PFS was defined as the time from treatment initiation to the date of occurrence of the event Follow up of patients who did not experience was censored at the date of last evaluation Duration of follow up was estimated using the Schemper and Smith method18 All statistical analyses were done using SAS software V94Evolution of innate immune cells was analyzed using GraphPAD PRISM software values at W4 and at W6 were described and compared with baseline by using Friedman test followed by Dunn's multiple comparisons testStudy oversightThe study was conducted in accordance with the protocol Good Clinical Practice guidelines and the provisions of the Declaration of Helsinki All patients provided written informed consentRESULTSPatient populationNineteen patients with advanced melanoma were treated at Gustave Roussy in the Mel Ipi Rx phase trial between August and July online supplementary table S1 The database lock occurred on April Nine patients received the four doses of ipilimumab and two patients received maintenance ipilimumab one and two cycles respectively All patients received the combined radiotherapy at week in three fractions Thirteen patients were enrolled at dose level Gy and six patients at dose level Gy Demographic data of patients are summarized in table Overall patients presented visceral metastases M1c had elevated level of lactate dehydrogenase LDH tumors were BRAF mutated and none had a history of brain metastases All patients had received systemic therapy previously Prior therapy included chemotherapy in patients BRAF inhibitors in patients radiotherapy in patient and surgery in patients respectively and all of them were immune checkpoint inhibitor naïve Irradiation was delivered on subcutaneous lesions in patients and patients in the cohorts treated with and Gy respectively and on lymph nodes in patients and patients in the cohorts treated with and Gy respectively online supplementary table S2 The average irradiated tumor volumes were relatively homogeneous with a median of mm IQR “SafetyAll patients presented at least one AE of any grade These AEs were felt to be probably related to ipilimumab The role of concurrent radiotherapy on these AEs was difficult Table Patient baseline characteristicsCharacteristics SexMaleFemaleAge yearsMedianRangeMutation BRAF statusNon mutantMutantUnknownLactate dehydrogenaseNormalElevatedM staging extent of metastasesM0M1aM1bM1cBrain metastasesNoYesSite of irradiated lesionsLymph nodesSubcutaneous nodulesVisceral ansPrevious treatments No prior systemic treatmentIpilimumabChemotherapyAnti BRAFRadiotherapySurgeryOverall populationN19 No “ The normal range for lactate dehydrogenase is UIL All patients were naïve to immune checkpoint inhibitorsto assess However no radiation induced necrosis or local symptoms were observed inside the radiation field AEs of all grades are summarized in table regardless of their attributionAsthenia was the most commonly reported AE of any grade It occurred in patients Among them nine patients were treated in the Gy cohort and five patients in the Gy cohort The median time to onset of asthenia was Q1 Q34“ weeks and the median duration was Q1 Q34“ weeks The other most common AEs of Boutros a0C et a0al J Immunother Cancer 20208e000627 101136jitc2020000627 0cTable Adverse events of all grade safety populationNo of patients with at least one adverse eventsFatigueDiarrheaDisease related painFeverPainNauseavomitingAnorexiaConstipationColitisPruritusWeight lossAnemiaEdema of limbsDyspneaSkin eruptionVitiligoHypereosinophiliaAlanineaspartate aminotransferase increasedCoughLymphedemaLevel dose N13 Level dose N6 TotalN19 any grade included diarrhea disease related pain fever nausea and vomitingThirteen patients discontinued the study owing to treatment related AEs nine patients in the cohort receiving Gy and four patients in the cohort receiving Gy Nine of these patients had AEs of grade Multiple AEs of grade occurred in some patients AEs of grade included colitis n2 hepatitis n2 asthenia n2 thyroid disorders n1 DRESS drug rash with eosinophilia and systemic symptoms syndrome n1 and nauseavomiting n1 Online supplementary table S3 presents the grade and grade events for each dose level for all events There were no treatment related deathsFour of the patients who discontinued the study did not have grade or AEs Indeed three patients had grade colitis associated with either contraindication to corticosteroids because of uncontrolled diabetes mellitus or complete remission after four cycles of ipilimumab leading to regular follow up without maintenance ipilimumab One patient had a grade asthenia after four cycles of ipilimumab leading to regular follow up without maintenance ipilimumab accessDose escalation and DLTsEighteen patients were evaluable for DLTs in this study patients in the cohort treated with Gy and patients in the cohort treated with Gy Among them four patients experienced DLTs All the DLTs occurred outside the radiation fieldIn the cohort treated with Gy of evaluable patients experienced DLTs after two cycles of ipilimumab combined with radiotherapy Indeed one of these patients presented grade hepatitis and the other one presented grade colitis with grade hypokalemia grade anorexia and grade thyroid disorders In both patients the three fractions of radiotherapy on Monday Wednesday and Friday were delivered on week DLTs led to permanent ipilimumab discontinuation in both patientsIn the cohort treated with Gy two of the six evaluable patients experienced a DLT after two and three cycles of ipilimumab respectively combined with radiotherapy One of these patients presented grade hepatitis and the other one presented grade colitis with unusually normal macroscopic colonoscopy but a total villous atrophy mimicking celiac disease on biopsies In both patients the radiotherapy fractions on Monday Wednesday and Friday were delivered on week DLTs led to permanent ipilimumab discontinuation in both patientsThe MTD of radiotherapy was thus Gy when combined with ipilimumab mgkg in the present design Consequently the RPTD of radiotherapy administered in combination with ipilimumab at mgkg in patients with metastatic melanoma was GyClinical outcomesAt the time of analysis the median duration of follow up was years Q145 Q368 The median overall survival CI was estimated at years “ The median PFS CI was “ Figure shows Kaplan Meier median overall survival and PFS curves with CI According to irRC the best response within the trial was CR for two patients both in the cohort receiving Gy PR for three patients two patients in the cohort receiving Gy and one patient in the cohort receiving Gy and SD for seven patients six patients in the cohort receiving Gy and one patient in the cohort receiving Gy giving an ORR of and a clinical benefit rate of at week online supplementary table S2 The initial melanoma staging of the patients who had CR was M1a for one patient multiple subcutaneous nodules and M1c multiple subcutaneous nodules and lymph nodes associated with an elevated LDH for the other patient The initial melanoma staging of the patients who had PR was M1a for one patient multiple subcutaneous nodules and M1c for two patients one of these patients had a subcutaneous nodule a lymph node and elevated LDH The other patient had multiple lymph nodes and bone metastasisBoutros a0C et a0al J Immunother Cancer 20208e000627 101136jitc2020000627 0c access Figure A Waterfall plot of patients according to the variation of tumor growth rate ΔTGR between reference before treatment and experimental period on treatment For each patient specific ΔTGR of irradiated and non irradiated lesions are represented ΔTGR on treatment lesions at the first evaluation are bigger than at baseline ΔTGR of non irradiated lesions was superior to the irradiated lesion B Changes of the sum of diameters of the target lesions irradiated and non irradiated respectively at months in compared with the baselineVariation of tumor growth rate across lesionsTGR variation for irradiated and non irradiated lesions before and after the treatment was evaluable for patients figure A total of non irradiated lesions with up to lesions for one patient were evaluated for the REF and EXP TGRnon irr median IQR “ non irradiated lesions by patient and irradiated lesions for the REF and EXP TGRirr one patient had two irradiated lesions Median reference and experimental period were IQR “ and IQR “ months respectively The sum of diameters of lesions at the three evaluation times prebaseline baseline first evaluation and corresponding TGR are presented in table The EXP TGR on treatment was not correlated with the REF TGR before treatment Spearman™s rho011 p073 Decrease of the TGR was ˆ’ of the tumor sizemonths IQR “ to “ for irradiated lesions and ˆ’ month IQR “ to “ for non irradiated lesions although the difference was not significantly different p082 Response Figure Kaplan Meier median overall survival A and progression free survival B curves with CINo pseudoprogression was observed among the patients treated in this study nor radiation induced necrosis or edema Of note three patients were not evaluable for response because they progressed and died before the radiologic assessment scheduled in this studyTreatments administered after the ˜Mel Ipi Rx™ study in patients with disease progression are summarized in online supplementary figure S3Boutros a0C et a0al J Immunother Cancer 20208e000627 101136jitc2020000627 0cTable Characteristics of lesions evaluated for tumor growth rate TGRPatients nLesions n sumLesions n median IQRSum of diameters prebaseline median IQRSum of diameters baseline median IQRSum of diameters at first evaluation median IQRREFperiod median IQREXPperiod median IQRREF TGR median IQREXP TGR median IQRTGRdiff median IQR accessWilcoxon p valueOverallIrradiated lesionsNon irradiated lesions to to to to to to to to to to to ˆ’ ˆ’ to to ˆ’ to ˆ’ ˆ’ to ˆ’ˆ’ ˆ’ to ˆ’ to to to ˆ’ ˆ’ to ˆ’ˆ’ ˆ’ to ˆ’REF TGR corresponds to TGR before the start of the treatment EXP TGR corresponds to TGR between the start of the treatment and the first evaluation at monthsof non irradiated lesions seemed more representative of patient outcomes vs irradiated lesion EXP TGRnon irr was significantly higher in patients with PD Although the number of patients was too low for statistical test p values are shown for information in table figure and online supplementary figure S4Immune analysesWe analyzed immune parameters in the blood that have been previously described to be changed during ipilimumab treatment to determine if radiotherapy could modify ipilimumab pharmacodynamics19 For CD4 T cell counts and as expected ipilimumab alone W4 could favor accumulation of TEM Treg and ICOSCD4 T cells Interestingly at W6 after ipilimumabradiotherapy only TEM and ICOSCD4 T cells remained significantly increased suggesting that combination favored accumulation of activated memory CD4 T cells rather than Treg cells For CD8 T cell counts no accumulation could be observed at W4 while augmentation of TCM and TEMRA could be depicted between W4 and W6 suggesting that adjunction of radiotherapy to ipilimumab was more prone to boost these CD8 T populations online supplementary figure S2 High fold change in CD8 from baseline to week was significantly correlated with PFS p00223 but not significantly correlated with overall survival p02355 online supplementary figure S5Innate immune cells and NLR absolute neutrophils count ANC divided by the number of lymphocytes or dNLR ANC divided by the number of white blood cellsˆ’ANC have been shown to have a prognostic role in patients treated with immunotherapy and even might represent a predictive biomarker of response We took advantage of standard blood tests to determine if Table Univariate analysis of tumor growth rate TGRAll lesions n37Progressive disease WilcoxonNop valueYesIrradiated lesion n13Progressive diseaseNoYesWilcoxon p valueNon irradiated lesions n24Progressive diseaseNoWilcoxon p valueYesSum of diameters prebaseline meanSum of diameters baseline meanSum of diameters at first evaluation meanREF TGR meanExp TGR meanΔTGR meanˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’TGR is evaluated in percentage per months ΔTGR EXP TGR “ REF TGR A negative value corresponds to a slowdown of the tumor growthBoutros a0C et a0al J Immunother Cancer 20208e000627 101136jitc2020000627 0c access neutrophils monocytes NLR or dNLR at baseline W4 and W6 could correlate with the prognosis in our study ANC or monocytes did not correlate with the prognosis of patients while absolute count of lymphocytes significantly increased at W6 compared with baseline in only patients with a clinical benefit CRPRSD Both NLR and dNLR were significantly lower at W6 only in patients with clinical benefit online supplementary figure S6 Note that we did not found an association with the dose of radiotherapy data not shownDISCUSSIONIn this dose escalation phase study four patients experienced DLTs All the DLTs occurred outside the radiation field Therefore it was difficult to assess the role of concurrent radiotherapy on the DLTs The MTD of radiotherapy combined with ipilimumab at mgkg was Gy The RPTD of radiotherapy administered in combination with ipilimumab at mgkg in patients with metastatic melanoma was Gy A hypofractionated radiation regimen higher doses per fraction was used in this study It is usually preferred for melanoma which displays low alphabeta ratio Our three fractions hypofractionated regimen is in line with the radiation standard in the metastatic setting Moreover it has been shown recently that radiation doses per session inferior to Gy combine more favorably with immunotherapy through interferon type induction21 Of note our study is the only one that combines radiotherapy and high dose of ipilimumab mgkgThe incidence of treatment related AEs was high with this combination in our study but numerically similar to the incidence reported previously with ipilimumab monotherapy at mgkg Grade or AEs occurred in of patients in our study whereas they occurred in “ of patients treated with ipilimumab monotherapy at mgkg2 The overall AE spectrum of the combination in this study was consistent with previous findings with the drug™s immune based mechanism of action The high rate of AEs might be partially attributed to the high dose of ipilimumab The dose of mgkg was chosen based on data from the randomized phase trial that compared various Answer:

Thyroid_Cancer

"Combination of thermally ablative focused ultrasound with gemcitabine controls breast cancer via adaptive immunityNatasha D Sheybani1 Alexandra R Witter2 Eric A Thim1 Hideo Yagita3 Timothy N J Bullock Richard J Price To cite Sheybani a0ND Witter a0AR Thim a0EA et a0al Combination of thermally ablative focused ultrasound with gemcitabine controls breast cancer via adaptive immunity Journal for ImmunoTherapy of Cancer 20208e001008 101136jitc2020001008 –º Additional material is published online only To view please visit the journal online http dx jitc NDS and ARW contributed equallyAccepted July Authors or their employers Re use permitted under CC BY NC No commercial re use See rights and permissions Published by BMJ1Biomedical Engineering University of Virginia Charlottesville Virginia USA2Pathology University of Virginia Charlottesville Virginia USA3Department of Immunology Juntendo University Graduate School of Medicine Bunkyo ku Tokyo Japan4Radiology Medical Imaging University of Virginia Charlottesville Virginia USACorrespondence toDr Richard J Price rprice virginia eduDr Timothy N J Bullock tb5v virginia eduBackground Triple negative breast cancer TNBC remains recalcitrant to most targeted therapy approaches However recent clinical studies suggest that inducing tumor damage can render TNBC responsive to immunotherapy We therefore tested a strategy for immune sensitization of murine TNBC 4T1 tumors through combination of focused ultrasound FUS thermal ablation and a chemotherapy gemcitabine GEM known to attenuate myeloid derived suppressor cells MDSCsMethods We applied a sparse scan thermally ablative FUS regimen at the tumor site in combination with systemically administered GEM We used flow cytometry analysis to investigate the roles of monotherapy and combinatorial therapy in mediating local and systemic immunity We also tested this combination in Rag1ˆ’ˆ’ mice or T cell depleted wild type mice to determine the essentiality of adaptive immunity Further we layered Programmed cell death protein PD1 blockade onto this combination to evaluate its impact on tumor outgrowth and survivalResults The immune modulatory effect of FUS monotherapy was insufficient to promote a robust T cell response against 4T1 consistent with the dominant MDSC driven immunosuppression evident in this model The combination of FUSGEM significantly constrained primary TNBC tumor outgrowth and extended overall survival of mice Tumor control correlated with increased circulating antigen experienced T cells and was entirely dependent on T cell mediated immunity The ability of FUSGEM to control primary tumor outgrowth was moderately enhanced by either neoadjuvant or adjuvant treatment with anti PD1Conclusion Thermally ablative FUS in combination with GEM restricts primary tumor outgrowth improves survival and enhances immunogenicity in a murine metastatic TNBC model This treatment strategy promises a novel option for potentiating the role of FUS in immunotherapy of metastatic TNBC and is worthy of future clinical evaluationTrial registration numbers NCT03237572 and NCT04116320BACKGROUNDMetastatic breast cancer BrCa particularly the triple negative breast cancer TNBC phenotype is resistant to most chemical and molecularly targeted therapeutic approaches Interestingly TNBC is often infiltrated with immune cells and the presence of these cells has been shown to have a favorable prognosis in patients treated with neoadjuvant chemotherapy1 Early studies in the use of immunotherapies targeting the PD1Programmed death ligand PD L1 checkpoint inhibitory axis showed some efficacy2“ in TNBC compared with other BrCa subtypes which are generally recalcitrant to checkpoint blockade Activity in the TNBC subtype may be related to the relatively high immune infiltration and correlated with the higher mutational burden observed in TNBC Greater immunotherapy efficacy in TNBC has been recently observed with the use of antibodies targeting the PD1PD L1 checkpoint inhibitory axis in combination with Nab paclitaxel5 This outcome suggests that inducing tumor damage augments antitumor immunity either by promoting antigen availability or disrupting the immunosuppressive tumor microenvironment TME found in TNBCAmong the potential networks in TNBC that could constrain the activity of antitumor immunity is the presence of immunosuppressive myeloid cell subsets These have the capacity to impair adaptive immunity and promote tumor growth and metastasis Among these cell types myeloid derived suppressor cells MDSCs prevail as a heterogeneous population of immature myeloid cells which serve the eponymous role of suppressing the antitumor immune response limiting both T cell activation and effector functions6 Increased levels of this cell type have been demonstrated in tumor tissues of patients with primary BrCa while those with metastatic disease bear the highest abundance of circulating MDSCs8 Studies have Sheybani a0ND et a0al J Immunother Cancer 20208e001008 101136jitc2020001008 0c access shown that approaches that either stimulate myeloid cells with inflammatory mediators or eliminate MDSC can improve antitumor immunity9“To this end the central premise put forth in this study is that focused ultrasound FUS”a safe noninvasive and nonionizing strategy for localized acoustic energy deposition into tissues”can synergize with immunotherapy in a murine model of metastatic TNBC FUS is capable of rapidly heating tumors to thermally ablative temperatures Its extracorporeal application obviates the need for catheterization injection or implantation FUS can be targeted with millimeter precision under MRI or ultrasound guidance thereby allowing for thermal damage and destruction of tumor tissue without compromising healthy intervening or peripheral tissues The bioeffects of FUS hold distinct implications for tumor antigenicity immune cell activation and trafficking13 Thermally active FUS regimes have elicited antitumor immune responses in implantable models of melanoma15 pancreatic16 prostate17“ colon20 kidney21 and BrCa23 Pertaining to the challenge of myeloid cell immunosuppression in TNBC thermally ablative FUS has been shown to induce the expression of heat shock proteins24 and proinflammatory cytokines including interleukin IL12 interferonÎ IFNÎ and tumor necrosis factorα TNFα from a variety of cancer cell lines and after in vivo treatment of tumors26 Whether the ability of FUS to induce these inflammatory mediators is sufficient to overcome myeloid suppression in the context of BrCa is currently under debate with some studies showing activation of antigen presenting cells and T cell recruitment in patients with BrCa treated with thermally ablative FUS28 while others show that additional innate stimuli are needed to support antitumor immunity23 Notably some studies have suggested that a sparse scan thermal ablation regimen more effectively recruits and activates dendritic cells DCs and antitumor immunity than total thermal ablation perhaps by limiting thermal denaturation of tumor antigens and innate stimuli31Based on the improved myeloid cell maturation that occurs with sparse scan regimens we herein tested the ability of a sparse scan partial thermal ablation FUS regimen as a monotherapy to promote antitumor immunity in an aggressive syngeneic model of metastatic murine TNBC with extensive granulocytic MDSC involvement that is recalcitrant to anti PD1 While some activity is evident with the partial ablation approach significantly greater control was achieved by targeting MDSC inhibition in combination with thermally ablative FUS This control was completely dependent on the adaptive immune responseMoreover we demonstrate that layering anti PD1 immune checkpoint blockade onto this combinatorial regimen moderately improves tumor growth restriction These data suggest that in disease settings where myeloid allied approaches to attenuate myeloid immunosuppression may be employed to reveal the full immunotherapeutic immunosuppression predominates potential of thermally ablative FUS Once immunosuppressive myeloid cells are accounted for FUS treatment can promote adaptive immunity that in turn potentiates immune checkpoint blockadeMETHODSCell line maintenance4T1 and E0771 cell lines were maintained in RPMI L glut or Dulbecco™s Modified Eagle™s Medium DMEM gL D glucose L glutamine respectively supplemented with Fetal Bovine Serum FBS at °C and CO2 Thawed cells were cultured for up to three passages and maintained in logarithmic growth phase for all experiments Cells tested negative for mycoplasmaEight week old to week old female BALBc or C57Bl6 mice were obtained from NCI Charles River NCI CRL or The Jackson Laboratory Female BALBc Rag1ˆ’ˆ’ mice were obtained from The Jackson Laboratory 4T1 or E0771 cells × were subcutaneously implanted into the right flank of mice Mice were housed on a hour12 hour lightdark cycle and supplied food ad libitum Tumor outgrowth was monitored via digital caliper measurements Tumor volume was calculated as follows volume length×width22 Approximately days 4T1 or days E0771 following tumor implantation mice were randomized into groups in a manner that ensured matching mean starting tumor volume across experimental groupsIn vivo ultrasoundguided FUS partial thermal ablationMice were treated with FUS either days 4T1 cohorts or days E0771 postimplantation On treatment day mice were anesthetized with intraperitoneal injection of ketamine mgkg Zoetis and dexdomitor mgkg Pfizer in sterilized saline Mouse flanks were shaved and depilated following which ultrasound guided FUS thermal ablation was performed using one of the two systems System and treatment details are provided in online supplementary materials and methods Mice that did not receive FUS treatment consistently underwent anesthesia and depilation of the flank Additionally these mice underwent a ˜sham™ treatment consisting of exposure to the °C degassed water bath exposure for min Following ˜sham™ or FUS treatment all mice were moved to a heating pad and given Antisedan for anesthesia reversal and recoveryGemcitabine therapyGemcitabine GEM mgmouse in µL volume Mylan diluted in saline and filter sterilized through a µm syringe filter was administered intraperitoneally once a week on the day of FUS treatment following which administration was repeated for an additional weeks Administration of GEM doses was based on existing literature demonstrating the use of GEM for inhibition of MDSCs in 4T112 The initial dose of GEM was administered immediately prior to ˜sham™ or FUS treatment Sheybani a0ND et a0al J Immunother Cancer 20208e001008 101136jitc2020001008 0cMice that did not receive GEM received an intraperitoneal injection of ˜vehicle™ treatment µL of sterile saline at the time points specifiedPD1 blockade therapyFor checkpoint inhibitor therapy the rat anti mouse PD1 antibody αPD1 RMP114 diluted in sterilized saline was administered intraperitoneally every days for a total of five doses µg per mouse Treatment was initiated on day ˜early αPD1™ or day ˜delayed αPD1™T cell depletionsT cell depletion antibodies”anti CD8 clone Bio X Cell and anti CD4 GK15 clone Bio X Cell”were diluted in sterilized saline and administered intraperitoneally every to days starting at day days post FUS for a total of seven doses µg of each antibody for a total µg per mouseImmunohistochemistryOn day sham or FUS exposed tumors were excised and fixed in neutral buffered formalin Sigma Fixed tumors were paraffin embedded sectioned and stained for hematoxylin and eosin Digital images of stained slides were acquired using the Vectra Automated Quantitative Pathology Imaging System Akoya Biosciences Whole slide screening and image capture were subsequently performed using Phenochart Akoya BiosciencesFlow cytometryMice were bled at days and via tail vein and samples were RBC lysed Hybri Max Sigma and stained for flow cytometry analysis At days post tumor implantation tissues were obtained from euthanized tumor bearing animals for immune response assessment In order to gain resolution into tissue resident versus vascular immune cell populations mice were injected intravenously with rat anti mouse CD45 FITC clone F11 BD Biosciences min prior to euthanasia 4T1 tumors spleens cardiac blood axillary and brachial tumor draining lymph nodes tumor DLNs pooled and nondraining inguinal lymph nodes were harvested processed and stained for flow cytometry analysis Additional details are provided in online supplementary materials and methodsSamples were acquired on an Attune NxT flow cytometer ThermoFisher Scientific and data were analyzed with FlowJo TreeStar or FCS Express De Novo Software A representative gating strategy for granulocytic myeloid derived suppressor cell G MDSC and CD44 T cells is provided in online supplementary figure Statistical analysisAll statistical analyses were performed in GraphPad Prism GraphPad Software A detailed description of statistical methods for each experiment is provided in the corresponding figure legend accessAnimal study approvalAll animal work was performed under a protocol approved by the Animal Care and Use Committee at the University of Virginia and conformed to the National Institutes of Health guidelines for the use of animals in researchRESULTSPartial thermal ablation of established TNBC tumors promotes peripheral DC activation but has limited impact on the presence of T cells and other myeloid cell subsetsTo achieve partial thermal ablation of 4T1 tumors we used an ultrasound guided FUS system equipped with a single element therapeutic transducer driven at MHz figure 1A online supplementary figure A grid of sonications was overlaid on the ultrasound visible tumor and ablated in a raster pattern under B mode ultrasound guidance figure 1B“C The exceptionally small focus of this system rendered a low ablation fraction “ of total tumor volume Immediately following ablation tumors displayed evidence of coagulative necrosis in the ablated zone with surrounding periablative margins figure 1D One week following FUS partial thermal ablation tumors and secondary lymphoid ans were excised for immunological characterization by flow cytometry figure 1B FUS partial thermal ablation of 4T1 tumors conferred a significant increase fold in the absolute number of CD11c hi DCs within the axillary tumor draining lymph node aDLN of mice figure 1E While this was accompanied by a nearly threefold elevation in the absolute number of CD86 DCs within the aDLN figure 1F the percentage of DCs expressing CD86 did not change figure 1G Increased numbers of DCs”and CD86 DCs in particular”suggest FUS is promoting the maturation or trafficking of these cells in the DLNs where they could encounter and activate T cells However this did not translate to tumor growth restriction data not shown We also did not observe significant differences in the absolute number of activated T cells in 4T1 tumors figure 1H or DLNs data not shown following FUS exposure suggesting limitations in the ability of FUS activated DC to further drive an antitumor T cell responseImmune profiling by flow cytometry revealed that irrespective of FUS exposure of the intratumoral CD45 immune cell population is comprised of CD11b myeloid cells figure 1I Similarly approximately of the circulating immune cell population in 4T1 tumor bearing mice is comprised of myeloid cells a striking fold elevation in circulating myeloid burden compared with naive mice online supplementary figure Notably Ly6G granulocytic myeloid derived suppressor cells G MDSCs significantly dominated the immune cell repertoire within 4T1 tumors relative to other myeloid including F480 macrophages Ly6C cell subsets monocytic myeloid derived suppressor cells M MDSCs and CD11c hi DCs figure 1J FUS partial thermal ablation did not significantly alter the absolute number per Sheybani a0ND et a0al J Immunother Cancer 20208e001008 101136jitc2020001008 0c access Figure Partial thermal ablation of established TNBC tumors promotes peripheral DC activation but has limited impact on the presence of T cells and other myeloid cell subsets A Design overview of a custom ultrasound guided FUS system consisting of a MHz single element transducer orthogonally co registered to an MHz linear ultrasound imaging array The tumor bearing flank of each anesthetized mouse was acoustically coupled to ultrasound transducers via degassed water bath maintained at °C ˜Sham™ mice were similarly positioned but did not undergo sonications B Schematic illustration of FUS partial thermal ablation scheme and study layout for evaluation of immune sequelae in 4T1 tumor bearing mice A grid of sonications was applied in a raster pattern onto the B mode ultrasound visible tumor In total two planes of sonication spaced mm apart were applied to each tumor Grid points were spaced mm apart within a single plane One week following thermal ablation tumors and secondary lymphoid ans were excised for sham n6 or FUS treated n5 mice and processed for flow cytometry C Representative B mode ultrasound images of ectopic 4T1 tumors either before top or during bottom FUS exposure Sonication grid depicting targets red points is superimposed on B mode image during treatment Subsequent to thermal ablation hyperechoic signatures yellow arrow are occasionally observed D Representative HE staining of either sham 4T1 tumors or those resected immediately following FUS partial thermal ablation Zoomed insets depict the transition from necrotic to intact tumor tissue within the periablative zone scale bars400 µm and µm on left and right inset respectively E Absolute number of CD11c hi DCs in the axillary tumor draining lymph node aDLN of 4T1 tumor bearing mice p00136 vs sham F Absolute number of CD86 CD11c hi DCs in the aDLN p00063 vs sham G Percentage of CD86 subset out of total CD11c hi DCs within aDLN H Absolute number of intratumoral CD44 CD8 and CD44 CD4 T cells and regulatory T cells Tregs per gram tumor I Percentage of CD11b myeloid cells out of total CD45 immune cells across tumor spleen aDLN inguinal DLN iDLN and nontumor draining axillary and inguinal LNs nDLNs p005 vs all other groups irrespective of FUS exposure specifically tumor vs spleen p00226 tumor spleen vs all other ans p00001 J Absolute number of intratumoral myeloid cells CD11c hi DCs F480 macrophages Ly6C monocytic myeloid derived suppressor cells M MDSCs Ly6G granulocytic myeloid derived suppressor cells G MDSCs per gram 4T1 tumor p00001 vs all other cell types irrespective of FUS exposure All data represented as mean±SEM Significance assessed by unpaired t test F“H or two way analysis of variance followed by Tukey multiple comparison correction I“K ˜ns™not significant DCs dendritic cells FUS focused ultrasound HIFU high intensityfocused ultrasoundSheybani a0ND et a0al J Immunother Cancer 20208e001008 101136jitc2020001008 0cgram tumor of these myeloid cell subsets These observations led us to formulate the hypothesis that widespread immunosuppressive mechanisms associated with the 4T1 TME must be addressed in order to facilitate the T cell response to FUSFUS partial thermal ablation in combination with GEM constrains primary TNBC tumor outgrowth and extends overall survivalOur observation of the overwhelming MDSC burden following 4T1 tumor implantation warranted implementation of an allied therapeutic strategy in order to counter this immunosuppressive barrier To this end we tested a combinatorial paradigm incorporating GEM a myelosuppressive chemotherapy demonstrated to inhibit MDSCs transiently in the 4T1 model without consequence to T cell phenotype or function12To evaluate the efficacy of FUS and GEM in combination we used a preclinical ultrasound guided FUS system to achieve partial thermal ablation of established 4T1 tumors 14d after tumor implantation average tumor volume of mm3 In combination with the single session of FUS thermal ablation we initiated GEM therapy mgmouse which was then readministered weekly for a total of three GEM doses figure 2A Combinatorial therapy synergized to produce significant constraint of 4T1 tumor outgrowth compared with sham and monotherapy groups figure 2B“CBy termination of treatments at day 4T1 tumors exposed to FUSGEM combination saw nearly × and × reductions in average volume compared with sham or GEM exposed tumors respectively figure 2B Two dimensional tumor projections at day postimplantation saw a nearly fold reduction in area from sham to combinatorial therapy setting figure 2D“E In a fraction of mice treated with FUSGEM we observed complete regression of 4T1 tumors although transient figure 2C tumor outgrowth eventually rebounded after termination of treatments 4T1 tumor bearing mice receiving FUSGEM treatment additionally saw the greatest extension in overall survival with and increases in median survival time compared with sham and GEM groups respectively HRs and for FUSGEM relative to sham and GEM groups respectively figure 2F We additionally observed that FUSGEM significantly constrained outgrowth in a separate C57Bl6 metastatic mammary carcinoma model E0771 online supplementary figure To further the clinical relevancy of these findings we applied this combinatorial strategy with the research grade analog of a clinical ultrasound guided FUS system Theraclion Echopulse that is already CE marked for applications in breast fibroadenoma thyroidparathyroid gland and varicose vein ablation and currently in use for multiple clinical trials leveraging FUS thermal ablation in combination with cancer immunotherapy We observed that partial thermal ablation using the Theraclion visualization and treatment unit MHz in combination accesswith GEM controlled 4T1 tumor outgrowth to a degree comparable with that observed with the custom in house system online supplementary figure These findings lend credence to the notion that the impact of combining GEM with FUS may be conserved across partial thermal ablation regimens Moreover they demonstrate that the efficacy of FUS partial thermal ablation in combination with GEM can be recapitulated on a system with a larger focus and in line image guidance that is currently in use clinicallyCombination of FUS partial thermal ablation with GEM increases the levels of circulating T cellsLymphocytes”in particular CD8 and CD4 T cells”play an important role in responding to tumor antigen and generating a durable antitumor response Based on the extended protective effect observed in mice treated with FUSGEM flow cytometry analysis was performed to evaluate the contribution of T cells in generating systemic and local tumor control We sampled the circulating immune cell repertoire in 4T1 tumor bearing mice via serial tail bleeds days and prior to readministration of GEM and a terminal cardiac bleed at the time of spleen harvest day figure 3A Combinatorial therapy significantly elevated absolute number of CD8 and CD4 T cells in the circulation at days and figure 3B“C and E“F Moreover a trend threefold to fivefold increase in circulating T cells was noted in the FUS group relative to sham figure 3B“C and E“F From days to systemic CD44 expressing antigen experienced T cell populations both CD8 and CD4 saw a steady significant increase after combinatorial therapy figure 3D and G A similar modest trend was noted for the FUS monotherapy group relative to sham and GEM figure 3D and G These changes were concordant with a decrease in circulating myeloid CD11b cells in GEM recipient groups demonstrating the ability of GEM to partially alleviate circulating myeloid burden figure 3HSplenomegaly is a common signature that arises in parallel with the leukemoid reaction to 4T1 tumors that is the expansion of immunosuppressive myeloid cells during tumor progression We observed that combinatorial therapy most significantly reverses splenomegaly online supplementary figure 6A“B Consistent with this observation immunological characterization of spleens revealed a significant decrease in CD11b myeloid cells”a “ reduction in FUSGEM spleens relative to sham or monotherapy figure 3I While there appeared to be a trend toward more CD11b cells in the monotherapy groups compared with the sham this difference was not significant and there was no difference between these groups in terms of absolute CD11b cell numbers within the spleen data not shown The decrease in myeloid cells in the combination treatment group was accompanied by a significant corresponding elevation in lymphocytes in the spleen following FUSGEM treatment Relative to these sham and GEM groups combination therapy elevated splenic CD8 T lymphocytes by fold and Sheybani a0ND et a0al J Immunother Cancer 20208e001008 101136jitc2020001008 0c access Figure Combination of focused ultrasound FUS partial thermal ablation with gemcitabine GEM constrains primary triple negative breast cancer outgrowth and extends overall survival A Overview of experimental design for evaluation combination of FUS with serial GEM treatment in murine mammary carcinoma B Average 4T1 tumor outgrowth in sham n7 FUS monotherapy n5 GEM monotherapy n10 and combinatorial FUSGEM therapy groups n10 Data are represented up to select time points corresponding with mouse dropout due to humane endpoints All data represented as mean±SEM Significance assessed on outgrowth up to day by repeated measures mixed effects model implementing restricted maximum likelihood method followed by Tukey multiple comparison correction p005 vs all other groups specifically sham vs FUSGEM p00001 FUS vs FUSGEM p00001 shamGEM vs FUSGEM p00026 C 4T1 tumor outgrowth from individual mice in sham FUS shamGEM or FUSGEM groups Data represent outgrowth from initiation of treatments at day up to removal of mouse from study for meeting a humane endpoint D Representative images of 4T1 tumors excised at day Scale bar1 cm E Quantification of 2D tumor areas from images in previous panel F Kaplan Meier curve depicting overall survival of sham treatment n9 FUS monotherapy n6 GEM monotherapy n10 and combinatorial FUSGEM therapy n10 recipient mice Significance assessed by log rank Mantel Cox test p005 vs all other groups specifically sham vs FUS p02154 sham vs FUSGEM p00001 sham vs shamGEM p00050 FUS vs FUSGEM p00021 FUS vs shamGEM p00312 FUSGEM vs shamGEM p00041Sheybani a0ND et a0al J Immunother Cancer 20208e001008 101136jitc2020001008 0c accessFigure Combination of focused ultrasound FUS partial thermal ablation with gemcitabine GEM increases the levels of circulating T cells A Overview of experimental design to understand the impact of FUS andor GEM treatment on circulating immune cells B“C Absolute number of circulating CD8 T cells at day B and day C D Percentage of circulating CD8 T cells expressing CD44 from days to E“F Absolute number of circulating CD4 T cells at day E and day F G Percentage of circulating CD4 T cells expressing CD44 from days to H Percentage of CD11b myeloid cells out of total CD45 immune cell in circulation from days to I“K Percentage of myeloid cells I CD8 T cells J and CD4 T cells K out of total CD452 immune cells All data represented as mean±SEM All data representative of sham n6“ FUS monotherapy n4“ GEM monotherapy n9 and combinatorial FUSGEM therapy n6“ groups Significance assessed by analysis of variance followed by Tukey multiple comparison correction for B C E F or Fisher™s least significant difference LSD without multiple comparisons correction for I“K Significance for D G and H assessed by repeated measures mixed effects model implementing restricted maximum likelihood method followed by Fisher™s LSD without multiple comparisons correction p005 vs all other groups unless otherwise indicated p001 p0001 vs groups indicatedSheybani a0ND et a0al J Immunother Cancer 20208e001008 101136jitc2020001008 0c access fold figure 3J and CD4 T lymphocytes by fold and fold figure 3K These elevations were accompanied by a modest increase in percentage of Foxp3 regulatory T cells Tregs online supplementary figure 6E Additionally increases in percentage of NK and B cells were noted twofold to fivefold online supplementary figure 6C“D These findings indicate that combinatorial therapy with FUSGEM promotes a systemic lymphocyte response that is discrete from the effects of either intervention alone which may account for reduced mortality associated with pulmonary metastasesCombinatorial FUSGEM therapy does not promote robust local antitumor T cell responsesGiven the robust systemic immune signatures within the blood and spleen following FUSGEM we assayed 4T1 tumors at a time point within the window of tumor growth restriction and subsequent to termination of treatments ie day to interrogate whether tumor control correlates with an increase in the effector functions of the intratumoral T cell response figure 4A Approximately hours prior to euthanasia mice received intravenous brefeldin A injection to inhibit cytokine secretion for subsequent intracellular cytokine staining by flow cytometry Immune characterization of tumors at days postimplantation”that is days subsequent to final GEM administration”revealed no significant changes in absolute number of antigen experienced CD44 CD8 or CD4 T lymphocytes figure 4B“C Moreover the polyfunctionality of these T cells as denoted by IFNÎ and granzyme B expression was not significantly altered figure 4D“E However intratumoral functional changes were noted in the myeloid compartment GEM monotherapy modestly increased IL 12p40 production by DCs fold but this was not conserved in the combinatorial therapy group figure 4F Moreover while FUS monotherapy generated a trend in elevated TNFα production by intratumoral G MDSCs GEM recipient groups saw a significant increase threefold relative to sham figure 4G These findings indicate that changes in the myeloid compartment in response to monotherapy and combination therapy may contribute to tumor control but are unlikely to drive the protective response entirely Interestingly intratumoral T cell representation correlates poorly with circulating lymphocytes suggesting a transitory immune response that either cannot be fully characterized at this time point or is hampered by additional modes of immunosuppressionProtection conferred by combination of FUS and GEM is dependent on adaptive immunitySince our findings revealed no obvious advantage or function of adaptive immunity in the local TME we next investigated the overarching role of the adaptive immune system in protection offered by combinatorial therapy with FUSGEM To this end we utilized an Rag1ˆ’ˆ’ model that is deficient in T and B cells to address the hypothesis that mature T andor B cells play a role in the observed response Wild type WT or Rag1ˆ’ˆ’ mice bearing 4T1 tumors were randomized into groups in a manner that preserved similarity in average initial tumor volumes Mice were subsequently treated with either GEM monotherapy or the combination of FUSGEM The tumor growth inhibition offered by FUSGEM was entirely lost in Rag1ˆ’ˆ’ mice relative to their WT counterparts with average 4T1 tumor volume in Rag1ˆ’ˆ’ mice being over fivefold higher than that of WT mice on terminati

cancer223

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: "Combination of thermally ablative focused ultrasound with gemcitabine controls breast cancer via adaptive immunityNatasha D Sheybani1 Alexandra R Witter2 Eric A Thim1 Hideo Yagita3 Timothy N J Bullock Richard J Price To cite Sheybani a0ND Witter a0AR Thim a0EA et a0al Combination of thermally ablative focused ultrasound with gemcitabine controls breast cancer via adaptive immunity Journal for ImmunoTherapy of Cancer 20208e001008 101136jitc2020001008 –º Additional material is published online only To view please visit the journal online http dx jitc NDS and ARW contributed equallyAccepted July Authors or their employers Re use permitted under CC BY NC No commercial re use See rights and permissions Published by BMJ1Biomedical Engineering University of Virginia Charlottesville Virginia USA2Pathology University of Virginia Charlottesville Virginia USA3Department of Immunology Juntendo University Graduate School of Medicine Bunkyo ku Tokyo Japan4Radiology Medical Imaging University of Virginia Charlottesville Virginia USACorrespondence toDr Richard J Price rprice virginia eduDr Timothy N J Bullock tb5v virginia eduBackground Triple negative breast cancer TNBC remains recalcitrant to most targeted therapy approaches However recent clinical studies suggest that inducing tumor damage can render TNBC responsive to immunotherapy We therefore tested a strategy for immune sensitization of murine TNBC 4T1 tumors through combination of focused ultrasound FUS thermal ablation and a chemotherapy gemcitabine GEM known to attenuate myeloid derived suppressor cells MDSCsMethods We applied a sparse scan thermally ablative FUS regimen at the tumor site in combination with systemically administered GEM We used flow cytometry analysis to investigate the roles of monotherapy and combinatorial therapy in mediating local and systemic immunity We also tested this combination in Rag1ˆ’ˆ’ mice or T cell depleted wild type mice to determine the essentiality of adaptive immunity Further we layered Programmed cell death protein PD1 blockade onto this combination to evaluate its impact on tumor outgrowth and survivalResults The immune modulatory effect of FUS monotherapy was insufficient to promote a robust T cell response against 4T1 consistent with the dominant MDSC driven immunosuppression evident in this model The combination of FUSGEM significantly constrained primary TNBC tumor outgrowth and extended overall survival of mice Tumor control correlated with increased circulating antigen experienced T cells and was entirely dependent on T cell mediated immunity The ability of FUSGEM to control primary tumor outgrowth was moderately enhanced by either neoadjuvant or adjuvant treatment with anti PD1Conclusion Thermally ablative FUS in combination with GEM restricts primary tumor outgrowth improves survival and enhances immunogenicity in a murine metastatic TNBC model This treatment strategy promises a novel option for potentiating the role of FUS in immunotherapy of metastatic TNBC and is worthy of future clinical evaluationTrial registration numbers NCT03237572 and NCT04116320BACKGROUNDMetastatic breast cancer BrCa particularly the triple negative breast cancer TNBC phenotype is resistant to most chemical and molecularly targeted therapeutic approaches Interestingly TNBC is often infiltrated with immune cells and the presence of these cells has been shown to have a favorable prognosis in patients treated with neoadjuvant chemotherapy1 Early studies in the use of immunotherapies targeting the PD1Programmed death ligand PD L1 checkpoint inhibitory axis showed some efficacy2“ in TNBC compared with other BrCa subtypes which are generally recalcitrant to checkpoint blockade Activity in the TNBC subtype may be related to the relatively high immune infiltration and correlated with the higher mutational burden observed in TNBC Greater immunotherapy efficacy in TNBC has been recently observed with the use of antibodies targeting the PD1PD L1 checkpoint inhibitory axis in combination with Nab paclitaxel5 This outcome suggests that inducing tumor damage augments antitumor immunity either by promoting antigen availability or disrupting the immunosuppressive tumor microenvironment TME found in TNBCAmong the potential networks in TNBC that could constrain the activity of antitumor immunity is the presence of immunosuppressive myeloid cell subsets These have the capacity to impair adaptive immunity and promote tumor growth and metastasis Among these cell types myeloid derived suppressor cells MDSCs prevail as a heterogeneous population of immature myeloid cells which serve the eponymous role of suppressing the antitumor immune response limiting both T cell activation and effector functions6 Increased levels of this cell type have been demonstrated in tumor tissues of patients with primary BrCa while those with metastatic disease bear the highest abundance of circulating MDSCs8 Studies have Sheybani a0ND et a0al J Immunother Cancer 20208e001008 101136jitc2020001008 0c access shown that approaches that either stimulate myeloid cells with inflammatory mediators or eliminate MDSC can improve antitumor immunity9“To this end the central premise put forth in this study is that focused ultrasound FUS”a safe noninvasive and nonionizing strategy for localized acoustic energy deposition into tissues”can synergize with immunotherapy in a murine model of metastatic TNBC FUS is capable of rapidly heating tumors to thermally ablative temperatures Its extracorporeal application obviates the need for catheterization injection or implantation FUS can be targeted with millimeter precision under MRI or ultrasound guidance thereby allowing for thermal damage and destruction of tumor tissue without compromising healthy intervening or peripheral tissues The bioeffects of FUS hold distinct implications for tumor antigenicity immune cell activation and trafficking13 Thermally active FUS regimes have elicited antitumor immune responses in implantable models of melanoma15 pancreatic16 prostate17“ colon20 kidney21 and BrCa23 Pertaining to the challenge of myeloid cell immunosuppression in TNBC thermally ablative FUS has been shown to induce the expression of heat shock proteins24 and proinflammatory cytokines including interleukin IL12 interferonÎ IFNÎ and tumor necrosis factorα TNFα from a variety of cancer cell lines and after in vivo treatment of tumors26 Whether the ability of FUS to induce these inflammatory mediators is sufficient to overcome myeloid suppression in the context of BrCa is currently under debate with some studies showing activation of antigen presenting cells and T cell recruitment in patients with BrCa treated with thermally ablative FUS28 while others show that additional innate stimuli are needed to support antitumor immunity23 Notably some studies have suggested that a sparse scan thermal ablation regimen more effectively recruits and activates dendritic cells DCs and antitumor immunity than total thermal ablation perhaps by limiting thermal denaturation of tumor antigens and innate stimuli31Based on the improved myeloid cell maturation that occurs with sparse scan regimens we herein tested the ability of a sparse scan partial thermal ablation FUS regimen as a monotherapy to promote antitumor immunity in an aggressive syngeneic model of metastatic murine TNBC with extensive granulocytic MDSC involvement that is recalcitrant to anti PD1 While some activity is evident with the partial ablation approach significantly greater control was achieved by targeting MDSC inhibition in combination with thermally ablative FUS This control was completely dependent on the adaptive immune responseMoreover we demonstrate that layering anti PD1 immune checkpoint blockade onto this combinatorial regimen moderately improves tumor growth restriction These data suggest that in disease settings where myeloid allied approaches to attenuate myeloid immunosuppression may be employed to reveal the full immunotherapeutic immunosuppression predominates potential of thermally ablative FUS Once immunosuppressive myeloid cells are accounted for FUS treatment can promote adaptive immunity that in turn potentiates immune checkpoint blockadeMETHODSCell line maintenance4T1 and E0771 cell lines were maintained in RPMI L glut or Dulbecco™s Modified Eagle™s Medium DMEM gL D glucose L glutamine respectively supplemented with Fetal Bovine Serum FBS at °C and CO2 Thawed cells were cultured for up to three passages and maintained in logarithmic growth phase for all experiments Cells tested negative for mycoplasmaEight week old to week old female BALBc or C57Bl6 mice were obtained from NCI Charles River NCI CRL or The Jackson Laboratory Female BALBc Rag1ˆ’ˆ’ mice were obtained from The Jackson Laboratory 4T1 or E0771 cells × were subcutaneously implanted into the right flank of mice Mice were housed on a hour12 hour lightdark cycle and supplied food ad libitum Tumor outgrowth was monitored via digital caliper measurements Tumor volume was calculated as follows volume length×width22 Approximately days 4T1 or days E0771 following tumor implantation mice were randomized into groups in a manner that ensured matching mean starting tumor volume across experimental groupsIn vivo ultrasoundguided FUS partial thermal ablationMice were treated with FUS either days 4T1 cohorts or days E0771 postimplantation On treatment day mice were anesthetized with intraperitoneal injection of ketamine mgkg Zoetis and dexdomitor mgkg Pfizer in sterilized saline Mouse flanks were shaved and depilated following which ultrasound guided FUS thermal ablation was performed using one of the two systems System and treatment details are provided in online supplementary materials and methods Mice that did not receive FUS treatment consistently underwent anesthesia and depilation of the flank Additionally these mice underwent a ˜sham™ treatment consisting of exposure to the °C degassed water bath exposure for min Following ˜sham™ or FUS treatment all mice were moved to a heating pad and given Antisedan for anesthesia reversal and recoveryGemcitabine therapyGemcitabine GEM mgmouse in µL volume Mylan diluted in saline and filter sterilized through a µm syringe filter was administered intraperitoneally once a week on the day of FUS treatment following which administration was repeated for an additional weeks Administration of GEM doses was based on existing literature demonstrating the use of GEM for inhibition of MDSCs in 4T112 The initial dose of GEM was administered immediately prior to ˜sham™ or FUS treatment Sheybani a0ND et a0al J Immunother Cancer 20208e001008 101136jitc2020001008 0cMice that did not receive GEM received an intraperitoneal injection of ˜vehicle™ treatment µL of sterile saline at the time points specifiedPD1 blockade therapyFor checkpoint inhibitor therapy the rat anti mouse PD1 antibody αPD1 RMP114 diluted in sterilized saline was administered intraperitoneally every days for a total of five doses µg per mouse Treatment was initiated on day ˜early αPD1™ or day ˜delayed αPD1™T cell depletionsT cell depletion antibodies”anti CD8 clone Bio X Cell and anti CD4 GK15 clone Bio X Cell”were diluted in sterilized saline and administered intraperitoneally every to days starting at day days post FUS for a total of seven doses µg of each antibody for a total µg per mouseImmunohistochemistryOn day sham or FUS exposed tumors were excised and fixed in neutral buffered formalin Sigma Fixed tumors were paraffin embedded sectioned and stained for hematoxylin and eosin Digital images of stained slides were acquired using the Vectra Automated Quantitative Pathology Imaging System Akoya Biosciences Whole slide screening and image capture were subsequently performed using Phenochart Akoya BiosciencesFlow cytometryMice were bled at days and via tail vein and samples were RBC lysed Hybri Max Sigma and stained for flow cytometry analysis At days post tumor implantation tissues were obtained from euthanized tumor bearing animals for immune response assessment In order to gain resolution into tissue resident versus vascular immune cell populations mice were injected intravenously with rat anti mouse CD45 FITC clone F11 BD Biosciences min prior to euthanasia 4T1 tumors spleens cardiac blood axillary and brachial tumor draining lymph nodes tumor DLNs pooled and nondraining inguinal lymph nodes were harvested processed and stained for flow cytometry analysis Additional details are provided in online supplementary materials and methodsSamples were acquired on an Attune NxT flow cytometer ThermoFisher Scientific and data were analyzed with FlowJo TreeStar or FCS Express De Novo Software A representative gating strategy for granulocytic myeloid derived suppressor cell G MDSC and CD44 T cells is provided in online supplementary figure Statistical analysisAll statistical analyses were performed in GraphPad Prism GraphPad Software A detailed description of statistical methods for each experiment is provided in the corresponding figure legend accessAnimal study approvalAll animal work was performed under a protocol approved by the Animal Care and Use Committee at the University of Virginia and conformed to the National Institutes of Health guidelines for the use of animals in researchRESULTSPartial thermal ablation of established TNBC tumors promotes peripheral DC activation but has limited impact on the presence of T cells and other myeloid cell subsetsTo achieve partial thermal ablation of 4T1 tumors we used an ultrasound guided FUS system equipped with a single element therapeutic transducer driven at MHz figure 1A online supplementary figure A grid of sonications was overlaid on the ultrasound visible tumor and ablated in a raster pattern under B mode ultrasound guidance figure 1B“C The exceptionally small focus of this system rendered a low ablation fraction “ of total tumor volume Immediately following ablation tumors displayed evidence of coagulative necrosis in the ablated zone with surrounding periablative margins figure 1D One week following FUS partial thermal ablation tumors and secondary lymphoid ans were excised for immunological characterization by flow cytometry figure 1B FUS partial thermal ablation of 4T1 tumors conferred a significant increase fold in the absolute number of CD11c hi DCs within the axillary tumor draining lymph node aDLN of mice figure 1E While this was accompanied by a nearly threefold elevation in the absolute number of CD86 DCs within the aDLN figure 1F the percentage of DCs expressing CD86 did not change figure 1G Increased numbers of DCs”and CD86 DCs in particular”suggest FUS is promoting the maturation or trafficking of these cells in the DLNs where they could encounter and activate T cells However this did not translate to tumor growth restriction data not shown We also did not observe significant differences in the absolute number of activated T cells in 4T1 tumors figure 1H or DLNs data not shown following FUS exposure suggesting limitations in the ability of FUS activated DC to further drive an antitumor T cell responseImmune profiling by flow cytometry revealed that irrespective of FUS exposure of the intratumoral CD45 immune cell population is comprised of CD11b myeloid cells figure 1I Similarly approximately of the circulating immune cell population in 4T1 tumor bearing mice is comprised of myeloid cells a striking fold elevation in circulating myeloid burden compared with naive mice online supplementary figure Notably Ly6G granulocytic myeloid derived suppressor cells G MDSCs significantly dominated the immune cell repertoire within 4T1 tumors relative to other myeloid including F480 macrophages Ly6C cell subsets monocytic myeloid derived suppressor cells M MDSCs and CD11c hi DCs figure 1J FUS partial thermal ablation did not significantly alter the absolute number per Sheybani a0ND et a0al J Immunother Cancer 20208e001008 101136jitc2020001008 0c access Figure Partial thermal ablation of established TNBC tumors promotes peripheral DC activation but has limited impact on the presence of T cells and other myeloid cell subsets A Design overview of a custom ultrasound guided FUS system consisting of a MHz single element transducer orthogonally co registered to an MHz linear ultrasound imaging array The tumor bearing flank of each anesthetized mouse was acoustically coupled to ultrasound transducers via degassed water bath maintained at °C ˜Sham™ mice were similarly positioned but did not undergo sonications B Schematic illustration of FUS partial thermal ablation scheme and study layout for evaluation of immune sequelae in 4T1 tumor bearing mice A grid of sonications was applied in a raster pattern onto the B mode ultrasound visible tumor In total two planes of sonication spaced mm apart were applied to each tumor Grid points were spaced mm apart within a single plane One week following thermal ablation tumors and secondary lymphoid ans were excised for sham n6 or FUS treated n5 mice and processed for flow cytometry C Representative B mode ultrasound images of ectopic 4T1 tumors either before top or during bottom FUS exposure Sonication grid depicting targets red points is superimposed on B mode image during treatment Subsequent to thermal ablation hyperechoic signatures yellow arrow are occasionally observed D Representative HE staining of either sham 4T1 tumors or those resected immediately following FUS partial thermal ablation Zoomed insets depict the transition from necrotic to intact tumor tissue within the periablative zone scale bars400 µm and µm on left and right inset respectively E Absolute number of CD11c hi DCs in the axillary tumor draining lymph node aDLN of 4T1 tumor bearing mice p00136 vs sham F Absolute number of CD86 CD11c hi DCs in the aDLN p00063 vs sham G Percentage of CD86 subset out of total CD11c hi DCs within aDLN H Absolute number of intratumoral CD44 CD8 and CD44 CD4 T cells and regulatory T cells Tregs per gram tumor I Percentage of CD11b myeloid cells out of total CD45 immune cells across tumor spleen aDLN inguinal DLN iDLN and nontumor draining axillary and inguinal LNs nDLNs p005 vs all other groups irrespective of FUS exposure specifically tumor vs spleen p00226 tumor spleen vs all other ans p00001 J Absolute number of intratumoral myeloid cells CD11c hi DCs F480 macrophages Ly6C monocytic myeloid derived suppressor cells M MDSCs Ly6G granulocytic myeloid derived suppressor cells G MDSCs per gram 4T1 tumor p00001 vs all other cell types irrespective of FUS exposure All data represented as mean±SEM Significance assessed by unpaired t test F“H or two way analysis of variance followed by Tukey multiple comparison correction I“K ˜ns™not significant DCs dendritic cells FUS focused ultrasound HIFU high intensityfocused ultrasoundSheybani a0ND et a0al J Immunother Cancer 20208e001008 101136jitc2020001008 0cgram tumor of these myeloid cell subsets These observations led us to formulate the hypothesis that widespread immunosuppressive mechanisms associated with the 4T1 TME must be addressed in order to facilitate the T cell response to FUSFUS partial thermal ablation in combination with GEM constrains primary TNBC tumor outgrowth and extends overall survivalOur observation of the overwhelming MDSC burden following 4T1 tumor implantation warranted implementation of an allied therapeutic strategy in order to counter this immunosuppressive barrier To this end we tested a combinatorial paradigm incorporating GEM a myelosuppressive chemotherapy demonstrated to inhibit MDSCs transiently in the 4T1 model without consequence to T cell phenotype or function12To evaluate the efficacy of FUS and GEM in combination we used a preclinical ultrasound guided FUS system to achieve partial thermal ablation of established 4T1 tumors 14d after tumor implantation average tumor volume of mm3 In combination with the single session of FUS thermal ablation we initiated GEM therapy mgmouse which was then readministered weekly for a total of three GEM doses figure 2A Combinatorial therapy synergized to produce significant constraint of 4T1 tumor outgrowth compared with sham and monotherapy groups figure 2B“CBy termination of treatments at day 4T1 tumors exposed to FUSGEM combination saw nearly × and × reductions in average volume compared with sham or GEM exposed tumors respectively figure 2B Two dimensional tumor projections at day postimplantation saw a nearly fold reduction in area from sham to combinatorial therapy setting figure 2D“E In a fraction of mice treated with FUSGEM we observed complete regression of 4T1 tumors although transient figure 2C tumor outgrowth eventually rebounded after termination of treatments 4T1 tumor bearing mice receiving FUSGEM treatment additionally saw the greatest extension in overall survival with and increases in median survival time compared with sham and GEM groups respectively HRs and for FUSGEM relative to sham and GEM groups respectively figure 2F We additionally observed that FUSGEM significantly constrained outgrowth in a separate C57Bl6 metastatic mammary carcinoma model E0771 online supplementary figure To further the clinical relevancy of these findings we applied this combinatorial strategy with the research grade analog of a clinical ultrasound guided FUS system Theraclion Echopulse that is already CE marked for applications in breast fibroadenoma thyroidparathyroid gland and varicose vein ablation and currently in use for multiple clinical trials leveraging FUS thermal ablation in combination with cancer immunotherapy We observed that partial thermal ablation using the Theraclion visualization and treatment unit MHz in combination accesswith GEM controlled 4T1 tumor outgrowth to a degree comparable with that observed with the custom in house system online supplementary figure These findings lend credence to the notion that the impact of combining GEM with FUS may be conserved across partial thermal ablation regimens Moreover they demonstrate that the efficacy of FUS partial thermal ablation in combination with GEM can be recapitulated on a system with a larger focus and in line image guidance that is currently in use clinicallyCombination of FUS partial thermal ablation with GEM increases the levels of circulating T cellsLymphocytes”in particular CD8 and CD4 T cells”play an important role in responding to tumor antigen and generating a durable antitumor response Based on the extended protective effect observed in mice treated with FUSGEM flow cytometry analysis was performed to evaluate the contribution of T cells in generating systemic and local tumor control We sampled the circulating immune cell repertoire in 4T1 tumor bearing mice via serial tail bleeds days and prior to readministration of GEM and a terminal cardiac bleed at the time of spleen harvest day figure 3A Combinatorial therapy significantly elevated absolute number of CD8 and CD4 T cells in the circulation at days and figure 3B“C and E“F Moreover a trend threefold to fivefold increase in circulating T cells was noted in the FUS group relative to sham figure 3B“C and E“F From days to systemic CD44 expressing antigen experienced T cell populations both CD8 and CD4 saw a steady significant increase after combinatorial therapy figure 3D and G A similar modest trend was noted for the FUS monotherapy group relative to sham and GEM figure 3D and G These changes were concordant with a decrease in circulating myeloid CD11b cells in GEM recipient groups demonstrating the ability of GEM to partially alleviate circulating myeloid burden figure 3HSplenomegaly is a common signature that arises in parallel with the leukemoid reaction to 4T1 tumors that is the expansion of immunosuppressive myeloid cells during tumor progression We observed that combinatorial therapy most significantly reverses splenomegaly online supplementary figure 6A“B Consistent with this observation immunological characterization of spleens revealed a significant decrease in CD11b myeloid cells”a “ reduction in FUSGEM spleens relative to sham or monotherapy figure 3I While there appeared to be a trend toward more CD11b cells in the monotherapy groups compared with the sham this difference was not significant and there was no difference between these groups in terms of absolute CD11b cell numbers within the spleen data not shown The decrease in myeloid cells in the combination treatment group was accompanied by a significant corresponding elevation in lymphocytes in the spleen following FUSGEM treatment Relative to these sham and GEM groups combination therapy elevated splenic CD8 T lymphocytes by fold and Sheybani a0ND et a0al J Immunother Cancer 20208e001008 101136jitc2020001008 0c access Figure Combination of focused ultrasound FUS partial thermal ablation with gemcitabine GEM constrains primary triple negative breast cancer outgrowth and extends overall survival A Overview of experimental design for evaluation combination of FUS with serial GEM treatment in murine mammary carcinoma B Average 4T1 tumor outgrowth in sham n7 FUS monotherapy n5 GEM monotherapy n10 and combinatorial FUSGEM therapy groups n10 Data are represented up to select time points corresponding with mouse dropout due to humane endpoints All data represented as mean±SEM Significance assessed on outgrowth up to day by repeated measures mixed effects model implementing restricted maximum likelihood method followed by Tukey multiple comparison correction p005 vs all other groups specifically sham vs FUSGEM p00001 FUS vs FUSGEM p00001 shamGEM vs FUSGEM p00026 C 4T1 tumor outgrowth from individual mice in sham FUS shamGEM or FUSGEM groups Data represent outgrowth from initiation of treatments at day up to removal of mouse from study for meeting a humane endpoint D Representative images of 4T1 tumors excised at day Scale bar1 cm E Quantification of 2D tumor areas from images in previous panel F Kaplan Meier curve depicting overall survival of sham treatment n9 FUS monotherapy n6 GEM monotherapy n10 and combinatorial FUSGEM therapy n10 recipient mice Significance assessed by log rank Mantel Cox test p005 vs all other groups specifically sham vs FUS p02154 sham vs FUSGEM p00001 sham vs shamGEM p00050 FUS vs FUSGEM p00021 FUS vs shamGEM p00312 FUSGEM vs shamGEM p00041Sheybani a0ND et a0al J Immunother Cancer 20208e001008 101136jitc2020001008 0c accessFigure Combination of focused ultrasound FUS partial thermal ablation with gemcitabine GEM increases the levels of circulating T cells A Overview of experimental design to understand the impact of FUS andor GEM treatment on circulating immune cells B“C Absolute number of circulating CD8 T cells at day B and day C D Percentage of circulating CD8 T cells expressing CD44 from days to E“F Absolute number of circulating CD4 T cells at day E and day F G Percentage of circulating CD4 T cells expressing CD44 from days to H Percentage of CD11b myeloid cells out of total CD45 immune cell in circulation from days to I“K Percentage of myeloid cells I CD8 T cells J and CD4 T cells K out of total CD452 immune cells All data represented as mean±SEM All data representative of sham n6“ FUS monotherapy n4“ GEM monotherapy n9 and combinatorial FUSGEM therapy n6“ groups Significance assessed by analysis of variance followed by Tukey multiple comparison correction for B C E F or Fisher™s least significant difference LSD without multiple comparisons correction for I“K Significance for D G and H assessed by repeated measures mixed effects model implementing restricted maximum likelihood method followed by Fisher™s LSD without multiple comparisons correction p005 vs all other groups unless otherwise indicated p001 p0001 vs groups indicatedSheybani a0ND et a0al J Immunother Cancer 20208e001008 101136jitc2020001008 0c access fold figure 3J and CD4 T lymphocytes by fold and fold figure 3K These elevations were accompanied by a modest increase in percentage of Foxp3 regulatory T cells Tregs online supplementary figure 6E Additionally increases in percentage of NK and B cells were noted twofold to fivefold online supplementary figure 6C“D These findings indicate that combinatorial therapy with FUSGEM promotes a systemic lymphocyte response that is discrete from the effects of either intervention alone which may account for reduced mortality associated with pulmonary metastasesCombinatorial FUSGEM therapy does not promote robust local antitumor T cell responsesGiven the robust systemic immune signatures within the blood and spleen following FUSGEM we assayed 4T1 tumors at a time point within the window of tumor growth restriction and subsequent to termination of treatments ie day to interrogate whether tumor control correlates with an increase in the effector functions of the intratumoral T cell response figure 4A Approximately hours prior to euthanasia mice received intravenous brefeldin A injection to inhibit cytokine secretion for subsequent intracellular cytokine staining by flow cytometry Immune characterization of tumors at days postimplantation”that is days subsequent to final GEM administration”revealed no significant changes in absolute number of antigen experienced CD44 CD8 or CD4 T lymphocytes figure 4B“C Moreover the polyfunctionality of these T cells as denoted by IFNÎ and granzyme B expression was not significantly altered figure 4D“E However intratumoral functional changes were noted in the myeloid compartment GEM monotherapy modestly increased IL 12p40 production by DCs fold but this was not conserved in the combinatorial therapy group figure 4F Moreover while FUS monotherapy generated a trend in elevated TNFα production by intratumoral G MDSCs GEM recipient groups saw a significant increase threefold relative to sham figure 4G These findings indicate that changes in the myeloid compartment in response to monotherapy and combination therapy may contribute to tumor control but are unlikely to drive the protective response entirely Interestingly intratumoral T cell representation correlates poorly with circulating lymphocytes suggesting a transitory immune response that either cannot be fully characterized at this time point or is hampered by additional modes of immunosuppressionProtection conferred by combination of FUS and GEM is dependent on adaptive immunitySince our findings revealed no obvious advantage or function of adaptive immunity in the local TME we next investigated the overarching role of the adaptive immune system in protection offered by combinatorial therapy with FUSGEM To this end we utilized an Rag1ˆ’ˆ’ model that is deficient in T and B cells to address the hypothesis that mature T andor B cells play a role in the observed response Wild type WT or Rag1ˆ’ˆ’ mice bearing 4T1 tumors were randomized into groups in a manner that preserved similarity in average initial tumor volumes Mice were subsequently treated with either GEM monotherapy or the combination of FUSGEM The tumor growth inhibition offered by FUSGEM was entirely lost in Rag1ˆ’ˆ’ mice relative to their WT counterparts with average 4T1 tumor volume in Rag1ˆ’ˆ’ mice being over fivefold higher than that of WT mice on terminati Answer:

Thyroid_Cancer

"rapeutic plasma exchange clears circulating soluble PD L1 and PD L1 positive extracellular vesicles Elizabeth Ann L Enninga2 Fabrice Lucien Matteoni3 Jacob J Orme Heather Dale4 Edwin Burgstaler4 Susan M Harrington3 Matthew K Ball4 Aaron S Mansfield1 Sean S Park5 Mathew S Block1 Svetomir N Markovic1 Yiyi Yan1 Haidong Dong3 Roxana S Dronca6 Jeffrey L Winters4To cite Orme a0JJ Enninga a0EAL Lucien Matteoni a0F et a0al Therapeutic plasma exchange clears circulating soluble PD L1 and PD L1 positive extracellular vesicles Journal for ImmunoTherapy of Cancer 20208e001113 101136jitc2020001113 –º Additional material is published online only To view please visit the journal online http dx jitc Accepted June Authors or their employers Re use permitted under CC BY NC No commercial re use See rights and permissions Published by BMJ1Division of Medical Oncology Mayo Clinic Rochester Minnesota USA2Department of Obstetrics and Gynecology Mayo Clinic Rochester Minnesota USA3Department of Urology Mayo Clinic Rochester Minnesota USA4Department of Laboratory Medicine and Pathology Mayo Clinic Rochester Minnesota USA5Department of Radiation Oncology Mayo Clinic Rochester Minnesota USA6Department of Hematology and Oncology Mayo Clinic Florida Jacksonville Florida USACorrespondence toDr Jacob J Orme orme jacob mayo eduBackground Trans acting programmed death ligand PD L1 derives from malignant cells in three known forms High levels of secreted splice variant PD L1 sPD L1 ADAM10ADAM17 shed sPD L1 and PD L1 positive extracellular vesicles evPD L1 each predict poor prognosis and limited response to PD L1 checkpoint inhibitors in cancer To our knowledge no clinical intervention has reduced any of these circulating forms of extracellular PD L1 Here we explore therapeutic plasma exchange TPE as a treatment to reduce circulating extracellular PD L1Results In patients with melanoma sPD L1 levels above ngmL predicted inferior overall survival In patients undergoing TPE for non malignant indications each TPE session removed a mean sPD L1 and evPD L1 detectable in plasma TPE also reduced total and ADAM10 positive extracellular vesiclesConclusion Here we report the first known clinical intervention to remove either sPD L1 or evPD L1 from plasma in vivo TPE reduces plasma sPD L1 and evPD L1 in vivo and may have a role in treatment with immunotherapy TPE may also prove useful in patients with other extracellular vesicle related conditionsINTRODUCTIONOvercoming initial or acquired resistance to programmed death ligand PD L1 immune checkpoint inhibitors is a major area of unmet need for many cancers1 Although the full scope of mechanisms of resistance to these therapies has yet to be determined different forms of tumor derived extracellular PD L1 have been linked to resistance in multiple clinical studies2“in Malignant cells produce trans acting extracellular PD L1 three distinct forms First tumor cells transcribe and secrete soluble PD L1 sPD L1 splice variants4 Second enzymes ADAM10 and ADAM17 shed sPD L1 ectodomain directly from the tumor cell surface6 Both forms of sPD L1 carry known homodimerization domains and can be detected by ELISA sPD L1 can outcompete PD L1 inhibitors kill CD8 T cells and limit the ability of healthy peripheral blood mononuclear cells to kill tumor cells in vitro6 In a third form tumors generate extracellular vesicles EVs bearing surface PD L18 PD L1 positive EVs evPD L1 exhibit similar properties to sPD L1 in systemic circulation9 Each type of trans acting extracellular PD L1 correlates with poor survival in multiple clinical trials online supplementary table While broad spectrum pharmacological inhibitors and genetic manipulation have been shown to reduce release of these forms of PD L1 in culture or animal models none are suitable for clinical use To date we know of no reported clinical intervention that safely and reliably eliminates any of these forms of immunosuppressive systemic extracellular PD L1Therapeutic plasma exchange TPE is a procedure in which blood is passed through an apheresis machine separating plasma from cellular components Removed plasma is discarded and replaced with either colloid eg albumin or crystalloid and colloid solutions Unlike dialysis which removes small ions by diffusion TPE removes plasma restricted substances like antibodies that are too large for rapid diffusion On average each TPE session removes approximately “ of large non cellular plasma restricted intravascular components10It is unknown whether sPD L1 approximately kDa or PD L1 positive EVs “ nm are plasma restricted non diffusing and unbound If so we hypothesized that TPE could efficiently remove these substances from patient blood Such an intervention could if effective improve response to immunotherapyOrme a0JJ et a0al J Immunother Cancer 20208e001113 101136jitc2020001113 0c access Figure Soluble programmed death ligand PD L1 suppresses antitumor immunity and predicts overall survival in patients with melanoma A A model of three known tumor derived extracellular PD L1 forms” evPD L1 ADAM10ADAM17 cleaved soluble PD L1 sPD L1 ectodomain and secreted splice variant sPD L1”that downregulate antitumor immunity and prevent response to PDL1 inhibition B A Kaplan Meier plot shows significantly worse overall survival for patients with melanoma exhibiting high ‰¥ ngmL versus low ngmL plasma sPD L1 levels p0005 C Patients with melanoma exhibited a higher mean plasma sPD L1 level ngmL in comparison to healthy controls ngmL p0001RESULTSsPDL1 levels predict overall survival in patients with melanomaEach form of extracellular PD L1 acts in trans as a systemic immunosuppressant through PD1 signaling figure 1A online supplementary table “ To confirm the clinical impact of plasma sPD L1 we measured sPD L1 levels in a retrospective cohort of patients with melanoma Exploratory analysis of overall survival OS determined a working cut off value of sPD L1 ‰¥ ngmL and baseline characteristics at the time of entry into study were similar online supplementary table Patients with high plasma sPD L1 levels experienced inferior median OS compared with patients with low plasma sPD L1 levels figure 1B vs months p0005 In comparison to healthy age matched controls patients with melanoma exhibited higher mean plasma sPD L1 figure 1C ngmL vs ngmL p0001 In a multivariate Cox proportional hazards analysis high sPD L1 prior to treatment predicted worse survival HR CI to p0025 when accounting for advanced age not significant sex not significant late stage p0002 and high serum LDH p001 online supplementary table TPE significantly reduces plasma sPDL1 levelsWe hypothesized that TPE may remove extracellular PD L1 in its various forms figure 2A To address this question we prospectively enrolled patients undergoing planned TPE figure 2B Twenty eight patients met inclusion criteria of which provided informed consent Baseline patient characteristics are in table One patient was excluded for biotin containing supplement use as biotin interferes with the established sPD L1 detection assay The remaining patients underwent plasma exchange and sample collection before and after the procedure as described Discarded plasma samples from the TPE device waste bag for each session were also collected sPD L1 was measured in each sample and most patients undergoing TPE exhibited sPD L1 levels above the clinically relevant ngmL cut off from the retrospective melanoma studyMost patients undergoing TPE did not have an active cancer diagnosis Baseline sPD L1 levels in all patients were compared with matched normal controls and patients with melanoma online supplementary fig and some patients exhibited sPD L1 above the clinically significant cut off level determined in the retrospective melanoma cohort Patients with high baseline sPD L1 levels were significantly more anemic than patients with lower baseline sPD L1 even when controlling for the higher number of female subjects in the high sPD L1 group female only mean Hgb vs p004 male only mean Hgb vs p003 Groups were otherwise similar TPE significantly reduced plasma sPD L1 levels in patients receiving albumin only ie no Orme a0JJ et a0al J Immunother Cancer 20208e001113 101136jitc2020001113 0c accessFigure Therapeutic plasma exchange TPE significantly reduces plasma soluble programmed death ligand sPD L1 levels A A model of the TPE procedure in which patient plasma is separated and replaced to extract non cellular substances confined to the plasma B A diagram of the present study in which patients undergo plasma exchange C All plasma levels of sPD L1 immediately prior to pre and after post TPE using albumin replacement fluid are plotted TPE significantly reduced sPD L1 levels in patient plasma by Wilcoxon signed rank test p00001 D In a typical timeline patient sPD L1 levels are reduced by each successive session of TPE gray bars See also table a0 online supplementary figures “FFP replacement fluid figure 2C p00001 Removed sPD L1 was detected in matching plasma samples from the TPE procedure waste bag Each TPE session removed a mean of detectable plasma sPD L1 mean regeneration of sPD L1 between sessions was table TPE sessions were usually separated by “ daysincluding sessions A representative individual patient treatment course showing sPD L1 reduction over four successive TPE sessions is also shown figure 2D All individual patient TPE courses involving donated human blood products eg fresh frozen plasma or FFP are shown in online supplementary fig Pre TPE and post TPE sPD L1 levels for all sessions are also shown online supplementary fig TPE significantly reduced plasma sPD L1 even when sessions requiring donated FFP were included p00001FFP is sometimes given during TPE for patients with increased risk of bleeding We observed that some patients receiving FFP with low baseline sPD L1 experienced rapid increases in sPD L1 levels after TPE presumably passively acquired from donor plasma as this was not observed in patients receiving albumin replacement alone sPD L1 was not detected in the discarded plasma from the procedure for these patients We observed a mild association between post FFP infusion rises in sPD L1 levels and the blood type of the recipient mainly in patients with O type blood Individuals with group Oˆ’ blood are universal recipients of FFP products and universal donors of cellular products due to a lack of ABO group antigens and the presence of preformed anti A and anti B antibodies respectively Recipients of FFP usually receive a mixture of compatible plasma from multiple donors To determine whether blood type in FFP donors is associated with FFP sPD L1 content we measured sPD L1 by ELISA in plasma from multiple FFP donors online supplementary fig O negative plasma donors showed higher sPD L1 levels than donors with most other blood typesTPE efficiently reduces plasma EV levels in vivoWe postulated that TPE may remove PD L1 positive EVs evPD L1 from patient blood To address this question we measured total EV levels and evPD L1 in each sample by flow cytometry We also determined the impact of TPE on platelet derived CD61 positive EVs one of the most abundant EV Orme a0JJ et a0al J Immunother Cancer 20208e001113 101136jitc2020001113 0c access Table Patient baseline characteristicsCharacteristicHigh sPDL1 n17Low sPDL1 n7StatisticStarting sPD L1Age yearsGender FActive cancer YesImmunotherapyNoneAtezolizumabPembrolizumabPlasma exchange indicationCNS demyelination myelitis MS NMO myelopathyImmune encephalitisMyasthenia gravisParaneoplastic syndrome encephalitis neuropathy pemphigusParaproteinemia Waldenström cryoglobulinemia kappa gammopathySusac syndromeTransplant rejection heart kidneyPre TPE white cell countPre TPE hemoglobinPre TPE creatinine to to to to to to to to to to F1223619 p0001F122035 p0558X2070 p0404 X20 p0967 X2288 p0237  X2288 p0315  F122078 p0385F122860 p0008F122382 p0063Patients undergoing therapeutic plasma exchange TPE are compared by starting soluble programmed death ligand sPD L1 level above or below survival cut off established in patients with melanoma ngmL For categorical variables n is given For continuous variables mean quartiles is givenKruskal Wallis PearsonCNS central nervous system MS multiple sclerosis NMO neuromyelitis opticasubpopulations in blood CD61 is a platelet marker and ADAM10 positive low density EVs ADAM10 has been implicated in exosome loading and pathogenesis11“TPE significantly reduced total plasma particle concentration figure 3A average per exchange p00001 TPE sessions requiring FFP or other human blood product were excluded from analysis leaving session pairs PD L1 positive evPD L1 and ADAM10 positive EVs were Table Soluble programmed death ligand sPD L1 reduction and regeneration per exchange Reduction per exchangen44Mean SDMedian min max Regeneration between exchangesMean SDMedian min maxRegeneration per cycle pgmLMean SDMedian min max ˆ’ n44 ˆ’ ˆ’38k 154kFor each exchange not requiring FFP percent sPD L1 reduction and regeneration between each exchange is calculated n44FFP fresh frozen plasmasignificantly reduced by TPE figure 3BC p0028 and p00001 respectively and were detected in waste plasma data not shown Each TPE session using albumin based replacement fluid with pre TPE levels above one million removed a mean of detectable PD L1 positive EVs from patients online supplementary table Platelet derived CD61 positive EVs while abundant were not significantly reduced by plasma exchange figure 3DIndividual patient courses showing total plasma PD L1 positive ADAM10 positive and CD61 positive EV levels before and after each TPE session are shown in online supplementary fig with exemplary nanoflow plots in online supplementary fig Three successive TPE sessions consistently depleted total PD L1 positive and ADAM10 positive but not CD61 positive EVs These trends were less pronounced when sessions in which patients received donor FFP were included online supplementary fig In normal control FFP donors blood type did not correlate with plasma EV concentrations online supplementary fig DISCUSSIONExtracellular PD L1”in the form of splice variant sPD L1 ADAM10ADAM17 cleaved sPD L1 ectodomain or evPD L1 positive EVs evPD L1”mediates resistance to PD L1 inhibitors4“ These forms are resistant to clinically tested Orme a0JJ et a0al J Immunother Cancer 20208e001113 101136jitc2020001113 0c accessFigure Plasma exchange efficiently reduces total programmed death ligand PD L1 positive and ADAM10 positive extracellular vesicle EV levels in vivo Plasma levels of total EVs immediately prior to pre and after post therapeutic plasma exchange TPE are plotted TPE significantly reduced A total p00001 B PD L1 positive p0028 and C ADAM10 positive p00001 but not D CD61 positive EVs p094 by Wilcoxon signed rank test See also online supplementary figures “ and online supplementary table combinations of chemotherapy and immunotherapies in vitro and in animal models and are associated with poor prognosis in many cancer types In the present study we found that TPE reliably reduces sPD L1 and evPD L1 This reduction was most pronounced over approximately three consecutive single plasma volume treatment sessions Given the dramatic reduction in sPD L1 and evPD L1 TPE may provide a novel approach to combating these mechanisms of resistanceWhile promising the present study was limited to patients receiving TPE mainly for non oncological indications over a short time horizon One patient in the study had melanoma receiving pembrolizumab and exhibited high pre TPE evPD L1 that was reduced on treatment Another patient had a uterine neuroendocrine tumor receiving atezolizumab and exhibited high pre TPE sPD L1 that was reduced on treatment The purpose of TPE in all cases however was to blunt paraneoplastic autoimmunity or treat some other coexisting autoimmune disorder”not the underlying malignancy Neither of these patients experienced improvement in their autoimmunity after TPE The source of sPD L1 and evPD L1 in these cases is uncertain as no assay currently differentiates tumor derived and non tumor derived PD L1 We observed that these forms of immunosuppressive extracellular PD L1 exist naturally although at lower levels in healthy subjects than in patients with cancer suggesting a potentially beneficial immunoregulatory role While we observed some regeneration for both sPD L1 and evPD L1 between TPE sessions it is unknown to what degree malignant cells may regenerate and maintain extracellular PD L1 homeostasis Relatedly it is uncertain how other plasma substances removed by TPE may affect response to immunotherapy or more broadly cancer immunity overall Nor is it known at what level sPD L1 andor PD L1 positive EV removal would become clinically relevant These facets will be tested in future studiesImmunotherapy resistance is widespread and costly In most instances PD L1 inhibitors such as pembrolizumab nivolumab atezolizumab durvalumab and avelumab are used in situations in which less than half of tumors are expected to respond Of patients that benefit many do not experience a sustained durable response These treatments represent a major investment the cost of PD L1 checkpoint blockade commonly reaches several hundred thousand dollars over the course of therapyTo our knowledge this is the first report of an intervention to achieve consistent rapid reduction in either sPD L1 or PD L1 positive EVs in a clinical setting TPE is safe and commonly prescribed Thus preimmunotherapy TPE may combat immunotherapy resistance In light of the heavy investment that anti PD L1 therapy entails the added cost of TPE in selected patients may be practical14 While the durability of extracellular PD L1 reduction in malignancy will be explored in future studies the present study suggests that this approach warrants further investigationOrme a0JJ et a0al J Immunother Cancer 20208e001113 101136jitc2020001113 0c access Beyond evPD L1 this is also to our knowledge the first known intervention to reliably deplete EVs in a clinical setting EVs have been implicated in oncogenesis and metastasis through miRNA carriage and direct protein signaling independent of PD L115 Beyond cancer EVs have also been implicated in autoimmunity17 agingneurodegeneration18 infection19 obesity20 and heart disease21 The selective removal of ADAM10 positive ie likely immune derived versus CD61 positive ie likely platelet derived EVs in this study suggests flexible selective EV depletion may be both possible and expedient in other indications The present study is only a proof of concept and additional exploratory studies in these areas are necessaryIn summary TPE reduces extracellular forms of PD L1 associated with PD L1 checkpoint inhibitor resistance Future studies will explore the potential role of TPE in improving cancer immunotherapyMETHODSRetrospective melanoma outcomes study designIn a retrospective analysis baseline blood samples from patients with melanoma prior to treatment in one of three clinical trials by the North Central Cancer Treatment Group N057e22 N077523 and N087924 between and were tested for sPD L1 of patients were diagnosed with cutaneous melanoma and none received immunotherapy treatments Blood from healthy volunteers undergoing blood donation at Mayo Clinic was also testedProspective TPE study designIn an investigator initiated label single center observational study adults undergoing TPE were approached from December through March In consenting subjects samples of whole blood immediately prior to TPE and on completion of the procedure were collected in ACD vacutainers BD In each case the first mL of blood was discarded to avoid contamination after which an mL sample was obtained in sequence Plasma was isolated by centrifugation A postprocedure blood sample was obtained after completion of the procedure In addition matching samples from discarded plasma from the procedure waste bag were collected Samples were obtained from up to four consecutive procedures for each patient If a patient underwent fewer than four TPE procedures samples were obtained from as many procedures as possiblePatients included were adults able to give consent and undergoing TPE for a variety of hematological neurological and renal diseases as indicated by published guidelines from the American Society for Apheresis ASFA or according to the medical judgment of the referring physicians25 Patients taking biotin supplements were excluded from the study due to biotin interference with the sPD L1 ELISA assay Procedures were performed using centrifugation based cell separators either the Fenwal Amicus Fresenius KABI USA LLC Lake Zurich Illinois USA or the Spectra Optia Terumo BCT Lakewood Colorado USA For each patient a single plasma volume was exchanged using either peripheral intravenous preferred or central lines for vascular access For this study due to the possibility of sPD L1 or PD L1 positive EVs present in donor plasma only TPE sessions using no donor plasma ie fresh frozen plasma FFP in the replacement fluid were included in calculations Anticoagulation consisted of either mL of acid citrate dextrose solution A ACD A or mL of ACD A with units of unfractionated heparin Anticoagulant to blood ratios were when ACD A was used and when ACD Aheparin was used Patients did not receive routine electrolyte replacement but mL of calcium gluconate was administered by slow intravenous push for signs and symptoms of hypocalcemia related to the ACD A anticoagulant in one patientELISAELISA was performed as previously published26 Both secreted splice variant and shed sPD L1 are reliably detected by this ELISA In brief paired mouse IgG2 monoclonal antibody clones H1A and B11 against extracellular human PD L1 were utilized in a capture detection plate assay using biotinylation and HRP streptavidin detection This assay is specific for sPD L1 and does not exhibit cross reactivity to other B7 H homologues nor to evPD L1 Concentrations were determined by optical density measurements along a known standard curve of recombinant human PD L1 ELISAs were performed by team members who were blinded to the identity of the samplesFlow cytometryFlow cytometry for EVs was performed as previously published27 In brief plasma samples were centrifuged twice at 2000g to deplete platelets Resultant platelet free plasma were analyzed using an A60 Micro Plus Nanoscale Flow Cytometer Apogee FlowSystems gating for mid intensity light angle light scatter and markers of interest Anti PD L1 Genentech atezolizumab ADAM10 RD Systems clone and CD61 BioLegend clone VI PL2 antibodies were conjugated to fluorophores Life Technologies Alexa647 PE phycoerythrin and Alexa488 and titrated prior to use Nanoscale flow cytometer calibration was performed using a standard reference bead mix as previously published Flow cytometry was performed by team members blinded to the identity of the samplesStatistical analysisAll statistical analyses were performed using R Statistical Software R Foundation Retrospective progression free survival was analyzed using Kaplan Meier and Cox proportional hazards modeling Optimal cut off values for sPD L1 levels were determined using the greyzoneSurv package for R Wilcoxon signed rank test was used to compare paired pre TPE and post TPE patient sample sPD L1 and EV levels as indicated Baseline clinical characteristics for the study were compared by Kruskal Wallis test for continuous variables and Pearson™s χ2 test for discrete variables as indicated Orme a0JJ et a0al J Immunother Cancer 20208e001113 101136jitc2020001113 0cOtherwise groups were compared by unpaired two sided Student™s t test Figures comprising box plots show quartile values and individual data points Mean values and CI are indicated in corresponding online supplementary figures and tables P was considered statistically significant In figures p values are denoted with with and with Twitter Jacob J Orme JakeOrmeMDPhDAcknowledgements Statistical guidance was provided generously by Nathan Foster of the Mayo Clinic Center for Clinical and Translational Science Some illustrations were created using Servier Medical Art templates which are licensed under a Creative Commons Attribution Unported License https smart servier com Additional illustrations were provided by Mayo Clinic Media Services The authors thank Daniel Summerfield MD MS for use of his likeness in Fig 2AContributors JO originated hypotheses designed the study oversaw experiments performed analyses and wrote the article EALE performed retrospective melanoma cohort analysis FL M performed nanoflow cytometry HD and EB oversaw and performed TPE study enrollment sample collectionprocessing and blinding SMH performed ELISAs MB AM SP MB SNM YY HD RD and JLW helped develop hypotheses provided clinical samples and reagents and contributed support and oversightFunding R21 5R21CA19787802 Role of Bim and soluble B7 H1 in monitoring T cell responses to anti PD1 therapy in melanoma HD and RD L30 CA23154101 Soluble B7H1 as a PD1 Checkpoint œRemote Control in Cancer JJO U10 CA180790 EE K12 CA090628 YY Richard M Schulze Family Foundation HD and RDCompeting interests Intellectual property has been filed addressing discoveries disclosed in this manuscript The authors report no other relevant conflicts of interestPatient consent for publication ObtainedEthics approval All research protocols involving human subjects were approved by Mayo Clinic™s Institutional Review Board and all human subjects gave written informed consentProvenance and peer review Not commissioned externally peer reviewedData availability statement Data are available in a public access repository All data will be available for download at the Science Framework at https osf io qtskd access This is an access article distributed in accordance with the Creative Commons Attribution Non Commercial CC BY NC license which permits others to distribute remix adapt build upon this work non commercially and license their derivative works on different terms provided the original work is properly cited appropriate credit is given any changes made indicated and the use is non commercial See http creativecommons licenses by nc ORCID iDJacob J a0Orme http orcid REFERENCES O'Donnell JS Long GV Scolyer RA et a0al Resistance to PD1PDL1 checkpoint inhibition Cancer Treat Rev “ Ando K Hamada K Watanabe M et a0al Plasma levels of soluble PD L1 correlate with tumor regression in patients with lung and gastric cancer treated with immune checkpoint inhibitors Anticancer Res “ Fan Y Che X Qu J et a0al Exosomal PD L1 retains immunosuppressive activity and is associated with gastric cancer prognosis Ann Surg Oncol “ Zhou J Mahoney KM Giobbie Hurder A et a0al Soluble PD L1 as a biomarker in malignant melanoma treated with checkpoint blockade Cancer Immunol Res “ access Mahoney KMet a0al œA secreted PD L1 splice variant that covalently dimerizes and mediates immunosuppression Cancer Immunol Immunother “ Orme JJ Jazieh KA Xie T et a0al ADAM10 and ADAM17 cleave PD L1 to mediate PD L1 inhibitor resistance OncoImmunology Romero Y Wise R Zolkiewska A Proteolytic processing of PD L1 by ADAM proteases in breast cancer cells Cancer Immunol Immunother “ Chen G Huang AC Zhang W et a0al Exosomal PD L1 contributes to immunosuppression and is associated with anti PD1 response Nature “ Poggio M Hu T Pai C C et a0al Suppression of exosomal PD L1 induces systemic anti tumor immunity and memory Cell “ Derksen RH Schuurman HJ Meyling FH et a0al The efficacy of plasma exchange in the removal of plasma components J Lab Clin Med “ Berckmans RJ Nieuwland R Böing AN et a0al Cell Derived microparticles circulate in healthy humans and support low grade thrombin generation Thromb Haemost “ Crescitelli R Lässer C Jang SC et a0al Subpopulations of extracellular vesicles from human metastatic melanoma tissue identified by quantitative proteomics after optimized isolation J Extracell Vesicles Kowal J Arras G Colombo M et a0al Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes Proc Natl Acad Sci U S A 2016113E968“ Winters JL Brown D Hazard E et a0al Cost minimization analysis of the direct costs of tpe and IVIg in the treatment of Guillain Barré syndrome BMC Health Serv Res “ Lee JC Zhao J T Gundara J et a0al Papillary thyroid cancer derived exosomes contain miRNA 146b and miRNA222 J Surg Res “ Yang J Wei F Schafer C et a0al Detection of tumor cell specific mRNA and protein in exosome like microvesicles from blood and saliva PLoS One 20149e110641 Nakao R Hasegawa H Ochiai K et a0al Outer membrane vesicles of Porphyromonas gingivalis elicit a mucosal immune response PLoS One 20116e26163 Thompson AGet a0al œExtracellular vesicles in neurodegenerative disease pathogenesis to biomarkers Nature Reviews Neurology Nature Publishing Group “ Marcilla A Martin Jaular L Trelis M et a0al Extracellular vesicles in parasitic diseases J Extracell Vesicles Huang Doran I Zhang CY Vidal Puig A œExtracellular Vesicles Novel Mediators of Cell Communication In Metabolic Disease Trends in Endocrinology and Metabolism Elsevier Inc “ Boulanger CM Loyer X Rautou PE et a0al œExtracellular vesicles in coronary artery disease Nature Reviews Cardiology Nature Publishing Group “ Kottschade LA Suman VJ Amatruda T et a0al A phase II trial of nab paclitaxel ABI007 and carboplatin in patients with unresectable stage IV melanoma a North Central Cancer Treatment Group Study N057E1 Cancer “ Kottschade LA Suman VJ Perez DG et a0al A randomized phase study of temozolomide and bevacizumab or nab paclitaxel carboplatin and bevacizumab in patients with unresectable stage IV melanoma a North Central Cancer Treatment Group study N0775 Cancer “ McWilliams RR Allred JB Slostad JA et a0al NCCTG N0879 Alliance A randomized phase cooperative group trial of carboplatin paclitaxel and bevacizumab ± everolimus for metastatic melanoma Cancer “ Padmanabhan A Connelly Smith L Aqui N et a0al Guidelines on the Use of Therapeutic Apheresis in Clinical Practice Evidence Based Approach from the Writing Committee of the American Society for Apheresis The Eighth Special Issue J Clin Apher “ Frigola X Inman BA Lohse CM et a0al Identification of a soluble form of B7 H1 that retains immunosuppressive activity and is associated with aggressive renal cell carcinoma Clin Cancer Res “ Gomes J Lucien F Cooper TT et a0al Analytical considerations in nanoscale flow cytometry of extracellular vesicles to achieve data linearity Thromb Haemost “Orme a0JJ et a0al J Immunother Cancer 20208e001113 101136jitc2020001113 0c"

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You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: "rapeutic plasma exchange clears circulating soluble PD L1 and PD L1 positive extracellular vesicles Elizabeth Ann L Enninga2 Fabrice Lucien Matteoni3 Jacob J Orme Heather Dale4 Edwin Burgstaler4 Susan M Harrington3 Matthew K Ball4 Aaron S Mansfield1 Sean S Park5 Mathew S Block1 Svetomir N Markovic1 Yiyi Yan1 Haidong Dong3 Roxana S Dronca6 Jeffrey L Winters4To cite Orme a0JJ Enninga a0EAL Lucien Matteoni a0F et a0al Therapeutic plasma exchange clears circulating soluble PD L1 and PD L1 positive extracellular vesicles Journal for ImmunoTherapy of Cancer 20208e001113 101136jitc2020001113 –º Additional material is published online only To view please visit the journal online http dx jitc Accepted June Authors or their employers Re use permitted under CC BY NC No commercial re use See rights and permissions Published by BMJ1Division of Medical Oncology Mayo Clinic Rochester Minnesota USA2Department of Obstetrics and Gynecology Mayo Clinic Rochester Minnesota USA3Department of Urology Mayo Clinic Rochester Minnesota USA4Department of Laboratory Medicine and Pathology Mayo Clinic Rochester Minnesota USA5Department of Radiation Oncology Mayo Clinic Rochester Minnesota USA6Department of Hematology and Oncology Mayo Clinic Florida Jacksonville Florida USACorrespondence toDr Jacob J Orme orme jacob mayo eduBackground Trans acting programmed death ligand PD L1 derives from malignant cells in three known forms High levels of secreted splice variant PD L1 sPD L1 ADAM10ADAM17 shed sPD L1 and PD L1 positive extracellular vesicles evPD L1 each predict poor prognosis and limited response to PD L1 checkpoint inhibitors in cancer To our knowledge no clinical intervention has reduced any of these circulating forms of extracellular PD L1 Here we explore therapeutic plasma exchange TPE as a treatment to reduce circulating extracellular PD L1Results In patients with melanoma sPD L1 levels above ngmL predicted inferior overall survival In patients undergoing TPE for non malignant indications each TPE session removed a mean sPD L1 and evPD L1 detectable in plasma TPE also reduced total and ADAM10 positive extracellular vesiclesConclusion Here we report the first known clinical intervention to remove either sPD L1 or evPD L1 from plasma in vivo TPE reduces plasma sPD L1 and evPD L1 in vivo and may have a role in treatment with immunotherapy TPE may also prove useful in patients with other extracellular vesicle related conditionsINTRODUCTIONOvercoming initial or acquired resistance to programmed death ligand PD L1 immune checkpoint inhibitors is a major area of unmet need for many cancers1 Although the full scope of mechanisms of resistance to these therapies has yet to be determined different forms of tumor derived extracellular PD L1 have been linked to resistance in multiple clinical studies2“in Malignant cells produce trans acting extracellular PD L1 three distinct forms First tumor cells transcribe and secrete soluble PD L1 sPD L1 splice variants4 Second enzymes ADAM10 and ADAM17 shed sPD L1 ectodomain directly from the tumor cell surface6 Both forms of sPD L1 carry known homodimerization domains and can be detected by ELISA sPD L1 can outcompete PD L1 inhibitors kill CD8 T cells and limit the ability of healthy peripheral blood mononuclear cells to kill tumor cells in vitro6 In a third form tumors generate extracellular vesicles EVs bearing surface PD L18 PD L1 positive EVs evPD L1 exhibit similar properties to sPD L1 in systemic circulation9 Each type of trans acting extracellular PD L1 correlates with poor survival in multiple clinical trials online supplementary table While broad spectrum pharmacological inhibitors and genetic manipulation have been shown to reduce release of these forms of PD L1 in culture or animal models none are suitable for clinical use To date we know of no reported clinical intervention that safely and reliably eliminates any of these forms of immunosuppressive systemic extracellular PD L1Therapeutic plasma exchange TPE is a procedure in which blood is passed through an apheresis machine separating plasma from cellular components Removed plasma is discarded and replaced with either colloid eg albumin or crystalloid and colloid solutions Unlike dialysis which removes small ions by diffusion TPE removes plasma restricted substances like antibodies that are too large for rapid diffusion On average each TPE session removes approximately “ of large non cellular plasma restricted intravascular components10It is unknown whether sPD L1 approximately kDa or PD L1 positive EVs “ nm are plasma restricted non diffusing and unbound If so we hypothesized that TPE could efficiently remove these substances from patient blood Such an intervention could if effective improve response to immunotherapyOrme a0JJ et a0al J Immunother Cancer 20208e001113 101136jitc2020001113 0c access Figure Soluble programmed death ligand PD L1 suppresses antitumor immunity and predicts overall survival in patients with melanoma A A model of three known tumor derived extracellular PD L1 forms” evPD L1 ADAM10ADAM17 cleaved soluble PD L1 sPD L1 ectodomain and secreted splice variant sPD L1”that downregulate antitumor immunity and prevent response to PDL1 inhibition B A Kaplan Meier plot shows significantly worse overall survival for patients with melanoma exhibiting high ‰¥ ngmL versus low ngmL plasma sPD L1 levels p0005 C Patients with melanoma exhibited a higher mean plasma sPD L1 level ngmL in comparison to healthy controls ngmL p0001RESULTSsPDL1 levels predict overall survival in patients with melanomaEach form of extracellular PD L1 acts in trans as a systemic immunosuppressant through PD1 signaling figure 1A online supplementary table “ To confirm the clinical impact of plasma sPD L1 we measured sPD L1 levels in a retrospective cohort of patients with melanoma Exploratory analysis of overall survival OS determined a working cut off value of sPD L1 ‰¥ ngmL and baseline characteristics at the time of entry into study were similar online supplementary table Patients with high plasma sPD L1 levels experienced inferior median OS compared with patients with low plasma sPD L1 levels figure 1B vs months p0005 In comparison to healthy age matched controls patients with melanoma exhibited higher mean plasma sPD L1 figure 1C ngmL vs ngmL p0001 In a multivariate Cox proportional hazards analysis high sPD L1 prior to treatment predicted worse survival HR CI to p0025 when accounting for advanced age not significant sex not significant late stage p0002 and high serum LDH p001 online supplementary table TPE significantly reduces plasma sPDL1 levelsWe hypothesized that TPE may remove extracellular PD L1 in its various forms figure 2A To address this question we prospectively enrolled patients undergoing planned TPE figure 2B Twenty eight patients met inclusion criteria of which provided informed consent Baseline patient characteristics are in table One patient was excluded for biotin containing supplement use as biotin interferes with the established sPD L1 detection assay The remaining patients underwent plasma exchange and sample collection before and after the procedure as described Discarded plasma samples from the TPE device waste bag for each session were also collected sPD L1 was measured in each sample and most patients undergoing TPE exhibited sPD L1 levels above the clinically relevant ngmL cut off from the retrospective melanoma studyMost patients undergoing TPE did not have an active cancer diagnosis Baseline sPD L1 levels in all patients were compared with matched normal controls and patients with melanoma online supplementary fig and some patients exhibited sPD L1 above the clinically significant cut off level determined in the retrospective melanoma cohort Patients with high baseline sPD L1 levels were significantly more anemic than patients with lower baseline sPD L1 even when controlling for the higher number of female subjects in the high sPD L1 group female only mean Hgb vs p004 male only mean Hgb vs p003 Groups were otherwise similar TPE significantly reduced plasma sPD L1 levels in patients receiving albumin only ie no Orme a0JJ et a0al J Immunother Cancer 20208e001113 101136jitc2020001113 0c accessFigure Therapeutic plasma exchange TPE significantly reduces plasma soluble programmed death ligand sPD L1 levels A A model of the TPE procedure in which patient plasma is separated and replaced to extract non cellular substances confined to the plasma B A diagram of the present study in which patients undergo plasma exchange C All plasma levels of sPD L1 immediately prior to pre and after post TPE using albumin replacement fluid are plotted TPE significantly reduced sPD L1 levels in patient plasma by Wilcoxon signed rank test p00001 D In a typical timeline patient sPD L1 levels are reduced by each successive session of TPE gray bars See also table a0 online supplementary figures “FFP replacement fluid figure 2C p00001 Removed sPD L1 was detected in matching plasma samples from the TPE procedure waste bag Each TPE session removed a mean of detectable plasma sPD L1 mean regeneration of sPD L1 between sessions was table TPE sessions were usually separated by “ daysincluding sessions A representative individual patient treatment course showing sPD L1 reduction over four successive TPE sessions is also shown figure 2D All individual patient TPE courses involving donated human blood products eg fresh frozen plasma or FFP are shown in online supplementary fig Pre TPE and post TPE sPD L1 levels for all sessions are also shown online supplementary fig TPE significantly reduced plasma sPD L1 even when sessions requiring donated FFP were included p00001FFP is sometimes given during TPE for patients with increased risk of bleeding We observed that some patients receiving FFP with low baseline sPD L1 experienced rapid increases in sPD L1 levels after TPE presumably passively acquired from donor plasma as this was not observed in patients receiving albumin replacement alone sPD L1 was not detected in the discarded plasma from the procedure for these patients We observed a mild association between post FFP infusion rises in sPD L1 levels and the blood type of the recipient mainly in patients with O type blood Individuals with group Oˆ’ blood are universal recipients of FFP products and universal donors of cellular products due to a lack of ABO group antigens and the presence of preformed anti A and anti B antibodies respectively Recipients of FFP usually receive a mixture of compatible plasma from multiple donors To determine whether blood type in FFP donors is associated with FFP sPD L1 content we measured sPD L1 by ELISA in plasma from multiple FFP donors online supplementary fig O negative plasma donors showed higher sPD L1 levels than donors with most other blood typesTPE efficiently reduces plasma EV levels in vivoWe postulated that TPE may remove PD L1 positive EVs evPD L1 from patient blood To address this question we measured total EV levels and evPD L1 in each sample by flow cytometry We also determined the impact of TPE on platelet derived CD61 positive EVs one of the most abundant EV Orme a0JJ et a0al J Immunother Cancer 20208e001113 101136jitc2020001113 0c access Table Patient baseline characteristicsCharacteristicHigh sPDL1 n17Low sPDL1 n7StatisticStarting sPD L1Age yearsGender FActive cancer YesImmunotherapyNoneAtezolizumabPembrolizumabPlasma exchange indicationCNS demyelination myelitis MS NMO myelopathyImmune encephalitisMyasthenia gravisParaneoplastic syndrome encephalitis neuropathy pemphigusParaproteinemia Waldenström cryoglobulinemia kappa gammopathySusac syndromeTransplant rejection heart kidneyPre TPE white cell countPre TPE hemoglobinPre TPE creatinine to to to to to to to to to to F1223619 p0001F122035 p0558X2070 p0404 X20 p0967 X2288 p0237  X2288 p0315  F122078 p0385F122860 p0008F122382 p0063Patients undergoing therapeutic plasma exchange TPE are compared by starting soluble programmed death ligand sPD L1 level above or below survival cut off established in patients with melanoma ngmL For categorical variables n is given For continuous variables mean quartiles is givenKruskal Wallis PearsonCNS central nervous system MS multiple sclerosis NMO neuromyelitis opticasubpopulations in blood CD61 is a platelet marker and ADAM10 positive low density EVs ADAM10 has been implicated in exosome loading and pathogenesis11“TPE significantly reduced total plasma particle concentration figure 3A average per exchange p00001 TPE sessions requiring FFP or other human blood product were excluded from analysis leaving session pairs PD L1 positive evPD L1 and ADAM10 positive EVs were Table Soluble programmed death ligand sPD L1 reduction and regeneration per exchange Reduction per exchangen44Mean SDMedian min max Regeneration between exchangesMean SDMedian min maxRegeneration per cycle pgmLMean SDMedian min max ˆ’ n44 ˆ’ ˆ’38k 154kFor each exchange not requiring FFP percent sPD L1 reduction and regeneration between each exchange is calculated n44FFP fresh frozen plasmasignificantly reduced by TPE figure 3BC p0028 and p00001 respectively and were detected in waste plasma data not shown Each TPE session using albumin based replacement fluid with pre TPE levels above one million removed a mean of detectable PD L1 positive EVs from patients online supplementary table Platelet derived CD61 positive EVs while abundant were not significantly reduced by plasma exchange figure 3DIndividual patient courses showing total plasma PD L1 positive ADAM10 positive and CD61 positive EV levels before and after each TPE session are shown in online supplementary fig with exemplary nanoflow plots in online supplementary fig Three successive TPE sessions consistently depleted total PD L1 positive and ADAM10 positive but not CD61 positive EVs These trends were less pronounced when sessions in which patients received donor FFP were included online supplementary fig In normal control FFP donors blood type did not correlate with plasma EV concentrations online supplementary fig DISCUSSIONExtracellular PD L1”in the form of splice variant sPD L1 ADAM10ADAM17 cleaved sPD L1 ectodomain or evPD L1 positive EVs evPD L1”mediates resistance to PD L1 inhibitors4“ These forms are resistant to clinically tested Orme a0JJ et a0al J Immunother Cancer 20208e001113 101136jitc2020001113 0c accessFigure Plasma exchange efficiently reduces total programmed death ligand PD L1 positive and ADAM10 positive extracellular vesicle EV levels in vivo Plasma levels of total EVs immediately prior to pre and after post therapeutic plasma exchange TPE are plotted TPE significantly reduced A total p00001 B PD L1 positive p0028 and C ADAM10 positive p00001 but not D CD61 positive EVs p094 by Wilcoxon signed rank test See also online supplementary figures “ and online supplementary table combinations of chemotherapy and immunotherapies in vitro and in animal models and are associated with poor prognosis in many cancer types In the present study we found that TPE reliably reduces sPD L1 and evPD L1 This reduction was most pronounced over approximately three consecutive single plasma volume treatment sessions Given the dramatic reduction in sPD L1 and evPD L1 TPE may provide a novel approach to combating these mechanisms of resistanceWhile promising the present study was limited to patients receiving TPE mainly for non oncological indications over a short time horizon One patient in the study had melanoma receiving pembrolizumab and exhibited high pre TPE evPD L1 that was reduced on treatment Another patient had a uterine neuroendocrine tumor receiving atezolizumab and exhibited high pre TPE sPD L1 that was reduced on treatment The purpose of TPE in all cases however was to blunt paraneoplastic autoimmunity or treat some other coexisting autoimmune disorder”not the underlying malignancy Neither of these patients experienced improvement in their autoimmunity after TPE The source of sPD L1 and evPD L1 in these cases is uncertain as no assay currently differentiates tumor derived and non tumor derived PD L1 We observed that these forms of immunosuppressive extracellular PD L1 exist naturally although at lower levels in healthy subjects than in patients with cancer suggesting a potentially beneficial immunoregulatory role While we observed some regeneration for both sPD L1 and evPD L1 between TPE sessions it is unknown to what degree malignant cells may regenerate and maintain extracellular PD L1 homeostasis Relatedly it is uncertain how other plasma substances removed by TPE may affect response to immunotherapy or more broadly cancer immunity overall Nor is it known at what level sPD L1 andor PD L1 positive EV removal would become clinically relevant These facets will be tested in future studiesImmunotherapy resistance is widespread and costly In most instances PD L1 inhibitors such as pembrolizumab nivolumab atezolizumab durvalumab and avelumab are used in situations in which less than half of tumors are expected to respond Of patients that benefit many do not experience a sustained durable response These treatments represent a major investment the cost of PD L1 checkpoint blockade commonly reaches several hundred thousand dollars over the course of therapyTo our knowledge this is the first report of an intervention to achieve consistent rapid reduction in either sPD L1 or PD L1 positive EVs in a clinical setting TPE is safe and commonly prescribed Thus preimmunotherapy TPE may combat immunotherapy resistance In light of the heavy investment that anti PD L1 therapy entails the added cost of TPE in selected patients may be practical14 While the durability of extracellular PD L1 reduction in malignancy will be explored in future studies the present study suggests that this approach warrants further investigationOrme a0JJ et a0al J Immunother Cancer 20208e001113 101136jitc2020001113 0c access Beyond evPD L1 this is also to our knowledge the first known intervention to reliably deplete EVs in a clinical setting EVs have been implicated in oncogenesis and metastasis through miRNA carriage and direct protein signaling independent of PD L115 Beyond cancer EVs have also been implicated in autoimmunity17 agingneurodegeneration18 infection19 obesity20 and heart disease21 The selective removal of ADAM10 positive ie likely immune derived versus CD61 positive ie likely platelet derived EVs in this study suggests flexible selective EV depletion may be both possible and expedient in other indications The present study is only a proof of concept and additional exploratory studies in these areas are necessaryIn summary TPE reduces extracellular forms of PD L1 associated with PD L1 checkpoint inhibitor resistance Future studies will explore the potential role of TPE in improving cancer immunotherapyMETHODSRetrospective melanoma outcomes study designIn a retrospective analysis baseline blood samples from patients with melanoma prior to treatment in one of three clinical trials by the North Central Cancer Treatment Group N057e22 N077523 and N087924 between and were tested for sPD L1 of patients were diagnosed with cutaneous melanoma and none received immunotherapy treatments Blood from healthy volunteers undergoing blood donation at Mayo Clinic was also testedProspective TPE study designIn an investigator initiated label single center observational study adults undergoing TPE were approached from December through March In consenting subjects samples of whole blood immediately prior to TPE and on completion of the procedure were collected in ACD vacutainers BD In each case the first mL of blood was discarded to avoid contamination after which an mL sample was obtained in sequence Plasma was isolated by centrifugation A postprocedure blood sample was obtained after completion of the procedure In addition matching samples from discarded plasma from the procedure waste bag were collected Samples were obtained from up to four consecutive procedures for each patient If a patient underwent fewer than four TPE procedures samples were obtained from as many procedures as possiblePatients included were adults able to give consent and undergoing TPE for a variety of hematological neurological and renal diseases as indicated by published guidelines from the American Society for Apheresis ASFA or according to the medical judgment of the referring physicians25 Patients taking biotin supplements were excluded from the study due to biotin interference with the sPD L1 ELISA assay Procedures were performed using centrifugation based cell separators either the Fenwal Amicus Fresenius KABI USA LLC Lake Zurich Illinois USA or the Spectra Optia Terumo BCT Lakewood Colorado USA For each patient a single plasma volume was exchanged using either peripheral intravenous preferred or central lines for vascular access For this study due to the possibility of sPD L1 or PD L1 positive EVs present in donor plasma only TPE sessions using no donor plasma ie fresh frozen plasma FFP in the replacement fluid were included in calculations Anticoagulation consisted of either mL of acid citrate dextrose solution A ACD A or mL of ACD A with units of unfractionated heparin Anticoagulant to blood ratios were when ACD A was used and when ACD Aheparin was used Patients did not receive routine electrolyte replacement but mL of calcium gluconate was administered by slow intravenous push for signs and symptoms of hypocalcemia related to the ACD A anticoagulant in one patientELISAELISA was performed as previously published26 Both secreted splice variant and shed sPD L1 are reliably detected by this ELISA In brief paired mouse IgG2 monoclonal antibody clones H1A and B11 against extracellular human PD L1 were utilized in a capture detection plate assay using biotinylation and HRP streptavidin detection This assay is specific for sPD L1 and does not exhibit cross reactivity to other B7 H homologues nor to evPD L1 Concentrations were determined by optical density measurements along a known standard curve of recombinant human PD L1 ELISAs were performed by team members who were blinded to the identity of the samplesFlow cytometryFlow cytometry for EVs was performed as previously published27 In brief plasma samples were centrifuged twice at 2000g to deplete platelets Resultant platelet free plasma were analyzed using an A60 Micro Plus Nanoscale Flow Cytometer Apogee FlowSystems gating for mid intensity light angle light scatter and markers of interest Anti PD L1 Genentech atezolizumab ADAM10 RD Systems clone and CD61 BioLegend clone VI PL2 antibodies were conjugated to fluorophores Life Technologies Alexa647 PE phycoerythrin and Alexa488 and titrated prior to use Nanoscale flow cytometer calibration was performed using a standard reference bead mix as previously published Flow cytometry was performed by team members blinded to the identity of the samplesStatistical analysisAll statistical analyses were performed using R Statistical Software R Foundation Retrospective progression free survival was analyzed using Kaplan Meier and Cox proportional hazards modeling Optimal cut off values for sPD L1 levels were determined using the greyzoneSurv package for R Wilcoxon signed rank test was used to compare paired pre TPE and post TPE patient sample sPD L1 and EV levels as indicated Baseline clinical characteristics for the study were compared by Kruskal Wallis test for continuous variables and Pearson™s χ2 test for discrete variables as indicated Orme a0JJ et a0al J Immunother Cancer 20208e001113 101136jitc2020001113 0cOtherwise groups were compared by unpaired two sided Student™s t test Figures comprising box plots show quartile values and individual data points Mean values and CI are indicated in corresponding online supplementary figures and tables P was considered statistically significant In figures p values are denoted with with and with Twitter Jacob J Orme JakeOrmeMDPhDAcknowledgements Statistical guidance was provided generously by Nathan Foster of the Mayo Clinic Center for Clinical and Translational Science Some illustrations were created using Servier Medical Art templates which are licensed under a Creative Commons Attribution Unported License https smart servier com Additional illustrations were provided by Mayo Clinic Media Services The authors thank Daniel Summerfield MD MS for use of his likeness in Fig 2AContributors JO originated hypotheses designed the study oversaw experiments performed analyses and wrote the article EALE performed retrospective melanoma cohort analysis FL M performed nanoflow cytometry HD and EB oversaw and performed TPE study enrollment sample collectionprocessing and blinding SMH performed ELISAs MB AM SP MB SNM YY HD RD and JLW helped develop hypotheses provided clinical samples and reagents and contributed support and oversightFunding R21 5R21CA19787802 Role of Bim and soluble B7 H1 in monitoring T cell responses to anti PD1 therapy in melanoma HD and RD L30 CA23154101 Soluble B7H1 as a PD1 Checkpoint œRemote Control in Cancer JJO U10 CA180790 EE K12 CA090628 YY Richard M Schulze Family Foundation HD and RDCompeting interests Intellectual property has been filed addressing discoveries disclosed in this manuscript The authors report no other relevant conflicts of interestPatient consent for publication ObtainedEthics approval All research protocols involving human subjects were approved by Mayo Clinic™s Institutional Review Board and all human subjects gave written informed consentProvenance and peer review Not commissioned externally peer reviewedData availability statement Data are available in a public access repository All data will be available for download at the Science Framework at https osf io qtskd access This is an access article distributed in accordance with the Creative Commons Attribution Non Commercial CC BY NC license which permits others to distribute remix adapt build upon this work non commercially and license their derivative works on different terms provided the original work is properly cited appropriate credit is given any changes made indicated and the use is non commercial See http creativecommons licenses by nc ORCID iDJacob J a0Orme http orcid REFERENCES O'Donnell JS Long GV Scolyer RA et a0al Resistance to PD1PDL1 checkpoint inhibition Cancer Treat Rev “ Ando K Hamada K Watanabe M et a0al Plasma levels of soluble PD L1 correlate with tumor regression in patients with lung and gastric cancer treated with immune checkpoint inhibitors Anticancer Res “ Fan Y Che X Qu J et a0al Exosomal PD L1 retains immunosuppressive activity and is associated with gastric cancer prognosis Ann Surg Oncol “ Zhou J Mahoney KM Giobbie Hurder A et a0al Soluble PD L1 as a biomarker in malignant melanoma treated with checkpoint blockade Cancer Immunol Res “ access Mahoney KMet a0al œA secreted PD L1 splice variant that covalently dimerizes and mediates immunosuppression Cancer Immunol Immunother “ Orme JJ Jazieh KA Xie T et a0al ADAM10 and ADAM17 cleave PD L1 to mediate PD L1 inhibitor resistance OncoImmunology Romero Y Wise R Zolkiewska A Proteolytic processing of PD L1 by ADAM proteases in breast cancer cells Cancer Immunol Immunother “ Chen G Huang AC Zhang W et a0al Exosomal PD L1 contributes to immunosuppression and is associated with anti PD1 response Nature “ Poggio M Hu T Pai C C et a0al Suppression of exosomal PD L1 induces systemic anti tumor immunity and memory Cell “ Derksen RH Schuurman HJ Meyling FH et a0al The efficacy of plasma exchange in the removal of plasma components J Lab Clin Med “ Berckmans RJ Nieuwland R Böing AN et a0al Cell Derived microparticles circulate in healthy humans and support low grade thrombin generation Thromb Haemost “ Crescitelli R Lässer C Jang SC et a0al Subpopulations of extracellular vesicles from human metastatic melanoma tissue identified by quantitative proteomics after optimized isolation J Extracell Vesicles Kowal J Arras G Colombo M et a0al Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes Proc Natl Acad Sci U S A 2016113E968“ Winters JL Brown D Hazard E et a0al Cost minimization analysis of the direct costs of tpe and IVIg in the treatment of Guillain Barré syndrome BMC Health Serv Res “ Lee JC Zhao J T Gundara J et a0al Papillary thyroid cancer derived exosomes contain miRNA 146b and miRNA222 J Surg Res “ Yang J Wei F Schafer C et a0al Detection of tumor cell specific mRNA and protein in exosome like microvesicles from blood and saliva PLoS One 20149e110641 Nakao R Hasegawa H Ochiai K et a0al Outer membrane vesicles of Porphyromonas gingivalis elicit a mucosal immune response PLoS One 20116e26163 Thompson AGet a0al œExtracellular vesicles in neurodegenerative disease pathogenesis to biomarkers Nature Reviews Neurology Nature Publishing Group “ Marcilla A Martin Jaular L Trelis M et a0al Extracellular vesicles in parasitic diseases J Extracell Vesicles Huang Doran I Zhang CY Vidal Puig A œExtracellular Vesicles Novel Mediators of Cell Communication In Metabolic Disease Trends in Endocrinology and Metabolism Elsevier Inc “ Boulanger CM Loyer X Rautou PE et a0al œExtracellular vesicles in coronary artery disease Nature Reviews Cardiology Nature Publishing Group “ Kottschade LA Suman VJ Amatruda T et a0al A phase II trial of nab paclitaxel ABI007 and carboplatin in patients with unresectable stage IV melanoma a North Central Cancer Treatment Group Study N057E1 Cancer “ Kottschade LA Suman VJ Perez DG et a0al A randomized phase study of temozolomide and bevacizumab or nab paclitaxel carboplatin and bevacizumab in patients with unresectable stage IV melanoma a North Central Cancer Treatment Group study N0775 Cancer “ McWilliams RR Allred JB Slostad JA et a0al NCCTG N0879 Alliance A randomized phase cooperative group trial of carboplatin paclitaxel and bevacizumab ± everolimus for metastatic melanoma Cancer “ Padmanabhan A Connelly Smith L Aqui N et a0al Guidelines on the Use of Therapeutic Apheresis in Clinical Practice Evidence Based Approach from the Writing Committee of the American Society for Apheresis The Eighth Special Issue J Clin Apher “ Frigola X Inman BA Lohse CM et a0al Identification of a soluble form of B7 H1 that retains immunosuppressive activity and is associated with aggressive renal cell carcinoma Clin Cancer Res “ Gomes J Lucien F Cooper TT et a0al Analytical considerations in nanoscale flow cytometry of extracellular vesicles to achieve data linearity Thromb Haemost “Orme a0JJ et a0al J Immunother Cancer 20208e001113 101136jitc2020001113 0c" Answer:

Thyroid_Cancer

"The coronavirus disease COVID19 is now a worldwide challenge for public health Among million patients about present mild to moderate disease but studies dedicate to these patients are actually scarce The aim of our study is to clarify the characteristics of laboratory test index of COVID19 patient with moderate symptoms during the first wave of the pandemic in Wuhan ChinaMethods In this retrospective cohort study we included adult inpatients with confirmed moderate disease of COVID19 from the Affiliated Hospital of Jianghan University during February and early March All of these patients were recovered from COVID19 and discharged from hospital Demographic clinical and laboratory data of admission and discharge were extracted from electronic medical records and analyzed using SPSS as well as among young middle age and elderly peopleResults The median age of this cohort of patients was years And the median hospitalization time was days Common clinical manifestations included fever cough asthenia and shortness of breath On admission laboratory results showed normal or increased neutrophil ratio low lymphocyte count decreased hemoglobin level and increased inflammatory indicators erythrocyte sedimentation rate ESR and Creactive protein CRP and some patients were complicated with coagulation disorder and myocardial damage Furthermore patients older than years had statistically higher CRP ESR and fibrinogen level As the health condition was improved at discharge the median level of most laboratory results were in the normal range except hemoglobin and related blood cell count as well as Manuscript submitted July accepted July Published online August aWuhan Institute of Biomedical Sciences School of Medicine Jianghan University Wuhan ChinabThe Affiliated Hospital of Jianghan University Wuhan ChinacThe two authors contributed equallydCorresponding Author Binlian Sun Wuhan Institute of Biomedical Sciences School of Medicine Jianghan University Wuhan China Email binlian17jhuneducn 1014740jocmr4293inflammatory indicator ESR And patients older than years showed slower recovery on coagulation parameters when compared to younger patientsConclusions The severe acute respiratory syndrome coronavirus SARSCoV2 infection induces a controllable inflammatory response in moderate disease of COVID19 in Wuhan China Since patients older than years had higher inflammatory state and more dysregulated coagulation condition it might be essential to closely assess their illnessKeywords COVID19 Moderate disease Clinical feature Laboratory findings InflammatoryIntroductionThe novel severe acute respiratory syndrome coronavirus SARSCoV2 was first identified in Wuhan China in December [ ] and now spread all over the world Its infection in human mainly appears as acute respiratory syndrome sometimes along with digestive and nervous disorders [ ] The outbreak of this coronavirus disease COVID19 was declared a pandemic by the World Health anization WHO on March Globally as of June there have been confirmed cases including deaths []A large cohort study showed the spectrum of COVID19 that among patients had mild symptom had severe disease and were in critical condition [] Naturally most studies about COVID19 focus on the severe and critical cases although more than of COVID19 patients show mild to moderate symptoms It should be equally important to understand the average degree of SARSCoV2 infection especially if this virus would become another endemic virus in communities like the influenza virusOur study describes patients that were admitted in a designated hospital in Wuhan the Affiliated Hospital of Jianghan University the sixth hospital of Wuhan city during February and early March These patients showed moderate symptom of COVID19 and were confirmed both by s The authors Journal compilation J Clin Med Res and Elmer Press Inc„¢ wwwjocmrThis is distributed under the terms of the Creative Commons Attribution NonCommercial International License which permits unrestricted noncommercial use distribution and reproduction in any medium provided the original work is properly cited 0cLiu et alJ Clin Med Res SARSCoV2 RNA test and chest radiography And they were all discharged from hospital after recovery The aim of our study is to clarify the characteristics of laboratory test index of COVID19 patient with moderate symptoms during the first wave of the pandemic in Wuhan These findings may help us extend our understanding of the pathogenicity in SARSCoV2 infectionMaterials and MethodsPatientsThis retrospective cohort study included adult patients ‰¥ years old with confirmed COVID19 admitted to the Affiliated Hospital of Jianghan University the sixth hospital of Wuhan city in Wuhan from February to March According to WHO interim guidance [] patients with moderate disease of COVID19 on admission were enrolled in this study they showed clinical signs of pneumonia fever cough dyspnea fast breathing but no signs of severe pneumonia including oxygen saturation SpO2 ‰¥ on room air All the patients were confirmed by SARSCoV2 RNA test in respiratory secretions for twice as well as by groundglass opacities or bilateral pulmonary infiltration showed in chest computed tomography CT scan During hospitalization patients were kept in regular wards without intensive cares or invasive mechanical ventilation They received supportive therapy effective oxygen therapy antiviral agent and if necessary antibiotics Patients were discharged from hospital when the following criteria were met body temperature normal for more than days respiratory symptoms significantly improved pulmonary imaging significantly improved on CT scan and SARSCoV2 RNA tests showed negative for twice This study was approved by the Ethics Committee of School of Medicine of Jianghan University Wuhan China This study was conducted in compliance with the ethical standards of the responsible institution on human subjects as well as with the Helsinki DeclarationData collectionsThe laboratory tests including blood routine biochemistry coagulation parameters and cardiac injury biomarkers were performed in patients on admission and during the hospitalization The demographic and clinical information laboratory results and outcome data were finally collected from electronic medical recordsStatistical analysisTable Demographics and Clinical Characteristics of Patients on Admission N Female MaleAge median IQR yearSex Hospitalization days median IQR dayMedical history Hypertension Diabetes Heart disease Cancer Other respiratory disease Kidney disease Liver disease Neurological disease Thyroid disease OthersSymptoms Fever Cough Sputum production Nose obstruction rhinorrhea Shortness of breath Headache Chest pain Myalgia Asthenia Vomiting DiarrheaSigns Respiratory rate median IQR bpm Heart rate median IQR bpm Systolic pressure 140mm Hg SpO2 ‰ Data are median IQR n or nN IQR interquartile range SpO2 oxygen saturationsion IBMResultsContinuous variables were expressed as median interquartile range IQR and compared with the oneway analysis of variance ANOVA test between different age groups A twosided α of less than was considered statistically significant Statistical analyses were done using SPSS verPatient demographics and characteristicsA total of patients with moderate disease of COVID19 were recruited The demographic and clinical characteristics s The authors Journal compilation J Clin Med Res and Elmer Press Inc„¢ wwwjocmr 0cClinical Features of Moderate COVID19J Clin Med Res Table Summary of Laboratory Findings on Admission and at DischargeResults on admissionResults at dischargeLaboratory indexNormal rangeNeutrophil count — 109L Median IQR Patient †‘†“†‘†“†“†“ †“†“†‘†“†‘†‘†‘†‘†‘†‘†‘†‘†‘Median IQR NA Patient †‘†“†‘†“†“†“†“†“†‘†“†‘†‘ †‘†‘†‘†‘NA†‘†‘ Female MaleNeutrophil ratio Lymphocyte count — 109LLymphocyte ratio Red blood cell count — 1012L Hemoglobin gL Hematocrit Platelet count — 109L Female Male Female MalehsCRP mgLESR mmhDdimer mgLFibrinogen gLFDP mgLNTproBNPa pgmL years years years hscTnL ngmLCK ULLDH ULData are median IQR or nN aThe normal level of NTproBNP is increased with age Three normal ranges for different age groups are listed and results are presented accordingly †‘For increased blood level †“For decreased blood level hsCRP high sensitive Creactive protein ESR erythrocyte sedimentation rate FDP fibrinogen degradation product NTproBNP Nterminal prohormone brain natriuretic peptide hscTnL high sensitive cardiac troponin L CK creatine kinase LDH lactose dehydrogenase IQR interquartile range NA not applicableof these patients are shown in Table Of these patients females and males the median age at disease onset was years range years with patients older than years Most patients had fever and cough as their first symptoms some also had asthenia shortness of breath sputum production and diarrhea As for the underlying diseases hypertension heart disease and diabetes were the most common in medical histories of these patients On admission the vital signs of patients were also recorded Notably although the SpO2 of all the patients were ‰¥ at room air of patients were ‰ Patients received symptomatic and pneumonia treatments in hospital and the median of their hospitalization were days IQR Whole blood cell counting findingsThe whole blood cell counting was monitored for all the patients on admission and during their hospitalization median values showed in Table The white blood cell counts were generally in the normal range whereas of patients of patients showed lymphopenia lymphocyte count — 109L Table Among patients showed lymphopenia of had a decrease by less than lymphocyte count s The authors Journal compilation J Clin Med Res and Elmer Press Inc„¢ wwwjocmr 0cLiu et alJ Clin Med Res Figure Illustration of percentage changes of laboratory index according to number of patients Data are presented as number of patients that had changed level in laboratory assessments a Decreased lymphocyte count on admission and at discharge b Decreased hemoglobin level on admission and at discharge c Increased CRP level on admission and at discharge d Increased ESR level on admission and at discharge e Increased fibrinogen level on admission and at discharge f Increased Ddimer level on admission and at discharge g Increased FDP level on admission and at discharge In for the data collected on admission out for data collected at discharge CRP Creactive protein ESR erythrocyte sedimentation rate FDP fibrinogen degradation products The authors Journal compilation J Clin Med Res and Elmer Press Inc„¢ wwwjocmr 0cClinical Features of Moderate COVID19J Clin Med Res Table Statistically Significant Biomarkers on Admission and at Discharge With Different AgesCRP inESR inESR outFibrinogen inFDP outD dimer outCK n n n 944a 1073a 983a 53b 41a 082a 798bData are expressed with the median IQR P values comparing different age groups are from oneway ANOVA aP for group and group vs group has statistical difference bP for group vs group has statistical difference In for the data collected on admission out for data collected at discharge CRP Creactive protein ESR erythrocyte sedimentation rate FDP fibrinogen degradation product CK creatine kinase IQR interquartile range — 109L Fig 1a while five of of them showed a decrease by more than lymphocyte count — 109L Fig 1a Twentythree percent of of patients Table still had lymphopenia when they were discharged from hospital and the decrease was limited for most Fig 1a The change of neutrophil count affects fewer patients on admission of nine of patients showed neutrophilia neutrophil count — 109L Table and of patients had neutropenia neutrophil count — 109L Table However neutrophil ratio was augmented in of of patients neutrophil ratio Table and the maximum increase was about This ratio appeared to be in the normal range for most patients at discharge Therefore the main change in white blood cell counts for patients with moderate COVID19 was the decrease of lymphocyte And as the health condition improved at discharge lymphocyte count also gradually increased to normal levelThe data showed that more than of all patients had declined level of red blood cell count of hemoglobin of and hematocrit of Table and four female patients were in severe condition hemoglobin gL Fig 1b When discharged from hospital of of patients still had these decreases Table The four female patients mentioned above had their hemoglobin increased to over gL However one female of years had less than gL hemoglobin and that was less than earlier result Furthermore there were five more male patients had hemoglobin declined at discharge Fig 1b These results suggested that SARSCoV2 infection may be accompanied by oxygen transport defect in red blood cellsAs for the platelet counting on admission of patients seven of had decreased platelet level platelet count — 109L Table and of had increased level platelet count — 109L Table At discharge most patients showed platelet level in the normal range Table Inflammatory biomarkersInflammatory biomarkers were also examined on admission and during hospitalization such as erythrocytes sedimentation rate ESR Creactive protein CRP and procalcitonin PCT For most patients PCT levels were in the normal range data not shown For CRP the median value was mgL IQR among patients on admission Table and patients older than years had significantly higher level Table To be specific CRP levels in serum were increased CRP mgL Table in of patients of and of showed an increase by more than CRP mgL Fig 1c At discharge only of Table had elevated level of CRP and the increased level did not exceed mgL except for one patient Fig 1cLevels of ESR were increased in of patients of ESR mmh Table and of Fig 1d had an increase exceeding mmh Similar to CRP patients older than years had statistically higher ESR level Table The median value of ESR on admission was mmh IQR while the value improved to mmh IQR at discharge Table There were still patients had ESR level more than mmh in serum Fig 1dCoagulation parametersThe coagulation parameters were examined on admission for patients Eighty percent of patients of Table had normal serum levels of Ddimer on admission Among the of patients of who had increased Ddimer level Ddimer mgL Table eight showed an increase by more than Ddimer 2mgL Fig 1f At discharge of patients seven of still had abnormal level of Ddimer though the results were very close to the normal range The fibrinogen level on the other hand had increased in of patients of fibrinogen mgL Table and decreased in of fibrinogen 2mgL Specifically patients of had an increase of fibrinogen level by more than fibrinogen mgL Fig 1e while most decreases were slight Furthermore when compared to patients younger than years fibrinogen level on admission was significantly higher in the patients older than years Table At discharge only seven patients still showed high level of fibrinogen Fig 1e Fibrinogen degradation product FDP was also s The authors Journal compilation J Clin Med Res and Elmer Press Inc„¢ wwwjocmr 0cLiu et alJ Clin Med Res cardial damage Since the outcome of this cohort is known as recovery and discharge from hospital we also collected the last available laboratory results Although all the patients met the discharge criteria for COVID19 some still had abnormal laboratory findings Especially we found the levels of the inflammatory biomarker ESR were still elevated for half of the patients tested Hence patients were asked to go back to the hospital for followup examination including the virus RNA test chest CT scan blood routine coagulation function and other biochemical indicators on both the 14th and 28th day after dischargePrevious studies have found that white blood cell WBC procalcitonin PCT aspartate aminotransferase AST lactose dehydrogenase LDH Cr hscTnL and Ddimer could indicate the progression of COVID19 on admission especially for the severe and mortal cases [ ] And in the moderate condition we analyzed most patients had these indicators in the normal range Therefore our findings are consistent with other studiesHowever these biomarkers were not all specific to COVID19 Similar to other virus infections including SARS [] and H1N1 influenza [] the neutrophil count was mostly normal in mild to moderate patients while lymphocyte count was significantly decreased As the major antiviral cells lymphocyte count declined down to — 109L in this cohort on admission And after receiving antiviral and supportive treatment the lymphocyte counts increased among all the patients at discharge Previous study found that lymphocyte counts would continually decrease in severe and critical COVID19 patients [] These results suggested that SARSCoV2 infection can suppress the level of lymphocyte Therefore closely monitoring lymphocyte counts could be one of the best methods when we evaluate the outcome of moderate patientsThe blood routine results also showed that about of patients had low level of hemoglobin red blood cell and hematocrit and the condition was not improved at discharge Previous studies also reported that of patients had hemoglobin below the normal range [] and the hemoglobin level of severe patients was lower although the difference was not statistically significant [] There might be two explanations First the inflammatory state caused by infection may interfere with erythrocytebone marrow metabolism and iron regulation [] and eventually result in a decline of hemoglobin and red blood cell Second patients could suffer from anemia for some time and appear more vulnerable to SARSCoV2 infection Once infected however correction of anemia could not benefit from all the symptoms of COVID19 and psychological stresses The decrease of functioning hemoglobin may contribute to hypoxia and further aggravate the disease in severe casesexamined and of patients had elevated level of FDP mgL Table Fig 1g on admission Twentyseven percent of patients of still showed increased amount of FDP at discharge Table Therefore fibrinogen and FDP were the most altered parameters in coagulation function from our analysisCardiac injury biomarkersCardiac injury biomarkers were monitored for patients on admission Among the patients assessed all had normal serum levels of high sensitive cardiac troponin L hscTnL ngmL The other cardiac biomarker examined was Nterminal prohormone brain natriuretic peptide NTproBNP and of patients of had elevated level NTproBNP and pgmL according to age Table on admission and five of had an increase by more than At discharge the NTproBNP level had decreased into the normal range except two male patients These two patients still had high level of NTproBNP more than pgmL however other cardiac biomarkers such as hscTnL and creatine kinaseMB CKMB were both in the normal range Considering their history of hypertension they were asked to reevaluate their heart function after the COVID19DiscussionAs an emerging infectious disease COVID19 is now a worldwide challenge for public health Among the million patients about present mild to moderate disease but studies dedicate to these patients are actually scarce Therefore we enrolled adult patients with moderate disease of COVID19 to elucidate the clinical manifestation and laboratory findingsOne of the risk factors of critical and mortal COVID19 is male with older age [] Considering the not severe condition of disease it is rational that female patients present more than male in this cohort Previous studies have found comorbidity such as diabetes and hypertension heart disease as other risk factors [] Similar to other study of patients had diabetes and had hypertension Similar to other studies in Asia [ ] patients mainly present fever cough asthenia and shortness of breath In Europe however a recent study showed that patients with mild to moderate disease mainly had headache loss of smell nasal obstruction and asthenia [] Considering the big differences it is possible that olfactory dysfunction might be neglected during the first wave of SARSCoV2 infection in Wuhan Also the virus is phylogenetically distinct from Asia type B to Europe type C [] which could contribute to different clinical outcomesThrough analysis of laboratory findings we found that patients with moderate disease of COVID19 had common characteristics on admission normal or increased neutrophil ratio low lymphocyte count decreased hemoglobin level increased inflammatory indicators ESR and CRP and some patients were complicated with coagulation disorder and myoExamination of inflammatory biomarkers found that PCT was in the normal range for moderate patients while CRP and ESR were increased in and of patients on admission respectively Other studies including mild to moderate disease showed heterogeneous results [] and found three of or of of patients had increased level of CRP [ ] Thus SARSCoV2 infection is involved with the disorder of inflammatory response Furthermore we find that the increase of both CRP and ESR were correlated with patient€™s age which may indicate that high inflammatory state s The authors Journal compilation J Clin Med Res and Elmer Press Inc„¢ wwwjocmr 0cClinical Features of Moderate COVID19J Clin Med Res and more severe pneumonia present more often in elderly people In addition the increase of CRP level was more transient as it returned to normal range in most people when they were discharged from hospital Nevertheless it took more time for ESR to return to normal level especially for patients older than years Certainly elder people would need more time to recover and to metabolize the extra inflammatory biomarkersAbnormal coagulation condition is known as associated with poor prognosis of COVID19 In particular serum level of Ddimer and FDP were significantly higher in lethal cases [] Other coronavirus infection such as SARS and Middle East respiratory syndrome MERS showed similar increased coagulation activities in severe cases [ ] The possible mechanism is that local inflammatory response induced by cytokine responses stimulates coagulation cascade and hemodynamic changes [ ] In patients with moderate COVID19 we found about and presented increased level in fibrinogen FDP and Ddimer on admission respectively And when compared to young people patients older than years tend to have higher levels of these coagulation parameters Certainly the hemodynamics and endothelial conditions are more complicate in elder people Furthermore SARSCoV2 infection as well as the inflammatory response it induced could deteriorate the existed dysregulation As the health condition improved at discharge these parameters decreased to normal ranges for most patients Yet again patients over years had significantly higher level of fibrin degradation products In the meantime we found that the platelet level had no obvious decrease in most patients with moderate disease Therefore as reported previously thrombocytopenia happens more often in severe and critical patients [] while abnormal coagulation condition is slight and temporary in moderate patientsThe receptor of SARSCoV2 angiotensinconverting enzyme ACE2 is expressed in myocardial cells and vascular endothelial cells [] It is possible that heart dysfunction is directly targeted by virus infection hence early evaluation and continued monitoring of cardiac damage are important [] We systematically examined the cardiac function biomarkers on admission CKMB and cTnL had no obvious change in these moderate patients while about of patients showed increased NTproBNP This percentage is much lower than previous reported of the severe cases that had elevated NTproBNP Furthermore we only found two patients still had NTproBNP higher than normal range at discharge indicating that moderate patients had little cardiac complicationThis study provides us more information about moderate COVID19 but still has some limitations First this is a relatively small singlecenter study Second due to the retrospective study design a few laboratory tests were not done in all patients In addition several patients were hospitalized during a short period that some tests were not reexamined before they were discharged Third we do not possess any viral kinetic data in these patients further studies are necessary to elucidate the correlation between viral load and laboratory changesIn this ongoing pandemic of infected people showed moderate disease [] it is important to understand more about this moderate spectrum of disease to settle into a longterm problem We conducted a cohort study of adult patients with moderate disease of COVID19 in Wuhan China Based on the clinical characteristics we conclude that SARSCoV2 infection inhibits the immune system of patients and induces a controllable inflammatory response in moderate COVID19 Elderly patients have higher inflammatory state and more dysregulated coagulation condition It is essential to closely assess their condition for a better clinical managementAcknowledgmentsThis study was supported by the Natural Science Foundation of China Hubei Province Department of Education Science and Technology Project B2019232 and the Wuhan Municipal Education Bureau Project Financial DisclosureThere are no financial conflicts of interest to discloseConflict of InterestAll authors declare no competing interestsInformed ConsentAll subjects provided written informed consentAuthor ContributionsYL XZ and BS designed and performed the study YL and BS drafted the manuscript and did critical editing XZ and RG were involved in data collection YL JQ QY JS and WL analyzed the data BS carefully supervised this manuscript preparation and writingData AvailabilityAll data used in the study are available from the corresponding author by request Although some data confidential in nature may only be provided with restrictionsReferences Zhu N Zhang D Wang W Li X Yang B Song J Zhao X et al A novel coronavirus from patients with pneumonia in China N Engl J Med Coronaviridae Study Group of the International Committee on Taxonomy of Viruses The species Severe acute respiratory syndromerelated coronavirus classifying 2019nCoV and naming it SARSCoV2 Nat Microbiol Huang C Wang Y Li X Ren L Zhao J Hu Y Zhang s The authors Journal compilation J Clin Med Res and Elmer Press Inc„¢ wwwjocmr 0cLiu et alJ Clin Med Res L et al Clinical features of patients infected with novel coronavirus in Wuhan China Lancet Wang D Hu B Hu C Zhu F Liu X Zhang J Wang B et al Clinical characteristics of hospitalized patients with novel coronavirusinfected pneumonia in Wuhan China JAMA WHO Coronavirus disease COVID19 Situation Report Available from wwwwhointdocsdefaultsourcecoronavirusesituationreports20200628covid19sitrep160pdfsfvrsn2fe1c658_2 Wu Z McGoogan JM Characteristics of and important lessons from the coronavirus disease COVID19 outbreak in China summary of a report of cases from the Chinese Center for Disease Control and Prevention JAMA WHO Clinical management of COVID19 Available from wwwwhointpublicationsdetailclinicalmanagementofcovid19 Liu K Chen Y Lin R Han K Clinical features of COVID19 in elderly patients A comparison with young and middleaged patients J Infect 2020806e14e18 Zheng Z Peng F Xu B Zhao J Liu H Peng J Li Q et al Risk factors of critical mortal COVID19 cases A systematic literature review and metaanalysis J Infect 2020812e16e25 Li LQ Huang T Wang YQ Wang ZP Liang Y Huang TB Zhang HY et al COVID19 patients' clinical characteristics discharge rate and fatality rate of metaanalysis J Med Virol Cao Y Liu X Xiong L Cai K Imaging and clinical features of patients with novel coronavirus SARSCoV2 A systematic review and metaanalysis J Med Virol Lechien JR ChiesaEstomba CM Place S Van Laethem Y Cabaraux P Mat Q Huet K et al Clinical and epidemiological characteristics of European patients with mildtomoderate coronavirus disease J Intern Med Forster P Forster L Renfrew C Forster M Phylogenetic network analysis of SARSCoV2 genomes Proc Natl Acad Sci U S A Zhou F Yu T Du R Fan G Liu Y Liu Z Xiang J et al Clinical course and risk factors for mortality of adult inpatients with COVID19 in Wuhan China a retrospective cohort study Lancet Velavan TP Meyer CG Mild versus severe COVID19 Laboratory markers Int J Infect Dis Lee N Hui D Wu A Chan P Cameron P Joynt GM Ahuja A et al A major outbreak of severe acute respiratory syndrome in Hong Kong N Engl J Med Cheng Y Zhao H Song P Zhang Z Chen J Zhou YH Dynamic changes of lymphocyte counts in adult patients with severe pandemic H1N1 influenza A J Infect Public Health Chen N Zhou M Dong X Qu J Gong F Han Y Qiu Y et al Epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in Wuhan China a descriptive study Lancet Cavezzi A Troiani E Corrao S COVID19 hemoglobin iron and hypoxia beyond inflammation A narrative review Clin Pract Zeng F Huang Y Guo Y Yin M Chen X Xiao L Deng G Association of inflammatory markers with the severity of COVID19 A metaanalysis Int J Infect Dis Chen G Wu D Guo W Cao Y Huang D Wang H Wang T et al Clinical and immunological features of severe and moderate coronavirus disease J Clin Invest Wang F Hou H Luo Y Tang G Wu S Huang M Liu W et al The laboratory tests and host immunity of COVID19 patients with different severity of illness JCI Insight Tang N Li D Wang X Sun Z Abnormal coagulation parameters are associated with poor prognosis in patients with novel coronavirus pneumonia J Thromb Haemost Wong RS Wu A To KF Lee N Lam CW Wong CK Chan PK et al Haematological manifestations in patients with severe acute respiratory syndrome retrospective analysis BMJ AlAbdallat MM Payne DC Alqasrawi S Rha B Tohme RA Abedi GR Al Nsour M et al Hospitalassociated outbreak of Middle East respiratory syndrome coronavirus a serologic epidemiologic and clinical description Clin Infect Dis Moore JB June CH Cytokine release syndrome in severe COVID19 Science Giannis D Ziogas IA Gianni P Coagulation disorders in coronavirus infected patients COVID19 SARSCoV1 MERSCoV and lessons from the past J Clin Virol Guan WJ Ni ZY Hu Y Liang WH Ou CQ He JX Liu L et al Clinical Characteristics of Coronavirus Disease in China N Engl J Med MendozaTorres E Oyarzun A MondacaRuff D Azocar A Castro PF Jalil JE Chiong M et al ACE2 and vasoactive peptides novel players in cardiovascularrenal remodeling and hypertension Ther Adv Cardiovasc Dis Guzik TJ Mohiddin SA Dimarco A Patel V Savvatis K MarelliBerg FM Madhur MS et al COVID19 and the cardiovascular system implications for risk assessment diagnosis and treatment options Cardiovasc Res s The authors Journal compilation J Clin Med Res and Elmer Press Inc„¢ wwwjocmr 0c"

cancer225

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: "The coronavirus disease COVID19 is now a worldwide challenge for public health Among million patients about present mild to moderate disease but studies dedicate to these patients are actually scarce The aim of our study is to clarify the characteristics of laboratory test index of COVID19 patient with moderate symptoms during the first wave of the pandemic in Wuhan ChinaMethods In this retrospective cohort study we included adult inpatients with confirmed moderate disease of COVID19 from the Affiliated Hospital of Jianghan University during February and early March All of these patients were recovered from COVID19 and discharged from hospital Demographic clinical and laboratory data of admission and discharge were extracted from electronic medical records and analyzed using SPSS as well as among young middle age and elderly peopleResults The median age of this cohort of patients was years And the median hospitalization time was days Common clinical manifestations included fever cough asthenia and shortness of breath On admission laboratory results showed normal or increased neutrophil ratio low lymphocyte count decreased hemoglobin level and increased inflammatory indicators erythrocyte sedimentation rate ESR and Creactive protein CRP and some patients were complicated with coagulation disorder and myocardial damage Furthermore patients older than years had statistically higher CRP ESR and fibrinogen level As the health condition was improved at discharge the median level of most laboratory results were in the normal range except hemoglobin and related blood cell count as well as Manuscript submitted July accepted July Published online August aWuhan Institute of Biomedical Sciences School of Medicine Jianghan University Wuhan ChinabThe Affiliated Hospital of Jianghan University Wuhan ChinacThe two authors contributed equallydCorresponding Author Binlian Sun Wuhan Institute of Biomedical Sciences School of Medicine Jianghan University Wuhan China Email binlian17jhuneducn 1014740jocmr4293inflammatory indicator ESR And patients older than years showed slower recovery on coagulation parameters when compared to younger patientsConclusions The severe acute respiratory syndrome coronavirus SARSCoV2 infection induces a controllable inflammatory response in moderate disease of COVID19 in Wuhan China Since patients older than years had higher inflammatory state and more dysregulated coagulation condition it might be essential to closely assess their illnessKeywords COVID19 Moderate disease Clinical feature Laboratory findings InflammatoryIntroductionThe novel severe acute respiratory syndrome coronavirus SARSCoV2 was first identified in Wuhan China in December [ ] and now spread all over the world Its infection in human mainly appears as acute respiratory syndrome sometimes along with digestive and nervous disorders [ ] The outbreak of this coronavirus disease COVID19 was declared a pandemic by the World Health anization WHO on March Globally as of June there have been confirmed cases including deaths []A large cohort study showed the spectrum of COVID19 that among patients had mild symptom had severe disease and were in critical condition [] Naturally most studies about COVID19 focus on the severe and critical cases although more than of COVID19 patients show mild to moderate symptoms It should be equally important to understand the average degree of SARSCoV2 infection especially if this virus would become another endemic virus in communities like the influenza virusOur study describes patients that were admitted in a designated hospital in Wuhan the Affiliated Hospital of Jianghan University the sixth hospital of Wuhan city during February and early March These patients showed moderate symptom of COVID19 and were confirmed both by s The authors Journal compilation J Clin Med Res and Elmer Press Inc„¢ wwwjocmrThis is distributed under the terms of the Creative Commons Attribution NonCommercial International License which permits unrestricted noncommercial use distribution and reproduction in any medium provided the original work is properly cited 0cLiu et alJ Clin Med Res SARSCoV2 RNA test and chest radiography And they were all discharged from hospital after recovery The aim of our study is to clarify the characteristics of laboratory test index of COVID19 patient with moderate symptoms during the first wave of the pandemic in Wuhan These findings may help us extend our understanding of the pathogenicity in SARSCoV2 infectionMaterials and MethodsPatientsThis retrospective cohort study included adult patients ‰¥ years old with confirmed COVID19 admitted to the Affiliated Hospital of Jianghan University the sixth hospital of Wuhan city in Wuhan from February to March According to WHO interim guidance [] patients with moderate disease of COVID19 on admission were enrolled in this study they showed clinical signs of pneumonia fever cough dyspnea fast breathing but no signs of severe pneumonia including oxygen saturation SpO2 ‰¥ on room air All the patients were confirmed by SARSCoV2 RNA test in respiratory secretions for twice as well as by groundglass opacities or bilateral pulmonary infiltration showed in chest computed tomography CT scan During hospitalization patients were kept in regular wards without intensive cares or invasive mechanical ventilation They received supportive therapy effective oxygen therapy antiviral agent and if necessary antibiotics Patients were discharged from hospital when the following criteria were met body temperature normal for more than days respiratory symptoms significantly improved pulmonary imaging significantly improved on CT scan and SARSCoV2 RNA tests showed negative for twice This study was approved by the Ethics Committee of School of Medicine of Jianghan University Wuhan China This study was conducted in compliance with the ethical standards of the responsible institution on human subjects as well as with the Helsinki DeclarationData collectionsThe laboratory tests including blood routine biochemistry coagulation parameters and cardiac injury biomarkers were performed in patients on admission and during the hospitalization The demographic and clinical information laboratory results and outcome data were finally collected from electronic medical recordsStatistical analysisTable Demographics and Clinical Characteristics of Patients on Admission N Female MaleAge median IQR yearSex Hospitalization days median IQR dayMedical history Hypertension Diabetes Heart disease Cancer Other respiratory disease Kidney disease Liver disease Neurological disease Thyroid disease OthersSymptoms Fever Cough Sputum production Nose obstruction rhinorrhea Shortness of breath Headache Chest pain Myalgia Asthenia Vomiting DiarrheaSigns Respiratory rate median IQR bpm Heart rate median IQR bpm Systolic pressure 140mm Hg SpO2 ‰ Data are median IQR n or nN IQR interquartile range SpO2 oxygen saturationsion IBMResultsContinuous variables were expressed as median interquartile range IQR and compared with the oneway analysis of variance ANOVA test between different age groups A twosided α of less than was considered statistically significant Statistical analyses were done using SPSS verPatient demographics and characteristicsA total of patients with moderate disease of COVID19 were recruited The demographic and clinical characteristics s The authors Journal compilation J Clin Med Res and Elmer Press Inc„¢ wwwjocmr 0cClinical Features of Moderate COVID19J Clin Med Res Table Summary of Laboratory Findings on Admission and at DischargeResults on admissionResults at dischargeLaboratory indexNormal rangeNeutrophil count — 109L Median IQR Patient †‘†“†‘†“†“†“ †“†“†‘†“†‘†‘†‘†‘†‘†‘†‘†‘†‘Median IQR NA Patient †‘†“†‘†“†“†“†“†“†‘†“†‘†‘ †‘†‘†‘†‘NA†‘†‘ Female MaleNeutrophil ratio Lymphocyte count — 109LLymphocyte ratio Red blood cell count — 1012L Hemoglobin gL Hematocrit Platelet count — 109L Female Male Female MalehsCRP mgLESR mmhDdimer mgLFibrinogen gLFDP mgLNTproBNPa pgmL years years years hscTnL ngmLCK ULLDH ULData are median IQR or nN aThe normal level of NTproBNP is increased with age Three normal ranges for different age groups are listed and results are presented accordingly †‘For increased blood level †“For decreased blood level hsCRP high sensitive Creactive protein ESR erythrocyte sedimentation rate FDP fibrinogen degradation product NTproBNP Nterminal prohormone brain natriuretic peptide hscTnL high sensitive cardiac troponin L CK creatine kinase LDH lactose dehydrogenase IQR interquartile range NA not applicableof these patients are shown in Table Of these patients females and males the median age at disease onset was years range years with patients older than years Most patients had fever and cough as their first symptoms some also had asthenia shortness of breath sputum production and diarrhea As for the underlying diseases hypertension heart disease and diabetes were the most common in medical histories of these patients On admission the vital signs of patients were also recorded Notably although the SpO2 of all the patients were ‰¥ at room air of patients were ‰ Patients received symptomatic and pneumonia treatments in hospital and the median of their hospitalization were days IQR Whole blood cell counting findingsThe whole blood cell counting was monitored for all the patients on admission and during their hospitalization median values showed in Table The white blood cell counts were generally in the normal range whereas of patients of patients showed lymphopenia lymphocyte count — 109L Table Among patients showed lymphopenia of had a decrease by less than lymphocyte count s The authors Journal compilation J Clin Med Res and Elmer Press Inc„¢ wwwjocmr 0cLiu et alJ Clin Med Res Figure Illustration of percentage changes of laboratory index according to number of patients Data are presented as number of patients that had changed level in laboratory assessments a Decreased lymphocyte count on admission and at discharge b Decreased hemoglobin level on admission and at discharge c Increased CRP level on admission and at discharge d Increased ESR level on admission and at discharge e Increased fibrinogen level on admission and at discharge f Increased Ddimer level on admission and at discharge g Increased FDP level on admission and at discharge In for the data collected on admission out for data collected at discharge CRP Creactive protein ESR erythrocyte sedimentation rate FDP fibrinogen degradation products The authors Journal compilation J Clin Med Res and Elmer Press Inc„¢ wwwjocmr 0cClinical Features of Moderate COVID19J Clin Med Res Table Statistically Significant Biomarkers on Admission and at Discharge With Different AgesCRP inESR inESR outFibrinogen inFDP outD dimer outCK n n n 944a 1073a 983a 53b 41a 082a 798bData are expressed with the median IQR P values comparing different age groups are from oneway ANOVA aP for group and group vs group has statistical difference bP for group vs group has statistical difference In for the data collected on admission out for data collected at discharge CRP Creactive protein ESR erythrocyte sedimentation rate FDP fibrinogen degradation product CK creatine kinase IQR interquartile range — 109L Fig 1a while five of of them showed a decrease by more than lymphocyte count — 109L Fig 1a Twentythree percent of of patients Table still had lymphopenia when they were discharged from hospital and the decrease was limited for most Fig 1a The change of neutrophil count affects fewer patients on admission of nine of patients showed neutrophilia neutrophil count — 109L Table and of patients had neutropenia neutrophil count — 109L Table However neutrophil ratio was augmented in of of patients neutrophil ratio Table and the maximum increase was about This ratio appeared to be in the normal range for most patients at discharge Therefore the main change in white blood cell counts for patients with moderate COVID19 was the decrease of lymphocyte And as the health condition improved at discharge lymphocyte count also gradually increased to normal levelThe data showed that more than of all patients had declined level of red blood cell count of hemoglobin of and hematocrit of Table and four female patients were in severe condition hemoglobin gL Fig 1b When discharged from hospital of of patients still had these decreases Table The four female patients mentioned above had their hemoglobin increased to over gL However one female of years had less than gL hemoglobin and that was less than earlier result Furthermore there were five more male patients had hemoglobin declined at discharge Fig 1b These results suggested that SARSCoV2 infection may be accompanied by oxygen transport defect in red blood cellsAs for the platelet counting on admission of patients seven of had decreased platelet level platelet count — 109L Table and of had increased level platelet count — 109L Table At discharge most patients showed platelet level in the normal range Table Inflammatory biomarkersInflammatory biomarkers were also examined on admission and during hospitalization such as erythrocytes sedimentation rate ESR Creactive protein CRP and procalcitonin PCT For most patients PCT levels were in the normal range data not shown For CRP the median value was mgL IQR among patients on admission Table and patients older than years had significantly higher level Table To be specific CRP levels in serum were increased CRP mgL Table in of patients of and of showed an increase by more than CRP mgL Fig 1c At discharge only of Table had elevated level of CRP and the increased level did not exceed mgL except for one patient Fig 1cLevels of ESR were increased in of patients of ESR mmh Table and of Fig 1d had an increase exceeding mmh Similar to CRP patients older than years had statistically higher ESR level Table The median value of ESR on admission was mmh IQR while the value improved to mmh IQR at discharge Table There were still patients had ESR level more than mmh in serum Fig 1dCoagulation parametersThe coagulation parameters were examined on admission for patients Eighty percent of patients of Table had normal serum levels of Ddimer on admission Among the of patients of who had increased Ddimer level Ddimer mgL Table eight showed an increase by more than Ddimer 2mgL Fig 1f At discharge of patients seven of still had abnormal level of Ddimer though the results were very close to the normal range The fibrinogen level on the other hand had increased in of patients of fibrinogen mgL Table and decreased in of fibrinogen 2mgL Specifically patients of had an increase of fibrinogen level by more than fibrinogen mgL Fig 1e while most decreases were slight Furthermore when compared to patients younger than years fibrinogen level on admission was significantly higher in the patients older than years Table At discharge only seven patients still showed high level of fibrinogen Fig 1e Fibrinogen degradation product FDP was also s The authors Journal compilation J Clin Med Res and Elmer Press Inc„¢ wwwjocmr 0cLiu et alJ Clin Med Res cardial damage Since the outcome of this cohort is known as recovery and discharge from hospital we also collected the last available laboratory results Although all the patients met the discharge criteria for COVID19 some still had abnormal laboratory findings Especially we found the levels of the inflammatory biomarker ESR were still elevated for half of the patients tested Hence patients were asked to go back to the hospital for followup examination including the virus RNA test chest CT scan blood routine coagulation function and other biochemical indicators on both the 14th and 28th day after dischargePrevious studies have found that white blood cell WBC procalcitonin PCT aspartate aminotransferase AST lactose dehydrogenase LDH Cr hscTnL and Ddimer could indicate the progression of COVID19 on admission especially for the severe and mortal cases [ ] And in the moderate condition we analyzed most patients had these indicators in the normal range Therefore our findings are consistent with other studiesHowever these biomarkers were not all specific to COVID19 Similar to other virus infections including SARS [] and H1N1 influenza [] the neutrophil count was mostly normal in mild to moderate patients while lymphocyte count was significantly decreased As the major antiviral cells lymphocyte count declined down to — 109L in this cohort on admission And after receiving antiviral and supportive treatment the lymphocyte counts increased among all the patients at discharge Previous study found that lymphocyte counts would continually decrease in severe and critical COVID19 patients [] These results suggested that SARSCoV2 infection can suppress the level of lymphocyte Therefore closely monitoring lymphocyte counts could be one of the best methods when we evaluate the outcome of moderate patientsThe blood routine results also showed that about of patients had low level of hemoglobin red blood cell and hematocrit and the condition was not improved at discharge Previous studies also reported that of patients had hemoglobin below the normal range [] and the hemoglobin level of severe patients was lower although the difference was not statistically significant [] There might be two explanations First the inflammatory state caused by infection may interfere with erythrocytebone marrow metabolism and iron regulation [] and eventually result in a decline of hemoglobin and red blood cell Second patients could suffer from anemia for some time and appear more vulnerable to SARSCoV2 infection Once infected however correction of anemia could not benefit from all the symptoms of COVID19 and psychological stresses The decrease of functioning hemoglobin may contribute to hypoxia and further aggravate the disease in severe casesexamined and of patients had elevated level of FDP mgL Table Fig 1g on admission Twentyseven percent of patients of still showed increased amount of FDP at discharge Table Therefore fibrinogen and FDP were the most altered parameters in coagulation function from our analysisCardiac injury biomarkersCardiac injury biomarkers were monitored for patients on admission Among the patients assessed all had normal serum levels of high sensitive cardiac troponin L hscTnL ngmL The other cardiac biomarker examined was Nterminal prohormone brain natriuretic peptide NTproBNP and of patients of had elevated level NTproBNP and pgmL according to age Table on admission and five of had an increase by more than At discharge the NTproBNP level had decreased into the normal range except two male patients These two patients still had high level of NTproBNP more than pgmL however other cardiac biomarkers such as hscTnL and creatine kinaseMB CKMB were both in the normal range Considering their history of hypertension they were asked to reevaluate their heart function after the COVID19DiscussionAs an emerging infectious disease COVID19 is now a worldwide challenge for public health Among the million patients about present mild to moderate disease but studies dedicate to these patients are actually scarce Therefore we enrolled adult patients with moderate disease of COVID19 to elucidate the clinical manifestation and laboratory findingsOne of the risk factors of critical and mortal COVID19 is male with older age [] Considering the not severe condition of disease it is rational that female patients present more than male in this cohort Previous studies have found comorbidity such as diabetes and hypertension heart disease as other risk factors [] Similar to other study of patients had diabetes and had hypertension Similar to other studies in Asia [ ] patients mainly present fever cough asthenia and shortness of breath In Europe however a recent study showed that patients with mild to moderate disease mainly had headache loss of smell nasal obstruction and asthenia [] Considering the big differences it is possible that olfactory dysfunction might be neglected during the first wave of SARSCoV2 infection in Wuhan Also the virus is phylogenetically distinct from Asia type B to Europe type C [] which could contribute to different clinical outcomesThrough analysis of laboratory findings we found that patients with moderate disease of COVID19 had common characteristics on admission normal or increased neutrophil ratio low lymphocyte count decreased hemoglobin level increased inflammatory indicators ESR and CRP and some patients were complicated with coagulation disorder and myoExamination of inflammatory biomarkers found that PCT was in the normal range for moderate patients while CRP and ESR were increased in and of patients on admission respectively Other studies including mild to moderate disease showed heterogeneous results [] and found three of or of of patients had increased level of CRP [ ] Thus SARSCoV2 infection is involved with the disorder of inflammatory response Furthermore we find that the increase of both CRP and ESR were correlated with patient€™s age which may indicate that high inflammatory state s The authors Journal compilation J Clin Med Res and Elmer Press Inc„¢ wwwjocmr 0cClinical Features of Moderate COVID19J Clin Med Res and more severe pneumonia present more often in elderly people In addition the increase of CRP level was more transient as it returned to normal range in most people when they were discharged from hospital Nevertheless it took more time for ESR to return to normal level especially for patients older than years Certainly elder people would need more time to recover and to metabolize the extra inflammatory biomarkersAbnormal coagulation condition is known as associated with poor prognosis of COVID19 In particular serum level of Ddimer and FDP were significantly higher in lethal cases [] Other coronavirus infection such as SARS and Middle East respiratory syndrome MERS showed similar increased coagulation activities in severe cases [ ] The possible mechanism is that local inflammatory response induced by cytokine responses stimulates coagulation cascade and hemodynamic changes [ ] In patients with moderate COVID19 we found about and presented increased level in fibrinogen FDP and Ddimer on admission respectively And when compared to young people patients older than years tend to have higher levels of these coagulation parameters Certainly the hemodynamics and endothelial conditions are more complicate in elder people Furthermore SARSCoV2 infection as well as the inflammatory response it induced could deteriorate the existed dysregulation As the health condition improved at discharge these parameters decreased to normal ranges for most patients Yet again patients over years had significantly higher level of fibrin degradation products In the meantime we found that the platelet level had no obvious decrease in most patients with moderate disease Therefore as reported previously thrombocytopenia happens more often in severe and critical patients [] while abnormal coagulation condition is slight and temporary in moderate patientsThe receptor of SARSCoV2 angiotensinconverting enzyme ACE2 is expressed in myocardial cells and vascular endothelial cells [] It is possible that heart dysfunction is directly targeted by virus infection hence early evaluation and continued monitoring of cardiac damage are important [] We systematically examined the cardiac function biomarkers on admission CKMB and cTnL had no obvious change in these moderate patients while about of patients showed increased NTproBNP This percentage is much lower than previous reported of the severe cases that had elevated NTproBNP Furthermore we only found two patients still had NTproBNP higher than normal range at discharge indicating that moderate patients had little cardiac complicationThis study provides us more information about moderate COVID19 but still has some limitations First this is a relatively small singlecenter study Second due to the retrospective study design a few laboratory tests were not done in all patients In addition several patients were hospitalized during a short period that some tests were not reexamined before they were discharged Third we do not possess any viral kinetic data in these patients further studies are necessary to elucidate the correlation between viral load and laboratory changesIn this ongoing pandemic of infected people showed moderate disease [] it is important to understand more about this moderate spectrum of disease to settle into a longterm problem We conducted a cohort study of adult patients with moderate disease of COVID19 in Wuhan China Based on the clinical characteristics we conclude that SARSCoV2 infection inhibits the immune system of patients and induces a controllable inflammatory response in moderate COVID19 Elderly patients have higher inflammatory state and more dysregulated coagulation condition It is essential to closely assess their condition for a better clinical managementAcknowledgmentsThis study was supported by the Natural Science Foundation of China Hubei Province Department of Education Science and Technology Project B2019232 and the Wuhan Municipal Education Bureau Project Financial DisclosureThere are no financial conflicts of interest to discloseConflict of InterestAll authors declare no competing interestsInformed ConsentAll subjects provided written informed consentAuthor ContributionsYL XZ and BS designed and performed the study YL and BS drafted the manuscript and did critical editing XZ and RG were involved in data collection YL JQ QY JS and WL analyzed the data BS carefully supervised this manuscript preparation and writingData AvailabilityAll data used in the study are available from the corresponding author by request Although some data confidential in nature may only be provided with restrictionsReferences Zhu N Zhang D Wang W Li X Yang B Song J Zhao X et al A novel coronavirus from patients with pneumonia in China N Engl J Med Coronaviridae Study Group of the International Committee on Taxonomy of Viruses The species Severe acute respiratory syndromerelated coronavirus classifying 2019nCoV and naming it SARSCoV2 Nat Microbiol Huang C Wang Y Li X Ren L Zhao J Hu Y Zhang s The authors Journal compilation J Clin Med Res and Elmer Press Inc„¢ wwwjocmr 0cLiu et alJ Clin Med Res L et al Clinical features of patients infected with novel coronavirus in Wuhan China Lancet Wang D Hu B Hu C Zhu F Liu X Zhang J Wang B et al Clinical characteristics of hospitalized patients with novel coronavirusinfected pneumonia in Wuhan China JAMA WHO Coronavirus disease COVID19 Situation Report Available from wwwwhointdocsdefaultsourcecoronavirusesituationreports20200628covid19sitrep160pdfsfvrsn2fe1c658_2 Wu Z McGoogan JM Characteristics of and important lessons from the coronavirus disease COVID19 outbreak in China summary of a report of cases from the Chinese Center for Disease Control and Prevention JAMA WHO Clinical management of COVID19 Available from wwwwhointpublicationsdetailclinicalmanagementofcovid19 Liu K Chen Y Lin R Han K Clinical features of COVID19 in elderly patients A comparison with young and middleaged patients J Infect 2020806e14e18 Zheng Z Peng F Xu B Zhao J Liu H Peng J Li Q et al Risk factors of critical mortal COVID19 cases A systematic literature review and metaanalysis J Infect 2020812e16e25 Li LQ Huang T Wang YQ Wang ZP Liang Y Huang TB Zhang HY et al COVID19 patients' clinical characteristics discharge rate and fatality rate of metaanalysis J Med Virol Cao Y Liu X Xiong L Cai K Imaging and clinical features of patients with novel coronavirus SARSCoV2 A systematic review and metaanalysis J Med Virol Lechien JR ChiesaEstomba CM Place S Van Laethem Y Cabaraux P Mat Q Huet K et al Clinical and epidemiological characteristics of European patients with mildtomoderate coronavirus disease J Intern Med Forster P Forster L Renfrew C Forster M Phylogenetic network analysis of SARSCoV2 genomes Proc Natl Acad Sci U S A Zhou F Yu T Du R Fan G Liu Y Liu Z Xiang J et al Clinical course and risk factors for mortality of adult inpatients with COVID19 in Wuhan China a retrospective cohort study Lancet Velavan TP Meyer CG Mild versus severe COVID19 Laboratory markers Int J Infect Dis Lee N Hui D Wu A Chan P Cameron P Joynt GM Ahuja A et al A major outbreak of severe acute respiratory syndrome in Hong Kong N Engl J Med Cheng Y Zhao H Song P Zhang Z Chen J Zhou YH Dynamic changes of lymphocyte counts in adult patients with severe pandemic H1N1 influenza A J Infect Public Health Chen N Zhou M Dong X Qu J Gong F Han Y Qiu Y et al Epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in Wuhan China a descriptive study Lancet Cavezzi A Troiani E Corrao S COVID19 hemoglobin iron and hypoxia beyond inflammation A narrative review Clin Pract Zeng F Huang Y Guo Y Yin M Chen X Xiao L Deng G Association of inflammatory markers with the severity of COVID19 A metaanalysis Int J Infect Dis Chen G Wu D Guo W Cao Y Huang D Wang H Wang T et al Clinical and immunological features of severe and moderate coronavirus disease J Clin Invest Wang F Hou H Luo Y Tang G Wu S Huang M Liu W et al The laboratory tests and host immunity of COVID19 patients with different severity of illness JCI Insight Tang N Li D Wang X Sun Z Abnormal coagulation parameters are associated with poor prognosis in patients with novel coronavirus pneumonia J Thromb Haemost Wong RS Wu A To KF Lee N Lam CW Wong CK Chan PK et al Haematological manifestations in patients with severe acute respiratory syndrome retrospective analysis BMJ AlAbdallat MM Payne DC Alqasrawi S Rha B Tohme RA Abedi GR Al Nsour M et al Hospitalassociated outbreak of Middle East respiratory syndrome coronavirus a serologic epidemiologic and clinical description Clin Infect Dis Moore JB June CH Cytokine release syndrome in severe COVID19 Science Giannis D Ziogas IA Gianni P Coagulation disorders in coronavirus infected patients COVID19 SARSCoV1 MERSCoV and lessons from the past J Clin Virol Guan WJ Ni ZY Hu Y Liang WH Ou CQ He JX Liu L et al Clinical Characteristics of Coronavirus Disease in China N Engl J Med MendozaTorres E Oyarzun A MondacaRuff D Azocar A Castro PF Jalil JE Chiong M et al ACE2 and vasoactive peptides novel players in cardiovascularrenal remodeling and hypertension Ther Adv Cardiovasc Dis Guzik TJ Mohiddin SA Dimarco A Patel V Savvatis K MarelliBerg FM Madhur MS et al COVID19 and the cardiovascular system implications for risk assessment diagnosis and treatment options Cardiovasc Res s The authors Journal compilation J Clin Med Res and Elmer Press Inc„¢ wwwjocmr 0c" Answer:

Thyroid_Cancer

range of diseases including malignancies and autoimmune disordersIts high eï¬ectivenessprice ratio also won extensive application in ophthalmology On the other hand although MTX has anexcellent pharmacological efficacy MTX associated side eï¬ects in clinical use which vary from patient to patient are nonnegligible ere is no comparatively systematic review on MTX associated side eï¬ects and its risk factors is review aimed toreveal novel clinical approaches of MTX and its adverse eï¬ects in order to provide a reference for ophthalmic scholars in clinicalapplication of MTX IntroductionMethotrexate MTX is an antifolate metabolite that inhibitsDNA synthesis repair and cellular replication It was firstlyused as one of the essential treatments of pediatric leukemia[ ] According to previous studies MTX has also beenused to treat rheumatoid arthritis RA and psoriasis as anti‚ammatory and immunomodulatory agent [] as MTXcould not only optimize the efficacy of biological diseasemodifying antirheumatic drugs DMARDs [ ] but alsomake the therapeutic goals via lower doses in comparisonwith other conventional synthetic DMARDs [] Figure shows the pathway of folate in DNA synthesis the cellularpathway of MTX and how MTX works inside the cell Whileimmediate and lowdose MTX is used to treat nonmalignantand immunemediated disorders highdose MTX HDMTX more than mgm2week is widely used to treatmalignancies Until now HDMTX with or without radiation therapy is still the backbone of most modern chemotherapy regimens [] as well as the prevention ofsystemiccentral nervous system CNS lymphoma recurrence at a dose of gm2 per week []MTX has also been widely applied in ophthalmic diseases systemically and locally Recently published spay more attention to new clinical applications routes ofadministration and newly discovered side eï¬ects which arefoci of this review Clinical Applications in OphthalmologyAs one of the known corticosteroidssparing agents MTXhas been widely used in the treatment of anterior intermediate posterior or pan uveitis scleritis and ocularmucous membrane pemphigoid [] as well as advancedproliferative diabetic retinopathy [] However followedresearches reveal that MTX works with a significant difference in eï¬ectiveness ratings by anatomic location of‚ammation [] with treatment success achieved mostcommonly in patients with anterior uveitis and scleritis []In the treatment of noninfectious intraocular ‚ammationoral and intravenous are the most common routes with ausual dose range of mg to mg weekly e typical doseobserved was mgweek [ ] which is in the range oflowdose MTX e median time to achieve the success of 0cJournal of OphthalmologyFigure e cellular pathway of folate and MTX Dietary folate enters the cells through RFC1 as well as MTX In lowdose MTX treatmentMTX inhibits enzymes of the folate pathway Ultimately MTX leads to an increase in intracellular adenosine level which would cause anti‚ammatory eï¬ects RFC1 � reduced folate carrier ABC family � adenosine triphosphatebinding cassette ABC family DHF � dihydrofolate THF � tetrahydrofolate GGH � cglutamyl hydrolase FPG � folylpolyglutamate synthase MTXPG � methotrexate polyglutamate DHFR � dihydrofolate reductase MTHFR � methylene tetrahydrofolate reductase AICAR � aminoimidazole carboxamideribonucleotide ATIC � AICAR transformylasetreatment defined as control of ‚ammation with theability to taper corticosteroids to mg or less daily rangesfrom months to months for MTX [ ]Intravitreal MTX injection with or without systemicchemotherapy and radiotherapy has already been used totreat primary intraocular lymphoma patients [ “]According to Larkin [] intravitreal MTX injectioncould achieve remission in a proportion of patients withprimary intraocular lymphoma What is particularly noteworthy is that although MTX has a slow rate of onset ofeï¬ect when it was used to treat intraocular lymphoma viaintravitreal injection it prolonged local remission of oculardisease even with an aggressively growing tumor []erefore it has been taken as a relatively firstline choice forthe treatment of recurrent intraocular lymphoma [] although the treatment for primary intraocular lymphoma islacking solid justification because of the limited retrospectiveand prospective case series [] Local treatment via intraocular injection provides a consistent therapeutic MTXconcentration to reduce the systemic MTX associated sideeï¬ects [] erefore intraocular MTX injection is worthtrying especially for unilateral ocular diseases New Approaches of Applications of MTX MTX Used against Epithelial Downgrowth Previousstudies have already demonstrated safety of intravitrealMTX [] It has been used to treat intraocular lymphomaand proliferative vitreoretinopathy because ofits antiproliferative properties [] ere is a novel use of intravitreal MTX for recurrent epithelial downgrowth which wasnot treated by surgical and medical methods Lambert et al[] administered intravitreal MTX to patients with refractory proliferative membrane after cataract surgery whilemembrane peel and endolaser treatment failed e injectionof MTX was administered alone based on previous protocols and the presumed halflife of drugs in vitreous cavityAfter injections totally there was no membrane recurrence is case suggests that intravitreal MTX plays a role intreatment against epithelial downgrowth MTX Used in Conventional erapyResistant DiseasesGenerally the antivascular endothelial growth factor antiVEGF therapy has dramatically improved the prognosis ofneovascular agerelated macular degeneration nAMDHowever there are still some patients who remain refractoryto antiVEGF therapy which is termed as treatmentresistant nAMD As there is evidence that MTX has eï¬ects ininterrupting the angiogenesis cascade at various levels []Kurup oï¬ered intravitreal MTX to patients who wererefractive to standard antiVEGF therapy [] Although itwas an oï¬label use the patients™ visual acuity improved atfollowup visit while ophthalmic imaging examinationsshowed significantly reduced cystoid macular edema usFolatefolic acidRFC 1Folatefolic acidDHFTHFDHFR5MTHFMTHFRPurine and pyridine synthesis DNA synthesisMTXMTXABC familyFPGGGHMTXFGAICAR5FormylAICARATICAdenosine †‘AdenosineCell membraneCell membrane“““ 0cJournal of Ophthalmologypatients who are refractory to traditional antiVEGF therapymight benefit from intravitreal injection of MTXis approach is not alone Khalil [] had μg01 ml of MTX intravitreal injection once monthlyadministrated to adult Behcet™s disease BD patientssuï¬ering from BDassociated ocular ‚ammation withposterior segment involvement eir results prove thatintravitreal MTX improves visual acuity reduces posteriorsegment manifestations associated with Behcet™s disease andallows the reduction of corticosteroids and immunosuppressive drugs [] ese results also supported Taylor andassociates who conducted trials on patients with unilateral uveitis andor cystoid macular edema [] eirclinical trials suggest that intravitreal MTX may help patients with uveitisassociated posterior segment involvementto regain normal anatomical structure and then allowed thereduction of immunosuppressive therapy The Pharmacogenetics of MTXWith molecular sequencing and highthroughput technology large numbers of genetic polymorphisms can now bedetected accurately and rapidly [] Researchers pay moreattention to pharmacogenetics the study of genetic polymorphisms in drugmetabolizing enzymes and the translation of inherited diï¬erences to diï¬erences in drug eï¬ects[] e genes encoding transporting proteins and metabolizing enzymes for MTX are also known to harborfunctionally significant SNPs e SNPs may ‚uence theefficacy of MTX and have been suggested as potential riskfactors for enhanced MTX toxicity even in lowdose regimens based on previous researches []e research of pharmacogenetics of MTX could bedivided into genetic polymorphisms aï¬ecting MTX transport and SNPs that‚uence enzymes in the cellularpathway of MTX []Once taken MTX enters the cell through an activetransport which is mediated by the reduced folate carrier RFC1 e loss of RFC1 gene expression might lead toeï¬ects of uptake and intracellular levels of MTX A G80ASNP of RFC1 was proposed [] making a decreasing [] orincreasing [ ] eï¬ect on intracellular level of MTXerefore a significant association between RFC1 SNPs andMTX toxicity should be considered Chango state thatthese SNPs strongly impact the overall MTX associated sideeï¬ects by resulting in altered cellular MTX concentrationbut with no ‚uence on MTX efficacy [] However someresearchers argue that these SNPs have no definite eï¬ect[] us it remains controversial whether SNPs of RFC1aï¬ect the transport of MTX Moreover Pglycoprotein amembrane transporter that has ‚uences on the dispositionand bioavailability of MTX [] was studied SNPs ofABCB1 including C3435T SNP and C1236T SNP werebelieved to have eï¬ects on the expression of Pglycoprotein[] Gervasini speculate the C1236T SNP of ABCB1aï¬ects the administered doses of MTX and the incidence ofhematological toxicity [] However just like G80A SNPthere are disputes about the ‚uences of these SNPs asdiï¬erent studies had diï¬erent outcomes []Metabolizing enzymes were also being analyzed giventhe critical role of transporters in disposition of MTX and itsactive products as well as the folate metabolism MTXpharmacogenetics mostly focused on the SNPs in theMTHFR gene e present study shows that genetic polymorphisms in the folate metabolic pathway and in MTXtransporters ‚uence the toxicity but not the efficacy of thelowdose MTX treatment in patients with autoimmunediseases [] For example C677T and A1298C are knownin MTHFR gene to result in a lower enzyme activity []Windsor and associates reported that MTHFR A1298C andC677T were associated with MTX related nephrotoxicityand anemia [] ese SNPs might be associated withdecreased activity of methylenetetrahydrofolate reductaseelevated plasma homocysteine levels and altered distribution of folate [] us patients with this genotype weremore vulnerable to potential MTX induced toxicity sincethese reactions above may lead to slower folate metabolismand slower cell repair [] Weisman used univariatelogistic regression to reveal that the MTHFR C677T alsoincreases the occurrence of side eï¬ects in central nervoussystem manifested as headache and lethargy [] HoweverLambrecht argued that MTHFR C667T was not apredictive factor for toxicity [] Berkani found noassociation between A1298C polymorphism and MTXtoxicity [] Interestingly Grabar claimed that thepatients with MTHFR 1298C genotype have a lower risk forMTX toxicity than the carriers of MTHFR 1298A allele []To date the study of pharmacogenetics of MTX continues An increasing number of SNPs have been found to bepossibly associated with the efficacy and toxicity of MTXe newly discovered genotypes include C347G in ATICand ²UTR 28bp repeat and ²UTR 6bp deletion inTYMS which may ‚uence both efficacy and toxicity ofMTX similarly factors that may aï¬ect MTX associatedtoxicity are for example A2756G in MS and A66G in MTRR[ ] e genes and their SNPs that might beassociated with the eï¬ects and side eï¬ects of MTX aresummarized in Table Growing evidences suggest that asingle genetic factor is unlikely to adequately predict theefficacy and toxicity of MTX in polygenic disease such as RAand autoimmune associated ocular disease Given the impactof MTX in several metabolic pathways a complex of multiple risk genotypes examination would help to predict theefficacy of MTX and to identify patients who may haveadverse eï¬ects from MTX administrationTaken together the efficacy and toxicity of MTX mayremain associated with the genetic markers in the patientserefore although this remains a controversial subject it isreasonable to believe that pharmacogenetics may be able topredict who is at risk of MTX associated adverse eï¬ects andmay help in maximizing the benefitrisk ratio of MTX The Side Effects of MTXe doselimiting toxicity of MTX mainly includes hepatotoxicity and nephrotoxicity [“] but mortality hasoften been reported due to either pneumonitis or secondaryinfections [] 0cJournal of OphthalmologyTable Summary of genes and their SNPs which might have possible clinical eï¬ects towards MTXTransportingproteinsABCfamilyGeneRFC1 [“]ABCB1 [ ]ABCC1 []ABCC2 []ABCC4 []MetabolizingenzymesMTHFR [“]ATIC [“]TYMS [ ]GGH [ ]DHFR [ ]MS [ ]MTRR [ ]SNPsG80AC3435TC1236Trs246240Srs3784862A2412GG1249AG1058AC934AC677TA1298CC347G²UTR 28bp²UTR 6bprepeatdeletionC452TC401TT721AC830TA2756GA66GSome experts divided MTX associated pulmonarycomplications into ‚ammatory infectious and lymphoproliferative [] In the authors™ opinion all MTX relatedside eï¬ects can be classified into these three categoriesaccording to the pharmacological eï¬ects of MTXMajor adverse events for MTX are related to the folateantagonism and primarily aï¬ect highly proliferative tissuessuch as bone marrow and gastrointestinal mucosa []Given the immunosuppression eï¬ect of MTX pancyt iawas one of the most frequent severe toxicities of methotrexate [] Meanwhile the risk of developing an infectiousprocess is increased all along the treatment and the severityof the infected disease would be worsen [ ] includingcommon bacterial infections herpes zoster eruptions andopportunistic infections According to previous studies therisk is larger than that with other disease modifying antirheumatic nonbiological drugs DMARDsSecondly the MTX acts as the hapten [] and is likely toreact directly with nucleophilic groups present in proteins ieto combine with endogenous protein [ ] e proteinadducts thus act as an antigenic signal to direct the eï¬ector armof the immune response [] e provoked immune responsesare most commonly type I immediate hypersensitivity andtype III immune complex reactions [] Hypersensitivitypneumonitis is the most common severe and unpredictablecomplication with a mortality of up to almost []Moreover a few studies have shown that longterm MTXuse can lead to lymphoproliferative disorders LPDs in bothnodal sites and extra nodal sites such as the skin lungsepipharynx thyroid gland nasal cavity spleen and kidneysespecially for patients who are positive for EBV infection[“] e reported frequency of EBV positive in MTXassociated LPDs patients is “ [] Although themechanism of onset is not fully understood it is believedPossible clinical eï¬ectsIncreasingdecreasing intracellular MTX levelAï¬ecting efficacy of MTXAï¬ecting the distribution of MTX and incidence of hematological toxicityAssociation with MTX related toxicityLeading to accumulation of MTX to nephrotoxic levelsAssociation with MTX related gastrointestinal toxicityAssociation with MTX related hepatotoxicityAssociation with MTX related hematological toxicityAï¬ecting the toxicity but not the efficacy by resulting in a lower enzymeactivity association with related nephrotoxicity anemia and neurologicside eï¬ectsAï¬ecting efficacy and toxicity of MTXAï¬ecting efficacy and toxicity of MTXAï¬ecting efficacy of MTXAï¬ecting efficacy of MTXAï¬ecting efficacy of MTXAssociation with MTX associated toxicityAssociation with MTX associated toxicitythat the combination of immunodeficiency and the immunosuppressive eï¬ect of MTX has been implicated in thepathogenesis of MTX associated LPDs e World HealthOrganization WHO has classified MTX associated LPDs aslymphoid neoplasms whether iatrogenic or immunodeficiency associated diseases [ ] MTX associated LPDsoften take a spontaneous remission which tends to completemostly within weeks after the discontinuation of MTX[] But there are a few reports showing that the lymphoidneoplasms occur even after stopping using MTX [] e Eï¬ects of Administration Routes Generally the sideeï¬ects of MTX depend on the route of administration Dosedependent [] gastrointestinal side eï¬ects are the mostfrequent side events with orally administered MTX as oraladministration is the most common delivery method[ ] More than of MTX is excreted by therenal system thus MTX associated nephrotoxicity is common among patients taking MTX Fortunately the resolution usually occurs after discontinuation of therapy andsalvage treatment with highdose corticosteroids[]erefore to achieve treatment with less side eï¬ects theappropriate route of administration and dose of MTX arenecessary During the treatment monitoring of patients™general condition mattersAdverse eï¬ects of intravitreal injections of MTX occuronly within the eyeincluding hyperemia keratopathycataract iridocyclitis vitreous hemorrhage retinal detachment maculopathy and endophthalmitis []Splitting doses of MTX rather than intravenous administration is a new attempt to avoid MTX associated sideeï¬ects MTX is split and given twice or thrice in a week toachieve higher bioavailability and better clinical response 0cJournal of Ophthalmology[ ] thus providing us with a novel method of oraladministration of MTX with less adverse eï¬ects Is LowDose MTX Safer Based on clinical cases observation side eï¬ects which can lead to discontinuation ofMTX are rare during the typical ophthalmology treatmentbecause of the lower dose of MTX required [] e application of lowdose MTX regimen has also become one ofthe main therapies of a variety of immunemediated diseasesbecause of its efficacy and an acceptable safety profile asmost lowdose MTX associated toxicity has been describedin case reports and relatively small case series []However although welltolerated and mostly reversibleeven a lowdose regimen of MTX can result in clinicallysignificant toxicity with substantial death rates about according to Kivity™s cohort study [] e lowdose MTXassociated severe adverse eï¬ects include major centralnervous system complications [] mucositis pulmonaryinvolvement hepatotoxicity [] and myelosuppression Is MTX Safe to the Pregnant and Fetuses As one of thelipidsoluble and low molecular weight drugs MTX could bereadily transferred across the placental membrane duringpregnancy and adversely aï¬ect the fetus [] In addition MTXmight take longer time for elimination in fetal tissues []Regarding pharmacogenetics mutations caused by MTXlead to severe decrease of the expression of folate and nucleobaseenzymes which are critical for cellular homeostasis [] Inpractice MTX aï¬ected formation of the blastocyst and causeddysmorphic features and neurologic defects in early pregnancyleading to malformations in some cases [] Multiple congenitalabnormalities have been observed after weekly MTX treatmentsat a mg dose during the first months of pregnancy [] evenfetal death [] Verberne had reviewed cases of congenitalanomalies after in utero exposure to MTX and proved that somecongenital anomalies such as microcephaly craniosynostosistetralogy of Fallot pulmonary valve atresia limb reductiondefects and syndactyly were truly part of the œfetal methotrexatesyndrome [] Administration of MTX in childhood mightalso cause manifestations including visual defect [] andSmith“Magenis syndrome [] among patients with specificmutations us special care should be taken with pregnantpatients and children in particular e Risk Factors of MTX Associated Side Eï¬ects e mostcommon risk facts of MTX induced adverse eï¬ects areadvanced age age years and underlying disease incaused by MTX administration with100 g alcohol concluding renal andor hepatic insufficiency and lung diseaseespecially patients with chronic hepatitis B and diabetesmellitus [“] Patients with a history of alcohol intakemight have a greater risk of liver fibrosis and hepatotoxicitysumption per week [] Also preexisting hypoalbuminemiaand past use of any of the DMARDs and protonpumpinhibitors have been described in studies to increase theincidence of MTX induced side eï¬ects [ ] Moreovertaking drugs that may interact with MTX at the same timemight also be dangerous these drugs include salicylatescotrimoxazole chloramphenicol sulfonamides cyclosporine and pyrimethamine [] Although no significantprotective eï¬ect of folate supplementation on MTX relatedtoxicity has been found [] the folate deficiency is anotherreason for the side eï¬ects based on clinical cases []Heidari found that MTX administration elevatedkidney ROS levels decreased tissue antioxidant capacityincreased lipid peroxidation and depleted renal glutathionestores eir research data indicate that MTX caused tissuedamage and organ dysfunction through oxidative stresserefore they proposed that patients with preexistingmitochondrial defects might be vulnerable to MTX inducedrenal injury []e use of highdose MTX HDMTX is also the riskfactor of adverse eï¬ects MTX induced liver fibrosis is morelikely to become morphologically evident with high cumulative doses possibly largely exceeding to mg[ ] and the side eï¬ects caused by omeprazole use inthe past were found in cancer patients receiving HDMTXtreatment []e distribution of MTX in vivo also plays a role in MTXrelated side eï¬ects As MTX tends to accumulate in theextravascular compartment patients with pleural eï¬usionascites and massive edema should get extra caution due tothe risk of toxicity from reabsorption of extravascular fluid[]Another noteworthy risk factor is UV UV recall phenomenon also known as MTX associated UV reactivationhas been reported [ ] It is reactivity of sunburn areaswithin to days of the treatment with MTX [ ]According to Adams and associates this phenomenon mightbe due to the immune response by uncontrolled sunburninduced ‚ammation released by MTX [] Patients whopreviously suï¬ered sunburns deserve more detailed monitoring when methotrexate is needed Is Folate Supplementation Necessary for OphthalmicPatients To prevent MTX associated side eï¬ectsit iscommon to take folate [as either folic acid FA or folinicacid FLA] in clinic [ ] However there is noconsistent and evidencebased guideline for folate supplementation in ophthalmic patientsFolate and folic acid play significant roles in the de novosynthesis of purines and thymidylate which are required forDNA replication and repair [] Funk and associates founda significant reduction of circulating folate concentrations in of patients receiving MTX treatment [] Patientstreated with highdose MTX HDMTX got routine folatesupplementation to reduce HDMTX associated side eï¬ects[“] After a systematic literature review of HDMTXtherapy and folate supplementation Van der Beek et al[] found lower incidence of MTX associated adverseeï¬ects in regimens with higher cumulative doses and earlieradministration of folate supplementation in similar HDMTX dosage studies Folate supplementation in patientswith lowdose methotrexate is also being studied Ortiz et al[] had proved the protectivefolateeï¬ectof 0cJournal of Ophthalmologysupplementation by conducting a Cochrane review including more than patients taking lowdose MTX Untilnow folate supplementation had been proved to prevent andimprove MTX associated eï¬ects including gastrointestinalrespiratory and neurologic side eï¬ects [ ] Mori et alsupported the protective eï¬ect by demonstrating that patients treated with lowdose MTX without folate supplements were significantly associated with the development ofmyelosuppression and pancyt ia []However Arabelovic and associates™ preliminary studyshowed a significant increase of MTX dose needed []since folic acid fortification enriched cereal grain productswere fully implemented in the USA and Canada [] isconveyed a message to us that high dose of folate supplementation might have ‚uence on the efficacy of MTXAlDabagh found that the reduction in efficacy ofMTX cannot be ignored while folate supplementation didmake a significant reduction in associated adverse eï¬ects[] Salim declared the decreasing ‚uence betweenthe anti‚ammatory eï¬ect of MTX and folate supplementation by carrying out a doubleblind clinical trial []Chladek had conducted an labeltwowaycrossover study supporting the opinion above [] Additionally because of the unequal distribution of folic acidand MTX in organs and tissues [] MTX discontinuationis more common for some MTX associated side eï¬ects inophthalmic clinic [] rather than higher dosage of folatesupplementationere are no ophthalmic studies to demonstrate theprotective eï¬ects of folic acid supplementation us although the folate supplementation is widely used amongpatients treated with lowdose MTX [ ] the necessityand standardized dosage of folate supplementation in specific patients [] as well as the MTXfolate interactionstill warrant further studies DiscussionMethotrexate as one of the alternative pharmacologicalsteroidsparing immunosuppressive agentsis becomingmore and more popular as the preferred treatment in severalautoimmune conditions requiring longterm immunosuppression [] Lowdose MTX has anti‚ammatory andimmunomodulatory properties by increasing levels of intracellular and extracellular adenosine [] which is thefoundation of ophthalmic MTX treatment e standardizedand recommended administration of ophthalmic MTXtreatment is once a week starting with a dose of mg andescalating every to weeks up to “ mgweek whennecessary [ ] In patients with insufficient response toMTX alone cyclosporin with or without azathioprine wasadded []To avoid side eï¬ects split doses of MTX administrationand folate supplementation are gradually being used inophthalmic clinic Prescription of to mg of folatesupplementation has a significant role in MTX safety []but the higher dosage is less applied even with higher dose ofMTX [] Prophylactic folate supplementation is notnecessary in most patients [] ere is also research toconvey that ml100 g or above dosage of fish oil is aseï¬ective as folinic acid in therapeutic potential in preventingbone loss during MTX chemotherapy [] For some resistant andor mortal adverse eï¬ects the discontinuation ofMTX will work instantlyWith the increasing longterm use of MTX it is importantto monitor patients™ blood examination results including bloodroutine and liver and renal functions As pancyt ia can be alate manifestation [] elevation of urea creatinine aminotransferases and albumin as well as electrolytes disturbancesmay result in MTX associated liver and renal side eï¬ects []Plasma MTX level is not a reliable predictor for adverse eventsin MTX therapy [] On the contrary circulating folate levelsand folate polyglutamate distribution change sensitively withMTX exposure and exogenous folate supply [] and could beused as a biomarker of MTX efficacy [] It should be notedthat as erythrocytes have a halflife of approximately daysthe results of blood examinations might reflect both pretreatment and posttreatment status which need to be analyzedcarefully []Numerous studies had been conducted to prove thatMTX could be used as a welltolerated safe and eï¬ectivefirstline treatment Hence the MTX administration shouldnot continue to be stigmatized as a œcancer drug or to bediscouraged because of associated adverse eï¬ects Contrarily the indication and the routes of administration areabout to gradually widenConflicts of Intereste authors declare that they have no conflicts of interestReferences[] R Q H Kloos R Pieters C van den Bos œe eï¬ect ofasparaginase therapy on methotrexate toxicity and efficacy inchildren with acute lymphoblastic leukemia Leukemia Lymphoma vol no pp “ [] R K Bath N K Brar F A Forouhar and G Y Wu œAreview of methotrexateassociated hepatotoxicity Journal ofDigestive Diseases vol no pp “ [] W Wang H Zhou and L Liu œSide eï¬ects of methotrexatetherapy for rheumatoid arthritis a systematic review European Journal of Medicinal Chemistry vol pp “[] J Smolen and R Landew´e œEULAR recommendations forthe management of rheumatoid arthritis with synthetic andbiological diseasemodifying antirheumatic drugs update Annals of the Rheumatic Diseases vol no pp “ [] J A Singh K G Saag S L Bridges œ Americancollege of rheumatology guideline for the treatment ofrheumatoid arthritis Arthritis Rheumatology vol no pp “ [] M Holdhoï¬ P Ambady A Abdelaziz œHighdosemethotrexate with or without rituximab in newly diagnosedprimary CNS lymphoma Neurology vol no pp “ [] J Pe™er J M Rowe S Frenkel and E J Dann œTesticularlymphoma intraocularvitreoretinal lymphoma and brainlymphoma involvement of three immunoprivileged sites in 0cJournal of Ophthalmologyone patient American Journal of Hematology vol no pp “ [] S Gangaputra C W Newcomb T L Liesegang et alœSystemic immunosuppressive therapy for eye diseases cohort study methotrexate for ocular ‚ammatory diseasesOphthalmology vol no pp “ [] P W Hardwig J S Pulido J C Erie K H Baratz andH Buettner œIntraocular methotrexate in ocular diseasesother than primary central nervous system lymphomaAmerican Journal of Ophthalmology vol no pp “ [] E Esterberg and N R Acharya œCorticosteroidsparingtherapy practice patterns among uveitis specialists Journalof Ophthalmic Inflammation and Infection vol no pp “ [] K Durrani F R Zakka M Ahmed M MemonS S Siddique and C S Foster œSystemic therapy withconventional and novel immunomodulatory agents for ocular ‚ammatory disease Survey of Ophthalmology vol no pp “ [] S S Gangaputra C W Newcomb M M Joï¬e et alœComparison between methotrexate and mycophenolatemofetil monotherapy for the control of noninfectious ocular‚ammatory diseases American Journal of Ophthalmologyvol pp “ [] V K Ayuso E L van de Winkel A Rothova and J Helenade Boer œRelapse rate of uveitis postmethotrexate treatmentin juvenile idiopathic arthritis American Journal of Ophthalmology vol no pp “ [] S R Rathinam M Babu R undikandy œA randomized clinical trial comparing methotrexate and mycophenolate mofetil for noninfectious uveitis Ophthalmologyvol no pp “ [] A Galor D A Jabs H A Leder œComparison ofantimetabolite drugs as corticosteroidsparing therapy for‚ammation Ophthalmologynoninfectiousvol no pp “ ocular[] M D de Smet V S Vancs D Kohler D Solomon andC C Chan œIntravitreal chemotherapy for the treatment ofrecurrent intraocular lymphoma British Journal of Ophthalmology vol no pp “ [] E Kim C Kim J Lee and Y Cho œA case of primaryintraocular lymphoma treated by intravitreal methotrexateKorean Journal of Ophthalmology vol no pp “[] J Smith J T Rosenbaum D J Wilson œRole ofintravitreal methotrexate in the management of primarycentral nervous system lymphoma with ocular involvementhistorical image Ophthalmology vol no pp “ [] CC Chan and D J Wallace œIntraocular lymphomaupdate on diagnosis and management Cancer Controlvol no pp “ [] K L Larkin U S Saboo G M Comer œUse ofintravitreal rituximab for treatment of vitreoretinallymphoma British Journal of Ophthalmology vol no pp “ [] C P Fox E H Phillips J Smith œGuidelines for thediagnosis and management of primary central nervoussystem diï¬use large Bcell lymphoma British Journal ofHaematology vol no pp “ [] A Sadaka R Sisk J Osher O Toygar M Duncan andinfusion forœIntravitreal methotrexateC Riemannproliferative vitreoretinopathy Clinical Ophthalmologyvol pp “ [] N G Lambert DJ Wilson D M Albert andW D Chamberlain œIntravitreal methotrexate for recurrentepithelial downgrowth JAMA Ophthalmology vol no p [] A M Joussen F E Kruse HE V¨olcker and B KirchhofœTopical application of methotrexate for inhibition of cornealangiogenesis Graefe™s Archive for Clinical and ExperimentalOphthalmology vol no pp “ [] S K Kurup C Gee and C M Greven œIntravitrealmethotrexate in therapeutically resistant exudative agerelated macular degeneration Acta Ophthalmologica vol no pp e145“e146 [] H E M Khalil H A Raafat N A Azab H E Haroun andH A Elgendi œe role of intraocular methotrexate in themanagement of uveitis and posterior segment involvementin Behçet™s disease patients e Egyptian Rheumatologistvol no pp “ [] S R J Taylor A Banker A Schlaen œIntraocularmethotrexate can induce extended remission in some patients in noninfectious uveitis Re

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You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: range of diseases including malignancies and autoimmune disordersIts high eï¬ectivenessprice ratio also won extensive application in ophthalmology On the other hand although MTX has anexcellent pharmacological efficacy MTX associated side eï¬ects in clinical use which vary from patient to patient are nonnegligible ere is no comparatively systematic review on MTX associated side eï¬ects and its risk factors is review aimed toreveal novel clinical approaches of MTX and its adverse eï¬ects in order to provide a reference for ophthalmic scholars in clinicalapplication of MTX IntroductionMethotrexate MTX is an antifolate metabolite that inhibitsDNA synthesis repair and cellular replication It was firstlyused as one of the essential treatments of pediatric leukemia[ ] According to previous studies MTX has also beenused to treat rheumatoid arthritis RA and psoriasis as anti‚ammatory and immunomodulatory agent [] as MTXcould not only optimize the efficacy of biological diseasemodifying antirheumatic drugs DMARDs [ ] but alsomake the therapeutic goals via lower doses in comparisonwith other conventional synthetic DMARDs [] Figure shows the pathway of folate in DNA synthesis the cellularpathway of MTX and how MTX works inside the cell Whileimmediate and lowdose MTX is used to treat nonmalignantand immunemediated disorders highdose MTX HDMTX more than mgm2week is widely used to treatmalignancies Until now HDMTX with or without radiation therapy is still the backbone of most modern chemotherapy regimens [] as well as the prevention ofsystemiccentral nervous system CNS lymphoma recurrence at a dose of gm2 per week []MTX has also been widely applied in ophthalmic diseases systemically and locally Recently published spay more attention to new clinical applications routes ofadministration and newly discovered side eï¬ects which arefoci of this review Clinical Applications in OphthalmologyAs one of the known corticosteroidssparing agents MTXhas been widely used in the treatment of anterior intermediate posterior or pan uveitis scleritis and ocularmucous membrane pemphigoid [] as well as advancedproliferative diabetic retinopathy [] However followedresearches reveal that MTX works with a significant difference in eï¬ectiveness ratings by anatomic location of‚ammation [] with treatment success achieved mostcommonly in patients with anterior uveitis and scleritis []In the treatment of noninfectious intraocular ‚ammationoral and intravenous are the most common routes with ausual dose range of mg to mg weekly e typical doseobserved was mgweek [ ] which is in the range oflowdose MTX e median time to achieve the success of 0cJournal of OphthalmologyFigure e cellular pathway of folate and MTX Dietary folate enters the cells through RFC1 as well as MTX In lowdose MTX treatmentMTX inhibits enzymes of the folate pathway Ultimately MTX leads to an increase in intracellular adenosine level which would cause anti‚ammatory eï¬ects RFC1 � reduced folate carrier ABC family � adenosine triphosphatebinding cassette ABC family DHF � dihydrofolate THF � tetrahydrofolate GGH � cglutamyl hydrolase FPG � folylpolyglutamate synthase MTXPG � methotrexate polyglutamate DHFR � dihydrofolate reductase MTHFR � methylene tetrahydrofolate reductase AICAR � aminoimidazole carboxamideribonucleotide ATIC � AICAR transformylasetreatment defined as control of ‚ammation with theability to taper corticosteroids to mg or less daily rangesfrom months to months for MTX [ ]Intravitreal MTX injection with or without systemicchemotherapy and radiotherapy has already been used totreat primary intraocular lymphoma patients [ “]According to Larkin [] intravitreal MTX injectioncould achieve remission in a proportion of patients withprimary intraocular lymphoma What is particularly noteworthy is that although MTX has a slow rate of onset ofeï¬ect when it was used to treat intraocular lymphoma viaintravitreal injection it prolonged local remission of oculardisease even with an aggressively growing tumor []erefore it has been taken as a relatively firstline choice forthe treatment of recurrent intraocular lymphoma [] although the treatment for primary intraocular lymphoma islacking solid justification because of the limited retrospectiveand prospective case series [] Local treatment via intraocular injection provides a consistent therapeutic MTXconcentration to reduce the systemic MTX associated sideeï¬ects [] erefore intraocular MTX injection is worthtrying especially for unilateral ocular diseases New Approaches of Applications of MTX MTX Used against Epithelial Downgrowth Previousstudies have already demonstrated safety of intravitrealMTX [] It has been used to treat intraocular lymphomaand proliferative vitreoretinopathy because ofits antiproliferative properties [] ere is a novel use of intravitreal MTX for recurrent epithelial downgrowth which wasnot treated by surgical and medical methods Lambert et al[] administered intravitreal MTX to patients with refractory proliferative membrane after cataract surgery whilemembrane peel and endolaser treatment failed e injectionof MTX was administered alone based on previous protocols and the presumed halflife of drugs in vitreous cavityAfter injections totally there was no membrane recurrence is case suggests that intravitreal MTX plays a role intreatment against epithelial downgrowth MTX Used in Conventional erapyResistant DiseasesGenerally the antivascular endothelial growth factor antiVEGF therapy has dramatically improved the prognosis ofneovascular agerelated macular degeneration nAMDHowever there are still some patients who remain refractoryto antiVEGF therapy which is termed as treatmentresistant nAMD As there is evidence that MTX has eï¬ects ininterrupting the angiogenesis cascade at various levels []Kurup oï¬ered intravitreal MTX to patients who wererefractive to standard antiVEGF therapy [] Although itwas an oï¬label use the patients™ visual acuity improved atfollowup visit while ophthalmic imaging examinationsshowed significantly reduced cystoid macular edema usFolatefolic acidRFC 1Folatefolic acidDHFTHFDHFR5MTHFMTHFRPurine and pyridine synthesis DNA synthesisMTXMTXABC familyFPGGGHMTXFGAICAR5FormylAICARATICAdenosine †‘AdenosineCell membraneCell membrane“““ 0cJournal of Ophthalmologypatients who are refractory to traditional antiVEGF therapymight benefit from intravitreal injection of MTXis approach is not alone Khalil [] had μg01 ml of MTX intravitreal injection once monthlyadministrated to adult Behcet™s disease BD patientssuï¬ering from BDassociated ocular ‚ammation withposterior segment involvement eir results prove thatintravitreal MTX improves visual acuity reduces posteriorsegment manifestations associated with Behcet™s disease andallows the reduction of corticosteroids and immunosuppressive drugs [] ese results also supported Taylor andassociates who conducted trials on patients with unilateral uveitis andor cystoid macular edema [] eirclinical trials suggest that intravitreal MTX may help patients with uveitisassociated posterior segment involvementto regain normal anatomical structure and then allowed thereduction of immunosuppressive therapy The Pharmacogenetics of MTXWith molecular sequencing and highthroughput technology large numbers of genetic polymorphisms can now bedetected accurately and rapidly [] Researchers pay moreattention to pharmacogenetics the study of genetic polymorphisms in drugmetabolizing enzymes and the translation of inherited diï¬erences to diï¬erences in drug eï¬ects[] e genes encoding transporting proteins and metabolizing enzymes for MTX are also known to harborfunctionally significant SNPs e SNPs may ‚uence theefficacy of MTX and have been suggested as potential riskfactors for enhanced MTX toxicity even in lowdose regimens based on previous researches []e research of pharmacogenetics of MTX could bedivided into genetic polymorphisms aï¬ecting MTX transport and SNPs that‚uence enzymes in the cellularpathway of MTX []Once taken MTX enters the cell through an activetransport which is mediated by the reduced folate carrier RFC1 e loss of RFC1 gene expression might lead toeï¬ects of uptake and intracellular levels of MTX A G80ASNP of RFC1 was proposed [] making a decreasing [] orincreasing [ ] eï¬ect on intracellular level of MTXerefore a significant association between RFC1 SNPs andMTX toxicity should be considered Chango state thatthese SNPs strongly impact the overall MTX associated sideeï¬ects by resulting in altered cellular MTX concentrationbut with no ‚uence on MTX efficacy [] However someresearchers argue that these SNPs have no definite eï¬ect[] us it remains controversial whether SNPs of RFC1aï¬ect the transport of MTX Moreover Pglycoprotein amembrane transporter that has ‚uences on the dispositionand bioavailability of MTX [] was studied SNPs ofABCB1 including C3435T SNP and C1236T SNP werebelieved to have eï¬ects on the expression of Pglycoprotein[] Gervasini speculate the C1236T SNP of ABCB1aï¬ects the administered doses of MTX and the incidence ofhematological toxicity [] However just like G80A SNPthere are disputes about the ‚uences of these SNPs asdiï¬erent studies had diï¬erent outcomes []Metabolizing enzymes were also being analyzed giventhe critical role of transporters in disposition of MTX and itsactive products as well as the folate metabolism MTXpharmacogenetics mostly focused on the SNPs in theMTHFR gene e present study shows that genetic polymorphisms in the folate metabolic pathway and in MTXtransporters ‚uence the toxicity but not the efficacy of thelowdose MTX treatment in patients with autoimmunediseases [] For example C677T and A1298C are knownin MTHFR gene to result in a lower enzyme activity []Windsor and associates reported that MTHFR A1298C andC677T were associated with MTX related nephrotoxicityand anemia [] ese SNPs might be associated withdecreased activity of methylenetetrahydrofolate reductaseelevated plasma homocysteine levels and altered distribution of folate [] us patients with this genotype weremore vulnerable to potential MTX induced toxicity sincethese reactions above may lead to slower folate metabolismand slower cell repair [] Weisman used univariatelogistic regression to reveal that the MTHFR C677T alsoincreases the occurrence of side eï¬ects in central nervoussystem manifested as headache and lethargy [] HoweverLambrecht argued that MTHFR C667T was not apredictive factor for toxicity [] Berkani found noassociation between A1298C polymorphism and MTXtoxicity [] Interestingly Grabar claimed that thepatients with MTHFR 1298C genotype have a lower risk forMTX toxicity than the carriers of MTHFR 1298A allele []To date the study of pharmacogenetics of MTX continues An increasing number of SNPs have been found to bepossibly associated with the efficacy and toxicity of MTXe newly discovered genotypes include C347G in ATICand ²UTR 28bp repeat and ²UTR 6bp deletion inTYMS which may ‚uence both efficacy and toxicity ofMTX similarly factors that may aï¬ect MTX associatedtoxicity are for example A2756G in MS and A66G in MTRR[ ] e genes and their SNPs that might beassociated with the eï¬ects and side eï¬ects of MTX aresummarized in Table Growing evidences suggest that asingle genetic factor is unlikely to adequately predict theefficacy and toxicity of MTX in polygenic disease such as RAand autoimmune associated ocular disease Given the impactof MTX in several metabolic pathways a complex of multiple risk genotypes examination would help to predict theefficacy of MTX and to identify patients who may haveadverse eï¬ects from MTX administrationTaken together the efficacy and toxicity of MTX mayremain associated with the genetic markers in the patientserefore although this remains a controversial subject it isreasonable to believe that pharmacogenetics may be able topredict who is at risk of MTX associated adverse eï¬ects andmay help in maximizing the benefitrisk ratio of MTX The Side Effects of MTXe doselimiting toxicity of MTX mainly includes hepatotoxicity and nephrotoxicity [“] but mortality hasoften been reported due to either pneumonitis or secondaryinfections [] 0cJournal of OphthalmologyTable Summary of genes and their SNPs which might have possible clinical eï¬ects towards MTXTransportingproteinsABCfamilyGeneRFC1 [“]ABCB1 [ ]ABCC1 []ABCC2 []ABCC4 []MetabolizingenzymesMTHFR [“]ATIC [“]TYMS [ ]GGH [ ]DHFR [ ]MS [ ]MTRR [ ]SNPsG80AC3435TC1236Trs246240Srs3784862A2412GG1249AG1058AC934AC677TA1298CC347G²UTR 28bp²UTR 6bprepeatdeletionC452TC401TT721AC830TA2756GA66GSome experts divided MTX associated pulmonarycomplications into ‚ammatory infectious and lymphoproliferative [] In the authors™ opinion all MTX relatedside eï¬ects can be classified into these three categoriesaccording to the pharmacological eï¬ects of MTXMajor adverse events for MTX are related to the folateantagonism and primarily aï¬ect highly proliferative tissuessuch as bone marrow and gastrointestinal mucosa []Given the immunosuppression eï¬ect of MTX pancyt iawas one of the most frequent severe toxicities of methotrexate [] Meanwhile the risk of developing an infectiousprocess is increased all along the treatment and the severityof the infected disease would be worsen [ ] includingcommon bacterial infections herpes zoster eruptions andopportunistic infections According to previous studies therisk is larger than that with other disease modifying antirheumatic nonbiological drugs DMARDsSecondly the MTX acts as the hapten [] and is likely toreact directly with nucleophilic groups present in proteins ieto combine with endogenous protein [ ] e proteinadducts thus act as an antigenic signal to direct the eï¬ector armof the immune response [] e provoked immune responsesare most commonly type I immediate hypersensitivity andtype III immune complex reactions [] Hypersensitivitypneumonitis is the most common severe and unpredictablecomplication with a mortality of up to almost []Moreover a few studies have shown that longterm MTXuse can lead to lymphoproliferative disorders LPDs in bothnodal sites and extra nodal sites such as the skin lungsepipharynx thyroid gland nasal cavity spleen and kidneysespecially for patients who are positive for EBV infection[“] e reported frequency of EBV positive in MTXassociated LPDs patients is “ [] Although themechanism of onset is not fully understood it is believedPossible clinical eï¬ectsIncreasingdecreasing intracellular MTX levelAï¬ecting efficacy of MTXAï¬ecting the distribution of MTX and incidence of hematological toxicityAssociation with MTX related toxicityLeading to accumulation of MTX to nephrotoxic levelsAssociation with MTX related gastrointestinal toxicityAssociation with MTX related hepatotoxicityAssociation with MTX related hematological toxicityAï¬ecting the toxicity but not the efficacy by resulting in a lower enzymeactivity association with related nephrotoxicity anemia and neurologicside eï¬ectsAï¬ecting efficacy and toxicity of MTXAï¬ecting efficacy and toxicity of MTXAï¬ecting efficacy of MTXAï¬ecting efficacy of MTXAï¬ecting efficacy of MTXAssociation with MTX associated toxicityAssociation with MTX associated toxicitythat the combination of immunodeficiency and the immunosuppressive eï¬ect of MTX has been implicated in thepathogenesis of MTX associated LPDs e World HealthOrganization WHO has classified MTX associated LPDs aslymphoid neoplasms whether iatrogenic or immunodeficiency associated diseases [ ] MTX associated LPDsoften take a spontaneous remission which tends to completemostly within weeks after the discontinuation of MTX[] But there are a few reports showing that the lymphoidneoplasms occur even after stopping using MTX [] e Eï¬ects of Administration Routes Generally the sideeï¬ects of MTX depend on the route of administration Dosedependent [] gastrointestinal side eï¬ects are the mostfrequent side events with orally administered MTX as oraladministration is the most common delivery method[ ] More than of MTX is excreted by therenal system thus MTX associated nephrotoxicity is common among patients taking MTX Fortunately the resolution usually occurs after discontinuation of therapy andsalvage treatment with highdose corticosteroids[]erefore to achieve treatment with less side eï¬ects theappropriate route of administration and dose of MTX arenecessary During the treatment monitoring of patients™general condition mattersAdverse eï¬ects of intravitreal injections of MTX occuronly within the eyeincluding hyperemia keratopathycataract iridocyclitis vitreous hemorrhage retinal detachment maculopathy and endophthalmitis []Splitting doses of MTX rather than intravenous administration is a new attempt to avoid MTX associated sideeï¬ects MTX is split and given twice or thrice in a week toachieve higher bioavailability and better clinical response 0cJournal of Ophthalmology[ ] thus providing us with a novel method of oraladministration of MTX with less adverse eï¬ects Is LowDose MTX Safer Based on clinical cases observation side eï¬ects which can lead to discontinuation ofMTX are rare during the typical ophthalmology treatmentbecause of the lower dose of MTX required [] e application of lowdose MTX regimen has also become one ofthe main therapies of a variety of immunemediated diseasesbecause of its efficacy and an acceptable safety profile asmost lowdose MTX associated toxicity has been describedin case reports and relatively small case series []However although welltolerated and mostly reversibleeven a lowdose regimen of MTX can result in clinicallysignificant toxicity with substantial death rates about according to Kivity™s cohort study [] e lowdose MTXassociated severe adverse eï¬ects include major centralnervous system complications [] mucositis pulmonaryinvolvement hepatotoxicity [] and myelosuppression Is MTX Safe to the Pregnant and Fetuses As one of thelipidsoluble and low molecular weight drugs MTX could bereadily transferred across the placental membrane duringpregnancy and adversely aï¬ect the fetus [] In addition MTXmight take longer time for elimination in fetal tissues []Regarding pharmacogenetics mutations caused by MTXlead to severe decrease of the expression of folate and nucleobaseenzymes which are critical for cellular homeostasis [] Inpractice MTX aï¬ected formation of the blastocyst and causeddysmorphic features and neurologic defects in early pregnancyleading to malformations in some cases [] Multiple congenitalabnormalities have been observed after weekly MTX treatmentsat a mg dose during the first months of pregnancy [] evenfetal death [] Verberne had reviewed cases of congenitalanomalies after in utero exposure to MTX and proved that somecongenital anomalies such as microcephaly craniosynostosistetralogy of Fallot pulmonary valve atresia limb reductiondefects and syndactyly were truly part of the œfetal methotrexatesyndrome [] Administration of MTX in childhood mightalso cause manifestations including visual defect [] andSmith“Magenis syndrome [] among patients with specificmutations us special care should be taken with pregnantpatients and children in particular e Risk Factors of MTX Associated Side Eï¬ects e mostcommon risk facts of MTX induced adverse eï¬ects areadvanced age age years and underlying disease incaused by MTX administration with100 g alcohol concluding renal andor hepatic insufficiency and lung diseaseespecially patients with chronic hepatitis B and diabetesmellitus [“] Patients with a history of alcohol intakemight have a greater risk of liver fibrosis and hepatotoxicitysumption per week [] Also preexisting hypoalbuminemiaand past use of any of the DMARDs and protonpumpinhibitors have been described in studies to increase theincidence of MTX induced side eï¬ects [ ] Moreovertaking drugs that may interact with MTX at the same timemight also be dangerous these drugs include salicylatescotrimoxazole chloramphenicol sulfonamides cyclosporine and pyrimethamine [] Although no significantprotective eï¬ect of folate supplementation on MTX relatedtoxicity has been found [] the folate deficiency is anotherreason for the side eï¬ects based on clinical cases []Heidari found that MTX administration elevatedkidney ROS levels decreased tissue antioxidant capacityincreased lipid peroxidation and depleted renal glutathionestores eir research data indicate that MTX caused tissuedamage and organ dysfunction through oxidative stresserefore they proposed that patients with preexistingmitochondrial defects might be vulnerable to MTX inducedrenal injury []e use of highdose MTX HDMTX is also the riskfactor of adverse eï¬ects MTX induced liver fibrosis is morelikely to become morphologically evident with high cumulative doses possibly largely exceeding to mg[ ] and the side eï¬ects caused by omeprazole use inthe past were found in cancer patients receiving HDMTXtreatment []e distribution of MTX in vivo also plays a role in MTXrelated side eï¬ects As MTX tends to accumulate in theextravascular compartment patients with pleural eï¬usionascites and massive edema should get extra caution due tothe risk of toxicity from reabsorption of extravascular fluid[]Another noteworthy risk factor is UV UV recall phenomenon also known as MTX associated UV reactivationhas been reported [ ] It is reactivity of sunburn areaswithin to days of the treatment with MTX [ ]According to Adams and associates this phenomenon mightbe due to the immune response by uncontrolled sunburninduced ‚ammation released by MTX [] Patients whopreviously suï¬ered sunburns deserve more detailed monitoring when methotrexate is needed Is Folate Supplementation Necessary for OphthalmicPatients To prevent MTX associated side eï¬ectsit iscommon to take folate [as either folic acid FA or folinicacid FLA] in clinic [ ] However there is noconsistent and evidencebased guideline for folate supplementation in ophthalmic patientsFolate and folic acid play significant roles in the de novosynthesis of purines and thymidylate which are required forDNA replication and repair [] Funk and associates founda significant reduction of circulating folate concentrations in of patients receiving MTX treatment [] Patientstreated with highdose MTX HDMTX got routine folatesupplementation to reduce HDMTX associated side eï¬ects[“] After a systematic literature review of HDMTXtherapy and folate supplementation Van der Beek et al[] found lower incidence of MTX associated adverseeï¬ects in regimens with higher cumulative doses and earlieradministration of folate supplementation in similar HDMTX dosage studies Folate supplementation in patientswith lowdose methotrexate is also being studied Ortiz et al[] had proved the protectivefolateeï¬ectof 0cJournal of Ophthalmologysupplementation by conducting a Cochrane review including more than patients taking lowdose MTX Untilnow folate supplementation had been proved to prevent andimprove MTX associated eï¬ects including gastrointestinalrespiratory and neurologic side eï¬ects [ ] Mori et alsupported the protective eï¬ect by demonstrating that patients treated with lowdose MTX without folate supplements were significantly associated with the development ofmyelosuppression and pancyt ia []However Arabelovic and associates™ preliminary studyshowed a significant increase of MTX dose needed []since folic acid fortification enriched cereal grain productswere fully implemented in the USA and Canada [] isconveyed a message to us that high dose of folate supplementation might have ‚uence on the efficacy of MTXAlDabagh found that the reduction in efficacy ofMTX cannot be ignored while folate supplementation didmake a significant reduction in associated adverse eï¬ects[] Salim declared the decreasing ‚uence betweenthe anti‚ammatory eï¬ect of MTX and folate supplementation by carrying out a doubleblind clinical trial []Chladek had conducted an labeltwowaycrossover study supporting the opinion above [] Additionally because of the unequal distribution of folic acidand MTX in organs and tissues [] MTX discontinuationis more common for some MTX associated side eï¬ects inophthalmic clinic [] rather than higher dosage of folatesupplementationere are no ophthalmic studies to demonstrate theprotective eï¬ects of folic acid supplementation us although the folate supplementation is widely used amongpatients treated with lowdose MTX [ ] the necessityand standardized dosage of folate supplementation in specific patients [] as well as the MTXfolate interactionstill warrant further studies DiscussionMethotrexate as one of the alternative pharmacologicalsteroidsparing immunosuppressive agentsis becomingmore and more popular as the preferred treatment in severalautoimmune conditions requiring longterm immunosuppression [] Lowdose MTX has anti‚ammatory andimmunomodulatory properties by increasing levels of intracellular and extracellular adenosine [] which is thefoundation of ophthalmic MTX treatment e standardizedand recommended administration of ophthalmic MTXtreatment is once a week starting with a dose of mg andescalating every to weeks up to “ mgweek whennecessary [ ] In patients with insufficient response toMTX alone cyclosporin with or without azathioprine wasadded []To avoid side eï¬ects split doses of MTX administrationand folate supplementation are gradually being used inophthalmic clinic Prescription of to mg of folatesupplementation has a significant role in MTX safety []but the higher dosage is less applied even with higher dose ofMTX [] Prophylactic folate supplementation is notnecessary in most patients [] ere is also research toconvey that ml100 g or above dosage of fish oil is aseï¬ective as folinic acid in therapeutic potential in preventingbone loss during MTX chemotherapy [] For some resistant andor mortal adverse eï¬ects the discontinuation ofMTX will work instantlyWith the increasing longterm use of MTX it is importantto monitor patients™ blood examination results including bloodroutine and liver and renal functions As pancyt ia can be alate manifestation [] elevation of urea creatinine aminotransferases and albumin as well as electrolytes disturbancesmay result in MTX associated liver and renal side eï¬ects []Plasma MTX level is not a reliable predictor for adverse eventsin MTX therapy [] On the contrary circulating folate levelsand folate polyglutamate distribution change sensitively withMTX exposure and exogenous folate supply [] and could beused as a biomarker of MTX efficacy [] It should be notedthat as erythrocytes have a halflife of approximately daysthe results of blood examinations might reflect both pretreatment and posttreatment status which need to be analyzedcarefully []Numerous studies had been conducted to prove thatMTX could be used as a welltolerated safe and eï¬ectivefirstline treatment Hence the MTX administration shouldnot continue to be stigmatized as a œcancer drug or to bediscouraged because of associated adverse eï¬ects Contrarily the indication and the routes of administration areabout to gradually widenConflicts of Intereste authors declare that they have no conflicts of interestReferences[] R Q H Kloos R Pieters C van den Bos œe eï¬ect ofasparaginase therapy on methotrexate toxicity and efficacy inchildren with acute lymphoblastic leukemia Leukemia Lymphoma vol no pp “ [] R K Bath N K Brar F A Forouhar and G Y Wu œAreview of methotrexateassociated hepatotoxicity Journal ofDigestive Diseases vol no pp “ [] W Wang H Zhou and L Liu œSide eï¬ects of methotrexatetherapy for rheumatoid arthritis a systematic review European Journal of Medicinal Chemistry vol pp “[] J Smolen and R Landew´e œEULAR recommendations forthe management of rheumatoid arthritis with synthetic andbiological diseasemodifying antirheumatic drugs update Annals of the Rheumatic Diseases vol no pp “ [] J A Singh K G Saag S L Bridges œ Americancollege of rheumatology guideline for the treatment ofrheumatoid arthritis Arthritis Rheumatology vol no pp “ [] M Holdhoï¬ P Ambady A Abdelaziz œHighdosemethotrexate with or without rituximab in newly diagnosedprimary CNS lymphoma Neurology vol no pp “ [] J Pe™er J M Rowe S Frenkel and E J Dann œTesticularlymphoma intraocularvitreoretinal lymphoma and brainlymphoma involvement of three immunoprivileged sites in 0cJournal of Ophthalmologyone patient American Journal of Hematology vol no pp “ [] S Gangaputra C W Newcomb T L Liesegang et alœSystemic immunosuppressive therapy for eye diseases cohort study methotrexate for ocular ‚ammatory diseasesOphthalmology vol no pp “ [] P W Hardwig J S Pulido J C Erie K H Baratz andH Buettner œIntraocular methotrexate in ocular diseasesother than primary central nervous system lymphomaAmerican Journal of Ophthalmology vol no pp “ [] E Esterberg and N R Acharya œCorticosteroidsparingtherapy practice patterns among uveitis specialists Journalof Ophthalmic Inflammation and Infection vol no pp “ [] K Durrani F R Zakka M Ahmed M MemonS S Siddique and C S Foster œSystemic therapy withconventional and novel immunomodulatory agents for ocular ‚ammatory disease Survey of Ophthalmology vol no pp “ [] S S Gangaputra C W Newcomb M M Joï¬e et alœComparison between methotrexate and mycophenolatemofetil monotherapy for the control of noninfectious ocular‚ammatory diseases American Journal of Ophthalmologyvol pp “ [] V K Ayuso E L van de Winkel A Rothova and J Helenade Boer œRelapse rate of uveitis postmethotrexate treatmentin juvenile idiopathic arthritis American Journal of Ophthalmology vol no pp “ [] S R Rathinam M Babu R undikandy œA randomized clinical trial comparing methotrexate and mycophenolate mofetil for noninfectious uveitis Ophthalmologyvol no pp “ [] A Galor D A Jabs H A Leder œComparison ofantimetabolite drugs as corticosteroidsparing therapy for‚ammation Ophthalmologynoninfectiousvol no pp “ ocular[] M D de Smet V S Vancs D Kohler D Solomon andC C Chan œIntravitreal chemotherapy for the treatment ofrecurrent intraocular lymphoma British Journal of Ophthalmology vol no pp “ [] E Kim C Kim J Lee and Y Cho œA case of primaryintraocular lymphoma treated by intravitreal methotrexateKorean Journal of Ophthalmology vol no pp “[] J Smith J T Rosenbaum D J Wilson œRole ofintravitreal methotrexate in the management of primarycentral nervous system lymphoma with ocular involvementhistorical image Ophthalmology vol no pp “ [] CC Chan and D J Wallace œIntraocular lymphomaupdate on diagnosis and management Cancer Controlvol no pp “ [] K L Larkin U S Saboo G M Comer œUse ofintravitreal rituximab for treatment of vitreoretinallymphoma British Journal of Ophthalmology vol no pp “ [] C P Fox E H Phillips J Smith œGuidelines for thediagnosis and management of primary central nervoussystem diï¬use large Bcell lymphoma British Journal ofHaematology vol no pp “ [] A Sadaka R Sisk J Osher O Toygar M Duncan andinfusion forœIntravitreal methotrexateC Riemannproliferative vitreoretinopathy Clinical Ophthalmologyvol pp “ [] N G Lambert DJ Wilson D M Albert andW D Chamberlain œIntravitreal methotrexate for recurrentepithelial downgrowth JAMA Ophthalmology vol no p [] A M Joussen F E Kruse HE V¨olcker and B KirchhofœTopical application of methotrexate for inhibition of cornealangiogenesis Graefe™s Archive for Clinical and ExperimentalOphthalmology vol no pp “ [] S K Kurup C Gee and C M Greven œIntravitrealmethotrexate in therapeutically resistant exudative agerelated macular degeneration Acta Ophthalmologica vol no pp e145“e146 [] H E M Khalil H A Raafat N A Azab H E Haroun andH A Elgendi œe role of intraocular methotrexate in themanagement of uveitis and posterior segment involvementin Behçet™s disease patients e Egyptian Rheumatologistvol no pp “ [] S R J Taylor A Banker A Schlaen œIntraocularmethotrexate can induce extended remission in some patients in noninfectious uveitis Re Answer:

Thyroid_Cancer

"coronavirus disease COVID19 caused by severe acute respiratory syndrome coronavirus SARSCoV2 emerged in China Currently it is breaking out globally and posing a serious threat to public health The typically clinical characteristics of COVID19 patients were fever and respiratory symptoms and a proportion of patients were accompanied by extrapulmonary symptoms including cardiac injury kidney injury liver injury digestive tract injury and neurological symptoms Angiotensin converting enzyme ACE2 has been proven to be a major receptor for SARSCoV2 and could mediate virus entry into cells And transmembrane protease serine TMPRSS2 could cleave the spike S protein of SARSCoV2 which facilitates the fusion of SARSCoV2 and cellular membranes The mRNA expressions of both ACE2 and TMPRSS2 were observed in the heart digestive tract liver kidney brain and other ans SARSCoV2 may have a capacity to infect extrapulmonary ans due to the expressions of ACE2 and TMPRSS2 in the cells and tissues of these ans It seems that there is a potential involvement of ACE2 and TMPRSS2 expressions in the virus infection of extrapulmonary ans and the manifestation of symptoms related to these ans in patients with COVID19 Here we revealed the expressions of ACE2 and TMPRSS2 in extrapulmonary ans and we also summarized the clinical manifestation and the management of extrapulmonary complications in patients with COVID19 Introduction Since the late a novel coronavirus officially named as severe acute respiratory syndrome coronavirus SARSCoV2 was identified as the pathogen to cause pneumonia [] As a member of the Betacoronavirus genus SARSCoV2 has genomic nucleotides identity with human severe acute respiratory syndrome coronavirus SARSCoV and shares amino acid sequence identity with SARSCoV [] The World Health anization WHO named the disease caused by SARSCoV2 as coronavirus disease COVID19 Until April the virus has swept through countries more than million cases with COVID19 have been confirmed and more than cases died which has been posing significant threats to public health SARSCoV2 can cause respiratory diseases and may lead to acute respiratory distress syndrome ARDS multiple an failure and even death in severe cases [] In addition to typical symptoms such as cough and fever some patients developed the symptoms in multiple systems such as cardiovascular system digestive system and Abbreviations ACE2 Angiotensin converting enzyme AKI Acute kidney injury ALI Acute liver injury ALP Alkaline phosphatase ALS Artificial liver system ALT Alanine aminotransferase AMI Acute myocardial infarction ARDS Acute respiratory distress syndrome AST Aspartate aminotransferase AT2 Alveolar cells BUN Blood urea nitrogen CCLE Cancer Cell Line Encyclopedia CNS Central nervous system COVID19 Coronavirus disease GEO Gene Expression Omnibus GGT Gammaglutamyltransferase GI Gastrointestinal injury GTEx GenotypeTissue Expression ICU Intensive care unit MCS Mechanical circulatory support NP Nucleoprotein PCI Percutaneous coronary intervention SARSCoV2 Severe acute respiratory syndrome coronavirus Scr Serum creatinine ScRNASeq Single cell RNA sequencing STEMI STelevation myocardial infarction TBIL Total bilirubin TEM Transmission electronic microscope TMPRSS2 Transmembrane protease serine VV venousvenous WHO World Health anization Corresponding authors at Department of Infectious Disease and Institute of Hepatology Qingdao Municipal Hospital Qingdao University Digestive Disease Key Laboratory of Qingdao Qingdao China Email addresses xinyongning9812163com Y Xin zlk0823163com L Zhuang 101016jbiopha2020110678 Received June Received in revised form August Accepted August BiomedicinePharmacotherapy1312020110678Availableonline24August2020075333222020TheAuthorsPublishedbyElsevierMassonSASThisisan accessundertheCCBYNCNDlicensehttpcreativecommonslicensesbyncnd40 0cexpressed not only in the cells and tissues of lung but also in extrapulmonary ans [“] Fig In this section the expression levels of ACE2 and TMPRSS2 in extrapulmonary ans including heart kidney liver digestive tract brain and other ans were reviewed Heart M Dong nervous system in the early stages of COVID19 which brings more challenges to the timely diagnosis of patients [] Angiotensin converting enzyme ACE2 as a metalloproteinase is a carboxyterminal dipeptidyl peptidase [] The primary physiological role of ACE2 is involved in the regulation of vasoconstriction and blood pressure [“] Transmembrane protease serine type2 TMPRSS2 belonging to the type II transmembrane serine protease family could cleave the coronavirus spike S protein [“] It was demonstrated that ACE2 and TMPRSS2 were crucial for the entry of SARSCoV and SARSCoV2 into the host cells [] Cell entry of SARSCoV2 depends on binding of the S protein to the specific cellular receptor and S protein priming by host cell proteases As shown in Fig each S protein of SARSCoV2 consists of two subunits a globular S1 domain at the Nterminal region and the membraneproximal S2 domain SARSCoV2 utilizes receptorbinding domain within the S1 domain to bind to the cellular receptor ACE2 which could trigger the effects of TMPRSS2 on the cleavage of protein S at the S1 and S2 sites and priming cell membrane fusion for viral entry [] As receptors and mediators of virus entry are important for determining viral host and an the route of SARSCoV2 infection and the infected an may depend on the expression and distribution of ACE2 and TMPRSS2 [] Studies have shown that ACE2 and TMPRSS2 are expressed not only in lung tissues but also in extrapulmonary ans including heart kidney liver colon esophagus brain gallbladder and testis suggesting that SARSCoV2 may also affect extrapulmonary ans [“] In this review the distributions of ACE2 and TMPRSS2 in extrapulmonary ans and the characteristics and clinical managements of extrapulmonary an injury caused by SARSCoV2 were summarized We believe that this will be important in understanding on the infection of extrapulmonary ans in patients with COVID19 The mRNA expressions of ACE2 and TMPRSS2 in extrapulmonary ans The mRNA expressions of ACE2 in different human ans were analyzed and the results showed that ACE2 was expressed in the heart [] Furthermore Chen analyzed the feature of ACE2 expressions among cardiac cell types and found that ACE2 was specifically expressed in pericyte [] Moreover RNA sequencing from patients with failing hearts and normal donors revealed that myocardial ACE2 expressions were significantly increased in patients with heart failure which was further validated at the protein level by proteomics profiling from heart failure and normal donors [] Another study also showed that the expression of ACE2 in heart tissues of patients with underlying heart disease was higher than that in normal heart tissues [] These two studies suggested that the expression of ACE2 in heart tissue of patients with underlying heart disease was higher than that in normal heart tissue Guo et al analyzed the mRNA expression of TMPRSS2 from the GenotypeTissue Expression GTEx database and the results showed that TMPRSS2 is also expressed in the heart [] By singlecell RNA sequencing scRNASeq to profile the gene expression landscapes of cardiac cells from human embryos Qi revealed that the cardiomyocytes from the heart contain ACE2expressed cells and TMPRSS2expressed cells and the cardiovascular progenitor cells and cells TMPRSS2expressed cells respectively [] These data showed that both ACE2 and TMPRSS2 were expressed in the heart contain ACE2expressed Kidney Studying the viral susceptibility of extrapulmonary ans is important for a deeper understanding for the pathogenesis of SARSCoV infection Studies have shown that ACE2 and TMPRSS2 were Expression analysis from the GTEx database showed that kidney displayed the fifth high expression of ACE2 [] To investigate the expression of ACE2 in kidney Lin analyzed the public singlecell transcriptome dataset of normal kidneys from healthy donors the Fig Entry of SARSCoV2 into host cells SARSCoV2 infected the host cells by the spike protein of the virus and the functions of ACE2 and TMPRSS2 in host cells BiomedicinePharmacotherapy13120201106782 0cM Dong Fig Tissue distributions of ACE2 and TMPRSS2 in human A“B the schematic diagram of the expressions of ACE2 A and TMPRSS2 B in multiple human tissues The colour strength is corresponding to the gene expression level ACE2 and TMPRSS2 were expressed in the brain and heart ACE2 expression is expressed at a relative low level in hepatocytes and mainly located in cholangiocytes while TMPRSS2 is expressed in the hepatocytes and cholangiocytes ACE2 and TMPRSS2 were highly expressed in kidney and intestinal epithelial cells Both ACE2 and TMPRSS2 were also expressed in the esophagus stomach nose testis pancreas breast prostate and thyroid results showed that the ACE2 was distributed across multiple cell types and was mostly enriched in proximal tubule cells [] Fan et al confirmed the specific ACE2 expression in tubular cells from the Gene Expression Omnibus GEO dataset while it was not observed in immune cells and glomerular parietal epithelial cells RNA and protein expression data of ACE2 in different human tissues and cancer cell lines were obtained from three online datasets including the Cancer Cell Line Encyclopedia CCLE GTEx database and the Human Protein Atlas dataset and the results indicated that both mRNA and protein expression levels of ACE2 were relatively high in kidney cells especially in renal tubular cells [] Meanwhile Suryawanshi analyzed the data of kidney tissues in scRNASeq datasets and found that either proximal tubular cells or tubular progenitor cells in the kidney coexpressed ACE2 and TMPRSS2 [] The data of the scRNAseq from GEO dataset GSE134355 showed that ACE2 and TMPRSS2 expression levels were high in nephron epithelial cells epithelial cells endothelial cells and mesangial cells of the kidney [] Recently Pan also found that the TMPRSS2 gene was coexpressed with ACE2 in kidney podocytes [] These data showed that both ACE2 and TMPRSS2 were highly expressed in tissues and cells of kidney Liver Chai et al analyzed the scRNAseq data from GEO database GSE124395 to evaluate ACE2 gene expression in liver the results showed that ACE2 was highly expressed in cholangiocytes which level was about times higher than that in hepatocytes [] The GTEx database also showed that both ACE2 and TMPRSS2 were expressed in the liver [] Zhou identified that TMPRSS2 is highly expressed in hepatocytes from Human Cell Atlas database [] Recently Wen indicated that ACE2 and TMPRSS2 are specifically coexpression in TROP2 liver progenitors of human liver tissue using scRNA sequencing [] These data indicate that ACE2 expression is expressed at a relative low level in hepatocytes and mainly located in cholangiocytes while TMPRSS2 is expressed in hepatocytes Digestive tract A previous study showed that ACE2 could be found in the upper esophagus and it could be detected in stratified epithelial cells and absorptive enterocytes of the ileum and colon [] Quantitative mRNA expression profiling of ACE2 across human tissues by Harmer showed that ACE2 was expressed at a high level in gastrointestinal tissues [] Zhang et al analyzed datasets with singlecell transcriptomes of esophagus gastric ileum colon and lung and the data showed that ACE2 was not only highly expressed in the type II alveolar cells AT2 of lung but also in the stratified epithelial cells ileum absorptive enterocytes cells and colon enterocytes [] Similarly the immunofluorescent staining of esophagus stomach duodenum and rectum showed that ACE2 was stained mainly in the cytoplasm of gastrointestinal epithelial cells [] Besides the scRNAseq data showed that ACE2 was significantly elevated in the proximal and distal enterocytes [] Guo et al suggested that TMPRSS2 was highly expressed in almost all ans of the digestive tract including colon stomach small intestine and esophagus [] Using published scRNAseq data and seven inhouse normal colon samples Lee reported that the coexpressions of ACE2 and TMPRSS2 transcripts were mainly observed in the small intestine and colon [] The highest expressions of TMPRSS2 and ACE2 were found in enterocytes among the intestinal cell types []These data showed that TMPRSS2 and AEC2 are highly expressed in the digestive tract Nervous system Analysis using the GTEx database showed that both TMPRSS2 and ACE2 are expressed at relatively low levels in the brain cortex [] Chen found that ACE2 was relatively highly expressed in some important brain areas such as the substantia nigra and brain ventricles using seven brain transcriptome databases [] ACE2 was expressed at high level in the piriform cortex of human brain and its expression could also be detected in many neurons including both excitatory and inhibitory neurons and some nonneuron cells including astrocytes and oligodendrocytes in human middle temporal gyrus and posterior cingulate cortex [] Qi analyzed the scRNAseq data of substantia nigra BiomedicinePharmacotherapy13120201106783 0cClinical classification of acute cardiac injury NonICUcases ICU cases NonICUcases ICUcases Nonsevere cases Severecases1965 Recoveredcases Died cases NonICUcases ICUcases Survivor cases Nonsurvivor cases Chen [] Hong [] Zhou [] China Korea China M Dong and cortex of brain from GEO database the results showed that both ACE2 and TMPRSS2 were expressed in the oligodendrocyte precursor cells and the astrocytes of the substantia nigra and cortex [] There are limited reports on the expressions of ACE2 and TMPRSS2 in peripheral nervous system Brann analyzed the ACE2 and TMPRSS2 expression in different cell type from human scRNAseq dataset GSE139522 and found that neither olfactory sensory neurons nor olfactory bulb neurons expressed these two genes while ACE2 and TMPRSS2 were expressed in the nonneuronal cells including the sustentacular cells and olfactory bulb pericytes [] These data showed that ACE2 and TMPRSS2 could also be coexpressed in the nervous system Other ans or tissues Table Characteristics of acute cardiac injury after SARScid0 CoV2 infection Study Basic heart disease Acute cardiac injury Country Subject China Wang [] China NA Huang [] Li [] China Moreover ACE2 and TMPRSS2 were also reported to be coexpressed in some other ans [] It has been revealed that both ACE2 and TMPRSS2 are expressed in testis by scRNA sequencing and expression profile analysis indicating that testicular cells might be the potential targets of SARSCoV2 Another report revealed that multiple kinds of cells in the nose including nasal brushing epithelial cells nasal turbinate epithelial cells and nasal airway epithelial cells contained ACE2expressed and TMPRSS2expressed cell clusters [] Moreover ACE2 and TMPRSS2 were also expressed in pancreas breast prostate and thyroid [] and these ans might also be the targets of SARSCov2 Infection of SARSCoV2 and extrapulmonary an injury of patients with COVID19 SARSCoV2 infection and cardiac injury Recently autopsy analysis by Fox revealed that the histopathology of the heart was consistent with the typical pattern of viral myocarditis [] SARSCoV2 RNA was detected in the cardiac tissues of the patients with COVID19 [] These data suggested that SARSCoV2 may directly infect heart The epidemiology of COVID19 reported that cardiac injury was one of the most severe an damages [] The clinical manifestations of cardiac injury in COVID19 patients are complex and could present with heart failure arrhythmias or acute myocardial infarction AMI [ ] Inciardi reported the first case who had the symptom of heart failure at first and later the patient was positive for SARSCoV2 using nucleic acid test [] Cardiac injury is a common symptom in patients with COVID19 Shi reported that patients with COVID19 had cardiac injury [] Moreover there were patients with acute cardiac injury in a cohort including COVID19 patients and of patients with acute cardiac injury in the intensive care unit ICU [] Furthermore a study by Wang showed that there were patients with acute cardiac injury and patients presented with arrhythmia of COVID19 patients while acute cardiac injury was observed in of patients with CIVID19 in the ICU [] These cases suggested that SARSCoV2 may cause serious heart damage which should be widespreadly concerned Furthermore acute cardiac injury is more prevalent in severe cases with COVID19 [“] Table And it has been reported that COVID19 patients with cardiac injury had higher mortality than those without cardiac injury [] In this review we also summarized the possible relationship between basic heart disease and further cardiac injury [“] Table In a cohort of COVID19 patients from Renmin Hospital of Wuhan University China Shi demonstrated that cardiac injury occurred in patients during hospitalization of which had basic heart disease including coronary heart disease and chronic heart failure And only patients with basic heart disease of COVID19 patients without cardiac injury [] Similarly Liu suggested that patients with basic heart disease in COVID19 patients had Table Comorbidity with cardiac injury in COVID19 patients with basic heart disease Subjects with COVID19 Proportion of basic heart disease Patients with Cardiacinjury Study Shi [] Liu [] Xu [] Ma [] Guo [] Patients with basic heart disease With cardiac injury Without cardiac injury cardiac injury compared with patients with basic cardiovascular diseases of COVID19 patients without cardiac injury [] Other studies also indicated that the patients with basic cardiovascular disease are more likely to present heat injury in COVID19 patients [ ] In view of the points above COVID19 patients with underlying cardiac conditions seem to have higher rates of cardiac injury SARSCoV2 infection and kidney injury Recently autopsy analysis on six COVID19 patients showed that varying degrees of acute tubular necrosis were observed in all the renal specimens Nucleoprotein NP antigens and NP positive inclusion body of SARSCoV2 could be seen in kidney tissues from all the samples Moreover viruslike ps were seen in kidney tissues by transmission electronic microscope TEM [] Su analyzed kidney abnormalities in autopsies of patients with COVID19 and found that diffuse proximal tubular damage with the loss of brush border were BiomedicinePharmacotherapy13120201106784 0c SARSCoV2 infection and liver injury M Dong observed Further investigation showed that diffuse necrosis can be seen under the light microscope and electron microscopic examination also showed the clusters of coronavirus ps with distinctive spikes in the tubular epithelium and podocytes [] It was reported that both NP antigens and RNA of SARSCoV2 were detected in urine of COVID19 patients [] These data coincide with the finding of the SARSCoV2 invasion in kidney Collectively SARSCoV2 could directly infect human renal tubules and lead to kidney damage Recent studies have shown that the incidence of acute kidney injury AKI in COVID19 patients ranged from “ and higher frequency of renal function damage with elevated blood urea nitrogen BUN or serum creatinine Scr was observed in COVID19 patients [ ““] Table A study of patients with COVID19 indicated that levels of BUN and Scr were increased in and patients with COVID19 respectively And routine urine tests were performed on patients among which patients were positive for urinary protein and patients were positive for hematuria [] Another study also showed that about patients with COVID19 had abnormal renal function [] Moreover COVID19 patients with more severe disease progression have higher rates of AKI Huang and colleagues reported that of patients with AKI in the ICU were observed and none of the patients who did not require care in the ICU suffered AKI [] Xu found that the fatality rate was obviously higher in COVID19 patients with AKI than those without renal injury [] Furthermore in another study investigating patients with COVID19 at hospital admission more severe patients had higher rates of AKI and the Cox regression analysis also suggested that COVID19 patients who developed AKI had a significantly higher mortality risk [] Therefore AKI is more prevalent in severe cases with COVID19 An autopsy report of a 50yearold patient with COVID19 showed moderate microvesicular steatosis and mild lobular activity in liver tissues [] Moreover Zhao used human liver ductal anoids as a tool to investigate the SARSCoV2 infection and the tissue damage induced by SARSCoV2 ex vivo and the results showed that the expression of SARSCoV2 NP was easily detected in the patchy areas of the hepatic duct indicating that liver ductal anoids were susceptible to SARSCoV2 infection [] In addition SARSCoV2 infection could disrupt the barrier and bile acid transporting functions of cholangiocytes which indicated that SARSCoV2 might directly induce cholangiocyte injury and consequently bile acid accumulation [] In view of the points above liver damage in the COVID19 patients might be directly caused by the viral infection Abnormal liver functions were frequently reported in COVID19 patients [] Epidemiologic studies showed that almost half of the patients had differing degrees of liver damage [“ “] Table Chen reported that out of patients had elevated alanine aminotransferase ALT patients had elevated aspartate aminotransferase AST and had elevated total bilirubin TBIL in Wuhan Jinyintan Hospital Wuhan China [] Similarly a nationwide study involving patients with COVID19 in China showed that more than of patients had elevated ALT and AST and of patients had elevated TBIL [] It was revealed that the levels of direct bilirubin indirect bilirubin ALT alkaline phosphatase ALP and gammaglutamyltransferase GGT were significantly higher in males than that in females with COVID19 and multivariate logistic regression analysis showed that male was an important independent risk factor for predicting acute liver injury ALI in COVID19 patients [] These data indicated that male patients with COVID19 may be more susceptible to liver injury Furthermore Table Characteristics of acute kidney injury after SARScid0 CoV2 infection Study Chen Wang Huang Guan Xu Preexisting kidney conditions NA NA NA [] [] [] [] [] Country China China China China China Subject Li [] Chen [] Hong [] Cheng [] Xiao [] Richardson [] Wan Li Qian Pei [] [] [] [] China China Korea China China America China China China China NA NA NA NA NA NA Scr Serum creatinine BUN Blood urea nitrogen AKI Acute kidney injury Abnormal renal functional indices Scr BUN NA Scr Scr Scr Scr BUN NA Scr BUN Scr NA NA NA Scr BUN Scr NA AKI NA Clinical classification of AKI NA NonICU cases ICU cases ICU cases Nonsevere cases Severe cases Mild cases Severe cases Critical ill cases Nonsevere cases Severe cases Recovered cases Died cases NonICU cases ICU cases NA Nonsevere cases Severe cases Cured cases In hospital cases Died cases Mild cases Severe cases Nonsevere cases Severe cases NA Moderate cases Severe cases Critically ill cases BiomedicinePharmacotherapy13120201106785 0cM Dong Table Characteristics of liver injury after SARScid0 CoV2 infection Study Country Subject China China China China China China China Korea America China China Chen [] Wang Huang Guan [] [] [] Xu Li [] [] Chen Hong [] [] Richardson et Wan Li al [] [] [] Qian [] China NA Patients with preexisting liverconditions NA NA NA Patients with abnormal liver functional indices ALT AST TBIL NA AST AST ALT TBIL ALT AST ALT AST TBIL ALT AST ALT AST TBIL AST ALT AST ALT AST TBIL ALT AST Abnormal liver functional indices in the Nonsevere patients AST ALT NA NA TBIL NA Abnormal liver functional indices in the severe patients ALT NA TBIL NA AST NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Nonsevere patients include patients without ICU care and recovered patients Severe patients include patients with ICU care and death ALT alanine aminotransferase AST aspartate aminotransferase TBIL total bilirubin multiple studies found that AST ALT and TBIL were significantly higher in patients treated in the ICU than that in nonICU patients [] Li suggested that among the patients with abnormal liver function moderate and severe types of patients were more likely to have liver injury and respectively [] Fu analyzed the relationship between ALI and mortality risk in COVID19 patients and the results showed that ALI is more common in the critically ill patients and ALI at the early stage increased death risk of COVID19 patients [] Together abnormal liver functions might be associated with the severity of patients with COVID19 SARSCoV2 infection and digestive tract injury Epithelial cells of the esophagus stomach duodenum and rectum in one COVID19 patient tested positive for SARSCoV2 RNA and the staining of viral NP was also visualized in the cytoplasm of epithelial cells in stomach duodenum and rectum [] Moreover minimally invasive autopsies were performed on three patients died of COVID19 and the results showed that some epithelial cells of the gastrointestinal mucosa were degenerated necrotic and detached [] These studies strongly supported that SARSCoV2 may directly infect the epithelial cells of digestive tract Table SARScid0 CoV2 detection in gastrointestinal specimens Study Subject [] [] Xiao Zhang Tan [] Xing Young Holshue Lescure Tang Wang Xu [] [] [] [] [] [] [] Gastrointestinal samples Stool Anal swabs Rectal swab Stool Stool Stool Stool Stool Stool Rectal swabs Tested positive in gastrointestinal specimens The positive time in gastrointestinal specimens days NA 6cid0 1cid0 5cid0 NA 3cid0 Positive time for Gastrointestinal samples after respiratory samples were negative days NA NA 8cid0 NA NA NA NA NA 2cid0 BiomedicinePharmacotherapy13120201106786 0cM Dong Multiple studies have identified that the SARSCoV2 RNA was detected in anal swabs [] rectal swabs [] and stool specimens [ ] of COVID19 patients It has been demonstrated that SARSCoV2 RNA could be detected in feces from more than half of COVID19 patients [] In another study Xing reported that SARSCoV2 RNA was detected in the feces of three pediatric cases with COVID19 in Qingdao China and the persistence of SARSCoV2 in the digestive tract lasted for 6cid0 days [] The possibility of fecaloral transmission of SARSCoV2 infection needs to be taken into account Furthermore as shown in Table long duration of SARSCoV2 detection in digestive tract by RTPCR has been reported and viral RNA remained detectable in the digestive tract for 2cid0 days after nucleic acid turned negative in respiratory samples [“] The studies suggested that SARSCoV2 could be detected from respiratory tract specimens during the early period to digestive tract specimens during the late period and viral nucleic acid tests in both the respiratory and digestive tract are necessary to confirm the complete clearance of virus Some COVID19 patients presented gastrointestinal symptoms such as diarrhea nausea vomiting and abdominal pain [] Holshue reported the first case of COVID19 patient in the USA which had nausea and vomiting before admission [] Multiple studies found that gastrointestinal symptoms including diarrhea “ nausea and vomiting “ and abdominal pain “ were common at presentation in COVID19 patients [ ““] Table In a cohort of patients with COVID19 in Wuhan China gastrointestinal symptoms were described in up to [] Moreover Sun showed that critically ill patients with COVID19 had gastrointestinal injury GI during hospital stay and the survival curves showed that the mortalities of patients with GI was greater than that of patients without GI [] Jin also found that the rate of the severe type was markedly higher in COVID19 patients with GI symptoms than that in those without GI symptoms [] These data suggested that GI is one of the common extrapulmonary an injuries in COVID19 patients and may be related to the severity of the disease On the other hand many studies showed that patients with COVID19 could present initially with the typical gastrointestinal sympto

cancer227

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: "coronavirus disease COVID19 caused by severe acute respiratory syndrome coronavirus SARSCoV2 emerged in China Currently it is breaking out globally and posing a serious threat to public health The typically clinical characteristics of COVID19 patients were fever and respiratory symptoms and a proportion of patients were accompanied by extrapulmonary symptoms including cardiac injury kidney injury liver injury digestive tract injury and neurological symptoms Angiotensin converting enzyme ACE2 has been proven to be a major receptor for SARSCoV2 and could mediate virus entry into cells And transmembrane protease serine TMPRSS2 could cleave the spike S protein of SARSCoV2 which facilitates the fusion of SARSCoV2 and cellular membranes The mRNA expressions of both ACE2 and TMPRSS2 were observed in the heart digestive tract liver kidney brain and other ans SARSCoV2 may have a capacity to infect extrapulmonary ans due to the expressions of ACE2 and TMPRSS2 in the cells and tissues of these ans It seems that there is a potential involvement of ACE2 and TMPRSS2 expressions in the virus infection of extrapulmonary ans and the manifestation of symptoms related to these ans in patients with COVID19 Here we revealed the expressions of ACE2 and TMPRSS2 in extrapulmonary ans and we also summarized the clinical manifestation and the management of extrapulmonary complications in patients with COVID19 Introduction Since the late a novel coronavirus officially named as severe acute respiratory syndrome coronavirus SARSCoV2 was identified as the pathogen to cause pneumonia [] As a member of the Betacoronavirus genus SARSCoV2 has genomic nucleotides identity with human severe acute respiratory syndrome coronavirus SARSCoV and shares amino acid sequence identity with SARSCoV [] The World Health anization WHO named the disease caused by SARSCoV2 as coronavirus disease COVID19 Until April the virus has swept through countries more than million cases with COVID19 have been confirmed and more than cases died which has been posing significant threats to public health SARSCoV2 can cause respiratory diseases and may lead to acute respiratory distress syndrome ARDS multiple an failure and even death in severe cases [] In addition to typical symptoms such as cough and fever some patients developed the symptoms in multiple systems such as cardiovascular system digestive system and Abbreviations ACE2 Angiotensin converting enzyme AKI Acute kidney injury ALI Acute liver injury ALP Alkaline phosphatase ALS Artificial liver system ALT Alanine aminotransferase AMI Acute myocardial infarction ARDS Acute respiratory distress syndrome AST Aspartate aminotransferase AT2 Alveolar cells BUN Blood urea nitrogen CCLE Cancer Cell Line Encyclopedia CNS Central nervous system COVID19 Coronavirus disease GEO Gene Expression Omnibus GGT Gammaglutamyltransferase GI Gastrointestinal injury GTEx GenotypeTissue Expression ICU Intensive care unit MCS Mechanical circulatory support NP Nucleoprotein PCI Percutaneous coronary intervention SARSCoV2 Severe acute respiratory syndrome coronavirus Scr Serum creatinine ScRNASeq Single cell RNA sequencing STEMI STelevation myocardial infarction TBIL Total bilirubin TEM Transmission electronic microscope TMPRSS2 Transmembrane protease serine VV venousvenous WHO World Health anization Corresponding authors at Department of Infectious Disease and Institute of Hepatology Qingdao Municipal Hospital Qingdao University Digestive Disease Key Laboratory of Qingdao Qingdao China Email addresses xinyongning9812163com Y Xin zlk0823163com L Zhuang 101016jbiopha2020110678 Received June Received in revised form August Accepted August BiomedicinePharmacotherapy1312020110678Availableonline24August2020075333222020TheAuthorsPublishedbyElsevierMassonSASThisisan accessundertheCCBYNCNDlicensehttpcreativecommonslicensesbyncnd40 0cexpressed not only in the cells and tissues of lung but also in extrapulmonary ans [“] Fig In this section the expression levels of ACE2 and TMPRSS2 in extrapulmonary ans including heart kidney liver digestive tract brain and other ans were reviewed Heart M Dong nervous system in the early stages of COVID19 which brings more challenges to the timely diagnosis of patients [] Angiotensin converting enzyme ACE2 as a metalloproteinase is a carboxyterminal dipeptidyl peptidase [] The primary physiological role of ACE2 is involved in the regulation of vasoconstriction and blood pressure [“] Transmembrane protease serine type2 TMPRSS2 belonging to the type II transmembrane serine protease family could cleave the coronavirus spike S protein [“] It was demonstrated that ACE2 and TMPRSS2 were crucial for the entry of SARSCoV and SARSCoV2 into the host cells [] Cell entry of SARSCoV2 depends on binding of the S protein to the specific cellular receptor and S protein priming by host cell proteases As shown in Fig each S protein of SARSCoV2 consists of two subunits a globular S1 domain at the Nterminal region and the membraneproximal S2 domain SARSCoV2 utilizes receptorbinding domain within the S1 domain to bind to the cellular receptor ACE2 which could trigger the effects of TMPRSS2 on the cleavage of protein S at the S1 and S2 sites and priming cell membrane fusion for viral entry [] As receptors and mediators of virus entry are important for determining viral host and an the route of SARSCoV2 infection and the infected an may depend on the expression and distribution of ACE2 and TMPRSS2 [] Studies have shown that ACE2 and TMPRSS2 are expressed not only in lung tissues but also in extrapulmonary ans including heart kidney liver colon esophagus brain gallbladder and testis suggesting that SARSCoV2 may also affect extrapulmonary ans [“] In this review the distributions of ACE2 and TMPRSS2 in extrapulmonary ans and the characteristics and clinical managements of extrapulmonary an injury caused by SARSCoV2 were summarized We believe that this will be important in understanding on the infection of extrapulmonary ans in patients with COVID19 The mRNA expressions of ACE2 and TMPRSS2 in extrapulmonary ans The mRNA expressions of ACE2 in different human ans were analyzed and the results showed that ACE2 was expressed in the heart [] Furthermore Chen analyzed the feature of ACE2 expressions among cardiac cell types and found that ACE2 was specifically expressed in pericyte [] Moreover RNA sequencing from patients with failing hearts and normal donors revealed that myocardial ACE2 expressions were significantly increased in patients with heart failure which was further validated at the protein level by proteomics profiling from heart failure and normal donors [] Another study also showed that the expression of ACE2 in heart tissues of patients with underlying heart disease was higher than that in normal heart tissues [] These two studies suggested that the expression of ACE2 in heart tissue of patients with underlying heart disease was higher than that in normal heart tissue Guo et al analyzed the mRNA expression of TMPRSS2 from the GenotypeTissue Expression GTEx database and the results showed that TMPRSS2 is also expressed in the heart [] By singlecell RNA sequencing scRNASeq to profile the gene expression landscapes of cardiac cells from human embryos Qi revealed that the cardiomyocytes from the heart contain ACE2expressed cells and TMPRSS2expressed cells and the cardiovascular progenitor cells and cells TMPRSS2expressed cells respectively [] These data showed that both ACE2 and TMPRSS2 were expressed in the heart contain ACE2expressed Kidney Studying the viral susceptibility of extrapulmonary ans is important for a deeper understanding for the pathogenesis of SARSCoV infection Studies have shown that ACE2 and TMPRSS2 were Expression analysis from the GTEx database showed that kidney displayed the fifth high expression of ACE2 [] To investigate the expression of ACE2 in kidney Lin analyzed the public singlecell transcriptome dataset of normal kidneys from healthy donors the Fig Entry of SARSCoV2 into host cells SARSCoV2 infected the host cells by the spike protein of the virus and the functions of ACE2 and TMPRSS2 in host cells BiomedicinePharmacotherapy13120201106782 0cM Dong Fig Tissue distributions of ACE2 and TMPRSS2 in human A“B the schematic diagram of the expressions of ACE2 A and TMPRSS2 B in multiple human tissues The colour strength is corresponding to the gene expression level ACE2 and TMPRSS2 were expressed in the brain and heart ACE2 expression is expressed at a relative low level in hepatocytes and mainly located in cholangiocytes while TMPRSS2 is expressed in the hepatocytes and cholangiocytes ACE2 and TMPRSS2 were highly expressed in kidney and intestinal epithelial cells Both ACE2 and TMPRSS2 were also expressed in the esophagus stomach nose testis pancreas breast prostate and thyroid results showed that the ACE2 was distributed across multiple cell types and was mostly enriched in proximal tubule cells [] Fan et al confirmed the specific ACE2 expression in tubular cells from the Gene Expression Omnibus GEO dataset while it was not observed in immune cells and glomerular parietal epithelial cells RNA and protein expression data of ACE2 in different human tissues and cancer cell lines were obtained from three online datasets including the Cancer Cell Line Encyclopedia CCLE GTEx database and the Human Protein Atlas dataset and the results indicated that both mRNA and protein expression levels of ACE2 were relatively high in kidney cells especially in renal tubular cells [] Meanwhile Suryawanshi analyzed the data of kidney tissues in scRNASeq datasets and found that either proximal tubular cells or tubular progenitor cells in the kidney coexpressed ACE2 and TMPRSS2 [] The data of the scRNAseq from GEO dataset GSE134355 showed that ACE2 and TMPRSS2 expression levels were high in nephron epithelial cells epithelial cells endothelial cells and mesangial cells of the kidney [] Recently Pan also found that the TMPRSS2 gene was coexpressed with ACE2 in kidney podocytes [] These data showed that both ACE2 and TMPRSS2 were highly expressed in tissues and cells of kidney Liver Chai et al analyzed the scRNAseq data from GEO database GSE124395 to evaluate ACE2 gene expression in liver the results showed that ACE2 was highly expressed in cholangiocytes which level was about times higher than that in hepatocytes [] The GTEx database also showed that both ACE2 and TMPRSS2 were expressed in the liver [] Zhou identified that TMPRSS2 is highly expressed in hepatocytes from Human Cell Atlas database [] Recently Wen indicated that ACE2 and TMPRSS2 are specifically coexpression in TROP2 liver progenitors of human liver tissue using scRNA sequencing [] These data indicate that ACE2 expression is expressed at a relative low level in hepatocytes and mainly located in cholangiocytes while TMPRSS2 is expressed in hepatocytes Digestive tract A previous study showed that ACE2 could be found in the upper esophagus and it could be detected in stratified epithelial cells and absorptive enterocytes of the ileum and colon [] Quantitative mRNA expression profiling of ACE2 across human tissues by Harmer showed that ACE2 was expressed at a high level in gastrointestinal tissues [] Zhang et al analyzed datasets with singlecell transcriptomes of esophagus gastric ileum colon and lung and the data showed that ACE2 was not only highly expressed in the type II alveolar cells AT2 of lung but also in the stratified epithelial cells ileum absorptive enterocytes cells and colon enterocytes [] Similarly the immunofluorescent staining of esophagus stomach duodenum and rectum showed that ACE2 was stained mainly in the cytoplasm of gastrointestinal epithelial cells [] Besides the scRNAseq data showed that ACE2 was significantly elevated in the proximal and distal enterocytes [] Guo et al suggested that TMPRSS2 was highly expressed in almost all ans of the digestive tract including colon stomach small intestine and esophagus [] Using published scRNAseq data and seven inhouse normal colon samples Lee reported that the coexpressions of ACE2 and TMPRSS2 transcripts were mainly observed in the small intestine and colon [] The highest expressions of TMPRSS2 and ACE2 were found in enterocytes among the intestinal cell types []These data showed that TMPRSS2 and AEC2 are highly expressed in the digestive tract Nervous system Analysis using the GTEx database showed that both TMPRSS2 and ACE2 are expressed at relatively low levels in the brain cortex [] Chen found that ACE2 was relatively highly expressed in some important brain areas such as the substantia nigra and brain ventricles using seven brain transcriptome databases [] ACE2 was expressed at high level in the piriform cortex of human brain and its expression could also be detected in many neurons including both excitatory and inhibitory neurons and some nonneuron cells including astrocytes and oligodendrocytes in human middle temporal gyrus and posterior cingulate cortex [] Qi analyzed the scRNAseq data of substantia nigra BiomedicinePharmacotherapy13120201106783 0cClinical classification of acute cardiac injury NonICUcases ICU cases NonICUcases ICUcases Nonsevere cases Severecases1965 Recoveredcases Died cases NonICUcases ICUcases Survivor cases Nonsurvivor cases Chen [] Hong [] Zhou [] China Korea China M Dong and cortex of brain from GEO database the results showed that both ACE2 and TMPRSS2 were expressed in the oligodendrocyte precursor cells and the astrocytes of the substantia nigra and cortex [] There are limited reports on the expressions of ACE2 and TMPRSS2 in peripheral nervous system Brann analyzed the ACE2 and TMPRSS2 expression in different cell type from human scRNAseq dataset GSE139522 and found that neither olfactory sensory neurons nor olfactory bulb neurons expressed these two genes while ACE2 and TMPRSS2 were expressed in the nonneuronal cells including the sustentacular cells and olfactory bulb pericytes [] These data showed that ACE2 and TMPRSS2 could also be coexpressed in the nervous system Other ans or tissues Table Characteristics of acute cardiac injury after SARScid0 CoV2 infection Study Basic heart disease Acute cardiac injury Country Subject China Wang [] China NA Huang [] Li [] China Moreover ACE2 and TMPRSS2 were also reported to be coexpressed in some other ans [] It has been revealed that both ACE2 and TMPRSS2 are expressed in testis by scRNA sequencing and expression profile analysis indicating that testicular cells might be the potential targets of SARSCoV2 Another report revealed that multiple kinds of cells in the nose including nasal brushing epithelial cells nasal turbinate epithelial cells and nasal airway epithelial cells contained ACE2expressed and TMPRSS2expressed cell clusters [] Moreover ACE2 and TMPRSS2 were also expressed in pancreas breast prostate and thyroid [] and these ans might also be the targets of SARSCov2 Infection of SARSCoV2 and extrapulmonary an injury of patients with COVID19 SARSCoV2 infection and cardiac injury Recently autopsy analysis by Fox revealed that the histopathology of the heart was consistent with the typical pattern of viral myocarditis [] SARSCoV2 RNA was detected in the cardiac tissues of the patients with COVID19 [] These data suggested that SARSCoV2 may directly infect heart The epidemiology of COVID19 reported that cardiac injury was one of the most severe an damages [] The clinical manifestations of cardiac injury in COVID19 patients are complex and could present with heart failure arrhythmias or acute myocardial infarction AMI [ ] Inciardi reported the first case who had the symptom of heart failure at first and later the patient was positive for SARSCoV2 using nucleic acid test [] Cardiac injury is a common symptom in patients with COVID19 Shi reported that patients with COVID19 had cardiac injury [] Moreover there were patients with acute cardiac injury in a cohort including COVID19 patients and of patients with acute cardiac injury in the intensive care unit ICU [] Furthermore a study by Wang showed that there were patients with acute cardiac injury and patients presented with arrhythmia of COVID19 patients while acute cardiac injury was observed in of patients with CIVID19 in the ICU [] These cases suggested that SARSCoV2 may cause serious heart damage which should be widespreadly concerned Furthermore acute cardiac injury is more prevalent in severe cases with COVID19 [“] Table And it has been reported that COVID19 patients with cardiac injury had higher mortality than those without cardiac injury [] In this review we also summarized the possible relationship between basic heart disease and further cardiac injury [“] Table In a cohort of COVID19 patients from Renmin Hospital of Wuhan University China Shi demonstrated that cardiac injury occurred in patients during hospitalization of which had basic heart disease including coronary heart disease and chronic heart failure And only patients with basic heart disease of COVID19 patients without cardiac injury [] Similarly Liu suggested that patients with basic heart disease in COVID19 patients had Table Comorbidity with cardiac injury in COVID19 patients with basic heart disease Subjects with COVID19 Proportion of basic heart disease Patients with Cardiacinjury Study Shi [] Liu [] Xu [] Ma [] Guo [] Patients with basic heart disease With cardiac injury Without cardiac injury cardiac injury compared with patients with basic cardiovascular diseases of COVID19 patients without cardiac injury [] Other studies also indicated that the patients with basic cardiovascular disease are more likely to present heat injury in COVID19 patients [ ] In view of the points above COVID19 patients with underlying cardiac conditions seem to have higher rates of cardiac injury SARSCoV2 infection and kidney injury Recently autopsy analysis on six COVID19 patients showed that varying degrees of acute tubular necrosis were observed in all the renal specimens Nucleoprotein NP antigens and NP positive inclusion body of SARSCoV2 could be seen in kidney tissues from all the samples Moreover viruslike ps were seen in kidney tissues by transmission electronic microscope TEM [] Su analyzed kidney abnormalities in autopsies of patients with COVID19 and found that diffuse proximal tubular damage with the loss of brush border were BiomedicinePharmacotherapy13120201106784 0c SARSCoV2 infection and liver injury M Dong observed Further investigation showed that diffuse necrosis can be seen under the light microscope and electron microscopic examination also showed the clusters of coronavirus ps with distinctive spikes in the tubular epithelium and podocytes [] It was reported that both NP antigens and RNA of SARSCoV2 were detected in urine of COVID19 patients [] These data coincide with the finding of the SARSCoV2 invasion in kidney Collectively SARSCoV2 could directly infect human renal tubules and lead to kidney damage Recent studies have shown that the incidence of acute kidney injury AKI in COVID19 patients ranged from “ and higher frequency of renal function damage with elevated blood urea nitrogen BUN or serum creatinine Scr was observed in COVID19 patients [ ““] Table A study of patients with COVID19 indicated that levels of BUN and Scr were increased in and patients with COVID19 respectively And routine urine tests were performed on patients among which patients were positive for urinary protein and patients were positive for hematuria [] Another study also showed that about patients with COVID19 had abnormal renal function [] Moreover COVID19 patients with more severe disease progression have higher rates of AKI Huang and colleagues reported that of patients with AKI in the ICU were observed and none of the patients who did not require care in the ICU suffered AKI [] Xu found that the fatality rate was obviously higher in COVID19 patients with AKI than those without renal injury [] Furthermore in another study investigating patients with COVID19 at hospital admission more severe patients had higher rates of AKI and the Cox regression analysis also suggested that COVID19 patients who developed AKI had a significantly higher mortality risk [] Therefore AKI is more prevalent in severe cases with COVID19 An autopsy report of a 50yearold patient with COVID19 showed moderate microvesicular steatosis and mild lobular activity in liver tissues [] Moreover Zhao used human liver ductal anoids as a tool to investigate the SARSCoV2 infection and the tissue damage induced by SARSCoV2 ex vivo and the results showed that the expression of SARSCoV2 NP was easily detected in the patchy areas of the hepatic duct indicating that liver ductal anoids were susceptible to SARSCoV2 infection [] In addition SARSCoV2 infection could disrupt the barrier and bile acid transporting functions of cholangiocytes which indicated that SARSCoV2 might directly induce cholangiocyte injury and consequently bile acid accumulation [] In view of the points above liver damage in the COVID19 patients might be directly caused by the viral infection Abnormal liver functions were frequently reported in COVID19 patients [] Epidemiologic studies showed that almost half of the patients had differing degrees of liver damage [“ “] Table Chen reported that out of patients had elevated alanine aminotransferase ALT patients had elevated aspartate aminotransferase AST and had elevated total bilirubin TBIL in Wuhan Jinyintan Hospital Wuhan China [] Similarly a nationwide study involving patients with COVID19 in China showed that more than of patients had elevated ALT and AST and of patients had elevated TBIL [] It was revealed that the levels of direct bilirubin indirect bilirubin ALT alkaline phosphatase ALP and gammaglutamyltransferase GGT were significantly higher in males than that in females with COVID19 and multivariate logistic regression analysis showed that male was an important independent risk factor for predicting acute liver injury ALI in COVID19 patients [] These data indicated that male patients with COVID19 may be more susceptible to liver injury Furthermore Table Characteristics of acute kidney injury after SARScid0 CoV2 infection Study Chen Wang Huang Guan Xu Preexisting kidney conditions NA NA NA [] [] [] [] [] Country China China China China China Subject Li [] Chen [] Hong [] Cheng [] Xiao [] Richardson [] Wan Li Qian Pei [] [] [] [] China China Korea China China America China China China China NA NA NA NA NA NA Scr Serum creatinine BUN Blood urea nitrogen AKI Acute kidney injury Abnormal renal functional indices Scr BUN NA Scr Scr Scr Scr BUN NA Scr BUN Scr NA NA NA Scr BUN Scr NA AKI NA Clinical classification of AKI NA NonICU cases ICU cases ICU cases Nonsevere cases Severe cases Mild cases Severe cases Critical ill cases Nonsevere cases Severe cases Recovered cases Died cases NonICU cases ICU cases NA Nonsevere cases Severe cases Cured cases In hospital cases Died cases Mild cases Severe cases Nonsevere cases Severe cases NA Moderate cases Severe cases Critically ill cases BiomedicinePharmacotherapy13120201106785 0cM Dong Table Characteristics of liver injury after SARScid0 CoV2 infection Study Country Subject China China China China China China China Korea America China China Chen [] Wang Huang Guan [] [] [] Xu Li [] [] Chen Hong [] [] Richardson et Wan Li al [] [] [] Qian [] China NA Patients with preexisting liverconditions NA NA NA Patients with abnormal liver functional indices ALT AST TBIL NA AST AST ALT TBIL ALT AST ALT AST TBIL ALT AST ALT AST TBIL AST ALT AST ALT AST TBIL ALT AST Abnormal liver functional indices in the Nonsevere patients AST ALT NA NA TBIL NA Abnormal liver functional indices in the severe patients ALT NA TBIL NA AST NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Nonsevere patients include patients without ICU care and recovered patients Severe patients include patients with ICU care and death ALT alanine aminotransferase AST aspartate aminotransferase TBIL total bilirubin multiple studies found that AST ALT and TBIL were significantly higher in patients treated in the ICU than that in nonICU patients [] Li suggested that among the patients with abnormal liver function moderate and severe types of patients were more likely to have liver injury and respectively [] Fu analyzed the relationship between ALI and mortality risk in COVID19 patients and the results showed that ALI is more common in the critically ill patients and ALI at the early stage increased death risk of COVID19 patients [] Together abnormal liver functions might be associated with the severity of patients with COVID19 SARSCoV2 infection and digestive tract injury Epithelial cells of the esophagus stomach duodenum and rectum in one COVID19 patient tested positive for SARSCoV2 RNA and the staining of viral NP was also visualized in the cytoplasm of epithelial cells in stomach duodenum and rectum [] Moreover minimally invasive autopsies were performed on three patients died of COVID19 and the results showed that some epithelial cells of the gastrointestinal mucosa were degenerated necrotic and detached [] These studies strongly supported that SARSCoV2 may directly infect the epithelial cells of digestive tract Table SARScid0 CoV2 detection in gastrointestinal specimens Study Subject [] [] Xiao Zhang Tan [] Xing Young Holshue Lescure Tang Wang Xu [] [] [] [] [] [] [] Gastrointestinal samples Stool Anal swabs Rectal swab Stool Stool Stool Stool Stool Stool Rectal swabs Tested positive in gastrointestinal specimens The positive time in gastrointestinal specimens days NA 6cid0 1cid0 5cid0 NA 3cid0 Positive time for Gastrointestinal samples after respiratory samples were negative days NA NA 8cid0 NA NA NA NA NA 2cid0 BiomedicinePharmacotherapy13120201106786 0cM Dong Multiple studies have identified that the SARSCoV2 RNA was detected in anal swabs [] rectal swabs [] and stool specimens [ ] of COVID19 patients It has been demonstrated that SARSCoV2 RNA could be detected in feces from more than half of COVID19 patients [] In another study Xing reported that SARSCoV2 RNA was detected in the feces of three pediatric cases with COVID19 in Qingdao China and the persistence of SARSCoV2 in the digestive tract lasted for 6cid0 days [] The possibility of fecaloral transmission of SARSCoV2 infection needs to be taken into account Furthermore as shown in Table long duration of SARSCoV2 detection in digestive tract by RTPCR has been reported and viral RNA remained detectable in the digestive tract for 2cid0 days after nucleic acid turned negative in respiratory samples [“] The studies suggested that SARSCoV2 could be detected from respiratory tract specimens during the early period to digestive tract specimens during the late period and viral nucleic acid tests in both the respiratory and digestive tract are necessary to confirm the complete clearance of virus Some COVID19 patients presented gastrointestinal symptoms such as diarrhea nausea vomiting and abdominal pain [] Holshue reported the first case of COVID19 patient in the USA which had nausea and vomiting before admission [] Multiple studies found that gastrointestinal symptoms including diarrhea “ nausea and vomiting “ and abdominal pain “ were common at presentation in COVID19 patients [ ““] Table In a cohort of patients with COVID19 in Wuhan China gastrointestinal symptoms were described in up to [] Moreover Sun showed that critically ill patients with COVID19 had gastrointestinal injury GI during hospital stay and the survival curves showed that the mortalities of patients with GI was greater than that of patients without GI [] Jin also found that the rate of the severe type was markedly higher in COVID19 patients with GI symptoms than that in those without GI symptoms [] These data suggested that GI is one of the common extrapulmonary an injuries in COVID19 patients and may be related to the severity of the disease On the other hand many studies showed that patients with COVID19 could present initially with the typical gastrointestinal sympto Answer:

Thyroid_Cancer

"Severe COVID19 has a high mortality rate Comprehensive pathological descriptions of COVID19 are scarce and limited in scope We aimed to describe the histopathological findings and viral tropism in patients who died of severe COVID19Methods In this case series patients were considered eligible if they were older than years with premortem diagnosis of severe acute respiratory syndrome coronavirus infection and COVID19 listed clinically as the direct cause of death Between March and April full postmortem examinations were done on nine patients with confirmed COVID19 including sampling of all major ans A limited autopsy was done on one additional patient Histochemical and immunohistochemical analyses were done and histopathological findings were reported by subspecialist pathologists Viral quantitative RTPCR analysis was done on tissue samples from a subset of patientsFindings The median age at death of our cohort of ten patients was years IQR “ Thrombotic features were observed in at least one major an in all full autopsies predominantly in the lung eight [] of nine patients heart five [] and kidney four [] Diffuse alveolar damage was the most consistent lung finding all ten patients however anisation was noted in patients with a longer clinical course We documented lymphocyte depletion particularly CD8positive T cells in haematological ans and haemophagocytosis Evidence of acute tubular injury was noted in all nine patients examined Major unexpected findings were acute pancreatitis two [] of nine patients adrenal microinfarction three [] pericarditis two [] disseminated mucormycosis one [] of ten patients aortic dissection one [] of nine patients and marantic endocarditis one [] Viral genomes were detected outside of the respiratory tract in four of five patients The presence of subgenomic viral RNA transcripts provided evidence of active viral replication outside the respiratory tract in three of five patientsInterpretation Our series supports clinical data showing that the four dominant interrelated pathological processes in severe COVID19 are diffuse alveolar damage thrombosis haemophagocytosis and immune cell depletion Additionally we report here several novel autopsy findings including pancreatitis pericarditis adrenal microinfarction secondary disseminated mucormycosis and brain microglial activation which require additional investigation to understand their role in COVID19Funding Imperial Biomedical Research Centre Wellcome Trust Biotechnology and Biological Sciences Research CouncilCopyright The Authors Published by Elsevier Ltd This is an Access under the CC BY licenseIntroductionIn the UK the death toll from severe COVID19 is among the highest worldwide1 Severe COVID19 is characterised by respiratory failure with socalled cytokine storm occurring in some patient subsets2 Pathological correlates are required to understand the pathophysiology of COVID19 Autopsybased histopathological analysis is crucial in this respect In anticipation of the COVID19 pandemic our group produced national guidelines for autopsy performance in suspected COVID19 cases3COVID19 is caused by infection with severe acute respiratory syndrome coronavirus SARSCoV245 Although SARSCoV2 and its predecessor SARSCoV causing severe acute respiratory syndrome [SARS] are toll similar on a molecular and clinical level COVID19 has a lower death rate for COVID19 vs for SARS and a substantially higher death deaths worldwide from COVID19 as of Aug vs from SARS than SARS due to a higher basic reproduction number1 The postmortem findings in patients with SARSCoV infection included diffuse alveolar damage DAD splenic and nodal lymphocyte depletion haemophagocytosis renal acute tubular injury cerebral oedema microthrombosis and adrenalitis with necrosis with intracellular SARSCoV detected in the lungs kidney brain and haematological ans6 Various autopsy series on COVID19 have begun to emerge in the literature7“ Here we document the major pathological Lancet Microbe Published Online August 101016 S2666524720301154Department of Cellular Pathology Northwest London Pathology B Hanley MBBCh Prof K N Naresh MD C Roufosse PhD J Weir FRCPath Prof R Goldin MD P Viola MD M Osborn FRCPath and Department of Hepatology P Manousou PhD Imperial College London NHS Trust London UK Centre for Haematology B Hanley Prof K N Naresh and Centre for Inflammatory Diseases C Roufosse Department of Immunology and Inflammation Department of Infectious Disease Prof G S Cooke FRCP A Abdolrasouli PhD O C Swann MRes L Baillon BSc R Penn MSc Prof W S Barclay PhD and Department of Metabolism Prof M Thursz MD Faculty of Medicine Imperial College London London UK Department of Histopathology Royal Brompton and Harefield NHS Foundation Trust and National Heart and Lung Institute Imperial College London London UK Prof A G Nicholson DM Renal and Transplant Centre Hammersmith Hospital Imperial College Healthcare NHS London UK R Corbett PhD Department of Neuropathology Kings College Hospital London UK Prof S AlSarraj FRCPath Death Investigation Committee Royal College of Pathologists London UK M Osborn and Nightingale NHS Hospital London UK M Osborn Correspondence to Dr Brian Hanley Department of Cellular Pathology Northwest London Pathology Charing Cross Hospital campus London W6 8NA UK bhanleyimperialacukwwwthelancetcommicrobe Published online August 101016S2666524720301154 s 0cResearch in contextEvidence before this studyCOVID19 is a new disease and comprehensive descriptions of the histopathological findings at autopsy are scarce We reviewed the literature available on COVID19 autopsy findings up to and including May For this we searched PubMed and Google Scholar databases with no language restrictions using the search terms œCOVID19 œSARSCoV2 œhistology œautopsy and œpostmortemAdded value of this studyOur series focused on providing a comprehensive description of the histopathological findings in patients with severe fatal COVID19 and correlating these findings with data on viral tropism The most prominent findings included diffuse alveolar damage thrombosis haemophagocytosis and immune cell depletion Several novel autopsy findings in patients with COVID19 were also described including pancreatitis pericarditis adrenal microinfarction secondary disseminated mucormycosis and brain microglial activationImplications of all the available evidenceOur study supports the existing clinical and autopsy literature that identified diffuse alveolar damage thrombosis immune cell depletion and macrophage activation as the most prominent pathological features in COVID19 Other factors including acute kidney injury pancreatitis pericarditis secondary fungal infections and preexisting liver disease require further investigation The presence of ongoing viral replication in late stage COVID19 supports the continued use of antiviral therapy even at a point in illness when immunopathology is dominantSee Online for appendixfindings of ten postmortem examinations done on patients with clinically confirmed COVID19MethodsPatient selectionFor this study eligible patients were older than years with premortem SARSCoV2 infection and COVID19 listed clinically as the direct cause of death under part on the Medical Certificate of Cause of Death [MCCD] Consent was obtained for all included patients according to the Human Tissue Authority codes of practice by a member of the Trust Core PostMortem Consent Team Consent rate was · ten of patients Exclusion criteria included extended postmortem interval before autopsy days and patients with COVID19 contributing but not directly leading to death under part of the MCCD Patients were from Imperial College National Health Service NHS Trust nine patients London UK and Royal Brompton Harefield Foundation NHS Trust one patient London UK Premortem SARSCoV2 infection was identified using the Coronavirus Typing multiplextandem PCR HighPlex System Aus Diagnostics Chesham UK Ethical approval for this project was provided by the Imperial College Healthcare Tissue Bank R20012Autopsy proceduresFull autopsies were done on nine patients PM1“ and one patient underwent percutaneous biopsy sampling heart lungs pancreas kidneys and liver using percutaneous biopsy under ultrasound guidance PM10 Full postmortem examinations included standard sampling and were done according to Royal College of Pathologists guidelines3 Eight different regions of the brain were sampled for each full neuropathological examination All tissue samples were fixed in formalin for a minimum of h before embedding Histochemical stains and immunohistochemistry were applied according to local protocols appendix p ans were reviewed by subspecialist pathologists in lung AGN and PV haem ato pathology and immune pathology KNN liver RG gastrointestinal MO neuropathology SAS and renal pathology CR Integrated interpretation was done by a subspecialty autopsy pathologist BH and MO All cases were reviewed independently by at least two pathologistsPCR proceduresFresh tissue for quantitative RTPCR qRTPCR analysis was processed within the biosafety level facilities at St Mary™s Hospital London UK approved by the UK Health and Safety Executive and in accordance with local rules at Imperial College London Total RNA was obtained from fresh tissue samples by use of TRIzolchloroform extraction followed by precipitation and purification using the RNeasy kit Qiagen Hilden Germany qRTPCR against E gene RdRp and subgenomic RNA was done as described elsewhere1617 In patient PM5 total fungal genomic DNA was extracted from four to five ribbon slices of a formalinfixed paraffinembedded lung tissue block Purified DNA was amplified with PCR for panfungal and Mucoralesspecific targetsStatistical analysisAll data was analysed using SPSS software version and expressed using median IQR and percentageRole of the funding sourceThe funder of the study had no role in study design data collection data analysis data interpretation or writing of the The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publicationResultsBetween March and April ten patients were included in the study The median age at death was years wwwthelancetcommicrobe Published online August 101016S2666524720301154s 0cIQR “ Seven of ten patients were men three were women and most patients were White or Asian nine [] Hypertension four [] patients and chronic obstructive pulmonary disease three [] were the most common contributing factors to death according to MCCD All ten patients developed fever and had at least two respiratory symptoms or signs cough shortness of breath reduced oxygen saturations or pleuritic chest pain during their early presentation Of eight patients assessed for inflammatory markers all had elevated inflammatory markers These features were either apparent upon presentation to hospital eight [] of ten patients or developed in an inpatient two [] patients PM8 and PM9 Most patients died within weeks of symptom onset seven [] patients and were not intubated or ventilated six [] patients Four patients were intubated during their presentation PM2 for days PM5 for days PM6 for less than day and PM7 for days The median bodymass index BMI was in the obese range · IQR ·“· and more patients were obese according to BMI at post mortem five [] of nine than indicated on the MCCD one [] of ten The median interval between death and postmortem examination was days IQR ·“· although the limited post mortem had a shorter interval less than h after death Detailed clinical case vignettes are available in the appendix p and clinical data are summarised also in the appendix p All patients had DAD six showed purely exudative phase DAD and four showed a mixture of exudative and anising DAD appendix p figure Three of four patients with anisingphase DAD had spent a substantial period on a ventilator days days and days Florid acute bronchopneumonia and ventilatorassociated pneumonia were not noted in this series although mild interstitial neutrophilic inflammation three [] of ten patients and patchy acute bronchopneumonia three [] patients were observed Interstitial macrophages were prominent Macrophages were accompanied by scattered plasma cells Mild or moderate lymphocyte inflammation was present in all ten patients although focal lymphocyte cuffing of small vessels was noted in six patients We noted that lymphocytes in the lung were predominantly CD4positive T cells CD56positive natural killer cells were rarely found Occasionally a patient had small aggregates of small B cells Chronic bronchiolitis was seen in most patients nine [] of ten No granulomas or viral inclusion were seen Invasive mucormycosis was noted in one patient PM5 figure and confirmed with Mucoralesspecific PCR The mucormycosis was vasculocentric and disseminated involving the hilar lymph nodes heart brain and kidney in the same patientMacroscopic two [] of nine patients and microscopic eight [] of nine pulmonary thromboemboli were frequent observations appendix p figure Both fibrinrich and plateletrich thrombi were identified in smallsized and mediumsized vessels and within the capillaries in alveolar septa figure External examination findings of deep venous thrombosis were not noted Very focal lymphocytic vasculitis was identified in one patientThrombotic features were universal in this cohort and all nine patients who underwent a full autopsy had at least one microthrombosis or macrothrombosis in a major an One of nine patients had a macroscopic acute coronary thrombosis in the right coronary artery whereas five patients had thrombi in the microcirculation of the heart on histological analysis Coronary artery disease was negligible or mild in most patients seven [] of nine Acute myocardial ischaemic damage h old was noted in the patient with an acute coronary artery thrombus figure 2A PM1 A mottled myocardium and subendocardial contraction band necrosis was noted in a ACEBDF 03m µm µmFigure Pulmonary pathological findings in patients with COVID19A Macroscopic subpleural petechial haemorrhage in a 24yearold man PM6 B Hyaline membranes indicative of exudative phase diffuse alveolar damage in a 79yearold woman PM9 at 20x magnification C CD61 immunohistochemical staining indicating plateletrich microthrombosis in alveolar capillaries PM6 D Squamous metaplasia in a 61yearold man PM1 with exudative phase diffuse alveolar damage at × magnification E Interstitial multinucleated giant cells in a 79yearold man PM7 with anising phase diffuse alveolar damage at × magnification the top right insert is of multinucleated giant cells showing positive CD68 staining indicative of macrophage lineage the bottom left insert shows absence of staining for cytokeratins F Periodic acid Schiff staining indicating wide irregular aseptate and ribbonlike hyphae with angle branching and a vasculocentric pattern indicative of mucormycosis in a 22yearold man PM5 the insert is a Grocott silver stain highlighting mucormycosis at 20x magnificationwwwthelancetcommicrobe Published online August 101016S2666524720301154 s 0csecond patient PM2 whether the contraction band necrosis was related to ischaemia or inotropic medication received in the intensive care unit is uncertain appendix p PM1 and PM2 were the two patients with the highest active viral load detected in the heart A single patient had a right atrial thrombus Pericarditis was ACEGBDFH µm µm 03m µmFigure Thrombotic features identified at autopsy in patients with COVID19A Macroscopic right coronary artery thrombosis arrow in a 61yearold man PM1 with exudative phase diffuse alveolar damage B Macroscopic pulmonary thromboembolism arrow in a 97yearold man PM8 C Thrombus in the lung of a 79yearold woman PM9 on haematoxylin and eosin staining at 20X magnification the insert shows CD61 immunohistochemistry indicating moderate staining for platelets D Plateletrich thrombus in the mediumsized vessels surrounding the heart in a 61yearold man PM1 the insert shows strong CD61 staining for platelets Periodic acid Schiff staining showing a glomerular microaneurysm arrow E and microthrombi within glomerular capillary loops arrow F at 40X magnification indicative of thrombotic microangiopathy in a 97yearold man PM8 Macroscopic splenic G and hepatic H infarction in a 22year old man PM5identified in two patients one patient showed florid fibrinous pericarditis containing fungal hyphae PM5 while the other showed only microscopic acute pericarditis appendix p figure The median heart weight was high g and four of nine patients had left ventricular hypertrophy Nonbacterial thrombotic marantic endocarditis was noted in one patient PM5 with no known history or autopsy findings consistent with malignancy or chronic disorder associated with nonbacterial thrombotic marantic endocarditis appendix p figure PM5 had disseminated mucormycosis and numerous other thrombotic features appendix p Cardiac amyloidosis and right atrial thrombosis were identified in one of ten patients PM8 appendix p Lymphocyte depletion involving specific compartments and increased phagocytosis were prominent findings appendix p figure Increased phagocytosis of other cells was identified in the sinusoidal macrophages of the red pulp of the spleen in four [] of seven patients sinus histiocytes of hilar lymph nodes in three [] of six and bone marrow four [] of eight Phagocytosis was identified in at least one of these ans in six of nine patients Bone marrow haemophagocytosis was prominent in two patients PM4 and PM8 and focal in two patients PM7 and PM9 Depletion of periarteriolar Tcell sheaths within the white pulp was observed figure Red pulp was generally congested showing reduced numbers of CD8positive T cells Plasma cells were variably prominent and sinusoidal histiocytes showed phagocytosis of red blood cells and other cells to varying extents Both IgMpositive and IgGpositive plasma cells were identified and they were polytypic for lightchain expression figure Lymph nodes showed preservation of follicles and relative depletion of paracortical areas Medullary areas showed prominence of plasma cells and histiocytes were prominent in the sinuses Bone marrow samples showed reactive changes with trilineage hyperplasia and prominence of plasma cells and histiocytes were a common finding A necrotising granuloma was noted in a single hilar lymph node in one patient and acidfast bacilli were noted on Ziehl Neelson staining appendix p All spleen and lymphoid material examined with immuno histochemistry were negative for EpsteinBarr virus and cytomegalovirusPancreatitis was noted in two of eight patients PM5 was a 22yearold man with frank necrotichaemorrhagic pancreatitis and secondary mucor mycosis figure No fungal hyphae were noted in the pancreas PM8 was a 97yearold man who showed no substantial macroscopic pancreatitis although micro scopic acute inflammation within the pancreas and periadrenal fat necrosis was noted figure A third of patients three [] of nine showed patchy areas of infarcttype adrenocortical necrosis with one patient showing anising microthrombi in adrenal vessels figure No wwwthelancetcommicrobe Published online August 101016S2666524720301154s 0cadrenalitis was noted Two of nine patients showed chronic inflammation in the thyroid with follicular epithelial cell disruption however the significance of this finding is uncertainMedian combined kidney weight was within normal range at g IQR “ Salient renal pathology findings were acute tubular injury in all nine patients underlying moderate cortical scarring of uncertain cause in one patient glomerular microaneurysm and thrombi in one patient figure and rare thrombi in interlobular arteries in four patients PM6 24yearold man of arterial intimal thickening than expected for that age appendix p We observed no evidence of focal and segmental glomerulosclerosis diabetic glomerulopathy or glomerulonephritisdegree had a higher Large droplet fatty change was seen in most patients seven [] of eight Cirrhosis or bridging hepatic fibrosis were noted in three patients No liver thrombosis was identified histologically but one patient showed macroscopic liver infarction figure The median liver weight was g IQR “ and three of nine patients showed hepatomegaly liver weighing g Two patients PM4 and PM7 showed marked autolysis and were not included in analysisModerate to intense microglial activation was the most prominent pathological feature in the CNS five [] of five patients Mild Tcell infiltration was noted around blood vessels and capillaries in all five patients but B cells were absent We found ischaemic changes of variable extent in the neurons of the cortex and in the white matter detected by BAPP β amyloid precursor protein stain However no necrosis of brain tissue or extensive infiltration of inflammatory cells in brain parenchyma or meninges was observed on histological examination although one of nine patients showed macroscopic haemorrhagic transformation in a large recent cerebral infarction in the distribution of the middle cerebral arteryTissues from five patients were analysed for presence of viral genomes against E gene and indications of viral replication against subgenomic RNA transcripts by qRTPCR Viral RNA was present in respiratory tract samples including lung of all five cases analysed In addition two of three patients had detectable viral RNA in the nasal epithelium and four of five patients in the trachea Evidence of viral genomes outside the respiratory tract was found for all five patients but the distribution and viral loads varied case by case figure 5A Viral genomes were also detected using a different qRTPCR targeted at RdRp gene and patterns were consistent between the two sets of primers data not shown A third primer set that detected subgenomic RNA indicated virus replication in all tissues examined with variation between patients in levels and distribution figure 5BACE µmBDF µm µmFigure Other notable autopsy findings in patients with COVID19A Contained aortic dissection green arrow and fibrinous pericarditis red arrow in a 22yearold man PM5 insert is a haematoxylin and eosin stain image of the pericardium showing fibrinous pericarditis 10X magnification B Adrenocortical microinfarcts in a 79yearold woman PM9 with reendothelialising thrombus in small adrenal vessels highlighted by CD34 insert bottom left and haematoxylin and eosin insert top right Marantic endocarditis C highlight with haematoxylin and eosin staining bottom left at 10x magnification and necrotising haemorrhagic pancreatitis D in a 22yearold man PM5 with COVID19 and a secondary fungal lung infection E Periodic acid Schiff staining showing a granular cast arrow indicative of acute tubular injury in a 24yearold man PM6 20X magnification F Microscopic acute pancreatitis on haematoxylin and eosin staining in a 97yearold man PM8 20X magnificationDiscussionIn this series we have described the major pathological findings identified at autopsy in ten patients who died of severe COVID19 The most consistent findings were DAD thrombosis haemophagocytosis and immune cell depletion although unexpected pathologies that are probably related to SARSCoV2 infection were also identifiedDAD was the most consistent and prominent feature in our series and others78 The specific phase of DAD probably represents the degree and chronicity of the offending insult SARSCoV2 infection in relation to the time of death This is similar to previous coronavirus epidemics6 The conclusion by Copin and colleagues7 that COVID19related lung injury œis not diffuse alveolar damage might relate to their sampling strategy and wwwthelancetcommicrobe Published online August 101016S2666524720301154 s 0cADBC µm µm µm µm µmEF µm µm µmFigure Pathological findings in haematological ans in patients with COVID19Tcell depletion in the spleen of a 79yearold woman PM9 with COVID19 haematoxylin and eosin staining of the spleen at 10X magnification A CD20 staining of spleen indicating presence of B cells B 10X magnification with the insert showing the same region at higher power 20X magnification and CD3 staining of spleen indicating depletion of T cells C 10X magnification with the insert showing the same region at higher power 20X magnification Bone marrow phagocytosis in a 97yearold man PM8 with COVID19 haematoxylin and eosin staining of a well preserved bone marrow with an arrow indicating presence of phagocytosis D 40X magnification and CD68PGM1 staining of bone marrow indicating presence of phagocytosis 20X [E] and 40x [F] magnificationchronicity five patients had spent approximately weeks on a ventilator Barton and colleagues8 described prominent acute bronchopneumonia as the major finding in one of two patients although the authors acknowledge that this was probably affected by aspiration in their patient with muscular dystrophy Reports of lung histology in early COVID19 also suggest a degree of lymphocytic pneumonia although DAD is probably superimposed on this over time in the majority of fatal cases7 Pulmonary macrophage infiltration and multinucleated giant cell reactions are prominent similar to other series8“ Definite evidence of in COVID19 will require quantitative analysis comparing tissues from COVID19 patients with DAD associated with other conditions and unaffected tissues Several cases of invasive pulmonary aspergillosis have been reported in patients with severe COVID19 pneumonia18 To our knowledge this is the first description of histologically proven mucormycosis in patients with COVID19 and suggests that other human fungal pathogens including members of Mucoromycotina can complicate COVID19associated infectionslymphocyte depletion tissuerelated Numerous clinical features including raised serum Ddimer concentrations raised procalcitonin concentrations and imaging findings suggest thrombosis is prominent in patients with COVID192 Thrombotic features were universal among patients who underwent full autopsies all nine patients had thrombi in at least one major an and have been noted to be prominent in other COVID19 autopsy series15 In a retrospective study of autopsies in patients with acute respiratory distress syndrome and DAD of various causes only showed thrombi within the small vessels of the lung despite sampling of every lobe of the lung19 Another study used postmortem angiography and identified thrombi in nearly all cases of acute respiratory distress syndrome from various causes20 Whether thrombosis in COVID19 is more common than in other causes of DAD remains uncertain however our data support thrombosis as being a striking feature in these patients A study suggested endotheliitis as a prominent feature in patients with severe COVID1910 but this was not a prominent feature in our patients Importantly limited post mortem or postmortem crosssectional imaging are likely to underrepresent the true extent of thrombosis particularly microthrombosis and its impact on patient death The extent of cardiomegaly fibrointimal thickening of renal blood vessels and obesity in our series supports a contribution of hypertension beyond that noted clinically only four patients had hypertension documented on the MCCDA raised cytokine profile has been documented in a subset of patients with severe COVID192 Consistent with this haematological ans in our series showed prominent phagocytosis in several patients which has not been documented in previous series21 Of the four patients with bone marrow haemophagocytosis one patient PM7 showed mild transaminitis hyperbilirubinaemia elevated serum ferritin concentrations and fever of ·°C however most clinical data were insufficient to assess the presence of haemophagocytic lympho histiocytosis wwwthelancetcommicrobe Published online August 101016S2666524720301154s 0cA substantial feature in COVID19 is lymphocyte depletion and this is supported in our series by the spleen and lymph node findings When compared with those with mild disease patients with severe COVID19 tend to have a higher neutrophil to lymphocyte ratio and higher CD4positive to CD8positive Tcell ratio22 Additionally a negative correlation exists between peripheral blood lymphocyte count and viral copy number22 We have corroborated this evidence by documenting a low number of T cells especially CD8positive T cells and FOXP3positive regulatory T cells in the spleen and lymph nodes in severe fatal COVID19 Notably normal plasma cell both IgM and IgG positive response was present in haematological ans in most patientsThe extent to which anspecific pathologies relate to direct viral replication or consequent immunological and cardiovascular complications is of clinical relevance We report here evidence of viral genomic RNA outside the respiratory tract This finding is in agreement with several previous studies that have identified viral genomes by qRTPCR in postmortem tissues including the colon14 spleen14 liver1423 skin24 heart23 and brain25 We also report detection of subgenomic RNA a product that is only produced in actively infected cells A report identified low viral load in the brain of three of patients with COVID19 but could not detect the virus in subsequent immunohistochemistry and concluded that the viral genomes might have been present in the blood25 Although we cannot exclude that the RNAs detected in our series were similarly carried to the site of sampling in blood the distribution of RNA in different tissues varied widely between postmortem casesPM3 and PM4 appear to have died earlier in the disease course days after symptom onset and had higher viral loads in the respiratory tract than other patients whereas PM3 and PM4 died after long stays in intensive care units and had either lower overall viral RNA PM5 or higher viral RNA outside the respiratory tract PM2 PM1 and PM2

cancer228

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: "Severe COVID19 has a high mortality rate Comprehensive pathological descriptions of COVID19 are scarce and limited in scope We aimed to describe the histopathological findings and viral tropism in patients who died of severe COVID19Methods In this case series patients were considered eligible if they were older than years with premortem diagnosis of severe acute respiratory syndrome coronavirus infection and COVID19 listed clinically as the direct cause of death Between March and April full postmortem examinations were done on nine patients with confirmed COVID19 including sampling of all major ans A limited autopsy was done on one additional patient Histochemical and immunohistochemical analyses were done and histopathological findings were reported by subspecialist pathologists Viral quantitative RTPCR analysis was done on tissue samples from a subset of patientsFindings The median age at death of our cohort of ten patients was years IQR “ Thrombotic features were observed in at least one major an in all full autopsies predominantly in the lung eight [] of nine patients heart five [] and kidney four [] Diffuse alveolar damage was the most consistent lung finding all ten patients however anisation was noted in patients with a longer clinical course We documented lymphocyte depletion particularly CD8positive T cells in haematological ans and haemophagocytosis Evidence of acute tubular injury was noted in all nine patients examined Major unexpected findings were acute pancreatitis two [] of nine patients adrenal microinfarction three [] pericarditis two [] disseminated mucormycosis one [] of ten patients aortic dissection one [] of nine patients and marantic endocarditis one [] Viral genomes were detected outside of the respiratory tract in four of five patients The presence of subgenomic viral RNA transcripts provided evidence of active viral replication outside the respiratory tract in three of five patientsInterpretation Our series supports clinical data showing that the four dominant interrelated pathological processes in severe COVID19 are diffuse alveolar damage thrombosis haemophagocytosis and immune cell depletion Additionally we report here several novel autopsy findings including pancreatitis pericarditis adrenal microinfarction secondary disseminated mucormycosis and brain microglial activation which require additional investigation to understand their role in COVID19Funding Imperial Biomedical Research Centre Wellcome Trust Biotechnology and Biological Sciences Research CouncilCopyright The Authors Published by Elsevier Ltd This is an Access under the CC BY licenseIntroductionIn the UK the death toll from severe COVID19 is among the highest worldwide1 Severe COVID19 is characterised by respiratory failure with socalled cytokine storm occurring in some patient subsets2 Pathological correlates are required to understand the pathophysiology of COVID19 Autopsybased histopathological analysis is crucial in this respect In anticipation of the COVID19 pandemic our group produced national guidelines for autopsy performance in suspected COVID19 cases3COVID19 is caused by infection with severe acute respiratory syndrome coronavirus SARSCoV245 Although SARSCoV2 and its predecessor SARSCoV causing severe acute respiratory syndrome [SARS] are toll similar on a molecular and clinical level COVID19 has a lower death rate for COVID19 vs for SARS and a substantially higher death deaths worldwide from COVID19 as of Aug vs from SARS than SARS due to a higher basic reproduction number1 The postmortem findings in patients with SARSCoV infection included diffuse alveolar damage DAD splenic and nodal lymphocyte depletion haemophagocytosis renal acute tubular injury cerebral oedema microthrombosis and adrenalitis with necrosis with intracellular SARSCoV detected in the lungs kidney brain and haematological ans6 Various autopsy series on COVID19 have begun to emerge in the literature7“ Here we document the major pathological Lancet Microbe Published Online August 101016 S2666524720301154Department of Cellular Pathology Northwest London Pathology B Hanley MBBCh Prof K N Naresh MD C Roufosse PhD J Weir FRCPath Prof R Goldin MD P Viola MD M Osborn FRCPath and Department of Hepatology P Manousou PhD Imperial College London NHS Trust London UK Centre for Haematology B Hanley Prof K N Naresh and Centre for Inflammatory Diseases C Roufosse Department of Immunology and Inflammation Department of Infectious Disease Prof G S Cooke FRCP A Abdolrasouli PhD O C Swann MRes L Baillon BSc R Penn MSc Prof W S Barclay PhD and Department of Metabolism Prof M Thursz MD Faculty of Medicine Imperial College London London UK Department of Histopathology Royal Brompton and Harefield NHS Foundation Trust and National Heart and Lung Institute Imperial College London London UK Prof A G Nicholson DM Renal and Transplant Centre Hammersmith Hospital Imperial College Healthcare NHS London UK R Corbett PhD Department of Neuropathology Kings College Hospital London UK Prof S AlSarraj FRCPath Death Investigation Committee Royal College of Pathologists London UK M Osborn and Nightingale NHS Hospital London UK M Osborn Correspondence to Dr Brian Hanley Department of Cellular Pathology Northwest London Pathology Charing Cross Hospital campus London W6 8NA UK bhanleyimperialacukwwwthelancetcommicrobe Published online August 101016S2666524720301154 s 0cResearch in contextEvidence before this studyCOVID19 is a new disease and comprehensive descriptions of the histopathological findings at autopsy are scarce We reviewed the literature available on COVID19 autopsy findings up to and including May For this we searched PubMed and Google Scholar databases with no language restrictions using the search terms œCOVID19 œSARSCoV2 œhistology œautopsy and œpostmortemAdded value of this studyOur series focused on providing a comprehensive description of the histopathological findings in patients with severe fatal COVID19 and correlating these findings with data on viral tropism The most prominent findings included diffuse alveolar damage thrombosis haemophagocytosis and immune cell depletion Several novel autopsy findings in patients with COVID19 were also described including pancreatitis pericarditis adrenal microinfarction secondary disseminated mucormycosis and brain microglial activationImplications of all the available evidenceOur study supports the existing clinical and autopsy literature that identified diffuse alveolar damage thrombosis immune cell depletion and macrophage activation as the most prominent pathological features in COVID19 Other factors including acute kidney injury pancreatitis pericarditis secondary fungal infections and preexisting liver disease require further investigation The presence of ongoing viral replication in late stage COVID19 supports the continued use of antiviral therapy even at a point in illness when immunopathology is dominantSee Online for appendixfindings of ten postmortem examinations done on patients with clinically confirmed COVID19MethodsPatient selectionFor this study eligible patients were older than years with premortem SARSCoV2 infection and COVID19 listed clinically as the direct cause of death under part on the Medical Certificate of Cause of Death [MCCD] Consent was obtained for all included patients according to the Human Tissue Authority codes of practice by a member of the Trust Core PostMortem Consent Team Consent rate was · ten of patients Exclusion criteria included extended postmortem interval before autopsy days and patients with COVID19 contributing but not directly leading to death under part of the MCCD Patients were from Imperial College National Health Service NHS Trust nine patients London UK and Royal Brompton Harefield Foundation NHS Trust one patient London UK Premortem SARSCoV2 infection was identified using the Coronavirus Typing multiplextandem PCR HighPlex System Aus Diagnostics Chesham UK Ethical approval for this project was provided by the Imperial College Healthcare Tissue Bank R20012Autopsy proceduresFull autopsies were done on nine patients PM1“ and one patient underwent percutaneous biopsy sampling heart lungs pancreas kidneys and liver using percutaneous biopsy under ultrasound guidance PM10 Full postmortem examinations included standard sampling and were done according to Royal College of Pathologists guidelines3 Eight different regions of the brain were sampled for each full neuropathological examination All tissue samples were fixed in formalin for a minimum of h before embedding Histochemical stains and immunohistochemistry were applied according to local protocols appendix p ans were reviewed by subspecialist pathologists in lung AGN and PV haem ato pathology and immune pathology KNN liver RG gastrointestinal MO neuropathology SAS and renal pathology CR Integrated interpretation was done by a subspecialty autopsy pathologist BH and MO All cases were reviewed independently by at least two pathologistsPCR proceduresFresh tissue for quantitative RTPCR qRTPCR analysis was processed within the biosafety level facilities at St Mary™s Hospital London UK approved by the UK Health and Safety Executive and in accordance with local rules at Imperial College London Total RNA was obtained from fresh tissue samples by use of TRIzolchloroform extraction followed by precipitation and purification using the RNeasy kit Qiagen Hilden Germany qRTPCR against E gene RdRp and subgenomic RNA was done as described elsewhere1617 In patient PM5 total fungal genomic DNA was extracted from four to five ribbon slices of a formalinfixed paraffinembedded lung tissue block Purified DNA was amplified with PCR for panfungal and Mucoralesspecific targetsStatistical analysisAll data was analysed using SPSS software version and expressed using median IQR and percentageRole of the funding sourceThe funder of the study had no role in study design data collection data analysis data interpretation or writing of the The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publicationResultsBetween March and April ten patients were included in the study The median age at death was years wwwthelancetcommicrobe Published online August 101016S2666524720301154s 0cIQR “ Seven of ten patients were men three were women and most patients were White or Asian nine [] Hypertension four [] patients and chronic obstructive pulmonary disease three [] were the most common contributing factors to death according to MCCD All ten patients developed fever and had at least two respiratory symptoms or signs cough shortness of breath reduced oxygen saturations or pleuritic chest pain during their early presentation Of eight patients assessed for inflammatory markers all had elevated inflammatory markers These features were either apparent upon presentation to hospital eight [] of ten patients or developed in an inpatient two [] patients PM8 and PM9 Most patients died within weeks of symptom onset seven [] patients and were not intubated or ventilated six [] patients Four patients were intubated during their presentation PM2 for days PM5 for days PM6 for less than day and PM7 for days The median bodymass index BMI was in the obese range · IQR ·“· and more patients were obese according to BMI at post mortem five [] of nine than indicated on the MCCD one [] of ten The median interval between death and postmortem examination was days IQR ·“· although the limited post mortem had a shorter interval less than h after death Detailed clinical case vignettes are available in the appendix p and clinical data are summarised also in the appendix p All patients had DAD six showed purely exudative phase DAD and four showed a mixture of exudative and anising DAD appendix p figure Three of four patients with anisingphase DAD had spent a substantial period on a ventilator days days and days Florid acute bronchopneumonia and ventilatorassociated pneumonia were not noted in this series although mild interstitial neutrophilic inflammation three [] of ten patients and patchy acute bronchopneumonia three [] patients were observed Interstitial macrophages were prominent Macrophages were accompanied by scattered plasma cells Mild or moderate lymphocyte inflammation was present in all ten patients although focal lymphocyte cuffing of small vessels was noted in six patients We noted that lymphocytes in the lung were predominantly CD4positive T cells CD56positive natural killer cells were rarely found Occasionally a patient had small aggregates of small B cells Chronic bronchiolitis was seen in most patients nine [] of ten No granulomas or viral inclusion were seen Invasive mucormycosis was noted in one patient PM5 figure and confirmed with Mucoralesspecific PCR The mucormycosis was vasculocentric and disseminated involving the hilar lymph nodes heart brain and kidney in the same patientMacroscopic two [] of nine patients and microscopic eight [] of nine pulmonary thromboemboli were frequent observations appendix p figure Both fibrinrich and plateletrich thrombi were identified in smallsized and mediumsized vessels and within the capillaries in alveolar septa figure External examination findings of deep venous thrombosis were not noted Very focal lymphocytic vasculitis was identified in one patientThrombotic features were universal in this cohort and all nine patients who underwent a full autopsy had at least one microthrombosis or macrothrombosis in a major an One of nine patients had a macroscopic acute coronary thrombosis in the right coronary artery whereas five patients had thrombi in the microcirculation of the heart on histological analysis Coronary artery disease was negligible or mild in most patients seven [] of nine Acute myocardial ischaemic damage h old was noted in the patient with an acute coronary artery thrombus figure 2A PM1 A mottled myocardium and subendocardial contraction band necrosis was noted in a ACEBDF 03m µm µmFigure Pulmonary pathological findings in patients with COVID19A Macroscopic subpleural petechial haemorrhage in a 24yearold man PM6 B Hyaline membranes indicative of exudative phase diffuse alveolar damage in a 79yearold woman PM9 at 20x magnification C CD61 immunohistochemical staining indicating plateletrich microthrombosis in alveolar capillaries PM6 D Squamous metaplasia in a 61yearold man PM1 with exudative phase diffuse alveolar damage at × magnification E Interstitial multinucleated giant cells in a 79yearold man PM7 with anising phase diffuse alveolar damage at × magnification the top right insert is of multinucleated giant cells showing positive CD68 staining indicative of macrophage lineage the bottom left insert shows absence of staining for cytokeratins F Periodic acid Schiff staining indicating wide irregular aseptate and ribbonlike hyphae with angle branching and a vasculocentric pattern indicative of mucormycosis in a 22yearold man PM5 the insert is a Grocott silver stain highlighting mucormycosis at 20x magnificationwwwthelancetcommicrobe Published online August 101016S2666524720301154 s 0csecond patient PM2 whether the contraction band necrosis was related to ischaemia or inotropic medication received in the intensive care unit is uncertain appendix p PM1 and PM2 were the two patients with the highest active viral load detected in the heart A single patient had a right atrial thrombus Pericarditis was ACEGBDFH µm µm 03m µmFigure Thrombotic features identified at autopsy in patients with COVID19A Macroscopic right coronary artery thrombosis arrow in a 61yearold man PM1 with exudative phase diffuse alveolar damage B Macroscopic pulmonary thromboembolism arrow in a 97yearold man PM8 C Thrombus in the lung of a 79yearold woman PM9 on haematoxylin and eosin staining at 20X magnification the insert shows CD61 immunohistochemistry indicating moderate staining for platelets D Plateletrich thrombus in the mediumsized vessels surrounding the heart in a 61yearold man PM1 the insert shows strong CD61 staining for platelets Periodic acid Schiff staining showing a glomerular microaneurysm arrow E and microthrombi within glomerular capillary loops arrow F at 40X magnification indicative of thrombotic microangiopathy in a 97yearold man PM8 Macroscopic splenic G and hepatic H infarction in a 22year old man PM5identified in two patients one patient showed florid fibrinous pericarditis containing fungal hyphae PM5 while the other showed only microscopic acute pericarditis appendix p figure The median heart weight was high g and four of nine patients had left ventricular hypertrophy Nonbacterial thrombotic marantic endocarditis was noted in one patient PM5 with no known history or autopsy findings consistent with malignancy or chronic disorder associated with nonbacterial thrombotic marantic endocarditis appendix p figure PM5 had disseminated mucormycosis and numerous other thrombotic features appendix p Cardiac amyloidosis and right atrial thrombosis were identified in one of ten patients PM8 appendix p Lymphocyte depletion involving specific compartments and increased phagocytosis were prominent findings appendix p figure Increased phagocytosis of other cells was identified in the sinusoidal macrophages of the red pulp of the spleen in four [] of seven patients sinus histiocytes of hilar lymph nodes in three [] of six and bone marrow four [] of eight Phagocytosis was identified in at least one of these ans in six of nine patients Bone marrow haemophagocytosis was prominent in two patients PM4 and PM8 and focal in two patients PM7 and PM9 Depletion of periarteriolar Tcell sheaths within the white pulp was observed figure Red pulp was generally congested showing reduced numbers of CD8positive T cells Plasma cells were variably prominent and sinusoidal histiocytes showed phagocytosis of red blood cells and other cells to varying extents Both IgMpositive and IgGpositive plasma cells were identified and they were polytypic for lightchain expression figure Lymph nodes showed preservation of follicles and relative depletion of paracortical areas Medullary areas showed prominence of plasma cells and histiocytes were prominent in the sinuses Bone marrow samples showed reactive changes with trilineage hyperplasia and prominence of plasma cells and histiocytes were a common finding A necrotising granuloma was noted in a single hilar lymph node in one patient and acidfast bacilli were noted on Ziehl Neelson staining appendix p All spleen and lymphoid material examined with immuno histochemistry were negative for EpsteinBarr virus and cytomegalovirusPancreatitis was noted in two of eight patients PM5 was a 22yearold man with frank necrotichaemorrhagic pancreatitis and secondary mucor mycosis figure No fungal hyphae were noted in the pancreas PM8 was a 97yearold man who showed no substantial macroscopic pancreatitis although micro scopic acute inflammation within the pancreas and periadrenal fat necrosis was noted figure A third of patients three [] of nine showed patchy areas of infarcttype adrenocortical necrosis with one patient showing anising microthrombi in adrenal vessels figure No wwwthelancetcommicrobe Published online August 101016S2666524720301154s 0cadrenalitis was noted Two of nine patients showed chronic inflammation in the thyroid with follicular epithelial cell disruption however the significance of this finding is uncertainMedian combined kidney weight was within normal range at g IQR “ Salient renal pathology findings were acute tubular injury in all nine patients underlying moderate cortical scarring of uncertain cause in one patient glomerular microaneurysm and thrombi in one patient figure and rare thrombi in interlobular arteries in four patients PM6 24yearold man of arterial intimal thickening than expected for that age appendix p We observed no evidence of focal and segmental glomerulosclerosis diabetic glomerulopathy or glomerulonephritisdegree had a higher Large droplet fatty change was seen in most patients seven [] of eight Cirrhosis or bridging hepatic fibrosis were noted in three patients No liver thrombosis was identified histologically but one patient showed macroscopic liver infarction figure The median liver weight was g IQR “ and three of nine patients showed hepatomegaly liver weighing g Two patients PM4 and PM7 showed marked autolysis and were not included in analysisModerate to intense microglial activation was the most prominent pathological feature in the CNS five [] of five patients Mild Tcell infiltration was noted around blood vessels and capillaries in all five patients but B cells were absent We found ischaemic changes of variable extent in the neurons of the cortex and in the white matter detected by BAPP β amyloid precursor protein stain However no necrosis of brain tissue or extensive infiltration of inflammatory cells in brain parenchyma or meninges was observed on histological examination although one of nine patients showed macroscopic haemorrhagic transformation in a large recent cerebral infarction in the distribution of the middle cerebral arteryTissues from five patients were analysed for presence of viral genomes against E gene and indications of viral replication against subgenomic RNA transcripts by qRTPCR Viral RNA was present in respiratory tract samples including lung of all five cases analysed In addition two of three patients had detectable viral RNA in the nasal epithelium and four of five patients in the trachea Evidence of viral genomes outside the respiratory tract was found for all five patients but the distribution and viral loads varied case by case figure 5A Viral genomes were also detected using a different qRTPCR targeted at RdRp gene and patterns were consistent between the two sets of primers data not shown A third primer set that detected subgenomic RNA indicated virus replication in all tissues examined with variation between patients in levels and distribution figure 5BACE µmBDF µm µmFigure Other notable autopsy findings in patients with COVID19A Contained aortic dissection green arrow and fibrinous pericarditis red arrow in a 22yearold man PM5 insert is a haematoxylin and eosin stain image of the pericardium showing fibrinous pericarditis 10X magnification B Adrenocortical microinfarcts in a 79yearold woman PM9 with reendothelialising thrombus in small adrenal vessels highlighted by CD34 insert bottom left and haematoxylin and eosin insert top right Marantic endocarditis C highlight with haematoxylin and eosin staining bottom left at 10x magnification and necrotising haemorrhagic pancreatitis D in a 22yearold man PM5 with COVID19 and a secondary fungal lung infection E Periodic acid Schiff staining showing a granular cast arrow indicative of acute tubular injury in a 24yearold man PM6 20X magnification F Microscopic acute pancreatitis on haematoxylin and eosin staining in a 97yearold man PM8 20X magnificationDiscussionIn this series we have described the major pathological findings identified at autopsy in ten patients who died of severe COVID19 The most consistent findings were DAD thrombosis haemophagocytosis and immune cell depletion although unexpected pathologies that are probably related to SARSCoV2 infection were also identifiedDAD was the most consistent and prominent feature in our series and others78 The specific phase of DAD probably represents the degree and chronicity of the offending insult SARSCoV2 infection in relation to the time of death This is similar to previous coronavirus epidemics6 The conclusion by Copin and colleagues7 that COVID19related lung injury œis not diffuse alveolar damage might relate to their sampling strategy and wwwthelancetcommicrobe Published online August 101016S2666524720301154 s 0cADBC µm µm µm µm µmEF µm µm µmFigure Pathological findings in haematological ans in patients with COVID19Tcell depletion in the spleen of a 79yearold woman PM9 with COVID19 haematoxylin and eosin staining of the spleen at 10X magnification A CD20 staining of spleen indicating presence of B cells B 10X magnification with the insert showing the same region at higher power 20X magnification and CD3 staining of spleen indicating depletion of T cells C 10X magnification with the insert showing the same region at higher power 20X magnification Bone marrow phagocytosis in a 97yearold man PM8 with COVID19 haematoxylin and eosin staining of a well preserved bone marrow with an arrow indicating presence of phagocytosis D 40X magnification and CD68PGM1 staining of bone marrow indicating presence of phagocytosis 20X [E] and 40x [F] magnificationchronicity five patients had spent approximately weeks on a ventilator Barton and colleagues8 described prominent acute bronchopneumonia as the major finding in one of two patients although the authors acknowledge that this was probably affected by aspiration in their patient with muscular dystrophy Reports of lung histology in early COVID19 also suggest a degree of lymphocytic pneumonia although DAD is probably superimposed on this over time in the majority of fatal cases7 Pulmonary macrophage infiltration and multinucleated giant cell reactions are prominent similar to other series8“ Definite evidence of in COVID19 will require quantitative analysis comparing tissues from COVID19 patients with DAD associated with other conditions and unaffected tissues Several cases of invasive pulmonary aspergillosis have been reported in patients with severe COVID19 pneumonia18 To our knowledge this is the first description of histologically proven mucormycosis in patients with COVID19 and suggests that other human fungal pathogens including members of Mucoromycotina can complicate COVID19associated infectionslymphocyte depletion tissuerelated Numerous clinical features including raised serum Ddimer concentrations raised procalcitonin concentrations and imaging findings suggest thrombosis is prominent in patients with COVID192 Thrombotic features were universal among patients who underwent full autopsies all nine patients had thrombi in at least one major an and have been noted to be prominent in other COVID19 autopsy series15 In a retrospective study of autopsies in patients with acute respiratory distress syndrome and DAD of various causes only showed thrombi within the small vessels of the lung despite sampling of every lobe of the lung19 Another study used postmortem angiography and identified thrombi in nearly all cases of acute respiratory distress syndrome from various causes20 Whether thrombosis in COVID19 is more common than in other causes of DAD remains uncertain however our data support thrombosis as being a striking feature in these patients A study suggested endotheliitis as a prominent feature in patients with severe COVID1910 but this was not a prominent feature in our patients Importantly limited post mortem or postmortem crosssectional imaging are likely to underrepresent the true extent of thrombosis particularly microthrombosis and its impact on patient death The extent of cardiomegaly fibrointimal thickening of renal blood vessels and obesity in our series supports a contribution of hypertension beyond that noted clinically only four patients had hypertension documented on the MCCDA raised cytokine profile has been documented in a subset of patients with severe COVID192 Consistent with this haematological ans in our series showed prominent phagocytosis in several patients which has not been documented in previous series21 Of the four patients with bone marrow haemophagocytosis one patient PM7 showed mild transaminitis hyperbilirubinaemia elevated serum ferritin concentrations and fever of ·°C however most clinical data were insufficient to assess the presence of haemophagocytic lympho histiocytosis wwwthelancetcommicrobe Published online August 101016S2666524720301154s 0cA substantial feature in COVID19 is lymphocyte depletion and this is supported in our series by the spleen and lymph node findings When compared with those with mild disease patients with severe COVID19 tend to have a higher neutrophil to lymphocyte ratio and higher CD4positive to CD8positive Tcell ratio22 Additionally a negative correlation exists between peripheral blood lymphocyte count and viral copy number22 We have corroborated this evidence by documenting a low number of T cells especially CD8positive T cells and FOXP3positive regulatory T cells in the spleen and lymph nodes in severe fatal COVID19 Notably normal plasma cell both IgM and IgG positive response was present in haematological ans in most patientsThe extent to which anspecific pathologies relate to direct viral replication or consequent immunological and cardiovascular complications is of clinical relevance We report here evidence of viral genomic RNA outside the respiratory tract This finding is in agreement with several previous studies that have identified viral genomes by qRTPCR in postmortem tissues including the colon14 spleen14 liver1423 skin24 heart23 and brain25 We also report detection of subgenomic RNA a product that is only produced in actively infected cells A report identified low viral load in the brain of three of patients with COVID19 but could not detect the virus in subsequent immunohistochemistry and concluded that the viral genomes might have been present in the blood25 Although we cannot exclude that the RNAs detected in our series were similarly carried to the site of sampling in blood the distribution of RNA in different tissues varied widely between postmortem casesPM3 and PM4 appear to have died earlier in the disease course days after symptom onset and had higher viral loads in the respiratory tract than other patients whereas PM3 and PM4 died after long stays in intensive care units and had either lower overall viral RNA PM5 or higher viral RNA outside the respiratory tract PM2 PM1 and PM2 Answer:

Thyroid_Cancer

"Optimizing Telemedicine Encounters for Oral and Maxillofacial Surgeons During the COVID19 Pandemic Hwi Sean Moon DDS MD1 Tim T Wang BA23 Karthik Rajasekaran MD4 Ryan Brewster BA5 Rabie M Shanti DMD MD46 Neeraj Panchal DDS MD MA7 1Resident Department of Oral Maxillofacial Surgery University of Pennsylvania Philadelphia PA 2DMD Candidate School of Dental Medicine University of Pennsylvania Philadelphia PA 3MPH Candidate Perelman School of Medicine University of Pennsylvania Philadelphia PA 4Assistant Professor of Otorhinolaryngology University of Pennsylvania Philadelphia PA 5MD Candidate Stanford University School of Medicine Stanford University Stanford CA 6Assistant Professor of Oral and Maxillofacial Surgery University of Pennsylvania Philadelphia PA 7Assistant Professor and Section Chief of Oral and Maxillofacial Surgery Philadelphia Veterans Affairs Medical Center Penn Presbyterian Medical Center University of Pennsylvania School of Dental Medicine Philadelphia PA Corresponding Author Neeraj Panchal DDS MD MA Tel Mailing Address N 39th St Philadelphia PA Email Address npanchalupennedu Disclosures None to report Abstract Word Count Manuscript Word Count Number of References Number of Figurestables Number of Supplements 0c The COVID19 pandemic has changed conventional medical practice patterns across all health disciplines including oral and maxillofacial surgery practices The use of telemedicine has rapidly expanded to uphold safety strategies of physical distancing and disease transmission reduction while maintaining uninterrupted care of patients To date there are no specific guidelines to optimize telemedicine encounters in the oral and maxillofacial surgery practice The goal of this paper is to provide best practices for both oral and maxillofacial surgeons and their patients to effectively utilize telemedicine for the duration of COVID19 and beyond Statement of Clinical Relevance The goal of this paper is to provide best practices for both oral and maxillofacial surgeons and their patients to effectively utilize telemedicine for the duration of COVID19 and beyond INTRODUCTION The COVID19 pandemic has disrupted society in a multitude of ways Healthcare is no exception the SARSCoV virus™ rapid transmission and high hospitalization rate have strained the availability of medical resources including personal protective equipment PPE respiratory ventilators and hospital beds[“] The virus also poses a major threat to healthcare personnel whose risk of exposure are compounded by the aforementioned PPE shortages[“] In response the American Association of Oral and Maxillofacial Surgeons AAOMS recommended delaying elective surgeries in accordance with the Centers for Disease Control and Prevention™s calls to 0c postpone elective medical and dental procedures[“] Similarly four out of five dental offices have closed for all except emergency procedures[] In the face of these challenges the medical and dental communities have remained steadfast in caring for patients with nonelective health needs and innovating alternate ways to deliver care One of the most important and popular alterations in the delivery of care is the increased utilization of telemedicine which allows surgeons and patients to connect virtually[ ] This has enabled patients to access muchneeded medical care while preserving PPE and minimizing exposure to pathogens Though studies have found telemedicine to decrease costs and save time without compromising patient satisfaction it was not widely used in healthcare before the COVID19 pandemic[ ] Similarly teledentistry was deemed to be œin its infancy by the founder of the American Teledentistry Association in [“] Nevertheless telemedicine has shown promise and has been incorporated into the workflow of various oral and maxillofacial surgery institutions and practices across the country Virtual visits are particularly useful in triaging patients For example patients with dentoalveolar infections can meet virtually with surgeons and receive prescriptions for appropriate analgesics and antibiotics without going to the emergency department Also patients with oral lesions can take images and show their surgeon before their inperson visit to expedite the diagnosis and treatment planning workflow This enables patients to access timely attention of providers while lightening the load on the healthcare system by reducing the number of inperson visits 0c Associated with the recent rise in telemedicine™s popularity is a learning curve for both surgeons and patients The incorporation of technology and the shift to virtual visits can be jarring for the patientsurgeon relationship and must be navigated thoughtfully While there have been helpful telehealth guides for surgeons and patients in other surgical specialties we do not know of any such guidelines for oral and maxillofacial surgery[ ] As such we detail best practices for both oral and maxillofacial surgeons OMS and their patients to effectively utilize telemedicine for the duration of COVID19 and beyond GENERAL CONSIDERATIONS FOR TELEMEDICINE In accordance with the AAOMS White Paper on ˜Telehealth and Remote Treatment™ virtual management of any oral and maxillofacial surgical condition should only be provided by appropriately licensed oral and maxillofacial surgeons as regulated by the state law[] The delivery of patient care through telemedicine must continue to follow evidencebased guidelines to ensure quality and safety for all patients All providers must comply with the latest telehealth requirements outlined by the United State Health and Human Services to protect patient privacy and comply with the Health Insurance Portability and Accountability Act HIPAA[] Furthermore providers are ethically obligated to inform all patients about the potential benefits limitations and risks of telemedicine[] Patients requiring emergency or urgent services must be directed to the nearest hospital 0c SETTING UP FOR TELEMEDICINE While there are several modalities to conduct a telemedicine encounter we strongly recommend a live synchronous twoway interaction between the patient and the OMS incorporating both audio and visual telecommunications tools This can be achieved with a desktop computer laptop or smartphone The United States Census Bureau reports that approximately percent of households have computers or smartphones and percent have broadband internet subscriptions[] Of these options though we recommend using a desktop or a large screen laptop with a highresolution camera over smartphones even if the latter meets the minimum technical requirements Ideally telemedicine visits can offer a clinic experience that closely simulates inperson encounters Trained administrative staff members should call patients beforehand to discuss the virtual setup and basic expectations for the visit Prasad et al created Figure which exemplifies a patient informational handout with graphic illustrations detailing the set up as well as key examination steps that patients may be asked to perform during the encounter [] In the following we will detail key aspects and considerations for both OMS and patients to maximize their telehealth visits 0c Insurance Coverage and Billing In an effort to reduce the burden posted on healthcare entities and facilitate mitigation efforts the Centers for Medicare Medicaid Services CMS broadened access for Telemedicine coverage with private payers following suit CMS expansion included voiceonly visits which is critical for patients without access to a smartphone or computer video capabilities Furthermore CMS allowed for parity of payment for Telemedicine visits and inperson visits so providers can bill Medicaid and Medicare at the same rate as they would for an inperson visit[] This new policy is especially relevant for older patients covered by Medicare for they are generally at higher risk for COVID19 complications[] Therefore before telehealth encounters providers should confirm if the patient™s insurance plan has Telemedicine coverage and also whether the insurance plan waives all copays for nonCOVID19 related visits to avoid a scenario where a patient receives a bill unknowingly The American Association of Oral and Maxillofacial Surgeons provide additional detailed information about telehealth billing relevant for OMSs including updated links to AMA and ADA billing codes on its website at httpswwwaaomsorgpracticeresourcestelehealthfaqs 0c Professionalism and provider attire The visit should replicate the same level of professionalism as that of inperson appointments Providers should dress professionally as they would in their office or hospitalbased practice Before the patient comes in providers should review the patient™s relevant medical records and chief complaints to save time and maximize the efficiency of the visit Also in the interest of both time and professionalism OMS and patients should both be mindful to start the visit on time During the visit OMS should communicate with patients to maintain transparency For instance if an OMS needs to document something during the visit they should respectfully inform the patient of the task to prevent any misunderstanding At the end of the visit it is important for OMS to summarize what they accomplished during the visit and provide a clear plan for appropriate next steps Physical background When possible OMS and patients should conduct virtual encounters in welllit spaces Lighting specifically can have a profound effect on video quality As such overhead lights can be helpful while lights behind the person should be avoided Care should also be taken to prevent other sources of potential disruptions such as background noise or visual distractions It may be helpful for OMS to evaluate their surroundings from the perspective of their patients 0c Technological background It is important for OMS to test their video and audio quality before visits to anticipate any potential technical difficulties that may interrupt the encounter A strong WiFi internet connection is preferred over cellular data to ensure a stable signal With the exception of electronic health records OMS should close any unnecessary programs or internet browser tabs to preserve internet bandwidth In addition OMS should be mindful that their patients may have varying internet speeds As such OMS should give approximately seconds of lag time after patients stop speaking to allow all of their words to come through completely Patient and camera position If possible patients should sit upright on a chair in front of a computer placed on top of a desk They should sit close enough to the camera so that their entire head and neck area are within the video frame The camera should be at approximately eyelevel for both OMS and patients to maintain eye contact and remain engaged during the virtual encounter For patients using a smartphone the device can be propped up at a to 90degree angle from the table surface to allow patients to free both of their hands for physical exam tasks Patient clothing Patients must be notified of the appropriate clothing well in advance prior to the appointment Ideally clothing should allow patients™ entire head and neck regions to be visualized while maintaining patient comfort and professionalism Any hat or scarves should be removed if at all possible maintaining appropriate cultural and religious norms Patient items Prior to the appointment patients should prepare the following items which can help aid OMS in visualization and retraction during the virtual physical exam Most of these items are commonplace inexpensive and available at the patient™s home 0c Flashlight A flashlight or a penlight can enhance visualization of certain obscure head and neck structures particularly those in the oral cavity The builtin flashlight of a smartphone can also be used Ruler A ruler or a measuring tape can be used to measure the patient™s maximal mouth opening and mandibular range of motion Napkins A napkin can be used to touch any intraoral landmarks and also to clean up after any inadvertent salivation from the virtual physical exam Spoon A spoon can be used to retract the cheeks or depress the tongue to evaluate structures of the oropharynx such as the soft palate and tonsillar pillars Cheek retractors A fun way for patients to achieve cheek retraction can be to use the plastic props from the board game œSpeak Out which was created in [] This game provides horseshoeshaped plastic retractors shown in Figure that can be placed along the patient™s upper and lower lips to lateralize the cheeks thus allowing handsfree visualization of the dentition and oral cavity soft tissue Patient assistant If available patients should ask a family member or friend to accompany them to the telehealth visit The assistant can help patients perform the physical tasks required during the virtual physical exam Assistants can also help position the web camera to improve the provider™s view In fact these assistants are essentially mandatory for pediatric patients or patients with disabilities Feedback It is a good practice for providers to seek feedback from patients after a visit This will help OMS hone their telehealth skills and ensure that their patients are receiving an appropriate quality of care 0c THE VIRTUAL HISTORY AND PHYSICAL Thorough patient assessment proper medical documentation and appropriate diagnostic testing are critical components of OMS practice that enable proper diagnosis and treatment planning OMS should obtain patients™ medical histories in a similar manner as they would in their offices New patients must be asked for comprehensive histories that include chief complaint history of present illness past medical history past surgical history dental history medications allergies pertinent family history social history and complete review of systems For patients of record their medical histories should be updated to reflect their current chief complaint All patients should also be screened with an up to date COVID19 questionnaire If an infection is suspected OMS should refer patients to their primary care physicians or local emergency department depending on the severity of symptoms for appropriate workup Vitals should be obtained if the patient has access to a thermometer blood pressure cuff pulse oximeter or weighing scale Even without any of these devices patients can still measure their pulse by applying two fingers on the patient™s carotid counting the number of beats per minute Also patients can calculate their respiratory rate by observing the number of chest rises in one minute Finally oxygen saturation can be measured with certain mobile health applications though OMS should not solely rely on their results for major medical decisions Finally patients with fever body temperature F warrants further work up in an emergency setting for a differential diagnosis that includes COVID19 infection The virtual physical exam will be limited to a head and neck exam and a cranial nerve exam While inspection and palpation are the basis of a focused physical examination in oral and maxillofacial surgery OMS must learn to work together with patients to achieve the same goals 0c virtually Patients must perform maneuvers on themselves with the OMS™s guidance To this end a printed stepbystep schematic as illustrated in Figure can be helpful for patients to receive before the visit During the visit the OMS can reinforce the diagram with clear verbal instructions that avoids medical jargon The exam itself must be conducted systematically with a topdown outsidein approach as is typical in an oral and maxillofacial surgery practice The exam can be further divided into head and neck subsites The OMS should ask for specific symptoms related to each subsite and carefully inspect for any abnormalities while guiding the patient or the patient™s assistant through the exam The following section offers additional details and considerations for each subsite Head The OMS should ask about any history of head trauma The head is assessed to ensure that it is normocephalic and atraumatic Face The OMS should ask the patient about any facial pain swelling weakness numbness or history of trauma to the region Then OMS can start the facial exam by asking the patient to lean close to the camera First the face is examined for any skin lesions along the forehead eyelids external ears nose malar region vermilion of the lips and the chin Patients™ left and right sides of the face should be compared for any gross asymmetry or deformities OMS can then guide patients through palpating their own face for any bony discontinuity or soft tissue swelling Patients can also tap their own face with two fingers to reveal any tenderness in the sinuses Regarding the eyes OMS can assess if the pupils are equal The extraocular muscles along with the oculomotor supratrochlear and the abducens nerves can be tested by having the 0c patient look up down left and right without moving the head The sensory portion of the trigeminal nerve can be tested by asking patients to close their eyes and slide both of their index fingers horizontally along the ipsilateral forehead ophthalmic branch cheek maxillary branch lip and chin mandibular branch The branches of the facial nerves can be tested by asking patients to raise their eyebrows close their eyes tightly puff out their cheeks smile widely and show their bottom teeth Temporomandibular joint TMJ The OMS should ask the patient about any facial jaw or ear pain trismus difficulty mastication clicking or locking of the joint Then the TMJ exam begins by asking patients to palpate their mandibular condyles and muscles of mastication to look for any tender spots Providers can ask patients to open and close their mouth while palpating the condyles to feel for any clicks or crepitus Also maximal interincisal opening can be roughly estimated by the number of fingerbreadth or precisely measured using a ruler The mandibular range of motion can be assessed or measured in protrusive and lateral excursive positions Neck The OMS should ask for any difficulty breathing dysphagia sore throat odynophagia hoarseness or new neck swelling The neck exam begins with inspection looking for any asymmetry or tracheal deviation Patients can be asked to turn the head from side to side look upwards and shrug the shoulders to assess the spinal accessory nerves OMS should ask the patient™s assistant if possible to stand right behind the patient and palpate the patient™s neck Using their fingertips on both hands the assistant can palpate the neck in a unidirectional manner from superior to inferior and then from lateral to medial Ask them to note any palpable bumps or tender spots It is particularly important to palpate the lateral neck for enlarged lymph nodes 0c Lastly OMS can identify the thyroid by asking patients to swallow while palpating the appropriate area on the neck to rule out thyromegaly Oral cavity and oropharynx The OMS should ask for any oral pain oral swelling or sores tongue numbness difficulty with tongue movement or dry mouth Examination of the oral cavity exam can be challenging because intraoral structures can be difficult to retract and illuminate The aforementioned cheek retractors whether from a board game or makeshift spoons can be helpful for retraction of soft tissue and visualization In addition patients™ friends and family can help a great deal by adjusting the camera while also properly angling an additional light source For each intraoral structure the OMS must carefully inspect for ulcers raised lesions abnormal white leukoplakic or bright red erythroplakic lesions In general OMS can best visualize structures near or at the level of the maxilla when patients lift their heads up to degrees Likewise structures near or at level of the mandible are best observed with the patient dropping the chin approximately degrees OMS may find it useful to practice these examination techniques on their own cameras before the visit Patients should be recommended to wash their hands or to use gloves before touching any intraoral landmarks The exam begins by sliding the patient™s index finger along the maxillary and mandibular vestibule to look for any swelling or fluctuance With the cheeks retracted the patient can palpate their buccal and labial mucosa using the thumb and index finger with one 0c finger compressing along the face extraorally When possible palpation should be bidigital Next the patient can use their index figures to palpate the tuberosity retromolar trigone and the hard palate for tenderness or irregularities The tongue is the most common site for oral cancer and must be thoroughly examined The dorsal surface of the tongue should be examined by asking the patient to fully protrude their tongue Providers should also ask patients to move their protruded tongues to the left and right to inspect the lateral tongue and ensure the function of the hypoglossal nerves The ventral tongue and the floor of the mouth can be observed by asking patients to touch the tip of their tongue to their hard palate The tongue can then be palpated for lumps or masses Next the sublingual and submandibular glands can be palpated for symmetry and lack of elevation by the patient with their extended index fingers on the floor of the mouth The examination of the oropharynx is mostly limited in virtual encounters Nevertheless the soft palate tonsils and uvula can be partially visualized with the patient™s mouth wide open and using a spoon to depress the tongue Although unpleasant the glossopharyngeal and vagus nerves can be tested by gently touching the soft palate using a spoon to induce a gag reflex Dentition If the patient is dentate the provider should ask about dental pain sensitivity loosening of teeth bleeding or sore gums or malocclusion The patient™s dentition can be evaluated after retracting soft tissue as described previously Dental caries missing teeth periodontal disease gingival lesions or swelling can be readily identified Mobility of teeth can be assessed by using the patient™s thumb and index finger In edentulous patients the alveolar 0c ridge should be examined for any abnormalities as part of the aforementioned oral soft tissue exam CONCLUSION The COVID19 pandemic has catalyzed an exponential increase in telemedicine usage Telemedicine helps patients maintain access to care conserves limited medical resources and protects both OMS and patients from pathogen exposure Nevertheless there is an expected learning curve that accompanies such a paradigm shift in the delivery of care As such this paper provides a guide of best practices to aid both OMS and patients to navigate this promising electronic tool In addition we provide an accessible schematic handout that can be given to patients before a telehealth appointment to help them prepare for the visit for both setting up and performing physical exam procedures Because telemedicine may have a role in oral and maxillofacial surgical care even after this pandemic we are optimistic that these best practices can be helpful and relevant for the present situation and beyond 0c FIGURE LEGEND Figure Patient informational handout with graphic illustrations detailing the set up as well as key examination steps that patients may be asked to perform during the telemedicine encounter Reprinted from [] 0c Figure Horseshoeshaped plastic lipcheek retractors REFERENCES Li Q Guan X Wu P et al Early Transmission Dynamics in Wuhan China of Novel CoronavirusInfected Pneumonia N Engl J Med “ httpsdoiorg101056NEJMoa2001316 Feng S Shen C Xia N et al Rational use of face masks in the COVID19 pandemic Lancet Respir Med httpsdoiorg101016S221326002030134X Li R Rivers C Tan Q et al Estimated Demand for US Hospital Inpatient and Intensive Care Unit Beds for Patients With COVID19 Based on Comparisons With Wuhan and Guangzhou China JAMA Netw Open 3e208297“e208297 httpsdoiorg101001jamanetworkopen20208297 White DB Lo B A Framework for Rationing Ventilators and Critical Care Beds During the COVID19 Pandemic JAMA httpsdoiorg101001jama20205046 Heinzerling A Stuckey MJ Scheuer T et al Transmission of COVID19 to Health Care Personnel During Exposures to a Hospitalized Patient Solano County California 0c February MMWR Morb Mortal Wkly Rep “ httpsdoiorg1015585mmwrmm6915e5 Ng K Poon BH Kiat Puar TH et al COVID19 and the Risk to Health Care Workers A Case Report Ann Intern Med httpsdoiorg107326L200175 Halepas S Ferneini EM A Pinch of Prevention is Worth a Pound of Cure Proactive Dentistry in the Wake of COVID19 Journal of Oral and Maxillofacial Surgery httpsdoiorg101016jjoms202003036 AAOMS Member Alert COVID19 Update Healthcare Facilities Preparing for Community Transmission In Centers for Disease Control and Prevention httpswwwcdcgovcoronavirus2019ncovhcpguidancehcfhtml Accessed May CDC Guidance for Providing Dental Care During COVID19 In Centers for Disease Control and Prevention httpswwwcdcgovoralhealthinfectioncontrolstatementCOVIDhtml Accessed May Carey M Second week of HPI polling shows dentists™ response to COVID19 American Dental Association Hollander JE Carr BG Virtually Perfect Telemedicine for Covid19 New England Journal of Medicine “ httpsdoiorg101056NEJMp2003539 Prasad A Brewster R Newman JG Rajasekaran K Optimizing your telemedicine visit during the COVID19 pandemic Practice guidelines for patients with head and neck cancer Head Neck na httpsdoiorg101002hed26197 Russo JE McCool RR Davies L VA Telemedicine An Analysis of Cost and Time Savings Telemedicine and eHealth “ httpsdoiorg101089tmj20150055 Cain SM Moore R Sturm L et al Clinical assessment and management of general surgery patients via synchronous telehealth Journal of Telemedicine and Telecare httpsdoiorg1011771357633X16636245 Teledental Practice and Teledental Encounters An American Association of Teledentistry Position Paper Jampani ND Nutalapati R Dontula BSK Boyapati R Applications of teledentistry A literature review and update J Int Soc Prev Community Dent “ httpsdoiorg1041032231076297695 Wicklund E Dentists Use Telehealth to Improve Access to Care And Fight a Phobia In mHealthIntelligence httpsmhealthintelligencecomnewsdentistsusetelehealthtoimproveaccesstocareandfightaphobia Accessed May 0c Smith WR Atala AJ Terlecki RP et al Implementation Guide for Rapid Integration of an Outpatient Telemedicine Program during the COVID19 Pandemic Journal of the American College of Surgeons httpsdoiorg101016jjamcollsurg202004030 AAOMS White Paper on Telehealth and Remote Treatment Notification of Enforcement Discretion for Telehealth Remote Communications During the COVID19 Nationwide Public Health Emergency In HHSgov httpswwwhhsgovhipaaforprofessionalsspecialtopicsemergencypreparednessnotificationenforcementdiscretiontelehealthindexhtml Accessed May Ethical Practice in Telemedicine In American Medical Association httpswwwamaassnorgdeliveringcareethicsethicalpracticetelemedicine Accessed May Ryan C Computer and Internet Use in the United States United States Census Bureau Additional BackgroundSweeping Regulatory Changes to Help US Healthcare System Address COVID19 Patient Surge CMS In CMSgov httpswwwcmsgovnewsroomfactsheetsadditionalbackgroundsweepingregulatorychangeshelpushealthcaresystemaddresscovid19patient Accessed Jun Medicaid Learning Network Telehealth Services Hasbro Brings Mouth Piece Challenge to the Masses with New SPEAK OUT Game In Business Wire httpswwwbusinesswirecomnewshome20160624005633enHasbroBringsMouthPieceChallengeMassesNew Accessed May 0c"

cancer229

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: "Optimizing Telemedicine Encounters for Oral and Maxillofacial Surgeons During the COVID19 Pandemic Hwi Sean Moon DDS MD1 Tim T Wang BA23 Karthik Rajasekaran MD4 Ryan Brewster BA5 Rabie M Shanti DMD MD46 Neeraj Panchal DDS MD MA7 1Resident Department of Oral Maxillofacial Surgery University of Pennsylvania Philadelphia PA 2DMD Candidate School of Dental Medicine University of Pennsylvania Philadelphia PA 3MPH Candidate Perelman School of Medicine University of Pennsylvania Philadelphia PA 4Assistant Professor of Otorhinolaryngology University of Pennsylvania Philadelphia PA 5MD Candidate Stanford University School of Medicine Stanford University Stanford CA 6Assistant Professor of Oral and Maxillofacial Surgery University of Pennsylvania Philadelphia PA 7Assistant Professor and Section Chief of Oral and Maxillofacial Surgery Philadelphia Veterans Affairs Medical Center Penn Presbyterian Medical Center University of Pennsylvania School of Dental Medicine Philadelphia PA Corresponding Author Neeraj Panchal DDS MD MA Tel Mailing Address N 39th St Philadelphia PA Email Address npanchalupennedu Disclosures None to report Abstract Word Count Manuscript Word Count Number of References Number of Figurestables Number of Supplements 0c The COVID19 pandemic has changed conventional medical practice patterns across all health disciplines including oral and maxillofacial surgery practices The use of telemedicine has rapidly expanded to uphold safety strategies of physical distancing and disease transmission reduction while maintaining uninterrupted care of patients To date there are no specific guidelines to optimize telemedicine encounters in the oral and maxillofacial surgery practice The goal of this paper is to provide best practices for both oral and maxillofacial surgeons and their patients to effectively utilize telemedicine for the duration of COVID19 and beyond Statement of Clinical Relevance The goal of this paper is to provide best practices for both oral and maxillofacial surgeons and their patients to effectively utilize telemedicine for the duration of COVID19 and beyond INTRODUCTION The COVID19 pandemic has disrupted society in a multitude of ways Healthcare is no exception the SARSCoV virus™ rapid transmission and high hospitalization rate have strained the availability of medical resources including personal protective equipment PPE respiratory ventilators and hospital beds[“] The virus also poses a major threat to healthcare personnel whose risk of exposure are compounded by the aforementioned PPE shortages[“] In response the American Association of Oral and Maxillofacial Surgeons AAOMS recommended delaying elective surgeries in accordance with the Centers for Disease Control and Prevention™s calls to 0c postpone elective medical and dental procedures[“] Similarly four out of five dental offices have closed for all except emergency procedures[] In the face of these challenges the medical and dental communities have remained steadfast in caring for patients with nonelective health needs and innovating alternate ways to deliver care One of the most important and popular alterations in the delivery of care is the increased utilization of telemedicine which allows surgeons and patients to connect virtually[ ] This has enabled patients to access muchneeded medical care while preserving PPE and minimizing exposure to pathogens Though studies have found telemedicine to decrease costs and save time without compromising patient satisfaction it was not widely used in healthcare before the COVID19 pandemic[ ] Similarly teledentistry was deemed to be œin its infancy by the founder of the American Teledentistry Association in [“] Nevertheless telemedicine has shown promise and has been incorporated into the workflow of various oral and maxillofacial surgery institutions and practices across the country Virtual visits are particularly useful in triaging patients For example patients with dentoalveolar infections can meet virtually with surgeons and receive prescriptions for appropriate analgesics and antibiotics without going to the emergency department Also patients with oral lesions can take images and show their surgeon before their inperson visit to expedite the diagnosis and treatment planning workflow This enables patients to access timely attention of providers while lightening the load on the healthcare system by reducing the number of inperson visits 0c Associated with the recent rise in telemedicine™s popularity is a learning curve for both surgeons and patients The incorporation of technology and the shift to virtual visits can be jarring for the patientsurgeon relationship and must be navigated thoughtfully While there have been helpful telehealth guides for surgeons and patients in other surgical specialties we do not know of any such guidelines for oral and maxillofacial surgery[ ] As such we detail best practices for both oral and maxillofacial surgeons OMS and their patients to effectively utilize telemedicine for the duration of COVID19 and beyond GENERAL CONSIDERATIONS FOR TELEMEDICINE In accordance with the AAOMS White Paper on ˜Telehealth and Remote Treatment™ virtual management of any oral and maxillofacial surgical condition should only be provided by appropriately licensed oral and maxillofacial surgeons as regulated by the state law[] The delivery of patient care through telemedicine must continue to follow evidencebased guidelines to ensure quality and safety for all patients All providers must comply with the latest telehealth requirements outlined by the United State Health and Human Services to protect patient privacy and comply with the Health Insurance Portability and Accountability Act HIPAA[] Furthermore providers are ethically obligated to inform all patients about the potential benefits limitations and risks of telemedicine[] Patients requiring emergency or urgent services must be directed to the nearest hospital 0c SETTING UP FOR TELEMEDICINE While there are several modalities to conduct a telemedicine encounter we strongly recommend a live synchronous twoway interaction between the patient and the OMS incorporating both audio and visual telecommunications tools This can be achieved with a desktop computer laptop or smartphone The United States Census Bureau reports that approximately percent of households have computers or smartphones and percent have broadband internet subscriptions[] Of these options though we recommend using a desktop or a large screen laptop with a highresolution camera over smartphones even if the latter meets the minimum technical requirements Ideally telemedicine visits can offer a clinic experience that closely simulates inperson encounters Trained administrative staff members should call patients beforehand to discuss the virtual setup and basic expectations for the visit Prasad et al created Figure which exemplifies a patient informational handout with graphic illustrations detailing the set up as well as key examination steps that patients may be asked to perform during the encounter [] In the following we will detail key aspects and considerations for both OMS and patients to maximize their telehealth visits 0c Insurance Coverage and Billing In an effort to reduce the burden posted on healthcare entities and facilitate mitigation efforts the Centers for Medicare Medicaid Services CMS broadened access for Telemedicine coverage with private payers following suit CMS expansion included voiceonly visits which is critical for patients without access to a smartphone or computer video capabilities Furthermore CMS allowed for parity of payment for Telemedicine visits and inperson visits so providers can bill Medicaid and Medicare at the same rate as they would for an inperson visit[] This new policy is especially relevant for older patients covered by Medicare for they are generally at higher risk for COVID19 complications[] Therefore before telehealth encounters providers should confirm if the patient™s insurance plan has Telemedicine coverage and also whether the insurance plan waives all copays for nonCOVID19 related visits to avoid a scenario where a patient receives a bill unknowingly The American Association of Oral and Maxillofacial Surgeons provide additional detailed information about telehealth billing relevant for OMSs including updated links to AMA and ADA billing codes on its website at httpswwwaaomsorgpracticeresourcestelehealthfaqs 0c Professionalism and provider attire The visit should replicate the same level of professionalism as that of inperson appointments Providers should dress professionally as they would in their office or hospitalbased practice Before the patient comes in providers should review the patient™s relevant medical records and chief complaints to save time and maximize the efficiency of the visit Also in the interest of both time and professionalism OMS and patients should both be mindful to start the visit on time During the visit OMS should communicate with patients to maintain transparency For instance if an OMS needs to document something during the visit they should respectfully inform the patient of the task to prevent any misunderstanding At the end of the visit it is important for OMS to summarize what they accomplished during the visit and provide a clear plan for appropriate next steps Physical background When possible OMS and patients should conduct virtual encounters in welllit spaces Lighting specifically can have a profound effect on video quality As such overhead lights can be helpful while lights behind the person should be avoided Care should also be taken to prevent other sources of potential disruptions such as background noise or visual distractions It may be helpful for OMS to evaluate their surroundings from the perspective of their patients 0c Technological background It is important for OMS to test their video and audio quality before visits to anticipate any potential technical difficulties that may interrupt the encounter A strong WiFi internet connection is preferred over cellular data to ensure a stable signal With the exception of electronic health records OMS should close any unnecessary programs or internet browser tabs to preserve internet bandwidth In addition OMS should be mindful that their patients may have varying internet speeds As such OMS should give approximately seconds of lag time after patients stop speaking to allow all of their words to come through completely Patient and camera position If possible patients should sit upright on a chair in front of a computer placed on top of a desk They should sit close enough to the camera so that their entire head and neck area are within the video frame The camera should be at approximately eyelevel for both OMS and patients to maintain eye contact and remain engaged during the virtual encounter For patients using a smartphone the device can be propped up at a to 90degree angle from the table surface to allow patients to free both of their hands for physical exam tasks Patient clothing Patients must be notified of the appropriate clothing well in advance prior to the appointment Ideally clothing should allow patients™ entire head and neck regions to be visualized while maintaining patient comfort and professionalism Any hat or scarves should be removed if at all possible maintaining appropriate cultural and religious norms Patient items Prior to the appointment patients should prepare the following items which can help aid OMS in visualization and retraction during the virtual physical exam Most of these items are commonplace inexpensive and available at the patient™s home 0c Flashlight A flashlight or a penlight can enhance visualization of certain obscure head and neck structures particularly those in the oral cavity The builtin flashlight of a smartphone can also be used Ruler A ruler or a measuring tape can be used to measure the patient™s maximal mouth opening and mandibular range of motion Napkins A napkin can be used to touch any intraoral landmarks and also to clean up after any inadvertent salivation from the virtual physical exam Spoon A spoon can be used to retract the cheeks or depress the tongue to evaluate structures of the oropharynx such as the soft palate and tonsillar pillars Cheek retractors A fun way for patients to achieve cheek retraction can be to use the plastic props from the board game œSpeak Out which was created in [] This game provides horseshoeshaped plastic retractors shown in Figure that can be placed along the patient™s upper and lower lips to lateralize the cheeks thus allowing handsfree visualization of the dentition and oral cavity soft tissue Patient assistant If available patients should ask a family member or friend to accompany them to the telehealth visit The assistant can help patients perform the physical tasks required during the virtual physical exam Assistants can also help position the web camera to improve the provider™s view In fact these assistants are essentially mandatory for pediatric patients or patients with disabilities Feedback It is a good practice for providers to seek feedback from patients after a visit This will help OMS hone their telehealth skills and ensure that their patients are receiving an appropriate quality of care 0c THE VIRTUAL HISTORY AND PHYSICAL Thorough patient assessment proper medical documentation and appropriate diagnostic testing are critical components of OMS practice that enable proper diagnosis and treatment planning OMS should obtain patients™ medical histories in a similar manner as they would in their offices New patients must be asked for comprehensive histories that include chief complaint history of present illness past medical history past surgical history dental history medications allergies pertinent family history social history and complete review of systems For patients of record their medical histories should be updated to reflect their current chief complaint All patients should also be screened with an up to date COVID19 questionnaire If an infection is suspected OMS should refer patients to their primary care physicians or local emergency department depending on the severity of symptoms for appropriate workup Vitals should be obtained if the patient has access to a thermometer blood pressure cuff pulse oximeter or weighing scale Even without any of these devices patients can still measure their pulse by applying two fingers on the patient™s carotid counting the number of beats per minute Also patients can calculate their respiratory rate by observing the number of chest rises in one minute Finally oxygen saturation can be measured with certain mobile health applications though OMS should not solely rely on their results for major medical decisions Finally patients with fever body temperature F warrants further work up in an emergency setting for a differential diagnosis that includes COVID19 infection The virtual physical exam will be limited to a head and neck exam and a cranial nerve exam While inspection and palpation are the basis of a focused physical examination in oral and maxillofacial surgery OMS must learn to work together with patients to achieve the same goals 0c virtually Patients must perform maneuvers on themselves with the OMS™s guidance To this end a printed stepbystep schematic as illustrated in Figure can be helpful for patients to receive before the visit During the visit the OMS can reinforce the diagram with clear verbal instructions that avoids medical jargon The exam itself must be conducted systematically with a topdown outsidein approach as is typical in an oral and maxillofacial surgery practice The exam can be further divided into head and neck subsites The OMS should ask for specific symptoms related to each subsite and carefully inspect for any abnormalities while guiding the patient or the patient™s assistant through the exam The following section offers additional details and considerations for each subsite Head The OMS should ask about any history of head trauma The head is assessed to ensure that it is normocephalic and atraumatic Face The OMS should ask the patient about any facial pain swelling weakness numbness or history of trauma to the region Then OMS can start the facial exam by asking the patient to lean close to the camera First the face is examined for any skin lesions along the forehead eyelids external ears nose malar region vermilion of the lips and the chin Patients™ left and right sides of the face should be compared for any gross asymmetry or deformities OMS can then guide patients through palpating their own face for any bony discontinuity or soft tissue swelling Patients can also tap their own face with two fingers to reveal any tenderness in the sinuses Regarding the eyes OMS can assess if the pupils are equal The extraocular muscles along with the oculomotor supratrochlear and the abducens nerves can be tested by having the 0c patient look up down left and right without moving the head The sensory portion of the trigeminal nerve can be tested by asking patients to close their eyes and slide both of their index fingers horizontally along the ipsilateral forehead ophthalmic branch cheek maxillary branch lip and chin mandibular branch The branches of the facial nerves can be tested by asking patients to raise their eyebrows close their eyes tightly puff out their cheeks smile widely and show their bottom teeth Temporomandibular joint TMJ The OMS should ask the patient about any facial jaw or ear pain trismus difficulty mastication clicking or locking of the joint Then the TMJ exam begins by asking patients to palpate their mandibular condyles and muscles of mastication to look for any tender spots Providers can ask patients to open and close their mouth while palpating the condyles to feel for any clicks or crepitus Also maximal interincisal opening can be roughly estimated by the number of fingerbreadth or precisely measured using a ruler The mandibular range of motion can be assessed or measured in protrusive and lateral excursive positions Neck The OMS should ask for any difficulty breathing dysphagia sore throat odynophagia hoarseness or new neck swelling The neck exam begins with inspection looking for any asymmetry or tracheal deviation Patients can be asked to turn the head from side to side look upwards and shrug the shoulders to assess the spinal accessory nerves OMS should ask the patient™s assistant if possible to stand right behind the patient and palpate the patient™s neck Using their fingertips on both hands the assistant can palpate the neck in a unidirectional manner from superior to inferior and then from lateral to medial Ask them to note any palpable bumps or tender spots It is particularly important to palpate the lateral neck for enlarged lymph nodes 0c Lastly OMS can identify the thyroid by asking patients to swallow while palpating the appropriate area on the neck to rule out thyromegaly Oral cavity and oropharynx The OMS should ask for any oral pain oral swelling or sores tongue numbness difficulty with tongue movement or dry mouth Examination of the oral cavity exam can be challenging because intraoral structures can be difficult to retract and illuminate The aforementioned cheek retractors whether from a board game or makeshift spoons can be helpful for retraction of soft tissue and visualization In addition patients™ friends and family can help a great deal by adjusting the camera while also properly angling an additional light source For each intraoral structure the OMS must carefully inspect for ulcers raised lesions abnormal white leukoplakic or bright red erythroplakic lesions In general OMS can best visualize structures near or at the level of the maxilla when patients lift their heads up to degrees Likewise structures near or at level of the mandible are best observed with the patient dropping the chin approximately degrees OMS may find it useful to practice these examination techniques on their own cameras before the visit Patients should be recommended to wash their hands or to use gloves before touching any intraoral landmarks The exam begins by sliding the patient™s index finger along the maxillary and mandibular vestibule to look for any swelling or fluctuance With the cheeks retracted the patient can palpate their buccal and labial mucosa using the thumb and index finger with one 0c finger compressing along the face extraorally When possible palpation should be bidigital Next the patient can use their index figures to palpate the tuberosity retromolar trigone and the hard palate for tenderness or irregularities The tongue is the most common site for oral cancer and must be thoroughly examined The dorsal surface of the tongue should be examined by asking the patient to fully protrude their tongue Providers should also ask patients to move their protruded tongues to the left and right to inspect the lateral tongue and ensure the function of the hypoglossal nerves The ventral tongue and the floor of the mouth can be observed by asking patients to touch the tip of their tongue to their hard palate The tongue can then be palpated for lumps or masses Next the sublingual and submandibular glands can be palpated for symmetry and lack of elevation by the patient with their extended index fingers on the floor of the mouth The examination of the oropharynx is mostly limited in virtual encounters Nevertheless the soft palate tonsils and uvula can be partially visualized with the patient™s mouth wide open and using a spoon to depress the tongue Although unpleasant the glossopharyngeal and vagus nerves can be tested by gently touching the soft palate using a spoon to induce a gag reflex Dentition If the patient is dentate the provider should ask about dental pain sensitivity loosening of teeth bleeding or sore gums or malocclusion The patient™s dentition can be evaluated after retracting soft tissue as described previously Dental caries missing teeth periodontal disease gingival lesions or swelling can be readily identified Mobility of teeth can be assessed by using the patient™s thumb and index finger In edentulous patients the alveolar 0c ridge should be examined for any abnormalities as part of the aforementioned oral soft tissue exam CONCLUSION The COVID19 pandemic has catalyzed an exponential increase in telemedicine usage Telemedicine helps patients maintain access to care conserves limited medical resources and protects both OMS and patients from pathogen exposure Nevertheless there is an expected learning curve that accompanies such a paradigm shift in the delivery of care As such this paper provides a guide of best practices to aid both OMS and patients to navigate this promising electronic tool In addition we provide an accessible schematic handout that can be given to patients before a telehealth appointment to help them prepare for the visit for both setting up and performing physical exam procedures Because telemedicine may have a role in oral and maxillofacial surgical care even after this pandemic we are optimistic that these best practices can be helpful and relevant for the present situation and beyond 0c FIGURE LEGEND Figure Patient informational handout with graphic illustrations detailing the set up as well as key examination steps that patients may be asked to perform during the telemedicine encounter Reprinted from [] 0c Figure Horseshoeshaped plastic lipcheek retractors REFERENCES Li Q Guan X Wu P et al Early Transmission Dynamics in Wuhan China of Novel CoronavirusInfected Pneumonia N Engl J Med “ httpsdoiorg101056NEJMoa2001316 Feng S Shen C Xia N et al Rational use of face masks in the COVID19 pandemic Lancet Respir Med httpsdoiorg101016S221326002030134X Li R Rivers C Tan Q et al Estimated Demand for US Hospital Inpatient and Intensive Care Unit Beds for Patients With COVID19 Based on Comparisons With Wuhan and Guangzhou China JAMA Netw Open 3e208297“e208297 httpsdoiorg101001jamanetworkopen20208297 White DB Lo B A Framework for Rationing Ventilators and Critical Care Beds During the COVID19 Pandemic JAMA httpsdoiorg101001jama20205046 Heinzerling A Stuckey MJ Scheuer T et al Transmission of COVID19 to Health Care Personnel During Exposures to a Hospitalized Patient Solano County California 0c February MMWR Morb Mortal Wkly Rep “ httpsdoiorg1015585mmwrmm6915e5 Ng K Poon BH Kiat Puar TH et al COVID19 and the Risk to Health Care Workers A Case Report Ann Intern Med httpsdoiorg107326L200175 Halepas S Ferneini EM A Pinch of Prevention is Worth a Pound of Cure Proactive Dentistry in the Wake of COVID19 Journal of Oral and Maxillofacial Surgery httpsdoiorg101016jjoms202003036 AAOMS Member Alert COVID19 Update Healthcare Facilities Preparing for Community Transmission In Centers for Disease Control and Prevention httpswwwcdcgovcoronavirus2019ncovhcpguidancehcfhtml Accessed May CDC Guidance for Providing Dental Care During COVID19 In Centers for Disease Control and Prevention httpswwwcdcgovoralhealthinfectioncontrolstatementCOVIDhtml Accessed May Carey M Second week of HPI polling shows dentists™ response to COVID19 American Dental Association Hollander JE Carr BG Virtually Perfect Telemedicine for Covid19 New England Journal of Medicine “ httpsdoiorg101056NEJMp2003539 Prasad A Brewster R Newman JG Rajasekaran K Optimizing your telemedicine visit during the COVID19 pandemic Practice guidelines for patients with head and neck cancer Head Neck na httpsdoiorg101002hed26197 Russo JE McCool RR Davies L VA Telemedicine An Analysis of Cost and Time Savings Telemedicine and eHealth “ httpsdoiorg101089tmj20150055 Cain SM Moore R Sturm L et al Clinical assessment and management of general surgery patients via synchronous telehealth Journal of Telemedicine and Telecare httpsdoiorg1011771357633X16636245 Teledental Practice and Teledental Encounters An American Association of Teledentistry Position Paper Jampani ND Nutalapati R Dontula BSK Boyapati R Applications of teledentistry A literature review and update J Int Soc Prev Community Dent “ httpsdoiorg1041032231076297695 Wicklund E Dentists Use Telehealth to Improve Access to Care And Fight a Phobia In mHealthIntelligence httpsmhealthintelligencecomnewsdentistsusetelehealthtoimproveaccesstocareandfightaphobia Accessed May 0c Smith WR Atala AJ Terlecki RP et al Implementation Guide for Rapid Integration of an Outpatient Telemedicine Program during the COVID19 Pandemic Journal of the American College of Surgeons httpsdoiorg101016jjamcollsurg202004030 AAOMS White Paper on Telehealth and Remote Treatment Notification of Enforcement Discretion for Telehealth Remote Communications During the COVID19 Nationwide Public Health Emergency In HHSgov httpswwwhhsgovhipaaforprofessionalsspecialtopicsemergencypreparednessnotificationenforcementdiscretiontelehealthindexhtml Accessed May Ethical Practice in Telemedicine In American Medical Association httpswwwamaassnorgdeliveringcareethicsethicalpracticetelemedicine Accessed May Ryan C Computer and Internet Use in the United States United States Census Bureau Additional BackgroundSweeping Regulatory Changes to Help US Healthcare System Address COVID19 Patient Surge CMS In CMSgov httpswwwcmsgovnewsroomfactsheetsadditionalbackgroundsweepingregulatorychangeshelpushealthcaresystemaddresscovid19patient Accessed Jun Medicaid Learning Network Telehealth Services Hasbro Brings Mouth Piece Challenge to the Masses with New SPEAK OUT Game In Business Wire httpswwwbusinesswirecomnewshome20160624005633enHasbroBringsMouthPieceChallengeMassesNew Accessed May 0c" Answer:

Thyroid_Cancer

" IBDFecal calprotectinEndoscopic activityIBD noninvasive managementThe term IBD is usually used for referring to a group of ‚ammatory gastrointestinal diseases mainly Crohn'sdisease and ulcerative colitis Accordingly IBD arises as a result of inappropriate immune response to intestinalcommensal anisms among genetically susceptible individuals Performing colonoscopy and histopathologicevaluation on an ‚amed bowel biopsy specimen are currently considered as gold standards for diagnosis andmanagement of IBD Correspondingly these techniques are known to be invasive and costly In recent decadesfecal calprotectin as a biomarker has received much attention for the diagnosis and noninvasive managementof IBD Up to now many studies have investigated the efficacy of fecal calprotectin in the areas of IBD diï¬erentiation from IBS prediction of endoscopic and histologic activities of IBD and prediction of disease recurrenceAlthough some of these studies have reported promising results some others have shown significant limitationsTherefore in this paper we reviewed the most interesting ones of these studies after a brief discussion of thelaboratory measurement of fecal calprotectin Moreover we attempted to provide an answer for the question ofwhether fecalcalprotectin could be considered as a potential surrogate marker for colonoscopy IntroductionInflammatory bowel disease IBD is a long life disease with remission and relapse periods IBD arises as a result of inappropriateimmune response to intestinal commensal anisms in individualswith genetic predisposition and consequently causes ‚ammation andintestinal ulcers [] In addition IBD has a complex pathogenesis andmany factors such as dysbiosis oxidative stress and epigenetics thatmay also be involved in disease pathogenesis [“] Ulcerative colitisUC and Crohn's diseases CD are known as two main forms of IBDAccordingly these diseases cause intestinal ulcers and some annoyingsymptoms such as diarrhea abdominal pain and rectal bleeding Occasionally the severity of these symptoms is very high which can leadpatients to be hospitalized In this regard therapeutic approaches totreat these diseases mainly focus on prolonging remission and are almost similar however diï¬erential diagnosis can also help to treat thedisease in a more eï¬ective way For example 5ASA which is acommon drug in the treatment of IBD is less eï¬ective on maintainingremission in CD patients On the other hand antibiotic therapy is notrecommended for the treatment UC but it can be eï¬ective on CD patients [][] Diï¬erential diagnosis is a serious challenge because CDand UC have significant similarities in terms of their clinical endoscopic and histological features However there are some diï¬erencesbetween UC and CD which are summarized in Table1 In addition tointestinal complications UC and CD also have significant extraintestinal manifestations For example it was shown that UC is significantly associated with Primary sclerosing cholangitis and CD is alsoassociated with cholelithiasis especially in cases that the ileum is involved [] Furthermore CD can cause fistulas to the urinary systemwhich leads intestinal bacteria to enter the urethra and recurrent urinary tract infections [] Both CD and UC can cause several disorderssuch as arthritis Erythema nodosum pyoderma gangrenosum andanemia which are known as the most important extraintestinal manifestations of IBD [][] The latest statistics showed that the globalŽ Corresponding author at Department of Clinical Biochemistry and Laboratory Medicine Faculty of Medicine Tabriz University of Medical Sciences DaneshgahStreet PO Box Tabriz IranEmail address vagharimtbzmedacir M VaghariTabari101016jcca202008025Received July Received in revised form August Accepted August Available online August Elsevier BV All rights reserved 0cF KhakiKhatibi et alTable1Clinical endoscopic and histological features of CD and UCClinical FeaturesFeaturesRectal bleedingAbdominal painFeverMucus defectionIntestinal obstructionPerineal diseasePostoperative recurrenceASCA positiveANCA positiveEndoscopic FeaturesCDOccasionallyFrequentlyFrequentlyOccasionallyYESYESYESFrequentlyNot commonUCFrequentlyOccasionallyNot commonFrequentlyNONONONot commonFrequentlyFeaturesCDUCLocationMucosal involvementDepth of ulcerationfistulaCobblestone appearanceAphthous ulcerationMucosal friabilityHistological featuresFeaturesGranulomasCrypt abscessesPatchinessAny part of GI tractDiscontinuousDeepYesYESFrequentlyNot commonCDFrequentlyNot commonFrequentlyColon and rectumContinuoussuperficialNONOOccasionallyFrequentlyUCRareFrequentlyNot commonprevalence of IBD currently is on the rise and it is not an exaggerationif we consider it as a global serious health problem [] According to areport published in IBD has the highest prevalence rate inEurope and its prevalence in the newly industrialized countries of AsiaAfrica and South America also appears to be increased over the pastthree decades []Unfortunately the peak of the disease is at the young age of“ years old [] therefore in addition to the suï¬ering from ‚icts on the patients it also has many negative eï¬ects on societyMoreover many financial burdens are annually imposed on countriesfor controlling and treating this chronic disease The invasive diagnosticand therapeutic measures are currently undertaken to diagnose andmanage IBD which are unpleasant for patients as well as having thehigh associated costs Now the gold standard method for diagnosingIBD and monitoring patient status is performing colonoscopy examination and histopathologic evaluation which are invasive measures[] Therefore in recent years many studies have been conducted tofind a suitable laboratory marker with sufficient sensitivity and specificity for the purpose of diagnosing and noninvasive management ofIBD A high proportion of these studies have investigated the efficacy offecal calprotectin in diagnosing and monitoring patients Althoughsome of these studies reported auspicious results there are still somedoubts on the eï¬ectiveness of fecal calprotectin on diagnosing andmonitoring IBD patients So in this review we addressed the advantages and limitations of fecal calprotectin for the diagnosis andmanagement of IBD The role of fecal calprotectin in diagnosis and management ofIBDThe efficacy of fecal calprotectin as an laboratory marker in various areas of IBD diagnosis and management have been studied including IBD diï¬erentiation from irritable bowel syndrome IBS evaluation of endoscopic activity of the disease evaluation of histologicalactivity of the disease and prediction of disease recurrence andClinica Chimica Acta “response to treatment In following after a brief introduction andmentioning the important points regarding laboratory measurement offecal calprotectin we reviewed the most interesting findings in all ofthe abovementioned areas Calprotectin A clinically valuable proteinCalprotectin is an antimicrobial protein mainly secreted by neutrophils This protein competes with bacteria over zinc thus kills thebacteria However this is not the only contribution that it has to antimicrobial activity Moreover this protein has many potential clinicalapplications such as the elevated serum levels that have been observedunder various immunological and immunopathological conditionsSerum calprotectin levels rapidly increase in response to bacterial infections in the kidney and heart or during transplant rejection At theearly stages of ‚ammation of the lung serum calprotectin can also beconsidered as a reliable marker besides plasma levels of calprotectinappear to be useful in reflecting disease activity in ‚ammation of thejoints [] In addition it has been demonstrated that serum calprotectin levels are increased in patients with bacterial sepsis so it can beconsidered as a reliable biomarker [] In Neonatal Sepsis the serumlevel of calprotectin increases as well as a sensitivity of and aspecificity of that have been reported for serum calprotectin indiagnosis of Neonatal Sepsis [] It has been recently shown thatserum calprotectin levels increase in patients with aneurysmal subarachnoid hemorrhage and higher levels in the first of onset areassociated with a poor prognosis at the first three months [] Serumcalprotectin levels also increase in patients with rheumatoid arthritisand even in patients with a moderate to high disease activity who havenormal or low CRP levels so they appear to be more efficient at reflecting disease activity []Some studies have also investigated the efficacy of serum calprotectin in the diagnosis of cancers Correspondingly in one of thesestudies it was shown that serum calprotectin levels significantly increased in patients with laryngeal carcinoma compared with healthyindividuals and those with benign laryngeal pathologies Moreover inthis study a direct relationship was also observed between serum levelsof calprotectin and stage of cancer [] Another study showed that theserum level of calprotectin increased in patients with papillary thyroidcarcinoma but it significantly decreased after operation [] Alsoregarding the efficacy of serum and saliva calprotectin for the diagnosisof IBD impressive results have been reported [][] A study onpatients with IBD both UC and CD have shown that serum calprotectinlevels were directly correlated with fecal calprotectin levels and weremore potent in IBD diagnosis compared to CRP and albumin This studyalso indicated that the combination of serum calprotectin with CRP oralbumin can be helpfulin the prediction of treatment escalationespecially in patients with CD [] However no significant correlationwas observed between serum calprotectin and fecal calprotectin levelsin patients with CD and UC as well as a slight correlation betweenserum calprotectin level and CRP that was observed only in patientswith UC [] Another study showed that the serum level of calprotectin was significantly higher in patients with CD compared to healthyindividuals In addition although a significant correlation was observedwith the clinical activity of the disease no significant correlation wasfound between the level serum calprotectin and endoscopic activity ofthe disease [] The efficacy of salivary calprotectin in the diagnosisof IBD has also been studied which showed that salivary calprotectinsignificantly increased in patients with IBD compared to healthy individuals In this study AUC values for unstimulated saliva and stimulated saliva to distinguish IBD patients from healthy individualswere reported to be and respectively [] However thepopularity of calprotectin is mainly due to the use of fecal calprotectinin the diagnosis and management of IBD that is discussed in the following 0cF KhakiKhatibi et alClinica Chimica Acta “ Laboratory measurement and reference intervalFecal calprotectin is a stable protein that remains stable for “ daysat room temperature [] This property is an excellent advantage for alaboratory marker Also it seems that keeping the specimen at refrigerated temperature °C can increase the stability of fecal calprotectin [] However evidence has been obtained regarding thatthe stability of this protein decreases after staying for three days atroom temperature On the other hand it is not also recommended tokeep samples in the refrigerator for more than days [] It seemsthat fecal calprotectin remains stable up to one year at ˆ’ °C []Measurement of fecal calprotectin can be done both qualitatively andquantitatively Accordingly in the qualitative measurement monoclonal antibodies are used to detect fecal calprotectin and the positiveresults are characterized by the appearance of colored lines on the testcassette However in the qualitative one only positive or negative results are reported and despite of sensitivity test specificity in theevaluation of disease activity was reported to be only It seems thatthe main application of this test is to diï¬erentiate healthy individualsfrom IBD patients rapidly however some studies have shown that it isnot accurate enough in this case as well [][] Nevertheless asignificant concordance has been reported between home test resultsIBDoc and fecal calprotectin laboratory measurement results whenQuantum Blue calprotectin ELISA kit was used Notably the agreements between results were and depending on the selectedcutoï¬s [] Several commercial kits are also available for fecal calprotectin qualitative test known as rapid calprotectin These tests reportpositive results ranged from to µgg There are also severalcommercial kits that can be used for the quantitative measurement offecal calprotectin These kits are usually designed in terms of the ELISAmethod and some have a measurement range between and µgg Moreover the chemiluminescence immunoassays CLIAmethod can also detect values between and µgg Fluoro enzyme immunoassays FEIA and particle enhanced turbidimetric immunoassays PETIA can also be used for the measurement of fecalcalprotectin In this regard one of the most serious challenges to thelaboratory evaluation of fecal calprotectin is the determination of theupper limit in healthy individuals Among healthy adults there is asignificant agreement on µgg as an upper limit One study suggested values up to µgg in people over years old and up to µgg in children aged between and years old as referenceranges of fecal calprotectin in healthy individuals []Fecal calprotectin levels appear to be higher in healthy infants andchildren under four years old than in adults and further studies areneeded in this regard to determine the acceptable upper limit for diagnosis of pediatric IBD [] Table lists the median levels of fecalcalprotectin in healthy individuals with diï¬erent ages reported in somestudies According to these reports age can aï¬ect fecal calprotectinlevels Fecal calprotectin and IBD diagnosisOnly a small percentage of patients complaining of abdominal painand diarrhea have IBD In many cases IBS as a functional gastrointestinal disorder is known as the cause of such clinical symptomsPatients with IBS have normal colonoscopy results while IBD patientsindicate abnormal colonoscopy results and have intestinal ulcersUnfortunately the significant prevalence of IBS and the overlap between clinical symptoms and IBD can increase the colonoscopy rateTherefore a noninvasive diagnostic marker can be very helpful in thisregard Notably the first evidence of the efficacy of fecal calprotectin inthe diagnosis of IBD was obtained in the 1990s Røseth et al in proposed a method for measuring Calprotectin in stool specimens []One of the first and most interesting studies regarding fecal calprotectinutility in IBD diagnosis was the study by Røseth et al published in In this study patients with ulcerative colitis were studied and according to their results fecal calprotectin levels are higher in patientswith ulcerative colitis compared to healthy controls This study havealso shown that even patients with low disease activities had higherlevels of fecal calprotectin compared to healthy individuals []Subsequent studies somehow confirmed and complemented the findings of this study In another study published in AUC values of CI “ were reported for fecal calprotectin in thediagnosis of colorectal ‚ammation [] Moreover in a study onchildren with IBD it was shown that the level of fecal calprotectin washigher in these patients compared to healthy children so it can beconcluded that it is also directly correlated with ESR levels [] In astudy published in Kolho et al reported AUC values of CI “ for fecal calprotectin in the diagnosis of pediatric IBD [] In a study on patients with Crohn disease a sensitivity of and a specificity of at cutoï¬ of μgg have been reportedfor fecal calprotectin in diagnosis of the disease [] The results of ourrecent study along with other studies showed that fecal calprotectin ispreferred over traditional ‚ammatory biomarkers such as CRP andESR in the diagnosis of IBD [][] Diamanti et al reported a sensitivity of and a specificity of for fecal calprotectin at a cutoï¬ of μgg in IBD diagnosis [] In our recent study a sensitivityof and a specificity of at a cutoï¬ of μgg were observed for fecal calprotectin in the diagnosis of IBD however oursample size was and the majority of patients were in the active phaseof the disease []In another study conducted on patients with ulcerative colitis asensitivity of and a specificity of at cutoï¬ of μgg havebeen reported in this regard [] In one study it was shown that fecalcalprotectin in cutoï¬ of μgg is able to distinguish patients withIBD from patients without IBD patients with diseases other than IBDpatients with IBS and healthy persons with sensitivity and specificity [] Caviglia et al in their study reported a sensitivity of and a specificity of at a cutoï¬ of μgg for fecalcalprotectin in diï¬erentiating between IBS and IBD [] Howeversome studies have reported significantly lower values Accordingly in astudy on patients with ulcerative colitis Kalantari et al reported asensitivity of and a specificity of at a cutoï¬ of μgg []Besides there is a considerable agreement between fecal calprotectinand capsule endoscopy findings in patients with Crohn's disease Asensitivity of and a specificity of have also been reported at acutoï¬ of mgkg for fecal calprotectin in predicting CE findings anddiagnosis of Crohn's disease [] In another study lower sensitivityand specificity rates sensitivity specificity were reportedfor fecal calprotectin in this regard [] Furthermore in one studythat examined the efficacy of fecal calprotectin in predicting wirelesscapsule endoscopy findings a sensitivity of and a specificity ofTable Reported median levels of fecal calprotectin in healthy individuals of diï¬erent agesAgesMedian levels of fecal calprotectin range µggNumber of subjectsUsed kitUp to monthChildren “ yearsChildren “ yearsAdultsOver years “ “ “ “ “Bühlmann ELISABühlmann ELISACALPRO® Calprotectin ELISA Test ALPPhiCalPhicalReference[][][][][] 0cF KhakiKhatibi et al were reported for this biomarker at μgg in the diagnosis ofsmall bowel ‚ammation in Crohn's disease [] Given these findings it seems that fecal calprotectin has no ideal sensitivity and specificity for the diagnosis of IBD where the small intestine is involvedBesides there are some preanalytical limitations which are explainedin the next sections Therefore optimistically speaking fecal calprotectin measurement can eliminate the need for colonoscopy Howeverin a metaanalysis performed to evaluate the efficacy of fecal calprotectin and some other ‚ammatory markers to diï¬erentiate betweenIBD and IBS the probability of IBD was less than at fecal calprotectin values lower than µgg or CRP values lower than mgdL[] Therefore it seems that fecal calprotectin can be helpful at leastin ruling out the possibility of IBD in patients with IBSlike symptoms aswell as reducing the rate of colonoscopy Moreover it should be notedthat although a systematic review has reported pooled sensitivity andspecificity above for fecal calprotectin to diï¬erentiate between IBDand IBS it emphasized more on the possibility of falsepositive resultsin low cutoï¬ points [] Hence performing extensive studies indiï¬erent countries on the healthy population and the IBD patient is beneeded to determine a suitable cutoï¬ with maximum sensitivity andspecificity and minimum falsepositive resultsTable summarizes the results of various clinical investigationsregarding fecal calprotectin utility in the diï¬erential diagnosis of IBDfrom IBS and Table4 summarizes some metaanalysis results in thisregard As shown in Table the most important limitation of the majority of clinical studies conducted to date is the small sample size Alarge global study may be helpful in providing a more precise evaluation of fecal calprotectin clinical value in discrimination between IBDand nonIBD diseases Fecal calprotectin and endoscopic and histologic activity evaluationUndoubtedly one of the most serious challenges in the managementof IBD is evaluating the endoscopic and histologic activities of thedisease Nowadays colonoscopy and histopathologic examinations arethe routine tools for the assessment of mucosal healing in patients withIBD As noted earlier several scoring systems have been devised toscore disease activity based on the findings of colonoscopy and histopathologic examinations In recent years many promising results havebeen reported regarding the correlation between these scores and fecalcalprotectin levels In addition many studies have been performed inthe last decade all of which cannot be reviewed in this article The firstevidence of a link between fecal calprotectin and disease endoscopicactivity was obtained in the late 1990s In one of the first studiesRoseth et al found a significant correlation between fecal calprotectinlevels and endoscopic and histologic activities in patients with ulcerative colitis [] Furthermore in another study they observed that IBDpatients who were in remission clinically and had normal fecal calprotectin levels less than mgL had normal colonoscopy results[] These interesting findings indicate that fecal calprotectin can beconsidered as a biomarker in the evaluation of endoscopic activity andClinica Chimica Acta “Table4summarized results of some metaanalysis regarding the utility of fecal calprotectin in discrimination between patients with IBD and without IBDSample sizePooled SensitivityPooled SpecificityReferences[][][][][]mucosal healing in IBD patients Also these studies were the startingpoint of extensive studies that have been conducted up to now In astudy conducted on patients with Crohn's disease Sipponen et alinvestigated the sensitivity and specificity of fecal calprotectin in predicting endoscopic activity of Crohn's disease [] Correspondinglythe researchers used the Crohn's Disease Endoscopic Index of SeverityCDEIS scoring system in their study to evaluate the endoscopic activity of Crohn's disease As a result they found that there was a significant correlation between the endoscopic activity of the disease andthe level of fecal calprotectin Besides the findings of this study demonstrated that fecal calprotectin at µgg cutoï¬ can predict theendoscopic activity of Crohn's disease with sensitivity and specificity In another study CDEIS and Mayo Disease Activity IndexMDAI were used to evaluate the endoscopic activity of Crohn's diseaseand ulcerative colitis respectively According to the results of thatstudy on IBD patients there was a significant correlation between fecalcalprotectin levels and disease endoscopic activity [] Another studyshowed that fecal calprotectin is more strongly correlated with theendoscopic activity of the disease in ulcerative colitis compared to theRachmilewitz clinical activity index In addition in this study theoverall accuracy of fecal calprotectin for endoscopically active diseaseidentification was obtained as []Some studies have also shown the superiority of fecal calprotectinover traditional ‚ammatory markers like CRP Besides one studyfound that fecal calprotectin was more strongly correlated with theSimple Endoscopic Score for Crohn's disease SESCD compared to theCRP and even Crohn's disease activity index CDAI [] The modifiedBaron Index was also used in another study to evaluate the endoscopicactivity of ulcerative colitis As a result it was shown that calprotectinis more strongly correlated with the endoscopic activity of ulcerativecolitis compared to CRP and clinical activity of the disease [] In thisregard similar results were also observed in our recent study in whichthe Ulcerative Colitis Endoscopic Index of Severity UCEIS and SESCDwere used [] Therefore fecal calprotectin appears to be superior totraditional ‚ammatory markers in the prediction of IBD endoscopicactivity The high values of sensitivity and specificity that were mentioned earlier have raised the hope that using fecal calprotectin canreduce colonoscopy rate for patients™ monitoring However severalrecent studies have reported some significantly lower values Accordingly in a recent study in which Mayo Endoscopic Score [MES] wasused to evaluate the endoscopic activity of ulcerative colitis aTable Summary of the results of some studies regarding the utility of fecal calprotectin in discrimination between patients with IBD and without IBDNumber of IBD patientsAge groupLocationCut oï¬SensitivitySpecificity CD and UC CD and UC CD and UC and unclassified68CD and UC CD and UC and unclassified CD and UC CD and UC UC CD UCAdultsAdultsAdultsBoth adult and pediatricpediatricAdultspediatricAdultsAdultsBoth adult and pediatricTaiwanChinaItalySpainFinlandIranItalyIranDenmarkIndia48µgg µgg150µgg150µgg595µgg784µgg160µgg164µgg150µgg188µggAUCReferences[][][]SPSrefidbib60[][][][][][][] 0cF KhakiKhatibi et alClinica Chimica Acta “Table Summary of the results of some studies regarding the correlation of fecal calprotectin with endoscopic activity in IBD patientsAge groupStudylocationUsedendoscopicactivity indexCorrelationcoefficientrReferenceNumberof IBDpatients CD UC UC CD UCAdultsAdultsAdultsAdultsAdultsFinlandIranSwitzerlandSwitzerlandSwitzerland ModifiedCDEISUCEISRachmilewitzSESCD UC CDAdultsAdults UC CD UC CD CD UC UCAdultsAdultsAdultsAdultsAdultsAdultsAdultsBaron ScoreRachmilewitzSESCDGermanyUSA andCanadaJapanItalyItalyBrazilFranceFranceSouth Korea UCEISMattsSESCDMayo scoreSESCDCDEISMayo score[][][][][][][][][][][][][][]sensitivity of and a specificity of were reported for fecalcalprotectin at µgg to diï¬erentiate active endoscopic from inactiveMES or from MES or [] In another study the sensitivityand specificity of fecal calprotectin at a cutoï¬ of µgg for diï¬erentiating MES ‰¤ in patients with ulcerative colitis were and respectively [] Overall as presented in Table several studiesperformed in diï¬erent countries reported the correlation between fecalcalprotectin and IBD endoscopic activity Although some of these studies reported a strong correlation some others reported a relativelyweak correlation As noted earlier there are significant diï¬erencesbetween the reports on the sensitivity and specificity of fecal calprotectin to predict the endoscopic activity of IBD Undoubtedly a widerange of factors from sample size and the inclusionexclusion criteriato preanalysis variables and indexes used to evaluate the endoscopicactivity may also contribute to these diï¬erences However fecal calprotectin does not appear to be a very reliable marker for the predictionof IBD endoscopic activity so currently it seems a bit optimistic toconsider fecal calprotectin as a reliable alternative for colonoscopy Inthis regard further studies are still needed However under some certain circumstances such as pregnancy or pandemics the use of fecalcalprotectin to evaluate IBD endoscopic activity can be helpfulPregnant patients with IBD have serious limitations for colonoscopyexamination and it has been recommended that colonoscopy should beonly performed in the second trimester of pregnancy and where there isa strong indication [] Therefore noninvasive markers such as fecalcalprotectin can be helpful during pregnancy In one study physicianglobal assessment [PGA] which is a clinical symptombased criterionwas used to evaluate IBD activity and subsequently the associationbetween fecal calprotectin and this criterion was investigated in pregnant women with IBD The results of this study showed a significantcorrelation between fecal calprotectin and PGA levels at prepregnancyduring pregnancy and postpartum stages [] In another study asignificant association was reported between fecal calprotectin levelsand clinical activity of IBD in pregnant women Moreover it was shownthat stool calprotectin at a cutoï¬ of mgkg had a sensitivity between and as well as a specificity between and in the assessment of IBD clinical activity at diï¬erent stages ofpregnancy [] A recently published systematic review has also confirmed the conclusions obtained from these studies [] According tothese results it seems that fecal calprotectin is not aï¬ected by physiological changes during pregnancy however it is significantly correlatedwith IBD clinical activity during pregnancy Therefore from the viewpoint of relatively acceptable sensitivity and specificity in predictingthe endoscopic activity of IBD fecal calprotectin may be considered as anoninvasive biomarker for the evaluation of IBD endoscopic activity inpregnant women In addition under pandemic conditions fecal calprotectin can be very helpful Following the COVID19 pandemicwhich began in late and is still ongoing healthcare systems indiï¬erent countries were forced to impose significant limitations oncolonoscopy Therefore noninvasive IBD management and fecal calprotectin as a noninvasive laboratory marker have become moreimportant than before The combination between disease clinical activity and fecal calprotectin has been recommended as a noninvasiveapproach that can help in making decisions on treatment duringCOVID19 pandemic [] Therefore it seems that fecal calprotectincan be considered as an alternative for colonoscopy used for IBD endoscopic activity evaluation during pandemic Fecal calprotectin appears to be associated with IBD histologic activity as well Given thedifficulty in the evaluation of the histologic activity of Crohn's disease[] some studies have been focused on the ulcerative colitis andmany scoring systems have been devised so far Correspondingly thesesystems score the disease's histologic activity based on histologic observationsTherefore for this purpose a biopsy of the intestinal tissue is required which can be prepared by colonoscopy and then sent to thelaboratory In this regard one of these histologic scoring systems isRobert™s score that was used in one of our recent studies where weobserved a significant correlation between the level of fecal calprotectinand the histologic activity of ulcerative colitis which was calculatedbased on the Robert™s scoring system [] Theede et al also used themodified Harpaz Index and performed some interesting studies in thisregard In one of their studies fecal calprotectin was found to be significantly associated with the histologic activity of the ulcerative colitisand it was shown that it could predict histological mucosal healingAUC CI95 “ Sensitivity Specificity andCutoï¬ mgkg [] In another study on patients with endoscopically inactive ulcerative colitis Mayo endoscopic score the researchers showed that patients with ulcerative colitis who were inendoscopic remission but had histologically active disease had higherlevels of fecal calprotectin compared to patients with no histologicallyactive disease versus mgkg P Also despite thehigh specificity the sensitivity of fecal calprotectin in theprediction of score of histological activity was achieved as at mgkg [] In a recent study the Geboes

cancer230

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: " IBDFecal calprotectinEndoscopic activityIBD noninvasive managementThe term IBD is usually used for referring to a group of ‚ammatory gastrointestinal diseases mainly Crohn'sdisease and ulcerative colitis Accordingly IBD arises as a result of inappropriate immune response to intestinalcommensal anisms among genetically susceptible individuals Performing colonoscopy and histopathologicevaluation on an ‚amed bowel biopsy specimen are currently considered as gold standards for diagnosis andmanagement of IBD Correspondingly these techniques are known to be invasive and costly In recent decadesfecal calprotectin as a biomarker has received much attention for the diagnosis and noninvasive managementof IBD Up to now many studies have investigated the efficacy of fecal calprotectin in the areas of IBD diï¬erentiation from IBS prediction of endoscopic and histologic activities of IBD and prediction of disease recurrenceAlthough some of these studies have reported promising results some others have shown significant limitationsTherefore in this paper we reviewed the most interesting ones of these studies after a brief discussion of thelaboratory measurement of fecal calprotectin Moreover we attempted to provide an answer for the question ofwhether fecalcalprotectin could be considered as a potential surrogate marker for colonoscopy IntroductionInflammatory bowel disease IBD is a long life disease with remission and relapse periods IBD arises as a result of inappropriateimmune response to intestinal commensal anisms in individualswith genetic predisposition and consequently causes ‚ammation andintestinal ulcers [] In addition IBD has a complex pathogenesis andmany factors such as dysbiosis oxidative stress and epigenetics thatmay also be involved in disease pathogenesis [“] Ulcerative colitisUC and Crohn's diseases CD are known as two main forms of IBDAccordingly these diseases cause intestinal ulcers and some annoyingsymptoms such as diarrhea abdominal pain and rectal bleeding Occasionally the severity of these symptoms is very high which can leadpatients to be hospitalized In this regard therapeutic approaches totreat these diseases mainly focus on prolonging remission and are almost similar however diï¬erential diagnosis can also help to treat thedisease in a more eï¬ective way For example 5ASA which is acommon drug in the treatment of IBD is less eï¬ective on maintainingremission in CD patients On the other hand antibiotic therapy is notrecommended for the treatment UC but it can be eï¬ective on CD patients [][] Diï¬erential diagnosis is a serious challenge because CDand UC have significant similarities in terms of their clinical endoscopic and histological features However there are some diï¬erencesbetween UC and CD which are summarized in Table1 In addition tointestinal complications UC and CD also have significant extraintestinal manifestations For example it was shown that UC is significantly associated with Primary sclerosing cholangitis and CD is alsoassociated with cholelithiasis especially in cases that the ileum is involved [] Furthermore CD can cause fistulas to the urinary systemwhich leads intestinal bacteria to enter the urethra and recurrent urinary tract infections [] Both CD and UC can cause several disorderssuch as arthritis Erythema nodosum pyoderma gangrenosum andanemia which are known as the most important extraintestinal manifestations of IBD [][] The latest statistics showed that the globalŽ Corresponding author at Department of Clinical Biochemistry and Laboratory Medicine Faculty of Medicine Tabriz University of Medical Sciences DaneshgahStreet PO Box Tabriz IranEmail address vagharimtbzmedacir M VaghariTabari101016jcca202008025Received July Received in revised form August Accepted August Available online August Elsevier BV All rights reserved 0cF KhakiKhatibi et alTable1Clinical endoscopic and histological features of CD and UCClinical FeaturesFeaturesRectal bleedingAbdominal painFeverMucus defectionIntestinal obstructionPerineal diseasePostoperative recurrenceASCA positiveANCA positiveEndoscopic FeaturesCDOccasionallyFrequentlyFrequentlyOccasionallyYESYESYESFrequentlyNot commonUCFrequentlyOccasionallyNot commonFrequentlyNONONONot commonFrequentlyFeaturesCDUCLocationMucosal involvementDepth of ulcerationfistulaCobblestone appearanceAphthous ulcerationMucosal friabilityHistological featuresFeaturesGranulomasCrypt abscessesPatchinessAny part of GI tractDiscontinuousDeepYesYESFrequentlyNot commonCDFrequentlyNot commonFrequentlyColon and rectumContinuoussuperficialNONOOccasionallyFrequentlyUCRareFrequentlyNot commonprevalence of IBD currently is on the rise and it is not an exaggerationif we consider it as a global serious health problem [] According to areport published in IBD has the highest prevalence rate inEurope and its prevalence in the newly industrialized countries of AsiaAfrica and South America also appears to be increased over the pastthree decades []Unfortunately the peak of the disease is at the young age of“ years old [] therefore in addition to the suï¬ering from ‚icts on the patients it also has many negative eï¬ects on societyMoreover many financial burdens are annually imposed on countriesfor controlling and treating this chronic disease The invasive diagnosticand therapeutic measures are currently undertaken to diagnose andmanage IBD which are unpleasant for patients as well as having thehigh associated costs Now the gold standard method for diagnosingIBD and monitoring patient status is performing colonoscopy examination and histopathologic evaluation which are invasive measures[] Therefore in recent years many studies have been conducted tofind a suitable laboratory marker with sufficient sensitivity and specificity for the purpose of diagnosing and noninvasive management ofIBD A high proportion of these studies have investigated the efficacy offecal calprotectin in diagnosing and monitoring patients Althoughsome of these studies reported auspicious results there are still somedoubts on the eï¬ectiveness of fecal calprotectin on diagnosing andmonitoring IBD patients So in this review we addressed the advantages and limitations of fecal calprotectin for the diagnosis andmanagement of IBD The role of fecal calprotectin in diagnosis and management ofIBDThe efficacy of fecal calprotectin as an laboratory marker in various areas of IBD diagnosis and management have been studied including IBD diï¬erentiation from irritable bowel syndrome IBS evaluation of endoscopic activity of the disease evaluation of histologicalactivity of the disease and prediction of disease recurrence andClinica Chimica Acta “response to treatment In following after a brief introduction andmentioning the important points regarding laboratory measurement offecal calprotectin we reviewed the most interesting findings in all ofthe abovementioned areas Calprotectin A clinically valuable proteinCalprotectin is an antimicrobial protein mainly secreted by neutrophils This protein competes with bacteria over zinc thus kills thebacteria However this is not the only contribution that it has to antimicrobial activity Moreover this protein has many potential clinicalapplications such as the elevated serum levels that have been observedunder various immunological and immunopathological conditionsSerum calprotectin levels rapidly increase in response to bacterial infections in the kidney and heart or during transplant rejection At theearly stages of ‚ammation of the lung serum calprotectin can also beconsidered as a reliable marker besides plasma levels of calprotectinappear to be useful in reflecting disease activity in ‚ammation of thejoints [] In addition it has been demonstrated that serum calprotectin levels are increased in patients with bacterial sepsis so it can beconsidered as a reliable biomarker [] In Neonatal Sepsis the serumlevel of calprotectin increases as well as a sensitivity of and aspecificity of that have been reported for serum calprotectin indiagnosis of Neonatal Sepsis [] It has been recently shown thatserum calprotectin levels increase in patients with aneurysmal subarachnoid hemorrhage and higher levels in the first of onset areassociated with a poor prognosis at the first three months [] Serumcalprotectin levels also increase in patients with rheumatoid arthritisand even in patients with a moderate to high disease activity who havenormal or low CRP levels so they appear to be more efficient at reflecting disease activity []Some studies have also investigated the efficacy of serum calprotectin in the diagnosis of cancers Correspondingly in one of thesestudies it was shown that serum calprotectin levels significantly increased in patients with laryngeal carcinoma compared with healthyindividuals and those with benign laryngeal pathologies Moreover inthis study a direct relationship was also observed between serum levelsof calprotectin and stage of cancer [] Another study showed that theserum level of calprotectin increased in patients with papillary thyroidcarcinoma but it significantly decreased after operation [] Alsoregarding the efficacy of serum and saliva calprotectin for the diagnosisof IBD impressive results have been reported [][] A study onpatients with IBD both UC and CD have shown that serum calprotectinlevels were directly correlated with fecal calprotectin levels and weremore potent in IBD diagnosis compared to CRP and albumin This studyalso indicated that the combination of serum calprotectin with CRP oralbumin can be helpfulin the prediction of treatment escalationespecially in patients with CD [] However no significant correlationwas observed between serum calprotectin and fecal calprotectin levelsin patients with CD and UC as well as a slight correlation betweenserum calprotectin level and CRP that was observed only in patientswith UC [] Another study showed that the serum level of calprotectin was significantly higher in patients with CD compared to healthyindividuals In addition although a significant correlation was observedwith the clinical activity of the disease no significant correlation wasfound between the level serum calprotectin and endoscopic activity ofthe disease [] The efficacy of salivary calprotectin in the diagnosisof IBD has also been studied which showed that salivary calprotectinsignificantly increased in patients with IBD compared to healthy individuals In this study AUC values for unstimulated saliva and stimulated saliva to distinguish IBD patients from healthy individualswere reported to be and respectively [] However thepopularity of calprotectin is mainly due to the use of fecal calprotectinin the diagnosis and management of IBD that is discussed in the following 0cF KhakiKhatibi et alClinica Chimica Acta “ Laboratory measurement and reference intervalFecal calprotectin is a stable protein that remains stable for “ daysat room temperature [] This property is an excellent advantage for alaboratory marker Also it seems that keeping the specimen at refrigerated temperature °C can increase the stability of fecal calprotectin [] However evidence has been obtained regarding thatthe stability of this protein decreases after staying for three days atroom temperature On the other hand it is not also recommended tokeep samples in the refrigerator for more than days [] It seemsthat fecal calprotectin remains stable up to one year at ˆ’ °C []Measurement of fecal calprotectin can be done both qualitatively andquantitatively Accordingly in the qualitative measurement monoclonal antibodies are used to detect fecal calprotectin and the positiveresults are characterized by the appearance of colored lines on the testcassette However in the qualitative one only positive or negative results are reported and despite of sensitivity test specificity in theevaluation of disease activity was reported to be only It seems thatthe main application of this test is to diï¬erentiate healthy individualsfrom IBD patients rapidly however some studies have shown that it isnot accurate enough in this case as well [][] Nevertheless asignificant concordance has been reported between home test resultsIBDoc and fecal calprotectin laboratory measurement results whenQuantum Blue calprotectin ELISA kit was used Notably the agreements between results were and depending on the selectedcutoï¬s [] Several commercial kits are also available for fecal calprotectin qualitative test known as rapid calprotectin These tests reportpositive results ranged from to µgg There are also severalcommercial kits that can be used for the quantitative measurement offecal calprotectin These kits are usually designed in terms of the ELISAmethod and some have a measurement range between and µgg Moreover the chemiluminescence immunoassays CLIAmethod can also detect values between and µgg Fluoro enzyme immunoassays FEIA and particle enhanced turbidimetric immunoassays PETIA can also be used for the measurement of fecalcalprotectin In this regard one of the most serious challenges to thelaboratory evaluation of fecal calprotectin is the determination of theupper limit in healthy individuals Among healthy adults there is asignificant agreement on µgg as an upper limit One study suggested values up to µgg in people over years old and up to µgg in children aged between and years old as referenceranges of fecal calprotectin in healthy individuals []Fecal calprotectin levels appear to be higher in healthy infants andchildren under four years old than in adults and further studies areneeded in this regard to determine the acceptable upper limit for diagnosis of pediatric IBD [] Table lists the median levels of fecalcalprotectin in healthy individuals with diï¬erent ages reported in somestudies According to these reports age can aï¬ect fecal calprotectinlevels Fecal calprotectin and IBD diagnosisOnly a small percentage of patients complaining of abdominal painand diarrhea have IBD In many cases IBS as a functional gastrointestinal disorder is known as the cause of such clinical symptomsPatients with IBS have normal colonoscopy results while IBD patientsindicate abnormal colonoscopy results and have intestinal ulcersUnfortunately the significant prevalence of IBS and the overlap between clinical symptoms and IBD can increase the colonoscopy rateTherefore a noninvasive diagnostic marker can be very helpful in thisregard Notably the first evidence of the efficacy of fecal calprotectin inthe diagnosis of IBD was obtained in the 1990s Røseth et al in proposed a method for measuring Calprotectin in stool specimens []One of the first and most interesting studies regarding fecal calprotectinutility in IBD diagnosis was the study by Røseth et al published in In this study patients with ulcerative colitis were studied and according to their results fecal calprotectin levels are higher in patientswith ulcerative colitis compared to healthy controls This study havealso shown that even patients with low disease activities had higherlevels of fecal calprotectin compared to healthy individuals []Subsequent studies somehow confirmed and complemented the findings of this study In another study published in AUC values of CI “ were reported for fecal calprotectin in thediagnosis of colorectal ‚ammation [] Moreover in a study onchildren with IBD it was shown that the level of fecal calprotectin washigher in these patients compared to healthy children so it can beconcluded that it is also directly correlated with ESR levels [] In astudy published in Kolho et al reported AUC values of CI “ for fecal calprotectin in the diagnosis of pediatric IBD [] In a study on patients with Crohn disease a sensitivity of and a specificity of at cutoï¬ of μgg have been reportedfor fecal calprotectin in diagnosis of the disease [] The results of ourrecent study along with other studies showed that fecal calprotectin ispreferred over traditional ‚ammatory biomarkers such as CRP andESR in the diagnosis of IBD [][] Diamanti et al reported a sensitivity of and a specificity of for fecal calprotectin at a cutoï¬ of μgg in IBD diagnosis [] In our recent study a sensitivityof and a specificity of at a cutoï¬ of μgg were observed for fecal calprotectin in the diagnosis of IBD however oursample size was and the majority of patients were in the active phaseof the disease []In another study conducted on patients with ulcerative colitis asensitivity of and a specificity of at cutoï¬ of μgg havebeen reported in this regard [] In one study it was shown that fecalcalprotectin in cutoï¬ of μgg is able to distinguish patients withIBD from patients without IBD patients with diseases other than IBDpatients with IBS and healthy persons with sensitivity and specificity [] Caviglia et al in their study reported a sensitivity of and a specificity of at a cutoï¬ of μgg for fecalcalprotectin in diï¬erentiating between IBS and IBD [] Howeversome studies have reported significantly lower values Accordingly in astudy on patients with ulcerative colitis Kalantari et al reported asensitivity of and a specificity of at a cutoï¬ of μgg []Besides there is a considerable agreement between fecal calprotectinand capsule endoscopy findings in patients with Crohn's disease Asensitivity of and a specificity of have also been reported at acutoï¬ of mgkg for fecal calprotectin in predicting CE findings anddiagnosis of Crohn's disease [] In another study lower sensitivityand specificity rates sensitivity specificity were reportedfor fecal calprotectin in this regard [] Furthermore in one studythat examined the efficacy of fecal calprotectin in predicting wirelesscapsule endoscopy findings a sensitivity of and a specificity ofTable Reported median levels of fecal calprotectin in healthy individuals of diï¬erent agesAgesMedian levels of fecal calprotectin range µggNumber of subjectsUsed kitUp to monthChildren “ yearsChildren “ yearsAdultsOver years “ “ “ “ “Bühlmann ELISABühlmann ELISACALPRO® Calprotectin ELISA Test ALPPhiCalPhicalReference[][][][][] 0cF KhakiKhatibi et al were reported for this biomarker at μgg in the diagnosis ofsmall bowel ‚ammation in Crohn's disease [] Given these findings it seems that fecal calprotectin has no ideal sensitivity and specificity for the diagnosis of IBD where the small intestine is involvedBesides there are some preanalytical limitations which are explainedin the next sections Therefore optimistically speaking fecal calprotectin measurement can eliminate the need for colonoscopy Howeverin a metaanalysis performed to evaluate the efficacy of fecal calprotectin and some other ‚ammatory markers to diï¬erentiate betweenIBD and IBS the probability of IBD was less than at fecal calprotectin values lower than µgg or CRP values lower than mgdL[] Therefore it seems that fecal calprotectin can be helpful at leastin ruling out the possibility of IBD in patients with IBSlike symptoms aswell as reducing the rate of colonoscopy Moreover it should be notedthat although a systematic review has reported pooled sensitivity andspecificity above for fecal calprotectin to diï¬erentiate between IBDand IBS it emphasized more on the possibility of falsepositive resultsin low cutoï¬ points [] Hence performing extensive studies indiï¬erent countries on the healthy population and the IBD patient is beneeded to determine a suitable cutoï¬ with maximum sensitivity andspecificity and minimum falsepositive resultsTable summarizes the results of various clinical investigationsregarding fecal calprotectin utility in the diï¬erential diagnosis of IBDfrom IBS and Table4 summarizes some metaanalysis results in thisregard As shown in Table the most important limitation of the majority of clinical studies conducted to date is the small sample size Alarge global study may be helpful in providing a more precise evaluation of fecal calprotectin clinical value in discrimination between IBDand nonIBD diseases Fecal calprotectin and endoscopic and histologic activity evaluationUndoubtedly one of the most serious challenges in the managementof IBD is evaluating the endoscopic and histologic activities of thedisease Nowadays colonoscopy and histopathologic examinations arethe routine tools for the assessment of mucosal healing in patients withIBD As noted earlier several scoring systems have been devised toscore disease activity based on the findings of colonoscopy and histopathologic examinations In recent years many promising results havebeen reported regarding the correlation between these scores and fecalcalprotectin levels In addition many studies have been performed inthe last decade all of which cannot be reviewed in this article The firstevidence of a link between fecal calprotectin and disease endoscopicactivity was obtained in the late 1990s In one of the first studiesRoseth et al found a significant correlation between fecal calprotectinlevels and endoscopic and histologic activities in patients with ulcerative colitis [] Furthermore in another study they observed that IBDpatients who were in remission clinically and had normal fecal calprotectin levels less than mgL had normal colonoscopy results[] These interesting findings indicate that fecal calprotectin can beconsidered as a biomarker in the evaluation of endoscopic activity andClinica Chimica Acta “Table4summarized results of some metaanalysis regarding the utility of fecal calprotectin in discrimination between patients with IBD and without IBDSample sizePooled SensitivityPooled SpecificityReferences[][][][][]mucosal healing in IBD patients Also these studies were the startingpoint of extensive studies that have been conducted up to now In astudy conducted on patients with Crohn's disease Sipponen et alinvestigated the sensitivity and specificity of fecal calprotectin in predicting endoscopic activity of Crohn's disease [] Correspondinglythe researchers used the Crohn's Disease Endoscopic Index of SeverityCDEIS scoring system in their study to evaluate the endoscopic activity of Crohn's disease As a result they found that there was a significant correlation between the endoscopic activity of the disease andthe level of fecal calprotectin Besides the findings of this study demonstrated that fecal calprotectin at µgg cutoï¬ can predict theendoscopic activity of Crohn's disease with sensitivity and specificity In another study CDEIS and Mayo Disease Activity IndexMDAI were used to evaluate the endoscopic activity of Crohn's diseaseand ulcerative colitis respectively According to the results of thatstudy on IBD patients there was a significant correlation between fecalcalprotectin levels and disease endoscopic activity [] Another studyshowed that fecal calprotectin is more strongly correlated with theendoscopic activity of the disease in ulcerative colitis compared to theRachmilewitz clinical activity index In addition in this study theoverall accuracy of fecal calprotectin for endoscopically active diseaseidentification was obtained as []Some studies have also shown the superiority of fecal calprotectinover traditional ‚ammatory markers like CRP Besides one studyfound that fecal calprotectin was more strongly correlated with theSimple Endoscopic Score for Crohn's disease SESCD compared to theCRP and even Crohn's disease activity index CDAI [] The modifiedBaron Index was also used in another study to evaluate the endoscopicactivity of ulcerative colitis As a result it was shown that calprotectinis more strongly correlated with the endoscopic activity of ulcerativecolitis compared to CRP and clinical activity of the disease [] In thisregard similar results were also observed in our recent study in whichthe Ulcerative Colitis Endoscopic Index of Severity UCEIS and SESCDwere used [] Therefore fecal calprotectin appears to be superior totraditional ‚ammatory markers in the prediction of IBD endoscopicactivity The high values of sensitivity and specificity that were mentioned earlier have raised the hope that using fecal calprotectin canreduce colonoscopy rate for patients™ monitoring However severalrecent studies have reported some significantly lower values Accordingly in a recent study in which Mayo Endoscopic Score [MES] wasused to evaluate the endoscopic activity of ulcerative colitis aTable Summary of the results of some studies regarding the utility of fecal calprotectin in discrimination between patients with IBD and without IBDNumber of IBD patientsAge groupLocationCut oï¬SensitivitySpecificity CD and UC CD and UC CD and UC and unclassified68CD and UC CD and UC and unclassified CD and UC CD and UC UC CD UCAdultsAdultsAdultsBoth adult and pediatricpediatricAdultspediatricAdultsAdultsBoth adult and pediatricTaiwanChinaItalySpainFinlandIranItalyIranDenmarkIndia48µgg µgg150µgg150µgg595µgg784µgg160µgg164µgg150µgg188µggAUCReferences[][][]SPSrefidbib60[][][][][][][] 0cF KhakiKhatibi et alClinica Chimica Acta “Table Summary of the results of some studies regarding the correlation of fecal calprotectin with endoscopic activity in IBD patientsAge groupStudylocationUsedendoscopicactivity indexCorrelationcoefficientrReferenceNumberof IBDpatients CD UC UC CD UCAdultsAdultsAdultsAdultsAdultsFinlandIranSwitzerlandSwitzerlandSwitzerland ModifiedCDEISUCEISRachmilewitzSESCD UC CDAdultsAdults UC CD UC CD CD UC UCAdultsAdultsAdultsAdultsAdultsAdultsAdultsBaron ScoreRachmilewitzSESCDGermanyUSA andCanadaJapanItalyItalyBrazilFranceFranceSouth Korea UCEISMattsSESCDMayo scoreSESCDCDEISMayo score[][][][][][][][][][][][][][]sensitivity of and a specificity of were reported for fecalcalprotectin at µgg to diï¬erentiate active endoscopic from inactiveMES or from MES or [] In another study the sensitivityand specificity of fecal calprotectin at a cutoï¬ of µgg for diï¬erentiating MES ‰¤ in patients with ulcerative colitis were and respectively [] Overall as presented in Table several studiesperformed in diï¬erent countries reported the correlation between fecalcalprotectin and IBD endoscopic activity Although some of these studies reported a strong correlation some others reported a relativelyweak correlation As noted earlier there are significant diï¬erencesbetween the reports on the sensitivity and specificity of fecal calprotectin to predict the endoscopic activity of IBD Undoubtedly a widerange of factors from sample size and the inclusionexclusion criteriato preanalysis variables and indexes used to evaluate the endoscopicactivity may also contribute to these diï¬erences However fecal calprotectin does not appear to be a very reliable marker for the predictionof IBD endoscopic activity so currently it seems a bit optimistic toconsider fecal calprotectin as a reliable alternative for colonoscopy Inthis regard further studies are still needed However under some certain circumstances such as pregnancy or pandemics the use of fecalcalprotectin to evaluate IBD endoscopic activity can be helpfulPregnant patients with IBD have serious limitations for colonoscopyexamination and it has been recommended that colonoscopy should beonly performed in the second trimester of pregnancy and where there isa strong indication [] Therefore noninvasive markers such as fecalcalprotectin can be helpful during pregnancy In one study physicianglobal assessment [PGA] which is a clinical symptombased criterionwas used to evaluate IBD activity and subsequently the associationbetween fecal calprotectin and this criterion was investigated in pregnant women with IBD The results of this study showed a significantcorrelation between fecal calprotectin and PGA levels at prepregnancyduring pregnancy and postpartum stages [] In another study asignificant association was reported between fecal calprotectin levelsand clinical activity of IBD in pregnant women Moreover it was shownthat stool calprotectin at a cutoï¬ of mgkg had a sensitivity between and as well as a specificity between and in the assessment of IBD clinical activity at diï¬erent stages ofpregnancy [] A recently published systematic review has also confirmed the conclusions obtained from these studies [] According tothese results it seems that fecal calprotectin is not aï¬ected by physiological changes during pregnancy however it is significantly correlatedwith IBD clinical activity during pregnancy Therefore from the viewpoint of relatively acceptable sensitivity and specificity in predictingthe endoscopic activity of IBD fecal calprotectin may be considered as anoninvasive biomarker for the evaluation of IBD endoscopic activity inpregnant women In addition under pandemic conditions fecal calprotectin can be very helpful Following the COVID19 pandemicwhich began in late and is still ongoing healthcare systems indiï¬erent countries were forced to impose significant limitations oncolonoscopy Therefore noninvasive IBD management and fecal calprotectin as a noninvasive laboratory marker have become moreimportant than before The combination between disease clinical activity and fecal calprotectin has been recommended as a noninvasiveapproach that can help in making decisions on treatment duringCOVID19 pandemic [] Therefore it seems that fecal calprotectincan be considered as an alternative for colonoscopy used for IBD endoscopic activity evaluation during pandemic Fecal calprotectin appears to be associated with IBD histologic activity as well Given thedifficulty in the evaluation of the histologic activity of Crohn's disease[] some studies have been focused on the ulcerative colitis andmany scoring systems have been devised so far Correspondingly thesesystems score the disease's histologic activity based on histologic observationsTherefore for this purpose a biopsy of the intestinal tissue is required which can be prepared by colonoscopy and then sent to thelaboratory In this regard one of these histologic scoring systems isRobert™s score that was used in one of our recent studies where weobserved a significant correlation between the level of fecal calprotectinand the histologic activity of ulcerative colitis which was calculatedbased on the Robert™s scoring system [] Theede et al also used themodified Harpaz Index and performed some interesting studies in thisregard In one of their studies fecal calprotectin was found to be significantly associated with the histologic activity of the ulcerative colitisand it was shown that it could predict histological mucosal healingAUC CI95 “ Sensitivity Specificity andCutoï¬ mgkg [] In another study on patients with endoscopically inactive ulcerative colitis Mayo endoscopic score the researchers showed that patients with ulcerative colitis who were inendoscopic remission but had histologically active disease had higherlevels of fecal calprotectin compared to patients with no histologicallyactive disease versus mgkg P Also despite thehigh specificity the sensitivity of fecal calprotectin in theprediction of score of histological activity was achieved as at mgkg [] In a recent study the Geboes Answer:

Thyroid_Cancer

"SARSCoV2 infection associated respiratory disease COVID19 has evolved into a pandemic but being anew form of virus pathogenesis of disease causation is not fully understood and drugs and vaccinesagainst this virus are still being tested so that no effective drugs or vaccines have been advised byregulatory authority In this context the Ministry of AYUSH Government of India has recommended˜Ayush Kwath™ to improve the immunity and combat the infection Our objective of this literature reviewis to review the role of immunity in pathogenesis of COVID19 and role of Ayush Kwath against the virusand regulation of immunity Current review was conducted using a search of available literature onCOVID19 and immunity Vyadhikshamatwa Ayurveda and COVID19 Rasayana Coronavirus SARSCoV immunomodulatory effects of medicinal plants TulsiHoly BasilOcimum sanctum DalchiniCinnamonCinnamomum zeylanicum SunthiGingerZingiber officinale and MarichBlack PepperPiper nigrum Ayurveda being an ancient science have both medicinal and cultural values and had stimulated our kitchenand ‚uenced what we ate in different seasons and the remedies we used for common ailments Herbssuch as Tulsi Marich Sunthi Dalchini are the most commonly used and easily available drugs in homeThus Ayush Kwath due to its immunemodulatory antiviral antioxidant anti‚ammatory antiplatelet antiatherosclerotic hepatoprotective renoprotective properties seems to be effective inimmunoregulation for controlling viral infections like COVID19 Further preclinical and clinical trialsneed to be done for the evaluation of safety and efficacy of this polyherbal formulation The Authors Published by Elsevier BV on behalf of Institute of Transdisciplinary Health Sciencesand Technology and World Ayurveda Foundation This is an access under the CC BYNCNDlicense httpcreativecommonslicensesbyncnd40 IntroductionCOVID19 also known as severe acute respiratory syndromecorona virus SARSCoV2 is an infectious disease believed to beoriginated from bats and transmitted to human beings [] Being anew form of virus pathogenesis of disease causation is not fullyunderstood and drugs and vaccines against this virus are still beingtested so that no effective drugs or vaccines have been advised byregulatory authority Not only for Coronavirus have many otherviruses also lack preventive vaccines and effective antiviral medications Studies have explored that these viruses can form drugresistant mutants which decrease the existing drug™s efficacy Sothese viruses can be a threat to the mankind for long time [] Corresponding authorEmail shankargautammohpgovnpPeer review under responsibility of Transdisciplinary University BangaloreHigh mortality among immunecompromised and those withsome underlying pathology implies that the factors that improveimmunity can prevent serious manifestations due to COVID19infection [] Many herbal products are found to have immunemodulatory and antiviral property so their discovery can be amilestone in the prevention and control of COVID19 [] In thiscontext the Government of India has recommended to take ˜AyushKwath™ in order to boost the immunity As this is a new formulationthis needs to be validated scientifically We have made an attemptto review the immunepathogenesis of COVID19 and the role ofeach herb over it Immunopathogenesis of COVID19The ˜S™ protein of coronavirus can bind to host cells through theACE2 receptor found in the oral and nasal mucosa [] Other siteswhere ACE2 receptors are found are lungs stomach intestinebladder heart and kidney [] Variable presentation of disease in101016jjaim202008003 The Authors Published by Elsevier BV on behalf of Institute of Transdisciplinary Health Sciences and Technology and World Ayurveda Foundation This isan access under the CC BYNCND license httpcreativecommonslicensesbyncnd40Please cite this as Gautam S Immunity against COVID19 Potential role of Ayush Kwath J Ayurveda Integr Med 101016jjaim202008003 0cS Gautam Journal of Ayurveda and Integrative Medicine xxx xxxx xxxdifferent age groups serious manifestations that are seen morecommonly in immunecompromised old aged and in those withunderlying pathology many asymptomatic cases in pediatric agegroup and presence of lymph ia in the majority of the casesthese factors implies that immunity has a vital role in the pathogenesis of COVID19 [16e8] It is assumed that our immune systemhas lack of memory against such a virus that gave it an edge overhumans []Viruses cause cell destruction mainly in two ways direct cytopathic effects ofthe virus and immune response mediateddestruction [] COVID19 cannot lyse the cells directly as the majorpathway of cell destruction is due to immunemediated destruction[] It has been mentioned that unlike adults less vigorous cellmediated immune response in alveoli of children results in beingasymptomatic in the majority of cases []The pathogenesis can be split into two stages Nonsevere andSevere [] Nonsevere stageThe virus fuses with the host cell membrane and enters insidethe host cell through airway epithelium [] The virus propagates and multiplies inside the host cell and can reach lower airwayand alveoli In adults with good innate cellular and humoral immunity propagation of virus can be limited and viral load reachingalveoli can be reduced thus recovery can take place within 2e3weeks with mild symptoms []Humoral immunity prevents the viruses to enter new cells whilecellmediated immunity targets on eradicating virusinfected cells[] In this stage a strong immune system can be helpful inpreventing the propagation of the virus thus reducing the severityof the disease [] Severe stageOnce the immune system is breached the virus propagates andreaches the lower respiratory tract and alveoli Then the virus canpenetrate alveoli and reaches systemic circulation causing viremia[] The virus binds to multiple ans having ACE2 receptor protein During this stage cellmediated immunity becomes robustand starts releasing various pro‚ammatory cytokines IFNaIFNg IL1B IL6 IL12 IL18 IL33 TNFa etc and chemokinesCCL2 CCL3 CCL5 CXCL8 CXCL9 CXCL10 etc causing damage tomultiple ans known as Cytokine storm [] We may need tosuppress the ‚ammation for improvement during this severestage []Tocilizumab and anti‚ammatory interleukin IL10 are proposed to have a therapeutic role in the reduction of severity and mortality of COVID19[] As increased risk of thromboembolic phenomena is alsofound to be associated with COVID19 prophylactic antithromboticmedications are advised during this stage []IL6 receptor antagonist Ayurveda purview Disease conceptIt seems that most early cases had a history of contact with theoriginal market for seafood but the disease has now advanced to betransmitted through human to human contact [] Thus this disease can be considered as Communicableboth contagious and infectious diseases In Ayurveda epidemics are discussed under theterm of Janapadodhwamsa [ CSVi ] by Charaka and Marakaby Sushruta [ SSSoo ] The symptoms like fever coughbreathing difficulty headache and vomiting resemble with clinicalfeatures of SARS [ SSSoo ] Dalhana in his commentary hasmentioned that symptoms like anosmia cough catarrh will occurafter the entry of contaminated air through the nasal ingwhich is similar to typical clinical features of COVID19 [ SSSoo] Furthermore this disease can be classified as Adidaivika BalaPravritta Vyadhi ABPV Sansargaja Upsargaja and Aupasargic RogaABPV are those diseases arising due to causes that cannot becontrolled by human intelligence Upasargaja Vyadhi are thosefeverlike diseases that manifest due to close contact with diseasedpersons [ SSSoo ] whereas Sansargaja Vyadhi resides withpeople who are cursed by almighty god ie due to ‚uence ofinvisible forcesforces behind human control [ SSSoo ]Aupasargic Vyadhi is defined in two different ways by Sushruta oneas a disease which spreads from one person to another person [SSNi ] and another as ˜¦Upadravasangyah™ ie complications or associated diseases that manifest after primary disease [SSSoo ] Susruta mentions the diseases like Jwara Kusthaskin diseases Shosha tuberculosis Netrabhisyandi conjunctivitis and other Aupasargika roga alike communicable diseasescan be spread through Prasanga intimate relationship GatraSansparsha direct contact Nishwasa breathing or airborneSahabhojana eating together Sahashayana sleeping togethersharing and using of others™ clothes ornaments ointments etc [SSNi ]Agantuja Vyadhi Ì´ diseases of exogenous origin occurs due tophysicalexternal factors like Bhuta Visha Vayu Agni and Praharatrauma etc without any involvement of Vataadi Dosha initiallyhowever in later stage dosha are involved in the disease process[ CSSoo1145] Cakrapaṇidatta clarifies that Bhuta meansVisakṛimi or a virulent anism [ CSSa1121] Krimi may beSahaja natural or Vaikarika pathogenic anisms that may bevisible macroscopic or invisible to the naked eye microscopic[ CSVi ]Thus it is difficult to correlate this disease with specific Ayurveda terminology but while interpreting the disease on the basis ofSamprapti by considering the causative agent and the clinical features like fever Jwara cough Kasa anorexia Aruchi fatigueTandra generalized body ache or myalgia Angamarda andTiredness it can be contemplated as an Agantuka Vyadhi whichlater on due to the involvement of dosha develops to Nija Vyadhi asKaphaVatolvana Hina Pitta Sannipataja Jwara Severe Vata andKapha with mild Pitta [ CSSoo CSChi ] Whiletalking about the pathogenesis of fever in Ayurveda Charakamentioned that when Vataadi dosha either singly or in Sansristatwo dosha or in Sannipataja all three dosha got aggravated thenit enters Amashaya and mixed with Rasa Dhatu causing obstructionof Rasavaha and Swedavaha Srotas resulting in the destruction ofAgni Agni then spreads out from its Sthana to whole over the bodycausing the febrile condition [ CSNi120 CSChi3129]Immunity concept in AyurvedaStrength health lifespan and vital breath are dependent on thecondition of Agni [ CSSoo ] Charaka has mentioned theterm Vyadhikshamatwa and states that during certain conditions ordue to certain factors even unwholesome unhealthy food doesnot produce disease immediately all unwholesome diet are notequally harmful all dosha are not equally powerful all persons arenot capable of resisting diseases [ CSSoo ] This suggeststhat the body™s immune system plays a crucial role in diseasedevelopment The equilibrium state of Dhatu is called Swasthya [CSSoo ] The person who is desirous to be healthy should adopthealthy practices related to diet conduct and activities [ CSSooImmunity can be considered in Ayurveda as] ThusPlease cite this as Gautam S Immunity against COVID19 Potential role of Ayush Kwath J Ayurveda Integr Med 101016jjaim202008003 0cS Gautam Journal of Ayurveda and Integrative Medicine xxx xxxx xxxVyadhikshamatwa and Oja which depends on the condition of AgniDosha and DhatuThere are three factors Aahara Swapna and Brahmacharya dietsound sleep and celibacy that support the life with which the bodywill be endowed with strength complexion and development tilllife span [ CSSoo ] Bala Ì´ StrengthImmunity is of threetypescongenital time affected and acquired Congenital is thatwhich is developed naturally in the body and mind time affected isdue to seasonal variation and age factor and acquired one is produced by the proper application of diet and exercise [ CSSoo ] Thus not only diet but also performing yoga or exercises withproper methods by giving rest in between exercises as Rasayanatherapy will increase acquired strength [ CSSoo1136] Oja isalso called Bala is the essence of all Sapta Dhatu being located inHridaya combines with Rasa and circulates through the Dhamaniand performs Tarpana or Prinanam of the whole body [ SSSoo CS1774] The equilibrium state of Kapha promotesstrength that™s why normal Kapha is called Oja [ CSSoo ]Normal pure blood promotes strength complexion health andlifespan [ CSSoo ] While dealing with Sannipataja JwaraSusruta in Uttarsthana mentioned Abhinyasa Jwara also called asHataujasa Jwara indicating the loss or deranged condition of Oja[ SSUtt ]The word ˜Rasayana Rasa þ ayana™ refers to nutrition and itstransportation in the body for attaining excellent Rasadi Dhatuswhich leads to gain longevity freedom from disorders optimumstrength of physique and sense ans [ CSChi ]Rasayana promotes nutrition by explicitly enriching the nutritionalvalue of Rasa by enhancing Agni ie digestion metabolism andabsorption by Srotashodhana Consequently any medication thatimproves Rasa™s consistency would enhance the health of all bodytissues Role of Ayurveda and traditional medicineEvery society has its own medical system which is deeplyrooted in its culture and guided by its philosophy of life Beingculturally and linguistically diverse countries there developedseveral types of traditional medicines TM based on practices skilltraditional knowledge based on beliefs theories and experiencesindigenous to different cultures Ayurveda Traditional Chinesemedicine TCM Ancient Egyptian medicine Sowa Rigpa etc system of medicine remain the most ancient yet living traditions inSouth East Asia Western Pacific Eastern Mediterranean Africaregion Up to percent of the population in some Asian and African countries depend on TM for primary health care PHC needs[] Still there is a high trend of using many herbs in religious andcultural works therapeutically for common ailments and as spicesfor foods according to occasion specific and seasonal regimes Ayurveda and TM have made a significant contribution to the prevention and alleviation of various communicable and noncommunicable diseases for thousands of years A long history ofusing many herbal remedies and experiences passed from generation to generation has resulted in people relying on herbal remedies and some simple home remedies for common diseases can beused even by illiterate citizens The selfcare an integral part ofPHC with home remedies using various herbs is the most commontreatment for India Nepal Bhutan and China for different flucommon cold fever GI disorders etc Prevention of smallpox inChina has been an epochmaking effort in the period of mankind™spreventive care One observational study found that the prevalencebetween the total number of COVID19 cases per million populationand the grams of spice supply per capita per day is clearly interrelated Most nations with lower spice intake per capita reportedmore COVID19 cases per million population and vice versa []Nevertheless with the invention of drugs many herbal remediesused historically have become modern medicines Few notableexamples include morphine digoxin artemisinin and colchicine Asmany herbs are found to have immunomodulatory role and possessantiviral activity many people are being optimistic over the traditional system of Medicine Ayurveda and TCM have descriptions ofimmunomodulation along with antiviral treatments even targeted to the coronavirus family []The key factor for COVID19 to occur and evolve is the interaction between the virus and an individual™s immune system [] Asmedicinal plants enhance NK cell activity inhibit activated transcription factor ATF2 downregulate Th17related cytokinesincluding transcription factor RORc IL17A and Th2related cytokines including IL5 IL13 and IL6 inhibit GATA3 IL4 IL6 IL1bRt IL17A TNFa expression and increase the secretions of ILit shows that natural products have potentimmunemodulatory and immuneboosting effects that may behelpful during the infection course by increasing innate immuneresponse to infections []INFg etc Ayush KwathConsidering the importance of immunity boosting measures inthe wake of COVID19 outbreak the Ministry of AYUSH Government of India with the interest of health promotion of the massesrecommends ˜Ayush Kwath™ or ˜Ayush Kudineer™ or ˜Ayush Joshanda™which comprises of four medicinal herbs Table [] Theherbs like holy basil cinnamon ginger black pepper are highlyavailable accessible and widely used in the kitchen and areconvenient to educate and train about its use to community healthworkers community and even to all public that they can have costeffective treatment with herbal home remedies This will help topromote immunity and to lower the gatherings at hospitals andpharmacies in this pandemic This type of public health measurewould eventually promote ˜health for all™ with the theme ˜ourhealth in our own hands™ making responsible to each and everypeople by active involvement in their own health instead of relyingon mass distribution of some medicine As people leave theirhomes to earn a living this herbal decoction will ensure broadaccess to health care The WHO SEARO adopted a resolution torevitalize PHC through health systems strengthening to achievehealth for all with the emphasis on health promotion and diseaseprevention [] This Kwath is not just a mechanical mixtureinvented for the COVID19 pandemic but it is a revival of healthtraditionMethod of preparation and useTake all the ingredients in dry form as per standards laid downin Ayurvedic Pharmacopoeia and make coarse powder Make sachets or tea bags each of g of powder or mg tablet of aqueousextract to be consumed like tea or hot drink by dissolving in mlof boiled water once or twice daily Gud JaggeryDraksha Resinsandor Lemon Juice can be added while consuming the formulation TulsiMany invitro animal and human experimental scientificstudies showed that due to presence of eugenol phenolic compoundslinoleic acid etc compounds Tulsi has antimicrobialincluding antibacterial antiviral antimalarial antidiarrhealantioxidantcardioprotectiveimmunemodulatory properties and is thus recommended as a treatmentfor a range of diseases including features like cough fever asthmaanxiety diarrhea gastric cardiac and genitourinary disorders[32e36] Due to its anti‚ammatory and antioxidant properties itanti‚ammatoryhepatoprotectiverenoprotectiveanalgesicantipyreticPlease cite this as Gautam S Immunity against COVID19 Potential role of Ayush Kwath J Ayurveda Integr Med 101016jjaim202008003 0cS Gautam Journal of Ayurveda and Integrative Medicine xxx xxxx xxxTable Contents and properties of Ayush KwathSN NameScientific name Parts used Main chemicalRasaVirya AyurvedicSansthanika KarmaProportion RemarksTulsiOcimumsanctum LinnLeavesconstituentsVolatile oil PhenolAldehyde EugenolAscorbic acid Linoleicacid CaroteneDosha karmaKatu Tikta Ushna KaphavatashamakaPittabardhakaDalchini CinnamomumStem Bark CinnamaldehydezeylanicumBreyncuminaldehydeEugenolKatu TiktaMadhuraUshna KaphavatashamakaPitta vardhakaSunthiZingiberofficinale RoscRhizomeZingiberene Zingiberol KatuUshna KaphavatashamakaVedanahara DeepanaPachana AnulomanaKrimighna HridhyaRaktashodhakaKasahara SwasaharaKshayanashakaMutrala VishaghnaJwaraghna esp useful inVatashlaishmikaVishama and Jirna JwaraDeepana PachanaVajikaranaVataanulomanaYakridutejaka GrahiHriyottejakaOjovardhakaRaktashodhakaShelshmaharaYakshmanashakaMutrajananaDeepana PachanaVrishyaShoolaprashamanaRaktashodhakaHridhyottejakaShothahara KaphaghnaSwasahara JwaraghnaAampachana partsPrabhava Specialaction Krimighna parts partsPrabhava KrimighnaContraindicationsPandu KushthaMutrakriccharaktapitta Grishma andSharada Ritu MarichPiper nigrumLinnFruitPiperine PiperidinePiperettine andChavicineKatuUshna Kaphashamaka Deepana Pachana partYakriduttejakaVatanulomanaKrimighnaHriddhyottejakaKaphaghnaKaphamissarakaJwaraghna espVishamjwarapratibandhakaprotects against toxic chemicalinduced injury enhance the antioxidant enzymes and protect cellular anelles and membranes byclearing damaged free radicals []The compounds such as ursolic acid carnosol rosmarinic acidcirsilineol apigenin eugenol and cirsimaritin present in O sanctumincrease haemoglobin concentration enhance SRBC agglutinin titers decrease cyclooxygenase CoX2 and lipoxygenase LOX5enzymes activity suppress NFkB classical pathway up regulationof IL2 IFNg and TNFa down regulation of IL1b and produce ofSRBC antigenspecific antibodies which represent a major defensemechanism to assess Tcelldependent antibody responses ie Tulsiby enhancing immune response boost the defense mechanismagainst the infection [38e40] Several studies have shown that Tulsiaqueous and methanol extract of leaf and seed oil besidesimproving vital capacity also is an immunemodulator and regulator as it enhances immune response by increasing Thelper andNK cells phagocytic activity and index with the rise in lymphocytecount neutrophil count and antibody titer []In an acute toxicity study it did not produce any hazardoussymptoms or CNS and ANS toxicities or death and did not show anychange in water and food consumption body weight and hematological and biochemical profiles [] DalchiniIt is a potent immune system booster and is used in variousailments like flu indigestion edema cough etc [] Cinnamonbark contains cinnamaldehyde benzaldehyde cuminaldehyde andterpenes [] In one study cinnamon at high dose mgkgshowed immunestimulant activity as it significantly increased thephagocytic index serum immunoglobulin levels and antibody titerand decreased the percentage reductions in neutrophil countCinnamon low dose mgkg increased serum immunoglobulinlevels only This showed that high dose increases both cell mediated and humoral immunity whereas low dose showed effect onlyon humoral immunity [] The studies also suggest that cinnamaldehyde can act as a strong regulator of monocytemacrophagemediated immune responses by inhibition of PI3K PDK1 and NFkBactivation of signaling components In addition to this by theactivation of CD29 and CD43 it blocked cell migration cellecelladhesion induced but not cellfibronectin adhesion and it wasable to suppress both the production of nitric oxide NO and upregulation of surface levels of costimulatory molecules CD69 andCD80 and pattern recognition receptors TLR2 and CR3 []Cinnamon bark decrease systemic levels ofIFNg withoutaltering the levels of IL4 or IL2 inhibit antiCD3 AbstimulatedIFNg and IL4 at the mRNA and secreted protein levels enhance IL protein secretion at the cellular level which helps to decrease celldeath inhibit IL2mRNA expression inhibit antiCD3induced p38JNK ERK12 and STAT4 activation but not IkBa degradation orSTAT6 and ultimately alter the ‚ammatory responses in T cellsThis shows the immunemodulatory effect of cinnamon on cytokine secretion and the involvement of intracellular signaling molecules in activated T cells It also causes a reduction in the subG1Please cite this as Gautam S Immunity against COVID19 Potential role of Ayush Kwath J Ayurveda Integr Med 101016jjaim202008003 0cS Gautam Journal of Ayurveda and Integrative Medicine xxx xxxx xxxphase accompanied by an increased ratio of apoptotic cells tonecrotic cells [] The constituents like cinnamaldehyde cinnamophilin etc are found to be a thromboxane A2 receptor antagonistanticoagulative antiatherosclerotic and thus prevents unnecessary clumping of platelets and atherosclerotic CVD []In a systematic review of its adverse events relatively few selflimiting adverse effects were reported like allergic reactions andgastrointestinal disorders on clinical trials case reports and caseseries The evidence available show that cinnamon is safe for use asspice in daily diets or as a medication [] However its use fortherapeutic reasons in high doses or for prolonged periods cancause some adverse effects and should be observed clinically SunthiAn alcohol extract increases the immunological status of micewith increased phagocytosis by macrophages whereas crudeextract was also shown to increase humoral and cellmediatedimmune responses [] The bioactive compounds of ginger suchas nevirapine bsitosterol 6gingediol germacrene methyl6shogaol 6gingerol alinalool 6shogaol gingerdion zingibereneetc are known to inhibit viral replication among these the mostpotent inhibitors of reverse transcriptase RT enzyme is bsitosterol which is predicted to be used as nonnucleoside reversetranscriptase NNRTIs HIV1 inhibitors [] It is reported thatGinger contains TNFa which is also known as an anti‚uenzacytokine [] The rhizome of Ginger and its main componentslike gingerols shogaols etc inhibit prostaglandin and leukotrienebiosynthesis inhibit cyclooxygenase and lipoxygenase activitiesinhibits the synthesis of pro‚ammatory cytokines such as IL1TNFa and IL8 without any significant effect in IL6 levelsinhibit the excessive production of NO PGE TNFa and IL1beta reduce the elevated expression of NFkB and TNFa downregulate ‚ammatory iNOS and COX2 gene expression inhibitthromboxane synthetase raise levels of prostacyclin without aconcomitant rise in PGE or PGE alpha inhibit platelet aggregation decrease agerelated oxidative stress markers and enhancefibrinolysis [53e58]The concentration of IgM and eosinophil count in nonsmokerswas significantly increased in a comparative study of the effect ofginger extract among male smokers and nonsmokers whereas theconcentration of hemoglobin and lymphocyte count in smokerswas strongly increased This indicates that in nonsmokers gingerresults in a stronger antibody response or humoral immunity thanin smokers []According to Ayurveda it is contraindicated to be used in a fewdiseases Kushtha Pandu Mutrakriccha Raktapitta and in Grishmasummer and Sharada autumn Ritu There are few minor adverseeffects recorded that did not need care such as mild gastrointestinal symptoms sleepiness mild diarrhea during prior few days oftreatment It is also explained that ginger has the ability to induceheartburn and as a gastric irritant with doses above g [] Duringpregnancy ginger did not pose a major risk for side effects oradverse events [] MarichIt has been also found to increase bioavailability thus enhancethe therapeutic efficacy of many drugs vaccines and nutrients andhave immunemodulatory antioxidant antiplatelets antihypertensive antiasthmatic antipyretic analgesic anticarcinogenicanti‚ammatory antidiarrheal antispasmodic anxiolytic antidepressants hepatoprotective antiulcer antithyroids antiapoptotic antimetastatic antimutagenic antibacterial antifungal[62e65] The extract and itsand antiamoebic propertiesconstituents like piperine regulate the balance of the cytokinesproduction of Th1 Th2 Th17 and Treg cells reduce the accumulation of ‚ammatory cells inhibit the expressions of GATA3 IL4IL6 IL1b Rt IL17A and TNFa increase INFg and IL10 secretions in BALF Bronchoalveolar lavage fluid and increasemacrophage activation and T and B cell proliferation []Beside this Marich possess cytotoxic activity suppresses thelevels of total IgE antiOVA IgE antiOVA IgG1 and histaminerelease in serum ameliorates fibrosis and ltration of ‚ammatory cells inhibits the allergic responses inhibitsTh2Th17 responses and mast cells activation inhibits NFkB cFos cAMPresponse elementbinding CREB and activated transcription factor ATF2 suppresses PMAinduced MMP9 expression inhibitsPKCaextracellular signalregulated kinase ERK and reducesNFkBAP1 activation In addition piperine also inhibits the Pglycoprotein Pgp and CYP3A4 functions [67e69] Piper nigrum isfound to have dose dependent antifertility effects on mice [] DiscussionAccording to Ayurveda therapeutics is of two types Swasthasyorjaskarawhich promotes strength immunity in the healthyand Roganutwhich alleviates disorders Both of these groupsperform both of these functions but Rasayana and Vajikarana aremostly used for promotive treatment CSChi [] AyushKwath has both immune promoting and disease alleviating properties which can be achieved by various treatment modalities likeRasayana Satwawajaya Yuktivyapashraya Vyadhi Viparitarthakarichikitsa etcThe Katu and Tikta Rasa Usna Virya and Deepana PachanaYakriduttejaka properties of Ayush Kwath help to improve Agni andSrotosodhana improves microcirculation and tissue perforationthus promotes proper digestion metabolism and absorption andacts as Rasayana for the development of preceding Dhatu andfinally form Oja Oja itself acts as immunity to prevent diseaseImmunity is dependent on the condition of Agni Ayush Kwath withits Agni promoting and Kaphashamaka properties balance Kaphaand with Raktashodhaka Hridhya Krimighna properties purify theblood It is already mentioned that natural Kapha and pure bloodpromote Oja and Bala respectively Krimighna is the Prabhavaspecial action of Tulsi and Sunthi which directly acts againstpathogens The properties like Jwaraghna esp VatashlaishmikaVishama Kasahara Swasahara Kshayanashaka ShoolaprashamanaSwothahara Kaphaghna Hridayaottejaka Yakridutejaka have directrole to alleviate various clinical signs symptoms and complicationsAs this disease is considered as KaphaVatolvana Hina PittaSannipataja Jwara the Kapha Vata Shamaka properties of AyushKwath can play a significant role in balancing the vitiated doshasAfter six days of Jwara Charaka suggests the decoction of Pachanadrugs in the case of Amdosha and Shamaniya drugs in Niramadosha[ CSChi ] This shows that Yuktivyapashraya and Vyadhiviparita chikitsa can be done even after the involvement of Dosha inlater stages Ayush Kwath has potential psychoneuroimmunemechanisms via evidence of a reduction in depression anxietyand stress in controlled trials and shows meaning response as it is aspecific remedy for cough and respiratory problems this shows therole of Satvawajaya Chikitsa in its management []Immunity plays a key role in the pathogenesis of COVID19 bothduring the early nonsevere stage and during the severe stage ofthe disease The earlystage strong immune response may preventthe propagation and spread of viruses inside the body thusreducing the severity of cases and early termination of infectionWhile during later stage strong cellmediated immunity of thebody against the virus itself can be a factor responsible for graveconsequences due to cytokine storm The target during the earlyPlease cite this as Gautam S Immunity against COVID19 Potential role of Ayush Kwath J Ayurveda Integr Med 101016jjaim202008003 0cS Gautam Journal of Ayurveda and Integrative Medicine xxx xxxx xxxlikesteroidsand IL6 receptorstage should be to reduce viral propagation while at a later stageshould be to reduce the ‚ammatory response of the immunesystem Medicinal herbs with immune booster property can be anoption during the early nonsevere stage while herbs with anti‚ammatory and antithrombotic properties can be an optionduring a later or severe stage Cytokine storm that is believed as amajor factor responsible for complications and death of COVID19patients has been found to be reduced with anti‚ammatorydrugsantagonists Anti‚ammatory interleukins IL10 in modern medicine [] Therole of medicinal herbs with anti‚ammatory property on thecytokine storm is still lacking in research Like anti‚ammatoryinterleukins and IL6 receptor antagonist Tocilizumab IL thatare proposed in modern medicine to have a therapeutic role in thereduction of severity and mortality of COVID19 Cinnamon barkthat is found to decrease INFg and IL4 Its antiatheroscleroticanticoagulative and antiplate

cancer231

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: "SARSCoV2 infection associated respiratory disease COVID19 has evolved into a pandemic but being anew form of virus pathogenesis of disease causation is not fully understood and drugs and vaccinesagainst this virus are still being tested so that no effective drugs or vaccines have been advised byregulatory authority In this context the Ministry of AYUSH Government of India has recommended˜Ayush Kwath™ to improve the immunity and combat the infection Our objective of this literature reviewis to review the role of immunity in pathogenesis of COVID19 and role of Ayush Kwath against the virusand regulation of immunity Current review was conducted using a search of available literature onCOVID19 and immunity Vyadhikshamatwa Ayurveda and COVID19 Rasayana Coronavirus SARSCoV immunomodulatory effects of medicinal plants TulsiHoly BasilOcimum sanctum DalchiniCinnamonCinnamomum zeylanicum SunthiGingerZingiber officinale and MarichBlack PepperPiper nigrum Ayurveda being an ancient science have both medicinal and cultural values and had stimulated our kitchenand ‚uenced what we ate in different seasons and the remedies we used for common ailments Herbssuch as Tulsi Marich Sunthi Dalchini are the most commonly used and easily available drugs in homeThus Ayush Kwath due to its immunemodulatory antiviral antioxidant anti‚ammatory antiplatelet antiatherosclerotic hepatoprotective renoprotective properties seems to be effective inimmunoregulation for controlling viral infections like COVID19 Further preclinical and clinical trialsneed to be done for the evaluation of safety and efficacy of this polyherbal formulation The Authors Published by Elsevier BV on behalf of Institute of Transdisciplinary Health Sciencesand Technology and World Ayurveda Foundation This is an access under the CC BYNCNDlicense httpcreativecommonslicensesbyncnd40 IntroductionCOVID19 also known as severe acute respiratory syndromecorona virus SARSCoV2 is an infectious disease believed to beoriginated from bats and transmitted to human beings [] Being anew form of virus pathogenesis of disease causation is not fullyunderstood and drugs and vaccines against this virus are still beingtested so that no effective drugs or vaccines have been advised byregulatory authority Not only for Coronavirus have many otherviruses also lack preventive vaccines and effective antiviral medications Studies have explored that these viruses can form drugresistant mutants which decrease the existing drug™s efficacy Sothese viruses can be a threat to the mankind for long time [] Corresponding authorEmail shankargautammohpgovnpPeer review under responsibility of Transdisciplinary University BangaloreHigh mortality among immunecompromised and those withsome underlying pathology implies that the factors that improveimmunity can prevent serious manifestations due to COVID19infection [] Many herbal products are found to have immunemodulatory and antiviral property so their discovery can be amilestone in the prevention and control of COVID19 [] In thiscontext the Government of India has recommended to take ˜AyushKwath™ in order to boost the immunity As this is a new formulationthis needs to be validated scientifically We have made an attemptto review the immunepathogenesis of COVID19 and the role ofeach herb over it Immunopathogenesis of COVID19The ˜S™ protein of coronavirus can bind to host cells through theACE2 receptor found in the oral and nasal mucosa [] Other siteswhere ACE2 receptors are found are lungs stomach intestinebladder heart and kidney [] Variable presentation of disease in101016jjaim202008003 The Authors Published by Elsevier BV on behalf of Institute of Transdisciplinary Health Sciences and Technology and World Ayurveda Foundation This isan access under the CC BYNCND license httpcreativecommonslicensesbyncnd40Please cite this as Gautam S Immunity against COVID19 Potential role of Ayush Kwath J Ayurveda Integr Med 101016jjaim202008003 0cS Gautam Journal of Ayurveda and Integrative Medicine xxx xxxx xxxdifferent age groups serious manifestations that are seen morecommonly in immunecompromised old aged and in those withunderlying pathology many asymptomatic cases in pediatric agegroup and presence of lymph ia in the majority of the casesthese factors implies that immunity has a vital role in the pathogenesis of COVID19 [16e8] It is assumed that our immune systemhas lack of memory against such a virus that gave it an edge overhumans []Viruses cause cell destruction mainly in two ways direct cytopathic effects ofthe virus and immune response mediateddestruction [] COVID19 cannot lyse the cells directly as the majorpathway of cell destruction is due to immunemediated destruction[] It has been mentioned that unlike adults less vigorous cellmediated immune response in alveoli of children results in beingasymptomatic in the majority of cases []The pathogenesis can be split into two stages Nonsevere andSevere [] Nonsevere stageThe virus fuses with the host cell membrane and enters insidethe host cell through airway epithelium [] The virus propagates and multiplies inside the host cell and can reach lower airwayand alveoli In adults with good innate cellular and humoral immunity propagation of virus can be limited and viral load reachingalveoli can be reduced thus recovery can take place within 2e3weeks with mild symptoms []Humoral immunity prevents the viruses to enter new cells whilecellmediated immunity targets on eradicating virusinfected cells[] In this stage a strong immune system can be helpful inpreventing the propagation of the virus thus reducing the severityof the disease [] Severe stageOnce the immune system is breached the virus propagates andreaches the lower respiratory tract and alveoli Then the virus canpenetrate alveoli and reaches systemic circulation causing viremia[] The virus binds to multiple ans having ACE2 receptor protein During this stage cellmediated immunity becomes robustand starts releasing various pro‚ammatory cytokines IFNaIFNg IL1B IL6 IL12 IL18 IL33 TNFa etc and chemokinesCCL2 CCL3 CCL5 CXCL8 CXCL9 CXCL10 etc causing damage tomultiple ans known as Cytokine storm [] We may need tosuppress the ‚ammation for improvement during this severestage []Tocilizumab and anti‚ammatory interleukin IL10 are proposed to have a therapeutic role in the reduction of severity and mortality of COVID19[] As increased risk of thromboembolic phenomena is alsofound to be associated with COVID19 prophylactic antithromboticmedications are advised during this stage []IL6 receptor antagonist Ayurveda purview Disease conceptIt seems that most early cases had a history of contact with theoriginal market for seafood but the disease has now advanced to betransmitted through human to human contact [] Thus this disease can be considered as Communicableboth contagious and infectious diseases In Ayurveda epidemics are discussed under theterm of Janapadodhwamsa [ CSVi ] by Charaka and Marakaby Sushruta [ SSSoo ] The symptoms like fever coughbreathing difficulty headache and vomiting resemble with clinicalfeatures of SARS [ SSSoo ] Dalhana in his commentary hasmentioned that symptoms like anosmia cough catarrh will occurafter the entry of contaminated air through the nasal ingwhich is similar to typical clinical features of COVID19 [ SSSoo] Furthermore this disease can be classified as Adidaivika BalaPravritta Vyadhi ABPV Sansargaja Upsargaja and Aupasargic RogaABPV are those diseases arising due to causes that cannot becontrolled by human intelligence Upasargaja Vyadhi are thosefeverlike diseases that manifest due to close contact with diseasedpersons [ SSSoo ] whereas Sansargaja Vyadhi resides withpeople who are cursed by almighty god ie due to ‚uence ofinvisible forcesforces behind human control [ SSSoo ]Aupasargic Vyadhi is defined in two different ways by Sushruta oneas a disease which spreads from one person to another person [SSNi ] and another as ˜¦Upadravasangyah™ ie complications or associated diseases that manifest after primary disease [SSSoo ] Susruta mentions the diseases like Jwara Kusthaskin diseases Shosha tuberculosis Netrabhisyandi conjunctivitis and other Aupasargika roga alike communicable diseasescan be spread through Prasanga intimate relationship GatraSansparsha direct contact Nishwasa breathing or airborneSahabhojana eating together Sahashayana sleeping togethersharing and using of others™ clothes ornaments ointments etc [SSNi ]Agantuja Vyadhi Ì´ diseases of exogenous origin occurs due tophysicalexternal factors like Bhuta Visha Vayu Agni and Praharatrauma etc without any involvement of Vataadi Dosha initiallyhowever in later stage dosha are involved in the disease process[ CSSoo1145] Cakrapaṇidatta clarifies that Bhuta meansVisakṛimi or a virulent anism [ CSSa1121] Krimi may beSahaja natural or Vaikarika pathogenic anisms that may bevisible macroscopic or invisible to the naked eye microscopic[ CSVi ]Thus it is difficult to correlate this disease with specific Ayurveda terminology but while interpreting the disease on the basis ofSamprapti by considering the causative agent and the clinical features like fever Jwara cough Kasa anorexia Aruchi fatigueTandra generalized body ache or myalgia Angamarda andTiredness it can be contemplated as an Agantuka Vyadhi whichlater on due to the involvement of dosha develops to Nija Vyadhi asKaphaVatolvana Hina Pitta Sannipataja Jwara Severe Vata andKapha with mild Pitta [ CSSoo CSChi ] Whiletalking about the pathogenesis of fever in Ayurveda Charakamentioned that when Vataadi dosha either singly or in Sansristatwo dosha or in Sannipataja all three dosha got aggravated thenit enters Amashaya and mixed with Rasa Dhatu causing obstructionof Rasavaha and Swedavaha Srotas resulting in the destruction ofAgni Agni then spreads out from its Sthana to whole over the bodycausing the febrile condition [ CSNi120 CSChi3129]Immunity concept in AyurvedaStrength health lifespan and vital breath are dependent on thecondition of Agni [ CSSoo ] Charaka has mentioned theterm Vyadhikshamatwa and states that during certain conditions ordue to certain factors even unwholesome unhealthy food doesnot produce disease immediately all unwholesome diet are notequally harmful all dosha are not equally powerful all persons arenot capable of resisting diseases [ CSSoo ] This suggeststhat the body™s immune system plays a crucial role in diseasedevelopment The equilibrium state of Dhatu is called Swasthya [CSSoo ] The person who is desirous to be healthy should adopthealthy practices related to diet conduct and activities [ CSSooImmunity can be considered in Ayurveda as] ThusPlease cite this as Gautam S Immunity against COVID19 Potential role of Ayush Kwath J Ayurveda Integr Med 101016jjaim202008003 0cS Gautam Journal of Ayurveda and Integrative Medicine xxx xxxx xxxVyadhikshamatwa and Oja which depends on the condition of AgniDosha and DhatuThere are three factors Aahara Swapna and Brahmacharya dietsound sleep and celibacy that support the life with which the bodywill be endowed with strength complexion and development tilllife span [ CSSoo ] Bala Ì´ StrengthImmunity is of threetypescongenital time affected and acquired Congenital is thatwhich is developed naturally in the body and mind time affected isdue to seasonal variation and age factor and acquired one is produced by the proper application of diet and exercise [ CSSoo ] Thus not only diet but also performing yoga or exercises withproper methods by giving rest in between exercises as Rasayanatherapy will increase acquired strength [ CSSoo1136] Oja isalso called Bala is the essence of all Sapta Dhatu being located inHridaya combines with Rasa and circulates through the Dhamaniand performs Tarpana or Prinanam of the whole body [ SSSoo CS1774] The equilibrium state of Kapha promotesstrength that™s why normal Kapha is called Oja [ CSSoo ]Normal pure blood promotes strength complexion health andlifespan [ CSSoo ] While dealing with Sannipataja JwaraSusruta in Uttarsthana mentioned Abhinyasa Jwara also called asHataujasa Jwara indicating the loss or deranged condition of Oja[ SSUtt ]The word ˜Rasayana Rasa þ ayana™ refers to nutrition and itstransportation in the body for attaining excellent Rasadi Dhatuswhich leads to gain longevity freedom from disorders optimumstrength of physique and sense ans [ CSChi ]Rasayana promotes nutrition by explicitly enriching the nutritionalvalue of Rasa by enhancing Agni ie digestion metabolism andabsorption by Srotashodhana Consequently any medication thatimproves Rasa™s consistency would enhance the health of all bodytissues Role of Ayurveda and traditional medicineEvery society has its own medical system which is deeplyrooted in its culture and guided by its philosophy of life Beingculturally and linguistically diverse countries there developedseveral types of traditional medicines TM based on practices skilltraditional knowledge based on beliefs theories and experiencesindigenous to different cultures Ayurveda Traditional Chinesemedicine TCM Ancient Egyptian medicine Sowa Rigpa etc system of medicine remain the most ancient yet living traditions inSouth East Asia Western Pacific Eastern Mediterranean Africaregion Up to percent of the population in some Asian and African countries depend on TM for primary health care PHC needs[] Still there is a high trend of using many herbs in religious andcultural works therapeutically for common ailments and as spicesfor foods according to occasion specific and seasonal regimes Ayurveda and TM have made a significant contribution to the prevention and alleviation of various communicable and noncommunicable diseases for thousands of years A long history ofusing many herbal remedies and experiences passed from generation to generation has resulted in people relying on herbal remedies and some simple home remedies for common diseases can beused even by illiterate citizens The selfcare an integral part ofPHC with home remedies using various herbs is the most commontreatment for India Nepal Bhutan and China for different flucommon cold fever GI disorders etc Prevention of smallpox inChina has been an epochmaking effort in the period of mankind™spreventive care One observational study found that the prevalencebetween the total number of COVID19 cases per million populationand the grams of spice supply per capita per day is clearly interrelated Most nations with lower spice intake per capita reportedmore COVID19 cases per million population and vice versa []Nevertheless with the invention of drugs many herbal remediesused historically have become modern medicines Few notableexamples include morphine digoxin artemisinin and colchicine Asmany herbs are found to have immunomodulatory role and possessantiviral activity many people are being optimistic over the traditional system of Medicine Ayurveda and TCM have descriptions ofimmunomodulation along with antiviral treatments even targeted to the coronavirus family []The key factor for COVID19 to occur and evolve is the interaction between the virus and an individual™s immune system [] Asmedicinal plants enhance NK cell activity inhibit activated transcription factor ATF2 downregulate Th17related cytokinesincluding transcription factor RORc IL17A and Th2related cytokines including IL5 IL13 and IL6 inhibit GATA3 IL4 IL6 IL1bRt IL17A TNFa expression and increase the secretions of ILit shows that natural products have potentimmunemodulatory and immuneboosting effects that may behelpful during the infection course by increasing innate immuneresponse to infections []INFg etc Ayush KwathConsidering the importance of immunity boosting measures inthe wake of COVID19 outbreak the Ministry of AYUSH Government of India with the interest of health promotion of the massesrecommends ˜Ayush Kwath™ or ˜Ayush Kudineer™ or ˜Ayush Joshanda™which comprises of four medicinal herbs Table [] Theherbs like holy basil cinnamon ginger black pepper are highlyavailable accessible and widely used in the kitchen and areconvenient to educate and train about its use to community healthworkers community and even to all public that they can have costeffective treatment with herbal home remedies This will help topromote immunity and to lower the gatherings at hospitals andpharmacies in this pandemic This type of public health measurewould eventually promote ˜health for all™ with the theme ˜ourhealth in our own hands™ making responsible to each and everypeople by active involvement in their own health instead of relyingon mass distribution of some medicine As people leave theirhomes to earn a living this herbal decoction will ensure broadaccess to health care The WHO SEARO adopted a resolution torevitalize PHC through health systems strengthening to achievehealth for all with the emphasis on health promotion and diseaseprevention [] This Kwath is not just a mechanical mixtureinvented for the COVID19 pandemic but it is a revival of healthtraditionMethod of preparation and useTake all the ingredients in dry form as per standards laid downin Ayurvedic Pharmacopoeia and make coarse powder Make sachets or tea bags each of g of powder or mg tablet of aqueousextract to be consumed like tea or hot drink by dissolving in mlof boiled water once or twice daily Gud JaggeryDraksha Resinsandor Lemon Juice can be added while consuming the formulation TulsiMany invitro animal and human experimental scientificstudies showed that due to presence of eugenol phenolic compoundslinoleic acid etc compounds Tulsi has antimicrobialincluding antibacterial antiviral antimalarial antidiarrhealantioxidantcardioprotectiveimmunemodulatory properties and is thus recommended as a treatmentfor a range of diseases including features like cough fever asthmaanxiety diarrhea gastric cardiac and genitourinary disorders[32e36] Due to its anti‚ammatory and antioxidant properties itanti‚ammatoryhepatoprotectiverenoprotectiveanalgesicantipyreticPlease cite this as Gautam S Immunity against COVID19 Potential role of Ayush Kwath J Ayurveda Integr Med 101016jjaim202008003 0cS Gautam Journal of Ayurveda and Integrative Medicine xxx xxxx xxxTable Contents and properties of Ayush KwathSN NameScientific name Parts used Main chemicalRasaVirya AyurvedicSansthanika KarmaProportion RemarksTulsiOcimumsanctum LinnLeavesconstituentsVolatile oil PhenolAldehyde EugenolAscorbic acid Linoleicacid CaroteneDosha karmaKatu Tikta Ushna KaphavatashamakaPittabardhakaDalchini CinnamomumStem Bark CinnamaldehydezeylanicumBreyncuminaldehydeEugenolKatu TiktaMadhuraUshna KaphavatashamakaPitta vardhakaSunthiZingiberofficinale RoscRhizomeZingiberene Zingiberol KatuUshna KaphavatashamakaVedanahara DeepanaPachana AnulomanaKrimighna HridhyaRaktashodhakaKasahara SwasaharaKshayanashakaMutrala VishaghnaJwaraghna esp useful inVatashlaishmikaVishama and Jirna JwaraDeepana PachanaVajikaranaVataanulomanaYakridutejaka GrahiHriyottejakaOjovardhakaRaktashodhakaShelshmaharaYakshmanashakaMutrajananaDeepana PachanaVrishyaShoolaprashamanaRaktashodhakaHridhyottejakaShothahara KaphaghnaSwasahara JwaraghnaAampachana partsPrabhava Specialaction Krimighna parts partsPrabhava KrimighnaContraindicationsPandu KushthaMutrakriccharaktapitta Grishma andSharada Ritu MarichPiper nigrumLinnFruitPiperine PiperidinePiperettine andChavicineKatuUshna Kaphashamaka Deepana Pachana partYakriduttejakaVatanulomanaKrimighnaHriddhyottejakaKaphaghnaKaphamissarakaJwaraghna espVishamjwarapratibandhakaprotects against toxic chemicalinduced injury enhance the antioxidant enzymes and protect cellular anelles and membranes byclearing damaged free radicals []The compounds such as ursolic acid carnosol rosmarinic acidcirsilineol apigenin eugenol and cirsimaritin present in O sanctumincrease haemoglobin concentration enhance SRBC agglutinin titers decrease cyclooxygenase CoX2 and lipoxygenase LOX5enzymes activity suppress NFkB classical pathway up regulationof IL2 IFNg and TNFa down regulation of IL1b and produce ofSRBC antigenspecific antibodies which represent a major defensemechanism to assess Tcelldependent antibody responses ie Tulsiby enhancing immune response boost the defense mechanismagainst the infection [38e40] Several studies have shown that Tulsiaqueous and methanol extract of leaf and seed oil besidesimproving vital capacity also is an immunemodulator and regulator as it enhances immune response by increasing Thelper andNK cells phagocytic activity and index with the rise in lymphocytecount neutrophil count and antibody titer []In an acute toxicity study it did not produce any hazardoussymptoms or CNS and ANS toxicities or death and did not show anychange in water and food consumption body weight and hematological and biochemical profiles [] DalchiniIt is a potent immune system booster and is used in variousailments like flu indigestion edema cough etc [] Cinnamonbark contains cinnamaldehyde benzaldehyde cuminaldehyde andterpenes [] In one study cinnamon at high dose mgkgshowed immunestimulant activity as it significantly increased thephagocytic index serum immunoglobulin levels and antibody titerand decreased the percentage reductions in neutrophil countCinnamon low dose mgkg increased serum immunoglobulinlevels only This showed that high dose increases both cell mediated and humoral immunity whereas low dose showed effect onlyon humoral immunity [] The studies also suggest that cinnamaldehyde can act as a strong regulator of monocytemacrophagemediated immune responses by inhibition of PI3K PDK1 and NFkBactivation of signaling components In addition to this by theactivation of CD29 and CD43 it blocked cell migration cellecelladhesion induced but not cellfibronectin adhesion and it wasable to suppress both the production of nitric oxide NO and upregulation of surface levels of costimulatory molecules CD69 andCD80 and pattern recognition receptors TLR2 and CR3 []Cinnamon bark decrease systemic levels ofIFNg withoutaltering the levels of IL4 or IL2 inhibit antiCD3 AbstimulatedIFNg and IL4 at the mRNA and secreted protein levels enhance IL protein secretion at the cellular level which helps to decrease celldeath inhibit IL2mRNA expression inhibit antiCD3induced p38JNK ERK12 and STAT4 activation but not IkBa degradation orSTAT6 and ultimately alter the ‚ammatory responses in T cellsThis shows the immunemodulatory effect of cinnamon on cytokine secretion and the involvement of intracellular signaling molecules in activated T cells It also causes a reduction in the subG1Please cite this as Gautam S Immunity against COVID19 Potential role of Ayush Kwath J Ayurveda Integr Med 101016jjaim202008003 0cS Gautam Journal of Ayurveda and Integrative Medicine xxx xxxx xxxphase accompanied by an increased ratio of apoptotic cells tonecrotic cells [] The constituents like cinnamaldehyde cinnamophilin etc are found to be a thromboxane A2 receptor antagonistanticoagulative antiatherosclerotic and thus prevents unnecessary clumping of platelets and atherosclerotic CVD []In a systematic review of its adverse events relatively few selflimiting adverse effects were reported like allergic reactions andgastrointestinal disorders on clinical trials case reports and caseseries The evidence available show that cinnamon is safe for use asspice in daily diets or as a medication [] However its use fortherapeutic reasons in high doses or for prolonged periods cancause some adverse effects and should be observed clinically SunthiAn alcohol extract increases the immunological status of micewith increased phagocytosis by macrophages whereas crudeextract was also shown to increase humoral and cellmediatedimmune responses [] The bioactive compounds of ginger suchas nevirapine bsitosterol 6gingediol germacrene methyl6shogaol 6gingerol alinalool 6shogaol gingerdion zingibereneetc are known to inhibit viral replication among these the mostpotent inhibitors of reverse transcriptase RT enzyme is bsitosterol which is predicted to be used as nonnucleoside reversetranscriptase NNRTIs HIV1 inhibitors [] It is reported thatGinger contains TNFa which is also known as an anti‚uenzacytokine [] The rhizome of Ginger and its main componentslike gingerols shogaols etc inhibit prostaglandin and leukotrienebiosynthesis inhibit cyclooxygenase and lipoxygenase activitiesinhibits the synthesis of pro‚ammatory cytokines such as IL1TNFa and IL8 without any significant effect in IL6 levelsinhibit the excessive production of NO PGE TNFa and IL1beta reduce the elevated expression of NFkB and TNFa downregulate ‚ammatory iNOS and COX2 gene expression inhibitthromboxane synthetase raise levels of prostacyclin without aconcomitant rise in PGE or PGE alpha inhibit platelet aggregation decrease agerelated oxidative stress markers and enhancefibrinolysis [53e58]The concentration of IgM and eosinophil count in nonsmokerswas significantly increased in a comparative study of the effect ofginger extract among male smokers and nonsmokers whereas theconcentration of hemoglobin and lymphocyte count in smokerswas strongly increased This indicates that in nonsmokers gingerresults in a stronger antibody response or humoral immunity thanin smokers []According to Ayurveda it is contraindicated to be used in a fewdiseases Kushtha Pandu Mutrakriccha Raktapitta and in Grishmasummer and Sharada autumn Ritu There are few minor adverseeffects recorded that did not need care such as mild gastrointestinal symptoms sleepiness mild diarrhea during prior few days oftreatment It is also explained that ginger has the ability to induceheartburn and as a gastric irritant with doses above g [] Duringpregnancy ginger did not pose a major risk for side effects oradverse events [] MarichIt has been also found to increase bioavailability thus enhancethe therapeutic efficacy of many drugs vaccines and nutrients andhave immunemodulatory antioxidant antiplatelets antihypertensive antiasthmatic antipyretic analgesic anticarcinogenicanti‚ammatory antidiarrheal antispasmodic anxiolytic antidepressants hepatoprotective antiulcer antithyroids antiapoptotic antimetastatic antimutagenic antibacterial antifungal[62e65] The extract and itsand antiamoebic propertiesconstituents like piperine regulate the balance of the cytokinesproduction of Th1 Th2 Th17 and Treg cells reduce the accumulation of ‚ammatory cells inhibit the expressions of GATA3 IL4IL6 IL1b Rt IL17A and TNFa increase INFg and IL10 secretions in BALF Bronchoalveolar lavage fluid and increasemacrophage activation and T and B cell proliferation []Beside this Marich possess cytotoxic activity suppresses thelevels of total IgE antiOVA IgE antiOVA IgG1 and histaminerelease in serum ameliorates fibrosis and ltration of ‚ammatory cells inhibits the allergic responses inhibitsTh2Th17 responses and mast cells activation inhibits NFkB cFos cAMPresponse elementbinding CREB and activated transcription factor ATF2 suppresses PMAinduced MMP9 expression inhibitsPKCaextracellular signalregulated kinase ERK and reducesNFkBAP1 activation In addition piperine also inhibits the Pglycoprotein Pgp and CYP3A4 functions [67e69] Piper nigrum isfound to have dose dependent antifertility effects on mice [] DiscussionAccording to Ayurveda therapeutics is of two types Swasthasyorjaskarawhich promotes strength immunity in the healthyand Roganutwhich alleviates disorders Both of these groupsperform both of these functions but Rasayana and Vajikarana aremostly used for promotive treatment CSChi [] AyushKwath has both immune promoting and disease alleviating properties which can be achieved by various treatment modalities likeRasayana Satwawajaya Yuktivyapashraya Vyadhi Viparitarthakarichikitsa etcThe Katu and Tikta Rasa Usna Virya and Deepana PachanaYakriduttejaka properties of Ayush Kwath help to improve Agni andSrotosodhana improves microcirculation and tissue perforationthus promotes proper digestion metabolism and absorption andacts as Rasayana for the development of preceding Dhatu andfinally form Oja Oja itself acts as immunity to prevent diseaseImmunity is dependent on the condition of Agni Ayush Kwath withits Agni promoting and Kaphashamaka properties balance Kaphaand with Raktashodhaka Hridhya Krimighna properties purify theblood It is already mentioned that natural Kapha and pure bloodpromote Oja and Bala respectively Krimighna is the Prabhavaspecial action of Tulsi and Sunthi which directly acts againstpathogens The properties like Jwaraghna esp VatashlaishmikaVishama Kasahara Swasahara Kshayanashaka ShoolaprashamanaSwothahara Kaphaghna Hridayaottejaka Yakridutejaka have directrole to alleviate various clinical signs symptoms and complicationsAs this disease is considered as KaphaVatolvana Hina PittaSannipataja Jwara the Kapha Vata Shamaka properties of AyushKwath can play a significant role in balancing the vitiated doshasAfter six days of Jwara Charaka suggests the decoction of Pachanadrugs in the case of Amdosha and Shamaniya drugs in Niramadosha[ CSChi ] This shows that Yuktivyapashraya and Vyadhiviparita chikitsa can be done even after the involvement of Dosha inlater stages Ayush Kwath has potential psychoneuroimmunemechanisms via evidence of a reduction in depression anxietyand stress in controlled trials and shows meaning response as it is aspecific remedy for cough and respiratory problems this shows therole of Satvawajaya Chikitsa in its management []Immunity plays a key role in the pathogenesis of COVID19 bothduring the early nonsevere stage and during the severe stage ofthe disease The earlystage strong immune response may preventthe propagation and spread of viruses inside the body thusreducing the severity of cases and early termination of infectionWhile during later stage strong cellmediated immunity of thebody against the virus itself can be a factor responsible for graveconsequences due to cytokine storm The target during the earlyPlease cite this as Gautam S Immunity against COVID19 Potential role of Ayush Kwath J Ayurveda Integr Med 101016jjaim202008003 0cS Gautam Journal of Ayurveda and Integrative Medicine xxx xxxx xxxlikesteroidsand IL6 receptorstage should be to reduce viral propagation while at a later stageshould be to reduce the ‚ammatory response of the immunesystem Medicinal herbs with immune booster property can be anoption during the early nonsevere stage while herbs with anti‚ammatory and antithrombotic properties can be an optionduring a later or severe stage Cytokine storm that is believed as amajor factor responsible for complications and death of COVID19patients has been found to be reduced with anti‚ammatorydrugsantagonists Anti‚ammatory interleukins IL10 in modern medicine [] Therole of medicinal herbs with anti‚ammatory property on thecytokine storm is still lacking in research Like anti‚ammatoryinterleukins and IL6 receptor antagonist Tocilizumab IL thatare proposed in modern medicine to have a therapeutic role in thereduction of severity and mortality of COVID19 Cinnamon barkthat is found to decrease INFg and IL4 Its antiatheroscleroticanticoagulative and antiplate Answer:

Thyroid_Cancer

" The advent of new cancer therapies alongside expected growth and ageing of the population better survival rates and associated costs of care is uncovering aneed to more clearly define and integrate supportive care services across the whole spectrum of the disease The current focus of cancer care is on initialdiagnosis and treatment and end of life care The Multinational Association of Supportive Care in Cancer defines supportive care as ˜the prevention andmanagement of the adverse effects of cancer and its treatment™ This encompasses the entire cancer journey and necessitates involvement and integration ofmost clinical specialties Optimal supportive care can assist in accurate diagnosis and management and ultimately improve outcomes A national strategy toimplement supportive care is needed to acknowledge evolving oncology practice changing disease patterns and the changing patient demographic“ The Royal College of Radiologists Published by Elsevier Ltd All rights reservedKey words Beyond cancer chronic cancer definition living with supportive careStatement of Search Strategies UsedA series of searches were constructed and carried out viaPubMed EMBASE and MEDLINE This generally consisted ofusing phrase searching due to the specificity of the subjectOnce concepts were established the authors used Booleanoperators to combine the concepts together and retrieve themost relevant papers Once a set of results were retrieved theauthors scanned each of the s using and titlefields to identify key papers Fulltext access to papers weresourced via the Christie Library and Knowledge ServiceIntroductionSupportive Care Makes Excellent Cancer Care PossibleMultinational Association of Supportive Care in CancerwwwmasccAdvances in diagnosis surgery radiotherapy and newdrugs have led to improvements in cancer survival PeopleAuthor for correspondence R Berman The Christie NHS FoundationTrust Wilmslow Road Manchester M20 4BX UK Tel ¾447710509402Email address RichardBermanchristienhsuk R Bermannow live nearly six times longer after their cancer diagnosisthan was the case years ago [] Half of people diagnosedwith cancer in England and Wales survive their disease for years or more [] Currently in England around million people are living with a diagnosis of cancer and thisnumber is increasing by over a year The total figure is setrise to over million by []Many more cancer patients are being treated closer todeath with novel less toxic high efficacy anticancer therapeutic agents developing with increasing pace within thelast decade The advent of molecular targeted agents forexample has brought new benefits as well as challenges tomodern cancer therapy potentially blurring the distinctionbetween active and palliative interventions []Yet despite this significant progress a large proportion ofpatients with cancer still experience morbidity and symptoms resulting from the cancer andor its treatment []Increases in cancer incidence [] emergency care hospitalisations [] earlier intensive care unit admissions [] andtreatment costs [] have all added to the global burden ofcancer care The disease is becoming a major economicexpenditure for all developed countries [] In the UK andin the USA cancer care costs are substantial and expected to101016jclon20200702009366555“ The Royal College of Radiologists Published by Elsevier Ltd All rights reservedPlease cite this as Berman R Supportive Care An Indispensable Component of Modern Oncology Clinical Oncology 101016jclon202007020 0cR Berman Clinical Oncology xxx xxxx xxxrise significantly in the future due to growth and aging ofthe population and improvements in survival as well astrends in treatment patterns and costs of care followingcancer diagnosis []cancerandcancerManagingtreatmentrelatedmorbidity is therefore a significant public health andeconomic challenge The coronavirus pandemic has deepened this challenge with many cancer outpatient visitsbeing replaced by telephone consultations and deferral ofsome routine therapy tests and procedures This has placedadditional pressures on an already fragile and vulnerablepopulation [] Patients and carers are experiencing moreuncertainty and anxiety associated with COVID19 A recentstudy found that although patients continue to feel wellsupported by their healthcare teams they have concernsabout the longerterm impact of changes to aspects of theirtreatment Patients and carers are no longer able to accessother support services in the way that they had previouslysuch as hospices and peer support groups []There is a growing body of evidence that timely access tosupportive treatments can lead to improvements in qualityof life and survival as well as benefitting the health economy [15e17] The development of a broad multiprofessionalbasis for the study and expansion of supportive carethrough the Multinational Association of Supportive Care inCancer MASCC has been an important step in fostering thegrowth of an evidence base [] MASCC's success has undoubtedly been underpinned by successful integration ofoncological and nononcological specialties []However variations in the definition of supportive careallocation of resources and a lack of clarity on who shouldlead onprovide services means that a clinical model forsupportive care in cancer does not yet exist [] Most specialties whilst they overlap other specialties are based on acore of knowledge or skill that is specific to that specialty[] Supportive care is currently provided by a patchwork ofdifferent medical specialties and is unique because it traverses the entire spectrum of the disease Figure fromdiagnosis through to survivorship or end of life care Theneed for ˜supportive oncology™ to become a specialty in itsown right is borne out not just by the progress in its development in the UK and abroad but by the unmet supportivecare need [] amplified by the rising incidence of cancerworldwide with many patients living longer with incurableillness because of more effective cancer treatments [] Asignificant next step would be to produce an evidencebasednational strategy for supportive care implemented throughappointment of supportive care lead clinicians within eachUK cancer centre This alongside support from the medicalRoyal Colleges and NHS England would be fundamental indeveloping a sustainable clinical modelPerhaps working as a distinct branch of oncology ˜specialists™ in supportive care medicine should have the skillsand resources to manage a broad range of effects associatedwith longterm cancers and cancer survival This paperexplores areas that are showing promise in this development and identifies key next steps needed to recognisesupportive care as an indispensable component of modernoncologyDefinition of Supportive CareThe Inuit may or may not have words for snow butsupportive care seems to have that number of definitions orconnotations [] Supportive care has been used as aeuphemism for palliative care and ˜early palliative care™[] and research suggests that a change in name frompalliative care to supportive care results in more and earlierreferrals to hospitalbased services [] Palliative care is anintegral component of supportive care but supportive careis much more than palliative care or even ˜early palliativecare™MASCC defines supportive care as ˜the prevention andmanagement of the adverse effects of cancer and its treatment This includes management of physical and psychological symptoms and side effects across the continuum ofthe cancer experience from diagnosis through treatment toposttreatment care Enhancing rehabilitation secondarycancer prevention survivorship and endoflife care areintegral to supportive care™ []Strategy for Implementation of SupportiveCare Within Cancer CareThe potential benefits of supportive care includedecreased morbidity improved quality of life and potentially decreased mortality ie secondary to optimal cancertreatment the potential benefits for healthcare servicesinclude decreased utilisation of healthcare resources andimproved treatment outcomes [] Indeed supportivecare offers patients more than many ˜palliative™ oncologicaltreatments and should be considered an essential not justan optional extraCurrently many cancer centres in the UK have supportivecare services either as a result of NHS England's EnhancedSupportive Care ESC Programme discussed below andFig The supportive care umbrellaPlease cite this as Berman R Supportive Care An Indispensable Component of Modern Oncology Clinical Oncology 101016jclon202007020 0cR Berman Clinical Oncology xxx xxxx xxxrelated Commissioning for Quality and Innovation CQUIN[] or as a result of local initiatives However the format ofthese teams is variable as is the patient cohort ie restrictedto specific cancer diagnoses and the interventions offeredie often restricted to symptom controlThus a national strategy is required to standardise supportive care services in relevant settings This needs to beevidencebased and ensure equity of care for all cancer patients irrespective of their cancer diagnosis or stage Thestrategy needs to address the current situation but alsoacknowledge evolving oncology practice ie new treatmentswith new toxicities changing disease patterns ie cancer as˜chronic disease™ and changing patient characteristicsIt needs to address education and training discussedbelow and be supported by benchmarking of servicesincluding inspections of clinical services incorporatingpatient feedback Investment will be required to standardise supportive care services and research fundingshould be allocated to determine the optimal model of careas well as the effectivenesscost effectiveness of the individual components of the servicesImplications for TrainingSupportive care encompasses the entire cancer journeyand so necessitates the involvement of most clinical specialties and many nonclinical services Figure Indeedmodern supportive care cannot be provided by a singleclinical specialty alone However as with other cancermultidisciplinary teams a dedicated ˜core team™ is neededto manage everyday problems with timely input from an˜extended team™ if the need arises Importantly the coreteam needs specificongoing education and training inprinciples of supportive careIt is also important to recognise that although manysupportive care services may have evolved from palliativecare services palliative care healthcare professionalsgenerally have limited formal training in supportive careand it is often not appropriate to extrapolate dataexperience from patients with advanced cancer to patientsreceiving anticancer treatment or cancer survivors Forexample the management of nausea and vomiting inadvanced cancer [] is very different from the management of chemotherapyinduced nausea and vomiting []The development of specialist supportive care servicesmust be supported by the educationtraining of the wideroncology workforce in the principles of supportive careand the management of common symptomsproblemsIndeed specialist supportive care services will only ever beable to see the ˜tip of the iceberg™ and so will need to focuson more complex problems and ones requiring specialistinterventions Moreover for example it is much moreappropriate for the team that gives the oncological intervention to manage the adverse effects of that oncologicalinterventionThus supportive care needs to be incorporated into thecurricula of all healthcare professionals involved in cancercare including primary care physicians AppropriateFig The extended supportive care teamcontinuing professional development opportunities need tobe developed for these groups Patients and their familiesneed access to appropriate educational resources in order tofacilitate rapidsuccessful treatment of the complications ofthe cancer andor the cancer treatmentEnhanced Supportive Care Programme eNHS EnglandNHS England promoted early development of supportivecare within some cancer centres via the ESC CQUIN programme CQUIN is the framework supporting improvements in the quality of services and the creation of newimproved patterns of care [] ESC CQUIN was developedby The Christie NHS Foundation Trust and was based uponsix key principles for the implementation and delivery ofsupportive care Figure [] The programme developedthrough recognition of what specialist palliative care professionals working alongside other cancer care disciplinescould offer across the whole cancer pathway e and throughrecognition of barriers to achieving earlier involvement[] Palliative care and supportive care are often differentlyanised across locations on the basis of resources andtraditions In some centres the two are anised as oneservice whereas in others they are completely separate[] The ESC programme required rebranding a closercollaboration with oncology and referral within weeks ofdiagnosis of incurable cancerNHS England's Specialised Commissioning ImprovingValue Team worked with commissioners and clinical teamsin ESC development Fourteen cancer centres took part inthe ESC CQUIN over a 3year period 2016e2019 Aninterim evaluation of the scheme took place in OctoberPlease cite this as Berman R Supportive Care An Indispensable Component of Modern Oncology Clinical Oncology 101016jclon202007020 0cR Berman Clinical Oncology xxx xxxx xxx The programme was associated with a variety ofpositive outcomes including timelier referral of patientswith supportive care needs improved symptom controlimproved quality of life reduced 30day mortality fromchemotherapyimproved overall survival and reducedhealthcare costs [] ESC's principles of early referral andintervention may have impacted positively on these outcomes by better supporting patients who decide to proceedwith chemotherapy as well as those who decide not toproceedA limitation of the ESC CQUIN related to variation inservice delivery model across the centres Further robustresearch needs to be undertaken to determine the ˜optimal™approach for delivery of supportive care services withincancer centres and in other settingsDeveloping the Research and Evidence inSupportive CareWhen the American Society for Clinical Oncology ASCOcelebrated its 50th anniversaryit listed the five topachievements in oncology over that period Prominentlylisted was the development of highly effective antiemetictreatment [] What has been the impact of this keyadvancement in cancer supportive care and how did we getthere Does this progress guide us in improving other areasin supportive careThe impact of preventing emesis is broad and largeNausea and vomiting affect all aspects of daily living thequality of life benefits of antiemetic prevention have beendocumented Economically this advance allowed nearly allchemotherapy to be given on an outpatient basis ratherthan requiring hospitalisation This also allows people tohave less disruption and to remain with their families whilepursuing normal activitiesThese improvements are the result of thoughtful andlogical research Principles of this research included thefollowing which can be applied to many supportive caresettings i an understanding of appropriate physiology[] ii establishment of good clinical methodology []and iii evidence that affecting specific neurotransmitterpathways resulted in major clinical benefit [] As aresult of this work 80e90 of patients can be spared emesisin difficult settings as opposed to in the pastAs we enter an era where chemotherapy is progressivelyless used new areas for supportive care emerge Are weprepared to understand in depth unanticipated challengesin supportive care Can we prevent dermatological toxicities with tyrosine kinase inhibitormediated molecularlytargeted approaches through better understanding of themechanisms of these agents and skin physiology Can wepredict who is likely to have autoimmune sideeffects withcheck point inhibitors []Skills in caring for patients with cancer and methods oftreating malignancy continue to improve The advancesmade in preventing chemotherapyinduced nausea andvomiting provide a model that can influence approaches tomany other aspects of supportive care in cancerInterface with Acute Oncology eAmbulatory Supportive CareAdvances in cancer management continue to improvepatient outcomes This has expectedly been associatedwith an increase in emergency presentations with disease or treatmentrelated complications The challengesof emergency oncology presentations have led to an interest in developing optimal care models for meetingpatients' needs [] Cancer patients seeking emergencycare generally have higher admission rateslongerFig NHS England ESC CQUIN programme principles of ESCPlease cite this as Berman R Supportive Care An Indispensable Component of Modern Oncology Clinical Oncology 101016jclon202007020 0cR Berman Clinical Oncology xxx xxxx xxxlengths of stay and higher mortality than noncancerpatients []Ambulatory care is recognised as a key tenet in ensuringthe safety and sustainability of acute care services Thefundamental basis for ambulatory care is that patientspresenting with acute illnesses can be stratified as low riskfor developing complications and therefore do not requiretraditional inpatient care []Individualised management of acute cancer presentations is a key issue for emergency oncology services sothat it can mirror routine cancer care [] There are anincreasing number of acute cancer presentations that can berisk assessed for care in an emergency ambulatory settingThese include lowrisk febrile neutropeniacancerincidental pulmonaryassociated deep vein thrombosisembolism chemotherapyrelated acute kidney injurychemotherapyinduced nausea and vomiting indwellingline infections acute management of pain crises malignanthypercalcaemiaabnormalitiesasymptomatic brain metastases and malignant pleuraleffusion [43e46]and otherelectrolyteAmbulatory models offer the opportunity to integratepalliative care and supportive care with oncology and acuteservices This facilitates improved access for patients toexpertise in cancer care and immediate management of thecomplications of cancer treatment with the goal of preventing downstream complications and future emergencypresentations For example ambulatory enhanced supportive care models have shown utility in the managementof lowrisk febrile neutropenia []Modelling of ambulatory emergency oncology serviceswithin integrated supportive care services is therefore keyin the provision of highquality personalised and sustainable emergency oncology careThe Importance of Supportive Care inExperimental Cancer MedicineExperimentalcancer medicine trialsECMTs arefundamental to the development of novel cancer therapiesThe primary aims of ECMTs are to identify treatmentrelated toxicities and determine the recommended drugdose [] These trials are increasingly complex []intensive with risks of toxicity for patients but there is agrowing recognition that they are a valid therapeutic option []ECMTs have strict eligibility criteria with the need forpatients to have a performance status of or indicatinghigh levels of day to day functioning [] However thesepatients typically have advanced disease multiple previouslines of treatment and therefore a high associated symptom burden [] Hui [] found that patients referredfor ECMTs have a similar symptom burden to those whowere not despite the perception of higher levels of fitness Ahigh symptom burden has also been associated with earlydiscontinuation from trials [] highlighting the potentialrole for supportive care Br 13edart [] suggested thatthis patient group is more likely to accept increased toxicityto facilitate continued access to trial drugs In one studyECMT patients stated that they would still participate in atrial despite the potential risk of serious toxicities and a chance of death []Research suggests that ECMT patients are less inclined toaccept traditional palliative care due to a general andsometimes unrealistic optimism regarding trial participation[] alongside the perception that palliative care is onlyapplicable at the end of life [] However supportive carepractices within the early phase trials setting have the potential to reduce the impact of symptom burden and adverseevents on patients [] potentially increasing trial recruitment and the length of time patients spend on an experimental therapy Evidence in an ongoing study by Ferrell et al[] indicates that additional support can improve thequality of life for this patient group On top of the benefit topatients of access to additional therapies prolonged exposure to trial drugs supports research through increasednumbers of evaluable patients aiding efficient and accurateassessment of novel therapies Thus there is growing evidence for the role of supportive care for ECMT patients withthe need for increased research to assess potential benefitsand identify optimal routes for its deliveryLearning from Other CountriesImplementation of Supportive Care inFranceWith the aim of increasing and improving communityinvestment in supportive care MASCC is promoting severaldifferent approaches to engage countries such as 0f The creation of accreditation for hospitals withdedicated supportive care units 0f Promotion of MASCC and collaboration with localassociations at MASCC meetings 0f Special links with these associations such as jointmembershipsFrance committed to the supportive care approach at theend of the 1990s and as part of its first cancer plan in The French Speaking Association for Supportive Care inCancer AFSOS affiliated to MASCC was created in with the objectives ofaccompaniment 0f Promoting knowledge and execution of supportivecare in oncology 0f Sharing experience with all professionals involved insymptomsthethroughout all phases of the disease 0f Identifying and understanding the impact of thetransferability and interdependency between disciplines facilitating key aspects obstacles interestsand limitations of work 0f Heightening ethical awareness among medical staffand careofAFSOS has set up a research committee with four strategic priority directions healthcare anisation crossPlease cite this as Berman R Supportive Care An Indispensable Component of Modern Oncology Clinical Oncology 101016jclon202007020 0cR Berman Clinical Oncology xxx xxxx xxxdisciplinary meetings and departments supportive careunits dedicated teams management of cancer symptomsand treatments health behaviour and human and socialsciencesIts actions are targeted towards institutions eg TheFrench National Cancer Institute Ministry of Health professionals guidance and symposia on specific topics such asemesis or nutritional disorders as well as patients andtheir specific associations through a patientfacing website a roadshow truck crisscrossing France and an inventoryof supportive care resources AFSOS has developed nationalmeetings devoted to physicians and nurses physiotherapists or other health caregivers Guideline resources with atoolkit app are discussed during a specific 2day event andupdated every years AFSOS is involved in promoting international collaboration with other MASCCaffiliated societies eg Network Italiano Cure di Supporto in Oncologia[NICSO] and the Japanese Association of Supportive Care inCancer [JASCC]This French national mobilisation has led many regionalteams to get involved in cancer safety management projectsfor the benefit of patients and their relatives and can becopied in other countriesof these is poor in the UK [] and there are a number ofreasons why this may be even lower in an oncology setting[] The risk of poor bone health and fracture is increasingly recognised across a number of malignancies forexample a recent large Danish registry study showedincreased risk of fragility fracture in adults with haematological malignancy with the largest risk in the first 2e4years following initiation of treatment [] Given thedevastating nature of fractures there is much supportivecare work to be done to identify and treat at risk patientsand manage fragility fractures effectively across the spectrum of the cancer journeyEndocrinologists have had a traditional role in cancersurvivorship [] For example managing the longtermeffects of brain radiotherapy on the pituitary gland inchildhood brain tumour survivors As the prognosis foradult brain tumour survivors improves similar issues mayarise [] More recently endocrine toxicities such ashypophysitis and insulindeficient diabetes caused byimmunotherapy treatments are also keeping endocrinologists busy [] in collaboration with acute oncology []This will be become an even more complex issue asimmunotherapy moves into the adjuvant arena with expertinput into decision making algorithms crucial []Interface with Other Specialities egEndocrinology and DiabetesDiscussionOptimal supportive care of cancer patients requires inputfrom a range of specialties outside of oncology to assistaccurate diagnosis and management and ultimatelyimprove outcomesUp to of inpatients with cancer have diabetes or areat risk of diabetes from the treatments they receive []The importance of this is increasingly recognised patientswith diabetes and cancer have an increased length of hospital stay [] and mortality [] Although there iscurrently a lack of data demonstrating that improving glycaemic control reduces mortality for cancer patients it iscertainly true that effective and timely management ofhyperglycaemia improves quality of life and reduces inpatient length of stay but this requires specialist input from adiabetes teamSimilarly up to of inpatients with cancer experiencehyponatremia commonly secondary to syndrome of inappropriate antidiuretic hormone secretion although in theera of immunotherapy cortisol deficiency is an importantand increasing cause which can be fatal if missed []Untreated hyponatremia can delay oncology treatmentsand extend the length of hospital stay [] Diagnosis andmanagement of hyponatremia is poorly managed in general and the oncology population are no exception [] Weconsider expert supportive care input into the managementof hyponatremia in oncology patients to be essential inimproving this situationFractures particularly those of the hip and spine aredevastating with up to mortality at year following hipfracture and significant ongoing morbidity Vertebral fractures are highly predictive of further fracture but reportingThe current focus of cancer care is on initial diagnosisand treatment and the last year of life end of life care []However a large proportion of patients with cancer experience debilitating morbidity and complex symptomsresulting from cancer andor its treatment across the entirecancer journey Supportive care has been shown to improvequality of life symptom burden and survival as well asbenefitting the health economy [15e17] Thus supportivecare should be an integral component of modern oncologymanagement and should involve input from a range ofspecialties within and outside of oncology Furthermore itscontinued development perhaps most effectively as a subspecialty of oncology is essential in supporting advances inoncology and the changing demographic of the cancerpopulationConflicts of interestR Berman is a director of Supportive Care UK Ltd Thisis outside the scope of the submitted workReferences[] Macmillan Cancer Support Living after diagnosis mediancancer survival times Available at wwwmacmillanukdocumentsaboutusnewsroomlivingaftercancermediancancersurvivaltimespdf[] Cancer Research UK Cancer survival statistics Available atwwwcancerresearchukhealthprofessionalcancerstatisticssurvivalheadingZeroPlease cite this as Berman R Supportive Care An Indispensable Component of Modern Oncology Clinical Oncology 101016jclon202007020 0cR Berman Clinical Oncology xxx xxxx xxx[] National Cancer Survivorship Initiative NCSI Living withand beyond cancer taking action to improve outcomesAvailableassetspublishingservicegovukgovernmentuploadssystemuploadsattachment_datafile1810549333TSO2900664NCSI_Report_FINALpdfat[] Clarke G Johnston S Corrie P Kuhn I Barclay S Withdrawal ofanticancer therapy in advanced disease a systematic literature review BMC Cancer [] Klastersky J Supportive care do we need a model Curr OpinOncol [] Global Burden of Disease Cancer Collaboration The GlobalBurden of Cancer JAMA Oncol 20151505e527[] National Audit Office Delivering the cancer reform strategyAvailable at wwwnaoukreportdeliveringthecancerreformstrategy[] Mokart D Pastores SM Darmon M Has survival increased incancer patients admitted to the ICU Yes Intens Care Med2014401570e1572[] National Institutes of Health Cancer trends progress report e update National Institutes of Health [] Sullivan R Peppercorn J Sikora K Zalcberg J Meropol NJAmir E Delivering affordable cancer care in highincomecountries Lancet Oncol 201112933e980[] Yabroff Y Lund J Kepka D Mariotto A Economic burden ofcancer in the US estimates projections and future CancerEpidemiol Biomarkers Prev 201120102006e2014[] Aggarwal A Sullivan R Affordability of cancer care in theUnited Kingdom e is it time to introduce user chargesJ Cancer Policy 2014231e39[] Saini K Heras B Castro J Venkitaraman R Poelman MSrinivasan G Effect of the COVID19 pandemic on cancertreatment and research Lancet Haem 202076e432ee435[] Radcliffe E Khan A Wright D Berman R Demain S RestorickBanks S Understanding the importance of selfmanagement support in people living with cancer reportReport on the impact of COVID19 in press[] Monnery D Benson S Griffiths A Cadwallader C HamptonMatthews J Coackley A Multiprofessionaldeliveredenhanced supportive care improves quality of life for patientswith incurable cancer Int J Palliat Nurs 20182410510e514[] Basch E Deal AM Dueck AC Scher HI Kris MG Hudis C et alOverall survival results of a trial assessing patientreportedoutcomes for symptom monitoring during routine cancertreatment JAMA 20173182197e198[] Cooksley T Campbell G AlSayed T LaMola L Berman RA novel approach to improving ambulatory outpatient management of low risk febrile neutropenia an Enhanced Supportive Care ESC clinic Support Care Cancer 2937e2940[] Klastersky J Christel F Editorial Supportive care in cancerpatients a constantly evolving field Curr Opin Oncol 314257e258[] Cooksley T Rice T Emergency oncology development current position and future direction in the USA and UK SupportCare Cancer 2017253e7[] Guly H Preface A history of accident and emergency medicine1948e2004 London Palgrave Macmillan [] Hui D Hannon BL Zimmerman C Bruera E Improving patientand caregiver outcomes in oncology teambased timely andtargeted palliative care CA Cancer J Clin 201868356e376[] Whelan TJ Mohide EA Willan AR Arnold A Tew M Sellick S The supportive care needs of newly diagnosed cancerpatients attending a regional cancer center Cancer 8081518e1524[] Hui D De La Cruz M Mori M Parsons HA Kwon JH TorresVigil I Concepts and definitions for œsupportive careœbest supportive care œpalliative care and œhospice care inthe published literature dictionaries and textbooks SupportCare Cancer 201321659e685[] Boyd K Moine S Murray SA Bowman D Brun N Shouldpalliative care be rebranded B

cancer232

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: " The advent of new cancer therapies alongside expected growth and ageing of the population better survival rates and associated costs of care is uncovering aneed to more clearly define and integrate supportive care services across the whole spectrum of the disease The current focus of cancer care is on initialdiagnosis and treatment and end of life care The Multinational Association of Supportive Care in Cancer defines supportive care as ˜the prevention andmanagement of the adverse effects of cancer and its treatment™ This encompasses the entire cancer journey and necessitates involvement and integration ofmost clinical specialties Optimal supportive care can assist in accurate diagnosis and management and ultimately improve outcomes A national strategy toimplement supportive care is needed to acknowledge evolving oncology practice changing disease patterns and the changing patient demographic“ The Royal College of Radiologists Published by Elsevier Ltd All rights reservedKey words Beyond cancer chronic cancer definition living with supportive careStatement of Search Strategies UsedA series of searches were constructed and carried out viaPubMed EMBASE and MEDLINE This generally consisted ofusing phrase searching due to the specificity of the subjectOnce concepts were established the authors used Booleanoperators to combine the concepts together and retrieve themost relevant papers Once a set of results were retrieved theauthors scanned each of the s using and titlefields to identify key papers Fulltext access to papers weresourced via the Christie Library and Knowledge ServiceIntroductionSupportive Care Makes Excellent Cancer Care PossibleMultinational Association of Supportive Care in CancerwwwmasccAdvances in diagnosis surgery radiotherapy and newdrugs have led to improvements in cancer survival PeopleAuthor for correspondence R Berman The Christie NHS FoundationTrust Wilmslow Road Manchester M20 4BX UK Tel ¾447710509402Email address RichardBermanchristienhsuk R Bermannow live nearly six times longer after their cancer diagnosisthan was the case years ago [] Half of people diagnosedwith cancer in England and Wales survive their disease for years or more [] Currently in England around million people are living with a diagnosis of cancer and thisnumber is increasing by over a year The total figure is setrise to over million by []Many more cancer patients are being treated closer todeath with novel less toxic high efficacy anticancer therapeutic agents developing with increasing pace within thelast decade The advent of molecular targeted agents forexample has brought new benefits as well as challenges tomodern cancer therapy potentially blurring the distinctionbetween active and palliative interventions []Yet despite this significant progress a large proportion ofpatients with cancer still experience morbidity and symptoms resulting from the cancer andor its treatment []Increases in cancer incidence [] emergency care hospitalisations [] earlier intensive care unit admissions [] andtreatment costs [] have all added to the global burden ofcancer care The disease is becoming a major economicexpenditure for all developed countries [] In the UK andin the USA cancer care costs are substantial and expected to101016jclon20200702009366555“ The Royal College of Radiologists Published by Elsevier Ltd All rights reservedPlease cite this as Berman R Supportive Care An Indispensable Component of Modern Oncology Clinical Oncology 101016jclon202007020 0cR Berman Clinical Oncology xxx xxxx xxxrise significantly in the future due to growth and aging ofthe population and improvements in survival as well astrends in treatment patterns and costs of care followingcancer diagnosis []cancerandcancerManagingtreatmentrelatedmorbidity is therefore a significant public health andeconomic challenge The coronavirus pandemic has deepened this challenge with many cancer outpatient visitsbeing replaced by telephone consultations and deferral ofsome routine therapy tests and procedures This has placedadditional pressures on an already fragile and vulnerablepopulation [] Patients and carers are experiencing moreuncertainty and anxiety associated with COVID19 A recentstudy found that although patients continue to feel wellsupported by their healthcare teams they have concernsabout the longerterm impact of changes to aspects of theirtreatment Patients and carers are no longer able to accessother support services in the way that they had previouslysuch as hospices and peer support groups []There is a growing body of evidence that timely access tosupportive treatments can lead to improvements in qualityof life and survival as well as benefitting the health economy [15e17] The development of a broad multiprofessionalbasis for the study and expansion of supportive carethrough the Multinational Association of Supportive Care inCancer MASCC has been an important step in fostering thegrowth of an evidence base [] MASCC's success has undoubtedly been underpinned by successful integration ofoncological and nononcological specialties []However variations in the definition of supportive careallocation of resources and a lack of clarity on who shouldlead onprovide services means that a clinical model forsupportive care in cancer does not yet exist [] Most specialties whilst they overlap other specialties are based on acore of knowledge or skill that is specific to that specialty[] Supportive care is currently provided by a patchwork ofdifferent medical specialties and is unique because it traverses the entire spectrum of the disease Figure fromdiagnosis through to survivorship or end of life care Theneed for ˜supportive oncology™ to become a specialty in itsown right is borne out not just by the progress in its development in the UK and abroad but by the unmet supportivecare need [] amplified by the rising incidence of cancerworldwide with many patients living longer with incurableillness because of more effective cancer treatments [] Asignificant next step would be to produce an evidencebasednational strategy for supportive care implemented throughappointment of supportive care lead clinicians within eachUK cancer centre This alongside support from the medicalRoyal Colleges and NHS England would be fundamental indeveloping a sustainable clinical modelPerhaps working as a distinct branch of oncology ˜specialists™ in supportive care medicine should have the skillsand resources to manage a broad range of effects associatedwith longterm cancers and cancer survival This paperexplores areas that are showing promise in this development and identifies key next steps needed to recognisesupportive care as an indispensable component of modernoncologyDefinition of Supportive CareThe Inuit may or may not have words for snow butsupportive care seems to have that number of definitions orconnotations [] Supportive care has been used as aeuphemism for palliative care and ˜early palliative care™[] and research suggests that a change in name frompalliative care to supportive care results in more and earlierreferrals to hospitalbased services [] Palliative care is anintegral component of supportive care but supportive careis much more than palliative care or even ˜early palliativecare™MASCC defines supportive care as ˜the prevention andmanagement of the adverse effects of cancer and its treatment This includes management of physical and psychological symptoms and side effects across the continuum ofthe cancer experience from diagnosis through treatment toposttreatment care Enhancing rehabilitation secondarycancer prevention survivorship and endoflife care areintegral to supportive care™ []Strategy for Implementation of SupportiveCare Within Cancer CareThe potential benefits of supportive care includedecreased morbidity improved quality of life and potentially decreased mortality ie secondary to optimal cancertreatment the potential benefits for healthcare servicesinclude decreased utilisation of healthcare resources andimproved treatment outcomes [] Indeed supportivecare offers patients more than many ˜palliative™ oncologicaltreatments and should be considered an essential not justan optional extraCurrently many cancer centres in the UK have supportivecare services either as a result of NHS England's EnhancedSupportive Care ESC Programme discussed below andFig The supportive care umbrellaPlease cite this as Berman R Supportive Care An Indispensable Component of Modern Oncology Clinical Oncology 101016jclon202007020 0cR Berman Clinical Oncology xxx xxxx xxxrelated Commissioning for Quality and Innovation CQUIN[] or as a result of local initiatives However the format ofthese teams is variable as is the patient cohort ie restrictedto specific cancer diagnoses and the interventions offeredie often restricted to symptom controlThus a national strategy is required to standardise supportive care services in relevant settings This needs to beevidencebased and ensure equity of care for all cancer patients irrespective of their cancer diagnosis or stage Thestrategy needs to address the current situation but alsoacknowledge evolving oncology practice ie new treatmentswith new toxicities changing disease patterns ie cancer as˜chronic disease™ and changing patient characteristicsIt needs to address education and training discussedbelow and be supported by benchmarking of servicesincluding inspections of clinical services incorporatingpatient feedback Investment will be required to standardise supportive care services and research fundingshould be allocated to determine the optimal model of careas well as the effectivenesscost effectiveness of the individual components of the servicesImplications for TrainingSupportive care encompasses the entire cancer journeyand so necessitates the involvement of most clinical specialties and many nonclinical services Figure Indeedmodern supportive care cannot be provided by a singleclinical specialty alone However as with other cancermultidisciplinary teams a dedicated ˜core team™ is neededto manage everyday problems with timely input from an˜extended team™ if the need arises Importantly the coreteam needs specificongoing education and training inprinciples of supportive careIt is also important to recognise that although manysupportive care services may have evolved from palliativecare services palliative care healthcare professionalsgenerally have limited formal training in supportive careand it is often not appropriate to extrapolate dataexperience from patients with advanced cancer to patientsreceiving anticancer treatment or cancer survivors Forexample the management of nausea and vomiting inadvanced cancer [] is very different from the management of chemotherapyinduced nausea and vomiting []The development of specialist supportive care servicesmust be supported by the educationtraining of the wideroncology workforce in the principles of supportive careand the management of common symptomsproblemsIndeed specialist supportive care services will only ever beable to see the ˜tip of the iceberg™ and so will need to focuson more complex problems and ones requiring specialistinterventions Moreover for example it is much moreappropriate for the team that gives the oncological intervention to manage the adverse effects of that oncologicalinterventionThus supportive care needs to be incorporated into thecurricula of all healthcare professionals involved in cancercare including primary care physicians AppropriateFig The extended supportive care teamcontinuing professional development opportunities need tobe developed for these groups Patients and their familiesneed access to appropriate educational resources in order tofacilitate rapidsuccessful treatment of the complications ofthe cancer andor the cancer treatmentEnhanced Supportive Care Programme eNHS EnglandNHS England promoted early development of supportivecare within some cancer centres via the ESC CQUIN programme CQUIN is the framework supporting improvements in the quality of services and the creation of newimproved patterns of care [] ESC CQUIN was developedby The Christie NHS Foundation Trust and was based uponsix key principles for the implementation and delivery ofsupportive care Figure [] The programme developedthrough recognition of what specialist palliative care professionals working alongside other cancer care disciplinescould offer across the whole cancer pathway e and throughrecognition of barriers to achieving earlier involvement[] Palliative care and supportive care are often differentlyanised across locations on the basis of resources andtraditions In some centres the two are anised as oneservice whereas in others they are completely separate[] The ESC programme required rebranding a closercollaboration with oncology and referral within weeks ofdiagnosis of incurable cancerNHS England's Specialised Commissioning ImprovingValue Team worked with commissioners and clinical teamsin ESC development Fourteen cancer centres took part inthe ESC CQUIN over a 3year period 2016e2019 Aninterim evaluation of the scheme took place in OctoberPlease cite this as Berman R Supportive Care An Indispensable Component of Modern Oncology Clinical Oncology 101016jclon202007020 0cR Berman Clinical Oncology xxx xxxx xxx The programme was associated with a variety ofpositive outcomes including timelier referral of patientswith supportive care needs improved symptom controlimproved quality of life reduced 30day mortality fromchemotherapyimproved overall survival and reducedhealthcare costs [] ESC's principles of early referral andintervention may have impacted positively on these outcomes by better supporting patients who decide to proceedwith chemotherapy as well as those who decide not toproceedA limitation of the ESC CQUIN related to variation inservice delivery model across the centres Further robustresearch needs to be undertaken to determine the ˜optimal™approach for delivery of supportive care services withincancer centres and in other settingsDeveloping the Research and Evidence inSupportive CareWhen the American Society for Clinical Oncology ASCOcelebrated its 50th anniversaryit listed the five topachievements in oncology over that period Prominentlylisted was the development of highly effective antiemetictreatment [] What has been the impact of this keyadvancement in cancer supportive care and how did we getthere Does this progress guide us in improving other areasin supportive careThe impact of preventing emesis is broad and largeNausea and vomiting affect all aspects of daily living thequality of life benefits of antiemetic prevention have beendocumented Economically this advance allowed nearly allchemotherapy to be given on an outpatient basis ratherthan requiring hospitalisation This also allows people tohave less disruption and to remain with their families whilepursuing normal activitiesThese improvements are the result of thoughtful andlogical research Principles of this research included thefollowing which can be applied to many supportive caresettings i an understanding of appropriate physiology[] ii establishment of good clinical methodology []and iii evidence that affecting specific neurotransmitterpathways resulted in major clinical benefit [] As aresult of this work 80e90 of patients can be spared emesisin difficult settings as opposed to in the pastAs we enter an era where chemotherapy is progressivelyless used new areas for supportive care emerge Are weprepared to understand in depth unanticipated challengesin supportive care Can we prevent dermatological toxicities with tyrosine kinase inhibitormediated molecularlytargeted approaches through better understanding of themechanisms of these agents and skin physiology Can wepredict who is likely to have autoimmune sideeffects withcheck point inhibitors []Skills in caring for patients with cancer and methods oftreating malignancy continue to improve The advancesmade in preventing chemotherapyinduced nausea andvomiting provide a model that can influence approaches tomany other aspects of supportive care in cancerInterface with Acute Oncology eAmbulatory Supportive CareAdvances in cancer management continue to improvepatient outcomes This has expectedly been associatedwith an increase in emergency presentations with disease or treatmentrelated complications The challengesof emergency oncology presentations have led to an interest in developing optimal care models for meetingpatients' needs [] Cancer patients seeking emergencycare generally have higher admission rateslongerFig NHS England ESC CQUIN programme principles of ESCPlease cite this as Berman R Supportive Care An Indispensable Component of Modern Oncology Clinical Oncology 101016jclon202007020 0cR Berman Clinical Oncology xxx xxxx xxxlengths of stay and higher mortality than noncancerpatients []Ambulatory care is recognised as a key tenet in ensuringthe safety and sustainability of acute care services Thefundamental basis for ambulatory care is that patientspresenting with acute illnesses can be stratified as low riskfor developing complications and therefore do not requiretraditional inpatient care []Individualised management of acute cancer presentations is a key issue for emergency oncology services sothat it can mirror routine cancer care [] There are anincreasing number of acute cancer presentations that can berisk assessed for care in an emergency ambulatory settingThese include lowrisk febrile neutropeniacancerincidental pulmonaryassociated deep vein thrombosisembolism chemotherapyrelated acute kidney injurychemotherapyinduced nausea and vomiting indwellingline infections acute management of pain crises malignanthypercalcaemiaabnormalitiesasymptomatic brain metastases and malignant pleuraleffusion [43e46]and otherelectrolyteAmbulatory models offer the opportunity to integratepalliative care and supportive care with oncology and acuteservices This facilitates improved access for patients toexpertise in cancer care and immediate management of thecomplications of cancer treatment with the goal of preventing downstream complications and future emergencypresentations For example ambulatory enhanced supportive care models have shown utility in the managementof lowrisk febrile neutropenia []Modelling of ambulatory emergency oncology serviceswithin integrated supportive care services is therefore keyin the provision of highquality personalised and sustainable emergency oncology careThe Importance of Supportive Care inExperimental Cancer MedicineExperimentalcancer medicine trialsECMTs arefundamental to the development of novel cancer therapiesThe primary aims of ECMTs are to identify treatmentrelated toxicities and determine the recommended drugdose [] These trials are increasingly complex []intensive with risks of toxicity for patients but there is agrowing recognition that they are a valid therapeutic option []ECMTs have strict eligibility criteria with the need forpatients to have a performance status of or indicatinghigh levels of day to day functioning [] However thesepatients typically have advanced disease multiple previouslines of treatment and therefore a high associated symptom burden [] Hui [] found that patients referredfor ECMTs have a similar symptom burden to those whowere not despite the perception of higher levels of fitness Ahigh symptom burden has also been associated with earlydiscontinuation from trials [] highlighting the potentialrole for supportive care Br 13edart [] suggested thatthis patient group is more likely to accept increased toxicityto facilitate continued access to trial drugs In one studyECMT patients stated that they would still participate in atrial despite the potential risk of serious toxicities and a chance of death []Research suggests that ECMT patients are less inclined toaccept traditional palliative care due to a general andsometimes unrealistic optimism regarding trial participation[] alongside the perception that palliative care is onlyapplicable at the end of life [] However supportive carepractices within the early phase trials setting have the potential to reduce the impact of symptom burden and adverseevents on patients [] potentially increasing trial recruitment and the length of time patients spend on an experimental therapy Evidence in an ongoing study by Ferrell et al[] indicates that additional support can improve thequality of life for this patient group On top of the benefit topatients of access to additional therapies prolonged exposure to trial drugs supports research through increasednumbers of evaluable patients aiding efficient and accurateassessment of novel therapies Thus there is growing evidence for the role of supportive care for ECMT patients withthe need for increased research to assess potential benefitsand identify optimal routes for its deliveryLearning from Other CountriesImplementation of Supportive Care inFranceWith the aim of increasing and improving communityinvestment in supportive care MASCC is promoting severaldifferent approaches to engage countries such as 0f The creation of accreditation for hospitals withdedicated supportive care units 0f Promotion of MASCC and collaboration with localassociations at MASCC meetings 0f Special links with these associations such as jointmembershipsFrance committed to the supportive care approach at theend of the 1990s and as part of its first cancer plan in The French Speaking Association for Supportive Care inCancer AFSOS affiliated to MASCC was created in with the objectives ofaccompaniment 0f Promoting knowledge and execution of supportivecare in oncology 0f Sharing experience with all professionals involved insymptomsthethroughout all phases of the disease 0f Identifying and understanding the impact of thetransferability and interdependency between disciplines facilitating key aspects obstacles interestsand limitations of work 0f Heightening ethical awareness among medical staffand careofAFSOS has set up a research committee with four strategic priority directions healthcare anisation crossPlease cite this as Berman R Supportive Care An Indispensable Component of Modern Oncology Clinical Oncology 101016jclon202007020 0cR Berman Clinical Oncology xxx xxxx xxxdisciplinary meetings and departments supportive careunits dedicated teams management of cancer symptomsand treatments health behaviour and human and socialsciencesIts actions are targeted towards institutions eg TheFrench National Cancer Institute Ministry of Health professionals guidance and symposia on specific topics such asemesis or nutritional disorders as well as patients andtheir specific associations through a patientfacing website a roadshow truck crisscrossing France and an inventoryof supportive care resources AFSOS has developed nationalmeetings devoted to physicians and nurses physiotherapists or other health caregivers Guideline resources with atoolkit app are discussed during a specific 2day event andupdated every years AFSOS is involved in promoting international collaboration with other MASCCaffiliated societies eg Network Italiano Cure di Supporto in Oncologia[NICSO] and the Japanese Association of Supportive Care inCancer [JASCC]This French national mobilisation has led many regionalteams to get involved in cancer safety management projectsfor the benefit of patients and their relatives and can becopied in other countriesof these is poor in the UK [] and there are a number ofreasons why this may be even lower in an oncology setting[] The risk of poor bone health and fracture is increasingly recognised across a number of malignancies forexample a recent large Danish registry study showedincreased risk of fragility fracture in adults with haematological malignancy with the largest risk in the first 2e4years following initiation of treatment [] Given thedevastating nature of fractures there is much supportivecare work to be done to identify and treat at risk patientsand manage fragility fractures effectively across the spectrum of the cancer journeyEndocrinologists have had a traditional role in cancersurvivorship [] For example managing the longtermeffects of brain radiotherapy on the pituitary gland inchildhood brain tumour survivors As the prognosis foradult brain tumour survivors improves similar issues mayarise [] More recently endocrine toxicities such ashypophysitis and insulindeficient diabetes caused byimmunotherapy treatments are also keeping endocrinologists busy [] in collaboration with acute oncology []This will be become an even more complex issue asimmunotherapy moves into the adjuvant arena with expertinput into decision making algorithms crucial []Interface with Other Specialities egEndocrinology and DiabetesDiscussionOptimal supportive care of cancer patients requires inputfrom a range of specialties outside of oncology to assistaccurate diagnosis and management and ultimatelyimprove outcomesUp to of inpatients with cancer have diabetes or areat risk of diabetes from the treatments they receive []The importance of this is increasingly recognised patientswith diabetes and cancer have an increased length of hospital stay [] and mortality [] Although there iscurrently a lack of data demonstrating that improving glycaemic control reduces mortality for cancer patients it iscertainly true that effective and timely management ofhyperglycaemia improves quality of life and reduces inpatient length of stay but this requires specialist input from adiabetes teamSimilarly up to of inpatients with cancer experiencehyponatremia commonly secondary to syndrome of inappropriate antidiuretic hormone secretion although in theera of immunotherapy cortisol deficiency is an importantand increasing cause which can be fatal if missed []Untreated hyponatremia can delay oncology treatmentsand extend the length of hospital stay [] Diagnosis andmanagement of hyponatremia is poorly managed in general and the oncology population are no exception [] Weconsider expert supportive care input into the managementof hyponatremia in oncology patients to be essential inimproving this situationFractures particularly those of the hip and spine aredevastating with up to mortality at year following hipfracture and significant ongoing morbidity Vertebral fractures are highly predictive of further fracture but reportingThe current focus of cancer care is on initial diagnosisand treatment and the last year of life end of life care []However a large proportion of patients with cancer experience debilitating morbidity and complex symptomsresulting from cancer andor its treatment across the entirecancer journey Supportive care has been shown to improvequality of life symptom burden and survival as well asbenefitting the health economy [15e17] Thus supportivecare should be an integral component of modern oncologymanagement and should involve input from a range ofspecialties within and outside of oncology Furthermore itscontinued development perhaps most effectively as a subspecialty of oncology is essential in supporting advances inoncology and the changing demographic of the cancerpopulationConflicts of interestR Berman is a director of Supportive Care UK Ltd Thisis outside the scope of the submitted workReferences[] Macmillan Cancer Support Living after diagnosis mediancancer survival times Available at wwwmacmillanukdocumentsaboutusnewsroomlivingaftercancermediancancersurvivaltimespdf[] Cancer Research UK Cancer survival statistics Available atwwwcancerresearchukhealthprofessionalcancerstatisticssurvivalheadingZeroPlease cite this as Berman R Supportive Care An Indispensable Component of Modern Oncology Clinical Oncology 101016jclon202007020 0cR Berman Clinical Oncology xxx xxxx xxx[] National Cancer Survivorship Initiative NCSI Living withand beyond cancer taking action to improve outcomesAvailableassetspublishingservicegovukgovernmentuploadssystemuploadsattachment_datafile1810549333TSO2900664NCSI_Report_FINALpdfat[] Clarke G Johnston S Corrie P Kuhn I Barclay S Withdrawal ofanticancer therapy in advanced disease a systematic literature review BMC Cancer [] Klastersky J Supportive care do we need a model Curr OpinOncol [] Global Burden of Disease Cancer Collaboration The GlobalBurden of Cancer JAMA Oncol 20151505e527[] National Audit Office Delivering the cancer reform strategyAvailable at wwwnaoukreportdeliveringthecancerreformstrategy[] Mokart D Pastores SM Darmon M Has survival increased incancer patients admitted to the ICU Yes Intens Care Med2014401570e1572[] National Institutes of Health Cancer trends progress report e update National Institutes of Health [] Sullivan R Peppercorn J Sikora K Zalcberg J Meropol NJAmir E Delivering affordable cancer care in highincomecountries Lancet Oncol 201112933e980[] Yabroff Y Lund J Kepka D Mariotto A Economic burden ofcancer in the US estimates projections and future CancerEpidemiol Biomarkers Prev 201120102006e2014[] Aggarwal A Sullivan R Affordability of cancer care in theUnited Kingdom e is it time to introduce user chargesJ Cancer Policy 2014231e39[] Saini K Heras B Castro J Venkitaraman R Poelman MSrinivasan G Effect of the COVID19 pandemic on cancertreatment and research Lancet Haem 202076e432ee435[] Radcliffe E Khan A Wright D Berman R Demain S RestorickBanks S Understanding the importance of selfmanagement support in people living with cancer reportReport on the impact of COVID19 in press[] Monnery D Benson S Griffiths A Cadwallader C HamptonMatthews J Coackley A Multiprofessionaldeliveredenhanced supportive care improves quality of life for patientswith incurable cancer Int J Palliat Nurs 20182410510e514[] Basch E Deal AM Dueck AC Scher HI Kris MG Hudis C et alOverall survival results of a trial assessing patientreportedoutcomes for symptom monitoring during routine cancertreatment JAMA 20173182197e198[] Cooksley T Campbell G AlSayed T LaMola L Berman RA novel approach to improving ambulatory outpatient management of low risk febrile neutropenia an Enhanced Supportive Care ESC clinic Support Care Cancer 2937e2940[] Klastersky J Christel F Editorial Supportive care in cancerpatients a constantly evolving field Curr Opin Oncol 314257e258[] Cooksley T Rice T Emergency oncology development current position and future direction in the USA and UK SupportCare Cancer 2017253e7[] Guly H Preface A history of accident and emergency medicine1948e2004 London Palgrave Macmillan [] Hui D Hannon BL Zimmerman C Bruera E Improving patientand caregiver outcomes in oncology teambased timely andtargeted palliative care CA Cancer J Clin 201868356e376[] Whelan TJ Mohide EA Willan AR Arnold A Tew M Sellick S The supportive care needs of newly diagnosed cancerpatients attending a regional cancer center Cancer 8081518e1524[] Hui D De La Cruz M Mori M Parsons HA Kwon JH TorresVigil I Concepts and definitions for œsupportive careœbest supportive care œpalliative care and œhospice care inthe published literature dictionaries and textbooks SupportCare Cancer 201321659e685[] Boyd K Moine S Murray SA Bowman D Brun N Shouldpalliative care be rebranded B Answer:

Thyroid_Cancer

"Cardiac arrhythmias Atrial fibrillation Sudden cardiac death Long QT syndrome Torsade des pointes Ventricular tachycardia Ventricular fibrillation As the coronavirus COVID19 pandemic marches unrelentingly more patients with cardiac arrhyth mias are emerging due to the effects of the virus on the respiratory and cardiovascular CV systems and the systemic ‚ammation that it incurs and also as a result of the proarrhythmic effects of COVID19 pharmacotherapies and other drug interactions and the associated autonomic imbalance that enhance ar rhythmogenicity The most worrisome of all arrhythmogenic mechanisms is the QT prolonging effect of various antiCOVID pharmacotherapies that can lead to polymorphic ventricular tachycardia in the form of torsade des pointes and sudden cardiac death It is therefore imperative to monitor the QT interval dur ing treatment however conventional approaches to such monitoring increase the transmission risk for the staff and strain the health system Hence there is dire need for contactless monitoring and teleme try for inpatients especially those admitted to the intensive care unit as well as for outpatients needing continued management In this context recent technological advances have ushered in a new era in im plementing digital health monitoring tools that circumvent these obstacles All these issues are herein discussed and a large body of recent relevant data are reviewed Elsevier Inc All rights reserved Introduction The ongoing pandemic of coronavirus disease COVID19 has created a global tumult [] According to current data of patients with COVID19 infection are afflicted by acute my ocardial injury with an attendant higher mortality rate compared with those without cardiac injury commensurate with the degree of cardiac troponin cTn elevation [“] Furthermore of patients develop cardiac arrhythmias Table including malig nant ventricular arrhythmias VAs [ ] with a higher prevalence noted in patients admitted to the intensive care unit ICU [] Importantly clinically stable patients may have a low preva Abbreviations AAD antiarrhythmic drug AF atrial fibrillation APCs atrial pre mature complexes AZM azithromycin COVID19 coronavirus CQ chloro quine cTn cardiac troponin CV cardiovascular CYP cytochrome P450 ECG elec trocardiogram HCQ hydroxychloroquine ICU intensive care unit LQTS long QT syndrome NSVT nonsustained ventricular tachycardia OOHCA outofhospital cardiac arrest SCD sudden cardiac death TdP torsade des pointes VAs ventricular arrhythmias VF ventricular fibrillation VPCs ventricular premature complexes VT ventricular tachycardia ˆ—Corresponding author Email address asmotenetgr AS Manolis lence of arrhythmias [] however critically ill patients are at much higher risk for cardiac arrhythmias [] Cardiac arrhythmias including lifethreatening VAs may be the consequence of direct effects of COVID19 infection but also of the deleterious effects of systemic illness and the adverse reactions to drugs employed in the treatment of this pandemic Table Fig [ “ ] A recent study indicated that among patients with ± years men African Ameri COVID19 mean age can receiving care in the ICU there were cardiac arrests incident atrial fibrillation AF episodes bradyarrhythmias and nonsustained ventricular tachycardias NSVTs [] Arrhythmias occurring in patients admitted to the ICU included cardiac arrests all events of cardiac arrest occurred in this group AF odds ratioOR vs those not in the ICU and NSVT OR Car diac arrests were associated with acute inhospital mortality Among patients with confirmed COVID19 ex hibited myocardial injury as indicated by elevated cardiac troponin T cTnT levels [] During hospitalization patients de veloped ventricular tachycardia VTventricular fibrillation VF patients with elevated cTnT levels had more frequent VAs vs p compared with those with normal cTnT levels A recent singleday snapshot survey of stable patients with 101016jtcm202008002 Elsevier Inc All rights reserved Please cite this as AS Manolis AA Manolis and TA Manolis COVID19 infection and cardiac arrhythmias Trends in Cardiovascular Medicine 101016jtcm202008002 0cJID TCM IN PRESS [m5G August ] AS Manolis AA Manolis and TA Manolis Trends in Cardiovascular Medicine xxx xxxx xxx Table Cardiac Arrhythmias Occurring in Patients with COVID19 Infection Sinus tachycardia Sinus bradycardia Conduction disturbances AVBBBB Atrial premature complexes Atrial fibrillation Supraventricular tachycardia Ventricular premature complexes Nonsustained ventricular tachycardia Polymorphic ventricular tachycardia Torsade des pointes Sustained ventricular tachycardia Ventricular fibrillation Pulseless electrical activity AVB atrioventricular block BBB bundle branch block Table Mechanisms of Arrhythmogenicity in Patients with COVID19 Infection Acute myocardial injury Myocarditis Hypoxia Systemic ‚ammation Autonomic imbalance SNS overactivity virusinduced vagal nerve injury Electrolyte abnormalities QT prolonging drugs antiCOVID pharmacotherapies AADs other agents Drugdrug interactions Cardiovascular comorbidities hypertension coronary artery disease cardiomyopathy AADs antiarrhythmic drugs SNS In this review we present current data about the whole spec trum of cardiac arrhythmias encountered in patients with COVID infection either attributable to the effect of the virus itself on the cardiovascular CV and the respiratory system andor to the effects of the treatments that these patients receive in combina tion with autonomic imbalance that is incurred by this unrelenting pandemic Acute myocardial injury and arrhythmias As mentioned in patients with evidence of acute myocardial injury the prevalence of cardiac arrhythmias is higher compared to patients without myocardial injury [] In a recent retrospec tive cohort study among patients with severe COVID19 had a cTnI level measured upon hospital admission of whom had positive results showing cardiac injury [] In patients with cardiac injury mortality was higher compared to patients without cardiac injury vs p Arrhythmias were found in of the patients with cardiac injury includ ing patients with VT or VF all of whom died [] A recent meta analysis of studies including COVID19 patients showed that patients with cardiac injury and newly occurring arrhythmias were at higher risk of developing severe disease or requiring ICU admission relative riskRR p [] sympathetic nervous system Sinus tachycardia Sinus tachycardia is the most common rhythm disturbance in patients with COVID19 infection due to multiple reasons such as fever respiratory insufficiencyhypoxemia hemodynamic compro mise fearanxiety pain and several other physical and emotional symptoms [] Bradycardiaconduction disturbances According to a retrospective series of patients transient si nus bradycardia lasting days is a possible manifestation of COVID19 hence another reason for close monitoring [] There may be many reasons for bradycardia but severe hypoxia ‚am matory injury of the sinus node by circulating cytokines and exag gerated response to medications are possible triggers Interestingly bradycardia has been suggested as a warning sign of the onset of a serious cytokine storm Fig The schema illustrates the various arrhythmias encountered in patients with COVID19 infection as a consequence of the virus infection affecting the heart and lung andor producing systemic ‚ammation the adverse proarrhythmic effects of COVID therapies and the drugdrug interactions that may occur see text for long QT discussion AF interval PEA pulseless electrical activity SB sud sinus tachycardia sympathetic nervous system STach den cardiac death SNS ventricular arrhythmias VF TdP ventricular fibril lation VT atrioventricular block LQT torsade des pointes VAs sinus bradycardia SCD atrial fibrillation AVB ventricular tachycardia COVID19 showed a incidence of arrhythmias limited to AF in and supraventricular tachycardia SVT in patients [] A Heart Rhythm Society HRS online survey of electrophysiology professionals n indicated that AF was the most commonly reported tachyarrhythmia whereas severe sinus bradycardia and complete heart block were the most common brad yarrhythmias in hospitalized patients with COVID19 [] Ven tricular tachycardiaVF arrest and pulseless electrical activity were reported by and of respondents respectively A meta analysis of retrospective cohort studies comprising pa tients with COVID19 showed that the pooled incidence was for cardiac arrhythmia for cardiac arrest [] p According to a retrospective cohort study of COVID19 pa tients multivariable logistic regression indicated that among other ECG abnormalities the presence of one or more atrial premature contractions APCs odds ratio OR a right bun dle branch block RBBB or intraventricular block IVB OR p increased the odds of death [] Another study analyzing the ECGs of COVID19 patients showed that abnormal PR interval behavior paradoxical prolonga tion or lack of shortening with increasing heart rate was associ and need ated with increased risk of death vs p for endotracheal intubation vs p compared to patients with PR interval shortening [] Atrial fibrillation According to a recent survey of electrophysiology professionals atrial fibrillation AF was the most commonly encountered car diac arrhythmia observed in patients with COVID19 infection [] Several mechanisms could be involved in the pathogenesis of AF in these patients virusinduced cardiac injury that could lead to perimyocarditis hypoxemia frequently occurring in these patients systemic infection common occurrence of the COVID19 infection Please cite this as AS Manolis AA Manolis and TA Manolis COVID19 infection and cardiac arrhythmias Trends in Cardiovascular Medicine 101016jtcm202008002 0cJID TCM IN PRESS [m5G August ] AS Manolis AA Manolis and TA Manolis Trends in Cardiovascular Medicine xxx xxxx xxx in older patients who are already susceptible to AF and sympa thetic nervous system overactivity could all account for such a high incidence of this arrhythmia in this particular population [ ] Guidance on acute management of AF In cases of AF associated hemodynamic compromise as done in all cases of hemodynamically unstable arrhythmias synchronized direct cur rent cardioversion should be used to restore sinus rhythm [] In all other cases one needs to initially proceed with a rate control strategy with use of a betablocker when there is no contraindi cation eg bronchospasm acute heart failure a calcium channel blocker in the absence of heart failure andor digoxin In cases of heart failure digoxin andor amiodarone may be used to achieve rate control For newonset AF within the last hours restoring sinus rhythm is the next target This can be achieved with use of class IA IC or III antiarrhythmic drugs AADs with the selection of the appropriate agent made as based on the presence where only amiodarone seems to be relatively safe or absence of under lying structural heart disease where all other options are available with the caveats being detailed in the discussion that follows be low also taking into account drug interactions with COVID phar macotherapies that are in use A major concern in using specific AADs relates to the baseline measurement of the QT interval and the coadministration of QTprolonging drugs see discussion be low Most importantly all AF patients should be receiving prophy lactic anticoagulation therapy with intravenous heparin Impact of national lockdown on newonset AF diagnosis An other aspect of the impact of national COVID19 lockdowns on the diagnosis of AF has been recently reported by a Danish study [] Using Danish registries the number of patients receiving a new onset AF diagnosis during the first months of and was compared A lower incidence of newonset AF during the weeks of lockdown was noted compared with the corresponding weeks in incidence rate ratios RRs for the weeks and There was a drop in total numbers vs [] Patients diagnosed during lockdown were younger and with a lower CHA2DS2VASc score while history of cancer heart fail ure and vascular disease were more prevalent During lockdown patients with newonset AF suffered an ischemic stroke and died compared with and pa tients during the corresponding period respectively The au thors concluded that following a national lockdown in Denmark a drop in registered newonset AF cases was observed indicat ing that the risk of undiagnosed AF patients developing complica tions could potentially translate into poorer outcomes in patients with AF during the COVID19 pandemic Ventricular arrhythmias In the setting of acute myocardial injury and acute myocarditis in patients with COVID19 infection various and serious ventricu lar arrhythmias VAs may occur [] Other important triggers in clude the severe respiratory insufficiency and the systemic ‚am mation incurred by COVID19 infection as well as the proarrhyth mic effects of COVID therapies and other drug interactions and also the autonomic imbalance superimposed in patients afflicted by the disease [ ] Furthermore hypoxemia which is common in these patients and electrolyte disturbances occurring for various reasons in this group of patients may aggravate arrhythmogenic ity Depending on preexisting or currently emerging CV disease various VAs may be encountered including ventricular premature complexes VPCs nonsustained VT NSVT and sustained VTVF Special attention is required for the development of polymorphic VT in the form of torsade des pointes TdP in the setting of QT prolongation either preexisting or acquired and induced by drugs especially when combination therapies are employed that are po tentially proarrhythmic [] Acute myocardial injury noted in of COVID19 patients can be the inciting factor for various VAs [ ] Among pa tients with confirmed COVID19 malignant VAs VTVF developed in patients during hospitalization patients with ele vated cTn levels had more frequent malignant arrhythmias vs [] A recent retrospective cohort study of patients with severe COVID19 indicated that among having a cTn level measured on admission with showing cardiac in jury arrhythmias developed in of the patients in cluding patients with VT or VF all of whom died [] Critically ill COVID19 patients often have comorbidities that can increase the risk for malignant VAs These include electrolyte abnormalities hypokalemia hypomagnesemia fever an ‚am matory state and most importantly COVID19 pharmacotherapies that are potentially proarrhythmic as they prolong the QT interval and may thus trigger TdP and sudden cardiac death SCD [] On the other hand the acute myocardial injury induced by the virus could also independently prolong the QT interval According to a recent report of a Kawasakilike syndrome temporally associated with COVID19 infection in children among whom myocardi tis was diagnosed in patients left ventricular ejection fractionLVEF range “ of these patients displayed im portant ECG changes that included QT interval prolongation and occasional VAs not attributable to any QTprolonging drug [] Inhospital cardiac arrest As mentioned among patients hospitalized with COVID19 infection all cardiac arrests occurred among patients admitted to the ICU [] In a retrospective cohort study inhospital VTVF occurred in of patients with cardiac injury all of whom died [] Outofhospital cardiac arrest OOHCA A recent Italian study compared all the consecutive outofhospital cardiac arrests OOHCA in the months following the first documented case of COVID19 in the region with those which occurred in the same time frame in [] The cumulative incidence of COVID19 from February to April in the study territory was COVID19100 inhabitants and the cumulative incidence of OOHCA was cases100 inhabitants with a increase as compared with OOHCAs in vs in p The authors concluded that the increase in OOHCAs in is significantly correlated to the COVID19 pandemic and is coupled with a reduction in shortterm outcome A French study comparing the OOHCAs of the pandemic period to the mean of the total over weeks in the non pandemic period indicated that the maximum weekly OOHCA in cidence increased from to per million inhabitants p before returning to normal in the final weeks of the pandemic period [] There was a higher rate of OOHCA at home less bystander cardiopulmonary resus vs p citation vs p and shockable rhythm vs and longer delays to intervention median p min vs min p The proportion of OOHCA patients ad mitted alive decreased from to p in the pan demic period After adjustment for confounders the pandemic pe riod remained significantly associated with lower survival rate at hospital admission odds ratio p COVID19 infection accounted for about one third of the increase in OOHCA incidence during the pandemic Druginduced prolongation of QTc interval and torsade des pointes Several agents employed for treating COVID19 infection may prolong the QT interval and lead to polymorphic VT in the form of TdP Table Chloroquinehydroxychloroquine and azithromycin which have been recently used for potential prophylaxis or treat ment for COVID19 infection are listed as definite causes of TdP Please cite this as AS Manolis AA Manolis and TA Manolis COVID19 infection and cardiac arrhythmias Trends in Cardiovascular Medicine 101016jtcm202008002 0cJID TCM IN PRESS [m5G August ] AS Manolis AA Manolis and TA Manolis Trends in Cardiovascular Medicine xxx xxxx xxx Table QTProlonging Drugs in COVID19 Infection Antibiotics Antiviral agents Anesthetics Antiemetics Antiarrhythmics Antipsychotics ChloroquineHydroxychloroquine Macrolides Azithromycin Quinolones LopinavirRitonavir Favipiravir Tocilizumab Fingolimod Propofol Domperidone Class IA Class III Haloperidol at crediblemeds [] According with the FDA azithromycin other macrolides and fluoroquinolones can cause lethal arrhyth mias as a potential consequence of QTinterval prolongation [] Chloroquine hydroxychloroquine Chloroquine CQ and hydroxychloroquine HCQ have been used for treatment and prophylaxis of malaria while they have also been employed for treatment of amebiasis that is occurring out side the gastrointestinal tract rheumatoid arthritis and lupus ery thematosus These agents were also found to have antiviral effects and have been proposed for the treatment of COVID19 infection [] However both these agents can be proarrhythmic by pro longing the QT interval and potentially initiating lifethreatening VAs including TdP they can also cause QRS widening Chloroquine interacts with multiple cardiac ion channels including the human etheragogorelated gene hERG potassium channel a reduction in hERG channel potassium current is the main cause of acquired druginduced long QT syndrome Recent experimental data indi cated that HCQ markedly increases the action potential dispersion and results in the development of repolarization alternans and ini tiates polymorphic VT [] Preliminary findings from a recent study suggested that the QTc prolonging effect of CQ is dosedependent [] Among pa tients enrolled with COVID19 infection were allocated to highdosage group ie mg CQ bid for days and to lowdosage group ie mg bid on day and qd for days Lethality until day was in the highdosage group of and in the lowdosage group of The highdosage group presented more instance of QTc interval ms compared with the lowdosage group Respiratory secre tion at day was negative in only of patients The authors suggested that the higher CQ dosage should not be rec ommended for critically ill patients with COVID19 because of its potential safety hazards especially when taken concurrently with azithromycin and oseltamivir A recent disproportionality analysis of HCQassociated CV ad verse reactions using the FDA adverse event reporting system FAERS database of datasets and patient records indicated that HCQ was associated with higher reporting odds ratios ROR of TdP ROR CI to complete atrioventricular AV block ROR CI to and QT prolongation ROR CI to [] QT prolongation and TdP are more frequent with high doses for a comparatively short period and represent the most common HCQassociated side effects A systematic review of data on COVID19 patients showed that of COVID19 patients treated with CQHCQ developed QT prolongation [] Ventricular arrhythmias developed in COVID patients from a group of treated with highdose CQ The authors suggest daily ECG monitoring and other risk mitigation strategies to be adopted in order to prevent possible arrhythmic sideeffects Macrolide antibiotics Azithromycin AZM also can cause modest QT interval pro longation but not through potent hERG channel blockade rather when used chronically through an increase in peak and late cardiac sodium current to cause potential loading of cardiomyocytes with sodium and calcium to produce calcium overload Advanced age and female gender are considered risk factors [] Azithromycin can also provoke nonpause“dependent polymorphic VT in the ab sence of QT prolongation [ ] After reviewing the data of AZM regarding risk of QT prolonga tion and associated TdP the FDA revised AZM product labels ad vising against its use in patients with known risk factors such as QTinterval prolongation hypokalemia hypomagnesemia bradycar dia or use of certain QTprolonging antiarrhythmic agents includ ing class IA eg quinidine and procainamide and class III eg dofetilide amiodarone and sotalol agents [] Antiviral agents The combined antiviral regimen of ritonavirlopinavir approved for human immunodeficiency virus HIV infection was also con sidered to be able to suppress SARSCoV2 replication [ ] Lopinavir is metabolized by the hepatic cytochrome P450 sys tem CYP3A [] it also inhibits drug transporters such as Pglycoprotein Pgp [] Thus ritonavirlopinavir may increase plasma concentrations of drugs primarily metabolized by CYP3A or substrates of these drug transporters Ritonavirlopinavir may require dose reductions or avoidance of CYP3Amediated drugs such as rivaroxaban and apixaban Ritonavirlopinavir has also been shown to cause QT and PR interval prolongation or occasionally second or thirddegree AV block particularly in patients with un derlying structural heart disease and preexisting conduction sys tem abnormalities [] Due to its competitive inhibition of the RNAdependent RNA polymerase favipiravir is being evaluated in treating patients with COVID19 alone or in combination therapies the risk for QT inter val prolongation by favipiravir is considered to be low [] Other agents Fingolimod is an immunomodulator and immuno suppressant which reduces lymphocyte migration and is used in the treatment of multiple sclerosis [] it has been proposed as a potential adjuvant therapeutic agent against COVID19 [] Fin golimod has Ltype calcium channel blockade effect causing pro longation of PR RR and QT interval It also activates acetylcholine dependent potassium channels IKach in sinoatrial node causing dosedependent bradycardia [] Thus fingolimod increases the risk of bradycardia and heart block through Ltype calcium channel and IKach blockade [] Combined therapies Treatments employed for COVID19 may increase arrhythmia risk particularly the risk for VAs through drug interactions Drug combinations can lead to greater prolongation of cellular action potential duration analogous to QT prolongation compared with single drug therapies [] The combination effect can result from both pharmacokinetic and pharmacodynamic drug interactions Importantly females with preexisting CV disease seem to be more susceptible to druginduced arrhythmias compared to males with CV disease or healthy persons of either gender Please cite this as AS Manolis AA Manolis and TA Manolis COVID19 infection and cardiac arrhythmias Trends in Cardiovascular Medicine 101016jtcm202008002 0cJID TCM IN PRESS [m5G August ] AS Manolis AA Manolis and TA Manolis Trends in Cardiovascular Medicine xxx xxxx xxx Table Measures to Prevent Arrhythmias in Patients with COVID19 Infection ¢ Withhold QT prolonging drugs in patients with baseline QTc ¢ Withdraw QTprolonging drugs when QTc increases to ¢ Do not use chloroquinehydroxychloroquine azithromycin other macrolides fluoroquinolones lopinavirritonavir or favipiravir in patients with known risk factors such as prolonged QTc hypokalemia hypomagnesemia bradycardia or concomitant use of certain QTprolonging antiarrhythmic drugs including class IA eg quinidine and procainamide and class III eg dofetilide amiodarone and sotalol agents ¢ Maintain K ¢ Monitor QTc via ECG or preferably via telemetry monitor or smart phone measurements ms compared to baseline measurement ms or if QTc is prolonged by ms or with known LQTS mEqL and Mg level to level to mgdL An online survey of electrophysiology professionals revealed that of respondents reported having to discontinue therapy with HCQ AZM due to significant QTc prolongation and reported cases of TdP in patients on HCQCQ and AZM [] Amiodarone was the most common antiarrhythmic drug used for VA management Among COVID19 positive suspected patients stud ± years male received AZM HCQ ied age ± and received both drugs [] Baseline mean QTc was ± ms p with medications Sig ms and increased to ± ms vs nificant prolongation was observed only in men ± ms in women p of patients reached critical ms or QTc QTc prolongation maximum QTc ‰¥ ms Changes in ‰¥ ms if QRS QTc were highest with the combination compared to either drug ± with much greater prolongation with combination vs AZM vs ‰¥ ms or QTc increase of No patients manifested TdP ‰¥ ms if QRS ± ms p ± ms p ± ms vs Another recent cohort study of patients treated for COVID with CQHCQ reported that patients received CQ received HCQ and also received AZM [] Although the maximum QTc during treatment was signifi cantly longer in the combination group vs the monotherapy group TdP was not ob served in the entire population and there were no arrhythmogenic deaths reported A study of COVID patients receiving combined HCQAZM therapy indicated longer QTcinterval than before ther ‰¥ ms had apy vs ms p higher values of transaminases p compared with those with ms [] At h Holter ECG monitoring COVID19 QTc patient and no control had No patients showed R on T VPCs Analysis of h QTc dynamics revealed that COVID19 patients had higher QTc values than controls with no significant hourly variability Therapy with HCQ and AZM pro longs QTc interval in patients with COVID19 particularly in those with high levels of transaminases ‰¥ run of NSVT p patients with a QTc Interestingly in nonCOVID patients a retrospective cohort study identified only two SCDVA events among com bination users CQHCQ plus AZM [] However the doses were lower in this study compared to doses used in COVIDpatients drugs were not used acutely in a hospital setting as currently done for COVID patients fewer cardiac patients received the drugs all suggesting an attenuated risk for cardiac arrhythmias in this par ticular cohort Nevertheless when all measures and precautions are taken Table the incidence of QT prolongation and the TdPevent rate may remain low In a recent study of patients with COVID ± years male HCQAZM was infection mean age ‰¤ 480ms and potassium level initiated only if baseline QTc was mmolL [] Two patients were not eligible for drug ± ms initiation QTc ± ms after h of combined therapy The and increased to treatment had to be stopped because of significant QTc prolonga tion in patients No druginduced TdP nor death was ob served In this specific population HCQAZM could not be initiated or had to be interrupted in ‰¥ ms Baseline average QTc was of the cases QTc monitoring Congenital long QT syndrome LQTS with a prevalence of in the general population may often be asymptomatic and if an ECG has not been recorded it will remain unknown to the affected person and the first manifestation may be SCD usually triggered by a drug [] Furthermore silent genetic variants or œforme fruste  of congenital LQTS encountered in of people may render a person vulnerable to QT prolongation TdP and SCD [] Therefore a large number of healthy individuals will be at an increased risk of a druginduced LQTS Data suggest that in African Americans may be at a higher risk of druginduced TdP during the COVID19 pandemic due to clustering of intrinsic genetic susceptibility ie exclusive oc currence of the proarrhythmic ion channel variant pSer1103Tyr SCN5A acquired risk factors eg electrolyte disturbances and QTcprolonging drug use and COVID19“specific risk factors eg profound hypoxemia and cytokine storm [] A heart ratecorrected QT QTc interval is measured with use ˆšof various formulas among which the Bazett™s correction formula is most commonly used QTc QT RRsec QTc is defined as pro longed when it exceeds ms in males and ms in females as measured preferably in lead II or V on a standard 12lead ECG [] A prolonged QTc predisposes to polymorphic VT in the form of TdP that may degenerate into VF and SCD For the wideQRS adjusted QTc methods that have been suggested include the JT ad justment obtained as QTc“QRS [] or subtracting of the QRS duration from the measured QT [] For patients receiving QTprolonging drugs it is imperative to monitor the QTc interval during treatment Table Traditionally this can be accomplished by obtaining a 12lead ECG however in the era of the COVID19 pandemic this poses a certain risk and puts considerable strain on medical personnel and the health sys tem [] Many telemetry systems are equipped with features of real time QTc monitoring and could be used in hospitalized pa tients and those managed in the ICU setting In addition smart phone heart monitors are also capable of providing remote accu rate QTc measurements [] In this context AliveCor has recently received clearance from the FDA to market the KardiaMobile6L device a previously FDAapproved device for AF detection for QTc monitoring of COVID19 patients treated with QT prolonging drugs such as CQHCQ [] Similarly the Apple Watch ECG an F

cancer233

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: "Cardiac arrhythmias Atrial fibrillation Sudden cardiac death Long QT syndrome Torsade des pointes Ventricular tachycardia Ventricular fibrillation As the coronavirus COVID19 pandemic marches unrelentingly more patients with cardiac arrhyth mias are emerging due to the effects of the virus on the respiratory and cardiovascular CV systems and the systemic ‚ammation that it incurs and also as a result of the proarrhythmic effects of COVID19 pharmacotherapies and other drug interactions and the associated autonomic imbalance that enhance ar rhythmogenicity The most worrisome of all arrhythmogenic mechanisms is the QT prolonging effect of various antiCOVID pharmacotherapies that can lead to polymorphic ventricular tachycardia in the form of torsade des pointes and sudden cardiac death It is therefore imperative to monitor the QT interval dur ing treatment however conventional approaches to such monitoring increase the transmission risk for the staff and strain the health system Hence there is dire need for contactless monitoring and teleme try for inpatients especially those admitted to the intensive care unit as well as for outpatients needing continued management In this context recent technological advances have ushered in a new era in im plementing digital health monitoring tools that circumvent these obstacles All these issues are herein discussed and a large body of recent relevant data are reviewed Elsevier Inc All rights reserved Introduction The ongoing pandemic of coronavirus disease COVID19 has created a global tumult [] According to current data of patients with COVID19 infection are afflicted by acute my ocardial injury with an attendant higher mortality rate compared with those without cardiac injury commensurate with the degree of cardiac troponin cTn elevation [“] Furthermore of patients develop cardiac arrhythmias Table including malig nant ventricular arrhythmias VAs [ ] with a higher prevalence noted in patients admitted to the intensive care unit ICU [] Importantly clinically stable patients may have a low preva Abbreviations AAD antiarrhythmic drug AF atrial fibrillation APCs atrial pre mature complexes AZM azithromycin COVID19 coronavirus CQ chloro quine cTn cardiac troponin CV cardiovascular CYP cytochrome P450 ECG elec trocardiogram HCQ hydroxychloroquine ICU intensive care unit LQTS long QT syndrome NSVT nonsustained ventricular tachycardia OOHCA outofhospital cardiac arrest SCD sudden cardiac death TdP torsade des pointes VAs ventricular arrhythmias VF ventricular fibrillation VPCs ventricular premature complexes VT ventricular tachycardia ˆ—Corresponding author Email address asmotenetgr AS Manolis lence of arrhythmias [] however critically ill patients are at much higher risk for cardiac arrhythmias [] Cardiac arrhythmias including lifethreatening VAs may be the consequence of direct effects of COVID19 infection but also of the deleterious effects of systemic illness and the adverse reactions to drugs employed in the treatment of this pandemic Table Fig [ “ ] A recent study indicated that among patients with ± years men African Ameri COVID19 mean age can receiving care in the ICU there were cardiac arrests incident atrial fibrillation AF episodes bradyarrhythmias and nonsustained ventricular tachycardias NSVTs [] Arrhythmias occurring in patients admitted to the ICU included cardiac arrests all events of cardiac arrest occurred in this group AF odds ratioOR vs those not in the ICU and NSVT OR Car diac arrests were associated with acute inhospital mortality Among patients with confirmed COVID19 ex hibited myocardial injury as indicated by elevated cardiac troponin T cTnT levels [] During hospitalization patients de veloped ventricular tachycardia VTventricular fibrillation VF patients with elevated cTnT levels had more frequent VAs vs p compared with those with normal cTnT levels A recent singleday snapshot survey of stable patients with 101016jtcm202008002 Elsevier Inc All rights reserved Please cite this as AS Manolis AA Manolis and TA Manolis COVID19 infection and cardiac arrhythmias Trends in Cardiovascular Medicine 101016jtcm202008002 0cJID TCM IN PRESS [m5G August ] AS Manolis AA Manolis and TA Manolis Trends in Cardiovascular Medicine xxx xxxx xxx Table Cardiac Arrhythmias Occurring in Patients with COVID19 Infection Sinus tachycardia Sinus bradycardia Conduction disturbances AVBBBB Atrial premature complexes Atrial fibrillation Supraventricular tachycardia Ventricular premature complexes Nonsustained ventricular tachycardia Polymorphic ventricular tachycardia Torsade des pointes Sustained ventricular tachycardia Ventricular fibrillation Pulseless electrical activity AVB atrioventricular block BBB bundle branch block Table Mechanisms of Arrhythmogenicity in Patients with COVID19 Infection Acute myocardial injury Myocarditis Hypoxia Systemic ‚ammation Autonomic imbalance SNS overactivity virusinduced vagal nerve injury Electrolyte abnormalities QT prolonging drugs antiCOVID pharmacotherapies AADs other agents Drugdrug interactions Cardiovascular comorbidities hypertension coronary artery disease cardiomyopathy AADs antiarrhythmic drugs SNS In this review we present current data about the whole spec trum of cardiac arrhythmias encountered in patients with COVID infection either attributable to the effect of the virus itself on the cardiovascular CV and the respiratory system andor to the effects of the treatments that these patients receive in combina tion with autonomic imbalance that is incurred by this unrelenting pandemic Acute myocardial injury and arrhythmias As mentioned in patients with evidence of acute myocardial injury the prevalence of cardiac arrhythmias is higher compared to patients without myocardial injury [] In a recent retrospec tive cohort study among patients with severe COVID19 had a cTnI level measured upon hospital admission of whom had positive results showing cardiac injury [] In patients with cardiac injury mortality was higher compared to patients without cardiac injury vs p Arrhythmias were found in of the patients with cardiac injury includ ing patients with VT or VF all of whom died [] A recent meta analysis of studies including COVID19 patients showed that patients with cardiac injury and newly occurring arrhythmias were at higher risk of developing severe disease or requiring ICU admission relative riskRR p [] sympathetic nervous system Sinus tachycardia Sinus tachycardia is the most common rhythm disturbance in patients with COVID19 infection due to multiple reasons such as fever respiratory insufficiencyhypoxemia hemodynamic compro mise fearanxiety pain and several other physical and emotional symptoms [] Bradycardiaconduction disturbances According to a retrospective series of patients transient si nus bradycardia lasting days is a possible manifestation of COVID19 hence another reason for close monitoring [] There may be many reasons for bradycardia but severe hypoxia ‚am matory injury of the sinus node by circulating cytokines and exag gerated response to medications are possible triggers Interestingly bradycardia has been suggested as a warning sign of the onset of a serious cytokine storm Fig The schema illustrates the various arrhythmias encountered in patients with COVID19 infection as a consequence of the virus infection affecting the heart and lung andor producing systemic ‚ammation the adverse proarrhythmic effects of COVID therapies and the drugdrug interactions that may occur see text for long QT discussion AF interval PEA pulseless electrical activity SB sud sinus tachycardia sympathetic nervous system STach den cardiac death SNS ventricular arrhythmias VF TdP ventricular fibril lation VT atrioventricular block LQT torsade des pointes VAs sinus bradycardia SCD atrial fibrillation AVB ventricular tachycardia COVID19 showed a incidence of arrhythmias limited to AF in and supraventricular tachycardia SVT in patients [] A Heart Rhythm Society HRS online survey of electrophysiology professionals n indicated that AF was the most commonly reported tachyarrhythmia whereas severe sinus bradycardia and complete heart block were the most common brad yarrhythmias in hospitalized patients with COVID19 [] Ven tricular tachycardiaVF arrest and pulseless electrical activity were reported by and of respondents respectively A meta analysis of retrospective cohort studies comprising pa tients with COVID19 showed that the pooled incidence was for cardiac arrhythmia for cardiac arrest [] p According to a retrospective cohort study of COVID19 pa tients multivariable logistic regression indicated that among other ECG abnormalities the presence of one or more atrial premature contractions APCs odds ratio OR a right bun dle branch block RBBB or intraventricular block IVB OR p increased the odds of death [] Another study analyzing the ECGs of COVID19 patients showed that abnormal PR interval behavior paradoxical prolonga tion or lack of shortening with increasing heart rate was associ and need ated with increased risk of death vs p for endotracheal intubation vs p compared to patients with PR interval shortening [] Atrial fibrillation According to a recent survey of electrophysiology professionals atrial fibrillation AF was the most commonly encountered car diac arrhythmia observed in patients with COVID19 infection [] Several mechanisms could be involved in the pathogenesis of AF in these patients virusinduced cardiac injury that could lead to perimyocarditis hypoxemia frequently occurring in these patients systemic infection common occurrence of the COVID19 infection Please cite this as AS Manolis AA Manolis and TA Manolis COVID19 infection and cardiac arrhythmias Trends in Cardiovascular Medicine 101016jtcm202008002 0cJID TCM IN PRESS [m5G August ] AS Manolis AA Manolis and TA Manolis Trends in Cardiovascular Medicine xxx xxxx xxx in older patients who are already susceptible to AF and sympa thetic nervous system overactivity could all account for such a high incidence of this arrhythmia in this particular population [ ] Guidance on acute management of AF In cases of AF associated hemodynamic compromise as done in all cases of hemodynamically unstable arrhythmias synchronized direct cur rent cardioversion should be used to restore sinus rhythm [] In all other cases one needs to initially proceed with a rate control strategy with use of a betablocker when there is no contraindi cation eg bronchospasm acute heart failure a calcium channel blocker in the absence of heart failure andor digoxin In cases of heart failure digoxin andor amiodarone may be used to achieve rate control For newonset AF within the last hours restoring sinus rhythm is the next target This can be achieved with use of class IA IC or III antiarrhythmic drugs AADs with the selection of the appropriate agent made as based on the presence where only amiodarone seems to be relatively safe or absence of under lying structural heart disease where all other options are available with the caveats being detailed in the discussion that follows be low also taking into account drug interactions with COVID phar macotherapies that are in use A major concern in using specific AADs relates to the baseline measurement of the QT interval and the coadministration of QTprolonging drugs see discussion be low Most importantly all AF patients should be receiving prophy lactic anticoagulation therapy with intravenous heparin Impact of national lockdown on newonset AF diagnosis An other aspect of the impact of national COVID19 lockdowns on the diagnosis of AF has been recently reported by a Danish study [] Using Danish registries the number of patients receiving a new onset AF diagnosis during the first months of and was compared A lower incidence of newonset AF during the weeks of lockdown was noted compared with the corresponding weeks in incidence rate ratios RRs for the weeks and There was a drop in total numbers vs [] Patients diagnosed during lockdown were younger and with a lower CHA2DS2VASc score while history of cancer heart fail ure and vascular disease were more prevalent During lockdown patients with newonset AF suffered an ischemic stroke and died compared with and pa tients during the corresponding period respectively The au thors concluded that following a national lockdown in Denmark a drop in registered newonset AF cases was observed indicat ing that the risk of undiagnosed AF patients developing complica tions could potentially translate into poorer outcomes in patients with AF during the COVID19 pandemic Ventricular arrhythmias In the setting of acute myocardial injury and acute myocarditis in patients with COVID19 infection various and serious ventricu lar arrhythmias VAs may occur [] Other important triggers in clude the severe respiratory insufficiency and the systemic ‚am mation incurred by COVID19 infection as well as the proarrhyth mic effects of COVID therapies and other drug interactions and also the autonomic imbalance superimposed in patients afflicted by the disease [ ] Furthermore hypoxemia which is common in these patients and electrolyte disturbances occurring for various reasons in this group of patients may aggravate arrhythmogenic ity Depending on preexisting or currently emerging CV disease various VAs may be encountered including ventricular premature complexes VPCs nonsustained VT NSVT and sustained VTVF Special attention is required for the development of polymorphic VT in the form of torsade des pointes TdP in the setting of QT prolongation either preexisting or acquired and induced by drugs especially when combination therapies are employed that are po tentially proarrhythmic [] Acute myocardial injury noted in of COVID19 patients can be the inciting factor for various VAs [ ] Among pa tients with confirmed COVID19 malignant VAs VTVF developed in patients during hospitalization patients with ele vated cTn levels had more frequent malignant arrhythmias vs [] A recent retrospective cohort study of patients with severe COVID19 indicated that among having a cTn level measured on admission with showing cardiac in jury arrhythmias developed in of the patients in cluding patients with VT or VF all of whom died [] Critically ill COVID19 patients often have comorbidities that can increase the risk for malignant VAs These include electrolyte abnormalities hypokalemia hypomagnesemia fever an ‚am matory state and most importantly COVID19 pharmacotherapies that are potentially proarrhythmic as they prolong the QT interval and may thus trigger TdP and sudden cardiac death SCD [] On the other hand the acute myocardial injury induced by the virus could also independently prolong the QT interval According to a recent report of a Kawasakilike syndrome temporally associated with COVID19 infection in children among whom myocardi tis was diagnosed in patients left ventricular ejection fractionLVEF range “ of these patients displayed im portant ECG changes that included QT interval prolongation and occasional VAs not attributable to any QTprolonging drug [] Inhospital cardiac arrest As mentioned among patients hospitalized with COVID19 infection all cardiac arrests occurred among patients admitted to the ICU [] In a retrospective cohort study inhospital VTVF occurred in of patients with cardiac injury all of whom died [] Outofhospital cardiac arrest OOHCA A recent Italian study compared all the consecutive outofhospital cardiac arrests OOHCA in the months following the first documented case of COVID19 in the region with those which occurred in the same time frame in [] The cumulative incidence of COVID19 from February to April in the study territory was COVID19100 inhabitants and the cumulative incidence of OOHCA was cases100 inhabitants with a increase as compared with OOHCAs in vs in p The authors concluded that the increase in OOHCAs in is significantly correlated to the COVID19 pandemic and is coupled with a reduction in shortterm outcome A French study comparing the OOHCAs of the pandemic period to the mean of the total over weeks in the non pandemic period indicated that the maximum weekly OOHCA in cidence increased from to per million inhabitants p before returning to normal in the final weeks of the pandemic period [] There was a higher rate of OOHCA at home less bystander cardiopulmonary resus vs p citation vs p and shockable rhythm vs and longer delays to intervention median p min vs min p The proportion of OOHCA patients ad mitted alive decreased from to p in the pan demic period After adjustment for confounders the pandemic pe riod remained significantly associated with lower survival rate at hospital admission odds ratio p COVID19 infection accounted for about one third of the increase in OOHCA incidence during the pandemic Druginduced prolongation of QTc interval and torsade des pointes Several agents employed for treating COVID19 infection may prolong the QT interval and lead to polymorphic VT in the form of TdP Table Chloroquinehydroxychloroquine and azithromycin which have been recently used for potential prophylaxis or treat ment for COVID19 infection are listed as definite causes of TdP Please cite this as AS Manolis AA Manolis and TA Manolis COVID19 infection and cardiac arrhythmias Trends in Cardiovascular Medicine 101016jtcm202008002 0cJID TCM IN PRESS [m5G August ] AS Manolis AA Manolis and TA Manolis Trends in Cardiovascular Medicine xxx xxxx xxx Table QTProlonging Drugs in COVID19 Infection Antibiotics Antiviral agents Anesthetics Antiemetics Antiarrhythmics Antipsychotics ChloroquineHydroxychloroquine Macrolides Azithromycin Quinolones LopinavirRitonavir Favipiravir Tocilizumab Fingolimod Propofol Domperidone Class IA Class III Haloperidol at crediblemeds [] According with the FDA azithromycin other macrolides and fluoroquinolones can cause lethal arrhyth mias as a potential consequence of QTinterval prolongation [] Chloroquine hydroxychloroquine Chloroquine CQ and hydroxychloroquine HCQ have been used for treatment and prophylaxis of malaria while they have also been employed for treatment of amebiasis that is occurring out side the gastrointestinal tract rheumatoid arthritis and lupus ery thematosus These agents were also found to have antiviral effects and have been proposed for the treatment of COVID19 infection [] However both these agents can be proarrhythmic by pro longing the QT interval and potentially initiating lifethreatening VAs including TdP they can also cause QRS widening Chloroquine interacts with multiple cardiac ion channels including the human etheragogorelated gene hERG potassium channel a reduction in hERG channel potassium current is the main cause of acquired druginduced long QT syndrome Recent experimental data indi cated that HCQ markedly increases the action potential dispersion and results in the development of repolarization alternans and ini tiates polymorphic VT [] Preliminary findings from a recent study suggested that the QTc prolonging effect of CQ is dosedependent [] Among pa tients enrolled with COVID19 infection were allocated to highdosage group ie mg CQ bid for days and to lowdosage group ie mg bid on day and qd for days Lethality until day was in the highdosage group of and in the lowdosage group of The highdosage group presented more instance of QTc interval ms compared with the lowdosage group Respiratory secre tion at day was negative in only of patients The authors suggested that the higher CQ dosage should not be rec ommended for critically ill patients with COVID19 because of its potential safety hazards especially when taken concurrently with azithromycin and oseltamivir A recent disproportionality analysis of HCQassociated CV ad verse reactions using the FDA adverse event reporting system FAERS database of datasets and patient records indicated that HCQ was associated with higher reporting odds ratios ROR of TdP ROR CI to complete atrioventricular AV block ROR CI to and QT prolongation ROR CI to [] QT prolongation and TdP are more frequent with high doses for a comparatively short period and represent the most common HCQassociated side effects A systematic review of data on COVID19 patients showed that of COVID19 patients treated with CQHCQ developed QT prolongation [] Ventricular arrhythmias developed in COVID patients from a group of treated with highdose CQ The authors suggest daily ECG monitoring and other risk mitigation strategies to be adopted in order to prevent possible arrhythmic sideeffects Macrolide antibiotics Azithromycin AZM also can cause modest QT interval pro longation but not through potent hERG channel blockade rather when used chronically through an increase in peak and late cardiac sodium current to cause potential loading of cardiomyocytes with sodium and calcium to produce calcium overload Advanced age and female gender are considered risk factors [] Azithromycin can also provoke nonpause“dependent polymorphic VT in the ab sence of QT prolongation [ ] After reviewing the data of AZM regarding risk of QT prolonga tion and associated TdP the FDA revised AZM product labels ad vising against its use in patients with known risk factors such as QTinterval prolongation hypokalemia hypomagnesemia bradycar dia or use of certain QTprolonging antiarrhythmic agents includ ing class IA eg quinidine and procainamide and class III eg dofetilide amiodarone and sotalol agents [] Antiviral agents The combined antiviral regimen of ritonavirlopinavir approved for human immunodeficiency virus HIV infection was also con sidered to be able to suppress SARSCoV2 replication [ ] Lopinavir is metabolized by the hepatic cytochrome P450 sys tem CYP3A [] it also inhibits drug transporters such as Pglycoprotein Pgp [] Thus ritonavirlopinavir may increase plasma concentrations of drugs primarily metabolized by CYP3A or substrates of these drug transporters Ritonavirlopinavir may require dose reductions or avoidance of CYP3Amediated drugs such as rivaroxaban and apixaban Ritonavirlopinavir has also been shown to cause QT and PR interval prolongation or occasionally second or thirddegree AV block particularly in patients with un derlying structural heart disease and preexisting conduction sys tem abnormalities [] Due to its competitive inhibition of the RNAdependent RNA polymerase favipiravir is being evaluated in treating patients with COVID19 alone or in combination therapies the risk for QT inter val prolongation by favipiravir is considered to be low [] Other agents Fingolimod is an immunomodulator and immuno suppressant which reduces lymphocyte migration and is used in the treatment of multiple sclerosis [] it has been proposed as a potential adjuvant therapeutic agent against COVID19 [] Fin golimod has Ltype calcium channel blockade effect causing pro longation of PR RR and QT interval It also activates acetylcholine dependent potassium channels IKach in sinoatrial node causing dosedependent bradycardia [] Thus fingolimod increases the risk of bradycardia and heart block through Ltype calcium channel and IKach blockade [] Combined therapies Treatments employed for COVID19 may increase arrhythmia risk particularly the risk for VAs through drug interactions Drug combinations can lead to greater prolongation of cellular action potential duration analogous to QT prolongation compared with single drug therapies [] The combination effect can result from both pharmacokinetic and pharmacodynamic drug interactions Importantly females with preexisting CV disease seem to be more susceptible to druginduced arrhythmias compared to males with CV disease or healthy persons of either gender Please cite this as AS Manolis AA Manolis and TA Manolis COVID19 infection and cardiac arrhythmias Trends in Cardiovascular Medicine 101016jtcm202008002 0cJID TCM IN PRESS [m5G August ] AS Manolis AA Manolis and TA Manolis Trends in Cardiovascular Medicine xxx xxxx xxx Table Measures to Prevent Arrhythmias in Patients with COVID19 Infection ¢ Withhold QT prolonging drugs in patients with baseline QTc ¢ Withdraw QTprolonging drugs when QTc increases to ¢ Do not use chloroquinehydroxychloroquine azithromycin other macrolides fluoroquinolones lopinavirritonavir or favipiravir in patients with known risk factors such as prolonged QTc hypokalemia hypomagnesemia bradycardia or concomitant use of certain QTprolonging antiarrhythmic drugs including class IA eg quinidine and procainamide and class III eg dofetilide amiodarone and sotalol agents ¢ Maintain K ¢ Monitor QTc via ECG or preferably via telemetry monitor or smart phone measurements ms compared to baseline measurement ms or if QTc is prolonged by ms or with known LQTS mEqL and Mg level to level to mgdL An online survey of electrophysiology professionals revealed that of respondents reported having to discontinue therapy with HCQ AZM due to significant QTc prolongation and reported cases of TdP in patients on HCQCQ and AZM [] Amiodarone was the most common antiarrhythmic drug used for VA management Among COVID19 positive suspected patients stud ± years male received AZM HCQ ied age ± and received both drugs [] Baseline mean QTc was ± ms p with medications Sig ms and increased to ± ms vs nificant prolongation was observed only in men ± ms in women p of patients reached critical ms or QTc QTc prolongation maximum QTc ‰¥ ms Changes in ‰¥ ms if QRS QTc were highest with the combination compared to either drug ± with much greater prolongation with combination vs AZM vs ‰¥ ms or QTc increase of No patients manifested TdP ‰¥ ms if QRS ± ms p ± ms p ± ms vs Another recent cohort study of patients treated for COVID with CQHCQ reported that patients received CQ received HCQ and also received AZM [] Although the maximum QTc during treatment was signifi cantly longer in the combination group vs the monotherapy group TdP was not ob served in the entire population and there were no arrhythmogenic deaths reported A study of COVID patients receiving combined HCQAZM therapy indicated longer QTcinterval than before ther ‰¥ ms had apy vs ms p higher values of transaminases p compared with those with ms [] At h Holter ECG monitoring COVID19 QTc patient and no control had No patients showed R on T VPCs Analysis of h QTc dynamics revealed that COVID19 patients had higher QTc values than controls with no significant hourly variability Therapy with HCQ and AZM pro longs QTc interval in patients with COVID19 particularly in those with high levels of transaminases ‰¥ run of NSVT p patients with a QTc Interestingly in nonCOVID patients a retrospective cohort study identified only two SCDVA events among com bination users CQHCQ plus AZM [] However the doses were lower in this study compared to doses used in COVIDpatients drugs were not used acutely in a hospital setting as currently done for COVID patients fewer cardiac patients received the drugs all suggesting an attenuated risk for cardiac arrhythmias in this par ticular cohort Nevertheless when all measures and precautions are taken Table the incidence of QT prolongation and the TdPevent rate may remain low In a recent study of patients with COVID ± years male HCQAZM was infection mean age ‰¤ 480ms and potassium level initiated only if baseline QTc was mmolL [] Two patients were not eligible for drug ± ms initiation QTc ± ms after h of combined therapy The and increased to treatment had to be stopped because of significant QTc prolonga tion in patients No druginduced TdP nor death was ob served In this specific population HCQAZM could not be initiated or had to be interrupted in ‰¥ ms Baseline average QTc was of the cases QTc monitoring Congenital long QT syndrome LQTS with a prevalence of in the general population may often be asymptomatic and if an ECG has not been recorded it will remain unknown to the affected person and the first manifestation may be SCD usually triggered by a drug [] Furthermore silent genetic variants or œforme fruste  of congenital LQTS encountered in of people may render a person vulnerable to QT prolongation TdP and SCD [] Therefore a large number of healthy individuals will be at an increased risk of a druginduced LQTS Data suggest that in African Americans may be at a higher risk of druginduced TdP during the COVID19 pandemic due to clustering of intrinsic genetic susceptibility ie exclusive oc currence of the proarrhythmic ion channel variant pSer1103Tyr SCN5A acquired risk factors eg electrolyte disturbances and QTcprolonging drug use and COVID19“specific risk factors eg profound hypoxemia and cytokine storm [] A heart ratecorrected QT QTc interval is measured with use ˆšof various formulas among which the Bazett™s correction formula is most commonly used QTc QT RRsec QTc is defined as pro longed when it exceeds ms in males and ms in females as measured preferably in lead II or V on a standard 12lead ECG [] A prolonged QTc predisposes to polymorphic VT in the form of TdP that may degenerate into VF and SCD For the wideQRS adjusted QTc methods that have been suggested include the JT ad justment obtained as QTc“QRS [] or subtracting of the QRS duration from the measured QT [] For patients receiving QTprolonging drugs it is imperative to monitor the QTc interval during treatment Table Traditionally this can be accomplished by obtaining a 12lead ECG however in the era of the COVID19 pandemic this poses a certain risk and puts considerable strain on medical personnel and the health sys tem [] Many telemetry systems are equipped with features of real time QTc monitoring and could be used in hospitalized pa tients and those managed in the ICU setting In addition smart phone heart monitors are also capable of providing remote accu rate QTc measurements [] In this context AliveCor has recently received clearance from the FDA to market the KardiaMobile6L device a previously FDAapproved device for AF detection for QTc monitoring of COVID19 patients treated with QT prolonging drugs such as CQHCQ [] Similarly the Apple Watch ECG an F Answer:

Thyroid_Cancer

structures assigned to the products were concordant with the microanalytical andspectral data Compounds 4e18 were screened for their ability to induce the antioxidant enzyme NADPH quinone oxidoreductase NQO1 in cells a classical target for transcription factor nuclear factorerythroid268diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioN345trimethoxyphenyl acetamide showed the most potent NQO1inducer activity in vitro Compound had low toxicity in mice LD50 ¼ mgkg It also reduced thedamaging effects of gamma radiation as assessed by the levels of Nrf2 NQO1 reactive oxygen speciesROS and malondialdehyde MDA in liver tissues In addition compound showed amelioration in thecomplete blood count of irradiated mice and enhanced survival over a period of days followingirradiation Molecular docking of inside the Nrf2binding site of Kelchlike ECH associated protein Keap1 the main negative regulator of Nrf2 showed the same binding interactions as that of the cocrystallized ligand considering the binding possibilities and energy scores These findings suggest thatcompound could be considered as a promising antioxidant and radiomodulatory agent The Authors Published by Elsevier Masson SAS This is an access under the CC BYlicense httpcreativecommonslicensesby40 IntroductionThe extensive use of radiotherapy and the damage caused to thesurrounding normal ans have provoked researchers to find newstrategies to protect normal tissues from radiation hazards []The risk of injury from radiation can diminish the value of radiotherapy and contribute to complications for longterm cancer survivors [] Ionizing radiation interrupts cell functions throughradiolysis of water and the production of reactive oxygen speciesROS or reactive nitrogen species RNS [] Excessive productionof ROS and RNS promotes oxidative stress which can affect allcellular components including single or double DNA strand breaks Corresponding authorEmail address mmsghorabyahoocom MM Ghorab[] This ROSmediated toxicity can lead to mutations and consequently cause cardiovascular neurological toxicities and sexualdysfunction as well as cancer [7e10] In order to reduce theseradiationinduced side effects radioprotective drugs are used []Also the use of multitarget antioxidants that act as radioprotectorscan help limit normal tissue damage caused by ionizing radiation[12e14]Nuclear factor erythroid 2related factor Nrf2 is a transcription factor that regulates the expression of various antioxidantproteins to protect against oxidative damage in the cell [] Theabundance of Nrf2 is negatively regulated by Kelchlike ECH associated protein Keap1 a substrate adaptor for a Cullin3Rbx1ubiquitin ligase that binds and continuously targets Nrf2 for ubiquitination and proteasomal degradation [16e18] Under conditionsof oxidative stress redoxsensitive cysteine sensors of Keap1 aremodified leading to loss of its ability to target Nrf2 for degradation101016jejmech2020112467 The Authors Published by Elsevier Masson SAS This is an access under the CC BY license httpcreativecommonslicensesby40 0cAM Soliman European Journal of Medicinal Chemistry consequently Nrf2 transports into the nucleus where it initiates thetranscription of its downstream target genes such as NADPHquinone oxidoreductase1 NQO1 []Quinazolinone is a strategic scaffold that has a wide range ofpharmacological activities such as antioxidant anti‚ammatoryand anticancer activities [20e23] Sulfonamides in addition totheir use as antibiotics [24e27] have many pharmacological activities and can be used as antiviral [] anti‚ammatory []antioxidant [] and anticancer agents [32e35] These versatilepharmacological activities make the two chemical classes excellentcandidates for developing new multitarget agents through a slightalteration in the structure that might lead to diversity in the biological activity []In addition numerous studies haverevealed iodine to be a potent antioxidant with higher potency thanthat of ascorbic acid [] Iodine can act as an electron donor that 0fquenches ROS such as OHand H2O2 [] or decreases thedamaging effects of ROS thus increasing the total antioxidant status in human serum []In this context it seemed of interest to search for new compounds with the ability to scavenge ROS and protect cells A seriesof new 68diiodoquinazolin43Hone conjugated to benzenesulfonamide was synthesized by the introduction of the sulfonamide group at the N3 of quinazolinone with the incorporation ofvarying acetamide terminal aimed at exploring the potential antioxidant and radioprotective activity The antioxidant potential ofthe target compounds was first measured using a quantitative androbust NQO1 inducer activity bioassay in cells Acute toxicity studyfor the most active compound was then performed in vivo A nontoxic dose was subsequently selected to investigate the potentialprotective effect against wholebody gamma irradiationinducedoxidative stress in experimental mice All groups were observed days after irradiation for survival and weight changes Additionally molecular docking was performed inside the Nrf2bindingsite of Keap1 to gain insights into the molecular interactions andpossible mode of action Results and discussion Chemistry wasreactionofpreparedfrom theScheme shows the synthesis of thioacetamide quinazolinonebenzenesulfonamide derivatives 5e18 The starting material 68diiodo2mercapto4 oxoquinazolin34Hyl benzenesulfonamideisothiocyanatobenzenesulfonamide [] and 2amino35diiodobenzoic acid The coupling of with the 2chloroNsubstituted acetamide in dry acetone and anhydrous K2CO3 yieldedthe corresponding 268diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioNsubstituted acetamide 5e18 IRspectra of 5e18 displayed additional NH CH2 aliphatic and CObands at their specified regions 1H NMR spectra of 5e18 revealedthe acetamide group through the presence of two singlets one at417e431 ppm referring to the CH2and the other at966e1121 ppm attributed to the NH protons with the disappearance of SH singlet of at ppm 13C NMR of 5e18 exhibited twosignals peculiar to the CH2 and CO carbons 1H NMR spectra of 6e8displayed singlets at and ppm assigned to the CH3group at the ortho meta and parapositions of the phenyl group 13CNMR of 6e8 showed signals at and ppm for theCH3 group 1H NMR spectra of 9e11 revealed triplets at and ppm attributed to the CH3 ethyl and quartet at and ppm referring to the CH2 ethyl at the ortho meta and parapositions 13C NMR of 9e11 showed two signals at due to CH3 ethyl and due to the CH2 ethylgroups respectively 1H NMR spectra of revealed singlet at ppm attributed to the OCH3 protons while 13C NMR of showed a signal at ppm due to the OCH3 carbon 1H NMRspectra of revealed triplet at ppm and quartet at ppmdue to the ethoxy group 1H NMR spectra of revealed a singlet at ppm due to the 2OCH3 protons while revealed two singletsat and ppm due to the 3OCH3 protons IR of 16e18 showedNO2 bands Biological activityIn vitro screeningThe antioxidant activity of compounds 4e18 was screened usingthe NQO1 inducer activity assay The Concentration of the novelcompounds to Double the specific enzyme activity of NQO1 CDvalue was used as a measure of inducer potency and results obtained are presented in Fig Table Evaluation of the NQO1inducer activity showed that compounds and wereinactive whereas compounds and had activityhowever CD value was not reached Compounds CD ¼ mMand CD ¼ mM showed concentrationdependent induceractivity These diiodoquinazolinones represent a new chemicalclass of NQO1 inducers thus adding to the existing knowledge ofthe diversity of the many chemical scaffolds that have been reported to induce this antioxidant enzyme The classical NQO1 inducers are primarily oxidants and electrophiles or othercompounds that react or are metabolized to products that reactand chemically modify cysteine sensors of Keap1 [] A newgeneration of NQO1 inducers is also emerging that of noncovalentsmallmolecule modulators of the Keap1eNrf2 proteinproteininteraction [44e46] Because our diiodoquinazolinones havesome common features with the Keap1eNrf2 proteinproteininteraction inhibitors in this study we tested the potential abilityof these compounds to directly disrupt the binding of Keap1 to Nrf2by molecular modeling see section In vivo evaluation Determination of toxicity lethal dose fifty LD50 of compound The most promising compound was investigatedin vivo for acute toxicity LD50 in albino mice and the value wasfound to be mgkg body weight ip Subsequently onetenthof this dose was selected as the therapeutic dose for further evaluation of the potential radioprotective effects of compound Evaluation of the radiomodulatory effect of compound inmice Four groups of mice were used the first group served ascontrol the second group was irradiated at a dose of Gy as a singledose the third group was injected ip with compound only for consecutive days and the last group received compound thenexposed to Gy of gamma radiation After days from irradiationfive mice were checked for liver and hematopoietic system toxicities The residual mice in all groups were monitored over daysto evaluate the survival rate and body weight changes The effect of compound on radiationinduced livertoxicity Gamma radiationinduced hepatic oxidative stress asshown by a significant increase in hepatic levels of nuclear Nrf213fold NQO1 32fold ROS 15fold and the lipid peroxidation product malondialdehyde MDA 2fold as compared to nonirradiated control mice This was in agreement with other studies[]Ionizing radiation is believed to induce damage through thegeneration of ROS resulting in an imbalance in the oxidantantioxidant ratio in cells [] In the current experiment the presenceof ROSmediated damage was confirmed by the increase in MDAlevels in irradiated liver in addition to the increase in the expression of the enzymatic antioxidant system Moreover these results 0cAM Soliman European Journal of Medicinal Chemistry Scheme The synthetic pathways for the development of the diiodoquinazolinone derivatives 4e18support the notion that Nrf2 is an initial regulator of cellular responses to radiation exposure [] Once Nrf2 translocates to thenucleus it induces expression of endogenous antioxidant enzymessuch as NQO1 [] a flavoprotein involved in cellular protectionagainst oxidative stress []Treatment of nonirradiated mice with compound led to anincrease in NQO1 and ROS levels and a decrease in Nrf2 with nosignificant change in MDA level as compared to normal nonirradiated mice Fig A significant increase in Nrf2 levels aswell decrease in the levels of NQO1 ROS and MDA was observed in irradiated mice livers treated with compound when compared to the group subjected to radiation aloneFig Moreover treatment with compound improved bothsurvival and body weight of the animals following irradiation 0cAM Soliman European Journal of Medicinal Chemistry Additionally it has been reported that Nrf2 modifies ROS production partly by regulating NQO1 expression [] On the other handthe NQO1 levels were significantly higher than the nonirradiatedcontrols in agreement with the cell culture results this studyNotably the increased levels of ROS in nonirradiated mice treatedwith compound are consistent with the increased levels of ROSfollowing genetic Nrf2 activation by Keap1 knockdown []Importantly however the increased ROS production that accompanies NQO1 induction does not lead to damage as evidenced bythe lack of increase in the levels of MDA this study The effect of compound on the hematopoietic systemTo examine the possible role of compound in protecting thehematopoietic system against irradiation we measured the peripheral blood cell counts of red blood cells RBCs white bloodcells WBCs hemoglobin HGB and platelets PLT The irradiatedmice exhibited a significant decrease in RBCs WBCs HGB and PLTcompared with the control group Fig These results are mainlyattributed to the fact that irradiation causes the formation of freeradicals which initiate a chain of events leading to the decline in thelevels of hematological parameters [] Indeed it has been wellestablished that gamma irradiation induces RBC injury includingmorphological and quantitative changes of RBCs These alternations may be partly attributed to radiationinduced oxidative stressin RBCs Exposure to radiation results in the formation of reactiveoxygen species ROS and reactive nitrogen species RNS as well asDNA damage which can then lead to severe injury to the hematopoietic system [] This is in harmony with Wang []who stated that injury to the hematopoietic system is the mostcommon injury induced by irradiation This was attributed to theeffect of ionizing radiation on hematopoietic stem cells and hematopoietic progenitor cells which are principally responsible forhematopoietic recovery Treatment of irradiated mice with compound ameliorated the decrease in peripheral blood cellsparticularly RBCs HGB and PLT Hence the antioxidant propertiesof compound may contribute to the amelioration of RBC countsand HGB in irradiated mice This is consistent with other studies forantioxidants effects on the hematopoietic system [] Thismight be explained through the promotion effect of radioprotectorsto proliferate hematopoietic stem cells and they also could increasethe levels of leukocyte growth factors [] Besides severalpotent radioprotectors protect various membrane systems as wellas hematopoietic stem cells from peroxidative damages thatFig Concentration dependence of the NQO1 inducer activity of compounds 4e18Fig without affecting the liver weight Fig as compared toirradiated mice The present results indicate that compound hasan antioxidant capacity as the treatment of irradiated mice with prevents oxidative stress reducing the increase in lipid peroxidation markers and maintaining the expression of Nrf2 comparedwith the irradiated group suggesting improved hepatic antioxidantcapacity Hence compound validated its radiomodulatory andantioxidant effect through its main structure quinazolinone andsulfonamide that goes in line with Soliman [] Also thisfinding was reinforced by Cuadrado and his colleagues whoemphasized the importance of therapeutic targeting for Nrf2because of its resourceful cytoprotective roles against a plethora ofdiseases that are associated with oxidative stress []At the same time it was found that NQO1 expression levels ofirradiated mice treated with were significantly lower ascompared to vehicletreated irradiated ones but still significantlyhigher than normal levels Interestingly the levels of NQO1 in allexperimental groups correlate with the levels of ROS suggestingROS involvement in the NQO1 induction The lower levels of NQO1and ROS in the irradiated group that also received could be theresults of increased antioxidant capacity due to Nrf2 activation []Table NQO1 inducer activity and CD values of compounds 4e18Conc mMCompound noCDaNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNR means not recordeda CD values are the averages of three independent experiments each with eight replicate wells of cells and SD for each data point was within of the value 0cAM Soliman European Journal of Medicinal Chemistry Fig Effect of compound on A Nrf2 B NQO1 C ROS and D MDA levels in liver of nonirradiated control and irradiated mice after days of irradiation The results wereexpressed as mean ± SE Statistical analysis was carried out by oneway ANOVA followed by Bonferroni€™s multiple comparison test significantly different from control group significantly different from irradiated group at p n ¼ happened after irradiation so it could protect blood componentsagainst irradiation [] Taken all together these results demonstrate the protective effect of compound against gammaradiation Molecular dockingMolecular docking was performed to assess the ability ofcompound to block the Kelch domain of Keap1 Through its Kelchdomain Keap1 binds to Nrf2 promoting its degradation resultingin low cytoprotective gene levels [] The PDB file 4IQK was obtained from the Protein Data Bank The binding site of Kelch domainhas been reported to have five subpockets P1 P2 P3 P4 and P5[] P1 and P2 are positively charged pockets that contain thearginine triad Arg Arg and Arg This triad is crucialfor the selectivity of the molecular recognition together with a 0cAM Soliman European Journal of Medicinal Chemistry Fig A Survival percent and B Body weight changes of control irradiated compound and compound þ irradiated mice through days after irradiation Theresults were expressed as mean ± SE n ¼ Statistical analysis was carried out byKaplanMeier method followed by the ManteleCox test for survival analysis Bodyweight changes between groups were analyzed by twoway ANOVA followed byBonferroni€™s post test significantly different from control group significantlydifferent from irradiated group at p group of hydrophobic residues contributes to the stability of thecomplex P1 is formed by residues Arg Ile Gly Phe Arg and Ser P2 is formed by Ser Arg Asn andAsn P3 is a neutrally charged pocket composed of Gly Ser Ala Gly Ser and Gly P4 is formed by Tyr Gln and Tyr whereas P5 is formed by Tyr and Phe The main interactions observed by the cocrystallized ligand NN0naphthalene14diylbis4methoxybenzenesulfonamidearetwo cationpi interaction with Arg piepi interaction with Tyr and two hydrogen bonds with Ser and Ser with S ¼ kcalmol Fig Compound showed the same key interactions exhibited by the cocrystallized ligand Compound S ¼ kcalmol RMSD ¼ has adopted a conformationallowing the presence of two cationpi interaction between Arg and the aromatic rings in addition to a hydrogen bond with themethoxy group Fig three hydrogen bonds made by ser andArg towards the methoxy groups and another hydrogen bondbetween Leu and NH2 group of the sulfonamide Superimposition between compound and the cocrystallized ligand showedthat they adopt the same orientation inside the binding site Fig Finally compound possessing the highest NQO1 inducer activityCD ¼ mM in this series showed the same interactions and thesame orientation of the native ligand inside the receptor indicatinga possible correlation between those multiple interactions and thenoted higher potency Based on the abovementioned resultscompound could possibly bind to Keap1 and disrupt its interaction with Nrf2The results from this study complement previous reportsshowing that the classical electrophilic Nrf2 activator sulforaphaneprotects cells including human retinal pigment epithelial cellskeratinocytes and mouse leukemia cells against oxidative damageFig Effect of compound on relative liver weight in nonirradiated control andirradiated mice after days of irradiation The results were expressed as mean ± SEn ¼ Statistical analysis was carried out by oneway ANOVA followed by Bonferroni€™s multiple comparison test There were no significant differences between groupscaused by oxidative stressors of four different types namelymenadione tertbutyl hydroperoxide 4hydroxynonenal and peroxynitrite as well as by exposure to ultraviolet radiation []Furthermore unlike the effects of most direct antioxidants theindirect antioxidant effect of sulforaphane which results from Nrf2activation persists for several days after sulforaphane is no longerpresent in the cell culture medium This is because direct antioxidants such as ascorbic acid tocopherols carotenoids and polyphenols which neutralize ROS and other chemical oxidants areconsumed in these reactions whereas Nrf2 activation results intranscriptional upregulation of antioxidant defences which aremediated by proteins with long halflives often several days Thenew compounds generated in the current study have an additionaladvantage in that they are nonelectrophilic and are therefore expected to have a broader therapeutic window compared to electrophilic Nrf2 activators This is supported by the very low toxicityof compound in mice Taken together these results demonstratethe powerful effect of Nrf2 activation and induction of NQO1 inprotecting cells and animals against high levels of ROS and preventing ROSmediated damage This is of particular relevance toprotecting the hematopoietic system which is highly sensitive toROS Conclusiontheacetamide268diiodo4oxo34sulfamoylphenyl3In summary a hybridization strategy was adopted using theiodinated quinazolinone scaffold and sulfonamide moiety to producedihydroquinazolin2ylthioNsubstitutedderivatives 5e18 Different substitutions were introduced to theacetamide group to study the structureactivity relationship All thecompounds were screened for their antioxidant potential using theNQO1 inducer activity assay The 345trimethoxyphenyl derivative showed the highestinducer activity in this seriesCD ¼ mM and had low toxicity LD50 ¼ mgkg Treatmentof gammairradiated mice with compound lowered oxidativestress as evidenced by the lower levels of MDA ROS and NQO1 inliver Furthermore compound ameliorated the complete bloodpicture of irradiated mice as well as enhanced the survival of mice 0cAM Soliman European Journal of Medicinal Chemistry Fig Effect of compound on A RBCs B WBCs C HGB concentration and D PLT counts in nonirradiated control and irradiated mice after days of irradiation The resultswere expressed as mean ± SE n ¼ Statistical analysis was carried out by oneway ANOVA followed by Bonferroni€™s multiple comparison test significantly different fromcontrol group significantly different from irradiated groupover a period of days postirradiation Molecular docking of inside the active site of Keap1 confirmed that it binds in the samemanner as that of the cocrystallized ligands The inducer activity ofcompound in upregulating NQO1 strongly suggests that it couldbe used as a lead antioxidant and radiomodulatory agent for furtheroptimization of the quinazolinone scaffold Materials and methods ChemistryAll chemicals were purchased from SigmaAldrich and are of ARgrade Melting points were determined in capillary on aGallen Kamp melting point apparatus Sanyo Gallen Kamp UKThin layer chromatography using precoated silica gel plates Kieselgel mm F254 Merck Germany was performed with asolvent system of chloroformmethanol to detect the spots byIR spectra KBr disc were recorded using an FTIRUV lightspectrophotometer Perkin Elmer USA NMR spectra were scannedon NMR spectrophotometer Bruker AXS Inc Switzerland operating at MHz for 1H and MHz for 13C Mass spectra wererecorded on the ISQ LT Thermo Scientific GCMS model Massachusetts USA Chemical shifts are expressed in dvalues ppmrelative to TMS as an internal standard using DMSO€‘d6 as a solventElemental analyses were done on a model CHNSO analyserPerkin Elmer USA All the values were within ± of thetheoretical values 8diiodo2mercapto4 oxoquinazolin34Hylbenzenesulfonamide A mixture of 2amino35diiodobenzoic acid g mol and isothiocyanatobenzenesulfonamide g mol in absolute ethanol mL containing drops of triethylamine was refluxed for h The solid product formed wascollected by filtration and crystallized from ethanol to give 00 NH2 Yield mp 0eC IR KBr ʋ cm 0cAM Soliman European Journal of Medicinal Chemistry the NN0naphthalene14diylbis4Fig 2D and 3D interaction poses ofmethoxybenzenesulfonamide showing cationp pp interaction and hydrogenbonds with the key amino acids inside the binding pocket arom CO CN SO2 1H NMRDMSO€‘d6 d ppm s 1H d 2H J ¼ Hz AB d2H J ¼ Hz AB d 1H J ¼ Hz d 1H J ¼ Hz s2H 13C NMR DMSO€‘d6 d ppm Anal Calcd for C14H9I2N3O3S2 C H N Found C H N 34Dihydroquinazolinsulfonamide derivatives General procedure A mixture of g mol and chloroNsubstituted acetamide derivatives mol in dryacetone mL and anhydrous K2CO3 g mol was stirredat room temperature for h filtered and the solid product formedwas crystallized from dioxane to give 5e18 268Diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioNphenylacetamide Yield 00 NH NH2 mp 0eC IR KBr ʋ cmarom aliph 2CO CN SO2 1H NMR DMSO€‘d6 d ppm s 2H 703e730 m 3H760e783 m 4H s 2H d 2H J ¼ Hz AB d1H J ¼ Hz d 1H J ¼ Hz s 1H 13C NMR DMSO€‘d6d ppm þ ] [Mþ1 MS mz [] [M] [] Anal Calcd for C22H16I2N4O4S2 C Fig 2D and 3D interaction pose of compound showing cationp pp interactionsinside the binding pocket of 4IQKH N Found C H N 268Diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioNotolylacetamide Yield 00 NH NH2 mp 0eC IR KBr ʋ cmarom aliph 2CO CN SO2 1H NMR DMSO€‘d6 d ppm s 3H s 2H ddd 1H J ¼ Hz 730e755 m 3H d 2H J ¼ HzAB s 2H d 2H J ¼ Hz AB d 1H J ¼ Hz d 1H J ¼ Hz s 1H 13C NMR DMSO€‘d6 d ppm Anal Calcd for C23H18I2N4O4S2 C H N Found C H N 268Diiodo 4oxo34sulfamoylphenyl34 Yielddihydroquinazolin2ylthioNmtolylacetamide 00 NH NH2 mp 0eC IR KBr ʋ cm arom aliph 2CO CN SO2 1H NMR DMSO€‘d6 d ppm s 3H s 2H 0cAM Soliman European Journal of Medicinal Chemistry Hz 721e748 m 2H 770e804 m 5H s 2H d 1HJ ¼ Hz d 1H J ¼ Hz s 1H 13C NMR DMSO€‘d6 dppm Anal CalcdforC24H20I2N4O4S2 C H N Found C H N acetamide 268Diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioN4ethylphenyl 00 Yield mp 0eC IR KBr ʋ cmNH NH2 arom aliph 2CO CN SO2 1H NMR DMSO€‘d6 d ppm t 3H J ¼ Hz q 2H J ¼ Hz s 2H d 2H J ¼ Hz AB d 2H J ¼ Hz AB d 2H J ¼ Hz 780e805 m 4H d 1H J ¼ Hz d 1H J ¼ Hz s 1H 13C NMRDMSO€‘d6 d ppm Anal Calcd forC24H20I2N4O4S2 C H N Found C H N 268Diiodo 4oxo34sulfamoylphenyl3 dihydroquinazolin2ylthioN 4methoxyphenyl acetamide 00 Yield mp 0eC IR KBr ʋ cmNH NH2 arom aliph 2CO CN SO2 1H NMR DMSO€‘d6 d ppm s 3H s 2H d 2H J ¼ Hz AB d 2H J ¼ Hz AB d2H J ¼ Hz AB d 2H J ¼ Hz s 2H d 1HJ ¼ Hz d 1H J ¼ Hz s 1H 13C NMR DMSO€‘d6d ppm Anal Calcdfor C23H18I2N4O5S2 C H N Found C H N acetamide 268Diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioN4ethoxyphenyl 00 Yield mp 0eC IR KBr ʋ cmNH NH2 arom aliph 2CO CN SO2 1H NMR DMSO€‘d6 d ppm t 3H J ¼ Hz q 2H J ¼ Hz s 2H d 2H J ¼ Hz AB d 2H J ¼ Hz AB d 2H J ¼ Hz AB 803e810 m4H d 1H J ¼ Hz d 1H J ¼ Hz s 1H 13CNMR DMSO€‘d6 d ppm Anal Calcdfor C24H20I2N4O5S2 C H N Found C H N 268Diiodo4oxo34sulfamoylphenyl34acetamidedihydroquinazolin2ylthioN35dimethoxyphenyl 00 Yield mp 0eC IR KBr ʋ cm NH NH2 arom aliph 2CO CN SO2 1H NMR DMSO€‘d6 d ppm s6H s 2H dd 1H J ¼ Hz dd 2H J ¼ Hz d 2H J ¼ Hz AB d 2H J ¼ Hz AB s2H d 1H J ¼ Hz d 1H J ¼ Hz s 1H 13CNMR DMSO€‘d6 d ppm Anal CalcdforC24H20I2N4O6S2 C H N Found C H N 268Diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioN345trimethoxyphenyl acetamideFig Superimposition of compound magenta and the cocrystallized ligand redshowed that they adopt the same orientation inside the receptor For interpretation ofthe references to color in this figure legend the reader is referred to the Web version ofthis m 1H 731e756 m 3H d 2H J ¼ Hz AB d2H J ¼ Hz AB s 2H d 1H J ¼ Hz d 1HJ ¼ Hz s 1H 13C NMR DMSO€‘d6 d ppm Anal Calcd for C23H18I2N4O4S2 C H N Found C H N 268Diiodo4oxo34sulfamoylphenyl34Yielddihydroquinazolin2ylthioNptolylacetamide 00 NH NH2 mp 0eC IR KBr ʋ cm arom aliph 2CO CN SO2 1H NMR DMSO€‘d6 d ppm s 3H s 2H d 2H J ¼ Hz AB m 2H d 2H J ¼ Hz AB d 2H J ¼ Hz AB s 2H d 1H J ¼ Hz d 1HJ ¼ Hz s 1H 13C NMR DMSO€‘d6 d ppm þ MS mz [] [M ] [] Anal Calcd forC23H18I2N4O4S2 C H N Found C H N acetamide 268Diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioN2ethylphenyl 00 NH Yield mp 0eC IR KBr ʋ cmNH2 arom aliph 2CO1619 CN SO2 1H NMR DMSO€‘d6 d ppm t 3H J ¼ Hz q 2H J ¼ Hz s 2H dd 1H J ¼ Hz m 1H ddd 1H J ¼ Hz dd 1H J ¼ Hz d 2H J ¼ Hz AB d 2H J ¼ Hz AB m 2H d1H J ¼ Hz d 1H J ¼ Hz s 1H 13C NMRDMSO€‘d6 d ppm MS mz []þ ] [Mþ1 ] [] Anal Calcd for [MC24H20I2N4O4S2 C H N Found C H N 268Diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioN3ethylphenyl 00 Yield mp 0eC IR KBr ʋ cmNH NH2 arom aliph 2CO CN SO2 1H NMR DMSO€‘d6 d ppm t 3H J ¼ Hz q 2H J ¼ Hz s 2H ddd 1H J ¼ acetamide 0c 00 Yield mp 0eC IR KBr ʋ cm NH NH2 arom aliph 2CO CN SO2 1H NMR DMSO€‘d6 d ppm s 6H s 3H s 2H d 2H J ¼ Hz d 2HJ ¼ Hz AB s 2H d 2H J ¼ Hz d 1HJ ¼ Hz AB d 1H J ¼ Hz s 1H 13C NMRDMSO€‘d6 d ppm MS mz [] þ[M ] [] Anal Calcd for C25H22I2N4O7S2 C H N Found C H N 268Diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioN2methyl4nitrophenyl acetamide 00 Yield mp 0eC IR KBr ʋ cm NH NH2 arom aliph 2CO CN NO2 SO2 1H NMR DMSO€‘d6 dppm s 3H s 2H d 1H J ¼ Hz d 2HJ ¼ Hz AB 790e805 m 6H d 1H J ¼ Hz d 1HJ ¼ Hz s 1H 13C NMR DMSO€‘d6 d ppm Anal Calcd for C23H17I2N5O6S2 C H N Found C H N N2methyl6nitrophenyl 8Diiodo4oxo34sulfamoylphenyl3 dihydroquinazolin2ylthioacet 00 amide Yield mp 0eC IR KBr ʋ cm NH NH2 arom aliph 2CO CN NO2 SO2 1H NMRDMSO€‘d6 d ppm s 3H s 2H dd 1H J ¼ Hz dd 1H J ¼ Hz d 2H J ¼ Hz 801e810m 5H d 1H J ¼ Hz d 1H J ¼ Hz s 1H13C NMR DMSO€‘d6 d ppm MS mz [] þ[M ] [] Anal Calcd for C23H17I2N5O6S2 C H N Found C H N 8Diiodo4oxo34sulfamoylphenyl3 dihydroquinazolin2ylthio N24dinitrophenyl acetamide 00 Yield mp 0eC IR KBr ʋ cmNH NH2 arom aliph 2CO CN NO2 SO2 1H NMR DMSO€‘d6 dppm s 2H d 2H J ¼ Hz AB d 1H J ¼ Hz800e804 m 2H d 2H J ¼ Hz AB d 1H J ¼ Hz830e834 m 3H s 1H 13C NMR DMSO€‘d6 d ppm Anal Calcd for C22H14I2N6O8S2 C H N Found C H N Biological evaluation NQO1 in vitro inducer activityHepa1c1c7 murine hepatoma cells were grown in a humidifiedatmosphere at 0eC CO2 The cells were tested routinely toensure that they were mycoplasmafree The aminimum essentialmedium aMEM supplemented with vv heat andcharcoalinactivated g100 mL min at 0eC fetal bovineserum was used For evaluation of the potential NQO1 inducer activity cells 104well were grown in transparent flatbottomplastic 96well plates for h after which the cell culture medium was replaced with fresh medium containing each inducerdissolved in DMSO and diluted in the medium and theAM Soliman European Journal of Medicinal Chemistry cells were grown for further h Three replicates of each treatment of eight serial dilutions of inducers were used The final DMSOconcentration in the cell culture medium was maintained at vv in all wells Cell lysates were prepared in digitonin and thespecific activity of NQO1 was determined using menadione as asubstrate as described [] Briefly the cell culture medium wasremoved from each well and the cells were washed three timeswith mL of phosphate buffered saline PBS and subsequentlylysed in mL of digitonin suspension in the presence of EDTA for min with shaking Of the cell lysate mL was transferred to t

cancer234

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: structures assigned to the products were concordant with the microanalytical andspectral data Compounds 4e18 were screened for their ability to induce the antioxidant enzyme NADPH quinone oxidoreductase NQO1 in cells a classical target for transcription factor nuclear factorerythroid268diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioN345trimethoxyphenyl acetamide showed the most potent NQO1inducer activity in vitro Compound had low toxicity in mice LD50 ¼ mgkg It also reduced thedamaging effects of gamma radiation as assessed by the levels of Nrf2 NQO1 reactive oxygen speciesROS and malondialdehyde MDA in liver tissues In addition compound showed amelioration in thecomplete blood count of irradiated mice and enhanced survival over a period of days followingirradiation Molecular docking of inside the Nrf2binding site of Kelchlike ECH associated protein Keap1 the main negative regulator of Nrf2 showed the same binding interactions as that of the cocrystallized ligand considering the binding possibilities and energy scores These findings suggest thatcompound could be considered as a promising antioxidant and radiomodulatory agent The Authors Published by Elsevier Masson SAS This is an access under the CC BYlicense httpcreativecommonslicensesby40 IntroductionThe extensive use of radiotherapy and the damage caused to thesurrounding normal ans have provoked researchers to find newstrategies to protect normal tissues from radiation hazards []The risk of injury from radiation can diminish the value of radiotherapy and contribute to complications for longterm cancer survivors [] Ionizing radiation interrupts cell functions throughradiolysis of water and the production of reactive oxygen speciesROS or reactive nitrogen species RNS [] Excessive productionof ROS and RNS promotes oxidative stress which can affect allcellular components including single or double DNA strand breaks Corresponding authorEmail address mmsghorabyahoocom MM Ghorab[] This ROSmediated toxicity can lead to mutations and consequently cause cardiovascular neurological toxicities and sexualdysfunction as well as cancer [7e10] In order to reduce theseradiationinduced side effects radioprotective drugs are used []Also the use of multitarget antioxidants that act as radioprotectorscan help limit normal tissue damage caused by ionizing radiation[12e14]Nuclear factor erythroid 2related factor Nrf2 is a transcription factor that regulates the expression of various antioxidantproteins to protect against oxidative damage in the cell [] Theabundance of Nrf2 is negatively regulated by Kelchlike ECH associated protein Keap1 a substrate adaptor for a Cullin3Rbx1ubiquitin ligase that binds and continuously targets Nrf2 for ubiquitination and proteasomal degradation [16e18] Under conditionsof oxidative stress redoxsensitive cysteine sensors of Keap1 aremodified leading to loss of its ability to target Nrf2 for degradation101016jejmech2020112467 The Authors Published by Elsevier Masson SAS This is an access under the CC BY license httpcreativecommonslicensesby40 0cAM Soliman European Journal of Medicinal Chemistry consequently Nrf2 transports into the nucleus where it initiates thetranscription of its downstream target genes such as NADPHquinone oxidoreductase1 NQO1 []Quinazolinone is a strategic scaffold that has a wide range ofpharmacological activities such as antioxidant anti‚ammatoryand anticancer activities [20e23] Sulfonamides in addition totheir use as antibiotics [24e27] have many pharmacological activities and can be used as antiviral [] anti‚ammatory []antioxidant [] and anticancer agents [32e35] These versatilepharmacological activities make the two chemical classes excellentcandidates for developing new multitarget agents through a slightalteration in the structure that might lead to diversity in the biological activity []In addition numerous studies haverevealed iodine to be a potent antioxidant with higher potency thanthat of ascorbic acid [] Iodine can act as an electron donor that 0fquenches ROS such as OHand H2O2 [] or decreases thedamaging effects of ROS thus increasing the total antioxidant status in human serum []In this context it seemed of interest to search for new compounds with the ability to scavenge ROS and protect cells A seriesof new 68diiodoquinazolin43Hone conjugated to benzenesulfonamide was synthesized by the introduction of the sulfonamide group at the N3 of quinazolinone with the incorporation ofvarying acetamide terminal aimed at exploring the potential antioxidant and radioprotective activity The antioxidant potential ofthe target compounds was first measured using a quantitative androbust NQO1 inducer activity bioassay in cells Acute toxicity studyfor the most active compound was then performed in vivo A nontoxic dose was subsequently selected to investigate the potentialprotective effect against wholebody gamma irradiationinducedoxidative stress in experimental mice All groups were observed days after irradiation for survival and weight changes Additionally molecular docking was performed inside the Nrf2bindingsite of Keap1 to gain insights into the molecular interactions andpossible mode of action Results and discussion Chemistry wasreactionofpreparedfrom theScheme shows the synthesis of thioacetamide quinazolinonebenzenesulfonamide derivatives 5e18 The starting material 68diiodo2mercapto4 oxoquinazolin34Hyl benzenesulfonamideisothiocyanatobenzenesulfonamide [] and 2amino35diiodobenzoic acid The coupling of with the 2chloroNsubstituted acetamide in dry acetone and anhydrous K2CO3 yieldedthe corresponding 268diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioNsubstituted acetamide 5e18 IRspectra of 5e18 displayed additional NH CH2 aliphatic and CObands at their specified regions 1H NMR spectra of 5e18 revealedthe acetamide group through the presence of two singlets one at417e431 ppm referring to the CH2and the other at966e1121 ppm attributed to the NH protons with the disappearance of SH singlet of at ppm 13C NMR of 5e18 exhibited twosignals peculiar to the CH2 and CO carbons 1H NMR spectra of 6e8displayed singlets at and ppm assigned to the CH3group at the ortho meta and parapositions of the phenyl group 13CNMR of 6e8 showed signals at and ppm for theCH3 group 1H NMR spectra of 9e11 revealed triplets at and ppm attributed to the CH3 ethyl and quartet at and ppm referring to the CH2 ethyl at the ortho meta and parapositions 13C NMR of 9e11 showed two signals at due to CH3 ethyl and due to the CH2 ethylgroups respectively 1H NMR spectra of revealed singlet at ppm attributed to the OCH3 protons while 13C NMR of showed a signal at ppm due to the OCH3 carbon 1H NMRspectra of revealed triplet at ppm and quartet at ppmdue to the ethoxy group 1H NMR spectra of revealed a singlet at ppm due to the 2OCH3 protons while revealed two singletsat and ppm due to the 3OCH3 protons IR of 16e18 showedNO2 bands Biological activityIn vitro screeningThe antioxidant activity of compounds 4e18 was screened usingthe NQO1 inducer activity assay The Concentration of the novelcompounds to Double the specific enzyme activity of NQO1 CDvalue was used as a measure of inducer potency and results obtained are presented in Fig Table Evaluation of the NQO1inducer activity showed that compounds and wereinactive whereas compounds and had activityhowever CD value was not reached Compounds CD ¼ mMand CD ¼ mM showed concentrationdependent induceractivity These diiodoquinazolinones represent a new chemicalclass of NQO1 inducers thus adding to the existing knowledge ofthe diversity of the many chemical scaffolds that have been reported to induce this antioxidant enzyme The classical NQO1 inducers are primarily oxidants and electrophiles or othercompounds that react or are metabolized to products that reactand chemically modify cysteine sensors of Keap1 [] A newgeneration of NQO1 inducers is also emerging that of noncovalentsmallmolecule modulators of the Keap1eNrf2 proteinproteininteraction [44e46] Because our diiodoquinazolinones havesome common features with the Keap1eNrf2 proteinproteininteraction inhibitors in this study we tested the potential abilityof these compounds to directly disrupt the binding of Keap1 to Nrf2by molecular modeling see section In vivo evaluation Determination of toxicity lethal dose fifty LD50 of compound The most promising compound was investigatedin vivo for acute toxicity LD50 in albino mice and the value wasfound to be mgkg body weight ip Subsequently onetenthof this dose was selected as the therapeutic dose for further evaluation of the potential radioprotective effects of compound Evaluation of the radiomodulatory effect of compound inmice Four groups of mice were used the first group served ascontrol the second group was irradiated at a dose of Gy as a singledose the third group was injected ip with compound only for consecutive days and the last group received compound thenexposed to Gy of gamma radiation After days from irradiationfive mice were checked for liver and hematopoietic system toxicities The residual mice in all groups were monitored over daysto evaluate the survival rate and body weight changes The effect of compound on radiationinduced livertoxicity Gamma radiationinduced hepatic oxidative stress asshown by a significant increase in hepatic levels of nuclear Nrf213fold NQO1 32fold ROS 15fold and the lipid peroxidation product malondialdehyde MDA 2fold as compared to nonirradiated control mice This was in agreement with other studies[]Ionizing radiation is believed to induce damage through thegeneration of ROS resulting in an imbalance in the oxidantantioxidant ratio in cells [] In the current experiment the presenceof ROSmediated damage was confirmed by the increase in MDAlevels in irradiated liver in addition to the increase in the expression of the enzymatic antioxidant system Moreover these results 0cAM Soliman European Journal of Medicinal Chemistry Scheme The synthetic pathways for the development of the diiodoquinazolinone derivatives 4e18support the notion that Nrf2 is an initial regulator of cellular responses to radiation exposure [] Once Nrf2 translocates to thenucleus it induces expression of endogenous antioxidant enzymessuch as NQO1 [] a flavoprotein involved in cellular protectionagainst oxidative stress []Treatment of nonirradiated mice with compound led to anincrease in NQO1 and ROS levels and a decrease in Nrf2 with nosignificant change in MDA level as compared to normal nonirradiated mice Fig A significant increase in Nrf2 levels aswell decrease in the levels of NQO1 ROS and MDA was observed in irradiated mice livers treated with compound when compared to the group subjected to radiation aloneFig Moreover treatment with compound improved bothsurvival and body weight of the animals following irradiation 0cAM Soliman European Journal of Medicinal Chemistry Additionally it has been reported that Nrf2 modifies ROS production partly by regulating NQO1 expression [] On the other handthe NQO1 levels were significantly higher than the nonirradiatedcontrols in agreement with the cell culture results this studyNotably the increased levels of ROS in nonirradiated mice treatedwith compound are consistent with the increased levels of ROSfollowing genetic Nrf2 activation by Keap1 knockdown []Importantly however the increased ROS production that accompanies NQO1 induction does not lead to damage as evidenced bythe lack of increase in the levels of MDA this study The effect of compound on the hematopoietic systemTo examine the possible role of compound in protecting thehematopoietic system against irradiation we measured the peripheral blood cell counts of red blood cells RBCs white bloodcells WBCs hemoglobin HGB and platelets PLT The irradiatedmice exhibited a significant decrease in RBCs WBCs HGB and PLTcompared with the control group Fig These results are mainlyattributed to the fact that irradiation causes the formation of freeradicals which initiate a chain of events leading to the decline in thelevels of hematological parameters [] Indeed it has been wellestablished that gamma irradiation induces RBC injury includingmorphological and quantitative changes of RBCs These alternations may be partly attributed to radiationinduced oxidative stressin RBCs Exposure to radiation results in the formation of reactiveoxygen species ROS and reactive nitrogen species RNS as well asDNA damage which can then lead to severe injury to the hematopoietic system [] This is in harmony with Wang []who stated that injury to the hematopoietic system is the mostcommon injury induced by irradiation This was attributed to theeffect of ionizing radiation on hematopoietic stem cells and hematopoietic progenitor cells which are principally responsible forhematopoietic recovery Treatment of irradiated mice with compound ameliorated the decrease in peripheral blood cellsparticularly RBCs HGB and PLT Hence the antioxidant propertiesof compound may contribute to the amelioration of RBC countsand HGB in irradiated mice This is consistent with other studies forantioxidants effects on the hematopoietic system [] Thismight be explained through the promotion effect of radioprotectorsto proliferate hematopoietic stem cells and they also could increasethe levels of leukocyte growth factors [] Besides severalpotent radioprotectors protect various membrane systems as wellas hematopoietic stem cells from peroxidative damages thatFig Concentration dependence of the NQO1 inducer activity of compounds 4e18Fig without affecting the liver weight Fig as compared toirradiated mice The present results indicate that compound hasan antioxidant capacity as the treatment of irradiated mice with prevents oxidative stress reducing the increase in lipid peroxidation markers and maintaining the expression of Nrf2 comparedwith the irradiated group suggesting improved hepatic antioxidantcapacity Hence compound validated its radiomodulatory andantioxidant effect through its main structure quinazolinone andsulfonamide that goes in line with Soliman [] Also thisfinding was reinforced by Cuadrado and his colleagues whoemphasized the importance of therapeutic targeting for Nrf2because of its resourceful cytoprotective roles against a plethora ofdiseases that are associated with oxidative stress []At the same time it was found that NQO1 expression levels ofirradiated mice treated with were significantly lower ascompared to vehicletreated irradiated ones but still significantlyhigher than normal levels Interestingly the levels of NQO1 in allexperimental groups correlate with the levels of ROS suggestingROS involvement in the NQO1 induction The lower levels of NQO1and ROS in the irradiated group that also received could be theresults of increased antioxidant capacity due to Nrf2 activation []Table NQO1 inducer activity and CD values of compounds 4e18Conc mMCompound noCDaNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNR means not recordeda CD values are the averages of three independent experiments each with eight replicate wells of cells and SD for each data point was within of the value 0cAM Soliman European Journal of Medicinal Chemistry Fig Effect of compound on A Nrf2 B NQO1 C ROS and D MDA levels in liver of nonirradiated control and irradiated mice after days of irradiation The results wereexpressed as mean ± SE Statistical analysis was carried out by oneway ANOVA followed by Bonferroni€™s multiple comparison test significantly different from control group significantly different from irradiated group at p n ¼ happened after irradiation so it could protect blood componentsagainst irradiation [] Taken all together these results demonstrate the protective effect of compound against gammaradiation Molecular dockingMolecular docking was performed to assess the ability ofcompound to block the Kelch domain of Keap1 Through its Kelchdomain Keap1 binds to Nrf2 promoting its degradation resultingin low cytoprotective gene levels [] The PDB file 4IQK was obtained from the Protein Data Bank The binding site of Kelch domainhas been reported to have five subpockets P1 P2 P3 P4 and P5[] P1 and P2 are positively charged pockets that contain thearginine triad Arg Arg and Arg This triad is crucialfor the selectivity of the molecular recognition together with a 0cAM Soliman European Journal of Medicinal Chemistry Fig A Survival percent and B Body weight changes of control irradiated compound and compound þ irradiated mice through days after irradiation Theresults were expressed as mean ± SE n ¼ Statistical analysis was carried out byKaplanMeier method followed by the ManteleCox test for survival analysis Bodyweight changes between groups were analyzed by twoway ANOVA followed byBonferroni€™s post test significantly different from control group significantlydifferent from irradiated group at p group of hydrophobic residues contributes to the stability of thecomplex P1 is formed by residues Arg Ile Gly Phe Arg and Ser P2 is formed by Ser Arg Asn andAsn P3 is a neutrally charged pocket composed of Gly Ser Ala Gly Ser and Gly P4 is formed by Tyr Gln and Tyr whereas P5 is formed by Tyr and Phe The main interactions observed by the cocrystallized ligand NN0naphthalene14diylbis4methoxybenzenesulfonamidearetwo cationpi interaction with Arg piepi interaction with Tyr and two hydrogen bonds with Ser and Ser with S ¼ kcalmol Fig Compound showed the same key interactions exhibited by the cocrystallized ligand Compound S ¼ kcalmol RMSD ¼ has adopted a conformationallowing the presence of two cationpi interaction between Arg and the aromatic rings in addition to a hydrogen bond with themethoxy group Fig three hydrogen bonds made by ser andArg towards the methoxy groups and another hydrogen bondbetween Leu and NH2 group of the sulfonamide Superimposition between compound and the cocrystallized ligand showedthat they adopt the same orientation inside the binding site Fig Finally compound possessing the highest NQO1 inducer activityCD ¼ mM in this series showed the same interactions and thesame orientation of the native ligand inside the receptor indicatinga possible correlation between those multiple interactions and thenoted higher potency Based on the abovementioned resultscompound could possibly bind to Keap1 and disrupt its interaction with Nrf2The results from this study complement previous reportsshowing that the classical electrophilic Nrf2 activator sulforaphaneprotects cells including human retinal pigment epithelial cellskeratinocytes and mouse leukemia cells against oxidative damageFig Effect of compound on relative liver weight in nonirradiated control andirradiated mice after days of irradiation The results were expressed as mean ± SEn ¼ Statistical analysis was carried out by oneway ANOVA followed by Bonferroni€™s multiple comparison test There were no significant differences between groupscaused by oxidative stressors of four different types namelymenadione tertbutyl hydroperoxide 4hydroxynonenal and peroxynitrite as well as by exposure to ultraviolet radiation []Furthermore unlike the effects of most direct antioxidants theindirect antioxidant effect of sulforaphane which results from Nrf2activation persists for several days after sulforaphane is no longerpresent in the cell culture medium This is because direct antioxidants such as ascorbic acid tocopherols carotenoids and polyphenols which neutralize ROS and other chemical oxidants areconsumed in these reactions whereas Nrf2 activation results intranscriptional upregulation of antioxidant defences which aremediated by proteins with long halflives often several days Thenew compounds generated in the current study have an additionaladvantage in that they are nonelectrophilic and are therefore expected to have a broader therapeutic window compared to electrophilic Nrf2 activators This is supported by the very low toxicityof compound in mice Taken together these results demonstratethe powerful effect of Nrf2 activation and induction of NQO1 inprotecting cells and animals against high levels of ROS and preventing ROSmediated damage This is of particular relevance toprotecting the hematopoietic system which is highly sensitive toROS Conclusiontheacetamide268diiodo4oxo34sulfamoylphenyl3In summary a hybridization strategy was adopted using theiodinated quinazolinone scaffold and sulfonamide moiety to producedihydroquinazolin2ylthioNsubstitutedderivatives 5e18 Different substitutions were introduced to theacetamide group to study the structureactivity relationship All thecompounds were screened for their antioxidant potential using theNQO1 inducer activity assay The 345trimethoxyphenyl derivative showed the highestinducer activity in this seriesCD ¼ mM and had low toxicity LD50 ¼ mgkg Treatmentof gammairradiated mice with compound lowered oxidativestress as evidenced by the lower levels of MDA ROS and NQO1 inliver Furthermore compound ameliorated the complete bloodpicture of irradiated mice as well as enhanced the survival of mice 0cAM Soliman European Journal of Medicinal Chemistry Fig Effect of compound on A RBCs B WBCs C HGB concentration and D PLT counts in nonirradiated control and irradiated mice after days of irradiation The resultswere expressed as mean ± SE n ¼ Statistical analysis was carried out by oneway ANOVA followed by Bonferroni€™s multiple comparison test significantly different fromcontrol group significantly different from irradiated groupover a period of days postirradiation Molecular docking of inside the active site of Keap1 confirmed that it binds in the samemanner as that of the cocrystallized ligands The inducer activity ofcompound in upregulating NQO1 strongly suggests that it couldbe used as a lead antioxidant and radiomodulatory agent for furtheroptimization of the quinazolinone scaffold Materials and methods ChemistryAll chemicals were purchased from SigmaAldrich and are of ARgrade Melting points were determined in capillary on aGallen Kamp melting point apparatus Sanyo Gallen Kamp UKThin layer chromatography using precoated silica gel plates Kieselgel mm F254 Merck Germany was performed with asolvent system of chloroformmethanol to detect the spots byIR spectra KBr disc were recorded using an FTIRUV lightspectrophotometer Perkin Elmer USA NMR spectra were scannedon NMR spectrophotometer Bruker AXS Inc Switzerland operating at MHz for 1H and MHz for 13C Mass spectra wererecorded on the ISQ LT Thermo Scientific GCMS model Massachusetts USA Chemical shifts are expressed in dvalues ppmrelative to TMS as an internal standard using DMSO€‘d6 as a solventElemental analyses were done on a model CHNSO analyserPerkin Elmer USA All the values were within ± of thetheoretical values 8diiodo2mercapto4 oxoquinazolin34Hylbenzenesulfonamide A mixture of 2amino35diiodobenzoic acid g mol and isothiocyanatobenzenesulfonamide g mol in absolute ethanol mL containing drops of triethylamine was refluxed for h The solid product formed wascollected by filtration and crystallized from ethanol to give 00 NH2 Yield mp 0eC IR KBr ʋ cm 0cAM Soliman European Journal of Medicinal Chemistry the NN0naphthalene14diylbis4Fig 2D and 3D interaction poses ofmethoxybenzenesulfonamide showing cationp pp interaction and hydrogenbonds with the key amino acids inside the binding pocket arom CO CN SO2 1H NMRDMSO€‘d6 d ppm s 1H d 2H J ¼ Hz AB d2H J ¼ Hz AB d 1H J ¼ Hz d 1H J ¼ Hz s2H 13C NMR DMSO€‘d6 d ppm Anal Calcd for C14H9I2N3O3S2 C H N Found C H N 34Dihydroquinazolinsulfonamide derivatives General procedure A mixture of g mol and chloroNsubstituted acetamide derivatives mol in dryacetone mL and anhydrous K2CO3 g mol was stirredat room temperature for h filtered and the solid product formedwas crystallized from dioxane to give 5e18 268Diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioNphenylacetamide Yield 00 NH NH2 mp 0eC IR KBr ʋ cmarom aliph 2CO CN SO2 1H NMR DMSO€‘d6 d ppm s 2H 703e730 m 3H760e783 m 4H s 2H d 2H J ¼ Hz AB d1H J ¼ Hz d 1H J ¼ Hz s 1H 13C NMR DMSO€‘d6d ppm þ ] [Mþ1 MS mz [] [M] [] Anal Calcd for C22H16I2N4O4S2 C Fig 2D and 3D interaction pose of compound showing cationp pp interactionsinside the binding pocket of 4IQKH N Found C H N 268Diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioNotolylacetamide Yield 00 NH NH2 mp 0eC IR KBr ʋ cmarom aliph 2CO CN SO2 1H NMR DMSO€‘d6 d ppm s 3H s 2H ddd 1H J ¼ Hz 730e755 m 3H d 2H J ¼ HzAB s 2H d 2H J ¼ Hz AB d 1H J ¼ Hz d 1H J ¼ Hz s 1H 13C NMR DMSO€‘d6 d ppm Anal Calcd for C23H18I2N4O4S2 C H N Found C H N 268Diiodo 4oxo34sulfamoylphenyl34 Yielddihydroquinazolin2ylthioNmtolylacetamide 00 NH NH2 mp 0eC IR KBr ʋ cm arom aliph 2CO CN SO2 1H NMR DMSO€‘d6 d ppm s 3H s 2H 0cAM Soliman European Journal of Medicinal Chemistry Hz 721e748 m 2H 770e804 m 5H s 2H d 1HJ ¼ Hz d 1H J ¼ Hz s 1H 13C NMR DMSO€‘d6 dppm Anal CalcdforC24H20I2N4O4S2 C H N Found C H N acetamide 268Diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioN4ethylphenyl 00 Yield mp 0eC IR KBr ʋ cmNH NH2 arom aliph 2CO CN SO2 1H NMR DMSO€‘d6 d ppm t 3H J ¼ Hz q 2H J ¼ Hz s 2H d 2H J ¼ Hz AB d 2H J ¼ Hz AB d 2H J ¼ Hz 780e805 m 4H d 1H J ¼ Hz d 1H J ¼ Hz s 1H 13C NMRDMSO€‘d6 d ppm Anal Calcd forC24H20I2N4O4S2 C H N Found C H N 268Diiodo 4oxo34sulfamoylphenyl3 dihydroquinazolin2ylthioN 4methoxyphenyl acetamide 00 Yield mp 0eC IR KBr ʋ cmNH NH2 arom aliph 2CO CN SO2 1H NMR DMSO€‘d6 d ppm s 3H s 2H d 2H J ¼ Hz AB d 2H J ¼ Hz AB d2H J ¼ Hz AB d 2H J ¼ Hz s 2H d 1HJ ¼ Hz d 1H J ¼ Hz s 1H 13C NMR DMSO€‘d6d ppm Anal Calcdfor C23H18I2N4O5S2 C H N Found C H N acetamide 268Diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioN4ethoxyphenyl 00 Yield mp 0eC IR KBr ʋ cmNH NH2 arom aliph 2CO CN SO2 1H NMR DMSO€‘d6 d ppm t 3H J ¼ Hz q 2H J ¼ Hz s 2H d 2H J ¼ Hz AB d 2H J ¼ Hz AB d 2H J ¼ Hz AB 803e810 m4H d 1H J ¼ Hz d 1H J ¼ Hz s 1H 13CNMR DMSO€‘d6 d ppm Anal Calcdfor C24H20I2N4O5S2 C H N Found C H N 268Diiodo4oxo34sulfamoylphenyl34acetamidedihydroquinazolin2ylthioN35dimethoxyphenyl 00 Yield mp 0eC IR KBr ʋ cm NH NH2 arom aliph 2CO CN SO2 1H NMR DMSO€‘d6 d ppm s6H s 2H dd 1H J ¼ Hz dd 2H J ¼ Hz d 2H J ¼ Hz AB d 2H J ¼ Hz AB s2H d 1H J ¼ Hz d 1H J ¼ Hz s 1H 13CNMR DMSO€‘d6 d ppm Anal CalcdforC24H20I2N4O6S2 C H N Found C H N 268Diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioN345trimethoxyphenyl acetamideFig Superimposition of compound magenta and the cocrystallized ligand redshowed that they adopt the same orientation inside the receptor For interpretation ofthe references to color in this figure legend the reader is referred to the Web version ofthis m 1H 731e756 m 3H d 2H J ¼ Hz AB d2H J ¼ Hz AB s 2H d 1H J ¼ Hz d 1HJ ¼ Hz s 1H 13C NMR DMSO€‘d6 d ppm Anal Calcd for C23H18I2N4O4S2 C H N Found C H N 268Diiodo4oxo34sulfamoylphenyl34Yielddihydroquinazolin2ylthioNptolylacetamide 00 NH NH2 mp 0eC IR KBr ʋ cm arom aliph 2CO CN SO2 1H NMR DMSO€‘d6 d ppm s 3H s 2H d 2H J ¼ Hz AB m 2H d 2H J ¼ Hz AB d 2H J ¼ Hz AB s 2H d 1H J ¼ Hz d 1HJ ¼ Hz s 1H 13C NMR DMSO€‘d6 d ppm þ MS mz [] [M ] [] Anal Calcd forC23H18I2N4O4S2 C H N Found C H N acetamide 268Diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioN2ethylphenyl 00 NH Yield mp 0eC IR KBr ʋ cmNH2 arom aliph 2CO1619 CN SO2 1H NMR DMSO€‘d6 d ppm t 3H J ¼ Hz q 2H J ¼ Hz s 2H dd 1H J ¼ Hz m 1H ddd 1H J ¼ Hz dd 1H J ¼ Hz d 2H J ¼ Hz AB d 2H J ¼ Hz AB m 2H d1H J ¼ Hz d 1H J ¼ Hz s 1H 13C NMRDMSO€‘d6 d ppm MS mz []þ ] [Mþ1 ] [] Anal Calcd for [MC24H20I2N4O4S2 C H N Found C H N 268Diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioN3ethylphenyl 00 Yield mp 0eC IR KBr ʋ cmNH NH2 arom aliph 2CO CN SO2 1H NMR DMSO€‘d6 d ppm t 3H J ¼ Hz q 2H J ¼ Hz s 2H ddd 1H J ¼ acetamide 0c 00 Yield mp 0eC IR KBr ʋ cm NH NH2 arom aliph 2CO CN SO2 1H NMR DMSO€‘d6 d ppm s 6H s 3H s 2H d 2H J ¼ Hz d 2HJ ¼ Hz AB s 2H d 2H J ¼ Hz d 1HJ ¼ Hz AB d 1H J ¼ Hz s 1H 13C NMRDMSO€‘d6 d ppm MS mz [] þ[M ] [] Anal Calcd for C25H22I2N4O7S2 C H N Found C H N 268Diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioN2methyl4nitrophenyl acetamide 00 Yield mp 0eC IR KBr ʋ cm NH NH2 arom aliph 2CO CN NO2 SO2 1H NMR DMSO€‘d6 dppm s 3H s 2H d 1H J ¼ Hz d 2HJ ¼ Hz AB 790e805 m 6H d 1H J ¼ Hz d 1HJ ¼ Hz s 1H 13C NMR DMSO€‘d6 d ppm Anal Calcd for C23H17I2N5O6S2 C H N Found C H N N2methyl6nitrophenyl 8Diiodo4oxo34sulfamoylphenyl3 dihydroquinazolin2ylthioacet 00 amide Yield mp 0eC IR KBr ʋ cm NH NH2 arom aliph 2CO CN NO2 SO2 1H NMRDMSO€‘d6 d ppm s 3H s 2H dd 1H J ¼ Hz dd 1H J ¼ Hz d 2H J ¼ Hz 801e810m 5H d 1H J ¼ Hz d 1H J ¼ Hz s 1H13C NMR DMSO€‘d6 d ppm MS mz [] þ[M ] [] Anal Calcd for C23H17I2N5O6S2 C H N Found C H N 8Diiodo4oxo34sulfamoylphenyl3 dihydroquinazolin2ylthio N24dinitrophenyl acetamide 00 Yield mp 0eC IR KBr ʋ cmNH NH2 arom aliph 2CO CN NO2 SO2 1H NMR DMSO€‘d6 dppm s 2H d 2H J ¼ Hz AB d 1H J ¼ Hz800e804 m 2H d 2H J ¼ Hz AB d 1H J ¼ Hz830e834 m 3H s 1H 13C NMR DMSO€‘d6 d ppm Anal Calcd for C22H14I2N6O8S2 C H N Found C H N Biological evaluation NQO1 in vitro inducer activityHepa1c1c7 murine hepatoma cells were grown in a humidifiedatmosphere at 0eC CO2 The cells were tested routinely toensure that they were mycoplasmafree The aminimum essentialmedium aMEM supplemented with vv heat andcharcoalinactivated g100 mL min at 0eC fetal bovineserum was used For evaluation of the potential NQO1 inducer activity cells 104well were grown in transparent flatbottomplastic 96well plates for h after which the cell culture medium was replaced with fresh medium containing each inducerdissolved in DMSO and diluted in the medium and theAM Soliman European Journal of Medicinal Chemistry cells were grown for further h Three replicates of each treatment of eight serial dilutions of inducers were used The final DMSOconcentration in the cell culture medium was maintained at vv in all wells Cell lysates were prepared in digitonin and thespecific activity of NQO1 was determined using menadione as asubstrate as described [] Briefly the cell culture medium wasremoved from each well and the cells were washed three timeswith mL of phosphate buffered saline PBS and subsequentlylysed in mL of digitonin suspension in the presence of EDTA for min with shaking Of the cell lysate mL was transferred to t Answer:

Thyroid_Cancer

" COVID19 pathways for brain andheart injury in comorbidity patients A role of medical imaging and artificial intelligencebased COVIDseverity classification A review Computers in Biology and Medicine 101016jcompbiomed2020103960 0cThis is a PDF file of an that has undergone enhancements after acceptance such as the additionof a cover page and metadata and formatting for readability but it is not yet the definitive version ofrecord This version will undergo additional copyediting typesetting and review before it is publishedin its final form but we are providing this version to give early visibility of the Please note thatduring the production process errors may be discovered which could affect the content and all legaldisclaimers that apply to the journal pertain Published by Elsevier Ltd 0cCOVID19 Pathways for Brain and Heart Injury in Comorbidity Patients A role of Medical Imaging and Artificial Intelligencebased COVID severity Classification A Review Jasjit S Suri PhD MBA FAIMBE FAIUM FAPVS1 Anudeep Puvvula MBBS FCD12 Mainak Biswas PhD3 Misha Majhail14 Luca Saba MD5 Gavino Faa MD6 Inder M Singh MD7 Ronald Oberleitner BS MBA8Monika Turk MD PhD9 Paramjit S Chadha BE1 Amer M Johri MD FASE10 J Miguel Sanches PhD11 Narendra N Khanna MD DM FACC12 Klaudija Viskovic MD PhD13 Sophie Mavrogeni MD PhD FESC14 John R Laird MD FACC FACP15 Gyan Pareek MD16 Martin Miner MD17 David W Sobel MD16 Antonella Balestrieri MD5 Petros P Sfikakis MD18 Gee Tsoulfas MD19 Athanasios Protogerou MD PhD20 Durga Prasanna Misra MD FRCP21 Vikas Agarwal MD FRCP21 Gee D Kitas MD PhD FRCP22 Puneet Ahluwalia MS MCh24 Raghu Kolluri MD25 Jagjit Teji MD26 Mustafa AlMaini MD27 Ann Agbakoba MD28 Surinder K Dhanjil MSc FSVU29 Meyypan Sockalingam MBBS FRCR30 Ajit Saxena MD12 Andrew Nicolaides MS PhD FRCS31 Aditya Sharma MD32 Vijay Rathore MD33 Janet N A Ajuluchukwu MD34 Mostafa Fatemi PhD FAIMBE FIEEE FASA FAIUM35 Azra Alizad MD FAIMBE FAIUM36 Vijay Viswanathan MD PhD37 P K Krishnan MD38 Subbaram Naidu PhD Life FIEEE39 1Stroke Monitoring and Diagnostic Division AtheroPoint„¢ Roseville CA USA 2Annu™s Hospitals for Skin and Diabetes Nellore AP INDIA 3JIS University Kolkata West Bengal INDIA 4Oakmont High School and AtheroPoint„¢ Roseville CA USA 5Department of Radiology Azienda Ospedaliero Universitaria Cagliari ITALY 6Department of Pathology AOU of Cagliari Italy 7Stroke Monitoring and Diagnostic Division AtheroPoint„¢ Roseville CA USA 8Behavior Imaging USA 9The HanseWissenschaftskolleg Institute for Advanced Study Delmenhorst GERMANY 10Department of Medicine Division of CardiologyQueen™s University Kingston Ontario CANADA 11Institute of Systems and Robotics Instituto Superior Tecnico Lisboa PORTUGAL 12Department of Cardiology Indraprastha APOLLO Hospitals New Delhi INDIA 13University Hospital for Infectious Diseases Zagreb CROTIA 14Cardiology Clinic Onassis Cardiac Surgery Center Athens GREECE 15Heart and Vascular Institute Adventist Health St Helena St Helena CA USA 16Minimally Invasive Urology Institute Brown University Providence Rhode Island USA 17Men™s Health Center Miriam Hospital Providence Rhode Island USA 18Rheumatology Unit National Kapodistrian University of Athens GREECE 19Aristoteleion University of Thessaloniki Thessaloniki GREECE 20National Kapodistrian University of Athens GREECE 21Sanjay Gandhi Postgraduate Institute of Medical Sciences Lucknow UP INDIA 22Academic Affairs Dudley Group NHS Foundation Trust Dudley UK 23Arthritis Research UK Epidemiology Unit Manchester University Manchester UK 24Max Institute of Cancer Care Max Superspeciality Hospital New Delhi INDIA 25OhioHealth Heart and Vascular Ohio USA 26Ann and Robert H Lurie Children™s Hospital of Chicago Chicago USA 27Allergy Clinical Immunology and Rheumatology Institute Toronto CANADA 28University of Lagos NIGERIA 29AtheroPoint LLC CA USA 30MV Center of Diabetes Chennai INDIA 31Vascular Screening and Diagnostic Centre and University of Nicosia Medical School CYPRUS Journal Preproof 0c32Division of Cardiovascular Medicine University of Virginia Charlottesville VA USA 33Nephrology Department Kaiser Permanente Sacramento CA USA 34Department of Medicine Lagos University Teaching Hospital Lagos NIGERIA 35Dept of Physiology Biomedical Engg Mayo Clinic College of Medicine and Science MN USA 36Dept of Radiology Mayo Clinic College of Medicine and Science MN USA 37MV Hospital for Diabetes and Professor M Viswanathan Diabetes Research Centre Chennai INDIA 38Neurology Department Fortis Hospital Bangalore INDIA 39Electrical Engineering Department University of Minnesota Duluth MN USA Corresponding Author Dr Jasjit S Suri PhD MBA FAIMBE FAIUM FAPVS Fellow American Institute of Medical and Biological Engineering Fellow American Institute of Ultrasound in Medicine Fellow Asia Pacific Vascular Society Stroke Monitoring and Diagnosis Division AtheroPoint„¢ Roseville Roseville CA USA Phone Email jasjitsuriatheropointcom Journal Preproof 0cCOVID19 Pathways for Brain and Heart Injury in Comorbidity Patients A role of Medical Imaging and Artificial Intelligencebased COVID severity Classification A Review Abstract Artificial intelligence AI has penetrated the field of medicine particularly the field of radiology Since its emergence the highly virulent coronavirus disease COVID19 has infected over million people leading to over deaths as of July 1st Since the outbreak began almost s about COVID19 have been published pubmedncbinlmnihgov however few have explored the role of imaging and artificial intelligence in COVID19 patients”specifically those with comorbidities This paper begins by presenting the four pathways that can lead to heart and brain injuries following a COVID19 infection Our survey also offers insights into the role that imaging can play in the treatment of comorbid patients based on probabilities derived from COVID19 symptom statistics Such symptoms include myocardial injury hypoxia plaque rupture arrhythmias venous thromboembolism coronary thrombosis encephalitis ischemia inflammation and lung injury At its core this study considers the role of imagebased AI which can be used to characterize the tissues of a COVID19 patient and classify the severity of their infection Imagebased AI is more important than ever as the pandemic surges and countries worldwide grapple with limited medical resources for detection and diagnosis We conclude that imaging and AIbased tissue characterization when considered alongside COVID19 symptoms and their pretest probabilities offer a compelling solution for assessing the risk of comorbid patients These methods show the potential to become an integral part of tracking and improving the healthcare system both during the pandemic and beyond Keywords COVID19 comorbidity pathophysiology heart brain lung imaging artificial intelligence risk assessment Journal Preproof 0c Introduction In December a novel coronavirus referred to as œsevere acute respiratory distress syndrome coronavirus  SARSCoV2 [] appeared in Wuhan the capital of Hubei Province in PR China The disease caused by the virus was initially named œnovel coronavirus pneumonia NCP by the Chinese government but was subsequently renamed œcoronavirus disease  COVID19 by the World Health anization WHO On January 30th it was declared a public health emergency of international concern PHEIC [] It is believed that SARSCoV2 is primarily transmitted through saliva droplets or nasal discharge [] The first evidence of humantohuman transmission was found by Jasper FukWoo Chan et al in their study at The University of Hong KongShenzhen Hospital [] Due to its contagiousness Ro27 the virus has reached epidemic levels affecting countries and causing over million infections and more than deaths as of July 1st [] shown in Figure Recent literature suggests that patients with preexisting diseases are likely to experience severe complications from COVID19 [] In one study on admitted diabetic and nondiabetic COVID19 patients the mortality rate vs and the rate of admission to the intensive care unit ICU vs were significantly higher for diabetic patients Diabetic patients also experienced severe inflammatory responses and cardiac hepatic and renal coagulopathy [] The prevalence of heart and brain injuries was also higher in COVID19 patients with concomitant chronic conditions like diabetes kidney disease dyslipidemia hypertension [] chronic obstructive pulmonary disease COPD and cardiovascular diseases [] Recent studies have shown that SARSCoV2 invades the thin lining of the epithelial cells of the arteries leading to atherosclerosis [] and arterial inflammatory disease”one of the major causes of cardiovascular diseases CVDs which also causes heart and brain injuries [ ] This could be due to a reduced expression of angiotensinconverting enzyme ACE2 causing endothelial dysfunction which in turn aggravates existing Journal Preproof 0catherosclerosis [ ] It has also been observed that comorbid patients when subjected to imagescreening show mild to severe pretest probability PTP for COVID19 [] The conventional cardiovascular risk factors CCVRF in these comorbid patients appear strongly correlated either to their heart imaging or to surrogate biomarkers of coronary artery disease such as carotid artery disease Both imaging and biomarkers could be helpful in severity predictions for COVID19 [] Figure illustrates the associations between SARSCoV2 and other comorbidities such as diabetes as well as the comparative survival rates for COVID19 patients with and without diabetes Figure World map showing COVID19 spread over countries courtesy John Hopkins University ACE2 expression causes scars in the vessels and can even rupture the walls of the arteries [] For this reason CCVRF should be considered alongside imaging in patients who present with COVID19 and many comorbidities [] The second stage is the one at which a patient is most severely affected by Journal Preproof 0cCOVID19 and has the highest probability of cardiac injury or release of troponin T TnT Imaging has been shown to offer benefits in monitoring the tissue scars caused by COVID19 [] Figure a Association of SARSCoV2 with other comorbidities and b comparison of the mortality rate of diabetic and nondiabetic COVID19 patients reproduced with permission [] Multiple modalities can be utilized to determine whether a patient has the sequelae of COVID19 including magnetic resonance imaging [] computed tomography [] and ultrasound [] The advantage of these imaging modalities is the visual access they provide to the scar tissue caused by the disease A disadvantage however is their inability to provide a œrisk assessment The application of artificial intelligence AI can enhance the information provided by these imaging modalities resulting in a more accurate characterization of the tissue and the disease process [] In fact the combination of AI and medical imaging has been shown to improve diagnosis and risk stratification speed up patient evaluation enhance disease monitoring and accelerate early intervention [ ] Thus this review will focus on the use of AIbased tissue characterization of images of comorbid patients affected by COVID19 The layout of this paper is as follows Section presents the pathophysiology of the four pathways leading to brain and heart injury Section summarizes the evidence related to the use of Journal Preproof 0cimaging during the COVID19 pandemic Section elaborates on the use of AIbased tissue characterization for risk assessment Finally the paper concludes with a critical discussion The Pathophysiology of SARCoV2 Leading to Brain and Heart Injury Several studies suggest that SARSCoV2 uses the ACE2 receptor to gain access to cells by binding to the SPIKE protein ˜S™ protein on their surface [] see Figure ACE2 and angiotensinconverting enzyme ACE1 are homolog carboxypeptidase enzymes that have different vital functions in the reninangiotensinaldosterone system RAAS pathway [] ACE2 is widely expressed in myocardial cells [] type pneumocytes enterocytes and astrocytes in the brain [ ] Thus it is recognized as a cause of extrapulmonary complications Figure shows the overall picture of how SARSCoV2 causes brain and heart injuries via four different pathways These include i the neuronal pathway ii the hypoxia pathway iii the RAAS pathway and iv the immune pathway We will discuss these pathways and the injuries they lead to which may manifest as viral encephalitis infectious toxic encephalopathy or acute cerebrovascular disease i The Neuronal Pathway Figure the pathway I Recent epidemiological studies have demonstrated similarities at the genomic level between SARSCoV1 MERS and SARSCoV2 [ ] Meanwhile previous experimental studies have shown that beta coronaviruses in general”such as SARSCoV1 and MERS”can spread into and directly infect the brain when inhaled as droplets via the nasal epithelium [ ] Figure depicts the olfactory nerve and the olfactory bulb []”labeled as œa and œb respectively”on the image of the sagittal brain in the neuronal pathway Based on recent reports we are aware that patients infected by SARSCoV2 show symptoms of dysgeusia loss of taste and anosmia loss of smell [ ] Bohmwald et al further validate that coronaviruses that infect through the olfactory nerve and bulb can reach the brain and cerebrovascular fluid CSF within seven days Additionally these viruses have been observed to cause inflammation and demyelination [] The Journal Preproof 0cauthors demonstrated in an experimental study of mice that removing the olfactory bulb from the pathway can lead to the restriction of CoV in the central nervous system CNS [] Based on this evidence we believe that the neuronal pathway is one possible track for SARSCoV2 ii The Hypoxia Pathway Figure pathway II In this pathway decreased levels of ACE2 proliferate in the lung parenchyma cells after the coronavirus has passed through causing exaggerated neutrophils accumulation enhanced vascular permeability and the formation of diffuse alveolar and interstitial exudates This ultimately leads to pulmonary edema and acute respiratory distress syndrome ARDS [] ARDS is characterized by severe abnormalities in blood gas composition resulting from an oxygen and carbon dioxide mismatch which leads to low blood oxygen levels [ ] This ongoing hypoxia can lead to myocardial ischemia and heart injury [ ] see Figure pathway IIA Hypoxia in the brain increases anaerobic metabolism in the mitochondria of the brain cells [] leading to cerebral vasodilatation edema and impaired flow This can result in cerebral ischemia and acute cerebrovascular diseases such as acute ischemic stroke [ ] see Figure pathway IIB iii The RAAS Pathway after SARSCoV2 Figure pathway III The RAAS pathway is crucial in regulating blood pressure as well as the balance of fluid and electrolytes Any disturbance in this pathway can trigger the pathogenesis of cardiovascular diseases [] Before a SARSCoV2 infection triggers the RAAS Angiotensin I Ang I cleaves to Angiotensin II Ang II via ACE1 Ang II causes vasospasm It is also a proinflammatory agent with prothrombotic and proliferative effects that are detrimental to vascular tone and hemostasis [ ] Thus as a counterregulatory mechanism ACE2 degrades Ang II and generates Ang which counteracts the negative impacts of Ang II [ ] Both ACE2 and Ang have cardiocerebral vascular protective effects [] After the triggering of SARSCoV2 infection it results are in the deregulation of RAAS causing heart and brain injury in two different pathways The main culprit is an increase in Ang II which is caused by the decrease in ACE2 levels Figure pathway IIIA First an increase in Ang II levels stimulates the adrenal cortex of the kidney resulting in an increased production of aldosterone Aldosterone is a steroid hormone that causes sodium and water Journal Preproof 0creabsorption to increase at the distal tubule and collecting duct of the nephron [] This reabsorption increases blood volume and causes an elevation in blood pressure which results in the endothelial dysfunction that causes brain and heart injury [] The second effect of an increase in Ang II levels ie as a consequence of decreased ACE2 levels is endothelial dysfunction leading to intimal damage in the arterial walls [] which can be seen during the imaging of the arterial wall see Figure pathway IIIB This pathway can also trigger a cytokine storm as high levels of Ang II can cause an increase in proinflammatory cytokines see the bridge line between the RAAS and immune pathways iv The Immune Pathway Figure pathway IV Several recent studies have reported SARSCov2 viral pneumonia [ ] having an exaggerated inflammatory response known as a œcytokine storm This response appears to present at advanced stages of severe COVID19 with increased levels of inflammatory cytokines leading to multiplean failure [] The rise in inflammatory markers”including IL6 IL7 IL12 IL15 IL22 TNFα and CXCL10”results in the destabilization of plaque This in turn can cause plaque rupture resulting in heart and brain injury [ ] The Role of Imaging in Comorbid Patients with COVID19 As the previous section discussed COVID19 uses four pathways ie neuronal hypoxia RAAS and immune to cause critical heart and brain injuries in patients with comorbidities The prevalence of myocardial injury and brain injury caused by COVID19 [ ] points to a need for increased use of medical imaging to expedite assessments differential diagnoses and patient management [ ] with proper safety measures [] The seriousness of a patient™s COVID19 symptoms helps to determine which imaging modality is appropriate portable or nonportable and invasive or noninvasive BMode ultrasound imaging is portable and can be used for lowrisk patients Meanwhile Xray magnetic resonance imaging [] and computed tomography [] are nonportable and can be used for mediumrisk patients Intravascular ultrasound IVUS [] and ventriculography are invasive imaging modalities used in highly critical cases [ ] Amongst all the imaging modalities ultrasound is noteworthy because it is radiationfree portable quick repeatable inexpensive Journal Preproof 0cand can be performed in isolation thus lowering the chance of spreading the COVID19 infection [ ] There are several examples of medical imaging that have led to proper treatment and healthcare management during the pandemic ultimately reducing the mortality rate Xray imaging of the chest has demonstrated irregular patchy hazy reticular and widespread groundglass opacities indicating the progression of COVID19 at various stages this information can support the healthcare team in developing the most appropriate treatment plan [] Chest CT scans of COVID19 patients revealed in almost of the patients that the disease was affecting at least one of the five lobes of their lungs [] Chest MRI scans of COVID19 patients showed pulmonary tissue consolidation in six diffusionrestricted regions in six and lung damage in seven [] Meanwhile heart MRI studies of recovered patients showed that of the patients had myocardial edema At the same time late gadolinium enhancement was found in implying that COVID19“related cardiac injury is longstanding and requires frequent monitoring even after recovery [] In a different study MR scans of a COVID19 patient revealed myocardial inflammation signifying myocardial injury due to a cytokine storm related to the SARSCoV2 infection as discussed in Section Pathway IV [] Several studies have also evaluated the effects of COVID19 on the brain In one MRI scans revealed hemorrhagic rim enhancing lesions within the bilateral thalami medial temporal lobes and subinsular regions [] shown in Figure In another brain MRI scans were completed for patients of which produced abnormal findings [] Additionally evidence of liver injury and gall bladder abnormality was found in a joint CT and ultrasound study of the abdomen [] Recent MRI scans of COVID19 patients™ olfactory bulbs have revealed the cause of olfactory function loss to be the interaction between SARSCoV2 and the ACE2 protein expressed by the olfactory epithelium which leads to inflammatory obstruction [] Journal Preproof 0cFigure We have shown in four pathways how COVID19 can cause Brain and heart injury Brain image in pathway I httpdebugliescom20200123olfactorydisturbanceshaveimplicationsinmentalandemotionalwellbeinghealth Courtesy of Debug Lies Invasive imaging is another option for diagnosing COVID19 patients who have critical comorbidities In one such study IVUS along with stenting was performed with precautions on a COVID19 patient with myocardial infarction [] shown in Figure A detailed discussion of precautions is included in section In another study takotsubo syndrome a form of myocardial injury triggered by COVID19 was detected using ventriculography [] In various studies the medical imaging of COVID19 patients had been crucial to ascertaining the extent of tissue damage and critical infection although there were no visible symptoms [ ] Therefore medical imaging is the preferred way to ascertain the extent of cardiac and brain tissue damage throughout the lifetime of COVID19 patients COVID19 Journal Preproof 0cpatients with comorbidities are especially vulnerable and so they need to be screened through medical imaging from the first day of their diagnosis Medical imaging can help patients with deep vein thrombosis DVT as they are highly susceptible to severe tissue damage from COVID19 A study showed that COVID19 patients suffering from DVT had a worse prognosis than patients without DVT Patients with DVT were admitted to the ICU more frequently discharged less frequently and suffered more deaths than those without DVT [] Figure MRI scan of COVID19 patient showing hemorrhage MRI images demonstrate T2 FLAIR hyperintensity within the bilateral medial temporal lobes and thalami A B E F with evidence of hemorrhage indicated by hypointense signal intensity on susceptibilityweighted images C G and rim enhancement on postcontrast images D H reproduced with permission [] Journal Preproof 0c Figure Application of chest CT and IVUS for a COVID19 patient suffering from myocardial infarction a Chest CT scan with viral pneumonia showing fibrinous focal exudative changes b when the patient complained of chest pain the ECG report showed the STsegments elevations in V1V5 lead c d CAG radiology that the proximal segment of LAD was occluded e f The blood flow of LAD restoration after DESs was implanted g the dissection distal shown by IVUS to the stent in LAD from “ o'clock h the low echogenic shadow with scattered higher echogenic flicker indicating a thrombus i after a DES was implanted and the stent was well expanded the dissection could not be seen j The thrombus disappearance after the intervention reproduced with permission [] Journal Preproof 0c Although medical imaging can be very useful to patients and doctors alike the exponential pandemic curve inadequate medical facilities [] and a limited number of radiologists make the assessment diagnosis and management processes challenging tedious and errorprone Therefore although medical imaging can make diagnoses faster as stated above it will be of limited use Given this fact new age techniques such as artificial intelligence AI [] applications in medical imaging for tissue characterization can make computeraided assessments and diagnoses faster The main reason for this is that the AI can be scaled up to match the pandemic curve thereby meeting the immediate demands of medical image diagnoses during the COVID19 pandemic AIbased tests can categorize the nature of a patient™s risk in one of the categories namely norisk low lowmedium LM highmedium HM lowhigh LH or highhigh HH risk depending on the patient™s symptoms and their severity [ ] as shown in Figure The imaging modality also varies with the degree of risk as follows no imaging for norisk portable imaging for low and LM risk nonportable imaging for HM and LH and invasive imaging for HH A probability PTP is performed to accurately interpret diagnostic results to categorize the patient into one of the four groups [] After that for norisk patients nonimaging biomarkers can be collected for risk assessment using AIbased data protocols For lowrisk patients portable 2D3D imaging such as ultrasound is used whereas nonportable and invasive 2D3D imaging such as MRICTXRayechocardiography can be used for LM patients For HH patients invasive imaging techniques such as IVUS and ventriculography can be used Based on the data provided by various 2D3D scans AIbased medical imaging is applied for risk assessment Further treatment is then planned based on this imaging process In the next section deep learning DLbased medical imaging is proposed for medical imaging scans particularly ultrasounds for COVID19 patients Machine Learning and Deep Learning for Tissue Characterization Using AI and associated technologies in healthcare can significantly slow down diagnosis times Journal Preproof 0cespecially during the COVID19 pandemic as patient numbers are continually growing and there are few specialists available [] Figure Role of AIbased risk assessment on COVID19 patients having comorbidity Journal Preproof 0cAlthough some caution must be exercised regarding its fullscale deployment [] its overall usefulness in healthcare management during times of crisis cannot be ignored [ ] In general AI in healthcare refers to all artificial intelligencebased technologies that make educated decisions regarding a patient™s diagnosis monitoring treatment and management The importance of AI has specifically increased many folds when imaging comes into play mainly because of large volumetric data sizes and the extensive need to characterize and quantify the disease via lesion images [] Tissue imaging and its characterization is of prime importance since it has a direct influence on decisions related to COVID19 severity for a patient [] The main benefit of AI methods is that the machines can be used to train by mimicking the physician™s cognitive actions and such trained models can be used to predict the disease™s severity in asymptomatic patients Within a short period several machine learning MLbased techniques used the power of AI to manage COVID19 [ ] ML and DL Architectures ML Architecture ML is a twostage process In stage I different features are extracted from the lesion COVID images the extractions are then operated on by an ML statistical model called a training system to generate offline coefficients These coefficients are then transformed by the test lesion images which yield an intelligent classification or inference A typical ML system for predicting risk class is shown in Figure CUSIP is an imagebased phenotype that uses the event equivalent gold standard EEGS [ ] model DL Architecture DL refers to a visual cortex that imitates multiple layers of a neural network applied directly to tissue images to extract features and for characterization purposes [] The convolution neural network CNN [] shown in Figure is one such DL network architecture that is widely used to characterize medical images It performs a series of convolution maxpooling operations to extract features and perform characterizations ML and DL both follow the supervised learning Journal Preproof 0capproach by which models are trained using offline data Figure Typical lowcost machine learning architecture utilizing EEGS model As discussed in previous sections there are four pathways through which a COVID19 infection leads to heart and brain injuries AI can be used via medical imaging to detect the extent of tissue damage in these pathways and help medical professionals to develop an effective treatment plan for patients There are several instances in which the AI paradigm has been used for tissue characterization based on Journal Preproof 0cmedical images during the pandemic as well as during standard times Some uses of AI are described anwise following a proposed model for characterizing DLbased tissues Figure A convolution neural network courtesy of AtheroPoint„¢ CA USA ML Architecture used for Tissue Characterization for Stroke Risk Stratification There are two types of tissue characterization that can be carried out using AI i MLbased [ ] and ii DLbased [] Various MLbased technologies have been developed to classify symptomatic and asymptomatic plaque from ultrasound images For example an MLbased technique based on support vector machines SVM was developed to characterize the symptomatic and asymptomatic plaques of carotid scans that indicated the presence of plaque [ ] SVM classifiers work by determining the maximum margin between two data clusters First a texture analysis [] is used to extract the features ie standard deviation entropy symmetry and run percentage in

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You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: " COVID19 pathways for brain andheart injury in comorbidity patients A role of medical imaging and artificial intelligencebased COVIDseverity classification A review Computers in Biology and Medicine 101016jcompbiomed2020103960 0cThis is a PDF file of an that has undergone enhancements after acceptance such as the additionof a cover page and metadata and formatting for readability but it is not yet the definitive version ofrecord This version will undergo additional copyediting typesetting and review before it is publishedin its final form but we are providing this version to give early visibility of the Please note thatduring the production process errors may be discovered which could affect the content and all legaldisclaimers that apply to the journal pertain Published by Elsevier Ltd 0cCOVID19 Pathways for Brain and Heart Injury in Comorbidity Patients A role of Medical Imaging and Artificial Intelligencebased COVID severity Classification A Review Jasjit S Suri PhD MBA FAIMBE FAIUM FAPVS1 Anudeep Puvvula MBBS FCD12 Mainak Biswas PhD3 Misha Majhail14 Luca Saba MD5 Gavino Faa MD6 Inder M Singh MD7 Ronald Oberleitner BS MBA8Monika Turk MD PhD9 Paramjit S Chadha BE1 Amer M Johri MD FASE10 J Miguel Sanches PhD11 Narendra N Khanna MD DM FACC12 Klaudija Viskovic MD PhD13 Sophie Mavrogeni MD PhD FESC14 John R Laird MD FACC FACP15 Gyan Pareek MD16 Martin Miner MD17 David W Sobel MD16 Antonella Balestrieri MD5 Petros P Sfikakis MD18 Gee Tsoulfas MD19 Athanasios Protogerou MD PhD20 Durga Prasanna Misra MD FRCP21 Vikas Agarwal MD FRCP21 Gee D Kitas MD PhD FRCP22 Puneet Ahluwalia MS MCh24 Raghu Kolluri MD25 Jagjit Teji MD26 Mustafa AlMaini MD27 Ann Agbakoba MD28 Surinder K Dhanjil MSc FSVU29 Meyypan Sockalingam MBBS FRCR30 Ajit Saxena MD12 Andrew Nicolaides MS PhD FRCS31 Aditya Sharma MD32 Vijay Rathore MD33 Janet N A Ajuluchukwu MD34 Mostafa Fatemi PhD FAIMBE FIEEE FASA FAIUM35 Azra Alizad MD FAIMBE FAIUM36 Vijay Viswanathan MD PhD37 P K Krishnan MD38 Subbaram Naidu PhD Life FIEEE39 1Stroke Monitoring and Diagnostic Division AtheroPoint„¢ Roseville CA USA 2Annu™s Hospitals for Skin and Diabetes Nellore AP INDIA 3JIS University Kolkata West Bengal INDIA 4Oakmont High School and AtheroPoint„¢ Roseville CA USA 5Department of Radiology Azienda Ospedaliero Universitaria Cagliari ITALY 6Department of Pathology AOU of Cagliari Italy 7Stroke Monitoring and Diagnostic Division AtheroPoint„¢ Roseville CA USA 8Behavior Imaging USA 9The HanseWissenschaftskolleg Institute for Advanced Study Delmenhorst GERMANY 10Department of Medicine Division of CardiologyQueen™s University Kingston Ontario CANADA 11Institute of Systems and Robotics Instituto Superior Tecnico Lisboa PORTUGAL 12Department of Cardiology Indraprastha APOLLO Hospitals New Delhi INDIA 13University Hospital for Infectious Diseases Zagreb CROTIA 14Cardiology Clinic Onassis Cardiac Surgery Center Athens GREECE 15Heart and Vascular Institute Adventist Health St Helena St Helena CA USA 16Minimally Invasive Urology Institute Brown University Providence Rhode Island USA 17Men™s Health Center Miriam Hospital Providence Rhode Island USA 18Rheumatology Unit National Kapodistrian University of Athens GREECE 19Aristoteleion University of Thessaloniki Thessaloniki GREECE 20National Kapodistrian University of Athens GREECE 21Sanjay Gandhi Postgraduate Institute of Medical Sciences Lucknow UP INDIA 22Academic Affairs Dudley Group NHS Foundation Trust Dudley UK 23Arthritis Research UK Epidemiology Unit Manchester University Manchester UK 24Max Institute of Cancer Care Max Superspeciality Hospital New Delhi INDIA 25OhioHealth Heart and Vascular Ohio USA 26Ann and Robert H Lurie Children™s Hospital of Chicago Chicago USA 27Allergy Clinical Immunology and Rheumatology Institute Toronto CANADA 28University of Lagos NIGERIA 29AtheroPoint LLC CA USA 30MV Center of Diabetes Chennai INDIA 31Vascular Screening and Diagnostic Centre and University of Nicosia Medical School CYPRUS Journal Preproof 0c32Division of Cardiovascular Medicine University of Virginia Charlottesville VA USA 33Nephrology Department Kaiser Permanente Sacramento CA USA 34Department of Medicine Lagos University Teaching Hospital Lagos NIGERIA 35Dept of Physiology Biomedical Engg Mayo Clinic College of Medicine and Science MN USA 36Dept of Radiology Mayo Clinic College of Medicine and Science MN USA 37MV Hospital for Diabetes and Professor M Viswanathan Diabetes Research Centre Chennai INDIA 38Neurology Department Fortis Hospital Bangalore INDIA 39Electrical Engineering Department University of Minnesota Duluth MN USA Corresponding Author Dr Jasjit S Suri PhD MBA FAIMBE FAIUM FAPVS Fellow American Institute of Medical and Biological Engineering Fellow American Institute of Ultrasound in Medicine Fellow Asia Pacific Vascular Society Stroke Monitoring and Diagnosis Division AtheroPoint„¢ Roseville Roseville CA USA Phone Email jasjitsuriatheropointcom Journal Preproof 0cCOVID19 Pathways for Brain and Heart Injury in Comorbidity Patients A role of Medical Imaging and Artificial Intelligencebased COVID severity Classification A Review Abstract Artificial intelligence AI has penetrated the field of medicine particularly the field of radiology Since its emergence the highly virulent coronavirus disease COVID19 has infected over million people leading to over deaths as of July 1st Since the outbreak began almost s about COVID19 have been published pubmedncbinlmnihgov however few have explored the role of imaging and artificial intelligence in COVID19 patients”specifically those with comorbidities This paper begins by presenting the four pathways that can lead to heart and brain injuries following a COVID19 infection Our survey also offers insights into the role that imaging can play in the treatment of comorbid patients based on probabilities derived from COVID19 symptom statistics Such symptoms include myocardial injury hypoxia plaque rupture arrhythmias venous thromboembolism coronary thrombosis encephalitis ischemia inflammation and lung injury At its core this study considers the role of imagebased AI which can be used to characterize the tissues of a COVID19 patient and classify the severity of their infection Imagebased AI is more important than ever as the pandemic surges and countries worldwide grapple with limited medical resources for detection and diagnosis We conclude that imaging and AIbased tissue characterization when considered alongside COVID19 symptoms and their pretest probabilities offer a compelling solution for assessing the risk of comorbid patients These methods show the potential to become an integral part of tracking and improving the healthcare system both during the pandemic and beyond Keywords COVID19 comorbidity pathophysiology heart brain lung imaging artificial intelligence risk assessment Journal Preproof 0c Introduction In December a novel coronavirus referred to as œsevere acute respiratory distress syndrome coronavirus  SARSCoV2 [] appeared in Wuhan the capital of Hubei Province in PR China The disease caused by the virus was initially named œnovel coronavirus pneumonia NCP by the Chinese government but was subsequently renamed œcoronavirus disease  COVID19 by the World Health anization WHO On January 30th it was declared a public health emergency of international concern PHEIC [] It is believed that SARSCoV2 is primarily transmitted through saliva droplets or nasal discharge [] The first evidence of humantohuman transmission was found by Jasper FukWoo Chan et al in their study at The University of Hong KongShenzhen Hospital [] Due to its contagiousness Ro27 the virus has reached epidemic levels affecting countries and causing over million infections and more than deaths as of July 1st [] shown in Figure Recent literature suggests that patients with preexisting diseases are likely to experience severe complications from COVID19 [] In one study on admitted diabetic and nondiabetic COVID19 patients the mortality rate vs and the rate of admission to the intensive care unit ICU vs were significantly higher for diabetic patients Diabetic patients also experienced severe inflammatory responses and cardiac hepatic and renal coagulopathy [] The prevalence of heart and brain injuries was also higher in COVID19 patients with concomitant chronic conditions like diabetes kidney disease dyslipidemia hypertension [] chronic obstructive pulmonary disease COPD and cardiovascular diseases [] Recent studies have shown that SARSCoV2 invades the thin lining of the epithelial cells of the arteries leading to atherosclerosis [] and arterial inflammatory disease”one of the major causes of cardiovascular diseases CVDs which also causes heart and brain injuries [ ] This could be due to a reduced expression of angiotensinconverting enzyme ACE2 causing endothelial dysfunction which in turn aggravates existing Journal Preproof 0catherosclerosis [ ] It has also been observed that comorbid patients when subjected to imagescreening show mild to severe pretest probability PTP for COVID19 [] The conventional cardiovascular risk factors CCVRF in these comorbid patients appear strongly correlated either to their heart imaging or to surrogate biomarkers of coronary artery disease such as carotid artery disease Both imaging and biomarkers could be helpful in severity predictions for COVID19 [] Figure illustrates the associations between SARSCoV2 and other comorbidities such as diabetes as well as the comparative survival rates for COVID19 patients with and without diabetes Figure World map showing COVID19 spread over countries courtesy John Hopkins University ACE2 expression causes scars in the vessels and can even rupture the walls of the arteries [] For this reason CCVRF should be considered alongside imaging in patients who present with COVID19 and many comorbidities [] The second stage is the one at which a patient is most severely affected by Journal Preproof 0cCOVID19 and has the highest probability of cardiac injury or release of troponin T TnT Imaging has been shown to offer benefits in monitoring the tissue scars caused by COVID19 [] Figure a Association of SARSCoV2 with other comorbidities and b comparison of the mortality rate of diabetic and nondiabetic COVID19 patients reproduced with permission [] Multiple modalities can be utilized to determine whether a patient has the sequelae of COVID19 including magnetic resonance imaging [] computed tomography [] and ultrasound [] The advantage of these imaging modalities is the visual access they provide to the scar tissue caused by the disease A disadvantage however is their inability to provide a œrisk assessment The application of artificial intelligence AI can enhance the information provided by these imaging modalities resulting in a more accurate characterization of the tissue and the disease process [] In fact the combination of AI and medical imaging has been shown to improve diagnosis and risk stratification speed up patient evaluation enhance disease monitoring and accelerate early intervention [ ] Thus this review will focus on the use of AIbased tissue characterization of images of comorbid patients affected by COVID19 The layout of this paper is as follows Section presents the pathophysiology of the four pathways leading to brain and heart injury Section summarizes the evidence related to the use of Journal Preproof 0cimaging during the COVID19 pandemic Section elaborates on the use of AIbased tissue characterization for risk assessment Finally the paper concludes with a critical discussion The Pathophysiology of SARCoV2 Leading to Brain and Heart Injury Several studies suggest that SARSCoV2 uses the ACE2 receptor to gain access to cells by binding to the SPIKE protein ˜S™ protein on their surface [] see Figure ACE2 and angiotensinconverting enzyme ACE1 are homolog carboxypeptidase enzymes that have different vital functions in the reninangiotensinaldosterone system RAAS pathway [] ACE2 is widely expressed in myocardial cells [] type pneumocytes enterocytes and astrocytes in the brain [ ] Thus it is recognized as a cause of extrapulmonary complications Figure shows the overall picture of how SARSCoV2 causes brain and heart injuries via four different pathways These include i the neuronal pathway ii the hypoxia pathway iii the RAAS pathway and iv the immune pathway We will discuss these pathways and the injuries they lead to which may manifest as viral encephalitis infectious toxic encephalopathy or acute cerebrovascular disease i The Neuronal Pathway Figure the pathway I Recent epidemiological studies have demonstrated similarities at the genomic level between SARSCoV1 MERS and SARSCoV2 [ ] Meanwhile previous experimental studies have shown that beta coronaviruses in general”such as SARSCoV1 and MERS”can spread into and directly infect the brain when inhaled as droplets via the nasal epithelium [ ] Figure depicts the olfactory nerve and the olfactory bulb []”labeled as œa and œb respectively”on the image of the sagittal brain in the neuronal pathway Based on recent reports we are aware that patients infected by SARSCoV2 show symptoms of dysgeusia loss of taste and anosmia loss of smell [ ] Bohmwald et al further validate that coronaviruses that infect through the olfactory nerve and bulb can reach the brain and cerebrovascular fluid CSF within seven days Additionally these viruses have been observed to cause inflammation and demyelination [] The Journal Preproof 0cauthors demonstrated in an experimental study of mice that removing the olfactory bulb from the pathway can lead to the restriction of CoV in the central nervous system CNS [] Based on this evidence we believe that the neuronal pathway is one possible track for SARSCoV2 ii The Hypoxia Pathway Figure pathway II In this pathway decreased levels of ACE2 proliferate in the lung parenchyma cells after the coronavirus has passed through causing exaggerated neutrophils accumulation enhanced vascular permeability and the formation of diffuse alveolar and interstitial exudates This ultimately leads to pulmonary edema and acute respiratory distress syndrome ARDS [] ARDS is characterized by severe abnormalities in blood gas composition resulting from an oxygen and carbon dioxide mismatch which leads to low blood oxygen levels [ ] This ongoing hypoxia can lead to myocardial ischemia and heart injury [ ] see Figure pathway IIA Hypoxia in the brain increases anaerobic metabolism in the mitochondria of the brain cells [] leading to cerebral vasodilatation edema and impaired flow This can result in cerebral ischemia and acute cerebrovascular diseases such as acute ischemic stroke [ ] see Figure pathway IIB iii The RAAS Pathway after SARSCoV2 Figure pathway III The RAAS pathway is crucial in regulating blood pressure as well as the balance of fluid and electrolytes Any disturbance in this pathway can trigger the pathogenesis of cardiovascular diseases [] Before a SARSCoV2 infection triggers the RAAS Angiotensin I Ang I cleaves to Angiotensin II Ang II via ACE1 Ang II causes vasospasm It is also a proinflammatory agent with prothrombotic and proliferative effects that are detrimental to vascular tone and hemostasis [ ] Thus as a counterregulatory mechanism ACE2 degrades Ang II and generates Ang which counteracts the negative impacts of Ang II [ ] Both ACE2 and Ang have cardiocerebral vascular protective effects [] After the triggering of SARSCoV2 infection it results are in the deregulation of RAAS causing heart and brain injury in two different pathways The main culprit is an increase in Ang II which is caused by the decrease in ACE2 levels Figure pathway IIIA First an increase in Ang II levels stimulates the adrenal cortex of the kidney resulting in an increased production of aldosterone Aldosterone is a steroid hormone that causes sodium and water Journal Preproof 0creabsorption to increase at the distal tubule and collecting duct of the nephron [] This reabsorption increases blood volume and causes an elevation in blood pressure which results in the endothelial dysfunction that causes brain and heart injury [] The second effect of an increase in Ang II levels ie as a consequence of decreased ACE2 levels is endothelial dysfunction leading to intimal damage in the arterial walls [] which can be seen during the imaging of the arterial wall see Figure pathway IIIB This pathway can also trigger a cytokine storm as high levels of Ang II can cause an increase in proinflammatory cytokines see the bridge line between the RAAS and immune pathways iv The Immune Pathway Figure pathway IV Several recent studies have reported SARSCov2 viral pneumonia [ ] having an exaggerated inflammatory response known as a œcytokine storm This response appears to present at advanced stages of severe COVID19 with increased levels of inflammatory cytokines leading to multiplean failure [] The rise in inflammatory markers”including IL6 IL7 IL12 IL15 IL22 TNFα and CXCL10”results in the destabilization of plaque This in turn can cause plaque rupture resulting in heart and brain injury [ ] The Role of Imaging in Comorbid Patients with COVID19 As the previous section discussed COVID19 uses four pathways ie neuronal hypoxia RAAS and immune to cause critical heart and brain injuries in patients with comorbidities The prevalence of myocardial injury and brain injury caused by COVID19 [ ] points to a need for increased use of medical imaging to expedite assessments differential diagnoses and patient management [ ] with proper safety measures [] The seriousness of a patient™s COVID19 symptoms helps to determine which imaging modality is appropriate portable or nonportable and invasive or noninvasive BMode ultrasound imaging is portable and can be used for lowrisk patients Meanwhile Xray magnetic resonance imaging [] and computed tomography [] are nonportable and can be used for mediumrisk patients Intravascular ultrasound IVUS [] and ventriculography are invasive imaging modalities used in highly critical cases [ ] Amongst all the imaging modalities ultrasound is noteworthy because it is radiationfree portable quick repeatable inexpensive Journal Preproof 0cand can be performed in isolation thus lowering the chance of spreading the COVID19 infection [ ] There are several examples of medical imaging that have led to proper treatment and healthcare management during the pandemic ultimately reducing the mortality rate Xray imaging of the chest has demonstrated irregular patchy hazy reticular and widespread groundglass opacities indicating the progression of COVID19 at various stages this information can support the healthcare team in developing the most appropriate treatment plan [] Chest CT scans of COVID19 patients revealed in almost of the patients that the disease was affecting at least one of the five lobes of their lungs [] Chest MRI scans of COVID19 patients showed pulmonary tissue consolidation in six diffusionrestricted regions in six and lung damage in seven [] Meanwhile heart MRI studies of recovered patients showed that of the patients had myocardial edema At the same time late gadolinium enhancement was found in implying that COVID19“related cardiac injury is longstanding and requires frequent monitoring even after recovery [] In a different study MR scans of a COVID19 patient revealed myocardial inflammation signifying myocardial injury due to a cytokine storm related to the SARSCoV2 infection as discussed in Section Pathway IV [] Several studies have also evaluated the effects of COVID19 on the brain In one MRI scans revealed hemorrhagic rim enhancing lesions within the bilateral thalami medial temporal lobes and subinsular regions [] shown in Figure In another brain MRI scans were completed for patients of which produced abnormal findings [] Additionally evidence of liver injury and gall bladder abnormality was found in a joint CT and ultrasound study of the abdomen [] Recent MRI scans of COVID19 patients™ olfactory bulbs have revealed the cause of olfactory function loss to be the interaction between SARSCoV2 and the ACE2 protein expressed by the olfactory epithelium which leads to inflammatory obstruction [] Journal Preproof 0cFigure We have shown in four pathways how COVID19 can cause Brain and heart injury Brain image in pathway I httpdebugliescom20200123olfactorydisturbanceshaveimplicationsinmentalandemotionalwellbeinghealth Courtesy of Debug Lies Invasive imaging is another option for diagnosing COVID19 patients who have critical comorbidities In one such study IVUS along with stenting was performed with precautions on a COVID19 patient with myocardial infarction [] shown in Figure A detailed discussion of precautions is included in section In another study takotsubo syndrome a form of myocardial injury triggered by COVID19 was detected using ventriculography [] In various studies the medical imaging of COVID19 patients had been crucial to ascertaining the extent of tissue damage and critical infection although there were no visible symptoms [ ] Therefore medical imaging is the preferred way to ascertain the extent of cardiac and brain tissue damage throughout the lifetime of COVID19 patients COVID19 Journal Preproof 0cpatients with comorbidities are especially vulnerable and so they need to be screened through medical imaging from the first day of their diagnosis Medical imaging can help patients with deep vein thrombosis DVT as they are highly susceptible to severe tissue damage from COVID19 A study showed that COVID19 patients suffering from DVT had a worse prognosis than patients without DVT Patients with DVT were admitted to the ICU more frequently discharged less frequently and suffered more deaths than those without DVT [] Figure MRI scan of COVID19 patient showing hemorrhage MRI images demonstrate T2 FLAIR hyperintensity within the bilateral medial temporal lobes and thalami A B E F with evidence of hemorrhage indicated by hypointense signal intensity on susceptibilityweighted images C G and rim enhancement on postcontrast images D H reproduced with permission [] Journal Preproof 0c Figure Application of chest CT and IVUS for a COVID19 patient suffering from myocardial infarction a Chest CT scan with viral pneumonia showing fibrinous focal exudative changes b when the patient complained of chest pain the ECG report showed the STsegments elevations in V1V5 lead c d CAG radiology that the proximal segment of LAD was occluded e f The blood flow of LAD restoration after DESs was implanted g the dissection distal shown by IVUS to the stent in LAD from “ o'clock h the low echogenic shadow with scattered higher echogenic flicker indicating a thrombus i after a DES was implanted and the stent was well expanded the dissection could not be seen j The thrombus disappearance after the intervention reproduced with permission [] Journal Preproof 0c Although medical imaging can be very useful to patients and doctors alike the exponential pandemic curve inadequate medical facilities [] and a limited number of radiologists make the assessment diagnosis and management processes challenging tedious and errorprone Therefore although medical imaging can make diagnoses faster as stated above it will be of limited use Given this fact new age techniques such as artificial intelligence AI [] applications in medical imaging for tissue characterization can make computeraided assessments and diagnoses faster The main reason for this is that the AI can be scaled up to match the pandemic curve thereby meeting the immediate demands of medical image diagnoses during the COVID19 pandemic AIbased tests can categorize the nature of a patient™s risk in one of the categories namely norisk low lowmedium LM highmedium HM lowhigh LH or highhigh HH risk depending on the patient™s symptoms and their severity [ ] as shown in Figure The imaging modality also varies with the degree of risk as follows no imaging for norisk portable imaging for low and LM risk nonportable imaging for HM and LH and invasive imaging for HH A probability PTP is performed to accurately interpret diagnostic results to categorize the patient into one of the four groups [] After that for norisk patients nonimaging biomarkers can be collected for risk assessment using AIbased data protocols For lowrisk patients portable 2D3D imaging such as ultrasound is used whereas nonportable and invasive 2D3D imaging such as MRICTXRayechocardiography can be used for LM patients For HH patients invasive imaging techniques such as IVUS and ventriculography can be used Based on the data provided by various 2D3D scans AIbased medical imaging is applied for risk assessment Further treatment is then planned based on this imaging process In the next section deep learning DLbased medical imaging is proposed for medical imaging scans particularly ultrasounds for COVID19 patients Machine Learning and Deep Learning for Tissue Characterization Using AI and associated technologies in healthcare can significantly slow down diagnosis times Journal Preproof 0cespecially during the COVID19 pandemic as patient numbers are continually growing and there are few specialists available [] Figure Role of AIbased risk assessment on COVID19 patients having comorbidity Journal Preproof 0cAlthough some caution must be exercised regarding its fullscale deployment [] its overall usefulness in healthcare management during times of crisis cannot be ignored [ ] In general AI in healthcare refers to all artificial intelligencebased technologies that make educated decisions regarding a patient™s diagnosis monitoring treatment and management The importance of AI has specifically increased many folds when imaging comes into play mainly because of large volumetric data sizes and the extensive need to characterize and quantify the disease via lesion images [] Tissue imaging and its characterization is of prime importance since it has a direct influence on decisions related to COVID19 severity for a patient [] The main benefit of AI methods is that the machines can be used to train by mimicking the physician™s cognitive actions and such trained models can be used to predict the disease™s severity in asymptomatic patients Within a short period several machine learning MLbased techniques used the power of AI to manage COVID19 [ ] ML and DL Architectures ML Architecture ML is a twostage process In stage I different features are extracted from the lesion COVID images the extractions are then operated on by an ML statistical model called a training system to generate offline coefficients These coefficients are then transformed by the test lesion images which yield an intelligent classification or inference A typical ML system for predicting risk class is shown in Figure CUSIP is an imagebased phenotype that uses the event equivalent gold standard EEGS [ ] model DL Architecture DL refers to a visual cortex that imitates multiple layers of a neural network applied directly to tissue images to extract features and for characterization purposes [] The convolution neural network CNN [] shown in Figure is one such DL network architecture that is widely used to characterize medical images It performs a series of convolution maxpooling operations to extract features and perform characterizations ML and DL both follow the supervised learning Journal Preproof 0capproach by which models are trained using offline data Figure Typical lowcost machine learning architecture utilizing EEGS model As discussed in previous sections there are four pathways through which a COVID19 infection leads to heart and brain injuries AI can be used via medical imaging to detect the extent of tissue damage in these pathways and help medical professionals to develop an effective treatment plan for patients There are several instances in which the AI paradigm has been used for tissue characterization based on Journal Preproof 0cmedical images during the pandemic as well as during standard times Some uses of AI are described anwise following a proposed model for characterizing DLbased tissues Figure A convolution neural network courtesy of AtheroPoint„¢ CA USA ML Architecture used for Tissue Characterization for Stroke Risk Stratification There are two types of tissue characterization that can be carried out using AI i MLbased [ ] and ii DLbased [] Various MLbased technologies have been developed to classify symptomatic and asymptomatic plaque from ultrasound images For example an MLbased technique based on support vector machines SVM was developed to characterize the symptomatic and asymptomatic plaques of carotid scans that indicated the presence of plaque [ ] SVM classifiers work by determining the maximum margin between two data clusters First a texture analysis [] is used to extract the features ie standard deviation entropy symmetry and run percentage in Answer:

Thyroid_Cancer

"Ethnopharmacological relevance Tetrastigma hemsleyanum Diels et Gilg Themsleyanum a rare herbal plant distributed in subtropical areas of mainland China has become a focus of scientific attention in recent years because of its high traditional value including uses for treatment of children with fever pneumonia asthma rheumatism hepatitis menstrual disorders scrofula and pharynx pain Aim This systematic review aims to provide an insightful understanding of traditional uses chemical composition pharmacological effect and clinical application of T hemsleyanum and lay a foundation for the further study and for the utilization of T hemsleyanum resource Materials and methods A domestic and overseas literature search in known databases was conducted for published s using the relevant keywords Results One hundred and fortytwo chemical constituents identified from T hemsleyanum have been reported including flavonoids phenolic acids polysaccharide anic acids fatty acids terpenoids steroids amino acid and others Among these components flavonoids and polysaccharides were the representative active ingredients of T hemsleyanum which have been widely investigated Modern pharmacological studies have shown that these components exhibited various pharmacological activities such as antiinflammatory antioxidant antivirus antitumor antipyretic antihepatic injury immunomodulatory antibacterial etc Moreover different toxicological studies indicated that the clinical dosage of T hemsleyanum was safe and reliable Conclusions Modern pharmacological studies have well supported and clarified some traditional uses and T hemsleyanum has a good prospect for the development of new drugs due to these outstanding properties However the present findings did not provide an indepth evaluation of bioactivity of the extracts the composition of its active extracts was not clear Moreover they were insufficient to satisfactorily explain some mechanisms of action Data regarding many aspects of T hemsleyanum such as links between the traditional uses and bioactivities pharmacokinetics quality control standard and the clinical value of active compositions is still limited which need more attention Introduction Tetrastigma hemsleyanum Diels et Gilg T hemsleyanum mostly known as œSan ye qing is a kind of folk plant Because of its slow growth it usually takes “ years to meet the requirements of commercial medicinal materials so it is a precious perennial medicinal resource It mainly grows in the eastern central southern and south western provinces of China such as Zhejiang Jiangsu Guangxi Fujian and Yunnan provinces Peng and Wang T hemsleyanum is known worldwide as sources of phytotherapeutics which have been used for the treatment of conditions related to inflammatory and immune response and been recorded based on clinical trials or the use of animal models Xu As an edible plant the leaves of T hemsleyanum consumed as a functional tea or dietary supplement for its health benefits such as improving the immune system of the body Sun while the aerial parts of T hemsleyanum developed as potential new traditional chinese medicine TCM preparations Guo Corresponding author Ningbo Research Institute of Zhejiang University Ningbo Zhejiang People™s Republic of China Email address px4142163com X Peng 101016jjep2020113247 Received May Received in revised form July Accepted August JournalofEthnopharmacology2642021113247Availableonline12August2020037887412020ElsevierBVAllrightsreserved 0cT Ji Abbreviations T hemsleyanum Tetrastigma hemsleyanum Diels et Gilg TCM UPLCESIQTOFMSMS Ultra high performance liquid Traditional Chinese Medicine chromatography tandem triple quadrupole time of flight mass spectrometry minimum inhibitory concentration glutathione malondialdehyde nuclear factorκB 5hydroxytryptamine norepinephrine dopamine prostaglandin E2 lipopolysaccharide tumor necrosis factoralpha interleukin1 beta interleukin MIC GSH MDA NFκB 5HT NE DA PGE2 MAPK mitogenactivated protein kinase LPS Celegans Caenorhabditis elegans TNFα IL1 IL6 IL12p40 interleukin subunit p40 sTNFR1 soluble TNF receptors IL10 IL1 IL4 iNOS TLR4 MD2 MyD88 myeloid differentiation protein JNK GPT GOT ALP SOD interleukin interleukin interleukin inducible NO synthase Tolllike receptor myeloid differentiation factor2 cJun Nterminal kinase glutamicpyruvic transaminase glutamicoxalacetic transaminase alkaline phosphatase superoxide dismutase and activities antiinflammatory The root tubers of T hemsleyanum are extensively used either alone or in combination with other herbal medicines in TCM clinics for the treatment of children with fever convulsion pneumonia asthma rheumatism hepatitis menstrual disorders scrofula and pharynx pain Sun Chen and Guo Therefore it was called as œnatural plant antibiotic according to its wide spectrum of prominent bactericidal In February T hemsleyanum was awarded as the new œeight famous kinds of TCM in Zhejiang province meant that it has become a key object of industrialization development of Zhejiang™s dominant large varieties of medicinal materials In COVID19 broke out and has caused more than deaths in China and infection cases have been reported in more than countries Hua Shi Xuan Fei mixture Approval number of Zhejiang medicine Z20200026000 which composed of T hemsleyanum has been approved by Zhejiang Provincial Drug Administration for clinical treatment of COVID19 Futhermore the modern pharmacological studies had shown that T hemsleyanum also had effects of antiinflammatory Ji antioxidant Hossain antivirus Ding antitumor Lin antipyretic Yang and Wang antihepatic injury Ma et al immunomodulatory Xu antibacterial Chen hypoglycemic Ru 2018ab etc Numerous reports have demonstrated that the biological activities of T hemsleyanum are attributed to its many chemical components Fu Wang has reported isolated alkaloids from the aerial parts of T hemsleyanum Wang Ru extracted a novel polysaccharide TDGP3 from is mainly alanine aminotransferase aspartate aminotransferase hyaluronan laminin total bilirubin total protein interferongamma immunoglobulin A secretory immunoglobulin A Epithelialmesenchymal transition ALT AST HA LN TBiLi TP IFNÎ IgA SIgA EMT MMPs matrix metalloproteinase TIMPs matrixmetallo proteinase Cytc CAT GSHPx glutathione peroxidase Tregs TGF COX2 Foxp3 PDL TAOC CCl4 CEF HVJ VSV A F S1 S2 PEF CFF EAF BAF Cytochrome c catalase regulatory T cells transforming growth factor beta cyclooxygenase forkheadwinged helix transcription factor gene population doubling time total antioxidant capacity carbon tetrachloride chicken embryo fibroblast Hemagglutinating virus of Japan vesicular stomatitis virus alkalicontaining extract of T hemsleyanum ketonecontaining extract of T hemsleyanum crude extract of T hemsleyanum crude extract of T hemsleyanum Petroleum ether extractions of T hemsleyanum ethanol extract Chloroform extractions of T hemsleyanum ethanol extract ethyl acetate extractions of T hemsleyanum ethanol extract nbutanol extractions of T hemsleyanum ethanol extract T hemsleyanum with a molecular weight of — Da by enzymolysisultrasonic assisted extraction method Ru 2019ab Large amounts of flavonoids were found in leaves aerial parts and root tubers of T hemsleyanum Xu 2014ab Deng Yu In addition T hemsleyanum also contains a variety of functional components such as anic acids Hu phenolic acids Liu minerals Fan amino acids Fu etc In recent years wild resources of T hemsleyanum have been overexploited and now are on the verge of extinction due to its multiple medicinal values coupled with the strict requirements of the growing environments In it was listed in the preferentially protected crop germplasm resources of Zhejiang province Based on our team™s preliminary research Peng Peng 2016ab Li we comprehensively summarized and analyzed the domestic and overseas research progress on traditional uses the bioactive components of T hemsleyanum pharmacological activities toxicology with the aim of providing guidance for indepth research and reference for its development and utilization Materials and methods The available information about the traditional uses phytochemicals and pharmacological properties of T hemsleyanum was searched via Web of Science Google Scholar PubMed Science Direct China National Knowledge Infrastructure CNKI and Springer search using Chinese or English as the retrieval languages The keywords used include T hemsleyanum root tubers of T hemsleyanum Radix Tetrastigma JournalofEthnopharmacology26420211132472 0cT Ji traditional uses phytochemistry bioactive components pharmacological activities toxicology and other related words All references were from experimental studies and published prior to April were reviewed All chemical structures were drawn using ChemDraw Pro software heatclearing were Botanical characteristics T hemsleyanum is a perennial grass climbing vine with longitudinal ribs glabrous or sparsely pilose It is usually grown in a cool and humid environment and the main soil type is yellow soil or yellow brown soil with rich humus The optimum pH is between and The root tubers are thick spindle shaped or elliptical and single or several are connected into a string of beads generally “ cm long and “ cm in diameter Fig The epidermis of the root tubers is tan and most of them are smooth a few of them have folds and lenticel like protuberances some of them have depressions in which there are residual tan roots hard and brittle with a flat and rough section The stem of T hemsleyanum is thin and weak with longitudinal rhombus rooting on the lower node Palmate compound leaves alternate leaflets are lanceolate oblong or ovate lanceolate The leaflets are “ cm long and “ cm wide with a tapered tip and a wedgeshaped or round base The flowers of T hemsleyanum are small yellow green and ovate The flowering stage of T hemsleyanum ranges from April to June and the fruit phase is normally from August to November When the flower withered it will form a small green round fruit with the size of millet When it is mature the fruit will turn from green to red the berries are spherical and soft spherical Traditional uses T hemsleyanum belonging to the family Vitaceae was firstly recorded in Ben Cao Gang Mu Ming Dynasty AD The aliases of Sanyeqing include Shi Hou Zi Shi Bao Zi Shi Lao Shu Lan Shan Hu Lei Dan Zi Po Shi Zhu Tu Jing Wan Sou Jia Feng San Ye Dui golden wire hanging gourd golden bell golden wire hanging potato etc The root tubers or whole grass of T hemsleyanum traditionally and ethnically used as a medicine for a long time it has been recorded in multiple hemsleyanum ancient books of TCM such as Zhi Wu Ming Shi Tu Kao Qing Dynasty Wu Jiangxi herbal medicine Common folk herbal medicine in Zhejiang All of these ancient works described the effects of toxicityremoving T dyspnearelieving promoting blood circulation and pain relief thus it can be applied to cure febrile convulsion pneumonia bronchitis pharyngitis sore throat acute and chronic hepatitis rheumatic arthralgia viral meningitis bruise eczema insect and snake bite poor joint flexure and extension irregular menstruation of women National compilation team of Chinese herbal medicine In the TCM culture the properties of T hemsleyanum was described as bitter and acrid in taste cool in nature which recorded in dictionaries of traditional Chinese medicine and Zhong Hua Ben Cao Shanghai Science and Technology Press The channel tropism was lung heart liver and kidney meridians Decocting with water or mashing for external application are the traditional possess methods of T hemsleyanum Considering its extensive traditional effects many prescriptions containing T hemsleyanum have been passed down from generation to generation and have been well supported and clarified by modern pharmacological studies Excitingly it has reported that Jinlian disinfection drink containing san ye qing combined with interferon can treat Covid19 He Jinqi Tablet made up of san ye qing astragalus and ginsenoside was used to treat cases of malignant tumor cases were completely relieved cases were partially relieved the total effective rate was Wei Moreover Zhonggan mixture including san ye qing could improve the quality of life and prolong the survival time of patients with stage III primary liver cancer Jiang and Gong In addition it has been used in the treatment of common gynecological diseases such as blood avalanche and leucorrhea Gao and it also has a good effect on measles complicated with pneumonia anal fissure chronic bronchitis and mosquito bites Ji Chemical compounds of Themsleyanum The chemical constituents of T hemsleyanum have been widely investigated Sun Sun Zeng Xu 2014ab Fu Fan Chen Ding 2015a Fig The aerial part A root tuber B and raw herb C of T hemsleyanum JournalofEthnopharmacology26420211132473 0cT Ji b Ding a total of one hundred and fortytwo compounds have been isolated and identified from T hemsleyanum until now The information about compound name molecular weight compound formula detection method analysis sample is summarized in Table Flavonoids and their glycosides Modern phytochemical studies have indicated that flavonoids are the representative and predominated class of constituents isolated from T hemsleyanum Lin Zhang Table To date fiftyone flavonoids and their glycosides have been extracted and identified from T hemsleyanum In this series compounds quercetin orientin vitexin isorhamnetin apigenin and kaempferol are the main types of skeleton some of their analogues can be identified from hydroxy moiety on C3² and C4™ on the B ring of flavonoid aglycone At present many modern analytical techniques have been used for qualitative and quantitative analysis of flavonoids Among them ultra high performance liquid chromatography tandem triple quadrupole time of flight mass spectrometry UPLCESIQTOFMS has become a powerful tool for identifying the complicated compounds due to its higher mass accuracy and resolution Our team used UPLCESIQTOFMS to identify chemical constituents from the aerial part of T hemsleyanum including flavonoids such as isoorientin quercetin kaempferol vitexin isovitexin kaempferol3glucoside etc Sun According to the report Liu total flavonoids of T hemsleyanum could protect the aged mice from acute lung injury through inhibiting the phosphorylation of mitogenactivated protein kinase MAPK and nuclear factorκB NFκB in lung tissue Moreover the flavonoids of T hemsleyanum had the activity of antilung cancer Wei Luteolin a flavonoid found in T hemsleyanum acted as an anticancer agent against various types of human malignancies such as lung breast glioblastoma prostate colon and pancreatic cancers Muhammad It is certain that T hemsleyanum flavonoids give a new vision for researchers to explore clinical anticancer drugs Polysaccharide Saccharide is another important active ingredient extracted from T hemsleyanum Shao Polysaccharide has great potential in clinical application because of its unique pharmacological activity However due to the complex structure of polysaccharide it is difficult and special to determine and synthesize their structures Guo Table The prescriptions and traditional uses of T hemsleyanum in China Prescriptions name Qingteng Fengshi Qufengshi Yaojiu Main composition Jiu Traditional use T hemsleyanum Parabarium chunianum Tsiang Zanthoxylum nitidum Roxb DC T hemsleyanum Deeringia amaranthoides Lam Merr Blumea aromatica Wall DC T hemsleyanum Deeringia amaranthoides Lam Merr Zanthoxylum nitidum Roxb DC Panax notoginseng Burk FH Chen T hemsleyanum Gypsum Lonicera japonica Thunb Houttuynia cordata Thunb Ophiopogon japonicus Linn f KerGawl T hemsleyanum T hemsleyanum Lysimachia christinae Hance Imperata cylindrica Citrus reticulata Blanco T hemsleyanum ginsenoside Astragalus propinquus Schischkin T hemsleyanum Nepeta cataria L Lonicera japonica Thunb Saposhnikovia divaricata Trucz Schischk Huatuo Fengtongbao capsule Sanyeqing Gypsum Decoction Sanyeqing Power Zhonggan mixture Jinqi Tablet Hua Shi Xuan Fei mixture extracted the polysaccharides from roots of T hemsleyanum RTP1 RTP2 and RTP3 were successively found by protein precipitation and purification Moreover further study indicated RTP31 was high purity polysaccharide with a molecular weight of kDa and it is mainly composed of kinds of monosaccharides arabinose galacturonic acid galactose and fructose the proportion is and respectively Ru 2018ab extracted a polysaccharide THP from T hemsleyanum with the average molecular weight estimated as kDa The results of study on the composition of polysaccharide showed that it was mainly composed of rhamnose arabinose mannose glucose galactose with the molar ratio of In Ru 2019ab successfully extracted polysaccharide THDP3 from T hemsleyanum with molecular weight of kDa which consists of rhamnose arabinose mannose glucose and galactose with molar ratio of Moreover TDGP3 mainly consists of †’4αDGalAp1†’ †’4DGalp1†’ and †’4αDGlcp1†’ residues as backbones and DManp1†’ †’36DManp1†’ and αDAraf1†’residues as branches Phenolic acids Phenolic acids refer to aromatic carboxylic acids with multiple phenolic groups substituted on one benzene ring As a secondary metabolite phenolic acids are widely found in many natural plants and have antiinflammatory antioxidant and lipid lowering effects Twenty three phenolic acids No52“ Table have been reported in the aerial parts of T hemsleyanum such as caffeic acid chlorogenic acid 1OgalloylDglucose protocatechol glucoside epigallocatechin 1caffeoylquinic acid 3caffeoylquinic acid 4caffeoylquinic acid 5caffeoylquinic acid 1pcoumaroylquinic acid 4pcoumaroylquinic acid and 5pcoumaroylquinic acid There were twentyone phenolic acids in the root tuber of T hemsleyanum some of which were the same as aerial parts Alkaloids Alkaloids are a group of basic anic compounds containing nitrogen that exist in nature Alkaloids are stored in small quantities in T hemsleyanum and the bioactivity investigations of those alkaloids are still rather rare Wang Fu extracted the aerial parts of T hemsleyanum with ethanol and then isolated ten alkaloids for the first time including seven indole alkaloids an amide a maleimide and Treatment of joint pain wind cold dampness arthralgia Treatment of arthralgia syndrome rheumarthritis rheumatoid arthritis scapulohumeral periarthritis Treatment of arthralgia syndrome rheumarthritis rheumatoid arthritis scapulohumeral periarthritis joint pain muscular constricture Treatment of infantile hyperpyretic convulsion Treatment of blood avalanche leucorrhea Treatment of liver cancer Treatment of malignant tumor Treatment of Covid19 Usage Oral administration “ mL once times a day Oral administration mL once times a day Oral administration capsules once times a day References Ministerial standard Ministerial standard Ministerial standard One dose a day decoct twice in water and take it “ times after mixing Oral administration Oral administration mL once times a day Oral administration capsules once times a day Oral administration mL once times a day Xu Gao Jiang and Gong Wei Zhejiang Provincial Drug Administration JournalofEthnopharmacology26420211132474 0cT Ji Detection Mode Analysis parts of sample Reference aerial part root tuber aerial part root tuber aerial part root tuber root tuber aerial part root tuber root tuber root tuber aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part root tuber root tuber root tuber aerial part root tuber root tuber aerial part aerial part aerial part aerial part aerial part aerial part aerial part root tuber aerial part root tuber root tuber aerial part root tuber root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part aerial part root tuber aerial part root tuber aerial part root tuber root tuber root tuber root tuber root tuber root tuber root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part root tuber aerial part aerial part aerial part aerial part root tuber root tuber aerial part root tuber aerial part root tuber aerial part root tuber Sun Sun Zeng Sun Sun Sun Zeng Zeng Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Zeng Sun Zeng Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Xu 2014b Sun Zeng Sun Zeng Sun Zeng Zeng Sun Sun Sun Sun Xu 2014b Sun Xu 2014b Sun Zeng Sun Xu 2014b Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Fu Sun Sun Xu 2014b Fan Xu 2014b Fan Sun continued on next page UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS Table Chemical constituents isolated from the different parts of T hemsleyanum Name Flavonoids and their glycosides quercetin quercitrin quercetin3Oglucoside quercetin3Orutinoside quercetin3galactoside quercetin3Oxylosylglucoside quercetin3Oxylosylglucose7Orhamnoside orientin orientin2²²Orhamnoside Isoorientin isoorientin2²²Orhamnoside isoorientin cid0 ²²Oxyloside vitexin vitexin2²²Orhamnoside vitexin2²²Oglucoside vitexin2²²Oarabinoside isovitexin isovitexin2²²Orhamnoside isovitexin2²²Oxyloside isorhamnetin isorhamnetin3rutinoside isorhamnetin3pyranoarabinose7glucosylrhamnoside apigenin apigenin7rhamnoside apigenin8Cxylosyl6Cglucoside apigenin6CαLarabinose8CDglucose eriodictyol eriodictyolOhexoside I eriodictyolOhexoside II luteolin luteolin6 8diChexoside catechin catechin glucopyranoside isomer epicatechin kaempferide kaempferol kaempferol3glucoside kaempferol3rutinoside kaempferol3sambubioside kaempferol3Oneohesperidin kaempferol3Orhamnoside kaempferol7Orhamnose3Oglucoside kaempferol3robinoside7rhamnoside kaempferol3rutinoside kaempferol3Ocarfuran7Orhamnosyl glucoside daidzein biochanin A procyanidin dimmer procyanidin B1 procyanidin B2 procyanidin trimer Phenolic acids and derivatives gallic acid protocatechuic acid caffeic acid dihydroxybenzoic acid hexoside 1caffeoylquinic acid 3caffeoylquinic acid 4caffeoylquinic acid 5caffeoylquinic acid 1pcoumaroylquinic acid 4pcoumaroylquinic acid 5pcoumaroylquinic acid phydroxybenzaldehyde pcoumaric acid ferulic acid hexoside salicylic acid chlorogenic acid neochlorogenic acid cryptochlorogenic acid protocatechualdehyde UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS 1HNMR13CNMR MS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS JournalofEthnopharmacology26420211132475 0cT Ji Table continued Name salicin2benzoate trihydroxycinnamoylquinic acid isomer protocatechuic acid hexoside apiosylglucosyl 4hydroxybenzoate 1OgalloylDglucose protocatechol glucoside epigallocatechin vanillic acid1Ofuran celery glucosyl ester protocatechuic acid1Ofuran celery glucosyl ester methoxyphenol1Ofuran glycosylOglucoside 2methoxy4methylbenzene1ofuracresyl glucoside oxyresveratrol dicaffeoylquinic acid 4hydroxycinnamic acid Alkaloids indole indole3carboxylic acid indole3propanoic acid 5hydroxyindole3carboxaldehyde 5hydroxyindole3carboxylic acid 6hydroxy3 4dihydro1oxocarboline hippophamide 4hydroxycinnamide pyrrole3propanoic acid Scid0 trolline Fatty acids trihydroxy octadecadienoic acid trihydroxy octadecenoic acid dihydroxy octadecenoic acid 9hydroxy1012octadecadienoic acid 9hydroxy octadecatrienoic acid hydroxyoctadecenoic acid hydroxyoctadecatrienoic acid Dihydroxyoctadecatrienoic acid dihydroartemisinin ethyl ether Trihydroxy octadecadienoic acid isomer hydroxyoxooctadecatrienoic acid octadecenedioic acid diMeester stearic acid linolenic acid linoleic acid palmitic acid oleic acid anic acids and derivatives malic acid quinic acid citric acid azelaic acid oxalic acid galactonic acid gallic acid succinic acid fumaric acid propanoic acid Terpenoids and steroids sitosterol daucosterol campesterol Stigmasterol 6Obenzoyl daucosterol ergosterol taraxerone Taraxerol αamyrine pteroside Z ganoderic acid H 3epipapyriferic acid oleanic acid Saponins Ginsenoside Rh1 Detection Mode UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS 1HNMR LCMS NMR UV MS NMR UV MS NMR UV MS NMR UV MS NMR UV MS NMR UV MS NMR UV MS NMR UV MS NMR UV MS NMR UV MS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS GCMS TCL HNMR CNMR MS GCMS GCMS IR HNMR EIMS IR HNMR MS IR HNMR MS IR HNMR MS IR EIMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS HNMR CNMR MS Analysis parts of sample root tuber root tuber root tuber root tuber aerial part aerial part root tuber aerial part root tuber root tuber root tuber root tuber root tuber root tuber root tuber root tuber aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts root tuber aerial part root tuber aerial part root tuber root tuber aerial part root tuber aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part root tuber root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part aerial part aerial part aerial part aerial part root tuber aerial part root tuber root tuber root tuber root tuber root tuber root tuber aerial part aerial part aerial part aerial part root tuber root tuber root tuber root tuber Reference Sun Sun Sun Sun Sun Sun Zeng Sun Xu 2014b Zeng Zeng Zeng Zeng Xu 2014b Xu 2014b Chen Fu Fu Fu Fu Fu Fu Fu Fu Fu Fu Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Chen Ding Sun Sun Guo Ru Ru Ru Ru Sun Sun Sun Ding UPLCESIQTOFMSMS root tuber Sun continued on next page JournalofEthnopharmacology26420211132476 0cT Ji Table continued Name Ginsenoside Rh2 Vinaginsenoside R1 Amino acid and derivatives Phenylalanine pyroglutamic acid glutimic acid hexose Tryptophan Lglutamic acid Detection Mode UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS Analysis parts of sample root tuber root tuber root tuber aerial part aerial part aerial part aerial part aerial part Reference Sun Sun Sun Sun Sun Sun Sun respectively a carboline By comparing with the spectral data of known compounds the alkaloids were indole3carboxylic acid indole3propanoic acid 5hydroxyindole3carboxaldehyde 5hydroxyindole3carboxylic acid 6hydroxy3 4dihydro1oxocarboline hippophamide 4hydroxycinnamide pyrrole3propanoic acid and Scid0 trolline The chemical structures were shown in Fig identified as indole a

cancer236

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: "Ethnopharmacological relevance Tetrastigma hemsleyanum Diels et Gilg Themsleyanum a rare herbal plant distributed in subtropical areas of mainland China has become a focus of scientific attention in recent years because of its high traditional value including uses for treatment of children with fever pneumonia asthma rheumatism hepatitis menstrual disorders scrofula and pharynx pain Aim This systematic review aims to provide an insightful understanding of traditional uses chemical composition pharmacological effect and clinical application of T hemsleyanum and lay a foundation for the further study and for the utilization of T hemsleyanum resource Materials and methods A domestic and overseas literature search in known databases was conducted for published s using the relevant keywords Results One hundred and fortytwo chemical constituents identified from T hemsleyanum have been reported including flavonoids phenolic acids polysaccharide anic acids fatty acids terpenoids steroids amino acid and others Among these components flavonoids and polysaccharides were the representative active ingredients of T hemsleyanum which have been widely investigated Modern pharmacological studies have shown that these components exhibited various pharmacological activities such as antiinflammatory antioxidant antivirus antitumor antipyretic antihepatic injury immunomodulatory antibacterial etc Moreover different toxicological studies indicated that the clinical dosage of T hemsleyanum was safe and reliable Conclusions Modern pharmacological studies have well supported and clarified some traditional uses and T hemsleyanum has a good prospect for the development of new drugs due to these outstanding properties However the present findings did not provide an indepth evaluation of bioactivity of the extracts the composition of its active extracts was not clear Moreover they were insufficient to satisfactorily explain some mechanisms of action Data regarding many aspects of T hemsleyanum such as links between the traditional uses and bioactivities pharmacokinetics quality control standard and the clinical value of active compositions is still limited which need more attention Introduction Tetrastigma hemsleyanum Diels et Gilg T hemsleyanum mostly known as œSan ye qing is a kind of folk plant Because of its slow growth it usually takes “ years to meet the requirements of commercial medicinal materials so it is a precious perennial medicinal resource It mainly grows in the eastern central southern and south western provinces of China such as Zhejiang Jiangsu Guangxi Fujian and Yunnan provinces Peng and Wang T hemsleyanum is known worldwide as sources of phytotherapeutics which have been used for the treatment of conditions related to inflammatory and immune response and been recorded based on clinical trials or the use of animal models Xu As an edible plant the leaves of T hemsleyanum consumed as a functional tea or dietary supplement for its health benefits such as improving the immune system of the body Sun while the aerial parts of T hemsleyanum developed as potential new traditional chinese medicine TCM preparations Guo Corresponding author Ningbo Research Institute of Zhejiang University Ningbo Zhejiang People™s Republic of China Email address px4142163com X Peng 101016jjep2020113247 Received May Received in revised form July Accepted August JournalofEthnopharmacology2642021113247Availableonline12August2020037887412020ElsevierBVAllrightsreserved 0cT Ji Abbreviations T hemsleyanum Tetrastigma hemsleyanum Diels et Gilg TCM UPLCESIQTOFMSMS Ultra high performance liquid Traditional Chinese Medicine chromatography tandem triple quadrupole time of flight mass spectrometry minimum inhibitory concentration glutathione malondialdehyde nuclear factorκB 5hydroxytryptamine norepinephrine dopamine prostaglandin E2 lipopolysaccharide tumor necrosis factoralpha interleukin1 beta interleukin MIC GSH MDA NFκB 5HT NE DA PGE2 MAPK mitogenactivated protein kinase LPS Celegans Caenorhabditis elegans TNFα IL1 IL6 IL12p40 interleukin subunit p40 sTNFR1 soluble TNF receptors IL10 IL1 IL4 iNOS TLR4 MD2 MyD88 myeloid differentiation protein JNK GPT GOT ALP SOD interleukin interleukin interleukin inducible NO synthase Tolllike receptor myeloid differentiation factor2 cJun Nterminal kinase glutamicpyruvic transaminase glutamicoxalacetic transaminase alkaline phosphatase superoxide dismutase and activities antiinflammatory The root tubers of T hemsleyanum are extensively used either alone or in combination with other herbal medicines in TCM clinics for the treatment of children with fever convulsion pneumonia asthma rheumatism hepatitis menstrual disorders scrofula and pharynx pain Sun Chen and Guo Therefore it was called as œnatural plant antibiotic according to its wide spectrum of prominent bactericidal In February T hemsleyanum was awarded as the new œeight famous kinds of TCM in Zhejiang province meant that it has become a key object of industrialization development of Zhejiang™s dominant large varieties of medicinal materials In COVID19 broke out and has caused more than deaths in China and infection cases have been reported in more than countries Hua Shi Xuan Fei mixture Approval number of Zhejiang medicine Z20200026000 which composed of T hemsleyanum has been approved by Zhejiang Provincial Drug Administration for clinical treatment of COVID19 Futhermore the modern pharmacological studies had shown that T hemsleyanum also had effects of antiinflammatory Ji antioxidant Hossain antivirus Ding antitumor Lin antipyretic Yang and Wang antihepatic injury Ma et al immunomodulatory Xu antibacterial Chen hypoglycemic Ru 2018ab etc Numerous reports have demonstrated that the biological activities of T hemsleyanum are attributed to its many chemical components Fu Wang has reported isolated alkaloids from the aerial parts of T hemsleyanum Wang Ru extracted a novel polysaccharide TDGP3 from is mainly alanine aminotransferase aspartate aminotransferase hyaluronan laminin total bilirubin total protein interferongamma immunoglobulin A secretory immunoglobulin A Epithelialmesenchymal transition ALT AST HA LN TBiLi TP IFNÎ IgA SIgA EMT MMPs matrix metalloproteinase TIMPs matrixmetallo proteinase Cytc CAT GSHPx glutathione peroxidase Tregs TGF COX2 Foxp3 PDL TAOC CCl4 CEF HVJ VSV A F S1 S2 PEF CFF EAF BAF Cytochrome c catalase regulatory T cells transforming growth factor beta cyclooxygenase forkheadwinged helix transcription factor gene population doubling time total antioxidant capacity carbon tetrachloride chicken embryo fibroblast Hemagglutinating virus of Japan vesicular stomatitis virus alkalicontaining extract of T hemsleyanum ketonecontaining extract of T hemsleyanum crude extract of T hemsleyanum crude extract of T hemsleyanum Petroleum ether extractions of T hemsleyanum ethanol extract Chloroform extractions of T hemsleyanum ethanol extract ethyl acetate extractions of T hemsleyanum ethanol extract nbutanol extractions of T hemsleyanum ethanol extract T hemsleyanum with a molecular weight of — Da by enzymolysisultrasonic assisted extraction method Ru 2019ab Large amounts of flavonoids were found in leaves aerial parts and root tubers of T hemsleyanum Xu 2014ab Deng Yu In addition T hemsleyanum also contains a variety of functional components such as anic acids Hu phenolic acids Liu minerals Fan amino acids Fu etc In recent years wild resources of T hemsleyanum have been overexploited and now are on the verge of extinction due to its multiple medicinal values coupled with the strict requirements of the growing environments In it was listed in the preferentially protected crop germplasm resources of Zhejiang province Based on our team™s preliminary research Peng Peng 2016ab Li we comprehensively summarized and analyzed the domestic and overseas research progress on traditional uses the bioactive components of T hemsleyanum pharmacological activities toxicology with the aim of providing guidance for indepth research and reference for its development and utilization Materials and methods The available information about the traditional uses phytochemicals and pharmacological properties of T hemsleyanum was searched via Web of Science Google Scholar PubMed Science Direct China National Knowledge Infrastructure CNKI and Springer search using Chinese or English as the retrieval languages The keywords used include T hemsleyanum root tubers of T hemsleyanum Radix Tetrastigma JournalofEthnopharmacology26420211132472 0cT Ji traditional uses phytochemistry bioactive components pharmacological activities toxicology and other related words All references were from experimental studies and published prior to April were reviewed All chemical structures were drawn using ChemDraw Pro software heatclearing were Botanical characteristics T hemsleyanum is a perennial grass climbing vine with longitudinal ribs glabrous or sparsely pilose It is usually grown in a cool and humid environment and the main soil type is yellow soil or yellow brown soil with rich humus The optimum pH is between and The root tubers are thick spindle shaped or elliptical and single or several are connected into a string of beads generally “ cm long and “ cm in diameter Fig The epidermis of the root tubers is tan and most of them are smooth a few of them have folds and lenticel like protuberances some of them have depressions in which there are residual tan roots hard and brittle with a flat and rough section The stem of T hemsleyanum is thin and weak with longitudinal rhombus rooting on the lower node Palmate compound leaves alternate leaflets are lanceolate oblong or ovate lanceolate The leaflets are “ cm long and “ cm wide with a tapered tip and a wedgeshaped or round base The flowers of T hemsleyanum are small yellow green and ovate The flowering stage of T hemsleyanum ranges from April to June and the fruit phase is normally from August to November When the flower withered it will form a small green round fruit with the size of millet When it is mature the fruit will turn from green to red the berries are spherical and soft spherical Traditional uses T hemsleyanum belonging to the family Vitaceae was firstly recorded in Ben Cao Gang Mu Ming Dynasty AD The aliases of Sanyeqing include Shi Hou Zi Shi Bao Zi Shi Lao Shu Lan Shan Hu Lei Dan Zi Po Shi Zhu Tu Jing Wan Sou Jia Feng San Ye Dui golden wire hanging gourd golden bell golden wire hanging potato etc The root tubers or whole grass of T hemsleyanum traditionally and ethnically used as a medicine for a long time it has been recorded in multiple hemsleyanum ancient books of TCM such as Zhi Wu Ming Shi Tu Kao Qing Dynasty Wu Jiangxi herbal medicine Common folk herbal medicine in Zhejiang All of these ancient works described the effects of toxicityremoving T dyspnearelieving promoting blood circulation and pain relief thus it can be applied to cure febrile convulsion pneumonia bronchitis pharyngitis sore throat acute and chronic hepatitis rheumatic arthralgia viral meningitis bruise eczema insect and snake bite poor joint flexure and extension irregular menstruation of women National compilation team of Chinese herbal medicine In the TCM culture the properties of T hemsleyanum was described as bitter and acrid in taste cool in nature which recorded in dictionaries of traditional Chinese medicine and Zhong Hua Ben Cao Shanghai Science and Technology Press The channel tropism was lung heart liver and kidney meridians Decocting with water or mashing for external application are the traditional possess methods of T hemsleyanum Considering its extensive traditional effects many prescriptions containing T hemsleyanum have been passed down from generation to generation and have been well supported and clarified by modern pharmacological studies Excitingly it has reported that Jinlian disinfection drink containing san ye qing combined with interferon can treat Covid19 He Jinqi Tablet made up of san ye qing astragalus and ginsenoside was used to treat cases of malignant tumor cases were completely relieved cases were partially relieved the total effective rate was Wei Moreover Zhonggan mixture including san ye qing could improve the quality of life and prolong the survival time of patients with stage III primary liver cancer Jiang and Gong In addition it has been used in the treatment of common gynecological diseases such as blood avalanche and leucorrhea Gao and it also has a good effect on measles complicated with pneumonia anal fissure chronic bronchitis and mosquito bites Ji Chemical compounds of Themsleyanum The chemical constituents of T hemsleyanum have been widely investigated Sun Sun Zeng Xu 2014ab Fu Fan Chen Ding 2015a Fig The aerial part A root tuber B and raw herb C of T hemsleyanum JournalofEthnopharmacology26420211132473 0cT Ji b Ding a total of one hundred and fortytwo compounds have been isolated and identified from T hemsleyanum until now The information about compound name molecular weight compound formula detection method analysis sample is summarized in Table Flavonoids and their glycosides Modern phytochemical studies have indicated that flavonoids are the representative and predominated class of constituents isolated from T hemsleyanum Lin Zhang Table To date fiftyone flavonoids and their glycosides have been extracted and identified from T hemsleyanum In this series compounds quercetin orientin vitexin isorhamnetin apigenin and kaempferol are the main types of skeleton some of their analogues can be identified from hydroxy moiety on C3² and C4™ on the B ring of flavonoid aglycone At present many modern analytical techniques have been used for qualitative and quantitative analysis of flavonoids Among them ultra high performance liquid chromatography tandem triple quadrupole time of flight mass spectrometry UPLCESIQTOFMS has become a powerful tool for identifying the complicated compounds due to its higher mass accuracy and resolution Our team used UPLCESIQTOFMS to identify chemical constituents from the aerial part of T hemsleyanum including flavonoids such as isoorientin quercetin kaempferol vitexin isovitexin kaempferol3glucoside etc Sun According to the report Liu total flavonoids of T hemsleyanum could protect the aged mice from acute lung injury through inhibiting the phosphorylation of mitogenactivated protein kinase MAPK and nuclear factorκB NFκB in lung tissue Moreover the flavonoids of T hemsleyanum had the activity of antilung cancer Wei Luteolin a flavonoid found in T hemsleyanum acted as an anticancer agent against various types of human malignancies such as lung breast glioblastoma prostate colon and pancreatic cancers Muhammad It is certain that T hemsleyanum flavonoids give a new vision for researchers to explore clinical anticancer drugs Polysaccharide Saccharide is another important active ingredient extracted from T hemsleyanum Shao Polysaccharide has great potential in clinical application because of its unique pharmacological activity However due to the complex structure of polysaccharide it is difficult and special to determine and synthesize their structures Guo Table The prescriptions and traditional uses of T hemsleyanum in China Prescriptions name Qingteng Fengshi Qufengshi Yaojiu Main composition Jiu Traditional use T hemsleyanum Parabarium chunianum Tsiang Zanthoxylum nitidum Roxb DC T hemsleyanum Deeringia amaranthoides Lam Merr Blumea aromatica Wall DC T hemsleyanum Deeringia amaranthoides Lam Merr Zanthoxylum nitidum Roxb DC Panax notoginseng Burk FH Chen T hemsleyanum Gypsum Lonicera japonica Thunb Houttuynia cordata Thunb Ophiopogon japonicus Linn f KerGawl T hemsleyanum T hemsleyanum Lysimachia christinae Hance Imperata cylindrica Citrus reticulata Blanco T hemsleyanum ginsenoside Astragalus propinquus Schischkin T hemsleyanum Nepeta cataria L Lonicera japonica Thunb Saposhnikovia divaricata Trucz Schischk Huatuo Fengtongbao capsule Sanyeqing Gypsum Decoction Sanyeqing Power Zhonggan mixture Jinqi Tablet Hua Shi Xuan Fei mixture extracted the polysaccharides from roots of T hemsleyanum RTP1 RTP2 and RTP3 were successively found by protein precipitation and purification Moreover further study indicated RTP31 was high purity polysaccharide with a molecular weight of kDa and it is mainly composed of kinds of monosaccharides arabinose galacturonic acid galactose and fructose the proportion is and respectively Ru 2018ab extracted a polysaccharide THP from T hemsleyanum with the average molecular weight estimated as kDa The results of study on the composition of polysaccharide showed that it was mainly composed of rhamnose arabinose mannose glucose galactose with the molar ratio of In Ru 2019ab successfully extracted polysaccharide THDP3 from T hemsleyanum with molecular weight of kDa which consists of rhamnose arabinose mannose glucose and galactose with molar ratio of Moreover TDGP3 mainly consists of †’4αDGalAp1†’ †’4DGalp1†’ and †’4αDGlcp1†’ residues as backbones and DManp1†’ †’36DManp1†’ and αDAraf1†’residues as branches Phenolic acids Phenolic acids refer to aromatic carboxylic acids with multiple phenolic groups substituted on one benzene ring As a secondary metabolite phenolic acids are widely found in many natural plants and have antiinflammatory antioxidant and lipid lowering effects Twenty three phenolic acids No52“ Table have been reported in the aerial parts of T hemsleyanum such as caffeic acid chlorogenic acid 1OgalloylDglucose protocatechol glucoside epigallocatechin 1caffeoylquinic acid 3caffeoylquinic acid 4caffeoylquinic acid 5caffeoylquinic acid 1pcoumaroylquinic acid 4pcoumaroylquinic acid and 5pcoumaroylquinic acid There were twentyone phenolic acids in the root tuber of T hemsleyanum some of which were the same as aerial parts Alkaloids Alkaloids are a group of basic anic compounds containing nitrogen that exist in nature Alkaloids are stored in small quantities in T hemsleyanum and the bioactivity investigations of those alkaloids are still rather rare Wang Fu extracted the aerial parts of T hemsleyanum with ethanol and then isolated ten alkaloids for the first time including seven indole alkaloids an amide a maleimide and Treatment of joint pain wind cold dampness arthralgia Treatment of arthralgia syndrome rheumarthritis rheumatoid arthritis scapulohumeral periarthritis Treatment of arthralgia syndrome rheumarthritis rheumatoid arthritis scapulohumeral periarthritis joint pain muscular constricture Treatment of infantile hyperpyretic convulsion Treatment of blood avalanche leucorrhea Treatment of liver cancer Treatment of malignant tumor Treatment of Covid19 Usage Oral administration “ mL once times a day Oral administration mL once times a day Oral administration capsules once times a day References Ministerial standard Ministerial standard Ministerial standard One dose a day decoct twice in water and take it “ times after mixing Oral administration Oral administration mL once times a day Oral administration capsules once times a day Oral administration mL once times a day Xu Gao Jiang and Gong Wei Zhejiang Provincial Drug Administration JournalofEthnopharmacology26420211132474 0cT Ji Detection Mode Analysis parts of sample Reference aerial part root tuber aerial part root tuber aerial part root tuber root tuber aerial part root tuber root tuber root tuber aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part root tuber root tuber root tuber aerial part root tuber root tuber aerial part aerial part aerial part aerial part aerial part aerial part aerial part root tuber aerial part root tuber root tuber aerial part root tuber root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part aerial part root tuber aerial part root tuber aerial part root tuber root tuber root tuber root tuber root tuber root tuber root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part root tuber aerial part aerial part aerial part aerial part root tuber root tuber aerial part root tuber aerial part root tuber aerial part root tuber Sun Sun Zeng Sun Sun Sun Zeng Zeng Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Zeng Sun Zeng Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Xu 2014b Sun Zeng Sun Zeng Sun Zeng Zeng Sun Sun Sun Sun Xu 2014b Sun Xu 2014b Sun Zeng Sun Xu 2014b Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Fu Sun Sun Xu 2014b Fan Xu 2014b Fan Sun continued on next page UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS Table Chemical constituents isolated from the different parts of T hemsleyanum Name Flavonoids and their glycosides quercetin quercitrin quercetin3Oglucoside quercetin3Orutinoside quercetin3galactoside quercetin3Oxylosylglucoside quercetin3Oxylosylglucose7Orhamnoside orientin orientin2²²Orhamnoside Isoorientin isoorientin2²²Orhamnoside isoorientin cid0 ²²Oxyloside vitexin vitexin2²²Orhamnoside vitexin2²²Oglucoside vitexin2²²Oarabinoside isovitexin isovitexin2²²Orhamnoside isovitexin2²²Oxyloside isorhamnetin isorhamnetin3rutinoside isorhamnetin3pyranoarabinose7glucosylrhamnoside apigenin apigenin7rhamnoside apigenin8Cxylosyl6Cglucoside apigenin6CαLarabinose8CDglucose eriodictyol eriodictyolOhexoside I eriodictyolOhexoside II luteolin luteolin6 8diChexoside catechin catechin glucopyranoside isomer epicatechin kaempferide kaempferol kaempferol3glucoside kaempferol3rutinoside kaempferol3sambubioside kaempferol3Oneohesperidin kaempferol3Orhamnoside kaempferol7Orhamnose3Oglucoside kaempferol3robinoside7rhamnoside kaempferol3rutinoside kaempferol3Ocarfuran7Orhamnosyl glucoside daidzein biochanin A procyanidin dimmer procyanidin B1 procyanidin B2 procyanidin trimer Phenolic acids and derivatives gallic acid protocatechuic acid caffeic acid dihydroxybenzoic acid hexoside 1caffeoylquinic acid 3caffeoylquinic acid 4caffeoylquinic acid 5caffeoylquinic acid 1pcoumaroylquinic acid 4pcoumaroylquinic acid 5pcoumaroylquinic acid phydroxybenzaldehyde pcoumaric acid ferulic acid hexoside salicylic acid chlorogenic acid neochlorogenic acid cryptochlorogenic acid protocatechualdehyde UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS 1HNMR13CNMR MS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS JournalofEthnopharmacology26420211132475 0cT Ji Table continued Name salicin2benzoate trihydroxycinnamoylquinic acid isomer protocatechuic acid hexoside apiosylglucosyl 4hydroxybenzoate 1OgalloylDglucose protocatechol glucoside epigallocatechin vanillic acid1Ofuran celery glucosyl ester protocatechuic acid1Ofuran celery glucosyl ester methoxyphenol1Ofuran glycosylOglucoside 2methoxy4methylbenzene1ofuracresyl glucoside oxyresveratrol dicaffeoylquinic acid 4hydroxycinnamic acid Alkaloids indole indole3carboxylic acid indole3propanoic acid 5hydroxyindole3carboxaldehyde 5hydroxyindole3carboxylic acid 6hydroxy3 4dihydro1oxocarboline hippophamide 4hydroxycinnamide pyrrole3propanoic acid Scid0 trolline Fatty acids trihydroxy octadecadienoic acid trihydroxy octadecenoic acid dihydroxy octadecenoic acid 9hydroxy1012octadecadienoic acid 9hydroxy octadecatrienoic acid hydroxyoctadecenoic acid hydroxyoctadecatrienoic acid Dihydroxyoctadecatrienoic acid dihydroartemisinin ethyl ether Trihydroxy octadecadienoic acid isomer hydroxyoxooctadecatrienoic acid octadecenedioic acid diMeester stearic acid linolenic acid linoleic acid palmitic acid oleic acid anic acids and derivatives malic acid quinic acid citric acid azelaic acid oxalic acid galactonic acid gallic acid succinic acid fumaric acid propanoic acid Terpenoids and steroids sitosterol daucosterol campesterol Stigmasterol 6Obenzoyl daucosterol ergosterol taraxerone Taraxerol αamyrine pteroside Z ganoderic acid H 3epipapyriferic acid oleanic acid Saponins Ginsenoside Rh1 Detection Mode UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS 1HNMR LCMS NMR UV MS NMR UV MS NMR UV MS NMR UV MS NMR UV MS NMR UV MS NMR UV MS NMR UV MS NMR UV MS NMR UV MS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS GCMS TCL HNMR CNMR MS GCMS GCMS IR HNMR EIMS IR HNMR MS IR HNMR MS IR HNMR MS IR EIMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS HNMR CNMR MS Analysis parts of sample root tuber root tuber root tuber root tuber aerial part aerial part root tuber aerial part root tuber root tuber root tuber root tuber root tuber root tuber root tuber root tuber aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts root tuber aerial part root tuber aerial part root tuber root tuber aerial part root tuber aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part root tuber root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part aerial part aerial part aerial part aerial part root tuber aerial part root tuber root tuber root tuber root tuber root tuber root tuber aerial part aerial part aerial part aerial part root tuber root tuber root tuber root tuber Reference Sun Sun Sun Sun Sun Sun Zeng Sun Xu 2014b Zeng Zeng Zeng Zeng Xu 2014b Xu 2014b Chen Fu Fu Fu Fu Fu Fu Fu Fu Fu Fu Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Chen Ding Sun Sun Guo Ru Ru Ru Ru Sun Sun Sun Ding UPLCESIQTOFMSMS root tuber Sun continued on next page JournalofEthnopharmacology26420211132476 0cT Ji Table continued Name Ginsenoside Rh2 Vinaginsenoside R1 Amino acid and derivatives Phenylalanine pyroglutamic acid glutimic acid hexose Tryptophan Lglutamic acid Detection Mode UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS Analysis parts of sample root tuber root tuber root tuber aerial part aerial part aerial part aerial part aerial part Reference Sun Sun Sun Sun Sun Sun Sun respectively a carboline By comparing with the spectral data of known compounds the alkaloids were indole3carboxylic acid indole3propanoic acid 5hydroxyindole3carboxaldehyde 5hydroxyindole3carboxylic acid 6hydroxy3 4dihydro1oxocarboline hippophamide 4hydroxycinnamide pyrrole3propanoic acid and Scid0 trolline The chemical structures were shown in Fig identified as indole a Answer:

Thyroid_Cancer

" Innovation Primary liver cancer PLC is a fatal disease that affects millions of livesworldwide PLC is the leading cause of cancerrelated deaths and theincidence rate is predicted to rise in the coming decades PLC can becategorized into three major histological subtypes hepatocellular carcinoma HCCintrahepatic cholangiocarcinoma ICC and combinedHCCICC These subtypes are distinct with respect to epidemiology clinicopathological features genetic alterations and clinical managementswhich are thoroughly summarized in this review The state of treatmentstrategies for each subtype including the currently approved drugs andthe potential novel therapies are also discussedKEYWORDS PRIMARY LIVER CANCER HEPATOCELLULAR CARCINOMA INTRAHEPATIC CHOLANGIOCARCINOMA COMBINED HCCICC PLC THERAPYIntroductionPrimary liver cancer PLC is a deadly malignancy with significant histological and biological heterogeneity and ranks as the fourth leading cause ofcancerrelated death worldwide12 Therefore it has become a major publichealthy challenge Over the past decades the morbidity and mortality associated with PLC have steadily risen According to Globocan's latest GlobalCancer Statistics Report cases of liver cancer were reported worldwide in accounting for of the total cancer cases in the sameperiod while deaths totaled accounting for of total cancerdeaths3 On the basis of annual projections the World Health anization estimates that patients will die from liver cancer in Incidenceand mortality of PLC differ widely between regions The highest incidenceof PLC was observed in East Asia and in subSaharan Africa4 In particularChina experiences the highest number of cases of PLC with a high incidencerate cases100000 inhabitants5PLC manifests as three subtypes hepatocellular carcinoma HCC intrahepatic cholangiocarcinoma ICC and combined HCCICC cHCCICCwhich differ notably in epidemiology clinicopathological morphology geneticalteration and appropriate therapeutic responses HCCs are primarily relatedto viral infection alcohol abuse and metabolic syndrome6 whereas ICCs aremainly associated with chronic liver ‚ammation and biliary tract diseases78 Risk factors for development of cHCCICC include overweightobsess nonalcoholic steatohepatitis and liver cirrhosis910 HCCs show asolid and trabecular pattern with local invasion restricted to the liver11“whereas ICCs are ductular papillary or solid tumor structures with highmetastasis to distal ans14“ cHCCICCs are the combination of theHCC and ICC phenotypes present in liver tissue and are classified into separate combined and mixed cHCCICC subclasses which are more aggressiveand have a poorer prognosis217“The three PLC subtypes have distinct genetic alterations and molecularpatterns HCCs are associated with genetic alterations in specific chromosomal regions and genes including TERT promoter mutation TP53 deletionand WNT signaling CTNNB1 and AXIN1 activation22“ ICCs show aunique mutational landscape with recurrent mutations with the genetic alterations in TP53 KRAS isocitrate dehydrogenase IDH and fibroblastgrowth factor receptor FGFR gene fusions30“ Combined cHCCICCsshow strong ICClike features whereas mixed cHCCICCs show HCClikefeatures3637 Understanding the molecular alterations that initiate variousPLC subtypes is of great importance for us to decipher the mechanisms oftumorigenesis Genetic alterations can be transformed into biomarkersthat may represent new therapeutic targets affectthe treatmentdecisions and ultimately improve the treatment of liver cancer patientsHCCs mainly respond to targeted therapy immunotherapy and antiviralagents while ICC patients benefit from classical chemotherapy targetedtherapy and immunotherapy Based on the pathological classification andthe molecular features of cHCCICCs combined cHCCICCs should betreated with similar therapies to ICCs whereas mixed cHCCICCs are treatedmore like HCCs In this review we systematically summarize the epidemiology pathogenesis genetic alteration and treatment for each subtype andcomprehensively describe current therapy drugs and the potential novel therapies for PLCEpidemiology and Risk Factors HCC HCC represents the major histologic subtype accounting for approximately of all cases of PLC The riskfactors for HCC includes hepatitis B virus HBVhepatitis C virus HCV infection aflatoxin B1 alcoholic abuse and nonalcoholic metabolic symptomssuch as diabetes and obesity6 According to the Global Burden of Diseasefrom to HBV and HCV accounted for liver cancer deaths alcohol for and other causes for deathsIn particular of all HCC cases worldwide are reported from China38 dueto the locally high prevalence of HBV infectionICC As the second most common liver carcinoma following HCC ICCaccounts for around of PLC cases with a high incidence of per population worldwide annually39 The most common risk factorsfor ICC are biliary tract diseasesincluding choledochal cysts cholelithiasis choledocholithiasisliver flukes viral hepatitis metabolic syndromeand other risk factors including tobacco and alcohol use and cirrhosis7Recently the incidence of ICC has been increasing more rapidly owing torisk factors8 including increasing chronic liver disease and environmentaltoxins and is found more often due to improved diagnostic tools andimagingcHCCICC cHCCICC presents as a heterogeneous tumor showing both hepatocyte and cholangiocyte differentiation and has a poor prognosis40cHCCICC is a rather rare tumor with an incidence rate less than Thepoor prognosis associated with cHCCICC is due to the limited treatment options and difficulty of diagnosis To date the largest cohort analysis whichincluded patients diagnosed with cHCCICC between and across registries41 reported that the incidence of cHCCICC in men andwomen was and per per year respectively with the averageage of years at diagnosis One and 5year causespecific survival rates forcHCCICC were and respectively with the median survival of months Among racial groups cHCCICCs are most common in Asianraces and Pacific Islanders Obesity nonalcoholic steatohepatitis and livercirrhosis were observed in some cHCCICC cohorts910 and are potentialrisk factors for cHCCICCClinicopathological Features HCC HCC shows a solid trabecular andpseudoglandular pattern with a high density of tumor cells It has three subtypes welldifferentiated HCC moderately differentiated HCC and poorlyllThe Innovation August 0cnoitavonnIehTReviewdifferentiated HCC11“ Welldifferentiated HCCs are often small less than cm in diameter and are composed of cells with a higher nuclear to cytoplasmic ratio arranged in a thin trabecular pattern with rare pseudoglandularstructures Moderately differentiated HCCs are usually larger tumors largerthan cm showing polygonal tumor cells in a thick trabecular arrangementwith a frequent pseudoglandular pattern Poorly differentiated HCCs arecomposed of pleomorphic tumor cells in a solid or compact growth patternICC ICC can be divided into two subtypes a small duct type that originatesfrom small intrahepatic ductules with no or minimal mucin production and alarge bile duct type that arises from large intrahepatic ducts proximal to thebifurcation of the right and left hepatic ducts with high mucin production ability14“ Further ICC shows three different growth patterns mass formingMF periductal ltrating PI and intraductal growth IG42 MF ICC is afirm multilobulated unencapsulated whitegray tumor owing to its extensivedesmoplastic stroma The PI subtype shows extensive ltration along theintrahepatic hilum structure and the IG subtype is usually restricted to tubeswith papillary structures MF ICC is the most common type associated with apoor prognosis while IG type is rare but has a favorable prognosis17cHCCICC Though the phenomenon of HCC and ICC being present in thesame liver was first described in cHCCICC was not systematicallydescribed until when it was classified into three subtypes dependingon the location of HCC and ICC type A separate type has separate nodulesof hepatocellular and bile duct carcinoma type B combined type showscontiguity with intermingling but with clearly defined areas type C mixedtype presents as intimate association without clear boundaries18 In another classification system with three subtypes was established type Icollision tumors is the simultaneous occurrence of both HCC and ICC inthe same patient type II transitional tumors is an identifiable intermediatetransition between HCC and ICC type III fibrolamellar tumors resemblesthe fibrolamellar variant of HCC but also contains mucinproducing pseudoglands19 Presently the World Health anization WHO classificationis commonly used in which cHCCICC is classified into two main types theclassic type and the stem cell SC type subtype with SC features with theSC type subdivided into three subtypes including the typical subtype intermediate subtype INT and cholangiocellular type43The lack of a unified classification system greatly adds to the difficulty forcHCCICC research and the clinicopathological characteristics of cHCCICCremain illdefined cHCCICC can exhibit stemprogenitor cell phenotypesconsisting of small cells with scant cytoplasm hyperchromatic nucleiembedded within a thick desmoplastic stroma a high nuclearcytoplasmicratio and increased mitotic activity1 In addition the immunohistochemistryhas identified stemnessrelated markers KRT19 CD56 EpCAM CD117CD113 OV6120 cHCCICC clinicopathologic characteristics include morefrequent multifocallesions more microvascular emboli and portal veinand lymph node invasion all of which indicate a poor prognosis21Genetic Alterations HCC Widescale genomic studies have revealedthat hundreds of somatic DNA alterations accrue in HCC including chromosome aberrations and mutations Highlevel DNA amplifications are enrichedin chromosome locations 6p21 and 11q13 in HCC44 which occur in “of cases Recently some oncogenic genes were identified in the regions offrequent DNA gain For example LINC01138 is an oncogenic long intergenicnoncoding RNA located in this region which has been identified as a driver ofHCC45 VEGFA and CCND1FGF19 have also identified in these regions andare potential therapeutic targets46 Loss of heterozygosity on chromosome8p is a frequent event in HCC47 These DNA alterations are often associatedwith cancer progression due to the deletion of tumor suppressor genesIntriguingly in these regions a variety of vulnerability genes have recentlybeen identified For example TSLNC8 was characterized as a tumor suppressor gene on chromosome 8p12 the region that shows allelic loss in HCC andwas shown to inhibit the proliferation and metastasis of HCC48 The geneticmutations of HCC have been well studied Mutations in the TERT promoteroccur in approximately of cases and cause recurrent viral insertion ofHBV49 Deletion mutations in TP53 are the most frequent genetic alterationsaccounting for about of cases22“ and are thought to be the initiatingevent driving the formation of precursor lesions Mutated genes in WNTsignaling CTNNB1 and AXIN1 and chromatin remodeling ARID1A accountfor approximately “ of cases22“ Accumulation of activating mutations in oncogenes including activation of AKT or mTOR and of the oxidativestress pathway activation occurs throughout tumor progression and couldbe potentially targeted with molecular therapies in the futureICC ICC shows a unique mutational landscape with recurrent mutationscompared with HCC It harbors the genetic alterations in TP53 KRASARID1A BAP1 IDH1 IDH2 PIK3CA SMARCB1 EPHA2 SMAD4 GNAS andPBRM1 as well as FGFR gene fusions30“ Gain of function of IDH1 andIDH2 mutation on R132 and R172 two hotpot codons was observed in“ of ICC cases32 Fusions amplifications translocations and rearrangements of FGFR genes are found in ICC and are closely related to theinitiation and progression of ICC50 The activating mutation of KRAS “ is another frequent genomic alteration in ICC315152 The KRAS mutationoften exists concurrently with FGFR2 fusions and IDH mutations suggestinga possible cooperative role in ICC pathogenesis5354 In addition recentstudies have shown that BRAF and Notch are considerably more prevalentin ICC and function in ICC pathogenesis55cHCCICC cHCCICCs are genetically complex tumors The combined subtype of cHCCICC shows strong ICClike features with the high expression ofEPCAM KRT19 PRDM5 and KRAS The mixed subtype of cHCCICC showsHCClike features with the high expression levels of AFP GPC3 APOE SALL4and AFP8136The most frequent mutation observed in cHCCICCs is TP53 with a strikingly high mutation frequency much higher than that in HCC “ and ICC “ Interestingly several studies have foundthat the disruption of Trp53 alone in livers of mice can induce the formationof cHCCICC3757 which further implies that TP53 may be the driver gene incHCCICC It is notable that Nestin a type VI intermediate filament IF proteinthat is commonly used as a neuroectodermal SC marker is highly expressedin cHCCICC and is strongly associated with poorer prognosis36 Hence Nestin may be a promising biomarker for cHCCICCChallenges and Limitations of Current Treatment Strategies ResectionTransplantation Local and Regional Therapies HCC The commonlyused staging system for HCC is the Barcelona Clinic Liver Cancer staging system Figure HCCs in the very early stage or intermediate stage can betreated with local regional therapies which include radiofrequency ablationRFA resection da Vinci surgery laparoscopic surgery or traditional surgery transplantation orthotopic liver transplantation piggyback transplantation split liver transplantation auxiliary liver transplantation percutaneousethanoltranscatheter arterial chemoembolizationTACE58injections PEI orICC Surgery is currently the only curative treatment for ICCs but only aminority of patients in early stages are considered candidates for resectionIn surgery ICC is usually treated with hepatic resection to achieve negativeresection margins59 For patients with locally unresectable ICC tumor ablation such as RFA or hepatic arterybased therapies like yttrium90 radioembolization appear promising59“cHCCICC An accurate diagnosis is of paramount importance for thetreatment of cHCCICC Currently major hepatectomy is the optimal management for cHCCICC65 The rarity of this cancer as well as the lack of biomarkers have made this cancer difficult to diagnosis and manage Surgicalresection remains the only curative option for patients with cHCCICCThe treatment options for cHCCICC are similar to those for HCC and ICCand include surgery radiation yttrium90 radioembolization chemotherapycombined radiation and chemotherapy combined surgery and chemotherapy and triple therapy surgery radiation and chemotherapy4166“ Arecently retrospective analysis from to of PLC patientsincluding cHCCICC HCC and ICC patients who underwentresection found that although cHCCICC is more poorly differentiated thanHCC and ICC it had a similar 5year survival rate and respectively and 3year recurrence rate respectively70Systemic Chemotherapy HCC Systemic chemotherapy has limited efficacy on HCC several clinicaltrials of chemotherapy have shownlow response rates and worse toxicity without a significant improvement inThe Innovation August wwwcellcomtheinnovation\x0cReviewFigure Barcelona Clinic Liver Cancer Staging Systemand Corresponding Treatment Options The schematic diagram illustrates therapeutic choice by which a treatmenttheoretically recommended for a different stage is the besttreatment option 1L firstline 2L secondline ECOGEastern Cooperative Oncology Group M metastasis stageN nodal stage PEI percutaneous ethanolinjection PSperformance status T tumor stage TACE transarterialchemoembolization TARE transarterialradioembolizationY90 Y90 radioembolizationTheInnovation[5FU]including gemcitabine and doxorubicinbasedthe overall survival OStreatment FOLFOX 5fluorouracilleucovorin oxaliplatin andPIAF cisplatininterferon alpha2bdoxorubicin5FU71“ This suggestsa limited role for traditional chemotherapy in the treatment of advanced HCCICC Current firstline standard of treatment for ICC is the combination ofgemcitabine and platinumderived chemotherapy Figure 2B With the poorprognosis the median survival of advanced ICC patients is less than one yearVery limited effective treatments are available for patients who progress onfirstline chemotherapy so there is a high medical demandFirstLine Treatment Effective molecular targeted therapy and immunotherapy is lacking so chemotherapy with gemcitabine platinum compoundsand fluoropyrimidines is still the mainstream of standard treatment for unresectable ICCThe primary chemotherapy for ICC is gemcitabine which was establishedas the firstline therapy for advanced biliary tract cancer BTC in In the randomized controlled ABC02 phase III clinical trial comparedthe benefit of gemcitabine plus cisplatin CisGem chemotherapy with thesingle agent gemcitabine75 This study showed an advantage for CisGemin OS months versus months hazard ratio [HR] confidence intervalPFS months versus months p This effectiveness wasconfirmed in a Japanese randomized phase II study BT22 median OS months versus months HR Based on these promising results CisGem is currently regarded as the standard of care in the firstlinetreatment for advanced cholangiocarcinoma[CI] “ and progressionfree survivalOther than cisplatin gemcitabine plus other agents such as oxaliplatin S1capecitabine bevacizumab and Nabpaclitaxel have also been considered asthe firstline choices for advanced cholangiocarcinoma based on the promising outcomes from several phase II or III trials77“A recent multicenter randomized phase III clinical trial NCT01470443showed that XELOX has the comparable efficacious effect to GEMOX interms of tumor response survival rate OS and PFS and safety Also XELOXhas an advantage of low hospital visits compared with GEMOX Thus XELOXcould be an alternative for cholangiocarcinoma therapiesSecondLine Treatment There is no established standard secondlinechemotherapy for advanced cholangiocarcinoma and all regimens haveshown limited efficacy with a median PFS of around months and medianOS of about months92FOLFOX Lfolinic acid 5FU and oxaliplatin is an optional secondlinetreatment option based on the randomized phase III multicenter labelABC06 study NCT01926236 FOLFOX showed increased benefit for median OS months and months and OS rate months and compared with months and for the control groupactive symptom control [ASC] arm92cHCCICC In contrastCurrently several phase II and III chemotherapy clinical trials are under wayTable Combined therapy with chemotherapy shows promise in the treatment of cholangiocarcinoma selective internal radiotherapy SIRT pluschemotherapy or hepatic arterialinfusion plus systemic chemotherapyboth had antitumor activity and are promising for the treatment of ICC9394to surgerybased treatments for resectablecHCCICC systemic therapy is the nonstandard option for advanced and unresectable cHCCICC based on the standard treatment strategy for the unresectable HCC or ICC Chemotherapy for advanced or unresectable cHCCICCis largely understudied with only a few case reports and some retrospectivestudies having been published91095“ Recently a multicenter retrospectiveanalysis has been conducted by Kobayashi and colleagues10According to dividedgroup treatment with gemcitabine plus cisplatinn 5FU plus cisplatin n sorafenib monotherapy n others n they found that patients with platinumcontaining treatment had longer OS time than those treated by sorafenib monotherapyshowing OS of months CI “ months CI “ months CI “ and months CI “respectivelyA similar conclusion was drawn in another retrospective study of cHCCICC patients with receiving gemcitabinebased therapygemcitabine platinum or gemcitabine 5FU or targeted agents sorafenib9 Median PFS favored gemcitabineplatinum and gemcitabine5FU and months respectively over sorafenib monotherapy monthsllThe Innovation August 0cnoitavonnIehTReviewABFigure Treatment Strategy for Advanced HCC and ICC The schematic illustration represents FDAapproved drugs for treatment of advanced HCC and ICC Firstlinedrugs for HCC include sorafenib lenvatinib atezolizumab plus bevacizumab tremelimumab plus durvalumab and donafenib whereas for ICC the combination ofgemcitabine and cisplatin is currently proposed as first line The bottom row represents corresponding secondline therapies that come in when patients are not suitable fortheir firstline therapyMolecular Targeted Therapy HCC FirstLine Drugs Sorafenib Sorafenib was the first US Food and Drug Administration FDAapproved firstline systemic targeted drug for advanced HCC It is an oral smallmoleculemultikinase inhibitor targeting VEGFR1 VEGFR2 VEGFR3 PDGFRb andRaf Two large international multicenter clinical trials SHARP and AsianPacific have proved that sorafenib can suppress tumor progression and prolong OS in patients with advanced HCC102103 These trials showed that sorafenib can increase PFS and OS by months in patients with advancedHCC in Western countries As the first generation of targeted drugs forHCC sorafenib has been used for over a decade During this time many patients have benefitedthough others quickly developed resistance tosorafenib104Lenvatinib Lenvatinib is becoming available for HCC patients whodevelop sorafenib resistance Lenvatinib is an oral tyrosine kinase inhibitorinhibiting VEGFR1“ FGFR1“ PDGFR RET and KIT In August theFDA approved lenvatinib for firstline treatment of patients with unresectableHCC after lenvatinib was proved to be noninferior to sorafenib in the phase REFLECT trial105Median OS in the lenvatinib arm and sorafenib arm was months and months HR CI respectively The adverse effectswere hypertension diarrhea and decreased appetite withlenvatinib and palmarplantar erythrodysesthesia diarrhea decreased weight hypertension and decreased appetite with sorafenibDonafenib Similar to sorafenib donafenib is a novel multikinase inhibitortargeting RAF kinase and various receptor tyrosine kinases RTKs includingVEGF receptor VEGFR and BRAF106 According to the report from International Conference of the American Society of Clinical Oncology CSCOdonafenib significantly improves OS over sorafenib versus monthswith fewer side effects and higher patient tolerance for advanced HCC patients in its phase IIIII label trial107 The grade and above adverse reaction rates for donafenib and sorafenib were and respectivelyThus donafenib was recommended as the firstline therapy in the CSCOguidelines for HCCSecondLine Drugs Regorafenib Regorafenib an oral multikinase inhibitor inhibits the activity of protein kinases involved in multiple biological processes such as tumorigenesis tumor angiogenesis distant metastasisand tumor immune escape These kinases include VEGFR “ TIE2RAF1 KIT RET RAF BRAF PDGFR FGFR and CSF1R The randomized doubleblind multicenter phase III clinical trial RESORCE showed that regorafenib significantly improves the OS of patients as compared with the placebofrom to months HR p Grade “ adverse eventswere reported in of patients receiving regorafenib and of patientsreceiving the placebo In regorafenib received FDA approval as the secondline drug for the treatment of patients with advanced HCC who fail torespond to the sorafenib treatmentCabozantinib Cabozantinib is an oral inhibitor and targets multiple kinasesincluding VEGFR2 cMET RET ROS1 TYRO3 MER KIT TRKBFLT3 TIE2 as well as the GAS6 receptor AXL109110 It was originallyapproved for medullary thyroid cancer in and advanced renal carcinoma in According to the randomized doubleblind multicenter phase clinical trial conducted across centers in countries median OS was months for patients receiving cabozantinib and months for patientstreated with placebo HR p Median PFS was monthsand months respectively Grade or adverse events occurred in of patients in the cabozantinib arm and in the placebo arm Theobserved hepatotoxicity can be mostly controlled through dose modifications Based on the encouraging results of prolonged OS and PFS cabozantinib received its FDA approval for HCC in Ramucirumab Ramucirumab is a completely human monoclonalantibody that can specifically inhibit VEGFR2112 For patients with alphafetoprotein R400 ngmL and who have been previously treated with sorafenib ramucirumab was approved as a monotherapy by the FDA on May The Innovation August wwwcellcomtheinnovation\x0cTable Systemic Therapies Currently or Promising Approved for Advanced HCC and ICCReviewTargetTherapy LineApproved YearTrialDrugsHCCSorafenib NexavarLenvatinib LenvimaRegorafenib StivargaNivolumab OpdivoVEGFR2 VEGFR3 PDGFRb RAF kinasesFGFR VEGFR PDGFRa RET KITTie2 VEGFR PDGFR FGFRPD1Cabozantinib CabometyxcMet VEGFR2 AXL RETPembrolizumab KeytrudaRamucirumab CYRAMZAPD1VEGFR2Nivolumab ipilimumab Opdivo YervoyPD1 CTLA4Atezolizumab bevacizumabTremelimumab durvalumabDonafenibApatinibICCGemcitabine cisplatinPemigatinib PemazyreIvosidenibPDL1VEGFPD1 CTLA4VEGFR BRAFVEGFR2chemotherapyFGFR1“IDH12TheInnovationpromisingpromisingpromisingpromisingSHARP AsianPacificREFLECTRESORCECHECKMATE040CELESTIALKEYNOTE224REACH2Cohort of CHECKMATE040IMbravel50NCT02519348NCT02645981NCT02329860ABC02FIGHT202promisingClarlDHyApproval was based on REACH NCT02435433 a randomized doubleblind multicenter phase III study of patients with AFP R400 ngmL whohad disease progression after sorafenib or were intolerant to sorafenib113More recently a study further confirmed the efficacy of ramucirumab inelderly patients with HCC and elevated AFP after sorafenib in REACH andREACH2 with a survival benefit observed across all age subgroups and atolerable safety profile supporting its value irrespective of age including forpatients R75 years114Apatinib Apatinib a tyrosine kinase inhibitor targeting VEGFR2 significantly prolonged OS and PFS in Chinese patients with advanced HCC whohad previously been treated with sorafenib andor chemotherapy accordingto the results of a randomized placebocontrolled phase III trial conducted in sites in China115 Median OS was almost months longer for patients whoreceived apatinib compared with patients receiving the placebo monthsversus months and median PFS was more than months longer months versus months115 The most common grade or worseadverse events occurred at a rate of in the apatinib arm and inthe placebo arm With the significantly prolonged OS and PFS and a manageable safety profile apatinib has potential to become a new secondline therapy for liver cancerNovel Therapeutic Targets Even with all these available treatments Table the median PFS for HCC patients remains less than a year Thus noveltreatment is still a critical unmet need for treatment of HCC Based on thegenomic profile and biomarkers reported in HCC several clinical trials targeting various pathways are currently ongoing Table Recently a firstinhuman phase I study NCT02508467 of fisogatinib BLU554 an orally bioavailable inhibitor of human FGFR4 demonstrated its antitumor activity in HCCand further validated that the aberrant FGF19FGFR4 signaling pathwaymay be a driver event116 In addition the TGFb1 receptor type I inhibitor galunisertib also showed an acceptable safety and prolonged OS outcome in combination with sorafenib in a phase II trial NCT01246986117118 Other potential candidatesincluding the cyclindependent kinase CDK inhibitorsregulating the cell cycle pathways ribociclib palbociclib119120 abemacicliband milciclib as well as the cMET inhibitors tepotinib121 and tivantinib122are being evaluated in HCC clinical trialsICC Moleculartargeted therapy controls tumor cell proliferationapoptosis adhesion and movement by inhibiting the surface molecules oftumor cell membranes and thereby inhibiting intracellular signaling pathways ICC genetic alterations primarily include FGFR IDH epidermal growthfactor EGFR and breast cancer type susceptible protein associated protein1 BAP1123“ Genetic alterations of these genes all have implicationsfor therapy At present a variety of molecular targeted drugs are in the clinicalresearch stage Table some of which have made progress in the treatment of ICC Table FGFR Inhibitors The most promising target therapy for cholangiocarcinoma identified in recent years is the inhibitor of the fibroblast growth factorFGF signaling pathway which consists of members labeled FGF1“FGF15 FGF19 called FGF1519 and four interacting transmembrane receptors FGFR1“ FGF signals regulate cell proliferation in which FGFR2fusions occurred in “ of ICC patients and are considered as a promising therapeutic target3351127128 Currently several FGFR inhibitors are being evaluated in clinical trials for cholangiocarcinomas with FGFR geneticaberrationsPemigatinib INCB054828 Pemigatinib is the first and only targeted therapy so far approved in by the FDA for the treatment of this rare cancerIt is a selective potent oral inhibitor of FGFR and Approval wasbased on findings from the phase II FIGHT202 trial NCT02924376 whichenrolled patients with locally advanced or metastatic cholangiocarcinoma with FGFR2 fusions or rearrangements cohort A other FGFFGFR genetic alterations cohort B or no FGFFGFR genetic alterations cohort CFor those in cohort A treatment with pemigatinib resulted in a median OSof months and median PFS of months The FIGHT202 study suggests that locally advanced or metastatic cholangiocarcinoma patientswith FGFR2 fusions or rearrangements may benefit from potent oralFGFR1 and inhibitor treatment Median PFS was months for patientswith FGFR2 alterations months for patients with other FGFFGFR alterations and months for those with no alterations in these genes MedianOS was months months and months for the respective cohorts130 With the promising results of phase II the phase III clinical trial ofpemigatinib is currently underway NCT03656536llThe Innovation August 0cnoitavonnIehTDrugTargeted TherapyCabozantinibLenvatinibDonafenibMilciclibPalbociclibRibociclibGalunisertib versus LY2157299 sorafenib versus placebo sorafenibImmunotherapyVEGFRVEGFRVEGFRCDK2CDK46CDK46TGFbToripalimab versus placeboNivolumab versus placeboNivolumab versus sorafenibPD1PD1PD1Hospices Civils de Lyonrecruitingphase Eisai Pharmaceuticals IndiaPvt Ltdnot yetrecruitingphase NCT03963206NCT04297254completedphase phase NCT02645981Suzhou ZelgenBiopharmaceuticalsTiziana LifeSciencesPfizeractive notrecruitingactive notrecruitingphase phase Texas Universityrecruitingphase Eli Lillyactive notrecruitingphase NCT03109886NCT01356628NCT02524119NCT02178358NCT03412773NCT03859128NCT03383458ReviewTable Selected Ongoing Systemic Therapy Clinical Trials for Advanced HCCTargetSponsorStatusPhaseEnrollmentTrial IdentifierTislelizumab versus sorafenibPD1BeiGeneactive notrecruitingphase Shanghai Junshi Biosciencerecruitingphase phase BristolMyers Squibbrecruitingphase BristolMyers Squibbactive notrecruitingphase NCT02576509Pembrolizumab versus placeboPD1Merck Sharp Dohmerecruitingphase AvelumabPDL1Seoul National UniversityHospitalactive notrecruitingphase Combined TherapyLenvatinib pembrolizumabversus lenvatinib placeboCS1003 lenvatinib versusplacebo lenvatinibVGFR PD1Merck Sharp Dohmeactive notrecruitingphase VGFR PD1CStone Pharmaceuticalsrecruitingphase Tislelizumab regorafenibversus placebo regorafenibVEGF PD1National Taiwan UniversityHospi

cancer237

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: " Innovation Primary liver cancer PLC is a fatal disease that affects millions of livesworldwide PLC is the leading cause of cancerrelated deaths and theincidence rate is predicted to rise in the coming decades PLC can becategorized into three major histological subtypes hepatocellular carcinoma HCCintrahepatic cholangiocarcinoma ICC and combinedHCCICC These subtypes are distinct with respect to epidemiology clinicopathological features genetic alterations and clinical managementswhich are thoroughly summarized in this review The state of treatmentstrategies for each subtype including the currently approved drugs andthe potential novel therapies are also discussedKEYWORDS PRIMARY LIVER CANCER HEPATOCELLULAR CARCINOMA INTRAHEPATIC CHOLANGIOCARCINOMA COMBINED HCCICC PLC THERAPYIntroductionPrimary liver cancer PLC is a deadly malignancy with significant histological and biological heterogeneity and ranks as the fourth leading cause ofcancerrelated death worldwide12 Therefore it has become a major publichealthy challenge Over the past decades the morbidity and mortality associated with PLC have steadily risen According to Globocan's latest GlobalCancer Statistics Report cases of liver cancer were reported worldwide in accounting for of the total cancer cases in the sameperiod while deaths totaled accounting for of total cancerdeaths3 On the basis of annual projections the World Health anization estimates that patients will die from liver cancer in Incidenceand mortality of PLC differ widely between regions The highest incidenceof PLC was observed in East Asia and in subSaharan Africa4 In particularChina experiences the highest number of cases of PLC with a high incidencerate cases100000 inhabitants5PLC manifests as three subtypes hepatocellular carcinoma HCC intrahepatic cholangiocarcinoma ICC and combined HCCICC cHCCICCwhich differ notably in epidemiology clinicopathological morphology geneticalteration and appropriate therapeutic responses HCCs are primarily relatedto viral infection alcohol abuse and metabolic syndrome6 whereas ICCs aremainly associated with chronic liver ‚ammation and biliary tract diseases78 Risk factors for development of cHCCICC include overweightobsess nonalcoholic steatohepatitis and liver cirrhosis910 HCCs show asolid and trabecular pattern with local invasion restricted to the liver11“whereas ICCs are ductular papillary or solid tumor structures with highmetastasis to distal ans14“ cHCCICCs are the combination of theHCC and ICC phenotypes present in liver tissue and are classified into separate combined and mixed cHCCICC subclasses which are more aggressiveand have a poorer prognosis217“The three PLC subtypes have distinct genetic alterations and molecularpatterns HCCs are associated with genetic alterations in specific chromosomal regions and genes including TERT promoter mutation TP53 deletionand WNT signaling CTNNB1 and AXIN1 activation22“ ICCs show aunique mutational landscape with recurrent mutations with the genetic alterations in TP53 KRAS isocitrate dehydrogenase IDH and fibroblastgrowth factor receptor FGFR gene fusions30“ Combined cHCCICCsshow strong ICClike features whereas mixed cHCCICCs show HCClikefeatures3637 Understanding the molecular alterations that initiate variousPLC subtypes is of great importance for us to decipher the mechanisms oftumorigenesis Genetic alterations can be transformed into biomarkersthat may represent new therapeutic targets affectthe treatmentdecisions and ultimately improve the treatment of liver cancer patientsHCCs mainly respond to targeted therapy immunotherapy and antiviralagents while ICC patients benefit from classical chemotherapy targetedtherapy and immunotherapy Based on the pathological classification andthe molecular features of cHCCICCs combined cHCCICCs should betreated with similar therapies to ICCs whereas mixed cHCCICCs are treatedmore like HCCs In this review we systematically summarize the epidemiology pathogenesis genetic alteration and treatment for each subtype andcomprehensively describe current therapy drugs and the potential novel therapies for PLCEpidemiology and Risk Factors HCC HCC represents the major histologic subtype accounting for approximately of all cases of PLC The riskfactors for HCC includes hepatitis B virus HBVhepatitis C virus HCV infection aflatoxin B1 alcoholic abuse and nonalcoholic metabolic symptomssuch as diabetes and obesity6 According to the Global Burden of Diseasefrom to HBV and HCV accounted for liver cancer deaths alcohol for and other causes for deathsIn particular of all HCC cases worldwide are reported from China38 dueto the locally high prevalence of HBV infectionICC As the second most common liver carcinoma following HCC ICCaccounts for around of PLC cases with a high incidence of per population worldwide annually39 The most common risk factorsfor ICC are biliary tract diseasesincluding choledochal cysts cholelithiasis choledocholithiasisliver flukes viral hepatitis metabolic syndromeand other risk factors including tobacco and alcohol use and cirrhosis7Recently the incidence of ICC has been increasing more rapidly owing torisk factors8 including increasing chronic liver disease and environmentaltoxins and is found more often due to improved diagnostic tools andimagingcHCCICC cHCCICC presents as a heterogeneous tumor showing both hepatocyte and cholangiocyte differentiation and has a poor prognosis40cHCCICC is a rather rare tumor with an incidence rate less than Thepoor prognosis associated with cHCCICC is due to the limited treatment options and difficulty of diagnosis To date the largest cohort analysis whichincluded patients diagnosed with cHCCICC between and across registries41 reported that the incidence of cHCCICC in men andwomen was and per per year respectively with the averageage of years at diagnosis One and 5year causespecific survival rates forcHCCICC were and respectively with the median survival of months Among racial groups cHCCICCs are most common in Asianraces and Pacific Islanders Obesity nonalcoholic steatohepatitis and livercirrhosis were observed in some cHCCICC cohorts910 and are potentialrisk factors for cHCCICCClinicopathological Features HCC HCC shows a solid trabecular andpseudoglandular pattern with a high density of tumor cells It has three subtypes welldifferentiated HCC moderately differentiated HCC and poorlyllThe Innovation August 0cnoitavonnIehTReviewdifferentiated HCC11“ Welldifferentiated HCCs are often small less than cm in diameter and are composed of cells with a higher nuclear to cytoplasmic ratio arranged in a thin trabecular pattern with rare pseudoglandularstructures Moderately differentiated HCCs are usually larger tumors largerthan cm showing polygonal tumor cells in a thick trabecular arrangementwith a frequent pseudoglandular pattern Poorly differentiated HCCs arecomposed of pleomorphic tumor cells in a solid or compact growth patternICC ICC can be divided into two subtypes a small duct type that originatesfrom small intrahepatic ductules with no or minimal mucin production and alarge bile duct type that arises from large intrahepatic ducts proximal to thebifurcation of the right and left hepatic ducts with high mucin production ability14“ Further ICC shows three different growth patterns mass formingMF periductal ltrating PI and intraductal growth IG42 MF ICC is afirm multilobulated unencapsulated whitegray tumor owing to its extensivedesmoplastic stroma The PI subtype shows extensive ltration along theintrahepatic hilum structure and the IG subtype is usually restricted to tubeswith papillary structures MF ICC is the most common type associated with apoor prognosis while IG type is rare but has a favorable prognosis17cHCCICC Though the phenomenon of HCC and ICC being present in thesame liver was first described in cHCCICC was not systematicallydescribed until when it was classified into three subtypes dependingon the location of HCC and ICC type A separate type has separate nodulesof hepatocellular and bile duct carcinoma type B combined type showscontiguity with intermingling but with clearly defined areas type C mixedtype presents as intimate association without clear boundaries18 In another classification system with three subtypes was established type Icollision tumors is the simultaneous occurrence of both HCC and ICC inthe same patient type II transitional tumors is an identifiable intermediatetransition between HCC and ICC type III fibrolamellar tumors resemblesthe fibrolamellar variant of HCC but also contains mucinproducing pseudoglands19 Presently the World Health anization WHO classificationis commonly used in which cHCCICC is classified into two main types theclassic type and the stem cell SC type subtype with SC features with theSC type subdivided into three subtypes including the typical subtype intermediate subtype INT and cholangiocellular type43The lack of a unified classification system greatly adds to the difficulty forcHCCICC research and the clinicopathological characteristics of cHCCICCremain illdefined cHCCICC can exhibit stemprogenitor cell phenotypesconsisting of small cells with scant cytoplasm hyperchromatic nucleiembedded within a thick desmoplastic stroma a high nuclearcytoplasmicratio and increased mitotic activity1 In addition the immunohistochemistryhas identified stemnessrelated markers KRT19 CD56 EpCAM CD117CD113 OV6120 cHCCICC clinicopathologic characteristics include morefrequent multifocallesions more microvascular emboli and portal veinand lymph node invasion all of which indicate a poor prognosis21Genetic Alterations HCC Widescale genomic studies have revealedthat hundreds of somatic DNA alterations accrue in HCC including chromosome aberrations and mutations Highlevel DNA amplifications are enrichedin chromosome locations 6p21 and 11q13 in HCC44 which occur in “of cases Recently some oncogenic genes were identified in the regions offrequent DNA gain For example LINC01138 is an oncogenic long intergenicnoncoding RNA located in this region which has been identified as a driver ofHCC45 VEGFA and CCND1FGF19 have also identified in these regions andare potential therapeutic targets46 Loss of heterozygosity on chromosome8p is a frequent event in HCC47 These DNA alterations are often associatedwith cancer progression due to the deletion of tumor suppressor genesIntriguingly in these regions a variety of vulnerability genes have recentlybeen identified For example TSLNC8 was characterized as a tumor suppressor gene on chromosome 8p12 the region that shows allelic loss in HCC andwas shown to inhibit the proliferation and metastasis of HCC48 The geneticmutations of HCC have been well studied Mutations in the TERT promoteroccur in approximately of cases and cause recurrent viral insertion ofHBV49 Deletion mutations in TP53 are the most frequent genetic alterationsaccounting for about of cases22“ and are thought to be the initiatingevent driving the formation of precursor lesions Mutated genes in WNTsignaling CTNNB1 and AXIN1 and chromatin remodeling ARID1A accountfor approximately “ of cases22“ Accumulation of activating mutations in oncogenes including activation of AKT or mTOR and of the oxidativestress pathway activation occurs throughout tumor progression and couldbe potentially targeted with molecular therapies in the futureICC ICC shows a unique mutational landscape with recurrent mutationscompared with HCC It harbors the genetic alterations in TP53 KRASARID1A BAP1 IDH1 IDH2 PIK3CA SMARCB1 EPHA2 SMAD4 GNAS andPBRM1 as well as FGFR gene fusions30“ Gain of function of IDH1 andIDH2 mutation on R132 and R172 two hotpot codons was observed in“ of ICC cases32 Fusions amplifications translocations and rearrangements of FGFR genes are found in ICC and are closely related to theinitiation and progression of ICC50 The activating mutation of KRAS “ is another frequent genomic alteration in ICC315152 The KRAS mutationoften exists concurrently with FGFR2 fusions and IDH mutations suggestinga possible cooperative role in ICC pathogenesis5354 In addition recentstudies have shown that BRAF and Notch are considerably more prevalentin ICC and function in ICC pathogenesis55cHCCICC cHCCICCs are genetically complex tumors The combined subtype of cHCCICC shows strong ICClike features with the high expression ofEPCAM KRT19 PRDM5 and KRAS The mixed subtype of cHCCICC showsHCClike features with the high expression levels of AFP GPC3 APOE SALL4and AFP8136The most frequent mutation observed in cHCCICCs is TP53 with a strikingly high mutation frequency much higher than that in HCC “ and ICC “ Interestingly several studies have foundthat the disruption of Trp53 alone in livers of mice can induce the formationof cHCCICC3757 which further implies that TP53 may be the driver gene incHCCICC It is notable that Nestin a type VI intermediate filament IF proteinthat is commonly used as a neuroectodermal SC marker is highly expressedin cHCCICC and is strongly associated with poorer prognosis36 Hence Nestin may be a promising biomarker for cHCCICCChallenges and Limitations of Current Treatment Strategies ResectionTransplantation Local and Regional Therapies HCC The commonlyused staging system for HCC is the Barcelona Clinic Liver Cancer staging system Figure HCCs in the very early stage or intermediate stage can betreated with local regional therapies which include radiofrequency ablationRFA resection da Vinci surgery laparoscopic surgery or traditional surgery transplantation orthotopic liver transplantation piggyback transplantation split liver transplantation auxiliary liver transplantation percutaneousethanoltranscatheter arterial chemoembolizationTACE58injections PEI orICC Surgery is currently the only curative treatment for ICCs but only aminority of patients in early stages are considered candidates for resectionIn surgery ICC is usually treated with hepatic resection to achieve negativeresection margins59 For patients with locally unresectable ICC tumor ablation such as RFA or hepatic arterybased therapies like yttrium90 radioembolization appear promising59“cHCCICC An accurate diagnosis is of paramount importance for thetreatment of cHCCICC Currently major hepatectomy is the optimal management for cHCCICC65 The rarity of this cancer as well as the lack of biomarkers have made this cancer difficult to diagnosis and manage Surgicalresection remains the only curative option for patients with cHCCICCThe treatment options for cHCCICC are similar to those for HCC and ICCand include surgery radiation yttrium90 radioembolization chemotherapycombined radiation and chemotherapy combined surgery and chemotherapy and triple therapy surgery radiation and chemotherapy4166“ Arecently retrospective analysis from to of PLC patientsincluding cHCCICC HCC and ICC patients who underwentresection found that although cHCCICC is more poorly differentiated thanHCC and ICC it had a similar 5year survival rate and respectively and 3year recurrence rate respectively70Systemic Chemotherapy HCC Systemic chemotherapy has limited efficacy on HCC several clinicaltrials of chemotherapy have shownlow response rates and worse toxicity without a significant improvement inThe Innovation August wwwcellcomtheinnovation\x0cReviewFigure Barcelona Clinic Liver Cancer Staging Systemand Corresponding Treatment Options The schematic diagram illustrates therapeutic choice by which a treatmenttheoretically recommended for a different stage is the besttreatment option 1L firstline 2L secondline ECOGEastern Cooperative Oncology Group M metastasis stageN nodal stage PEI percutaneous ethanolinjection PSperformance status T tumor stage TACE transarterialchemoembolization TARE transarterialradioembolizationY90 Y90 radioembolizationTheInnovation[5FU]including gemcitabine and doxorubicinbasedthe overall survival OStreatment FOLFOX 5fluorouracilleucovorin oxaliplatin andPIAF cisplatininterferon alpha2bdoxorubicin5FU71“ This suggestsa limited role for traditional chemotherapy in the treatment of advanced HCCICC Current firstline standard of treatment for ICC is the combination ofgemcitabine and platinumderived chemotherapy Figure 2B With the poorprognosis the median survival of advanced ICC patients is less than one yearVery limited effective treatments are available for patients who progress onfirstline chemotherapy so there is a high medical demandFirstLine Treatment Effective molecular targeted therapy and immunotherapy is lacking so chemotherapy with gemcitabine platinum compoundsand fluoropyrimidines is still the mainstream of standard treatment for unresectable ICCThe primary chemotherapy for ICC is gemcitabine which was establishedas the firstline therapy for advanced biliary tract cancer BTC in In the randomized controlled ABC02 phase III clinical trial comparedthe benefit of gemcitabine plus cisplatin CisGem chemotherapy with thesingle agent gemcitabine75 This study showed an advantage for CisGemin OS months versus months hazard ratio [HR] confidence intervalPFS months versus months p This effectiveness wasconfirmed in a Japanese randomized phase II study BT22 median OS months versus months HR Based on these promising results CisGem is currently regarded as the standard of care in the firstlinetreatment for advanced cholangiocarcinoma[CI] “ and progressionfree survivalOther than cisplatin gemcitabine plus other agents such as oxaliplatin S1capecitabine bevacizumab and Nabpaclitaxel have also been considered asthe firstline choices for advanced cholangiocarcinoma based on the promising outcomes from several phase II or III trials77“A recent multicenter randomized phase III clinical trial NCT01470443showed that XELOX has the comparable efficacious effect to GEMOX interms of tumor response survival rate OS and PFS and safety Also XELOXhas an advantage of low hospital visits compared with GEMOX Thus XELOXcould be an alternative for cholangiocarcinoma therapiesSecondLine Treatment There is no established standard secondlinechemotherapy for advanced cholangiocarcinoma and all regimens haveshown limited efficacy with a median PFS of around months and medianOS of about months92FOLFOX Lfolinic acid 5FU and oxaliplatin is an optional secondlinetreatment option based on the randomized phase III multicenter labelABC06 study NCT01926236 FOLFOX showed increased benefit for median OS months and months and OS rate months and compared with months and for the control groupactive symptom control [ASC] arm92cHCCICC In contrastCurrently several phase II and III chemotherapy clinical trials are under wayTable Combined therapy with chemotherapy shows promise in the treatment of cholangiocarcinoma selective internal radiotherapy SIRT pluschemotherapy or hepatic arterialinfusion plus systemic chemotherapyboth had antitumor activity and are promising for the treatment of ICC9394to surgerybased treatments for resectablecHCCICC systemic therapy is the nonstandard option for advanced and unresectable cHCCICC based on the standard treatment strategy for the unresectable HCC or ICC Chemotherapy for advanced or unresectable cHCCICCis largely understudied with only a few case reports and some retrospectivestudies having been published91095“ Recently a multicenter retrospectiveanalysis has been conducted by Kobayashi and colleagues10According to dividedgroup treatment with gemcitabine plus cisplatinn 5FU plus cisplatin n sorafenib monotherapy n others n they found that patients with platinumcontaining treatment had longer OS time than those treated by sorafenib monotherapyshowing OS of months CI “ months CI “ months CI “ and months CI “respectivelyA similar conclusion was drawn in another retrospective study of cHCCICC patients with receiving gemcitabinebased therapygemcitabine platinum or gemcitabine 5FU or targeted agents sorafenib9 Median PFS favored gemcitabineplatinum and gemcitabine5FU and months respectively over sorafenib monotherapy monthsllThe Innovation August 0cnoitavonnIehTReviewABFigure Treatment Strategy for Advanced HCC and ICC The schematic illustration represents FDAapproved drugs for treatment of advanced HCC and ICC Firstlinedrugs for HCC include sorafenib lenvatinib atezolizumab plus bevacizumab tremelimumab plus durvalumab and donafenib whereas for ICC the combination ofgemcitabine and cisplatin is currently proposed as first line The bottom row represents corresponding secondline therapies that come in when patients are not suitable fortheir firstline therapyMolecular Targeted Therapy HCC FirstLine Drugs Sorafenib Sorafenib was the first US Food and Drug Administration FDAapproved firstline systemic targeted drug for advanced HCC It is an oral smallmoleculemultikinase inhibitor targeting VEGFR1 VEGFR2 VEGFR3 PDGFRb andRaf Two large international multicenter clinical trials SHARP and AsianPacific have proved that sorafenib can suppress tumor progression and prolong OS in patients with advanced HCC102103 These trials showed that sorafenib can increase PFS and OS by months in patients with advancedHCC in Western countries As the first generation of targeted drugs forHCC sorafenib has been used for over a decade During this time many patients have benefitedthough others quickly developed resistance tosorafenib104Lenvatinib Lenvatinib is becoming available for HCC patients whodevelop sorafenib resistance Lenvatinib is an oral tyrosine kinase inhibitorinhibiting VEGFR1“ FGFR1“ PDGFR RET and KIT In August theFDA approved lenvatinib for firstline treatment of patients with unresectableHCC after lenvatinib was proved to be noninferior to sorafenib in the phase REFLECT trial105Median OS in the lenvatinib arm and sorafenib arm was months and months HR CI respectively The adverse effectswere hypertension diarrhea and decreased appetite withlenvatinib and palmarplantar erythrodysesthesia diarrhea decreased weight hypertension and decreased appetite with sorafenibDonafenib Similar to sorafenib donafenib is a novel multikinase inhibitortargeting RAF kinase and various receptor tyrosine kinases RTKs includingVEGF receptor VEGFR and BRAF106 According to the report from International Conference of the American Society of Clinical Oncology CSCOdonafenib significantly improves OS over sorafenib versus monthswith fewer side effects and higher patient tolerance for advanced HCC patients in its phase IIIII label trial107 The grade and above adverse reaction rates for donafenib and sorafenib were and respectivelyThus donafenib was recommended as the firstline therapy in the CSCOguidelines for HCCSecondLine Drugs Regorafenib Regorafenib an oral multikinase inhibitor inhibits the activity of protein kinases involved in multiple biological processes such as tumorigenesis tumor angiogenesis distant metastasisand tumor immune escape These kinases include VEGFR “ TIE2RAF1 KIT RET RAF BRAF PDGFR FGFR and CSF1R The randomized doubleblind multicenter phase III clinical trial RESORCE showed that regorafenib significantly improves the OS of patients as compared with the placebofrom to months HR p Grade “ adverse eventswere reported in of patients receiving regorafenib and of patientsreceiving the placebo In regorafenib received FDA approval as the secondline drug for the treatment of patients with advanced HCC who fail torespond to the sorafenib treatmentCabozantinib Cabozantinib is an oral inhibitor and targets multiple kinasesincluding VEGFR2 cMET RET ROS1 TYRO3 MER KIT TRKBFLT3 TIE2 as well as the GAS6 receptor AXL109110 It was originallyapproved for medullary thyroid cancer in and advanced renal carcinoma in According to the randomized doubleblind multicenter phase clinical trial conducted across centers in countries median OS was months for patients receiving cabozantinib and months for patientstreated with placebo HR p Median PFS was monthsand months respectively Grade or adverse events occurred in of patients in the cabozantinib arm and in the placebo arm Theobserved hepatotoxicity can be mostly controlled through dose modifications Based on the encouraging results of prolonged OS and PFS cabozantinib received its FDA approval for HCC in Ramucirumab Ramucirumab is a completely human monoclonalantibody that can specifically inhibit VEGFR2112 For patients with alphafetoprotein R400 ngmL and who have been previously treated with sorafenib ramucirumab was approved as a monotherapy by the FDA on May The Innovation August wwwcellcomtheinnovation\x0cTable Systemic Therapies Currently or Promising Approved for Advanced HCC and ICCReviewTargetTherapy LineApproved YearTrialDrugsHCCSorafenib NexavarLenvatinib LenvimaRegorafenib StivargaNivolumab OpdivoVEGFR2 VEGFR3 PDGFRb RAF kinasesFGFR VEGFR PDGFRa RET KITTie2 VEGFR PDGFR FGFRPD1Cabozantinib CabometyxcMet VEGFR2 AXL RETPembrolizumab KeytrudaRamucirumab CYRAMZAPD1VEGFR2Nivolumab ipilimumab Opdivo YervoyPD1 CTLA4Atezolizumab bevacizumabTremelimumab durvalumabDonafenibApatinibICCGemcitabine cisplatinPemigatinib PemazyreIvosidenibPDL1VEGFPD1 CTLA4VEGFR BRAFVEGFR2chemotherapyFGFR1“IDH12TheInnovationpromisingpromisingpromisingpromisingSHARP AsianPacificREFLECTRESORCECHECKMATE040CELESTIALKEYNOTE224REACH2Cohort of CHECKMATE040IMbravel50NCT02519348NCT02645981NCT02329860ABC02FIGHT202promisingClarlDHyApproval was based on REACH NCT02435433 a randomized doubleblind multicenter phase III study of patients with AFP R400 ngmL whohad disease progression after sorafenib or were intolerant to sorafenib113More recently a study further confirmed the efficacy of ramucirumab inelderly patients with HCC and elevated AFP after sorafenib in REACH andREACH2 with a survival benefit observed across all age subgroups and atolerable safety profile supporting its value irrespective of age including forpatients R75 years114Apatinib Apatinib a tyrosine kinase inhibitor targeting VEGFR2 significantly prolonged OS and PFS in Chinese patients with advanced HCC whohad previously been treated with sorafenib andor chemotherapy accordingto the results of a randomized placebocontrolled phase III trial conducted in sites in China115 Median OS was almost months longer for patients whoreceived apatinib compared with patients receiving the placebo monthsversus months and median PFS was more than months longer months versus months115 The most common grade or worseadverse events occurred at a rate of in the apatinib arm and inthe placebo arm With the significantly prolonged OS and PFS and a manageable safety profile apatinib has potential to become a new secondline therapy for liver cancerNovel Therapeutic Targets Even with all these available treatments Table the median PFS for HCC patients remains less than a year Thus noveltreatment is still a critical unmet need for treatment of HCC Based on thegenomic profile and biomarkers reported in HCC several clinical trials targeting various pathways are currently ongoing Table Recently a firstinhuman phase I study NCT02508467 of fisogatinib BLU554 an orally bioavailable inhibitor of human FGFR4 demonstrated its antitumor activity in HCCand further validated that the aberrant FGF19FGFR4 signaling pathwaymay be a driver event116 In addition the TGFb1 receptor type I inhibitor galunisertib also showed an acceptable safety and prolonged OS outcome in combination with sorafenib in a phase II trial NCT01246986117118 Other potential candidatesincluding the cyclindependent kinase CDK inhibitorsregulating the cell cycle pathways ribociclib palbociclib119120 abemacicliband milciclib as well as the cMET inhibitors tepotinib121 and tivantinib122are being evaluated in HCC clinical trialsICC Moleculartargeted therapy controls tumor cell proliferationapoptosis adhesion and movement by inhibiting the surface molecules oftumor cell membranes and thereby inhibiting intracellular signaling pathways ICC genetic alterations primarily include FGFR IDH epidermal growthfactor EGFR and breast cancer type susceptible protein associated protein1 BAP1123“ Genetic alterations of these genes all have implicationsfor therapy At present a variety of molecular targeted drugs are in the clinicalresearch stage Table some of which have made progress in the treatment of ICC Table FGFR Inhibitors The most promising target therapy for cholangiocarcinoma identified in recent years is the inhibitor of the fibroblast growth factorFGF signaling pathway which consists of members labeled FGF1“FGF15 FGF19 called FGF1519 and four interacting transmembrane receptors FGFR1“ FGF signals regulate cell proliferation in which FGFR2fusions occurred in “ of ICC patients and are considered as a promising therapeutic target3351127128 Currently several FGFR inhibitors are being evaluated in clinical trials for cholangiocarcinomas with FGFR geneticaberrationsPemigatinib INCB054828 Pemigatinib is the first and only targeted therapy so far approved in by the FDA for the treatment of this rare cancerIt is a selective potent oral inhibitor of FGFR and Approval wasbased on findings from the phase II FIGHT202 trial NCT02924376 whichenrolled patients with locally advanced or metastatic cholangiocarcinoma with FGFR2 fusions or rearrangements cohort A other FGFFGFR genetic alterations cohort B or no FGFFGFR genetic alterations cohort CFor those in cohort A treatment with pemigatinib resulted in a median OSof months and median PFS of months The FIGHT202 study suggests that locally advanced or metastatic cholangiocarcinoma patientswith FGFR2 fusions or rearrangements may benefit from potent oralFGFR1 and inhibitor treatment Median PFS was months for patientswith FGFR2 alterations months for patients with other FGFFGFR alterations and months for those with no alterations in these genes MedianOS was months months and months for the respective cohorts130 With the promising results of phase II the phase III clinical trial ofpemigatinib is currently underway NCT03656536llThe Innovation August 0cnoitavonnIehTDrugTargeted TherapyCabozantinibLenvatinibDonafenibMilciclibPalbociclibRibociclibGalunisertib versus LY2157299 sorafenib versus placebo sorafenibImmunotherapyVEGFRVEGFRVEGFRCDK2CDK46CDK46TGFbToripalimab versus placeboNivolumab versus placeboNivolumab versus sorafenibPD1PD1PD1Hospices Civils de Lyonrecruitingphase Eisai Pharmaceuticals IndiaPvt Ltdnot yetrecruitingphase NCT03963206NCT04297254completedphase phase NCT02645981Suzhou ZelgenBiopharmaceuticalsTiziana LifeSciencesPfizeractive notrecruitingactive notrecruitingphase phase Texas Universityrecruitingphase Eli Lillyactive notrecruitingphase NCT03109886NCT01356628NCT02524119NCT02178358NCT03412773NCT03859128NCT03383458ReviewTable Selected Ongoing Systemic Therapy Clinical Trials for Advanced HCCTargetSponsorStatusPhaseEnrollmentTrial IdentifierTislelizumab versus sorafenibPD1BeiGeneactive notrecruitingphase Shanghai Junshi Biosciencerecruitingphase phase BristolMyers Squibbrecruitingphase BristolMyers Squibbactive notrecruitingphase NCT02576509Pembrolizumab versus placeboPD1Merck Sharp Dohmerecruitingphase AvelumabPDL1Seoul National UniversityHospitalactive notrecruitingphase Combined TherapyLenvatinib pembrolizumabversus lenvatinib placeboCS1003 lenvatinib versusplacebo lenvatinibVGFR PD1Merck Sharp Dohmeactive notrecruitingphase VGFR PD1CStone Pharmaceuticalsrecruitingphase Tislelizumab regorafenibversus placebo regorafenibVEGF PD1National Taiwan UniversityHospi Answer:

Thyroid_Cancer

" new coronavirus SARSCoV2 has determined a pneumonia outbreak in China Wuhan Hubei Province in December called COVID19 disease In addition to the personto person transmission dynamic of the novel respiratory virus it has been recently studied the role of environmental factors in accelerate SARSCoV2 spread and its lethality The time being air pollution has been identified as the largest environmental cause of disease and premature death in the world It affects body™s immunity making people more vulnerable to pathogens The hypothesis that air pollution resulting from a combination of factors such as meteorological data level of industrialization as well as regional topography can acts both as a carrier of the infection and as a worsening factor of the health impact of COVID19 disease has been raised recently With this review we want to provide an update state of art relating the role of air pollution in particular PM25 PM10 and NO2 in COVID19 spread and lethality The Authors who first investigated this association often used different research methods or not all include confounding factors whenever possible In addition to date incidence data are underestimated in all countries and to a lesser extent also mortality data For this reason the cases included in the reviewed studies cannot be considered conclusive Although it determines important limitations for direct comparison of results and more studies are needed to strengthen scientific evidences and support firm s major findings are consistent highlighting the important contribution of PM25 and NO2 as triggering of the COVID19 spread and lethality and with a less extent also PM10 although the potential effect of airborne virus exposure it has not been still demonstrated Introduction A new coronavirus SARSCoV2 has determined a pneumonia outbreak in China Wuhan Hubei Province in December called COVID19 disease The scientific community has come together to implement pharmaceutical and nonpharmaceutical intervention measures designed to contain SARSCoV2 global spread Nevertheless on March 11th WHO™s Directeneral announced that COVID19 can be characterized as a pandemic SARSCoV2 is primarily transmitted from persontoperson through close contact approximately m by aerosol respiratory droplets smaller than μm in diameter wwwwhoint Indoor environments might be especially hazardous because of their reduced ventilation Morawska lack ultraviolet light which rapidly inactivates the virus and because it can become less diluted than it would in outdoor environments Schuit It is also known how the virus can survive and being infectious in aerosols for hours and on surface up to days van Doremalen et al similarly with transmission dynamics known for SARSCoV1 associated with nosocomial spread and superspreading events Chen et al 2020ab Beyond the causality it is uncertain even if certain demographics of the population are more vulnerable to SARSCoV2 infection Based on recent reports male gender advancing age and comorbidities seem to be correlated with death and severe illness Harris et al Furthermore COVID19 seems to be associated with an increasing rate of thromboembolic events in hospitalized patients Llitjos Mechanisms of social and economic interactions are additionally supposed to be involved in the diffusion dynamic of COVID19 in the diverse parts of the world or of the same country such as the living conditions the healthrelated behaviour KhalatbariSoltani et al Corresponding author Email address ccopatunictit C Copat 101016jenvres2020110129 Received July Received in revised form August Accepted August EnvironmentalResearch1912020110129Availableonline24August2020001393512020ElsevierIncAllrightsreserved 0cC Copat the commercial exchanges Bontempi 2020a or the migration scale index H Chen It seems that these diffusion dynamics have particularly affected the COVID19 spread at the early stage Among the environmental parameters some climate condition such as temperature humidity sunlight and wind revealed a reduction of the COVID19 spread S Chen Coccia 2020a and air pollution seems to have a role in airborne transmission of SARSCoV2 and severity of COVID19 Domingo Nevertheless to better understand COVID19™s diffusion patterns an interdisciplinary multidimensional approach should be encourage in order to develop firm s Bontempi Air pollution has been identified as the largest environmental cause of disease and premature death in the world GBD Ambient particulate matter PM induces its proinflammatory and thrombogenic effects through the generation of oxidative stress by its chemical compounds and metals Li Signorelli The recent identification of environmentally persistent free radicals EPFRs in the PM resulting from a mixture of combustion sources theorize its role in the increase of disease severity of lower respiratory tract infections LRTI Jaligama Scientific evidences support that short and longterm exposures to ambient air pollutants are associated with a broad of adverse health outcomes Ferrante and Conti Fiore such as higher mortality rates greater hospital admissions and increased outpatient visits Bremner Cohen Dehghani Dockery It has markedly detrimental consequences on asthma bronchitis pneumonia and COPD Dick Perng and Chen Raji Vignal Yarahmadi et al where bacteria and viruses are the most accepted causative factors that harm airway stability driving to infection exacerbation Furthermore air pollution represents an aggravating factor for infection diseases caused by some viral infections Domingo and Rovira such as respiratory syncytial virus RSV influenza A and B para influenza virus type pneumonia and influenzalike illness Carugno Croft Fukuda Huang Huh Liang Lin Silva Somayaji It determines an increase in the rate of hospitalizations and access to emergency department visits Studies related to the epidemic of SARSCoV coronavirus identified in November from the Guangdong province of southern China reported similar associations Cai Cui Kan Several Authors suggest that outdoor air pollution resulting from a combination of factors such as meteorological data level of industrialization as well as regional topography could operate both as a carrier of the infection and as a worsening factor of the COVID19 severity Conticini Frontera Isaifan Martelletti and Martelletti This association is getting stronger thanks to the results of the numerous studies that have been launched all over the world and summarized with this review Most of the reviewed studies support that chronic exposure to air pollution might led people more susceptible to COVID19 disease leading to widespread COVID19 spread and lethality Nevertheless as suggested by Bontempi 2020b the potential effect of airborne virus exposure due to PM10 remain unclear With this review we want to provide an updated state of art of the recently epidemiological studies dealing with understanding the role of air pollution in particular PM25 PM10 and NO2 in COVID19 spread and lethality Fig PRISMA Flow Diagram of identification screening and inclusion of studies EnvironmentalResearch19120201101292 0cC Copat Method We have conducted a systematic review of the literature concerning the relationship between some air pollutants PM25 PM10 and NO2 and COVID19 outbreak The research was performed in compliance with the PRISMA criteria Preferred Reporting Items for Systematic Reviews and MetaAnalyses and the Flow Diagram is showed in Fig The research was conducted between April and July 6th in PubMed database It was used the Advanced Search Builder and the keywords were searched in [Title OR Abstract] We have filtered only research articles published in English language and selected the following keywords Air pollution and COVID19 or SARSCOV2 Particulate matter or PM and COVID19 or SARSCOV2 Nitrogen dioxide or NO2 and COVID19 or SARSCOV2 We choose as inclusion criteria all the available epidemiological studies aimed to identify any temporal and spatial association between reported COVID19 cases andor deaths and air pollution data related to PM25 PM10 and NO2 thus excluding any Letter Opinion Commentary Review or nonrelevant articles We obtained a total of eligible published research articles in their final version and paper in its preprint version For some of them we chose to include only principal findings that clearly fit the aim this review Particulate Matter and COVID19 Atmospheric particulate matter PM is originated by a wide range of anthropogenic and natural sources Kim It consists of a heterogeneous mixture of solid and liquid particles suspended in air that varies continuously in size and chemical composition including nitrates sulphates elemental and anic carbon anic compounds biological compounds and metals WHO It has been associated with increased respiratory morbidity and mortality Liu especially in susceptible people due to cardiorespiratory events including asthma chronic obstructive pulmonary disease and thrombosis Li Rhee In vitro and in vivo studies highlighted its role in the exacerbation of respiratory viral infections Becker and Soukup Recently the research group of Setti gave first preliminary evidence that SARSCoV2 RNA can be present on outdoor particulate matter thus suggesting that in conditions of atmospheric stability and high concentrations of PM it could represent a potential early indicator of COVID19 although it does not give information regarding COVID19 progression or severity Several observations report a significant association between ambient concentrations of PM25 Adhikari and Yin Bashir Fattorini and Regoli Frontera Jiang Li VasquezApestegui Wu Yao Zhu Zoran 2020a and PM10 Bashir Coccia 2020b Fattorini and Regoli Jiang Li Yao Zhu Zoran 2020a with COVID19 pandemic across the most affected countries China Italy and USA see Table First evidences on the temporal association between air pollution and COVID19 were reported in China where the outbreak was first identified Zhu explored the relationship between particulate matter and the viral infection caused by the novel coronavirus in cities in China The Authors included over of dailyconfirmed new cases in the whole of China between January 23rd and February 29th They applied a Generalized Additive Model GAM to examine the effects of meteorological factors and air pollution on COVID19 incidence applying a movingaverage approach to capture the cumulative lag effect of ambient air pollution and considering population size and density as potential confounders They observed that the effect of PM25 on daily confirmed cases was greater than PM10 In particular they found that a 10μgm3 increase lag0“ in PM25 and PM10 was associated with a CI to and CI to increase in the daily counts of COVID19 confirmed cases respectively Jiang focused their attention on three most affected cities of China Wuhan XiaoGan and HuangGang collecting data of daily cases and ambient air pollutant from Jan 25th to Feb 29th The Authors by applying a multivariate Poisson regression revealed a significant temporal association between PM25 increased and COVID19 incidence in all the considered cities especially in HuangGang Wuhan RR CI “ XiaoGan RR CI “ HuangGang RR CI “ Conversely an increase in PM10 concentrations was associated with a decrease of COVID19 incidence These results were partially confirmed by findings of Li who conducted a simple linear regression to compare COVID19 incidence with PM concentrations in Wuhan and XiaoGan from Jan 26th to Feb 29th in They found that an increase in PM25 was correlated with an increase of COVID19 incidence in both cities Wuhan R2 p XiaoGan R2 p while for PM10 only in XiaoGan R2 p The spatial distribution of particulate matter and case fatality rate CFR of COVID19 was studied by Yao in cities of China including Wuhan collecting data up to March 22nd First they found a significantly positive global spatial autocorrelation of COVID19 CFR Global Moran™s index I p highlighting a high CFR clustering located in Hubei Province With a multiple linear regression they adjusted their results for several effect modifiers and confounder factors such temperature relative humidity gross domestic product GDP per capita hospital beds per capita local indicators of spatial association LISA map values city size and population or proportion of people older than years It was found that for every μgm3 increase in PM25 and PM10 the CFR increased by “ and “ respectively and the risk estimates increased to “ and “ with every μgm3 increase in average concentrations of PM25 and PM10 in “ respectively Some studies describe the association between air pollution and COVID19 across Italy the second country of the world where the infection spread significantly at the beginning of the pandemic and suddenly has reached many other European countries The 28th of July Italy recorded more than total confirmed cases and deaths WHO most of which were distributed in the regions of Northern Italy especially the Lombardy It is recognized as one the most air polluted areas of Europe EEA where the frequent PM10 annual exceedances of the WHO threshold of μgm3 are responsible for attributable deaths per year corresponding to attributable community rates of deaths per inhabitants per year Baccini Bontempi 2020bfocused the attention on two of the most affected regions of Northern Italy Lombardy and Piedmont The Authors based on PM10 daily exceedances and COVID19 confirmed cases on March 12th thus before the Italian sanitary crisis observed that PM10 concentration was exceeded only few times among the Lombard cities that at the beginning of the epidemic were most affected On the contrary among some Piedmont cities suffering of severe PM10 pollution events COVID19 incidence was lower Based on their results the Authors concluded that COVID19 diffusion by airborne PM10 is hard to demonstrate nevertheless several research article revealed how PM in particular PM25 could had a role in accelerate and vast diffusion of COVID19 in Northern Italy For example Coccia 2020b by analyzed data on Italian province capitals and data of infected individuals up to April 7th revealed a relationship between air pollution of cities measured with days exceeding the limits set for PM10 in previous years and COVID19 diffusion In particular cities with more than days of PM10 exceedances showed a very high average number of infected individual about infected individuals on 7th April whereas cities having less than days of PM10 exceedances showed a lower average number of infected about infected individuals Frontera gave also evidences on the role of PM25 as a contributing factor of COVID19 outbreak in Northern Italy where EnvironmentalResearch19120201101293 0cC Copat Table Summary table reporting reviewed results on the association between COVID19 casesdeaths and air pollution PM25 PM10 and NO2 References Zhu Data analysis Generalized Additive Model GAM Aim Temporal association between daily confirmed cases and air pollution PM25 PM10 and NO2 Temporal association between daily confirmed cases and air pollution PM25 PM10 and NO2 Temporal association between daily confirmed cases and air pollution PM25 PM10 and NO2 Spatial association between fatality rate and air pollution PM25 and PM10 Spatial association between deaths counts and air pollution NO2 Temporal association between total cases daily confirmed cases and total deaths and air pollution PM25 and PM10 Temporal association between total cases daily confirmed cases and total deaths and air pollution NO2 Spatial description of PM10 exceedances versus COVID19 cases Multivariate Poisson regression Simple linear regression Multiple linear regression Descriptive analysis percentage of deaths in three NO2 μmol m2concentration range “ “ “ Pearson coefficient correlation Pearson coefficient correlation Descriptive analysis Number of days of PM10 exceeding μgm3 and COVID19 incidence Area of Study cities of China Period From Jan 23rd to Feb 29th Jiang Li Yao Ogen Zoran 2020a Zoran 2020b Bontempi 2020b From Jan 25th to Feb 29th From Jan 26th to Feb 29th in Data up to March 22nd Data up to the end of Feb From Jan 1st to Apr 30th From Jan 1st to Apr 30th From Feb 10th to March 12th Wuhan XiaoGan and HuangGang China Wuhan and XiaoGan cities of China administrative regions in Italy Spain France and Germany Milan Italy Milan Italy provinces of Lombardy Italy provinces of Piedmont Italy Coccia 2020b Data up to April 7th Italian Provinces Fattorini and Regoli Data up to April 27th Italian provinces PM25 A 10μgm3 PM25 increase lag0“ was associated with a increase of daily confirmed new cases PM10 A 10μgm3 PM10 increase lag0“ was associated with a increase of daily confirmed new cases Wuhan RR CI1032“ XiaoGan RR CI “ HuangGang RR CI “ Wuhan R2 p XiaoGan R2 p Wuhan RR CI “ XiaoGan RR CI “ HuangGang RR CI “ Wuhan R2 p XiaoGan R2 p χ2 p A μgm3 increase in PM25 was associated with a “ increase in fatality rate χ2 p A μgm3 increase in PM10 was associated with a “ increase in fatality rate NO2 A 10μgm3 NO2 increase lag0“ was associated with a increase in daily confirmed new cases Wuhan RR CI “ XiaoGan RR CI “ HuangGang no association found Wuhan R2 p XiaoGan R2 p of fatality cases are associated with NO2 μmolm2 R cid0 R R cid0 R cid0 R R cid0 R cid0 R cid0 R cid0 Lombardy PM10 exceeding between and COVID19 incidence between and Piedmont PM10 exceeding between and COVID19 incidence between and COVID19 in North Italy has a high association with air pollution of cities measured with days exceeding the limits set for PM10 R2 p R2 p continued on next page Hierarchical multiple regression model Pearson regression coefficient analysis R2 p Spatial association between confirmed cases and air pollution PM10 Spatial association between total confirmed cases and air pollution PM25 PM10 and NO2 EnvironmentalResearch19120201101294 0cC Copat Table continued References Frontera Frontera Wu Adhikari and Yin Bashir Bashir VasquezApestegui VasquezApestegui VasquezApestegui Period Data up to 31st March Data up to 31st March Data up to April 04th From March 1st to Apr 20th From March 4th to April 24th From March 4th to April 24th Data up to June 12th Data up to June 12th Data up to June 12th Area of Study Italian regions Italian regions counties in the USA Queens county New York USA California California districts of Lima Perù districts of Lima Perù districts of Lima Perù Aim Spatial association between total confirmed cases and air pollution PM25 Spatial association between deaths and air pollution PM25 Prediction of risk of COVID19 deaths in the long term average exposure to fine particulate matter PM25 Temporal association between daily confirmed cases and total deaths and air pollution PM25 Association between confirmed cases and air pollution PM25 PM10 and NO2 Association between deaths and air pollution PM25 PM10 and NO2 Spatial association between total confirmed cases and air pollution PM25 Spatial association between deaths and air pollution PM25 Spatial association between case fatality rate and air pollution PM25 Data analysis Pearson regression coefficient analysis PM25 R2 p PM10 Pearson regression coefficient analysis R2 p Longterm exposure increase of μgm3 in PM25 is associated with a increase in the COVID19 death rate Estimate on cases values cid0 CI “ Estimate on deaths value cid0 CI “ Kendall r cid0 Spearman r cid0 Zeroinflated negative binomia models Negative binomial regression model Spearman and Kendall correlation tests Spearman and Kendall correlation tests NO2 Kendall r cid0 Spearman r cid0 Kendall r cid0 Spearman r cid0 Kendall r cid0 Spearman r cid0 Kendall r cid0 Spearman r cid0 Kendall r cid0 Spearman r cid0 Multivariate regression model Crude coefficient p Multivariate regression model Crude coefficient p Multivariate regression model Crude coefficient cid0 p mortality was found significantly higher than less polluted Italian regions By collecting data up to March 31st for all Italian regions and performing a Pearson correlation analysis they found a strong positive association both with the total number of confirmed cases R and deaths R other than with hospitalized cases R The Italian situation was further highlighted by the study of Fattorini and Regoli in Italian provinces They explored the spatial association between air pollution and COVID19 cases with data up to April 27th By applying the Pearson regression coefficient analysis they revealed a positive association both with PM25 and PM10 R2 p and R2 p respectively A focus on the most affected city of Italy Milan was conducted by Zoran 2020a This city is located in the Po valley basin known hotspot for atmospheric pollution at the continental scale EEA The Authors performed a temporal association between COVID19 Total cases Daily New positive cases and Total Deaths and particulate matter from Jan 1st and Apr 30th by applying a Person correlation In accordance with other studied they found a positive association between daily confirmed cases and PM25 R and PM10 R although they did not consider any delay time from infection to COVID19 onset Nevertheless they found a negative association between total cases and total deaths and particulate matter but the assumption of a temporal linear correlation may be inaccurate because the above mentioned variables could have more complex nonlinear relationships To date the USA have more than million confirmed cases and thousand deaths WHO Here ambient concentrations of PM and O3 were found responsible to cause between and premature deaths Fann The association between air pollutants and COVID19 cases and deaths was studied by Bashir in the state of California from March 4th to April 24th corresponding to the beginning of the COVID19 outbreak in USA Based on their significant correlation found the Authors state that a limited human exposure to these pollutants will contribute to defeating COVID19 This seems unclear because they found a negative correlation with PM25 and PM10 EnvironmentalResearch19120201101295 0cC Copat by applying both the Kendall rank correlation and Spearman™s one and it is not clear if they normalized COVID19 cases by population size and if they performed a day by day association or a spatial association across the country A focus on the Queen county New York USA was provided by Adhikari and Yin They retrieved data of PM daily concentrations from two ground monitoring stations and collected data of confirmed COVID19 cases and numbers of related deaths from USAFacts in the period from March to April The Authors elaborated their data with a negative binomial regression model and considered the cumulative lag effect of PM25 on disease outcomes over the past days They found a significant negative association among PM25 and new daily confirmed COVID19 cases cid0 CI “ and deaths cid0 CI “ Low PM concentrations in this area of study mean μgm3 are likely to have played a less central role in the spread of infection than in other areas such as Italy where PM25 monthly concentrations reached values higher than μgm3 Fattorini and Regoli Frontera or in China where PM25 monthly concentrations reached values higher than μgm3 Zhu Jiang As said by the Authors other gaseous pollutants such as NO2 and SO2 could have influenced transmission and pathogenesis of COVID19 In the United States Wu investigated whether longterm average exposure to fine particulate matter PM25 increases the risk of COVID19 deaths by considering approximately counties in the United States of the population With an exposure prediction model the Authors calculated the county level longterm exposure to PM25 averaged for to and collected COVID19 deaths counts up to April 04th They conducted a strong and robust statistical analysis with zeroinflated negative binomial mixed models adjusting their results by several potential confounders such as sociodemographic socioeconomic behavioural and meteorological factors They found that a small longterm exposure increase of only μgm3 in PM25 is associated with a increase in the COVID19 death rate confidence interval CI VasquezApestegui recently reported first evidences on the spatial relationship between particulate matter and COVID19 outbreak from Latin America The Authors described the situation occurred in districts of Lima located in the second most affected country of Latin America Peru In particular by applying a multivariate regression model they evaluated the association between the population exposure to PM25 concentrations in the previous years “ and cases deaths and casefatality rates of COVID19 with data up to June 12th A significant association has been found both with cases and deaths Crude coefficient with p and with p respectively but not with case fatality rate All these studies highlight the role of PM in triggers of the COVID19 disease and how government measures targeting to sustainable growth such as the reduction of urban and industrial emissions could have a positive impact on the prevention of health outcomes reducing mortality rate as well the burden on health care systems Nitrogen dioxide NO2 and COVID19 induced lung damage Hence viral infection becomes more common after exposure to NO2 Zhu Furthermore NO2 is associated with other several health effects such as elevated risks for asthma allergic rhinitis and eczema in children To increase of outpatient visits and hospitalizations due to bronchitis and asthma exacerbation Bahrami Asl Kowalska increase of chronic obstructive pulmonary disease COPD Ghanbari Ghozikali Pfeffer and increase of pulmonary heart disease related mortality Chen A recent study explored the possible role of NO2 in interference in Angiotensin converting enzyme ACE2 The expression of ACE2 is high on lung alveolar epithelial cells and it is the human cell receptor of virus agent of COVID19 Alifano First observations report an association between ambient concentrations of NO2 and COVID19 pandemic across Europe China and USA Bashir Fattorini and Regoli Jiang Li et al Ogen Zhu et al Zoran et al 2020b Conversely to the other papers findings of Zoran 2020b and Bashir provides different findings reporting no association or a negative one between NO2 and daily deaths counts In China Zhu by applying the same method explained for PM observed that a 10μgm3 increase lag0“ in NO2 is associated with a CI “ increase in the daily counts of COVID19 confirmed cases in cities of China These findings are confirmed by Jiang and Li et a who applied the same method described for PM Jiang revealed a significant positive association between NO2 and COVID19 both in Wuhan and XiaoGan Wuhan RR CI1053“ XiaoGan RR CI “ but did not found any significant association in HuangGang Li found a significant linear correlation both in Wuhan R2 p and XiaoGan R2 p Ogen presented evidences on the relationship between exposure to NO2 including the months of January and February shortly before the COVID19 spread in Europe and novel coronavirus fatality in the most affected European countries concluding that longterm exposure to NO2 may be a potential contributor to mortality caused by SARSCoV2 He collected data concerning the number of fatality cases from administrative regions in Italy Spain France and Germany and correlated mortality with tropospheric NO2 concentrations measured by the Sentinel5 Precursor spaceborne satellite The major tropospheric NO2 hotspot identified was located in the Northern Italy In all European regions considered gas concentrations ranged between and μmolm2 with airflows directed downwards Results showed that out of the fatality cases by March were in five regions located in north Italy and central Spain Furthermore by analysing mortality trends it was revealed that the highest percentage of deaths occurred in regions where the maximum NO2 concentration was above μmolm2 with a significant decrease where the maximum concentration was between and μmolm2 and below μmolm2 The methodology used by Ogen cannot support a longterm exposure investigation Surely a validation of the satellite measure with those of the ground ™ones the adjustment of the results according to the different population size of each country could have made their results more robust Nevertheless the study provide new insights for future investigation The Italian situation was further studied by Fattorini and Regoli who collected data of COVID19 incidence up to April 27th from Italian provinces They revealed a strong spatial corr

cancer238

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: " new coronavirus SARSCoV2 has determined a pneumonia outbreak in China Wuhan Hubei Province in December called COVID19 disease In addition to the personto person transmission dynamic of the novel respiratory virus it has been recently studied the role of environmental factors in accelerate SARSCoV2 spread and its lethality The time being air pollution has been identified as the largest environmental cause of disease and premature death in the world It affects body™s immunity making people more vulnerable to pathogens The hypothesis that air pollution resulting from a combination of factors such as meteorological data level of industrialization as well as regional topography can acts both as a carrier of the infection and as a worsening factor of the health impact of COVID19 disease has been raised recently With this review we want to provide an update state of art relating the role of air pollution in particular PM25 PM10 and NO2 in COVID19 spread and lethality The Authors who first investigated this association often used different research methods or not all include confounding factors whenever possible In addition to date incidence data are underestimated in all countries and to a lesser extent also mortality data For this reason the cases included in the reviewed studies cannot be considered conclusive Although it determines important limitations for direct comparison of results and more studies are needed to strengthen scientific evidences and support firm s major findings are consistent highlighting the important contribution of PM25 and NO2 as triggering of the COVID19 spread and lethality and with a less extent also PM10 although the potential effect of airborne virus exposure it has not been still demonstrated Introduction A new coronavirus SARSCoV2 has determined a pneumonia outbreak in China Wuhan Hubei Province in December called COVID19 disease The scientific community has come together to implement pharmaceutical and nonpharmaceutical intervention measures designed to contain SARSCoV2 global spread Nevertheless on March 11th WHO™s Directeneral announced that COVID19 can be characterized as a pandemic SARSCoV2 is primarily transmitted from persontoperson through close contact approximately m by aerosol respiratory droplets smaller than μm in diameter wwwwhoint Indoor environments might be especially hazardous because of their reduced ventilation Morawska lack ultraviolet light which rapidly inactivates the virus and because it can become less diluted than it would in outdoor environments Schuit It is also known how the virus can survive and being infectious in aerosols for hours and on surface up to days van Doremalen et al similarly with transmission dynamics known for SARSCoV1 associated with nosocomial spread and superspreading events Chen et al 2020ab Beyond the causality it is uncertain even if certain demographics of the population are more vulnerable to SARSCoV2 infection Based on recent reports male gender advancing age and comorbidities seem to be correlated with death and severe illness Harris et al Furthermore COVID19 seems to be associated with an increasing rate of thromboembolic events in hospitalized patients Llitjos Mechanisms of social and economic interactions are additionally supposed to be involved in the diffusion dynamic of COVID19 in the diverse parts of the world or of the same country such as the living conditions the healthrelated behaviour KhalatbariSoltani et al Corresponding author Email address ccopatunictit C Copat 101016jenvres2020110129 Received July Received in revised form August Accepted August EnvironmentalResearch1912020110129Availableonline24August2020001393512020ElsevierIncAllrightsreserved 0cC Copat the commercial exchanges Bontempi 2020a or the migration scale index H Chen It seems that these diffusion dynamics have particularly affected the COVID19 spread at the early stage Among the environmental parameters some climate condition such as temperature humidity sunlight and wind revealed a reduction of the COVID19 spread S Chen Coccia 2020a and air pollution seems to have a role in airborne transmission of SARSCoV2 and severity of COVID19 Domingo Nevertheless to better understand COVID19™s diffusion patterns an interdisciplinary multidimensional approach should be encourage in order to develop firm s Bontempi Air pollution has been identified as the largest environmental cause of disease and premature death in the world GBD Ambient particulate matter PM induces its proinflammatory and thrombogenic effects through the generation of oxidative stress by its chemical compounds and metals Li Signorelli The recent identification of environmentally persistent free radicals EPFRs in the PM resulting from a mixture of combustion sources theorize its role in the increase of disease severity of lower respiratory tract infections LRTI Jaligama Scientific evidences support that short and longterm exposures to ambient air pollutants are associated with a broad of adverse health outcomes Ferrante and Conti Fiore such as higher mortality rates greater hospital admissions and increased outpatient visits Bremner Cohen Dehghani Dockery It has markedly detrimental consequences on asthma bronchitis pneumonia and COPD Dick Perng and Chen Raji Vignal Yarahmadi et al where bacteria and viruses are the most accepted causative factors that harm airway stability driving to infection exacerbation Furthermore air pollution represents an aggravating factor for infection diseases caused by some viral infections Domingo and Rovira such as respiratory syncytial virus RSV influenza A and B para influenza virus type pneumonia and influenzalike illness Carugno Croft Fukuda Huang Huh Liang Lin Silva Somayaji It determines an increase in the rate of hospitalizations and access to emergency department visits Studies related to the epidemic of SARSCoV coronavirus identified in November from the Guangdong province of southern China reported similar associations Cai Cui Kan Several Authors suggest that outdoor air pollution resulting from a combination of factors such as meteorological data level of industrialization as well as regional topography could operate both as a carrier of the infection and as a worsening factor of the COVID19 severity Conticini Frontera Isaifan Martelletti and Martelletti This association is getting stronger thanks to the results of the numerous studies that have been launched all over the world and summarized with this review Most of the reviewed studies support that chronic exposure to air pollution might led people more susceptible to COVID19 disease leading to widespread COVID19 spread and lethality Nevertheless as suggested by Bontempi 2020b the potential effect of airborne virus exposure due to PM10 remain unclear With this review we want to provide an updated state of art of the recently epidemiological studies dealing with understanding the role of air pollution in particular PM25 PM10 and NO2 in COVID19 spread and lethality Fig PRISMA Flow Diagram of identification screening and inclusion of studies EnvironmentalResearch19120201101292 0cC Copat Method We have conducted a systematic review of the literature concerning the relationship between some air pollutants PM25 PM10 and NO2 and COVID19 outbreak The research was performed in compliance with the PRISMA criteria Preferred Reporting Items for Systematic Reviews and MetaAnalyses and the Flow Diagram is showed in Fig The research was conducted between April and July 6th in PubMed database It was used the Advanced Search Builder and the keywords were searched in [Title OR Abstract] We have filtered only research articles published in English language and selected the following keywords Air pollution and COVID19 or SARSCOV2 Particulate matter or PM and COVID19 or SARSCOV2 Nitrogen dioxide or NO2 and COVID19 or SARSCOV2 We choose as inclusion criteria all the available epidemiological studies aimed to identify any temporal and spatial association between reported COVID19 cases andor deaths and air pollution data related to PM25 PM10 and NO2 thus excluding any Letter Opinion Commentary Review or nonrelevant articles We obtained a total of eligible published research articles in their final version and paper in its preprint version For some of them we chose to include only principal findings that clearly fit the aim this review Particulate Matter and COVID19 Atmospheric particulate matter PM is originated by a wide range of anthropogenic and natural sources Kim It consists of a heterogeneous mixture of solid and liquid particles suspended in air that varies continuously in size and chemical composition including nitrates sulphates elemental and anic carbon anic compounds biological compounds and metals WHO It has been associated with increased respiratory morbidity and mortality Liu especially in susceptible people due to cardiorespiratory events including asthma chronic obstructive pulmonary disease and thrombosis Li Rhee In vitro and in vivo studies highlighted its role in the exacerbation of respiratory viral infections Becker and Soukup Recently the research group of Setti gave first preliminary evidence that SARSCoV2 RNA can be present on outdoor particulate matter thus suggesting that in conditions of atmospheric stability and high concentrations of PM it could represent a potential early indicator of COVID19 although it does not give information regarding COVID19 progression or severity Several observations report a significant association between ambient concentrations of PM25 Adhikari and Yin Bashir Fattorini and Regoli Frontera Jiang Li VasquezApestegui Wu Yao Zhu Zoran 2020a and PM10 Bashir Coccia 2020b Fattorini and Regoli Jiang Li Yao Zhu Zoran 2020a with COVID19 pandemic across the most affected countries China Italy and USA see Table First evidences on the temporal association between air pollution and COVID19 were reported in China where the outbreak was first identified Zhu explored the relationship between particulate matter and the viral infection caused by the novel coronavirus in cities in China The Authors included over of dailyconfirmed new cases in the whole of China between January 23rd and February 29th They applied a Generalized Additive Model GAM to examine the effects of meteorological factors and air pollution on COVID19 incidence applying a movingaverage approach to capture the cumulative lag effect of ambient air pollution and considering population size and density as potential confounders They observed that the effect of PM25 on daily confirmed cases was greater than PM10 In particular they found that a 10μgm3 increase lag0“ in PM25 and PM10 was associated with a CI to and CI to increase in the daily counts of COVID19 confirmed cases respectively Jiang focused their attention on three most affected cities of China Wuhan XiaoGan and HuangGang collecting data of daily cases and ambient air pollutant from Jan 25th to Feb 29th The Authors by applying a multivariate Poisson regression revealed a significant temporal association between PM25 increased and COVID19 incidence in all the considered cities especially in HuangGang Wuhan RR CI “ XiaoGan RR CI “ HuangGang RR CI “ Conversely an increase in PM10 concentrations was associated with a decrease of COVID19 incidence These results were partially confirmed by findings of Li who conducted a simple linear regression to compare COVID19 incidence with PM concentrations in Wuhan and XiaoGan from Jan 26th to Feb 29th in They found that an increase in PM25 was correlated with an increase of COVID19 incidence in both cities Wuhan R2 p XiaoGan R2 p while for PM10 only in XiaoGan R2 p The spatial distribution of particulate matter and case fatality rate CFR of COVID19 was studied by Yao in cities of China including Wuhan collecting data up to March 22nd First they found a significantly positive global spatial autocorrelation of COVID19 CFR Global Moran™s index I p highlighting a high CFR clustering located in Hubei Province With a multiple linear regression they adjusted their results for several effect modifiers and confounder factors such temperature relative humidity gross domestic product GDP per capita hospital beds per capita local indicators of spatial association LISA map values city size and population or proportion of people older than years It was found that for every μgm3 increase in PM25 and PM10 the CFR increased by “ and “ respectively and the risk estimates increased to “ and “ with every μgm3 increase in average concentrations of PM25 and PM10 in “ respectively Some studies describe the association between air pollution and COVID19 across Italy the second country of the world where the infection spread significantly at the beginning of the pandemic and suddenly has reached many other European countries The 28th of July Italy recorded more than total confirmed cases and deaths WHO most of which were distributed in the regions of Northern Italy especially the Lombardy It is recognized as one the most air polluted areas of Europe EEA where the frequent PM10 annual exceedances of the WHO threshold of μgm3 are responsible for attributable deaths per year corresponding to attributable community rates of deaths per inhabitants per year Baccini Bontempi 2020bfocused the attention on two of the most affected regions of Northern Italy Lombardy and Piedmont The Authors based on PM10 daily exceedances and COVID19 confirmed cases on March 12th thus before the Italian sanitary crisis observed that PM10 concentration was exceeded only few times among the Lombard cities that at the beginning of the epidemic were most affected On the contrary among some Piedmont cities suffering of severe PM10 pollution events COVID19 incidence was lower Based on their results the Authors concluded that COVID19 diffusion by airborne PM10 is hard to demonstrate nevertheless several research article revealed how PM in particular PM25 could had a role in accelerate and vast diffusion of COVID19 in Northern Italy For example Coccia 2020b by analyzed data on Italian province capitals and data of infected individuals up to April 7th revealed a relationship between air pollution of cities measured with days exceeding the limits set for PM10 in previous years and COVID19 diffusion In particular cities with more than days of PM10 exceedances showed a very high average number of infected individual about infected individuals on 7th April whereas cities having less than days of PM10 exceedances showed a lower average number of infected about infected individuals Frontera gave also evidences on the role of PM25 as a contributing factor of COVID19 outbreak in Northern Italy where EnvironmentalResearch19120201101293 0cC Copat Table Summary table reporting reviewed results on the association between COVID19 casesdeaths and air pollution PM25 PM10 and NO2 References Zhu Data analysis Generalized Additive Model GAM Aim Temporal association between daily confirmed cases and air pollution PM25 PM10 and NO2 Temporal association between daily confirmed cases and air pollution PM25 PM10 and NO2 Temporal association between daily confirmed cases and air pollution PM25 PM10 and NO2 Spatial association between fatality rate and air pollution PM25 and PM10 Spatial association between deaths counts and air pollution NO2 Temporal association between total cases daily confirmed cases and total deaths and air pollution PM25 and PM10 Temporal association between total cases daily confirmed cases and total deaths and air pollution NO2 Spatial description of PM10 exceedances versus COVID19 cases Multivariate Poisson regression Simple linear regression Multiple linear regression Descriptive analysis percentage of deaths in three NO2 μmol m2concentration range “ “ “ Pearson coefficient correlation Pearson coefficient correlation Descriptive analysis Number of days of PM10 exceeding μgm3 and COVID19 incidence Area of Study cities of China Period From Jan 23rd to Feb 29th Jiang Li Yao Ogen Zoran 2020a Zoran 2020b Bontempi 2020b From Jan 25th to Feb 29th From Jan 26th to Feb 29th in Data up to March 22nd Data up to the end of Feb From Jan 1st to Apr 30th From Jan 1st to Apr 30th From Feb 10th to March 12th Wuhan XiaoGan and HuangGang China Wuhan and XiaoGan cities of China administrative regions in Italy Spain France and Germany Milan Italy Milan Italy provinces of Lombardy Italy provinces of Piedmont Italy Coccia 2020b Data up to April 7th Italian Provinces Fattorini and Regoli Data up to April 27th Italian provinces PM25 A 10μgm3 PM25 increase lag0“ was associated with a increase of daily confirmed new cases PM10 A 10μgm3 PM10 increase lag0“ was associated with a increase of daily confirmed new cases Wuhan RR CI1032“ XiaoGan RR CI “ HuangGang RR CI “ Wuhan R2 p XiaoGan R2 p Wuhan RR CI “ XiaoGan RR CI “ HuangGang RR CI “ Wuhan R2 p XiaoGan R2 p χ2 p A μgm3 increase in PM25 was associated with a “ increase in fatality rate χ2 p A μgm3 increase in PM10 was associated with a “ increase in fatality rate NO2 A 10μgm3 NO2 increase lag0“ was associated with a increase in daily confirmed new cases Wuhan RR CI “ XiaoGan RR CI “ HuangGang no association found Wuhan R2 p XiaoGan R2 p of fatality cases are associated with NO2 μmolm2 R cid0 R R cid0 R cid0 R R cid0 R cid0 R cid0 R cid0 Lombardy PM10 exceeding between and COVID19 incidence between and Piedmont PM10 exceeding between and COVID19 incidence between and COVID19 in North Italy has a high association with air pollution of cities measured with days exceeding the limits set for PM10 R2 p R2 p continued on next page Hierarchical multiple regression model Pearson regression coefficient analysis R2 p Spatial association between confirmed cases and air pollution PM10 Spatial association between total confirmed cases and air pollution PM25 PM10 and NO2 EnvironmentalResearch19120201101294 0cC Copat Table continued References Frontera Frontera Wu Adhikari and Yin Bashir Bashir VasquezApestegui VasquezApestegui VasquezApestegui Period Data up to 31st March Data up to 31st March Data up to April 04th From March 1st to Apr 20th From March 4th to April 24th From March 4th to April 24th Data up to June 12th Data up to June 12th Data up to June 12th Area of Study Italian regions Italian regions counties in the USA Queens county New York USA California California districts of Lima Perù districts of Lima Perù districts of Lima Perù Aim Spatial association between total confirmed cases and air pollution PM25 Spatial association between deaths and air pollution PM25 Prediction of risk of COVID19 deaths in the long term average exposure to fine particulate matter PM25 Temporal association between daily confirmed cases and total deaths and air pollution PM25 Association between confirmed cases and air pollution PM25 PM10 and NO2 Association between deaths and air pollution PM25 PM10 and NO2 Spatial association between total confirmed cases and air pollution PM25 Spatial association between deaths and air pollution PM25 Spatial association between case fatality rate and air pollution PM25 Data analysis Pearson regression coefficient analysis PM25 R2 p PM10 Pearson regression coefficient analysis R2 p Longterm exposure increase of μgm3 in PM25 is associated with a increase in the COVID19 death rate Estimate on cases values cid0 CI “ Estimate on deaths value cid0 CI “ Kendall r cid0 Spearman r cid0 Zeroinflated negative binomia models Negative binomial regression model Spearman and Kendall correlation tests Spearman and Kendall correlation tests NO2 Kendall r cid0 Spearman r cid0 Kendall r cid0 Spearman r cid0 Kendall r cid0 Spearman r cid0 Kendall r cid0 Spearman r cid0 Kendall r cid0 Spearman r cid0 Multivariate regression model Crude coefficient p Multivariate regression model Crude coefficient p Multivariate regression model Crude coefficient cid0 p mortality was found significantly higher than less polluted Italian regions By collecting data up to March 31st for all Italian regions and performing a Pearson correlation analysis they found a strong positive association both with the total number of confirmed cases R and deaths R other than with hospitalized cases R The Italian situation was further highlighted by the study of Fattorini and Regoli in Italian provinces They explored the spatial association between air pollution and COVID19 cases with data up to April 27th By applying the Pearson regression coefficient analysis they revealed a positive association both with PM25 and PM10 R2 p and R2 p respectively A focus on the most affected city of Italy Milan was conducted by Zoran 2020a This city is located in the Po valley basin known hotspot for atmospheric pollution at the continental scale EEA The Authors performed a temporal association between COVID19 Total cases Daily New positive cases and Total Deaths and particulate matter from Jan 1st and Apr 30th by applying a Person correlation In accordance with other studied they found a positive association between daily confirmed cases and PM25 R and PM10 R although they did not consider any delay time from infection to COVID19 onset Nevertheless they found a negative association between total cases and total deaths and particulate matter but the assumption of a temporal linear correlation may be inaccurate because the above mentioned variables could have more complex nonlinear relationships To date the USA have more than million confirmed cases and thousand deaths WHO Here ambient concentrations of PM and O3 were found responsible to cause between and premature deaths Fann The association between air pollutants and COVID19 cases and deaths was studied by Bashir in the state of California from March 4th to April 24th corresponding to the beginning of the COVID19 outbreak in USA Based on their significant correlation found the Authors state that a limited human exposure to these pollutants will contribute to defeating COVID19 This seems unclear because they found a negative correlation with PM25 and PM10 EnvironmentalResearch19120201101295 0cC Copat by applying both the Kendall rank correlation and Spearman™s one and it is not clear if they normalized COVID19 cases by population size and if they performed a day by day association or a spatial association across the country A focus on the Queen county New York USA was provided by Adhikari and Yin They retrieved data of PM daily concentrations from two ground monitoring stations and collected data of confirmed COVID19 cases and numbers of related deaths from USAFacts in the period from March to April The Authors elaborated their data with a negative binomial regression model and considered the cumulative lag effect of PM25 on disease outcomes over the past days They found a significant negative association among PM25 and new daily confirmed COVID19 cases cid0 CI “ and deaths cid0 CI “ Low PM concentrations in this area of study mean μgm3 are likely to have played a less central role in the spread of infection than in other areas such as Italy where PM25 monthly concentrations reached values higher than μgm3 Fattorini and Regoli Frontera or in China where PM25 monthly concentrations reached values higher than μgm3 Zhu Jiang As said by the Authors other gaseous pollutants such as NO2 and SO2 could have influenced transmission and pathogenesis of COVID19 In the United States Wu investigated whether longterm average exposure to fine particulate matter PM25 increases the risk of COVID19 deaths by considering approximately counties in the United States of the population With an exposure prediction model the Authors calculated the county level longterm exposure to PM25 averaged for to and collected COVID19 deaths counts up to April 04th They conducted a strong and robust statistical analysis with zeroinflated negative binomial mixed models adjusting their results by several potential confounders such as sociodemographic socioeconomic behavioural and meteorological factors They found that a small longterm exposure increase of only μgm3 in PM25 is associated with a increase in the COVID19 death rate confidence interval CI VasquezApestegui recently reported first evidences on the spatial relationship between particulate matter and COVID19 outbreak from Latin America The Authors described the situation occurred in districts of Lima located in the second most affected country of Latin America Peru In particular by applying a multivariate regression model they evaluated the association between the population exposure to PM25 concentrations in the previous years “ and cases deaths and casefatality rates of COVID19 with data up to June 12th A significant association has been found both with cases and deaths Crude coefficient with p and with p respectively but not with case fatality rate All these studies highlight the role of PM in triggers of the COVID19 disease and how government measures targeting to sustainable growth such as the reduction of urban and industrial emissions could have a positive impact on the prevention of health outcomes reducing mortality rate as well the burden on health care systems Nitrogen dioxide NO2 and COVID19 induced lung damage Hence viral infection becomes more common after exposure to NO2 Zhu Furthermore NO2 is associated with other several health effects such as elevated risks for asthma allergic rhinitis and eczema in children To increase of outpatient visits and hospitalizations due to bronchitis and asthma exacerbation Bahrami Asl Kowalska increase of chronic obstructive pulmonary disease COPD Ghanbari Ghozikali Pfeffer and increase of pulmonary heart disease related mortality Chen A recent study explored the possible role of NO2 in interference in Angiotensin converting enzyme ACE2 The expression of ACE2 is high on lung alveolar epithelial cells and it is the human cell receptor of virus agent of COVID19 Alifano First observations report an association between ambient concentrations of NO2 and COVID19 pandemic across Europe China and USA Bashir Fattorini and Regoli Jiang Li et al Ogen Zhu et al Zoran et al 2020b Conversely to the other papers findings of Zoran 2020b and Bashir provides different findings reporting no association or a negative one between NO2 and daily deaths counts In China Zhu by applying the same method explained for PM observed that a 10μgm3 increase lag0“ in NO2 is associated with a CI “ increase in the daily counts of COVID19 confirmed cases in cities of China These findings are confirmed by Jiang and Li et a who applied the same method described for PM Jiang revealed a significant positive association between NO2 and COVID19 both in Wuhan and XiaoGan Wuhan RR CI1053“ XiaoGan RR CI “ but did not found any significant association in HuangGang Li found a significant linear correlation both in Wuhan R2 p and XiaoGan R2 p Ogen presented evidences on the relationship between exposure to NO2 including the months of January and February shortly before the COVID19 spread in Europe and novel coronavirus fatality in the most affected European countries concluding that longterm exposure to NO2 may be a potential contributor to mortality caused by SARSCoV2 He collected data concerning the number of fatality cases from administrative regions in Italy Spain France and Germany and correlated mortality with tropospheric NO2 concentrations measured by the Sentinel5 Precursor spaceborne satellite The major tropospheric NO2 hotspot identified was located in the Northern Italy In all European regions considered gas concentrations ranged between and μmolm2 with airflows directed downwards Results showed that out of the fatality cases by March were in five regions located in north Italy and central Spain Furthermore by analysing mortality trends it was revealed that the highest percentage of deaths occurred in regions where the maximum NO2 concentration was above μmolm2 with a significant decrease where the maximum concentration was between and μmolm2 and below μmolm2 The methodology used by Ogen cannot support a longterm exposure investigation Surely a validation of the satellite measure with those of the ground ™ones the adjustment of the results according to the different population size of each country could have made their results more robust Nevertheless the study provide new insights for future investigation The Italian situation was further studied by Fattorini and Regoli who collected data of COVID19 incidence up to April 27th from Italian provinces They revealed a strong spatial corr Answer:

Thyroid_Cancer

" formulated a traditional Chinese medicine TCM prescription Hanshiyi Formula HSYF which was approved and promoted by the Wuhan Municipal Health Commission for treating mild and moderate coronavirus disease COVID19 We aimed to evaluate the effect of HSYF on the progression to severe disease in mild and moderate COVID19 patients We conducted a retrospective cohort study of patients with mild and moderate COVID19 in a quarantine station in Wuchang District Wuhan Using the realtime Internet information collection application and Centers for Disease Control for the Wuchang District patient data were collected through patient selfreports and followups HSYF intervention was defined as the exposure The primary outcome was the proportion of patients who progressed to a severe disease status and a stratification analysis was performed Univariate and multivariate regression analyses were performed to identify influencing factors that may affect the outcome Further we used pr sity score matching PSM to assess the effect of HSYF intervention on the conversion of mild and moderate to a severe disease status Totally mild and moderate COVID19 patients were enrolled including HSYF users exposed group and nonusers control group No cases in the exposed group and P cases in the control group progressed to severe disease and the difference between the two groups exposed groupcontrol group was cid0 [ confidence interval CI cid0 cid0 ] Univariate regression analysis revealed sex male age fever cough and fatigue as risk factors for progression to severe disease After PSM none of the HSYF users and P non users transitioned to severe disease and the difference between the two groups exposed groupcontrol group was cid0 [ CI cid0 cid0 ] Multivariate regression analysis revealed that sex male [OR CI “ P ] and age years [odds ratio OR CI “ P ] were independent risk factors for conversion to severe disease Therefore HSYF can significantly reduce the progression to severe disease in patients with mild and moderate COVID19 which may effectively prevent and treat the disease However further larger clinical studies are required to verify our results Corresponding authors Email addresses tina_yai126com J Tian yanshiyan0927sinacom S Yan wanghan4313163com H Wang novelzhangsinacom Y Zhang luoyuorz163com Y Zheng drwhrfoxmailcom H Wu leexiuyang126com X Li zhengzhigaozezheng163com Z Gao aiyanke163com Y Ai christiana55555163com X Gou tcmxpzl126com L Zhang hely3699163com L He lfm565sohucom F Lian 13601180524139com B Liu tongxiaolinvip163com X Tong Jiaxing Tian Shiyan Yan Han Wang and Ying Zhang contributed equally to this study 101016jphrs2020105127 Received April Received in revised form August Accepted August PharmacologicalResearch1612020105127Availableonline10August2020104366182020ElsevierLtdAllrightsreserved 0cJ Tian Introduction At the end of December a series of coronavirus disease COVID19 cases emerged in Wuhan Hubei Province China and the number of diagnosed cases have rapidly increased since then [] In the early stage of the epidemic a large number of patients with mild and moderate COVID19 were diagnosed and treated in quarantine stations Identifying effective treatments for early intervention can improve the prognosis of patients with mild and moderate COVID19 which can also help control the epidemic progression and reduce the medical burden Wuchang District is located at the center of Wuhan with a high population density It was one of the most severely affected areas with many confirmed COVID19 cases For a large number of patients with mild and moderate COVID19 in the quarantine station the Wuhan Municipal Health Commission adopted a combination of Chinese herbal medicines and used Internet data to tackle the pandemic Our study group formulated a traditional Chinese medicine TCM prescription named Hanshiyi Formula HSYF to prevent the exacerbation of the epidemic This was approved and promoted by the Wuhan Municipal Health Commission for the treatment of mild and moderate COVID19 patients in quarantine stations [] Data was collected from the realtime Internet information collection application Yuge medical system which was convenient for physicians to communicate with isolated patients in real time dynamically monitor changes in the patient™s condition observe the efficacy of HSYF in mild and moderate COVID19 patients and adjust the treatment plan if necessary In guiding the treatment of mild and moderate COVID19 patients we found that after HSYF intervention patients with mild and moderate disease experienced rapid resolution of symptoms and fewer patients transitioned to a severe disease status Therefore we compared the clinical data of mild and moderate COVID19 patients treated with HSYF with patients who were diagnosed but were not treated with HSYF Data were collected from the database of Wuhan Wuchang District Disease Control Center CDC as well as from the realtime Internet information collection application to evaluate the effect of HSYF on the proportion of COVID19 patients who progressed from a mild and moderate to severe disease status Methods Study design and participants In this retrospective cohort study conducted in the Wuchang District Wuhan data were collected from mild and moderate COVID19 patients in quarantine stations in Wuchang District Wuhan before March The exposed group included patients who were administered HSYF for more than days If there is no adverse effect or disease progression HSYF can be taken continuously until recovery The control group included patients who were diagnosed with COVID19 but were not administered HSYF including decoction granules etc at the same time Data on age sex disease duration medical history initial symptoms concomitant medication and outcomes of the two groups were collected and the difference in the proportion of mild to moderate COVID19 patients who transitioned to a severe status were analyzed Fig The diagnosis of COVID19 patients was made by the designated tertiary hospital in Wuchang District Wuhan The diagnostic criteria was according to the Diagnosis and Treatment Guideline for COVID19 released by the National Health Commission of the People™s Republic of China [] The study included patients who met the criteria of mild and moderate COVID19 the mild COVID19 patients usually presented mild clinical symptoms with no radiological manifestations of pneumonia the moderate COVID19 patients had fever and respiratory symptoms with radiological manifestations of pneumonia Severe and critical patients were excluded This study was approved by the institutional ethics board of Hubei Provincial Hospital of Traditional Chinese Medicine No HBZY2020 C0101 and was registered with chictrcn ChiCTR2000029601 Data collection Data from the exposure group was first provided by the patients who scanned the QR code on the medication box and entered data on the real time Internet information collection application After downloading and sorting the data if there were mistakes or lack of relevant information a medical staff member followed up the patients by telephone and if necessary contacted the staff of the community health service station to verify the information to ensure accuracy and completeness of the data Data from the control group were provided by the Wuhan Wuchang District CDC database Data of mild and moderate COVID19 patients diagnosed at the same time were screened and downloaded and a telephone followup was conducted by a medical staff member to ensure accuracy and completeness of the data All data were collected using a standard electronic database In order to ensure the accuracy of the data and avoid bias data were verified by two researches A and B and a third researcher C resolved any dispute between the first two researchers The results of the study were analyzed and reported in accordance with the STROBE guidelines Fig Flow chart of the observational research PharmacologicalResearch16120201051272 0cJ Tian Table Demographic and Patient Characteristics All patients Exposed group Control group Age Mean ± SD 014yr 1549yr 5064yr ‰¥65yr ‰¤48yr ＞48yr Sex Female Medical history Medical history Hypertension Coronary heart disease Diabetes Bronchial asthma Chronic obstructive pulmonary disease Hyperlipidemia Fatty liver Gallbladder disease Thyroid disease Stroke Chronic glomerulonephritis Cancer Hepatitis Tuberculosis Other diseases Initial symptoms Initial symptoms Fever Cough Diarrhea Fatigue Conjunctivitis Other symptoms Concomitant medication Antiviral treatment Oseltamivir Lopinavir Aciclovir Ribavirin Arbidol Other antiviral drugs Antibiotics Antibiotics Amoxicillin Cephalosporin Levofloxacin Moxifloxacin Clarithromycin Other antibiotics Chinese patent medicine Chinese patent medicine Lianhua Qingwen capsule Xiaochaihu granule Shuanghuanglian oral solution Huoxiang Zhengqi preparation Cough syrup Banlangen preparation Ganmao Qingre granule Other Chinese patent medicines ± ± ± Statistics Z10789 X210724 X2 X20817 X21796 X23145 X21788 X21023 X23933 X2＜ X2 X20318 X20186 X24649 X22578 X20812 X22638 X2＜ X25259 X20567 X2 X2 X2 X2 X2 X21049 X2 X20091 X27352 X2 X2 X2 X20250 X2 X21648 X20218 X27552 X210734 X20257 X22752 X27191 X23935 X24895 X2 X211160 X210579 X2 X27812 Pvalue ＜ ＜ ＜ ＜ ＜ ＜ ＜ ＜ ＜ ＜ ＜ ＜ ＜ ＜ ＜ ＜ Outcomes Statistical analysis The primary outcome of this study was the proportion of mild and moderate COVID19 patients who progressed to a severe disease status We also analysed factors that may affect the outcome including the patient™s age sex disease duration medical history initial symptoms concomitant medication and grouping Pr sity score matching PSM was used to further assess the effect of HSYF intervention on the conversion from mild and moderate COVID19 to a severe status Statistical analyses were conducted using SPSS software SPSS Inc Chicago IL United States Twosided tests were used and Pvalues were considered statistically significant Numerical variables are summarized as mean ±SD The data of the categorical variables are described as numbers and percentages The ttestWilcoxon ranksum test was used to compare the ages between the two groups The chisquare testFisher™s exact test was used to compare the sex medical history initial symptoms and concomitant PharmacologicalResearch16120201051273 0cJ Tian medication between the two groups to calculate the rate difference and confidence interval CI Using the proportion of patients who transitioned to a severe status as the dependent variable the effects of age sex disease duration medical history initial symptoms concomitant antiviral drugs antibiotics Chinese patent medicines and grouping were analyzed by univariate and multivariate logistic regression For factors with a statistical significance in the univariate regression analysis a logistic regression was performed to match the two groups of patients by pr sity score in a ratio and the difference in the primary outcome between the two groups was evaluated Results Demographic and patient characteristics By March we enrolled mild and moderate COVID19 patients from quarantine stations in the Wuchang District After further screening patients who refused followup patients who were not diagnosed and two patients who took other types of TCM prescriptions were excluded Finally patients were included in our study cohort including HSYF users and nonusers including decoction granules etcAmong all patients enrolled in this study were men and were women The median age of the patients was years old The median age of the exposed group was significantly lower than that of the control group P We found that of patients had a medical history which included hypertension fatty liver and diabetes A significantly higher proportion of patients in the exposed group had a history of bronchial asthma vs P and thyroid disease vs P compared with the control group while the proportion of patients with hyperlipidemia vs P was higher in the control group than the exposed group Among all patients had experienced initial COVID19 symptoms The number of patients with diarrhea in the exposed group was significantly higher than that in the control group vs respectively P and the number of patients with fever vs P cough vs P and fatigue vs P was significantly lower in the exposed group than in the control group The patients were administered multiple medications during the treatment There were no differences in the number of patients receiving antiviral drugs antibiotics and Chinese patent medicines between the two groups In terms of antiviral treatment ribavirin vs P and arbidol vs P were the most commonly administered medications in the exposed group while oseltamivir vs P and acyclovir vs P were the most commonly administered medication in the control group In terms of antibiotics amoxicillin vs P and moxifloxacin vs P were most commonly used in the exposed group while clarithromycin vs P were most commonly used in the control group In terms of Chinese patent medicines the patients in the exposed group were usually administered Xiaochaihu granules vs P Shuanghuanglian oral solution vs P cough syrup vs P Banlangen preparation vs P and Ganmao Qingre granules vs P while the patients in the control group were administered Lianhua Qingwen capsule vs P and Huoxiang Zhengqi preparation vs P as shown in Table The majority of onset times in the two groups were from January to February vs The proportion to severe status Our primary outcome was the proportion of COVID19 patients who progressed from mild and moderate COVID19 to a severe disease status There were no cases of progression to severe disease in the exposed group while cases P in the control group progressed to severe disease The difference between the two groups in terms of progression to severe disease exposed groupcontrol group was cid0 [ CI cid0 cid0 ] Stratification analysis showed that excluding diarrhea there were significant differences between the two groups in terms of sex age years medical history initial symptoms fever cough diarrhea fatigue concomitant medication etc P Table Efficacy evaluation The proportion to severe status The proportion to severe status Sex Male Female Age Age‰¤48yr Age＞48yr Underlying disease Yes No Initial symptoms Yes No Fever Yes No Cough Yes No Diarrhea Yes No Fatigue Yes No Chinese patent medicine Yes No All patients Exposed group Control group Statistics X2 X2 X28004 X29521 X2 X29251 X2 X2 X2 X27842 X26575 X211744 X2 X21915 X2 X2 X28085 Pvalue ＜ ＜ ＜ ＜ ＜ ＜ ＜ ＜ ＜ PharmacologicalResearch16120201051274 0cFactors OR95 CI Univariate analysis Pvalue Table Univariate and multivariate regression analysis J Tian therefore these factors were accounted for in the subsequent analysis Table The univariate logistic regression analysis showed that sex male age years fever cough and fatigue were risk factors for progression to a severe status We further performed a multivariate logistic regression analysis to adjust for the above risk factors The results showed that after adjusting for all factors sex male OR CI “ P and age years OR CI “ P were independent risk factors for progression to a severe disease status Table Considering the difference in sample size between the two groups and the imbalance in confounding factors the risk factors analyzed in the univariate logistic regression analysis were used as variables for PSM caliper 025σ and the number of patients in both groups were Among them no patients in the exposed group progressed to severe disease and seven patients in the control group progressed to severe disease P The difference between the two groups in terms of progression to severe disease exposed groupcontrol group was cid0 [ CI cid0 cid0 ] Table Discussion Age The global prevalence of COVID19 has brought tremendous medical pressure worldwide According to data from Johns Hopkins University in the United States by April the number of COVID19 cases exceeded million globally Furthermore the number of diagnosed cases in the United States were over and that in Italy and Spain were over with the number of newly diagnosed cases still rising [] With a large number of people in close contact with mildmoderate COVID19 patients all countries have adopted community control and home quarantine measures to prevent the spread of the epidemic However during home quarantine problems such as crossinfection and cluster outbreaks in the community have emerged due to the lack of strict community supervision as well as the need to purchase necessities and travel for hospital consultations In addition mild and moderate COVID19 patients who were under home quarantine lacked effective intervention strategies without the guidance from physicians which may have resulted in problems such as the progression from mild and moderate to severe disease [] In such patients an effective largescale community intervention strategy could reduce this conversion to severe disease and reduce the number of severe and critical COVID19 patients thus significantly improving and controlling the COVID19 epidemic For mild and moderate COVID19 patients antiviral antibiotic and symptomatic supportive treatments are most commonly used However most of the antiviral drugs used to treat COVID19 currently are based on previous treatments for severe acute respiratory syndrome SARS Middle East respiratory syndrome and influenza A and uncertainties on the efficacy and side effects of these drugs remain problematic One case report stated that a patient was cured with remdesivir [] In another clinical study lopinavirritonavir had no clinical benefit on the treatment of patients with severe COVID19 and adverse gastrointestinal events were identified [] Furthermore Abidol and lopinavirritonavir did not improve COVID19 symptoms or shorten the conversion of the viral nucleic acid to a negative result in a clinical study of patients [] The adverse reactions and side effects of related antiviral drugs such as hypocalcemia hemolytic anemia hypomagnesemia as well as those that emerged during the treatment of SARS by ribavirin cannot be ignored [] Hydroxychloroquine and chloroquine were urgently approved by the Food and Drug Administration to treat COVID19 but chloroquine drugs hydroxychloroquine and chloroquine phosphate can cause adverse reactions such as dizziness headache vertigo loss of appetite and nausea [] Our study showed that under the conditions of home quarantine TCM is effective in reducing the progression of mild and moderate COVID19 to severe disease which can serve as a reference for the Multivariate analysis Pvalue ＜ ＜ OR95 CI continued on next page SexRef female Medical history Ref no Fever Cough Diarrhea Fatigue PharmacologicalResearch16120201051275 0cJ Tian Table continued Factors Univariate analysis Pvalue OR95 CI Multivariate analysis Pvalue Antiviral treatment Ref no Antibiotics Ref no Chinese patent medicine Ref no Grouping Ref control group OR95 CI \u3000 The model includes age and sex The model includes age gender and medical history The model includes age sex medical history and initial symptoms The model includes age gender medical history initial symptoms and antiviral drugs The model includes age gender medical history initial symptoms antiviral drugs and antibiotics The model includes age gender medical history initial symptoms antiviral drugs antibiotics and Chinese patent medicine The model includes age gender medical history initial symptoms antiviral drugs antibiotics Chinese patent medicine and grouping prevention of the disease As a key point of COVID19 prognosis there have only been a few studies assessing the proportion of mild and moderate COVID19 patients who have progressed to a severe disease status currently In attempting to prevent and control the epidemic in China TCM has been widely used Some studies revealed that TCM can reduce the clinical symptoms of fever and cough in mild and moderate patients [] and improve relevant imaging indexes [] However these studies usually had small sample sizes and did not assess the proportion of mild and moderate patients who progressed to a severe disease status In this retrospective cohort study the results showed that the main influencing factors for the progression of mild and moderate COVID19 to severe disease included sex male age fever cough and fatigue which are consistent with other studies [“] After excluding the effects of the above factors we found that the HSYF intervention can effectively reduce the conversion of mild and moderate COVID19 to severe disease providing evidence on the effectiveness of TCM Thus TCM can be an option in addition to antibiotic therapy antiviral treatment and supportive oxygen therapy for the treatment of mild and moderate COVID19 patients HSYF administered in the study mainly contains the following Chinese materia medica Ma Huang Ephedrae Herba Shi Gao Gypsum fibrosum Xing Ren Armeniacae Semen Qiang Huo Notopterygii Rhizoma seu Radix Ting Li Zi LepidiiDescurainiae Semen Guan Zhong Cyrtomii Rhizoma Di Long Pheretima Xu Chang Qing Cynanchi paniculati Radix Huo Xiang Pogostemonis Herba Pei Lan Eupatorii Herba Cang Zhu Atractylodis Rhizoma Yun Ling Poria Sheng Bai Zhu Atractylodis macrocephalae Rhizoma Jiao Shan Zha Crataegi Fructus Jiao Shen Qu Massa medicate fermentata Jiao Mai Ya Hordei Fructus germinatus Hou Po Magnoliae officinalis Cortex Jiao Bing Lang Arecae Semen Wei Cao Guo Tsaoko Fructus and Sheng Jiang Zingiberis Rhizoma recens In TCM theory these prescriptions can improve the patients™ healthy qi and dispel evil factors Ma Huang Ephedrae Herba Shi Gao Gypsum fibrosum Xing Ren Armeniacae Semen Ting Li Zi LepidiiDescurainiae Semen focus on improving asthma symptoms in the lung Yun Ling Poria Sheng Bai Zhu Atractylodis macrocephalae Rhizoma Jiao Shan Zha Crataegi Fructus Jiao Shen Qu Massa medicate fermentata Jiao Mai Ya Hordei Fructus germinatus Hou Po Magnoliae officinalis Cortex Jiao Bing Lang Arecae Semen and Wei Cao Guo Tsaoko Fructus help strengthen the healthy qi Huo Xiang Pogostemonis Herba and Pei Lan Eupatorii Herba can dispel the evil factors Studies have shown that many Chinese components of HSYF can exert antiviral effects and improve respiratory symptoms In terms of the antiviral components methyl ephedrine Lephedrine and Dpseudoephedrine are present in Ma Huang Ephedrae Herba which can significantly inhibit the in vitro proliferation of influenza A H1N1 virus [] Patchouli alcohol in Huo Xiang Pogostemonis Herba can effectively inhibit the replication of H1N1 virus [] Hou Po Magnoliae officinalis Cortex can alter the cell cycle and promote H1N1 infected cells into the S phase [] the magnolol extract can inhibit the secretion of CD44 and CD54 reduce the levels of inflammatory factors IL1β IL6 and TNFα and relieve inflammation [] The active ingredients of Wei Cao Guo Tsaoko Fructus inhibit the binding region of severe acute respiratory syndrome coronavirus SARSCoV2 Sprotein to human ACE2 and control the replication of SARSCoV2 in the human body [] In addition to its antiviral effect Ma Huang Ephedrae Herba is a widely used Chinese herbal medicine for respiratory diseases and it also stimulates β receptors of the bronchial smooth muscle expands the bronchus exerts antiinflammatory effects [] and can stimulate central nervous system excitability and regulate body temperature [] This plays an important role in improving the symptoms of COVID19 including fever cough shortness of breath and other respiratory symptoms The clinical benefits and risks need to be balanced for the treatment of mild and moderate COVID19 The lack of understanding on the efficacy side effects and other risks associated with current antiviral treatments make it difficult to be used as a conventional intervention for mild and Table Pr sity score matching PSM Variables Mean ± SD MedianQ1Q3 Age SexRef female Fever Cough Fatigue Before PSM Exposed group n ± Control group n ± Pvalue ＜ ＜ ＜ ＜ After PSM Exposed group n ± Control group n ± Pvalue PharmacologicalResearch16120201051276 0cAppendix A Supplementary data J Tian moderate COVID19 patients However the results of our study provide evidence on the effectiveness of TCM for the treatment of mild and moderate COVID19 TCM treatment has the advantage of being widely used and inexpensive making it convenient for mild and moderate COVID19 patients isolated in the largescale community to quickly recover Furthermore our study used a realtime Internet information collection application to collect data which enabled physicians to communicate with the patients in real time and dynamically monitor changes in the patient™s condition Thus selfquarantined patients did not need to go to the hospital for a consultation which prevented cross infection Although the results of our study are significant and provides insight on the use of TCM treatment for mild and moderate COVID19 there are still some limitations Firstly our study was a retrospective study rather than a prospective randomized clinical trial RCT due to the current situation In the future we aim to conduct RCTs and experiments to further verify the efficacy and mechanism of HSYF Secondly as a retrospective cohort study some baseline factors between the exposed group and the control group were not completely accounted for and the age of the exposed group was significantly younger than that of the control group which may be related to our data collection method Using a realtime Internet information collection application is a more acceptable method in the younger population and imbalances in baseline data such as sex age and medical history could have introduced bias which may have affected the results of the study Nevertheless we have corrected these possible influencing factors during our analysis In order to improve the reliability of the study results a cohort study with a larger sample size or RCTs is necessary Conclusion [] National Health Commission of the People™s Republic of China National HSYF can significantly reduce the progression of mild and moderate COVID19 to a severe disease status which has a positive effect on the prevention and treatment of the disease However future clinical studies with a larger sample size are needed to further verify our results Funding This work was funded by the Special Project for Emergency of the Ministry of Science and Technology 2020YFC0845000 and the Traditional Chinese Medicine Special Project for COVID19 Emergency of National Administration of Traditional Chinese Medicine2020ZYLCYJ041 Declaration of Competing Interest Authors declare no competing interests Jiangsu Kanion Pharmaceutical Co Ltd provided the medications for the study while they did not participate in research design data collection data analysis data interpretation nor article writing Acknowledgements We acknowledge all volunteers and healthcare workers in the Wuchang community health service station Hubei Provincial Hospital of Traditional Chinese Medicine and Jiangsu Kanion Pharmaceutical Co Ltd Supplementary material related to this article can be found in the online version at 101016jphrs2020105127 References [] H Lu CW Stratton YW Tang Outbreak of pneumonia of unknown etiology in Wuhan China the mystery and the miracle J Med Virol “ [] Medical treatment group of Wuhan COVID prevention and control headquarters Notice Concerning the Recommendation of the Prescription of Traditional Chinese Medicine in the Treatment of COVID19 February [] XL Tong XY Li LH Zhao Discussion on traditional chinese medicine prevention and treatment strategies of coronavirus disease COVID19 from the perspective of œColddampness pestilence J Tradit Chin Med “ Administration of Traditional Chinese Medicine of the People™s Republic of China Guidance for Corona Virus Disease Prevention Control Diagnosis and Management People™s Medical Publishing House March [] World health anization Coronavirus disease COVID19 Situation dashboard whosprinklrcom [] A Kimball KM Hatfield M Arons

cancer239

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: " formulated a traditional Chinese medicine TCM prescription Hanshiyi Formula HSYF which was approved and promoted by the Wuhan Municipal Health Commission for treating mild and moderate coronavirus disease COVID19 We aimed to evaluate the effect of HSYF on the progression to severe disease in mild and moderate COVID19 patients We conducted a retrospective cohort study of patients with mild and moderate COVID19 in a quarantine station in Wuchang District Wuhan Using the realtime Internet information collection application and Centers for Disease Control for the Wuchang District patient data were collected through patient selfreports and followups HSYF intervention was defined as the exposure The primary outcome was the proportion of patients who progressed to a severe disease status and a stratification analysis was performed Univariate and multivariate regression analyses were performed to identify influencing factors that may affect the outcome Further we used pr sity score matching PSM to assess the effect of HSYF intervention on the conversion of mild and moderate to a severe disease status Totally mild and moderate COVID19 patients were enrolled including HSYF users exposed group and nonusers control group No cases in the exposed group and P cases in the control group progressed to severe disease and the difference between the two groups exposed groupcontrol group was cid0 [ confidence interval CI cid0 cid0 ] Univariate regression analysis revealed sex male age fever cough and fatigue as risk factors for progression to severe disease After PSM none of the HSYF users and P non users transitioned to severe disease and the difference between the two groups exposed groupcontrol group was cid0 [ CI cid0 cid0 ] Multivariate regression analysis revealed that sex male [OR CI “ P ] and age years [odds ratio OR CI “ P ] were independent risk factors for conversion to severe disease Therefore HSYF can significantly reduce the progression to severe disease in patients with mild and moderate COVID19 which may effectively prevent and treat the disease However further larger clinical studies are required to verify our results Corresponding authors Email addresses tina_yai126com J Tian yanshiyan0927sinacom S Yan wanghan4313163com H Wang novelzhangsinacom Y Zhang luoyuorz163com Y Zheng drwhrfoxmailcom H Wu leexiuyang126com X Li zhengzhigaozezheng163com Z Gao aiyanke163com Y Ai christiana55555163com X Gou tcmxpzl126com L Zhang hely3699163com L He lfm565sohucom F Lian 13601180524139com B Liu tongxiaolinvip163com X Tong Jiaxing Tian Shiyan Yan Han Wang and Ying Zhang contributed equally to this study 101016jphrs2020105127 Received April Received in revised form August Accepted August PharmacologicalResearch1612020105127Availableonline10August2020104366182020ElsevierLtdAllrightsreserved 0cJ Tian Introduction At the end of December a series of coronavirus disease COVID19 cases emerged in Wuhan Hubei Province China and the number of diagnosed cases have rapidly increased since then [] In the early stage of the epidemic a large number of patients with mild and moderate COVID19 were diagnosed and treated in quarantine stations Identifying effective treatments for early intervention can improve the prognosis of patients with mild and moderate COVID19 which can also help control the epidemic progression and reduce the medical burden Wuchang District is located at the center of Wuhan with a high population density It was one of the most severely affected areas with many confirmed COVID19 cases For a large number of patients with mild and moderate COVID19 in the quarantine station the Wuhan Municipal Health Commission adopted a combination of Chinese herbal medicines and used Internet data to tackle the pandemic Our study group formulated a traditional Chinese medicine TCM prescription named Hanshiyi Formula HSYF to prevent the exacerbation of the epidemic This was approved and promoted by the Wuhan Municipal Health Commission for the treatment of mild and moderate COVID19 patients in quarantine stations [] Data was collected from the realtime Internet information collection application Yuge medical system which was convenient for physicians to communicate with isolated patients in real time dynamically monitor changes in the patient™s condition observe the efficacy of HSYF in mild and moderate COVID19 patients and adjust the treatment plan if necessary In guiding the treatment of mild and moderate COVID19 patients we found that after HSYF intervention patients with mild and moderate disease experienced rapid resolution of symptoms and fewer patients transitioned to a severe disease status Therefore we compared the clinical data of mild and moderate COVID19 patients treated with HSYF with patients who were diagnosed but were not treated with HSYF Data were collected from the database of Wuhan Wuchang District Disease Control Center CDC as well as from the realtime Internet information collection application to evaluate the effect of HSYF on the proportion of COVID19 patients who progressed from a mild and moderate to severe disease status Methods Study design and participants In this retrospective cohort study conducted in the Wuchang District Wuhan data were collected from mild and moderate COVID19 patients in quarantine stations in Wuchang District Wuhan before March The exposed group included patients who were administered HSYF for more than days If there is no adverse effect or disease progression HSYF can be taken continuously until recovery The control group included patients who were diagnosed with COVID19 but were not administered HSYF including decoction granules etc at the same time Data on age sex disease duration medical history initial symptoms concomitant medication and outcomes of the two groups were collected and the difference in the proportion of mild to moderate COVID19 patients who transitioned to a severe status were analyzed Fig The diagnosis of COVID19 patients was made by the designated tertiary hospital in Wuchang District Wuhan The diagnostic criteria was according to the Diagnosis and Treatment Guideline for COVID19 released by the National Health Commission of the People™s Republic of China [] The study included patients who met the criteria of mild and moderate COVID19 the mild COVID19 patients usually presented mild clinical symptoms with no radiological manifestations of pneumonia the moderate COVID19 patients had fever and respiratory symptoms with radiological manifestations of pneumonia Severe and critical patients were excluded This study was approved by the institutional ethics board of Hubei Provincial Hospital of Traditional Chinese Medicine No HBZY2020 C0101 and was registered with chictrcn ChiCTR2000029601 Data collection Data from the exposure group was first provided by the patients who scanned the QR code on the medication box and entered data on the real time Internet information collection application After downloading and sorting the data if there were mistakes or lack of relevant information a medical staff member followed up the patients by telephone and if necessary contacted the staff of the community health service station to verify the information to ensure accuracy and completeness of the data Data from the control group were provided by the Wuhan Wuchang District CDC database Data of mild and moderate COVID19 patients diagnosed at the same time were screened and downloaded and a telephone followup was conducted by a medical staff member to ensure accuracy and completeness of the data All data were collected using a standard electronic database In order to ensure the accuracy of the data and avoid bias data were verified by two researches A and B and a third researcher C resolved any dispute between the first two researchers The results of the study were analyzed and reported in accordance with the STROBE guidelines Fig Flow chart of the observational research PharmacologicalResearch16120201051272 0cJ Tian Table Demographic and Patient Characteristics All patients Exposed group Control group Age Mean ± SD 014yr 1549yr 5064yr ‰¥65yr ‰¤48yr ＞48yr Sex Female Medical history Medical history Hypertension Coronary heart disease Diabetes Bronchial asthma Chronic obstructive pulmonary disease Hyperlipidemia Fatty liver Gallbladder disease Thyroid disease Stroke Chronic glomerulonephritis Cancer Hepatitis Tuberculosis Other diseases Initial symptoms Initial symptoms Fever Cough Diarrhea Fatigue Conjunctivitis Other symptoms Concomitant medication Antiviral treatment Oseltamivir Lopinavir Aciclovir Ribavirin Arbidol Other antiviral drugs Antibiotics Antibiotics Amoxicillin Cephalosporin Levofloxacin Moxifloxacin Clarithromycin Other antibiotics Chinese patent medicine Chinese patent medicine Lianhua Qingwen capsule Xiaochaihu granule Shuanghuanglian oral solution Huoxiang Zhengqi preparation Cough syrup Banlangen preparation Ganmao Qingre granule Other Chinese patent medicines ± ± ± Statistics Z10789 X210724 X2 X20817 X21796 X23145 X21788 X21023 X23933 X2＜ X2 X20318 X20186 X24649 X22578 X20812 X22638 X2＜ X25259 X20567 X2 X2 X2 X2 X2 X21049 X2 X20091 X27352 X2 X2 X2 X20250 X2 X21648 X20218 X27552 X210734 X20257 X22752 X27191 X23935 X24895 X2 X211160 X210579 X2 X27812 Pvalue ＜ ＜ ＜ ＜ ＜ ＜ ＜ ＜ ＜ ＜ ＜ ＜ ＜ ＜ ＜ ＜ Outcomes Statistical analysis The primary outcome of this study was the proportion of mild and moderate COVID19 patients who progressed to a severe disease status We also analysed factors that may affect the outcome including the patient™s age sex disease duration medical history initial symptoms concomitant medication and grouping Pr sity score matching PSM was used to further assess the effect of HSYF intervention on the conversion from mild and moderate COVID19 to a severe status Statistical analyses were conducted using SPSS software SPSS Inc Chicago IL United States Twosided tests were used and Pvalues were considered statistically significant Numerical variables are summarized as mean ±SD The data of the categorical variables are described as numbers and percentages The ttestWilcoxon ranksum test was used to compare the ages between the two groups The chisquare testFisher™s exact test was used to compare the sex medical history initial symptoms and concomitant PharmacologicalResearch16120201051273 0cJ Tian medication between the two groups to calculate the rate difference and confidence interval CI Using the proportion of patients who transitioned to a severe status as the dependent variable the effects of age sex disease duration medical history initial symptoms concomitant antiviral drugs antibiotics Chinese patent medicines and grouping were analyzed by univariate and multivariate logistic regression For factors with a statistical significance in the univariate regression analysis a logistic regression was performed to match the two groups of patients by pr sity score in a ratio and the difference in the primary outcome between the two groups was evaluated Results Demographic and patient characteristics By March we enrolled mild and moderate COVID19 patients from quarantine stations in the Wuchang District After further screening patients who refused followup patients who were not diagnosed and two patients who took other types of TCM prescriptions were excluded Finally patients were included in our study cohort including HSYF users and nonusers including decoction granules etcAmong all patients enrolled in this study were men and were women The median age of the patients was years old The median age of the exposed group was significantly lower than that of the control group P We found that of patients had a medical history which included hypertension fatty liver and diabetes A significantly higher proportion of patients in the exposed group had a history of bronchial asthma vs P and thyroid disease vs P compared with the control group while the proportion of patients with hyperlipidemia vs P was higher in the control group than the exposed group Among all patients had experienced initial COVID19 symptoms The number of patients with diarrhea in the exposed group was significantly higher than that in the control group vs respectively P and the number of patients with fever vs P cough vs P and fatigue vs P was significantly lower in the exposed group than in the control group The patients were administered multiple medications during the treatment There were no differences in the number of patients receiving antiviral drugs antibiotics and Chinese patent medicines between the two groups In terms of antiviral treatment ribavirin vs P and arbidol vs P were the most commonly administered medications in the exposed group while oseltamivir vs P and acyclovir vs P were the most commonly administered medication in the control group In terms of antibiotics amoxicillin vs P and moxifloxacin vs P were most commonly used in the exposed group while clarithromycin vs P were most commonly used in the control group In terms of Chinese patent medicines the patients in the exposed group were usually administered Xiaochaihu granules vs P Shuanghuanglian oral solution vs P cough syrup vs P Banlangen preparation vs P and Ganmao Qingre granules vs P while the patients in the control group were administered Lianhua Qingwen capsule vs P and Huoxiang Zhengqi preparation vs P as shown in Table The majority of onset times in the two groups were from January to February vs The proportion to severe status Our primary outcome was the proportion of COVID19 patients who progressed from mild and moderate COVID19 to a severe disease status There were no cases of progression to severe disease in the exposed group while cases P in the control group progressed to severe disease The difference between the two groups in terms of progression to severe disease exposed groupcontrol group was cid0 [ CI cid0 cid0 ] Stratification analysis showed that excluding diarrhea there were significant differences between the two groups in terms of sex age years medical history initial symptoms fever cough diarrhea fatigue concomitant medication etc P Table Efficacy evaluation The proportion to severe status The proportion to severe status Sex Male Female Age Age‰¤48yr Age＞48yr Underlying disease Yes No Initial symptoms Yes No Fever Yes No Cough Yes No Diarrhea Yes No Fatigue Yes No Chinese patent medicine Yes No All patients Exposed group Control group Statistics X2 X2 X28004 X29521 X2 X29251 X2 X2 X2 X27842 X26575 X211744 X2 X21915 X2 X2 X28085 Pvalue ＜ ＜ ＜ ＜ ＜ ＜ ＜ ＜ ＜ PharmacologicalResearch16120201051274 0cFactors OR95 CI Univariate analysis Pvalue Table Univariate and multivariate regression analysis J Tian therefore these factors were accounted for in the subsequent analysis Table The univariate logistic regression analysis showed that sex male age years fever cough and fatigue were risk factors for progression to a severe status We further performed a multivariate logistic regression analysis to adjust for the above risk factors The results showed that after adjusting for all factors sex male OR CI “ P and age years OR CI “ P were independent risk factors for progression to a severe disease status Table Considering the difference in sample size between the two groups and the imbalance in confounding factors the risk factors analyzed in the univariate logistic regression analysis were used as variables for PSM caliper 025σ and the number of patients in both groups were Among them no patients in the exposed group progressed to severe disease and seven patients in the control group progressed to severe disease P The difference between the two groups in terms of progression to severe disease exposed groupcontrol group was cid0 [ CI cid0 cid0 ] Table Discussion Age The global prevalence of COVID19 has brought tremendous medical pressure worldwide According to data from Johns Hopkins University in the United States by April the number of COVID19 cases exceeded million globally Furthermore the number of diagnosed cases in the United States were over and that in Italy and Spain were over with the number of newly diagnosed cases still rising [] With a large number of people in close contact with mildmoderate COVID19 patients all countries have adopted community control and home quarantine measures to prevent the spread of the epidemic However during home quarantine problems such as crossinfection and cluster outbreaks in the community have emerged due to the lack of strict community supervision as well as the need to purchase necessities and travel for hospital consultations In addition mild and moderate COVID19 patients who were under home quarantine lacked effective intervention strategies without the guidance from physicians which may have resulted in problems such as the progression from mild and moderate to severe disease [] In such patients an effective largescale community intervention strategy could reduce this conversion to severe disease and reduce the number of severe and critical COVID19 patients thus significantly improving and controlling the COVID19 epidemic For mild and moderate COVID19 patients antiviral antibiotic and symptomatic supportive treatments are most commonly used However most of the antiviral drugs used to treat COVID19 currently are based on previous treatments for severe acute respiratory syndrome SARS Middle East respiratory syndrome and influenza A and uncertainties on the efficacy and side effects of these drugs remain problematic One case report stated that a patient was cured with remdesivir [] In another clinical study lopinavirritonavir had no clinical benefit on the treatment of patients with severe COVID19 and adverse gastrointestinal events were identified [] Furthermore Abidol and lopinavirritonavir did not improve COVID19 symptoms or shorten the conversion of the viral nucleic acid to a negative result in a clinical study of patients [] The adverse reactions and side effects of related antiviral drugs such as hypocalcemia hemolytic anemia hypomagnesemia as well as those that emerged during the treatment of SARS by ribavirin cannot be ignored [] Hydroxychloroquine and chloroquine were urgently approved by the Food and Drug Administration to treat COVID19 but chloroquine drugs hydroxychloroquine and chloroquine phosphate can cause adverse reactions such as dizziness headache vertigo loss of appetite and nausea [] Our study showed that under the conditions of home quarantine TCM is effective in reducing the progression of mild and moderate COVID19 to severe disease which can serve as a reference for the Multivariate analysis Pvalue ＜ ＜ OR95 CI continued on next page SexRef female Medical history Ref no Fever Cough Diarrhea Fatigue PharmacologicalResearch16120201051275 0cJ Tian Table continued Factors Univariate analysis Pvalue OR95 CI Multivariate analysis Pvalue Antiviral treatment Ref no Antibiotics Ref no Chinese patent medicine Ref no Grouping Ref control group OR95 CI \u3000 The model includes age and sex The model includes age gender and medical history The model includes age sex medical history and initial symptoms The model includes age gender medical history initial symptoms and antiviral drugs The model includes age gender medical history initial symptoms antiviral drugs and antibiotics The model includes age gender medical history initial symptoms antiviral drugs antibiotics and Chinese patent medicine The model includes age gender medical history initial symptoms antiviral drugs antibiotics Chinese patent medicine and grouping prevention of the disease As a key point of COVID19 prognosis there have only been a few studies assessing the proportion of mild and moderate COVID19 patients who have progressed to a severe disease status currently In attempting to prevent and control the epidemic in China TCM has been widely used Some studies revealed that TCM can reduce the clinical symptoms of fever and cough in mild and moderate patients [] and improve relevant imaging indexes [] However these studies usually had small sample sizes and did not assess the proportion of mild and moderate patients who progressed to a severe disease status In this retrospective cohort study the results showed that the main influencing factors for the progression of mild and moderate COVID19 to severe disease included sex male age fever cough and fatigue which are consistent with other studies [“] After excluding the effects of the above factors we found that the HSYF intervention can effectively reduce the conversion of mild and moderate COVID19 to severe disease providing evidence on the effectiveness of TCM Thus TCM can be an option in addition to antibiotic therapy antiviral treatment and supportive oxygen therapy for the treatment of mild and moderate COVID19 patients HSYF administered in the study mainly contains the following Chinese materia medica Ma Huang Ephedrae Herba Shi Gao Gypsum fibrosum Xing Ren Armeniacae Semen Qiang Huo Notopterygii Rhizoma seu Radix Ting Li Zi LepidiiDescurainiae Semen Guan Zhong Cyrtomii Rhizoma Di Long Pheretima Xu Chang Qing Cynanchi paniculati Radix Huo Xiang Pogostemonis Herba Pei Lan Eupatorii Herba Cang Zhu Atractylodis Rhizoma Yun Ling Poria Sheng Bai Zhu Atractylodis macrocephalae Rhizoma Jiao Shan Zha Crataegi Fructus Jiao Shen Qu Massa medicate fermentata Jiao Mai Ya Hordei Fructus germinatus Hou Po Magnoliae officinalis Cortex Jiao Bing Lang Arecae Semen Wei Cao Guo Tsaoko Fructus and Sheng Jiang Zingiberis Rhizoma recens In TCM theory these prescriptions can improve the patients™ healthy qi and dispel evil factors Ma Huang Ephedrae Herba Shi Gao Gypsum fibrosum Xing Ren Armeniacae Semen Ting Li Zi LepidiiDescurainiae Semen focus on improving asthma symptoms in the lung Yun Ling Poria Sheng Bai Zhu Atractylodis macrocephalae Rhizoma Jiao Shan Zha Crataegi Fructus Jiao Shen Qu Massa medicate fermentata Jiao Mai Ya Hordei Fructus germinatus Hou Po Magnoliae officinalis Cortex Jiao Bing Lang Arecae Semen and Wei Cao Guo Tsaoko Fructus help strengthen the healthy qi Huo Xiang Pogostemonis Herba and Pei Lan Eupatorii Herba can dispel the evil factors Studies have shown that many Chinese components of HSYF can exert antiviral effects and improve respiratory symptoms In terms of the antiviral components methyl ephedrine Lephedrine and Dpseudoephedrine are present in Ma Huang Ephedrae Herba which can significantly inhibit the in vitro proliferation of influenza A H1N1 virus [] Patchouli alcohol in Huo Xiang Pogostemonis Herba can effectively inhibit the replication of H1N1 virus [] Hou Po Magnoliae officinalis Cortex can alter the cell cycle and promote H1N1 infected cells into the S phase [] the magnolol extract can inhibit the secretion of CD44 and CD54 reduce the levels of inflammatory factors IL1β IL6 and TNFα and relieve inflammation [] The active ingredients of Wei Cao Guo Tsaoko Fructus inhibit the binding region of severe acute respiratory syndrome coronavirus SARSCoV2 Sprotein to human ACE2 and control the replication of SARSCoV2 in the human body [] In addition to its antiviral effect Ma Huang Ephedrae Herba is a widely used Chinese herbal medicine for respiratory diseases and it also stimulates β receptors of the bronchial smooth muscle expands the bronchus exerts antiinflammatory effects [] and can stimulate central nervous system excitability and regulate body temperature [] This plays an important role in improving the symptoms of COVID19 including fever cough shortness of breath and other respiratory symptoms The clinical benefits and risks need to be balanced for the treatment of mild and moderate COVID19 The lack of understanding on the efficacy side effects and other risks associated with current antiviral treatments make it difficult to be used as a conventional intervention for mild and Table Pr sity score matching PSM Variables Mean ± SD MedianQ1Q3 Age SexRef female Fever Cough Fatigue Before PSM Exposed group n ± Control group n ± Pvalue ＜ ＜ ＜ ＜ After PSM Exposed group n ± Control group n ± Pvalue PharmacologicalResearch16120201051276 0cAppendix A Supplementary data J Tian moderate COVID19 patients However the results of our study provide evidence on the effectiveness of TCM for the treatment of mild and moderate COVID19 TCM treatment has the advantage of being widely used and inexpensive making it convenient for mild and moderate COVID19 patients isolated in the largescale community to quickly recover Furthermore our study used a realtime Internet information collection application to collect data which enabled physicians to communicate with the patients in real time and dynamically monitor changes in the patient™s condition Thus selfquarantined patients did not need to go to the hospital for a consultation which prevented cross infection Although the results of our study are significant and provides insight on the use of TCM treatment for mild and moderate COVID19 there are still some limitations Firstly our study was a retrospective study rather than a prospective randomized clinical trial RCT due to the current situation In the future we aim to conduct RCTs and experiments to further verify the efficacy and mechanism of HSYF Secondly as a retrospective cohort study some baseline factors between the exposed group and the control group were not completely accounted for and the age of the exposed group was significantly younger than that of the control group which may be related to our data collection method Using a realtime Internet information collection application is a more acceptable method in the younger population and imbalances in baseline data such as sex age and medical history could have introduced bias which may have affected the results of the study Nevertheless we have corrected these possible influencing factors during our analysis In order to improve the reliability of the study results a cohort study with a larger sample size or RCTs is necessary Conclusion [] National Health Commission of the People™s Republic of China National HSYF can significantly reduce the progression of mild and moderate COVID19 to a severe disease status which has a positive effect on the prevention and treatment of the disease However future clinical studies with a larger sample size are needed to further verify our results Funding This work was funded by the Special Project for Emergency of the Ministry of Science and Technology 2020YFC0845000 and the Traditional Chinese Medicine Special Project for COVID19 Emergency of National Administration of Traditional Chinese Medicine2020ZYLCYJ041 Declaration of Competing Interest Authors declare no competing interests Jiangsu Kanion Pharmaceutical Co Ltd provided the medications for the study while they did not participate in research design data collection data analysis data interpretation nor article writing Acknowledgements We acknowledge all volunteers and healthcare workers in the Wuchang community health service station Hubei Provincial Hospital of Traditional Chinese Medicine and Jiangsu Kanion Pharmaceutical Co Ltd Supplementary material related to this article can be found in the online version at 101016jphrs2020105127 References [] H Lu CW Stratton YW Tang Outbreak of pneumonia of unknown etiology in Wuhan China the mystery and the miracle J Med Virol “ [] Medical treatment group of Wuhan COVID prevention and control headquarters Notice Concerning the Recommendation of the Prescription of Traditional Chinese Medicine in the Treatment of COVID19 February [] XL Tong XY Li LH Zhao Discussion on traditional chinese medicine prevention and treatment strategies of coronavirus disease COVID19 from the perspective of œColddampness pestilence J Tradit Chin Med “ Administration of Traditional Chinese Medicine of the People™s Republic of China Guidance for Corona Virus Disease Prevention Control Diagnosis and Management People™s Medical Publishing House March [] World health anization Coronavirus disease COVID19 Situation dashboard whosprinklrcom [] A Kimball KM Hatfield M Arons Answer:

Thyroid_Cancer

Large cell neuroendocrine carcinoma Descending colon Colonic neuroendocrine neoplasm Colonic adenocarcinoma Neuroendocrine neoplasms are most often found in the small intestine rectum appendix and stomach The colon excluding the appendix and the cecum is a rare location for these neoplasms and often gives rise to highly proliferative poorly differentiated tumors with aggressive features and dismal prognosis A 32yearold male presents with a large cell neuroendocrine carcinoma arising from an unusual location the descending colon The pa tient™s clinical and imaging characteristics resembles those seen in the much more common neoplasm colonic adenocarcinoma Computed tomography and In111 octreotide scan are limited in diagnosing large cell neuroendocrine carcinoma Pathologic correlation of a sur gical specimen is required to make the correct diagnosis The Authors Published by Elsevier Inc on behalf of University of Washington This is an access under the CC BYNCND license httpcreativecommonslicensesbyncnd40 Introduction Neuroendocrine neoplasms NENs are uncommon slow growing neoplasms of the neuroendocrine system that most frequently occur in the ileum the rectum “ the appendix “ and the stomach “ [ ] The colon excluding the appendix and the cecum is a rare ori gin for NENs with a reported incidence of per per sons [] The overall median age at diagnosis is years [] Colonic NENs are not normally associated with hereditary tu mor syndromes such as multiple endocrine neoplasia type [] Colonic NENs are typically poorly differentiated on histol ogy and often appear as a large mass with aggressive growth rapid dissemination and distant metastases at the time of diagnosis [“] Once metastasized the prognosis is dismal with a median survival of months [] Patients with NENs typically present with nonspecific com plaints such as bleeding diarrhea abdominal pain gastroin testinal blood loss or weight loss [ ] Carcinoid syndrome is more often seen in patients with gastric or small intesti nal NENs with liver metastasis In contrast carcinoid syn drome is rare in patients with colonic NENs because these tumors rarely contain serotonin or secrete serotonin precur sors [ ] Urine levels of the serotonin metabolite 5HIAA are not significantly elevated in patients with colonic NENs [] Serum chromogranin A may be elevated in of all gas trointestinal NENs and correlate with tumor burden [] How ever its diagnostic accuracy can be lower for poorly differen tiated NENs [ ] Also it may be falsely elevated in proton œ Competing Interests The authors have declared that no competing interests exist ˆ—Corresponding author Email address alanmlarkinhospitalcom A Mo 101016jradcr202007045 The Authors Published by Elsevier Inc on behalf of University of Washington This is an access under the CC BYNCND license httpcreativecommonslicensesbyncnd40 0c R a d i o l o g y C a s e R e p o r t s “ Fig “ Axial a and coronal b images of CT with IV contrast with multiphasic acquisition Irregular circumferential wall thickening of the descending colon — cm heterogeneously with a contiguous enhancing mass — pump inhibitor use atrophic gastritis impaired renal func tion rheumatoid arthritis inflammatory bowel disease and nonneuroendocrine neoplasms such as prostate cancer ovar ian cancer breast cancer and colorectal cancer [] The crosssectional imaging features of colonic NENs include irregular circumferential wall thickening or large polypoidal mass with lymphadenopathy closely resembling those of colonic adenocarcinoma [ ] Metastasis to the liver is common and appear as hypervascular lesions that demonstrate moderatetointense homogenous or pe ripheral rim enhancement during hepatic arterial phase on multiphasic computed tomography or magnetic resonance imaging [ ] In111 octreotide scintigraphy utilizes a synthetic somatostatin analog to characterize NENs [] Neuroendocrine tumors containing somatostatin receptors demonstrate increased radiotracer uptake We present a rare case of large cell neuroendocrine carcinoma in the descend ing colon with metastasis in the liver which demonstrates clinical and imaging features closely resembling those of metastatic colonic adenocarcinoma Case presentation A 32yearold male with a past medical history of depression and schizophrenia presented with constant left abdominal pain radiating down to the hip and groin No pertinent surgi cal or family history was noted The patient admitted to daily use of alcohol and tobacco and denied recreational drug use The review of systems was positive for fatigue intermittent bloodstreak stools and unintentional weight loss of lbs The patient denied fever night sweats decreased appetite nausea vomiting diarrhea cutaneous flushing sweating or bronchospasm The physical exam was unremarkable except for tenderness over the left flank and mid abdomen Computed tomography of the abdomen and pelvis with IV contrast revealed irregular circumferential wall thickening of — cm het the descending colon with a contiguous erogeneously enhancing mass Fig Innumerable hypoat tenuating lesions with hypovascular peripheral enhancement — Fig “ Axial images of CT of the abdomen and pelvis at the level of the right portal vein Noncontrasted axial CT image a demonstrates innumerable illdefined hypoattenuating masses throughout the liver suspicious for metastatic lesions Contrasted axial CT image on arterial phase b demonstrates the same masses with peripheral rim enhancement and a hypoattenuating center The masses remain hypodense to the background hepatic parenchyma Each mass contains a faint hypoattenuating and nonenhancing halo Contrasted axial CT image on portal venous phase c and delayed phase d demonstrate persistent peripheral enhancement with no rapid washout were observed throughout the hepatic parenchyma Fig No skeletal metastasis was appreciated In111 octreotide scan demonstrated multiple phot ic lesions within the liver Fig After an unsuccessful attempt with colonoscopy left hemi colectomy and surgical pathology were pursued Surgical pathology of the colonic mass revealed poorly differentiated large cell neuroendocrine carcinoma with tumoral invasion into the visceral peritoneum and positive of lymph nodes Fig Additionally interventional radiology was consulted for CTguided biopsy of the liver lesions Tissue biopsy of the hepatic lesions confirmed metastasis from the colonic mass Evaluation of 5hydroxyindoleacetic acid 5HIAA in a hour urine specimen was within normal limits at 4mg24h cisplatin The patient was treated with intravenous mgm etoposide for first for weeks in combination with mgm days Discussion Colonic NENs demonstrate similar crosssectional imag ing features as colorectal adenocarcinomas [ ] Both 0cR a d i o l o g y C a s e R e p o r t s “ demonstrating moderatetointense homogenous or periph eral rim enhancement during arterial phase [ ] Hyper vascular hepatic metastasis are nonspecific and can be com monly seen in melanoma renal cell carcinoma choriocarci noma and thyroid carcinoma [] However in the setting of a patient with a colonic mass hypervascular hepatic metastatic lesions may be the differentiating imaging feature to sug gest colonic NENs rather than adenocarcinoma In our case the hepatic metastatic lesions demonstrated hypovascular peripheral enhancement that resemble those typically seen with colonic adenocarcinoma as opposed to those seen with colonic NENs We postulate that the appearance of these le sions may be attributed to the fibrogenic nature of NENs [ ] and central necrosis due to the poor cell differentiation of the mass In111 octreotide scintigraphy is commonly used in diag nosis of NENs with a sensitivity of for all NENs [] It utilizes a synthetic somatostatin analog that binds to somato statin transmembrane receptors which are expressed in of all NENs [] However poorly differentiated NENs may sometimes express fewer somatostatin receptors or may even completely lack them all together producing less reli able results [ ] In our case In111 octreotide scintigraphy demonstrates multiple phot ic lesions in the liver This may be secondary to the absence or fewer numbers of somato statin receptors in poorly differentiated NENs Our patient presents with abdominal pain fatigue weight loss and hematochezia without the characteristic symptoms of carcinoid syndrome despite having substantial hepatic Fig “ hours delayed anterior projection of the chest In111 octreotide scintigraphy demonstrates multiple phot ic lesions within the liver feature irregular circumferential wall thickening or polypoid intramural mass with areas of central necrosis and degener ation [ ] Also both malignancies often metastasize to the liver [] Colonic adenocarcinomas often produce hypo vascular hepatic metastatic lesions In contrast of gas trointestinal NENs metastases feature hypervascular lesions Fig “ HE stain scan power view shows sheetlike growth pattern of tumor cells involving whole layer of colon A On higher magnification B tumor cells show solid growth pattern Note the vesicular nuclei with saltandpepper chromatin Immunohistochemical staining for chromogranin A C and synaptophysin D reveals diffuse positive in tumor cells 0c R a d i o l o g y C a s e R e p o r t s “ metastasis Also laboratory analysis of urine 5HIAA is un remarkable CT and In111 octreotide scan are limited in diagnosing large cell neuroendocrine carcinoma Ultimately the correct diagnosis is made through immunohistochemical evaluation of the surgical pathologic specimen R E F E R E N C E S [] Turaga KK Kvols LK Recent progress in the understanding diagnosis and treatment of gastroenteropancreatic neuroendocrine tumors CA Cancer J Clinic “ [] Koenig A Krug S Mueller D Barth PJ Koenig U Scharf M et al Clinicopathological hallmarks and biomarkers of colorectal neuroendocrine neoplasms PloS One 20171212e0188876 [] Yao JC Hassan M Phan A Dagohoy C Leary C Mares JE et al One hundred years after ˜carcinoid™ epidemiology of and prognostic factors for neuroendocrine tumors in cases in the United States J Clin Orthodont “ [] Caplin M Sundin A Nillson O Richard PB Klaus JK Fahrettin K et al ENETS consensus guidelines for the management of patients with digestive neuroendocrine neoplasms colorectal neuroendocrine neoplasms Neuroendocrinology “ [] Grabowski P Sch¶nfelder J AhnertHilger G Foss HD Heine B Schindler I et al Expression of Neuroendocrine Markers A Signature of Human Undifferentiated Carcinoma of the Colon and Rectum Virchows Archiv Int J Pathol “ [] Chang S Choi D Lee SJ Lee WJ Park MH Kim SW et al Neuroendocrine neoplasms of the gastrointestinal tract classification pathologic basis and imaging features Radiographics “ [] Ganeshan Dhakshina Bhosale Priya Yang Thomas Kundra Vikas Imaging features of carcinoid tumors of the gastrointestinal tract AJR Am J Roentgenol “ [] Kaltsas GA Besser GM Grossman AB The diagnosis and medical management of advanced neuroendocrine tumors Endocr Rev “ [] Cimitan M Buonadonna A Cannizzaro R Canzonieri V Borsatti E Ruffo R De Apollonia L Somatostatin receptor scintigraphy versus chromogranin a assay in the management of patients with neuroendocrine tumors of different types clinical role Ann Oncol “ [] Gut P Czarnywojtek A Fischbach J B ˛aczyk M Ziemnicka K Wrotkowska E et al Chromogranin a unspecific neuroendocrine marker Clinical utility and potential diagnostic pitfalls Arch Med Sci AMS “ [] Levy AD Sobin LH From the archives of the AFIP gastrointestinal carcinoids imaging features with clinicopathologic comparison Radiographics “ [] Sahani DV Bonaffini PA Castillo CFD Blake MA Gastroenteropancreatic neuroendocrine tumors role of imaging in diagnosis and management Radiology “ [] Bader TR Semelka RC Chiu VC Armao DM Woosley JT MRI of carcinoid tumors spectrum of appearances in the gastrointestinal tract and liver J Magn Reson Imaging JMRI “ [] Intenzo CM Jabbour S Lin HC Miller JL Kim SM Capuzzi DM et al œScintigraphic imaging of body neuroendocrine tumors Radiographics “ [] Namasivayam S Martin DR Saini S Imaging of liver metastases MRI Cancer Imaging “ [] Laskaratos FM Rombouts K Caplin M Toumpanakis C Thirlwell C Mandair D Neuroendocrine tumors and fibrosis an unsolved mystery Cancer “ 0c'

cancer240

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: Large cell neuroendocrine carcinoma Descending colon Colonic neuroendocrine neoplasm Colonic adenocarcinoma Neuroendocrine neoplasms are most often found in the small intestine rectum appendix and stomach The colon excluding the appendix and the cecum is a rare location for these neoplasms and often gives rise to highly proliferative poorly differentiated tumors with aggressive features and dismal prognosis A 32yearold male presents with a large cell neuroendocrine carcinoma arising from an unusual location the descending colon The pa tient™s clinical and imaging characteristics resembles those seen in the much more common neoplasm colonic adenocarcinoma Computed tomography and In111 octreotide scan are limited in diagnosing large cell neuroendocrine carcinoma Pathologic correlation of a sur gical specimen is required to make the correct diagnosis The Authors Published by Elsevier Inc on behalf of University of Washington This is an access under the CC BYNCND license httpcreativecommonslicensesbyncnd40 Introduction Neuroendocrine neoplasms NENs are uncommon slow growing neoplasms of the neuroendocrine system that most frequently occur in the ileum the rectum “ the appendix “ and the stomach “ [ ] The colon excluding the appendix and the cecum is a rare ori gin for NENs with a reported incidence of per per sons [] The overall median age at diagnosis is years [] Colonic NENs are not normally associated with hereditary tu mor syndromes such as multiple endocrine neoplasia type [] Colonic NENs are typically poorly differentiated on histol ogy and often appear as a large mass with aggressive growth rapid dissemination and distant metastases at the time of diagnosis [“] Once metastasized the prognosis is dismal with a median survival of months [] Patients with NENs typically present with nonspecific com plaints such as bleeding diarrhea abdominal pain gastroin testinal blood loss or weight loss [ ] Carcinoid syndrome is more often seen in patients with gastric or small intesti nal NENs with liver metastasis In contrast carcinoid syn drome is rare in patients with colonic NENs because these tumors rarely contain serotonin or secrete serotonin precur sors [ ] Urine levels of the serotonin metabolite 5HIAA are not significantly elevated in patients with colonic NENs [] Serum chromogranin A may be elevated in of all gas trointestinal NENs and correlate with tumor burden [] How ever its diagnostic accuracy can be lower for poorly differen tiated NENs [ ] Also it may be falsely elevated in proton œ Competing Interests The authors have declared that no competing interests exist ˆ—Corresponding author Email address alanmlarkinhospitalcom A Mo 101016jradcr202007045 The Authors Published by Elsevier Inc on behalf of University of Washington This is an access under the CC BYNCND license httpcreativecommonslicensesbyncnd40 0c R a d i o l o g y C a s e R e p o r t s “ Fig “ Axial a and coronal b images of CT with IV contrast with multiphasic acquisition Irregular circumferential wall thickening of the descending colon — cm heterogeneously with a contiguous enhancing mass — pump inhibitor use atrophic gastritis impaired renal func tion rheumatoid arthritis inflammatory bowel disease and nonneuroendocrine neoplasms such as prostate cancer ovar ian cancer breast cancer and colorectal cancer [] The crosssectional imaging features of colonic NENs include irregular circumferential wall thickening or large polypoidal mass with lymphadenopathy closely resembling those of colonic adenocarcinoma [ ] Metastasis to the liver is common and appear as hypervascular lesions that demonstrate moderatetointense homogenous or pe ripheral rim enhancement during hepatic arterial phase on multiphasic computed tomography or magnetic resonance imaging [ ] In111 octreotide scintigraphy utilizes a synthetic somatostatin analog to characterize NENs [] Neuroendocrine tumors containing somatostatin receptors demonstrate increased radiotracer uptake We present a rare case of large cell neuroendocrine carcinoma in the descend ing colon with metastasis in the liver which demonstrates clinical and imaging features closely resembling those of metastatic colonic adenocarcinoma Case presentation A 32yearold male with a past medical history of depression and schizophrenia presented with constant left abdominal pain radiating down to the hip and groin No pertinent surgi cal or family history was noted The patient admitted to daily use of alcohol and tobacco and denied recreational drug use The review of systems was positive for fatigue intermittent bloodstreak stools and unintentional weight loss of lbs The patient denied fever night sweats decreased appetite nausea vomiting diarrhea cutaneous flushing sweating or bronchospasm The physical exam was unremarkable except for tenderness over the left flank and mid abdomen Computed tomography of the abdomen and pelvis with IV contrast revealed irregular circumferential wall thickening of — cm het the descending colon with a contiguous erogeneously enhancing mass Fig Innumerable hypoat tenuating lesions with hypovascular peripheral enhancement — Fig “ Axial images of CT of the abdomen and pelvis at the level of the right portal vein Noncontrasted axial CT image a demonstrates innumerable illdefined hypoattenuating masses throughout the liver suspicious for metastatic lesions Contrasted axial CT image on arterial phase b demonstrates the same masses with peripheral rim enhancement and a hypoattenuating center The masses remain hypodense to the background hepatic parenchyma Each mass contains a faint hypoattenuating and nonenhancing halo Contrasted axial CT image on portal venous phase c and delayed phase d demonstrate persistent peripheral enhancement with no rapid washout were observed throughout the hepatic parenchyma Fig No skeletal metastasis was appreciated In111 octreotide scan demonstrated multiple phot ic lesions within the liver Fig After an unsuccessful attempt with colonoscopy left hemi colectomy and surgical pathology were pursued Surgical pathology of the colonic mass revealed poorly differentiated large cell neuroendocrine carcinoma with tumoral invasion into the visceral peritoneum and positive of lymph nodes Fig Additionally interventional radiology was consulted for CTguided biopsy of the liver lesions Tissue biopsy of the hepatic lesions confirmed metastasis from the colonic mass Evaluation of 5hydroxyindoleacetic acid 5HIAA in a hour urine specimen was within normal limits at 4mg24h cisplatin The patient was treated with intravenous mgm etoposide for first for weeks in combination with mgm days Discussion Colonic NENs demonstrate similar crosssectional imag ing features as colorectal adenocarcinomas [ ] Both 0cR a d i o l o g y C a s e R e p o r t s “ demonstrating moderatetointense homogenous or periph eral rim enhancement during arterial phase [ ] Hyper vascular hepatic metastasis are nonspecific and can be com monly seen in melanoma renal cell carcinoma choriocarci noma and thyroid carcinoma [] However in the setting of a patient with a colonic mass hypervascular hepatic metastatic lesions may be the differentiating imaging feature to sug gest colonic NENs rather than adenocarcinoma In our case the hepatic metastatic lesions demonstrated hypovascular peripheral enhancement that resemble those typically seen with colonic adenocarcinoma as opposed to those seen with colonic NENs We postulate that the appearance of these le sions may be attributed to the fibrogenic nature of NENs [ ] and central necrosis due to the poor cell differentiation of the mass In111 octreotide scintigraphy is commonly used in diag nosis of NENs with a sensitivity of for all NENs [] It utilizes a synthetic somatostatin analog that binds to somato statin transmembrane receptors which are expressed in of all NENs [] However poorly differentiated NENs may sometimes express fewer somatostatin receptors or may even completely lack them all together producing less reli able results [ ] In our case In111 octreotide scintigraphy demonstrates multiple phot ic lesions in the liver This may be secondary to the absence or fewer numbers of somato statin receptors in poorly differentiated NENs Our patient presents with abdominal pain fatigue weight loss and hematochezia without the characteristic symptoms of carcinoid syndrome despite having substantial hepatic Fig “ hours delayed anterior projection of the chest In111 octreotide scintigraphy demonstrates multiple phot ic lesions within the liver feature irregular circumferential wall thickening or polypoid intramural mass with areas of central necrosis and degener ation [ ] Also both malignancies often metastasize to the liver [] Colonic adenocarcinomas often produce hypo vascular hepatic metastatic lesions In contrast of gas trointestinal NENs metastases feature hypervascular lesions Fig “ HE stain scan power view shows sheetlike growth pattern of tumor cells involving whole layer of colon A On higher magnification B tumor cells show solid growth pattern Note the vesicular nuclei with saltandpepper chromatin Immunohistochemical staining for chromogranin A C and synaptophysin D reveals diffuse positive in tumor cells 0c R a d i o l o g y C a s e R e p o r t s “ metastasis Also laboratory analysis of urine 5HIAA is un remarkable CT and In111 octreotide scan are limited in diagnosing large cell neuroendocrine carcinoma Ultimately the correct diagnosis is made through immunohistochemical evaluation of the surgical pathologic specimen R E F E R E N C E S [] Turaga KK Kvols LK Recent progress in the understanding diagnosis and treatment of gastroenteropancreatic neuroendocrine tumors CA Cancer J Clinic “ [] Koenig A Krug S Mueller D Barth PJ Koenig U Scharf M et al Clinicopathological hallmarks and biomarkers of colorectal neuroendocrine neoplasms PloS One 20171212e0188876 [] Yao JC Hassan M Phan A Dagohoy C Leary C Mares JE et al One hundred years after ˜carcinoid™ epidemiology of and prognostic factors for neuroendocrine tumors in cases in the United States J Clin Orthodont “ [] Caplin M Sundin A Nillson O Richard PB Klaus JK Fahrettin K et al ENETS consensus guidelines for the management of patients with digestive neuroendocrine neoplasms colorectal neuroendocrine neoplasms Neuroendocrinology “ [] Grabowski P Sch¶nfelder J AhnertHilger G Foss HD Heine B Schindler I et al Expression of Neuroendocrine Markers A Signature of Human Undifferentiated Carcinoma of the Colon and Rectum Virchows Archiv Int J Pathol “ [] Chang S Choi D Lee SJ Lee WJ Park MH Kim SW et al Neuroendocrine neoplasms of the gastrointestinal tract classification pathologic basis and imaging features Radiographics “ [] Ganeshan Dhakshina Bhosale Priya Yang Thomas Kundra Vikas Imaging features of carcinoid tumors of the gastrointestinal tract AJR Am J Roentgenol “ [] Kaltsas GA Besser GM Grossman AB The diagnosis and medical management of advanced neuroendocrine tumors Endocr Rev “ [] Cimitan M Buonadonna A Cannizzaro R Canzonieri V Borsatti E Ruffo R De Apollonia L Somatostatin receptor scintigraphy versus chromogranin a assay in the management of patients with neuroendocrine tumors of different types clinical role Ann Oncol “ [] Gut P Czarnywojtek A Fischbach J B ˛aczyk M Ziemnicka K Wrotkowska E et al Chromogranin a unspecific neuroendocrine marker Clinical utility and potential diagnostic pitfalls Arch Med Sci AMS “ [] Levy AD Sobin LH From the archives of the AFIP gastrointestinal carcinoids imaging features with clinicopathologic comparison Radiographics “ [] Sahani DV Bonaffini PA Castillo CFD Blake MA Gastroenteropancreatic neuroendocrine tumors role of imaging in diagnosis and management Radiology “ [] Bader TR Semelka RC Chiu VC Armao DM Woosley JT MRI of carcinoid tumors spectrum of appearances in the gastrointestinal tract and liver J Magn Reson Imaging JMRI “ [] Intenzo CM Jabbour S Lin HC Miller JL Kim SM Capuzzi DM et al œScintigraphic imaging of body neuroendocrine tumors Radiographics “ [] Namasivayam S Martin DR Saini S Imaging of liver metastases MRI Cancer Imaging “ [] Laskaratos FM Rombouts K Caplin M Toumpanakis C Thirlwell C Mandair D Neuroendocrine tumors and fibrosis an unsolved mystery Cancer “ 0c' Answer:

Thyroid_Cancer

"Handling EditorAdrian CovaciKeywordsNuclear accidentsHealth surveillancePreparednessCommunicationStakeholdersRecommendationsSerious accidents at nuclear power plants have been rare but theirstories can teach us how to prevent or mitigate the eï¬ects of futurenuclear catastrophes The accidents at the Fukushima Daiichi nuclearpower plant and Chernobyl nuclear power plant occurred years and years ago respectively and there are still lessons to learn from themregarding numerous issues including radiation exposure assessmentand medical followup of emergency responders evacuees and residents decisions to lift evacuation orders and communication withresponders and stakeholders Bazyka Callen and Homma Lester Soï¬er Some of the lessons from theseaccidents have been extensively reviewed and taken into considerationby national and international anizations such as the InternationalAtomic Energy Agency the International Commission on RadiologicalProtection and the World Health anisation and are reflected inpublished literature Bennett Carr Clarke IAEA 2015a 2015b Nisbet SGDSN This hasallowed the development of various recommendations and guidancedocuments targeting specific issues of radiation protection training andcommunication and socioeconomic aspects in order to prepare andimprove decision making processes in the early and intermediatephases eg Carr IAEA 2015b Nisbet However the majority of these texts focus on technical issues andare directed towards radiation protection experts rather than for thesupport of aï¬ected populations The traditional approaches of emergency response and recovery including evacuation relocation andhealth surveillance are largely based on dose levels Although manyrecognise the importance of psychosocial or human factors it has beendifficult to adapt the approaches to better address the social economicethical and psychological factors These include the health and welfareeï¬ects that may arise from the accident from the concerns about thepresence of radiation in the environment from the mitigation actionstaken and from the information mixed or absent provided to the population Changes in the ethical and legal requirements for personaldata collection use and storage raise additional challenges particularlyin the area of health surveillance and epidemiologyAbbreviations COVID19 Coronavirus Disease EJP CONCERT European Joint Programme for the Integration of Radiation Protection Research OPERRA Project for the European Radiation Research Area SGDSN Secrtariat gnral de la dfense et de la scurit nationale France SHAMISEN NuclearEmergency Situations Improvement of Medical and Health Surveillance SHAMISEN SINGS SHAMISEN Stakeholder INvolvement in Generating Science UN UnitedNations UNDRR United Nations Office for Disaster Risk Reduction101016jenvint2020106000Received July Accepted July Published by Elsevier Ltd This is an access under the CC BYNCND license httpcreativecommonslicensesBYNCND40 0c Main components of the SHAMISEN projectThe SHAMISEN project started in late at a time when somedeleterious eï¬ects of evacuation and ultrasound thyroid screening inFukushima had started to be reported The project therefore aimed toreview the lessons learned from major nuclear accidents in particularfrom experiences of populations aï¬ected by the Chernobyl andFukushima accidents to develop recommendations for medical andhealth surveillance of populations aï¬ected by previous and future radiation accidents The ultimate motivation was to minimise the negative impacts of the accident and improve the health of aï¬ected populations The holistic WHO definition of health was used in this contextie œa state of complete physical mental and social wellbeing and notmerely the absence of disease or rmity WHO The Recommendations were to address in particular the following complementary aspects dose assessment supporting all phases of an accident including emergency response clinical decisionmaking recoveryactions and health surveillance improvement of living conditions ofaï¬ected populations engaging them and responding to their needs andminimising unnecessary anxiety and health surveillance and wherefeasible improvement of estimates of radiationinduced risk for radiation protection and communication with aï¬ected populationsTo achieve this and recognising the need for a holistic approach toaccident management and health surveillance SHAMISEN brought together a team of researchers from institutions including RadiationProtection Authorities Universities Research Centres and Associationsin Europe and Japan with complementary experience and a long trackrecord in post accidental management dosimetry radiation protectionmedical followup and screening population health surveillance healtheconomics epidemiology ethics and sociology of radiation protectionThe project also drew upon additional expertise from Belarus RussiaUkraine Japan and the UK as well as from outside of the radiationresearch field and established contacts with major international anizations including the World Health anisation and the NuclearEnergy Agency of the anisation for Economic Cooperation andDevelopmentDetails of the SHAMISEN Project are provided in the paper by Ohbaand collaborators Ohba this issue Briefly the approach involved in particular challengingevaluating the eï¬ectiveness of measures taken after Chernobyl or Fukushima accidents in particularcid129 Systematic thyroid screening with ultrasound for early detection ofpotential thyroid cancer casescid129 Criteria for evacuation and their consequencescid129 Measures taken to contribute to the wellbeing of aï¬ected populations and develop a radiological protection culture and resilience inaï¬ected populationscid129 Challenges and good practice in communication and training withthe objective of regaining the trust of the population and engagingthem in retaking control of their livescid129 Role of ethics in disaster preparedness response and health surveillance autonomy and dignity respect of privacy beneficence¦cid129 Role of health professionals in the diï¬erent phases of the accidentmanagementcid129 Cost eï¬ectiveness of the measures takenAll of this was brought together to develop practical recommendations for preparedness and the diï¬erent phases of the accident SHAMISEN results and the way forwardAlthough the SHAMISEN project was developed during a limitedperiod of months in response to the second call of the EuropeanOPERRA project a series of key results has been achieved The mainresults are several topical reports and a set of recommendations dividedinto five main topics eg Evacuation Communication and trainingEnvironment International Dosimetry Health surveillance Epidemiology These topics focusspecifically on the health surveillance of people following a nuclearaccident combining natural and social sciences values and practice tohelp health professionals decisionmakers and local stakeholders to setup protective actions and health programmes responding to the concernof aï¬ected populations Therefore the SHAMISEN recommendationsare not intended to cover all aspects of emergency and recovery response and preparednessThe formulation of the recommendations is generic enough to beapplied in diï¬erent countries recognising that cultural diï¬erences willbe important The structure describes the general context and the mainreasons for developing each recommendation provides explanations onhow to develop it and indicates who would be involved in the development of the recommendation Depending on the context specificarrangements have to be made for the implementation of these recommendations during the diï¬erent phases eg preparedness earlyand intermediate longterm recoveryThe recommendations provide advice on the values to be consideredwhen addressing the issue at stake and what type of tools and protocolsare needed rather than the tools themselves Due to the duration of theproject it was not manageable to develop them However they providea significant input for further developments for practical tools in different domains and identify the main expectations from stakeholderswith regards to health surveillance in postaccident situationsDeveloped as a research project it was not the intention to specifyabsolute dose criteria for the implementation of actions Of coursediscussions on the feedback experience from the management of theChernobyl and Fukushima accidents point out some challenges associated with the use of specific dose criteria but the spirit of the recommendations is to provide indications and guidance for the decisionmakers and health professionals with regard to the choice to be madeon the adoption of dose criteria for the diï¬erent actions to be implementedBesides the management of the direct radiation induced health effects the report underlines the need to develop a multidisciplinaryapproach to identify measure assess and alleviate psychological andother indirect health impacts of socioeconomic and social upheavals ofthe consequences of the accident For this purpose it is recommendedto promote the engagement process of local stakeholders since thepreparedness phase targeting the overall wellbeing of populations withdue considerations of the ethical principles of respect for autonomydignity and justiceThis special issue of Environmental International combines a series ofscientific papers and is an opportunity to emphasize the main analysesdeveloped during the SHAMISEN project combining advanced scientific research analyses of feedback experience from the Chernobyl andFukushima accidents and applying a multidisciplinary approachAlthough the topics presented in this special issue have already beenaddressed in general in several papers the originality of the approachadopted in the SHAMISEN project provides new insights for healthsurveillance issuesIt is worth mentioning that following the SHAMISEN project otherresearch projects have been launched A first series of projects arededicated to the development of Apps with and for citizens as recommended in the SHAMISEN project This has notably been done withthe European research project SHAMISEN SINGS as part of the EJPCONCERT as well as with an ongoing project developed by FukushimaMedical University In addition several projects are currently underdevelopment in diï¬erent countries and at the European level thatpromote a citizen science approach for addressing health and radiological monitoringAs an example of the multidisciplinary approach the SHAMISENproject has identified a series of recommendations calling for furthercooperation with diï¬erent European Research Platforms combininglow doses eï¬ects dosimetry radioecology emergency and recoverymanagement social sciences and humanities and medical research 0cEnvironment International and Thierry SchneideraŽ Deborah Oughtonb Elisabeth Cardiscdea CEPN Nuclear Protection Evaluation Centre Rue de la Redoute FontenayauxRoses Franceb Centre for Environmental Radioactivity CERAD NMBU „«s Norwayc Barcelona Institute for Global Health ISGlobal Barcelona Spaind Pompeu Fabra University Barcelona Spaine Spanish Consortium for Research and Public Health CIBERESP Institutode Salud Carlos III Madrid SpainEmail address thierryschneidercepnassofr T SchneiderReferencesBazyka D Belyi D Chumak A Lessons from chornobyl considerations forstrengthening radiation emergency preparedness in Ukraine Radiat Prot Dosim “ 101093rpdncw196Bennett B Repacholi M Carr Z Eds Health eï¬ects of the Chernobyl accidentand special health care programmes Report of the UN Chernobyl Forum expert groupœHealth WHO Press GenevaCallen J Homma T Lessons learned in protection of the public for the accident atthe Fukushima Daiichi nuclear power plant Health Phys “ 101097HP0000000000000666Carr Z Clarke M Akl EA Schneider R Murith C Li C ParrishSprowl J StenkeL CuiPing L Bertrand S Miller C Using the grade approach to supportthe development of recommendations for public health interventions in radiationemergencies Radiat Prot Dosim “ 101093rpdncw234Clarke R Valentin J International Commission on Radiological Protection Task Group ICRP publication Application of the Commission™s Recommendations forthe protection of people in emergency exposure situations Ann ICRP “101016jicrp200905004Croua¯l P Camps J Raskob W Schneider T NERIS Roadmap on medium andlongterm research on preparedness for nuclear and radiological emergency responseand recovery Version eunerisnetlibrarysra259nerisroadmap2020htmlIAEA 2015a The Fukushima Daiichi Accident No Technical Volume IAEAInternational Atomic Energy Agency Vienna Austria wwwiaeapublications10962thefukushimadaiichiaccidentIAEA 2015b Preparedness and Response for a Nuclear or Radiological Emergency NoGSR7 Safety Standards Series IAEA International Atomic Energy AgencyVienna Austria httpwwwpubiaeaMTCDPublicationsPDFP_1708_webpdfImpens N Salomaa S Second joint roadmap for radiation protection researchDeliverable No EJPCONCERT European Joint Programme for the Integrationof Radiation Protection Research H2020 wwwconcerth2020euDocumentashxdtwebfileListsDeliverablesAttachments206D37_Second20joint20roadmap_draft_reviewed_20052020_approved03062020pdfguid01b5ac77b2ec4cda9c98917dba396f0fLester MS Public information during a nuclear power plant accident lessonslearned from Three Mile Island Bull N Y Acad Med “Nisbet AF Jones A Turcanu C Camps J Andersson KG H¤nninen RRavantaara A Solatie D Kostiainen E Julien T Pupin V Ollagnon HPapachristodoulou C Ioannides K Oughton D Generic Handbook forAssisting in the Management of Contaminated Food Production Systems in Europefollowing a radiological emergency v2 No CAT1TN0901 EURANOS eunerisnetlibraryhandbookshtmlOhba T Liutsko L Schneider T Tanigawa K Fattibene P Laurier D Sarukhan ABarquinero J Kesminiene A Skuterud L Cardis E this issue The SHAMISENProject challenging historical recommendations for preparedness response andfollowup of nuclear accidents lessons learnt from Chernobyl and FukushimaSGDSN National response plan Major nuclear or radiological accident httpwwwgouvernementfrsitesdefaultfilesrisquespdfnational_plan_nuclear_radiological_accidentspdfSoï¬er Y Schwartz D Goldberg A Henenfeld M BarDayan Y Populationevacuations in industrial accidents a review of the literature about four major eventsPrehosp Disaster Med “UNDRR The Sendai Framework for Disaster Risk Reduction “ UnitedNations Office for Disaster Risk Reduction wwwundrrpublicationsendaiframeworkdisasterriskreduction20152030WHO WHO Definition of Health World Health anisation Geneva httpwwwwhointaboutdefinitionenprinthtmlThese recommendations have already been considered in the development of the European joint roadmap for radiation protection researchImpens and Salomaa and of the strategic research agenda ofdiï¬erent European platforms notably NERIS on emergency and recovery Croua¯l The results of the SHAMISEN project have been presented and discussed in several national and international workshops and meetingsRecommendations are being disseminated to decision makers and radiation protection authorities for translation into strategy and policy aswell as to scientific medical and nonexpert audiences They are nowreferred and used as basis of the reflections and the initiatives of national and international anizations for both preparedness NuclearEnergy Agency World Health anisation International Commissionon Radiological Protection ICRP National committee for postaccident management CODIRPA in France and the management of theFukushima situation with a key role of the Japanese partners involvedin the SHAMISEN project Fukushima Medical University NagasakiUniversity Hiroshima UniversityMore broadly the approach adopted in the SHAMISEN project andits results may contribute to address other hazards including naturaldisasters industrial accidents or even pandemic crisis Similarities canbe emphasized with the Sendai Framework for Disaster Risk Reduction“ UNDRR adopted at the 3rd UN World Conference in This framework underlines the importance of improving theunderstanding of disaster risk better addressing vulnerability and hazard characteristics strengthening risk governance reinforcing accountability for risk management with development of preparednessinvolvement of stakeholders and due considerations of resilience ofhealth infrastructureFinally the pandemic crisis of COVID19 highlights a series of issuesquite similar to those addressed in the SHAMISEN project confinementversus evacuation psychosocial aspects communication and dialogueanisation of the transition phases and of course the preparation ofhealth surveillance strategies and structures of epidemiological studiesThese diï¬erent issues would benefit from crosscomparison analysisand the s presented in this special issue could certainly contributeto the reflectionDeclaration of Competing InterestThe authors declare that they have no known competing financialinterests or personal relationships that could have appeared to ‚uence the work reported in this paperAcknowledgmentsSHAMISEN was supported by the EURATOM European AtomicEnergy Community program of the European Commission in the framework of the OPERRA Project for the European RadiationResearch Area project FP7 grant agreement No The authors are grateful to all partners experts and stakeholderscontributed to theandorwho participated in the projectRecommendationsISGlobal acknowledges supportfrom the Spanish Ministry ofScience Innovation and Universities through the œCentro de ExcelenciaSevero Ochoa “ Program CEX2018000806S and supportfrom the Generalitat de Catalunya through the CERCA Program NMBUacknowledges the support of the Research Council of Norway RCNthrough its Centres of Excellence funding scheme project numberŽ Corresponding author 0c"

cancer241

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: "Handling EditorAdrian CovaciKeywordsNuclear accidentsHealth surveillancePreparednessCommunicationStakeholdersRecommendationsSerious accidents at nuclear power plants have been rare but theirstories can teach us how to prevent or mitigate the eï¬ects of futurenuclear catastrophes The accidents at the Fukushima Daiichi nuclearpower plant and Chernobyl nuclear power plant occurred years and years ago respectively and there are still lessons to learn from themregarding numerous issues including radiation exposure assessmentand medical followup of emergency responders evacuees and residents decisions to lift evacuation orders and communication withresponders and stakeholders Bazyka Callen and Homma Lester Soï¬er Some of the lessons from theseaccidents have been extensively reviewed and taken into considerationby national and international anizations such as the InternationalAtomic Energy Agency the International Commission on RadiologicalProtection and the World Health anisation and are reflected inpublished literature Bennett Carr Clarke IAEA 2015a 2015b Nisbet SGDSN This hasallowed the development of various recommendations and guidancedocuments targeting specific issues of radiation protection training andcommunication and socioeconomic aspects in order to prepare andimprove decision making processes in the early and intermediatephases eg Carr IAEA 2015b Nisbet However the majority of these texts focus on technical issues andare directed towards radiation protection experts rather than for thesupport of aï¬ected populations The traditional approaches of emergency response and recovery including evacuation relocation andhealth surveillance are largely based on dose levels Although manyrecognise the importance of psychosocial or human factors it has beendifficult to adapt the approaches to better address the social economicethical and psychological factors These include the health and welfareeï¬ects that may arise from the accident from the concerns about thepresence of radiation in the environment from the mitigation actionstaken and from the information mixed or absent provided to the population Changes in the ethical and legal requirements for personaldata collection use and storage raise additional challenges particularlyin the area of health surveillance and epidemiologyAbbreviations COVID19 Coronavirus Disease EJP CONCERT European Joint Programme for the Integration of Radiation Protection Research OPERRA Project for the European Radiation Research Area SGDSN Secrtariat gnral de la dfense et de la scurit nationale France SHAMISEN NuclearEmergency Situations Improvement of Medical and Health Surveillance SHAMISEN SINGS SHAMISEN Stakeholder INvolvement in Generating Science UN UnitedNations UNDRR United Nations Office for Disaster Risk Reduction101016jenvint2020106000Received July Accepted July Published by Elsevier Ltd This is an access under the CC BYNCND license httpcreativecommonslicensesBYNCND40 0c Main components of the SHAMISEN projectThe SHAMISEN project started in late at a time when somedeleterious eï¬ects of evacuation and ultrasound thyroid screening inFukushima had started to be reported The project therefore aimed toreview the lessons learned from major nuclear accidents in particularfrom experiences of populations aï¬ected by the Chernobyl andFukushima accidents to develop recommendations for medical andhealth surveillance of populations aï¬ected by previous and future radiation accidents The ultimate motivation was to minimise the negative impacts of the accident and improve the health of aï¬ected populations The holistic WHO definition of health was used in this contextie œa state of complete physical mental and social wellbeing and notmerely the absence of disease or rmity WHO The Recommendations were to address in particular the following complementary aspects dose assessment supporting all phases of an accident including emergency response clinical decisionmaking recoveryactions and health surveillance improvement of living conditions ofaï¬ected populations engaging them and responding to their needs andminimising unnecessary anxiety and health surveillance and wherefeasible improvement of estimates of radiationinduced risk for radiation protection and communication with aï¬ected populationsTo achieve this and recognising the need for a holistic approach toaccident management and health surveillance SHAMISEN brought together a team of researchers from institutions including RadiationProtection Authorities Universities Research Centres and Associationsin Europe and Japan with complementary experience and a long trackrecord in post accidental management dosimetry radiation protectionmedical followup and screening population health surveillance healtheconomics epidemiology ethics and sociology of radiation protectionThe project also drew upon additional expertise from Belarus RussiaUkraine Japan and the UK as well as from outside of the radiationresearch field and established contacts with major international anizations including the World Health anisation and the NuclearEnergy Agency of the anisation for Economic Cooperation andDevelopmentDetails of the SHAMISEN Project are provided in the paper by Ohbaand collaborators Ohba this issue Briefly the approach involved in particular challengingevaluating the eï¬ectiveness of measures taken after Chernobyl or Fukushima accidents in particularcid129 Systematic thyroid screening with ultrasound for early detection ofpotential thyroid cancer casescid129 Criteria for evacuation and their consequencescid129 Measures taken to contribute to the wellbeing of aï¬ected populations and develop a radiological protection culture and resilience inaï¬ected populationscid129 Challenges and good practice in communication and training withthe objective of regaining the trust of the population and engagingthem in retaking control of their livescid129 Role of ethics in disaster preparedness response and health surveillance autonomy and dignity respect of privacy beneficence¦cid129 Role of health professionals in the diï¬erent phases of the accidentmanagementcid129 Cost eï¬ectiveness of the measures takenAll of this was brought together to develop practical recommendations for preparedness and the diï¬erent phases of the accident SHAMISEN results and the way forwardAlthough the SHAMISEN project was developed during a limitedperiod of months in response to the second call of the EuropeanOPERRA project a series of key results has been achieved The mainresults are several topical reports and a set of recommendations dividedinto five main topics eg Evacuation Communication and trainingEnvironment International Dosimetry Health surveillance Epidemiology These topics focusspecifically on the health surveillance of people following a nuclearaccident combining natural and social sciences values and practice tohelp health professionals decisionmakers and local stakeholders to setup protective actions and health programmes responding to the concernof aï¬ected populations Therefore the SHAMISEN recommendationsare not intended to cover all aspects of emergency and recovery response and preparednessThe formulation of the recommendations is generic enough to beapplied in diï¬erent countries recognising that cultural diï¬erences willbe important The structure describes the general context and the mainreasons for developing each recommendation provides explanations onhow to develop it and indicates who would be involved in the development of the recommendation Depending on the context specificarrangements have to be made for the implementation of these recommendations during the diï¬erent phases eg preparedness earlyand intermediate longterm recoveryThe recommendations provide advice on the values to be consideredwhen addressing the issue at stake and what type of tools and protocolsare needed rather than the tools themselves Due to the duration of theproject it was not manageable to develop them However they providea significant input for further developments for practical tools in different domains and identify the main expectations from stakeholderswith regards to health surveillance in postaccident situationsDeveloped as a research project it was not the intention to specifyabsolute dose criteria for the implementation of actions Of coursediscussions on the feedback experience from the management of theChernobyl and Fukushima accidents point out some challenges associated with the use of specific dose criteria but the spirit of the recommendations is to provide indications and guidance for the decisionmakers and health professionals with regard to the choice to be madeon the adoption of dose criteria for the diï¬erent actions to be implementedBesides the management of the direct radiation induced health effects the report underlines the need to develop a multidisciplinaryapproach to identify measure assess and alleviate psychological andother indirect health impacts of socioeconomic and social upheavals ofthe consequences of the accident For this purpose it is recommendedto promote the engagement process of local stakeholders since thepreparedness phase targeting the overall wellbeing of populations withdue considerations of the ethical principles of respect for autonomydignity and justiceThis special issue of Environmental International combines a series ofscientific papers and is an opportunity to emphasize the main analysesdeveloped during the SHAMISEN project combining advanced scientific research analyses of feedback experience from the Chernobyl andFukushima accidents and applying a multidisciplinary approachAlthough the topics presented in this special issue have already beenaddressed in general in several papers the originality of the approachadopted in the SHAMISEN project provides new insights for healthsurveillance issuesIt is worth mentioning that following the SHAMISEN project otherresearch projects have been launched A first series of projects arededicated to the development of Apps with and for citizens as recommended in the SHAMISEN project This has notably been done withthe European research project SHAMISEN SINGS as part of the EJPCONCERT as well as with an ongoing project developed by FukushimaMedical University In addition several projects are currently underdevelopment in diï¬erent countries and at the European level thatpromote a citizen science approach for addressing health and radiological monitoringAs an example of the multidisciplinary approach the SHAMISENproject has identified a series of recommendations calling for furthercooperation with diï¬erent European Research Platforms combininglow doses eï¬ects dosimetry radioecology emergency and recoverymanagement social sciences and humanities and medical research 0cEnvironment International and Thierry SchneideraŽ Deborah Oughtonb Elisabeth Cardiscdea CEPN Nuclear Protection Evaluation Centre Rue de la Redoute FontenayauxRoses Franceb Centre for Environmental Radioactivity CERAD NMBU „«s Norwayc Barcelona Institute for Global Health ISGlobal Barcelona Spaind Pompeu Fabra University Barcelona Spaine Spanish Consortium for Research and Public Health CIBERESP Institutode Salud Carlos III Madrid SpainEmail address thierryschneidercepnassofr T SchneiderReferencesBazyka D Belyi D Chumak A Lessons from chornobyl considerations forstrengthening radiation emergency preparedness in Ukraine Radiat Prot Dosim “ 101093rpdncw196Bennett B Repacholi M Carr Z Eds Health eï¬ects of the Chernobyl accidentand special health care programmes Report of the UN Chernobyl Forum expert groupœHealth WHO Press GenevaCallen J Homma T Lessons learned in protection of the public for the accident atthe Fukushima Daiichi nuclear power plant Health Phys “ 101097HP0000000000000666Carr Z Clarke M Akl EA Schneider R Murith C Li C ParrishSprowl J StenkeL CuiPing L Bertrand S Miller C Using the grade approach to supportthe development of recommendations for public health interventions in radiationemergencies Radiat Prot Dosim “ 101093rpdncw234Clarke R Valentin J International Commission on Radiological Protection Task Group ICRP publication Application of the Commission™s Recommendations forthe protection of people in emergency exposure situations Ann ICRP “101016jicrp200905004Croua¯l P Camps J Raskob W Schneider T NERIS Roadmap on medium andlongterm research on preparedness for nuclear and radiological emergency responseand recovery Version eunerisnetlibrarysra259nerisroadmap2020htmlIAEA 2015a The Fukushima Daiichi Accident No Technical Volume IAEAInternational Atomic Energy Agency Vienna Austria wwwiaeapublications10962thefukushimadaiichiaccidentIAEA 2015b Preparedness and Response for a Nuclear or Radiological Emergency NoGSR7 Safety Standards Series IAEA International Atomic Energy AgencyVienna Austria httpwwwpubiaeaMTCDPublicationsPDFP_1708_webpdfImpens N Salomaa S Second joint roadmap for radiation protection researchDeliverable No EJPCONCERT European Joint Programme for the Integrationof Radiation Protection Research H2020 wwwconcerth2020euDocumentashxdtwebfileListsDeliverablesAttachments206D37_Second20joint20roadmap_draft_reviewed_20052020_approved03062020pdfguid01b5ac77b2ec4cda9c98917dba396f0fLester MS Public information during a nuclear power plant accident lessonslearned from Three Mile Island Bull N Y Acad Med “Nisbet AF Jones A Turcanu C Camps J Andersson KG H¤nninen RRavantaara A Solatie D Kostiainen E Julien T Pupin V Ollagnon HPapachristodoulou C Ioannides K Oughton D Generic Handbook forAssisting in the Management of Contaminated Food Production Systems in Europefollowing a radiological emergency v2 No CAT1TN0901 EURANOS eunerisnetlibraryhandbookshtmlOhba T Liutsko L Schneider T Tanigawa K Fattibene P Laurier D Sarukhan ABarquinero J Kesminiene A Skuterud L Cardis E this issue The SHAMISENProject challenging historical recommendations for preparedness response andfollowup of nuclear accidents lessons learnt from Chernobyl and FukushimaSGDSN National response plan Major nuclear or radiological accident httpwwwgouvernementfrsitesdefaultfilesrisquespdfnational_plan_nuclear_radiological_accidentspdfSoï¬er Y Schwartz D Goldberg A Henenfeld M BarDayan Y Populationevacuations in industrial accidents a review of the literature about four major eventsPrehosp Disaster Med “UNDRR The Sendai Framework for Disaster Risk Reduction “ UnitedNations Office for Disaster Risk Reduction wwwundrrpublicationsendaiframeworkdisasterriskreduction20152030WHO WHO Definition of Health World Health anisation Geneva httpwwwwhointaboutdefinitionenprinthtmlThese recommendations have already been considered in the development of the European joint roadmap for radiation protection researchImpens and Salomaa and of the strategic research agenda ofdiï¬erent European platforms notably NERIS on emergency and recovery Croua¯l The results of the SHAMISEN project have been presented and discussed in several national and international workshops and meetingsRecommendations are being disseminated to decision makers and radiation protection authorities for translation into strategy and policy aswell as to scientific medical and nonexpert audiences They are nowreferred and used as basis of the reflections and the initiatives of national and international anizations for both preparedness NuclearEnergy Agency World Health anisation International Commissionon Radiological Protection ICRP National committee for postaccident management CODIRPA in France and the management of theFukushima situation with a key role of the Japanese partners involvedin the SHAMISEN project Fukushima Medical University NagasakiUniversity Hiroshima UniversityMore broadly the approach adopted in the SHAMISEN project andits results may contribute to address other hazards including naturaldisasters industrial accidents or even pandemic crisis Similarities canbe emphasized with the Sendai Framework for Disaster Risk Reduction“ UNDRR adopted at the 3rd UN World Conference in This framework underlines the importance of improving theunderstanding of disaster risk better addressing vulnerability and hazard characteristics strengthening risk governance reinforcing accountability for risk management with development of preparednessinvolvement of stakeholders and due considerations of resilience ofhealth infrastructureFinally the pandemic crisis of COVID19 highlights a series of issuesquite similar to those addressed in the SHAMISEN project confinementversus evacuation psychosocial aspects communication and dialogueanisation of the transition phases and of course the preparation ofhealth surveillance strategies and structures of epidemiological studiesThese diï¬erent issues would benefit from crosscomparison analysisand the s presented in this special issue could certainly contributeto the reflectionDeclaration of Competing InterestThe authors declare that they have no known competing financialinterests or personal relationships that could have appeared to ‚uence the work reported in this paperAcknowledgmentsSHAMISEN was supported by the EURATOM European AtomicEnergy Community program of the European Commission in the framework of the OPERRA Project for the European RadiationResearch Area project FP7 grant agreement No The authors are grateful to all partners experts and stakeholderscontributed to theandorwho participated in the projectRecommendationsISGlobal acknowledges supportfrom the Spanish Ministry ofScience Innovation and Universities through the œCentro de ExcelenciaSevero Ochoa “ Program CEX2018000806S and supportfrom the Generalitat de Catalunya through the CERCA Program NMBUacknowledges the support of the Research Council of Norway RCNthrough its Centres of Excellence funding scheme project numberŽ Corresponding author 0c" Answer:

Thyroid_Cancer

"analyse the quality of information included in websites aimed at the public on COVID19Methods Yahoo Google and Bing search engines were browsed using selected keywords on COVID19first websites from each search engine for each keyword were evaluated Validated tools wereThe used to assess readability [Flesch Reading Ease Score FRES] usability and reliability LIDA tool andquality DISCERN instrument Nonparametric tests were used for statistical analysesResults Eightyfour eligible sites were analysed The median FRES score was range 00 Themedian LIDA usability and reliability scores were range 00 and 37range14 00 respectively Alow overall LIDA score was recorded for n of the websites The median DISCERN score 14 was found in websites The DISCERNwas range “ The DISCERN score of score was significantly associated with LIDA usability and reliability scores p and the FRES scorep Conclusion The majority of websites on COVID19 for the public had moderate to low scores with regardsto readability usability reliability and qualityPractice Implications Prompt strategies should be implemented to standardize online health informationon COVID19 during this pandemic to ensure the general public has access to good quality reliableinformationï¾ Elsevier BV All rights reserved Introductionfor The coronavirus COVID19 pandemic has become the greatestglobal health crisis of the st century [] During this pandemicthe demand information on COVID19 has skyrocketedInformation such as latest news updates on the pandemic itssymptoms prevention and mechanism of transmission are highlysought by the public [] On the other hand free access toinformation especially through social media which is accessed bythe majority [] has led to an increase in misinformation and panicassociated with COVID19 [] Although high quality healthinformation is known to be related to lower stress levels andbetter psychological health [] previous studies have shown thatonline information on many medical disorders to be of substandard quality []A previous study done on websites related to COVID19 hasreported substandard quality information that could potentiallymislead the public [] However this study has used a limitedsearch strategy and had not assessed some important areasincluding usability and reliability of the information Thereforethis topic remains a knowledge gap in COVID19 research []Therefore we conducted this study to analyse the current COVID websites targeting the general public in terms of qualityusability readability and reliability using a wide search strategyand validated instruments MethodsAbbreviations USA United States of America FRES Flesch reading ease scoreHONcode Health on the net Code of Conduct SPSS Statistical package for socialsciences NICE National Institute for Health and Care Excellence WHO WorldHealth anization Corresponding author at Department of Surgery Faculty of MedicineUniversity of Colombo PO Box Kynsey Road Colombo Western ProvinceSri LankaEmail addresses ravindrijayasinghegmailcom R Jayasingheranasigmcgmailcom S Ranasinghe umeshejayagmailcom U Jayarajahsanjeewasrgcmbaclk S SeneviratneYahoo Google and Bing were searched using the keywordsœsevere acute respiratory synœnovel coronavirusSARSCoV2 and œcoronavirus The searchdrome coronavirus2 œCOVID19was performed during the first week of May The details ofthe search strategy and the piloting process are provided in thesupplementary material File S1 []Two independent investigators with previous experience ofconducting similar studies assessed the selected websites []Prior to the assessment a pilot run was conducted to ensure101016jpec20200800107383991ï¾ Elsevier BV All rights reservedPlease cite this in press as R Jayasinghe et al Quality of online information for the general public on COVID19 Patient Educ Couns 101016jpec202008001 0cModelGPEC No of Pages R Jayasinghe et al Patient Education and Counseling xxx xxx“xxxuniformity and accuracy The information on symptoms investigations public health measures and available treatmentmodalities were collected The accuracy of the content wasassessed using the national institute for health and care excellenceNICE guidelines on COVID19 [] A rating was given as all ornone based on congruence with the guidelinesValidated instruments were used to assess the quality ofwebsites Readability was assessed using the Flesch Reading EaseScore FRES [] The LIDA Instrument 2007Version12 wasused to analyse the content and the design of the websites usingthe usability and reliability domains [] The quality of thecontent was assessed using the DISCERN questionnaire which has questions in two separate groups [] The detailed assessmentcriteria and the scoring system is included in the supplementarymaterial File S1A website was classified as governmental if it was maintainedby the country™s public health authority If managed by privateinstitutions nongovernmental anizations or voluntary institutions independent from the government they were consideredas nongovernmental The online healthrelated websites arestandardized in terms of their credibility and reliability by onlinecertification sites We chose the Health on the Net code of conductHONcode which is the oldest and widely used out of the qualityevaluation tools available []Data analysis was performed using SPSS Version20 softwareand the associations were determined with non parametric testsA pvalue of was considered statistically significant ResultsOf the retrieved websites were excluded and thein the analysis Theremaining websites were characteristics of the websites are mentioned in Table included Half were governmental websites and only n were HON accredited websites The median FRES was range 00 10th12th grade readability level which is classified asfairly difficult to read Only three websites had a readabilityscore of above equivalent to 7th grade which is therecommended standardThe overall median LIDA score was range “ whilethe median LIDA usability and reliability scores were range 00 and range 00 respectively The median DISCERNscore was range “ which classifies websites as being ofœfair quality Excellent “ Good “ Fair “Poor “ Very poor “ However the top websitesTable A were of excellent qualityTable Website characteristicsWebsite Characteristics Frequency PercentageUsability Governmental websites Notforprofit and private websites HONcode accredited Readability score of above equivalent to 7th grade Readability Date of publication stated References mentioned Disclosure statement by authors Infographics Moderate Low Moderate 00 Low score Moderate 00 Low Used Textonly Reliability Table Correlation between DISCERN scores and other factorsDISCERN SCORELow Mean High 00 Mean Range 00 00 00 00 Range 00 00 00 00 N P valueP0001P0001P0001P P P LIDA Usability LIDA Reliability LIDA Overall FRES Score Government HON Certification No N No Yes Yes Significant correlations were observed between the DISCERNscore and the overall LIDA score as well as LIDA usability andreliability scores Table p HONcode certified websitessites obtained significantly higher DISCERN scores p Pertaining to the currency of information only publishers stated the date of the publication Most websites n did not declare the sources of evidence This was furtherestablished by the median reliability score of Nevertheless the authors have included a disclosure statement in mostn websitesœlowFigures A1 and A2 summarize the rating of websites onindividual criteria assessed by the DISCERN tool The specificinformation provided regarding COVID19 is shown in Fig More than half of the websites failed to discuss the treatmentoptions available n benefits or risks n and effects of no treatment n Furthermore potentialcomplications and prognosis were stated only in and websites respectively Discussion and conclusion DiscussionThis study has shown that still most of the websites on onlinehealth information on COVID19 are of suboptimal quality exceptfor a few credible sources of goodquality health informationNevertheless the websites ranked among the top according tothe DISCERN score Table A2 had high scores indicating thepotential for publishing credible highquality information onlinewhich would benefit the publicin turn causes panic which ranges Misinformation is a major concern during this pandemic aspeople fail to spend adequate time to critically analyse the onlineinformation This fromhoarding medical supplies to panic shopping and using drugswithout prescription with negative social and medical consequences [] Therefore measures implemented to ensure the qualityand accuracy of online information by the responsible authoritiesmay help negate these adverse consequencesinformation Stating the methods of content production with names of thecontributing authors may help increase the credibility of onlinehealth information while displaying the date of the publicationprovides an idea of the currency of the information Absence ofin over half of the websites was a majorsuch drawback especially for COVID19 where new information isgenerated almost daily Health authorities should therefore ensurethat the patient information websites provide the above information and certify websites based on such details so that the publiccan get information from trusted sources []Most users of the worldwide web only have an average level ofeducation and reading skills [] Guidance from the NationalPlease cite this in press as R Jayasinghe et al Quality of online information for the general public on COVID19 Patient Educ Couns 101016jpec202008001 0cModelGPEC No of Pages R Jayasinghe et al Patient Education and Counseling xxx xxx“xxx Fig Website characteristics evaluated outside the DISCERN toolInstitute of Health NIH had shown that the readability should bebelow the level of seventh grade for the lay public to adequatelyunderstand the content [] However the median readabilitylevel was found to be equivalent to 10th12th grade readabilitySuch complexities with the readability of information mayincrease the risk of misunderstandings or misinterpretation Usingshort sentences in writing using the active voice using 12point orlarger font size using illustrations and nontextual media asappropriate and accompanying explanations with examples wouldbe helpful to overcome this problem []So far only a limited number of studies have been done to assessthe quality of health information websites related to COVID19 Thestudy by CuanBaltazar et al prior to February reported poorquality information with approximately of included websiteswith low DISCERN scores [] Our study done three months latershows similar results with only a minimal improvement in thequality of information Furthermore the Cuan Baltazar study hadseveral limitations which includes the limited search strategy andnoninclusion of key quality parameters including readabilityFurthermore out of the sites they had includedwere online news sites that are not considered as patientinformation websites In that study the HONcode seal waspresent only in n websites whereas in our study ofthe sites were HONcode certifiedThere were several limitations in this study Although mostpopular search engines were used in this study under defaultsettings they may produce variable results depending on manyfactors including geographical location and popularity of websitesat a given point of time The algorithms unique to those searchengines are subjected to constant change and therefore the exactresults of our study may not be reproducible However we believethe general patterns observed in our study are validproviding health information to the general public are to be ofsubstandard quality Practice implicationsTo improve the credibility of the content the websites shouldstate methods of content production and display the date of thepublication to give an idea about the currency of the informationTo improve the readability of the content the websites shouldincorporate more nontextual media write in short sentencesusing the activevoice and use larger font sizes The patientinformation websites should display scores of reliability qualityand readability as a guidance for its users Furthermore it is vitalfor medical regulatory authorities and the government to imposeregulations to ensure quality and to prevent the spread ofmisinformationAvailability of data and materialsOn reasonable request from the corresponding author the dataused in the above study can be made availableEthics approval and consent to participateUnnecessary in this type of studyInformed consent and patient detailsNot applicable in this type of studyFundingNone ConclusionCRediT authorship contribution statementThis study has shown the quality readability usability andreliability of the information on COVID19 on majority of websitesRavindri Jayasinghe Conceptualization Methodology Datacuration Writing original draft Visualization InvestigationPlease cite this in press as R Jayasinghe et al Quality of online information for the general public on COVID19 Patient Educ Couns 101016jpec202008001 0cModelGPEC No of Pages R Jayasinghe et al Patient Education and Counseling xxx xxx“xxxValidation Formal analysis Resources Sonali RanasingheConceptualization Methodology Data curation Writing originaldraft Visualization Investigation Validation Formal analysisResources Umesh Jayarajah Conceptualization MethodologyData curation Writing original draft Visualization InvestigationValidation Formal analysis Resources Sanjeewa SeneviratneConceptualization Methodology Writing review editingSupervision Project administrationDeclaration of Competing InterestThe authors report no declarations of interestAcknowledgementsNone declaredAppendix A Supplementary dataSupplementary material related to this can be found in101016jversion at online the pec202008001References[] IJ Bes do Nascimento N Cacic HM Abdulazeem Novel Coronavirusinfection COVID19 in humans a scoping review and metaanalysis JClinical Med [] HT Le DN Nguyen AS Beydoun XTT Le TT Nguyen QT Pham NTK Ta QT Nguyen AN Nguyen MT Hoang Demand for health information onCOVID19 among Vietnamese Int J Environ Res Public Health [] C Wang R Pan X Wan Y Tan L Xu RS McIntyre FN Choo B Tran R Ho VK Sharma A longitudinal study on the mental health of general populationduring the COVID19 epidemic in China Brain Behav Immun [] CS Ho CY Chee RC Ho Mental health strategies to combat the psychologicalimpact of COVID19 beyond paranoia and panic Ann Acad Med Singapore “[] C Wang R Pan X Wan Y Tan L Xu CS Ho RC Ho Immediate psychologicalresponses and associated factors during the initial stage of the coronavirus disease COVID19 epidemic among the general population inChina Int J Environ Res Public Health [] RH Waidyasekera U Jayarajah DN Samarasekera Quality and scientificaccuracy of patientoriented information on the internet on minimallyinvasive surgery for colorectal cancer Health Policy Technol “[] R Jayasinghe S Ranasinghe U Jayarajah S Seneviratne Quality of patientoriented webbased information on oesophageal cancer J Cancer Educ In press[] JY CuanBaltazar MJ MuñozPerez C RobledoVega MF PÃrezZepeda ESotoVega Misinformation of COVID19 on the internet Infodemiology studyJMIR Public Health Surveill 2020e18444[] BX Tran GH Ha LH Nguyen GT Vu MT Hoang HT Le CA Latkin CS HoR Ho Studies of novel coronavirus disease COVID19 pandemic a globalanalysis of literature Int J Environ Res Public Health [] G Eysenbach C Köhler How do consumers search for and appraise healthinformation on the world wide web Qualitative study using focus groupsusability tests and indepth interviews Brit Med J “[] AS Prasanth U Jayarajah R Mohanappirian SA Seneviratne Assessment ofthe quality of patientoriented information over internet on testicular cancerBMC Cancer [] V Udayanga U Jayarajah SD Colonne SA Seneviratne Quality of thepatientoriented information on thyroid cancer in the internet Health PolicyTechnol [] National Institute for Health and Care Excellence Coronavirus COVID19 Accessed April wwwniceukcovid19[] Readable The Flesch Reading Ease and FleschKincaid Grade Level Accessed February readablecomblogthefleschreadingeaseandfleschkincaidgradelevel[] Minervation The Minervation Validation Instrument for Healthcare WebsitesLIDA Tool Accessed February httpwwwminervationcomwpcontentuploads201104MinervationLIDAinstrumentv12pdf[] Minervation Is the Lida Website Assessment Tool Valid Accessed February httpwwwminervationcomdoeslidawork[] Discern Online The DISCERN Instrument Accessed February httpwwwdiscernukdiscern_instrumentphp[] E Fahy R Hardikar A Fox S Mackay Quality of patient health information onthe Internet reviewing a complex and evolving landscape Australas Med J “[] J Kluger As Disinfectant Use Soars to Fight Coronavirus So Do AccidentalPoisonings Accessed April timecom5824316coronavirusdisinfectantpoisoning[] BX Tran AK Dang PK Thai HT Le XTT Le TTT Do TH Nguyen HQPham HT Phan GT Vu Coverage of health information by different sourcesin communities implication for COVID19 epidemic response Int J EnvironRes Public Health [] National Institutes of Health How to Write EasyToRead Health Materials Accessed April wwwscribdcomdocument261199628HowtoWriteEasyToReadHealthMaterialsMedlinePlus[] DM D™Alessandro P Kingsley J JohnsonWest The readability of pediatricpatient education materials on the world wide web Arch Pediatr AdolescMed “Please cite this in press as R Jayasinghe et al Quality of online information for the general public on COVID19 Patient Educ Couns 101016jpec202008001 0c"

cancer242

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: "analyse the quality of information included in websites aimed at the public on COVID19Methods Yahoo Google and Bing search engines were browsed using selected keywords on COVID19first websites from each search engine for each keyword were evaluated Validated tools wereThe used to assess readability [Flesch Reading Ease Score FRES] usability and reliability LIDA tool andquality DISCERN instrument Nonparametric tests were used for statistical analysesResults Eightyfour eligible sites were analysed The median FRES score was range 00 Themedian LIDA usability and reliability scores were range 00 and 37range14 00 respectively Alow overall LIDA score was recorded for n of the websites The median DISCERN score 14 was found in websites The DISCERNwas range “ The DISCERN score of score was significantly associated with LIDA usability and reliability scores p and the FRES scorep Conclusion The majority of websites on COVID19 for the public had moderate to low scores with regardsto readability usability reliability and qualityPractice Implications Prompt strategies should be implemented to standardize online health informationon COVID19 during this pandemic to ensure the general public has access to good quality reliableinformationï¾ Elsevier BV All rights reserved Introductionfor The coronavirus COVID19 pandemic has become the greatestglobal health crisis of the st century [] During this pandemicthe demand information on COVID19 has skyrocketedInformation such as latest news updates on the pandemic itssymptoms prevention and mechanism of transmission are highlysought by the public [] On the other hand free access toinformation especially through social media which is accessed bythe majority [] has led to an increase in misinformation and panicassociated with COVID19 [] Although high quality healthinformation is known to be related to lower stress levels andbetter psychological health [] previous studies have shown thatonline information on many medical disorders to be of substandard quality []A previous study done on websites related to COVID19 hasreported substandard quality information that could potentiallymislead the public [] However this study has used a limitedsearch strategy and had not assessed some important areasincluding usability and reliability of the information Thereforethis topic remains a knowledge gap in COVID19 research []Therefore we conducted this study to analyse the current COVID websites targeting the general public in terms of qualityusability readability and reliability using a wide search strategyand validated instruments MethodsAbbreviations USA United States of America FRES Flesch reading ease scoreHONcode Health on the net Code of Conduct SPSS Statistical package for socialsciences NICE National Institute for Health and Care Excellence WHO WorldHealth anization Corresponding author at Department of Surgery Faculty of MedicineUniversity of Colombo PO Box Kynsey Road Colombo Western ProvinceSri LankaEmail addresses ravindrijayasinghegmailcom R Jayasingheranasigmcgmailcom S Ranasinghe umeshejayagmailcom U Jayarajahsanjeewasrgcmbaclk S SeneviratneYahoo Google and Bing were searched using the keywordsœsevere acute respiratory synœnovel coronavirusSARSCoV2 and œcoronavirus The searchdrome coronavirus2 œCOVID19was performed during the first week of May The details ofthe search strategy and the piloting process are provided in thesupplementary material File S1 []Two independent investigators with previous experience ofconducting similar studies assessed the selected websites []Prior to the assessment a pilot run was conducted to ensure101016jpec20200800107383991ï¾ Elsevier BV All rights reservedPlease cite this in press as R Jayasinghe et al Quality of online information for the general public on COVID19 Patient Educ Couns 101016jpec202008001 0cModelGPEC No of Pages R Jayasinghe et al Patient Education and Counseling xxx xxx“xxxuniformity and accuracy The information on symptoms investigations public health measures and available treatmentmodalities were collected The accuracy of the content wasassessed using the national institute for health and care excellenceNICE guidelines on COVID19 [] A rating was given as all ornone based on congruence with the guidelinesValidated instruments were used to assess the quality ofwebsites Readability was assessed using the Flesch Reading EaseScore FRES [] The LIDA Instrument 2007Version12 wasused to analyse the content and the design of the websites usingthe usability and reliability domains [] The quality of thecontent was assessed using the DISCERN questionnaire which has questions in two separate groups [] The detailed assessmentcriteria and the scoring system is included in the supplementarymaterial File S1A website was classified as governmental if it was maintainedby the country™s public health authority If managed by privateinstitutions nongovernmental anizations or voluntary institutions independent from the government they were consideredas nongovernmental The online healthrelated websites arestandardized in terms of their credibility and reliability by onlinecertification sites We chose the Health on the Net code of conductHONcode which is the oldest and widely used out of the qualityevaluation tools available []Data analysis was performed using SPSS Version20 softwareand the associations were determined with non parametric testsA pvalue of was considered statistically significant ResultsOf the retrieved websites were excluded and thein the analysis Theremaining websites were characteristics of the websites are mentioned in Table included Half were governmental websites and only n were HON accredited websites The median FRES was range 00 10th12th grade readability level which is classified asfairly difficult to read Only three websites had a readabilityscore of above equivalent to 7th grade which is therecommended standardThe overall median LIDA score was range “ whilethe median LIDA usability and reliability scores were range 00 and range 00 respectively The median DISCERNscore was range “ which classifies websites as being ofœfair quality Excellent “ Good “ Fair “Poor “ Very poor “ However the top websitesTable A were of excellent qualityTable Website characteristicsWebsite Characteristics Frequency PercentageUsability Governmental websites Notforprofit and private websites HONcode accredited Readability score of above equivalent to 7th grade Readability Date of publication stated References mentioned Disclosure statement by authors Infographics Moderate Low Moderate 00 Low score Moderate 00 Low Used Textonly Reliability Table Correlation between DISCERN scores and other factorsDISCERN SCORELow Mean High 00 Mean Range 00 00 00 00 Range 00 00 00 00 N P valueP0001P0001P0001P P P LIDA Usability LIDA Reliability LIDA Overall FRES Score Government HON Certification No N No Yes Yes Significant correlations were observed between the DISCERNscore and the overall LIDA score as well as LIDA usability andreliability scores Table p HONcode certified websitessites obtained significantly higher DISCERN scores p Pertaining to the currency of information only publishers stated the date of the publication Most websites n did not declare the sources of evidence This was furtherestablished by the median reliability score of Nevertheless the authors have included a disclosure statement in mostn websitesœlowFigures A1 and A2 summarize the rating of websites onindividual criteria assessed by the DISCERN tool The specificinformation provided regarding COVID19 is shown in Fig More than half of the websites failed to discuss the treatmentoptions available n benefits or risks n and effects of no treatment n Furthermore potentialcomplications and prognosis were stated only in and websites respectively Discussion and conclusion DiscussionThis study has shown that still most of the websites on onlinehealth information on COVID19 are of suboptimal quality exceptfor a few credible sources of goodquality health informationNevertheless the websites ranked among the top according tothe DISCERN score Table A2 had high scores indicating thepotential for publishing credible highquality information onlinewhich would benefit the publicin turn causes panic which ranges Misinformation is a major concern during this pandemic aspeople fail to spend adequate time to critically analyse the onlineinformation This fromhoarding medical supplies to panic shopping and using drugswithout prescription with negative social and medical consequences [] Therefore measures implemented to ensure the qualityand accuracy of online information by the responsible authoritiesmay help negate these adverse consequencesinformation Stating the methods of content production with names of thecontributing authors may help increase the credibility of onlinehealth information while displaying the date of the publicationprovides an idea of the currency of the information Absence ofin over half of the websites was a majorsuch drawback especially for COVID19 where new information isgenerated almost daily Health authorities should therefore ensurethat the patient information websites provide the above information and certify websites based on such details so that the publiccan get information from trusted sources []Most users of the worldwide web only have an average level ofeducation and reading skills [] Guidance from the NationalPlease cite this in press as R Jayasinghe et al Quality of online information for the general public on COVID19 Patient Educ Couns 101016jpec202008001 0cModelGPEC No of Pages R Jayasinghe et al Patient Education and Counseling xxx xxx“xxx Fig Website characteristics evaluated outside the DISCERN toolInstitute of Health NIH had shown that the readability should bebelow the level of seventh grade for the lay public to adequatelyunderstand the content [] However the median readabilitylevel was found to be equivalent to 10th12th grade readabilitySuch complexities with the readability of information mayincrease the risk of misunderstandings or misinterpretation Usingshort sentences in writing using the active voice using 12point orlarger font size using illustrations and nontextual media asappropriate and accompanying explanations with examples wouldbe helpful to overcome this problem []So far only a limited number of studies have been done to assessthe quality of health information websites related to COVID19 Thestudy by CuanBaltazar et al prior to February reported poorquality information with approximately of included websiteswith low DISCERN scores [] Our study done three months latershows similar results with only a minimal improvement in thequality of information Furthermore the Cuan Baltazar study hadseveral limitations which includes the limited search strategy andnoninclusion of key quality parameters including readabilityFurthermore out of the sites they had includedwere online news sites that are not considered as patientinformation websites In that study the HONcode seal waspresent only in n websites whereas in our study ofthe sites were HONcode certifiedThere were several limitations in this study Although mostpopular search engines were used in this study under defaultsettings they may produce variable results depending on manyfactors including geographical location and popularity of websitesat a given point of time The algorithms unique to those searchengines are subjected to constant change and therefore the exactresults of our study may not be reproducible However we believethe general patterns observed in our study are validproviding health information to the general public are to be ofsubstandard quality Practice implicationsTo improve the credibility of the content the websites shouldstate methods of content production and display the date of thepublication to give an idea about the currency of the informationTo improve the readability of the content the websites shouldincorporate more nontextual media write in short sentencesusing the activevoice and use larger font sizes The patientinformation websites should display scores of reliability qualityand readability as a guidance for its users Furthermore it is vitalfor medical regulatory authorities and the government to imposeregulations to ensure quality and to prevent the spread ofmisinformationAvailability of data and materialsOn reasonable request from the corresponding author the dataused in the above study can be made availableEthics approval and consent to participateUnnecessary in this type of studyInformed consent and patient detailsNot applicable in this type of studyFundingNone ConclusionCRediT authorship contribution statementThis study has shown the quality readability usability andreliability of the information on COVID19 on majority of websitesRavindri Jayasinghe Conceptualization Methodology Datacuration Writing original draft Visualization InvestigationPlease cite this in press as R Jayasinghe et al Quality of online information for the general public on COVID19 Patient Educ Couns 101016jpec202008001 0cModelGPEC No of Pages R Jayasinghe et al Patient Education and Counseling xxx xxx“xxxValidation Formal analysis Resources Sonali RanasingheConceptualization Methodology Data curation Writing originaldraft Visualization Investigation Validation Formal analysisResources Umesh Jayarajah Conceptualization MethodologyData curation Writing original draft Visualization InvestigationValidation Formal analysis Resources Sanjeewa SeneviratneConceptualization Methodology Writing review editingSupervision Project administrationDeclaration of Competing InterestThe authors report no declarations of interestAcknowledgementsNone declaredAppendix A Supplementary dataSupplementary material related to this can be found in101016jversion at online the pec202008001References[] IJ Bes do Nascimento N Cacic HM Abdulazeem Novel Coronavirusinfection COVID19 in humans a scoping review and metaanalysis JClinical Med [] HT Le DN Nguyen AS Beydoun XTT Le TT Nguyen QT Pham NTK Ta QT Nguyen AN Nguyen MT Hoang Demand for health information onCOVID19 among Vietnamese Int J Environ Res Public Health [] C Wang R Pan X Wan Y Tan L Xu RS McIntyre FN Choo B Tran R Ho VK Sharma A longitudinal study on the mental health of general populationduring the COVID19 epidemic in China Brain Behav Immun [] CS Ho CY Chee RC Ho Mental health strategies to combat the psychologicalimpact of COVID19 beyond paranoia and panic Ann Acad Med Singapore “[] C Wang R Pan X Wan Y Tan L Xu CS Ho RC Ho Immediate psychologicalresponses and associated factors during the initial stage of the coronavirus disease COVID19 epidemic among the general population inChina Int J Environ Res Public Health [] RH Waidyasekera U Jayarajah DN Samarasekera Quality and scientificaccuracy of patientoriented information on the internet on minimallyinvasive surgery for colorectal cancer Health Policy Technol “[] R Jayasinghe S Ranasinghe U Jayarajah S Seneviratne Quality of patientoriented webbased information on oesophageal cancer J Cancer Educ In press[] JY CuanBaltazar MJ MuñozPerez C RobledoVega MF PÃrezZepeda ESotoVega Misinformation of COVID19 on the internet Infodemiology studyJMIR Public Health Surveill 2020e18444[] BX Tran GH Ha LH Nguyen GT Vu MT Hoang HT Le CA Latkin CS HoR Ho Studies of novel coronavirus disease COVID19 pandemic a globalanalysis of literature Int J Environ Res Public Health [] G Eysenbach C Köhler How do consumers search for and appraise healthinformation on the world wide web Qualitative study using focus groupsusability tests and indepth interviews Brit Med J “[] AS Prasanth U Jayarajah R Mohanappirian SA Seneviratne Assessment ofthe quality of patientoriented information over internet on testicular cancerBMC Cancer [] V Udayanga U Jayarajah SD Colonne SA Seneviratne Quality of thepatientoriented information on thyroid cancer in the internet Health PolicyTechnol [] National Institute for Health and Care Excellence Coronavirus COVID19 Accessed April wwwniceukcovid19[] Readable The Flesch Reading Ease and FleschKincaid Grade Level Accessed February readablecomblogthefleschreadingeaseandfleschkincaidgradelevel[] Minervation The Minervation Validation Instrument for Healthcare WebsitesLIDA Tool Accessed February httpwwwminervationcomwpcontentuploads201104MinervationLIDAinstrumentv12pdf[] Minervation Is the Lida Website Assessment Tool Valid Accessed February httpwwwminervationcomdoeslidawork[] Discern Online The DISCERN Instrument Accessed February httpwwwdiscernukdiscern_instrumentphp[] E Fahy R Hardikar A Fox S Mackay Quality of patient health information onthe Internet reviewing a complex and evolving landscape Australas Med J “[] J Kluger As Disinfectant Use Soars to Fight Coronavirus So Do AccidentalPoisonings Accessed April timecom5824316coronavirusdisinfectantpoisoning[] BX Tran AK Dang PK Thai HT Le XTT Le TTT Do TH Nguyen HQPham HT Phan GT Vu Coverage of health information by different sourcesin communities implication for COVID19 epidemic response Int J EnvironRes Public Health [] National Institutes of Health How to Write EasyToRead Health Materials Accessed April wwwscribdcomdocument261199628HowtoWriteEasyToReadHealthMaterialsMedlinePlus[] DM D™Alessandro P Kingsley J JohnsonWest The readability of pediatricpatient education materials on the world wide web Arch Pediatr AdolescMed “Please cite this in press as R Jayasinghe et al Quality of online information for the general public on COVID19 Patient Educ Couns 101016jpec202008001 0c" Answer:

Thyroid_Cancer

"Interferon COVID19SARSCOV2IranIn this study efficacy and safety of interferon Interferon beta1-b in the treatment of patients with severe COVID19 wereevaluatedAmong an label randomized clinical trial adult patients ‰¥ years old with severe COVID19 wererandomly assigned to the IFN group or the control group Patients in the IFN group received Interferon beta1-b mcg subcutaneously every other day for two consecutive weeks along with the national protocol medicationswhile in the control group patients received only the national protocol medications lopinavirritonavir oratazanavirritonavir plus hydroxychloroquine for “ days The primary outcome of the study was time toclinical improvement Secondary outcomes were inhospital complications and 28daymortalityBetween April and May patients were enrolled and finally patients in each group completed the study Time to clinical improvment in the IFN group was significantly shorter than the control group[“ vs “ days respectively p HR CI “] At day the percentageof discharged patients was and in the IFN and control groups respectively OR CI“ p ICU admission rate in the control group was significantly higher than the IFN group vs p The duration of hospitalization and ICU stay were not significantly differentbetween the groups Allcause 28day mortality was and in the IFN and control groups respectively p Interferon beta1-b was effective in shortening the time to clinical improvement without serious adverse events inpatients with severe COVID19 Furthermore admission in ICU and need for invasive mechanical ventilationdecreased following administration of Interferon beta1-b Although 28day mortality was lower in the IFN group furtherrandomized clinical trials with large sample size are needed for exact estimation of survival benefit of Interferon beta1-b IntroductionCoronavirus disease CoVID19 was reported from Wuhan forthe first time in late December Causing severe acute respiratorysyndrome coronavirus 2SARSCoV2 [] it rapidly spread throughoutthe world to the extent that the World Health anization WHOstated it as pandemic in March [] Until July more than million confirmed cases of CoVID19 were reported worldwideFurthermore more than deaths were recorded []Until now there is no definite antiviral treatment for CoVID19 andattempts continue for finding effective treatments worldwide Howeverfrom the beginning of the pandemic various treatments such as antiretrovirals antimalaria agents favipiravir remdesivir and corticosteroids immunoglobulin and cytokine blockers as adjunctive therapieswere suggested for the treatment of CoVID19 [] Except for the remdesivir which has had acceptable results the efficacy of other drugshas not been significant on the outcomes of the patients with CoVID19[“]Interferons IFNs have a key role in defense against viral infectionsas a component of innate immune system [] Invitro activity of IFN Ž Corresponding author at Department of Pharmacotherapy Tehran University of Medical Sciences Tehran IranEmail addresses khalilihtumsacir Khalilihsinatumsacir H Khalili101016jintimp2020106903Received July Received in revised form July Accepted August Available online August Elsevier BV All rights reserved 0cInternational Immunopharmacology prophylaxis deep vein thrombosis treatment of electrolyte disordersand antibiotic therapy were considered according to the hospital protocols The duration of the study was two weeks A 4week followupperiod was considered for all patientsPatients™ demographic data baseline diseases symptoms at the timeof disease presentation vital signs and laboratory data at the time ofhospital admission were recorded Patients were daily monitored interms of changes in the vital signs hemodynamic parameters oxygenation status laboratory data and treatment strategies Clinical statusof the patients was assessed by the sixcategory ordinal scale at days and of the randomization [] Need for supplemental oxygentherapy and also invasive or noninvasive respiratory supports wereevaluated regularly OutcomesTime to clinical improvement was considered as primary outcome ofstudy Clinical improvement was defined as improvement of at leasttwo points from the baseline status on the sixcategory ordinal scale[] This scale contains the subsequent categories death hospital admission requiring invasive mechanical ventilation hospitaladmission requiring noninvasive positive pressure ventilation hospital admission requiring oxygen hospital admission not requiring oxygen discharge Secondary outcomes were clinical statusof patients at day and ICU admission and intubation rateslength of hospitalization and ICU stay and 28day mortalitySide effects related to IFN therapy and other adverse events duringthe study period were monitored and recorded as the safety outcomesCategorization of adverse events was done according to the commonterminology criteria for adverse events CTCAE National Institutes ofHealth and National Cancer Institute Also serious complications during the hospitalization course such asacute respiratory distress syndrome ARDS nosocomial infectionsseptic shock acute kidney injury AKI and acute hepatic injury AHIwere considered Statistical analysis and randomizationContinuous variables are demonstrated as median interquartilerange IQR and categorical variables as frequencies and percentagesContinuous variables were compared between the groups by MannWhitney U test The Fisher™s exact test was applied for comparison ofcategorical variablesThe Hazard Ratio HR and CI for clinical improvement wereestimated by Cox proportional hazards regression analysis The effect ofischemic heart disease lymphocyte count Aspartate aminotransferaseAST and Creactive protein CRP on the primary outcome was evaluated by the adjusted Cox regression models as potential confoundingfactors Time to clinical improvement was estimated by KaplanMeierplot and compared with a logrank test All statistical analysis was doneby SPSS software version Time to clinical improvement was estimated to be approximately days and sample size was calculated by following equationH Rahmani et alhas been shown against severe acute respiratory syndrome coronavirusSARSCoV and Middle East respiratory syndrome coronavirus MERSCoV [“] Although IFN was used less than IFN α for the treatment of SARSCOV and MERSCoV in human studies it was effective inthe treatment of MERSCoV in retrospective studies and case series[“] The efficacy of Interferon beta1-b is being assessed in the treatment ofMERS in a randomized clinical trial [] According to the presence ofthis evidence IFN was considered as a promising option for thetreatment of CoVID19In this label randomized clinical trial efficacy and safety ofInterferon beta1-b in the treatment of patients with severe CoVID19 were assessed Materials and methods Study designThis label randomized clinical trial was designed to evaluatethe efficacy and safety of Interferon beta1-b in the treatment of patients withCoVID19 Patients with severe CoVID19 who were hospitalized duringApril to May in Imam Khomeini Hospital Center one ofthe largest referral hospitals in Tehran Iran were includedThe protocol of the study was approved by Ethics Committee ofTehran UniversityReferencenumberIRTUMSVCRREC13981053 Furthermore the study was registeredas a clinical trial register ID IRCT20100228003449N27 The studyprotocol was described for participants and written informed consentswere obtained from all patients or their firstdegree family members Eligibility criteriaof MedicalSciencesSARSCoV2 in patients™ nasopharyngeal swabs was detected usingRealTime Polymerase Chain Reaction RTPCR Total RNA extractionwas done applying Viral Nucleic Acid Extraction kit Cat No YVN50YVN100 from RBC Bioscience Taipei Taiwan The Novel Coronavirus2019nCOV Nucleic Acid Diagnostic Kit PCRFluorescence Probingof Sansure Biotech S3102E Changsha China was used for RTPCRAdult patients ‰¥ years old with positive PCR and clinicalsymptomssigns of pneumonia including dyspnea cough and feverperipheral oxygen saturation SPO2 ‰ in ambient air or arterialinspired oxygen PaO2oxygen partial pressure to fractionalFiO2 or SPO2FiO2 and lung involvement in chestimaging were included These criteria indicated severe form of thedisease [] At baseline patients with serious allergic reactions to IFNhistory of suicide thoughts and attempts alanine amino transferaseALT × the upper limit of the normal range uncontrolled underlying diseases such as neuropsychiatric disorders thyroid disorderscardiovascular diseases and also pregnant and lactating women werenot includedRecruitment was considered during the first 48hour of the hospitaladmission During the study period patients who received less than doses of Interferon beta1-b were excluded If patients were discharged beforefulfilment of the treatment course the treatment was applied at home ProceduresEligible patients were recruited in the IFN group or the controlgroup according to the permuted block randomization Patients in theIFN group received Interferon beta1-b along with the national protocol medications while in the control group patients received only the nationalprotocol medications Interferon beta1-b Ziferon® Zist Daru Daneh Co Iranwas administrated as mcg subcutaneously every other day for twoconsecutive weeks The national protocol consisted lopinavirritonavir mg BD or atazanavirritonavir mg daily plushydroxychloroquine mg BD in first day and then mg BD for“ days Other supportive cares such as fluid therapy stress ulcernkzz1122211222nn121512005015Z19612Z1041 0cH Rahmani et alInternational Immunopharmacology Fig Consort flowchart of the studyAccording to the above equation at least patients in each groupwere expected to make a difference of days in time to clinical improvement with power of Patients were randomly recruited to the IFN group or the control group The method of randomizationwas the permuted block randomization patients per block A biostatistician who was not involved in patients™ care did this process Results PatientsA total of patients were screened Of them patients did nothave the eligibility criteria of study and patients were referred fromanother hospital Three and four patients withdrew the consent duringthe study in the IFN group and control groups respectively Four patients did not adhere to IFN injection after second or third dose Alsothree patients in the control group were enrolled in another trialFinally patients in each group completed the study Fig The median IQR age of patients was “ years and of them were male No significant difference in terms of the patients™demographic data was detected between the groups The most commoncomorbidities were hypertension diabetes mellitus and ischemic heartdisease Dyspnea fever and cough were the most frequent symptoms atthe time of hospital admission The median IQR time from onset of thesymptoms to hospital admission was “ and “ days in the IFNgroup and control groups respectively The time from onset of thesymptoms to randomization was not statistically significant betweenthe groups All of patients required respiratory support at the time ofrandomization Oxygenation through facemask was required for morethan percent of patients None of the patients in both groups wereintubated at baseline Table Vital signs and laboratory data of patients at the time of recruitment were comparable between the groupsTable During the hospitalization course oxygen saturation droppedin and of patients in the IFN and control groups respectively All of those patients were intubated At least one antibioticwas administrated for and of patients in the IFN groupand control groups respectively Methylprednisolone was administeredfor of patients in the IFN group and of patients in the4Power085 0cH Rahmani et alTable Baseline characteristics of patientsParameter Median IQR or n AgeSexMaleFemaleComorbid conditions nHypertensionDiabetes mellitusIschemic heart diseaseAsthmaCOPDMalignancyTransplantationSymptoms at admission nDyspneaFeverCoughChillsDuration of symptoms before admissionmedian IQR daysTime from symptom onset torandomization median IQR daysSix category scale at day of intervention3hospital admission requiring highflownasal cannula or noninvasivemechanical ventilationsupplemental oxygen hospital admission requiringInterferon groupn “ ““Control groupn “ ““control group The dose of methylprednisolone was mg daily for days Methylprednisolone was considered during the cytokine orhyperinflammation phase days “ of onset of the symptoms Approximately and of patients in the INF and control groupsneeded vasopressors during the hospitalization course respectivelyTable Primary outcomesThe time to clinical improvement in the IFN group was significantlyshorter than the control group [“ vs “ days respectivelyp ] Table Moreover the Cox proportional hazards regression analysis showed that time difference to clinical improvement wasstatistically significant between the groups HR CI“ Fig Then the model was adjusted for the confoundingInternational Immunopharmacology Interferon groupn Control groupn Table Respiratory support and medicationsParameter n Respiratory supportNasal cannulaFace maskNIPPVIMVAntibiotics mer em piperacillintazobactam ceftriaxone FQsvancomycin azithromycin andColistin n CorticosteroidsVitamin CVasopressorsDiphenhydramineCardiovascular drugsStatinsARBsBetablockersACEIsNIPPV noninvasive positive pressure ventilation IMV invasive mechanicalventilation FQs fluoroquinolones ARB Angiotensin Π Receptor Blocker ACEIangiotensin converting enzyme inhibitorfactors and similar results were seen HR CI “ Secondary outcomesAccording to the six category scale and of patientswere discharged in the IFN and the control groups at day respectivelyOR CI “ p Only one patient in thecontrol group died at day Also at this time and patients wereintubated in the IFN and control groups respectively At day thepercentage of discharged patients reached to and in theIFN and control groups respectively OR CI “p Furthermore the number of deaths increased to and patients the IFN and control groups respectively Finally at day ofinclusion the proportion of discharged patients were in the IFNgroup and in the control group OR CI “p At this time ICU admission rate in the control group wassignificantly higher than the IFN group vs p Moreover more patients in the control group needed invasive mechanical ventilation compared with the IFN group but the rate was notstatistically different p Although length of hospitalization wasTable Patients™ vital signs and laboratory data at the time of hospital admissionParameter Median IQRTemperature °CHeart rate beats minuteRespiratory rate breathsminSystolic blood pressure mm HgSPO2 Laboratory dataWhite Blood Cell cells μlAcute Lymphocyte count cellsμlHemoglobin gdlPlatelet count cells × 103μlBlood Urea Nitrogen mgdlCreatinine mgdlAspartate aminotransferase ulAlanine aminotransferase ulAlkaline phosphatase ulTotal bilirubinmgdlCreactive protein mgdlErythrocyte sedimentation rate mmhLactate dehydrogenase ulInterferon group n ““““““““““““““““““Control group n ““““““““““““““““““ 0cH Rahmani et alInternational Immunopharmacology Table Outcomes and complicationsParameter Median IQR or n Time to clinical response medianIQR daysICU admission n Intubation requirementLength of stay in ICU days median IQR daysLength of stay in hospital days median IQR daysAllcause mortality at day Six category scale at day of intervention Death Hospital admission requiring invasive mechanical ventilation Hospital admission requiring highflow nasal cannula or noninvasive mechanical ventilation Hospital admission requiring supplemental oxygen Hospital admission not requiring supplemental oxygen DischargeSix category scale at day of intervention Death Hospital admission requiring invasive mechanical ventilation Hospital admission requiring highflow nasal cannula or noninvasive mechanical ventilation Hospital admission requiring supplemental oxygen Hospital admission not requiring supplemental oxygen DischargeSix category scale at day of intervention Death Hospital admission requiring invasive mechanical ventilation Hospital admission requiring highflow nasal cannula or noninvasive mechanical ventilation Hospital admission requiring supplemental oxygen Hospital admission not requiring supplemental oxygen DischargeInterferon group n “““Control group n “ ““pvalueOR95 CI“““shorter [ “ days in the IFN group vs “ days in thecontrol group p ] but length of ICU stay was not significantlydifferent between the groups Allcause 28day mortality was and in the IFN and control groups respectively p Table Safety outcomesA total of and common adverse events were recorded duringthe study period in the IFN and control groups respectively Moreovernumber of serious adverse events was in the IFN group and in thecontrol group The incidence of grade or of adverse events washigher in the control group than the IFN group As it was expected IFNrelated common adverse effects injection site reactions and flulikesyndrome occurred only in the IFN group More patients in the controlgroup experienced ARDS secondary infections septic shock AKI andAHI compared with patients in the IFN group Table Nosocomial infections were detected in patients and patientsin the INF and control groups respectively Bloodstream infection withstaphylococcus aureus was detected in a patient in the INF group Threepatients in the control group experienced ventilator associated pneumonia with klebsiella pneumonia in two patients and acinetobacterbaumannii in another patient Other patients in the control group hadbloodstream infection with staphylococcus aureus DiscussionThis is first randomized clinical trial that evaluated efficacy andsafety of IFN subtype 1b in patients with severe COVID19 In thisstudy Interferon beta1-b as addon therapy significantly shortened the time toclinical response increased the discharge rate at day and decreasedneed for ICU admission in these patients However duration of hospitalization intubation rate length of ICU stay and allcause 28daymortality were not significantly changed Incidence rates of commonand serious adverse events were higher in the control group comparedwith the IFN group The sample size was calculated to assess effect ofInterferon beta1-b on time to clinical improvement in hospitalized patients withCOVID19 However the sample size might not have enough power todifferentiate effects of Interferon beta1-b on the secondary endpointsIFN is a subtype of the type INFs that is released by the lymphocytes as the first cytokine following exposure to viruses It activatesinterferonstimulated genes ISGs after binding to the receptors Theantiviral effects of IFNs are regulated through these genes InadequateIFN response caused uncontrolled viral replication raised viral load andled to poor outcomes in SARSCoV infection A strong IFN responsefollowing infection with SARSCoV2 was detected [“] Expressionof ISGs significantly increased in patients with CoVID19 [] In evaluation of transcriptional responses in various models in vitro ex vivoand in vivo BalancoMelo et al showed that the levels of IFN I andIFN III decreased in SARSCoV2 infection In in vitro model expressions of IFN I and IFN III were not detected in A549 cells as adenocarcinomic human lung cell line infected with SARSCoV2 Of notemoderate increase in the expression of ISGs was observed Next stepthe cells were treated by IFN that caused substantially reduction inthe viral replication Furthermore in ex vivo model the levels of IFN Iand IFN III were undetectable following infection of human bronchialepithelial cells with SARSCoV2 Finally in vivo assessment was considered Postmortem lungtissue samples were extracted from patientswith COVID19 and related transcriptional responses were comparedwith samples from the healthy individuals Similar to previous modelsmodest expressions of ISGs were detected but not about IFNs It is interesting that in all of the models robust cytokine and inflammatoryresponses were noticed []In the study of Yuan et al the antiviral activity of agents including hostbased IFNs IFN 1a Interferon beta1-b pegylated IFN α2a andIFN γ1B and virus targeting antivirals remdesivir and lopinavir wereassessed EC50 of these agents was determined according the plaquereduction assay The most potent IFNs were Interferon beta1-b EC50 IUml and IFN 1a EC50 IUml The EC50 values for remdesivirand lopinavir were determined as and µM respectively TheCC50 values of IFNs remdesivir and lopinavir were IUml µM and µM respectively Among IFNs the most reductive effects on viral load belonged to IFN 1a and Interferon beta1-bHowever Interferon beta1-b showed highest potency and selectivity indexagainst SARSCOV2 []In a randomized clinical trial and patients were recruited in 0cH Rahmani et alInternational Immunopharmacology Fig KaplanMeier plot for estimation of time to clinical improvementthe combination and control groups respectively Patients in the combination group received Interferon beta1-b lopinavirritonavir and ribavirinwhile those in the control group received only lopinavirritonavir Theprimary outcome was defined as the time to reach a negative RTPCR ofrespiratory secretions for SARSCoV2 The time to resolution of thesymptoms was considered as one of the secondary outcomes Themedian time to achieving a negative RTPCR was significantly shorterin the combination group compared to the control group vs days Moreover resolution of the symptoms occurred notably fasterin the combination group than the control group vs days []Similar with our study Interferon beta1-b was started in the viral phase ofCOVID19 ie within first days of onset of the symptoms In our studymedian time from onset of the symptoms to randomization was daysIn both studies first dose of Interferon beta1-b was administered within to h of hospital admission Initiation of antiviral agents as soon aspossible following onset of the symptoms is critical in control of viralreplication and prevention of tissue viral invasion The efficacy of antivirals significantly decreased after establishment of the cytokines release phase in COVID19 [] Due to resource limitations evaluation of viral clearance was not possible in our study No patient died inHung et al study while in our study approximately and ofpatients died in the IFN and control groups respectively Regardingcomparison of the results it should be considered that Hung et alevaluated Interferon beta1-b efficacy in patients with mild to moderate COVID while in our only study patients with severe COVID19 were included Moreover considering severity of the disease incidence rates ofthe serious complications during the hospitalization course were muchhigher in our studyEstebanez et al evaluated the efficacy of Interferon beta1-b in patientswith COVID19 Of them and patients were assigned to the IFNand control groups respectively Inhospital mortality was considered asthe primary outcome of study The mortality rate was statistically significant in the control group than the IFN group vs [] Retrospective design and lack of matching of the groups in termsof receiving other antivirals should be considered when interpreting theresultsIn a case series characteristics and outcomes of five patients withsevere COVID19 who were treated with Interferon beta1-b lopinavirritonavirand hydroxychloroquine were described The antiviral regimen appliedfor these patients was similar to our study Treatment was successful in patients while clinical status of patients deteriorated during thetreatment course All patients received corticosteroids Furthermore allpatients were initially admitted in another hospital and later transferred to the referral hospital [] Clinical outcomes of patients mighthad been affected during lag time of the transfer Moreover patientswere different in terms of the clinical presentations and managementstrategies So definite role of Interferon beta1-b in treatment of these patientscannot be assessedPayandemehr et al evaluated the efficacy of IFN 1a in patientswith moderate to severe COVID19 during a singlearm labelclinical trial All patients received IFN 1a along with hydroxychloroquine lopinavirritonavir and oseltamivir In this study only No at riskInterferon 0Control 0cH Rahmani et alTable Summary of the adverse events during the study periodParameter n Control group n Interferon groupn Any gradeAny gradeGrade or Common adverse events““““““““““““““Grade or “Leukocytosis“Leuk iaLymph ia“Thrombocyt ia“Thrombocytosis“AnemiaHyperkalemia“Hypokalemia“Hyponatremia“Increased creatinine“Increased aspartateaminotransferase“Nausea“Diarrhea“Abdominal pain“Injection site reaction“Flulike syndromeSerious adverse eventsARDS“Nosocomial infection“Septic shock“Acute kidney injury“Acute hepatic injuryARDS acute respiratory distress syndromepatients needed ICU admission and only one death occurred in thehospital Fifteen of the discharged patients were followed for days Noside effects were detected while in our study some patients experiencedcommon adverse effects such as injection site reactions and flulikesyndrome It might be due to receiving concomitant antipyretics andanalgesics that masked these reactions Furthermore main outcomes ofthe study were not welldefined in the method section Duration of thefollowup was only days []The efficacy of IFN 1a in patients with COVID19 was assessed inanother study In this noncontrolled prospective trial patients wereenrolled Five doses of mcg of IFN 1a were administrated subcutaneously on alternate days for these patients The patients also received hydroxychloroquine and lopinavirritonavir for days Theprimary outcome of the study was symptom alleviation during 14dayperiod Within days all patients became afebrile The resolution ofother symptoms gradually occurred [] The oxygenation status andtypes of respiratory supports were not exactly defined In general highflow nasal cannula was applied for most patients and three patientsreceived noninvasive positive pressure ventilation NIPPV No seriousadverse events were detected and none of the patients died Rate of ICUadmission and requirement for invasive mechanical ventilation werenot reported in this study Accounting these limitations absence ofcontrol group and small sample size the interpretation of the resultsshould be done with cautionIn another study efficacy and safety of IFN 1a were evaluated inpatients with severe COVID19 in an label randomized clinicaltrial Fortytwo and patients were recruited to the IFN and controlgroups respectively Time to clinical response based on the six ordinarycategory scale was primary endpoint of this study Following twoweektreatment with IFN 1a time to clinical response was not statisticallydifferent between the groups On day the numbers of dischargedpatients were significantly higher in the IFN group compared with thecontrol group vs Early administration of IFN 1asignificantly reduced the mortality rate compared with late administration [] Absence of followup PCR and chest imaging along withthe small sample size were the major limitations of the studyOur study suffered from some limitations Follow up chest imagingInternational Immunopharmacology or virological assessment was not possible due to resources limitationstherefore the effect of Interferon beta1-b on viral clearance was not determinedSmall sample size did not allow accurate estimation of survival benefitof Interferon beta1-bIn conclusion Interferon beta1-b was effective in shortening the time toclinical improvement without serious adverse events in patients withsevere COVID19 Furthermore ICU admission rate and need for invasive mechanical ventilation significantly reduced by administrationof Interferon beta1-b Although compared with the control group Interferon beta1-b reduced duration of hospitalization length of ICU stay intubation rateand 28day mortality were not statistically different between thegroups Further randomized clinical trials with enough sample size areneeded to accurately estimate survival benefit of Interferon beta1-bCRediT authorship contribution statementHamid Rahmani Data Curation Formal analysis InvestigationWriting original draft Effat DavoudiMonfared Data CurationAnahid Nourian Data Curation Hossein Khalili ConceptualizationMethodology Supervision Writing review editing NooshinHajizadeh Project Administration Narjes zarei Jalalabadi ProjectAdministration Mohammad Reza Fazeli Resources MonirehGhazaeian Resources Mir Saeed Yekaninejad Formal analysisDeclaration of Competing InterestThe authors declare that they have no known competing financialinterests or personal relationships that could have appeared to influence the work reported in this paperAcknowledgementWe would like to thank the nurses and other staffs of ImamKhomeini Hospital Complex for their kind supports and also Ms AvaKhalili for English proofreading the manuscriptFundingThe authors did not receive any fund for this workAppendix A Supplementary materialSupplementary data to this article can be found online at 101016jintimp2020106903References[] L Wang Y Wang D Ye Q Liu Review of the novel coronavirus SARSCoV[] JH Beigel KM Tomashek LE Dodd AK Mehta BS Zingman AC Kalil based on current evidence Int J Antimicrob Agents 101016jijantimicag2020105948[] D Cucinotta M Vanelli WHO Declares COVID19 a Pandemic Acta Biomed “ 1023750abmv91i19397[] Johns Hopkins Coronavirus Resource Center Home Page July coronavirusjhuedumaphtml[] JM Sanders ML Monogue TZ Jodlowski JB Cutrell Pharmacologic treatmentsfor coronavirus disease COVID19 a review JAMA 101001jama20206019E Hohmann HY Chu A Luetkemeyer S Kline DL de Castilla RW FinbergK Dierberg V Tapson L Hsieh TF Patterson R Paredes DA SweeneyWR Short G Touloumi DC Lye N Ohmagari MD Oh GM RuizPalaciosT Benfield G Fatkenheuer MG Kortepeter RL Atmar CB Creech J LundgrenAG Babiker S Pett JD Neaton TH Burgess T Bonnett M GreenM Makowski A Osinusi S Nayak HC Lane Remdesivir for the treatment ofCovid19 preliminary report N Engl J Med 101056NEJMoa2007764DK Manson C Kubin RG Barr ME Sobieszczyk NW Schluger Observationalstudy of hydroxychloroquine in hospitalized patients with Covid19 N Engl JMed “ 101056NEJMoa2012410[] J Geleris Y Sun J Platt J Zucker M Baldwin G Hripcsak A Labella[] B Cao Y Wang D Wen W Liu J Wang G Fan L Ruan B Song Y Cai M Wei 0c[] Z Zhou L Ren L Zhang J Zhong Y Xiao Z Jia L Gou J Yang C Wang 101016jantiviral2020104791S Jiang D Yang G Zhang H Li F Chen Y Xu M Chen Z Gao J Yang J DongB Liu X Zhang W Wang K He Q Jin M Li J Wang Heightened innate immuneresponses in the respiratory tract of COVID19 patients

cancer243

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: "Interferon COVID19SARSCOV2IranIn this study efficacy and safety of interferon Interferon beta1-b in the treatment of patients with severe COVID19 wereevaluatedAmong an label randomized clinical trial adult patients ‰¥ years old with severe COVID19 wererandomly assigned to the IFN group or the control group Patients in the IFN group received Interferon beta1-b mcg subcutaneously every other day for two consecutive weeks along with the national protocol medicationswhile in the control group patients received only the national protocol medications lopinavirritonavir oratazanavirritonavir plus hydroxychloroquine for “ days The primary outcome of the study was time toclinical improvement Secondary outcomes were inhospital complications and 28daymortalityBetween April and May patients were enrolled and finally patients in each group completed the study Time to clinical improvment in the IFN group was significantly shorter than the control group[“ vs “ days respectively p HR CI “] At day the percentageof discharged patients was and in the IFN and control groups respectively OR CI“ p ICU admission rate in the control group was significantly higher than the IFN group vs p The duration of hospitalization and ICU stay were not significantly differentbetween the groups Allcause 28day mortality was and in the IFN and control groups respectively p Interferon beta1-b was effective in shortening the time to clinical improvement without serious adverse events inpatients with severe COVID19 Furthermore admission in ICU and need for invasive mechanical ventilationdecreased following administration of Interferon beta1-b Although 28day mortality was lower in the IFN group furtherrandomized clinical trials with large sample size are needed for exact estimation of survival benefit of Interferon beta1-b IntroductionCoronavirus disease CoVID19 was reported from Wuhan forthe first time in late December Causing severe acute respiratorysyndrome coronavirus 2SARSCoV2 [] it rapidly spread throughoutthe world to the extent that the World Health anization WHOstated it as pandemic in March [] Until July more than million confirmed cases of CoVID19 were reported worldwideFurthermore more than deaths were recorded []Until now there is no definite antiviral treatment for CoVID19 andattempts continue for finding effective treatments worldwide Howeverfrom the beginning of the pandemic various treatments such as antiretrovirals antimalaria agents favipiravir remdesivir and corticosteroids immunoglobulin and cytokine blockers as adjunctive therapieswere suggested for the treatment of CoVID19 [] Except for the remdesivir which has had acceptable results the efficacy of other drugshas not been significant on the outcomes of the patients with CoVID19[“]Interferons IFNs have a key role in defense against viral infectionsas a component of innate immune system [] Invitro activity of IFN Ž Corresponding author at Department of Pharmacotherapy Tehran University of Medical Sciences Tehran IranEmail addresses khalilihtumsacir Khalilihsinatumsacir H Khalili101016jintimp2020106903Received July Received in revised form July Accepted August Available online August Elsevier BV All rights reserved 0cInternational Immunopharmacology prophylaxis deep vein thrombosis treatment of electrolyte disordersand antibiotic therapy were considered according to the hospital protocols The duration of the study was two weeks A 4week followupperiod was considered for all patientsPatients™ demographic data baseline diseases symptoms at the timeof disease presentation vital signs and laboratory data at the time ofhospital admission were recorded Patients were daily monitored interms of changes in the vital signs hemodynamic parameters oxygenation status laboratory data and treatment strategies Clinical statusof the patients was assessed by the sixcategory ordinal scale at days and of the randomization [] Need for supplemental oxygentherapy and also invasive or noninvasive respiratory supports wereevaluated regularly OutcomesTime to clinical improvement was considered as primary outcome ofstudy Clinical improvement was defined as improvement of at leasttwo points from the baseline status on the sixcategory ordinal scale[] This scale contains the subsequent categories death hospital admission requiring invasive mechanical ventilation hospitaladmission requiring noninvasive positive pressure ventilation hospital admission requiring oxygen hospital admission not requiring oxygen discharge Secondary outcomes were clinical statusof patients at day and ICU admission and intubation rateslength of hospitalization and ICU stay and 28day mortalitySide effects related to IFN therapy and other adverse events duringthe study period were monitored and recorded as the safety outcomesCategorization of adverse events was done according to the commonterminology criteria for adverse events CTCAE National Institutes ofHealth and National Cancer Institute Also serious complications during the hospitalization course such asacute respiratory distress syndrome ARDS nosocomial infectionsseptic shock acute kidney injury AKI and acute hepatic injury AHIwere considered Statistical analysis and randomizationContinuous variables are demonstrated as median interquartilerange IQR and categorical variables as frequencies and percentagesContinuous variables were compared between the groups by MannWhitney U test The Fisher™s exact test was applied for comparison ofcategorical variablesThe Hazard Ratio HR and CI for clinical improvement wereestimated by Cox proportional hazards regression analysis The effect ofischemic heart disease lymphocyte count Aspartate aminotransferaseAST and Creactive protein CRP on the primary outcome was evaluated by the adjusted Cox regression models as potential confoundingfactors Time to clinical improvement was estimated by KaplanMeierplot and compared with a logrank test All statistical analysis was doneby SPSS software version Time to clinical improvement was estimated to be approximately days and sample size was calculated by following equationH Rahmani et alhas been shown against severe acute respiratory syndrome coronavirusSARSCoV and Middle East respiratory syndrome coronavirus MERSCoV [“] Although IFN was used less than IFN α for the treatment of SARSCOV and MERSCoV in human studies it was effective inthe treatment of MERSCoV in retrospective studies and case series[“] The efficacy of Interferon beta1-b is being assessed in the treatment ofMERS in a randomized clinical trial [] According to the presence ofthis evidence IFN was considered as a promising option for thetreatment of CoVID19In this label randomized clinical trial efficacy and safety ofInterferon beta1-b in the treatment of patients with severe CoVID19 were assessed Materials and methods Study designThis label randomized clinical trial was designed to evaluatethe efficacy and safety of Interferon beta1-b in the treatment of patients withCoVID19 Patients with severe CoVID19 who were hospitalized duringApril to May in Imam Khomeini Hospital Center one ofthe largest referral hospitals in Tehran Iran were includedThe protocol of the study was approved by Ethics Committee ofTehran UniversityReferencenumberIRTUMSVCRREC13981053 Furthermore the study was registeredas a clinical trial register ID IRCT20100228003449N27 The studyprotocol was described for participants and written informed consentswere obtained from all patients or their firstdegree family members Eligibility criteriaof MedicalSciencesSARSCoV2 in patients™ nasopharyngeal swabs was detected usingRealTime Polymerase Chain Reaction RTPCR Total RNA extractionwas done applying Viral Nucleic Acid Extraction kit Cat No YVN50YVN100 from RBC Bioscience Taipei Taiwan The Novel Coronavirus2019nCOV Nucleic Acid Diagnostic Kit PCRFluorescence Probingof Sansure Biotech S3102E Changsha China was used for RTPCRAdult patients ‰¥ years old with positive PCR and clinicalsymptomssigns of pneumonia including dyspnea cough and feverperipheral oxygen saturation SPO2 ‰ in ambient air or arterialinspired oxygen PaO2oxygen partial pressure to fractionalFiO2 or SPO2FiO2 and lung involvement in chestimaging were included These criteria indicated severe form of thedisease [] At baseline patients with serious allergic reactions to IFNhistory of suicide thoughts and attempts alanine amino transferaseALT × the upper limit of the normal range uncontrolled underlying diseases such as neuropsychiatric disorders thyroid disorderscardiovascular diseases and also pregnant and lactating women werenot includedRecruitment was considered during the first 48hour of the hospitaladmission During the study period patients who received less than doses of Interferon beta1-b were excluded If patients were discharged beforefulfilment of the treatment course the treatment was applied at home ProceduresEligible patients were recruited in the IFN group or the controlgroup according to the permuted block randomization Patients in theIFN group received Interferon beta1-b along with the national protocol medications while in the control group patients received only the nationalprotocol medications Interferon beta1-b Ziferon® Zist Daru Daneh Co Iranwas administrated as mcg subcutaneously every other day for twoconsecutive weeks The national protocol consisted lopinavirritonavir mg BD or atazanavirritonavir mg daily plushydroxychloroquine mg BD in first day and then mg BD for“ days Other supportive cares such as fluid therapy stress ulcernkzz1122211222nn121512005015Z19612Z1041 0cH Rahmani et alInternational Immunopharmacology Fig Consort flowchart of the studyAccording to the above equation at least patients in each groupwere expected to make a difference of days in time to clinical improvement with power of Patients were randomly recruited to the IFN group or the control group The method of randomizationwas the permuted block randomization patients per block A biostatistician who was not involved in patients™ care did this process Results PatientsA total of patients were screened Of them patients did nothave the eligibility criteria of study and patients were referred fromanother hospital Three and four patients withdrew the consent duringthe study in the IFN group and control groups respectively Four patients did not adhere to IFN injection after second or third dose Alsothree patients in the control group were enrolled in another trialFinally patients in each group completed the study Fig The median IQR age of patients was “ years and of them were male No significant difference in terms of the patients™demographic data was detected between the groups The most commoncomorbidities were hypertension diabetes mellitus and ischemic heartdisease Dyspnea fever and cough were the most frequent symptoms atthe time of hospital admission The median IQR time from onset of thesymptoms to hospital admission was “ and “ days in the IFNgroup and control groups respectively The time from onset of thesymptoms to randomization was not statistically significant betweenthe groups All of patients required respiratory support at the time ofrandomization Oxygenation through facemask was required for morethan percent of patients None of the patients in both groups wereintubated at baseline Table Vital signs and laboratory data of patients at the time of recruitment were comparable between the groupsTable During the hospitalization course oxygen saturation droppedin and of patients in the IFN and control groups respectively All of those patients were intubated At least one antibioticwas administrated for and of patients in the IFN groupand control groups respectively Methylprednisolone was administeredfor of patients in the IFN group and of patients in the4Power085 0cH Rahmani et alTable Baseline characteristics of patientsParameter Median IQR or n AgeSexMaleFemaleComorbid conditions nHypertensionDiabetes mellitusIschemic heart diseaseAsthmaCOPDMalignancyTransplantationSymptoms at admission nDyspneaFeverCoughChillsDuration of symptoms before admissionmedian IQR daysTime from symptom onset torandomization median IQR daysSix category scale at day of intervention3hospital admission requiring highflownasal cannula or noninvasivemechanical ventilationsupplemental oxygen hospital admission requiringInterferon groupn “ ““Control groupn “ ““control group The dose of methylprednisolone was mg daily for days Methylprednisolone was considered during the cytokine orhyperinflammation phase days “ of onset of the symptoms Approximately and of patients in the INF and control groupsneeded vasopressors during the hospitalization course respectivelyTable Primary outcomesThe time to clinical improvement in the IFN group was significantlyshorter than the control group [“ vs “ days respectivelyp ] Table Moreover the Cox proportional hazards regression analysis showed that time difference to clinical improvement wasstatistically significant between the groups HR CI“ Fig Then the model was adjusted for the confoundingInternational Immunopharmacology Interferon groupn Control groupn Table Respiratory support and medicationsParameter n Respiratory supportNasal cannulaFace maskNIPPVIMVAntibiotics mer em piperacillintazobactam ceftriaxone FQsvancomycin azithromycin andColistin n CorticosteroidsVitamin CVasopressorsDiphenhydramineCardiovascular drugsStatinsARBsBetablockersACEIsNIPPV noninvasive positive pressure ventilation IMV invasive mechanicalventilation FQs fluoroquinolones ARB Angiotensin Π Receptor Blocker ACEIangiotensin converting enzyme inhibitorfactors and similar results were seen HR CI “ Secondary outcomesAccording to the six category scale and of patientswere discharged in the IFN and the control groups at day respectivelyOR CI “ p Only one patient in thecontrol group died at day Also at this time and patients wereintubated in the IFN and control groups respectively At day thepercentage of discharged patients reached to and in theIFN and control groups respectively OR CI “p Furthermore the number of deaths increased to and patients the IFN and control groups respectively Finally at day ofinclusion the proportion of discharged patients were in the IFNgroup and in the control group OR CI “p At this time ICU admission rate in the control group wassignificantly higher than the IFN group vs p Moreover more patients in the control group needed invasive mechanical ventilation compared with the IFN group but the rate was notstatistically different p Although length of hospitalization wasTable Patients™ vital signs and laboratory data at the time of hospital admissionParameter Median IQRTemperature °CHeart rate beats minuteRespiratory rate breathsminSystolic blood pressure mm HgSPO2 Laboratory dataWhite Blood Cell cells μlAcute Lymphocyte count cellsμlHemoglobin gdlPlatelet count cells × 103μlBlood Urea Nitrogen mgdlCreatinine mgdlAspartate aminotransferase ulAlanine aminotransferase ulAlkaline phosphatase ulTotal bilirubinmgdlCreactive protein mgdlErythrocyte sedimentation rate mmhLactate dehydrogenase ulInterferon group n ““““““““““““““““““Control group n ““““““““““““““““““ 0cH Rahmani et alInternational Immunopharmacology Table Outcomes and complicationsParameter Median IQR or n Time to clinical response medianIQR daysICU admission n Intubation requirementLength of stay in ICU days median IQR daysLength of stay in hospital days median IQR daysAllcause mortality at day Six category scale at day of intervention Death Hospital admission requiring invasive mechanical ventilation Hospital admission requiring highflow nasal cannula or noninvasive mechanical ventilation Hospital admission requiring supplemental oxygen Hospital admission not requiring supplemental oxygen DischargeSix category scale at day of intervention Death Hospital admission requiring invasive mechanical ventilation Hospital admission requiring highflow nasal cannula or noninvasive mechanical ventilation Hospital admission requiring supplemental oxygen Hospital admission not requiring supplemental oxygen DischargeSix category scale at day of intervention Death Hospital admission requiring invasive mechanical ventilation Hospital admission requiring highflow nasal cannula or noninvasive mechanical ventilation Hospital admission requiring supplemental oxygen Hospital admission not requiring supplemental oxygen DischargeInterferon group n “““Control group n “ ““pvalueOR95 CI“““shorter [ “ days in the IFN group vs “ days in thecontrol group p ] but length of ICU stay was not significantlydifferent between the groups Allcause 28day mortality was and in the IFN and control groups respectively p Table Safety outcomesA total of and common adverse events were recorded duringthe study period in the IFN and control groups respectively Moreovernumber of serious adverse events was in the IFN group and in thecontrol group The incidence of grade or of adverse events washigher in the control group than the IFN group As it was expected IFNrelated common adverse effects injection site reactions and flulikesyndrome occurred only in the IFN group More patients in the controlgroup experienced ARDS secondary infections septic shock AKI andAHI compared with patients in the IFN group Table Nosocomial infections were detected in patients and patientsin the INF and control groups respectively Bloodstream infection withstaphylococcus aureus was detected in a patient in the INF group Threepatients in the control group experienced ventilator associated pneumonia with klebsiella pneumonia in two patients and acinetobacterbaumannii in another patient Other patients in the control group hadbloodstream infection with staphylococcus aureus DiscussionThis is first randomized clinical trial that evaluated efficacy andsafety of IFN subtype 1b in patients with severe COVID19 In thisstudy Interferon beta1-b as addon therapy significantly shortened the time toclinical response increased the discharge rate at day and decreasedneed for ICU admission in these patients However duration of hospitalization intubation rate length of ICU stay and allcause 28daymortality were not significantly changed Incidence rates of commonand serious adverse events were higher in the control group comparedwith the IFN group The sample size was calculated to assess effect ofInterferon beta1-b on time to clinical improvement in hospitalized patients withCOVID19 However the sample size might not have enough power todifferentiate effects of Interferon beta1-b on the secondary endpointsIFN is a subtype of the type INFs that is released by the lymphocytes as the first cytokine following exposure to viruses It activatesinterferonstimulated genes ISGs after binding to the receptors Theantiviral effects of IFNs are regulated through these genes InadequateIFN response caused uncontrolled viral replication raised viral load andled to poor outcomes in SARSCoV infection A strong IFN responsefollowing infection with SARSCoV2 was detected [“] Expressionof ISGs significantly increased in patients with CoVID19 [] In evaluation of transcriptional responses in various models in vitro ex vivoand in vivo BalancoMelo et al showed that the levels of IFN I andIFN III decreased in SARSCoV2 infection In in vitro model expressions of IFN I and IFN III were not detected in A549 cells as adenocarcinomic human lung cell line infected with SARSCoV2 Of notemoderate increase in the expression of ISGs was observed Next stepthe cells were treated by IFN that caused substantially reduction inthe viral replication Furthermore in ex vivo model the levels of IFN Iand IFN III were undetectable following infection of human bronchialepithelial cells with SARSCoV2 Finally in vivo assessment was considered Postmortem lungtissue samples were extracted from patientswith COVID19 and related transcriptional responses were comparedwith samples from the healthy individuals Similar to previous modelsmodest expressions of ISGs were detected but not about IFNs It is interesting that in all of the models robust cytokine and inflammatoryresponses were noticed []In the study of Yuan et al the antiviral activity of agents including hostbased IFNs IFN 1a Interferon beta1-b pegylated IFN α2a andIFN γ1B and virus targeting antivirals remdesivir and lopinavir wereassessed EC50 of these agents was determined according the plaquereduction assay The most potent IFNs were Interferon beta1-b EC50 IUml and IFN 1a EC50 IUml The EC50 values for remdesivirand lopinavir were determined as and µM respectively TheCC50 values of IFNs remdesivir and lopinavir were IUml µM and µM respectively Among IFNs the most reductive effects on viral load belonged to IFN 1a and Interferon beta1-bHowever Interferon beta1-b showed highest potency and selectivity indexagainst SARSCOV2 []In a randomized clinical trial and patients were recruited in 0cH Rahmani et alInternational Immunopharmacology Fig KaplanMeier plot for estimation of time to clinical improvementthe combination and control groups respectively Patients in the combination group received Interferon beta1-b lopinavirritonavir and ribavirinwhile those in the control group received only lopinavirritonavir Theprimary outcome was defined as the time to reach a negative RTPCR ofrespiratory secretions for SARSCoV2 The time to resolution of thesymptoms was considered as one of the secondary outcomes Themedian time to achieving a negative RTPCR was significantly shorterin the combination group compared to the control group vs days Moreover resolution of the symptoms occurred notably fasterin the combination group than the control group vs days []Similar with our study Interferon beta1-b was started in the viral phase ofCOVID19 ie within first days of onset of the symptoms In our studymedian time from onset of the symptoms to randomization was daysIn both studies first dose of Interferon beta1-b was administered within to h of hospital admission Initiation of antiviral agents as soon aspossible following onset of the symptoms is critical in control of viralreplication and prevention of tissue viral invasion The efficacy of antivirals significantly decreased after establishment of the cytokines release phase in COVID19 [] Due to resource limitations evaluation of viral clearance was not possible in our study No patient died inHung et al study while in our study approximately and ofpatients died in the IFN and control groups respectively Regardingcomparison of the results it should be considered that Hung et alevaluated Interferon beta1-b efficacy in patients with mild to moderate COVID while in our only study patients with severe COVID19 were included Moreover considering severity of the disease incidence rates ofthe serious complications during the hospitalization course were muchhigher in our studyEstebanez et al evaluated the efficacy of Interferon beta1-b in patientswith COVID19 Of them and patients were assigned to the IFNand control groups respectively Inhospital mortality was considered asthe primary outcome of study The mortality rate was statistically significant in the control group than the IFN group vs [] Retrospective design and lack of matching of the groups in termsof receiving other antivirals should be considered when interpreting theresultsIn a case series characteristics and outcomes of five patients withsevere COVID19 who were treated with Interferon beta1-b lopinavirritonavirand hydroxychloroquine were described The antiviral regimen appliedfor these patients was similar to our study Treatment was successful in patients while clinical status of patients deteriorated during thetreatment course All patients received corticosteroids Furthermore allpatients were initially admitted in another hospital and later transferred to the referral hospital [] Clinical outcomes of patients mighthad been affected during lag time of the transfer Moreover patientswere different in terms of the clinical presentations and managementstrategies So definite role of Interferon beta1-b in treatment of these patientscannot be assessedPayandemehr et al evaluated the efficacy of IFN 1a in patientswith moderate to severe COVID19 during a singlearm labelclinical trial All patients received IFN 1a along with hydroxychloroquine lopinavirritonavir and oseltamivir In this study only No at riskInterferon 0Control 0cH Rahmani et alTable Summary of the adverse events during the study periodParameter n Control group n Interferon groupn Any gradeAny gradeGrade or Common adverse events““““““““““““““Grade or “Leukocytosis“Leuk iaLymph ia“Thrombocyt ia“Thrombocytosis“AnemiaHyperkalemia“Hypokalemia“Hyponatremia“Increased creatinine“Increased aspartateaminotransferase“Nausea“Diarrhea“Abdominal pain“Injection site reaction“Flulike syndromeSerious adverse eventsARDS“Nosocomial infection“Septic shock“Acute kidney injury“Acute hepatic injuryARDS acute respiratory distress syndromepatients needed ICU admission and only one death occurred in thehospital Fifteen of the discharged patients were followed for days Noside effects were detected while in our study some patients experiencedcommon adverse effects such as injection site reactions and flulikesyndrome It might be due to receiving concomitant antipyretics andanalgesics that masked these reactions Furthermore main outcomes ofthe study were not welldefined in the method section Duration of thefollowup was only days []The efficacy of IFN 1a in patients with COVID19 was assessed inanother study In this noncontrolled prospective trial patients wereenrolled Five doses of mcg of IFN 1a were administrated subcutaneously on alternate days for these patients The patients also received hydroxychloroquine and lopinavirritonavir for days Theprimary outcome of the study was symptom alleviation during 14dayperiod Within days all patients became afebrile The resolution ofother symptoms gradually occurred [] The oxygenation status andtypes of respiratory supports were not exactly defined In general highflow nasal cannula was applied for most patients and three patientsreceived noninvasive positive pressure ventilation NIPPV No seriousadverse events were detected and none of the patients died Rate of ICUadmission and requirement for invasive mechanical ventilation werenot reported in this study Accounting these limitations absence ofcontrol group and small sample size the interpretation of the resultsshould be done with cautionIn another study efficacy and safety of IFN 1a were evaluated inpatients with severe COVID19 in an label randomized clinicaltrial Fortytwo and patients were recruited to the IFN and controlgroups respectively Time to clinical response based on the six ordinarycategory scale was primary endpoint of this study Following twoweektreatment with IFN 1a time to clinical response was not statisticallydifferent between the groups On day the numbers of dischargedpatients were significantly higher in the IFN group compared with thecontrol group vs Early administration of IFN 1asignificantly reduced the mortality rate compared with late administration [] Absence of followup PCR and chest imaging along withthe small sample size were the major limitations of the studyOur study suffered from some limitations Follow up chest imagingInternational Immunopharmacology or virological assessment was not possible due to resources limitationstherefore the effect of Interferon beta1-b on viral clearance was not determinedSmall sample size did not allow accurate estimation of survival benefitof Interferon beta1-bIn conclusion Interferon beta1-b was effective in shortening the time toclinical improvement without serious adverse events in patients withsevere COVID19 Furthermore ICU admission rate and need for invasive mechanical ventilation significantly reduced by administrationof Interferon beta1-b Although compared with the control group Interferon beta1-b reduced duration of hospitalization length of ICU stay intubation rateand 28day mortality were not statistically different between thegroups Further randomized clinical trials with enough sample size areneeded to accurately estimate survival benefit of Interferon beta1-bCRediT authorship contribution statementHamid Rahmani Data Curation Formal analysis InvestigationWriting original draft Effat DavoudiMonfared Data CurationAnahid Nourian Data Curation Hossein Khalili ConceptualizationMethodology Supervision Writing review editing NooshinHajizadeh Project Administration Narjes zarei Jalalabadi ProjectAdministration Mohammad Reza Fazeli Resources MonirehGhazaeian Resources Mir Saeed Yekaninejad Formal analysisDeclaration of Competing InterestThe authors declare that they have no known competing financialinterests or personal relationships that could have appeared to influence the work reported in this paperAcknowledgementWe would like to thank the nurses and other staffs of ImamKhomeini Hospital Complex for their kind supports and also Ms AvaKhalili for English proofreading the manuscriptFundingThe authors did not receive any fund for this workAppendix A Supplementary materialSupplementary data to this article can be found online at 101016jintimp2020106903References[] L Wang Y Wang D Ye Q Liu Review of the novel coronavirus SARSCoV[] JH Beigel KM Tomashek LE Dodd AK Mehta BS Zingman AC Kalil based on current evidence Int J Antimicrob Agents 101016jijantimicag2020105948[] D Cucinotta M Vanelli WHO Declares COVID19 a Pandemic Acta Biomed “ 1023750abmv91i19397[] Johns Hopkins Coronavirus Resource Center Home Page July coronavirusjhuedumaphtml[] JM Sanders ML Monogue TZ Jodlowski JB Cutrell Pharmacologic treatmentsfor coronavirus disease COVID19 a review JAMA 101001jama20206019E Hohmann HY Chu A Luetkemeyer S Kline DL de Castilla RW FinbergK Dierberg V Tapson L Hsieh TF Patterson R Paredes DA SweeneyWR Short G Touloumi DC Lye N Ohmagari MD Oh GM RuizPalaciosT Benfield G Fatkenheuer MG Kortepeter RL Atmar CB Creech J LundgrenAG Babiker S Pett JD Neaton TH Burgess T Bonnett M GreenM Makowski A Osinusi S Nayak HC Lane Remdesivir for the treatment ofCovid19 preliminary report N Engl J Med 101056NEJMoa2007764DK Manson C Kubin RG Barr ME Sobieszczyk NW Schluger Observationalstudy of hydroxychloroquine in hospitalized patients with Covid19 N Engl JMed “ 101056NEJMoa2012410[] J Geleris Y Sun J Platt J Zucker M Baldwin G Hripcsak A Labella[] B Cao Y Wang D Wen W Liu J Wang G Fan L Ruan B Song Y Cai M Wei 0c[] Z Zhou L Ren L Zhang J Zhong Y Xiao Z Jia L Gou J Yang C Wang 101016jantiviral2020104791S Jiang D Yang G Zhang H Li F Chen Y Xu M Chen Z Gao J Yang J DongB Liu X Zhang W Wang K He Q Jin M Li J Wang Heightened innate immuneresponses in the respiratory tract of COVID19 patients Answer:

Thyroid_Cancer

"Pluripotent stem cellsDirected differentiationIn vitro disease modellingLungAirwayMechanical cuesContentsChronic lung diseases remain major healthcare burdens for which the only curative treatment is lung transplantation In vitro human models are promising platforms for identifying and testing novel compounds to potentiallydecrease this burden Directed differentiation of pluripotent stem cells is an important strategy to generate lungcells to create such models Current lung directed differentiation protocols are limited as they do not recapitulate the diversity of respiratory epithelium generate consistent or sufficient cell numbers for drug discoveryplatforms and establish the histologic tissuelevel anization critical for modeling lung function In this review we describe how lung development has formed the basis for directed differentiation protocols and discussthe utility of available protocols for lung epithelial cell generation and drug development We further highlighttissue engineering strategies for manipulating biophysical signals during directed differentiation such that futureprotocols can recapitulate both chemical and physical cues present during lung development Elsevier BV All rights reservedOverview of key developmental stages Lung anogenesis molecularly defining lung fate in the embryo Branching morphogenesis and other mechanical cues generated during lung development Introduction Human embryology as a blueprint for lung directed differentiation Directed differentiation of lung epithelia inspired by embryology Mouse embryonic stem cell derived lung epithelia Modeling airway and lung diseases for drug discovery Opportunities to exploit mechanical cues for improving directed differentiation protocols in the future Micropatterning in 2D Stem cell behaviour on substrate topographies Micropatterning in 3D anoid systemsSubstrate textureHuman pluripotent stem cellderived lung epithelia Creation of human proximal lung epithelia Comparisons of proximal airway directed differentiation protocols Creation of human distal lung epitheliaComparisons of distal lung directed differentiation protocols Limitations of current directed differentiation protocols Ž Correspondence to G Karoubi Latner Thoracic Surgery Research Laboratories Toronto General Hospital College St Toronto ON M5G 1L7 CanadaŽŽ Correspondence to A P McGuigan Institute for Biomaterials and Biomedical Engineering University of Toronto College Street Toronto ON M5S 3G9 CanadaEmail addresses golnazkaroubiuhnresearchca G Karoubi alisonmcguiganutorontoca AP McGuigan101016jaddr2020080050169409X Elsevier BV All rights reservedPlease cite this as R Varma JP Soleas TK Waddell Current strategies and opportunities to manufacture cells for modelinghuman lungs Adv Drug Deliv Rev 101016jaddr202008005 0cR Varma Advanced Drug Delivery Reviews xxx xxxAcknowledgementsReferencesFuture outlook IntroductionEndstage lung disease is the third leading cause of morbidity andmortality worldwide [] and produces a significant burden onhealthcare systems due to extensive resource expenditures for diseasemanagement and as lung transplantation is the only curative treatmentoption Such diseases include acute respiratory distress syndromechronic obstructive pulmonary disease cystic fibrosis and pulmonaryfibrosis Chronic pulmonary diseases result in million global deathsper year [] Patients who receive transplants face continued complications associated with chronic immunosuppression and graft rejectionwith the transplant survival rates at and years being and respectively [] Furthermore since lungs function as an important barrier between the internal and the external environments they are a critical site for bacterial and viral infections and disease transmissionparticularly relevant given the current COVID19 pandemic There istherefore a critical need to better elucidate the mechanisms of infectiondisease progression host response and cellular repair in the lung to enable the development of novel targeted therapeutics for lung diseaseTissueengineered models have emerged as a technology to addressthis challenge and shown some success in drug identification and toxicology studies For example commercially available airway epithelialmodels such as EpiAirwayTM MatTek Life Sciences serve as convenientplatforms with airliquid interface culture capabilities for assessing theeffect of chemical and physical stimuli [“] Other examples includethe Alveolus LungChip and Airway LungChip systems Emulate Incoriginally developed in the Ingber laboratory which mimic theepithelialendothelial interface of the airway and provide a more dynamic platform for testing new anti‚ammatory compounds inasthma [] and new small molecule targets to decrease cancerassociated pulmonary edema [] More complex models have alsobeen reported which involve selfassembly of heterogeneous progenitor cells into 3D structures termed anoids [] These anoidmodels can recapitulate aspects of human lung development in termsof tissue structures and protein expression and therefore present apromising opportunity for drug screening []A challenge in developing such human in vitro lung models to screenfor drugs however is the requirement for large batches of similarhuman cells as a starting population for tissue manufacturing to ensureminimal heterogeneity between test wells [] Achieving this is especially challenging when using primary human lung cells which exhibitconsiderable heterogeneity across donors and have a limited ability togrow and differentiate reliably [] Furthermore primary cells areoften extracted from diseased donors which is not ideal for conductingcontrolled studies due to the wide range of therapeutic and environmental factors these cells have already been exposed to Directed differentiation of pluripotent populations has the potential to create vastnumbers of cells from either healthy or diseased patients It allows introduction of specific diseaseassociated mutations via CRISPRCas9gene editing to recapitulate and understand pathologies in a controlledmanner As such directed differentiation enables the generation of anattractive cell source for drug screening platforms and personalized disease models that may provide insight into tissue regeneration mechanisms [“]Directed differentiation protocols to manufacture specific cell populations from pluripotent stem cells PSCs have been developed to meetthe need for a homogeneous human cell source Older lung directed differentiation protocols from the late 2000s have been proven inefficientdue to the nonstandardized methods through which they derive lungendoderm from embryoid bodies [“] A series of more standardizedstepwise protocols have since emerged in the last decade that provideavenues for developing airway and lung epithelia albeit with variableefficiencies [“] The first ‚uential directed differentiation protocol to produce lung epithelia used human PSCs in [] whichwas further supported by two prominent studies conducted usingmouse PSCs in [] These protocols have continued to be enhanced through adaptations related to the selection of growth factorsand small molecules the chronology of morphogen delivery as wellas innovations in enabling platforms such as cell sorting 3D cultureand singlecell analyses to efficiently derive normal and diseased lungepithelia from human PSCs [“] Despite such advancements limitations pertaining to heterogeneity in the resultingpopulations still exist which are likely attributed to variability across directed differentiation trials PSC cell lines or the persistence of contaminating cell populations belonging to other lineages While protocolshave progressed to some degree in differentiating proximal airwayand distal alveolar epithelia they remain limited Overall many unanswered questions remain with regards to the identity maturity andfunctionality of resulting cell types as well as their utility for tissue engineering and drug testing approaches Therefore these protocols mustbe optimized further to reliably produce large numbers of spatially relevant and functional lung and airway epithelial cells that appropriatelyrespond to both chemical and mechanical stimuli in the context of disease modeling and drug discoveryIn this review we discuss the directed differentiation protocols thatattempt to recapitulate lung development and disease and highlightpossible opportunities to enhance these protocols in the future Wefirst describe development of native lung tissue and the patterningevents that occur that differentiation models attempt to mimic andhighlight how human lung embryology has served as the blueprint tocreate the common pathway of lung directed differentiation protocolsWe then discuss the evolution of directed differentiation protocols tofind opportunities for creating specific populations of airway and lungepithelia through targeted manipulation of key signaling pathways in2D and 3D models We further describe how these models have beenused to recapitulate different airway and lung diseases Finally we discuss how tissue engineering and biophysical cues using biomaterialscan be utilized during lung directed differentiation to mimic patterningcues present in development to augment current differentiationprotocols Human embryology as a blueprintdifferentiationforlung directed Overview of key developmental stagesDirected differentiation protocols have been designed to mimicin vivo human lung development [] Indeed in vitro models of lungdevelopment have provided unique insight into human lung development [] As human lung development has been described at greatlength in earlier reviews [] we provide a brief overview as followsschematically represented in Fig During early embryogenesis at days post fertilization a process called gastrulation begins with the appearance of a structure called primitive streak through which cells migrate to form the primary embryonic germ layers definitive endodermmesoderm and ectoderm [“] Definitive endoderm expandsthereby forming the primitive gut tube comprised of three endodermalregions foregut midgut and hindgut [] This is when lungPlease cite this as R Varma JP Soleas TK Waddell Current strategies and opportunities to manufacture cells for modelinghuman lungs Adv Drug Deliv Rev 101016jaddr202008005 0cR Varma Advanced Drug Delivery Reviews xxx xxxFig Schematic of human lung development from an epithelial perspectivedevelopment begins at approximately four weeks into embryonic lifewith the outgrowth of foregut endoderm [] and continues througheight years of postnatal life [] There are five stages to lungdevelopment Embryonic weeks “ The future lung buds emerge from the ventral side of the primitive foregut endoderm into the surroundingmesenchyme and develop into embryonic lung buds with early trachea and bronchi [“] Pseudoglandular weeks “ Branching of the airway continuesleading to formation of conducting and terminal bronchioles whilethe proximal airway epithelium begins to develop [] Canalicular weeks “ Development of the respiratory or gasexchanging airways is initiated primitive alveoli form and the future distal epithelium begins to thin as distal epithelial markers areexpressed [] Saccular weeks “ Emergence of sacshaped distal airwayswhich develop crests with muscle and elastin to create indentationsThese distal airways extend to form alveoli by weeks [] The developing epithelium and vasculature within the future alveolus continue to merge closer together to facilitate future gas exchange andfurther differentiation of alveolar epithelial cells AEC I and II takesplace Alveolar periods week “ years True alveoli are seen in week and the majority of alveolarization takes place through sacculeseptation a process by which the sacshaped distal airways changetheir internal architecture and create thin walls intraluminallySeptation leads to an increase in surface area of the gas exchangingportion of the developing lung and prepares the fetus to breath airduring this stage [] Lung anogenesis molecularly defining lung fate in the embryoDuring the embryonic period early lung is genetically defined by theexpression of transcription factor NK2 Homeobox NKX21 and Srybox SOX2 [“] During human lung development it has beenfound that the lung buds and branches given off during thepseudoglandular period are mostly SOX2SOX9 [] BothSOX2 and SOX9 are individual markers of the early proximal or distal lineage respectively [“] Over the course of the canalicular and saccular periods of development weeks “ these double positivepopulations downregulate one SOX protein and maintain expressionof the other as these cells mature towards proximal or distal lineages[] The proximal airway closer to the mouth is comprised of apseudostratified columnar epithelium that is responsible for theconducting airway function debris and pathogen removal ciliatedcells mucus production goblet cells prevention of airway ‚ammation club cells and humidification of air as it passes through to the distal lung compartment [“] The squamous distal epitheliumcomposed of alveolar epithelial cells AEC I and II facilitates the respiratory function of the lung as air in the epithelial compartment is broughtinto close apposition to blood from the pulmonary vasculature it alsosecretes surfactants which play an immunologic role and decrease thesurface tension present at the airliquid interface thereby preventing alveolar collapse [] In humans a number of cell types are found in theproximal airway each identified with specific markers Table This includes basal cells tumor protein p63P63 keratinKRT5 nerve growthfactor receptorNGFR integrin α6ITGA6 integrin β4ITGB4 ciliatedcells Forkhead BoxJ1FOXJ1 acetylated tubulinAcTUB goblet cellsmucin 5ACMUC5AC mucin 5BMUC5B club cells club cell secretoryproteinCCSP or SCGB1A1 and pulmonary neuroendocrine cellsPNECs synaptophysinSYP chromogranin ACHGA On the otherhand homeodomainonly protein HOPX identifies the distal lungalong with AEC I cells T1α podoplaninPDPN aquaporin 5AQP5while AEC II cells are recognized via surfactant protein B SPC prosurfactant protein C proSPC or SPC and HT2280 []One mechanism by which lung epithelia begin to mature is based onchemokine secretions from the surrounding mesenchyme and the developing heart field which are well reviewed here [] Key players including fibroblast growth factors FGFs [“] WNTs [“]and bone morphogenetic proteins BMPs [“] are known to inducethe differentiation of early lung progenitors in a controlled manner Forexample in mouse it has been found that FGF10 plays a role in bud outgrowth [] and drives lung progenitors towards a distal fate []through canonical WNT signaling [] Proximal epithelia developbecause they are located further away from distally located FGF reservoirs in the mesenchyme in a mechanism that appears dependent onconcentration gradients [] BMP4 plays a key role in lung bud formation from foregut endoderm and establishment of both dorsoventralback to front and proximodistal top to bottom patterning in the nascent lung [] BMP4 is also present at high levels in distal bud tips andepithelia including AEC II cells [] however its inhibition promotesa proximal fate and along with BMP2 inhibition ciliated cell development [] Branching morphogenesis and other mechanical cues generated duringlung developmentWhile the cell fate of early proximal and distal lineages is directedthrough chemical signals the lung epithelium itself undergoes markedPlease cite this as R Varma JP Soleas TK Waddell Current strategies and opportunities to manufacture cells for modelinghuman lungs Adv Drug Deliv Rev 101016jaddr202008005 0cR Varma Advanced Drug Delivery Reviews xxx xxxTable Epithelial populations in native human airways and lungsRegionProximal AirwayDistal LungCell TypeAssociated Markers for Cell CharacterizationCiliated CellGoblet CellClub CellBasal CellAlveolar epithelial cell type I AEC IAlveolar epithelial cell type II AEC IIFOXJ1 AcTUBMUC5AC MUC5BCCSP SCGB1A1 SCGB3A2P63 KRT5 NGFR ITGA6 ITGB4HOPX PDPN AQP5SPB SPC HT2280Cell Proportions in Native Lung“ []“ []“ []“ [] [] []changes in architecture a process known as branching morphogenesis[] From the simple tube of the anterior foregut endoderm to thecomplex tubular structure of the adult a highly stereotyped mechanismof branching morphogenesis facilitates the outgrowth division placement and structure of lung airway [] Branching morphogenesis ofthe lung is driven by three simple and iteratively used processes domain branching planar and orthogonal bifurcation [] The first formof branching is domain branching along a primary branch buds formin a linear and sequential fashion from proximal to distal The nextform of branching is planar bifurcation in which the tip of the formingtube bifurcates to create two new tips which subsequently elongateand bifurcate again creating four tips The last process of branching isknown as orthogonal bifurcation In this process the initial planar bifurcation is followed by a rotation around the planar axis which createstwo new tips through bifurcation A critical gene in this processSprouty has been found to attenuate Erk12 signaling thereby alteringthe orientation of cell division and future tube elongation [] Othercritical genes and regulatory networks associated with FGF signalingalso contribute to controlling the periodicity of the branched network[] Although elements such as domain specification bifurcation rotation and branch generation remain largely undetermined [] newtechnologies involving highresolution live imaging tension sensingand forcemapping are opening paths to further explore and explainthe branching morphogenesis phenomenon []The early structure of the lung gives rise to a striking architecturalseparation of future SOX2 proximal lineages and SOX9 distal lineages at least in mice [] The diameter of tube generated duringbranching morphogenesis in the pseudoglandular and canalicularstages has a small degree of variance within each stage as measuredfrom electron micrograph sources of fetal human tissue [] This suggests that the branching program is rigorous in its control of lung structure and that tubes themselves may have instructive potential on thedeveloping epithelia Once the basic an structure has formed thelung continues to be exposed to mechanical cues as it continues to mature In several cases these cues have been shown to be essential forcorrect an function In utero the fetal lung is a secretory an thatonly converts to an absorptive one to prepare for breathing afterbirth through a change in the activity of chloride and sodium channelslate in development Fetal lung secretions result in a static fluid pressureof around cmH2O in the developing terminal sacs of the fetus whichpropels branching morphogenesis outwards into the developing thoracic cavity [] Lack of amniotic fluid in the developing lung alters the expression of distal epithelial markers and consequentlyresults in the creation of smaller than normal lungs pulmonary hypoplasia [] highlighting the importance of this mechanical pressureduring lung development In addition cyclic strain is generated fromfetal breathing movements FBM in utero that prime the airway foruse after birth FBM are detectable from the tenth week of pregnancyand begin as infrequent and erratic activity with long quiescent periodsAs development continues these quiescent periods decrease andsustained periods of fetal breathing occur These breathing movementsvary with the fetal sleep cycle and can be chemically tuned [] andalter the volume of terminal sacs by around [] againhighlighting the importance of mechanical signals ‚uencing lung development Finally a novel FGF10FGFR2dependenttensionalmechanism has been shown by which distal epithelial cells in the lungaccumulate motor proteins at the apex of the cell thereby becoming resistant to compression from increasing fluid pressure within the tubelumen Cells under this tension are more likely to become AEC II cellswhile those under compression become AEC I cells [] Interestinglywhile the above examples highlight the importance of specific mechanical signals in the growth development and differentiation of the lungPSC directed differentiation protocols of the lung are primarily based onmimicking the sequential chemical changes that occur during lungdevelopment Directed differentiation of lung epithelia inspired by embryologyEarly attempts to create lung epithelia from PSCs began in mouseand did not attempt to mimic the stepwise changes in chemical signaling that occur during development Rather groups focused on applyinglunglike physical cues such as airliquid interface [] These protocols while successful in generating NKX21 positive populationsalso produced contaminating cells expressing pluripotency markersOCT4 NANOG SSEA4 TRA160 TRA181 These early attempts solidified that further optimization particularly related to the chemical cuesapplied was needed to reliably create lung progenitors from pluripotentsources without remnant pluripotent contaminating cells More successful directed differentiation protocols were rationalized from the detailed understanding of the chemical changes during lung embryologyIn this section we describe in detail the different differentiation protocols currently available that evolved from this approach Mouse embryonic stem cell derived lung epitheliaAlthough mouse models do not fully recapitulate human lung development they have served as guides for earlier iterations of PSC directeddifferentiation protocols and have identified critical chemical cues forlung anogenesis Broadly speaking these protocols begin by drivingstem cells towards a definitive endoderm fate SOX17 and FOXA2mimicking the preembryonic period of human lung developmentweeks “ through high doses of the nodal activating molecule ActivinA [“] Foregut endoderm is then induced via transforminggrowth factor beta TGFβ inhibition either alone [] or with BMP inhibition [] for a short period a process called anteriorization as duringthe embryonic period of human lung development weeks “ Thisforegut endoderm FOXA2SOX2 is subsequently induced to generate NKX21 cells putative lung progenitors by stimulating theretinoic acid RA BMP WNT and FGF signaling pathways []These lung progenitors are further matured as demonstrated by increased NKX21 expression through application of corticosteroids[] In brief each protocol begins with PSCs guided through definitiveendoderm followed by anteriorization to foregut endoderm and subsequent ventralization to generate NKX21 cells [] These protocolsformed the basis and backbone for the creation of human lung epitheliafrom PSCsGiven the structural and cellular complexity of the lung it is reasonable that the earliest protocols focused on mouse However there aresubtle differences that highlight how human models are different interms of structure patterning and differentiation For example thePlease cite this as R Varma JP Soleas TK Waddell Current strategies and opportunities to manufacture cells for modelinghuman lungs Adv Drug Deliv Rev 101016jaddr202008005 0cR Varma Advanced Drug Delivery Reviews xxx xxxentire human conducting airway is comprised of a pseudostratified epithelium even at diameters less than 05mm [] In contrastconducting airways in mice only exhibit pseudostratified epitheliumwith accompanying submucosal glands and cartilage in the most proximal portion of the airway and transition directly into alveolar sacs []This difference in histology affects the residing cell populations as evidenced by the lack of basal cells in the lower portion of the proximal airways of mice [] Similarly mouse models suggest thatSOX2SOX9 progenitors are quite rare and their cell fate is ambiguous [] However evidence from directed differentiation of humanlung epithelia [] which has been confirmed in vivo []reveals that SOX2SOX9 progenitors are common in the developinglung buds and that branch tips of the pseudoglandular staged lunggive rise to both proximal and distal epithelia [] Moreover specificprotein markers have been found to differ in both timing and locationof expression between human and mouse models proSPC in mouseis expressed early and throughout the developing mouse epithelium[] while in human proSPC is rarely detected early in development and is only robustly found later in distal epithelia [] These examples highlight that while there are similarities development andpatterning of mouse and human lungs is different and these differencesrequire human models to be fully appreciated Human pluripotent stem cellderived lung epitheliaHuman PSC protocols have generally followed the same differentiation chronology as that of mouse directed differentiation wherein definitive endoderm anterior foregut endoderm and NKX21 lungprogenitors are produced sequentiallyDifferent groups have adhered to their own methods of generatingdefinitive endoderm which primarily involves exposing PSCs to highconcentrations of Activin A Slight variations such as introducing WNTagonism through WNT3a or CHIR99021 prior to [] or alongside[] Activin A or additional exposure to BMP4 and FGF2[] during this stage exist across protocols for differentially inducing primitive streak and its anteriorization towards producing definitive endoderm In addition the use of embryoid bodies which arelimited by user experience and technique has resulted in a widerange of production efficiencies for achieving this stage from CKITCXCR4EPCAM cells [] to CKIT CXCR4 cells []Recent advances in commercial products have led to development ofstandardized 2D culturebased media STEMdiff Definitive EndodermKit STEMCELL Technologies which allow reliable derivation of of definitive endoderm []Similarly generation of both anterior foregut endoderm andtoventralized lung progenitor populations has been subjectmuch investigation and modification Earlier work suggested thatSOX2FOXA2 ± in their case anterior foregut endoderm canonly be induced by subjecting definitive endoderm to TGFβ and BMP inhibition [] Subsequent studies however attempted to anteriorize definitive endoderm to foregut endoderm through TGFβ inhibition alone SOX2 a combination of endogenous WNT TGFβ and BMP inhibition not quantified [] and via Sonic Hedgehog SHH and FGF2signaling FOXA2 EPCAM [] A comparison of the lattertwo strategies demonstrated that SHH and FGF2 are insufficient inproducing reliable NKX21 lung progenitors [] possibly becauseFGF2 is involved in promoting thyroid lineages [] In general TGFβand BMP inhibition [] is the basis for currently applied endodermanteriorization strategies [“]Factors involved in early versions of ventralization in directed differentiation protocols included WNT3a FGF7 FGF10 BMP4 epidermalgrowth factor EGF and RA have now been reduced based on elimination studies [] As such CHIR99021 CHIR WNT agonist BMP4and RA are necessary and sufficient for producing lung progenitorsfrom anterior foregut endoderm derived from both mouse and humanPSCs [] Despite finding that FGF7 and FGF10 are nonessential for inducing NKX21 expression they continue to be used forventralization in some protocols [] Although each protocol differsin terms of the duration of each phase NKX21 lung progenitors aregenerally achieved by days with the exception of a study by deCarvalho in which they maintained their cultures for an additional days in FGF7 FGF10 and CHIR99021 to attain NKX21FOXA2 lung progenitors In all cases these lung progenitors are theneither sorted or directly guided towards proximal or distal progeny in2D or 3D culture systems Ideally products of directed differentiationprotocols should mimic the cell proportions present in human airwaysand lungs Table however current protocols have not progressedthat far While these protocols continue to be refined the percentageof select cell populations generated from these protocols have beensummarized in Table Creation of human proximal lung epitheliaProtocols to create proximal lung epithelia have focused on the production of the four major cells types present ciliated goblet club andbasal cells see Table for a summary of markers for each cell type Motivation for creating proximal epithelia in the field has primarily been todevelop patientspecific cystic fibrosis CF models [] andor toproduce epithelia with multiciliated cell populations for protocol validation [] A shift towards human PSCderived CF models hasbeen critical as mouse models do not accurately represent CF diseaseprogression and phenotypes seen in humans [“] As such thefirst evidence of human PSC proximalization using CF patientderivedPSCs was shown by Mou who exposed anterior foregut endodermto BMP4 GSK3iXV WNT agonist FGF2 and RAsupplemented B27 togenerate NKX21 cells by Day Although contaminatingneuroectodermal and distal lung NKX21SOX9 cells were presentday populations included proximal NKX21SOX2 progenitorsSubcutaneous implantation of this population in immunodeficientmice for days resulted in emergence of NKX21P63 cells howeverno mature epithelial markers for ciliated goblet and club cells werefoundWong employed a longer 2D differentiation approach to produce mature proximal airway epithelia in vitro Through a processthey called œproximal specification they generated day lung progenitors via low levels of BMP4 mimicking signaling gradients in theairway FGF7 and FGF10 which began expressing proximal genes Further culture with FGF7 FGF10 and FGF18 resulted in upregulated geneexpression of KRT5 P63 FOXJ1 SOX17 cystic fibrosis transmembraneconductance regulator CFTR and SCGB1A1 to a lesser extent alongwith low levels of distal SOX9 and SPC by day Protein expressionamounted to NKX21 panKRT P63 FOXJ1 cells These cells were subsequently matured in airliquid interface ALI culture for weeks week of submerged culture withFGF18 followed by weeks of ALI culture to generate CFTRpanKRT FOXJ1 with coexpressing CFTR and CFTRLHS28 cells The resulting epithelium ranged from being squamous to cuboidal with sparse pseudostratified regions implying thatthis protocol lacked specific maturation cues Contaminating thyroidthyroglobulin and PAX9 liver HNF4 and AFP and pancreaticPDX1 lineages were detected through quantitative PCR while percentages of goblet club and basal cell populations barring gene expression analysis were not evaluatedA similar 2D culture approach was employed by Firth to generate proximal lung progenitors which were subsequently matured intomulticiliated epithelia They optimized lower concentrations of BMP4required during the ventralization phase day “

cancer244

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: "Pluripotent stem cellsDirected differentiationIn vitro disease modellingLungAirwayMechanical cuesContentsChronic lung diseases remain major healthcare burdens for which the only curative treatment is lung transplantation In vitro human models are promising platforms for identifying and testing novel compounds to potentiallydecrease this burden Directed differentiation of pluripotent stem cells is an important strategy to generate lungcells to create such models Current lung directed differentiation protocols are limited as they do not recapitulate the diversity of respiratory epithelium generate consistent or sufficient cell numbers for drug discoveryplatforms and establish the histologic tissuelevel anization critical for modeling lung function In this review we describe how lung development has formed the basis for directed differentiation protocols and discussthe utility of available protocols for lung epithelial cell generation and drug development We further highlighttissue engineering strategies for manipulating biophysical signals during directed differentiation such that futureprotocols can recapitulate both chemical and physical cues present during lung development Elsevier BV All rights reservedOverview of key developmental stages Lung anogenesis molecularly defining lung fate in the embryo Branching morphogenesis and other mechanical cues generated during lung development Introduction Human embryology as a blueprint for lung directed differentiation Directed differentiation of lung epithelia inspired by embryology Mouse embryonic stem cell derived lung epithelia Modeling airway and lung diseases for drug discovery Opportunities to exploit mechanical cues for improving directed differentiation protocols in the future Micropatterning in 2D Stem cell behaviour on substrate topographies Micropatterning in 3D anoid systemsSubstrate textureHuman pluripotent stem cellderived lung epithelia Creation of human proximal lung epithelia Comparisons of proximal airway directed differentiation protocols Creation of human distal lung epitheliaComparisons of distal lung directed differentiation protocols Limitations of current directed differentiation protocols Ž Correspondence to G Karoubi Latner Thoracic Surgery Research Laboratories Toronto General Hospital College St Toronto ON M5G 1L7 CanadaŽŽ Correspondence to A P McGuigan Institute for Biomaterials and Biomedical Engineering University of Toronto College Street Toronto ON M5S 3G9 CanadaEmail addresses golnazkaroubiuhnresearchca G Karoubi alisonmcguiganutorontoca AP McGuigan101016jaddr2020080050169409X Elsevier BV All rights reservedPlease cite this as R Varma JP Soleas TK Waddell Current strategies and opportunities to manufacture cells for modelinghuman lungs Adv Drug Deliv Rev 101016jaddr202008005 0cR Varma Advanced Drug Delivery Reviews xxx xxxAcknowledgementsReferencesFuture outlook IntroductionEndstage lung disease is the third leading cause of morbidity andmortality worldwide [] and produces a significant burden onhealthcare systems due to extensive resource expenditures for diseasemanagement and as lung transplantation is the only curative treatmentoption Such diseases include acute respiratory distress syndromechronic obstructive pulmonary disease cystic fibrosis and pulmonaryfibrosis Chronic pulmonary diseases result in million global deathsper year [] Patients who receive transplants face continued complications associated with chronic immunosuppression and graft rejectionwith the transplant survival rates at and years being and respectively [] Furthermore since lungs function as an important barrier between the internal and the external environments they are a critical site for bacterial and viral infections and disease transmissionparticularly relevant given the current COVID19 pandemic There istherefore a critical need to better elucidate the mechanisms of infectiondisease progression host response and cellular repair in the lung to enable the development of novel targeted therapeutics for lung diseaseTissueengineered models have emerged as a technology to addressthis challenge and shown some success in drug identification and toxicology studies For example commercially available airway epithelialmodels such as EpiAirwayTM MatTek Life Sciences serve as convenientplatforms with airliquid interface culture capabilities for assessing theeffect of chemical and physical stimuli [“] Other examples includethe Alveolus LungChip and Airway LungChip systems Emulate Incoriginally developed in the Ingber laboratory which mimic theepithelialendothelial interface of the airway and provide a more dynamic platform for testing new anti‚ammatory compounds inasthma [] and new small molecule targets to decrease cancerassociated pulmonary edema [] More complex models have alsobeen reported which involve selfassembly of heterogeneous progenitor cells into 3D structures termed anoids [] These anoidmodels can recapitulate aspects of human lung development in termsof tissue structures and protein expression and therefore present apromising opportunity for drug screening []A challenge in developing such human in vitro lung models to screenfor drugs however is the requirement for large batches of similarhuman cells as a starting population for tissue manufacturing to ensureminimal heterogeneity between test wells [] Achieving this is especially challenging when using primary human lung cells which exhibitconsiderable heterogeneity across donors and have a limited ability togrow and differentiate reliably [] Furthermore primary cells areoften extracted from diseased donors which is not ideal for conductingcontrolled studies due to the wide range of therapeutic and environmental factors these cells have already been exposed to Directed differentiation of pluripotent populations has the potential to create vastnumbers of cells from either healthy or diseased patients It allows introduction of specific diseaseassociated mutations via CRISPRCas9gene editing to recapitulate and understand pathologies in a controlledmanner As such directed differentiation enables the generation of anattractive cell source for drug screening platforms and personalized disease models that may provide insight into tissue regeneration mechanisms [“]Directed differentiation protocols to manufacture specific cell populations from pluripotent stem cells PSCs have been developed to meetthe need for a homogeneous human cell source Older lung directed differentiation protocols from the late 2000s have been proven inefficientdue to the nonstandardized methods through which they derive lungendoderm from embryoid bodies [“] A series of more standardizedstepwise protocols have since emerged in the last decade that provideavenues for developing airway and lung epithelia albeit with variableefficiencies [“] The first ‚uential directed differentiation protocol to produce lung epithelia used human PSCs in [] whichwas further supported by two prominent studies conducted usingmouse PSCs in [] These protocols have continued to be enhanced through adaptations related to the selection of growth factorsand small molecules the chronology of morphogen delivery as wellas innovations in enabling platforms such as cell sorting 3D cultureand singlecell analyses to efficiently derive normal and diseased lungepithelia from human PSCs [“] Despite such advancements limitations pertaining to heterogeneity in the resultingpopulations still exist which are likely attributed to variability across directed differentiation trials PSC cell lines or the persistence of contaminating cell populations belonging to other lineages While protocolshave progressed to some degree in differentiating proximal airwayand distal alveolar epithelia they remain limited Overall many unanswered questions remain with regards to the identity maturity andfunctionality of resulting cell types as well as their utility for tissue engineering and drug testing approaches Therefore these protocols mustbe optimized further to reliably produce large numbers of spatially relevant and functional lung and airway epithelial cells that appropriatelyrespond to both chemical and mechanical stimuli in the context of disease modeling and drug discoveryIn this review we discuss the directed differentiation protocols thatattempt to recapitulate lung development and disease and highlightpossible opportunities to enhance these protocols in the future Wefirst describe development of native lung tissue and the patterningevents that occur that differentiation models attempt to mimic andhighlight how human lung embryology has served as the blueprint tocreate the common pathway of lung directed differentiation protocolsWe then discuss the evolution of directed differentiation protocols tofind opportunities for creating specific populations of airway and lungepithelia through targeted manipulation of key signaling pathways in2D and 3D models We further describe how these models have beenused to recapitulate different airway and lung diseases Finally we discuss how tissue engineering and biophysical cues using biomaterialscan be utilized during lung directed differentiation to mimic patterningcues present in development to augment current differentiationprotocols Human embryology as a blueprintdifferentiationforlung directed Overview of key developmental stagesDirected differentiation protocols have been designed to mimicin vivo human lung development [] Indeed in vitro models of lungdevelopment have provided unique insight into human lung development [] As human lung development has been described at greatlength in earlier reviews [] we provide a brief overview as followsschematically represented in Fig During early embryogenesis at days post fertilization a process called gastrulation begins with the appearance of a structure called primitive streak through which cells migrate to form the primary embryonic germ layers definitive endodermmesoderm and ectoderm [“] Definitive endoderm expandsthereby forming the primitive gut tube comprised of three endodermalregions foregut midgut and hindgut [] This is when lungPlease cite this as R Varma JP Soleas TK Waddell Current strategies and opportunities to manufacture cells for modelinghuman lungs Adv Drug Deliv Rev 101016jaddr202008005 0cR Varma Advanced Drug Delivery Reviews xxx xxxFig Schematic of human lung development from an epithelial perspectivedevelopment begins at approximately four weeks into embryonic lifewith the outgrowth of foregut endoderm [] and continues througheight years of postnatal life [] There are five stages to lungdevelopment Embryonic weeks “ The future lung buds emerge from the ventral side of the primitive foregut endoderm into the surroundingmesenchyme and develop into embryonic lung buds with early trachea and bronchi [“] Pseudoglandular weeks “ Branching of the airway continuesleading to formation of conducting and terminal bronchioles whilethe proximal airway epithelium begins to develop [] Canalicular weeks “ Development of the respiratory or gasexchanging airways is initiated primitive alveoli form and the future distal epithelium begins to thin as distal epithelial markers areexpressed [] Saccular weeks “ Emergence of sacshaped distal airwayswhich develop crests with muscle and elastin to create indentationsThese distal airways extend to form alveoli by weeks [] The developing epithelium and vasculature within the future alveolus continue to merge closer together to facilitate future gas exchange andfurther differentiation of alveolar epithelial cells AEC I and II takesplace Alveolar periods week “ years True alveoli are seen in week and the majority of alveolarization takes place through sacculeseptation a process by which the sacshaped distal airways changetheir internal architecture and create thin walls intraluminallySeptation leads to an increase in surface area of the gas exchangingportion of the developing lung and prepares the fetus to breath airduring this stage [] Lung anogenesis molecularly defining lung fate in the embryoDuring the embryonic period early lung is genetically defined by theexpression of transcription factor NK2 Homeobox NKX21 and Srybox SOX2 [“] During human lung development it has beenfound that the lung buds and branches given off during thepseudoglandular period are mostly SOX2SOX9 [] BothSOX2 and SOX9 are individual markers of the early proximal or distal lineage respectively [“] Over the course of the canalicular and saccular periods of development weeks “ these double positivepopulations downregulate one SOX protein and maintain expressionof the other as these cells mature towards proximal or distal lineages[] The proximal airway closer to the mouth is comprised of apseudostratified columnar epithelium that is responsible for theconducting airway function debris and pathogen removal ciliatedcells mucus production goblet cells prevention of airway ‚ammation club cells and humidification of air as it passes through to the distal lung compartment [“] The squamous distal epitheliumcomposed of alveolar epithelial cells AEC I and II facilitates the respiratory function of the lung as air in the epithelial compartment is broughtinto close apposition to blood from the pulmonary vasculature it alsosecretes surfactants which play an immunologic role and decrease thesurface tension present at the airliquid interface thereby preventing alveolar collapse [] In humans a number of cell types are found in theproximal airway each identified with specific markers Table This includes basal cells tumor protein p63P63 keratinKRT5 nerve growthfactor receptorNGFR integrin α6ITGA6 integrin β4ITGB4 ciliatedcells Forkhead BoxJ1FOXJ1 acetylated tubulinAcTUB goblet cellsmucin 5ACMUC5AC mucin 5BMUC5B club cells club cell secretoryproteinCCSP or SCGB1A1 and pulmonary neuroendocrine cellsPNECs synaptophysinSYP chromogranin ACHGA On the otherhand homeodomainonly protein HOPX identifies the distal lungalong with AEC I cells T1α podoplaninPDPN aquaporin 5AQP5while AEC II cells are recognized via surfactant protein B SPC prosurfactant protein C proSPC or SPC and HT2280 []One mechanism by which lung epithelia begin to mature is based onchemokine secretions from the surrounding mesenchyme and the developing heart field which are well reviewed here [] Key players including fibroblast growth factors FGFs [“] WNTs [“]and bone morphogenetic proteins BMPs [“] are known to inducethe differentiation of early lung progenitors in a controlled manner Forexample in mouse it has been found that FGF10 plays a role in bud outgrowth [] and drives lung progenitors towards a distal fate []through canonical WNT signaling [] Proximal epithelia developbecause they are located further away from distally located FGF reservoirs in the mesenchyme in a mechanism that appears dependent onconcentration gradients [] BMP4 plays a key role in lung bud formation from foregut endoderm and establishment of both dorsoventralback to front and proximodistal top to bottom patterning in the nascent lung [] BMP4 is also present at high levels in distal bud tips andepithelia including AEC II cells [] however its inhibition promotesa proximal fate and along with BMP2 inhibition ciliated cell development [] Branching morphogenesis and other mechanical cues generated duringlung developmentWhile the cell fate of early proximal and distal lineages is directedthrough chemical signals the lung epithelium itself undergoes markedPlease cite this as R Varma JP Soleas TK Waddell Current strategies and opportunities to manufacture cells for modelinghuman lungs Adv Drug Deliv Rev 101016jaddr202008005 0cR Varma Advanced Drug Delivery Reviews xxx xxxTable Epithelial populations in native human airways and lungsRegionProximal AirwayDistal LungCell TypeAssociated Markers for Cell CharacterizationCiliated CellGoblet CellClub CellBasal CellAlveolar epithelial cell type I AEC IAlveolar epithelial cell type II AEC IIFOXJ1 AcTUBMUC5AC MUC5BCCSP SCGB1A1 SCGB3A2P63 KRT5 NGFR ITGA6 ITGB4HOPX PDPN AQP5SPB SPC HT2280Cell Proportions in Native Lung“ []“ []“ []“ [] [] []changes in architecture a process known as branching morphogenesis[] From the simple tube of the anterior foregut endoderm to thecomplex tubular structure of the adult a highly stereotyped mechanismof branching morphogenesis facilitates the outgrowth division placement and structure of lung airway [] Branching morphogenesis ofthe lung is driven by three simple and iteratively used processes domain branching planar and orthogonal bifurcation [] The first formof branching is domain branching along a primary branch buds formin a linear and sequential fashion from proximal to distal The nextform of branching is planar bifurcation in which the tip of the formingtube bifurcates to create two new tips which subsequently elongateand bifurcate again creating four tips The last process of branching isknown as orthogonal bifurcation In this process the initial planar bifurcation is followed by a rotation around the planar axis which createstwo new tips through bifurcation A critical gene in this processSprouty has been found to attenuate Erk12 signaling thereby alteringthe orientation of cell division and future tube elongation [] Othercritical genes and regulatory networks associated with FGF signalingalso contribute to controlling the periodicity of the branched network[] Although elements such as domain specification bifurcation rotation and branch generation remain largely undetermined [] newtechnologies involving highresolution live imaging tension sensingand forcemapping are opening paths to further explore and explainthe branching morphogenesis phenomenon []The early structure of the lung gives rise to a striking architecturalseparation of future SOX2 proximal lineages and SOX9 distal lineages at least in mice [] The diameter of tube generated duringbranching morphogenesis in the pseudoglandular and canalicularstages has a small degree of variance within each stage as measuredfrom electron micrograph sources of fetal human tissue [] This suggests that the branching program is rigorous in its control of lung structure and that tubes themselves may have instructive potential on thedeveloping epithelia Once the basic an structure has formed thelung continues to be exposed to mechanical cues as it continues to mature In several cases these cues have been shown to be essential forcorrect an function In utero the fetal lung is a secretory an thatonly converts to an absorptive one to prepare for breathing afterbirth through a change in the activity of chloride and sodium channelslate in development Fetal lung secretions result in a static fluid pressureof around cmH2O in the developing terminal sacs of the fetus whichpropels branching morphogenesis outwards into the developing thoracic cavity [] Lack of amniotic fluid in the developing lung alters the expression of distal epithelial markers and consequentlyresults in the creation of smaller than normal lungs pulmonary hypoplasia [] highlighting the importance of this mechanical pressureduring lung development In addition cyclic strain is generated fromfetal breathing movements FBM in utero that prime the airway foruse after birth FBM are detectable from the tenth week of pregnancyand begin as infrequent and erratic activity with long quiescent periodsAs development continues these quiescent periods decrease andsustained periods of fetal breathing occur These breathing movementsvary with the fetal sleep cycle and can be chemically tuned [] andalter the volume of terminal sacs by around [] againhighlighting the importance of mechanical signals ‚uencing lung development Finally a novel FGF10FGFR2dependenttensionalmechanism has been shown by which distal epithelial cells in the lungaccumulate motor proteins at the apex of the cell thereby becoming resistant to compression from increasing fluid pressure within the tubelumen Cells under this tension are more likely to become AEC II cellswhile those under compression become AEC I cells [] Interestinglywhile the above examples highlight the importance of specific mechanical signals in the growth development and differentiation of the lungPSC directed differentiation protocols of the lung are primarily based onmimicking the sequential chemical changes that occur during lungdevelopment Directed differentiation of lung epithelia inspired by embryologyEarly attempts to create lung epithelia from PSCs began in mouseand did not attempt to mimic the stepwise changes in chemical signaling that occur during development Rather groups focused on applyinglunglike physical cues such as airliquid interface [] These protocols while successful in generating NKX21 positive populationsalso produced contaminating cells expressing pluripotency markersOCT4 NANOG SSEA4 TRA160 TRA181 These early attempts solidified that further optimization particularly related to the chemical cuesapplied was needed to reliably create lung progenitors from pluripotentsources without remnant pluripotent contaminating cells More successful directed differentiation protocols were rationalized from the detailed understanding of the chemical changes during lung embryologyIn this section we describe in detail the different differentiation protocols currently available that evolved from this approach Mouse embryonic stem cell derived lung epitheliaAlthough mouse models do not fully recapitulate human lung development they have served as guides for earlier iterations of PSC directeddifferentiation protocols and have identified critical chemical cues forlung anogenesis Broadly speaking these protocols begin by drivingstem cells towards a definitive endoderm fate SOX17 and FOXA2mimicking the preembryonic period of human lung developmentweeks “ through high doses of the nodal activating molecule ActivinA [“] Foregut endoderm is then induced via transforminggrowth factor beta TGFβ inhibition either alone [] or with BMP inhibition [] for a short period a process called anteriorization as duringthe embryonic period of human lung development weeks “ Thisforegut endoderm FOXA2SOX2 is subsequently induced to generate NKX21 cells putative lung progenitors by stimulating theretinoic acid RA BMP WNT and FGF signaling pathways []These lung progenitors are further matured as demonstrated by increased NKX21 expression through application of corticosteroids[] In brief each protocol begins with PSCs guided through definitiveendoderm followed by anteriorization to foregut endoderm and subsequent ventralization to generate NKX21 cells [] These protocolsformed the basis and backbone for the creation of human lung epitheliafrom PSCsGiven the structural and cellular complexity of the lung it is reasonable that the earliest protocols focused on mouse However there aresubtle differences that highlight how human models are different interms of structure patterning and differentiation For example thePlease cite this as R Varma JP Soleas TK Waddell Current strategies and opportunities to manufacture cells for modelinghuman lungs Adv Drug Deliv Rev 101016jaddr202008005 0cR Varma Advanced Drug Delivery Reviews xxx xxxentire human conducting airway is comprised of a pseudostratified epithelium even at diameters less than 05mm [] In contrastconducting airways in mice only exhibit pseudostratified epitheliumwith accompanying submucosal glands and cartilage in the most proximal portion of the airway and transition directly into alveolar sacs []This difference in histology affects the residing cell populations as evidenced by the lack of basal cells in the lower portion of the proximal airways of mice [] Similarly mouse models suggest thatSOX2SOX9 progenitors are quite rare and their cell fate is ambiguous [] However evidence from directed differentiation of humanlung epithelia [] which has been confirmed in vivo []reveals that SOX2SOX9 progenitors are common in the developinglung buds and that branch tips of the pseudoglandular staged lunggive rise to both proximal and distal epithelia [] Moreover specificprotein markers have been found to differ in both timing and locationof expression between human and mouse models proSPC in mouseis expressed early and throughout the developing mouse epithelium[] while in human proSPC is rarely detected early in development and is only robustly found later in distal epithelia [] These examples highlight that while there are similarities development andpatterning of mouse and human lungs is different and these differencesrequire human models to be fully appreciated Human pluripotent stem cellderived lung epitheliaHuman PSC protocols have generally followed the same differentiation chronology as that of mouse directed differentiation wherein definitive endoderm anterior foregut endoderm and NKX21 lungprogenitors are produced sequentiallyDifferent groups have adhered to their own methods of generatingdefinitive endoderm which primarily involves exposing PSCs to highconcentrations of Activin A Slight variations such as introducing WNTagonism through WNT3a or CHIR99021 prior to [] or alongside[] Activin A or additional exposure to BMP4 and FGF2[] during this stage exist across protocols for differentially inducing primitive streak and its anteriorization towards producing definitive endoderm In addition the use of embryoid bodies which arelimited by user experience and technique has resulted in a widerange of production efficiencies for achieving this stage from CKITCXCR4EPCAM cells [] to CKIT CXCR4 cells []Recent advances in commercial products have led to development ofstandardized 2D culturebased media STEMdiff Definitive EndodermKit STEMCELL Technologies which allow reliable derivation of of definitive endoderm []Similarly generation of both anterior foregut endoderm andtoventralized lung progenitor populations has been subjectmuch investigation and modification Earlier work suggested thatSOX2FOXA2 ± in their case anterior foregut endoderm canonly be induced by subjecting definitive endoderm to TGFβ and BMP inhibition [] Subsequent studies however attempted to anteriorize definitive endoderm to foregut endoderm through TGFβ inhibition alone SOX2 a combination of endogenous WNT TGFβ and BMP inhibition not quantified [] and via Sonic Hedgehog SHH and FGF2signaling FOXA2 EPCAM [] A comparison of the lattertwo strategies demonstrated that SHH and FGF2 are insufficient inproducing reliable NKX21 lung progenitors [] possibly becauseFGF2 is involved in promoting thyroid lineages [] In general TGFβand BMP inhibition [] is the basis for currently applied endodermanteriorization strategies [“]Factors involved in early versions of ventralization in directed differentiation protocols included WNT3a FGF7 FGF10 BMP4 epidermalgrowth factor EGF and RA have now been reduced based on elimination studies [] As such CHIR99021 CHIR WNT agonist BMP4and RA are necessary and sufficient for producing lung progenitorsfrom anterior foregut endoderm derived from both mouse and humanPSCs [] Despite finding that FGF7 and FGF10 are nonessential for inducing NKX21 expression they continue to be used forventralization in some protocols [] Although each protocol differsin terms of the duration of each phase NKX21 lung progenitors aregenerally achieved by days with the exception of a study by deCarvalho in which they maintained their cultures for an additional days in FGF7 FGF10 and CHIR99021 to attain NKX21FOXA2 lung progenitors In all cases these lung progenitors are theneither sorted or directly guided towards proximal or distal progeny in2D or 3D culture systems Ideally products of directed differentiationprotocols should mimic the cell proportions present in human airwaysand lungs Table however current protocols have not progressedthat far While these protocols continue to be refined the percentageof select cell populations generated from these protocols have beensummarized in Table Creation of human proximal lung epitheliaProtocols to create proximal lung epithelia have focused on the production of the four major cells types present ciliated goblet club andbasal cells see Table for a summary of markers for each cell type Motivation for creating proximal epithelia in the field has primarily been todevelop patientspecific cystic fibrosis CF models [] andor toproduce epithelia with multiciliated cell populations for protocol validation [] A shift towards human PSCderived CF models hasbeen critical as mouse models do not accurately represent CF diseaseprogression and phenotypes seen in humans [“] As such thefirst evidence of human PSC proximalization using CF patientderivedPSCs was shown by Mou who exposed anterior foregut endodermto BMP4 GSK3iXV WNT agonist FGF2 and RAsupplemented B27 togenerate NKX21 cells by Day Although contaminatingneuroectodermal and distal lung NKX21SOX9 cells were presentday populations included proximal NKX21SOX2 progenitorsSubcutaneous implantation of this population in immunodeficientmice for days resulted in emergence of NKX21P63 cells howeverno mature epithelial markers for ciliated goblet and club cells werefoundWong employed a longer 2D differentiation approach to produce mature proximal airway epithelia in vitro Through a processthey called œproximal specification they generated day lung progenitors via low levels of BMP4 mimicking signaling gradients in theairway FGF7 and FGF10 which began expressing proximal genes Further culture with FGF7 FGF10 and FGF18 resulted in upregulated geneexpression of KRT5 P63 FOXJ1 SOX17 cystic fibrosis transmembraneconductance regulator CFTR and SCGB1A1 to a lesser extent alongwith low levels of distal SOX9 and SPC by day Protein expressionamounted to NKX21 panKRT P63 FOXJ1 cells These cells were subsequently matured in airliquid interface ALI culture for weeks week of submerged culture withFGF18 followed by weeks of ALI culture to generate CFTRpanKRT FOXJ1 with coexpressing CFTR and CFTRLHS28 cells The resulting epithelium ranged from being squamous to cuboidal with sparse pseudostratified regions implying thatthis protocol lacked specific maturation cues Contaminating thyroidthyroglobulin and PAX9 liver HNF4 and AFP and pancreaticPDX1 lineages were detected through quantitative PCR while percentages of goblet club and basal cell populations barring gene expression analysis were not evaluatedA similar 2D culture approach was employed by Firth to generate proximal lung progenitors which were subsequently matured intomulticiliated epithelia They optimized lower concentrations of BMP4required during the ventralization phase day “ Answer:

Thyroid_Cancer

"Laryngology COVID19 Coronavirus Head and Neck Cancer Otolaryngology Key Points Journal Preproof 0c The landscape of ever evolving information about COVID19 during the pandemic has hindered the transition to normal clinical volume and efficiency COVID19 should not be a reason for delay in diagnosis or treatment with patients who have upper aerodigestive tract pathology or malignancy The approach to resection reconstruction and surveillance for patients with head and neck cancer may need to be altered to consider severity of disease patient comorbidity and prevalence of regional COVID19 infections amongst other factors In light of the significant number of prolonged intubations there may be an increase in the number of patients who develop early and late sequelae of treatment for COVID19 Tracheostomy should be performed in a safe and efficient manner when specific indications are met Synopsis This review explores the changes to practice associated with COVID19 for providers treating patients with head and neck cancer and laryngeal pathology The aim of the review is to highlight some of the challenges and considerations associated with treating this patient population during the pandemic Additionally it seeks to discuss some of the areas of concern related to ramping up clinical volume IntroductionHistoryDefinitionsBackground The downstream effects of COVID19 caused by severe acute respiratory syndrome coronavirus SARSCoV2 have now pervaded most aspects of society and have made an indelible mark on the way that medicine and specifically otolaryngology is being practiced Of note the Journal Preproof 0cdisease represents a threat to an aging population throughout the world but also has dangerous implications for providers Among the most œatrisk group of medical providers may be those within the fields of otolaryngology and ophthalmology An otolaryngologist was among one of the first providers to succumb to the illness in its early days as it spread through Wuhan China In light of the risk to both patients healthcare workers and society atlarge a push has been made to mitigate the risk of transmission within the field of Otolaryngology Head and Neck Surgery As of June 22nd there are a total of COVID19 cases reported worldwide with a total of deaths The United States has the highest number of cases at with the total number dead at Given the high mortality associated with the novel virus much of the world has enacted significant social distancing restrictions and facial covering mandates to curb the spread of the disease The origin of the virus is not well understood but it is thought that a bat or pangolin vector might have served as the primary reservoir The disease tends to be marked by fever of patients and cough of patients however a litany of other symptoms have also been described including gastrointestinal upset diarrhea shortness of breath headache loss of smelltaste among others Severe disease is characterized by an acute respiratory distress syndrome ARDS with a mortality for patients that require mechanical ventilation The disease has a slight male predominance at Severity of disease seems to correlate to age as patients who are aged have a mortality while those over present with a mortality approaching in early studies Journal Preproof 0cThe nasal cavity and nasopharynx seem to harbor the highest viral load concentration and thus the nasopharynx is the preferred location for acquisition of samples for diagnostic testing Nasal swabs oropharyngeal swabs bronchial alveolar lavage saliva and tracheal aspirates have also been suggested as possible testing sites The current preferred diagnostic assay is RTPCR which has a variable sensitivity of depending on the institution and type of test During the months of May and June many cities states and countries have focused on a return to normal activity and a ramp up of commercial activities During this time many otolaryngology practices have aimed to ramp up activity as well while employing telehealth social distancing and utilization of personal protective equipment PPE The American Academy of Otolaryngology recently published return to practice guidelines which are detailed below As the world continues to move forward during the COVID19 era considerations such as testing including preoperativepreprocedure COVID testing surgical triage clinic workflow and practice management continue to evolve as more information becomes available This review is intended to highlight some of the current recommendations for patient care within the Laryngology and Head and Neck Surgical Oncology scope of practice Discussion Laryngology Journal Preproof 0cAs cases continue to rise increased emphasis has been placed on protection for the provider in the clinical setting Over the last decade officebased management of many common laryngeal disorders has significantly expanded This includes but is not limited to officebased laser ablation of papilloma or dysplasia transoral or transcervical injection laryngoplasty for vocal fold paralysis and EMGguided injection of Botox for spasmodic dysphonia Given the high number of clinicbased aerosolgenerating procedures practiced by today™s laryngologists many providers have seen a marked reduction in their ability to treat patients and their clinical productivity Within the category of aerosolgenerating procedures is flexible fiberoptic laryngoscopy one of the most widely used diagnostic tools for all otolaryngologists and speech pathologists A consensus statement reported by Rameau et al from a virtual webinar attended by approximately participants in the American laryngology community recommended flexible laryngoscopy should be reserved for critical cases in which the findings may have an immediate impact on diagnosis or treatment œIndications include hemoptysis odynophagia limiting hydration and nutrition or airway compromise”notably secondary to infectious and malignant conditions Some have advocated for preclinic COVID testing prior to any aerosol generating procedure however given the high false negative rate of many available tests the use of universal personal protective precautions is recommended According to Givi and colleagues examinations should take place in negative pressure rooms if possible with avoidance of topical lidocaine spray although other groups recommend administration of a topical anesthetic to theoretically decrease the coughsneeze reflex unpublished online chats A substitute to standard aerosolized anesthesia may be pledgets soaked in lidocaine and Journal Preproof 0c oxymetazoline The group also suggests using videolaryngoscopy whenever possible to keep the practitioner and the patient farther apart Disposable laryngoscopes should be used whenever possible Most studies universally recommend the following personal protective equipment PPE N95 mask or powered airpurifying respirators PAPRs gloves gown eye shield or goggles and cap It has also been suggested that the patients wear a mask covering the mouth during flexible laryngoscopy to reduce aerosolization from phonatory maneuvers and in case of coughing or sneezing At this time transoral rigid and mirror laryngoscopy are discouraged unless flexible laryngoscopy cannot be performed due to the increased risk of gagging and coughing as well as the need for the patient to phonate with the mouth uncovered to allow visualization of the larynx Additionally universal masking is encouraged in all clinical spaces in accordance with many state policies Patients in the waiting rooms are encouraged to physically distance or wait in their car for a phone call prior to presenting for their appointment Crosby also suggests offering personal protective equipment for the friends and family accompanying the patient during laryngoscopy and other sites prevent friends and family from accompanying patients inside for the visit Some alternatives to flexible laryngoscopy have been raised including transcervical laryngeal ultrasound which has a reported concordance of in identifying vocal fold motion abnormalities Another key consideration for the laryngologist in the COVID19 era is the approach to sanitization and room turnover after aerosol generating procedures AGPs Laryngoscope turnover should include highlevel disinfection including the use of such chemical disinfectants as glutaraldehyde chlorine dioxide or orthophthaladehyde OPA Some authors recommend immediate placement of the scope after use into a covered receptacle for transport from the Journal Preproof 0cexamination room to the sterile processing areas After completion of laryngoscopy room sanitization with an EPAregistered hospitalgrade disinfectant is recommended with a hydrogen peroxide solution gL chlorine disinfectant or alcohol According to the CDC website it is unknown how long the air inside a particular examination room remains infectious and likely relates to the room size rapidity of air exchange patient factors like viral shedding amount of coughingsneezing and length of time patient was in the room The CDC suggests that rooms with air changeshour ACH take about and minutes to purify the air with and efficiency respectively As the number of air changeshour decreases the time between patients should be increased to allow for appropriate dissipation of theoretical infectious agents As such many hospitals have recommended a turnover time of 4x the time it takes to purify the air with efficiency which may be either minutes or minutes depending on the level of air turnover or could be no additional time if any additional HEPA filtration system and negative pressure has been added Limited data exists to support this approach for SARSCoV2 Laryngology patients are quite diverse with respect to their level of acuity Some patients require more urgent intervention while others may have their care deferred1221 Most guidelines advocate for a tiered approach to ramping up both clinicbased and surgical activity The American Academy of Otolaryngology published guidelines for ramping up clinical activity on May The AAO recommends limiting patient care to individuals with œtimesensitiveurgent and emergent medical conditions This approach is also echoed in the care of head and neck cancer patients see below According to the guidelines emergent conditions include œimpending airway obstruction due to infection neoplasm stenosis foreign body which may Journal Preproof 0cwarrant the following intervention œflexible and rigid laryngoscopy with intervention direct laryngoscopysuspension laryngoscopy bronchoscopy and tracheostomy Urgent conditions include œmoderate or impending airway obstruction progressive dysphonia progressive dysphagia glottic incompetence causing aspiration or impaired pulmonary toilet which warrant the previously described procedures in addition to œstroboscopy functional endoscopic evaluation of swallow esophagoscopy with or without intervention open airway procedures for cancer Time sensitive conditions include œT1 glottic carcinoma or carcinoma in situ stablemild dysphonia stable dysphagia which adds œtranscervical Botox injection to the above list of procedures Routine conditions which may be deferred for days or more include œmildmoderate dysplasia nonobstructive benignphonotraumatic lesions of the vocal folds glottic incompetence glottic incompetence with mild to moderate dysphonia gender affirmation globuscough without alarm signs Comparing acuity of patients also raises an important point about the subset of patients who are typically seen for benign phonotraumatic voice disorders Many live vocal performance venues have shut down concerts have been cancelled or postponed and some studies point to live singing as being a potential source of massive spread For this reason one might assume that the percentage of patients being seen for acute phonotraumatic voice disorders diminishes somewhat Conversely as patients continue to recover from hospitalizations related to COVID19 it is anticipated that there may be a number of patients with sequelae of prolonged intubation including posterior glottic stenosis vocal fold granulomas and trachealsubglottic stenosis Laryngeal surgery in the era of COVID has had to undergo some significant changes in the approach to patient triage surgical technique and management of the airway Preoperative Journal Preproof 0cevaluation of patients must weigh the risk of delaying surgery with the risk of complications related to COVID19 infection Lei et al studied a group of operative patients in whom all were COVID19 positive within the incubation period Mortality was for this group and required ICU admission25 Of note all patients in this study underwent surgery about days prior to demonstrating signs or symptoms of COVID19 pneumonia This suggests there is significant risk associated with elective surgery in seemingly asymptomatic patients who are infected with COVID19 For this reason many authors have suggested preoperative COVID testing although it is a subject of some debate Some advocate for a negative test within hours followed by selfquarantine until the time of surgery while others favor a negative test 48hrs from the time of surgery and a point of care negative test on the day of surgery17 This is not always possible given the limitations of the institution where the patient is undergoing surgery Just as discussed earlier with regard to personal protective equipment in clinic universal precautions should be taken including full PPE and all patients should be presumed positive Airway management in the COVID19 era has become a point of focus for quality improvement and safety groups Endotracheal intubation is cited as one of the procedures which seems to have the highest aerosol generating burden It is recommended that intubation be performed by the most experienced practitioner Additionally some recommend early intubation for patients that are high risk for decompensation while others have advocated delaying intubation in favor of noninvasive means of ventilation This may include high flow nasal cannula which actually has minimal dispersion of exhaled air if appropriately fitted according to Cheung It is recommended that flexible fiberoptic intubation be avoided whenever possible Journal Preproof 0cAdditionally excessive bagmask ventilation should be avoided due to the risk of dispersion of exhaled air Furthermore jet ventilation is considered particularly high risk and should be avoided if possible Management of the surgical airway and the topic of tracheostomy has been well represented in the recent literature During the SARS outbreak in open tracheostomy was the most common surgical procedure performed on infected patients Most studies seem to favor open tracheostomy over percutaneous tracheostomy however consideration may be given for percutaneous dilatation tracheostomy in some patients if the anatomy is favorable and the practitioner has sufficient expertise with the procedure Tay and colleagues advocate for use of PAPR during tracheostomy based on the experience of countries during the SARS crisis Other authors have suggested the use of an N95 mask appropriate eye protection gown double gloves and cap To decrease the risk of autocontamination some have recommended an infection control nurse be available to monitor donning and doffing procedures during tracheostomy Additional proposals include trach teams which may consist of a surgeon anesthetist and scrub nurse to increase efficiency and create an environment of consistent verbal and nonverbal communication especially important given the burdens of communicating through a mask or PAPR Portugal et al discuss a surgical safety checklist for performing tracheostomy in patients with COVID1932 This includes performing tracheostomy in the ICU whenever possible decreasing the number of personnel in the room and having a specific tracheostomy bundle in the ICU room to decrease the number of times providers and nurses need to break scrub to leave the room They also recommend donning inner gloves prior to gown and outer gloves after donning gown to maintain clean inner gloves for the removal and Journal Preproof 0cdisposal of the rest of the PPE Two universally agreed upon maneuvers include stopping ventilation prior to entrance into the airway and holding ventilation until after the tracheostomy tube cuff has been inflated Givi and colleagues suggest that a smaller tracheotomy cuffed may be preferred to decrease the spread of aerosolized particles Miles discusses the New York experience suggesting that for intubated patients the cuff pressure should be checked every hours with a goal of cm H2O greater pressure predisposes tracheal pressure necrosis33 The group also suggests delaying the timing of tracheostomy until days after onset of symptoms when feasible Finally some have advocated for the use of specific air containment setups including plastic draping smoke evacuator tubing or specifically designed negative pressure box The field of laryngology has had to undergo significant change in the setting of the COVID pandemic As the numbers of COVID19 patients have continued to increase during the month of June it is clear that practice of laryngology in the postCOVID era will need to be carefully ramped up to protect patients and providers alike Additionally one would expect a continued increase in the number of recovered patients being seen for sequelae of prolonged intubation Decisions to relax restrictions on flexible laryngoscopy and other AGPs will depend on the local incidence of COVID19 infection availability and accuracy of preprocedure testing sustainable supply of PPE the ability to properly sanitize rooms and ultimately development of an effective vaccine Head and Neck Surgical Oncology Journal Preproof 0cSimilar to laryngology the approach to head and neck surgical oncology continues to evolve as more information becomes available during the COVID era During the early weeks of the pandemic the aspect of cancer care most concerning to patients and providers involved potential delays in therapy Finley suggests that delaying cancer surgery should be done with extreme caution despite COVID1937 Additionally delays beyond weeks could significantly affect longterm outcomes and morbidity of treatment Among patients diagnosed with severe COVID19 requiring ICU admission patients with cancer deteriorated faster than noncancer patients Desai and colleagues discovered a higher risk of severe events in patients recently treated with chemotherapy or surgery in the past days compared to noncancer COVID16 patients38 Additionally patients with advanced stage cancer tended to have a higher rate of severe events when compared to early stage cancer Cancer patients undergoing active treatment are predisposed to COVID19 related complications and critically ill patients with cancer have a higher predisposition to death Head and neck cancer patients especially are considered a highrisk population for complications associated with COVID19 infection8 making safe coordination of care difficult but imperative Head and neck cancer patients are an atrisk group for a number of reasons Silverman points out that head and neck cancer patients tend to present with advanced age history of tobacco and alcohol abuse and cardiac and pulmonary comorbidities which are similarly found in COVID19 Risk of respiratory sequelae in patients who have received chemotherapy andor radiation therapy are high with increased rates of dysphagia aspiration and pneumonia Additionally head and neck cancer patients have an increased risk of respiratory infections and aspiration pneumonitis These factors may expedite deterioration to Journal Preproof 0csevere adverse events in patients with COVID19 Additionally head and neck patients who are actively receiving chemotherapy or immunotherapy may have depressed immune function malnutrition and older age For this reason the patients need to be carefully selected and comorbidities strongly considered when constructing a treatment plan for patients with head and neck cancer Within the United States mortality for patients of color African American and Latinx with COVID19 is significantly higher than for Caucasian patients40 Unfortunately this is a consequence of inequality within society and the healthcare system rather than a biological or pathological difference41 Correspondingly these communities also tend to present with more advanced disease and have significantly worse mortality compared to their Caucasian fellow citizens This pandemic has laid to bear some of the gross inequities within the American health care system and highlighted the need for equitable decision making for all patients with a diagnosis of head and neck cancer during the COVID19 era Another consideration for the head and neck cancer patient during the COVID19 era may include the financial burden and cost of survivorship associated with undergoing cancer treatment and financial hardship related to COVID19™s effect on the world economy and increasing levels of unemployment Given the significant job losses across the United States there is preliminary data to suggest that there will be at least million newly unemployed people who will also lose their insurance coverage in the wake of the pandemic Increased financial strain has been associated with decreased quality of life scores and subsequently mortality in head and neck cancer patients Journal Preproof 0c Recommendations for head and neck clinic are similar to what was previously discussed for laryngology Providers are expected to take universal precautions regardless of the patient™s COVID status Flexible fiberoptic laryngoscopy is considered a highrisk aerosol generating procedure Due to this laryngoscopy should be reserved for instances in which it is likely to change management One of the beneficial consequences of the COVID19 era is the increased access of care through the widespread adoption of telehealth clinics among most hospitals49 Providers may use telemedicine as an initial preoperative assessment or prescreen for patients that will later be seen in clinic or prior to surgery While telehealth is wonderful for obtaining a detailed history reviewing dataimaginglabs and discussing surgical optionsrisksbenefits a big drawback is the inability to perform a comprehensive head and neck physical exam Physical examination with or without fiberoptic laryngoscopy is important to define the extent of tumor and formulating an ablative and reconstructive plan Fortunately some workarounds include anatomic and physiologic imaging for ablative planning and CT angiography and virtual planning sessions for microvascular reconstruction Telemedicine also serves a vital role in triage of posttreatment head and neck cancer patients who may not be able to be seen as frequently due to the pandemic Telemedicine also serves a vital role in the coordination of care between multiple oncologic disciplines Dharmarajan and colleagues highlighted the University of Pittsburgh approach to a virtual multidisciplinary tumor board clinic MDC This strategy has been adopted by multiple institutions and works quite well to coordinate care between specialties In their study they found that of virtual tumor board participants preferred virtual multidisciplinary clinic to Journal Preproof 0cthe inperson format Additionally about of participants indicated that they would prefer to continue the virtual multidisciplinary format once in person meeting restrictions had been lifted Through multiple virtual meeting applications practitioners can share imaging and laryngoscopy which may assist with decision making for patients Similar to the guidelines published for laryngeal surgery the American Academy of Otolaryngology has published recommendations for ramping up clinical volume as it relates to triage for head and neck surgical oncology Setzen et al note that most head and neck cases fall within the urgent category The guidelines list emergent procedures as being tumor obstructing airway significant bleeding acute or impending neurological change and salivary gland or deep neck abscesses Urgent procedures within days include all head and neck squamous cell carcinomas of the upper aerodigestive tract benign tumors with impending complications or morbidity anaplastic thyroid cancer medullary thyroid cancer bulky differentiated thyroid cancer with regionaldistant metastasis locally aggressive or large nodules 4cm Bethesda high grade salivary malignancies skin cancers and parathyroid carcinomas with significant systemic effects Time sensitive procedures include lowrisk differentiated thyroid cancer lowgrade salivary neoplasms and slower growing basal cell carcinomas in favorable locations Routine procedures include benign thyroid pathology parathyroid disease without significant systemic effects benign salivary lesions and low risk skin cancers and posttreatment disorders Ranasinghe and colleagues recommend a tiered approach to surgical triage with more aggressive pathology being prioritized in a similar fashion to the AAO guidelines Similarly the review recommends considering alternatives to longduration microvascular reconstructive cases It is recommended that the focus shift to simplifying reconstruction and Journal Preproof 0creducing surgical duration when it™s feasible and appropriate However it is also acknowledged that this approach may lead to an increase in downstream complications Endocrine surgery is similarly tiered in a memo by Ashok Shaha which outlines a strategic approach to thyroid surgery during the pandemic Similar to other strategies anaplastic thyroid cancer medullary thyroid cancer and locally aggressive differentiated thyroid cancer specifically with impending concern for airway obstruction take precedent However some alternatives are also discussed for instance in patients with BRAF V600E mutations who may have surgery delayed while being treated with appropriate targeted therapies Additionally deescalation of surgical care is advocated for benign conditions like thyroid goiters that are nonobstructive and even papillary thyroid microcarcinoma which may be followed with serial ultrasonography until resource allocation has returned to preCOVID levels As institutions attempt to weigh the pros and cons of elective and essential surgery in the midst of the pandemic some authors have advocated for creating rating systems to allow for appropriate surgical triage during periods of limited clinical output and resource reallocation The medically necessary timesensitive MeNTS procedures scoring system aims to œethically and efficiently manage resource reallocation and risk to healthcare providers during the COVID19 pandemic51 The scoring system which uses procedural disease and patient factors within a 5point Likert scale to determine the potential risk of proceeding with surgery The cumulative MeNTS score may range between and with score above being considered within the high risk or resource heavy procedures either due to patient factors AgeComorbidities or procedure factors head and neck surgical site high anticipated blood loss ICU admission requirement Using scoring systems like MeNTS should help hospitals more Journal Preproof 0cappropriately and objectively triage elective and essential surgeries in the setting of a resurgence of caseslimiting of resources Given the significant lack of available knowledge regarding SARSCoV2 and its associated complications it is difficult to characterize risk for patients undergoing ablative and reconstructive head and neck surgery As mentioned earlier in asymptomatic COVID19 positive patients undergoing elective surgery mortality approached COVID19 has demonstrated myriad manifestations which might interfere with the success and management of patients undergoing head and neck surgery Tang demonstrated that coagulopathy was more common in patients with severe disease and nonsurviving COVID patients52 In this study Ddimer fibrin

cancer245

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: "Laryngology COVID19 Coronavirus Head and Neck Cancer Otolaryngology Key Points Journal Preproof 0c The landscape of ever evolving information about COVID19 during the pandemic has hindered the transition to normal clinical volume and efficiency COVID19 should not be a reason for delay in diagnosis or treatment with patients who have upper aerodigestive tract pathology or malignancy The approach to resection reconstruction and surveillance for patients with head and neck cancer may need to be altered to consider severity of disease patient comorbidity and prevalence of regional COVID19 infections amongst other factors In light of the significant number of prolonged intubations there may be an increase in the number of patients who develop early and late sequelae of treatment for COVID19 Tracheostomy should be performed in a safe and efficient manner when specific indications are met Synopsis This review explores the changes to practice associated with COVID19 for providers treating patients with head and neck cancer and laryngeal pathology The aim of the review is to highlight some of the challenges and considerations associated with treating this patient population during the pandemic Additionally it seeks to discuss some of the areas of concern related to ramping up clinical volume IntroductionHistoryDefinitionsBackground The downstream effects of COVID19 caused by severe acute respiratory syndrome coronavirus SARSCoV2 have now pervaded most aspects of society and have made an indelible mark on the way that medicine and specifically otolaryngology is being practiced Of note the Journal Preproof 0cdisease represents a threat to an aging population throughout the world but also has dangerous implications for providers Among the most œatrisk group of medical providers may be those within the fields of otolaryngology and ophthalmology An otolaryngologist was among one of the first providers to succumb to the illness in its early days as it spread through Wuhan China In light of the risk to both patients healthcare workers and society atlarge a push has been made to mitigate the risk of transmission within the field of Otolaryngology Head and Neck Surgery As of June 22nd there are a total of COVID19 cases reported worldwide with a total of deaths The United States has the highest number of cases at with the total number dead at Given the high mortality associated with the novel virus much of the world has enacted significant social distancing restrictions and facial covering mandates to curb the spread of the disease The origin of the virus is not well understood but it is thought that a bat or pangolin vector might have served as the primary reservoir The disease tends to be marked by fever of patients and cough of patients however a litany of other symptoms have also been described including gastrointestinal upset diarrhea shortness of breath headache loss of smelltaste among others Severe disease is characterized by an acute respiratory distress syndrome ARDS with a mortality for patients that require mechanical ventilation The disease has a slight male predominance at Severity of disease seems to correlate to age as patients who are aged have a mortality while those over present with a mortality approaching in early studies Journal Preproof 0cThe nasal cavity and nasopharynx seem to harbor the highest viral load concentration and thus the nasopharynx is the preferred location for acquisition of samples for diagnostic testing Nasal swabs oropharyngeal swabs bronchial alveolar lavage saliva and tracheal aspirates have also been suggested as possible testing sites The current preferred diagnostic assay is RTPCR which has a variable sensitivity of depending on the institution and type of test During the months of May and June many cities states and countries have focused on a return to normal activity and a ramp up of commercial activities During this time many otolaryngology practices have aimed to ramp up activity as well while employing telehealth social distancing and utilization of personal protective equipment PPE The American Academy of Otolaryngology recently published return to practice guidelines which are detailed below As the world continues to move forward during the COVID19 era considerations such as testing including preoperativepreprocedure COVID testing surgical triage clinic workflow and practice management continue to evolve as more information becomes available This review is intended to highlight some of the current recommendations for patient care within the Laryngology and Head and Neck Surgical Oncology scope of practice Discussion Laryngology Journal Preproof 0cAs cases continue to rise increased emphasis has been placed on protection for the provider in the clinical setting Over the last decade officebased management of many common laryngeal disorders has significantly expanded This includes but is not limited to officebased laser ablation of papilloma or dysplasia transoral or transcervical injection laryngoplasty for vocal fold paralysis and EMGguided injection of Botox for spasmodic dysphonia Given the high number of clinicbased aerosolgenerating procedures practiced by today™s laryngologists many providers have seen a marked reduction in their ability to treat patients and their clinical productivity Within the category of aerosolgenerating procedures is flexible fiberoptic laryngoscopy one of the most widely used diagnostic tools for all otolaryngologists and speech pathologists A consensus statement reported by Rameau et al from a virtual webinar attended by approximately participants in the American laryngology community recommended flexible laryngoscopy should be reserved for critical cases in which the findings may have an immediate impact on diagnosis or treatment œIndications include hemoptysis odynophagia limiting hydration and nutrition or airway compromise”notably secondary to infectious and malignant conditions Some have advocated for preclinic COVID testing prior to any aerosol generating procedure however given the high false negative rate of many available tests the use of universal personal protective precautions is recommended According to Givi and colleagues examinations should take place in negative pressure rooms if possible with avoidance of topical lidocaine spray although other groups recommend administration of a topical anesthetic to theoretically decrease the coughsneeze reflex unpublished online chats A substitute to standard aerosolized anesthesia may be pledgets soaked in lidocaine and Journal Preproof 0c oxymetazoline The group also suggests using videolaryngoscopy whenever possible to keep the practitioner and the patient farther apart Disposable laryngoscopes should be used whenever possible Most studies universally recommend the following personal protective equipment PPE N95 mask or powered airpurifying respirators PAPRs gloves gown eye shield or goggles and cap It has also been suggested that the patients wear a mask covering the mouth during flexible laryngoscopy to reduce aerosolization from phonatory maneuvers and in case of coughing or sneezing At this time transoral rigid and mirror laryngoscopy are discouraged unless flexible laryngoscopy cannot be performed due to the increased risk of gagging and coughing as well as the need for the patient to phonate with the mouth uncovered to allow visualization of the larynx Additionally universal masking is encouraged in all clinical spaces in accordance with many state policies Patients in the waiting rooms are encouraged to physically distance or wait in their car for a phone call prior to presenting for their appointment Crosby also suggests offering personal protective equipment for the friends and family accompanying the patient during laryngoscopy and other sites prevent friends and family from accompanying patients inside for the visit Some alternatives to flexible laryngoscopy have been raised including transcervical laryngeal ultrasound which has a reported concordance of in identifying vocal fold motion abnormalities Another key consideration for the laryngologist in the COVID19 era is the approach to sanitization and room turnover after aerosol generating procedures AGPs Laryngoscope turnover should include highlevel disinfection including the use of such chemical disinfectants as glutaraldehyde chlorine dioxide or orthophthaladehyde OPA Some authors recommend immediate placement of the scope after use into a covered receptacle for transport from the Journal Preproof 0cexamination room to the sterile processing areas After completion of laryngoscopy room sanitization with an EPAregistered hospitalgrade disinfectant is recommended with a hydrogen peroxide solution gL chlorine disinfectant or alcohol According to the CDC website it is unknown how long the air inside a particular examination room remains infectious and likely relates to the room size rapidity of air exchange patient factors like viral shedding amount of coughingsneezing and length of time patient was in the room The CDC suggests that rooms with air changeshour ACH take about and minutes to purify the air with and efficiency respectively As the number of air changeshour decreases the time between patients should be increased to allow for appropriate dissipation of theoretical infectious agents As such many hospitals have recommended a turnover time of 4x the time it takes to purify the air with efficiency which may be either minutes or minutes depending on the level of air turnover or could be no additional time if any additional HEPA filtration system and negative pressure has been added Limited data exists to support this approach for SARSCoV2 Laryngology patients are quite diverse with respect to their level of acuity Some patients require more urgent intervention while others may have their care deferred1221 Most guidelines advocate for a tiered approach to ramping up both clinicbased and surgical activity The American Academy of Otolaryngology published guidelines for ramping up clinical activity on May The AAO recommends limiting patient care to individuals with œtimesensitiveurgent and emergent medical conditions This approach is also echoed in the care of head and neck cancer patients see below According to the guidelines emergent conditions include œimpending airway obstruction due to infection neoplasm stenosis foreign body which may Journal Preproof 0cwarrant the following intervention œflexible and rigid laryngoscopy with intervention direct laryngoscopysuspension laryngoscopy bronchoscopy and tracheostomy Urgent conditions include œmoderate or impending airway obstruction progressive dysphonia progressive dysphagia glottic incompetence causing aspiration or impaired pulmonary toilet which warrant the previously described procedures in addition to œstroboscopy functional endoscopic evaluation of swallow esophagoscopy with or without intervention open airway procedures for cancer Time sensitive conditions include œT1 glottic carcinoma or carcinoma in situ stablemild dysphonia stable dysphagia which adds œtranscervical Botox injection to the above list of procedures Routine conditions which may be deferred for days or more include œmildmoderate dysplasia nonobstructive benignphonotraumatic lesions of the vocal folds glottic incompetence glottic incompetence with mild to moderate dysphonia gender affirmation globuscough without alarm signs Comparing acuity of patients also raises an important point about the subset of patients who are typically seen for benign phonotraumatic voice disorders Many live vocal performance venues have shut down concerts have been cancelled or postponed and some studies point to live singing as being a potential source of massive spread For this reason one might assume that the percentage of patients being seen for acute phonotraumatic voice disorders diminishes somewhat Conversely as patients continue to recover from hospitalizations related to COVID19 it is anticipated that there may be a number of patients with sequelae of prolonged intubation including posterior glottic stenosis vocal fold granulomas and trachealsubglottic stenosis Laryngeal surgery in the era of COVID has had to undergo some significant changes in the approach to patient triage surgical technique and management of the airway Preoperative Journal Preproof 0cevaluation of patients must weigh the risk of delaying surgery with the risk of complications related to COVID19 infection Lei et al studied a group of operative patients in whom all were COVID19 positive within the incubation period Mortality was for this group and required ICU admission25 Of note all patients in this study underwent surgery about days prior to demonstrating signs or symptoms of COVID19 pneumonia This suggests there is significant risk associated with elective surgery in seemingly asymptomatic patients who are infected with COVID19 For this reason many authors have suggested preoperative COVID testing although it is a subject of some debate Some advocate for a negative test within hours followed by selfquarantine until the time of surgery while others favor a negative test 48hrs from the time of surgery and a point of care negative test on the day of surgery17 This is not always possible given the limitations of the institution where the patient is undergoing surgery Just as discussed earlier with regard to personal protective equipment in clinic universal precautions should be taken including full PPE and all patients should be presumed positive Airway management in the COVID19 era has become a point of focus for quality improvement and safety groups Endotracheal intubation is cited as one of the procedures which seems to have the highest aerosol generating burden It is recommended that intubation be performed by the most experienced practitioner Additionally some recommend early intubation for patients that are high risk for decompensation while others have advocated delaying intubation in favor of noninvasive means of ventilation This may include high flow nasal cannula which actually has minimal dispersion of exhaled air if appropriately fitted according to Cheung It is recommended that flexible fiberoptic intubation be avoided whenever possible Journal Preproof 0cAdditionally excessive bagmask ventilation should be avoided due to the risk of dispersion of exhaled air Furthermore jet ventilation is considered particularly high risk and should be avoided if possible Management of the surgical airway and the topic of tracheostomy has been well represented in the recent literature During the SARS outbreak in open tracheostomy was the most common surgical procedure performed on infected patients Most studies seem to favor open tracheostomy over percutaneous tracheostomy however consideration may be given for percutaneous dilatation tracheostomy in some patients if the anatomy is favorable and the practitioner has sufficient expertise with the procedure Tay and colleagues advocate for use of PAPR during tracheostomy based on the experience of countries during the SARS crisis Other authors have suggested the use of an N95 mask appropriate eye protection gown double gloves and cap To decrease the risk of autocontamination some have recommended an infection control nurse be available to monitor donning and doffing procedures during tracheostomy Additional proposals include trach teams which may consist of a surgeon anesthetist and scrub nurse to increase efficiency and create an environment of consistent verbal and nonverbal communication especially important given the burdens of communicating through a mask or PAPR Portugal et al discuss a surgical safety checklist for performing tracheostomy in patients with COVID1932 This includes performing tracheostomy in the ICU whenever possible decreasing the number of personnel in the room and having a specific tracheostomy bundle in the ICU room to decrease the number of times providers and nurses need to break scrub to leave the room They also recommend donning inner gloves prior to gown and outer gloves after donning gown to maintain clean inner gloves for the removal and Journal Preproof 0cdisposal of the rest of the PPE Two universally agreed upon maneuvers include stopping ventilation prior to entrance into the airway and holding ventilation until after the tracheostomy tube cuff has been inflated Givi and colleagues suggest that a smaller tracheotomy cuffed may be preferred to decrease the spread of aerosolized particles Miles discusses the New York experience suggesting that for intubated patients the cuff pressure should be checked every hours with a goal of cm H2O greater pressure predisposes tracheal pressure necrosis33 The group also suggests delaying the timing of tracheostomy until days after onset of symptoms when feasible Finally some have advocated for the use of specific air containment setups including plastic draping smoke evacuator tubing or specifically designed negative pressure box The field of laryngology has had to undergo significant change in the setting of the COVID pandemic As the numbers of COVID19 patients have continued to increase during the month of June it is clear that practice of laryngology in the postCOVID era will need to be carefully ramped up to protect patients and providers alike Additionally one would expect a continued increase in the number of recovered patients being seen for sequelae of prolonged intubation Decisions to relax restrictions on flexible laryngoscopy and other AGPs will depend on the local incidence of COVID19 infection availability and accuracy of preprocedure testing sustainable supply of PPE the ability to properly sanitize rooms and ultimately development of an effective vaccine Head and Neck Surgical Oncology Journal Preproof 0cSimilar to laryngology the approach to head and neck surgical oncology continues to evolve as more information becomes available during the COVID era During the early weeks of the pandemic the aspect of cancer care most concerning to patients and providers involved potential delays in therapy Finley suggests that delaying cancer surgery should be done with extreme caution despite COVID1937 Additionally delays beyond weeks could significantly affect longterm outcomes and morbidity of treatment Among patients diagnosed with severe COVID19 requiring ICU admission patients with cancer deteriorated faster than noncancer patients Desai and colleagues discovered a higher risk of severe events in patients recently treated with chemotherapy or surgery in the past days compared to noncancer COVID16 patients38 Additionally patients with advanced stage cancer tended to have a higher rate of severe events when compared to early stage cancer Cancer patients undergoing active treatment are predisposed to COVID19 related complications and critically ill patients with cancer have a higher predisposition to death Head and neck cancer patients especially are considered a highrisk population for complications associated with COVID19 infection8 making safe coordination of care difficult but imperative Head and neck cancer patients are an atrisk group for a number of reasons Silverman points out that head and neck cancer patients tend to present with advanced age history of tobacco and alcohol abuse and cardiac and pulmonary comorbidities which are similarly found in COVID19 Risk of respiratory sequelae in patients who have received chemotherapy andor radiation therapy are high with increased rates of dysphagia aspiration and pneumonia Additionally head and neck cancer patients have an increased risk of respiratory infections and aspiration pneumonitis These factors may expedite deterioration to Journal Preproof 0csevere adverse events in patients with COVID19 Additionally head and neck patients who are actively receiving chemotherapy or immunotherapy may have depressed immune function malnutrition and older age For this reason the patients need to be carefully selected and comorbidities strongly considered when constructing a treatment plan for patients with head and neck cancer Within the United States mortality for patients of color African American and Latinx with COVID19 is significantly higher than for Caucasian patients40 Unfortunately this is a consequence of inequality within society and the healthcare system rather than a biological or pathological difference41 Correspondingly these communities also tend to present with more advanced disease and have significantly worse mortality compared to their Caucasian fellow citizens This pandemic has laid to bear some of the gross inequities within the American health care system and highlighted the need for equitable decision making for all patients with a diagnosis of head and neck cancer during the COVID19 era Another consideration for the head and neck cancer patient during the COVID19 era may include the financial burden and cost of survivorship associated with undergoing cancer treatment and financial hardship related to COVID19™s effect on the world economy and increasing levels of unemployment Given the significant job losses across the United States there is preliminary data to suggest that there will be at least million newly unemployed people who will also lose their insurance coverage in the wake of the pandemic Increased financial strain has been associated with decreased quality of life scores and subsequently mortality in head and neck cancer patients Journal Preproof 0c Recommendations for head and neck clinic are similar to what was previously discussed for laryngology Providers are expected to take universal precautions regardless of the patient™s COVID status Flexible fiberoptic laryngoscopy is considered a highrisk aerosol generating procedure Due to this laryngoscopy should be reserved for instances in which it is likely to change management One of the beneficial consequences of the COVID19 era is the increased access of care through the widespread adoption of telehealth clinics among most hospitals49 Providers may use telemedicine as an initial preoperative assessment or prescreen for patients that will later be seen in clinic or prior to surgery While telehealth is wonderful for obtaining a detailed history reviewing dataimaginglabs and discussing surgical optionsrisksbenefits a big drawback is the inability to perform a comprehensive head and neck physical exam Physical examination with or without fiberoptic laryngoscopy is important to define the extent of tumor and formulating an ablative and reconstructive plan Fortunately some workarounds include anatomic and physiologic imaging for ablative planning and CT angiography and virtual planning sessions for microvascular reconstruction Telemedicine also serves a vital role in triage of posttreatment head and neck cancer patients who may not be able to be seen as frequently due to the pandemic Telemedicine also serves a vital role in the coordination of care between multiple oncologic disciplines Dharmarajan and colleagues highlighted the University of Pittsburgh approach to a virtual multidisciplinary tumor board clinic MDC This strategy has been adopted by multiple institutions and works quite well to coordinate care between specialties In their study they found that of virtual tumor board participants preferred virtual multidisciplinary clinic to Journal Preproof 0cthe inperson format Additionally about of participants indicated that they would prefer to continue the virtual multidisciplinary format once in person meeting restrictions had been lifted Through multiple virtual meeting applications practitioners can share imaging and laryngoscopy which may assist with decision making for patients Similar to the guidelines published for laryngeal surgery the American Academy of Otolaryngology has published recommendations for ramping up clinical volume as it relates to triage for head and neck surgical oncology Setzen et al note that most head and neck cases fall within the urgent category The guidelines list emergent procedures as being tumor obstructing airway significant bleeding acute or impending neurological change and salivary gland or deep neck abscesses Urgent procedures within days include all head and neck squamous cell carcinomas of the upper aerodigestive tract benign tumors with impending complications or morbidity anaplastic thyroid cancer medullary thyroid cancer bulky differentiated thyroid cancer with regionaldistant metastasis locally aggressive or large nodules 4cm Bethesda high grade salivary malignancies skin cancers and parathyroid carcinomas with significant systemic effects Time sensitive procedures include lowrisk differentiated thyroid cancer lowgrade salivary neoplasms and slower growing basal cell carcinomas in favorable locations Routine procedures include benign thyroid pathology parathyroid disease without significant systemic effects benign salivary lesions and low risk skin cancers and posttreatment disorders Ranasinghe and colleagues recommend a tiered approach to surgical triage with more aggressive pathology being prioritized in a similar fashion to the AAO guidelines Similarly the review recommends considering alternatives to longduration microvascular reconstructive cases It is recommended that the focus shift to simplifying reconstruction and Journal Preproof 0creducing surgical duration when it™s feasible and appropriate However it is also acknowledged that this approach may lead to an increase in downstream complications Endocrine surgery is similarly tiered in a memo by Ashok Shaha which outlines a strategic approach to thyroid surgery during the pandemic Similar to other strategies anaplastic thyroid cancer medullary thyroid cancer and locally aggressive differentiated thyroid cancer specifically with impending concern for airway obstruction take precedent However some alternatives are also discussed for instance in patients with BRAF V600E mutations who may have surgery delayed while being treated with appropriate targeted therapies Additionally deescalation of surgical care is advocated for benign conditions like thyroid goiters that are nonobstructive and even papillary thyroid microcarcinoma which may be followed with serial ultrasonography until resource allocation has returned to preCOVID levels As institutions attempt to weigh the pros and cons of elective and essential surgery in the midst of the pandemic some authors have advocated for creating rating systems to allow for appropriate surgical triage during periods of limited clinical output and resource reallocation The medically necessary timesensitive MeNTS procedures scoring system aims to œethically and efficiently manage resource reallocation and risk to healthcare providers during the COVID19 pandemic51 The scoring system which uses procedural disease and patient factors within a 5point Likert scale to determine the potential risk of proceeding with surgery The cumulative MeNTS score may range between and with score above being considered within the high risk or resource heavy procedures either due to patient factors AgeComorbidities or procedure factors head and neck surgical site high anticipated blood loss ICU admission requirement Using scoring systems like MeNTS should help hospitals more Journal Preproof 0cappropriately and objectively triage elective and essential surgeries in the setting of a resurgence of caseslimiting of resources Given the significant lack of available knowledge regarding SARSCoV2 and its associated complications it is difficult to characterize risk for patients undergoing ablative and reconstructive head and neck surgery As mentioned earlier in asymptomatic COVID19 positive patients undergoing elective surgery mortality approached COVID19 has demonstrated myriad manifestations which might interfere with the success and management of patients undergoing head and neck surgery Tang demonstrated that coagulopathy was more common in patients with severe disease and nonsurviving COVID patients52 In this study Ddimer fibrin Answer:

Thyroid_Cancer

"protein protein interaction screen carried out to maphuman cellsurface receptorligandinteractions between proteins belongingto the immunoglobulin domainsuperfamily IgSF begins to unravel thecomplex network of cellsurfaceinteractions that allows cells to recognizeand respond to one another and theirdynamically changing environmentHighlightsd Human IgSF interactome reveals complex network of cellsurface protein interactionsd Phylogenetic homology analysis predicts proteinproteininteractionsd 18 previously unknown proteinprotein interactionsidentifiedd Deorphanization of receptors and new binding partners forwellstudied receptorsWojtowicz Cell “August ª Elsevier Inc101016jcell202007025ll 0cllResourceA Human IgSF CellSurface Interactome Reveals aComplex Network of ProteinProtein InteractionsWoj M Wojtowicz16 Jost Vielmetter26 Ricardo A Fernandes17 Dirk H Siepe137 Catharine L Eastman17Gregory B Chisholm2 Sarah Cox4 Heath Klock4 Paul W Anderson4 Sarah M Rue4 Jessica J Miller4 Scott M Glaser4Melisa L Bragstad4 Julie Vance4 Annie W Lam2 Scott A Lesley4 Kai Zinn2 and K Christopher Garcia13581Department of Molecular and Cellular Physiology Stanford University School of Medicine Stanford CA USA2Division of Biology and Biological Engineering California Institute of Technology Pasadena CA USA3Howard Hughes Medical Institute Stanford University School of Medicine Stanford CA USA4The Genomics Institute of the Novartis Research Foundation San Diego CA USA5Department of Structural Biology Stanford University School of Medicine Stanford CA USA6These authors contributed equally7These authors contributed equally8Lead ContactCorrespondence wojstanfordedu WMW kcgarciastanfordedu KCG101016jcell202007025SUMMARYCellsurface proteinprotein interactions PPIs mediate cellcell communication recognition and responses We executed an interactome screen of human cellsurface and secreted proteins most ofwhich are immunoglobulin superfamily IgSF proteins using a highthroughput automated ELISAbasedscreening platform employing a pooledprotein strategy to test all PPI combinations Screen resultsaugmented by phylogenetic homology analysis revealed 18 previously unreported PPIs We validated asubset using surface plasmon resonance and cell binding assays Observed PPIs reveal a large and complexnetwork of interactions both within and across biological systems We identified new PPIs for receptors withwellcharacterized ligands and binding partners for ˜˜orphan™™ receptors New PPIs include proteins expressed on multiple cell types and involved in diverse processes including immune and nervous systemdevelopment and function differentiationproliferation metabolism vascularization and reproductionThese PPIs provide a resource for further biological investigation into their functional relevance and may offernew therapeutic drug targetsINTRODUCTIONProteinprotein interactions PPIs at the cell surface allowcells to respond to one another and their environment in ahighly dynamic contextdependent and spatiotemporalmanner Wood and Wright The essential role playedby cellsurface PPIs is exemplified by estimates that 18 of genes in the human genome encode cellsurfaceproteins and 18 encode secreted proteins Fonseca At present a comprehensive human cellsurface interactomeis lacking Mapping extracellular PPIs has proved challengingbecause most cellsurface proteins are refractory to classicbiochemical screening techniques and cellsurface PPIs are underrepresented in affinity purificationmass spectrometrybaseddatasets Huttlin Additionally cell surfacePPIs often have fast binding kinetics spanning a broad rangeof affinities low nM to hundreds of mM van der Merwe and Barclay rendering them difficult to detect using standardbiochemical assaysIn recent years several assays have been developed thatallow detection of lowaffinity cellsurface PPIs by impartingavidity through clustering of secreted proteins and the extracellular domains ECDs of single transmembrane STM cellsurface proteins Clustering is achieved using multimerization domains and can increase binding signal up to 250fold Bushell Experimental platforms that utilize clusteringinclude several variations of ELISAbased binding assaysWojtowicz Bushell O¨ zkan BioPlex beads Li protein microarrays Sun cellsignaling assays Barrow cellsurface staining microarrays Turner and beadbasedassays Husain Multiple groups have shown thatELISAbased binding assays have a remarkably low falsepositive rate Wojtowicz Bushell So¨ llnerand Wright Martin Crosnier O¨ zkan Visser Ranaivoson Previously we conducted a screen for all 18 Drosophilacellsurface and secreted proteins containing three types of domains immunoglobulin Ig and Iglike fibronectin type III FN3Cell “ August ª Elsevier Inc 0clland leucinerich repeats LRRs O¨ zkan This screenreported over new PPIs including a previously unknownimmunoglobulin superfamily IgSF PPI network between members of the Dpr and DIP subfamilies Since we reported theDprDIP network functional studies have revealed that thisnetwork mediates neuronal wiring decisions in the fly brain andneuromuscular system for review see Honig and Shapiro Sanes and Zipursky In humans there are an estimated 18 secreted and STMproteins totaling 18 M putative PPIs Screening this vast number requires a highthroughput assay Here we developed ascreening platform that combines a highthroughput version ofthe ELISAbased extracellular interactome assay ECIA O¨ zkan with an automated pooledprotein strategy apECIA We performed a screen of human IgSF secreted andSTM cellsurface proteins excluding antibodies and T cell receptors along with other select proteins of interest The IgSFis the largest and most functionally diverse family in the cellsurface proteome Members include receptor tyrosine kinasesphosphatases costimulatory or coinhibitory immune receptors growth factor and adhesion receptors among many othersand are present in most if not all cell typesWe produced proteins and screened every possible PPI We observed PPIs of which are previously unreported New PPIs form a complexnetwork and include PPIs between phylogenetically related proteins within a subfamily different subfamilies and distantlyrelated proteins Screen results were combined with phylogenetic homology analysis PHA to predict additional PPIs between subfamily members using a nearestneighbor approachWe confirmed a subset of both screen and PHA predictedPPIs using surface plasmon resonance SPR and cell bindingassaysRESULTSSelection and Production of Proteins for PPI ScreeningTo identify human IgSF proteins we utilized the HUGO GeneNomenclature Committee HGNC Yates HumanProtein Atlas Uhle´ n and UniProt UniProt Consortium databases ECDs and secreted proteins for IgSF and nonIgSF proteins of interest were produced with˜˜bait™™ and ˜˜prey™™ multimerization domains into cell supernatantsFigures 1A and 1B Data S1 and S2 and expression wasconfirmed by western blot Data S3 Westerns revealed detectable levels of protein for of baits and of preys We andothers have observed that PPIs can be detected in the ECIA andother ELISAbased binding assays even when proteins are present at levels below the limit of detection by western O¨ zkan Visser Ranaivoson Assuch all bait and prey were included in the screen regardlessof whether protein was detectedDevelopment of an Automated PooledPrey ECIAPlatform apECIAECIA and other ELISAbased assays allow bait and preyproteins to be tested for binding directly from conditionedmedia B These assays test one baitprey pair per Cell “ August Resourcewell To increase throughput we pooled three preys andfollowing screening deconvoluted positive wells to identifyPPIs B Pooling experiments with a panel of knownPPIs showed binding for all 3fold diluted prey Figures 1CS1C and data not shown As baitprey pairs are testedin both orientations a falsenegative resulting from poolingcan be ˜˜rescued™™ by a positive resultin the converseorientation We reasoned thatthe advantages of poolingwhich reduces the number of binding reactions outweighedthe potentialincrease in false negatives To further improvethroughput we optimized a 384well format and developedan automated workflow using liquid handling robots TheapECIA platform allows testing of baitprey combinations per weekPPI Screen Reveals a Complex Network of PPIsFollowing screening deconvolution of positive wells R2 foldoverbackground was performed by retesting each preyindividually B Nine prey gave rise to large numbers ofwells with positive signals and were excluded as nonspecificbinders Figure S2 Following removal of nonspecific PPIs deconvolution revealed positive wells comprising uniquePPIs Data S4 In each case only one of the three deconvolutedprey yielded a positive signal To confirm binding the positiveprey was retested againstin triplicateEightyone percent of PPIs are between IgSF proteinsThe remaining include PPIs between IgSF and otherproteins present in the screen Figure S3its cognate baitAlmost half of the proteins tested were involved in a PPI Proteins not involved in PPIs may be misfolded have binding partners notincluded in the screenrequire coreceptors or fall outside the dynamic range of theassay false negatives which is determined by PPI affinityand bait and prey concentrations Figure S4F Confirming thesensitivity of the assay many bait or prey proteins expressedat very low levels still engaged in one or two PPIs FiguresS4A and S4B A small number of PPIs were observed withbait or prey proteins exhibiting undetectable levels in conditioned media Figures S4C“S4E To interrogate the dynamicrange of the assay we plotted prey AP levels for PPIs with reported affinities Figure S4F These data suggest thatforvery poorly expressed prey proteins Figures S1A S4A andS4B PPIs with KD mM are likely to be missed false negatives Figure S4FOf the PPIs are previously unreported basedon literature and PPI databases Data S4 The majority of PPIsform one large network comprising proteinsD Only proteins involving PPIs are not connectedto the network Different regions of the network are shown inFigure Ninetyeight proteins had one PPI had two to five PPIs and had PPIs EBecause of proteins exhibit binding we calculated theexpected frequency with which each protein will bind at least˜˜x™™ number of proteins up to the maximally observed PPIsFigure S2B and compared the expected and observed frequencies F The observed number of binding partnersis significantly greater than predicted by random chance ofPPIs for a network of this size 0cResourcellABCEDFFigure apECIA Screen Details and Overview of ResultsA Schematic depiction of a subset of proteins in library Pie graph of library distribution Full protein domain names at martemblde Sun B Left flow chart of screen HT highthroughput Right example plate from screen Schematic of ECIA and pooledprey strategy Illustration of screen matrixC ECIA of undiluted prey single prey versus 3fold diluted prey pooled prey Background subtracted data are represented as ±SD Bkgd backgroundD Network of PPIs observed in screen Inset the PPIs that are not connected to the network Node size is proportional to number of PPIs Siglec subfamilynodes are highlighted in colorE Pie graph of distribution of the number of binding partners observed in screen overlaid on the network in a degreesorted circular layoutF Observed versus expected frequency with which each protein will bind at least ˜˜x™™ number of proteins up to the maximally observed PPIs assuming randomchance of interactions p KolmogorovSmirnov [KS] testSee also Figures S1 S2 S3 and S4 and Data S1 S2 S3 and S4Cell “ August 0cllResource Cell “ August legend on next page 0cResourcellPhylogenetic Homology Analysis PHA to Predict PPIsPPIs often occur between phylogenetically related proteins bothwithin and between subfamilies We performed multiplesequence alignments to identify subfamily members within ourlibrary and generated phylogenetic trees Using a combinedapproach which we call apECIAPHA we analyzed screendata alongside the phylogenetic trees to predict additionalPPIs between subfamily members that may have been missedin the screenPPI Validation by Surface Plasmon ResonanceWe selected a subset of screen and PHA predicted PPIs to validate by SPR Bona fide PPIs are expected to display distinct association and dissociation which can be observed with highsensitivity by SPR To increase avidity and therefore sensitivityFc dimerized ECD analytes and ligands were used in most experiments Figure S5 This increase in sensitivity prevented us fromdetermining monomeric KD constants Binding profiles characteristic of PPIs exhibiting clear resonance signals above background negative control ligand and receptor and concentrationdependent binding curves were deemed indicative of aspecific ligandanalyte PPI Nonspecific PPIs generally exhibitdeviations from this behavior such as high background andnonlinear concentration responsesTwentyfour newly identified PPIs observed in our screen weretested by SPR Of these we observed specific ligandanalyteinteractions We additionally tested PHA predicted PPIs andobserved PPIs for Three additional PPIs were observed between homologous proteins in mouse and crossspecies humanmouse In total we SPR validated newly identifiedPPIs Table Data S5Combined apECIAPHA Approach Reveals Multiple DCCSubfamily PPIsThe netrin1 receptor DCC has wellcharacterized functions inthe nervous system Finci DCC is a dependence receptor and is implicated in colorectal and other cancers but itsroles in these cancers are not well understood Goldschneiderand Mehlen We observed DCC binding to insulinlikegrowth factorbinding proteinlike IGFBPL1 but not to insulinlike growth factorbinding protein IGFBP7 Figures 2I and3A Our phylogenetic tree revealed IGFBPL1 and IGFBP7 residein a cluster and share the highest amino acid sequence similarity among subfamily members As such we examined binding of DCC to both IGFBPL1 and IGFBP7 by SPR and observedbinding for both Figure 3CPHA pointed us to four proteins that cluster with DCC PUNCPUNC e11 neogenin NEO1 and protogenin PRTG Figure 3ATogetherthese proteins play roles in diverse processesincluding nervous system development myogenesis and angiogenesis inflammation and tissue regeneration leukocyte migration neural tube and mammary gland formation development ofbone and connective tissues and stem cell differentiation Salbaum Wilson and Key Takahashi Schievenbusch DakouaneGiudicelli PUNC is an ˜˜orphan™™ receptor expressed in the developing nervous system Salbaum PUNC e11 and PRTG bound intercellular adhesion molecule ICAM5Figure 3A a proteinexclusively expressed in the brain that functions in synapse formation stabilization and refinement Gahmberg Cleaved ICAM5 ECD exhibits immunosuppressive functionsthrough cytokine regulation and may play important roles inregulation of brain inflammation We confirmed binding ofPUNC e11 and PRTG to ICAM5 by SPR Figure 3C AlthoughPPIs with ICAM5 were not detected in the screen for the remaining DCC subfamily members we tested them by SPR andobserved binding for all three Figure 3CIn our screen one or more DCC subfamily members alsobound to WFIKKN2 a secreted protein that binds transforming growth factorbeta subfamily members and modulates theirpresentation to cells Monestier and Blanquet lactotransferrin LTF an ironbinding protein with antimicrobial activity Hao interleukin6 receptor subunit alpha IL6Ra a cytokine receptor Schaper and RoseJohn and ISLR2LINX which functions in nervous system developmentMandai Panza Abudureyimu We confirmed binding of all DCC subfamily members tothese proteins by SPR and to other proteins observed in ourscreen Figures 3C 3D and 3F These results demonstratethe value of using a combined apECIAPHA approach to identifyadditional PPIs beyond screen data resulting in the elucidationof a more complete network Figure 3FLARPTPR Subfamily PPIs with SALMsThe LARfamily of type IIA protein tyrosine phosphatase receptors LARPTPRs comprises PTPRF also known as LARFigure Select Regions of the Complex PPI NetworkA Select PPIs including four proteins not connected to the network CD146 CNTN1 NFASC and NrCAMB Region largely comprised of immune system proteinsC Region comprising PPIs both within and across biological systemsD Two regions highlighting subfamilyspecific type IIA and type IIB PTPR PPIs Type IIA PPIs with SALMs and IL1APs include PPIs observed in screen and PHApredicted PPIs validated by SPR Figures and S6E Region highlighting PPIs for ˜˜orphan™™ receptors ILDR1 and PUNCF Region showing a subset of LILR subfamily PPIsG Region showing a subset of Siglec PPIs with nonSiglecs CD33Siglec3H Region showing SiglecSiglec PPIs CD33Siglec3 MAGSiglec4a SNSiglec1I Region highlighting PPIs between nervous system proteins and with proteins in immune and reproductive systems Within this region multiple additional PHApredicted PPIs were validated by SPR Figure Table Because a network is composed of interconnected nodes some linkage proteins are present in more than one panel Colored nodes denote proteins and PPIsvalidated by additional experiments Green line previously reported PPI gray line previously unreported PPISee also Data S4Cell “ August 0cllResourceTable SPRValidated apECIA Screen and PHA Predicted PPIsTable ContinuedLigandFcAnalyteFcapECIATIE1hISLR2mIslr2SALM1SALM2SALM3SALM4SALM5IL1RAPL1IL1RAPL2DSCAMCTDSCAML1CTKIR2DL5IL6RaPSG7PSG9ICAM5WFIKKN2PDL1PDL2LTFTrkAhLEPamLepahLEPamLepaPTPRDPTPRFPTPRSPTPRDPTPRFPTPRSPTPRDPTPRFPTPRSPTPRDPTPRSPTPRDPTPRFPTPRSPTPRSFGFR3PTPRSPTPRMPTPRTPTPRMPTPRTPVRISLR2DCCDSCAMDSCAMDCCIL6RaISLR2NEO1PRTGPUNCPUNC e11DCCIL6RaISLR2PRTGPUNCPUNC e11PDL1PDL1DCCIL6RaNANANANANANAknownknownknownNANAknownknownknownknownNANANANANANANANANAknownNANAPHANANANANANANANANANANANANANANANANANANANANANANANANANANANANANAContinued on next page Cell “ August LigandFcAnalyteFcapECIAPHAIGFBPL1IGFBP7ISLR2NEO1PRTGPUNCPUNC e11DCCIL6RaISLR2NEO1PRTGPUNCPUNC e11DCCIL6RaISLR2NEO1PRTGPUNCPUNC e11NANANANANANANANANANANANANANANANANANANAECDFc ligand and ECDFc analyte PPIs observed by SPR apECIA denotes PPI was observed in screen PHA denotes PPI was predicted byphylogenetic homology analysis SPR conditions are included in DataS5 See also Figure S6ahLEP and mLEP monomer proteinPTPRD and PTPRS Figures 4A and 4B LARPTPRs playimportant roles in synaptic anization and function Nam Presynaptic LARPTPRs mediate transsynapticadhesion through PPIs with multiple postsynaptic ligandsMouse mutants in LARPTPRs and their ligands exhibit defectsin synapse structure and function Lie Several PPIs are known between specific LARPTPRs andSALM235 Li Choi GotoIto Figure 4A We observed these as well as additionalPPIs in our screen Figures 2D 4A and 4C No LARPTPR binding to SALM1 or SALM4 has been reported and we did notobserve binding in our screen PHA led us to test binding of allLARPTPRs to all SALMs by SPR With the exception ofPTPRFSALM4 we observed binding of all LARPTPRs to allSALMs Figures 4E and S6A PTPRSALM pairs exhibited differences in maximum response units RU a relative comparison ofbinding strength suggesting a spectrum of binding affinities mayexist among LARPTPRs and SALMs Figure 4FLARPTPR Subfamily PPIs with Interleukin1 ReceptorAccessory Proteins IL1APsTranssynaptic LARPTPR interactions with IL1RAP and IL1RAPL1 induce bidirectional pre and postsynaptic differentiation Yoshida IL1RAP is known to interactwith all three LARPTPRs and IL1RAPL1 with PTPRD Weobserved these PPIs and additionally binding of IL1RAPL1 toPTPRS Figure 2D IL1RAPL1 shares sequence similarity 0cllBResourceACDEFlegend on next pageCell “ August 0cllResourcewith IL1RAPL2 an orphan receptor expressed in the brain withno known biological function Boraschi In ourscreen we detected binding of IL1RAPL2 to PTPRD and PTPRSFigure 2D We tested both known and previously unknown PPIsby SPR and observed binding for all Figure S6B IL1RAPL2additionally interacted with fibroblast growth factor receptor FGFR3 in the screen and by SPR Figure S6BType IIB PTPR Subfamily PPIs with DSCAMsType IIB PTPRs are expressed in most tissues and regulatediverse processes including cell growth migration and differentiation immune cell development endothelial cell adhesion andmigration neuronal development and synapse formation andoncogenic transformation Lee Stoker The typeIIB subfamily is composed of PTPRK PTPRM and PTPRT Figures 4A and 4B and multiple binding partners are known Weobserved binding of all type IIB PTPRs with Down syndromecell adhesion molecule DSCAM and Down syndrome cell adhesion molecule like1 DSCAML1 Figures 4A and 4C proteinsthat play various roles in nervous system development Sanesand Zipursky The ECD of DSCAMs contains nine Iglike domains followedby four FN3 domains one Iglike domain and two FN3 domainsFigure 4B The Nterminal region engages in homophilic bindingand mediates binding to secreted chemoattractant and chemorepellent ligands netrin1 and slit1 respectively Wojtowicz Ly Alavi No binding partnerhas been reported for the Cterminal ECD region In our screenwe observed binding between the Cterminal regions of bothDSCAMs DSCAMCT and DSCAML1CT and all three typeIIA PTPRs Figure 4C We confirmed binding of DSCAMCTand DSCAML1CT to PTPRT and PTPRM by SPR PTPRK wasnot expressed at sufficient levels Figure 4D vascular endothelial growth factorNew PPIs between Immune System ProteinsNew PPIs were observed for wellstudied immune systemproteins as well as orphan receptors Figure 2B Two PPIswere detected for the orphan granulocyte receptor CEACAM4receptor VEGFR3 and programmed cell death ligand PDL2 CEACAM4 is a member of the carcinoembryonic antigenrelated cell adhesion molecule subfamily expressed inthe immune system epithelial and endothelial cells and brainWakabayashiNakao Zinn and O¨ zkan Inthe immune system CEACAMs play roles in immunity anddevelopment We also observed binding between CEACAM1and the immune checkpoint receptor programmed cell deathprotein PD1Interactions of PD1 with its ligands PDL1 and PDL2 inhibitT cell proliferation cytokine production and cytotoxic activityBardhan PDL2 competes with PDL1 for bindingto PD1 and can be expressed on tumor cells where it mayplay a role in tumor evasion Ghiotto Cheng Bardhan Using SPR we confirmed the knownPPI between PDL1 and PDL2 Lee and observedhomophilic binding of PDL1 a previously unknown PPI Figure S6C By comparison with PDL1 binding to PD1 which approaches saturation at 18 nM we infer that the affinity of PDL1 homophilic binding is lower than binding to PD1 This mayexplain why this PPI within the wellstudied PD1PDL1 axishas not been identified previously and highlights the value of using multimerized proteins to detect lowaffinity PPIsHomophilic and Heterophilic Siglec Subfamily PPIsEight of the proteins with the highest number of PPIs in ourscreen are members of the Siglec subfamily members proteins highly restricted to the immune system that have immunemodulatory effects on Tolllike receptor TLR signaling andplay important roles in self versus nonself discrimination Macauley Siglecs display differential expression on cellsand exhibit a broad spectrum of Siglecspecific recognition ofsialylated glycan profiles present on healthy cells inflamed ormalignant cells and pathogens We observed a network of homophilic and heterophilic PPIs among Siglecs Figure 2H DataS4 as well as PPIs with distantly related proteins a subset areshown in Figure 2G Data S4PPIs between Immune and Nervous System ProteinsThe signaling lymphocytic activation molecule subfamily SLAM members is expressed on most immune cells Dragovich andMor SLAMs function as both costimulatory and coinhibitory molecules in innate and adaptive immunity and playan integral role in autoimmune disorders SLAMF9 is an orphanreceptorWe observed SLAMF9 binding to bactericidal permeabilityincreasing protein BPI and IGSF10 Figure 2C BPI is a neutrophilderived antibiotic protein that participates in bacteria killingthrough its highly cationic Nterminal region Bu¨ low The Cterminal region of BPI exhibits no bactericidal activity andis believed to interact with other proteins and function in differentprocesses IgSF10 is an orphan receptor involved in the migration of gonadotropinreleasing hormone expressing GnRH neurons Howard IgSF10 has no known function in theFigure SPR Validation of DCC Subfamily PPIs Identified by apECIAPHA ApproachA Dendrograms of a subset of DCC subfamily PPIs showing the number of SPRvalidated PPIs observed in the apECIA screen turquoise and predicted by PHAmagentaB Dendrogram of a subset of PSG PPIsC SPR sensrams for DCC subfamily IL6Ra and ISLR2 analytes 2fold dilutions “ nM nM for PRTGWFIKKN2 due to incomplete chipregeneration at higher concentrations binding to various ligands TrkA and TIE1 negative controlsD SPR sensram for IL6Ra analyte 2fold dilutions “ nM binding to ISLR2 ligand PUNC negative controlE SPR sensrams for DSCAM and DCC analytes 2fold dilutions “ nM binding to PSG ligands TIE1 negative controlF Subnetwork of SPRvalidated PPIsRU resonance unitsSee also Figure S5 and Data S5 Cell “ August 0cResourcellABCEDFlegend on next pageCell “ August 0cllResourceimmune system and SLAMF9 function in the nervous system isunknown revealing a PPI between two orphan receptors fromdifferent biological systemsFigure 2F These PPIs present MOG and MPZ PPIs with LILRsas new candidates for neuronal regeneration studiesPPIs with PregnancySpecific Glycoproteins PSGsDuring pregnancy PSGs members are the most abundanttrophoblastic proteins in maternal blood and serve as markersfor trophoblast quality and embryo viability Moore and Dveksler The mechanisms of PSG action in pregnancy are not wellunderstood Studies suggest PSGs also have immunoregulatory proangiogenic and antiplatelet aggregation functionsWe observed several PSG binding partners includingplateletderived growth factor receptor alpha PDGFRAfibroblast growth factor receptor FGFR4 Ctype lectindomain family member A CLEC4A DCC and DSCAMFigures 2I and 3BObserved PSG interactions occur selectively and differentiallyto these binding partners Figure 2I Data S4 Using SPR weexamined binding of DCC and DSCAM to PSG7 and PSG9 Figure 3E We observed binding for DSCAM with PSG9 and bothDCC and DSCAM with PSG7 Interestingly DCC is expressedin human placenta DakouaneGiudicelli ThesePPIs present new candidate receptors for studying the role ofPSGs in pregnancy immunoregulation and angiogenesisLILR Subfamily PPIs with BTNL8 and MyelinationProteinsLeukocyte immunoglobulinlike receptors LILRs membersare a subfamily of activating LILRA and inhibitory LILRB receptors that exhibit immunomodulatory activity and function ininflammation regulation tolerance and differentiation Burshtynand Morcos We observed binding of multiple activatingand inhibitory LILRs to butyrophilin8 BTNL8 Figure 2F BTNLsare members of the extended B7 family of molecules and function as costimulatory or coinhibitory signals for T cell activationRhodes LILRs are also expressed on neurons and function in the regulation of development synaptic plasticity and axonal regeneration Hirayasu and Arase Myelin the protective insulatinglayer around axons inhibits neuronal regeneration following spinal injury Monje Three myelin proteins Nogo MAG andOMgp are known to interact with PirB in mouse one of only twomouse LILRB orthologs Atwal We observed bindingof multiple LILRs to two additional myelin proteins myelinoligodendrocyte glycoprotein MOG and myelin protein P0 MPZPVR Selectively Interacts with a KillerCellImmunoglobulinlike Receptor KIRThe polygenic Killercellimmunoglobulinlike receptors KIRsare a highly polymorphic subfamily of activating and inhibitoryproteins expressed on natural killer NK cells that regulate development maturation and activation Pende NK cellsinitially express a stochastic combination of KIRs that is refinedduring maturation to tune killing response threshold and ensureoptimal discrimination of target cells from healthy cells Weobserved binding of KIR2DL5 to poliovirus receptor PVR Figures 2C and 5A which validates a recently reported PPI Husain We confirmed KIR2DL5 binding to PVR by ECIAtitration analysis and SPR Figures 5C and 5DTo examine binding at the cell surface fluorescent tetramersof PVRFc and KIR2DL5Fc ECDFcSA647 were incubatedwith fulllength KIR2DL5 and PVRtransfected cells respectively Flow cytometry analysis revealed concentrationdependent binding of both ligands to cells expressing cognate fulllength receptor but not control cells Figure 5E PVRFc tetramers did not bind cells transfected with fulllength KIR2DL4and KIR2DL1 which share and sequence similaritywith KIR2DL5 respectively Figures 5B and 5F To furtherexamine the specificity of PVR for KIR2DL5 ECIA was performed using KIRs PVR bound specifically to KIR2DL5 Figure 5G Because KIR2DL5 has been associated with increasedvirus susceptibility and reduced antiviral response to therapythis specificity may have implications for the role of KIR2DL5 inimmunityTrkA Selectively Interacts with TIE1Highaffinity nerve growth factor receptor NTRK1 also knownas TrkA has multiple wellstudied functions in the nervous system Amatu In the immune system where its function is not well understood TrkA is expressed on monocytesmacrophages dendritic cells resting and activated B cells andneutrophils and erythroblasts Minnone In ourscreen we observed TrkA binding to tyrosineprotein kinase receptor TIE1 but not to TIE2 Figures 2A and 6A TIE1 is expressed on endothelial cells immature hematopoietic cells andplatelets and functions in complex with the angiopoietinTIE2pathway to inhibit angiogenesis Eklund We performed SPR and observed TrkA binding to TIE1 but not toFigure SPR Validation of Type IIA and Type IIB PTPRs with SALMs and DSCAMsA Left dendrogram highlighting phylogenetic clustering of SALMs DSCAMs type IIA PTPRs PTPRFSD and type IIB PTPRs PTPRTMK Right SPRvalidated PPIs green line previously known PPI turquoise line previously unknown PPI observed in screen magenta line previously unknown PHA predictedPPI Gray line screen PPI not SPR testedB Protein domain structures Ig immunoglobulinlike domain FN fibronectin type III domain MAM meprin A5 protein domain LRR leucinerich repeat TMtransmembrane NT Nterminal region of ECD CT Cterminal region of ECDC Screen data showing PPI specificity of PTPRFSD and PTPRTMK subfamilies Background subtracted OD nm data are represented as mean oftriplicate wellsD SPR sensrams for DSCAML1CT and DSCAMCT analytes 2fold dilutions “ nM binding to PTPRM and PTPRT ligandsE SPR sensrams for PTPRS analyte 2fold dilutions “ nM binding to SALMs and DSCAMCT ligandsF SPR max RU values for every pairwise combination of PTPRFSD analytes with SALM ligandsRU resonance unitsSee also Figures S5 and S6 and Data S5 Cell “ August 0cResourcellABCEDFGlegend on next pageCell “ August 0cllResourceTIE2 Figures 6B and S7A As a positive control for TIE2 weconfirmed binding to monomer angiopoietin1 Figure S7A Davis We next investigated whether TrkA can bind NGF and TIE1simultaneously We preincubated TrkA with NGF and comparedbinding of T

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You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: "protein protein interaction screen carried out to maphuman cellsurface receptorligandinteractions between proteins belongingto the immunoglobulin domainsuperfamily IgSF begins to unravel thecomplex network of cellsurfaceinteractions that allows cells to recognizeand respond to one another and theirdynamically changing environmentHighlightsd Human IgSF interactome reveals complex network of cellsurface protein interactionsd Phylogenetic homology analysis predicts proteinproteininteractionsd 18 previously unknown proteinprotein interactionsidentifiedd Deorphanization of receptors and new binding partners forwellstudied receptorsWojtowicz Cell “August ª Elsevier Inc101016jcell202007025ll 0cllResourceA Human IgSF CellSurface Interactome Reveals aComplex Network of ProteinProtein InteractionsWoj M Wojtowicz16 Jost Vielmetter26 Ricardo A Fernandes17 Dirk H Siepe137 Catharine L Eastman17Gregory B Chisholm2 Sarah Cox4 Heath Klock4 Paul W Anderson4 Sarah M Rue4 Jessica J Miller4 Scott M Glaser4Melisa L Bragstad4 Julie Vance4 Annie W Lam2 Scott A Lesley4 Kai Zinn2 and K Christopher Garcia13581Department of Molecular and Cellular Physiology Stanford University School of Medicine Stanford CA USA2Division of Biology and Biological Engineering California Institute of Technology Pasadena CA USA3Howard Hughes Medical Institute Stanford University School of Medicine Stanford CA USA4The Genomics Institute of the Novartis Research Foundation San Diego CA USA5Department of Structural Biology Stanford University School of Medicine Stanford CA USA6These authors contributed equally7These authors contributed equally8Lead ContactCorrespondence wojstanfordedu WMW kcgarciastanfordedu KCG101016jcell202007025SUMMARYCellsurface proteinprotein interactions PPIs mediate cellcell communication recognition and responses We executed an interactome screen of human cellsurface and secreted proteins most ofwhich are immunoglobulin superfamily IgSF proteins using a highthroughput automated ELISAbasedscreening platform employing a pooledprotein strategy to test all PPI combinations Screen resultsaugmented by phylogenetic homology analysis revealed 18 previously unreported PPIs We validated asubset using surface plasmon resonance and cell binding assays Observed PPIs reveal a large and complexnetwork of interactions both within and across biological systems We identified new PPIs for receptors withwellcharacterized ligands and binding partners for ˜˜orphan™™ receptors New PPIs include proteins expressed on multiple cell types and involved in diverse processes including immune and nervous systemdevelopment and function differentiationproliferation metabolism vascularization and reproductionThese PPIs provide a resource for further biological investigation into their functional relevance and may offernew therapeutic drug targetsINTRODUCTIONProteinprotein interactions PPIs at the cell surface allowcells to respond to one another and their environment in ahighly dynamic contextdependent and spatiotemporalmanner Wood and Wright The essential role playedby cellsurface PPIs is exemplified by estimates that 18 of genes in the human genome encode cellsurfaceproteins and 18 encode secreted proteins Fonseca At present a comprehensive human cellsurface interactomeis lacking Mapping extracellular PPIs has proved challengingbecause most cellsurface proteins are refractory to classicbiochemical screening techniques and cellsurface PPIs are underrepresented in affinity purificationmass spectrometrybaseddatasets Huttlin Additionally cell surfacePPIs often have fast binding kinetics spanning a broad rangeof affinities low nM to hundreds of mM van der Merwe and Barclay rendering them difficult to detect using standardbiochemical assaysIn recent years several assays have been developed thatallow detection of lowaffinity cellsurface PPIs by impartingavidity through clustering of secreted proteins and the extracellular domains ECDs of single transmembrane STM cellsurface proteins Clustering is achieved using multimerization domains and can increase binding signal up to 250fold Bushell Experimental platforms that utilize clusteringinclude several variations of ELISAbased binding assaysWojtowicz Bushell O¨ zkan BioPlex beads Li protein microarrays Sun cellsignaling assays Barrow cellsurface staining microarrays Turner and beadbasedassays Husain Multiple groups have shown thatELISAbased binding assays have a remarkably low falsepositive rate Wojtowicz Bushell So¨ llnerand Wright Martin Crosnier O¨ zkan Visser Ranaivoson Previously we conducted a screen for all 18 Drosophilacellsurface and secreted proteins containing three types of domains immunoglobulin Ig and Iglike fibronectin type III FN3Cell “ August ª Elsevier Inc 0clland leucinerich repeats LRRs O¨ zkan This screenreported over new PPIs including a previously unknownimmunoglobulin superfamily IgSF PPI network between members of the Dpr and DIP subfamilies Since we reported theDprDIP network functional studies have revealed that thisnetwork mediates neuronal wiring decisions in the fly brain andneuromuscular system for review see Honig and Shapiro Sanes and Zipursky In humans there are an estimated 18 secreted and STMproteins totaling 18 M putative PPIs Screening this vast number requires a highthroughput assay Here we developed ascreening platform that combines a highthroughput version ofthe ELISAbased extracellular interactome assay ECIA O¨ zkan with an automated pooledprotein strategy apECIA We performed a screen of human IgSF secreted andSTM cellsurface proteins excluding antibodies and T cell receptors along with other select proteins of interest The IgSFis the largest and most functionally diverse family in the cellsurface proteome Members include receptor tyrosine kinasesphosphatases costimulatory or coinhibitory immune receptors growth factor and adhesion receptors among many othersand are present in most if not all cell typesWe produced proteins and screened every possible PPI We observed PPIs of which are previously unreported New PPIs form a complexnetwork and include PPIs between phylogenetically related proteins within a subfamily different subfamilies and distantlyrelated proteins Screen results were combined with phylogenetic homology analysis PHA to predict additional PPIs between subfamily members using a nearestneighbor approachWe confirmed a subset of both screen and PHA predictedPPIs using surface plasmon resonance SPR and cell bindingassaysRESULTSSelection and Production of Proteins for PPI ScreeningTo identify human IgSF proteins we utilized the HUGO GeneNomenclature Committee HGNC Yates HumanProtein Atlas Uhle´ n and UniProt UniProt Consortium databases ECDs and secreted proteins for IgSF and nonIgSF proteins of interest were produced with˜˜bait™™ and ˜˜prey™™ multimerization domains into cell supernatantsFigures 1A and 1B Data S1 and S2 and expression wasconfirmed by western blot Data S3 Westerns revealed detectable levels of protein for of baits and of preys We andothers have observed that PPIs can be detected in the ECIA andother ELISAbased binding assays even when proteins are present at levels below the limit of detection by western O¨ zkan Visser Ranaivoson Assuch all bait and prey were included in the screen regardlessof whether protein was detectedDevelopment of an Automated PooledPrey ECIAPlatform apECIAECIA and other ELISAbased assays allow bait and preyproteins to be tested for binding directly from conditionedmedia B These assays test one baitprey pair per Cell “ August Resourcewell To increase throughput we pooled three preys andfollowing screening deconvoluted positive wells to identifyPPIs B Pooling experiments with a panel of knownPPIs showed binding for all 3fold diluted prey Figures 1CS1C and data not shown As baitprey pairs are testedin both orientations a falsenegative resulting from poolingcan be ˜˜rescued™™ by a positive resultin the converseorientation We reasoned thatthe advantages of poolingwhich reduces the number of binding reactions outweighedthe potentialincrease in false negatives To further improvethroughput we optimized a 384well format and developedan automated workflow using liquid handling robots TheapECIA platform allows testing of baitprey combinations per weekPPI Screen Reveals a Complex Network of PPIsFollowing screening deconvolution of positive wells R2 foldoverbackground was performed by retesting each preyindividually B Nine prey gave rise to large numbers ofwells with positive signals and were excluded as nonspecificbinders Figure S2 Following removal of nonspecific PPIs deconvolution revealed positive wells comprising uniquePPIs Data S4 In each case only one of the three deconvolutedprey yielded a positive signal To confirm binding the positiveprey was retested againstin triplicateEightyone percent of PPIs are between IgSF proteinsThe remaining include PPIs between IgSF and otherproteins present in the screen Figure S3its cognate baitAlmost half of the proteins tested were involved in a PPI Proteins not involved in PPIs may be misfolded have binding partners notincluded in the screenrequire coreceptors or fall outside the dynamic range of theassay false negatives which is determined by PPI affinityand bait and prey concentrations Figure S4F Confirming thesensitivity of the assay many bait or prey proteins expressedat very low levels still engaged in one or two PPIs FiguresS4A and S4B A small number of PPIs were observed withbait or prey proteins exhibiting undetectable levels in conditioned media Figures S4C“S4E To interrogate the dynamicrange of the assay we plotted prey AP levels for PPIs with reported affinities Figure S4F These data suggest thatforvery poorly expressed prey proteins Figures S1A S4A andS4B PPIs with KD mM are likely to be missed false negatives Figure S4FOf the PPIs are previously unreported basedon literature and PPI databases Data S4 The majority of PPIsform one large network comprising proteinsD Only proteins involving PPIs are not connectedto the network Different regions of the network are shown inFigure Ninetyeight proteins had one PPI had two to five PPIs and had PPIs EBecause of proteins exhibit binding we calculated theexpected frequency with which each protein will bind at least˜˜x™™ number of proteins up to the maximally observed PPIsFigure S2B and compared the expected and observed frequencies F The observed number of binding partnersis significantly greater than predicted by random chance ofPPIs for a network of this size 0cResourcellABCEDFFigure apECIA Screen Details and Overview of ResultsA Schematic depiction of a subset of proteins in library Pie graph of library distribution Full protein domain names at martemblde Sun B Left flow chart of screen HT highthroughput Right example plate from screen Schematic of ECIA and pooledprey strategy Illustration of screen matrixC ECIA of undiluted prey single prey versus 3fold diluted prey pooled prey Background subtracted data are represented as ±SD Bkgd backgroundD Network of PPIs observed in screen Inset the PPIs that are not connected to the network Node size is proportional to number of PPIs Siglec subfamilynodes are highlighted in colorE Pie graph of distribution of the number of binding partners observed in screen overlaid on the network in a degreesorted circular layoutF Observed versus expected frequency with which each protein will bind at least ˜˜x™™ number of proteins up to the maximally observed PPIs assuming randomchance of interactions p KolmogorovSmirnov [KS] testSee also Figures S1 S2 S3 and S4 and Data S1 S2 S3 and S4Cell “ August 0cllResource Cell “ August legend on next page 0cResourcellPhylogenetic Homology Analysis PHA to Predict PPIsPPIs often occur between phylogenetically related proteins bothwithin and between subfamilies We performed multiplesequence alignments to identify subfamily members within ourlibrary and generated phylogenetic trees Using a combinedapproach which we call apECIAPHA we analyzed screendata alongside the phylogenetic trees to predict additionalPPIs between subfamily members that may have been missedin the screenPPI Validation by Surface Plasmon ResonanceWe selected a subset of screen and PHA predicted PPIs to validate by SPR Bona fide PPIs are expected to display distinct association and dissociation which can be observed with highsensitivity by SPR To increase avidity and therefore sensitivityFc dimerized ECD analytes and ligands were used in most experiments Figure S5 This increase in sensitivity prevented us fromdetermining monomeric KD constants Binding profiles characteristic of PPIs exhibiting clear resonance signals above background negative control ligand and receptor and concentrationdependent binding curves were deemed indicative of aspecific ligandanalyte PPI Nonspecific PPIs generally exhibitdeviations from this behavior such as high background andnonlinear concentration responsesTwentyfour newly identified PPIs observed in our screen weretested by SPR Of these we observed specific ligandanalyteinteractions We additionally tested PHA predicted PPIs andobserved PPIs for Three additional PPIs were observed between homologous proteins in mouse and crossspecies humanmouse In total we SPR validated newly identifiedPPIs Table Data S5Combined apECIAPHA Approach Reveals Multiple DCCSubfamily PPIsThe netrin1 receptor DCC has wellcharacterized functions inthe nervous system Finci DCC is a dependence receptor and is implicated in colorectal and other cancers but itsroles in these cancers are not well understood Goldschneiderand Mehlen We observed DCC binding to insulinlikegrowth factorbinding proteinlike IGFBPL1 but not to insulinlike growth factorbinding protein IGFBP7 Figures 2I and3A Our phylogenetic tree revealed IGFBPL1 and IGFBP7 residein a cluster and share the highest amino acid sequence similarity among subfamily members As such we examined binding of DCC to both IGFBPL1 and IGFBP7 by SPR and observedbinding for both Figure 3CPHA pointed us to four proteins that cluster with DCC PUNCPUNC e11 neogenin NEO1 and protogenin PRTG Figure 3ATogetherthese proteins play roles in diverse processesincluding nervous system development myogenesis and angiogenesis inflammation and tissue regeneration leukocyte migration neural tube and mammary gland formation development ofbone and connective tissues and stem cell differentiation Salbaum Wilson and Key Takahashi Schievenbusch DakouaneGiudicelli PUNC is an ˜˜orphan™™ receptor expressed in the developing nervous system Salbaum PUNC e11 and PRTG bound intercellular adhesion molecule ICAM5Figure 3A a proteinexclusively expressed in the brain that functions in synapse formation stabilization and refinement Gahmberg Cleaved ICAM5 ECD exhibits immunosuppressive functionsthrough cytokine regulation and may play important roles inregulation of brain inflammation We confirmed binding ofPUNC e11 and PRTG to ICAM5 by SPR Figure 3C AlthoughPPIs with ICAM5 were not detected in the screen for the remaining DCC subfamily members we tested them by SPR andobserved binding for all three Figure 3CIn our screen one or more DCC subfamily members alsobound to WFIKKN2 a secreted protein that binds transforming growth factorbeta subfamily members and modulates theirpresentation to cells Monestier and Blanquet lactotransferrin LTF an ironbinding protein with antimicrobial activity Hao interleukin6 receptor subunit alpha IL6Ra a cytokine receptor Schaper and RoseJohn and ISLR2LINX which functions in nervous system developmentMandai Panza Abudureyimu We confirmed binding of all DCC subfamily members tothese proteins by SPR and to other proteins observed in ourscreen Figures 3C 3D and 3F These results demonstratethe value of using a combined apECIAPHA approach to identifyadditional PPIs beyond screen data resulting in the elucidationof a more complete network Figure 3FLARPTPR Subfamily PPIs with SALMsThe LARfamily of type IIA protein tyrosine phosphatase receptors LARPTPRs comprises PTPRF also known as LARFigure Select Regions of the Complex PPI NetworkA Select PPIs including four proteins not connected to the network CD146 CNTN1 NFASC and NrCAMB Region largely comprised of immune system proteinsC Region comprising PPIs both within and across biological systemsD Two regions highlighting subfamilyspecific type IIA and type IIB PTPR PPIs Type IIA PPIs with SALMs and IL1APs include PPIs observed in screen and PHApredicted PPIs validated by SPR Figures and S6E Region highlighting PPIs for ˜˜orphan™™ receptors ILDR1 and PUNCF Region showing a subset of LILR subfamily PPIsG Region showing a subset of Siglec PPIs with nonSiglecs CD33Siglec3H Region showing SiglecSiglec PPIs CD33Siglec3 MAGSiglec4a SNSiglec1I Region highlighting PPIs between nervous system proteins and with proteins in immune and reproductive systems Within this region multiple additional PHApredicted PPIs were validated by SPR Figure Table Because a network is composed of interconnected nodes some linkage proteins are present in more than one panel Colored nodes denote proteins and PPIsvalidated by additional experiments Green line previously reported PPI gray line previously unreported PPISee also Data S4Cell “ August 0cllResourceTable SPRValidated apECIA Screen and PHA Predicted PPIsTable ContinuedLigandFcAnalyteFcapECIATIE1hISLR2mIslr2SALM1SALM2SALM3SALM4SALM5IL1RAPL1IL1RAPL2DSCAMCTDSCAML1CTKIR2DL5IL6RaPSG7PSG9ICAM5WFIKKN2PDL1PDL2LTFTrkAhLEPamLepahLEPamLepaPTPRDPTPRFPTPRSPTPRDPTPRFPTPRSPTPRDPTPRFPTPRSPTPRDPTPRSPTPRDPTPRFPTPRSPTPRSFGFR3PTPRSPTPRMPTPRTPTPRMPTPRTPVRISLR2DCCDSCAMDSCAMDCCIL6RaISLR2NEO1PRTGPUNCPUNC e11DCCIL6RaISLR2PRTGPUNCPUNC e11PDL1PDL1DCCIL6RaNANANANANANAknownknownknownNANAknownknownknownknownNANANANANANANANANAknownNANAPHANANANANANANANANANANANANANANANANANANANANANANANANANANANANANAContinued on next page Cell “ August LigandFcAnalyteFcapECIAPHAIGFBPL1IGFBP7ISLR2NEO1PRTGPUNCPUNC e11DCCIL6RaISLR2NEO1PRTGPUNCPUNC e11DCCIL6RaISLR2NEO1PRTGPUNCPUNC e11NANANANANANANANANANANANANANANANANANANAECDFc ligand and ECDFc analyte PPIs observed by SPR apECIA denotes PPI was observed in screen PHA denotes PPI was predicted byphylogenetic homology analysis SPR conditions are included in DataS5 See also Figure S6ahLEP and mLEP monomer proteinPTPRD and PTPRS Figures 4A and 4B LARPTPRs playimportant roles in synaptic anization and function Nam Presynaptic LARPTPRs mediate transsynapticadhesion through PPIs with multiple postsynaptic ligandsMouse mutants in LARPTPRs and their ligands exhibit defectsin synapse structure and function Lie Several PPIs are known between specific LARPTPRs andSALM235 Li Choi GotoIto Figure 4A We observed these as well as additionalPPIs in our screen Figures 2D 4A and 4C No LARPTPR binding to SALM1 or SALM4 has been reported and we did notobserve binding in our screen PHA led us to test binding of allLARPTPRs to all SALMs by SPR With the exception ofPTPRFSALM4 we observed binding of all LARPTPRs to allSALMs Figures 4E and S6A PTPRSALM pairs exhibited differences in maximum response units RU a relative comparison ofbinding strength suggesting a spectrum of binding affinities mayexist among LARPTPRs and SALMs Figure 4FLARPTPR Subfamily PPIs with Interleukin1 ReceptorAccessory Proteins IL1APsTranssynaptic LARPTPR interactions with IL1RAP and IL1RAPL1 induce bidirectional pre and postsynaptic differentiation Yoshida IL1RAP is known to interactwith all three LARPTPRs and IL1RAPL1 with PTPRD Weobserved these PPIs and additionally binding of IL1RAPL1 toPTPRS Figure 2D IL1RAPL1 shares sequence similarity 0cllBResourceACDEFlegend on next pageCell “ August 0cllResourcewith IL1RAPL2 an orphan receptor expressed in the brain withno known biological function Boraschi In ourscreen we detected binding of IL1RAPL2 to PTPRD and PTPRSFigure 2D We tested both known and previously unknown PPIsby SPR and observed binding for all Figure S6B IL1RAPL2additionally interacted with fibroblast growth factor receptor FGFR3 in the screen and by SPR Figure S6BType IIB PTPR Subfamily PPIs with DSCAMsType IIB PTPRs are expressed in most tissues and regulatediverse processes including cell growth migration and differentiation immune cell development endothelial cell adhesion andmigration neuronal development and synapse formation andoncogenic transformation Lee Stoker The typeIIB subfamily is composed of PTPRK PTPRM and PTPRT Figures 4A and 4B and multiple binding partners are known Weobserved binding of all type IIB PTPRs with Down syndromecell adhesion molecule DSCAM and Down syndrome cell adhesion molecule like1 DSCAML1 Figures 4A and 4C proteinsthat play various roles in nervous system development Sanesand Zipursky The ECD of DSCAMs contains nine Iglike domains followedby four FN3 domains one Iglike domain and two FN3 domainsFigure 4B The Nterminal region engages in homophilic bindingand mediates binding to secreted chemoattractant and chemorepellent ligands netrin1 and slit1 respectively Wojtowicz Ly Alavi No binding partnerhas been reported for the Cterminal ECD region In our screenwe observed binding between the Cterminal regions of bothDSCAMs DSCAMCT and DSCAML1CT and all three typeIIA PTPRs Figure 4C We confirmed binding of DSCAMCTand DSCAML1CT to PTPRT and PTPRM by SPR PTPRK wasnot expressed at sufficient levels Figure 4D vascular endothelial growth factorNew PPIs between Immune System ProteinsNew PPIs were observed for wellstudied immune systemproteins as well as orphan receptors Figure 2B Two PPIswere detected for the orphan granulocyte receptor CEACAM4receptor VEGFR3 and programmed cell death ligand PDL2 CEACAM4 is a member of the carcinoembryonic antigenrelated cell adhesion molecule subfamily expressed inthe immune system epithelial and endothelial cells and brainWakabayashiNakao Zinn and O¨ zkan Inthe immune system CEACAMs play roles in immunity anddevelopment We also observed binding between CEACAM1and the immune checkpoint receptor programmed cell deathprotein PD1Interactions of PD1 with its ligands PDL1 and PDL2 inhibitT cell proliferation cytokine production and cytotoxic activityBardhan PDL2 competes with PDL1 for bindingto PD1 and can be expressed on tumor cells where it mayplay a role in tumor evasion Ghiotto Cheng Bardhan Using SPR we confirmed the knownPPI between PDL1 and PDL2 Lee and observedhomophilic binding of PDL1 a previously unknown PPI Figure S6C By comparison with PDL1 binding to PD1 which approaches saturation at 18 nM we infer that the affinity of PDL1 homophilic binding is lower than binding to PD1 This mayexplain why this PPI within the wellstudied PD1PDL1 axishas not been identified previously and highlights the value of using multimerized proteins to detect lowaffinity PPIsHomophilic and Heterophilic Siglec Subfamily PPIsEight of the proteins with the highest number of PPIs in ourscreen are members of the Siglec subfamily members proteins highly restricted to the immune system that have immunemodulatory effects on Tolllike receptor TLR signaling andplay important roles in self versus nonself discrimination Macauley Siglecs display differential expression on cellsand exhibit a broad spectrum of Siglecspecific recognition ofsialylated glycan profiles present on healthy cells inflamed ormalignant cells and pathogens We observed a network of homophilic and heterophilic PPIs among Siglecs Figure 2H DataS4 as well as PPIs with distantly related proteins a subset areshown in Figure 2G Data S4PPIs between Immune and Nervous System ProteinsThe signaling lymphocytic activation molecule subfamily SLAM members is expressed on most immune cells Dragovich andMor SLAMs function as both costimulatory and coinhibitory molecules in innate and adaptive immunity and playan integral role in autoimmune disorders SLAMF9 is an orphanreceptorWe observed SLAMF9 binding to bactericidal permeabilityincreasing protein BPI and IGSF10 Figure 2C BPI is a neutrophilderived antibiotic protein that participates in bacteria killingthrough its highly cationic Nterminal region Bu¨ low The Cterminal region of BPI exhibits no bactericidal activity andis believed to interact with other proteins and function in differentprocesses IgSF10 is an orphan receptor involved in the migration of gonadotropinreleasing hormone expressing GnRH neurons Howard IgSF10 has no known function in theFigure SPR Validation of DCC Subfamily PPIs Identified by apECIAPHA ApproachA Dendrograms of a subset of DCC subfamily PPIs showing the number of SPRvalidated PPIs observed in the apECIA screen turquoise and predicted by PHAmagentaB Dendrogram of a subset of PSG PPIsC SPR sensrams for DCC subfamily IL6Ra and ISLR2 analytes 2fold dilutions “ nM nM for PRTGWFIKKN2 due to incomplete chipregeneration at higher concentrations binding to various ligands TrkA and TIE1 negative controlsD SPR sensram for IL6Ra analyte 2fold dilutions “ nM binding to ISLR2 ligand PUNC negative controlE SPR sensrams for DSCAM and DCC analytes 2fold dilutions “ nM binding to PSG ligands TIE1 negative controlF Subnetwork of SPRvalidated PPIsRU resonance unitsSee also Figure S5 and Data S5 Cell “ August 0cResourcellABCEDFlegend on next pageCell “ August 0cllResourceimmune system and SLAMF9 function in the nervous system isunknown revealing a PPI between two orphan receptors fromdifferent biological systemsFigure 2F These PPIs present MOG and MPZ PPIs with LILRsas new candidates for neuronal regeneration studiesPPIs with PregnancySpecific Glycoproteins PSGsDuring pregnancy PSGs members are the most abundanttrophoblastic proteins in maternal blood and serve as markersfor trophoblast quality and embryo viability Moore and Dveksler The mechanisms of PSG action in pregnancy are not wellunderstood Studies suggest PSGs also have immunoregulatory proangiogenic and antiplatelet aggregation functionsWe observed several PSG binding partners includingplateletderived growth factor receptor alpha PDGFRAfibroblast growth factor receptor FGFR4 Ctype lectindomain family member A CLEC4A DCC and DSCAMFigures 2I and 3BObserved PSG interactions occur selectively and differentiallyto these binding partners Figure 2I Data S4 Using SPR weexamined binding of DCC and DSCAM to PSG7 and PSG9 Figure 3E We observed binding for DSCAM with PSG9 and bothDCC and DSCAM with PSG7 Interestingly DCC is expressedin human placenta DakouaneGiudicelli ThesePPIs present new candidate receptors for studying the role ofPSGs in pregnancy immunoregulation and angiogenesisLILR Subfamily PPIs with BTNL8 and MyelinationProteinsLeukocyte immunoglobulinlike receptors LILRs membersare a subfamily of activating LILRA and inhibitory LILRB receptors that exhibit immunomodulatory activity and function ininflammation regulation tolerance and differentiation Burshtynand Morcos We observed binding of multiple activatingand inhibitory LILRs to butyrophilin8 BTNL8 Figure 2F BTNLsare members of the extended B7 family of molecules and function as costimulatory or coinhibitory signals for T cell activationRhodes LILRs are also expressed on neurons and function in the regulation of development synaptic plasticity and axonal regeneration Hirayasu and Arase Myelin the protective insulatinglayer around axons inhibits neuronal regeneration following spinal injury Monje Three myelin proteins Nogo MAG andOMgp are known to interact with PirB in mouse one of only twomouse LILRB orthologs Atwal We observed bindingof multiple LILRs to two additional myelin proteins myelinoligodendrocyte glycoprotein MOG and myelin protein P0 MPZPVR Selectively Interacts with a KillerCellImmunoglobulinlike Receptor KIRThe polygenic Killercellimmunoglobulinlike receptors KIRsare a highly polymorphic subfamily of activating and inhibitoryproteins expressed on natural killer NK cells that regulate development maturation and activation Pende NK cellsinitially express a stochastic combination of KIRs that is refinedduring maturation to tune killing response threshold and ensureoptimal discrimination of target cells from healthy cells Weobserved binding of KIR2DL5 to poliovirus receptor PVR Figures 2C and 5A which validates a recently reported PPI Husain We confirmed KIR2DL5 binding to PVR by ECIAtitration analysis and SPR Figures 5C and 5DTo examine binding at the cell surface fluorescent tetramersof PVRFc and KIR2DL5Fc ECDFcSA647 were incubatedwith fulllength KIR2DL5 and PVRtransfected cells respectively Flow cytometry analysis revealed concentrationdependent binding of both ligands to cells expressing cognate fulllength receptor but not control cells Figure 5E PVRFc tetramers did not bind cells transfected with fulllength KIR2DL4and KIR2DL1 which share and sequence similaritywith KIR2DL5 respectively Figures 5B and 5F To furtherexamine the specificity of PVR for KIR2DL5 ECIA was performed using KIRs PVR bound specifically to KIR2DL5 Figure 5G Because KIR2DL5 has been associated with increasedvirus susceptibility and reduced antiviral response to therapythis specificity may have implications for the role of KIR2DL5 inimmunityTrkA Selectively Interacts with TIE1Highaffinity nerve growth factor receptor NTRK1 also knownas TrkA has multiple wellstudied functions in the nervous system Amatu In the immune system where its function is not well understood TrkA is expressed on monocytesmacrophages dendritic cells resting and activated B cells andneutrophils and erythroblasts Minnone In ourscreen we observed TrkA binding to tyrosineprotein kinase receptor TIE1 but not to TIE2 Figures 2A and 6A TIE1 is expressed on endothelial cells immature hematopoietic cells andplatelets and functions in complex with the angiopoietinTIE2pathway to inhibit angiogenesis Eklund We performed SPR and observed TrkA binding to TIE1 but not toFigure SPR Validation of Type IIA and Type IIB PTPRs with SALMs and DSCAMsA Left dendrogram highlighting phylogenetic clustering of SALMs DSCAMs type IIA PTPRs PTPRFSD and type IIB PTPRs PTPRTMK Right SPRvalidated PPIs green line previously known PPI turquoise line previously unknown PPI observed in screen magenta line previously unknown PHA predictedPPI Gray line screen PPI not SPR testedB Protein domain structures Ig immunoglobulinlike domain FN fibronectin type III domain MAM meprin A5 protein domain LRR leucinerich repeat TMtransmembrane NT Nterminal region of ECD CT Cterminal region of ECDC Screen data showing PPI specificity of PTPRFSD and PTPRTMK subfamilies Background subtracted OD nm data are represented as mean oftriplicate wellsD SPR sensrams for DSCAML1CT and DSCAMCT analytes 2fold dilutions “ nM binding to PTPRM and PTPRT ligandsE SPR sensrams for PTPRS analyte 2fold dilutions “ nM binding to SALMs and DSCAMCT ligandsF SPR max RU values for every pairwise combination of PTPRFSD analytes with SALM ligandsRU resonance unitsSee also Figures S5 and S6 and Data S5 Cell “ August 0cResourcellABCEDFGlegend on next pageCell “ August 0cllResourceTIE2 Figures 6B and S7A As a positive control for TIE2 weconfirmed binding to monomer angiopoietin1 Figure S7A Davis We next investigated whether TrkA can bind NGF and TIE1simultaneously We preincubated TrkA with NGF and comparedbinding of T Answer:

Thyroid_Cancer

"Pharyngitis Tonsillitis Deep learning Smartphone Automated diagnosis Telemedicine Purpose Severe pharyngitis is frequently associated with inflammations caused by streptococcal pharyngitis which can cause immunemediated and postinfectious complications The recent global pandemic of coronavirus disease COVID19 encourages the use of telemedicine for patients with respiratory symptoms This study therefore purposes automated detection of severe pharyngitis using a deep learning framework with selftaken throat images Methods A dataset composed of two classes of throat images with pharyngitis and normal throat images was collected Before the training classifier we constructed a cycle consistency generative adversarial network CycleGAN to augment the training dataset The ResNet50 Inceptionv3 and MobileNetv2 architectures were trained with transfer learning and validated using a randomly selected test dataset The performance of the models was evaluated based on the accuracy and area under the receiver operating characteristic curve ROC AUC Results The CycleGANbased synthetic images reflected the pragmatic characteristic features of pharyngitis Using the synthetic throat images the deep learning model demonstrated a significant improvement in the accuracy of the pharyngitis diagnosis ResNet50 with GANbased augmentation showed the best ROCAUC of for pharyngitis detection in the test dataset In the 4fold crossvalidation using the ResNet50 the highest detection accuracy and ROCAUC achieved were and respectively Conclusion The deep learning model for smartphonebased pharyngitis screening allows fast identification of severe pharyngitis with a potential of the timely diagnosis of pharyngitis In the recent pandemic of COVID19 this framework will help patients with upper respiratory symptoms to improve convenience in diagnosis and reduce transmission Introduction Diagnostic support in remote healthcare services has shown the ability to minimize the exposure of ill patients to healthcare providers and other patients [] The recent global pandemic of coronavirus disease COVID19 has encouraged the use of telemedicine for patients with upper respiratory symptoms [] Because smartphones have become ubiquitous many researchers are interested in utilizing them in telemedicine Deep learning technology can assist with patient examination using a smartphone when clinicians deal with limited information in a remote patient monitoring setting In particular a smartphone is useful to take a picture of the throat [] Therefore home monitoring using a smartphone will help in the diagnosis and treatment of patients with upper respiratory symptoms to improve convenience and to reduce Corresponding author Department of Ophthalmology Aerospace Medical Center Republic of Korea Air Force Danjaero Namilmyeon Cheongwongun Chungcheongbukdo Cheongju South Korea Corresponding author Epilepsy Center Neurological Institute Cleveland Clinic Euclid Ave Cleveland Ohio USA Email addresses eyetaekeunyoogmailcom TK Yoo jychoi717gmailcom JY Choi Tae Keun Yoo and Joon Yul Choi contributed equally to this work 101016jcompbiomed2020103980 Received June Received in revised form August Accepted August ComputersinBiologyandMedicine1252020103980Availableonline20August2020001048252020ElsevierLtdAllrightsreserved 0cimages were posted by users asking for medical advice via the social QA The search strategy was based on the key terms œsore throat  œpharyngitis œtonsillitis œexudative tonsillitis œtonsillopharyngitis  œthroat image and œsmartphone in Korean Japanese and English languages The most recent image from the database search was achieved on April Images that were not taken with smartphones were manually picked and excluded for this study Images with the characteristics of either pharyngitis or normal throat were manually classified by two clinicians and the ambiguous images were excluded to clarify the image domains Finally we collected the initial dataset with two classes including throat images with pharyngitis and normal throat images The dataset was randomly separated into training N validation N and test sets N to apply deep learning to an independent dataset Detailed data distribution and augmentation are described in Table Only the throat and tonsils images were used for the input data without further manipulation to reduce the intraclass variance Original images were extracted from the database in the PNG Portable Network Graphics format The images were resampled to a pixel resolution of — — in the PNG formatfor CycleGAN and deep learning models All procedures were performed in accordance with the ethical standards of the institutional and national research committee and the Helsinki declaration and its later amendments or comparable ethical standards This study did not require ethics committee approval instead the researchers used webbased and deidentified data All datasets for the development of the deep learning model are available at Mendeley Data repositories 10176328ynyhnj2kz TK Yoo transmission There have been several approaches adopting deep learning for automated diagnosis of several diseases using images captured by smartphones [] Pharyngitis which is diagnosed in more than million patients in the United States annually is a common condition associated with acute upper respiratory tract infection [] Pharyngitis is an inflammation of the back of the throat and tonsils Sore throat caused by pharyngitis is one of the main causes of medical visits for young patients [] The most common cause of acute pharyngitis is a selflimiting viral infection However Streptococcus pyogenes is the major bacterial infectious cause of pharyngitis and is responsible for an estimated “ of cases of sore throat [] Frequently severe pharyngitis with fever and exudative tonsillitis is associated with streptococcal pharyngitis which can cause immunemediated and postinfectious complications such as acute rheumatic fever [] Therefore timely diagnosis of pharyngitis for treatment is important to reduce symptoms fever and complications [] However many patients with upper respiratory infection ignore their symptoms in the early stage and medical visits do not routinely take place Moreover in recent days many patients hesitate to visit clinics because of the COVID19 outbreak The importance of a mobilebased monitoring system for patients with acute upper respiratory infections has been raised because of its applicability and effectiveness [] A previous study endeavored to collect throat images using additional equipment in conjunction with the smartphone and used the knearest neighbor algorithm in color distribution space to classify images with streptococcal tonsil [] However the need for additional equipment limited their effectiveness of this method in a realworld setting Moreover color distribution was unable to represent the characteristic features of pharyngitis Throat images exhibit variation in the size illumination and shape of the oral cavity Here we present a deep learning model with smartphonebased throat images facilitating the detection of severe pharyngitis using selftaken throat images Fig We performed automated detection of severe pharyngitis using a convolutional neural network CNN framework Methods Data collection The basic concept of our framework is throat examination using a selftaken smartphone image with computeraided diagnosis system which is similar to the previous dermatology study [] This study was performed using publicly accessible selftaken throat images on the web We collected throat images from webbased social QA systems including Naver Korea kinnavercom and Yahoo Japan chiebukuroyahoocojp The additional throat image datasets were extracted using the Google image search engine Most throat Data augmentation using GAN Because of the limited number of datasets and their imbalanced distribution data augmentation is required for deep learning training Basic data augmentation techniques such as flip translation rotation and brightness change have been applied to train deep learning models Several previous studies have attempted to train deep learning models using generative adversarial network GANbased synthetic images to increase the classification performance [] Inspired by previous works using a generative adversarial network we adopted the CycleGANbased data augmentation to increase the accuracy of diagnosis The cycle consistency in Fig allows CycleGAN to capture the characteristics of two image domains and automatically learn how these characteristics should be translated to transfer to domains without any paired datasets [] CycleGAN was developed to overcome the limitations of paired data when two generators and two discriminators are used It is considered to be a powerful technique that performs image domain transfer and face transfer [] Previous studies have demonstrated that CycleGAN can improve deep learning models by generating training situations to learn better decision boundaries between classes We built the CycleGAN augmentation model to increase the Fig Workflow of building a deep learning model for pharyngitis diagnosis using a smartphone ComputersinBiologyandMedicine12520201039802 0cTK Yoo Fig Schematics of the CycleGAN model generating new normal images and pathologic throat images with pharyngitis generalizability of the dataset and to improve the classification performance in the imbalanced dataset Before training the CycleGAN the throat images were augmented using linear transformation including left and right flip width and height translation from to random rotation from —¦ to —¦ zooming from to and random brightness change from to We defined these transformations as the basic augmentation step In this step normal throat and pharyngitis images were randomly sampled for the training set and normal throat and pharyngitis images were randomly sampled for the validation set Using the data with basic augmentation we trained the CycleGAN models to transform both normal to pharyngitis throat images and pharyngitis to normal images The trained CycleGAN model augmented the training set normal throat and pharyngitis images before augmentation and a total of throat images including normal and pharyngitis images were prepared to train the diagnostic classifier model after CycleGANbased augmentation It should be noted that supervised GAN techniques including conditional GAN and Pix2px were unable to be applied in this study because of a lack of paired normal and pharyngitis images To use a verified and predesigned image generator all the input images required resizing to a pixel resolution of — — which is the basic setup of a CycleGAN Therefore we used the default parameter settings that is the ADAM optimizer with a batch size of to optimize the GAN networks Development of CNN model We trained conventional deep learning models after data augmentation Because of a small image dataset in this study developing a custom deep learning method is challenging due to difficulty and time consuming eg a small training dataset can easily result in an over fitted model and low performance [] To overcome the problem of the small dataset transfer learning was widely used to train deep learning models using pretrained architectures [] This study also applied pretrained learning models to the classification task of throat images The last fully connected layer of the CNNs was only trained and the study used the pretrained conventional model as a feature extractor [] The CNN models including ResNet50 Inceptionv3 and MobileNet v2 were adopted to build binary classifiers [] These CNN models have been used successfully in many studies demonstrating stateoftheart performance with the saliency map [] The models were trained using the training set and the validation set was used to estimate how well the model had been trained We downloaded the CNN models which were pretrained on the ImageNet database and performed finetuning of the weights of the pretrained networks This process generally maintains the weights of some bottom layers to avoid overfitting and performs delicate modification of the highlevel features To use the images generated by CycleGAN the size of the input images for the deep learning models was set to a pixel resolution of — — and the images were resized for each pretrained model Most conventional deep learning models adopted a pixel resolution of — — or — — [] One deep learning research showed that the best performance was achieved at an image resolution of between — and — pixels for binary classification [] Therefore the resolution of our study was appropriate to detect pharyngitis in a binary decision The model was trained with epochs and a batch size of The ADAM optimizer with a learning rate of was also used with a crossentropy loss for all CNN models The crossentropy loss function is defined as Lcross entropy cid0ˆ‘Nipi logqi Where pi represents the ground truth value and qi represents predicted probability value from a classifier for the ith image The optimizer updated the network parameters to minimize the loss function In our experiments it tuned a fully connected layer of the CNN models For example the first layers of ResNet50 were left frozen and we trained the last fully connected layer using the training dataset which is described in Table the ADAM optimizer We chose the final classifier model which maximized the accuracy in the validation dataset To visualize the clusters to see if the classes are separable by a considerable margin the tdistributed stochastic neighbor embedding t SNE algorithm was executed using sampled instances The feature vectors from the last layer of the pretrained Inceptionv3 model were extracted to train the tSNE [] As there is a growing demand for explainable artificial intelligence methods in medicine [] we adopted githubcomjacobgilpytorch the GradCAM technique ComputersinBiologyandMedicine12520201039803 0cTK Yoo Table Throat image dataset and augmentation used in this study Number of validation set Number of training set Raw data Basic augmentation GANbased image synthesis augmentation Class Normal Pharyngitis Normal Pharyngitis Normal Pharyngitis Number of test set GAN generative adversarial network gradcam to generate the attention map [] The GradCAM visualizes the decisional areas of the CNN model using the gradients of any target flowing into the final convolutional network The heatmap has a low resolution and it was upsampled via bicubic interpolation Finally it produces heatmaps that highlight the area of interest and interprets the decision of the deep learning models The performance of the CNN models was evaluated based on the accuracy and area under the curve AUC of the receiver operating characteristic curve ROC and the precisionrecall curve PRC The Youden index which is an estimate of the optimal diagnostic threshold was adopted in this study [] After obtaining the sensitivity and specificity the Youden index was calculated at each cutoff point We selected the optimal value which maximized the Youden index These performance indexes are expressed as follows Accuracy TP TNTP TN FP FN Sensitivity Recall TPTP FNSpecificity Precision TNTN FPTPTP FP Youden index Sensitivity Specificity cid0 Where TP TN FP and FN denote true positives true negatives false positives and false negatives respectively We also performed a 4fold crossvalidation using the entire dataset to evaluate a generalized performance Google Colab Pro a cloud service for disseminating the deep learning research was adopted to implement the CycleGAN and CNN models [] Google Colab Pro provides a development environment using the Tensorflowbased deep learning libraries and a robust graphic processing unit GPU This enables the rapid processing of a heavy deep learning network without the need for a personal GPU [] The available hardware for each virtual machine varied by session but typically included top products of NVIDIA GPUs K80 T4 or P100 around “ GB of RAM and “ GB of free space on the virtual machine hard drive [] We used the Colab CycleGAN tutorial page to develop and validate the CycleGAN model and all of the code is available on the Tensorflow webpage wwwtensorflowtutorialsgene rativecyclegan We only modified the input pipeline to import our dataset The code of the CycleGAN and CNN models is presented in Supplementary Material Results We developed a deep learning model using GANbased augmentation in the challenging context of pharyngitis detection To build a balanced training dataset the CycleGAN models generated normal and pathologic throat images using the initial training dataset The color intensity distributions of the pharyngitis and normal throat images were not significantly different although most throat images had exudate regions Fig A The tSNE algorithm demonstrates that both groups are clustered and they are separable by a considerable margin Fig B The final CycleGAN model for the pharyngitis throat image was trained for epochs which required h After training normal throat images were translated into pathologic images and throat images with pharyngitis were translated into normal images Finally we constructed a balanced augmented training dataset including normal and pharyngitis images using CycleGAN The CycleGANbased synthetic images were realistic and reflected the characteristic features of pharyngitis Fig A The CycleGAN model synthesized white or gray patches and increased the redness on the throat wall and tonsils from normal images Fig B Generated throat images were reviewed by three clinicians including an otolaryngologist and an anesthesiologist All pharyngitis images generated were deemed by the three clinicians to show œmore pathologic and inflammatory results when compared to the original images This feature generation based on the CycleGAN model can be effective for generating a new sample to increase the intra class variation and generalizability The CNN models were trained using the final augmented dataset via the transfer learning technique Fig demonstrates the training process of the ResNet50 CNN model using the training and validation sets The training process for CNN took approximately h for epochs with finetuning for each training step After the 200th epoch the validation accuracy was not improved and the crossentropy of the validation result increased Therefore we considered that training for epochs was optimal in the ResNet50 model and selected the trained model at the 200th epoch Fig A shows the training validation and test datasets of CNN models with and without CycleGANbased augmentation The ROCAUCs of ResNet50 Inceptionv3 and MobileNetv2 without GAN based augmentation were and respectively Fig B We obtained the best ROCAUC of pharyngitis detection using ResNet50 with GANbased augmentation corresponding to Fig C At the optimal diagnostic cutoff value the ResNet50 model predicted pharyngitis with an accuracy of sensitivity of and specificity of The ROCAUC of Inceptionv3 and MobileNetv2 corresponded to and respectively The models with GAN based augmentation demonstrated superior performance in comparison with the models with only basic augmentation We also evaluated the performance of the models using PRCs Fig The PRCAUC also demonstrated that deep learning models with GANbased augmentation had better performance than those without GANbased augmentation ResNet50 with GANbased augmentation predicted pharyngitis with the highest PRCAUC of When a custom deep learning network without the transfer learning technique was trained the validation accuracy was lower than and was not improved during the training epochs Supplementary materials Table shows the performance of CNN models via 4fold cross validation in the entire dataset A similar tendency is observed for the average accuracy and AUC values in the crossvalidation The result shows that the highest detection accuracy and ROCAUC achieved were and respectively by using the ResNet50 Other CNN models showed lower performance than the ResNet50 with GANbased augmentation but there were no significant differences between CNN models in the 4fold crossvalidation A saliency map using the Grad CAM technique was generated to visualize the characteristic pathologic features of pharyngitis for the predicted evidence Fig A In normal throat images however some regions were highlighted Fig B When synthesized images were tested using the trained model synthesized exudates were highlighted correctly in the images as shown in Fig C Furthermore external clinical cases from previous literature were analyzed to investigate to show the capability of detecting pharyngitis of the framework developed in this study [] The representative cases with severe pharyngitis are shown in Fig One case presents an ComputersinBiologyandMedicine12520201039804 0cTK Yoo Fig Visual data exploration of pharyngitis and normal throat images A The mean redgreenblue RGB distributions B The feature space visualized using the tSNE technique For interpretation of the references to color in this figure legend the reader is referred to the Web version of this article Fig Data augmentation using CycleGAN to improve the diagnostic performance of pharyngitis A CycleGANbased augmentation for imbalanced data B Examples of pathologic throat images with pharyngitis generated by the CycleGAN Fig Training process of the ResNet50 CNN model for pharyngitis detection A Accuracy learning curves of the training and validation sets B Loss learning curves of the training and validation sets ComputersinBiologyandMedicine12520201039805 0cTK Yoo Fig Performance of pharyngitis detection deep learning models A Schematics of basic augmentation and GANbased augmentation B The receiver operating characteristic curves of deep learning models using basic augmentation C The receiver operating characteristic curves of deep learning models using GANbased augmentation Fig Precisionrecall curves of pharyngitis detection deep learning models A The deep learning models using basic augmentation B The deep learning models using GANbased augmentation Table Classification performance for severe pharyngitis detection in the 4fold crossvalidation using the developmental set AUC CI Accuracy CI Sensitivity CI Specificity CI Pvalue “ “ “ Basic augmentation ResNet50 Inceptionv3 MobileNetv2 GANbased image synthesis augmentation ResNet50 Inceptionv3 MobileNetv2 AUC area under the receiver operating characteristic curve CI confidence interval GAN generative adversarial network Comparison of receiver operating characteristics curves with the single best technique ResNet50 with GANbased image synthesis augmentation according to the Delong test “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ Reference 81yearold man with odynophagia Fig A and the other case presents a 41yearold diabetic man with throat pain Fig B The ResNet50 model was able to detect pharyngitis in both cases The GradCAM technique highlighted white patches on the throat wall as markers of severe pharyngitis in the deep learning model Discussion The current proofofconcept study investigated the possibility of deep learning using a smartphone for detecting pharyngitis A recentstudy demonstrated the ability of selftaken throat images to detect pharyngitis based on the knearest neighbor algorithm in a color space [] That study used additional equipment as well as a ComputersinBiologyandMedicine12520201039806 0cTK Yoo Fig Example of deep learning classification results with a saliency map using the GradCAM technique A Pharyngitis throat images B Normal throat images C Synthesized images smartphone to obtain throat images We utilized selftaken throat images without additional equipment using deep learning techniques for the detection of pharyngitis We showed that the trained GAN models were able to generate new realistic synthetic throat images which can improve the diagnostic accuracy The final deep learning model achieves high accuracy for automated diagnosis of pharyngitis using smartphone images Therefore this framework could be targeted toward patients with a sore throat who need screening for severe pharyngitis We believe that our study could be extended to computeraided diagnosis using images from an endoscope system in otolaryngology clinics similar to what has been done with colonoscope images using a deep learning model [] To the best of our knowledge no study has been performed to detect pharyngitis based on deep learning using smartphone images However it should be noted that this study is considered as only a preliminary and proofofconcept study because of its technical limitations due to using only Google Colab The current study framework is similar to that of Chamier which showed a deep learning framework using Google Drive and training and prediction on Google Colab [] Our proposed conceptual workflow is shown in Fig It needs the Flask servers and interfaces implemented using HTML and JavaScript to be applied in a real clinical setting This work could be part of a larger project to enable smartphonebased pharyngitis detection via a cloudbased applevel mobile data analysis We believe that a future appbased model can provide a robust solution for the costeffective and convenient screening of pharyngitis in a telemedicine setting We have highlighted the feasibility of a smartphonebased approach with deep learning to detect pharyngitis Our approach does not require Fig Classification results from the deep learning model applied to clinical cases with severe pharyngitis A An 81yearold man with odynophagia [] B A 41yearold diabetic man with throat pain [] Fig Example of a proposed smartphonebased system for pharyngitis detection ComputersinBiologyandMedicine12520201039807 0cexplored clinical datasets and webbased datasets and showed that the labeling of webbased images is often uncertain [] Therefore further clinical datasets with validation will be required to confirm the effectiveness of our framework Fourth because we collected only throat images via image search engines there was no metadata including gender race season or age According to a previous epidemiologic study these factors could affect the pharyngitis detection performance [] Fifth although the datasets were reviewed by authors images could be potentially duplicated in the dataset The duplicated images would affect the independence of the validation dataset We have shown the feasibility of deep learning for the detection of tonsil swelling and exudates in throat images The limitations of our study should be overcome by the availability of a sufficient number of throat images taken by a smartphone with a wellanized study protocol To validate the effectiveness of our framework a prospective study with many patients should be performed in a clinic once the app based framework is developed This will solve the problem of possible duplicated images in the dataset and the absence of metadata Conclusion TK Yoo additional equipment to collect throat images and we collected self taken images using a smartphone The use of webbased image capture of users from various devices and GANbased image augmentation may allow robust image processing While in the current study the implementation was performed on a desktop computer and the Colab™s remote server a smartphone application will also be possible to perform this identification for detecting pharyngitis The light deep learning models such as MobileNet can be executed on a smartphone without transmitting the images to a server [] Our result demonstrates that MobileNetv2 also has a high diagnostic performance similar to ResNet50 and Inceptionv3 In future research an increased amount of training data is needed to improve the detection accuracy using light models The utility of deep learning and smartphones may facilitate the widespread adoption of telemedicine and physical examination platforms via our approach Furthermore the presented framework enabled using a smartphone camera and deep learning techniques will help the patients in selfscreening for severe pharyngitis and may accelerate diagnostic support in remote healthcare services Because of the recent pandemic of COVID19 patients with respiratory symptoms need selfmonitoring to evaluate their pathologic status [] Advances in technology will require clinicians to embrace remote healthcare services The framework presented in this paper was designed for the timely diagnosis of pharyngitis for treatment Similar smartphoneimagebased diagnostic approaches have been introduced in several other medical image domains including skin diseases [] hematologic diseases [] oral diseases [] and eye diseases [] The main concern of deep learning models in this study

cancer247

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: "Pharyngitis Tonsillitis Deep learning Smartphone Automated diagnosis Telemedicine Purpose Severe pharyngitis is frequently associated with inflammations caused by streptococcal pharyngitis which can cause immunemediated and postinfectious complications The recent global pandemic of coronavirus disease COVID19 encourages the use of telemedicine for patients with respiratory symptoms This study therefore purposes automated detection of severe pharyngitis using a deep learning framework with selftaken throat images Methods A dataset composed of two classes of throat images with pharyngitis and normal throat images was collected Before the training classifier we constructed a cycle consistency generative adversarial network CycleGAN to augment the training dataset The ResNet50 Inceptionv3 and MobileNetv2 architectures were trained with transfer learning and validated using a randomly selected test dataset The performance of the models was evaluated based on the accuracy and area under the receiver operating characteristic curve ROC AUC Results The CycleGANbased synthetic images reflected the pragmatic characteristic features of pharyngitis Using the synthetic throat images the deep learning model demonstrated a significant improvement in the accuracy of the pharyngitis diagnosis ResNet50 with GANbased augmentation showed the best ROCAUC of for pharyngitis detection in the test dataset In the 4fold crossvalidation using the ResNet50 the highest detection accuracy and ROCAUC achieved were and respectively Conclusion The deep learning model for smartphonebased pharyngitis screening allows fast identification of severe pharyngitis with a potential of the timely diagnosis of pharyngitis In the recent pandemic of COVID19 this framework will help patients with upper respiratory symptoms to improve convenience in diagnosis and reduce transmission Introduction Diagnostic support in remote healthcare services has shown the ability to minimize the exposure of ill patients to healthcare providers and other patients [] The recent global pandemic of coronavirus disease COVID19 has encouraged the use of telemedicine for patients with upper respiratory symptoms [] Because smartphones have become ubiquitous many researchers are interested in utilizing them in telemedicine Deep learning technology can assist with patient examination using a smartphone when clinicians deal with limited information in a remote patient monitoring setting In particular a smartphone is useful to take a picture of the throat [] Therefore home monitoring using a smartphone will help in the diagnosis and treatment of patients with upper respiratory symptoms to improve convenience and to reduce Corresponding author Department of Ophthalmology Aerospace Medical Center Republic of Korea Air Force Danjaero Namilmyeon Cheongwongun Chungcheongbukdo Cheongju South Korea Corresponding author Epilepsy Center Neurological Institute Cleveland Clinic Euclid Ave Cleveland Ohio USA Email addresses eyetaekeunyoogmailcom TK Yoo jychoi717gmailcom JY Choi Tae Keun Yoo and Joon Yul Choi contributed equally to this work 101016jcompbiomed2020103980 Received June Received in revised form August Accepted August ComputersinBiologyandMedicine1252020103980Availableonline20August2020001048252020ElsevierLtdAllrightsreserved 0cimages were posted by users asking for medical advice via the social QA The search strategy was based on the key terms œsore throat  œpharyngitis œtonsillitis œexudative tonsillitis œtonsillopharyngitis  œthroat image and œsmartphone in Korean Japanese and English languages The most recent image from the database search was achieved on April Images that were not taken with smartphones were manually picked and excluded for this study Images with the characteristics of either pharyngitis or normal throat were manually classified by two clinicians and the ambiguous images were excluded to clarify the image domains Finally we collected the initial dataset with two classes including throat images with pharyngitis and normal throat images The dataset was randomly separated into training N validation N and test sets N to apply deep learning to an independent dataset Detailed data distribution and augmentation are described in Table Only the throat and tonsils images were used for the input data without further manipulation to reduce the intraclass variance Original images were extracted from the database in the PNG Portable Network Graphics format The images were resampled to a pixel resolution of — — in the PNG formatfor CycleGAN and deep learning models All procedures were performed in accordance with the ethical standards of the institutional and national research committee and the Helsinki declaration and its later amendments or comparable ethical standards This study did not require ethics committee approval instead the researchers used webbased and deidentified data All datasets for the development of the deep learning model are available at Mendeley Data repositories 10176328ynyhnj2kz TK Yoo transmission There have been several approaches adopting deep learning for automated diagnosis of several diseases using images captured by smartphones [] Pharyngitis which is diagnosed in more than million patients in the United States annually is a common condition associated with acute upper respiratory tract infection [] Pharyngitis is an inflammation of the back of the throat and tonsils Sore throat caused by pharyngitis is one of the main causes of medical visits for young patients [] The most common cause of acute pharyngitis is a selflimiting viral infection However Streptococcus pyogenes is the major bacterial infectious cause of pharyngitis and is responsible for an estimated “ of cases of sore throat [] Frequently severe pharyngitis with fever and exudative tonsillitis is associated with streptococcal pharyngitis which can cause immunemediated and postinfectious complications such as acute rheumatic fever [] Therefore timely diagnosis of pharyngitis for treatment is important to reduce symptoms fever and complications [] However many patients with upper respiratory infection ignore their symptoms in the early stage and medical visits do not routinely take place Moreover in recent days many patients hesitate to visit clinics because of the COVID19 outbreak The importance of a mobilebased monitoring system for patients with acute upper respiratory infections has been raised because of its applicability and effectiveness [] A previous study endeavored to collect throat images using additional equipment in conjunction with the smartphone and used the knearest neighbor algorithm in color distribution space to classify images with streptococcal tonsil [] However the need for additional equipment limited their effectiveness of this method in a realworld setting Moreover color distribution was unable to represent the characteristic features of pharyngitis Throat images exhibit variation in the size illumination and shape of the oral cavity Here we present a deep learning model with smartphonebased throat images facilitating the detection of severe pharyngitis using selftaken throat images Fig We performed automated detection of severe pharyngitis using a convolutional neural network CNN framework Methods Data collection The basic concept of our framework is throat examination using a selftaken smartphone image with computeraided diagnosis system which is similar to the previous dermatology study [] This study was performed using publicly accessible selftaken throat images on the web We collected throat images from webbased social QA systems including Naver Korea kinnavercom and Yahoo Japan chiebukuroyahoocojp The additional throat image datasets were extracted using the Google image search engine Most throat Data augmentation using GAN Because of the limited number of datasets and their imbalanced distribution data augmentation is required for deep learning training Basic data augmentation techniques such as flip translation rotation and brightness change have been applied to train deep learning models Several previous studies have attempted to train deep learning models using generative adversarial network GANbased synthetic images to increase the classification performance [] Inspired by previous works using a generative adversarial network we adopted the CycleGANbased data augmentation to increase the accuracy of diagnosis The cycle consistency in Fig allows CycleGAN to capture the characteristics of two image domains and automatically learn how these characteristics should be translated to transfer to domains without any paired datasets [] CycleGAN was developed to overcome the limitations of paired data when two generators and two discriminators are used It is considered to be a powerful technique that performs image domain transfer and face transfer [] Previous studies have demonstrated that CycleGAN can improve deep learning models by generating training situations to learn better decision boundaries between classes We built the CycleGAN augmentation model to increase the Fig Workflow of building a deep learning model for pharyngitis diagnosis using a smartphone ComputersinBiologyandMedicine12520201039802 0cTK Yoo Fig Schematics of the CycleGAN model generating new normal images and pathologic throat images with pharyngitis generalizability of the dataset and to improve the classification performance in the imbalanced dataset Before training the CycleGAN the throat images were augmented using linear transformation including left and right flip width and height translation from to random rotation from —¦ to —¦ zooming from to and random brightness change from to We defined these transformations as the basic augmentation step In this step normal throat and pharyngitis images were randomly sampled for the training set and normal throat and pharyngitis images were randomly sampled for the validation set Using the data with basic augmentation we trained the CycleGAN models to transform both normal to pharyngitis throat images and pharyngitis to normal images The trained CycleGAN model augmented the training set normal throat and pharyngitis images before augmentation and a total of throat images including normal and pharyngitis images were prepared to train the diagnostic classifier model after CycleGANbased augmentation It should be noted that supervised GAN techniques including conditional GAN and Pix2px were unable to be applied in this study because of a lack of paired normal and pharyngitis images To use a verified and predesigned image generator all the input images required resizing to a pixel resolution of — — which is the basic setup of a CycleGAN Therefore we used the default parameter settings that is the ADAM optimizer with a batch size of to optimize the GAN networks Development of CNN model We trained conventional deep learning models after data augmentation Because of a small image dataset in this study developing a custom deep learning method is challenging due to difficulty and time consuming eg a small training dataset can easily result in an over fitted model and low performance [] To overcome the problem of the small dataset transfer learning was widely used to train deep learning models using pretrained architectures [] This study also applied pretrained learning models to the classification task of throat images The last fully connected layer of the CNNs was only trained and the study used the pretrained conventional model as a feature extractor [] The CNN models including ResNet50 Inceptionv3 and MobileNet v2 were adopted to build binary classifiers [] These CNN models have been used successfully in many studies demonstrating stateoftheart performance with the saliency map [] The models were trained using the training set and the validation set was used to estimate how well the model had been trained We downloaded the CNN models which were pretrained on the ImageNet database and performed finetuning of the weights of the pretrained networks This process generally maintains the weights of some bottom layers to avoid overfitting and performs delicate modification of the highlevel features To use the images generated by CycleGAN the size of the input images for the deep learning models was set to a pixel resolution of — — and the images were resized for each pretrained model Most conventional deep learning models adopted a pixel resolution of — — or — — [] One deep learning research showed that the best performance was achieved at an image resolution of between — and — pixels for binary classification [] Therefore the resolution of our study was appropriate to detect pharyngitis in a binary decision The model was trained with epochs and a batch size of The ADAM optimizer with a learning rate of was also used with a crossentropy loss for all CNN models The crossentropy loss function is defined as Lcross entropy cid0ˆ‘Nipi logqi Where pi represents the ground truth value and qi represents predicted probability value from a classifier for the ith image The optimizer updated the network parameters to minimize the loss function In our experiments it tuned a fully connected layer of the CNN models For example the first layers of ResNet50 were left frozen and we trained the last fully connected layer using the training dataset which is described in Table the ADAM optimizer We chose the final classifier model which maximized the accuracy in the validation dataset To visualize the clusters to see if the classes are separable by a considerable margin the tdistributed stochastic neighbor embedding t SNE algorithm was executed using sampled instances The feature vectors from the last layer of the pretrained Inceptionv3 model were extracted to train the tSNE [] As there is a growing demand for explainable artificial intelligence methods in medicine [] we adopted githubcomjacobgilpytorch the GradCAM technique ComputersinBiologyandMedicine12520201039803 0cTK Yoo Table Throat image dataset and augmentation used in this study Number of validation set Number of training set Raw data Basic augmentation GANbased image synthesis augmentation Class Normal Pharyngitis Normal Pharyngitis Normal Pharyngitis Number of test set GAN generative adversarial network gradcam to generate the attention map [] The GradCAM visualizes the decisional areas of the CNN model using the gradients of any target flowing into the final convolutional network The heatmap has a low resolution and it was upsampled via bicubic interpolation Finally it produces heatmaps that highlight the area of interest and interprets the decision of the deep learning models The performance of the CNN models was evaluated based on the accuracy and area under the curve AUC of the receiver operating characteristic curve ROC and the precisionrecall curve PRC The Youden index which is an estimate of the optimal diagnostic threshold was adopted in this study [] After obtaining the sensitivity and specificity the Youden index was calculated at each cutoff point We selected the optimal value which maximized the Youden index These performance indexes are expressed as follows Accuracy TP TNTP TN FP FN Sensitivity Recall TPTP FNSpecificity Precision TNTN FPTPTP FP Youden index Sensitivity Specificity cid0 Where TP TN FP and FN denote true positives true negatives false positives and false negatives respectively We also performed a 4fold crossvalidation using the entire dataset to evaluate a generalized performance Google Colab Pro a cloud service for disseminating the deep learning research was adopted to implement the CycleGAN and CNN models [] Google Colab Pro provides a development environment using the Tensorflowbased deep learning libraries and a robust graphic processing unit GPU This enables the rapid processing of a heavy deep learning network without the need for a personal GPU [] The available hardware for each virtual machine varied by session but typically included top products of NVIDIA GPUs K80 T4 or P100 around “ GB of RAM and “ GB of free space on the virtual machine hard drive [] We used the Colab CycleGAN tutorial page to develop and validate the CycleGAN model and all of the code is available on the Tensorflow webpage wwwtensorflowtutorialsgene rativecyclegan We only modified the input pipeline to import our dataset The code of the CycleGAN and CNN models is presented in Supplementary Material Results We developed a deep learning model using GANbased augmentation in the challenging context of pharyngitis detection To build a balanced training dataset the CycleGAN models generated normal and pathologic throat images using the initial training dataset The color intensity distributions of the pharyngitis and normal throat images were not significantly different although most throat images had exudate regions Fig A The tSNE algorithm demonstrates that both groups are clustered and they are separable by a considerable margin Fig B The final CycleGAN model for the pharyngitis throat image was trained for epochs which required h After training normal throat images were translated into pathologic images and throat images with pharyngitis were translated into normal images Finally we constructed a balanced augmented training dataset including normal and pharyngitis images using CycleGAN The CycleGANbased synthetic images were realistic and reflected the characteristic features of pharyngitis Fig A The CycleGAN model synthesized white or gray patches and increased the redness on the throat wall and tonsils from normal images Fig B Generated throat images were reviewed by three clinicians including an otolaryngologist and an anesthesiologist All pharyngitis images generated were deemed by the three clinicians to show œmore pathologic and inflammatory results when compared to the original images This feature generation based on the CycleGAN model can be effective for generating a new sample to increase the intra class variation and generalizability The CNN models were trained using the final augmented dataset via the transfer learning technique Fig demonstrates the training process of the ResNet50 CNN model using the training and validation sets The training process for CNN took approximately h for epochs with finetuning for each training step After the 200th epoch the validation accuracy was not improved and the crossentropy of the validation result increased Therefore we considered that training for epochs was optimal in the ResNet50 model and selected the trained model at the 200th epoch Fig A shows the training validation and test datasets of CNN models with and without CycleGANbased augmentation The ROCAUCs of ResNet50 Inceptionv3 and MobileNetv2 without GAN based augmentation were and respectively Fig B We obtained the best ROCAUC of pharyngitis detection using ResNet50 with GANbased augmentation corresponding to Fig C At the optimal diagnostic cutoff value the ResNet50 model predicted pharyngitis with an accuracy of sensitivity of and specificity of The ROCAUC of Inceptionv3 and MobileNetv2 corresponded to and respectively The models with GAN based augmentation demonstrated superior performance in comparison with the models with only basic augmentation We also evaluated the performance of the models using PRCs Fig The PRCAUC also demonstrated that deep learning models with GANbased augmentation had better performance than those without GANbased augmentation ResNet50 with GANbased augmentation predicted pharyngitis with the highest PRCAUC of When a custom deep learning network without the transfer learning technique was trained the validation accuracy was lower than and was not improved during the training epochs Supplementary materials Table shows the performance of CNN models via 4fold cross validation in the entire dataset A similar tendency is observed for the average accuracy and AUC values in the crossvalidation The result shows that the highest detection accuracy and ROCAUC achieved were and respectively by using the ResNet50 Other CNN models showed lower performance than the ResNet50 with GANbased augmentation but there were no significant differences between CNN models in the 4fold crossvalidation A saliency map using the Grad CAM technique was generated to visualize the characteristic pathologic features of pharyngitis for the predicted evidence Fig A In normal throat images however some regions were highlighted Fig B When synthesized images were tested using the trained model synthesized exudates were highlighted correctly in the images as shown in Fig C Furthermore external clinical cases from previous literature were analyzed to investigate to show the capability of detecting pharyngitis of the framework developed in this study [] The representative cases with severe pharyngitis are shown in Fig One case presents an ComputersinBiologyandMedicine12520201039804 0cTK Yoo Fig Visual data exploration of pharyngitis and normal throat images A The mean redgreenblue RGB distributions B The feature space visualized using the tSNE technique For interpretation of the references to color in this figure legend the reader is referred to the Web version of this article Fig Data augmentation using CycleGAN to improve the diagnostic performance of pharyngitis A CycleGANbased augmentation for imbalanced data B Examples of pathologic throat images with pharyngitis generated by the CycleGAN Fig Training process of the ResNet50 CNN model for pharyngitis detection A Accuracy learning curves of the training and validation sets B Loss learning curves of the training and validation sets ComputersinBiologyandMedicine12520201039805 0cTK Yoo Fig Performance of pharyngitis detection deep learning models A Schematics of basic augmentation and GANbased augmentation B The receiver operating characteristic curves of deep learning models using basic augmentation C The receiver operating characteristic curves of deep learning models using GANbased augmentation Fig Precisionrecall curves of pharyngitis detection deep learning models A The deep learning models using basic augmentation B The deep learning models using GANbased augmentation Table Classification performance for severe pharyngitis detection in the 4fold crossvalidation using the developmental set AUC CI Accuracy CI Sensitivity CI Specificity CI Pvalue “ “ “ Basic augmentation ResNet50 Inceptionv3 MobileNetv2 GANbased image synthesis augmentation ResNet50 Inceptionv3 MobileNetv2 AUC area under the receiver operating characteristic curve CI confidence interval GAN generative adversarial network Comparison of receiver operating characteristics curves with the single best technique ResNet50 with GANbased image synthesis augmentation according to the Delong test “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ Reference 81yearold man with odynophagia Fig A and the other case presents a 41yearold diabetic man with throat pain Fig B The ResNet50 model was able to detect pharyngitis in both cases The GradCAM technique highlighted white patches on the throat wall as markers of severe pharyngitis in the deep learning model Discussion The current proofofconcept study investigated the possibility of deep learning using a smartphone for detecting pharyngitis A recentstudy demonstrated the ability of selftaken throat images to detect pharyngitis based on the knearest neighbor algorithm in a color space [] That study used additional equipment as well as a ComputersinBiologyandMedicine12520201039806 0cTK Yoo Fig Example of deep learning classification results with a saliency map using the GradCAM technique A Pharyngitis throat images B Normal throat images C Synthesized images smartphone to obtain throat images We utilized selftaken throat images without additional equipment using deep learning techniques for the detection of pharyngitis We showed that the trained GAN models were able to generate new realistic synthetic throat images which can improve the diagnostic accuracy The final deep learning model achieves high accuracy for automated diagnosis of pharyngitis using smartphone images Therefore this framework could be targeted toward patients with a sore throat who need screening for severe pharyngitis We believe that our study could be extended to computeraided diagnosis using images from an endoscope system in otolaryngology clinics similar to what has been done with colonoscope images using a deep learning model [] To the best of our knowledge no study has been performed to detect pharyngitis based on deep learning using smartphone images However it should be noted that this study is considered as only a preliminary and proofofconcept study because of its technical limitations due to using only Google Colab The current study framework is similar to that of Chamier which showed a deep learning framework using Google Drive and training and prediction on Google Colab [] Our proposed conceptual workflow is shown in Fig It needs the Flask servers and interfaces implemented using HTML and JavaScript to be applied in a real clinical setting This work could be part of a larger project to enable smartphonebased pharyngitis detection via a cloudbased applevel mobile data analysis We believe that a future appbased model can provide a robust solution for the costeffective and convenient screening of pharyngitis in a telemedicine setting We have highlighted the feasibility of a smartphonebased approach with deep learning to detect pharyngitis Our approach does not require Fig Classification results from the deep learning model applied to clinical cases with severe pharyngitis A An 81yearold man with odynophagia [] B A 41yearold diabetic man with throat pain [] Fig Example of a proposed smartphonebased system for pharyngitis detection ComputersinBiologyandMedicine12520201039807 0cexplored clinical datasets and webbased datasets and showed that the labeling of webbased images is often uncertain [] Therefore further clinical datasets with validation will be required to confirm the effectiveness of our framework Fourth because we collected only throat images via image search engines there was no metadata including gender race season or age According to a previous epidemiologic study these factors could affect the pharyngitis detection performance [] Fifth although the datasets were reviewed by authors images could be potentially duplicated in the dataset The duplicated images would affect the independence of the validation dataset We have shown the feasibility of deep learning for the detection of tonsil swelling and exudates in throat images The limitations of our study should be overcome by the availability of a sufficient number of throat images taken by a smartphone with a wellanized study protocol To validate the effectiveness of our framework a prospective study with many patients should be performed in a clinic once the app based framework is developed This will solve the problem of possible duplicated images in the dataset and the absence of metadata Conclusion TK Yoo additional equipment to collect throat images and we collected self taken images using a smartphone The use of webbased image capture of users from various devices and GANbased image augmentation may allow robust image processing While in the current study the implementation was performed on a desktop computer and the Colab™s remote server a smartphone application will also be possible to perform this identification for detecting pharyngitis The light deep learning models such as MobileNet can be executed on a smartphone without transmitting the images to a server [] Our result demonstrates that MobileNetv2 also has a high diagnostic performance similar to ResNet50 and Inceptionv3 In future research an increased amount of training data is needed to improve the detection accuracy using light models The utility of deep learning and smartphones may facilitate the widespread adoption of telemedicine and physical examination platforms via our approach Furthermore the presented framework enabled using a smartphone camera and deep learning techniques will help the patients in selfscreening for severe pharyngitis and may accelerate diagnostic support in remote healthcare services Because of the recent pandemic of COVID19 patients with respiratory symptoms need selfmonitoring to evaluate their pathologic status [] Advances in technology will require clinicians to embrace remote healthcare services The framework presented in this paper was designed for the timely diagnosis of pharyngitis for treatment Similar smartphoneimagebased diagnostic approaches have been introduced in several other medical image domains including skin diseases [] hematologic diseases [] oral diseases [] and eye diseases [] The main concern of deep learning models in this study Answer:

Thyroid_Cancer

"Immigrant statusfamily relationsACP contemplationACP discussionburial planninga b s t r a c tObjectives To examine how immigrant status and family relationships are associated with advance careplanning ACP engagement and endoflife EOL preference in burial planning among older ChineseAmericans the largest subgroup of Asian AmericansDesign Crosssectional surveySetting Communities in Honolulu Hawai™iParticipants Participants were older Chinese Americans aged years and olderMeasures Measures included ACP contemplation ACP discussion and EOL preference in burial planningimmigrant status family cohesion family conflict demographic information and health statusResults Results show that in comparison to foreignborn Chinese Americans USborn Chinese Americanswere more likely to have ACP contemplation [odds ratio OR confidence interval CI ] ACP discussion OR CI and preferences for burial plans at the end of life OR CI Family conflict increased the possibility of having ACP contemplation OR CI ACP discussion OR CI and EOL preference in burial planning OR CI whereas family cohesion was not associated with these study outcomesConclusions and Implications This study suggests that ACP should be adapted to be more culturallyappropriate especially in a time of coronavirus and xenophobia such as framing ACP as a tool to helpfamilies reduce stress while fulfilling filial obligations in order to ensure equitable access to ACP“ AMDA e The Society for PostAcute and LongTerm Care MedicineAdvance care planning ACP is a process of understanding andcommunicating individuals™ values goals and preferences regardingendoflife EOL care12 Contemplation of individuals™ EOL wishes anddiscussions with families can be as important as discussions withphysicians and completion of an advance directive in guiding care34ACP is a social process built on relationships and alleviation ofburden on others a means to prepare for death and a measure toexercise the ethical principle of patient autonomy5 Burial planningcan ensure individuals™ wishes are executed and relieve the burden ofloved ones to determine what the deceased would have wantedduring the time of grief In this sense burial planning is an importantThis study was supported by a research grant from the Rory Meyers College ofNursing at New York UniversityThe authors declare no conflicts of interest Address correspondence to Bei Wu PhD Rory Meyers College of Nursing NewYork University First Avenue Room New York NY USAEmail address beiwunyuedu B Wu101016jjamda20200604015258610“ AMDA e The Society for PostAcute and LongTerm Care Medicineelement of ACP6 Therefore it makes sense to examine ACP contemplation ACP discussion with family and EOL preference in burialplanning togetherACP can improve quality of EOL care for individuals including lessinhospital death and increased hospice use7 Despite the benefits ofACP the participation rate of ACP remains low especially among olderadults of racial and ethnic minorities Studies found that in the UnitedStates Blacks and Hispanics are less likely to have an EOL discussion adurable power of attorney and an advance directive than their Whitecounterparts89 but there is a lack of knowledge on ACP engagementamong Chinese Americans the largest subgroup of Asian Americansand the fastestgrowing minority group in the USA10Compared with nativeborn Chinese AmericansforeignbornChinese Americans may face more cultural and logistical challengesin ACP engagement because of their limited English proficiencygreater cultural burden in discussing death and dying and acceptingindividual autonomy and lack of ACP knowledge1112 In addition theeffectiveness of ACP may rely on the involvement knowledge and 0cY Pei JAMDA xxx 1e4cooperation of family members13 however because of the lack of richand comprehensive measures of family relationships in previousresearch on ACP few studies have examined the extent to whichfamily relationships ‚uence individuals™ ACP engagement To fill thisknowledge gap this study aimed to examine how immigrant statusand family relationships are associated with ACP contemplation ACPdiscussion with family and preference in burial planning among olderChinese AmericansMethodsDataData were derived from a survey conducted in Honolulu Hawai™iwhere approximately of the total population is composed ofChinese Americans and of the adult population are immigrants14We used snowball sampling and convenience sampling to identify andrecruit key informants from local Chinese groups social anizationsbusinesses and faithbased agencies based on their capacity ofaccessing Chinese communities and their willingness to assist inrecruiting Chinese older adults in the community We collaboratedwith key community leaders This is a common and effective strategyto recruit respondents from minority populations15 as random sampling is challenging because of the unfeasibility of constructing acompleted sampling frame cultural appropriatenesstime andexpense16 The inclusion criteria for the survey participants includedHonolulu residents aged years and older who selfidentified asChinese The detailed recruitment and data collection methods werereported in previous studies17 The participants provided informedconsent prior to the data collection This study was approved by theinstitutional review board at the university with which the secondauthor was affiliated A total of participants were recruited fromJanuary to September MeasuresDependent variables ACP engagement and EOL preference in burialplanningACP engagement includes ACP contemplation and ACP discussionACP contemplation and ACP discussion was assessed by asking respondents if they previously had thought about their endoflifecare plan with family and had discussed the plan with familyrespectivelyEOL preference in burial planning was measured by a hypothesizedquestion Respondents were asked whether formulating a burial planwas one of the most important things for them to consider if theywere diagnosed with a terminal illness and only had months to liveamong several other options Other mentioned options includedhaving religious beliefssupport alleviating pain reducing care andfinancial burden on family and extending their lifeIndependent variablesImmigrant status was measured by asking respondents whetherthey were US or foreignbornFamily relationships were measured by reliable and valid existingscalesdfamily cohesion and family conflict The index of familycohesion was assessed by asking respondents whether familymembers like to spend free time with each other family membersfeel very close to each other and family togetherness is veryimportant18Family conflict was measured using the 5item Family CulturalConflict scale which assesses cultural and intergenerational conflictperceived by respondents in their family19CovariatesSociodemographic variables included gender age marital statuseducation financial strain living arrangement and social activityparticipation Health need factors included selfrated health comorbidity a continuous variable that examines the existence of at least chronic conditions including heart diseases stroke cancer diabeteshypertension high cholesterol thyroid disease arthritis liverrelateddiseases and others disabilities in activities of daily living andpsychological distress Psychological distress was assessed by theKessler Psychological Distress Scale K1020AnalysisFirst we summarized the sample characteristics Then we usedlogistic regression models and calculated odds ratios ORs to testwhether immigrant status family cohesion and family conflict wereassociated with ACP engagement and EOL preferences All the analyses were conducted using Stata version The missing rates for ACP contemplation ACP discussion and EOLpreferences in burial planning were and respectively Toreduce sampling errors and attain more stable analytical results weconducted multiple imputations MIs for each model All thedependent variables were imputed and the imputed values wereretained in the analysis We used imputed data sets as there werehigh levels of missingness on the dependent variables21 For sensitivity analysis a dependent variable was imputed and imputed valueswere deleted for analysis MID The MID method produced ORs thatwere almost identical to those in the model where the imputed valueswere retainedResultsTable summarizes sample characteristics It shows that less thanhalf of the participants had ACP contemplation and ACP discussion Only had EOL preference in burial planning inthe hypothesized situationTable shows ORs with confidence intervals CIs from logisticregressions The USborn Chinese Americans were more likely to haveACP contemplation OR CI ACP discussion OR CI and preference in burial planning OR CI than the foreignborn Higher levels of familyconflict were associated with higher likelihood of ACP contemplationOR CI ACP discussion OR CI and preference in burial planning OR CI whereasfamily cohesion was not significantly related to these outcomesDiscussionThis study aimed to examine the roles of immigrant status andfamily relationships in the associations between ACP engagement andgiving EOL preferences to burial planning among older ChineseAmericans The USborn Chinese Americans were more likely to haveACP contemplation and ACP discussion than the foreignborn Thismay be because the foreignborn Chinese Americans have lower socioeconomic status less English proficiency lower levels of acculturation and less knowledge about ACP and the US healthcare systemthan their USborn counterparts111222 In addition these individuallevel differences may be mixed with other systemlevel barrierswithin the US healthcare system to worsen the disparities in ACPengagement23 For example Chinese American immigrants may havea stronger belief that family and society are held in higher regard thanindividuals and attribute a higher value to collectivism of family andsociety rather than patient autonomy in EOL decision making12Moreover because traditional Chinese culture expects children tocarry the role of protecting their parents™ health safety and generalwellbeing many Chinese children may construe this responsibility as 0cTable Characteristics of the Study Sample of Chinese Americans N ¼ or Mean SDCodingY Pei JAMDA xxx 1e4ACP engagementACP contemplationACP discussionEOL preferences in burial planningUSbornFamily RelationshipsFamily cohesionFamily conflictFemaleAgeMarriedEducationFinancial strainLiving aloneParticipation in social activitiesSelfrated health as excellentgoodNumber of chronic diseaseADL disabilityPsychological distressADL activities of daily living never and don™t want tonever but want toreluctant to yes never and don™t want tonever but want toreluctant to yes no yes no yes least cohesivee12 most cohesive least cultural conflicte10 most cultural conflict male female unmarried married not at alle3 a great deal no yes no yes no yes no help needed needs help least distressfule5 most distressfulmaking every effort to prolong their older parents™ life which maysometimes be in opposition to their parents™ own wishes24 Thesepotential factors surrounding older Chinese immigrants may helpexplain this population™s lack of engagement in ACP Healthcare providers in turn should pay closer attention to these factors in order tothoroughly evaluate patients™ EOL wishes It is noted that the USbornChinese Americans were far more likely to have preferences in burialplanning than the foreignborn The finding is consistent with a previous study in that decisions such as EOL care and funeral and burialpreplanning are impacted by similar factors25 Indeed EOL care decision making and burial planning are integrated processes at theend of life26 and burial plan is included in some advance directivedocuments in practice Future studies on ACP need to consider burialplanningSecond family cohesion was not associated with ACP contemplation ACP discussion and EOL preference in burial planning whereasfamily conflict increased the possibility of ACP contemplation ACPdiscussion and EOL preferences in burial planning The finding isinconsistent with previous study conducted among White olderadults revealing that the positive family relationship encourageswhereas problematic family relationship hinders ACP engagement27The inconsistency is likely due to the fact that Chinese Americansvalue family in the process of EOL decision making2829 The lack ofassociation between family cohesion and ACP engagement may bebecause older adults with higher levels of family cohesion have tobalance between the potential benefit and harm of ACP engagementOn the hand older Chinese Americans may have positive attitudesabout ACP engagement and believe that ACP engagement is importantand necessary because it allows them to witness their loved ones™death and dying experience12 On the other hand closeknit familialrelationships may make both older Chinese Americans and theirfamilies feel more uncomfortable to start a conversation on EOL carebecause discussions about death and dying are often considered ataboo in Chinese culture30 In this sense strong family ties may havelimited impact on ACP engagement An explanation for the significantrelationship between family conflict and ACP engagement could bethat higher levels of family conflict may indicate a greater need for ACPengagement This is because the members in these families are lesslikely to know about the EOL care preferences of older adults and betrusted in the EOL decision making13 These findings suggest thatculture may play an important role in the complex association between family relationships and ACP engagementSeveral limitations of the study deserve mentioning First thecrosssectional data from a small region limit our ability to generalizefindings to older Chinese Americans living in other parts of the UnitedStates as well as to make causalinferences Second the ACPengagement in our study only included ACP contemplation ACP discussion and preference in burial planning Future studies need toinclude more ACP options such as the completion of living wills oradvance directives and the selection of a durable power of attorneyfor health care to understand more about ACP engagement in ChineseAmerican families Third ACP knowledge is an important confoundingvariable for both immigrant status and ACP engagement Futurestudies on ACP engagement need to consider this variableConclusions and ImplicationsDespite these limitations this study sheds light on how immigrantstatus and family relationships shape ACP engagement among olderChinese AmericansIt is found that immigrant status decreaseswhereas family conflict increases the likelihood of having ACPcontemplation ACP discussion and preference in burial planningTable Factors Associated With Advance Care Planning Engagement Results of Logistic Regression N ¼ USborn ref ¼ foreignbornFamily relationshipsFamily cohesionFamily conflictACP ContemplationOR CI ACP DiscussionOR CI EOL Preferences inBurial Planning OR CI All models adjusted for age gender marital status education financial strain living alone social activity participation selfrated health number of chronic disease activitiesof daily living and psychological distress 0cY Pei JAMDA xxx 1e4Health care providers may consider patients™ immigrant status andfamily relationships to better serve ethnically diverse populationsGiven that cultural factors play an important role in ACP engagementACP should be adapted to be more culturally appropriate amongChinese Americans especially in a time of coronavirus and xenophobia such as framing ACP as a tool to help families reduce stresswhile fulfilling filial obligations in order to ensure equitable access toACPAcknowledgmentWe thank Katherine Wang for her editorial assistance We wouldalso like to thank the research team at the University of Hawaii fortheir data collectionReferences Rietjens JAC Sudore RL Connolly M Definition and recommendations foradvance care planning An international consensus supported by the EuropeanAssociation for Palliative Care Lancet Oncol 201718e543ee551 Sudore RL Lum HD You JJ Defining advance care planning for adults Aconsensus definition from a multidisciplinary Delphi panel J Pain SymptomManage 201753821e832e821 Sudore RL Schickedanz AD Landefeld CS Engagement in multiple steps ofthe advance care planning process A descriptive study of diverse older adultsJ Am Geriatr Soc 2008561006e1013 Sudore RL Knight SJ McMahan RD A novel website to prepare diverseolder adults for decision making and advance care planning A pilot studyJ Pain Symptom Manage 201447674e686 Dahlin C Giansiracusa D Communication in palliative care In Ferrell BCoyle N editors Textbook of Palliative Nursing New York NY Oxford University Press p 67e96 KataokaYahiro MR Conde FA Wong RS Advance care planning amongAsian Americans and Native Hawaiians receiving haemodialysis Int J PalliatNurs 20101632e40 Bischoff KE Sudore R Miao Y Advance care planning and the quality ofendoflife care in older adults J Am Geriatr Soc 201361209e214 Kale MS Ornstein KA Smith CB Kelley AS Endoflife discussions with olderadults J Am Geriatr Soc 2016641962e1967 Harrison KL Adrion ER Ritchie CS Low completion and disparities inadvance care planning activities among older Medicare beneficiaries JAMAIntern Med 20161761872e1875 Pew Research Center Key facts about Asian Americans a diverse and growingpopulation Available at wwwpewresearchfacttank20170908keyfactsaboutasianamericans Accessed November Gao X Sun F Ko E Knowledge of advance directive and perceptions ofendoflife care in ChineseAmerican elders The role of acculturation PalliatSupport Care 2015131677e1684 Lee MC Byon HD Hinderer K Alexander C Beliefs in advance care planningamong Chinese Americans Similarities and differences between the youngerand older generations Asian Pac Isl Nurs J 2017283e90 Parks SM Winter L Santana AJ Family factors in endoflife decisionJ Palliat Med making Family conflict and proxy relationship179e184 Tong M Sentell T Insights in public health Challenges investigating healthoutcomes in Chinese Americans using populationbased survey data Hawaii JMed Public Health Ibrahim S Sidani S Strategies to recruit minority persons A systematic reviewJ Immigr Minor Health 20145882e888 Spring M Westermeyer J Halcon L Sampling in difficult to access refugeeand immigrant communities J Nerv Ment Dis 2003191813e819 Zhang W Liu S Zhang K Wu B Neighborhood social cohesion resilience andpsychological wellbeing among Chinese older adults in Hawai™i Gerontologist201960229e238 Rivera FI Guarnaccia PJ MulvaneyDay N Family cohesion and its relationship to psychological distress among Latino groups Hisp J Behav Sci 30357e378 Alegria M Vila D Woo M Cultural relevance and equivalence in theNLAAS instrument Integrating etic and emic in the development of crosscultural measures for a psychiatric epidemiology and services study of LatinosInt J Methods Psychiatr Res 200413270e288 Kessler RC Andrews G Colpe LJ Short screening scales to monitor population prevalences and trends in nonspecific psychological distress PsycholMed 200232959e976 Johnson DR Young R Toward best practices in analyzing datasets withmissing data Comparisons and recommendations J Marriage Fam 926e945 Carr D The social stratification of older adults™ preparations for endoflifehealth care J Health Soc Behav 201253297e312 Shen MJ Prigerson HG Tergas AI Maciejewski PK Impact of immigrant statuson aggressive medical care counter to patients™ values near death amongadvanced cancer patients J Palliat Med 20192234e40 Bowman K Singer P Chinese seniors™ perspectives on endoflife decisions SocSci Med 200153455e464 Kelly CM Masters JL DeViney S Endoflife planning activities An integratedprocess Death Stud 201337529e551 Tanaka M Takahashi M Kawashima D Endoflife activities amongin Japan Omega Westport communitydwelling older adults Carr D Moorman SM Boerner K Endoflife planning in a family context Doesrelationship quality affect whether and with whom older adults planJ Gerontol B Psychol Sci Soc Sci 201368586e592 Hinderer KA Lee MC Chinese Americans™ attitudes toward advance directivesAn assessment of outcomes based on a nursingled intervention Appl Nurs Res20194991e96 YonashiroCho J Cote S Enguidanos S Knowledge about and perceptions ofadvance care planning and communication of ChineseAmerican older adultsJ Am Geriatr Soc 2016641884e1889 Chi HL Cataldo J Ho EY Rehm RS Can we talk about it now Recognizing theoptimal time to initiate endoflife care discussions with older ChineseAmericans and their families J Transcult Nurs 201829532e539 0c"

cancer248

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: "Immigrant statusfamily relationsACP contemplationACP discussionburial planninga b s t r a c tObjectives To examine how immigrant status and family relationships are associated with advance careplanning ACP engagement and endoflife EOL preference in burial planning among older ChineseAmericans the largest subgroup of Asian AmericansDesign Crosssectional surveySetting Communities in Honolulu Hawai™iParticipants Participants were older Chinese Americans aged years and olderMeasures Measures included ACP contemplation ACP discussion and EOL preference in burial planningimmigrant status family cohesion family conflict demographic information and health statusResults Results show that in comparison to foreignborn Chinese Americans USborn Chinese Americanswere more likely to have ACP contemplation [odds ratio OR confidence interval CI ] ACP discussion OR CI and preferences for burial plans at the end of life OR CI Family conflict increased the possibility of having ACP contemplation OR CI ACP discussion OR CI and EOL preference in burial planning OR CI whereas family cohesion was not associated with these study outcomesConclusions and Implications This study suggests that ACP should be adapted to be more culturallyappropriate especially in a time of coronavirus and xenophobia such as framing ACP as a tool to helpfamilies reduce stress while fulfilling filial obligations in order to ensure equitable access to ACP“ AMDA e The Society for PostAcute and LongTerm Care MedicineAdvance care planning ACP is a process of understanding andcommunicating individuals™ values goals and preferences regardingendoflife EOL care12 Contemplation of individuals™ EOL wishes anddiscussions with families can be as important as discussions withphysicians and completion of an advance directive in guiding care34ACP is a social process built on relationships and alleviation ofburden on others a means to prepare for death and a measure toexercise the ethical principle of patient autonomy5 Burial planningcan ensure individuals™ wishes are executed and relieve the burden ofloved ones to determine what the deceased would have wantedduring the time of grief In this sense burial planning is an importantThis study was supported by a research grant from the Rory Meyers College ofNursing at New York UniversityThe authors declare no conflicts of interest Address correspondence to Bei Wu PhD Rory Meyers College of Nursing NewYork University First Avenue Room New York NY USAEmail address beiwunyuedu B Wu101016jjamda20200604015258610“ AMDA e The Society for PostAcute and LongTerm Care Medicineelement of ACP6 Therefore it makes sense to examine ACP contemplation ACP discussion with family and EOL preference in burialplanning togetherACP can improve quality of EOL care for individuals including lessinhospital death and increased hospice use7 Despite the benefits ofACP the participation rate of ACP remains low especially among olderadults of racial and ethnic minorities Studies found that in the UnitedStates Blacks and Hispanics are less likely to have an EOL discussion adurable power of attorney and an advance directive than their Whitecounterparts89 but there is a lack of knowledge on ACP engagementamong Chinese Americans the largest subgroup of Asian Americansand the fastestgrowing minority group in the USA10Compared with nativeborn Chinese AmericansforeignbornChinese Americans may face more cultural and logistical challengesin ACP engagement because of their limited English proficiencygreater cultural burden in discussing death and dying and acceptingindividual autonomy and lack of ACP knowledge1112 In addition theeffectiveness of ACP may rely on the involvement knowledge and 0cY Pei JAMDA xxx 1e4cooperation of family members13 however because of the lack of richand comprehensive measures of family relationships in previousresearch on ACP few studies have examined the extent to whichfamily relationships ‚uence individuals™ ACP engagement To fill thisknowledge gap this study aimed to examine how immigrant statusand family relationships are associated with ACP contemplation ACPdiscussion with family and preference in burial planning among olderChinese AmericansMethodsDataData were derived from a survey conducted in Honolulu Hawai™iwhere approximately of the total population is composed ofChinese Americans and of the adult population are immigrants14We used snowball sampling and convenience sampling to identify andrecruit key informants from local Chinese groups social anizationsbusinesses and faithbased agencies based on their capacity ofaccessing Chinese communities and their willingness to assist inrecruiting Chinese older adults in the community We collaboratedwith key community leaders This is a common and effective strategyto recruit respondents from minority populations15 as random sampling is challenging because of the unfeasibility of constructing acompleted sampling frame cultural appropriatenesstime andexpense16 The inclusion criteria for the survey participants includedHonolulu residents aged years and older who selfidentified asChinese The detailed recruitment and data collection methods werereported in previous studies17 The participants provided informedconsent prior to the data collection This study was approved by theinstitutional review board at the university with which the secondauthor was affiliated A total of participants were recruited fromJanuary to September MeasuresDependent variables ACP engagement and EOL preference in burialplanningACP engagement includes ACP contemplation and ACP discussionACP contemplation and ACP discussion was assessed by asking respondents if they previously had thought about their endoflifecare plan with family and had discussed the plan with familyrespectivelyEOL preference in burial planning was measured by a hypothesizedquestion Respondents were asked whether formulating a burial planwas one of the most important things for them to consider if theywere diagnosed with a terminal illness and only had months to liveamong several other options Other mentioned options includedhaving religious beliefssupport alleviating pain reducing care andfinancial burden on family and extending their lifeIndependent variablesImmigrant status was measured by asking respondents whetherthey were US or foreignbornFamily relationships were measured by reliable and valid existingscalesdfamily cohesion and family conflict The index of familycohesion was assessed by asking respondents whether familymembers like to spend free time with each other family membersfeel very close to each other and family togetherness is veryimportant18Family conflict was measured using the 5item Family CulturalConflict scale which assesses cultural and intergenerational conflictperceived by respondents in their family19CovariatesSociodemographic variables included gender age marital statuseducation financial strain living arrangement and social activityparticipation Health need factors included selfrated health comorbidity a continuous variable that examines the existence of at least chronic conditions including heart diseases stroke cancer diabeteshypertension high cholesterol thyroid disease arthritis liverrelateddiseases and others disabilities in activities of daily living andpsychological distress Psychological distress was assessed by theKessler Psychological Distress Scale K1020AnalysisFirst we summarized the sample characteristics Then we usedlogistic regression models and calculated odds ratios ORs to testwhether immigrant status family cohesion and family conflict wereassociated with ACP engagement and EOL preferences All the analyses were conducted using Stata version The missing rates for ACP contemplation ACP discussion and EOLpreferences in burial planning were and respectively Toreduce sampling errors and attain more stable analytical results weconducted multiple imputations MIs for each model All thedependent variables were imputed and the imputed values wereretained in the analysis We used imputed data sets as there werehigh levels of missingness on the dependent variables21 For sensitivity analysis a dependent variable was imputed and imputed valueswere deleted for analysis MID The MID method produced ORs thatwere almost identical to those in the model where the imputed valueswere retainedResultsTable summarizes sample characteristics It shows that less thanhalf of the participants had ACP contemplation and ACP discussion Only had EOL preference in burial planning inthe hypothesized situationTable shows ORs with confidence intervals CIs from logisticregressions The USborn Chinese Americans were more likely to haveACP contemplation OR CI ACP discussion OR CI and preference in burial planning OR CI than the foreignborn Higher levels of familyconflict were associated with higher likelihood of ACP contemplationOR CI ACP discussion OR CI and preference in burial planning OR CI whereasfamily cohesion was not significantly related to these outcomesDiscussionThis study aimed to examine the roles of immigrant status andfamily relationships in the associations between ACP engagement andgiving EOL preferences to burial planning among older ChineseAmericans The USborn Chinese Americans were more likely to haveACP contemplation and ACP discussion than the foreignborn Thismay be because the foreignborn Chinese Americans have lower socioeconomic status less English proficiency lower levels of acculturation and less knowledge about ACP and the US healthcare systemthan their USborn counterparts111222 In addition these individuallevel differences may be mixed with other systemlevel barrierswithin the US healthcare system to worsen the disparities in ACPengagement23 For example Chinese American immigrants may havea stronger belief that family and society are held in higher regard thanindividuals and attribute a higher value to collectivism of family andsociety rather than patient autonomy in EOL decision making12Moreover because traditional Chinese culture expects children tocarry the role of protecting their parents™ health safety and generalwellbeing many Chinese children may construe this responsibility as 0cTable Characteristics of the Study Sample of Chinese Americans N ¼ or Mean SDCodingY Pei JAMDA xxx 1e4ACP engagementACP contemplationACP discussionEOL preferences in burial planningUSbornFamily RelationshipsFamily cohesionFamily conflictFemaleAgeMarriedEducationFinancial strainLiving aloneParticipation in social activitiesSelfrated health as excellentgoodNumber of chronic diseaseADL disabilityPsychological distressADL activities of daily living never and don™t want tonever but want toreluctant to yes never and don™t want tonever but want toreluctant to yes no yes no yes least cohesivee12 most cohesive least cultural conflicte10 most cultural conflict male female unmarried married not at alle3 a great deal no yes no yes no yes no help needed needs help least distressfule5 most distressfulmaking every effort to prolong their older parents™ life which maysometimes be in opposition to their parents™ own wishes24 Thesepotential factors surrounding older Chinese immigrants may helpexplain this population™s lack of engagement in ACP Healthcare providers in turn should pay closer attention to these factors in order tothoroughly evaluate patients™ EOL wishes It is noted that the USbornChinese Americans were far more likely to have preferences in burialplanning than the foreignborn The finding is consistent with a previous study in that decisions such as EOL care and funeral and burialpreplanning are impacted by similar factors25 Indeed EOL care decision making and burial planning are integrated processes at theend of life26 and burial plan is included in some advance directivedocuments in practice Future studies on ACP need to consider burialplanningSecond family cohesion was not associated with ACP contemplation ACP discussion and EOL preference in burial planning whereasfamily conflict increased the possibility of ACP contemplation ACPdiscussion and EOL preferences in burial planning The finding isinconsistent with previous study conducted among White olderadults revealing that the positive family relationship encourageswhereas problematic family relationship hinders ACP engagement27The inconsistency is likely due to the fact that Chinese Americansvalue family in the process of EOL decision making2829 The lack ofassociation between family cohesion and ACP engagement may bebecause older adults with higher levels of family cohesion have tobalance between the potential benefit and harm of ACP engagementOn the hand older Chinese Americans may have positive attitudesabout ACP engagement and believe that ACP engagement is importantand necessary because it allows them to witness their loved ones™death and dying experience12 On the other hand closeknit familialrelationships may make both older Chinese Americans and theirfamilies feel more uncomfortable to start a conversation on EOL carebecause discussions about death and dying are often considered ataboo in Chinese culture30 In this sense strong family ties may havelimited impact on ACP engagement An explanation for the significantrelationship between family conflict and ACP engagement could bethat higher levels of family conflict may indicate a greater need for ACPengagement This is because the members in these families are lesslikely to know about the EOL care preferences of older adults and betrusted in the EOL decision making13 These findings suggest thatculture may play an important role in the complex association between family relationships and ACP engagementSeveral limitations of the study deserve mentioning First thecrosssectional data from a small region limit our ability to generalizefindings to older Chinese Americans living in other parts of the UnitedStates as well as to make causalinferences Second the ACPengagement in our study only included ACP contemplation ACP discussion and preference in burial planning Future studies need toinclude more ACP options such as the completion of living wills oradvance directives and the selection of a durable power of attorneyfor health care to understand more about ACP engagement in ChineseAmerican families Third ACP knowledge is an important confoundingvariable for both immigrant status and ACP engagement Futurestudies on ACP engagement need to consider this variableConclusions and ImplicationsDespite these limitations this study sheds light on how immigrantstatus and family relationships shape ACP engagement among olderChinese AmericansIt is found that immigrant status decreaseswhereas family conflict increases the likelihood of having ACPcontemplation ACP discussion and preference in burial planningTable Factors Associated With Advance Care Planning Engagement Results of Logistic Regression N ¼ USborn ref ¼ foreignbornFamily relationshipsFamily cohesionFamily conflictACP ContemplationOR CI ACP DiscussionOR CI EOL Preferences inBurial Planning OR CI All models adjusted for age gender marital status education financial strain living alone social activity participation selfrated health number of chronic disease activitiesof daily living and psychological distress 0cY Pei JAMDA xxx 1e4Health care providers may consider patients™ immigrant status andfamily relationships to better serve ethnically diverse populationsGiven that cultural factors play an important role in ACP engagementACP should be adapted to be more culturally appropriate amongChinese Americans especially in a time of coronavirus and xenophobia such as framing ACP as a tool to help families reduce stresswhile fulfilling filial obligations in order to ensure equitable access toACPAcknowledgmentWe thank Katherine Wang for her editorial assistance We wouldalso like to thank the research team at the University of Hawaii fortheir data collectionReferences Rietjens JAC Sudore RL Connolly M Definition and recommendations foradvance care planning An international consensus supported by the EuropeanAssociation for Palliative Care Lancet Oncol 201718e543ee551 Sudore RL Lum HD You JJ Defining advance care planning for adults Aconsensus definition from a multidisciplinary Delphi panel J Pain SymptomManage 201753821e832e821 Sudore RL Schickedanz AD Landefeld CS Engagement in multiple steps ofthe advance care planning process A descriptive study of diverse older adultsJ Am Geriatr Soc 2008561006e1013 Sudore RL Knight SJ McMahan RD A novel website to prepare diverseolder adults for decision making and advance care planning A pilot studyJ Pain Symptom Manage 201447674e686 Dahlin C Giansiracusa D Communication in palliative care In Ferrell BCoyle N editors Textbook of Palliative Nursing New York NY Oxford University Press p 67e96 KataokaYahiro MR Conde FA Wong RS Advance care planning amongAsian Americans and Native Hawaiians receiving haemodialysis Int J PalliatNurs 20101632e40 Bischoff KE Sudore R Miao Y Advance care planning and the quality ofendoflife care in older adults J Am Geriatr Soc 201361209e214 Kale MS Ornstein KA Smith CB Kelley AS Endoflife discussions with olderadults J Am Geriatr Soc 2016641962e1967 Harrison KL Adrion ER Ritchie CS Low completion and disparities inadvance care planning activities among older Medicare beneficiaries JAMAIntern Med 20161761872e1875 Pew Research Center Key facts about Asian Americans a diverse and growingpopulation Available at wwwpewresearchfacttank20170908keyfactsaboutasianamericans Accessed November Gao X Sun F Ko E Knowledge of advance directive and perceptions ofendoflife care in ChineseAmerican elders The role of acculturation PalliatSupport Care 2015131677e1684 Lee MC Byon HD Hinderer K Alexander C Beliefs in advance care planningamong Chinese Americans Similarities and differences between the youngerand older generations Asian Pac Isl Nurs J 2017283e90 Parks SM Winter L Santana AJ Family factors in endoflife decisionJ Palliat Med making Family conflict and proxy relationship179e184 Tong M Sentell T Insights in public health Challenges investigating healthoutcomes in Chinese Americans using populationbased survey data Hawaii JMed Public Health Ibrahim S Sidani S Strategies to recruit minority persons A systematic reviewJ Immigr Minor Health 20145882e888 Spring M Westermeyer J Halcon L Sampling in difficult to access refugeeand immigrant communities J Nerv Ment Dis 2003191813e819 Zhang W Liu S Zhang K Wu B Neighborhood social cohesion resilience andpsychological wellbeing among Chinese older adults in Hawai™i Gerontologist201960229e238 Rivera FI Guarnaccia PJ MulvaneyDay N Family cohesion and its relationship to psychological distress among Latino groups Hisp J Behav Sci 30357e378 Alegria M Vila D Woo M Cultural relevance and equivalence in theNLAAS instrument Integrating etic and emic in the development of crosscultural measures for a psychiatric epidemiology and services study of LatinosInt J Methods Psychiatr Res 200413270e288 Kessler RC Andrews G Colpe LJ Short screening scales to monitor population prevalences and trends in nonspecific psychological distress PsycholMed 200232959e976 Johnson DR Young R Toward best practices in analyzing datasets withmissing data Comparisons and recommendations J Marriage Fam 926e945 Carr D The social stratification of older adults™ preparations for endoflifehealth care J Health Soc Behav 201253297e312 Shen MJ Prigerson HG Tergas AI Maciejewski PK Impact of immigrant statuson aggressive medical care counter to patients™ values near death amongadvanced cancer patients J Palliat Med 20192234e40 Bowman K Singer P Chinese seniors™ perspectives on endoflife decisions SocSci Med 200153455e464 Kelly CM Masters JL DeViney S Endoflife planning activities An integratedprocess Death Stud 201337529e551 Tanaka M Takahashi M Kawashima D Endoflife activities amongin Japan Omega Westport communitydwelling older adults Carr D Moorman SM Boerner K Endoflife planning in a family context Doesrelationship quality affect whether and with whom older adults planJ Gerontol B Psychol Sci Soc Sci 201368586e592 Hinderer KA Lee MC Chinese Americans™ attitudes toward advance directivesAn assessment of outcomes based on a nursingled intervention Appl Nurs Res20194991e96 YonashiroCho J Cote S Enguidanos S Knowledge about and perceptions ofadvance care planning and communication of ChineseAmerican older adultsJ Am Geriatr Soc 2016641884e1889 Chi HL Cataldo J Ho EY Rehm RS Can we talk about it now Recognizing theoptimal time to initiate endoflife care discussions with older ChineseAmericans and their families J Transcult Nurs 201829532e539 0c" Answer:

Thyroid_Cancer

"fundamental influences of sex and gender as modifiers of the major causes of death and morbidity We articulate how the genetic epigenetic and hormonal influences of biological sex influence physiology and disease and how the social constructs of gender affect the behaviour of the community clinicians and patients in the healthcare system and interact with pathobiology We aim to guide clinicians and researchers to consider sex and gender in their approach to diagnosis prevention and treatment of diseases as a necessary and fundamental step towards precision medicine which will benefit men™s and women™s healthIntroductionWhat clinicians know about the diagnosis treatment and prevention of disease originates from studies mostly done on male cells male mice and men1 Historically for multiple reasons including the purported safety of women and their offspring women of childbearing age were excluded from clinical trials As a result medical research and care have been centred on male physiology The assumption was that male and female cells and animals were biologically identical and evidencebased medicine was defined by clinical trials done predominantly in men1 In the US National Institutes of Health NIH mandated the inclusion of women in NIHfunded clinical trials but many investigators did not follow this mandate and many of those who did include women did not analyse the results by sex23 minimising the effectiveness of this policy Preclinical research and drug development studies have also predominantly used male animal models and cells4“ It is not surprising that a US Government Accountability Office report found that eight of the ten prescription drugs withdrawn from the market between and œposed greater health risks for women than for men Most funding agencies from Europe and North America have implemented policies to support and mandate researchers to consider sex and gender at all levels of medical research8 Still the field of sexbased biology and medicine is often viewed as a specialised area of interest rather than a central consideration in medical research Essential for the success of clinical care and translational science is awareness by clinicians and researchers that the diseases they are treating and studying are characterised by differences between women and men in epidemiology pathophysiology clinical manifestations psychological effects disease progression and response to treatmentThis Review explores the role of sex biological constructs and gender social constructs as modifiers of the most common causes of death and morbidity and articulates the genetic biological and environmental determinants that underlie these differences We aim to guide clinicians and researchers to better understand and harness the importance of sex and gender as genetic wwwthelancetcom Vol August biological and environmental modifiers of chronic disease Ultimately it is a necessary and fundamental step towards precision medicine that will benefit women and menSex as a genetic modifier of biology and diseaseSex differences in disease prevalence manifestation and response to treatment are rooted in the genetic differences between men and women Genetic sex differences start at conception when the ovum fuses with a sperm cell carrying an X or a Y chromosome resulting in an embryo carrying either XX or XY chromosomes This fundamental difference in chromosome complement eg genes outside the testisdetermining SRY gene generates ubiquitous sex differences in the molecular makeup of all male and female cells9 First the Y chromosome carries genes that exhibit subtle functional differences from their Xlinked homologues eg ZFY vs ZFX and UTY vs UTX and also carries genes with no homologue at all eg SRY In addition in men the X chromosome carries only maternal imprints ”ie epigenetic modifications made by the parent in generating the sex cells”which alter the expression of genes in the offspring As women have X chromosomes from both parents they carry maternal and paternal imprints which target a different set of genes Random inactivation of one of the X chromosomes in female cells which prevents sex differences in X chromosome gene dosage causes another degree of sex difference in gene expression As some of these Xlinked genes escape inactivation in women those genes are often expressed at higher levels in women than in men9 Sexspecific gene expression due to genomic Search strategy and selection criteriaWe searched PubMed for papers published in English between Jan and June using œsex or œgender and the name of the disease of interest as search terms Although we tried to cite seminal studies when necessary because of space limitation representative reviews were often selectedLancet “Diabetes Discovery SexBased Medicine Laboratory Section of Endocrinology John W Deming Department of Medicine Tulane University School of Medicine and Southeast Louisiana Veterans Health Care System Medical Center New Orleans LA USA Prof F MauvaisJarvis MD Barbra Streisand Women™s Heart Center CedarsSinai Smidt Heart Institute Los Angeles CA USA Prof N Bairey Merz MD National Heart Lung Institute Imperial College London London UK Prof P J Barnes MD Department of Pharmacology and Department of Neurology College of Medicine Center for Innovation in Brain Science University of Arizona Tucson AZ USA Prof R D Brinton PhD Department of Medical Epidemiology and Biostatistics and Center for Gender Medicine Karolinska Institutet Stockholm Sweden Prof JJ Carrero PhD Channing Division of Network Medicine and the Division of Pulmonary and Critical Care Medicine Department of Medicine Brigham and Women™s Hospital Harvard Medical School Boston MA USA D L DeMeo MD Neuroscience Institute and Department of Biology Geia State University Atlanta GA USA Prof G J DeVries PhD Department of Psychiatry University of Colorado School of Medicine Anschutz Medical Campus Aurora CO USA Prof C N Epperson MD Division of Oncology Department of Medicine Washington University School of Medicine St Louis MO USA Prof R Govindan MD W Harry Feinstone Department of Molecular Microbiology and Immunology The Johns Hopkins Bloomberg School of Public Health Baltimore MD USA Prof S L Klein PhD Department of Biomedical Metabolic and Neural Sciences University of Modena andReview 0cReggio Emilia Azienda OspedalieroUniversitaria di Modena Ospedale Civile di Baggiovara Modena Italy Prof A Lonardo MD Department of Psychiatry Department of Psychology and Department of Obstetrics Gynecology University of Illinois at Chicago Chicago IL USA Prof P M Maki PhD Department of Neurology McGovern Medical School University of Texas Health Science Center Houston TX USA Prof L D McCullough MD Berlin Institute of Gender Medicine Charit”Universittsmedizin Berlin Berlin Germany Prof V RegitzZagrosek MD Department of Cardiology University Hospital Z¼rich University of Z¼rich Switzerland Prof V RegitzZagrosek Center for Women™s Health Research Divisions of General Internal Medicine and Cardiology University of Colorado School of Medicine Aurora CO USAimprinting extends to autosomes as well and these imprinted genes exhibit sexspecific and tissuespecific expression in humans10 Thus fundamental sex differences deriving directly from genetic heterogeneity between the X and Y chromosome complements and parentoforigin inher itance exist at the molecular level in all human cells These sex differences persist throughout life and are independent of sex hormones figure Arguably the greatest source of differences between men and women comes from the Y chromosomal SRY gene which directs the development of a testis in men The ensuing developmental surge of testicular testosterone permanently masculinises the reproductive tract and the anisation of brain circuits affecting male behaviour at puberty1112 In humans the first surge occurs at the end of the first trimester of pregnancy Because it alters cellular gene expression and tissue structure in multiple ans of men via epigenetic mechanisms this testosterone surge is also paramount in programming sex differences in physiology and susceptibility to diseases that will manifest in adulthood After this initial testicular testosterone surge gonadal hormone concentrations remain low until puberty which triggers lasting sex differences in circulating oestrogens and testosterone concentrations After puberty cells with androgen or AFemale sexBRandom X chromosomeinactivation and escapeXXXXXXXXXXXXXXXXXXXY chromosome complementMale sexSRYCTesticular testosterone surgeFetal testisTestosteroneOHOGenetic diï¬erences of male and female cellsEpigenetic programming of male cellsFigure Genetic causes of sex differencesA Genetic sex differences start with cells carrying either XX or XY chromosome complement eg genes outside the testisdetermining SRY gene which generates ubiquitous sex differences in the molecular makeup of all male and female cells B Random inactivation of one X chromosome in female cells causes another level of sex differences in gene expression Some Xlinked genes escape inactivation in female individuals and have a higher expression in female than male individuals C The Y chromosomal SRY gene directs the development of a testis in male individuals which produces a surge of testicular testosterone at the end of pregnancy The testosterone surge programmes cellular gene expression and tissue structure in multiple ans of male individuals via epigenetic remodelling The combination of these genetic and developmental events programmes sex differences in physiology and susceptibility to diseases that will manifest in adulthoodoestrogen receptors will be affected differ ently in men and women The combination of all genetic and hormonal causes of sex differences aforementioned culminates in two different biological systems in men and women that translate into differences in disease predisposition manifestation and response to treatment Therefore sex is an important modifier of physiology and disease via genetic epigenetic and hormonal regulations figure Gender as a determinant of patients™ and doctors™ behaviour and as a modifier of health disease and medicineGender according to the Global Health definition refers to the socially constructed norms that impose and determine roles relationships and positional power for all people across their lifetime13 Gender interacts with sex the biological and physical characteristics that define women men and those with intersex identities13 Gender is not a binary term It includes the understanding that in many people traits of masculinity or femininity coexist and are expressed to different degrees Gender attributes are fluid more than two thirds of women and men report genderrelated characteristics traditionally attributed to the opposite sex1415 In transgender people gender identity differs with the sex they were assigned at birth So far transgender people have generally been underrepresented in clinical studies to date although this underrepresentation is changing Gender is an equally important variable as biological sex in human health and influences the behaviour of communities clinicians and patients1415 Gender roles represent the behavioural norms applied to men and women in society which influence individuals™ everyday actions expectations and experiences including diet perceived stress smoking and physical activity and affect health and disease susceptibility Gender identity describes the fluidity of how a person perceives oneself as a woman or a man which affects feelings and behaviours Gender relations refer to how we interact with or are treated by people on the basis of our ascribed gender Institutionalised gender reflects the distribution of power between men and women in the political educational and social institutions in society and shapes social norms that define perpetuate and often justify different expectations and opportunities for women and men1617 As such the distribution of genderrelated charac teristics within populations of men and women can influence health differently than biological sex Together these gender constructs determine access to health care helpseeking behaviours and individual use of the healthcare system Being perceived as a man or a woman triggers different responses from clinicians who might diagnose and suggest interventions differently according to gender As such gender largely determines the use of preventive measures and referral for or acceptance of invasive therapeutic strategies Genderrelated behaviours contribute to risk exposure and preventive behaviour in several wwwthelancetcom Vol August Review 0cdiseases This postulation is well exemplified in the cardiovascular field in which women often underestimate their risk compared with men and seek consultation later than men in the clinic for treatment of myocardial infarction1819 In the GENESISPRAXY prospective study mortality year after an acute coronary event was more strongly associated with gender than with biological sex1415 Similarly control of cardio vascular risk factors hypertension diabetes depres sive symptoms was better predicted by gender than by biological sex1415 Therefore including a gender dimension in clinical studies and practice will contribute to the understanding of different clinical manifestations and outcomes of diseases in women and men1617 Although beyond the scope of this Review it is also important to consider that regarding health and disease gender intersect with race or ethnicity and age20“ Sex and gender are fundamentally and frequently reciprocally interrelated in biology and disease24 Sex influences behaviours eg towards more aggressive or caring phenotypes On the other hand genderrelated behaviours eg smoking lifestyle perceived stress and pain and nutritional habits might produce epigenetic modifications that modulate gene expression and biological phenotypes Figure summarises how sex and gender are interrelated in biology and diseaseSex and gender differences in major chronic diseasesHaving established the importance of sex and gender in disease we will summarise their influences on the most common causes of death and debilitating diseases in the USA as an example figure Note that these sex and gender disparities are relevant to other highincome countries as well as lowincome and middleincome countries where the burden of these diseases becomes increasingly like those in highincome countries26 In most diseases efforts to separate the effects of sex and gender are still incomplete so that we just refer to the differences among women and men Because current knowledge about pathophysiology diagnosis and treatment of disease is primarily based on men as representative of the human species this Review focuses on how women differ from men We discuss some key aspects regarding the dimensions of men in a dedicated sectionHeart diseaseEpidemiology pathogenesis manifestations and diagnosisHeart disease is the leading cause of death in the USA In heart disease accounted for · of all deaths for men and for · of all deaths for women figure Ischaemic heart disease and heart failure are major contributors to heart disease mortality and have important sex and gender differences For example heart failure disproportionately contributes to coronary heart disease mortality in women27 potentially due to undiagnosed ischaemic heart disease in women The strength of the association with cardiovascular risk factors differ by sex Biological sexSex chromosomesEpigeneticeï¬ectsSex hormonesOHOHOHOBehaviour of patients and doctorsSocietyGender constructsLifestyleNutritional habitsExercisePerceived stressSmokingDiseasePathophysiologyManifestationResponse to treatmentDisease perceptionHelpseeking behaviourUse of health careDecision makingTherapeutic responseSex and gender diï¬erences in health disease and medicineFigure Interrelation between sex and gender in health diseases and medicineBiological sex causes sex differences through genetic and hormonal influences in disease pathophysiology clinical manifestations and response to treatment Sex also influences behaviours towards more aggressive or caring phenotypes On the other hand genderrelated behaviours eg smoking lifestyle perceived stress and nutritional habits produce epigenetic modifications that modulate the expression of biological sex Gender constructs determine patients™ perception of disease helpseeking behaviour and individual use of health care Gender constructs also influence decision making and trigger different therapeutic responses from providers biased by genderSystolic blood pressure and hypertension smoking and diabetes are associated with higher hazard ratios for myocardial infarction in women than in men28Ischaemic heart disease is the most recognised example for integrating the concept of gender and sex which shape divergent or distinct disease outcomes Compared with men women suffering from ischaemic heart disease are older this difference is historically believed to be due to the protection of endogenous oestrogens29 although contemporary study refutes this simplistic explanation30 and associations cannot be inferred to be causation Still women suffering from ischaemic heart disease are underdiagnosed3132 and less likely to have a prehospital diagnosis of myocardial infarction33“ The reasons for this disparity reflect the intersection between sex and gender First biological sex differences exist in the pathogenesis of ischaemic heart disease Whereas men are more likely to be affected by obstructive coronary artery disease of large vessels than women coronary microvascular dysfunction36 leading to chronic myocardial ischaemia without obstructive coronary artery disease has a higher prevalence in women than men37 A metaanalysis reported that following acute myocardial infarction both sexes most often presented with chest pain but compared with men women were more likely to present with pain between the shoulder blades nausea or vomiting and shortness of breathProf J G Regensteiner PhD Department of Medicine Department of Paediatrics and Department of Neuroscience Washington University School of Medicine St Louis MO USA Prof J B Rubin MD Center for the Study of Sex Differences in Health Aging and Disease Geetown University Washington DC USA Prof K Sandberg PhD Division of Gastroenterology Duke University Medical Center Durham NC USA A Suzuki MD and Durham VA Medical Center Durham NC USA A SuzukiCorrespondence to Prof Franck MauvaisJarvis Diabetes Discovery SexBased Medicine Laboratory Section of Endocrinology John W Deming Department of Medicine Tulane University School of Medicine New Orleans LA USA fmauvaistulaneeduwwwthelancetcom Vol August Review 0cMale individualsOther·Heart disease·protection might disappear after menopause45 By contrast testosterone induces adverse cardiac remod elling in the male heart44Chronic liver disease ·Influenza and pneumonia ·Suicide ·Alzheimer™s disease ·Type diabetes ·Stroke ·CPD ·Injuries ·Female individualsOther·Septicaemia ·Chronic kidney disease ·Influenza and pneumonia ·Type diabetes ·Injuries ·Alzheimer™s disease ·Stroke ·CPD ·Cancer·Heart disease·Cancer·Figure Percent distribution of the ten leading causes of death by sex USA Adapted from Heron25 CPDchronic pulmonary diseaseSecond a gender bias appears to be responsible for the absence of recognition of ischaemic heart disease presentation in women38 Men and women with ischaemic heart disease who score high on feminine roles and personality traits on questionnaires designed to ascertain aspects of gender are at an increased risk of recurrent ischaemic heart disease independent of female sex39Heart failure affects of adults aged years and older and more women than men in absolute numbers4041 Heart failure occurs at an older age and with less ischaemic causes in women than in men However hypertension and diabetes predispose older women to heart failure to a greater extent than men Heart failure with preserved ejection fraction a form of heart failure with normal systolic function is twice as prevalent in women as in men By contrast heart failure with reduced ejection fraction affects more men than women Women who have heart failure with preserved ejection fraction have smaller and stiffer hearts than men Inflammation and the resulting fibrosis play a sexspecific role in the pathogenesis of heart failure with preserved ejection fraction Under stress premenopausal women™s hearts develop less inflammation resulting in less fibrosis than men™s hearts4243 This difference is partially driven by sex hormones as oestrogens produce antiinflammatory actions on endothelial and immune cells and promote cardioprotective effects in premenopausal women44 This Response to treatmentCompared with men women suffering from ischaemic heart disease are less likely to receive evidencebased treatment3132 and when suffering from acute myocardial infarction they are less likely to receive reperfusion33“ An STelevation myocardial infarction registry revealed that compared with men women exhibit delayed reperfusion leading to higher mortality46 Women suffering from acute myocardial infarction treated by male emergency physicians have a higher mortality rate than those treated by female physicians38 Additionally male physicians are more effective at treating female patients with acute myocardial infarction when they work with female colleagues and when they have experience in treating female patients38 This treatment disparity between women and men can be corrected by improving emergency recognition of STelevation myocardial infarction in women and acceleration of percutaneous coronary intervention which equilibrates gender mortality47Guidelines for the treatment of heart failure are similar for women and men24 However evidence suggests that optimal survival in women occurs with lower doses of β blockers angiotensin receptor blockers and angiotensin converting enzyme inhibitors than in men48 Finally fewer women undergo heart transplantation than men although women are more frequently donors suggesting a referral bias could exist41CancersEpidemiology pathogenesis manifestations and diagnosisCancers are the second leading cause of death dominated by lung cancer accounting for · of deaths in men and · of deaths in women figure More men develop cancer than women49 With few exceptions eg meningioma thyroid cancer lung cancer in nonsmokers nonreproductive cancers exhibit a male predominance though for some cancers oropharynx larynx oesophagus and bladder the male versus female incidence ratios can be higher than A male predominance in cancers that affect both sexes is evident around the world in all races and at all ages50 Survival is also shorter for men than women across multiple cancer types The higher cancer risk in men is partially explained by gender constructs like dietary habits or risk behaviours such as smoking and alcohol consump tion51 It is unlikely to be the only cause After appropriate adjustment for these risk factors adult men still have a higher cancer risk than women52 Moreover a similar male bias in incidence and survival is seen in paediatric cancers before puberty and the adoption of highrisk cancerpromoting behaviours eg smoking53The universal male predominance in cancer incidence and differential outcomes argues for a fundamental role wwwthelancetcom Vol August Review 0cin inutero tumour suppressors of sex in addition to gender in cancer biology Sexspecific biology includes genetic differences XX vs XY chromosomes the incomplete Xinactivation in female individuals which results in biallelic expression of Xencoded female cells54 Y chromosomeencoded oncogenes such as the RNAbinding motif on Y chromosome in male cells55 and the chromatin remodelling effects of testicular testosterone in male cells56 These mechanisms have an influence on several of the hallmarks of cancer57 including metabolism growth regulation58 angiogenesis and immunity which all contribute to cancer predisposition59 A crucial example is the male predisposition to glioblastoma which is the most common form of brain cancer In glioblastoma there is a cellintrinsic predisposition of male astrocytes a subtype of glial cell to malignant transformation60 After puberty sex hormones produce additional epigenetic and acute effects on cells that further influence sex disparities in cancer For example the increased frequency and aggressive phenotype of hepatocellular carcinoma in male individuals has been linked to the stimulatory effects of androgens in male individuals and the protective effects of oestrogens in female individuals61 Importantly the biology of cancer is not the same across histological and genetic diagnostic groups or even within single histol ogical subtypes Thus the interaction between sex gender and cancer mechanisms cannot be expected to be constant Take colon cancer the second leading cause of cancerrelated death for example Although women have a lower overall incidence of colon cancer than men they have a higher incidence of rightsided colon cancers which have the worst outcomes62 Tumours from women with rightsided colon cancers exhibit a distinct molecular signature of energy metabolism compared with those of women with leftsided colon cancers63 This molecular signature is not observed when comparing tumours from men with rightsided colon cancers to men with leftsided colon cancers Thus overall the male predisposition to cancer is probably the consequence of genetic program ming of male cells and the effect of sex hormones after puberty interacting with genderspecific behaviours to establish cancer risksResponse to treatmentIn the future cancer prevention and treatment will be improved by sexspecific and genderspecific approaches For example immune checkpoint inhibitors can improve survival for men with advanced melanomas and nonsmallcell lung cancers more than for women64 The molecular subtyping of glioblastomas based on sexspecific transcriptomes has the potential to enhance chemotherapy in a sexspecific manner65 In colon cancer sex differences in xenobiotic metabolism regulatory networks might underlie greater treatment response in women than men and require modification of approaches for men with colon cancer66 Other elements of cancer metabolism are also ripe for novel sexspecific targeting in treatment Cellular nutrient partitioning is sexually dimorphic so approaches such as ketogenic diets or glutaminase inhibition might be associated with substantial sexspecific responses67 Furthermore data from models of development ageing and cancer all indicate that molecular pathways such as the enzyme phosphatidylinositol 3kinase and tumour suppressors such as p53 and retinoblastoma protein are sexually dimorphic and require sexspecific targeting545968Chronic pulmonary diseaseEpidemiology pathogenesis manifestations and diagnosisChronic pulmonary disease is the third leading cause of death for women · of deaths and the fourth for men · figure It is mostly accounted for by chronic obstructive pulmonary disease and to a lesser extent by asthma Chronic obstructive pulmonary disease is characterised by irreversible airflow limitation and is associated with previous exposure to smoking or air pollutants Women are overrepresented among individuals with chronic obstructive pulmonary disease especially among those with earlyonset disease or those who have never smoked69 Women are also twice as likely to have chronic obstructive pulmonary disease with chronic bronchitis and women with severe chronic obstructive pulmonary disease have as much emphysema as men countering the misconception of emphysema as a male form of chronic obstructive pulmonary disease70 The female lung is more susceptible to chronic obstructive pulmonary disease than the male lung and women develop symptoms of the disease at a younger age with less tobacco exposure than men71 The Genetic Epidemiology of Chronic Obstructive Pulmonary Disease COPDGene study72 suggests that earlyonset chronic obstructive pulmonary disease might originate in utero in susceptible women from alterations in lung development potentiated by maternal asthma and smoking genetic factors or hormonal influences Future studies should focus on the contribution of maternally inherited factors such as mitochondrial and X chromosome genes to understand disease pathogenesis It is important to consider gender constructs as well Smoking advertising campaigns targeting women rose in the 1960s and the resulting higher smoking rates influenced women™s risk for developing chronic obstructive pulmonary disease73 From a disease severity vantage point chronic obstructive pulmonary disease exacerbation rates are also higher in women than men especially at a younger age74 Additionally despite the burden of symptomatology and increased rates of hospitalisations and deaths women with chronic obstructive pulmonary disease are often misdiagnosed and disproportionally suffer from comorbid conditions including anxiety and depression Therefore physicians should consider chronic obstructive pulmonary disease in the differential diagnosis of women with pulmonary symptoms regardless of tobacco or pollutant exposure historieswwwthelancetcom Vol August Review 0cAsthma characterised by variable airflow obstruction and chronic airway inflammation also affects men and women differently Asthma is more prevalent in prepubertal boys than girls Regarding asthma both male biological sex lung development and atopy and male gender constructs related to outdoor play and indoor pet exposure are factors contributing to the devel opment of asthma and sex versus gender contributions could be difficult to separate75 F

cancer249

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: "fundamental influences of sex and gender as modifiers of the major causes of death and morbidity We articulate how the genetic epigenetic and hormonal influences of biological sex influence physiology and disease and how the social constructs of gender affect the behaviour of the community clinicians and patients in the healthcare system and interact with pathobiology We aim to guide clinicians and researchers to consider sex and gender in their approach to diagnosis prevention and treatment of diseases as a necessary and fundamental step towards precision medicine which will benefit men™s and women™s healthIntroductionWhat clinicians know about the diagnosis treatment and prevention of disease originates from studies mostly done on male cells male mice and men1 Historically for multiple reasons including the purported safety of women and their offspring women of childbearing age were excluded from clinical trials As a result medical research and care have been centred on male physiology The assumption was that male and female cells and animals were biologically identical and evidencebased medicine was defined by clinical trials done predominantly in men1 In the US National Institutes of Health NIH mandated the inclusion of women in NIHfunded clinical trials but many investigators did not follow this mandate and many of those who did include women did not analyse the results by sex23 minimising the effectiveness of this policy Preclinical research and drug development studies have also predominantly used male animal models and cells4“ It is not surprising that a US Government Accountability Office report found that eight of the ten prescription drugs withdrawn from the market between and œposed greater health risks for women than for men Most funding agencies from Europe and North America have implemented policies to support and mandate researchers to consider sex and gender at all levels of medical research8 Still the field of sexbased biology and medicine is often viewed as a specialised area of interest rather than a central consideration in medical research Essential for the success of clinical care and translational science is awareness by clinicians and researchers that the diseases they are treating and studying are characterised by differences between women and men in epidemiology pathophysiology clinical manifestations psychological effects disease progression and response to treatmentThis Review explores the role of sex biological constructs and gender social constructs as modifiers of the most common causes of death and morbidity and articulates the genetic biological and environmental determinants that underlie these differences We aim to guide clinicians and researchers to better understand and harness the importance of sex and gender as genetic wwwthelancetcom Vol August biological and environmental modifiers of chronic disease Ultimately it is a necessary and fundamental step towards precision medicine that will benefit women and menSex as a genetic modifier of biology and diseaseSex differences in disease prevalence manifestation and response to treatment are rooted in the genetic differences between men and women Genetic sex differences start at conception when the ovum fuses with a sperm cell carrying an X or a Y chromosome resulting in an embryo carrying either XX or XY chromosomes This fundamental difference in chromosome complement eg genes outside the testisdetermining SRY gene generates ubiquitous sex differences in the molecular makeup of all male and female cells9 First the Y chromosome carries genes that exhibit subtle functional differences from their Xlinked homologues eg ZFY vs ZFX and UTY vs UTX and also carries genes with no homologue at all eg SRY In addition in men the X chromosome carries only maternal imprints ”ie epigenetic modifications made by the parent in generating the sex cells”which alter the expression of genes in the offspring As women have X chromosomes from both parents they carry maternal and paternal imprints which target a different set of genes Random inactivation of one of the X chromosomes in female cells which prevents sex differences in X chromosome gene dosage causes another degree of sex difference in gene expression As some of these Xlinked genes escape inactivation in women those genes are often expressed at higher levels in women than in men9 Sexspecific gene expression due to genomic Search strategy and selection criteriaWe searched PubMed for papers published in English between Jan and June using œsex or œgender and the name of the disease of interest as search terms Although we tried to cite seminal studies when necessary because of space limitation representative reviews were often selectedLancet “Diabetes Discovery SexBased Medicine Laboratory Section of Endocrinology John W Deming Department of Medicine Tulane University School of Medicine and Southeast Louisiana Veterans Health Care System Medical Center New Orleans LA USA Prof F MauvaisJarvis MD Barbra Streisand Women™s Heart Center CedarsSinai Smidt Heart Institute Los Angeles CA USA Prof N Bairey Merz MD National Heart Lung Institute Imperial College London London UK Prof P J Barnes MD Department of Pharmacology and Department of Neurology College of Medicine Center for Innovation in Brain Science University of Arizona Tucson AZ USA Prof R D Brinton PhD Department of Medical Epidemiology and Biostatistics and Center for Gender Medicine Karolinska Institutet Stockholm Sweden Prof JJ Carrero PhD Channing Division of Network Medicine and the Division of Pulmonary and Critical Care Medicine Department of Medicine Brigham and Women™s Hospital Harvard Medical School Boston MA USA D L DeMeo MD Neuroscience Institute and Department of Biology Geia State University Atlanta GA USA Prof G J DeVries PhD Department of Psychiatry University of Colorado School of Medicine Anschutz Medical Campus Aurora CO USA Prof C N Epperson MD Division of Oncology Department of Medicine Washington University School of Medicine St Louis MO USA Prof R Govindan MD W Harry Feinstone Department of Molecular Microbiology and Immunology The Johns Hopkins Bloomberg School of Public Health Baltimore MD USA Prof S L Klein PhD Department of Biomedical Metabolic and Neural Sciences University of Modena andReview 0cReggio Emilia Azienda OspedalieroUniversitaria di Modena Ospedale Civile di Baggiovara Modena Italy Prof A Lonardo MD Department of Psychiatry Department of Psychology and Department of Obstetrics Gynecology University of Illinois at Chicago Chicago IL USA Prof P M Maki PhD Department of Neurology McGovern Medical School University of Texas Health Science Center Houston TX USA Prof L D McCullough MD Berlin Institute of Gender Medicine Charit”Universittsmedizin Berlin Berlin Germany Prof V RegitzZagrosek MD Department of Cardiology University Hospital Z¼rich University of Z¼rich Switzerland Prof V RegitzZagrosek Center for Women™s Health Research Divisions of General Internal Medicine and Cardiology University of Colorado School of Medicine Aurora CO USAimprinting extends to autosomes as well and these imprinted genes exhibit sexspecific and tissuespecific expression in humans10 Thus fundamental sex differences deriving directly from genetic heterogeneity between the X and Y chromosome complements and parentoforigin inher itance exist at the molecular level in all human cells These sex differences persist throughout life and are independent of sex hormones figure Arguably the greatest source of differences between men and women comes from the Y chromosomal SRY gene which directs the development of a testis in men The ensuing developmental surge of testicular testosterone permanently masculinises the reproductive tract and the anisation of brain circuits affecting male behaviour at puberty1112 In humans the first surge occurs at the end of the first trimester of pregnancy Because it alters cellular gene expression and tissue structure in multiple ans of men via epigenetic mechanisms this testosterone surge is also paramount in programming sex differences in physiology and susceptibility to diseases that will manifest in adulthood After this initial testicular testosterone surge gonadal hormone concentrations remain low until puberty which triggers lasting sex differences in circulating oestrogens and testosterone concentrations After puberty cells with androgen or AFemale sexBRandom X chromosomeinactivation and escapeXXXXXXXXXXXXXXXXXXXY chromosome complementMale sexSRYCTesticular testosterone surgeFetal testisTestosteroneOHOGenetic diï¬erences of male and female cellsEpigenetic programming of male cellsFigure Genetic causes of sex differencesA Genetic sex differences start with cells carrying either XX or XY chromosome complement eg genes outside the testisdetermining SRY gene which generates ubiquitous sex differences in the molecular makeup of all male and female cells B Random inactivation of one X chromosome in female cells causes another level of sex differences in gene expression Some Xlinked genes escape inactivation in female individuals and have a higher expression in female than male individuals C The Y chromosomal SRY gene directs the development of a testis in male individuals which produces a surge of testicular testosterone at the end of pregnancy The testosterone surge programmes cellular gene expression and tissue structure in multiple ans of male individuals via epigenetic remodelling The combination of these genetic and developmental events programmes sex differences in physiology and susceptibility to diseases that will manifest in adulthoodoestrogen receptors will be affected differ ently in men and women The combination of all genetic and hormonal causes of sex differences aforementioned culminates in two different biological systems in men and women that translate into differences in disease predisposition manifestation and response to treatment Therefore sex is an important modifier of physiology and disease via genetic epigenetic and hormonal regulations figure Gender as a determinant of patients™ and doctors™ behaviour and as a modifier of health disease and medicineGender according to the Global Health definition refers to the socially constructed norms that impose and determine roles relationships and positional power for all people across their lifetime13 Gender interacts with sex the biological and physical characteristics that define women men and those with intersex identities13 Gender is not a binary term It includes the understanding that in many people traits of masculinity or femininity coexist and are expressed to different degrees Gender attributes are fluid more than two thirds of women and men report genderrelated characteristics traditionally attributed to the opposite sex1415 In transgender people gender identity differs with the sex they were assigned at birth So far transgender people have generally been underrepresented in clinical studies to date although this underrepresentation is changing Gender is an equally important variable as biological sex in human health and influences the behaviour of communities clinicians and patients1415 Gender roles represent the behavioural norms applied to men and women in society which influence individuals™ everyday actions expectations and experiences including diet perceived stress smoking and physical activity and affect health and disease susceptibility Gender identity describes the fluidity of how a person perceives oneself as a woman or a man which affects feelings and behaviours Gender relations refer to how we interact with or are treated by people on the basis of our ascribed gender Institutionalised gender reflects the distribution of power between men and women in the political educational and social institutions in society and shapes social norms that define perpetuate and often justify different expectations and opportunities for women and men1617 As such the distribution of genderrelated charac teristics within populations of men and women can influence health differently than biological sex Together these gender constructs determine access to health care helpseeking behaviours and individual use of the healthcare system Being perceived as a man or a woman triggers different responses from clinicians who might diagnose and suggest interventions differently according to gender As such gender largely determines the use of preventive measures and referral for or acceptance of invasive therapeutic strategies Genderrelated behaviours contribute to risk exposure and preventive behaviour in several wwwthelancetcom Vol August Review 0cdiseases This postulation is well exemplified in the cardiovascular field in which women often underestimate their risk compared with men and seek consultation later than men in the clinic for treatment of myocardial infarction1819 In the GENESISPRAXY prospective study mortality year after an acute coronary event was more strongly associated with gender than with biological sex1415 Similarly control of cardio vascular risk factors hypertension diabetes depres sive symptoms was better predicted by gender than by biological sex1415 Therefore including a gender dimension in clinical studies and practice will contribute to the understanding of different clinical manifestations and outcomes of diseases in women and men1617 Although beyond the scope of this Review it is also important to consider that regarding health and disease gender intersect with race or ethnicity and age20“ Sex and gender are fundamentally and frequently reciprocally interrelated in biology and disease24 Sex influences behaviours eg towards more aggressive or caring phenotypes On the other hand genderrelated behaviours eg smoking lifestyle perceived stress and pain and nutritional habits might produce epigenetic modifications that modulate gene expression and biological phenotypes Figure summarises how sex and gender are interrelated in biology and diseaseSex and gender differences in major chronic diseasesHaving established the importance of sex and gender in disease we will summarise their influences on the most common causes of death and debilitating diseases in the USA as an example figure Note that these sex and gender disparities are relevant to other highincome countries as well as lowincome and middleincome countries where the burden of these diseases becomes increasingly like those in highincome countries26 In most diseases efforts to separate the effects of sex and gender are still incomplete so that we just refer to the differences among women and men Because current knowledge about pathophysiology diagnosis and treatment of disease is primarily based on men as representative of the human species this Review focuses on how women differ from men We discuss some key aspects regarding the dimensions of men in a dedicated sectionHeart diseaseEpidemiology pathogenesis manifestations and diagnosisHeart disease is the leading cause of death in the USA In heart disease accounted for · of all deaths for men and for · of all deaths for women figure Ischaemic heart disease and heart failure are major contributors to heart disease mortality and have important sex and gender differences For example heart failure disproportionately contributes to coronary heart disease mortality in women27 potentially due to undiagnosed ischaemic heart disease in women The strength of the association with cardiovascular risk factors differ by sex Biological sexSex chromosomesEpigeneticeï¬ectsSex hormonesOHOHOHOBehaviour of patients and doctorsSocietyGender constructsLifestyleNutritional habitsExercisePerceived stressSmokingDiseasePathophysiologyManifestationResponse to treatmentDisease perceptionHelpseeking behaviourUse of health careDecision makingTherapeutic responseSex and gender diï¬erences in health disease and medicineFigure Interrelation between sex and gender in health diseases and medicineBiological sex causes sex differences through genetic and hormonal influences in disease pathophysiology clinical manifestations and response to treatment Sex also influences behaviours towards more aggressive or caring phenotypes On the other hand genderrelated behaviours eg smoking lifestyle perceived stress and nutritional habits produce epigenetic modifications that modulate the expression of biological sex Gender constructs determine patients™ perception of disease helpseeking behaviour and individual use of health care Gender constructs also influence decision making and trigger different therapeutic responses from providers biased by genderSystolic blood pressure and hypertension smoking and diabetes are associated with higher hazard ratios for myocardial infarction in women than in men28Ischaemic heart disease is the most recognised example for integrating the concept of gender and sex which shape divergent or distinct disease outcomes Compared with men women suffering from ischaemic heart disease are older this difference is historically believed to be due to the protection of endogenous oestrogens29 although contemporary study refutes this simplistic explanation30 and associations cannot be inferred to be causation Still women suffering from ischaemic heart disease are underdiagnosed3132 and less likely to have a prehospital diagnosis of myocardial infarction33“ The reasons for this disparity reflect the intersection between sex and gender First biological sex differences exist in the pathogenesis of ischaemic heart disease Whereas men are more likely to be affected by obstructive coronary artery disease of large vessels than women coronary microvascular dysfunction36 leading to chronic myocardial ischaemia without obstructive coronary artery disease has a higher prevalence in women than men37 A metaanalysis reported that following acute myocardial infarction both sexes most often presented with chest pain but compared with men women were more likely to present with pain between the shoulder blades nausea or vomiting and shortness of breathProf J G Regensteiner PhD Department of Medicine Department of Paediatrics and Department of Neuroscience Washington University School of Medicine St Louis MO USA Prof J B Rubin MD Center for the Study of Sex Differences in Health Aging and Disease Geetown University Washington DC USA Prof K Sandberg PhD Division of Gastroenterology Duke University Medical Center Durham NC USA A Suzuki MD and Durham VA Medical Center Durham NC USA A SuzukiCorrespondence to Prof Franck MauvaisJarvis Diabetes Discovery SexBased Medicine Laboratory Section of Endocrinology John W Deming Department of Medicine Tulane University School of Medicine New Orleans LA USA fmauvaistulaneeduwwwthelancetcom Vol August Review 0cMale individualsOther·Heart disease·protection might disappear after menopause45 By contrast testosterone induces adverse cardiac remod elling in the male heart44Chronic liver disease ·Influenza and pneumonia ·Suicide ·Alzheimer™s disease ·Type diabetes ·Stroke ·CPD ·Injuries ·Female individualsOther·Septicaemia ·Chronic kidney disease ·Influenza and pneumonia ·Type diabetes ·Injuries ·Alzheimer™s disease ·Stroke ·CPD ·Cancer·Heart disease·Cancer·Figure Percent distribution of the ten leading causes of death by sex USA Adapted from Heron25 CPDchronic pulmonary diseaseSecond a gender bias appears to be responsible for the absence of recognition of ischaemic heart disease presentation in women38 Men and women with ischaemic heart disease who score high on feminine roles and personality traits on questionnaires designed to ascertain aspects of gender are at an increased risk of recurrent ischaemic heart disease independent of female sex39Heart failure affects of adults aged years and older and more women than men in absolute numbers4041 Heart failure occurs at an older age and with less ischaemic causes in women than in men However hypertension and diabetes predispose older women to heart failure to a greater extent than men Heart failure with preserved ejection fraction a form of heart failure with normal systolic function is twice as prevalent in women as in men By contrast heart failure with reduced ejection fraction affects more men than women Women who have heart failure with preserved ejection fraction have smaller and stiffer hearts than men Inflammation and the resulting fibrosis play a sexspecific role in the pathogenesis of heart failure with preserved ejection fraction Under stress premenopausal women™s hearts develop less inflammation resulting in less fibrosis than men™s hearts4243 This difference is partially driven by sex hormones as oestrogens produce antiinflammatory actions on endothelial and immune cells and promote cardioprotective effects in premenopausal women44 This Response to treatmentCompared with men women suffering from ischaemic heart disease are less likely to receive evidencebased treatment3132 and when suffering from acute myocardial infarction they are less likely to receive reperfusion33“ An STelevation myocardial infarction registry revealed that compared with men women exhibit delayed reperfusion leading to higher mortality46 Women suffering from acute myocardial infarction treated by male emergency physicians have a higher mortality rate than those treated by female physicians38 Additionally male physicians are more effective at treating female patients with acute myocardial infarction when they work with female colleagues and when they have experience in treating female patients38 This treatment disparity between women and men can be corrected by improving emergency recognition of STelevation myocardial infarction in women and acceleration of percutaneous coronary intervention which equilibrates gender mortality47Guidelines for the treatment of heart failure are similar for women and men24 However evidence suggests that optimal survival in women occurs with lower doses of β blockers angiotensin receptor blockers and angiotensin converting enzyme inhibitors than in men48 Finally fewer women undergo heart transplantation than men although women are more frequently donors suggesting a referral bias could exist41CancersEpidemiology pathogenesis manifestations and diagnosisCancers are the second leading cause of death dominated by lung cancer accounting for · of deaths in men and · of deaths in women figure More men develop cancer than women49 With few exceptions eg meningioma thyroid cancer lung cancer in nonsmokers nonreproductive cancers exhibit a male predominance though for some cancers oropharynx larynx oesophagus and bladder the male versus female incidence ratios can be higher than A male predominance in cancers that affect both sexes is evident around the world in all races and at all ages50 Survival is also shorter for men than women across multiple cancer types The higher cancer risk in men is partially explained by gender constructs like dietary habits or risk behaviours such as smoking and alcohol consump tion51 It is unlikely to be the only cause After appropriate adjustment for these risk factors adult men still have a higher cancer risk than women52 Moreover a similar male bias in incidence and survival is seen in paediatric cancers before puberty and the adoption of highrisk cancerpromoting behaviours eg smoking53The universal male predominance in cancer incidence and differential outcomes argues for a fundamental role wwwthelancetcom Vol August Review 0cin inutero tumour suppressors of sex in addition to gender in cancer biology Sexspecific biology includes genetic differences XX vs XY chromosomes the incomplete Xinactivation in female individuals which results in biallelic expression of Xencoded female cells54 Y chromosomeencoded oncogenes such as the RNAbinding motif on Y chromosome in male cells55 and the chromatin remodelling effects of testicular testosterone in male cells56 These mechanisms have an influence on several of the hallmarks of cancer57 including metabolism growth regulation58 angiogenesis and immunity which all contribute to cancer predisposition59 A crucial example is the male predisposition to glioblastoma which is the most common form of brain cancer In glioblastoma there is a cellintrinsic predisposition of male astrocytes a subtype of glial cell to malignant transformation60 After puberty sex hormones produce additional epigenetic and acute effects on cells that further influence sex disparities in cancer For example the increased frequency and aggressive phenotype of hepatocellular carcinoma in male individuals has been linked to the stimulatory effects of androgens in male individuals and the protective effects of oestrogens in female individuals61 Importantly the biology of cancer is not the same across histological and genetic diagnostic groups or even within single histol ogical subtypes Thus the interaction between sex gender and cancer mechanisms cannot be expected to be constant Take colon cancer the second leading cause of cancerrelated death for example Although women have a lower overall incidence of colon cancer than men they have a higher incidence of rightsided colon cancers which have the worst outcomes62 Tumours from women with rightsided colon cancers exhibit a distinct molecular signature of energy metabolism compared with those of women with leftsided colon cancers63 This molecular signature is not observed when comparing tumours from men with rightsided colon cancers to men with leftsided colon cancers Thus overall the male predisposition to cancer is probably the consequence of genetic program ming of male cells and the effect of sex hormones after puberty interacting with genderspecific behaviours to establish cancer risksResponse to treatmentIn the future cancer prevention and treatment will be improved by sexspecific and genderspecific approaches For example immune checkpoint inhibitors can improve survival for men with advanced melanomas and nonsmallcell lung cancers more than for women64 The molecular subtyping of glioblastomas based on sexspecific transcriptomes has the potential to enhance chemotherapy in a sexspecific manner65 In colon cancer sex differences in xenobiotic metabolism regulatory networks might underlie greater treatment response in women than men and require modification of approaches for men with colon cancer66 Other elements of cancer metabolism are also ripe for novel sexspecific targeting in treatment Cellular nutrient partitioning is sexually dimorphic so approaches such as ketogenic diets or glutaminase inhibition might be associated with substantial sexspecific responses67 Furthermore data from models of development ageing and cancer all indicate that molecular pathways such as the enzyme phosphatidylinositol 3kinase and tumour suppressors such as p53 and retinoblastoma protein are sexually dimorphic and require sexspecific targeting545968Chronic pulmonary diseaseEpidemiology pathogenesis manifestations and diagnosisChronic pulmonary disease is the third leading cause of death for women · of deaths and the fourth for men · figure It is mostly accounted for by chronic obstructive pulmonary disease and to a lesser extent by asthma Chronic obstructive pulmonary disease is characterised by irreversible airflow limitation and is associated with previous exposure to smoking or air pollutants Women are overrepresented among individuals with chronic obstructive pulmonary disease especially among those with earlyonset disease or those who have never smoked69 Women are also twice as likely to have chronic obstructive pulmonary disease with chronic bronchitis and women with severe chronic obstructive pulmonary disease have as much emphysema as men countering the misconception of emphysema as a male form of chronic obstructive pulmonary disease70 The female lung is more susceptible to chronic obstructive pulmonary disease than the male lung and women develop symptoms of the disease at a younger age with less tobacco exposure than men71 The Genetic Epidemiology of Chronic Obstructive Pulmonary Disease COPDGene study72 suggests that earlyonset chronic obstructive pulmonary disease might originate in utero in susceptible women from alterations in lung development potentiated by maternal asthma and smoking genetic factors or hormonal influences Future studies should focus on the contribution of maternally inherited factors such as mitochondrial and X chromosome genes to understand disease pathogenesis It is important to consider gender constructs as well Smoking advertising campaigns targeting women rose in the 1960s and the resulting higher smoking rates influenced women™s risk for developing chronic obstructive pulmonary disease73 From a disease severity vantage point chronic obstructive pulmonary disease exacerbation rates are also higher in women than men especially at a younger age74 Additionally despite the burden of symptomatology and increased rates of hospitalisations and deaths women with chronic obstructive pulmonary disease are often misdiagnosed and disproportionally suffer from comorbid conditions including anxiety and depression Therefore physicians should consider chronic obstructive pulmonary disease in the differential diagnosis of women with pulmonary symptoms regardless of tobacco or pollutant exposure historieswwwthelancetcom Vol August Review 0cAsthma characterised by variable airflow obstruction and chronic airway inflammation also affects men and women differently Asthma is more prevalent in prepubertal boys than girls Regarding asthma both male biological sex lung development and atopy and male gender constructs related to outdoor play and indoor pet exposure are factors contributing to the devel opment of asthma and sex versus gender contributions could be difficult to separate75 F Answer:

Thyroid_Cancer

"production process errors may bediscovered which could affect the content and all legal disclaimers that apply to the journalpertain Published by Elsevier 0cSARSCoV2 another kind of liver aggressor how does it do that Sonia A LozanoSepulveda1 Kame GalanHuerta Natalia Mart­nezAcu±a Daniel ArellanosSoto and Ana Mar­a RivasEstilla1 1Department of Biochemistry and Molecular Medicine School of Medicine and Hospital Universitario œDr Jose E Gonzalez Autonomous University of Nuevo Len Monterrey Ana Mar­a RivasEstilla Department of Biochemistry and Molecular Medicine School of Medicine and Hospital Universitario œDr Jose E Gonzalez Autonomous University of NL Mxico Corresponding author Nuevo Leon Ave Francisco I Madero y Ave Gonzalitos sn Col Mitras Centro Journal Preproofbut complications such as pneumonia respiratory distress syndrome and multian Monterrey Nuevo Len Mxico Telfax Email amrivas1yahooca Abstract Clinical manifestations of SARSCoV2 infection include more frequently fever and cough failure can occur in persons with additional comorbidities Liver dysfunction is one of the most striking affections among patients suggesting that SARSCoV2 may represent a new king of liver aggressor However the molecular process underlying this phenomenon is 0cstill unclear In this work we overview the most recent findings between the molecular biology of the virus pathogenic mechanisms and its relationship to liver disease observed in patients Abbreviations AaDO2 ACE2 AIH ALT AST COVID19 GGT GI GTEx Alveolararterial Oxygen Gradient Angiotensinconverting enzyme Autoimmune Hepatitis Alanine transaminase Aspartate aminotransferase Coronavirus infectious disease Gamma glutamyl transpeptidase Gastrointestinal GenotypeTissue Expression Metabolicassociated fatty liver disease Nonstructural proteins Reading Frame Journal Preproofcomplex disease in many severely ill patients In other infected subjects an infection is Keywords SARSCoV2 liver liver impairment COVID19 ACE2 MAFLD nsp ORF Introduction SARSCoV2 is the etiological agent of the disease known as COVID19 which causes a disease characterized by pneumonia cough fever occasional diarrhea headache cardiac injury and in some patients™ liver alterations COVID19 has been found to be an extremely reported to be so severe that it can lead to a disproportionate and mortal reaction of the immune system known as a cytokine storm All of these factors make COVID19 highly unpredictable it is what specialists call a multisystem disease 0cAround the world cases of liver dysfunction denoted by elevated hepatic enzymes in serum such as AST aspartate aminotransferase and ALT alanine transaminase have been documented among patients infected with SARSCoV2 There is still no certainty whether the COVID19related liver damagedysfunction is due mainly to the viral replication per se drugs toxicity or other coexisting comorbidities whether sexrelated difference could help to explain why infected men are more healthy or harmful relationship between the liver and its viral aggressor In this paper we describe a brief overview of the implications for researchers in the field of It is important to understand how liver function can be altered by direct infection with this predisposed to develop COVID19associated liver dysfunction than infected women to analyze if there is any genetic predisposition related to impaired liver function during the œrespiratory virus which mechanisms of viral pathogenesis are involved to evaluate disease and of course the crosstalk between viral and cellular proteins that mediate this Journal Preproofliver disease of the most recent findings between the molecular biology of the virus This emerging viral illness is typically characterized by fever dry cough myalgia headache sore throat diarrhea and may be aggravated with shortness of breath and respiratory failure [] The angiotensinconverting enzyme ACE2 the functional receptor of the spike glycoprotein of SARSCoV2 is widely distributed in the anism Historically Hamming and colleagues reported ACE2 expression in the surface of lung alveolar epithelial cells enterocytes of the small intestine arterial and venous endothelial pathogenic mechanisms and its relationship to liver disease observed in patients Clinical characteristics and liver injury in patients with COVID19 0ccells and arterial smooth muscle cells [] Posterior transcriptomic and proteomic analyses confirmed their findings and added high ACE2 expression in adipose tissue bone marrow duodenum endometrium heart kidney small intestine smooth muscle testis and thyroid [] ACE2 is also expressed in liver but in lesser extent One of the most worrisome severe cases of COVID19 [] Regarding the gastrointestinal GI tract and liver over COVID19associated liver injury is defined as any liver damage that occurs during disease progression andor COVID19 treatment in patients with or without a history of previous complications is the unusual formation of blood clots in many patients with COVID19 even those who were receiving anticoagulants Researchers at Mount Sinai in New York published studies suggesting that clots in the lungs play an important role in the most of COVID19 patients develop GI symptoms such as anorexia diarrhea nausea and vomiting and a significant proportion present with altered liver function tests [] Journal PreproofDecreased albumin levels are associated with severe infection and poor prognosis Still AST elevation is the most common abnormality in patients presenting with COVID19 observed more frequently in men and is mainly documented in more severe cases [] liver disease In general the incidence of increased liver biochemical markers in hospitalized patients with COVID19 mainly AST and ALT and slightly elevated bilirubin varies between to of cases [] The increase in liver enzymes is there are no reports of acute or subacute liver failure in patients with COVID19 The largest cohort study that included cases of COVID19 from China showed that had preexisting chronic liver disease Lei and colleagues reported that impaired liver function was related to mortality in COVID19 patients [] Elevated AST was more frequent and significant than the increase of ALT in severe 0chospitalized patients Moreover elevated AST was shown to be associated with highest mortality risk [] In the study reported by Yijin Wang [] they found that of COVID patients had elevated AST activity The median levels of ALT were UL vs UL respectively AST were UL vs UL respectively in abnormal and normal aminotransferase groups Liver enzymes abnormality were associated with disease severity protein levels In addition they found by ultrastructural examination of coronavirus ps in hepatocytes in COVID19 cases SARSCoV2 infected hepatocytes displayed as well as a series of laboratory tests including higher Alveolararterial Oxygen Gradient AaDO2 higher gamma glutamyl transpeptidase GGT lower albumin and lower total conspicuous mitochondrial swelling endoplasmic reticulum dilatation and glycogen granule decreased Histological findings showed apoptosis and binuclear hepatocytes Journal Preproofshowed a disease course similar to that reported in non immunosuppressed population coronavirus the interaction of its proteins with cellular proteins and consequently the immunosuppressive therapy for autoimmune hepatitis AIH developing COVID19 Taken together both ultrastructural and histological evidence indicated a typical lesion of viral infection [] All these findings by different reports demonstrates that SARSCoV2 infection in liver is a crucial cause of hepatic impairment in COVID19 patients However alteration of cellular metabolism that give rise to systematic alterations and metabolic Report from Alessio Gerussi et al[] demonstrated that patients under today the cellular and molecular mechanisms that are altered by infection with this changes are still unknown 0cMolecular biology of SARSCov2 Coronaviruses are enveloped viruses that contain a positively polarized unsegmented RNA genome belonging to the Coronaviridae family and the order of Nidovirales they are distributed in humans and other mammals[] The size of the SARSCoV2 virions is approximately to nm in diameter [“] SARSCoV2 has a genome that consists polymerase RdRp which is nsp12 and is responsible of the replication and transcription of the virus[] which are encoded by the various genetic loci on the genome [] At the center of the virion lies a nucleocapsid composed of the genomic RNA and the nucleocapsid protein[] The virus glycoprotein S consists of two subunits S1 which is at the amino terminus and that encodes for structural proteins and a larger region that encodes two reading frames ORF 1a and 1b which together encode for the nonstructural virus proteins from nucleocapsid protein which is within the phospholipid bilayer and nonstructural proteins nsp1 to nsp16 []The virions have a structural Sspike protein outer spiky glycoprotein Mmembrane protein a type III transmembrane glycoprotein Nof nucleotides [] encoding amino acids and it is composed of a region Journal Preproofvirus [] as well as protein M which is a type III transmembrane glycoprotein[] and participates in the cellular membrane rearrangements for the replication and transcription complexes[] Among the encoded proteins is an RNAdependent RNA provides the receptor binding site and S2 which is at the carboxyl terminus responsible for membrane fusion [] The envelope protein E has a role in the assembly and release of the Nonstructural proteins have several functions during de viral cycle For example nsp 0cThe virus enters the cell by endocytosis through the interaction between envelope glycoprotein S with the cell receptor ACE2 and with the participation of the type II transmembrane serine protease TMPRSS2 [] Once it enters the cell the N protein with viral genome are released within the cytoplasm then cellular proteases degrade the capsid and the virus genome is left free Next the polyprotein containing the viral proteins that are How does the virus select which cells to infect Viruses can infect only certain species of hosts and only permissive cells within that host Permissive cells make all the necessary proteins and viral factors to allow virus to replicate Once a virus gets inside a cell it hijacks the cellular processes to produce virally encoded proteins that will replicate the virus™s genetic material []Viral replication may cause exocytosis[] will translate into viral proteins this entire process will occur in the cell cytoplasm The processed to form the replication complex is translated and then the complementary strand of negative sense pregenomic RNA is synthesized to be used as a template to replicate the structural proteins that will be synthesized in the endoplasmic reticulum membrane to assembly the viral p and finish the cycle through the release of the viruses by positive sense viral genome[] Furthermore the replication and transcription complex will lead to a series of smaller positive sense subgenomic RNAs these are the ones that Journal PreproofBoth SARSCoV and the new SARSCoV2 are very similar in structure and pathogenicity but the major structural protein S protein is slightly different between them[] Compared to other beta coronaviruses the presence of a furinlike cleavage site in SARSCoV2 enables the S protein priming and facilitates an increase on the efficiency of the spread of SARSCoV2 as is reported wide world [] 0cbiochemical changes producing cell damage called cytopathic effects Like other coronaviruses SARSCoV2 requires cellular receptors to initiate its internalization to the cells that carry these factors []Li SARSCoV2 uses the angiotensinconverting enzyme ACE2 as a host cell receptor SARSCoV2 spike S protein binds ACE2 with significantly high affinity[] In addition the main host protease that suggested to promote the pathogenesis of this coronavirus [] program httpsportalgdccancergov They compared ACE2 expression levels across human tissues between males and females and between younger and older persons in these individual tissues Furthermore other reports have analyzed the correlations between ACE2 In order to provide insights into the mechanism of SARSCoV2 infection Li [] analyzed the expression of ACE2 in various normal human tissues using the datasets from the GenotypeTissue Expression GTEx project and The Cancer Genome Atlas TCGA transmembrane serine protease[] Other host proteases such as furin have also been mediates Sprotein activation on primary target cells and initial viral entry is the type II Journal Preproofreported by Li ACE2 expression levels showed no significant difference between have no significant association with sex age or race Is the liver a direct target for SARSCoV2 males and females between younger and older persons or between Asian and nonAsian races This finding suggests that the infection risk of SARSCoV2 and SARSCoV may expression levels and immune signature enrichment levels in individual tissues As As we expected because the systemic manifestations of COVID19 it has been reported that SARSCoV2 has an anotropism beyond the respiratory tract including the kidneys 0cliver heart and brain and possibly that anotropism influences the course of Covid19 disease and aggravates preexisting conditions The ACE2 protein is found at high levels in the GI tract as the colon biliary system and liver [] On the other hand it is well documented a SARSCoV2 RNA shedding in the GI tract [] supporting its tropism for architecture express ACE TMPRSS1 receptors [] The presence of these two host factors in the liver suggests that a direct viral cytopathic effect occurs Also in SARS infection the presence of viral RNA in liver tissue was documented but not as extensively as the new coronavirus Data published by Gordon [] suggest that mitochondrial proteins interact directly with the virus which helps to understand the potential mechanism by which elevated AST the GI tract and liver cells and these may be sites of active viral replication and either direct or indirect tissue injury Indeed a large part of the cells distributed in the liver Journal Preproofeffect the exacerbated inflammatory response in COVID19 may play a central role in profiles are detected in these patients [] Furthermore in addition to this intracellular More recently [] identified the clinical and laboratory characteristics of COVID19 patients with abnormal liver transaminases and they reported that SARSCoV2 is able to which high levels of IL6 have been reported [] which are involved in both infect liver cells and cause liver impairment by direct cytopathic effect inflammatory and repair responses in liver disease Mechanisms of liver pathogenicity 0cIf SARSCoV2 replication has direct adverse effects on liver function it is still unknown Findings in liver biopsy of patients killed by COVID19 showed moderate micro vesicular steatosis and mild portal and necroinflammatory activity[] This seems to indicate that a direct injury occurred while the infection that could have been directly caused by SARSCoV2 Another possibility is that a druginduced liver injury occurred To date there are the following possible mechanisms Figure infection The massive release of cytokines by the immune system in response to the viral infection can result in a cytokine storm and symptoms of sepsis that are the cause of death in of fatal COVID19 cases [] In these cases uncontrolled inflammation induces multian damage leading including liver failure Biomarkers of inflammation such as Creactive protein PCR serum ferritin LDH Ddimer IL6 IL2 are have been found to be significantly elevated in Immune damage from exacerbated inflammation in response to SARSCoV2 Journal Preproofpathogenesis of SARS CoV “related liver disease more studies should be liver is a potential target for direct infection with this virus To understand the performed for evidence of viral mechanisms of replication in different cell anelles as cytoplasm endoplasmic reticulum Golgi apparatus and lipidrafts CoV2 enters cells through the ACE2 molecule which is abundantly expressed in the liver and in particular in bile epithelial cells [] Based on this expression the Direct cytopathic effect due to active viral replication in various liver cells SARScritically ill patients with COVID19 [] into hepatocytes and liver histology characterization It is also important to know in cells their capacity to efficiently produce both infectious and defective non 0cinfective whole virions There are not yet enough data to know the viral dynamics in the different tissues and the associated pathogenesis Anoxia respiratory failure is one of the main characteristics of COVID19 Anoxic hypoxic hepatitis is common in patients with severe symptoms [] Reactivation of preexisting liver disease Patients with preexisting chronic liver medications such as tocilizumab and baricitinib used to combat the adverse immune cholestatic liver disease various studies such as lopinavir ritonavir remdesivir chloroquine tocilizumab uminefovir traditional Chinese medicine so it is important to consider that they could be potentially hepatotoxic in some patients [] Druginduced liver damage DILI Initial clinical guidelines recommended antiviral agents for COVID19 so a variety of drugs have been administered in disease may be more susceptible to liver damage from SARSCoV2 Biological Journal Preproof Genetic factors Genetics may well be one of the determining factors in some reaction may also cause HBV reactivation [] and induce eventual impairment of liver function in those patients with HBV On the other hand it is still unknown whether SARSCoV2 infection exacerbates cholestasis in people with underlying patients who become seriously ill with COVID19 but until now we cannot be sure It is possible for example that the genetic variation that makes an individual more susceptible to high blood pressure or diabetes also makes him more vulnerable to the virus It will be important to find out what role genetic factors predisposing to liver steatosis have and their sensitivity to severe symptoms of COVID19 Ji and 0ccolleagues showed that subjects with metabolicassociated fatty liver disease MAFLD have a higher risk of COVID19 severity disease and abnormal liver blood tests than patients without MAFLD [] In contrast Louise Biquard [] demonstrated that MAFLD is not associated with changes in liver expression blood test abnormalities reported by Ji and colleagues is thus likely not explained by Concluding remarks The scenario is not yet complete which does not allow us to establish or understand the natural history of the disease and the participation or commitment of the liver in this disease Certainly the application of new technological platforms such as singlecell increased hepatic SARSCoV2 uptake Still several contradictory reports will help of genes implicated in SARSCoV2 infection The observed persistence of liver to find the real role of genetic factors in the evolution of this disease Journal Preprooftranscriptomic assays will allow us to quickly know the commitment of each cell type in affected ans and the meaning of viral dynamics in the various affected systems including the liver However as we have already learned from the old hepatotropic viruses history still is ongoing and we have much to learn and understand about the virologic characteristics of this emerging RNA virus that allow us to develop specific antivirals such as the case of HCV and the vaccine to decrease the impact of this œacute infection Declarations of interest none Ethical approval Not required 0c References [] Chen N Zhou M Dong X Qu J Gong F Han Y Epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in Wuhan China httpsdoi101002path1570 [] Wang D Eraslan B Wieland T Hallstr¶m B Hopf T Zolg DP A deep proteome and transcriptome abundance atlas of healthy human tissues Mol Syst [] Hamming I Timens W Bulthuis MLC Lely AT Navis GJ van Goor H Tissue distribution of ACE2 protein the functional receptor for SARS coronavirus A first step in understanding SARS pathogenesis J Pathol “ a descriptive study Lancet “ httpsdoi101016S0140Journal Preproofa manifestation of COVID19 Rev Gastroenterol Mxico English Ed Biol 201915e8503 httpsdoi1015252msb20188503 Patients with COVID19 J Am Coll Cardiol httpsdoi101016jjacc202005001 [] Paranjpe I Fuster V Lala A Russak A Glicksberg BS Levin MA Association of Treatment Dose Anticoagulation with InHospital Survival Among Hospitalized [] Schmulson M D¡valos MF Berumen J Beware Gastrointestinal symptoms can be httpsdoi101016jrgmxen202004001 [] Cai Q Huang D Yu H Zhu Z Xia Z Su Y COVID19 Abnormal liver function tests J Hepatol httpsdoi101016jjhep202004006 0c[] Siddiqi HK Mehra MR COVID19 illness in native and immunosuppressed states A clinicaltherapeutic staging proposal J Hear Lung Transplant Off Publ Int Soc Hear Transplant “ httpsdoi101016jhealun202003012 [] Feng G Zheng KI Yan QQ Rios RS Targher G Byrne CD COVID19 and [] between markers of liver injury and mortality in COVID19 in China Hepatology httpsdoi101002hep31301 [] de la Rica R Bes M Aranda M del Castillo A Socias A Payeras A Low Transl Hepatol “ httpsdoi1014218JCTH202000018 albumin levels are associated with poorer outcomes in a case series of COVID19 patients in Spain a retrospective cohort study MedRxiv Liver Dysfunction Current Insights and Emergent Therapeutic Strategies J Clin Lei F Liu YM Zhou F Qin JJ Zhang P Zhu L Longitudinal association Journal Preproofmortality of adult inpatients with COVID19 in Wuhan China a retrospective cohort study Lancet “ httpsdoi101016S0140liver directly contributes to hepatic impairment in patients with COVID19 J Hepatol httpsdoi101016jjhep202005002 httpsdoi1011012020050720094987 [] Zhou F Yu T Du R Fan G Liu Y Liu Z Clinical course and risk factors for [] Wang Y Liu S Liu H Li W Lin F Jiang L SARSCoV2 infection of the [] Gerussi A Rigamonti C Elia C Cazzagon N Floreani A Pozzi R Coronavirus Disease COVID19 in autoimmune hepatitis a lesson from 0cimmunosuppressed patients Hepatol Commun 2020na httpsdoi101002hep41557 [] Richman D Whitley R Hayden F Clinical virology 4th ed ASM Press [] Ksiazek TG Erdman D Goldsmith CS Zaki SR Peret T Emery S A Novel “ httpsdoi101056NEJMoa030781 [] Kuiken T Fouchier RAM Schutten M Rimmelzwaan GF van Amerongen G van respiratory syndrome Lancet “ httpsdoi101016S0140[] Drosten C G¼nther S Preiser W Van der Werf S Brodt HR Becker S Identification of a novel coronavirus in patients with severe acute respiratory Riel D Newly discovered coronavirus as the primary cause of severe acute Coronavirus Associated with Severe Acute Respiratory Syndrome N Engl J Med Journal Preproofsyndrome N Engl J Med “ httpsdoi101056NEJMoa030747 outbreak associated with a new coronavirus of probable bat origin Nature “ httpsdoi101038s4158602020127 [] Mortola E Roy P Efficient assembly and release of SARS coronaviruslike ps by a heterologous expression system FEBS Lett “ httpsdoi101016jfebslet200409009 [] Fehr A Perlman S Coronaviruses Methods and Protocols Methods in Molecular Biology Chapter Coronaviruses an overview of their replication and [] Zhou P Yang XL Lou Wang XGG Hu B Zhang L Zhang W A pneumonia 0cpathogenesis Springer Berlin Heidelberg [] Belouzard S Millet JK Licitra BN Whittaker GR Mechanisms of coronavirus cell entry mediated by the viral spike protein Viruses “ httpsdoi103390v4061011 [] Vennema H Godeke GJ Rossen JW Voorhout WF Horzinek MC Opstelten DJ et [] Snijder EJ Decroly E Ziebuhr J The Nonstructural Proteins Directing Coronavirus [] Neuman BW Buchmeier MJ Supramolecular Architecture of the Coronavirus P Adv Virus Res “ httpsdoi101016bsaivir201608005 [] van der Hoeven B Oudshoorn D Koster AJ Snijder EJ Kikkert M Barcena M [] Neuman BW Kiss G Kunding AH Bhella D Baksh MF Connelly S A structural analysis of M protein in coronavirus assembly and morphology J Struct Biol “ httpsdoi101016jjsb201011021 expression of viral envelope protein genes EMBO J “ al Nucleocapsidindependent assembly of coronaviruslike ps by coJournal PreproofBiogenesis and architecture of arterivirus replication anelles Virus Res “ httpsdoi101016jvirusres201604001 RNA Synthesis and Processing vol 1st ed Elsevier Inc httpsdoi101016bsaivir201608008 [] Rabi F Al Zoubi M Kasasbeh G Salameh D AlNasser A SARSCoV2 and Coronavirus Disease what we know so far Pathogens [] Masters P The molecular biology of coronaviruses Adv Virus Res “ 0c [] Shang J Ye G Shi K Wan Y Luo C Aihara H Structural basis of receptor recognition by SARSCoV2 Nature “ httpsdoi101038s415860202179y [] Rabaan AA AlAhmed SH Haque S Sah R Tiwari R Malik YS SARSCoV“ [] Li W Moore MJ Vasilieva N Sui J Wong SK Berne MA Angiotensin“ httpsdoi101038nature02145 [] Hoffmann M KleineWeber H P¶hlmann S A Multibasic Cleavage Site in the [] Cohen FS How Viruses Invade Cells Biophys J “ httpsdoi101016jbpj201602006 SARSCoV and MERSCOV A comparative overview Le Infez Med converting enzyme is a functional receptor for the SARS coronavirus Nature Journal PreproofEM structure of the 2019nCoV spike in the prefusion conformation Science “ httpsdoi101126scienceabb2507 Spike Protein of SARSCoV2 Is Essential for Infection of Human Lung Cells Mol Cell 202078779784e5 httpsdoi101016jmolcel202004022 [] Ziegler CGK Allon SJ Nyquist SK Mbano IM Miao VN Tzouanas CN SARSCoV2 Receptor ACE2 Is an InterferonStimulated Gene in Human Airway Epithelial Cells and Is Detected in Specific Cell Subsets across Tissues Cell “ httpsdoi101016jcell202004035 [] Wrapp D Wang N Corbett KS Goldsmith JA Hsieh CL Abiona O Cryo 0c[] Follis K York J Nunberg J Furin cleavage of the SARS coronavirus spike glycoprotein enhances cell“cell fusion but does not affect virion entry Virology “ httpsdoi101016jvirol200602003 [] Millet JK Whittaker GR Host cell proteases Critical determinants of coronavirus [] Li MYY Li L Zhang Y Wang XSS Expression of the SARSCoV2 cell receptor httpsdoi101186s4024902000662x [] Xu H Zhong L Deng J Peng J Dan H Zeng X High expression of ACE2 receptor of 2019nCoV on the epithelial cells of oral mucosa Int J Oral Sci httpsdoi101038s413680200074x tropism and pathogenesis Virus Res “ httpsdoi101016jvirusres201411021 gene ACE2 in a wide variety of human tissues Infect Dis Poverty 20209NA Journal PreproofCoV2 protein interaction map reveals targets for drug repurposing Nature Infection of SARSCoV2 Gastroenterology 202015818311833e3 httpsdoi101053jgastro202002055 httpsdoi101038s4158602022869 [] Xu L Liu J Lu M Yang D Zheng X Liver injury during highly pathogenic human coronavirus infections Liver Int Off J Int Assoc Study Liver “ httpsdoi101111liv14435 [] Coomes EA Haghbayan H Interleukin6 in COVID19 A Systematic Review and [] Xiao F Tang M Zheng X Liu Y Li X Shan H Evidence for Gastrointestinal [] Gordon DE Jang GM Bouhaddou M Xu J Obernier K White KM A SARS 0cMetaAnalysis MedRxiv httpsdoi1011012020033020048058 [] Xu Z Shi L Wang Y Zhang J Huang L Zhang C Pathological findings of COVID19 associated with acute respiratory distress syndrome Lancet Respir Med “ httpsdoi101016S221326002030076X [] Zhang B Zhou X Qiu Y Feng F Feng J Jia Y Clinical characteristics of [] Chen G Wu D Guo W Cao Y Huang D Wang H Clinical and [] Chai X Hu L Zhang Y Han W Lu Z Ke A Specific ACE2 Expression in Invest “ httpsdoi101172JCI137244 death cases with COVID19 MedRxiv httpsdoi1011012020022620028191 immunological features of severe and moderate coronavirus disease J Clin Journal PreproofCholangiocytes May Cause Liver Damage After 2019nCoV Infection BioRxiv patients MedRxiv httpsdoi1011012020040120047381 httpsdoi10110120200203931766 [] Herold T Jurinovic V Arnreich C Hellmuth JC von BergweltBaildon M Klein M Level of IL6 predicts respiratory failure in hospitalized symptomatic COVID[] Grein J Ohmagari N Shin D Diaz G Asperges E Castagna A Compassionate Use of Remdesivir for Patients with Severe Covid19 N Engl J Med “ httpsdoi101056nejmoa2007016 [] U S Food and Drug Administration Fact sheet for health care providers emergency 0cuse authorization EUA of hydroxychloroquine sulfate supplied from the strategic national stockpile for treatment of COVID19 in certain hospitalized patients [] Varona Prez J Rodriguez Chinesta JM Riesgo de reactivacin de la hepatitis B asociado al tratamiento con corticoides frente a SARSCoV2 COVID19 Rev Cl­nica Espa±ola httpsdoi101016jrce202004012 [] Ji D Qin E Xu J Zhang D Cheng G Wang Y Nonalcoholic fatty liver httpsdoi101016jjhep202003044 SARSCoV2 in metabolicassociated fatty liver disease J Hepatol diseases in patients with COVID19 A retrospective study J Hepatol Journal Preproof[] Biquard L Valla D Rautou PE No evidence for an increased liver uptake of httpsdoi101016jjhep202004035 0cFigure legends Journal PreproofFig1 Proposed mechanisms of liver pathogenicity of SARSCoV2 in infected cells SARS CoV2 Infection2 Cytokinestorm3 Drugeffects4 Hypoxia5 PreviousliverdamageBiochemicallabmarkersWhite bloodcellsGenomereleaseReplicationTranslationVirionassemblyViral proteinsMaturevirus release\uf0e9AaDO2MitochondrialproteinsHypoxicisquemicliverinjuryLIVER DAMAGELopinavirRitonavirRemdesivirChloroquineTocilizumabOxidativeimbalanceSteatosisACE2S proteinCytopathiceffect\uf0e9GMCSF\uf0e9IL6\uf0e9IL1β\uf0e9IL2\uf0e9IL8\uf0e9CCL2\uf0e9CCL3\uf0e9CCL5\uf0e9CXCL \uf0e9ALT\uf0e9AST\uf0eaAlbumin\uf0e9PCR\uf0e9LDH\uf0e9Ddimer\uf0e9Ferritin\uf0e9Bilirubin 0c"

cancer250

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: "production process errors may bediscovered which could affect the content and all legal disclaimers that apply to the journalpertain Published by Elsevier 0cSARSCoV2 another kind of liver aggressor how does it do that Sonia A LozanoSepulveda1 Kame GalanHuerta Natalia Mart­nezAcu±a Daniel ArellanosSoto and Ana Mar­a RivasEstilla1 1Department of Biochemistry and Molecular Medicine School of Medicine and Hospital Universitario œDr Jose E Gonzalez Autonomous University of Nuevo Len Monterrey Ana Mar­a RivasEstilla Department of Biochemistry and Molecular Medicine School of Medicine and Hospital Universitario œDr Jose E Gonzalez Autonomous University of NL Mxico Corresponding author Nuevo Leon Ave Francisco I Madero y Ave Gonzalitos sn Col Mitras Centro Journal Preproofbut complications such as pneumonia respiratory distress syndrome and multian Monterrey Nuevo Len Mxico Telfax Email amrivas1yahooca Abstract Clinical manifestations of SARSCoV2 infection include more frequently fever and cough failure can occur in persons with additional comorbidities Liver dysfunction is one of the most striking affections among patients suggesting that SARSCoV2 may represent a new king of liver aggressor However the molecular process underlying this phenomenon is 0cstill unclear In this work we overview the most recent findings between the molecular biology of the virus pathogenic mechanisms and its relationship to liver disease observed in patients Abbreviations AaDO2 ACE2 AIH ALT AST COVID19 GGT GI GTEx Alveolararterial Oxygen Gradient Angiotensinconverting enzyme Autoimmune Hepatitis Alanine transaminase Aspartate aminotransferase Coronavirus infectious disease Gamma glutamyl transpeptidase Gastrointestinal GenotypeTissue Expression Metabolicassociated fatty liver disease Nonstructural proteins Reading Frame Journal Preproofcomplex disease in many severely ill patients In other infected subjects an infection is Keywords SARSCoV2 liver liver impairment COVID19 ACE2 MAFLD nsp ORF Introduction SARSCoV2 is the etiological agent of the disease known as COVID19 which causes a disease characterized by pneumonia cough fever occasional diarrhea headache cardiac injury and in some patients™ liver alterations COVID19 has been found to be an extremely reported to be so severe that it can lead to a disproportionate and mortal reaction of the immune system known as a cytokine storm All of these factors make COVID19 highly unpredictable it is what specialists call a multisystem disease 0cAround the world cases of liver dysfunction denoted by elevated hepatic enzymes in serum such as AST aspartate aminotransferase and ALT alanine transaminase have been documented among patients infected with SARSCoV2 There is still no certainty whether the COVID19related liver damagedysfunction is due mainly to the viral replication per se drugs toxicity or other coexisting comorbidities whether sexrelated difference could help to explain why infected men are more healthy or harmful relationship between the liver and its viral aggressor In this paper we describe a brief overview of the implications for researchers in the field of It is important to understand how liver function can be altered by direct infection with this predisposed to develop COVID19associated liver dysfunction than infected women to analyze if there is any genetic predisposition related to impaired liver function during the œrespiratory virus which mechanisms of viral pathogenesis are involved to evaluate disease and of course the crosstalk between viral and cellular proteins that mediate this Journal Preproofliver disease of the most recent findings between the molecular biology of the virus This emerging viral illness is typically characterized by fever dry cough myalgia headache sore throat diarrhea and may be aggravated with shortness of breath and respiratory failure [] The angiotensinconverting enzyme ACE2 the functional receptor of the spike glycoprotein of SARSCoV2 is widely distributed in the anism Historically Hamming and colleagues reported ACE2 expression in the surface of lung alveolar epithelial cells enterocytes of the small intestine arterial and venous endothelial pathogenic mechanisms and its relationship to liver disease observed in patients Clinical characteristics and liver injury in patients with COVID19 0ccells and arterial smooth muscle cells [] Posterior transcriptomic and proteomic analyses confirmed their findings and added high ACE2 expression in adipose tissue bone marrow duodenum endometrium heart kidney small intestine smooth muscle testis and thyroid [] ACE2 is also expressed in liver but in lesser extent One of the most worrisome severe cases of COVID19 [] Regarding the gastrointestinal GI tract and liver over COVID19associated liver injury is defined as any liver damage that occurs during disease progression andor COVID19 treatment in patients with or without a history of previous complications is the unusual formation of blood clots in many patients with COVID19 even those who were receiving anticoagulants Researchers at Mount Sinai in New York published studies suggesting that clots in the lungs play an important role in the most of COVID19 patients develop GI symptoms such as anorexia diarrhea nausea and vomiting and a significant proportion present with altered liver function tests [] Journal PreproofDecreased albumin levels are associated with severe infection and poor prognosis Still AST elevation is the most common abnormality in patients presenting with COVID19 observed more frequently in men and is mainly documented in more severe cases [] liver disease In general the incidence of increased liver biochemical markers in hospitalized patients with COVID19 mainly AST and ALT and slightly elevated bilirubin varies between to of cases [] The increase in liver enzymes is there are no reports of acute or subacute liver failure in patients with COVID19 The largest cohort study that included cases of COVID19 from China showed that had preexisting chronic liver disease Lei and colleagues reported that impaired liver function was related to mortality in COVID19 patients [] Elevated AST was more frequent and significant than the increase of ALT in severe 0chospitalized patients Moreover elevated AST was shown to be associated with highest mortality risk [] In the study reported by Yijin Wang [] they found that of COVID patients had elevated AST activity The median levels of ALT were UL vs UL respectively AST were UL vs UL respectively in abnormal and normal aminotransferase groups Liver enzymes abnormality were associated with disease severity protein levels In addition they found by ultrastructural examination of coronavirus ps in hepatocytes in COVID19 cases SARSCoV2 infected hepatocytes displayed as well as a series of laboratory tests including higher Alveolararterial Oxygen Gradient AaDO2 higher gamma glutamyl transpeptidase GGT lower albumin and lower total conspicuous mitochondrial swelling endoplasmic reticulum dilatation and glycogen granule decreased Histological findings showed apoptosis and binuclear hepatocytes Journal Preproofshowed a disease course similar to that reported in non immunosuppressed population coronavirus the interaction of its proteins with cellular proteins and consequently the immunosuppressive therapy for autoimmune hepatitis AIH developing COVID19 Taken together both ultrastructural and histological evidence indicated a typical lesion of viral infection [] All these findings by different reports demonstrates that SARSCoV2 infection in liver is a crucial cause of hepatic impairment in COVID19 patients However alteration of cellular metabolism that give rise to systematic alterations and metabolic Report from Alessio Gerussi et al[] demonstrated that patients under today the cellular and molecular mechanisms that are altered by infection with this changes are still unknown 0cMolecular biology of SARSCov2 Coronaviruses are enveloped viruses that contain a positively polarized unsegmented RNA genome belonging to the Coronaviridae family and the order of Nidovirales they are distributed in humans and other mammals[] The size of the SARSCoV2 virions is approximately to nm in diameter [“] SARSCoV2 has a genome that consists polymerase RdRp which is nsp12 and is responsible of the replication and transcription of the virus[] which are encoded by the various genetic loci on the genome [] At the center of the virion lies a nucleocapsid composed of the genomic RNA and the nucleocapsid protein[] The virus glycoprotein S consists of two subunits S1 which is at the amino terminus and that encodes for structural proteins and a larger region that encodes two reading frames ORF 1a and 1b which together encode for the nonstructural virus proteins from nucleocapsid protein which is within the phospholipid bilayer and nonstructural proteins nsp1 to nsp16 []The virions have a structural Sspike protein outer spiky glycoprotein Mmembrane protein a type III transmembrane glycoprotein Nof nucleotides [] encoding amino acids and it is composed of a region Journal Preproofvirus [] as well as protein M which is a type III transmembrane glycoprotein[] and participates in the cellular membrane rearrangements for the replication and transcription complexes[] Among the encoded proteins is an RNAdependent RNA provides the receptor binding site and S2 which is at the carboxyl terminus responsible for membrane fusion [] The envelope protein E has a role in the assembly and release of the Nonstructural proteins have several functions during de viral cycle For example nsp 0cThe virus enters the cell by endocytosis through the interaction between envelope glycoprotein S with the cell receptor ACE2 and with the participation of the type II transmembrane serine protease TMPRSS2 [] Once it enters the cell the N protein with viral genome are released within the cytoplasm then cellular proteases degrade the capsid and the virus genome is left free Next the polyprotein containing the viral proteins that are How does the virus select which cells to infect Viruses can infect only certain species of hosts and only permissive cells within that host Permissive cells make all the necessary proteins and viral factors to allow virus to replicate Once a virus gets inside a cell it hijacks the cellular processes to produce virally encoded proteins that will replicate the virus™s genetic material []Viral replication may cause exocytosis[] will translate into viral proteins this entire process will occur in the cell cytoplasm The processed to form the replication complex is translated and then the complementary strand of negative sense pregenomic RNA is synthesized to be used as a template to replicate the structural proteins that will be synthesized in the endoplasmic reticulum membrane to assembly the viral p and finish the cycle through the release of the viruses by positive sense viral genome[] Furthermore the replication and transcription complex will lead to a series of smaller positive sense subgenomic RNAs these are the ones that Journal PreproofBoth SARSCoV and the new SARSCoV2 are very similar in structure and pathogenicity but the major structural protein S protein is slightly different between them[] Compared to other beta coronaviruses the presence of a furinlike cleavage site in SARSCoV2 enables the S protein priming and facilitates an increase on the efficiency of the spread of SARSCoV2 as is reported wide world [] 0cbiochemical changes producing cell damage called cytopathic effects Like other coronaviruses SARSCoV2 requires cellular receptors to initiate its internalization to the cells that carry these factors []Li SARSCoV2 uses the angiotensinconverting enzyme ACE2 as a host cell receptor SARSCoV2 spike S protein binds ACE2 with significantly high affinity[] In addition the main host protease that suggested to promote the pathogenesis of this coronavirus [] program httpsportalgdccancergov They compared ACE2 expression levels across human tissues between males and females and between younger and older persons in these individual tissues Furthermore other reports have analyzed the correlations between ACE2 In order to provide insights into the mechanism of SARSCoV2 infection Li [] analyzed the expression of ACE2 in various normal human tissues using the datasets from the GenotypeTissue Expression GTEx project and The Cancer Genome Atlas TCGA transmembrane serine protease[] Other host proteases such as furin have also been mediates Sprotein activation on primary target cells and initial viral entry is the type II Journal Preproofreported by Li ACE2 expression levels showed no significant difference between have no significant association with sex age or race Is the liver a direct target for SARSCoV2 males and females between younger and older persons or between Asian and nonAsian races This finding suggests that the infection risk of SARSCoV2 and SARSCoV may expression levels and immune signature enrichment levels in individual tissues As As we expected because the systemic manifestations of COVID19 it has been reported that SARSCoV2 has an anotropism beyond the respiratory tract including the kidneys 0cliver heart and brain and possibly that anotropism influences the course of Covid19 disease and aggravates preexisting conditions The ACE2 protein is found at high levels in the GI tract as the colon biliary system and liver [] On the other hand it is well documented a SARSCoV2 RNA shedding in the GI tract [] supporting its tropism for architecture express ACE TMPRSS1 receptors [] The presence of these two host factors in the liver suggests that a direct viral cytopathic effect occurs Also in SARS infection the presence of viral RNA in liver tissue was documented but not as extensively as the new coronavirus Data published by Gordon [] suggest that mitochondrial proteins interact directly with the virus which helps to understand the potential mechanism by which elevated AST the GI tract and liver cells and these may be sites of active viral replication and either direct or indirect tissue injury Indeed a large part of the cells distributed in the liver Journal Preproofeffect the exacerbated inflammatory response in COVID19 may play a central role in profiles are detected in these patients [] Furthermore in addition to this intracellular More recently [] identified the clinical and laboratory characteristics of COVID19 patients with abnormal liver transaminases and they reported that SARSCoV2 is able to which high levels of IL6 have been reported [] which are involved in both infect liver cells and cause liver impairment by direct cytopathic effect inflammatory and repair responses in liver disease Mechanisms of liver pathogenicity 0cIf SARSCoV2 replication has direct adverse effects on liver function it is still unknown Findings in liver biopsy of patients killed by COVID19 showed moderate micro vesicular steatosis and mild portal and necroinflammatory activity[] This seems to indicate that a direct injury occurred while the infection that could have been directly caused by SARSCoV2 Another possibility is that a druginduced liver injury occurred To date there are the following possible mechanisms Figure infection The massive release of cytokines by the immune system in response to the viral infection can result in a cytokine storm and symptoms of sepsis that are the cause of death in of fatal COVID19 cases [] In these cases uncontrolled inflammation induces multian damage leading including liver failure Biomarkers of inflammation such as Creactive protein PCR serum ferritin LDH Ddimer IL6 IL2 are have been found to be significantly elevated in Immune damage from exacerbated inflammation in response to SARSCoV2 Journal Preproofpathogenesis of SARS CoV “related liver disease more studies should be liver is a potential target for direct infection with this virus To understand the performed for evidence of viral mechanisms of replication in different cell anelles as cytoplasm endoplasmic reticulum Golgi apparatus and lipidrafts CoV2 enters cells through the ACE2 molecule which is abundantly expressed in the liver and in particular in bile epithelial cells [] Based on this expression the Direct cytopathic effect due to active viral replication in various liver cells SARScritically ill patients with COVID19 [] into hepatocytes and liver histology characterization It is also important to know in cells their capacity to efficiently produce both infectious and defective non 0cinfective whole virions There are not yet enough data to know the viral dynamics in the different tissues and the associated pathogenesis Anoxia respiratory failure is one of the main characteristics of COVID19 Anoxic hypoxic hepatitis is common in patients with severe symptoms [] Reactivation of preexisting liver disease Patients with preexisting chronic liver medications such as tocilizumab and baricitinib used to combat the adverse immune cholestatic liver disease various studies such as lopinavir ritonavir remdesivir chloroquine tocilizumab uminefovir traditional Chinese medicine so it is important to consider that they could be potentially hepatotoxic in some patients [] Druginduced liver damage DILI Initial clinical guidelines recommended antiviral agents for COVID19 so a variety of drugs have been administered in disease may be more susceptible to liver damage from SARSCoV2 Biological Journal Preproof Genetic factors Genetics may well be one of the determining factors in some reaction may also cause HBV reactivation [] and induce eventual impairment of liver function in those patients with HBV On the other hand it is still unknown whether SARSCoV2 infection exacerbates cholestasis in people with underlying patients who become seriously ill with COVID19 but until now we cannot be sure It is possible for example that the genetic variation that makes an individual more susceptible to high blood pressure or diabetes also makes him more vulnerable to the virus It will be important to find out what role genetic factors predisposing to liver steatosis have and their sensitivity to severe symptoms of COVID19 Ji and 0ccolleagues showed that subjects with metabolicassociated fatty liver disease MAFLD have a higher risk of COVID19 severity disease and abnormal liver blood tests than patients without MAFLD [] In contrast Louise Biquard [] demonstrated that MAFLD is not associated with changes in liver expression blood test abnormalities reported by Ji and colleagues is thus likely not explained by Concluding remarks The scenario is not yet complete which does not allow us to establish or understand the natural history of the disease and the participation or commitment of the liver in this disease Certainly the application of new technological platforms such as singlecell increased hepatic SARSCoV2 uptake Still several contradictory reports will help of genes implicated in SARSCoV2 infection The observed persistence of liver to find the real role of genetic factors in the evolution of this disease Journal Preprooftranscriptomic assays will allow us to quickly know the commitment of each cell type in affected ans and the meaning of viral dynamics in the various affected systems including the liver However as we have already learned from the old hepatotropic viruses history still is ongoing and we have much to learn and understand about the virologic characteristics of this emerging RNA virus that allow us to develop specific antivirals such as the case of HCV and the vaccine to decrease the impact of this œacute infection Declarations of interest none Ethical approval Not required 0c References [] Chen N Zhou M Dong X Qu J Gong F Han Y Epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in Wuhan China httpsdoi101002path1570 [] Wang D Eraslan B Wieland T Hallstr¶m B Hopf T Zolg DP A deep proteome and transcriptome abundance atlas of healthy human tissues Mol Syst [] Hamming I Timens W Bulthuis MLC Lely AT Navis GJ van Goor H Tissue distribution of ACE2 protein the functional receptor for SARS coronavirus A first step in understanding SARS pathogenesis J Pathol “ a descriptive study Lancet “ httpsdoi101016S0140Journal Preproofa manifestation of COVID19 Rev Gastroenterol Mxico English Ed Biol 201915e8503 httpsdoi1015252msb20188503 Patients with COVID19 J Am Coll Cardiol httpsdoi101016jjacc202005001 [] Paranjpe I Fuster V Lala A Russak A Glicksberg BS Levin MA Association of Treatment Dose Anticoagulation with InHospital Survival Among Hospitalized [] Schmulson M D¡valos MF Berumen J Beware Gastrointestinal symptoms can be httpsdoi101016jrgmxen202004001 [] Cai Q Huang D Yu H Zhu Z Xia Z Su Y COVID19 Abnormal liver function tests J Hepatol httpsdoi101016jjhep202004006 0c[] Siddiqi HK Mehra MR COVID19 illness in native and immunosuppressed states A clinicaltherapeutic staging proposal J Hear Lung Transplant Off Publ Int Soc Hear Transplant “ httpsdoi101016jhealun202003012 [] Feng G Zheng KI Yan QQ Rios RS Targher G Byrne CD COVID19 and [] between markers of liver injury and mortality in COVID19 in China Hepatology httpsdoi101002hep31301 [] de la Rica R Bes M Aranda M del Castillo A Socias A Payeras A Low Transl Hepatol “ httpsdoi1014218JCTH202000018 albumin levels are associated with poorer outcomes in a case series of COVID19 patients in Spain a retrospective cohort study MedRxiv Liver Dysfunction Current Insights and Emergent Therapeutic Strategies J Clin Lei F Liu YM Zhou F Qin JJ Zhang P Zhu L Longitudinal association Journal Preproofmortality of adult inpatients with COVID19 in Wuhan China a retrospective cohort study Lancet “ httpsdoi101016S0140liver directly contributes to hepatic impairment in patients with COVID19 J Hepatol httpsdoi101016jjhep202005002 httpsdoi1011012020050720094987 [] Zhou F Yu T Du R Fan G Liu Y Liu Z Clinical course and risk factors for [] Wang Y Liu S Liu H Li W Lin F Jiang L SARSCoV2 infection of the [] Gerussi A Rigamonti C Elia C Cazzagon N Floreani A Pozzi R Coronavirus Disease COVID19 in autoimmune hepatitis a lesson from 0cimmunosuppressed patients Hepatol Commun 2020na httpsdoi101002hep41557 [] Richman D Whitley R Hayden F Clinical virology 4th ed ASM Press [] Ksiazek TG Erdman D Goldsmith CS Zaki SR Peret T Emery S A Novel “ httpsdoi101056NEJMoa030781 [] Kuiken T Fouchier RAM Schutten M Rimmelzwaan GF van Amerongen G van respiratory syndrome Lancet “ httpsdoi101016S0140[] Drosten C G¼nther S Preiser W Van der Werf S Brodt HR Becker S Identification of a novel coronavirus in patients with severe acute respiratory Riel D Newly discovered coronavirus as the primary cause of severe acute Coronavirus Associated with Severe Acute Respiratory Syndrome N Engl J Med Journal Preproofsyndrome N Engl J Med “ httpsdoi101056NEJMoa030747 outbreak associated with a new coronavirus of probable bat origin Nature “ httpsdoi101038s4158602020127 [] Mortola E Roy P Efficient assembly and release of SARS coronaviruslike ps by a heterologous expression system FEBS Lett “ httpsdoi101016jfebslet200409009 [] Fehr A Perlman S Coronaviruses Methods and Protocols Methods in Molecular Biology Chapter Coronaviruses an overview of their replication and [] Zhou P Yang XL Lou Wang XGG Hu B Zhang L Zhang W A pneumonia 0cpathogenesis Springer Berlin Heidelberg [] Belouzard S Millet JK Licitra BN Whittaker GR Mechanisms of coronavirus cell entry mediated by the viral spike protein Viruses “ httpsdoi103390v4061011 [] Vennema H Godeke GJ Rossen JW Voorhout WF Horzinek MC Opstelten DJ et [] Snijder EJ Decroly E Ziebuhr J The Nonstructural Proteins Directing Coronavirus [] Neuman BW Buchmeier MJ Supramolecular Architecture of the Coronavirus P Adv Virus Res “ httpsdoi101016bsaivir201608005 [] van der Hoeven B Oudshoorn D Koster AJ Snijder EJ Kikkert M Barcena M [] Neuman BW Kiss G Kunding AH Bhella D Baksh MF Connelly S A structural analysis of M protein in coronavirus assembly and morphology J Struct Biol “ httpsdoi101016jjsb201011021 expression of viral envelope protein genes EMBO J “ al Nucleocapsidindependent assembly of coronaviruslike ps by coJournal PreproofBiogenesis and architecture of arterivirus replication anelles Virus Res “ httpsdoi101016jvirusres201604001 RNA Synthesis and Processing vol 1st ed Elsevier Inc httpsdoi101016bsaivir201608008 [] Rabi F Al Zoubi M Kasasbeh G Salameh D AlNasser A SARSCoV2 and Coronavirus Disease what we know so far Pathogens [] Masters P The molecular biology of coronaviruses Adv Virus Res “ 0c [] Shang J Ye G Shi K Wan Y Luo C Aihara H Structural basis of receptor recognition by SARSCoV2 Nature “ httpsdoi101038s415860202179y [] Rabaan AA AlAhmed SH Haque S Sah R Tiwari R Malik YS SARSCoV“ [] Li W Moore MJ Vasilieva N Sui J Wong SK Berne MA Angiotensin“ httpsdoi101038nature02145 [] Hoffmann M KleineWeber H P¶hlmann S A Multibasic Cleavage Site in the [] Cohen FS How Viruses Invade Cells Biophys J “ httpsdoi101016jbpj201602006 SARSCoV and MERSCOV A comparative overview Le Infez Med converting enzyme is a functional receptor for the SARS coronavirus Nature Journal PreproofEM structure of the 2019nCoV spike in the prefusion conformation Science “ httpsdoi101126scienceabb2507 Spike Protein of SARSCoV2 Is Essential for Infection of Human Lung Cells Mol Cell 202078779784e5 httpsdoi101016jmolcel202004022 [] Ziegler CGK Allon SJ Nyquist SK Mbano IM Miao VN Tzouanas CN SARSCoV2 Receptor ACE2 Is an InterferonStimulated Gene in Human Airway Epithelial Cells and Is Detected in Specific Cell Subsets across Tissues Cell “ httpsdoi101016jcell202004035 [] Wrapp D Wang N Corbett KS Goldsmith JA Hsieh CL Abiona O Cryo 0c[] Follis K York J Nunberg J Furin cleavage of the SARS coronavirus spike glycoprotein enhances cell“cell fusion but does not affect virion entry Virology “ httpsdoi101016jvirol200602003 [] Millet JK Whittaker GR Host cell proteases Critical determinants of coronavirus [] Li MYY Li L Zhang Y Wang XSS Expression of the SARSCoV2 cell receptor httpsdoi101186s4024902000662x [] Xu H Zhong L Deng J Peng J Dan H Zeng X High expression of ACE2 receptor of 2019nCoV on the epithelial cells of oral mucosa Int J Oral Sci httpsdoi101038s413680200074x tropism and pathogenesis Virus Res “ httpsdoi101016jvirusres201411021 gene ACE2 in a wide variety of human tissues Infect Dis Poverty 20209NA Journal PreproofCoV2 protein interaction map reveals targets for drug repurposing Nature Infection of SARSCoV2 Gastroenterology 202015818311833e3 httpsdoi101053jgastro202002055 httpsdoi101038s4158602022869 [] Xu L Liu J Lu M Yang D Zheng X Liver injury during highly pathogenic human coronavirus infections Liver Int Off J Int Assoc Study Liver “ httpsdoi101111liv14435 [] Coomes EA Haghbayan H Interleukin6 in COVID19 A Systematic Review and [] Xiao F Tang M Zheng X Liu Y Li X Shan H Evidence for Gastrointestinal [] Gordon DE Jang GM Bouhaddou M Xu J Obernier K White KM A SARS 0cMetaAnalysis MedRxiv httpsdoi1011012020033020048058 [] Xu Z Shi L Wang Y Zhang J Huang L Zhang C Pathological findings of COVID19 associated with acute respiratory distress syndrome Lancet Respir Med “ httpsdoi101016S221326002030076X [] Zhang B Zhou X Qiu Y Feng F Feng J Jia Y Clinical characteristics of [] Chen G Wu D Guo W Cao Y Huang D Wang H Clinical and [] Chai X Hu L Zhang Y Han W Lu Z Ke A Specific ACE2 Expression in Invest “ httpsdoi101172JCI137244 death cases with COVID19 MedRxiv httpsdoi1011012020022620028191 immunological features of severe and moderate coronavirus disease J Clin Journal PreproofCholangiocytes May Cause Liver Damage After 2019nCoV Infection BioRxiv patients MedRxiv httpsdoi1011012020040120047381 httpsdoi10110120200203931766 [] Herold T Jurinovic V Arnreich C Hellmuth JC von BergweltBaildon M Klein M Level of IL6 predicts respiratory failure in hospitalized symptomatic COVID[] Grein J Ohmagari N Shin D Diaz G Asperges E Castagna A Compassionate Use of Remdesivir for Patients with Severe Covid19 N Engl J Med “ httpsdoi101056nejmoa2007016 [] U S Food and Drug Administration Fact sheet for health care providers emergency 0cuse authorization EUA of hydroxychloroquine sulfate supplied from the strategic national stockpile for treatment of COVID19 in certain hospitalized patients [] Varona Prez J Rodriguez Chinesta JM Riesgo de reactivacin de la hepatitis B asociado al tratamiento con corticoides frente a SARSCoV2 COVID19 Rev Cl­nica Espa±ola httpsdoi101016jrce202004012 [] Ji D Qin E Xu J Zhang D Cheng G Wang Y Nonalcoholic fatty liver httpsdoi101016jjhep202003044 SARSCoV2 in metabolicassociated fatty liver disease J Hepatol diseases in patients with COVID19 A retrospective study J Hepatol Journal Preproof[] Biquard L Valla D Rautou PE No evidence for an increased liver uptake of httpsdoi101016jjhep202004035 0cFigure legends Journal PreproofFig1 Proposed mechanisms of liver pathogenicity of SARSCoV2 in infected cells SARS CoV2 Infection2 Cytokinestorm3 Drugeffects4 Hypoxia5 PreviousliverdamageBiochemicallabmarkersWhite bloodcellsGenomereleaseReplicationTranslationVirionassemblyViral proteinsMaturevirus release\uf0e9AaDO2MitochondrialproteinsHypoxicisquemicliverinjuryLIVER DAMAGELopinavirRitonavirRemdesivirChloroquineTocilizumabOxidativeimbalanceSteatosisACE2S proteinCytopathiceffect\uf0e9GMCSF\uf0e9IL6\uf0e9IL1β\uf0e9IL2\uf0e9IL8\uf0e9CCL2\uf0e9CCL3\uf0e9CCL5\uf0e9CXCL \uf0e9ALT\uf0e9AST\uf0eaAlbumin\uf0e9PCR\uf0e9LDH\uf0e9Ddimer\uf0e9Ferritin\uf0e9Bilirubin 0c" Answer:

Thyroid_Cancer

"enzymatic cascade for posttranslational protein modification It includes the ubiquitinactivatingenzyme E1 ubiquitinconjugating enzyme E2 and ubiquitin ligase E3 RINGtype E3 ubiquitin ligases catalyse theposttranslational proteolytic and nonproteolytic functions in various physiological and pathological processes such as‚ammationassociated signal transduction Resulting from the diversity of substrates and functional mechanisms RINGtypeligases regulate microbe recognition and ‚ammation by being involved in multiple ‚ammatory signalling pathways Theseprocesses also occur in autoimmune diseases especially ‚ammatory bowel disease IBD To understand the importance ofRINGtype ligases in ‚ammation we have discussed their functional mechanisms in multiple ‚ammationassociatedpathways and correlation between RINGtype ligases and IBD Owing to the limited data on the biology of RINGtype ligasesthere is an urgent need to analyse their potential as biomarkers and therapeutic targets in IBD in the future IntroductionUbiquitination is a crucial part of a diverse range of physiological and pathological processes such as protein degradationand ‚ammationassociated signalling [ ] It is a threestep enzymatic process that consists of ubiquitinactivatingenzyme E1 ubiquitinconjugating enzyme E2 and ubiquitin ligase E3 [] E3 ligases transfer activated ubiquitin fromE2 to specific substrates thereby forming mono or polyubiquitinated proteins to activate proteasomemediated proteolysis signal transduction endocytosis etc [] E3 ligases arecrucial as they catalyse target ubiquitination and enable theformation of polyubiquitin chains that enhance the complexity of ubiquitination in physiological and pathological processes Although dysregulated ubiquitination is involved inthe development of various types of immune pathologieseg systemic lupus erythematosus rheumatoid arthritisand ‚ammatory bowel disease [IBD] [ ] there is limitedknowledge regarding the role of RINGtype ligases in‚ammationassociated pathways In this review we havefocused on IBD owing to its complex pathogenesis involvement in a wide range of etiological factors including dysregulated ubiquitination The term IBD is used for a group ofchronic autoimmune gastrointestinal disordersincludingmainly Crohn™s disease CD and ulcerative colitis UC []Chronicity of IBD often causes intestinal complications hospitalisation steroid dependency and surgery in diagnosedpatients [] Although significant progress has been made inunderstanding the nature of IBD the underlying interactingmechanism involving ubiquitination RINGtype ligases andonset of IBD remains to be fully understood Ubiquitination Mediated by RINGType LigasesHuman cells express more than E3 ubiquitin ligases thatare classified into three types based on their catalyticdomains RING HECT homologous to the E6AP carboxylterminus and a recently identified RBR RINGbetweenRINGRING type of E3 ligases [ ] The RING domainhas a crossbraced structure with two atoms of zinc that catalyse the direct transfer of ubiquitin from the E2Ubiquitin 0cMediators of Inflammationthioester to the substrate [] Figure Apart from catalysing monoubiquitination RINGtype E3 ligases also elongatehomotypic polyubiquitin chains with varying linkage specificities such as that on Lys48 during the proteasomal targeting ofsubstrates and Lys63 in signal transduction thereby modulating proteins for proteolytic and nonproteolytic activity []However their roles in catalysing other less common typesof ubiquitination including atypical homotypic eg Lys6Lys11 Lys27 Lys29 Lys33 and Met1 [] heterotypic andbranched polyubiquitination remain ambiguous The efficiency of RINGtype E3 ligases in ubiquitination depends onmultiple factors such as substrate modification phosphorylation of E2E3 enzymes autoubiquitination by E3 ligases andpseudosubstrate competition [] The role of the RINGtype ligases and their sophisticated functional mechanism ofubiquitination will be discussed in the following sections Signalling Pathways Regulated by RINGType LigaseslectinlikereceptorsRLRs Ctype Pathogen Recognition Under physiological conditionspattern recognition receptors PRRs comprise tolllikereceptors TLRs retinoic acid inducible gene RIG Ilikereceptorsandnucleotidebinding oligomerisation domainlike receptorsPRRs recognise pathogenassociated molecular patternsPAMPs and trigger the activation of downstream eï¬ectorsin innate immune responses [] For ‚ammatory diseasesthat are closely associated with microbiome dysbiosis such asIBD [ ] dysregulation of PRRs and relevant RINGtypeligases may be involved in pathogeninduced ‚ammation TLR Signalling Upon recognising a wide range ofmicrobial components such as lipopolysaccharides flagellinand microbial nucleic acids activated TLRs expressed onantigenpresenting cells trigger eï¬ector T cell responses in‚ammatory diseases [“] RINGtype ligases modulatethe activation of PAMPinduced TLRs By directly bindingwith TLR3 ring finger protein RNF170 catalyses theLys48linked ubiquitination and proteasomal degradationof TLR3 thereby suppressing TLR3mediated innate immunity in macrophages [] On the other hand FcÎ receptorFcÎR IIb an inhibitory FcR on antibodydependent monocyte phagocytosis is targeted by MARCH3 RNF173 forubiquitination and degradation in lipopolysaccharideinduced TLR4 activation [] RIGI Signalling RIGI is a cytoplasmic PRR that recognises viral RNA and triggers the activation of downstreamimmune responses that are associated with both viral infections and noninfectious autoimmune diseases such asenterocolitis [] The deficiency of RIGI aggravates virusinduced cell death in intestinal epithelial cells and inducessusceptibility to chemically induced colitis in mice suggesting the importance of RIGI signalling in intestinal antiviralimmune response [ ] E3 ligaseslike RNF122 andRNF125 mediate Lys48linked RIGI ubiquitination andproteasomal degradation leading to the reduced expressionof infection inducedpro‚ammatory cytokines includingIL6 and type I interferons α and [ ] In contrastindependent of its E3 ligase activity RNF123 binds with theCARD domain of RIGI and melanoma diï¬erentiationassociated gene to compete with the mutual downstreamadaptor mitochondrial antiviral signalling protein MAVSand inhibit RLRmediated antiviral response [] Unlikethe aforementioned RINGtype ligases that directly targetRLRs RNF114 negatively regulates RLR signalling by polyubiquitinating and inducing the proteasomal degradation ofMAVS []is essential Pro‚ammatory Pathways Nuclear Factor Kappa B NFκB Signalling The NFκBpathway is one of the most wellstudied pro‚ammatorypathways regulated by ubiquitination [] TRAF6 RNF85ubiquitinates the evolutionarily conserved signalling intermediate in TLR activation thatfor TLR4dependent NFκB activation [] RNF183 promotes NFκBsignalling by inducing the ubiquitindependent degradationof IκBα [] TRAF2 RNF117 and TRAF3 RNF118induce Lys48linked ubiquitination and proteasomal degradation of cRel and interferon regulatory factor therebyprohibiting the synthesis of pro‚ammatory cytokines inmacrophages [] MKRN2 RNF62 mediates the polyubiquitination and degradation of the p65 subunit of NFκBthereby inhibiting NFκB signalling [] RNF114 negativelyregulates NFκB signalling and T cell activation by ubiquitinating and stabilising NFκB signalling inhibitors A20 andIκBα [ ] It has also been reported that RNF20 downregulation decreases histone H2B monoubiquitination and leadsto the NFκBdependent transcription of pro‚ammatorycytokines such as IL6 and IL8 [] Nevertheless despitethe formation of heterodimers of RNF40 with RNF20RNF40 alone activates NFκB signalling and upregulatesNFκBdependent transcription by promoting IκB kinaseIKK phosphorylation and p65 nuclear translocation indicating the involvement of NFκBdependent transcriptionin the ubiquitination of substrates other than H2B []MARCH3 RNF173 mediates Lys48linked ubiquitinationand lysosomal degradation of IL1 receptor I and therebyinhibits IL1triggered NFκB activation [] Independentof its E3 ligase activity RNF8 inhibits TNFαinduced NFκBactivation by directly binding with the kinase domain of IKKand interfering with IKKα phosphorylation [] RNF11also exerts a noncanonical role in negatively regulating NFκB signalling RNF11 has high affinity for the E2 enzymeUbc13 and minimal E3 ligase activity that subsequently outcompetes E1 enzymes and other E3 enzymes such as TRAF6[] and impedes the activation of NFκB signalling [] MitogenActivated Protein Kinase MAPK SignallingMAPKs are another family of proteins closely related to‚ammationassociated pathologies such as IBD []Upon stimulation by TNFα TRAF2 is autoubiquitinatedon the Lys63 residue that enables its translocation to thecytoskeletal fraction and activates JNK signalling [ ]In vitro experiments have shown that JNK signalling issuppressed and enhanced in cells overexpressing RNF213 0cMediators of InflammationUbUbE2LysSubstrateRING E3 ligasePolyubiquitinationchain initiationUbE2SubstrateRING E3 ligaseMonoubiquitinationE2SubstrateRING E3 ligaseMultiple monoubiquitinationUbUbE2SubstrateRING E3 ligaseHomotypic polyubiquitinationmodification sites of ubiquitin include Lys6 Lys11 Lys27 Lys29 Lys33 Lys48Lys63 and Met1 SubstrateUb Ub Ub Ub UbChain elongationSubstrateUbUb Ub Ub UbUbUbHeterotypic polyubiquitinationSubstrateUbUbUb Ub UbE2SubstrateRING E3 ligaseBranched polyubiquitinationSubstrateUb Ub Ub Ub UbUb Ub Ub UbFigure Functional mechanisms of RINGtype E3 ligase Acting as a scaï¬olding RINGtype E3 ligase recruits a E2ubiquitin thioester and asubstrate and allows the lysine of substrate to attack the thioester for ubiquitin transfer RINGtype E3 ligases catalyse monoubiquitination ormultiple monoubiquitination by transferring a single ubiquitin to one or several residues of the substrate For polyubiquitination ubiquitinsform eight diï¬erent linkage types including Met1 Lys6 Lys11 Lys27 Lys29 Lys33 Lys48 and Lys63 Apart from homotypic chains RINGtype E3 ligases also catalyse heterotypic chains and branched ubiquitin chains by adopting multiple linkage types and branched topology inthe formation of polyubiquitin chainsand RNF186 respectively [ ] however the mechanismsinvolved in this regulation remain to be understood TRAF7RNF119 upregulatesthe kinase activity of mitogenactivated protein kinase via the WD40 domain and potentiates cell apoptosis via the RING finger domain []Similarly RNF13 mediates endoplasmic reticulum ERstressinduced JNK activation and subsequent cell apoptosisby binding with and promoting the phosphorylation ofthe ER stress sensor endoplasmic reticulum to nucleussignalling [] Janus Kinase JAKSignal Transducer and Activator ofTranscription STAT3 Signalling JAKSTAT3 is one of themajor pro‚ammatory signalling pathways that orchestrate 0cRINGtype ligaseRNF11RNF183RNF2RNF135RNF125TRAF6cCblZNRF2Table RINGtype ligases targeted by noncoding RNAs and the relevant pathwaysMediators of InflammationNoncoding RNAmiR19b3pmiR7miR1395pmiR4853pmiR15bmiR124miR146miR216alncRNA TTN antisense RNA acts as a ceRNA for miR1533pRelevant pathwayNFκB signallingNFκB signallingER stressinduced apoptosisMAPK signallingMAPKERK signallingRIGI signallingTLR signallingTLR signallingPI3KAKT signallingPI3KAKT signallingReference[][][][][][][][][][]the progression of ‚ammatory and autoimmune diseases[] A number of RINGtype ligases modulate JAKSTAT3signalling RNF6 and RNF38 function in catalysing theubiquitinationinduced proteasomal degradation of SH2containing protein tyrosine phosphatase thattargetsphosphorylated STAT3 thereby maintaining STAT3 phosphorylation and activating STAT3 signalling [ ] Incontrast TRAF6 promotes Lys63linked ubiquitination ofSTAT3 and represses STAT3mediated transcription ofdownstream ‚ammationrelated genes such as Creactiveprotein [] Interestingly RNF41 modulates the cell surfaceexpression of JAK2associated cytokine receptors by blocking the cleavage of receptors and enhancing receptor shedding in a ubiquitinationdependent manner [] Phosphatidylinositol 3Kinase PI3K Signalling PI3K isanother classical pathway involved in ‚ammation whereinRINGtype ligases are of crucialimportance MKRN1RNF61 functions in the positivefeedback regulation of sustained PI3KAKT activation upon stimulation by epidermalgrowth factor AKT activation phosphorylates and stabilisesE3 ligase MKRN1 that further ubiquitinates and degradesphosphatase and tensin homologue a PI3KAKT inhibitor[] MKRN2 RNF62 induces the ubiquitindependent degradation of the p85α subunit of PI3K and downregulated AKTphosphorylation suggesting a negative regulatory role ofMKRN2 in PI3KAKT signalling [] Downregulation ofUHRF1 RNF106 represses the phosphorylation of PI3Kand AKT which reveals an underlying interaction betweenUHRF1 and PI3KAKT signalling [] Transforming Growth Factor TGF Signalling TGF signalling functions in immunosuppression and inhibitingthe activity of eï¬ector T cells maintaining Treg diï¬erentiation reducing B cell responsiveness and inducing macrophage anergy [] RNF11 plays a dualrole in themodulation of TGF signalling By competing with Smad7for Smurf2 RNF11 is a positive regulator for TGF signalling and reduces the formation of Smad7Smurf2 complexesthat degrade TGF receptors [] RNF11 is also responsiblefor the ubiquitinationmediated stabilisation of Smad4 thatenhances Smad4dependent TGF signalling by directinteraction [] Notably RNF11 may negatively regulatesignallingbytheenablingformationTGFofSmurf2RNF11 complexes and inducing the ubiquitinationand degradation of the associated molecule with the SH3domain of STAM that promotes TGF signalling []PRAJA RNF70 mediates the ubiquitinationinduced proteasomal degradation of embryonic liver fodrin a Smad4adaptor protein thereby negatively regulating TGF signalling [] Autophagy Accumulating evidence reveals that autophagy contributes extensively to immune cell developmentand cell death and its dysregulation has been implicated inmany autoimmune diseases [] TRAF6 catalyses Lys63linked ubiquitination of BECN1 and stimulates TLRinduced autophagy in macrophages upon pro‚ammatorystimulation [ ] RNF166 has a novel role in antibacterialhost defence owing to its function in inducing the Lys29 andLys33linked ubiquitination of autophagy adaptor p62which mediates the recruitment of p62 to bacteria and initiates bacteria engulfment [] Noncoding RNAs Since dysregulated noncoding RNAsare involved in the progression of ‚ammatory diseases[] the posttranscriptional regulation of RINGtype ligasesby noncoding RNAs may play critical roles in potential‚ammationrelevant signalling pathways Until now quitea few microRNAs have been proved to posttranscriptionallyregulate RINGtype ligases by hampering translation orinducing mRNA degradation Table [ “] Nevertheless although the other two major types of noncodingRNAs long noncoding RNAs and circular RNAs also havediverse functions in ‚ammatory diseases eg competingendogenous RNA [ceRNA] transcription regulation andRNAbinding protein sponges [ ] to what extent theymodulate RINGtype ligases awaits further analysis RINGType Ligases in IBD Pathogenesis of IBD The pathogenesis of IBD has beenelucidated over the past years More than loci have beenimplicated in increased genetic risk for IBD that correlate withthe functioning of cellular processes such as innateadaptiveimmune responseintestinal mucosal barrier homeostasis 0cMediators of InflammationTable Roles of RINGtype ligases in IBD patients and animal models of colitisRINGtypeligaseRoleIBD patientsAnimal model of colitisReferenceRNF186ControversialIncrease risk of UC and ERstressinduced apoptosisAttenuate ER stress and maintain intestinalpermeability in DSSinduced mouse model[“]of colitisRNF20RNF40Anti‚ammatoryPro‚ammatoryDecrease in the colonic tissueProtect mice from DSSinduced colitis andfrom UC patientsmaintain intestinal barrier”Activate NFκB signalling in DSSinducedmouse model of colitis[][][][“][ ]RNF183Pro‚ammatoryIncrease in the colonic tissuefrom CD and UC patientscorrelate with endoscopicindex of disease severityUHRF1Anti‚ammatory”TRAF2Anti‚ammatoryIncrease in the colonic tissuefrom CD and UC patientsTRAF3Anti‚ammatoryTRAF5Anti‚ammatoryIncrease in the plasma andcolonic tissue from CD andUC patientsIncrease in the plasma andcolonic tissue from CD andUC patientsIncrease in TNBSinduced mouse modelof colitisRegulate TNFα expression in mice withDSSinduced colitis and zebrafishModulate the proliferation anddiï¬erentiation of Treg cellsModulate colonic microbiotacomposition pro‚ammatorycytokine expression and immunecell ltration in mice withDSSinduced colitisRegulate pro‚ammatory cytokineexpression and mitigate ‚ammatorydamage in DSSinduced mousemodel of colitis[ ]Inhibit IL17mediated pro‚ammatorypathway in TNBSinduced mousemodel of colitisControl pro‚ammatory cytokineexpression and protect mice againstexperimental colitis[“]and autophagy suggesting the involvement of multiple factorsin shaping the procolitogenic environment during the development of IBD [ ] IBD patients manifest with abnormalities in the composition of gut microbiota such as decreasedbacterial diversity increased proportion of harmful bacterialstrains and decreased proportion of protective probioticswhich trigger pro‚ammatory intestinal pathogenic immuneresponses and contribute to the pathogenesis of IBD [ ]Elevated levels of pro‚ammatory cytokines eg IL1 IL6and IL23 and activation of adaptive eg Th1 Th2 Th9and Th17 cells and innate immune cells eg neutrophilsand NK cells constitute a synergistic ‚ammatory networkthat induces intestinal mucosal ‚ammation and sustainedactivation of multiple pro‚ammatory signalling pathways[ ] IL1 familyinduced NFκB IL6induced STAT3MAPK and PI3K signalling pathways are pivotal in intestinal‚ammation [ ] TGF signalling can mitigateimmune cell hyperactivation but also causes the formation ofintestinal strictures in chronic intestinal ‚ammation []Autophagy is involved in the regulation of immune cell function thus defective autophagy also plays an important role inIBD pathogenesis [] A dysfunctional gut barrier and subsequent increased intestinal permeability are also consideredimportant etiologic factors in the development of IBD thatresult in the uncontrolled exchange of materials between theintestinal lumen and internal environment A compromisedgut barrier is often attributed to the pro‚ammatory stimulation and subsequent downregulation of sealing tight junctionproteins eg claudin5 and claudin8 and upregulation ofporeforming tight junction proteins eg claudin2 [“]Since multiple etiologic factors function in the pathogenesisof IBD in a synergistic manner further research is warrantedto understand the dynamics between these players Role of RINGType Ligases in IBD Despite the limitedknowledge on RINGtype ligases in IBD research suggestsa correlation between RINGtype ligases and IBD pathogenesis Table Genomewide association studies have identified RNF186 as one of the genes associated with susceptibilityto UC the diseasecoding variant of RNF186 involves analtered RING domain [ ] The truncated RNF186 lacking the second transmembrane domain is associated withprotecting individuals against developing UC by inhibitingthe ER localisation of RNF186 and subsequent Lys29 andLys63linked polyubiquitination of proapoptotic BCL2 interacting protein under ER stress [ ] Notably RNF186functions diï¬erently in a dextran sulfate sodium DSSinduced mouse model of colitis RNF186deficient mice 0cMediators of Inflammationdevelop more severe colitis during DSS administration andtheir colonic epithelial cell exhibits enhanced signs of ER stressand apoptosis [] RNF186 also modulates intestinal barrierfunction by mediating the Lys48linked ubiquitination of tightjunction protein occludin and RNF186 deficiency increasesintestinal permeability in RNF186 knockout mice [] ThusRNF186 targets diï¬erent substrates and has a complex association with gut ‚ammationApart from the reduced expression of RNF20 in the colonsamples from UC patients homozygous RNF20knockoutmice die due to embryonic lethality and heterozygous miceare susceptible to DSSinduced colitis with increased intestinal permeability suggesting the anti‚ammatory role ofRNF20 [] RNF40 knockout mice exhibit mitigated gut‚ammation upon treatment with DSS this can be attributed to the attenuated activation of NFκB signalling []The upregulation of RNF183 in the colonic tissue from IBDpatients and 246trinitrobenzenesulfonic acid TNBSinduced mouse model of colitis indicates its pro‚ammatory function probably by promoting the ubiquitinationinduced degradation of IκBα []Because UHRF1catalysed histone H3 monoubiquitination recruits and stimulates DNA methyltransferase toDNA methylation sites and thereby maintains DNA methylation UHRF1 participates in the epigenetic control of multiple genes such as TNFα [ ] Mice with macrophagesdeficient for UHRF1 manifest with TNFα overexpressionand aggravated DSSinduced colitis Also the loss of functionin UHRF1 reduces the methylation of the TNFα promoterin macrophages indicating the regulatory role of UHRF1 inthe mouse model of colitis [] Similarly an in vivo studyin zebrafish has revealed that loss in function of UHRF1 leadsto defects in the epigenetic regulation of TNFα promotermethylation and elicits elevated TNFα expression in ‚ammatory processes including intestinal epithelial cell apoptosis neutrophil recruitment and weakened intestinal barrierfunction [] However UHRF1 may eï¬ect diï¬erentlyamong subtypes of regulatory T Treg cells as UHRF1 maintains the proliferation and maturation of colonic Treg cellsbut inhibits the diï¬erentiation of peripheral induced Tregcells in the development of colitis [ ]Studies have shown the diverse roles involved with theupregulation of TRAFs including TRAF1235 in the bloodor colonic mucosa of IBD patients [ ] DSSinducedcolitis models of TRAF2 and TRAF3deficient mice revealsimilar functions of TRAF2 and TRAF3 as negative regulators of experimental colitis by decreasing pro‚ammatorycytokines and reducing the ltration of immune cells inthe colon [] In another study TRAF2deficient micedevelop severe spontaneous colitis and exhibit altered colonicmicrobiota composition indicating the anti‚ammatoryrole of TRAF2 in controlling colonic microbiota [] TRAF3also acts as a colitis regulator by binding with the IL17receptor and interfering with the IL17mediated pro‚ammatory pathway in mice with TNBSinduced colitis []Although TRAF5 RNF84 promotes the ubiquitinationand stabilisation of the retinoic acidrelated orphan receptorÎt that mediates pro‚ammatory Th17 cell diï¬erentiationand IL17AIL17F expression [ ] TRAF5deficientmice exhibit aggravated experimental colitis and upregulation of pro‚ammatory cytokines [] The complex function of TRAF5 needs further analysis DiscussionAs the importance of RINGtype E3 ligases is graduallyunveiled there are still problems to be solved Firstly apartfrom the canonical role of RINGtype ligases in modulatingkey signalling pathways and their downstream adaptors asE3 ubiquitin ligases some RINGtype ligases interfere withthe ubiquitination cascade by competition or direct interaction with other E3 ligases [] Owing to the variety ofRINGtype ligases and substrate specificity of E3 ligases thepotential competition among RINGtype ligases in regulatingimmune response remains to be fully understood Secondlydiï¬erences in RING E3 ligasemediated target ubiquitinationcan also be attributed to the variance in length and linkagetype of ubiquitin chains Although RINGtype ligases function in proteolytic degradation and signal transduction bycatalysing Lys48linked and Lys63linked ubiquitinationrespectively their roles in catalysing less common linkagetypes of homotypic polyubiquitin chains such as Lys11linked ubiquitination [] and the outcome of such polyubiquitination are still obscureRINGtype ligases form a sophisticated but importantubiquitination network wherein the expression and functionof RINGtype ligases are also ‚uenced reciprocally in physiological and pathological processes Understanding themechanisms employed by RINGtype ligases in modulating‚ammationassociated pathways by catalysing atypicallinkages and aï¬ecting signaltransduction may furtherexplain the interaction between RINGtype ligases and IBDSimilarly research on heterotypic polyubiquitin chains isalso important to unveil these underlying mechanismsIn this review we have highlighted the roles of RINGtype ligases in PAMP recognition and modulation of‚ammationassociated pathways that are crucial etiologicalfactors in the development of autoimmune diseases Accumulating evidence shows that many RINGtype ligases areinvolved in ‚ammationassociated pathways such as pro‚ammatory NFκB MAPK JAKSTAT3 and PI3K signalling and anti‚ammatory TGF signalling Subsequentlywe have discussed the role of RINGtype ligases in the pathogenesis of IBD via ‚ammationrelated pathways Patientswith IBD exhibit the diï¬erential expression of specific RINGtype ligases such as TRAFs However there are limited studies on the potential clinical value of RINGtype ligases inpredicting or treating IBD Thus far there have been a fewattempts to use RINGtype ligases as predictive biomarkersand therapeutic targets in treating cancer RNF43 modulatesWnt signalling and has been used to target colorectal cancerand pancreatic ductal carcinoma [ ] Neverthelessthe potential of RINGtype ligases in autoimmune diseasesespecially in IBD needs to be understood in greater detailfuture research on the expression profile ofThereforeRINGtype ligases in the gastrointestinaltract and thedetailed mechanisms is warranted 0cMediators of InflammationConflicts of InterestThe authors have no conflict of interest to discloseAuthors™ ContributionsThe guarantor of the article is Shenghong Zhang ShenghongZhang designed the study Shenghong Zhang Liguo ZhuYing Li and Longyuan Zhou wrote and revised the manuscript Guang Yang Ying Wang Jing Han and Li Li revisedthe important intellectual content of the manuscript All theauthors approved the final version Liguo Zhu Ying L andLongyuan Zhou contributed equally to the workAcknowledgmentsThis study was supported by the National Natural ScienceFoundation of China under Grants and Guangdong Science and Technology Department under Grant 2017A030306021 and FundamentalResearch Funds for the Central Universities under Grant19ykzd11References[] D Popovic D Vucic and I Dikic œUbiquitination in diseasepathogenesis and treatment Nature Medicine vol no pp “ [] D Aki Q Li H Li Y C Liu and J H Lee œImmune regulation by protein ubiquitination roles of the E3 ligases VHLand itch Protein Cell vol no pp “ [] C M Pickart œMechanisms underlying ubiquitinationAnnual Review of Biochemistry vol no pp “[] H Hu and S C Sun œUbiquitin signaling in immuneresponses Cell Research vol no pp “ [] R Hodson œInflammatory bowel disease Nature vol no p S97 [] L PeyrinBiroulet E V Loftus Jr J F Colombel and W JSandborn œThe natural history of adult Crohnʼs disease inpopulationbased cohorts The American Journal of Gastroenterology vol no pp “ [] N Zheng and N Shabek œUbiquitin ligases structure function and regulation Annual Review of Biochemistryvol no pp “ [] F E Morreale and H Walden œTypes of ubiquitin ligasesCell vol no pp “248e1 [] C E Berndsen and C Wolberger œNew insights into ubiquitin E3 ligase mechanism Nature Structural MolecularBiology vol no pp “ [] R J Deshaies and C A P Joazeiro œRING domain E3 ubiquitin ligases Annual Review of Biochemistry vol no pp “ [] Y Kulathu and D Komander œAtypical ubiquitylation theunexplored world of polyubiquitin beyond Lys48 and Lys63linkages Nature Reviews Molecular Cell Biology vol no pp “ [] S Akira œInnate immunity to pathogens diversity in receptors for microbial recognition Immunological Reviewsvol no pp “ [] X C Morgan T L Tickle H Sokol œDysfunction ofthe intestinal microbiome in ‚ammatory bowel diseaseand treatment Genome Biology vol no p R79[] R B Sartor and G D Wu œRoles for intestinal bacteriaviruses and fungi in pathogenesis of ‚ammatory bowel disapproaches Gastroenterologyeasesvol no pp “339e4 therapeuticand[] T Kawai and S Akira œThe role of patternrecognition receptors in innate immunity update on tolllike receptors NatureImmunology vol no pp “ [] J Q Chen P Szodoray and M Zeher œTolllike receptorpathways in autoimmune diseases Clinical Reviews inAllergy and Immunology vol no pp “ [] J H Park L PeyrinBiroulet M Eisenhut and J I ShinœIBD immunopathogenesis a comprehensive review of‚ammatory molecules Autoimmunity Reviews vol no pp “ [] X Song S Liu W Wang Z Ma X Cao and M Jiang œE3ubiquitin ligase RNF170 inhibits innate immune responsesby targeting and degrading TLR3 in murine cells Cellular Molecular Immunology vol no pp “ [] K Fatehchand L Ren S Elavazhagan œTolllikereceptorIIbFcÎRIIb via MARCH3 proteinmediated ubiquitinationThe Journal of Biological Chemistry vol no pp “ ligands downregulate FcÎ receptor[] T Matsumiya and D M Staï¬orini œFunction and regulationof retinoic acidinducible geneI Critical Reviews in Immunology vol no pp “ [] Y Hirata A H Broquet L Menchén and M F Kagno﬜Activation of innate immune defense mechanisms by signaling through RIGIIPS1 in intestinal epithelial cellsJournal of Immunology vol no pp “[] W Wang M Jiang S Liu œRNF122 suppresses antiviraltype I interferon production by targeting RIGI CARDs tomediate RIGI degradation Proceedings of the NationalAcademy of Sciences ofthe United States of Americavol no pp “ [] K Arimoto H Takahashi T Hishiki H Konishi T Fujitaand K Shimotohno œNegative regulation of the RIGI signaling by the ubiquitin ligase RNF125 Proceedings of theNational Academy of Sciences of the United States of Americavol no pp “ [] S Wang Y K Yang T Chen œRNF123 has an E3ligaseindependentreceptormediated antiviral signaling EMBO Reports vol no pp “ in RIGIlikefunction[] B Lin Q Ke H Li N S Pheifer D C Velliquette and D WLeaman œNegative regulation of the RLH signaling by the E3ubiquitin ligase RNF114 Cytokine vol pp “ [] J Chen and Z J Chen œRegulation of NFκB by ubiquitination Current Opinion in Immunology vol no pp “ [] Y Min M J Kim S Lee E Chun and K Y Lee œInhibitionof TRAF6 ubiquitinligase activity by PRDX1 leads to inhibition of NFKB activation and autophagy activation Autophagy vol no pp “ [] Q Yu S Zhang K Chao œE3 ubiquitin ligasein ‚ammatory bowelRNF183 is a novelregulator 0cMediators of Inflammationdisease Journal of Crohn's Colitis vol no pp “ [] J Jin Y Xiao H Hu œPro‚ammatory TLR signallingis regulated by a TRAF2dependent proteolysis mechanismin macrophages Nature Communications vol no article [] C Shin Y Ito S Ichikawa M Tokunaga K SakataSogawa and T Tanaka œMKRN2 is a novel ubiquitin E3ligase for the p65 subunit of NFκB and negatively regulates ‚ammatory responses Scientific Reports vol no article [] M S Rodriguez I Egaña F LopitzOtsoa œThe RINGubiquitin E3 RNF114 interacts with A20 and modulatesNFκB activity and Tcell activation Cell Death Diseasevol no article e1399 [] K Heyninck and R Beyaert œA20 inhibits NFkappaB activation by dual ubiquitinediting functions Trends in Biochemical Sciences vol no pp “ [] O Tarcic I S Pateras T Cooks œRNF20 links histoneH2B ubiquitylation with ‚ammation and ‚ammationassociated cancer Cell Reports vol no pp “ [] R L Kosinsky R L Chua M Qui œLoss of RNF40decreases NFκB activity in colorectal cancer cells andreduces colitis burden in mice Journal of Crohn's Colitisvol no pp “ [] H Lin D Gao M M Hu œMARCH3 attenuates IL1triggered ‚ammation by mediating K48linked polyubiquitination and degradation of IL1RI Proceedings of theNational Academy of Sciences of the United States of America

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You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: "enzymatic cascade for posttranslational protein modification It includes the ubiquitinactivatingenzyme E1 ubiquitinconjugating enzyme E2 and ubiquitin ligase E3 RINGtype E3 ubiquitin ligases catalyse theposttranslational proteolytic and nonproteolytic functions in various physiological and pathological processes such as‚ammationassociated signal transduction Resulting from the diversity of substrates and functional mechanisms RINGtypeligases regulate microbe recognition and ‚ammation by being involved in multiple ‚ammatory signalling pathways Theseprocesses also occur in autoimmune diseases especially ‚ammatory bowel disease IBD To understand the importance ofRINGtype ligases in ‚ammation we have discussed their functional mechanisms in multiple ‚ammationassociatedpathways and correlation between RINGtype ligases and IBD Owing to the limited data on the biology of RINGtype ligasesthere is an urgent need to analyse their potential as biomarkers and therapeutic targets in IBD in the future IntroductionUbiquitination is a crucial part of a diverse range of physiological and pathological processes such as protein degradationand ‚ammationassociated signalling [ ] It is a threestep enzymatic process that consists of ubiquitinactivatingenzyme E1 ubiquitinconjugating enzyme E2 and ubiquitin ligase E3 [] E3 ligases transfer activated ubiquitin fromE2 to specific substrates thereby forming mono or polyubiquitinated proteins to activate proteasomemediated proteolysis signal transduction endocytosis etc [] E3 ligases arecrucial as they catalyse target ubiquitination and enable theformation of polyubiquitin chains that enhance the complexity of ubiquitination in physiological and pathological processes Although dysregulated ubiquitination is involved inthe development of various types of immune pathologieseg systemic lupus erythematosus rheumatoid arthritisand ‚ammatory bowel disease [IBD] [ ] there is limitedknowledge regarding the role of RINGtype ligases in‚ammationassociated pathways In this review we havefocused on IBD owing to its complex pathogenesis involvement in a wide range of etiological factors including dysregulated ubiquitination The term IBD is used for a group ofchronic autoimmune gastrointestinal disordersincludingmainly Crohn™s disease CD and ulcerative colitis UC []Chronicity of IBD often causes intestinal complications hospitalisation steroid dependency and surgery in diagnosedpatients [] Although significant progress has been made inunderstanding the nature of IBD the underlying interactingmechanism involving ubiquitination RINGtype ligases andonset of IBD remains to be fully understood Ubiquitination Mediated by RINGType LigasesHuman cells express more than E3 ubiquitin ligases thatare classified into three types based on their catalyticdomains RING HECT homologous to the E6AP carboxylterminus and a recently identified RBR RINGbetweenRINGRING type of E3 ligases [ ] The RING domainhas a crossbraced structure with two atoms of zinc that catalyse the direct transfer of ubiquitin from the E2Ubiquitin 0cMediators of Inflammationthioester to the substrate [] Figure Apart from catalysing monoubiquitination RINGtype E3 ligases also elongatehomotypic polyubiquitin chains with varying linkage specificities such as that on Lys48 during the proteasomal targeting ofsubstrates and Lys63 in signal transduction thereby modulating proteins for proteolytic and nonproteolytic activity []However their roles in catalysing other less common typesof ubiquitination including atypical homotypic eg Lys6Lys11 Lys27 Lys29 Lys33 and Met1 [] heterotypic andbranched polyubiquitination remain ambiguous The efficiency of RINGtype E3 ligases in ubiquitination depends onmultiple factors such as substrate modification phosphorylation of E2E3 enzymes autoubiquitination by E3 ligases andpseudosubstrate competition [] The role of the RINGtype ligases and their sophisticated functional mechanism ofubiquitination will be discussed in the following sections Signalling Pathways Regulated by RINGType LigaseslectinlikereceptorsRLRs Ctype Pathogen Recognition Under physiological conditionspattern recognition receptors PRRs comprise tolllikereceptors TLRs retinoic acid inducible gene RIG Ilikereceptorsandnucleotidebinding oligomerisation domainlike receptorsPRRs recognise pathogenassociated molecular patternsPAMPs and trigger the activation of downstream eï¬ectorsin innate immune responses [] For ‚ammatory diseasesthat are closely associated with microbiome dysbiosis such asIBD [ ] dysregulation of PRRs and relevant RINGtypeligases may be involved in pathogeninduced ‚ammation TLR Signalling Upon recognising a wide range ofmicrobial components such as lipopolysaccharides flagellinand microbial nucleic acids activated TLRs expressed onantigenpresenting cells trigger eï¬ector T cell responses in‚ammatory diseases [“] RINGtype ligases modulatethe activation of PAMPinduced TLRs By directly bindingwith TLR3 ring finger protein RNF170 catalyses theLys48linked ubiquitination and proteasomal degradationof TLR3 thereby suppressing TLR3mediated innate immunity in macrophages [] On the other hand FcÎ receptorFcÎR IIb an inhibitory FcR on antibodydependent monocyte phagocytosis is targeted by MARCH3 RNF173 forubiquitination and degradation in lipopolysaccharideinduced TLR4 activation [] RIGI Signalling RIGI is a cytoplasmic PRR that recognises viral RNA and triggers the activation of downstreamimmune responses that are associated with both viral infections and noninfectious autoimmune diseases such asenterocolitis [] The deficiency of RIGI aggravates virusinduced cell death in intestinal epithelial cells and inducessusceptibility to chemically induced colitis in mice suggesting the importance of RIGI signalling in intestinal antiviralimmune response [ ] E3 ligaseslike RNF122 andRNF125 mediate Lys48linked RIGI ubiquitination andproteasomal degradation leading to the reduced expressionof infection inducedpro‚ammatory cytokines includingIL6 and type I interferons α and [ ] In contrastindependent of its E3 ligase activity RNF123 binds with theCARD domain of RIGI and melanoma diï¬erentiationassociated gene to compete with the mutual downstreamadaptor mitochondrial antiviral signalling protein MAVSand inhibit RLRmediated antiviral response [] Unlikethe aforementioned RINGtype ligases that directly targetRLRs RNF114 negatively regulates RLR signalling by polyubiquitinating and inducing the proteasomal degradation ofMAVS []is essential Pro‚ammatory Pathways Nuclear Factor Kappa B NFκB Signalling The NFκBpathway is one of the most wellstudied pro‚ammatorypathways regulated by ubiquitination [] TRAF6 RNF85ubiquitinates the evolutionarily conserved signalling intermediate in TLR activation thatfor TLR4dependent NFκB activation [] RNF183 promotes NFκBsignalling by inducing the ubiquitindependent degradationof IκBα [] TRAF2 RNF117 and TRAF3 RNF118induce Lys48linked ubiquitination and proteasomal degradation of cRel and interferon regulatory factor therebyprohibiting the synthesis of pro‚ammatory cytokines inmacrophages [] MKRN2 RNF62 mediates the polyubiquitination and degradation of the p65 subunit of NFκBthereby inhibiting NFκB signalling [] RNF114 negativelyregulates NFκB signalling and T cell activation by ubiquitinating and stabilising NFκB signalling inhibitors A20 andIκBα [ ] It has also been reported that RNF20 downregulation decreases histone H2B monoubiquitination and leadsto the NFκBdependent transcription of pro‚ammatorycytokines such as IL6 and IL8 [] Nevertheless despitethe formation of heterodimers of RNF40 with RNF20RNF40 alone activates NFκB signalling and upregulatesNFκBdependent transcription by promoting IκB kinaseIKK phosphorylation and p65 nuclear translocation indicating the involvement of NFκBdependent transcriptionin the ubiquitination of substrates other than H2B []MARCH3 RNF173 mediates Lys48linked ubiquitinationand lysosomal degradation of IL1 receptor I and therebyinhibits IL1triggered NFκB activation [] Independentof its E3 ligase activity RNF8 inhibits TNFαinduced NFκBactivation by directly binding with the kinase domain of IKKand interfering with IKKα phosphorylation [] RNF11also exerts a noncanonical role in negatively regulating NFκB signalling RNF11 has high affinity for the E2 enzymeUbc13 and minimal E3 ligase activity that subsequently outcompetes E1 enzymes and other E3 enzymes such as TRAF6[] and impedes the activation of NFκB signalling [] MitogenActivated Protein Kinase MAPK SignallingMAPKs are another family of proteins closely related to‚ammationassociated pathologies such as IBD []Upon stimulation by TNFα TRAF2 is autoubiquitinatedon the Lys63 residue that enables its translocation to thecytoskeletal fraction and activates JNK signalling [ ]In vitro experiments have shown that JNK signalling issuppressed and enhanced in cells overexpressing RNF213 0cMediators of InflammationUbUbE2LysSubstrateRING E3 ligasePolyubiquitinationchain initiationUbE2SubstrateRING E3 ligaseMonoubiquitinationE2SubstrateRING E3 ligaseMultiple monoubiquitinationUbUbE2SubstrateRING E3 ligaseHomotypic polyubiquitinationmodification sites of ubiquitin include Lys6 Lys11 Lys27 Lys29 Lys33 Lys48Lys63 and Met1 SubstrateUb Ub Ub Ub UbChain elongationSubstrateUbUb Ub Ub UbUbUbHeterotypic polyubiquitinationSubstrateUbUbUb Ub UbE2SubstrateRING E3 ligaseBranched polyubiquitinationSubstrateUb Ub Ub Ub UbUb Ub Ub UbFigure Functional mechanisms of RINGtype E3 ligase Acting as a scaï¬olding RINGtype E3 ligase recruits a E2ubiquitin thioester and asubstrate and allows the lysine of substrate to attack the thioester for ubiquitin transfer RINGtype E3 ligases catalyse monoubiquitination ormultiple monoubiquitination by transferring a single ubiquitin to one or several residues of the substrate For polyubiquitination ubiquitinsform eight diï¬erent linkage types including Met1 Lys6 Lys11 Lys27 Lys29 Lys33 Lys48 and Lys63 Apart from homotypic chains RINGtype E3 ligases also catalyse heterotypic chains and branched ubiquitin chains by adopting multiple linkage types and branched topology inthe formation of polyubiquitin chainsand RNF186 respectively [ ] however the mechanismsinvolved in this regulation remain to be understood TRAF7RNF119 upregulatesthe kinase activity of mitogenactivated protein kinase via the WD40 domain and potentiates cell apoptosis via the RING finger domain []Similarly RNF13 mediates endoplasmic reticulum ERstressinduced JNK activation and subsequent cell apoptosisby binding with and promoting the phosphorylation ofthe ER stress sensor endoplasmic reticulum to nucleussignalling [] Janus Kinase JAKSignal Transducer and Activator ofTranscription STAT3 Signalling JAKSTAT3 is one of themajor pro‚ammatory signalling pathways that orchestrate 0cRINGtype ligaseRNF11RNF183RNF2RNF135RNF125TRAF6cCblZNRF2Table RINGtype ligases targeted by noncoding RNAs and the relevant pathwaysMediators of InflammationNoncoding RNAmiR19b3pmiR7miR1395pmiR4853pmiR15bmiR124miR146miR216alncRNA TTN antisense RNA acts as a ceRNA for miR1533pRelevant pathwayNFκB signallingNFκB signallingER stressinduced apoptosisMAPK signallingMAPKERK signallingRIGI signallingTLR signallingTLR signallingPI3KAKT signallingPI3KAKT signallingReference[][][][][][][][][][]the progression of ‚ammatory and autoimmune diseases[] A number of RINGtype ligases modulate JAKSTAT3signalling RNF6 and RNF38 function in catalysing theubiquitinationinduced proteasomal degradation of SH2containing protein tyrosine phosphatase thattargetsphosphorylated STAT3 thereby maintaining STAT3 phosphorylation and activating STAT3 signalling [ ] Incontrast TRAF6 promotes Lys63linked ubiquitination ofSTAT3 and represses STAT3mediated transcription ofdownstream ‚ammationrelated genes such as Creactiveprotein [] Interestingly RNF41 modulates the cell surfaceexpression of JAK2associated cytokine receptors by blocking the cleavage of receptors and enhancing receptor shedding in a ubiquitinationdependent manner [] Phosphatidylinositol 3Kinase PI3K Signalling PI3K isanother classical pathway involved in ‚ammation whereinRINGtype ligases are of crucialimportance MKRN1RNF61 functions in the positivefeedback regulation of sustained PI3KAKT activation upon stimulation by epidermalgrowth factor AKT activation phosphorylates and stabilisesE3 ligase MKRN1 that further ubiquitinates and degradesphosphatase and tensin homologue a PI3KAKT inhibitor[] MKRN2 RNF62 induces the ubiquitindependent degradation of the p85α subunit of PI3K and downregulated AKTphosphorylation suggesting a negative regulatory role ofMKRN2 in PI3KAKT signalling [] Downregulation ofUHRF1 RNF106 represses the phosphorylation of PI3Kand AKT which reveals an underlying interaction betweenUHRF1 and PI3KAKT signalling [] Transforming Growth Factor TGF Signalling TGF signalling functions in immunosuppression and inhibitingthe activity of eï¬ector T cells maintaining Treg diï¬erentiation reducing B cell responsiveness and inducing macrophage anergy [] RNF11 plays a dualrole in themodulation of TGF signalling By competing with Smad7for Smurf2 RNF11 is a positive regulator for TGF signalling and reduces the formation of Smad7Smurf2 complexesthat degrade TGF receptors [] RNF11 is also responsiblefor the ubiquitinationmediated stabilisation of Smad4 thatenhances Smad4dependent TGF signalling by directinteraction [] Notably RNF11 may negatively regulatesignallingbytheenablingformationTGFofSmurf2RNF11 complexes and inducing the ubiquitinationand degradation of the associated molecule with the SH3domain of STAM that promotes TGF signalling []PRAJA RNF70 mediates the ubiquitinationinduced proteasomal degradation of embryonic liver fodrin a Smad4adaptor protein thereby negatively regulating TGF signalling [] Autophagy Accumulating evidence reveals that autophagy contributes extensively to immune cell developmentand cell death and its dysregulation has been implicated inmany autoimmune diseases [] TRAF6 catalyses Lys63linked ubiquitination of BECN1 and stimulates TLRinduced autophagy in macrophages upon pro‚ammatorystimulation [ ] RNF166 has a novel role in antibacterialhost defence owing to its function in inducing the Lys29 andLys33linked ubiquitination of autophagy adaptor p62which mediates the recruitment of p62 to bacteria and initiates bacteria engulfment [] Noncoding RNAs Since dysregulated noncoding RNAsare involved in the progression of ‚ammatory diseases[] the posttranscriptional regulation of RINGtype ligasesby noncoding RNAs may play critical roles in potential‚ammationrelevant signalling pathways Until now quitea few microRNAs have been proved to posttranscriptionallyregulate RINGtype ligases by hampering translation orinducing mRNA degradation Table [ “] Nevertheless although the other two major types of noncodingRNAs long noncoding RNAs and circular RNAs also havediverse functions in ‚ammatory diseases eg competingendogenous RNA [ceRNA] transcription regulation andRNAbinding protein sponges [ ] to what extent theymodulate RINGtype ligases awaits further analysis RINGType Ligases in IBD Pathogenesis of IBD The pathogenesis of IBD has beenelucidated over the past years More than loci have beenimplicated in increased genetic risk for IBD that correlate withthe functioning of cellular processes such as innateadaptiveimmune responseintestinal mucosal barrier homeostasis 0cMediators of InflammationTable Roles of RINGtype ligases in IBD patients and animal models of colitisRINGtypeligaseRoleIBD patientsAnimal model of colitisReferenceRNF186ControversialIncrease risk of UC and ERstressinduced apoptosisAttenuate ER stress and maintain intestinalpermeability in DSSinduced mouse model[“]of colitisRNF20RNF40Anti‚ammatoryPro‚ammatoryDecrease in the colonic tissueProtect mice from DSSinduced colitis andfrom UC patientsmaintain intestinal barrier”Activate NFκB signalling in DSSinducedmouse model of colitis[][][][“][ ]RNF183Pro‚ammatoryIncrease in the colonic tissuefrom CD and UC patientscorrelate with endoscopicindex of disease severityUHRF1Anti‚ammatory”TRAF2Anti‚ammatoryIncrease in the colonic tissuefrom CD and UC patientsTRAF3Anti‚ammatoryTRAF5Anti‚ammatoryIncrease in the plasma andcolonic tissue from CD andUC patientsIncrease in the plasma andcolonic tissue from CD andUC patientsIncrease in TNBSinduced mouse modelof colitisRegulate TNFα expression in mice withDSSinduced colitis and zebrafishModulate the proliferation anddiï¬erentiation of Treg cellsModulate colonic microbiotacomposition pro‚ammatorycytokine expression and immunecell ltration in mice withDSSinduced colitisRegulate pro‚ammatory cytokineexpression and mitigate ‚ammatorydamage in DSSinduced mousemodel of colitis[ ]Inhibit IL17mediated pro‚ammatorypathway in TNBSinduced mousemodel of colitisControl pro‚ammatory cytokineexpression and protect mice againstexperimental colitis[“]and autophagy suggesting the involvement of multiple factorsin shaping the procolitogenic environment during the development of IBD [ ] IBD patients manifest with abnormalities in the composition of gut microbiota such as decreasedbacterial diversity increased proportion of harmful bacterialstrains and decreased proportion of protective probioticswhich trigger pro‚ammatory intestinal pathogenic immuneresponses and contribute to the pathogenesis of IBD [ ]Elevated levels of pro‚ammatory cytokines eg IL1 IL6and IL23 and activation of adaptive eg Th1 Th2 Th9and Th17 cells and innate immune cells eg neutrophilsand NK cells constitute a synergistic ‚ammatory networkthat induces intestinal mucosal ‚ammation and sustainedactivation of multiple pro‚ammatory signalling pathways[ ] IL1 familyinduced NFκB IL6induced STAT3MAPK and PI3K signalling pathways are pivotal in intestinal‚ammation [ ] TGF signalling can mitigateimmune cell hyperactivation but also causes the formation ofintestinal strictures in chronic intestinal ‚ammation []Autophagy is involved in the regulation of immune cell function thus defective autophagy also plays an important role inIBD pathogenesis [] A dysfunctional gut barrier and subsequent increased intestinal permeability are also consideredimportant etiologic factors in the development of IBD thatresult in the uncontrolled exchange of materials between theintestinal lumen and internal environment A compromisedgut barrier is often attributed to the pro‚ammatory stimulation and subsequent downregulation of sealing tight junctionproteins eg claudin5 and claudin8 and upregulation ofporeforming tight junction proteins eg claudin2 [“]Since multiple etiologic factors function in the pathogenesisof IBD in a synergistic manner further research is warrantedto understand the dynamics between these players Role of RINGType Ligases in IBD Despite the limitedknowledge on RINGtype ligases in IBD research suggestsa correlation between RINGtype ligases and IBD pathogenesis Table Genomewide association studies have identified RNF186 as one of the genes associated with susceptibilityto UC the diseasecoding variant of RNF186 involves analtered RING domain [ ] The truncated RNF186 lacking the second transmembrane domain is associated withprotecting individuals against developing UC by inhibitingthe ER localisation of RNF186 and subsequent Lys29 andLys63linked polyubiquitination of proapoptotic BCL2 interacting protein under ER stress [ ] Notably RNF186functions diï¬erently in a dextran sulfate sodium DSSinduced mouse model of colitis RNF186deficient mice 0cMediators of Inflammationdevelop more severe colitis during DSS administration andtheir colonic epithelial cell exhibits enhanced signs of ER stressand apoptosis [] RNF186 also modulates intestinal barrierfunction by mediating the Lys48linked ubiquitination of tightjunction protein occludin and RNF186 deficiency increasesintestinal permeability in RNF186 knockout mice [] ThusRNF186 targets diï¬erent substrates and has a complex association with gut ‚ammationApart from the reduced expression of RNF20 in the colonsamples from UC patients homozygous RNF20knockoutmice die due to embryonic lethality and heterozygous miceare susceptible to DSSinduced colitis with increased intestinal permeability suggesting the anti‚ammatory role ofRNF20 [] RNF40 knockout mice exhibit mitigated gut‚ammation upon treatment with DSS this can be attributed to the attenuated activation of NFκB signalling []The upregulation of RNF183 in the colonic tissue from IBDpatients and 246trinitrobenzenesulfonic acid TNBSinduced mouse model of colitis indicates its pro‚ammatory function probably by promoting the ubiquitinationinduced degradation of IκBα []Because UHRF1catalysed histone H3 monoubiquitination recruits and stimulates DNA methyltransferase toDNA methylation sites and thereby maintains DNA methylation UHRF1 participates in the epigenetic control of multiple genes such as TNFα [ ] Mice with macrophagesdeficient for UHRF1 manifest with TNFα overexpressionand aggravated DSSinduced colitis Also the loss of functionin UHRF1 reduces the methylation of the TNFα promoterin macrophages indicating the regulatory role of UHRF1 inthe mouse model of colitis [] Similarly an in vivo studyin zebrafish has revealed that loss in function of UHRF1 leadsto defects in the epigenetic regulation of TNFα promotermethylation and elicits elevated TNFα expression in ‚ammatory processes including intestinal epithelial cell apoptosis neutrophil recruitment and weakened intestinal barrierfunction [] However UHRF1 may eï¬ect diï¬erentlyamong subtypes of regulatory T Treg cells as UHRF1 maintains the proliferation and maturation of colonic Treg cellsbut inhibits the diï¬erentiation of peripheral induced Tregcells in the development of colitis [ ]Studies have shown the diverse roles involved with theupregulation of TRAFs including TRAF1235 in the bloodor colonic mucosa of IBD patients [ ] DSSinducedcolitis models of TRAF2 and TRAF3deficient mice revealsimilar functions of TRAF2 and TRAF3 as negative regulators of experimental colitis by decreasing pro‚ammatorycytokines and reducing the ltration of immune cells inthe colon [] In another study TRAF2deficient micedevelop severe spontaneous colitis and exhibit altered colonicmicrobiota composition indicating the anti‚ammatoryrole of TRAF2 in controlling colonic microbiota [] TRAF3also acts as a colitis regulator by binding with the IL17receptor and interfering with the IL17mediated pro‚ammatory pathway in mice with TNBSinduced colitis []Although TRAF5 RNF84 promotes the ubiquitinationand stabilisation of the retinoic acidrelated orphan receptorÎt that mediates pro‚ammatory Th17 cell diï¬erentiationand IL17AIL17F expression [ ] TRAF5deficientmice exhibit aggravated experimental colitis and upregulation of pro‚ammatory cytokines [] The complex function of TRAF5 needs further analysis DiscussionAs the importance of RINGtype E3 ligases is graduallyunveiled there are still problems to be solved Firstly apartfrom the canonical role of RINGtype ligases in modulatingkey signalling pathways and their downstream adaptors asE3 ubiquitin ligases some RINGtype ligases interfere withthe ubiquitination cascade by competition or direct interaction with other E3 ligases [] Owing to the variety ofRINGtype ligases and substrate specificity of E3 ligases thepotential competition among RINGtype ligases in regulatingimmune response remains to be fully understood Secondlydiï¬erences in RING E3 ligasemediated target ubiquitinationcan also be attributed to the variance in length and linkagetype of ubiquitin chains Although RINGtype ligases function in proteolytic degradation and signal transduction bycatalysing Lys48linked and Lys63linked ubiquitinationrespectively their roles in catalysing less common linkagetypes of homotypic polyubiquitin chains such as Lys11linked ubiquitination [] and the outcome of such polyubiquitination are still obscureRINGtype ligases form a sophisticated but importantubiquitination network wherein the expression and functionof RINGtype ligases are also ‚uenced reciprocally in physiological and pathological processes Understanding themechanisms employed by RINGtype ligases in modulating‚ammationassociated pathways by catalysing atypicallinkages and aï¬ecting signaltransduction may furtherexplain the interaction between RINGtype ligases and IBDSimilarly research on heterotypic polyubiquitin chains isalso important to unveil these underlying mechanismsIn this review we have highlighted the roles of RINGtype ligases in PAMP recognition and modulation of‚ammationassociated pathways that are crucial etiologicalfactors in the development of autoimmune diseases Accumulating evidence shows that many RINGtype ligases areinvolved in ‚ammationassociated pathways such as pro‚ammatory NFκB MAPK JAKSTAT3 and PI3K signalling and anti‚ammatory TGF signalling Subsequentlywe have discussed the role of RINGtype ligases in the pathogenesis of IBD via ‚ammationrelated pathways Patientswith IBD exhibit the diï¬erential expression of specific RINGtype ligases such as TRAFs However there are limited studies on the potential clinical value of RINGtype ligases inpredicting or treating IBD Thus far there have been a fewattempts to use RINGtype ligases as predictive biomarkersand therapeutic targets in treating cancer RNF43 modulatesWnt signalling and has been used to target colorectal cancerand pancreatic ductal carcinoma [ ] Neverthelessthe potential of RINGtype ligases in autoimmune diseasesespecially in IBD needs to be understood in greater detailfuture research on the expression profile ofThereforeRINGtype ligases in the gastrointestinaltract and thedetailed mechanisms is warranted 0cMediators of InflammationConflicts of InterestThe authors have no conflict of interest to discloseAuthors™ ContributionsThe guarantor of the article is Shenghong Zhang ShenghongZhang designed the study Shenghong Zhang Liguo ZhuYing Li and Longyuan Zhou wrote and revised the manuscript Guang Yang Ying Wang Jing Han and Li Li revisedthe important intellectual content of the manuscript All theauthors approved the final version Liguo Zhu Ying L andLongyuan Zhou contributed equally to the workAcknowledgmentsThis study was supported by the National Natural ScienceFoundation of China under Grants and Guangdong Science and Technology Department under Grant 2017A030306021 and FundamentalResearch Funds for the Central Universities under Grant19ykzd11References[] D Popovic D Vucic and I Dikic œUbiquitination in diseasepathogenesis and treatment Nature Medicine vol no pp “ [] D Aki Q Li H Li Y C Liu and J H Lee œImmune regulation by protein ubiquitination roles of the E3 ligases VHLand itch Protein Cell vol no pp “ [] C M Pickart œMechanisms underlying ubiquitinationAnnual Review of Biochemistry vol no pp “[] H Hu and S C Sun œUbiquitin signaling in immuneresponses Cell Research vol no pp “ [] R Hodson œInflammatory bowel disease Nature vol no p S97 [] L PeyrinBiroulet E V Loftus Jr J F Colombel and W JSandborn œThe natural history of adult Crohnʼs disease inpopulationbased cohorts The American Journal of Gastroenterology vol no pp “ [] N Zheng and N Shabek œUbiquitin ligases structure function and regulation Annual Review of Biochemistryvol no pp “ [] F E Morreale and H Walden œTypes of ubiquitin ligasesCell vol no pp “248e1 [] C E Berndsen and C Wolberger œNew insights into ubiquitin E3 ligase mechanism Nature Structural MolecularBiology vol no pp “ [] R J Deshaies and C A P Joazeiro œRING domain E3 ubiquitin ligases Annual Review of Biochemistry vol no pp “ [] Y Kulathu and D Komander œAtypical ubiquitylation theunexplored world of polyubiquitin beyond Lys48 and Lys63linkages Nature Reviews Molecular Cell Biology vol no pp “ [] S Akira œInnate immunity to pathogens diversity in receptors for microbial recognition Immunological Reviewsvol no pp “ [] X C Morgan T L Tickle H Sokol œDysfunction ofthe intestinal microbiome in ‚ammatory bowel diseaseand treatment Genome Biology vol no p R79[] R B Sartor and G D Wu œRoles for intestinal bacteriaviruses and fungi in pathogenesis of ‚ammatory bowel disapproaches Gastroenterologyeasesvol no pp “339e4 therapeuticand[] T Kawai and S Akira œThe role of patternrecognition receptors in innate immunity update on tolllike receptors NatureImmunology vol no pp “ [] J Q Chen P Szodoray and M Zeher œTolllike receptorpathways in autoimmune diseases Clinical Reviews inAllergy and Immunology vol no pp “ [] J H Park L PeyrinBiroulet M Eisenhut and J I ShinœIBD immunopathogenesis a comprehensive review of‚ammatory molecules Autoimmunity Reviews vol no pp “ [] X Song S Liu W Wang Z Ma X Cao and M Jiang œE3ubiquitin ligase RNF170 inhibits innate immune responsesby targeting and degrading TLR3 in murine cells Cellular Molecular Immunology vol no pp “ [] K Fatehchand L Ren S Elavazhagan œTolllikereceptorIIbFcÎRIIb via MARCH3 proteinmediated ubiquitinationThe Journal of Biological Chemistry vol no pp “ ligands downregulate FcÎ receptor[] T Matsumiya and D M Staï¬orini œFunction and regulationof retinoic acidinducible geneI Critical Reviews in Immunology vol no pp “ [] Y Hirata A H Broquet L Menchén and M F Kagno﬜Activation of innate immune defense mechanisms by signaling through RIGIIPS1 in intestinal epithelial cellsJournal of Immunology vol no pp “[] W Wang M Jiang S Liu œRNF122 suppresses antiviraltype I interferon production by targeting RIGI CARDs tomediate RIGI degradation Proceedings of the NationalAcademy of Sciences ofthe United States of Americavol no pp “ [] K Arimoto H Takahashi T Hishiki H Konishi T Fujitaand K Shimotohno œNegative regulation of the RIGI signaling by the ubiquitin ligase RNF125 Proceedings of theNational Academy of Sciences of the United States of Americavol no pp “ [] S Wang Y K Yang T Chen œRNF123 has an E3ligaseindependentreceptormediated antiviral signaling EMBO Reports vol no pp “ in RIGIlikefunction[] B Lin Q Ke H Li N S Pheifer D C Velliquette and D WLeaman œNegative regulation of the RLH signaling by the E3ubiquitin ligase RNF114 Cytokine vol pp “ [] J Chen and Z J Chen œRegulation of NFκB by ubiquitination Current Opinion in Immunology vol no pp “ [] Y Min M J Kim S Lee E Chun and K Y Lee œInhibitionof TRAF6 ubiquitinligase activity by PRDX1 leads to inhibition of NFKB activation and autophagy activation Autophagy vol no pp “ [] Q Yu S Zhang K Chao œE3 ubiquitin ligasein ‚ammatory bowelRNF183 is a novelregulator 0cMediators of Inflammationdisease Journal of Crohn's Colitis vol no pp “ [] J Jin Y Xiao H Hu œPro‚ammatory TLR signallingis regulated by a TRAF2dependent proteolysis mechanismin macrophages Nature Communications vol no article [] C Shin Y Ito S Ichikawa M Tokunaga K SakataSogawa and T Tanaka œMKRN2 is a novel ubiquitin E3ligase for the p65 subunit of NFκB and negatively regulates ‚ammatory responses Scientific Reports vol no article [] M S Rodriguez I Egaña F LopitzOtsoa œThe RINGubiquitin E3 RNF114 interacts with A20 and modulatesNFκB activity and Tcell activation Cell Death Diseasevol no article e1399 [] K Heyninck and R Beyaert œA20 inhibits NFkappaB activation by dual ubiquitinediting functions Trends in Biochemical Sciences vol no pp “ [] O Tarcic I S Pateras T Cooks œRNF20 links histoneH2B ubiquitylation with ‚ammation and ‚ammationassociated cancer Cell Reports vol no pp “ [] R L Kosinsky R L Chua M Qui œLoss of RNF40decreases NFκB activity in colorectal cancer cells andreduces colitis burden in mice Journal of Crohn's Colitisvol no pp “ [] H Lin D Gao M M Hu œMARCH3 attenuates IL1triggered ‚ammation by mediating K48linked polyubiquitination and degradation of IL1RI Proceedings of theNational Academy of Sciences of the United States of America Answer:

Thyroid_Cancer

"Oral squamous cell carcinoma is one of the most common causes of malignancy‘associated death Early diag‘nosis of oral squamous cell carcinoma OSCC is important in patient treatment and prognostic evaluation Due to the lack of significant therapeutic benefit the ‘year survival rate has not improved Therefore effective novel markers are needed to improve diagnosis To determine novel promising diagnostic biomarkers for OSCC upregulated and downregulated differentially expressed genes were screened from OSCC tissues using an RNA microarray The results suggested that minichro‘mosome maintenance protein MCM5 mRNA was significantly overexpressed in OSCC tissues compared with that in adjacent normal tissues Moreover silencing of MCM5 expression an OSCC cell line SCC‘ significantly impaired proliferation and colony formation Furthermore negative regulation of the mRNA and protein expression of MCM5 and demonstrated that MCM5 served as a cancer‘promoting gene modulating OSCC cell proliferation through induced G2M phase arrest In this process the mRNA expression of cyclin E and cyclin‘dependent kinase was downregulated while p21 expression was upregu‘lated These results suggested that MCM5 may be an important pathogenic factor of OSCC High expression levels of MCM5 may serve as a marker for the early diagnosis of OSCC IntroductionOral squamous cell carcinoma OSCC affects individuals worldwide annually Presently the clinical treatment of OSCC is primarily surgery radiotherapy or chemotherapy Over the past decades the overall survival Correspondence to Dr Xiaofeng Wang Department of Stomatology China‘Japan Union Hospital of Jilin University Xiantai Changchun Jilin PR ChinaE‘mail wangxiaofengjlueducnContributed equallyKey words minichromosome maintenance protein cell cycle oral squamous cell carcinoma biomarker microarrayOS rate of patients with OSCC has not significantly improved with a ‘year survival rate of ‘ Insufficient sensitive and specific biomarkers may lead to the diagnosis of OSCC at advanced stages Therefore it is necessary to identify novel biomarkers for the early diagnosis and treatment of OSCCRecently with the continuous development of sequencing technology researchers can efficiently distinguish differentially expressed genes DEGs by transcriptome sequencing which allows screening of potential tumor markers or therapeutic drug targets For example a number of new potential tumor markers have been found in human malignancies such as breast cancer epithelial ovarian cancer and glioma Minichromosome maintenance protein MCM5 a member of mini‘chromosome maintenance family of proteins plays an important role in cell proliferation and DNA replica‘tion Some studies have confirmed that MCM5 is highly expressed in numerous human malignancies such as renal cell carcinoma pancreatic ductal adenocarcinoma cervical cancer and skin cancer Further studies have found that high expression of MCM5 is closely associ‘ated with the clinicopathological features of specific cancer types For example overexpression of MCM5 is significantly associated with overall survival rate OS in hepatocellular carcinoma Moreover increased expression of MCM5 is positively correlated with larger tumor size positive lymph node metastasis more advanced clinical stage higher histo‘logical grade deeper invasion depth and perineural invasion of OSCC However thus far the expression function and potential mechanisms of MCM5 in OSCC are still unclear Therefore the present study aimed to analyze the DEGs in OSCC using a microarray screen for MCM5 and further eval‘uate the possible functions of MCM5 in OSCC The present results may provide evidence to support the value of MCM5 as a biomarker or a therapeutic target of OSCC Materials and methodsTissue sampling Pairs of OSCC tissues and adjacent normal tissues were obtained from patients undergoing resec‘tion operations at the China‘Japan Union Hospital of Jilin University Changchun China Clinicopathological data were also collected No patient received preoperative treatment including radiotherapy or chemotherapy No other inclu‘sionexclusion criteria were used Matched normal OSCC 0cHAO MCM5 IS A POTENTIAL EARLY DIAGNOSTIC MARKER FOR ORAL SQUAMOUS CELL CARCINOMAtissues were obtained from a segment of the resected specimens ‘cm away from the tumor Pathological analysis was used to identify surgically resected specimens Pathological analysis was performed by our group with no specific diagnostic guide‘lines Three paired samples were obtained for transcriptome sequencing Then to confirm the reliability of sequencing data the samples size was increased using the remaining paired tissues and analyzed using quantitative qPCR All compari‘sons between OSCC tissues and adjacent normal tissues were performed simultaneously The Kaplan‘Meier analysis of OS and survival curves were from the Cancer Genome Atlas database TCGA wwwcancergovThe study was approved by the Ethics Committee of China‘Japan Union Hospital of Jilin University Written informed consent was obtained from all patients who participated in this studyTranscriptome sequencing and functional annotation analysis Total RNA extraction RNA library construction and transcriptome sequencing were performed at Sangon Biotech Co Ltd The biological relevance of unique genes in expression profiles of DEGs were screened according to the threshold values of log2fold‘change‰¥ and P005 Then the differentially expressed mRNAs were analyzed by Gene Ontology GO whose annotations were downloaded from Gene Ontology httpgeneontology UniProt sparqluniprot and NCBI wwwncbinlmnihgov Significant GO categories were identified using Fisher's exact test with a P005 which indicated that signifi‘cantly upregulated genes in the set of DEGs were assigned to a specific functional category more often than expected by chance Significant pathways of the DEGs were then analyzed and identified according to the Kyoto Encyclopedia of Genes and Genomes KEGG database wwwkeggjpCell lines The human tongue squamous cell carcinoma SCC‘ and CAL‘ were obtained from the American Type Culture Collection CAL‘ cells were cultured in DMEM medium with fetal bovine serum FBS Gibco Thermo Fisher Scientific Inc Uml penicillin and streptomycin at ˚C in a humidified atmosphere containing CO2 SCC‘ cells were cultured in MEM medium with FBS NEAA Uml penicillin and streptomycin at ˚C in a humidified atmosphere containing CO2MCM5‘specific siRNA and transfection Three MCM5 siRNA sequences were synthesized by Suzhou GenePharma Co Ltd The sequences were as follows '‘' siRNA‘ Forward CCG ACU ACU UGU ACA AGC ATT and reverse UGC UUG UAC AAG UAG UCG GTT siRNA‘ forward CCA AAU GUC UAU GAG GUC ATT and reverse UGA CCU CAU AGA CAU UUG GTT siRNA‘ forward GUC GUC UGU AUU GAC GAG UTT and reverse ACU CGU CAA UAC AGA CGA CTT and scrambled forward UUC UCC GAA CGU GUC ACG UTT and reverse ACG UGA CAC GUU CGG AGA ATT The mock was an untransfected empty vector serving as the controlSCC‘ cells 45x104 cellswell were cultured in ‘well plates overnight at ˚C Then cells were transfected with nM negative control siRNA or MCM5 siRNA using Lipofectamine® Transfection Reagent Invitrogen Thermo Fisher Scientific Inc according to the manufacturer's protocol After h transfection cells were collected and then RNA was extracted by TRIzol® regent Invitrogen Thermo Fisher Scientific Inc for further experiments as indicatedReverse transcription RTqPCR RNA extracted from tissues samples were reverse transcribed into cDNA using a GoScript Reverse Transcription System kit Monad httpwwwmonad‘biotechcom according to the manufacturer's instructions Relative mRNA expressions were quantified by qPCR using the QuantiTect SYBR Green PCR kit Roche Diagnostics and normalized to GAPDH using primers listed in Table I The cycling parameters were cycles of ˚C for sec ˚C for sec and ˚C for sec Relative mRNA levels were assessed by the comparative ‘ΔΔCq method All analyses of the samples were conducted in triplicateFor association of MCM5 expression levels with clinico‘pathologic features of OSCC the relative expression levels of MCM5 were evaluated using qPCR as aforementioned Relative mRNA levels of paired samples adjacent vs cancer tissues were assessed by the comparative ‘ΔΔCq method A ratio was considered to have high MCM5 expression whereas a ratio ‰¤ was considered to have low MCM5 expression Cell proliferation assay To analyze cell proliferation a Cell Counting Kit CCK‘ Dojindo Molecular Technologies Inc was used according to the manufacturer's instructions In total cells were cultured to each well of ‘well plates After h post siRNA transfection cells were incubated for and h CCK‘ reagent was added h prior to detec‘tion The OD was measured at nm using a microplate reader Bio‘Tek The experiment was performed three timesColony formation assay This assay was performed according to a previous study Briefly cells were cultured in ‘well plates at cellswell followed by culture in complete medium DMEM supplemented with FBS Uml peni‘cillin and streptomycin for weeks The colonies were fixed with methanol for min at room temperature and washed with PBS and stained with crystal violet at room tempera‘ture Beyotime Institute of Biotechnology solution for min A cell colony was defied as a group formation of at least cells Finally formed colonies were observed and images were captured under a light microscope at magnification x200 Cell migration analysis using scratching assays SCC‘ cells were cultured in a ‘well plate at 5x105 cellswell overnight at ˚C Then the cells were scratched and scraped with fresh DMEM Cells were observed and images were captured under a light microscope magnification x200 at h The width of the scratch was measured and referred to as Wbefore Then the cells were starved with no FBS and returned to the incubator for h at ˚C The width of the same scratch was measured and referred to as Wafter Migrating distance was calculated by subtracting Wafter from Wbefore The migration of the control was set as Western blot analysis The protein extractions from the cells were isolated using RIPA Lysis Buffer P0013B Beyotime Institute of Biotechnology Then ‘ µg protein was loaded per lane on a gel resolved using SDS‘PAGE and electroblotted onto 0cONCOLOGY LETTERS Table I Primers used for reverse transcription quantitative‘PCRmRNA MCM5 P21 CyclinE CDK2 GAPDH MCM5 minichromosome maintenance protein cyclin‘dependent kinase Forward primer '‘' GATCCTGGCATTTTCTACAG GGAGACTCTCAGGGTCGAAA TTCTTGAGCAACACCCTCTTCTGCAGCC GCTAGCAGACTTTGGACTAGCCAG AGAAGGCTGGGGCTCATTTG Reverse primer '‘'CCCTGTATTTGAAGGTGAAGGGATTAGGGCTTCCTCTTGGTCGCCATATACCGGTCAAAGAAATCTTGTGCCAGCTCGGTACCACAGGGTCAAGGGGCCATCCACAGTCTTCFigure Volcano plots and KEGG pathway analysis of differentially expressed genes between OSCC cancer tissue group and adjacent normal tissue group A Differences in gene expression profiles between OSCC cancer tissue group and adjacent normal tissue group The horizontal line corresponds to a ‘fold log2 scaled change up or down and the vertical line represents P005 The red points on the plot represent the differentially expressed genes with a ‘fold change upregulation while the green points represent downregulation with P005 B Top KEGG enrichment terms of DEG in OSCC The vertical axis represents the pathway category and the horizontal axis represents the enrichment score [‘lgP‘value] of the pathway LgP was the logarithm of P‘value and P005 was considered significant KEGG Kyoto Encyclopedia of Genes and Genomes OSCC oral squamous cell carcinoma DEG different expressed genes OSCC oral squamous cell carcinomaPVDF membranes Roche Diagnostics After blocking at ˚C for h non‘fat milk in PBS plus Tween the blots were incubated with primary antibodies against anti‘MCM5 D220960‘ BBI Life Sciences anti‘p21 Proteintech anti‘cyclin E ProteinTech Group Inc and anti‘β‘actin D16H11 CST Biological Reagents Co Ltd at ˚C for h Western blots were probed with secondary antibodies and detected using the Odyssey infrared imaging system LI‘COR BiosciencesCell cycle analysis SCC‘ cells were harvested and fixed with ethanol on ice for min and then washed with PBS to decant the ethanol solution Then the cells were suspended and stained by PI and RNase A treatment Cell cycle analysis was performed using a flow cytometer FACSARIA…¡ BD Biosciences The data was performed using CXP Analysis software Beckman Coulter Inc Statistical analysis All the data analysis was performed using SPSS version SPSS Inc The results are presented as mean SD Associations between MCM5 mRNA expres‘sion and clinicopathological factors were analyzed using the Pearson's χ2 test or Fisher's exact test The differences in MCM5 mRNA expression between carcinoma and adjacent normal tissues were evaluated by a paired t‘test One‘way ANOVA followed by Tukey's post hoc test was used to determine the differences between groups and unpaired t‘tests for the rest of the data The survival rate was calculated by the Kaplan‘Meier method and compared using the log‘rank test P005 was considered to indicate a statistically significant difference All experiments were performed in triplicateResultsRNA sequencing and functional annotation analysis To explore novel biomarkers for OSCC the RNAs derived from tissue samples by sequencing were detected Three matched primary OSCC tissues and adjacent normal tissues were randomly selected As shown in Fig 1A the aberrant expression of genes was detected in tissue samples To screen 0cHAO MCM5 IS A POTENTIAL EARLY DIAGNOSTIC MARKER FOR ORAL SQUAMOUS CELL CARCINOMATable II Twenty randomly selected differentially expressed genes between oral squamous cell carcinoma and adjacent tissue samples The genes selected randomly instead of listing based on rank or fold‘change in expressionGene ID ENSG00000160182 ENSG00000205592 ENSG00000171195 ENSG00000126549 ENSG00000090382 ENSG00000161798 ENSG00000161055 ENSG00000107562 ENSG00000214711 ENSG00000106066 ENSG00000184330 ENSG00000137745 ENSG00000243207 ENSG00000107159 ENSG00000183072 ENSG00000196611 ENSG00000171217 ENSG00000178445 ENSG00000100297 ENSG00000127564 Log2 fold‘change ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ Gene nameTFF1MUC19MUC7STATHLYZAQP5SCGB3A1CXCL12CAPN14CPVLS100A7AMMP13PPAN‘P2RY11CA9NKX2‘MMP1CLDN20GLDCMCM5PKMYT1P‘value 881x10‘ 468x10‘ 144x10‘ 140x10‘ 209x10‘ 528x10‘ 959x10‘ 604x10‘ 601x10‘ 846x10‘ 234x10‘ 753x10‘ 222x10‘ 161x10‘ 380x10‘ 154x10‘ 705x10‘ 751x10‘ 565x10‘ 240x10‘ Result Downregulation Downregulation Downregulation Downregulation Downregulation Downregulation Downregulation Downregulation Downregulation Downregulation Upregulation Upregulation Upregulation Upregulation Upregulation Upregulation Upregulation Upregulation Upregulation Upregulation the candidate biomarkers for diagnosing OSCC the DEGs were selected when changes in RNA expression were fold‘changes As shown in Fig 1A aberrant RNAs were significantly downregulated while RNAs were upregu‘lated In order to represent all differentially expressed genes twenty randomly selected dysregulated genes between OSCC and adjacent tissue samples are summarized in Table II The P‘value and log2 fold‘changes of all aberrant expression genes are shown in Table SI The DEGs were selected randomly instead of listing based on rank or fold‘change in expression To explore the role of differentially expressed RNAs in OSCC KEGG pathway analysis was performed Depending on the P‘value and enrichment signal pathways associ‘ated with OSCC were identified Table SII The top KEGG enrichment terms of DEGs are shown in Fig 1B including ˜cell cycle™ ˜pathways in cancer™ ˜cell cycle‘yeast™ ˜meiosis‘yeast™ and ˜cytokine‘cytokine receptor interac‘tion™ Among these it was reported that some genes such as MCM5 cell division cycle ‘related protein kinase and Cyclin‘dependent kinase homolog were primarily enriched in the ˜cell cycle™ pathway Some genes such as lysozyme C statherin and aquaporin‘ were enriched in the ˜saliva secretion™ pathway MCM5 which participated in cell cycle regulation and had high expression in OSCC was selected for further study and it was hypothesized that MCM5 might be a candidate tumor marker for OSCCValidation of MCM5 using RT‘qPCR To further verify the aforementioned expression profile data MCM5 expression levels were investigated using RT‘qPCR in tumor and Figure Relative expression levels of MCM5 mRNA in paired adjacent normal tissues and oral squamous cell carcinoma tissues using quantita‘tive PCR The relative expression data were analyzed by the ‘ΔΔCq method GAPDH was used as an internal control P005 P001 vs adjacent normal tissue MCM5 minichromosome maintenance proteinadjacent normal tissues As shown in Fig MCM5 mRNA was significantly upregulated in of tumor tissues compared with that in matched normal tissues These results showed that MCM5 was highly expressed in OSCC tissues which was in line with the sequencing data Association of MCM5 expression levels with clinicopathologic features of OSCC and survival analysis The results of the potential association between MCM5 expression and clinicopathological features in patients with OSCC are presented in Table III No significant association with MCM5 expression was found for age sex histological differentiation metastasisrecurrence and survival status P05 0cONCOLOGY LETTERS Value n MCM5 expression‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘Low n3 High n12 Table III Association between expression of MCM5 and clinicopathologic features of patients with oral squamous cell carcinoma Characteristic Age years ‰¥ Sex Male Female Histological differentiation Well and Moderate Poor MetastasisRecurrence Yes No Survival status Death Survival P‘valueA Kaplan‘Meier analysis of OS is shown in Fig Analysis of clinical data from TCGA showed that high MCM5 expres‘sion was no associated with a shorter OS in patients with OSCC log‘rank P062 These results suggested that MCM5 might not be a prognostic biomarker for OSCC Inhibitory effect of MCM5 in OSCC cell lines To determine the functional role of MCM5 first MCM5 expression was analyzed using RT‘qPCR in two OSCC cell lines Notably SCC‘ cells expressed significantly higher levels of MCM5 compared with Cal‘ cells P001 Fig 4A Considering that knockdown of MCM5 in the cell line with high MCM5 expres‘sion may bring about more significant changes the SCC‘ cell line was selected for further investigation of the functional role of MCM5 Three specific siRNA sequences were designed to inhibit MCM5 expression and transfected in SCC‘ cells and the impact on MCM5 expression was determined using RT‘qPCR As shown in Fig 4B siRNA1 siRNA2 and siRNA3 transfection decreased MCM5 expression by 651P001 P001 and P001 respectively compared with the negative control Then the efficiencies were confirmed using western blotting Fig 4C The inhibitory effect of siRNA1 and siRNA2 was significant but not found in siRNA3 The results were consistent with those of RNA expression siRNA1 trans‘fection reduced MCM5 expression significantly in SCC‘ cells Therefore siRNA1 was used for subsequent experiments Effect of MCM5 inhibition on proliferation colony formation and migration To determine whether MCM5 regulates cell cycle and modulates cell proliferation in OSCC the effect of inhibiting MCM5 expression on SCC‘ cell proliferation was investigated As shown in Fig 5A the results showed that downregulation of MCM5 had significant anti‘proliferative Figure Survival curves from The Cancer Genome Atlas datasets n518 containing high and low MCM5 expression levels MCM5 minichromosome maintenance protein HR hazard ratio TPM transcripts per millioneffect compared with the negative control P001 Colony formation assays were performed and the results revealed that compared with the number of colonies in the control group downregulation of MCM5 significantly reduced colony formation P001 Fig 5B and C To estimate the impact of MCM5 on OSCC migration scratching assays were conducted and inhibition of MCM5 showed no significant impact on the migration of SCC‘ cells P005 Fig 5D These results suggested that inhibiting MCM5 expression inhibited cell proliferation and colony formation but had no effect on migration in SCC‘ cells\x0cHAO MCM5 IS A POTENTIAL EARLY DIAGNOSTIC MARKER FOR ORAL SQUAMOUS CELL CARCINOMAFigure Inhibition of MCM5 expression in oral squamous cell lines A MCM5 expression in Cal‘ and SCC‘ cells B Efficiency of siRNA‘MCM5 was confirmed by reverse transcription quantitative‘PCR in SCC‘ cells C The efficiency of siRNA‘MCM5 was confirmed by western blot in SCC‘ cells MCM5 minichromosome maintenance protein si small interfering NC negative controlFigure Effects of MCM5 inhibition on antiproliferation colony formation and migration capacity in SCC‘ cells A Downregulation of MCM5 expression suppressed SCC‘ cell proliferation compared with the corresponding control at different time points B Downregulation of MCM5 expression inhibited SCC‘ cells colony formation compared with the corresponding control C Quantification of MCM5 inhibition on colony formation compared with the corresponding control D Cells scratching wounds observed by microscopy E Downregulation of MCM5 had no effect on the migration capacity of SCC‘ cells P001 vs NC MCM5 minichromosome maintenance protein si small interfering NC negative controlEffect of MCM5 inhibition on cell cycle To determine the potential mechanism for the observed proliferation inhibition of SCC‘ cells by MCM5 inhibition cell cycle analysis was performed using flow cytometry As shown in Fig 6A and B after MCM5 inhibition the number of cells were decreased in the G0G1 phase and the S phase but significantly increased in the G2M phase compared with the negative control These results indicated that MCM5 inhibi‘tion significantly induced G2M phase arrest compared with that in the control group\x0cONCOLOGY LETTERS Figure Effects of MCM5 inhibition on cell cycle regulation in SCC‘ cells A Flow cytometry assays were performed to analysis the cell cycle progres‘sion when SCC‘ cells transfected with siRNA‘MCM5 B The bar chart represented the percentage of cells in G0G1 S or G2M phase as indicated C Expression levels of cell cycle‘regulated genes detected by quantitative PCR and normalized to GAPDH D Expression levels of cell cycle‘regulated proteins determined using western blotting β‘actin was used as the loading control Data are presented as mean ± SD P005 P001 vs control group MCM5 minichromosome maintenance protein si small interfering NC negative control CDK2 cyclin‘dependent kinase To elucidate the mechanism underlying this effect the expressions levels of cyclin E cyclin‘dependent kinase CDK2 and p21 related to cell cycle arrest were deter‘mined using RT‘qPCR As shown in Fig 6D MCM5 inhibition decreased both cyclin E and CDK2 mRNA levels but increased the mRNA expression of p21 significantly Then cyclin E and p21 were selected to detect the protein levels using western blotting As shown in Fig 6C cyclin E levels decreased while p21 levels increased in MCM5‘downregulated SCC‘ cells which was consistent with the RT‘qPCR results DiscussionDespite notable progress in cancer research and treatment the survival rate of patients with OSCC has not significantly improved in the past few decades To date there are no effective tumor‘specific biomarkers for the early detection and prognosis prediction of OSCC Several studies have shown that DEGs serve an important role in the development of tumors in different cancer types ‘ However few studies have reported differentially expressed genes in OSCC The present screened genes that regulate the progression of oral cancer by gene expression profiling and found that genes were dysregulated of which DEGs were upregulated and were downregulated DEGs significantly affected GO terms and KEGG pathways MCM5 did not have one of the highest log2 fold‘change values and log10 qValues however MCM5 is one of the differentially expressed genes in cell cycle signaling pathway which was the most signifi‘cant enrichment of differentially expressed genes Therefore MCM5 which regulates the cell cycle was selected for further investigation However previously published studies on biomarkers in OSCC mainly focused on pathological studies The present study not only verified the over‘expression of MCM5 in OSCC but also confirmed using cell experiments that MCM5 affects cell proliferation by regulating the cell cycle MCM5 is a member of the MCM family of proteins and is a component of the starting complex for DNA synthesis MCM5 has been identified as a cell cycle biomarker of aber‘rant proliferation which is associated with the progression of various cancer types Previously MCM5 has been found to be overexpressed in numerous human malignancies such as esophageal thyroid and ovarian cancer For example increased MCM5 levels in urine sediment cells predicts the presence of bladder cancer Inhibition of transcription factor SOX‘ can inhibit the proliferation of skin melanocytes and MCM5 expression is significantly decreased following downregulation of SOX‘ Moreover high expression of MCM5 is associated with poor prog‘nosis and poor malignant status in patients with cervical adenocarcinoma It is well known that immunohistochemistry and western blot‘ting are necessary methods to evaluate protein expression 0cHAO MCM5 IS A POTENTIAL EARLY DIAGNOSTIC MARKER FOR ORAL SQUAMOUS CELL CARCINOMAHowever due to a limited number of tissue samples the present study did not have enough samples for simultaneous qPCR western blotting and immunohistochemistry detection Therefore this is a limitation of the present study However the study did perform qPCR to evaluate the expression of MCM5 at the mRNA level The results demonstrated that of patients with OSCC have high MCM5 expression which is consistent with the results of the aforementioned studies indicating that high MCM5 expression may play an important role in the pathogenesis of OSCC In addition other members of the MCM family of proteins have also served as biological markers of dysplasia and malignancy such as glioma cervical colorectal breast prostate and lung cancer ‘ Therefore some researchers even suggested that changes in MCM5 expression may be a sign of cell cycle disorders It is worth noting that some researchers reported that the high expression of MCM family members may be closely related to tumorigenesis and prognosis For example MCM2 MCM4 and MCM6 are overexpressed in breast cancer of high histological grades MCM7 expression is a potent prognostic marker in non‘small cell lung cancer while MCM5 may be an independent adverse prognostic marker in lung squamous cell carcinoma It is well known that the cell cycle is related to cell proliferation signaling path‘ways In most tumors an increase in the expression level of genes encoding proteins that regulate cell proliferation is observed The abnormal expression of cell‘cycle‘related genes is associated with infinite proliferation of tumors and poor prognosis Thus far only Yu reported the relationship between MCM5 and OSCC The study reported that overexpression of MCM5 in patients with OSCC was significantly associated with tumor site tumor size positive lymph node metastasis later clinical stage higher histological grade deeper infiltration depth and peripheral nerve infiltration However in the present study association between high expression of MCM5 with survival metastasis and poor histologic differentiation was not observed A Kaplan‘Meier analysis of the overall survival rate was not significantly changed in patients with high MCM5 expression compared with patients with low MCM5 expression It was speculated that due to the small number of samples that there were large differences between individuals Therefore in future research a larger sample size should be used to clarify the relationship between high expression of MCM5 and prognosis of OSCC Little is known about the role and potential function of MCM5 in OSCC In the present study a loss‘of‘function analysis was conducted and it was demonstrated that MCM5 participated in regulating cell cycle and cell proliferation in OSCC cells In fact inhibiting the expression of MCM5 in SCC‘ cells resulted in the downregulation of cyclin E and CDK expression and upregulation of p21 expression which ultimately led to G2M phase arrest in oral cancer cells These results further verified that MCM5 is highly expressed in patients with OSCC which promotes the proliferation of OSCC cells and regulates cell cycle In addition it was observed that MCM5 was not only expressed in SCC‘ cells but also expressed in CAL‘ cells Fig 4A Notably according to the ATCC the MCM5 gene had no mutations in either of the two cell lines indicating that the two cell lines selected in this study have similar genetic backgrounds and could be used for the study of MCM5 cell functions Considering MCM5‘knockdown experiments in SCC‘ cells with high MCM5 expression received more significant results SCC‘ cells were selected for follow‘up studies However analyzing the functional effects of MCM5‘knockdown in CAL27 cell lines may provide more information In addition both SCC‘ and CAL‘ cells are transformed cell lines In future investigations untransformed cell lines for multiple comparisons should be used to clarify the role of MCM5 in OSCC Surgical resection is currently the main method to treat OSCC However considering the particularity of the oral struc‘ture surgical resection will lead to a huge impact on patients' quality of life Therefore it is important to find effective diagnostic biomarkers for early detection or to develop targeted drugs for OSCC In the present study it was reported that MCM5 is overexpressed in OSCC and that MCM5 can affect cell proliferation by regulating cell cycle Therefore the results suggested that MCM5 might be one of the important patho‘genic factors of OSCC and is expected to be used as a potential tumor marker for OSCC target drugs The specific mechanism of action of MCM5 is still worthy of further investigationOverall the present study evaluated differentially expressed genes using sequencing patterns in OSCC tumor tissues and further validated MCM5 upregulated expression in OSCC tissues By knocking down MCM5 expression in SCC‘ cells it was revealed that cell proliferation and colony formation was significantly inhibited by inducing G2M phase arrest The results also suggested that during this process cyclin E and cell cycle‘related gene expression levels were decreased while p21 was significantly upregulated Therefore MCM5 may modulate OSCC cell proliferation by regulating the cell cycle MCM5 is an important pathogenic factor and might have important role as a potential diagnostic marker or drug target for OSCCAcknowledgementsNot applicableFunding This study was funded by the National Natural Science Foundation of China grant no the Bethune Project of Jilin University of China grant no 2018B02 the Education Department of Jilin Province grant no JJKH20190074KJ and Department of Science and Technology of Jilin Province grant no 20190103086JH and 20200201398JC Availability of data and materials The datasets used andor analyzed during the present study are available from the corresponding author on reasonable request The other datasets generated andor analyzed during the current study are available in The Cancer Genome Atlas wwwcancergov Gene Ontology httpgeneon‘tology UniProt sparqluniprot NCBI wwwncbinlmnihgov and Kyoto Encyclopedia of Genes and Genomes wwwkeggjp databases\x0cONCOLOGY LETTERS Authors' contributions HW CZ and CL performed the experiments HW MH and XW analyzed the data MH and HW wrote the manuscript MH and XW designed and supervised

cancer252

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: "Oral squamous cell carcinoma is one of the most common causes of malignancy‘associated death Early diag‘nosis of oral squamous cell carcinoma OSCC is important in patient treatment and prognostic evaluation Due to the lack of significant therapeutic benefit the ‘year survival rate has not improved Therefore effective novel markers are needed to improve diagnosis To determine novel promising diagnostic biomarkers for OSCC upregulated and downregulated differentially expressed genes were screened from OSCC tissues using an RNA microarray The results suggested that minichro‘mosome maintenance protein MCM5 mRNA was significantly overexpressed in OSCC tissues compared with that in adjacent normal tissues Moreover silencing of MCM5 expression an OSCC cell line SCC‘ significantly impaired proliferation and colony formation Furthermore negative regulation of the mRNA and protein expression of MCM5 and demonstrated that MCM5 served as a cancer‘promoting gene modulating OSCC cell proliferation through induced G2M phase arrest In this process the mRNA expression of cyclin E and cyclin‘dependent kinase was downregulated while p21 expression was upregu‘lated These results suggested that MCM5 may be an important pathogenic factor of OSCC High expression levels of MCM5 may serve as a marker for the early diagnosis of OSCC IntroductionOral squamous cell carcinoma OSCC affects individuals worldwide annually Presently the clinical treatment of OSCC is primarily surgery radiotherapy or chemotherapy Over the past decades the overall survival Correspondence to Dr Xiaofeng Wang Department of Stomatology China‘Japan Union Hospital of Jilin University Xiantai Changchun Jilin PR ChinaE‘mail wangxiaofengjlueducnContributed equallyKey words minichromosome maintenance protein cell cycle oral squamous cell carcinoma biomarker microarrayOS rate of patients with OSCC has not significantly improved with a ‘year survival rate of ‘ Insufficient sensitive and specific biomarkers may lead to the diagnosis of OSCC at advanced stages Therefore it is necessary to identify novel biomarkers for the early diagnosis and treatment of OSCCRecently with the continuous development of sequencing technology researchers can efficiently distinguish differentially expressed genes DEGs by transcriptome sequencing which allows screening of potential tumor markers or therapeutic drug targets For example a number of new potential tumor markers have been found in human malignancies such as breast cancer epithelial ovarian cancer and glioma Minichromosome maintenance protein MCM5 a member of mini‘chromosome maintenance family of proteins plays an important role in cell proliferation and DNA replica‘tion Some studies have confirmed that MCM5 is highly expressed in numerous human malignancies such as renal cell carcinoma pancreatic ductal adenocarcinoma cervical cancer and skin cancer Further studies have found that high expression of MCM5 is closely associ‘ated with the clinicopathological features of specific cancer types For example overexpression of MCM5 is significantly associated with overall survival rate OS in hepatocellular carcinoma Moreover increased expression of MCM5 is positively correlated with larger tumor size positive lymph node metastasis more advanced clinical stage higher histo‘logical grade deeper invasion depth and perineural invasion of OSCC However thus far the expression function and potential mechanisms of MCM5 in OSCC are still unclear Therefore the present study aimed to analyze the DEGs in OSCC using a microarray screen for MCM5 and further eval‘uate the possible functions of MCM5 in OSCC The present results may provide evidence to support the value of MCM5 as a biomarker or a therapeutic target of OSCC Materials and methodsTissue sampling Pairs of OSCC tissues and adjacent normal tissues were obtained from patients undergoing resec‘tion operations at the China‘Japan Union Hospital of Jilin University Changchun China Clinicopathological data were also collected No patient received preoperative treatment including radiotherapy or chemotherapy No other inclu‘sionexclusion criteria were used Matched normal OSCC 0cHAO MCM5 IS A POTENTIAL EARLY DIAGNOSTIC MARKER FOR ORAL SQUAMOUS CELL CARCINOMAtissues were obtained from a segment of the resected specimens ‘cm away from the tumor Pathological analysis was used to identify surgically resected specimens Pathological analysis was performed by our group with no specific diagnostic guide‘lines Three paired samples were obtained for transcriptome sequencing Then to confirm the reliability of sequencing data the samples size was increased using the remaining paired tissues and analyzed using quantitative qPCR All compari‘sons between OSCC tissues and adjacent normal tissues were performed simultaneously The Kaplan‘Meier analysis of OS and survival curves were from the Cancer Genome Atlas database TCGA wwwcancergovThe study was approved by the Ethics Committee of China‘Japan Union Hospital of Jilin University Written informed consent was obtained from all patients who participated in this studyTranscriptome sequencing and functional annotation analysis Total RNA extraction RNA library construction and transcriptome sequencing were performed at Sangon Biotech Co Ltd The biological relevance of unique genes in expression profiles of DEGs were screened according to the threshold values of log2fold‘change‰¥ and P005 Then the differentially expressed mRNAs were analyzed by Gene Ontology GO whose annotations were downloaded from Gene Ontology httpgeneontology UniProt sparqluniprot and NCBI wwwncbinlmnihgov Significant GO categories were identified using Fisher's exact test with a P005 which indicated that signifi‘cantly upregulated genes in the set of DEGs were assigned to a specific functional category more often than expected by chance Significant pathways of the DEGs were then analyzed and identified according to the Kyoto Encyclopedia of Genes and Genomes KEGG database wwwkeggjpCell lines The human tongue squamous cell carcinoma SCC‘ and CAL‘ were obtained from the American Type Culture Collection CAL‘ cells were cultured in DMEM medium with fetal bovine serum FBS Gibco Thermo Fisher Scientific Inc Uml penicillin and streptomycin at ˚C in a humidified atmosphere containing CO2 SCC‘ cells were cultured in MEM medium with FBS NEAA Uml penicillin and streptomycin at ˚C in a humidified atmosphere containing CO2MCM5‘specific siRNA and transfection Three MCM5 siRNA sequences were synthesized by Suzhou GenePharma Co Ltd The sequences were as follows '‘' siRNA‘ Forward CCG ACU ACU UGU ACA AGC ATT and reverse UGC UUG UAC AAG UAG UCG GTT siRNA‘ forward CCA AAU GUC UAU GAG GUC ATT and reverse UGA CCU CAU AGA CAU UUG GTT siRNA‘ forward GUC GUC UGU AUU GAC GAG UTT and reverse ACU CGU CAA UAC AGA CGA CTT and scrambled forward UUC UCC GAA CGU GUC ACG UTT and reverse ACG UGA CAC GUU CGG AGA ATT The mock was an untransfected empty vector serving as the controlSCC‘ cells 45x104 cellswell were cultured in ‘well plates overnight at ˚C Then cells were transfected with nM negative control siRNA or MCM5 siRNA using Lipofectamine® Transfection Reagent Invitrogen Thermo Fisher Scientific Inc according to the manufacturer's protocol After h transfection cells were collected and then RNA was extracted by TRIzol® regent Invitrogen Thermo Fisher Scientific Inc for further experiments as indicatedReverse transcription RTqPCR RNA extracted from tissues samples were reverse transcribed into cDNA using a GoScript Reverse Transcription System kit Monad httpwwwmonad‘biotechcom according to the manufacturer's instructions Relative mRNA expressions were quantified by qPCR using the QuantiTect SYBR Green PCR kit Roche Diagnostics and normalized to GAPDH using primers listed in Table I The cycling parameters were cycles of ˚C for sec ˚C for sec and ˚C for sec Relative mRNA levels were assessed by the comparative ‘ΔΔCq method All analyses of the samples were conducted in triplicateFor association of MCM5 expression levels with clinico‘pathologic features of OSCC the relative expression levels of MCM5 were evaluated using qPCR as aforementioned Relative mRNA levels of paired samples adjacent vs cancer tissues were assessed by the comparative ‘ΔΔCq method A ratio was considered to have high MCM5 expression whereas a ratio ‰¤ was considered to have low MCM5 expression Cell proliferation assay To analyze cell proliferation a Cell Counting Kit CCK‘ Dojindo Molecular Technologies Inc was used according to the manufacturer's instructions In total cells were cultured to each well of ‘well plates After h post siRNA transfection cells were incubated for and h CCK‘ reagent was added h prior to detec‘tion The OD was measured at nm using a microplate reader Bio‘Tek The experiment was performed three timesColony formation assay This assay was performed according to a previous study Briefly cells were cultured in ‘well plates at cellswell followed by culture in complete medium DMEM supplemented with FBS Uml peni‘cillin and streptomycin for weeks The colonies were fixed with methanol for min at room temperature and washed with PBS and stained with crystal violet at room tempera‘ture Beyotime Institute of Biotechnology solution for min A cell colony was defied as a group formation of at least cells Finally formed colonies were observed and images were captured under a light microscope at magnification x200 Cell migration analysis using scratching assays SCC‘ cells were cultured in a ‘well plate at 5x105 cellswell overnight at ˚C Then the cells were scratched and scraped with fresh DMEM Cells were observed and images were captured under a light microscope magnification x200 at h The width of the scratch was measured and referred to as Wbefore Then the cells were starved with no FBS and returned to the incubator for h at ˚C The width of the same scratch was measured and referred to as Wafter Migrating distance was calculated by subtracting Wafter from Wbefore The migration of the control was set as Western blot analysis The protein extractions from the cells were isolated using RIPA Lysis Buffer P0013B Beyotime Institute of Biotechnology Then ‘ µg protein was loaded per lane on a gel resolved using SDS‘PAGE and electroblotted onto 0cONCOLOGY LETTERS Table I Primers used for reverse transcription quantitative‘PCRmRNA MCM5 P21 CyclinE CDK2 GAPDH MCM5 minichromosome maintenance protein cyclin‘dependent kinase Forward primer '‘' GATCCTGGCATTTTCTACAG GGAGACTCTCAGGGTCGAAA TTCTTGAGCAACACCCTCTTCTGCAGCC GCTAGCAGACTTTGGACTAGCCAG AGAAGGCTGGGGCTCATTTG Reverse primer '‘'CCCTGTATTTGAAGGTGAAGGGATTAGGGCTTCCTCTTGGTCGCCATATACCGGTCAAAGAAATCTTGTGCCAGCTCGGTACCACAGGGTCAAGGGGCCATCCACAGTCTTCFigure Volcano plots and KEGG pathway analysis of differentially expressed genes between OSCC cancer tissue group and adjacent normal tissue group A Differences in gene expression profiles between OSCC cancer tissue group and adjacent normal tissue group The horizontal line corresponds to a ‘fold log2 scaled change up or down and the vertical line represents P005 The red points on the plot represent the differentially expressed genes with a ‘fold change upregulation while the green points represent downregulation with P005 B Top KEGG enrichment terms of DEG in OSCC The vertical axis represents the pathway category and the horizontal axis represents the enrichment score [‘lgP‘value] of the pathway LgP was the logarithm of P‘value and P005 was considered significant KEGG Kyoto Encyclopedia of Genes and Genomes OSCC oral squamous cell carcinoma DEG different expressed genes OSCC oral squamous cell carcinomaPVDF membranes Roche Diagnostics After blocking at ˚C for h non‘fat milk in PBS plus Tween the blots were incubated with primary antibodies against anti‘MCM5 D220960‘ BBI Life Sciences anti‘p21 Proteintech anti‘cyclin E ProteinTech Group Inc and anti‘β‘actin D16H11 CST Biological Reagents Co Ltd at ˚C for h Western blots were probed with secondary antibodies and detected using the Odyssey infrared imaging system LI‘COR BiosciencesCell cycle analysis SCC‘ cells were harvested and fixed with ethanol on ice for min and then washed with PBS to decant the ethanol solution Then the cells were suspended and stained by PI and RNase A treatment Cell cycle analysis was performed using a flow cytometer FACSARIA…¡ BD Biosciences The data was performed using CXP Analysis software Beckman Coulter Inc Statistical analysis All the data analysis was performed using SPSS version SPSS Inc The results are presented as mean SD Associations between MCM5 mRNA expres‘sion and clinicopathological factors were analyzed using the Pearson's χ2 test or Fisher's exact test The differences in MCM5 mRNA expression between carcinoma and adjacent normal tissues were evaluated by a paired t‘test One‘way ANOVA followed by Tukey's post hoc test was used to determine the differences between groups and unpaired t‘tests for the rest of the data The survival rate was calculated by the Kaplan‘Meier method and compared using the log‘rank test P005 was considered to indicate a statistically significant difference All experiments were performed in triplicateResultsRNA sequencing and functional annotation analysis To explore novel biomarkers for OSCC the RNAs derived from tissue samples by sequencing were detected Three matched primary OSCC tissues and adjacent normal tissues were randomly selected As shown in Fig 1A the aberrant expression of genes was detected in tissue samples To screen 0cHAO MCM5 IS A POTENTIAL EARLY DIAGNOSTIC MARKER FOR ORAL SQUAMOUS CELL CARCINOMATable II Twenty randomly selected differentially expressed genes between oral squamous cell carcinoma and adjacent tissue samples The genes selected randomly instead of listing based on rank or fold‘change in expressionGene ID ENSG00000160182 ENSG00000205592 ENSG00000171195 ENSG00000126549 ENSG00000090382 ENSG00000161798 ENSG00000161055 ENSG00000107562 ENSG00000214711 ENSG00000106066 ENSG00000184330 ENSG00000137745 ENSG00000243207 ENSG00000107159 ENSG00000183072 ENSG00000196611 ENSG00000171217 ENSG00000178445 ENSG00000100297 ENSG00000127564 Log2 fold‘change ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ Gene nameTFF1MUC19MUC7STATHLYZAQP5SCGB3A1CXCL12CAPN14CPVLS100A7AMMP13PPAN‘P2RY11CA9NKX2‘MMP1CLDN20GLDCMCM5PKMYT1P‘value 881x10‘ 468x10‘ 144x10‘ 140x10‘ 209x10‘ 528x10‘ 959x10‘ 604x10‘ 601x10‘ 846x10‘ 234x10‘ 753x10‘ 222x10‘ 161x10‘ 380x10‘ 154x10‘ 705x10‘ 751x10‘ 565x10‘ 240x10‘ Result Downregulation Downregulation Downregulation Downregulation Downregulation Downregulation Downregulation Downregulation Downregulation Downregulation Upregulation Upregulation Upregulation Upregulation Upregulation Upregulation Upregulation Upregulation Upregulation Upregulation the candidate biomarkers for diagnosing OSCC the DEGs were selected when changes in RNA expression were fold‘changes As shown in Fig 1A aberrant RNAs were significantly downregulated while RNAs were upregu‘lated In order to represent all differentially expressed genes twenty randomly selected dysregulated genes between OSCC and adjacent tissue samples are summarized in Table II The P‘value and log2 fold‘changes of all aberrant expression genes are shown in Table SI The DEGs were selected randomly instead of listing based on rank or fold‘change in expression To explore the role of differentially expressed RNAs in OSCC KEGG pathway analysis was performed Depending on the P‘value and enrichment signal pathways associ‘ated with OSCC were identified Table SII The top KEGG enrichment terms of DEGs are shown in Fig 1B including ˜cell cycle™ ˜pathways in cancer™ ˜cell cycle‘yeast™ ˜meiosis‘yeast™ and ˜cytokine‘cytokine receptor interac‘tion™ Among these it was reported that some genes such as MCM5 cell division cycle ‘related protein kinase and Cyclin‘dependent kinase homolog were primarily enriched in the ˜cell cycle™ pathway Some genes such as lysozyme C statherin and aquaporin‘ were enriched in the ˜saliva secretion™ pathway MCM5 which participated in cell cycle regulation and had high expression in OSCC was selected for further study and it was hypothesized that MCM5 might be a candidate tumor marker for OSCCValidation of MCM5 using RT‘qPCR To further verify the aforementioned expression profile data MCM5 expression levels were investigated using RT‘qPCR in tumor and Figure Relative expression levels of MCM5 mRNA in paired adjacent normal tissues and oral squamous cell carcinoma tissues using quantita‘tive PCR The relative expression data were analyzed by the ‘ΔΔCq method GAPDH was used as an internal control P005 P001 vs adjacent normal tissue MCM5 minichromosome maintenance proteinadjacent normal tissues As shown in Fig MCM5 mRNA was significantly upregulated in of tumor tissues compared with that in matched normal tissues These results showed that MCM5 was highly expressed in OSCC tissues which was in line with the sequencing data Association of MCM5 expression levels with clinicopathologic features of OSCC and survival analysis The results of the potential association between MCM5 expression and clinicopathological features in patients with OSCC are presented in Table III No significant association with MCM5 expression was found for age sex histological differentiation metastasisrecurrence and survival status P05 0cONCOLOGY LETTERS Value n MCM5 expression‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘Low n3 High n12 Table III Association between expression of MCM5 and clinicopathologic features of patients with oral squamous cell carcinoma Characteristic Age years ‰¥ Sex Male Female Histological differentiation Well and Moderate Poor MetastasisRecurrence Yes No Survival status Death Survival P‘valueA Kaplan‘Meier analysis of OS is shown in Fig Analysis of clinical data from TCGA showed that high MCM5 expres‘sion was no associated with a shorter OS in patients with OSCC log‘rank P062 These results suggested that MCM5 might not be a prognostic biomarker for OSCC Inhibitory effect of MCM5 in OSCC cell lines To determine the functional role of MCM5 first MCM5 expression was analyzed using RT‘qPCR in two OSCC cell lines Notably SCC‘ cells expressed significantly higher levels of MCM5 compared with Cal‘ cells P001 Fig 4A Considering that knockdown of MCM5 in the cell line with high MCM5 expres‘sion may bring about more significant changes the SCC‘ cell line was selected for further investigation of the functional role of MCM5 Three specific siRNA sequences were designed to inhibit MCM5 expression and transfected in SCC‘ cells and the impact on MCM5 expression was determined using RT‘qPCR As shown in Fig 4B siRNA1 siRNA2 and siRNA3 transfection decreased MCM5 expression by 651P001 P001 and P001 respectively compared with the negative control Then the efficiencies were confirmed using western blotting Fig 4C The inhibitory effect of siRNA1 and siRNA2 was significant but not found in siRNA3 The results were consistent with those of RNA expression siRNA1 trans‘fection reduced MCM5 expression significantly in SCC‘ cells Therefore siRNA1 was used for subsequent experiments Effect of MCM5 inhibition on proliferation colony formation and migration To determine whether MCM5 regulates cell cycle and modulates cell proliferation in OSCC the effect of inhibiting MCM5 expression on SCC‘ cell proliferation was investigated As shown in Fig 5A the results showed that downregulation of MCM5 had significant anti‘proliferative Figure Survival curves from The Cancer Genome Atlas datasets n518 containing high and low MCM5 expression levels MCM5 minichromosome maintenance protein HR hazard ratio TPM transcripts per millioneffect compared with the negative control P001 Colony formation assays were performed and the results revealed that compared with the number of colonies in the control group downregulation of MCM5 significantly reduced colony formation P001 Fig 5B and C To estimate the impact of MCM5 on OSCC migration scratching assays were conducted and inhibition of MCM5 showed no significant impact on the migration of SCC‘ cells P005 Fig 5D These results suggested that inhibiting MCM5 expression inhibited cell proliferation and colony formation but had no effect on migration in SCC‘ cells\x0cHAO MCM5 IS A POTENTIAL EARLY DIAGNOSTIC MARKER FOR ORAL SQUAMOUS CELL CARCINOMAFigure Inhibition of MCM5 expression in oral squamous cell lines A MCM5 expression in Cal‘ and SCC‘ cells B Efficiency of siRNA‘MCM5 was confirmed by reverse transcription quantitative‘PCR in SCC‘ cells C The efficiency of siRNA‘MCM5 was confirmed by western blot in SCC‘ cells MCM5 minichromosome maintenance protein si small interfering NC negative controlFigure Effects of MCM5 inhibition on antiproliferation colony formation and migration capacity in SCC‘ cells A Downregulation of MCM5 expression suppressed SCC‘ cell proliferation compared with the corresponding control at different time points B Downregulation of MCM5 expression inhibited SCC‘ cells colony formation compared with the corresponding control C Quantification of MCM5 inhibition on colony formation compared with the corresponding control D Cells scratching wounds observed by microscopy E Downregulation of MCM5 had no effect on the migration capacity of SCC‘ cells P001 vs NC MCM5 minichromosome maintenance protein si small interfering NC negative controlEffect of MCM5 inhibition on cell cycle To determine the potential mechanism for the observed proliferation inhibition of SCC‘ cells by MCM5 inhibition cell cycle analysis was performed using flow cytometry As shown in Fig 6A and B after MCM5 inhibition the number of cells were decreased in the G0G1 phase and the S phase but significantly increased in the G2M phase compared with the negative control These results indicated that MCM5 inhibi‘tion significantly induced G2M phase arrest compared with that in the control group\x0cONCOLOGY LETTERS Figure Effects of MCM5 inhibition on cell cycle regulation in SCC‘ cells A Flow cytometry assays were performed to analysis the cell cycle progres‘sion when SCC‘ cells transfected with siRNA‘MCM5 B The bar chart represented the percentage of cells in G0G1 S or G2M phase as indicated C Expression levels of cell cycle‘regulated genes detected by quantitative PCR and normalized to GAPDH D Expression levels of cell cycle‘regulated proteins determined using western blotting β‘actin was used as the loading control Data are presented as mean ± SD P005 P001 vs control group MCM5 minichromosome maintenance protein si small interfering NC negative control CDK2 cyclin‘dependent kinase To elucidate the mechanism underlying this effect the expressions levels of cyclin E cyclin‘dependent kinase CDK2 and p21 related to cell cycle arrest were deter‘mined using RT‘qPCR As shown in Fig 6D MCM5 inhibition decreased both cyclin E and CDK2 mRNA levels but increased the mRNA expression of p21 significantly Then cyclin E and p21 were selected to detect the protein levels using western blotting As shown in Fig 6C cyclin E levels decreased while p21 levels increased in MCM5‘downregulated SCC‘ cells which was consistent with the RT‘qPCR results DiscussionDespite notable progress in cancer research and treatment the survival rate of patients with OSCC has not significantly improved in the past few decades To date there are no effective tumor‘specific biomarkers for the early detection and prognosis prediction of OSCC Several studies have shown that DEGs serve an important role in the development of tumors in different cancer types ‘ However few studies have reported differentially expressed genes in OSCC The present screened genes that regulate the progression of oral cancer by gene expression profiling and found that genes were dysregulated of which DEGs were upregulated and were downregulated DEGs significantly affected GO terms and KEGG pathways MCM5 did not have one of the highest log2 fold‘change values and log10 qValues however MCM5 is one of the differentially expressed genes in cell cycle signaling pathway which was the most signifi‘cant enrichment of differentially expressed genes Therefore MCM5 which regulates the cell cycle was selected for further investigation However previously published studies on biomarkers in OSCC mainly focused on pathological studies The present study not only verified the over‘expression of MCM5 in OSCC but also confirmed using cell experiments that MCM5 affects cell proliferation by regulating the cell cycle MCM5 is a member of the MCM family of proteins and is a component of the starting complex for DNA synthesis MCM5 has been identified as a cell cycle biomarker of aber‘rant proliferation which is associated with the progression of various cancer types Previously MCM5 has been found to be overexpressed in numerous human malignancies such as esophageal thyroid and ovarian cancer For example increased MCM5 levels in urine sediment cells predicts the presence of bladder cancer Inhibition of transcription factor SOX‘ can inhibit the proliferation of skin melanocytes and MCM5 expression is significantly decreased following downregulation of SOX‘ Moreover high expression of MCM5 is associated with poor prog‘nosis and poor malignant status in patients with cervical adenocarcinoma It is well known that immunohistochemistry and western blot‘ting are necessary methods to evaluate protein expression 0cHAO MCM5 IS A POTENTIAL EARLY DIAGNOSTIC MARKER FOR ORAL SQUAMOUS CELL CARCINOMAHowever due to a limited number of tissue samples the present study did not have enough samples for simultaneous qPCR western blotting and immunohistochemistry detection Therefore this is a limitation of the present study However the study did perform qPCR to evaluate the expression of MCM5 at the mRNA level The results demonstrated that of patients with OSCC have high MCM5 expression which is consistent with the results of the aforementioned studies indicating that high MCM5 expression may play an important role in the pathogenesis of OSCC In addition other members of the MCM family of proteins have also served as biological markers of dysplasia and malignancy such as glioma cervical colorectal breast prostate and lung cancer ‘ Therefore some researchers even suggested that changes in MCM5 expression may be a sign of cell cycle disorders It is worth noting that some researchers reported that the high expression of MCM family members may be closely related to tumorigenesis and prognosis For example MCM2 MCM4 and MCM6 are overexpressed in breast cancer of high histological grades MCM7 expression is a potent prognostic marker in non‘small cell lung cancer while MCM5 may be an independent adverse prognostic marker in lung squamous cell carcinoma It is well known that the cell cycle is related to cell proliferation signaling path‘ways In most tumors an increase in the expression level of genes encoding proteins that regulate cell proliferation is observed The abnormal expression of cell‘cycle‘related genes is associated with infinite proliferation of tumors and poor prognosis Thus far only Yu reported the relationship between MCM5 and OSCC The study reported that overexpression of MCM5 in patients with OSCC was significantly associated with tumor site tumor size positive lymph node metastasis later clinical stage higher histological grade deeper infiltration depth and peripheral nerve infiltration However in the present study association between high expression of MCM5 with survival metastasis and poor histologic differentiation was not observed A Kaplan‘Meier analysis of the overall survival rate was not significantly changed in patients with high MCM5 expression compared with patients with low MCM5 expression It was speculated that due to the small number of samples that there were large differences between individuals Therefore in future research a larger sample size should be used to clarify the relationship between high expression of MCM5 and prognosis of OSCC Little is known about the role and potential function of MCM5 in OSCC In the present study a loss‘of‘function analysis was conducted and it was demonstrated that MCM5 participated in regulating cell cycle and cell proliferation in OSCC cells In fact inhibiting the expression of MCM5 in SCC‘ cells resulted in the downregulation of cyclin E and CDK expression and upregulation of p21 expression which ultimately led to G2M phase arrest in oral cancer cells These results further verified that MCM5 is highly expressed in patients with OSCC which promotes the proliferation of OSCC cells and regulates cell cycle In addition it was observed that MCM5 was not only expressed in SCC‘ cells but also expressed in CAL‘ cells Fig 4A Notably according to the ATCC the MCM5 gene had no mutations in either of the two cell lines indicating that the two cell lines selected in this study have similar genetic backgrounds and could be used for the study of MCM5 cell functions Considering MCM5‘knockdown experiments in SCC‘ cells with high MCM5 expression received more significant results SCC‘ cells were selected for follow‘up studies However analyzing the functional effects of MCM5‘knockdown in CAL27 cell lines may provide more information In addition both SCC‘ and CAL‘ cells are transformed cell lines In future investigations untransformed cell lines for multiple comparisons should be used to clarify the role of MCM5 in OSCC Surgical resection is currently the main method to treat OSCC However considering the particularity of the oral struc‘ture surgical resection will lead to a huge impact on patients' quality of life Therefore it is important to find effective diagnostic biomarkers for early detection or to develop targeted drugs for OSCC In the present study it was reported that MCM5 is overexpressed in OSCC and that MCM5 can affect cell proliferation by regulating cell cycle Therefore the results suggested that MCM5 might be one of the important patho‘genic factors of OSCC and is expected to be used as a potential tumor marker for OSCC target drugs The specific mechanism of action of MCM5 is still worthy of further investigationOverall the present study evaluated differentially expressed genes using sequencing patterns in OSCC tumor tissues and further validated MCM5 upregulated expression in OSCC tissues By knocking down MCM5 expression in SCC‘ cells it was revealed that cell proliferation and colony formation was significantly inhibited by inducing G2M phase arrest The results also suggested that during this process cyclin E and cell cycle‘related gene expression levels were decreased while p21 was significantly upregulated Therefore MCM5 may modulate OSCC cell proliferation by regulating the cell cycle MCM5 is an important pathogenic factor and might have important role as a potential diagnostic marker or drug target for OSCCAcknowledgementsNot applicableFunding This study was funded by the National Natural Science Foundation of China grant no the Bethune Project of Jilin University of China grant no 2018B02 the Education Department of Jilin Province grant no JJKH20190074KJ and Department of Science and Technology of Jilin Province grant no 20190103086JH and 20200201398JC Availability of data and materials The datasets used andor analyzed during the present study are available from the corresponding author on reasonable request The other datasets generated andor analyzed during the current study are available in The Cancer Genome Atlas wwwcancergov Gene Ontology httpgeneon‘tology UniProt sparqluniprot NCBI wwwncbinlmnihgov and Kyoto Encyclopedia of Genes and Genomes wwwkeggjp databases\x0cONCOLOGY LETTERS Authors' contributions HW CZ and CL performed the experiments HW MH and XW analyzed the data MH and HW wrote the manuscript MH and XW designed and supervised Answer:

Thyroid_Cancer

"Drug resistance leads to tumor relapse and further progression during chemotherapy in lung cancer Close homolog of L1 CHL1 has been identified as a tumor suppressor in most malignancies However to the best of our knowledge whether CHL1 mediates chemoresistance remains unknown The present study observed that CHL1 was significantly downregulated in cisplatin DDP‘resistant cells A549DDP and paclitaxel PTX‘resistant cells A549PTX compared with A549 cells When treated with or without DDP and PTX silencing of CHL1 in A549 cells promoted the cell survival rate and clone formation and decreased apoptosis Whereas overexpression of CHL1 in A549DDP and A549PTX cells impeded the cell survival and clone formation and promoted apoptosis Additionally CHL1 overexpression enhanced the chemosensitivity of A549DDP cells to DDP in vivo Notably the chemoresistance induced by CHL1 depletion was reversed by the Akt inhibitor SC66 in A549 cells The results of the present study demonstrated that CHL1 enhanced sensitivity of lung cancer cells by suppressing the Akt pathway which suggested that CHL1 may be a potential target for overcoming chemoresistance in lung cancerIntroductionLung cancer is the most common human malignancy accounting for of all cancer‘associated deaths worldwide during In addition its morbidity and mortality rank the highest among all malignant tumor types worldwide According to the differentiation degree and morphological Correspondence to Dr Rimao Huang Department of Cardiothoracic Surgery Xiangya Changde Hospital Moon Avenue West of Langzhou North Road Changde Hunan PR ChinaE‘mail xyhuangrm163comKey words lung cancer close homolog of cisplatin paclitaxel chemosensitivitycharacteristics of cancer cells lung cancer can be roughly clas‘sified into non‘small‘cell lung cancer NSCLC and small‘cell lung cancer Among patients with lung cancer nearly are diagnosed as NSCLC which manifests with earlier diffu‘sion and metastasis Currently resection chemotherapy radiotherapy and targeted therapy are the primary treatments for lung cancer For patients with advanced NSCLC or those who are clinically incapacitated for surgery chemo‘therapy is a remarkably important treatment Cisplatin DDP is widely applied in the treatment of several malignan‘cies and it exhibits a broad spectrum of antitumor effects by inducing DNA damage and hindering DNA damage repair Paclitaxel PTX another commonly used chemotherapeutic agent in the clinic targets the microtubule cytoskeleton and impedes cell division The majority of patients have a good initial response to chemotherapy agents however subse‘quent relapse is common and largely due to the emergence of drug resistance Thus chemoresistance is considered one of the main factors of poor prognosis in patients with advanced NSCLC Therefore there is an urgent need to investigate the target and mechanism of chemoresistance in NSCLCClose homolog of L1 CHL1 is a member of the L1 family of nerve cell adhesion molecules and is located on the 3q26 locus As a nerve cell adhesion molecule CHL1 serves an important role in the development regeneration and plasticity of the nervous system The absence or mutation of CHL1 can trigger 3p syndrome and schizophrenia The abnormal expression of CHL1 may lead to reduced working memory and social behavior mental damage and abnormal behavior CHL1 has been reported to be involved in carcinogenesis and progression in a variety of human cancers In esophageal squamous cell carcinoma ESCC CHL1 downregulation is associated with invasion lymph node metastasis and poor overall survival Functional studies revealed that CHL1 has anti‘proliferation and anti‘metastasis abilities The expression of CHL1 is downregulated by hypermethylation in human breast cancer and its negative expression contributes to breast tumorigenesis and progression In thyroid cancer and colonic adenocarcinoma CHL1 impedes cell proliferation and invasion and acts as a tumor suppressor In lung cancer HÓ§tzel evaluated CHL1 expression 0cCAI CHL1 ENHANCES THE CHEMOSENSITIVITY OF LUNG CANCER CELLSin NSCLC cases based on a tissue microarray and it was reported that CHL1 expression is associated with T stage in adenocarcinomas as well as with metastatic lymph node status and improved survival Additionally by analyzing the Gene Expression Omnibus dataset GSE21656 submitted by Sun microarray results demonstrated that CHL1 expression in DDP‘resistant H460 cells is significantly lower compared with that in parental cells suggesting that CHL1 may be involved in NSCLC chemoresistance however to the best of our knowledge the underlying mechanism remains unknownIn the present study the expression of CHL1 in DDP‘ and PTX‘resistant A549 cells and the parental cells was assessed Functional studies of CHL1 were performed to investigate its potential role in chemoresistanceMaterials and methodsData processing The human GSE21656 microarray dataset was downloaded from the NCBI Gene Expression Omnibus GEO database wwwncbinlmnihgovgeo The available dataset GSE21656 was based on the GPL6244 platform Affymetrix Human Gene ST Array Affymetrix Thermo Fisher Scientific Inc This data includes H460 cells and DDP‘resistant H460 cells sample and each cell has three repeats samples The online tool GEO2R httpwwwncbinlmnihgovgeogeo2r was used to determine the differen‘tially expressed genes in H460 and DDP‘resistant H460 cells P005 and log2fold‘change‰¥ were set as cut‘off standardsCell culture The human NSCLC cell line A549 the PTX‘resistant cell line A549PTX and the DDP‘resistant cells A549DDP were purchased from Procell Life Science Technology Co Ltd The cells were cultured in Ham's F‘12K medium supplemented with fetal bovine serum both purchased from Thermo Fisher Scientific Inc Uml penicillin and Uml strep‘tomycin cat no Thermo Fisher Scientific Inc in a ˚C humidified incubator with CO2Cell transfection The resistant cells A549PTX and A549DDP cells were transfected with µg CHL1 recombinant expres‘sion plasmid cat no HG10143‘NY Sino Biological Inc Empty vector pCMV3‘SP‘N‘HA was used as the control A549 cells were transfected with pmol small interfering siRNAs The siRNA sequence for CHL1 Guangzhou RiboBio Co Ltd were siRNA‘ '‘GGA GCU AAU UUG ACC AUA Utt‘' siRNA‘ '‘CAG CAA UAU UAG CGA GUA Utt‘' and scrambled control '‘UUC UCC GAA CGU GUC ACG Utt‘' Plasmids and siRNAs were transfected into cells using Lipofectamine® Thermo Fisher Scientific Inc following the manufacturer's instructions The time interval between transfection and subsequent experimentation was h For the rescue experiments the CHL1 silenced A549 cells were treated with the Akt inhibitor SC66 cat no S5313 Selleck Chemicals along with DDP µgml or PTX ngml both purchased from Selleck Chemicals for h at ˚CRNA extraction and reverse transcription‘quantitative PCR RT‘qPCR assay Total RNAs were isolated using TRIzol reagent Thermo Fisher Scientific Inc according to the manufacturer's instructions and the mixed DNAs were elim‘inated by DNase I New England Biolabs Inc First‘strand cDNA synthesis was conducted using the GoScriptTM kit Promega Corporation according to the manufacturer's instructions The reaction conditions for reverse transcription were ˚C for min ˚C for min and ˚C for min The SYBR Green Real‘Time PCR Master mix Thermo Fisher Scientific Inc was used to perform RT‘qPCR using a LightCycler480 system Roche Diagnostics GmbH The CHL1 primer sequences were as follows Forward '‘GGC TTG GTC TCT TGC TTT CC‘' and reverse '‘ATC TTC CCT CCC TTT GCA CG‘' and ‘actin forward '‘TTC CTT CCT GGG CAT GGA GTC ‘' and reverse '‘TCT TCA TTG TGC TGG GTG CC‘' The following thermocycling conditions were used for qPCR min at ˚C followed by cycles at ˚C for sec sec at ˚C and a final extension at ˚C for sec Each reaction was conducted in triplicate Relative expression levels were calculated using the ‘ΔΔCq method Cell viability Cell viability was detected by MTT assay A cell suspension µl was seeded into ‘well plates at a density of 1x104 cellswell and incubated overnight at ˚C The concentrations of DDP used to treat A549 cells were and µgml While the concentrations of PTX used to treat A549 cells were and ngml The concentrations of DDP used to treat A549DDP cells were and µgml While the concentrations of PTX used to treat A549PTX cells were and ngml After treating with different concentrations of DDP or PTX for h at ˚C µl MTT mgml solution was added to each well and incubated for h at ˚C Subsequently µl DMSO was added to each well to dissolve the blue formazan crystals and the absorbance was measured using a microplate reader BioTek Instruments Inc at nmClone formation assay A total of 1x103 cells were seeded into a ‘mm dish in triplicate and maintained in F‘12K medium with or without DDP or PTX at ˚C for h A total of weeks later cells were fixed in paraformalde‘hyde for min at room temperature and stained with crystal violet dye at room temperature for min The rate of colony formation was calculated using the following equation Number of coloniesnumber of seeded cells x100Flow cytometry Apoptosis was detected using a FITC Annexin V Apoptosis kit BD Pharmingen BD Biosciences according to the manufacturer's protocol Cells 1x105 were collected and washed twice with PBS prior to being suspended in µl binding buffer Subsequently cells were incubated with µl Annexin V‘FITC and µl propidium iodide in the dark for min at room temperature and apoptosis was analyzed using a CytoFlex flow cytometer Beckman Coulter Inc Data were analyzed using CytEXpert software Beckman Coulter Inc The ratio between early and late apoptosis was calculatedWestern blotting Cells were collected washed twice with PBS and lysed with RIPA lysis buffer Thermo Fisher Scientific Inc Proteins were isolated from the cell lysis buffer and 0cONCOLOGY LETTERS quantified using the Piercetm„¢ BCA Protein Assay kit cat no Thermo Fisher Scientific Inc with bovine serum album as a standard Equal amount of protein µg proteins were separated by SDS‘PAGE gel Next the proteins were transferred onto a polyvinylidene membrane Thermo Fisher Scientific Inc blocked with BSA Thermo Fisher Scientific Inc for h at ˚C and incubated overnight at ˚C with primary antibodies against CHL1 cat no ‘‘AP ProteinTech Inc multi‘drug resistance gene MDR1 cat no ‘‘AP ProteinTech Inc multidrug resistance‘associated protein MRP cat no ‘‘Ig ProteinTech Inc low‘density lipoprotein receptor‘related protein LRP cat no ‘‘AP ProteinTech Inc phosphorylated p‘Akt cat no ab38449 Abcam and Akt cat no ab227385 Abcam After washing three times with PBS the membrane was incubated with horseradish peroxide‘conjugated goat anti‘rabbit cat no ab6271 Abcam_or rabbit anti‘mouse cat no ab6728 Abcam secondary antibodies for h at room temperature and the blots were detected with enhanced chemiluminescence reagent Thermo Fisher Scientific Inc Protein expression was quantified using Image‘pro plus software Media Cybernetics IncAnimal experiments The animal experiments were approved by the Medical Ethics Committee of Xiangya Changde Hospital approval no and were performed in compliance with all regulatory institutional guidelines for animal welfare the National Institutes of Health Publications no ‘ A total of male BALBc‘nu mice ‘week‘old ± g Hunan SJA Laboratory Animals Center of the Chinese Academy of Sciences were used in this study All animals were kept at the SPF level laboratory at ‘ËšC a relative humidity of ‘ a h lightdark cycle and timesh of fresh air exchange All mice were given free access to food and water The bedding materials drinking water feeding cages and other items in contact with the animals were all autoclaved prior to use A549DDP cells 1x107 transfected with empty vector and CHL1 overexpression vector using Lipofectamine® reagent Thermo Fisher Scientific Inc were subcu‘taneously injected into the nude mice to establish xenograft models following anaesthesia with chloral hydrate mgkg Xenografts were allowed to grow to mm3 over weeks and the mice were randomly divided into four groups n3group as follows i vector group A549DDP cells transfected with empty vector and treated with µl saline solution ii vector‘DDP group A549DDP cells trans‘fected with empty vector and treated with mgkg DDP iii CHL1 group A549DDP cells transfected with CHL1 overexpression vector and treated with µl saline solu‘tion and iv CHL1‘DDP group A549DDP cells transfected with CHL1 overexpression vector and treated with mgkg DDP DDP was administered by intraperitoneal injection every days for weeks The mice were observed daily and the tumors were measured by a vernier caliper every days The tumor volumes were calculated as length x width22 A total of weeks post‘injection mice were euthanized with CO2 at volume displacement rate VDR per min using a programmable logic controller Barry‘Wehmiller Design Group Inc Mice were monitored continuously and once the mice were immobile except for breathing for min the VDR was provided at for min The animals remained in the euthanasia chamber for min and were then observed for an additional min Breathing and heart rate were monitored to determine deathStatistical analysis All experiments were performed in tripli‘cate and data are presented as the mean ± standard deviation All experiments were performed at least three times Paired Student's t‘test was performed for comparisons between two groups and one‘way analysis of variance followed by Tukey's multiple comparison post‘hoc analysis was performed for comparisons between multiple groups SPSS IBM Corp was used to perform the analysis P005 was considered to indicate a statistically significant differenceResultsCHL1 is downregulated in A549DDP and A549PTX‘resistant cells In order to investigate the mechanism of chemore‘sistance in lung cancer the lung adenocarcinoma cell line A549 the DDP‘resistant cells A549DDP and PTX‘resistant cells A549PTX were used in the present study Cells were exposed to different concentrations of DDP ‘ µgml and PTX ‘ ngml and MTT assay was used to detect the cell survival rate A549DDP and A549PTX cells demonstrated higher resistance to DDP and PTX compared with A549 cells Fig 1A The half maximal inhibitory concentration IC50 of DDP was significantly higher in A549DDP cells ± µgml compared with A549 cells ± µgml and the IC50 of PTX was significantly higher in A549PTX cells ± ngml compared with A549 cells ± ngml Fig 1B In addition the expression levels of the drug‘resistant markers MDR1 MRP and LRP were significantly higher in A549DDP and A549PTX cells compared with A549 cells Fig 1C Additionally the mRNA and protein expression levels of CHL1 were significantly lower in A549DDP and A549PTX cells compared with those in A549 cells Fig 1D and E and this was also observed in H460 DDP‘resistant cells obtained from the GEO dataset GSE21656 Fig 1F These results suggested that CHL1 may be involved in regulating DDP and PTX resistance in NSCLCKnockdown of CHL1 enhances resistance to DDP and PTX in A549 cells As CHL1 was upregulated in A549 cells CHL1 was silenced in A549 cells using siRNAs CHL1 expres‘sion was significantly reduced in the CHL1 siRNA groups compared with that of the scrambled control group Fig 2A As siRNA‘ demonstrated the greatest interference efficiency it was selected for use in the following experiments Notably CHL1‘knockdown enhanced the resistance to DDP and PTX in A549 cells Fig 2B and C Colony formation assay revealed that compared with the control group CHL1‘knockdown significantly increased the rate of colony formation in the absence of chemotherapeutics and enhanced the resistance to DDP and PTX Fig 2D Flow cytometry results demon‘strated significantly reduced apoptosis in CHL1‘knockdown cells after DDP and PTX treatment compared with that of the control group Fig 2E\x0cCAI CHL1 ENHANCES THE CHEMOSENSITIVITY OF LUNG CANCER CELLSFigure CHL1 is downregulated in DDP and PTX‘resistant A549 cells A Cell survival of A549 and A549‘resistant cells A549DDP and A549PTX treated with increasing concentrations of DDP and PTX as assessed by MTT assay B The IC50 values of DDP in A549DDP and A549 cells and the IC50 values of PTX in A549PTX and A549 cells P005 vs A549 cells C Western blotting demonstrated the expression of drug resistance‘related proteins MDR1 MRP and LRP in A549 cells and A549‘resistant cells A549DDP and A549PTX P005 vs A549 cells The protein and mRNA expression levels of CHL1 in A549 cells and A549‘resistant cells A549DDP and A549PTX were analysed by D western blotting and E reverse transcription‘quantitative PCR respectively P005 vs A549 cells F The mRNA expression of CHL1 in H460 and H460DDP cells in the GSE21656 dataset P005 vs H460 cells CHL1 close homolog of L1 DDP cisplatin PTX paclitaxel MDR1 multi‘drug resistance gene MRP multidrug resistance‘associated protein LRP low‘density lipoprotein receptor‘related protein IC50 half maximal inhibitory concentration CHL1 overexpression enhances the sensitivity of A549 resistant cells to DDP and PTX As CHL1 is downregu‘lated in A549DDP and A549PTX cells the present study successfully overexpressed CHL1 in these cells using CHL1 recombinant expression plasmids Fig 3A The results demonstrated that CHL1 overexpression alleviated the resis‘tance to DDP and PTX compared with that of the control group Fig 3B and C In addition CHL1 overexpression inhibited colony formation in the absence or presence of DDP and PTX Fig 3D Additionally flow cytometry results demonstrated that restoration of CHL1 expression promoted apoptosis in resistant cells following DDP and PTX treat‘ment Fig 3ETo further validate the effects of CHL1 overexpression on DDP or PTX sensitivity xenograft mice model experi‘ments were performed The results demonstrated that CHL1 overexpression or DDP treatment significantly impeded the tumor growth Fig 3F and decreased the tumor weight Fig 3G In addition CHL1 overexpression further aggra‘vated DDP‘mediated repression on tumor growth Fig 3F and G These data suggested that CHL1 overexpression suppressed tumor growth and enhanced the chemosensitivity in NSCLCCHL1 mediates chemosensitivity by inhibiting Akt activity Recently studies have confirmed that CHL1 inhibits Akt activity in ESCC and neuroblastoma cell lines Thus the present study investigated whether CHL1 mediates chemoresistance via the Akt pathway in NSCLC In A549 cells compared with the scrambled group CHL1‘knockdown elevated the expression of p‘Aktser473 Fig 4A By contrast restoring CHL1 expression in A549DDP and A549PTX cells inhibited the Akt phosphorylation compared with the control group Fig 4A suggesting CHL1 mediates chemosensitivity via the Akt pathway Subsequently CHL1‘silenced A549 cells were treated with the Akt inhibitor SC66 and it was demon‘strated that inhibiting Akt activity significantly reduced the promotive effects on cell survival Fig 4B and clone forma‘tion Fig 4C and the inhibitory effects on apoptosis Fig 4D induced by CHL1‘depletion These results confirmed that CHL1 mediates chemosensitivity in NSCLC by inhibiting the Akt pathway\x0cONCOLOGY LETTERS Figure CHL1‘knockdown increases A549 cell resistance to DDP and PTX A Western blotting was performed to validate the efficiency of transfection with CHL1 siRNAs P005 vs scramble MTT assays were performed to determine the survival rate of CHL1‘knockdown A549 cells treated with B ‘ µgml DDP or C ‘ ngml DDP D Colony formation assay of A549 cells transfected with CHL1 siRNA in the presence or absence of µgml DDP or ngml PTX E Flow cytometry analysis was used to detect apoptosis in A549 cells transfected with CHL1 siRNA in the presence or absence of µgml DDP or ngml PTX P005 P0001 CHL1 close homolog of L1 DDP cisplatin PTX paclitaxel si small interfering DiscussionThe results of the present study demonstrated that CHL1 was significantly downregulated in A549DDP and A549PTX cells compared with A549 cells The knockdown of CHL1 in A549 cells facilitated the cell survival and clone formation and decreased apoptosis when treated with or without DDP and PTX whereas CHL1 overexpression in A549DDP and A549PTX cells inhibited cell survival and clone formation and increased apoptosis The results of the present study also demonstrated that CHL1 enhances NSCLC chemosensitivity through inhibition of the Akt pathway These data suggested that CHL1 may be a promising target to improve the efficacy of chemosensitivity in NSCLCCHL1 belongs to the L1 family of nerve cell adhesion molecules it was initially cloned in mice and its expression in mouse development was analyzed by Senchenko Through cell‘cell interactions and mediating cell‘cell and cell‘matrix interactions CHL1 has an important effect on the development regeneration and plasticity of the nervous system Previous reports have demonstrated that CHL1 also participates in carcinogenesis ‘ CHL1 was observed to be significantly downregulated in up to types of tumor tissues compared with their adjacent normal tissues In most tumors CHL1 is a potential tumor suppressor gene whose silencing is associated with tumor growth invasion and metastasis ‘ For example knockdown of CHL1 expression results in enhanced cervical cancer cell invasion and migration A low expres‘sion of CHL1 in patients with neuroblastoma predicts a poor prognosis and enhancing CHL1 expression suppresses tumor progression In contrast CHL1 has been reported to promote cell proliferation metastasis and migration in human gliomas However to the best of our knowledge research on CHL1 and tumor chemoresistance has rarely been reported\x0cCAI CHL1 ENHANCES THE CHEMOSENSITIVITY OF LUNG CANCER CELLSFigure Overexpression of CHL1 increases the sensitivity of resistant A549 cells to DDP and PTX A Western blotting was performed to detect CHL1 expression in A549DDP and A549PTX cells transfected with CHL1 expression plasmids P005 vs vector Effect of CHL1 overexpression on resistant A549 cell survival rate when treated with B ‘ µgml DDP or C ‘ ngml PTX as determined by MTT assay D Colony formation assays demon‘strated the number of colonies of resistant A549 cells transfected with CHL1 expression plasmids in the presence or absence of µgml DDP or ngml PTX E Flow cytometry analysis was performed to assess apoptosis in resistant A549 cells transfected with CHL1 expression plasmids in the presence or absence of µgml DDP or ngml PTX CHL1 overexpression enhanced chemosensitivity of A549DDP cells to DDP in vivo which was demonstrated by the effect of DDP treatment or CHL1 overexpression on the F growth and G weight of xenografts derived from A549DDP cells P005 P001 CHL1 close homolog of L1 DDP cisplatin PTX paclitaxel The present study examined the differentially expressed genes in NSCLC DDP‘resistant cells in a GEO dataset CHL1 was demonstrated to be upregulated in DDP‘resistant cells compared with parental cells suggesting that CHL1 may be involved in NSCLC chemotherapy resistance Similarly a study that compared and analyzed the differentially expressed genes in chemosensitive tumors and chemoresistant ovarian adenocarcinomas tissues reported that the expression of CHL1 in chemotherapy‘sensitive tumor tissues is higher compared with that in drug‘resistant tissues suggesting that CHL1 may help to predict the efficacy of chemotherapy for ovarian cancer In addition aberrant methylation of CHL1 may be associated with the recurrence of colorectal cancer CRC following chemotherapy ‘azadC treatment restores ‘fluro‘uracil sensitivity in vitro which also suggests that CHL1 may be involved in CRC chemotherapy resistance The results of the present study demonstrated that CHL1 was down‘regulated in A549DDP cells Additionally as multiple drug resistance is a common characteristic another type of resistant cells A549TAX cells were also used in the current study The results also demonstrated that CHL1 was downregulated in A549PTX cells Compared with control cells overexpression of CHL1 significantly increased the sensitivity of cells resistant to DDP and PTX whereas knockdown of CHL1 expression in 0cONCOLOGY LETTERS Figure CHL1 mediates DDP and PTX sensitivity by inhibiting Akt activity A Western blotting was performed to detect the expression of p‘Akt and total Akt in CHL‘silenced and ‘restored cell models P005 vs scramble or vector B MTT assays were performed to detect cell survival rates of A549 cells treated with CHL1 siRNA and Akt inhibitor SC66 P005 C Colony formation assays were performed in A549 cells treated with CHL1 siRNA and the Akt inhibitor SC66 in the presence of DDP µgml or PTX ngml P005 vs si‘CHL1 D Apoptosis were measured in A549 cells treated with CHL1 siRNA and Akt inhibitor SC66 in the presence of DDP µgml and PTX ngml P005 vs si‘CHL1 CHL1 close homolog of L1 DDP cisplatin PTX paclitaxel si small interfering p‘ phosphorylated parent A549 cells displayed the opposite results To the best of our knowledge this study is the first study to suggest that CHL1 may be involved in chemosensitivity in lung cancer The concentration of DDP used in vivo is mgkg however this may not be in line with the concentrations that would be used in a clinical setting In a clinical trial the human initial dose was calculated from the no observed adverse effect levels NOAELs verified in animal experiments NOAEL is the maximum dose level without significant adverse reactions The NOAEL verified in animal experiments can be converted to a human equivalent dose according to the body surface area conversion which is based on the area standardization mgm2 proportional among different species In the present study the concentration of DDP used in vivo was not the NOAEL thus it was not consistent with the concentrations used in clinical settingsAkt is a serinethreonine protein kinase that is activated by phosphorylation As a key molecule of the PI3KAkt signaling pathway p‘Akt regulates cell survival cell growth cell motility and angiogenesis and prevents apoptosis Additionally Akt activation is associated with tumor chemore‘sistance The results of the present study demonstrated that compared with the control groups the expression of p‘Akt was increased in CHL1‘knockdown A549 cells and its expres‘sion was reduced in CHL1 overexpressed A549DDP and A549PTX cells When Akt activity was inhibited by the Akt inhibitor the sensitivity to DDP and PTX in CHL1‘knockdown A549 cells was restored This finding suggested that CHL1 enhanced the chemosensitivity of NSCLC by inhibiting the Akt pathway Considering numerous studies have confirmed that the Akt pathway mediates chemoresistance via regulation of ATP binding cassette ABC members ‘ the present study didn't further investigate the specific ABC members and mechanisms which was a of the limitation to the present study thus this research should be further investigated in vivoIn summary the present study demonstrated that CHL1 was downregulated in resistant cells A549DDP and A549PTX and upregulation of CHL1 enhanced the chemosensitivity of NSCLC via inhibiting the Akt pathway To the best of our knowledge this was the first study to confirm the function and 0cCAI CHL1 ENHANCES THE CHEMOSENSITIVITY OF LUNG CANCER CELLSmechanism of CHL1 in mediating chemosensitivity in cancer Thus the development of CHL1‘based therapeutic strategies may improve the efficacy of chemosensitivity in NSCLCAcknowledgementsThe authors of the present study would like to thank Mr Dingliang Li Xiangya Hospital Changsha China for his guidance and assistance in flow cytometric analysisFundingNo funding was receivedAvailability of data and materialsThe datasets used andor analyzed during the present study are available from the corresponding author upon reasonable requestAuthors' contributionsRH conceived and designed the present study XC BH YH and PL performed experiments and collected the data SL ZZ and ZH analyzed and interpreted the data ML and LZ analyzed the data and prepared the figure XC ML and LZ drafted the initial manuscript and revised it for intellectual content All authors read and approved the final manuscriptEthics approval and consent to participateThe animal experiments were approved by the Medical Ethics Committee of Xiangya Changde Hospital Changde China approval no Patient consent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsReferences Parascandola M and Xiao L Tobacco and the lung cancer epidemic in China Transl Lung Cancer Res Suppl S21‘S30 Chen W Cancer statistics Updated cancer burden in China Chin J Cancer Res Oser MG Niederst MJ Sequist LV and Engelman JA Transformation from non‘small‘cell lung cancer to small‘cell lung cancer Molecular drivers and cells of origin Lancet Oncol e165‘e172 Thatcher N Faivre‘Finn C Blackhall F Anderson H and Lorigan P Sequential platinum‘based chemotherapy‘thoracic radiotherapy in early stage non‘small cell lung cancer Clin Cancer Res Suppl S5051‘S5056 Yano T Okamoto T Fukuyama S and Maehara Y Therapeutic strategy for postoperative recurrence in patients with non‘small cell lung cancer World J Clin Oncol ‘ Fang Z Chen W Yuan Z Liu X and Jiang H LncRNA‘MALAT1 contributes to the cisplatin‘resistance of lung cancer by upregulating MRP1 and MDR1 via STAT3 activation Biomed Pharmacother ‘ Cai Y Dong ZY and Wang JY LncRNA NNT‘AS1 is a major mediator of cisplatin chemoresistance in non‘small cell lung cancer through MAPKSlug pathway Eur Rev Med Pharmacol Sci ‘ Han ML Zhao YF Tan CH Xiong YJ Wang WJ Wu F Fei Y Wang L and Liang ZQ Cathepsin L upregulation‘induced EMT phenotype is associated with the acquisition of cisplatin or paclitaxel resistance in A549 cells Acta Pharmacol Sin ‘ Liu J Meisner D Kwong E Wu XY and Johnston MR Translymphatic chemotherapy by intrapleural placement of gelatin sponge containing biodegradable Paclitaxel colloids controls lymphatic metastasis in lung cancer Cancer Res ‘ Hassan WA Yoshida R Kudoh S Kameyama H Hasegawa K Niimori‘Kita K and Ito T Notch1 controls cell chemoresistance in small cell lung carcinoma cells Thorac Cancer ‘ Tang H Jiang L Zhu C Liu R Wu Y Yan Q Liu M Jia Y Chen J Qin Y Loss of cell adhesion molecule L1 like promotes tumor growth and metastasis in esophageal squamous cell carcinoma Oncogene ‘ Liu H Focia PJ and He X Homophilic adhesion mechanism of neurofascin a member of the L1 family of neural cell adhesion molecules J Biol Chem ‘ Tassano E Biancheri R Denegri L Porta S Novara F Zuffardi O Gimelli G and Cuoco C Heterozygous deletion of CHL1 gene Detailed array‘CGH and clinical characterization of a new case and review of the literature Eur J Med Genet ‘ Morellini F Lepsveridze E Kahler B Dityatev A and Schachner M Reduced reactivity to novelty impaired social behavior and enhanced basal synaptic excitatory activity in perforant path projections to the dentate gyrus in young adult mice deficient in the neural cell adhesion molecule CHL1 Mol Cell Neurosci ‘ He LH Ma Q Shi YH Ge J Zhao HM Li SF and Tong ZS CHL1 is involved in human breast tumorigenesis and progres‘sion Biochem Biophys Res Commun ‘ Martín‘Sánchez E Mendaza S Ulazia‘Garmendia A Monreal‘Santesteban I Blanco‘Luquin I Córdoba A Vicente‘García F Pérez‘Janices N Escors D Megías D CHL1 hypermethylation as a potential biomarker of poor prog‘nosis in breast cancer Oncotarget ‘ Zhu H Fang J Zhang J Zhao Z Liu L Wang J Xi Q and Gu M miR‘ targets CHL1 and controls tumor growth and invasion in papillary thyroid carcinoma Biochem Biophys Res Commun ‘ Yu W Zhu K Wang Y Yu H and Guo J Overexpression of miR‘‘5p promotes proliferation and invasion of colon adeno‘carcinoma cells through targeting CHL1 Mol Med Hötzel J Melling N Müller J Polonski A Wolters‘Eisfeld G Izbicki JR Karstens KF and Tachezy M Protein expression of close homologue of L1 CHL1 is a marker for overall survival in non‘small cell lung cancer NSCLC J Canc

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You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: "Drug resistance leads to tumor relapse and further progression during chemotherapy in lung cancer Close homolog of L1 CHL1 has been identified as a tumor suppressor in most malignancies However to the best of our knowledge whether CHL1 mediates chemoresistance remains unknown The present study observed that CHL1 was significantly downregulated in cisplatin DDP‘resistant cells A549DDP and paclitaxel PTX‘resistant cells A549PTX compared with A549 cells When treated with or without DDP and PTX silencing of CHL1 in A549 cells promoted the cell survival rate and clone formation and decreased apoptosis Whereas overexpression of CHL1 in A549DDP and A549PTX cells impeded the cell survival and clone formation and promoted apoptosis Additionally CHL1 overexpression enhanced the chemosensitivity of A549DDP cells to DDP in vivo Notably the chemoresistance induced by CHL1 depletion was reversed by the Akt inhibitor SC66 in A549 cells The results of the present study demonstrated that CHL1 enhanced sensitivity of lung cancer cells by suppressing the Akt pathway which suggested that CHL1 may be a potential target for overcoming chemoresistance in lung cancerIntroductionLung cancer is the most common human malignancy accounting for of all cancer‘associated deaths worldwide during In addition its morbidity and mortality rank the highest among all malignant tumor types worldwide According to the differentiation degree and morphological Correspondence to Dr Rimao Huang Department of Cardiothoracic Surgery Xiangya Changde Hospital Moon Avenue West of Langzhou North Road Changde Hunan PR ChinaE‘mail xyhuangrm163comKey words lung cancer close homolog of cisplatin paclitaxel chemosensitivitycharacteristics of cancer cells lung cancer can be roughly clas‘sified into non‘small‘cell lung cancer NSCLC and small‘cell lung cancer Among patients with lung cancer nearly are diagnosed as NSCLC which manifests with earlier diffu‘sion and metastasis Currently resection chemotherapy radiotherapy and targeted therapy are the primary treatments for lung cancer For patients with advanced NSCLC or those who are clinically incapacitated for surgery chemo‘therapy is a remarkably important treatment Cisplatin DDP is widely applied in the treatment of several malignan‘cies and it exhibits a broad spectrum of antitumor effects by inducing DNA damage and hindering DNA damage repair Paclitaxel PTX another commonly used chemotherapeutic agent in the clinic targets the microtubule cytoskeleton and impedes cell division The majority of patients have a good initial response to chemotherapy agents however subse‘quent relapse is common and largely due to the emergence of drug resistance Thus chemoresistance is considered one of the main factors of poor prognosis in patients with advanced NSCLC Therefore there is an urgent need to investigate the target and mechanism of chemoresistance in NSCLCClose homolog of L1 CHL1 is a member of the L1 family of nerve cell adhesion molecules and is located on the 3q26 locus As a nerve cell adhesion molecule CHL1 serves an important role in the development regeneration and plasticity of the nervous system The absence or mutation of CHL1 can trigger 3p syndrome and schizophrenia The abnormal expression of CHL1 may lead to reduced working memory and social behavior mental damage and abnormal behavior CHL1 has been reported to be involved in carcinogenesis and progression in a variety of human cancers In esophageal squamous cell carcinoma ESCC CHL1 downregulation is associated with invasion lymph node metastasis and poor overall survival Functional studies revealed that CHL1 has anti‘proliferation and anti‘metastasis abilities The expression of CHL1 is downregulated by hypermethylation in human breast cancer and its negative expression contributes to breast tumorigenesis and progression In thyroid cancer and colonic adenocarcinoma CHL1 impedes cell proliferation and invasion and acts as a tumor suppressor In lung cancer HÓ§tzel evaluated CHL1 expression 0cCAI CHL1 ENHANCES THE CHEMOSENSITIVITY OF LUNG CANCER CELLSin NSCLC cases based on a tissue microarray and it was reported that CHL1 expression is associated with T stage in adenocarcinomas as well as with metastatic lymph node status and improved survival Additionally by analyzing the Gene Expression Omnibus dataset GSE21656 submitted by Sun microarray results demonstrated that CHL1 expression in DDP‘resistant H460 cells is significantly lower compared with that in parental cells suggesting that CHL1 may be involved in NSCLC chemoresistance however to the best of our knowledge the underlying mechanism remains unknownIn the present study the expression of CHL1 in DDP‘ and PTX‘resistant A549 cells and the parental cells was assessed Functional studies of CHL1 were performed to investigate its potential role in chemoresistanceMaterials and methodsData processing The human GSE21656 microarray dataset was downloaded from the NCBI Gene Expression Omnibus GEO database wwwncbinlmnihgovgeo The available dataset GSE21656 was based on the GPL6244 platform Affymetrix Human Gene ST Array Affymetrix Thermo Fisher Scientific Inc This data includes H460 cells and DDP‘resistant H460 cells sample and each cell has three repeats samples The online tool GEO2R httpwwwncbinlmnihgovgeogeo2r was used to determine the differen‘tially expressed genes in H460 and DDP‘resistant H460 cells P005 and log2fold‘change‰¥ were set as cut‘off standardsCell culture The human NSCLC cell line A549 the PTX‘resistant cell line A549PTX and the DDP‘resistant cells A549DDP were purchased from Procell Life Science Technology Co Ltd The cells were cultured in Ham's F‘12K medium supplemented with fetal bovine serum both purchased from Thermo Fisher Scientific Inc Uml penicillin and Uml strep‘tomycin cat no Thermo Fisher Scientific Inc in a ˚C humidified incubator with CO2Cell transfection The resistant cells A549PTX and A549DDP cells were transfected with µg CHL1 recombinant expres‘sion plasmid cat no HG10143‘NY Sino Biological Inc Empty vector pCMV3‘SP‘N‘HA was used as the control A549 cells were transfected with pmol small interfering siRNAs The siRNA sequence for CHL1 Guangzhou RiboBio Co Ltd were siRNA‘ '‘GGA GCU AAU UUG ACC AUA Utt‘' siRNA‘ '‘CAG CAA UAU UAG CGA GUA Utt‘' and scrambled control '‘UUC UCC GAA CGU GUC ACG Utt‘' Plasmids and siRNAs were transfected into cells using Lipofectamine® Thermo Fisher Scientific Inc following the manufacturer's instructions The time interval between transfection and subsequent experimentation was h For the rescue experiments the CHL1 silenced A549 cells were treated with the Akt inhibitor SC66 cat no S5313 Selleck Chemicals along with DDP µgml or PTX ngml both purchased from Selleck Chemicals for h at ˚CRNA extraction and reverse transcription‘quantitative PCR RT‘qPCR assay Total RNAs were isolated using TRIzol reagent Thermo Fisher Scientific Inc according to the manufacturer's instructions and the mixed DNAs were elim‘inated by DNase I New England Biolabs Inc First‘strand cDNA synthesis was conducted using the GoScriptTM kit Promega Corporation according to the manufacturer's instructions The reaction conditions for reverse transcription were ˚C for min ˚C for min and ˚C for min The SYBR Green Real‘Time PCR Master mix Thermo Fisher Scientific Inc was used to perform RT‘qPCR using a LightCycler480 system Roche Diagnostics GmbH The CHL1 primer sequences were as follows Forward '‘GGC TTG GTC TCT TGC TTT CC‘' and reverse '‘ATC TTC CCT CCC TTT GCA CG‘' and ‘actin forward '‘TTC CTT CCT GGG CAT GGA GTC ‘' and reverse '‘TCT TCA TTG TGC TGG GTG CC‘' The following thermocycling conditions were used for qPCR min at ˚C followed by cycles at ˚C for sec sec at ˚C and a final extension at ˚C for sec Each reaction was conducted in triplicate Relative expression levels were calculated using the ‘ΔΔCq method Cell viability Cell viability was detected by MTT assay A cell suspension µl was seeded into ‘well plates at a density of 1x104 cellswell and incubated overnight at ˚C The concentrations of DDP used to treat A549 cells were and µgml While the concentrations of PTX used to treat A549 cells were and ngml The concentrations of DDP used to treat A549DDP cells were and µgml While the concentrations of PTX used to treat A549PTX cells were and ngml After treating with different concentrations of DDP or PTX for h at ˚C µl MTT mgml solution was added to each well and incubated for h at ˚C Subsequently µl DMSO was added to each well to dissolve the blue formazan crystals and the absorbance was measured using a microplate reader BioTek Instruments Inc at nmClone formation assay A total of 1x103 cells were seeded into a ‘mm dish in triplicate and maintained in F‘12K medium with or without DDP or PTX at ˚C for h A total of weeks later cells were fixed in paraformalde‘hyde for min at room temperature and stained with crystal violet dye at room temperature for min The rate of colony formation was calculated using the following equation Number of coloniesnumber of seeded cells x100Flow cytometry Apoptosis was detected using a FITC Annexin V Apoptosis kit BD Pharmingen BD Biosciences according to the manufacturer's protocol Cells 1x105 were collected and washed twice with PBS prior to being suspended in µl binding buffer Subsequently cells were incubated with µl Annexin V‘FITC and µl propidium iodide in the dark for min at room temperature and apoptosis was analyzed using a CytoFlex flow cytometer Beckman Coulter Inc Data were analyzed using CytEXpert software Beckman Coulter Inc The ratio between early and late apoptosis was calculatedWestern blotting Cells were collected washed twice with PBS and lysed with RIPA lysis buffer Thermo Fisher Scientific Inc Proteins were isolated from the cell lysis buffer and 0cONCOLOGY LETTERS quantified using the Piercetm„¢ BCA Protein Assay kit cat no Thermo Fisher Scientific Inc with bovine serum album as a standard Equal amount of protein µg proteins were separated by SDS‘PAGE gel Next the proteins were transferred onto a polyvinylidene membrane Thermo Fisher Scientific Inc blocked with BSA Thermo Fisher Scientific Inc for h at ˚C and incubated overnight at ˚C with primary antibodies against CHL1 cat no ‘‘AP ProteinTech Inc multi‘drug resistance gene MDR1 cat no ‘‘AP ProteinTech Inc multidrug resistance‘associated protein MRP cat no ‘‘Ig ProteinTech Inc low‘density lipoprotein receptor‘related protein LRP cat no ‘‘AP ProteinTech Inc phosphorylated p‘Akt cat no ab38449 Abcam and Akt cat no ab227385 Abcam After washing three times with PBS the membrane was incubated with horseradish peroxide‘conjugated goat anti‘rabbit cat no ab6271 Abcam_or rabbit anti‘mouse cat no ab6728 Abcam secondary antibodies for h at room temperature and the blots were detected with enhanced chemiluminescence reagent Thermo Fisher Scientific Inc Protein expression was quantified using Image‘pro plus software Media Cybernetics IncAnimal experiments The animal experiments were approved by the Medical Ethics Committee of Xiangya Changde Hospital approval no and were performed in compliance with all regulatory institutional guidelines for animal welfare the National Institutes of Health Publications no ‘ A total of male BALBc‘nu mice ‘week‘old ± g Hunan SJA Laboratory Animals Center of the Chinese Academy of Sciences were used in this study All animals were kept at the SPF level laboratory at ‘ËšC a relative humidity of ‘ a h lightdark cycle and timesh of fresh air exchange All mice were given free access to food and water The bedding materials drinking water feeding cages and other items in contact with the animals were all autoclaved prior to use A549DDP cells 1x107 transfected with empty vector and CHL1 overexpression vector using Lipofectamine® reagent Thermo Fisher Scientific Inc were subcu‘taneously injected into the nude mice to establish xenograft models following anaesthesia with chloral hydrate mgkg Xenografts were allowed to grow to mm3 over weeks and the mice were randomly divided into four groups n3group as follows i vector group A549DDP cells transfected with empty vector and treated with µl saline solution ii vector‘DDP group A549DDP cells trans‘fected with empty vector and treated with mgkg DDP iii CHL1 group A549DDP cells transfected with CHL1 overexpression vector and treated with µl saline solu‘tion and iv CHL1‘DDP group A549DDP cells transfected with CHL1 overexpression vector and treated with mgkg DDP DDP was administered by intraperitoneal injection every days for weeks The mice were observed daily and the tumors were measured by a vernier caliper every days The tumor volumes were calculated as length x width22 A total of weeks post‘injection mice were euthanized with CO2 at volume displacement rate VDR per min using a programmable logic controller Barry‘Wehmiller Design Group Inc Mice were monitored continuously and once the mice were immobile except for breathing for min the VDR was provided at for min The animals remained in the euthanasia chamber for min and were then observed for an additional min Breathing and heart rate were monitored to determine deathStatistical analysis All experiments were performed in tripli‘cate and data are presented as the mean ± standard deviation All experiments were performed at least three times Paired Student's t‘test was performed for comparisons between two groups and one‘way analysis of variance followed by Tukey's multiple comparison post‘hoc analysis was performed for comparisons between multiple groups SPSS IBM Corp was used to perform the analysis P005 was considered to indicate a statistically significant differenceResultsCHL1 is downregulated in A549DDP and A549PTX‘resistant cells In order to investigate the mechanism of chemore‘sistance in lung cancer the lung adenocarcinoma cell line A549 the DDP‘resistant cells A549DDP and PTX‘resistant cells A549PTX were used in the present study Cells were exposed to different concentrations of DDP ‘ µgml and PTX ‘ ngml and MTT assay was used to detect the cell survival rate A549DDP and A549PTX cells demonstrated higher resistance to DDP and PTX compared with A549 cells Fig 1A The half maximal inhibitory concentration IC50 of DDP was significantly higher in A549DDP cells ± µgml compared with A549 cells ± µgml and the IC50 of PTX was significantly higher in A549PTX cells ± ngml compared with A549 cells ± ngml Fig 1B In addition the expression levels of the drug‘resistant markers MDR1 MRP and LRP were significantly higher in A549DDP and A549PTX cells compared with A549 cells Fig 1C Additionally the mRNA and protein expression levels of CHL1 were significantly lower in A549DDP and A549PTX cells compared with those in A549 cells Fig 1D and E and this was also observed in H460 DDP‘resistant cells obtained from the GEO dataset GSE21656 Fig 1F These results suggested that CHL1 may be involved in regulating DDP and PTX resistance in NSCLCKnockdown of CHL1 enhances resistance to DDP and PTX in A549 cells As CHL1 was upregulated in A549 cells CHL1 was silenced in A549 cells using siRNAs CHL1 expres‘sion was significantly reduced in the CHL1 siRNA groups compared with that of the scrambled control group Fig 2A As siRNA‘ demonstrated the greatest interference efficiency it was selected for use in the following experiments Notably CHL1‘knockdown enhanced the resistance to DDP and PTX in A549 cells Fig 2B and C Colony formation assay revealed that compared with the control group CHL1‘knockdown significantly increased the rate of colony formation in the absence of chemotherapeutics and enhanced the resistance to DDP and PTX Fig 2D Flow cytometry results demon‘strated significantly reduced apoptosis in CHL1‘knockdown cells after DDP and PTX treatment compared with that of the control group Fig 2E\x0cCAI CHL1 ENHANCES THE CHEMOSENSITIVITY OF LUNG CANCER CELLSFigure CHL1 is downregulated in DDP and PTX‘resistant A549 cells A Cell survival of A549 and A549‘resistant cells A549DDP and A549PTX treated with increasing concentrations of DDP and PTX as assessed by MTT assay B The IC50 values of DDP in A549DDP and A549 cells and the IC50 values of PTX in A549PTX and A549 cells P005 vs A549 cells C Western blotting demonstrated the expression of drug resistance‘related proteins MDR1 MRP and LRP in A549 cells and A549‘resistant cells A549DDP and A549PTX P005 vs A549 cells The protein and mRNA expression levels of CHL1 in A549 cells and A549‘resistant cells A549DDP and A549PTX were analysed by D western blotting and E reverse transcription‘quantitative PCR respectively P005 vs A549 cells F The mRNA expression of CHL1 in H460 and H460DDP cells in the GSE21656 dataset P005 vs H460 cells CHL1 close homolog of L1 DDP cisplatin PTX paclitaxel MDR1 multi‘drug resistance gene MRP multidrug resistance‘associated protein LRP low‘density lipoprotein receptor‘related protein IC50 half maximal inhibitory concentration CHL1 overexpression enhances the sensitivity of A549 resistant cells to DDP and PTX As CHL1 is downregu‘lated in A549DDP and A549PTX cells the present study successfully overexpressed CHL1 in these cells using CHL1 recombinant expression plasmids Fig 3A The results demonstrated that CHL1 overexpression alleviated the resis‘tance to DDP and PTX compared with that of the control group Fig 3B and C In addition CHL1 overexpression inhibited colony formation in the absence or presence of DDP and PTX Fig 3D Additionally flow cytometry results demonstrated that restoration of CHL1 expression promoted apoptosis in resistant cells following DDP and PTX treat‘ment Fig 3ETo further validate the effects of CHL1 overexpression on DDP or PTX sensitivity xenograft mice model experi‘ments were performed The results demonstrated that CHL1 overexpression or DDP treatment significantly impeded the tumor growth Fig 3F and decreased the tumor weight Fig 3G In addition CHL1 overexpression further aggra‘vated DDP‘mediated repression on tumor growth Fig 3F and G These data suggested that CHL1 overexpression suppressed tumor growth and enhanced the chemosensitivity in NSCLCCHL1 mediates chemosensitivity by inhibiting Akt activity Recently studies have confirmed that CHL1 inhibits Akt activity in ESCC and neuroblastoma cell lines Thus the present study investigated whether CHL1 mediates chemoresistance via the Akt pathway in NSCLC In A549 cells compared with the scrambled group CHL1‘knockdown elevated the expression of p‘Aktser473 Fig 4A By contrast restoring CHL1 expression in A549DDP and A549PTX cells inhibited the Akt phosphorylation compared with the control group Fig 4A suggesting CHL1 mediates chemosensitivity via the Akt pathway Subsequently CHL1‘silenced A549 cells were treated with the Akt inhibitor SC66 and it was demon‘strated that inhibiting Akt activity significantly reduced the promotive effects on cell survival Fig 4B and clone forma‘tion Fig 4C and the inhibitory effects on apoptosis Fig 4D induced by CHL1‘depletion These results confirmed that CHL1 mediates chemosensitivity in NSCLC by inhibiting the Akt pathway\x0cONCOLOGY LETTERS Figure CHL1‘knockdown increases A549 cell resistance to DDP and PTX A Western blotting was performed to validate the efficiency of transfection with CHL1 siRNAs P005 vs scramble MTT assays were performed to determine the survival rate of CHL1‘knockdown A549 cells treated with B ‘ µgml DDP or C ‘ ngml DDP D Colony formation assay of A549 cells transfected with CHL1 siRNA in the presence or absence of µgml DDP or ngml PTX E Flow cytometry analysis was used to detect apoptosis in A549 cells transfected with CHL1 siRNA in the presence or absence of µgml DDP or ngml PTX P005 P0001 CHL1 close homolog of L1 DDP cisplatin PTX paclitaxel si small interfering DiscussionThe results of the present study demonstrated that CHL1 was significantly downregulated in A549DDP and A549PTX cells compared with A549 cells The knockdown of CHL1 in A549 cells facilitated the cell survival and clone formation and decreased apoptosis when treated with or without DDP and PTX whereas CHL1 overexpression in A549DDP and A549PTX cells inhibited cell survival and clone formation and increased apoptosis The results of the present study also demonstrated that CHL1 enhances NSCLC chemosensitivity through inhibition of the Akt pathway These data suggested that CHL1 may be a promising target to improve the efficacy of chemosensitivity in NSCLCCHL1 belongs to the L1 family of nerve cell adhesion molecules it was initially cloned in mice and its expression in mouse development was analyzed by Senchenko Through cell‘cell interactions and mediating cell‘cell and cell‘matrix interactions CHL1 has an important effect on the development regeneration and plasticity of the nervous system Previous reports have demonstrated that CHL1 also participates in carcinogenesis ‘ CHL1 was observed to be significantly downregulated in up to types of tumor tissues compared with their adjacent normal tissues In most tumors CHL1 is a potential tumor suppressor gene whose silencing is associated with tumor growth invasion and metastasis ‘ For example knockdown of CHL1 expression results in enhanced cervical cancer cell invasion and migration A low expres‘sion of CHL1 in patients with neuroblastoma predicts a poor prognosis and enhancing CHL1 expression suppresses tumor progression In contrast CHL1 has been reported to promote cell proliferation metastasis and migration in human gliomas However to the best of our knowledge research on CHL1 and tumor chemoresistance has rarely been reported\x0cCAI CHL1 ENHANCES THE CHEMOSENSITIVITY OF LUNG CANCER CELLSFigure Overexpression of CHL1 increases the sensitivity of resistant A549 cells to DDP and PTX A Western blotting was performed to detect CHL1 expression in A549DDP and A549PTX cells transfected with CHL1 expression plasmids P005 vs vector Effect of CHL1 overexpression on resistant A549 cell survival rate when treated with B ‘ µgml DDP or C ‘ ngml PTX as determined by MTT assay D Colony formation assays demon‘strated the number of colonies of resistant A549 cells transfected with CHL1 expression plasmids in the presence or absence of µgml DDP or ngml PTX E Flow cytometry analysis was performed to assess apoptosis in resistant A549 cells transfected with CHL1 expression plasmids in the presence or absence of µgml DDP or ngml PTX CHL1 overexpression enhanced chemosensitivity of A549DDP cells to DDP in vivo which was demonstrated by the effect of DDP treatment or CHL1 overexpression on the F growth and G weight of xenografts derived from A549DDP cells P005 P001 CHL1 close homolog of L1 DDP cisplatin PTX paclitaxel The present study examined the differentially expressed genes in NSCLC DDP‘resistant cells in a GEO dataset CHL1 was demonstrated to be upregulated in DDP‘resistant cells compared with parental cells suggesting that CHL1 may be involved in NSCLC chemotherapy resistance Similarly a study that compared and analyzed the differentially expressed genes in chemosensitive tumors and chemoresistant ovarian adenocarcinomas tissues reported that the expression of CHL1 in chemotherapy‘sensitive tumor tissues is higher compared with that in drug‘resistant tissues suggesting that CHL1 may help to predict the efficacy of chemotherapy for ovarian cancer In addition aberrant methylation of CHL1 may be associated with the recurrence of colorectal cancer CRC following chemotherapy ‘azadC treatment restores ‘fluro‘uracil sensitivity in vitro which also suggests that CHL1 may be involved in CRC chemotherapy resistance The results of the present study demonstrated that CHL1 was down‘regulated in A549DDP cells Additionally as multiple drug resistance is a common characteristic another type of resistant cells A549TAX cells were also used in the current study The results also demonstrated that CHL1 was downregulated in A549PTX cells Compared with control cells overexpression of CHL1 significantly increased the sensitivity of cells resistant to DDP and PTX whereas knockdown of CHL1 expression in 0cONCOLOGY LETTERS Figure CHL1 mediates DDP and PTX sensitivity by inhibiting Akt activity A Western blotting was performed to detect the expression of p‘Akt and total Akt in CHL‘silenced and ‘restored cell models P005 vs scramble or vector B MTT assays were performed to detect cell survival rates of A549 cells treated with CHL1 siRNA and Akt inhibitor SC66 P005 C Colony formation assays were performed in A549 cells treated with CHL1 siRNA and the Akt inhibitor SC66 in the presence of DDP µgml or PTX ngml P005 vs si‘CHL1 D Apoptosis were measured in A549 cells treated with CHL1 siRNA and Akt inhibitor SC66 in the presence of DDP µgml and PTX ngml P005 vs si‘CHL1 CHL1 close homolog of L1 DDP cisplatin PTX paclitaxel si small interfering p‘ phosphorylated parent A549 cells displayed the opposite results To the best of our knowledge this study is the first study to suggest that CHL1 may be involved in chemosensitivity in lung cancer The concentration of DDP used in vivo is mgkg however this may not be in line with the concentrations that would be used in a clinical setting In a clinical trial the human initial dose was calculated from the no observed adverse effect levels NOAELs verified in animal experiments NOAEL is the maximum dose level without significant adverse reactions The NOAEL verified in animal experiments can be converted to a human equivalent dose according to the body surface area conversion which is based on the area standardization mgm2 proportional among different species In the present study the concentration of DDP used in vivo was not the NOAEL thus it was not consistent with the concentrations used in clinical settingsAkt is a serinethreonine protein kinase that is activated by phosphorylation As a key molecule of the PI3KAkt signaling pathway p‘Akt regulates cell survival cell growth cell motility and angiogenesis and prevents apoptosis Additionally Akt activation is associated with tumor chemore‘sistance The results of the present study demonstrated that compared with the control groups the expression of p‘Akt was increased in CHL1‘knockdown A549 cells and its expres‘sion was reduced in CHL1 overexpressed A549DDP and A549PTX cells When Akt activity was inhibited by the Akt inhibitor the sensitivity to DDP and PTX in CHL1‘knockdown A549 cells was restored This finding suggested that CHL1 enhanced the chemosensitivity of NSCLC by inhibiting the Akt pathway Considering numerous studies have confirmed that the Akt pathway mediates chemoresistance via regulation of ATP binding cassette ABC members ‘ the present study didn't further investigate the specific ABC members and mechanisms which was a of the limitation to the present study thus this research should be further investigated in vivoIn summary the present study demonstrated that CHL1 was downregulated in resistant cells A549DDP and A549PTX and upregulation of CHL1 enhanced the chemosensitivity of NSCLC via inhibiting the Akt pathway To the best of our knowledge this was the first study to confirm the function and 0cCAI CHL1 ENHANCES THE CHEMOSENSITIVITY OF LUNG CANCER CELLSmechanism of CHL1 in mediating chemosensitivity in cancer Thus the development of CHL1‘based therapeutic strategies may improve the efficacy of chemosensitivity in NSCLCAcknowledgementsThe authors of the present study would like to thank Mr Dingliang Li Xiangya Hospital Changsha China for his guidance and assistance in flow cytometric analysisFundingNo funding was receivedAvailability of data and materialsThe datasets used andor analyzed during the present study are available from the corresponding author upon reasonable requestAuthors' contributionsRH conceived and designed the present study XC BH YH and PL performed experiments and collected the data SL ZZ and ZH analyzed and interpreted the data ML and LZ analyzed the data and prepared the figure XC ML and LZ drafted the initial manuscript and revised it for intellectual content All authors read and approved the final manuscriptEthics approval and consent to participateThe animal experiments were approved by the Medical Ethics Committee of Xiangya Changde Hospital Changde China approval no Patient consent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsReferences Parascandola M and Xiao L Tobacco and the lung cancer epidemic in China Transl Lung Cancer Res Suppl S21‘S30 Chen W Cancer statistics Updated cancer burden in China Chin J Cancer Res Oser MG Niederst MJ Sequist LV and Engelman JA Transformation from non‘small‘cell lung cancer to small‘cell lung cancer Molecular drivers and cells of origin Lancet Oncol e165‘e172 Thatcher N Faivre‘Finn C Blackhall F Anderson H and Lorigan P Sequential platinum‘based chemotherapy‘thoracic radiotherapy in early stage non‘small cell lung cancer Clin Cancer Res Suppl S5051‘S5056 Yano T Okamoto T Fukuyama S and Maehara Y Therapeutic strategy for postoperative recurrence in patients with non‘small cell lung cancer World J Clin Oncol ‘ Fang Z Chen W Yuan Z Liu X and Jiang H LncRNA‘MALAT1 contributes to the cisplatin‘resistance of lung cancer by upregulating MRP1 and MDR1 via STAT3 activation Biomed Pharmacother ‘ Cai Y Dong ZY and Wang JY LncRNA NNT‘AS1 is a major mediator of cisplatin chemoresistance in non‘small cell lung cancer through MAPKSlug pathway Eur Rev Med Pharmacol Sci ‘ Han ML Zhao YF Tan CH Xiong YJ Wang WJ Wu F Fei Y Wang L and Liang ZQ Cathepsin L upregulation‘induced EMT phenotype is associated with the acquisition of cisplatin or paclitaxel resistance in A549 cells Acta Pharmacol Sin ‘ Liu J Meisner D Kwong E Wu XY and Johnston MR Translymphatic chemotherapy by intrapleural placement of gelatin sponge containing biodegradable Paclitaxel colloids controls lymphatic metastasis in lung cancer Cancer Res ‘ Hassan WA Yoshida R Kudoh S Kameyama H Hasegawa K Niimori‘Kita K and Ito T Notch1 controls cell chemoresistance in small cell lung carcinoma cells Thorac Cancer ‘ Tang H Jiang L Zhu C Liu R Wu Y Yan Q Liu M Jia Y Chen J Qin Y Loss of cell adhesion molecule L1 like promotes tumor growth and metastasis in esophageal squamous cell carcinoma Oncogene ‘ Liu H Focia PJ and He X Homophilic adhesion mechanism of neurofascin a member of the L1 family of neural cell adhesion molecules J Biol Chem ‘ Tassano E Biancheri R Denegri L Porta S Novara F Zuffardi O Gimelli G and Cuoco C Heterozygous deletion of CHL1 gene Detailed array‘CGH and clinical characterization of a new case and review of the literature Eur J Med Genet ‘ Morellini F Lepsveridze E Kahler B Dityatev A and Schachner M Reduced reactivity to novelty impaired social behavior and enhanced basal synaptic excitatory activity in perforant path projections to the dentate gyrus in young adult mice deficient in the neural cell adhesion molecule CHL1 Mol Cell Neurosci ‘ He LH Ma Q Shi YH Ge J Zhao HM Li SF and Tong ZS CHL1 is involved in human breast tumorigenesis and progres‘sion Biochem Biophys Res Commun ‘ Martín‘Sánchez E Mendaza S Ulazia‘Garmendia A Monreal‘Santesteban I Blanco‘Luquin I Córdoba A Vicente‘García F Pérez‘Janices N Escors D Megías D CHL1 hypermethylation as a potential biomarker of poor prog‘nosis in breast cancer Oncotarget ‘ Zhu H Fang J Zhang J Zhao Z Liu L Wang J Xi Q and Gu M miR‘ targets CHL1 and controls tumor growth and invasion in papillary thyroid carcinoma Biochem Biophys Res Commun ‘ Yu W Zhu K Wang Y Yu H and Guo J Overexpression of miR‘‘5p promotes proliferation and invasion of colon adeno‘carcinoma cells through targeting CHL1 Mol Med Hötzel J Melling N Müller J Polonski A Wolters‘Eisfeld G Izbicki JR Karstens KF and Tachezy M Protein expression of close homologue of L1 CHL1 is a marker for overall survival in non‘small cell lung cancer NSCLC J Canc Answer:

Thyroid_Cancer

Pralatrexate is a folate analogue inhibitor of dihydrofolate reductase exhibiting high affinity for reduced folate carrier1 with antineoplastic and immunosuppressive activities similar to methotrexate Despite advances in multimodality treatment strategies the survival rates for children with highrisk neuroblastoma have failed to improve Therefore the intense research continues in order to identify the ideal novel agent or combination of chemotherapy drugs to treat highrisk neuroblastomaMaterials and Methods Four human neuroblastoma cell lines were used to determine IC50 values of select chemotherapy agents Antiproliferative effects of pralatrexate were assessed by adherent and nonadherent colony formation assays Cell cycle arrest and apoptosis were measured by flow cytometry and immunoblotting PDX tissue culture was used to assess ex vivo efficacyResults Treatment with pralatrexate in all four neuroblastoma cell lines blocked cell growth in 2D and 3D culture conditions in a timedependent manner The potency of pralatrexate was tenfold stronger than methotrexate as measured by IC50 Pralatrexateinduced apoptosis was confirmed by caspase3 activation and PARP cleavage MYCN and SLC19A1 mRNA expressions were decreased with pralatrexate in MYCNamplified neuroblastoma cellsConclusions Pralatrexate demonstrated effective inhibition of cell growth and viability The higher potency of pralatrexate compared to methotrexate a drug with high levels of toxicity suggests pralatrexate may be a safer alternative to methotrexate as an effective chemotherapeutic agent in the treatment of patients with highrisk neuroblastomaINTRODUCTIONNeuroblastoma is a pediatric tumor derived from neural crest cells It is the most common pediatric solid tumor accounting for approximately of pediatric cancer deaths [] and it typically presents as a painless abdominal mass in infants and toddlers of to months of age [] Poor prognostic factors in children with neuroblastoma include age months at time of diagnosis unfavorable histology increased vascularization and MYCN amplification [] Despite intense research focused on the biology of neuroblastoma it remains one of the most enigmatic pediatric cancers in terms of its underlying molecular pathogenesis There has been only incremental improvement in the overall survival of children with highrisk neuroblastoma necessitating the search for a novel agent or combination of chemotherapy drugs []is key Altered metabolism to cancer cell proliferation Among the various metabolic pathways that are affected folate metabolism plays an important role Folate is essential for DNA synthesis and cell growth especially in rapidly dividing cells Inhibition of folate metabolism is the basis for many chemotherapy drugs In neuroblastoma folate mediated onecarbon metabolism is associated with aggressiveness and MYCN amplification Oncotargetwwwoncotargetcomwwwoncotargetcom Oncotarget Vol No pp 0cMethotrexate is a widely used [] A study by Lau in demonstrated higher folate requirements in MYCN amplified neuroblastoma cells compared to nonMYCN amplified cells [] They also showed that the increased folate uptake is mediated by reduced folate carrier1 RFC1 which is encoded by the gene SLC19A1 [] Previous studies have demonstrated that SLC19A1 is associated with MYCN amplification in neuroblastoma and that SLC19A1 is a direct transcriptional target of Nmyc [] The association between MYCN amplification and folate metabolism suggests the potential role of antifolate drugs in the treatment of neuroblastomasinhibitor of folate metabolism It inhibits dihydrofolate reductase DHFR and therefore disrupts purine and thymidylate biosynthesis leading to inhibited DNA replication and cell death However in the 1970s methotrexate was found to have high levels of toxicity combined with low treatment response rates in neuroblastoma patients and therefore it has not been clinically used for neuroblastoma treatment [] Pralatrexate is a folate analogue inhibitor of DHFR that exhibits high affinity for RFC1 [] and folylpolyglutamate synthetase FPGS Pralatrexate demonstrates antineoplastic and immunosuppressive properties that are similar to methotrexate It was FDA approved in the United States for treatment of relapsed or refractory peripheral Tcell lymphoma in [] A study by Serova in demonstrated decreased mRNA expression of SLC19A1 and SLC25A32 a mitochondrial folate carrier with pralatrexate treatment in several cancer cell lines [] As previously discussed SLC19A1 is downstream target of Nmyc in neuroblastoma The high affinity of pralatrexate for the SLC19A1 encoded RFC1 protein may demonstrate a potential role in the treatment of MYCNamplified neuroblastomaThe development of a new chemotherapeutic regimen is a long process that can take years to enter clinical trials and subsequently into bedside therapy Identifying alterative applications for previously FDAapproved drugs is a method that allows for quicker use in clinical practice [] Therefore we sought to evaluate current FDA approved antineoplastic drugs as potential novel treatment strategies for highrisk neuroblastoma and set out to assess the inhibitory role of pralatrexate on neuroblastoma cellsRESULTSThe IC50 of pralatrexate is tenfold less than methotrexateFour human neuroblastoma cell lines including MYCNamplified BE2C CHP212 and LAN1 as well as the nonMYCN amplified cell line SKNAS were treated with methotrexate or pralatrexate Cell growth was determined after h of continuous exposure to methotrexate or pralatrexate nM“ µM measured by Cell Titer Glo„¢ assay and the IC50 of each drug was calculated for each cell line using the Genedata Screener software Values shown are mean ± SD of three separate experiments As shown in A in all four cell lines the IC50 of pralatrexate was approximately tenfold less than the IC50 of methotrexate These data demonstrate that highrisk neuroblastoma cells have enhanced pralatrexate sensitivity compared to methotrexate and that pralatrexate inhibits both MYCN amplified and nonMYCN amplified neuroblastoma growth in the low nanomolar range in vitroPralatrexate inhibited neuroblastoma cell growthTo demonstrate the timing of growth inhibition we treated neuroblastoma cells with pralatrexate or nM and measured cellular viability over a time course of days B Significant cell growth inhibition was first noted by day in SKNAS and CHP212 cells and by day in LAN1 and BE2C cells This indicates that pralatrexate effectively inhibits the proliferative potential of neuroblastoma cells We further validated the effects of pralatrexate on neuroblastoma cells in vitro by quantifying colony growth in a 3D matrix hydrogel where cells grow and selfassemble into clusters 3D cultures are more physiologically relevant and better represent in vivo tissue BE2C and LAN1 cells are high colonyforming neuroblastoma cell lines [] Concurrent treatment with pralatrexate completely abolished the ability of BE2C and LAN1 cells to develop colonies in gel drops A Both BE2C and LAN1 cell lines treated with pralatrexate demonstrated a decreased colony count and a decrease in colony size compared to cells treated with DMSO Figure 2B and 2C The colonies were counted from three separate microscopic fields and their size was measured using the scale bar on each image using Image J These findings suggest that pralatrexate represses the tumorigenesis potential and tumor progression of neuroblastomaPralatrexate induced G1 phase cell cycle arrest apoptosis and decreased Nmyc expressionTo further test whether pralatrexate directly altered neuroblastoma cell proliferation we evaluated the cell cycle distribution of treated cells compared with control Cell cycle analysis was performed in BE2C cells treated with pralatrexate nM or control at days and after treatment We observed a significant but modest increase in the G1 phase of the cell cycle ranging from a “ increase in the G1 cell population demonstrating induction of G1 cell cycle arrest Figure 3A Given the dramatic decrease in cell viability observed between days and of pralatrexate treatment Figure 1B we hypothesized that pralatrexate may also induce apoptosis in neuroblastoma cells To confirm apoptosis Oncotargetwwwoncotargetcom\x0cin cells treated with pralatrexate Western blotting was performed BE2C and CHP212 cells were treated with increasing doses of pralatrexate and nM The protein expression of total and cleaved caspase3 as well as total and cleaved PARP were examined at each increasing dose of pralatrexate Figure 3B confirming the induction of apoptosis Apoptosis was also seen secondary to pralatrexate treatment in nonMYCN amplified cells SKNAS SKNSH and SHSY5Y Supplementary Figure Given the proliferating role of Nmyc in neuroblastoma tumorigenesis we next evaluated whether pralatrexate could alter the Nmyc expression Interestingly we found decreased Nmyc expression with increasing doses of pralatrexate in both BE2C and CHP212 cells Figure 3B demonstrating persistent defects in proliferative potential induced by pralatrexate in neuroblastomaPralatrexate decreased MYCN and SLC19A1 gene expressions compared to SLC25A32Previous studies demonstrated that the expression of SLC19A1 the gene encoding the RFC1 receptor is associated with MYCN amplification in neuroblastoma [] Pralatrexate is a folate analogue inhibitor with high affinity for RFC1 [] Therefore we sought to examine the effects of pralatrexate treatment on MYCN and SLC19A1 gene expression in BE2C and CHP212 cell lines compared to the effects in nonMYCN amplified cells BE2C and CHP212 cells were treated with and nM pralatrexate or DMSO and qPCR was performed As expected from the finding in Figure 3B pralatrexate treatment resulted in decreased MYCN expression in both BE2C and CHP212 cells Figure 4A and 4B Interestingly we also found that both BE2C and CHP212 cells treated with pralatrexate demonstrated decreased SLC19A1 expression but no difference in SLC25A32 expression compared to control cells Figure 4A and 4B Treatment of nonMYCN amplified cells SKNAS SKNSH and SHSY5Y did not affect SLC19A1 or SLC25A32 expression Supplementary Figure These findings may support the previous studies [ ] that SLC19A1 is a direct transcription target of MYCN in neuroblastomas and pralatrexate treatment affects SLC19A1 expression in MYCN amplified cells but not SLC25A32 expression In addition in both BE2C and CHP212 cells pralatrexate did not decrease FPGS mRNA expression Figure 4C and 4D Neither SLC25A32 or FPGS expression was affected in nonMYCN amplified cells treated with pralatrexate Supplementary Figure Interestingly in the BE2C cells and the nonMYCN amplified cells pralatrexate treatment led to an increase in DHFR expression This may imply treatment with pralatrexate selected for cells with increased DHFR expression and inherent pralatrexate resistance or a resultant upregulation of the DHFR gene with DHFR protein inhibition These findings would Figure IC50 of human neuroblastoma cell lines BE2C CHP212 and LAN1 MYCNamplified and SKNAS nonMYCNamplified A The IC50 doses of BE2C CHP212 LAN1 and SKNAS cells treated with methotrexate were and µM respectively The IC50 doses of BE2C CHP212 LAN1 and SKNAS cells treated with pralatrexate were and µM respectively Cell growth was determined after h of continuous exposure to methotrexate or pralatrexate nM“ µM measured by Cell Titer Glo„¢ assay and the IC50 of each drug was calculated for each cell line using the Genedata Screener software Values shown are mean ± SD of three separate experiments B Cell viability was assessed using the Cell Counting Kit8 assay Pralatrexate significantly inhibited cell viability in all four neuroblastoma cell lines as compared to DMSO control groupOncotargetwwwoncotargetcom\x0cbe consistent with findings by Serova et al in which pralatrexateresistant cells demonstrated increased DHFR protein expression []Pralatrexate treatment response in ex vivo neuroblastoma growthGiven these persistent in vitro findings we next evaluated whether pralatrexate could be evaluated ex vivo to guide treatment decisions for individual patients We used a neuroblastoma PDX model where tumors were dissected into 1mm3 pieces and cultured in duplicate on a presoaked gelatin sponge in 24well plates containing µL RPMI with FBS antibioticantimycotic solution mgmL hydrocortisone and mgmL insulin The size of these fragments is comparable to the size of a standard clinical tumor biopsy specimen The fragments were then cultured for days in the presence of pralatrexate nM using standard cell culture conditions Notably tumor tissues were significantly affected by ex vivo pralatrexate treatment and showed decreased Ki67 staining compared to tissues cultured in vehicle control treatment Figure These results suggest that a simple shortterm ex vivo treatment assay of a viable tumor specimen may aid in identifying neuroblastoma patients who are likely to gain benefit from pralatrexate treatment options in the futureDISCUSSIONHighrisk neuroblastoma remains quite difficult to cure necessitating the discovery of new chemotherapy agents to be used alone or in combination therapy Previous studies have reported an increased folate in MYCNamplified neuroblastoma cells demand mediated by the RFC1 receptor [] Additionally the gene encoding the RFC1 receptor SLC19A1 is a direct transcriptional target of Nmyc in neuroblastoma cells suggesting a role for antifolate drugs in the treatment of neuroblastoma Methotrexate has previously been studied in neuroblastoma however it was found to have a prohibitive toxicity and has not been used in neuroblastoma clinically In contrast pralatrexate a folate analogue inhibitor is similar to methotrexate with a more favorable side effect profile suggesting a potential role for the use of pralatrexate as a chemotherapeutic agent against neuroblastomaThe present study sought to determine the effects of treatment with pralatrexate on in vitro and ex vivo cell growth in four human neuroblastoma cell lines The IC50 of pralatrexate was found to be 10fold less than that of methotrexate This tenfold difference between pralatrexate and methotrexate was also found in colon breast and thyroid cancer cells by Serova et al in [] The decreased IC50 of pralatrexate allows for treatment with lower doses and a more tolerable sideeffect profile compared to methotrexate independent of MYCN amplificationPralatrexate not only induced celldeath via apoptosis but it also successfully inhibited neuroblastoma in vitro cell growth and proliferation in 2D and 3D cell cultures as well as in our PDX exvivo model By inhibiting the RFC1 receptor pralatrexate decreased the amount of folate entering cells and in turn decreased Figure Pralatrexate inhibited neuroblastoma colony growth A Representative images of light microscopy × magnification for BE2C and LAN1 cells after days of treatment with pralatrexate versus control Pralatrexate treatment decreased cell growth in both BE2C and LAN1 cells compared to control scale bar μm B Colony count and colony size for BE2C cells were analyzed and quantified mean ± SD p for nM pralatrexate treatment vs no drug C Colony count and colony size for LAN1 cells were analyzed and quantified mean ± SD p for nM pralatrexate treatment vs no drugOncotargetwwwoncotargetcom\x0cDNA synthesis This was demonstrated by the increased time spent in the G1phase of the cell cycle in cells treated with pralatrexate PDXs have been shown to parallel clinical outcome in various tumor types [] The major applications of neuroblastoma PDXs would be related to drug testing exploration of treatment resistance and biomarker discovery Combining PDXs and ex vivo culture will incorporate human tumor tissue in its native Figure Effects of pralatrexate treatment on caspase3 PARP and Nmyc protein expression A Cell cycle analysis with propidium iodide demonstrates enhanced G1 cell cycle arrest at and h following treatment Cell cycle analysis was completed with events per replicate mean ± SD p for nM pralatrexate treatment vs no drug B Treatment with increasing doses of pralatrexate induced apoptosis in BE2C and CHP212 cells Cells treated with pralatrexate demonstrated cleaved caspase3 protein expression when treated with and nM doses Cleaved PARP expression was noted after treatment with nM Treatment with pralatrexate decreased Nmyc protein expression in BE2C and CHP212 cells βactin was used as an internal controlOncotargetwwwoncotargetcom\x0c3D state and enable dynamic manipulation of the system minimizing animal experiments and costLau has shown that SLC19A1 is a downstream direct transcriptional target of Nmyc in neuroblastoma cells and that MYCNamplified cells have an increased folate dependence [] In our study pralatrexate also led to a decrease in expression of the RFC1 genes SLC19A1 and SLC25A32 The decrease in SLC19A1 was more pronounced compared to the mitochondrial folate receptor gene SC25A32 suggesting pralatrexate may be more specific to the cytosolic RFC1 receptor compared to the mitochondrial RFC1 receptor However further studies are necessary to investigate this relationship Meanwhile pralatrexate treatment led to a marked increase in DHFR expression in BE2C cells and a slight increase in CHP cells This is unlikely an upregulation of DHFR and more indicative of increased DHFR mRNA being harvested from pralatrexate resistance cells Similar results were found in a previous study on colon breast and thyroid cancer cells lines Serova found that pralatrexateresistant cells had increased DHFR protein expression [] The increase in DHFR expression may lead to an increase in the amount of DHFR protein requiring more than nM of pralatrexate to inhibit cell growth and proliferation However further studies surrounding the dose of pralatrexate and its relationship to DHFR gene expression are neededNeuroblastoma is a heterogenous tumor and further studies are needed to examine the effects of pralatrexate on additional cells lines Additionally the remaining cells that survived after pralatrexate treatment may represent pralatrexate resistant cells Future studies are needed to elucidate potential mechanisms of pralatrexate resistance such as increased DHFR gene expression as well as the relationship between pralatrexate and SLC19A1 versus other folate synthesis enzyme expressions Given pralatrexate is already an FDAapproved and in clinical use future clinical studies are needed to investigate the effects of pralatrexate treatment on neuroblastoma in vivoMATERIALS AND METHODSCells antibodies and reagentsThe neuroblastoma cell line LAN1 was a gift from Dr Robert C Seeger University of Southern California Los Angeles CA All other neuroblastoma cell lines BE2C CHP212 SKNAS SKNSH and SHSY5Y Figure Effects of pralatrexate treatment on MYCN SLC19A1 and SLC25A32 gene expressions After day of treatment the mRNA expression of MYCN SLC19A1 and SLC25A32 were measured by qPCR in A BE2C cells treated with nM of pralatrexate when compared with DMSO treated cells and in B CHP212 cells treated with nM of pralatrexate C D qPCR was performed on BE2C and CHP212 cells treated with pralatrexate to assess for mRNA expression of two keyenzymes in folate synthesies DHFR and FPGS in the treatment mean ± SD p for pralatrexate treatment vs no drugOncotargetwwwoncotargetcom\x0cwere purchased from the American Type Culture Collection ATCC Manassas VA Cells were maintained in RPMI with glutamine and FBS at °C in a humidified atmosphere consisting of CO2 and air Primary antibodies for Caspase3 Cat No PARP Cat No Nmyc Cat No were purchased from Cell Signaling Technology Danvers MA Ki67 Cat No was from Abcam Cambridge MA and βactin Cat No A2066 was from SigmaAldrich St Louis MO Methotrexate and pralatrexate were obtained from National Cancer InstituteDivision of Cancer Treatment and DiagnosisDevelopmental Therapeutics Program httpdtpcancergov and dissolved in dimethyl sulfoxide DMSO and further diluted in culture media to desired concentrations Neuroblastoma COGN415× patientderived xenograft PDX cells were obtained from the Childhood Cancer Repository maintained by the Children™s Oncology Group COG and Xenograft RepositoryDrug sensitivity and dose responsive curve assayFor cell viability screening cells [BE2C1500well LAN1 3000well CHP2124000well SKNAS 3000well] were plated and treated the following day with methotrexate and pralatrexate Cell growth was determined after h of continuous exposure to nM“ µM of methotrexate or pralatrexate using Cell Titer GloTM reagent Promega with luminescence measured using an EnVision multilabel plate reader PerkinElmer IncCell viability assayNeuroblastoma cells were seeded onto 96well plates permitted to attach overnight and were treated with either pralatrexate or nM or DMSO for days Cell viability measurements using the Cell Counting Kit8 Dojindo Molecular Technologies Inc Rockville MD were obtained daily3D colony formation assayBE2C or LAN1 cells were trypsinized embedded in μl of Cultrex® RGF BME Type matrix hydrogel Trevigen Gaithersburg MD and seeded in 48well plates cellswell RPMI medium containing FBS was added with pralatrexate treatment and incubated for days Colonies were photographed and the number and size were quantified The colonies were counted from three separate microscopic fields and their size was measured by the scale bar on each image using Image JCell cycle analysisCell cycle distribution was analyzed using flow cytometry with propidium iodide Sigma Aldrich BE2C cells were plated at equal numbers × cells and treated with either pralatrexate nM or DMSO At day and after treatment cells were washed and fixed in ethanol Fixed cells were incubated with mgmL RNAase for minutes at °C stained with propidium iodide mg mL and analyzed on a BD FACSCalibur BD Biosciences San Jose CAqPCR and immunoblottingTotal RNA was isolated and purified using a TRIzol® Reagent Thermo Scientific cDNA was synthesized using the qScript cDNA SuperMix QuantaBio RealFigure Ex vivo tissue culture model recapitulated antitumor response to pralatrexate Representative HE and Ki67 immunohistochemistry staining sections were obtained from a neuroblastoma PDX COGN415× treated ex vivo with nM pralatrexate or vehicle control for days and demonstrated poorly differentiated neuroblastoma cells and decreased Ki67 staining in pralatrexatetreated tumor compared to vehicle control × magnification scale bar μmOncotargetwwwoncotargetcom\x0creverse reverse time PCR and data collection were performed on a CFX96 instrument BioRad Data were normalized to an endogenous control βactin Specific target primers are MYCN forward 5ʹGCTTCTACCCGGACGAAGATG3ʹ reverse 5ʹCAG CTCGTTCTCAAGCAGCAT3ʹ SLC19A1 forward 5ʹAACAGGTCTGGGTTTTGTGC3ʹ 5ʹGTGCAGTATCATGCCCTGTG3ʹ SLC25A32 forward 5ʹATTGGTGGAAGCTGATTTGC3ʹ 5ʹTGGTCTGGATTTGGTCAACA3ʹ DHFR forward 5ʹCTCAAGGAACCTCCACAAGG3ʹ reverse 5ʹGTTTAAGATGGCCTGGGTGA3ʹ FPGS forward 5ʹGGGTGACCCTCAGACACAGT3ʹ reverse 5ʹGTCTTCAGGCCATAGCTTCG3ʹ Amplification was performed for cycles of s at °C s at °C and s at °C Whole cell lysates were collected using cell lysis buffer and equal amounts of protein were loaded on a NuPAGE “ BisTris gel followed by transfer onto PVDF membranes BioRad Hercules CA USA and probed with antibodiesEx vivo culture and immunohistochemistryCOGN415× patientderived xenograft cells were obtained from the Childhood Cancer Repository maintained by COG Clinical and genomic features of the tumors were detailed in a study by Harenza in [] Cells were suspended in Matrigel diluted with PBS and × cells were injected into the flank of NOD scid gamma mice at “ weeks of age UTSW Mouse Breeding Core All studies were approved by the Institutional Animal Care and Use Committee at University of Texas Southwestern Medical Center To keep cost down we used only female mice for passing PDX Mice were euthanized once tumors reached mm and tumors were dissected into 1mm3 pieces and cultured in duplicate on a presoaked gelatin sponge Johnson and Johnson in 24well plates containing µL RPMI with FBS antibioticantimycotic solution mgmL hydrocortisone and mgmL insulin SigmaAldrich Tissues were cultured at °C for days with either pralatrexate nM or vehicle control then formalinfixed and paraffin embedded COGN415× tissue sections were stained with hematoxylin and eosin or with an antibody against Ki67Statistical analysisAll results are shown as the mean value ± SD statistical analyses were performed using student ttest for comparisons between the groups A p value of was considered significant GraphPad™s Prism software was used for the statistical analysisAbbreviationsCOG Children™s Oncology Group DHFR dihydrofolate reductase DMSO dimethyl sulfoxide FPGS folylpolyglutamate synthetase PDX patientderived xenograft RFC1 reduced folate carrier1Author contributionsStudy concept and design RAC SL JQ DHC Acquisition of data RAC SL JQ Analysis and interpretation of data RAC SL JQ DHC Drafting of manuscript RAC SL JQ DHC Critical revision RAC SL JQ DHC RAC and SL contributed equally to this workACKNOWLEDGMENTSWe thank Karen Martin for her assistance in manuscript preparationCONFLICTS OF INTERESTThe authors declare no conflicts of interestFUNDINGThis work was supported by a grant from the National Institutes of Health R01 DK61470REFERENCES Colon NC Chung DH Neuroblastoma Adv Pediatr “ httpsdoiorg101016jyapd201103011 [PubMed] Park JR Eggert A Caron H Neuroblastoma biology prognosis and treatment Hematol Oncol Clin North Am “ httpsdoiorg101016jhoc200911011 [PubMed] Matthay KK Maris JM Schleiermacher G Nakagawara A Mackall CL Diller L Weiss WA Neuroblastoma Nat Rev Dis Primers httpsdoiorg101038nrdp201678 [PubMed] Sidarovich V De Mariano M Aveic S Pancher M Adami V Gatto P Pizzini S Pasini L Croce M Parodi F Cimmino F Avitabile M Emionite L A HighContent Screening of Anticancer Compounds Suggests the Multiple Tyrosine Kinase Inhibitor Ponatinib for Repurposing in Neuroblastoma Therapy Mol Cancer Ther “ httpsdoiorg10115815357163MCT170841 [PubMed] Schramm G Wiesberg S Diessl N Kranz AL Sagulenko V Oswald M Reinelt G Westermann F Eils R Konig R PathWave discovering patterns of differentially regulated enzymes in metabolic pathways Bioinformatics “ httpsdoiorg101093bioinformaticsbtq113 [PubMed] Lau DT Flemming CL Gherardi S Perini G Oberthuer A Fischer M Juraeva D Brors B Xue C Norris MD Marshall Oncotargetwwwoncotargetcom\x0cGM Haber M Fletcher JI et al MYCN amplification confers enhanced folate dependence and methotrexate sensitivity in neuroblastoma Oncotarget “ httpsdoiorg1018632oncotarget3732 [PubMed] O™Connor OA Amengual J Colbourn D Deng C Sawas A Pralatrexate a comprehensive update on pharmacology clinical activity and strategies to optimize use Leuk Lymphoma “ httpsdoiorg101080 [PubMed] Foss FM Evaluation of the pharmacokinetics preclinical and clinical efficacy of pralatrexate for the treatment of Tcell lymphoma Expert Opin Drug Metab Toxicol “ httpsdoiorg10151717425255201159540 [PubMed] Visentin M Unal ES Zhao R Goldman ID The membrane transport and polyglutamation of pralatrexate a newgeneration dihydrofolate reductase inhibitor Cancer Chemother Pharmacol “ httpsdoiorg101007s0028001322319 [PubMed] Serova M Bieche I Sablin MP Pronk GJ Vidaud M Cvitkovic E Faivre S Raymond E Single agent and combination studies of pralatrexate and molecular correlates of sensitivity Br J Cancer “ httpsdoiorg101038sjbjc6606063 [PubMed] Walton JD Kattan DR Thomas SK Spengler BA Guo HF Biedler JL Cheung NK Ross RA Characteristics of stem cells from human neuroblastoma cell lines and in tumors Neoplasia “ httpsdoiorg101593neo04310 [PubMed] Hidalgo M Amant F Biankin AV Budinska E Byrne AT Caldas C Clarke RB de Jong S Jonkers J Maelandsmo GM RomanRoman S Seoane J Trusolino L et al Patientderived xenograft models an emerging platform for translational cancer research Cancer Discov “ httpsdoiorg10115821598290CD14 [PubMed] Harenza JL Diamond MA Adams RN Song MM Davidson HL Hart LS Dent MH Fortina P Reynolds CP Maris JM Transcriptomic profiling of commonlyused neuroblastoma cell lines Sci Data httpsdoiorg101038sdata201733 [PubMed]Oncotargetwwwoncotargetcom\x0c'

cancer254

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: Pralatrexate is a folate analogue inhibitor of dihydrofolate reductase exhibiting high affinity for reduced folate carrier1 with antineoplastic and immunosuppressive activities similar to methotrexate Despite advances in multimodality treatment strategies the survival rates for children with highrisk neuroblastoma have failed to improve Therefore the intense research continues in order to identify the ideal novel agent or combination of chemotherapy drugs to treat highrisk neuroblastomaMaterials and Methods Four human neuroblastoma cell lines were used to determine IC50 values of select chemotherapy agents Antiproliferative effects of pralatrexate were assessed by adherent and nonadherent colony formation assays Cell cycle arrest and apoptosis were measured by flow cytometry and immunoblotting PDX tissue culture was used to assess ex vivo efficacyResults Treatment with pralatrexate in all four neuroblastoma cell lines blocked cell growth in 2D and 3D culture conditions in a timedependent manner The potency of pralatrexate was tenfold stronger than methotrexate as measured by IC50 Pralatrexateinduced apoptosis was confirmed by caspase3 activation and PARP cleavage MYCN and SLC19A1 mRNA expressions were decreased with pralatrexate in MYCNamplified neuroblastoma cellsConclusions Pralatrexate demonstrated effective inhibition of cell growth and viability The higher potency of pralatrexate compared to methotrexate a drug with high levels of toxicity suggests pralatrexate may be a safer alternative to methotrexate as an effective chemotherapeutic agent in the treatment of patients with highrisk neuroblastomaINTRODUCTIONNeuroblastoma is a pediatric tumor derived from neural crest cells It is the most common pediatric solid tumor accounting for approximately of pediatric cancer deaths [] and it typically presents as a painless abdominal mass in infants and toddlers of to months of age [] Poor prognostic factors in children with neuroblastoma include age months at time of diagnosis unfavorable histology increased vascularization and MYCN amplification [] Despite intense research focused on the biology of neuroblastoma it remains one of the most enigmatic pediatric cancers in terms of its underlying molecular pathogenesis There has been only incremental improvement in the overall survival of children with highrisk neuroblastoma necessitating the search for a novel agent or combination of chemotherapy drugs []is key Altered metabolism to cancer cell proliferation Among the various metabolic pathways that are affected folate metabolism plays an important role Folate is essential for DNA synthesis and cell growth especially in rapidly dividing cells Inhibition of folate metabolism is the basis for many chemotherapy drugs In neuroblastoma folate mediated onecarbon metabolism is associated with aggressiveness and MYCN amplification Oncotargetwwwoncotargetcomwwwoncotargetcom Oncotarget Vol No pp 0cMethotrexate is a widely used [] A study by Lau in demonstrated higher folate requirements in MYCN amplified neuroblastoma cells compared to nonMYCN amplified cells [] They also showed that the increased folate uptake is mediated by reduced folate carrier1 RFC1 which is encoded by the gene SLC19A1 [] Previous studies have demonstrated that SLC19A1 is associated with MYCN amplification in neuroblastoma and that SLC19A1 is a direct transcriptional target of Nmyc [] The association between MYCN amplification and folate metabolism suggests the potential role of antifolate drugs in the treatment of neuroblastomasinhibitor of folate metabolism It inhibits dihydrofolate reductase DHFR and therefore disrupts purine and thymidylate biosynthesis leading to inhibited DNA replication and cell death However in the 1970s methotrexate was found to have high levels of toxicity combined with low treatment response rates in neuroblastoma patients and therefore it has not been clinically used for neuroblastoma treatment [] Pralatrexate is a folate analogue inhibitor of DHFR that exhibits high affinity for RFC1 [] and folylpolyglutamate synthetase FPGS Pralatrexate demonstrates antineoplastic and immunosuppressive properties that are similar to methotrexate It was FDA approved in the United States for treatment of relapsed or refractory peripheral Tcell lymphoma in [] A study by Serova in demonstrated decreased mRNA expression of SLC19A1 and SLC25A32 a mitochondrial folate carrier with pralatrexate treatment in several cancer cell lines [] As previously discussed SLC19A1 is downstream target of Nmyc in neuroblastoma The high affinity of pralatrexate for the SLC19A1 encoded RFC1 protein may demonstrate a potential role in the treatment of MYCNamplified neuroblastomaThe development of a new chemotherapeutic regimen is a long process that can take years to enter clinical trials and subsequently into bedside therapy Identifying alterative applications for previously FDAapproved drugs is a method that allows for quicker use in clinical practice [] Therefore we sought to evaluate current FDA approved antineoplastic drugs as potential novel treatment strategies for highrisk neuroblastoma and set out to assess the inhibitory role of pralatrexate on neuroblastoma cellsRESULTSThe IC50 of pralatrexate is tenfold less than methotrexateFour human neuroblastoma cell lines including MYCNamplified BE2C CHP212 and LAN1 as well as the nonMYCN amplified cell line SKNAS were treated with methotrexate or pralatrexate Cell growth was determined after h of continuous exposure to methotrexate or pralatrexate nM“ µM measured by Cell Titer Glo„¢ assay and the IC50 of each drug was calculated for each cell line using the Genedata Screener software Values shown are mean ± SD of three separate experiments As shown in A in all four cell lines the IC50 of pralatrexate was approximately tenfold less than the IC50 of methotrexate These data demonstrate that highrisk neuroblastoma cells have enhanced pralatrexate sensitivity compared to methotrexate and that pralatrexate inhibits both MYCN amplified and nonMYCN amplified neuroblastoma growth in the low nanomolar range in vitroPralatrexate inhibited neuroblastoma cell growthTo demonstrate the timing of growth inhibition we treated neuroblastoma cells with pralatrexate or nM and measured cellular viability over a time course of days B Significant cell growth inhibition was first noted by day in SKNAS and CHP212 cells and by day in LAN1 and BE2C cells This indicates that pralatrexate effectively inhibits the proliferative potential of neuroblastoma cells We further validated the effects of pralatrexate on neuroblastoma cells in vitro by quantifying colony growth in a 3D matrix hydrogel where cells grow and selfassemble into clusters 3D cultures are more physiologically relevant and better represent in vivo tissue BE2C and LAN1 cells are high colonyforming neuroblastoma cell lines [] Concurrent treatment with pralatrexate completely abolished the ability of BE2C and LAN1 cells to develop colonies in gel drops A Both BE2C and LAN1 cell lines treated with pralatrexate demonstrated a decreased colony count and a decrease in colony size compared to cells treated with DMSO Figure 2B and 2C The colonies were counted from three separate microscopic fields and their size was measured using the scale bar on each image using Image J These findings suggest that pralatrexate represses the tumorigenesis potential and tumor progression of neuroblastomaPralatrexate induced G1 phase cell cycle arrest apoptosis and decreased Nmyc expressionTo further test whether pralatrexate directly altered neuroblastoma cell proliferation we evaluated the cell cycle distribution of treated cells compared with control Cell cycle analysis was performed in BE2C cells treated with pralatrexate nM or control at days and after treatment We observed a significant but modest increase in the G1 phase of the cell cycle ranging from a “ increase in the G1 cell population demonstrating induction of G1 cell cycle arrest Figure 3A Given the dramatic decrease in cell viability observed between days and of pralatrexate treatment Figure 1B we hypothesized that pralatrexate may also induce apoptosis in neuroblastoma cells To confirm apoptosis Oncotargetwwwoncotargetcom\x0cin cells treated with pralatrexate Western blotting was performed BE2C and CHP212 cells were treated with increasing doses of pralatrexate and nM The protein expression of total and cleaved caspase3 as well as total and cleaved PARP were examined at each increasing dose of pralatrexate Figure 3B confirming the induction of apoptosis Apoptosis was also seen secondary to pralatrexate treatment in nonMYCN amplified cells SKNAS SKNSH and SHSY5Y Supplementary Figure Given the proliferating role of Nmyc in neuroblastoma tumorigenesis we next evaluated whether pralatrexate could alter the Nmyc expression Interestingly we found decreased Nmyc expression with increasing doses of pralatrexate in both BE2C and CHP212 cells Figure 3B demonstrating persistent defects in proliferative potential induced by pralatrexate in neuroblastomaPralatrexate decreased MYCN and SLC19A1 gene expressions compared to SLC25A32Previous studies demonstrated that the expression of SLC19A1 the gene encoding the RFC1 receptor is associated with MYCN amplification in neuroblastoma [] Pralatrexate is a folate analogue inhibitor with high affinity for RFC1 [] Therefore we sought to examine the effects of pralatrexate treatment on MYCN and SLC19A1 gene expression in BE2C and CHP212 cell lines compared to the effects in nonMYCN amplified cells BE2C and CHP212 cells were treated with and nM pralatrexate or DMSO and qPCR was performed As expected from the finding in Figure 3B pralatrexate treatment resulted in decreased MYCN expression in both BE2C and CHP212 cells Figure 4A and 4B Interestingly we also found that both BE2C and CHP212 cells treated with pralatrexate demonstrated decreased SLC19A1 expression but no difference in SLC25A32 expression compared to control cells Figure 4A and 4B Treatment of nonMYCN amplified cells SKNAS SKNSH and SHSY5Y did not affect SLC19A1 or SLC25A32 expression Supplementary Figure These findings may support the previous studies [ ] that SLC19A1 is a direct transcription target of MYCN in neuroblastomas and pralatrexate treatment affects SLC19A1 expression in MYCN amplified cells but not SLC25A32 expression In addition in both BE2C and CHP212 cells pralatrexate did not decrease FPGS mRNA expression Figure 4C and 4D Neither SLC25A32 or FPGS expression was affected in nonMYCN amplified cells treated with pralatrexate Supplementary Figure Interestingly in the BE2C cells and the nonMYCN amplified cells pralatrexate treatment led to an increase in DHFR expression This may imply treatment with pralatrexate selected for cells with increased DHFR expression and inherent pralatrexate resistance or a resultant upregulation of the DHFR gene with DHFR protein inhibition These findings would Figure IC50 of human neuroblastoma cell lines BE2C CHP212 and LAN1 MYCNamplified and SKNAS nonMYCNamplified A The IC50 doses of BE2C CHP212 LAN1 and SKNAS cells treated with methotrexate were and µM respectively The IC50 doses of BE2C CHP212 LAN1 and SKNAS cells treated with pralatrexate were and µM respectively Cell growth was determined after h of continuous exposure to methotrexate or pralatrexate nM“ µM measured by Cell Titer Glo„¢ assay and the IC50 of each drug was calculated for each cell line using the Genedata Screener software Values shown are mean ± SD of three separate experiments B Cell viability was assessed using the Cell Counting Kit8 assay Pralatrexate significantly inhibited cell viability in all four neuroblastoma cell lines as compared to DMSO control groupOncotargetwwwoncotargetcom\x0cbe consistent with findings by Serova et al in which pralatrexateresistant cells demonstrated increased DHFR protein expression []Pralatrexate treatment response in ex vivo neuroblastoma growthGiven these persistent in vitro findings we next evaluated whether pralatrexate could be evaluated ex vivo to guide treatment decisions for individual patients We used a neuroblastoma PDX model where tumors were dissected into 1mm3 pieces and cultured in duplicate on a presoaked gelatin sponge in 24well plates containing µL RPMI with FBS antibioticantimycotic solution mgmL hydrocortisone and mgmL insulin The size of these fragments is comparable to the size of a standard clinical tumor biopsy specimen The fragments were then cultured for days in the presence of pralatrexate nM using standard cell culture conditions Notably tumor tissues were significantly affected by ex vivo pralatrexate treatment and showed decreased Ki67 staining compared to tissues cultured in vehicle control treatment Figure These results suggest that a simple shortterm ex vivo treatment assay of a viable tumor specimen may aid in identifying neuroblastoma patients who are likely to gain benefit from pralatrexate treatment options in the futureDISCUSSIONHighrisk neuroblastoma remains quite difficult to cure necessitating the discovery of new chemotherapy agents to be used alone or in combination therapy Previous studies have reported an increased folate in MYCNamplified neuroblastoma cells demand mediated by the RFC1 receptor [] Additionally the gene encoding the RFC1 receptor SLC19A1 is a direct transcriptional target of Nmyc in neuroblastoma cells suggesting a role for antifolate drugs in the treatment of neuroblastoma Methotrexate has previously been studied in neuroblastoma however it was found to have a prohibitive toxicity and has not been used in neuroblastoma clinically In contrast pralatrexate a folate analogue inhibitor is similar to methotrexate with a more favorable side effect profile suggesting a potential role for the use of pralatrexate as a chemotherapeutic agent against neuroblastomaThe present study sought to determine the effects of treatment with pralatrexate on in vitro and ex vivo cell growth in four human neuroblastoma cell lines The IC50 of pralatrexate was found to be 10fold less than that of methotrexate This tenfold difference between pralatrexate and methotrexate was also found in colon breast and thyroid cancer cells by Serova et al in [] The decreased IC50 of pralatrexate allows for treatment with lower doses and a more tolerable sideeffect profile compared to methotrexate independent of MYCN amplificationPralatrexate not only induced celldeath via apoptosis but it also successfully inhibited neuroblastoma in vitro cell growth and proliferation in 2D and 3D cell cultures as well as in our PDX exvivo model By inhibiting the RFC1 receptor pralatrexate decreased the amount of folate entering cells and in turn decreased Figure Pralatrexate inhibited neuroblastoma colony growth A Representative images of light microscopy × magnification for BE2C and LAN1 cells after days of treatment with pralatrexate versus control Pralatrexate treatment decreased cell growth in both BE2C and LAN1 cells compared to control scale bar μm B Colony count and colony size for BE2C cells were analyzed and quantified mean ± SD p for nM pralatrexate treatment vs no drug C Colony count and colony size for LAN1 cells were analyzed and quantified mean ± SD p for nM pralatrexate treatment vs no drugOncotargetwwwoncotargetcom\x0cDNA synthesis This was demonstrated by the increased time spent in the G1phase of the cell cycle in cells treated with pralatrexate PDXs have been shown to parallel clinical outcome in various tumor types [] The major applications of neuroblastoma PDXs would be related to drug testing exploration of treatment resistance and biomarker discovery Combining PDXs and ex vivo culture will incorporate human tumor tissue in its native Figure Effects of pralatrexate treatment on caspase3 PARP and Nmyc protein expression A Cell cycle analysis with propidium iodide demonstrates enhanced G1 cell cycle arrest at and h following treatment Cell cycle analysis was completed with events per replicate mean ± SD p for nM pralatrexate treatment vs no drug B Treatment with increasing doses of pralatrexate induced apoptosis in BE2C and CHP212 cells Cells treated with pralatrexate demonstrated cleaved caspase3 protein expression when treated with and nM doses Cleaved PARP expression was noted after treatment with nM Treatment with pralatrexate decreased Nmyc protein expression in BE2C and CHP212 cells βactin was used as an internal controlOncotargetwwwoncotargetcom\x0c3D state and enable dynamic manipulation of the system minimizing animal experiments and costLau has shown that SLC19A1 is a downstream direct transcriptional target of Nmyc in neuroblastoma cells and that MYCNamplified cells have an increased folate dependence [] In our study pralatrexate also led to a decrease in expression of the RFC1 genes SLC19A1 and SLC25A32 The decrease in SLC19A1 was more pronounced compared to the mitochondrial folate receptor gene SC25A32 suggesting pralatrexate may be more specific to the cytosolic RFC1 receptor compared to the mitochondrial RFC1 receptor However further studies are necessary to investigate this relationship Meanwhile pralatrexate treatment led to a marked increase in DHFR expression in BE2C cells and a slight increase in CHP cells This is unlikely an upregulation of DHFR and more indicative of increased DHFR mRNA being harvested from pralatrexate resistance cells Similar results were found in a previous study on colon breast and thyroid cancer cells lines Serova found that pralatrexateresistant cells had increased DHFR protein expression [] The increase in DHFR expression may lead to an increase in the amount of DHFR protein requiring more than nM of pralatrexate to inhibit cell growth and proliferation However further studies surrounding the dose of pralatrexate and its relationship to DHFR gene expression are neededNeuroblastoma is a heterogenous tumor and further studies are needed to examine the effects of pralatrexate on additional cells lines Additionally the remaining cells that survived after pralatrexate treatment may represent pralatrexate resistant cells Future studies are needed to elucidate potential mechanisms of pralatrexate resistance such as increased DHFR gene expression as well as the relationship between pralatrexate and SLC19A1 versus other folate synthesis enzyme expressions Given pralatrexate is already an FDAapproved and in clinical use future clinical studies are needed to investigate the effects of pralatrexate treatment on neuroblastoma in vivoMATERIALS AND METHODSCells antibodies and reagentsThe neuroblastoma cell line LAN1 was a gift from Dr Robert C Seeger University of Southern California Los Angeles CA All other neuroblastoma cell lines BE2C CHP212 SKNAS SKNSH and SHSY5Y Figure Effects of pralatrexate treatment on MYCN SLC19A1 and SLC25A32 gene expressions After day of treatment the mRNA expression of MYCN SLC19A1 and SLC25A32 were measured by qPCR in A BE2C cells treated with nM of pralatrexate when compared with DMSO treated cells and in B CHP212 cells treated with nM of pralatrexate C D qPCR was performed on BE2C and CHP212 cells treated with pralatrexate to assess for mRNA expression of two keyenzymes in folate synthesies DHFR and FPGS in the treatment mean ± SD p for pralatrexate treatment vs no drugOncotargetwwwoncotargetcom\x0cwere purchased from the American Type Culture Collection ATCC Manassas VA Cells were maintained in RPMI with glutamine and FBS at °C in a humidified atmosphere consisting of CO2 and air Primary antibodies for Caspase3 Cat No PARP Cat No Nmyc Cat No were purchased from Cell Signaling Technology Danvers MA Ki67 Cat No was from Abcam Cambridge MA and βactin Cat No A2066 was from SigmaAldrich St Louis MO Methotrexate and pralatrexate were obtained from National Cancer InstituteDivision of Cancer Treatment and DiagnosisDevelopmental Therapeutics Program httpdtpcancergov and dissolved in dimethyl sulfoxide DMSO and further diluted in culture media to desired concentrations Neuroblastoma COGN415× patientderived xenograft PDX cells were obtained from the Childhood Cancer Repository maintained by the Children™s Oncology Group COG and Xenograft RepositoryDrug sensitivity and dose responsive curve assayFor cell viability screening cells [BE2C1500well LAN1 3000well CHP2124000well SKNAS 3000well] were plated and treated the following day with methotrexate and pralatrexate Cell growth was determined after h of continuous exposure to nM“ µM of methotrexate or pralatrexate using Cell Titer GloTM reagent Promega with luminescence measured using an EnVision multilabel plate reader PerkinElmer IncCell viability assayNeuroblastoma cells were seeded onto 96well plates permitted to attach overnight and were treated with either pralatrexate or nM or DMSO for days Cell viability measurements using the Cell Counting Kit8 Dojindo Molecular Technologies Inc Rockville MD were obtained daily3D colony formation assayBE2C or LAN1 cells were trypsinized embedded in μl of Cultrex® RGF BME Type matrix hydrogel Trevigen Gaithersburg MD and seeded in 48well plates cellswell RPMI medium containing FBS was added with pralatrexate treatment and incubated for days Colonies were photographed and the number and size were quantified The colonies were counted from three separate microscopic fields and their size was measured by the scale bar on each image using Image JCell cycle analysisCell cycle distribution was analyzed using flow cytometry with propidium iodide Sigma Aldrich BE2C cells were plated at equal numbers × cells and treated with either pralatrexate nM or DMSO At day and after treatment cells were washed and fixed in ethanol Fixed cells were incubated with mgmL RNAase for minutes at °C stained with propidium iodide mg mL and analyzed on a BD FACSCalibur BD Biosciences San Jose CAqPCR and immunoblottingTotal RNA was isolated and purified using a TRIzol® Reagent Thermo Scientific cDNA was synthesized using the qScript cDNA SuperMix QuantaBio RealFigure Ex vivo tissue culture model recapitulated antitumor response to pralatrexate Representative HE and Ki67 immunohistochemistry staining sections were obtained from a neuroblastoma PDX COGN415× treated ex vivo with nM pralatrexate or vehicle control for days and demonstrated poorly differentiated neuroblastoma cells and decreased Ki67 staining in pralatrexatetreated tumor compared to vehicle control × magnification scale bar μmOncotargetwwwoncotargetcom\x0creverse reverse time PCR and data collection were performed on a CFX96 instrument BioRad Data were normalized to an endogenous control βactin Specific target primers are MYCN forward 5ʹGCTTCTACCCGGACGAAGATG3ʹ reverse 5ʹCAG CTCGTTCTCAAGCAGCAT3ʹ SLC19A1 forward 5ʹAACAGGTCTGGGTTTTGTGC3ʹ 5ʹGTGCAGTATCATGCCCTGTG3ʹ SLC25A32 forward 5ʹATTGGTGGAAGCTGATTTGC3ʹ 5ʹTGGTCTGGATTTGGTCAACA3ʹ DHFR forward 5ʹCTCAAGGAACCTCCACAAGG3ʹ reverse 5ʹGTTTAAGATGGCCTGGGTGA3ʹ FPGS forward 5ʹGGGTGACCCTCAGACACAGT3ʹ reverse 5ʹGTCTTCAGGCCATAGCTTCG3ʹ Amplification was performed for cycles of s at °C s at °C and s at °C Whole cell lysates were collected using cell lysis buffer and equal amounts of protein were loaded on a NuPAGE “ BisTris gel followed by transfer onto PVDF membranes BioRad Hercules CA USA and probed with antibodiesEx vivo culture and immunohistochemistryCOGN415× patientderived xenograft cells were obtained from the Childhood Cancer Repository maintained by COG Clinical and genomic features of the tumors were detailed in a study by Harenza in [] Cells were suspended in Matrigel diluted with PBS and × cells were injected into the flank of NOD scid gamma mice at “ weeks of age UTSW Mouse Breeding Core All studies were approved by the Institutional Animal Care and Use Committee at University of Texas Southwestern Medical Center To keep cost down we used only female mice for passing PDX Mice were euthanized once tumors reached mm and tumors were dissected into 1mm3 pieces and cultured in duplicate on a presoaked gelatin sponge Johnson and Johnson in 24well plates containing µL RPMI with FBS antibioticantimycotic solution mgmL hydrocortisone and mgmL insulin SigmaAldrich Tissues were cultured at °C for days with either pralatrexate nM or vehicle control then formalinfixed and paraffin embedded COGN415× tissue sections were stained with hematoxylin and eosin or with an antibody against Ki67Statistical analysisAll results are shown as the mean value ± SD statistical analyses were performed using student ttest for comparisons between the groups A p value of was considered significant GraphPad™s Prism software was used for the statistical analysisAbbreviationsCOG Children™s Oncology Group DHFR dihydrofolate reductase DMSO dimethyl sulfoxide FPGS folylpolyglutamate synthetase PDX patientderived xenograft RFC1 reduced folate carrier1Author contributionsStudy concept and design RAC SL JQ DHC Acquisition of data RAC SL JQ Analysis and interpretation of data RAC SL JQ DHC Drafting of manuscript RAC SL JQ DHC Critical revision RAC SL JQ DHC RAC and SL contributed equally to this workACKNOWLEDGMENTSWe thank Karen Martin for her assistance in manuscript preparationCONFLICTS OF INTERESTThe authors declare no conflicts of interestFUNDINGThis work was supported by a grant from the National Institutes of Health R01 DK61470REFERENCES Colon NC Chung DH Neuroblastoma Adv Pediatr “ httpsdoiorg101016jyapd201103011 [PubMed] Park JR Eggert A Caron H Neuroblastoma biology prognosis and treatment Hematol Oncol Clin North Am “ httpsdoiorg101016jhoc200911011 [PubMed] Matthay KK Maris JM Schleiermacher G Nakagawara A Mackall CL Diller L Weiss WA Neuroblastoma Nat Rev Dis Primers httpsdoiorg101038nrdp201678 [PubMed] Sidarovich V De Mariano M Aveic S Pancher M Adami V Gatto P Pizzini S Pasini L Croce M Parodi F Cimmino F Avitabile M Emionite L A HighContent Screening of Anticancer Compounds Suggests the Multiple Tyrosine Kinase Inhibitor Ponatinib for Repurposing in Neuroblastoma Therapy Mol Cancer Ther “ httpsdoiorg10115815357163MCT170841 [PubMed] Schramm G Wiesberg S Diessl N Kranz AL Sagulenko V Oswald M Reinelt G Westermann F Eils R Konig R PathWave discovering patterns of differentially regulated enzymes in metabolic pathways Bioinformatics “ httpsdoiorg101093bioinformaticsbtq113 [PubMed] Lau DT Flemming CL Gherardi S Perini G Oberthuer A Fischer M Juraeva D Brors B Xue C Norris MD Marshall Oncotargetwwwoncotargetcom\x0cGM Haber M Fletcher JI et al MYCN amplification confers enhanced folate dependence and methotrexate sensitivity in neuroblastoma Oncotarget “ httpsdoiorg1018632oncotarget3732 [PubMed] O™Connor OA Amengual J Colbourn D Deng C Sawas A Pralatrexate a comprehensive update on pharmacology clinical activity and strategies to optimize use Leuk Lymphoma “ httpsdoiorg101080 [PubMed] Foss FM Evaluation of the pharmacokinetics preclinical and clinical efficacy of pralatrexate for the treatment of Tcell lymphoma Expert Opin Drug Metab Toxicol “ httpsdoiorg10151717425255201159540 [PubMed] Visentin M Unal ES Zhao R Goldman ID The membrane transport and polyglutamation of pralatrexate a newgeneration dihydrofolate reductase inhibitor Cancer Chemother Pharmacol “ httpsdoiorg101007s0028001322319 [PubMed] Serova M Bieche I Sablin MP Pronk GJ Vidaud M Cvitkovic E Faivre S Raymond E Single agent and combination studies of pralatrexate and molecular correlates of sensitivity Br J Cancer “ httpsdoiorg101038sjbjc6606063 [PubMed] Walton JD Kattan DR Thomas SK Spengler BA Guo HF Biedler JL Cheung NK Ross RA Characteristics of stem cells from human neuroblastoma cell lines and in tumors Neoplasia “ httpsdoiorg101593neo04310 [PubMed] Hidalgo M Amant F Biankin AV Budinska E Byrne AT Caldas C Clarke RB de Jong S Jonkers J Maelandsmo GM RomanRoman S Seoane J Trusolino L et al Patientderived xenograft models an emerging platform for translational cancer research Cancer Discov “ httpsdoiorg10115821598290CD14 [PubMed] Harenza JL Diamond MA Adams RN Song MM Davidson HL Hart LS Dent MH Fortina P Reynolds CP Maris JM Transcriptomic profiling of commonlyused neuroblastoma cell lines Sci Data httpsdoiorg101038sdata201733 [PubMed]Oncotargetwwwoncotargetcom\x0c' Answer:

Thyroid_Cancer

"development of cancer is a problem that has accompanied mankind for years The growing number of cases emerging drug resistance and the need to reduce the serious side effects of pharmacotherapy are forcing scientists to better understand the complex mechanisms responsible for the initiation promotion and progression of the disease This paper discusses the modulation of the particular stages of carcinogenesis by selected physiological factors including acetylcholine ACh peroxisome proliferatoractivated receptors PPAR fatty acidbinding proteins FABPs Bruton™s tyrosine kinase Btk aquaporins AQPs insulinlike growth factor2 IGF2 and exosomes Understanding their role may contribute to the development of more effective and safer therapies based on new binding sitesINTRODUCTIONThe increasing prevalence of various types of cancers is a global phenomenon that has been occurring widely and affecting increasing numbers of people In over million new cases were reported and statistics show that every fifth man and every sixth woman is at risk for developing cancer [] Carcinogenesis is a multistage process that leads to cancer development It includes three main stages initiation promotion and progression and each is characterized by different processes During the promotion stage the genetic material of cells is damaged and if not detected by repair systems before the next division the change is transferred to newly formed cells Under the control of growth factors initiated cells may move on to the second stage of cancer formation“promotion During that stage mutated cells undergo multiple divisions their growth is uninhibited as they become insensitive to apoptotic signals As a result the tumor increases its mass but the cells remain within one an The formation of metastases occurs in the next phase“progression Metalloproteinases secreted by tumor cells destroy the extracellular matrix This allows them to enter the bloodstream and reach a new an at a considerable distance from the primary lesion with the stream of flowing blood The key step for the survival of migrating cells is adhesion to the attacked an and the formation of new blood vessels thanks to which they will receive the components necessary for development Angiogenesis the formation of new blood vessels is the last element of carcinogenesis and is enhanced by tumor cell derived factors metabolic changes and a decrease in available oxygen Each of these processes is controlled by different signal pathways via proteins that naturally occur in the body Figure Dysregulation of their expression and thus activity both to an excessive and insufficient degree ensures the continuity of carcinogenesis and increases the chances cancer cells survive in a host anism Understanding the role of individual substances and components of the body in the subsequent stages of neoplastic transformation provides a more complete picture of cancer pathogenesis This allows to develop new recommendations to improve the quality and safety of pharmacotherapy which has a direct effect on an improvement in the cancer patients™ quality of life It also provides the basis for research aimed at developing new compounds that are effective weapons in the fight against cancer Therefore there is no doubt that any actions that bring scientists closer to solving the problem of insufficient cancer therapy are sensible and much needed The purpose of this paper was to discuss the role of selected physiological factors in the various stages of neoplastic transformationOncotargetwwwoncotargetcomwwwoncotargetcom Oncotarget Vol No pp 0cCrucial mechanisms involved in carcinogenesisThe carcinogenic process is regulated by many molecular and cellular mechanisms The diversity of signaling pathways exploited during cancer initiation promotion and progression is immense Many of them are common in different cancer types but there are also clearly unique molecular and cellular signaling signatures specific for particular cancers [] Pathways such as AktPI3K RasMAPKERK Wntcatenin JAKSTAT occupy crucial roles in the regulation of cell proliferation apoptosis differentiation angiogenesis cell migration and invasion immunological activities and inflammationPI3K phosphatidylinositol 3kinase is a family of enzymes that after activation by growth factors cytokines and hormones are involved in the conversion of PIP2 phosphatidylinositol45bisphosphate into PIP3 phosphatidylinositol 345trisphosphate [] The latter in turn takes part in recruiting Akt into the cell membrane where it is phosphorylated and activates other agents such as mTOR NFkB Wnt or inhibits factors like Bad p27 to enhance carcinogenesis Dysregulation of this pathway occurs in endometrial [] bladder [] or gastric cancers [] The aforementioned PIP2 can be also transformed by phospholipase C into DAG and IP3 which then activate protein kinase C PKC [] One of the PKC substrates is Raf kinase which is an element of the RasMAPKERK pathway which is involved in the pathogenesis of ovary cancer [] or nonsmall cell lung cancer [] Ras proteins activate Raf which then phosphorylates kinases MEK12 Its final substrate is kinase ERK12 which after transportation into the nuclei regulates transcription factors essential for DNA synthesis and cell cycle progression They condition cell growth proliferation and viability [] Because of that targeting those two cascades as an anticancer therapy seems to be beneficial However it is important to notice that the existing crosstalk between both of the pathways can impede treatment and inhibition of one can strengthen the activity of the other Another component that is also connected with the MAPK family Figure Modulators of individual stages of carcinogenesis Inducers red frames inhibitors green frames M1rec muscarinic receptor M3rec muscarinic receptor Nrec nicotinic acetylcholine receptor COX2 cyclooxygenase2 PPARÎ peroxisome proliferatoractivated receptors gamma Btk Bruton™s tyrosine kinase FABP fatty acidbinding protein LFABP Ltype fatty acidbinding protein AQPs aquaporins IGF2 insulinlike growth factor IGFBP3 insulinlike growth factor binding protein IGFBP5 Insulin Like Growth Factor Binding Protein IGF2BP2 Insulin Like Growth Factor2 mRNA Binding Protein2 IGF2BP3 Insulin Like Growth Factor2 mRNA Binding Protein Oncotargetwwwoncotargetcom 0cis a signaling pathway conducted by p38MAPKs This subfamily contains four kinases which are activated mainly by inflammatory cytokines and stress factors p38MAPKs control many aspects of cell physiology such as cell cycle regulation differentiation or skeleton remodelling Additional p38MPAKs can act as tumor promotors by enhancing metalloproteinase and VEGF expression []Wntcatenin signaling plays essential roles in caricinogenesis Activation of canonical Wnt signaling results in the inhibition of GSK3 kinase dissociation of catenin proteins and its transfer to the nucleus where it regulates multiple downstream genes like cMyc and cyclin D [] This axis was found to be associated with several oncogenic events including tumor cell proliferation migration epithelial“mesenchymal transition and invasion An abnormally active WntBcatenin pathway is observed among others in gastric [] colon [] breast [] or adrenocortical cancer [] Another signaling pathway involved in the pathogenesis of many cancer types is that induced by the Jak kinases family It includes four kinases which after activation impact of the STAT molecules causing their translocation into the nuclei [] STAT a family that gathers seven forms of proteins is associated with cancer cell development progression metastasis survival and resistance to treatment [] STAT3 and STAT5 factors seem to be the most important agents in light of cancer progression They have an impact on p53 protein which leads to a disruption in cycle control and apoptosis and induces cell proliferation by the increased cMyc and cyclin D expression By the induction of VEGF gene expression they take part in enhanced angiogenesis and induction of genes such as survivin Bcl2 BclXL conditioning overall survival of the tumor cells Moreover pSTAT3 acts to negatively regulate neutrophils NK cells effector T cells and dendritic cells while positively regulating populations of MDSCs myeloidderived suppressor cells and regulatory T cell leads to a highly immunosuppressive TME tumor microenvironment []Multiple endogenous factors are involved in carcinogenesis which affects particular stages of carcinogenesis in various mechanisms Some of them interact directly with the DNA intracellular signalling molecules others by specific cellular receptors Among them are well known growth factors EGF TGFα and TGF FGF or reactive endogenously generated oxygen molecules as well as less often described agents like ACh PPAR FABPs Btk AQPs IGF2 or exosomes The latter have only recently gained in importance and are more widely studied and discussed therefore are considered in this articleAacetylcholine Ach and its receptorsOne of the factors involved in the carcinogenesis process is acetylcholine and its receptors both muscarinic inhibitors and nicotinic Their expression has been demonstrated in numerous types of cancer cells [“] It has also been proven that these cells contain acetylcholinesterase AChE an enzyme that enables cells to produce ACh in the absence of agonists from the external environment Cancer cells also have the ability to produce autoantibodies that stimulate one of the muscarinic receptor subtypes M3 These facts prompted more extensive research to explain the exact role of ACh and its receptors in the carcinogenesis process Figure Muscarinic receptors can be divided into five subtypes each of which is involved in tumor development through a different mechanism The role of muscarinic M3 receptor is the most widely described subtype It is involved in the pathogenesis of lung colon gastric and breast cancer In the course of colon cancer it has been observed that stimulation of the M3 receptor causes phosphorylation of the Akt and ERK12 kinases which are factors that through the ability to regulate the activity of pro and antiapoptotic proteins affect the intensification of cell proliferation survival and motility In addition coexpression of the M3 receptor and endothelial growth factor receptor EGFR as well as EGFR transactivation after M3 receptor stimulation has been demonstrated [ ] Moreover the administration of EGFR inhibits acetylcholineinduced ERK12 kinase phosphorylation which is reflected in a significant decrease in colon cancer cell proliferation This indicates that acetylcholine is involved in the formation of colon cancer but for its full action it is necessary to activate the EGFR receptor Thus indicating that enriching colon cancer treatment with EGFR inhibitors may improve patient outcomes Acetylcholine is also involved in the formation of vascularlike structures in the vicinity of a developing tumor guaranteeing the supply of essential nutrients to the proliferating cancer cells This is called vascular mimicry The basis of this process is the acetylcholinedependent regulation of metastasisassociated in colon cancer1 MACC1 oncogene expression via the M3RAMPKMACC1 signaling pathway [] High activity of this oncogene is associated with an increase in cell invasiveness intensified epithelial“mesenchymal transition more frequent metastases and hence worse treatment prognosis [“] The first subtype of the M receptor M1 is also involved in the tumor development process It activates the hedgehog pathway [ ] whose physiological role is to regulate the transcription factors affecting the oncogenic activity of Gli zinc finger transcription factors [ ] Excessive stimulation causes acceleration of carcinogenesis intensification of proliferation growth and survival of cancer cells as well as angiogenesis by affecting the expression of genes such as cyclin D antiapoptotic Bcl2 or VEGF Furthermore stimulation of this pathway promotes the creation of a microenvironment conducive to tumor growth through increased expression of extracellular matrix components [] Nicotinic Oncotargetwwwoncotargetcom 0cacetylcholine receptors are part of the ligandgated ion channels and are created from two types of subunits α and Receptors that contain an alpha7 or alpha9 subunit in their structure are characterized by a high degree of permeability to calcium ions thanks to which they are involved in the course of numerous processes based on signaling involving secondary messengers Thus after activating N receptors Ca2 inflows into the cell causing the secretion of growth factors exerting paracrine action on neighboring cells Simultaneously apoptosis is inhibited enabling further proliferation Studies conducted on a lung cancer cell line after exposure to cigarette smoke have shown that nicotine and nitrosamines which have a higher affinity for nicotinic acetylcholine receptors nAChRs than nicotine bind to the alpha7 receptors activating the RasERKMAPK and the JAK2STAT3PI3K signaling pathways [ ] The effect of this interaction is increased proliferation and migration of cancer cells and thus facilitated metastasis creation Moreover by affecting Badrenergic receptors nicotine intensifies COX2 expression which through the p38 MAPK and the JNK pathways leads to an increase in VEGF production a highly proangiogenic factor In addition the high activity of cyclooxygenase affects the inactivation of the PTEN protein [ ] whose biological role is silencing PI3KAkt pathway signaling This leads to cellular signal transition and induces effects that contribute to the enhanced growth and proliferation of cancer cells as well as inhibits the process of their death Under the influence of the Akt kinase proapoptotic transcription factors are inactivated including kinase9 mTOR kinase intensifying cell proliferation is activated and VEGF is activated This indicates the involvement of this pathway in processes that facilitate cancer cell survival Another mechanism contributing to cancer development in which cyclooxygenase is involved is the complex process involving the COX2PGE2EP4 axis as a result of which metalloproteinase expression is stimulated [“] MMP9 causes proteolytic activation of TGF which begins the induction of epithelialmesenchymal transformation EMT During this transition the cells acquire features that increase their invasiveness This leads to epithelial cell phenotype changes the connections between adjacent cells and between cells and the basement Figure Carcinogenic effect of acetylcholine Ach Intracellular signaling pathways activated by ACh via M1 and M3 receptors Induction of process is illustrated by arrows inhibition by horizontal lines M1 muscarinic receptor M3 muscarinic receptor N receptor nicotinic acetylcholine receptor Akt protein kinase B ERK extracellular signalregulated kinase JAK2 Janus kinase PI3K phosphoinositide 3kinase STAT3 signal transducer and activator of transcription proteins MAPK mitogenactivated protein kinaseOncotargetwwwoncotargetcom 0cmembrane are loosened the cytoskeleton is rearranged cells lose contact inhibition thus causing excessive proliferation Furthermore the microenvironment of the growing tumor also changes promoting features that facilitate the movement of the cancer cells from the primary focus to other regions of the body Through the EMT process cells acquire greater migration capacity and thus their invasiveness increases In this cellular process MMP9 also modulates the activity of integrins and other molecules that ensure cell adhesion [] and secrete soluble factors into the bloodstream that facilitate the implantation of migrating cells in ans distal to the primary tumor focus [] Thanks to these processes COX2 enables the formation of cancer metastasesPPAR peroxisome proliferatoractivated receptorsThe family of peroxisome proliferatoractivated receptors PPARs includes isoforms of these proteins that function as transcription factors Each type of steroid receptor differs in its location and affinity for ligands [] The best known is the function of the PPARÎ form Agonists of this receptor are a therapeutic option in the treatment of patients with diabetes Current research provides evidence that stimulation of PPARÎ receptors inhibits cell proliferation vessel formation and induces apoptosis Figure [ ] These properties make PPARÎ agonists attractive candidates for anticancer drugs To achieve full transcriptional activity heterodimerization of PPARÎ with the retinoid X receptor is necessary [ ] This interaction enables the promoter to bind with the target gene and regulate its expression An example of a gene against which PPARÎ acts as a repressor is the vascular endothelial growth factor VEGF gene [] Thus angiogenesis is inhibited and this mechanism has been demonstrated in studies on human hepatocellular carcinoma HCC cells [] For this cancer PPARÎ is also involved in inhibiting cell growth and attenuating cell migration and invasiveness By upregulating KLF4 Krüppellike factor expression and inhibiting cyclin D1 expression the cell cycle is inhibited Furthermore PPARÎ negatively affects the expression of transcription factor STAT3 thereby inhibiting proliferation survival and metastasis Lowering STAT3 levels also negatively affects the angiogenesis process by inhibiting VEGF depriving cancer cells of nutrients PPARÎ activation in HHC cells also impairs their metastatic ability [] This is associated with the inhibition of the expression of metalloproteinases MMP9 and MMP13 the increased expression of their inhibitor and Ecadherin a protein conditioning cell adhesion In addition PPARÎ increases plasminogen activator inhibitor expression thus inhibiting the breakdown of the extracellular matrix and basement membrane proteins which also reduces cancer cell invasiveness PPARÎ has also been shown to inhibit esophageal cancer [] The mechanism responsible for suppressing this tumor is MAPK signaling pathway inhibition This process requires TLR4 Tolllike receptor inhibition which allows PPARÎ agonists to be administered TLR4 is a procarcinogenic protein and affects among others changes in the tumor microenvironment and the severity of angiogenesis Therefore in addition to the indirect effect on the MAPK cascade its inhibition is an important element of tumor suppression Studies on esophageal cancer cells indicate that PPARÎ stimulation leads to a decrease in PCNA proliferating cell nuclear antigen factor expression which indicates inhibition of DNA replication [] In addition PPARÎ activation intensifies the process of apoptosis as evidenced by an increase in the expression of the active form of caspase and the Bax gene and a decrease in Bcl2 expression In nonsmallcell lung cancer PPARÎ stimulation inhibits the neoplastic process by regulating PTEN protein expression [] This protein is a tumor suppressor and its activation causes the dephosphorylation and inactivation of the PIP3 second messenger As a result Akt kinase activity is inhibited leading to a decrease in NFkB expression Due to PPARÎ stimulation by eicosapentaenoic acid EPA a relationship is sought between PPARÎ and COX2 activity that uses EPA as a substrate for prostanoid production The combination of PPARÎ agonists with COX2 inhibitors has demonstrated a synergistic tumorsuppressing effect in studies on nonsmallcell lung cancer Researchers indicate significantly reduced thromboxane TXA2 synthesis using combination therapy compared with monotherapy as the main mechanism of this phenomenon [] A decrease in its concentration limits signaling in four pathways ERK p38 MAPK JAK and catenin limiting tumor growth [] Furthermore it affects the arrest of the cell cycle in the G2M phase preventing further cell division and contributing to the reduction of the expression of survivin an antiapoptotic protein [] The described mechanisms affect the induction of the apoptosis process and inhibit cell cycle progression which limits cancer development The above examples indicate that peroxisome proliferatoractivated receptors play a significant role in inhibiting the development of various types of cancer which is why agonist compounds can be considered potential anticancer drugs However further comprehensive and extensive research is needed to identify potential applications and treatment regimensBtk Bruton™s tyrosine kinaseBruton™s tyrosine kinase Btk also plays an important role in carcinogenesis Figure It is a key point in signal transduction from the BCR receptor leading to the activation of the NFkB signaling pathway [] Btk affects phospholipase CÎ2 [] which after phosphorylation causes PIP2 hydrolysis The result Oncotargetwwwoncotargetcom 0cis IP3 and DAG DAG activates protein kinase C which stimulates NFkB pathway factors Finally genes conditioning cell survival are expressed Btk also contributes to inhibiting the apoptosis process through interacting with the Akt kinase [] The signaling cascade includes active PI3K which recruits Akt to the plasma membrane Under the influence of Btk its phosphorylation occurs and thus its activation Then the active Akt returns to the cytoplasm and induces antiapoptotic pathways dependent on NFkB among others In addition Btk as a kinase belonging to the Tec family affects intercellular signaling causing changes in the tumor microenvironment In most cancer cells the developing microenvironment leads to the inhibition of immune processes which results in protection and protects defective cells from natural defense mechanisms One of the most common mechanisms of this type is the effect of cancer cells on the increased differentiation of T lymphocytes towards Th2 cells The biological role of these cells is the secretion of interleukins that start a humoral response dependent on B lymphocytes Due to the increased differentiation of T lymphocytes in this direction the number of formed Th1 lymphocytes directly destroying cells containing the abnormal genome decreases The tumor microenvironment also interferes with normal dendritic cell activity It inhibits their ability to migrate and to present the antigen to immune response cells Some types of cancer show increased CXCL12 expression [] which attracts immature dendritic cells and prevents their differentiation In addition this cytokine via the CXCR4 receptor causes a reanization of the cytoskeleton [] and increases cell motility [] Tec family tyrosine kinases have also been shown to have the ability to activate CXCR4 which determines the growth survival and migration of tumor cells []FABPs fatty acidbinding proteinsThe fatty acids supplied to the cell are involved in its metabolism Due to their lipid nature they are Figure Carcinogenic effect of peroxisome proliferatoractivated receptors PPARÎ The induction of the process is illustrated by arrows the inhibition by the horizontal line PTEN phosphatase and tensin homolog deleted on chromosome ten PIP3 phosphatidylinositol 345trisphosphate Akt protein kinase B PCNA proliferating cell nuclear antigen TXA2 thromboxane A2 ERK extracellular signalregulated kinases p38MAPK P38 mitogenactivated protein kinases JAK Janusactivated kinases KLF4 Kruppellike factor STAT3 Signal transducer and activator of transcription MMP314 metalloproteinase or PAI Plasminogen activator inhibitor TRL4 tolllike receptor MAPK mitogenactivated protein kinaseOncotargetwwwoncotargetcom 0cunable to enter the aqueous environment of the cellular cytoplasm Solubilizing molecules ie fatty acidbinding proteins FABPs enable their transport to various cellular structures and thus become involved in the processes taking place in the cells including their growth reproduction and inflammatory processes In cells characterized by intensive lipid metabolism such as the liver intestine heart brain etc high transporter expression is observed which is associated with their physiological role in the cell Depending on the type of cancer both the overexpression and a decrease in FABPs can be seen FABP isoforms are not specific to one type of cancer and each plays a different role in the carcinogenesis process Figure In liver cancer LFABP was overexpressed and its high concentration was shown to correlate positively with VEGFA [“] The causes of this phenomenon are sought in the direct interaction between these two proteins which in effect provokes the activation of the SrcFAKcdc42 and the AktmTORP70S6K4EBP1 signaling pathways Activation of the SrcFAKcdc42 pathway increases tumor cell migration and LFABP plays a key role in this process [ ] Moreover by stimulating the second mentioned signaling cascade LFABP enhances VEGFA expression facilitating angiogenesis This process is regulated by HIF1α which additionally induces blood vessel formation [] Due to the high homology in their construction FABP3 and FABP4 isoforms were assigned to the same protein subfamily In addition to their physical features it has been observed that the same factors ie HIF1α VEGF increase their coexpression The physiological role of both proteins is to inhibit excessive cell proliferation and increase apoptosis Excessive FABP3 expression leads to an increase in reactive oxygen species and a decrease in the mitochondrial membrane potential [] resulting in the ing of mitochondrial megachannels which play a key role in initiating apoptosis FABP4 activates the apoptosis process by mediating the response to lipidinduced endoplasmic reticulum stress Due to the biological functions of both proteins their deficiency may be the cause of cancer progression When examining the level of FABP3 expression in embryonic tumor cells breast cancer cells [] and FABP4 in breast ovarian prostate bladder or liver cancer cells low levels of both proteins were observed which was associated with a worse prognosis in terms of overall survival Furthermore high FABP4 levels slowed the development of hepatocellular carcinoma and thus contributed to its reduction in size [] This effect is explained by the inhibition of STAT3 phosphorylation via the RaspSTAT3 signaling pathway and the inhibition of the Snail protein an accelerator of the epithelialmesenchymal transition [] However the increased FABP3 expression in tumors of the stomach brain small and nonsmallcell lung cancer seems paradoxical it has been shown to increase tumor aggressiveness and poorer patient prognosis [ ] Thus far the molecular mechanism responsible for the oncogenic potential of FABP3 and FABP4 is unknown Further research is needed to clarify the reasons these proteins promote the neoplastic process Another subtype of fatty acid binding proteins is FABP5 It is involved in the development of breast prostate liver stomach and colon cancers FABP5 has been shown to transport saturated and unsaturated longchain fatty acids that act as PPARδ ligands [ ] The nature of the supplied fatty acids determines the oncogenic activity if unsaturated fatty acid or a suppressor action is supplied to PPARδ in the case of saturated ligands [] When the Figure Carcinogenic effect of Bruton™s tyrosine kinase Btk The induction of the process is illustrated by arrows the inhibiton by the horizontal line Btk Bruton™s tyrosine kinase CXCR2 CXC chemokine receptor type CÎ2 phospholipase CÎ2Oncotargetwwwoncotargetcom 0cFABP5PPARδ pathway is stimulated the transcription of the genes responsible for cell growth and survival increases Moreover one of the target genes stimulated by PPARδ is the FABP5 gene which increases the expression of the transport protein in question [] In prostate cancer cells by supplying long chain fatty acids FABP5 leads to PPARÎ activation [“] Stimulation of these receptors results in increased expression of vascular endothelial growth factor VEGF thereby accelerating the angiogenesis process This mechanism plays a key role in the development of castrationresistant prostate cancer [“] In the case of colon cancer FABP5 contributes to its development by reducing p21 activity Overexpression of FABP5 increases the expression of cMYC which inhibits the action of a cell cycle inhibitor After silencing FABP5 activity a significant reduction in the invasive capacity of colon cancer cells CRC is observed [] The molecular basis of this interaction is not yet known at the moment High expression of monoacylglycerol lipase MAGL responsible for the production of free fatty acids is observed in highly malignant colon cancer cells [ ] This fact combined with the physiological role of FABP5 consisting of transporting free fatty acids to the appropriate cell compartments prompts to seek the answer to the question whether FABP5 and MAGL come into functional interaction with each other and if so how does this affect CRC progression Another FABP isoform that may be involved in colon cancer development is FABP6 [“] It transports bile acids between the epithelial cells of the large intestine which are transformed into secondary metabolites such as deoxycholic acid DCA and lithocholic acid via cellular metabolism A correlation was demonstrated between increased exposure of intestinal epithelial cells to DCA and an increase in reactive oxygen species inside them which is associated with oxidative stress development and DNA damage [“] This mechanism contributes to the formation of mutations that in the event of a replication of defective cells initiate their malignancy Due to their detergent properties bile acids can cause damage to the cell membrane Compensatory mechanisms lead to an intensified inflammatory response and increased proliferation which is qualified as early tumor development Furthermore bile acids activate the muscarinic M3 receptor [“] Its stimulation induces Figure Carcinogenic effect of fatty acid binding proteins FABPs The induction of the process is illustrated by arrows the inhibition by the horizontal lineSTAT3 signal transducer and activator of transcription Src protooncogene tyrosineprotein kinase Src FAK focal adhesion kinase cdc42 cell division control protein Homolog Akt protein kinase B mTOR mammalian target of rapamycin P70S6K p70 ribosomal protein S6 kinase 4EBP1 eukaryotic translation initiation factor 4E eIF4Ebinding protein VEGFA vascular endothelial growth factor1 M3 muscarinic receptor MMPs metalloproteinases PPARÎ peroxisome proliferatoractivated receptors Î PPARδ peroxisome proliferatoractivated receptors or δOncotargetwwwoncotargetcom 0cmetalloproteinase activity and the WntBcatenin signaling pathway which in turn increases the metastatic potential as well as the proliferation and survival of colon cancer cells Desp

cancer255

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: "development of cancer is a problem that has accompanied mankind for years The growing number of cases emerging drug resistance and the need to reduce the serious side effects of pharmacotherapy are forcing scientists to better understand the complex mechanisms responsible for the initiation promotion and progression of the disease This paper discusses the modulation of the particular stages of carcinogenesis by selected physiological factors including acetylcholine ACh peroxisome proliferatoractivated receptors PPAR fatty acidbinding proteins FABPs Bruton™s tyrosine kinase Btk aquaporins AQPs insulinlike growth factor2 IGF2 and exosomes Understanding their role may contribute to the development of more effective and safer therapies based on new binding sitesINTRODUCTIONThe increasing prevalence of various types of cancers is a global phenomenon that has been occurring widely and affecting increasing numbers of people In over million new cases were reported and statistics show that every fifth man and every sixth woman is at risk for developing cancer [] Carcinogenesis is a multistage process that leads to cancer development It includes three main stages initiation promotion and progression and each is characterized by different processes During the promotion stage the genetic material of cells is damaged and if not detected by repair systems before the next division the change is transferred to newly formed cells Under the control of growth factors initiated cells may move on to the second stage of cancer formation“promotion During that stage mutated cells undergo multiple divisions their growth is uninhibited as they become insensitive to apoptotic signals As a result the tumor increases its mass but the cells remain within one an The formation of metastases occurs in the next phase“progression Metalloproteinases secreted by tumor cells destroy the extracellular matrix This allows them to enter the bloodstream and reach a new an at a considerable distance from the primary lesion with the stream of flowing blood The key step for the survival of migrating cells is adhesion to the attacked an and the formation of new blood vessels thanks to which they will receive the components necessary for development Angiogenesis the formation of new blood vessels is the last element of carcinogenesis and is enhanced by tumor cell derived factors metabolic changes and a decrease in available oxygen Each of these processes is controlled by different signal pathways via proteins that naturally occur in the body Figure Dysregulation of their expression and thus activity both to an excessive and insufficient degree ensures the continuity of carcinogenesis and increases the chances cancer cells survive in a host anism Understanding the role of individual substances and components of the body in the subsequent stages of neoplastic transformation provides a more complete picture of cancer pathogenesis This allows to develop new recommendations to improve the quality and safety of pharmacotherapy which has a direct effect on an improvement in the cancer patients™ quality of life It also provides the basis for research aimed at developing new compounds that are effective weapons in the fight against cancer Therefore there is no doubt that any actions that bring scientists closer to solving the problem of insufficient cancer therapy are sensible and much needed The purpose of this paper was to discuss the role of selected physiological factors in the various stages of neoplastic transformationOncotargetwwwoncotargetcomwwwoncotargetcom Oncotarget Vol No pp 0cCrucial mechanisms involved in carcinogenesisThe carcinogenic process is regulated by many molecular and cellular mechanisms The diversity of signaling pathways exploited during cancer initiation promotion and progression is immense Many of them are common in different cancer types but there are also clearly unique molecular and cellular signaling signatures specific for particular cancers [] Pathways such as AktPI3K RasMAPKERK Wntcatenin JAKSTAT occupy crucial roles in the regulation of cell proliferation apoptosis differentiation angiogenesis cell migration and invasion immunological activities and inflammationPI3K phosphatidylinositol 3kinase is a family of enzymes that after activation by growth factors cytokines and hormones are involved in the conversion of PIP2 phosphatidylinositol45bisphosphate into PIP3 phosphatidylinositol 345trisphosphate [] The latter in turn takes part in recruiting Akt into the cell membrane where it is phosphorylated and activates other agents such as mTOR NFkB Wnt or inhibits factors like Bad p27 to enhance carcinogenesis Dysregulation of this pathway occurs in endometrial [] bladder [] or gastric cancers [] The aforementioned PIP2 can be also transformed by phospholipase C into DAG and IP3 which then activate protein kinase C PKC [] One of the PKC substrates is Raf kinase which is an element of the RasMAPKERK pathway which is involved in the pathogenesis of ovary cancer [] or nonsmall cell lung cancer [] Ras proteins activate Raf which then phosphorylates kinases MEK12 Its final substrate is kinase ERK12 which after transportation into the nuclei regulates transcription factors essential for DNA synthesis and cell cycle progression They condition cell growth proliferation and viability [] Because of that targeting those two cascades as an anticancer therapy seems to be beneficial However it is important to notice that the existing crosstalk between both of the pathways can impede treatment and inhibition of one can strengthen the activity of the other Another component that is also connected with the MAPK family Figure Modulators of individual stages of carcinogenesis Inducers red frames inhibitors green frames M1rec muscarinic receptor M3rec muscarinic receptor Nrec nicotinic acetylcholine receptor COX2 cyclooxygenase2 PPARÎ peroxisome proliferatoractivated receptors gamma Btk Bruton™s tyrosine kinase FABP fatty acidbinding protein LFABP Ltype fatty acidbinding protein AQPs aquaporins IGF2 insulinlike growth factor IGFBP3 insulinlike growth factor binding protein IGFBP5 Insulin Like Growth Factor Binding Protein IGF2BP2 Insulin Like Growth Factor2 mRNA Binding Protein2 IGF2BP3 Insulin Like Growth Factor2 mRNA Binding Protein Oncotargetwwwoncotargetcom 0cis a signaling pathway conducted by p38MAPKs This subfamily contains four kinases which are activated mainly by inflammatory cytokines and stress factors p38MAPKs control many aspects of cell physiology such as cell cycle regulation differentiation or skeleton remodelling Additional p38MPAKs can act as tumor promotors by enhancing metalloproteinase and VEGF expression []Wntcatenin signaling plays essential roles in caricinogenesis Activation of canonical Wnt signaling results in the inhibition of GSK3 kinase dissociation of catenin proteins and its transfer to the nucleus where it regulates multiple downstream genes like cMyc and cyclin D [] This axis was found to be associated with several oncogenic events including tumor cell proliferation migration epithelial“mesenchymal transition and invasion An abnormally active WntBcatenin pathway is observed among others in gastric [] colon [] breast [] or adrenocortical cancer [] Another signaling pathway involved in the pathogenesis of many cancer types is that induced by the Jak kinases family It includes four kinases which after activation impact of the STAT molecules causing their translocation into the nuclei [] STAT a family that gathers seven forms of proteins is associated with cancer cell development progression metastasis survival and resistance to treatment [] STAT3 and STAT5 factors seem to be the most important agents in light of cancer progression They have an impact on p53 protein which leads to a disruption in cycle control and apoptosis and induces cell proliferation by the increased cMyc and cyclin D expression By the induction of VEGF gene expression they take part in enhanced angiogenesis and induction of genes such as survivin Bcl2 BclXL conditioning overall survival of the tumor cells Moreover pSTAT3 acts to negatively regulate neutrophils NK cells effector T cells and dendritic cells while positively regulating populations of MDSCs myeloidderived suppressor cells and regulatory T cell leads to a highly immunosuppressive TME tumor microenvironment []Multiple endogenous factors are involved in carcinogenesis which affects particular stages of carcinogenesis in various mechanisms Some of them interact directly with the DNA intracellular signalling molecules others by specific cellular receptors Among them are well known growth factors EGF TGFα and TGF FGF or reactive endogenously generated oxygen molecules as well as less often described agents like ACh PPAR FABPs Btk AQPs IGF2 or exosomes The latter have only recently gained in importance and are more widely studied and discussed therefore are considered in this articleAacetylcholine Ach and its receptorsOne of the factors involved in the carcinogenesis process is acetylcholine and its receptors both muscarinic inhibitors and nicotinic Their expression has been demonstrated in numerous types of cancer cells [“] It has also been proven that these cells contain acetylcholinesterase AChE an enzyme that enables cells to produce ACh in the absence of agonists from the external environment Cancer cells also have the ability to produce autoantibodies that stimulate one of the muscarinic receptor subtypes M3 These facts prompted more extensive research to explain the exact role of ACh and its receptors in the carcinogenesis process Figure Muscarinic receptors can be divided into five subtypes each of which is involved in tumor development through a different mechanism The role of muscarinic M3 receptor is the most widely described subtype It is involved in the pathogenesis of lung colon gastric and breast cancer In the course of colon cancer it has been observed that stimulation of the M3 receptor causes phosphorylation of the Akt and ERK12 kinases which are factors that through the ability to regulate the activity of pro and antiapoptotic proteins affect the intensification of cell proliferation survival and motility In addition coexpression of the M3 receptor and endothelial growth factor receptor EGFR as well as EGFR transactivation after M3 receptor stimulation has been demonstrated [ ] Moreover the administration of EGFR inhibits acetylcholineinduced ERK12 kinase phosphorylation which is reflected in a significant decrease in colon cancer cell proliferation This indicates that acetylcholine is involved in the formation of colon cancer but for its full action it is necessary to activate the EGFR receptor Thus indicating that enriching colon cancer treatment with EGFR inhibitors may improve patient outcomes Acetylcholine is also involved in the formation of vascularlike structures in the vicinity of a developing tumor guaranteeing the supply of essential nutrients to the proliferating cancer cells This is called vascular mimicry The basis of this process is the acetylcholinedependent regulation of metastasisassociated in colon cancer1 MACC1 oncogene expression via the M3RAMPKMACC1 signaling pathway [] High activity of this oncogene is associated with an increase in cell invasiveness intensified epithelial“mesenchymal transition more frequent metastases and hence worse treatment prognosis [“] The first subtype of the M receptor M1 is also involved in the tumor development process It activates the hedgehog pathway [ ] whose physiological role is to regulate the transcription factors affecting the oncogenic activity of Gli zinc finger transcription factors [ ] Excessive stimulation causes acceleration of carcinogenesis intensification of proliferation growth and survival of cancer cells as well as angiogenesis by affecting the expression of genes such as cyclin D antiapoptotic Bcl2 or VEGF Furthermore stimulation of this pathway promotes the creation of a microenvironment conducive to tumor growth through increased expression of extracellular matrix components [] Nicotinic Oncotargetwwwoncotargetcom 0cacetylcholine receptors are part of the ligandgated ion channels and are created from two types of subunits α and Receptors that contain an alpha7 or alpha9 subunit in their structure are characterized by a high degree of permeability to calcium ions thanks to which they are involved in the course of numerous processes based on signaling involving secondary messengers Thus after activating N receptors Ca2 inflows into the cell causing the secretion of growth factors exerting paracrine action on neighboring cells Simultaneously apoptosis is inhibited enabling further proliferation Studies conducted on a lung cancer cell line after exposure to cigarette smoke have shown that nicotine and nitrosamines which have a higher affinity for nicotinic acetylcholine receptors nAChRs than nicotine bind to the alpha7 receptors activating the RasERKMAPK and the JAK2STAT3PI3K signaling pathways [ ] The effect of this interaction is increased proliferation and migration of cancer cells and thus facilitated metastasis creation Moreover by affecting Badrenergic receptors nicotine intensifies COX2 expression which through the p38 MAPK and the JNK pathways leads to an increase in VEGF production a highly proangiogenic factor In addition the high activity of cyclooxygenase affects the inactivation of the PTEN protein [ ] whose biological role is silencing PI3KAkt pathway signaling This leads to cellular signal transition and induces effects that contribute to the enhanced growth and proliferation of cancer cells as well as inhibits the process of their death Under the influence of the Akt kinase proapoptotic transcription factors are inactivated including kinase9 mTOR kinase intensifying cell proliferation is activated and VEGF is activated This indicates the involvement of this pathway in processes that facilitate cancer cell survival Another mechanism contributing to cancer development in which cyclooxygenase is involved is the complex process involving the COX2PGE2EP4 axis as a result of which metalloproteinase expression is stimulated [“] MMP9 causes proteolytic activation of TGF which begins the induction of epithelialmesenchymal transformation EMT During this transition the cells acquire features that increase their invasiveness This leads to epithelial cell phenotype changes the connections between adjacent cells and between cells and the basement Figure Carcinogenic effect of acetylcholine Ach Intracellular signaling pathways activated by ACh via M1 and M3 receptors Induction of process is illustrated by arrows inhibition by horizontal lines M1 muscarinic receptor M3 muscarinic receptor N receptor nicotinic acetylcholine receptor Akt protein kinase B ERK extracellular signalregulated kinase JAK2 Janus kinase PI3K phosphoinositide 3kinase STAT3 signal transducer and activator of transcription proteins MAPK mitogenactivated protein kinaseOncotargetwwwoncotargetcom 0cmembrane are loosened the cytoskeleton is rearranged cells lose contact inhibition thus causing excessive proliferation Furthermore the microenvironment of the growing tumor also changes promoting features that facilitate the movement of the cancer cells from the primary focus to other regions of the body Through the EMT process cells acquire greater migration capacity and thus their invasiveness increases In this cellular process MMP9 also modulates the activity of integrins and other molecules that ensure cell adhesion [] and secrete soluble factors into the bloodstream that facilitate the implantation of migrating cells in ans distal to the primary tumor focus [] Thanks to these processes COX2 enables the formation of cancer metastasesPPAR peroxisome proliferatoractivated receptorsThe family of peroxisome proliferatoractivated receptors PPARs includes isoforms of these proteins that function as transcription factors Each type of steroid receptor differs in its location and affinity for ligands [] The best known is the function of the PPARÎ form Agonists of this receptor are a therapeutic option in the treatment of patients with diabetes Current research provides evidence that stimulation of PPARÎ receptors inhibits cell proliferation vessel formation and induces apoptosis Figure [ ] These properties make PPARÎ agonists attractive candidates for anticancer drugs To achieve full transcriptional activity heterodimerization of PPARÎ with the retinoid X receptor is necessary [ ] This interaction enables the promoter to bind with the target gene and regulate its expression An example of a gene against which PPARÎ acts as a repressor is the vascular endothelial growth factor VEGF gene [] Thus angiogenesis is inhibited and this mechanism has been demonstrated in studies on human hepatocellular carcinoma HCC cells [] For this cancer PPARÎ is also involved in inhibiting cell growth and attenuating cell migration and invasiveness By upregulating KLF4 Krüppellike factor expression and inhibiting cyclin D1 expression the cell cycle is inhibited Furthermore PPARÎ negatively affects the expression of transcription factor STAT3 thereby inhibiting proliferation survival and metastasis Lowering STAT3 levels also negatively affects the angiogenesis process by inhibiting VEGF depriving cancer cells of nutrients PPARÎ activation in HHC cells also impairs their metastatic ability [] This is associated with the inhibition of the expression of metalloproteinases MMP9 and MMP13 the increased expression of their inhibitor and Ecadherin a protein conditioning cell adhesion In addition PPARÎ increases plasminogen activator inhibitor expression thus inhibiting the breakdown of the extracellular matrix and basement membrane proteins which also reduces cancer cell invasiveness PPARÎ has also been shown to inhibit esophageal cancer [] The mechanism responsible for suppressing this tumor is MAPK signaling pathway inhibition This process requires TLR4 Tolllike receptor inhibition which allows PPARÎ agonists to be administered TLR4 is a procarcinogenic protein and affects among others changes in the tumor microenvironment and the severity of angiogenesis Therefore in addition to the indirect effect on the MAPK cascade its inhibition is an important element of tumor suppression Studies on esophageal cancer cells indicate that PPARÎ stimulation leads to a decrease in PCNA proliferating cell nuclear antigen factor expression which indicates inhibition of DNA replication [] In addition PPARÎ activation intensifies the process of apoptosis as evidenced by an increase in the expression of the active form of caspase and the Bax gene and a decrease in Bcl2 expression In nonsmallcell lung cancer PPARÎ stimulation inhibits the neoplastic process by regulating PTEN protein expression [] This protein is a tumor suppressor and its activation causes the dephosphorylation and inactivation of the PIP3 second messenger As a result Akt kinase activity is inhibited leading to a decrease in NFkB expression Due to PPARÎ stimulation by eicosapentaenoic acid EPA a relationship is sought between PPARÎ and COX2 activity that uses EPA as a substrate for prostanoid production The combination of PPARÎ agonists with COX2 inhibitors has demonstrated a synergistic tumorsuppressing effect in studies on nonsmallcell lung cancer Researchers indicate significantly reduced thromboxane TXA2 synthesis using combination therapy compared with monotherapy as the main mechanism of this phenomenon [] A decrease in its concentration limits signaling in four pathways ERK p38 MAPK JAK and catenin limiting tumor growth [] Furthermore it affects the arrest of the cell cycle in the G2M phase preventing further cell division and contributing to the reduction of the expression of survivin an antiapoptotic protein [] The described mechanisms affect the induction of the apoptosis process and inhibit cell cycle progression which limits cancer development The above examples indicate that peroxisome proliferatoractivated receptors play a significant role in inhibiting the development of various types of cancer which is why agonist compounds can be considered potential anticancer drugs However further comprehensive and extensive research is needed to identify potential applications and treatment regimensBtk Bruton™s tyrosine kinaseBruton™s tyrosine kinase Btk also plays an important role in carcinogenesis Figure It is a key point in signal transduction from the BCR receptor leading to the activation of the NFkB signaling pathway [] Btk affects phospholipase CÎ2 [] which after phosphorylation causes PIP2 hydrolysis The result Oncotargetwwwoncotargetcom 0cis IP3 and DAG DAG activates protein kinase C which stimulates NFkB pathway factors Finally genes conditioning cell survival are expressed Btk also contributes to inhibiting the apoptosis process through interacting with the Akt kinase [] The signaling cascade includes active PI3K which recruits Akt to the plasma membrane Under the influence of Btk its phosphorylation occurs and thus its activation Then the active Akt returns to the cytoplasm and induces antiapoptotic pathways dependent on NFkB among others In addition Btk as a kinase belonging to the Tec family affects intercellular signaling causing changes in the tumor microenvironment In most cancer cells the developing microenvironment leads to the inhibition of immune processes which results in protection and protects defective cells from natural defense mechanisms One of the most common mechanisms of this type is the effect of cancer cells on the increased differentiation of T lymphocytes towards Th2 cells The biological role of these cells is the secretion of interleukins that start a humoral response dependent on B lymphocytes Due to the increased differentiation of T lymphocytes in this direction the number of formed Th1 lymphocytes directly destroying cells containing the abnormal genome decreases The tumor microenvironment also interferes with normal dendritic cell activity It inhibits their ability to migrate and to present the antigen to immune response cells Some types of cancer show increased CXCL12 expression [] which attracts immature dendritic cells and prevents their differentiation In addition this cytokine via the CXCR4 receptor causes a reanization of the cytoskeleton [] and increases cell motility [] Tec family tyrosine kinases have also been shown to have the ability to activate CXCR4 which determines the growth survival and migration of tumor cells []FABPs fatty acidbinding proteinsThe fatty acids supplied to the cell are involved in its metabolism Due to their lipid nature they are Figure Carcinogenic effect of peroxisome proliferatoractivated receptors PPARÎ The induction of the process is illustrated by arrows the inhibition by the horizontal line PTEN phosphatase and tensin homolog deleted on chromosome ten PIP3 phosphatidylinositol 345trisphosphate Akt protein kinase B PCNA proliferating cell nuclear antigen TXA2 thromboxane A2 ERK extracellular signalregulated kinases p38MAPK P38 mitogenactivated protein kinases JAK Janusactivated kinases KLF4 Kruppellike factor STAT3 Signal transducer and activator of transcription MMP314 metalloproteinase or PAI Plasminogen activator inhibitor TRL4 tolllike receptor MAPK mitogenactivated protein kinaseOncotargetwwwoncotargetcom 0cunable to enter the aqueous environment of the cellular cytoplasm Solubilizing molecules ie fatty acidbinding proteins FABPs enable their transport to various cellular structures and thus become involved in the processes taking place in the cells including their growth reproduction and inflammatory processes In cells characterized by intensive lipid metabolism such as the liver intestine heart brain etc high transporter expression is observed which is associated with their physiological role in the cell Depending on the type of cancer both the overexpression and a decrease in FABPs can be seen FABP isoforms are not specific to one type of cancer and each plays a different role in the carcinogenesis process Figure In liver cancer LFABP was overexpressed and its high concentration was shown to correlate positively with VEGFA [“] The causes of this phenomenon are sought in the direct interaction between these two proteins which in effect provokes the activation of the SrcFAKcdc42 and the AktmTORP70S6K4EBP1 signaling pathways Activation of the SrcFAKcdc42 pathway increases tumor cell migration and LFABP plays a key role in this process [ ] Moreover by stimulating the second mentioned signaling cascade LFABP enhances VEGFA expression facilitating angiogenesis This process is regulated by HIF1α which additionally induces blood vessel formation [] Due to the high homology in their construction FABP3 and FABP4 isoforms were assigned to the same protein subfamily In addition to their physical features it has been observed that the same factors ie HIF1α VEGF increase their coexpression The physiological role of both proteins is to inhibit excessive cell proliferation and increase apoptosis Excessive FABP3 expression leads to an increase in reactive oxygen species and a decrease in the mitochondrial membrane potential [] resulting in the ing of mitochondrial megachannels which play a key role in initiating apoptosis FABP4 activates the apoptosis process by mediating the response to lipidinduced endoplasmic reticulum stress Due to the biological functions of both proteins their deficiency may be the cause of cancer progression When examining the level of FABP3 expression in embryonic tumor cells breast cancer cells [] and FABP4 in breast ovarian prostate bladder or liver cancer cells low levels of both proteins were observed which was associated with a worse prognosis in terms of overall survival Furthermore high FABP4 levels slowed the development of hepatocellular carcinoma and thus contributed to its reduction in size [] This effect is explained by the inhibition of STAT3 phosphorylation via the RaspSTAT3 signaling pathway and the inhibition of the Snail protein an accelerator of the epithelialmesenchymal transition [] However the increased FABP3 expression in tumors of the stomach brain small and nonsmallcell lung cancer seems paradoxical it has been shown to increase tumor aggressiveness and poorer patient prognosis [ ] Thus far the molecular mechanism responsible for the oncogenic potential of FABP3 and FABP4 is unknown Further research is needed to clarify the reasons these proteins promote the neoplastic process Another subtype of fatty acid binding proteins is FABP5 It is involved in the development of breast prostate liver stomach and colon cancers FABP5 has been shown to transport saturated and unsaturated longchain fatty acids that act as PPARδ ligands [ ] The nature of the supplied fatty acids determines the oncogenic activity if unsaturated fatty acid or a suppressor action is supplied to PPARδ in the case of saturated ligands [] When the Figure Carcinogenic effect of Bruton™s tyrosine kinase Btk The induction of the process is illustrated by arrows the inhibiton by the horizontal line Btk Bruton™s tyrosine kinase CXCR2 CXC chemokine receptor type CÎ2 phospholipase CÎ2Oncotargetwwwoncotargetcom 0cFABP5PPARδ pathway is stimulated the transcription of the genes responsible for cell growth and survival increases Moreover one of the target genes stimulated by PPARδ is the FABP5 gene which increases the expression of the transport protein in question [] In prostate cancer cells by supplying long chain fatty acids FABP5 leads to PPARÎ activation [“] Stimulation of these receptors results in increased expression of vascular endothelial growth factor VEGF thereby accelerating the angiogenesis process This mechanism plays a key role in the development of castrationresistant prostate cancer [“] In the case of colon cancer FABP5 contributes to its development by reducing p21 activity Overexpression of FABP5 increases the expression of cMYC which inhibits the action of a cell cycle inhibitor After silencing FABP5 activity a significant reduction in the invasive capacity of colon cancer cells CRC is observed [] The molecular basis of this interaction is not yet known at the moment High expression of monoacylglycerol lipase MAGL responsible for the production of free fatty acids is observed in highly malignant colon cancer cells [ ] This fact combined with the physiological role of FABP5 consisting of transporting free fatty acids to the appropriate cell compartments prompts to seek the answer to the question whether FABP5 and MAGL come into functional interaction with each other and if so how does this affect CRC progression Another FABP isoform that may be involved in colon cancer development is FABP6 [“] It transports bile acids between the epithelial cells of the large intestine which are transformed into secondary metabolites such as deoxycholic acid DCA and lithocholic acid via cellular metabolism A correlation was demonstrated between increased exposure of intestinal epithelial cells to DCA and an increase in reactive oxygen species inside them which is associated with oxidative stress development and DNA damage [“] This mechanism contributes to the formation of mutations that in the event of a replication of defective cells initiate their malignancy Due to their detergent properties bile acids can cause damage to the cell membrane Compensatory mechanisms lead to an intensified inflammatory response and increased proliferation which is qualified as early tumor development Furthermore bile acids activate the muscarinic M3 receptor [“] Its stimulation induces Figure Carcinogenic effect of fatty acid binding proteins FABPs The induction of the process is illustrated by arrows the inhibition by the horizontal lineSTAT3 signal transducer and activator of transcription Src protooncogene tyrosineprotein kinase Src FAK focal adhesion kinase cdc42 cell division control protein Homolog Akt protein kinase B mTOR mammalian target of rapamycin P70S6K p70 ribosomal protein S6 kinase 4EBP1 eukaryotic translation initiation factor 4E eIF4Ebinding protein VEGFA vascular endothelial growth factor1 M3 muscarinic receptor MMPs metalloproteinases PPARÎ peroxisome proliferatoractivated receptors Î PPARδ peroxisome proliferatoractivated receptors or δOncotargetwwwoncotargetcom 0cmetalloproteinase activity and the WntBcatenin signaling pathway which in turn increases the metastatic potential as well as the proliferation and survival of colon cancer cells Desp Answer:

Thyroid_Cancer

"Lung carcinoma is a prominent cause of mortality among patients with cancer Previous studies have reported the vital role of long noncoding RNAs lncRNAs in the malignant progression of lung cancer lncRNA RP11284F219 was originally identified to be expressed in lung carcinoma but its specific function remains unknown Therefore the present study aimed to elucidate the role of lncRNA RP11284F219 in lung carcinoma progression The expression of RP11‘284F219 in lung cell lines and tissues was measured using reverse transcription‘quantitative PCR The endogenous expression of RP11284F219 was silenced using RNA interference and cell viabilities were measured with a Cell Counting Kit‘ assay The invasion and apoptosis of cells were determined via Transwell assays and flow cytometry respectively The protein expression levels were measured by western blotting An increased expression of RP11‘284F219 was identified in both lung carcinoma tissues and cells Knockdown of RP11‘284F219 in lung carcinoma cells inhibited cell proliferation and invasion but promoted cell apoptosis The present study identified the existence of a direct interaction between RP11‘284F219 and microRNA miRNAmiR6273p Mechanistically it was demonstrated that RP11284F219 promoted the proliferation and invasiveness of lung carcinoma cells in part via the regulation of miR6273p Furthermore cell division cycle and apoptosis regulator CCAR1 was identified as a target gene of miR6273p The in vivo tumor growth assay also demonstrated that the knockdown of RP11‘284F219 suppressed tumor growth upregulated miR6273p and downregulated Correspondence to Dr Yuan Wang Department of Medical Imaging The First Affiliated Hospital of Xi'an Jiaotong University West Yanta Road Xi'an Shaanxi PR ChinaEmail wangyuan8003126comAbbreviations CCAR1 cell division cycle and apoptosis regulator NSCLC non‘small cell lung cancer SCLC small cell lung cancerKey words RP11284F219 lung carcinoma proliferation invasion microRNA6273p CCARCCAR1 in the xenograft model of nude mice Thus the present findings indicated the tumor promoting functions of RP11284F219 in the progression of lung carcinoma and provided a novel lncRNAmiRNA axis as a target for the management of lung cancerIntroductionPulmonary malignancies including lung and bronchus cancer rank first and second among different cancer types in terms of mortality and morbidity respectively in both men and women Furthermore of lung cancer cases are categorized as nonsmall cell lung cancer NSCLC while the remaining are classified as SCLC Although diagnostic methods and therapeutic strategies based on traditional surgical excision chemotherapy and chest radiotherapy have continuously improved the prognosis of lung carcinoma remains at for an overall 5year survival Therefore an increased understanding of the malignant progression and studies on novel therapeutic targets for the improved management of this disease are essentialLong noncoding RNAs lncRNAs are nucleotides in length and have little or no protein coding capacity The mechanisms via which lncRNAs regulate gene expression are diverse and include regulating the transcription of target genes functioning as transcriptional precursors of small RNAs generating different splice variants via regulating mRNA splicing patterns modulating protein activity and subcellular localization and scaffolding for the assembly of multiple component complexes In recent years previous studies have reported that various human cancer types exhibit lncRNAs dysfunction and these lncRNAs are involved in different aspects of pathogenesis such as the proliferation metastasis and apoptosis of tumor cells In lung cancer lncRNA metastasisassociated lung adenocarcinoma transcript is found to be upregulated in patients with advanced lung adenocarcinoma and may serve as a prognostic marker to predict the survival outcome of patients with cancer lncRNA HOX transcript antisense RNA is also highly expressed in lung cancer and it enhances the aggressiveness of lymph node metastasis and indicates a short diseasefree survival in patients with NSCLC Furthermore studies have shown that the expression of lncRNA Urothelial carcinoma‘associated 0cLI RP11‘284F219 PROMOTES LUNG CARCINOMA PROLIFERATION AND INVASIONis significantly upregulated in NSCLC and may induce resistance to treatment of EGFR‘tyrosine kinase inhibitors by activating the AKTmTOR pathway lncRNA RP11284F219 was primarily discovered in a Pancancer transcriptomic analysis lncRNA RP11‘284F219 exists as a cluster of three annotated lncRNAs RP11284F219107 antisense to brevican which is a proteoglycan linked to invasiveness in glioma but lacks expression in squamous cell lung carcinomas However the specific function and the underlying mechanism of RP11284F219 in lung carcinoma remain unknownTo the best of our knowledge the present study demonstrated for the first time that lncRNA RP11284F219 was significantly upregulated in lung carcinoma tissues and cell lines and was involved in the carcinogenesis of lung cancer Together with microRNA miRNAmiR6273p and cell division cycle and apoptosis regulator CCAR1 the regulatory axis of RP11‘284F219miR‘‘3pCCAR1 exists both in the lung carcinoma cells in vitro and in the tumor growth model in vivo The present study aimed to investigate RP11284F219 function in lung carcinoma and demonstrate the molecular mechanism underlying the regulation process via the RP11‘284F219miR‘‘3pCCAR1 axisMaterials and methodsTissue samples and cell lines Between May and Jan paired tumor and adjacent healthy tissues were isolated from patients with lung carcinoma age range ‘ years nine male patients four female patients who were diagnosed and treated in First Affiliated Hospital of Xi'an Jiaotong University The samples were dissected during the surgery and immediately flash‘frozen in liquid nitrogen and transferred to ‘ËšC storage for further extraction of both RNA and protein All the tissue samples were obtained with written informed consent from the patients The protocol was approved by The First Affiliated Hospital of Xi'an Jiaotong University approval no A normal lung epithelial cell line BEAS2B and lung carcinoma cell lines NCIH460 NCIH1299 and A549 were purchased from American Type Culture Collection ATCC and cultured according to the ATCC guidelines 293T cells were purchased from Procell Life ScienceTechnology Co Ltd and cultured in DMEM supplemented with FBS cat no ‘ ATCC and 1X Penicillin‘streptomycin Thermo Fisher Scientific Inc BEAS2B cells were cultured in bronchial epithelial growth medium BEGM cat no CC‘ Clonetics Corporation according to the manufacturer's instructions NCI‘H460 and NCI‘H1299 cells were cultured in RPMI‘ medium cat no ‘ ATCC and A549 cells in F12K medium cat no ‘ ATCC supplemented with FBS cat no ATCC and 1X Penicillin‘streptomycin Thermo Fisher Scientific Inc All cells were culture at ˚C with CO2RNA extraction and reverse transcription‘quantitative PCR RT‘qPCR Total RNA from both tissue samples and cell lines were extracted using TRIzol® reagent Invitrogen Thermo Fisher Scientific Inc For each sample ng total RNA was reverse transcribed to synthesize the first‘strand cDNA using the PrimeScript RT reagent kit Takara Bio InccDNA samples were diluted times to perform the RT‘qPCR using SYBR Premix Ex Taq Takara Bio Inc on a CFX96 realtime PCR detection system BioRad Laboratories Inc Expression levels of mRNAs lncRNAs and miRNAs were normalized to GAPDH The primers used for RTqPCR analyses were as follows GAPDH forward '‘AAC GAC CCC TTC ATT GAC C‘' and reverse '‘TCC ACG ACA TAC TCA GCA CC‘' RP11‘284F219 forward '‘AGG ATT GGC ACT CAC TTC GG‘' and reverse '‘TCT CTC ACC ACG TCT GGT CT‘' and CCAR1 forward '‘CTG ATG GCT AGC CCT AGT ATG GA‘' and reverse '‘TGC CTT TCA TGC CCA CTA AAA ‘' The temperature protocol used to perform RT was ˚C for h followed by ˚C for min Thermal conditions of PCR reactions were Initial denaturation at ˚C for min followed by cycles for sec at ˚C and sec at ˚C The mRNA expression levels were determined using the 2ΔΔCq method Oligonucleotides and cell transfection The small interfering RNA siRNA synthetic negative control siNC RP11284F219 siRNAs siRP11284F219 miRNC miR‘‘3p mimics and miR‘‘3p inhibitor were purchased from Shanghai GenePharma Co LtdAll primer sequence information is presented in Table I At a density of 2x105 cellswell the cells were plated in 6well plates h before transfection and were transfected at confluency All of the oligonucleotides were transfected at a final concentration of nM using Lipofectamine® reagent Invitrogen Thermo Fisher Scientific Inc according to the manufacturer's instruction Cells were collected at h posttransfection for subsequent experimentsCell Counting Kit CCK‘ assay and EdU labeling of prolif‘erating cells A CCK‘ was used for cell proliferation assay the cells were seeded into ‘well plates 2x103 cellswell and observed for and days or indicated time points following the manufacturer's instructions Dojindo Molecular Technologies Inc The optical density was measured at nm using a spectrophotometer Thermo Fisher Scientific IncFor the EdU assay cells were incubated with µM EdU cat no ab219801 Abcam for h at ˚C and fixed with formaldehyde at room temperature for min After a brief washing with PBS click reagent was added into each well and incubated in the dark for min at room temperature Followed by PBS washing the cells were stained with µgml DAPI at room temperature for min Images were captured using a fluorescence microscope Nikon Corporation and measured using Adobe Photoshop software Adobe Systems Inc The EdU labeled cells were analyzed with MoFlo Astrios Beckman‘Coulter Inc Magnification x200Transwell assay and flow cytometry measurement of cell apoptosis Transwell assays were performed with a coating of Matrigel BD Biosciences mixed with culture medium mixed at ratio at ˚C for h A total of 1x105 cells in µl serum‘free medium were added to the upper layer of the Transwell chambers µm pore size Corning Inc and cultured for h The lower chamber contained the culture medium with FBS The migrated cells were fixed with 0cONCOLOGY REPORTS Table I Sequence of siRNAs and miRNA mimics and inhibitorsOligonucleotides si‘NC si‘RP11‘284F219 miR‘NC miR‘‘3p mimics miR‘‘3p inhibitor miR microRNA siRNA small interfering RNA NC negative controlSequence '†’'UUCUCCGAACGUGUCACGUTTUAUUGGCACCAAGGAUAGCUCGUUAAUCGGCUAUAAUACGCUCUUUUCUUUGAGACUCACUUCUUUUCUUUGAGACUCACU paraformaldehyde for min at room temperature stained with crystal violet for min at room temperature and images of six randomly selected fields in each well were captured under a light microscope Magnification x200Cellular apoptosis was detected using the Apoptosis Detection kit cat no KGF001 Nanjing KeyGen Biotech Co Ltd according to the manufacturer's instructions Cells were stained with fluorescein isothiocyanateconjugated annexin V and PI After incubated for min at ˚C in the dark µl 1X Binding Buffer was added to each tube and stained cells were analyzed using BD FACS Canto II flow cytometry FACS Calibur BD Biosciences Data were analyzed using FlowJo software version Tree Star IncLuciferase reporter assay The RP11284F219 wildtype wt or mutant mut '‘untranslated region '‘UTR and CCAR1 wt or mut '‘UTR sequences were cloned into the pmirGLO plasmid Youbio httpwwwyoubiocn cat no VT1439 The vectors µgml were co‘transfected with miR‘NC or miR6273p mimic nM and Renilla plasmids ngwell used as an internal control into cells seeded in a 48well plate 1x104well using Lipofectamine® reagent Invitrogen Thermo Fisher Scientific Inc Cell lysates were collected at h after transfection and the luciferase activities were detected with the DualLuciferase Reporter Assay system Promega Corporation according to the manufacturer's instructionsWestern blotting Cell were lysed using RIPA lysis buffer Sigma‘Aldrich Merck KGaA and protein concentrations were assessed with the BCA Protein Assay kit according to the manufacturer's instructions Beyotime Institute of Biotechnology Shanghai China Equal amounts µg of cell protein lysates were loaded and separated by SDS‘PAGE transferred to a PVDF membrane and blocked with non‘fat milk at room temperature for h The membranes were then incubated with CCAR1 primary antibody cat no ab70243 Abcam overnight at ˚C followed by incubation with goat anti‘mouse or goat anti‘rabbit IgG‘horseradish peroxidase conjugate secondary antibodies cat no ab205718 Abcam at room temperature for h GAPDH cat no ab181602 Abcam was used as loading control The signals were detected using the ECL system Protein Simple according to the manufacturer's instructionsIn vivo tumorigenicity analysis in mice Male BALBc nude mice age weeks weight ‘ g were obtained from Beijing Vital River Laboratory Animal Technology Co Ltd and housed at a room temperature of ˚C with a h lightdark cycle The mice were maintained in an individually ventilated cage system under specific pathogen‘free conditions temperature ˚C humidity and fed with sterile food and water free access To evaluate the effect of RP11‘284F219 knockdown on the growth of lung carcinoma in vivo 5x106 siNC or siRP11284F219 treated NCI‘H1299 cells in µl serum‘free medium were subcutaneously injected into each mouse n5 per group under anesthesia which was induced by isoflurane and maintained by isoflurane flow rate 1lmin The animals were monitored daily and the following criteria for humane endpoint was used Severe tumor burden mm in diameter difficulty breathing significant body‘weight loss and clinical signs such as prostration hypothermia and significant abdominal distension Tumors were measured on days and and the volumes were calculated using the formula a x b22 [the largest diameter a and the smallest diameter b] Then weeks after inoculation the mice were euthanized by CO2 inhalation CO2 flow rate of cage volume and the death of animals were confirmed by cessation of heartbeat The xenografts were imaged and weighedThe total RNA was then extracted from the xenografts as aforementioned Animal care and study were approved by the Institutional Animal Care and Use Committee of The First Affiliated Hospital of Xi'an Jiaotong University approval no Target prediction Potential target miRNAs of RP11284F219 were predicted using LncBase V2 httpcarolinaimisathena‘ innovationgrdiana_toolswebindexphprlncbasev2index The target genes of miR‘‘3p were predicted using three bioinformatics algorithms TargetScanV72 httpwwwtargetscanorgvert_72 and miRDB httpwwwmirdborgmininghtmlStatistics analysis Data were analyzed using the GraphPad Prism software GraphPad Software Inc and presented as the mean ± SD from ‰¥ independent experiments A two‘tailed unpaired Student's t‘test or one‘way ANOVA with Tukey's post‘hoc analysis were performed to evaluate the statistical significance P005 was considered to indicate a statistically significant difference\x0cLI RP11‘284F219 PROMOTES LUNG CARCINOMA PROLIFERATION AND INVASIONFigure RP11‘284F219 expression is upregulated in LC tissues and cell lines A Expression of RP11‘284F219 in LC tissues in comparison with adjacent healthy tissues was analyzed using RTqPCR P0001 vs adjacent tissues n13 B Expression of RP11‘284F219 in human lung carcinoma cell lines NCIH460 NCIH1299 and A549 compared with normal human lung epithelial cell line BEAS2B was analyzed using RTqPCR P005 P0001 vs BEAS‘2B n3 LC lung carcinoma RT‘qPCR reverse transcription‘quantitative PCRResultsExpression of RP11‘284F219 is upregulated in lung carci‘noma To investigate the potential role of RP11284F219 in lung carcinoma its expression was analyzed in tissue samples and matched adjacent healthy tissues from patients with lung carcinoma The results demonstrated that the expression of RP11‘284F219 was significantly upregulated in tumor tissues compared with healthy tissues Fig 1A The expression of RP11‘284F219 was also analyzed in human lung carcinoma cell lines NCIH460 NCIH1299 and A549 and normal human lung epithelial cell line BEAS2B Consistent with the findings in the tissue samples the expression of RP11‘284F219 was significantly increased in carcinoma cell lines compared with the normal epithelial cell line Fig 1B These results indicated that RP11284F219 may serve an oncogenic role in lung carcinomaKnockdown of RP11‘284F219 exerts anti‘oncogenic effects in lung carcinoma cells To study the specific role of RP11284F219 in lung carcinoma cells RP11284F219 siRNA was transfected into NCIH1299 and NCIH460 cells Fig 2A After transfection the proliferation of these cells was measured using CCK‘ and EdU assays Fig 2B‘D The results suggested that knocking down RP11‘284F219 significantly reduced the proliferation of lung carcinoma cells compared with the NC group Fig 2BD The invasiveness of si‘RP11‘284F219 transfected cells also significantly decreased as indicated by the data from the Transwell assay Fig 2F To further validate the invasive capability a RT‘qPCR assay was performed to detect the expression levels of invasion‘related genes and the results identified that both MMP2 and MMP9 were significantly decreased when RP11‘284F219 was downregulated Fig S1The results of flow cytometry measurement based apoptosis assay suggested that cells transfected with siRP11284F219 had a higher apoptotic rate compared with the siNC transfected group Fig 2E These data demonstrated the antitumor effects of RP11‘284F219 knockdown in lung carcinoma cells indicating an oncogenic role of RP11284F219RP11‘284F219 directly interacts with miR‘‘3p Based on the prediction of the online tool lncBase v2 from DIANA Prediction module httpcarolinaimisathena‘innovationgrdiana_toolswebindexphprlncbasev2index which was used to identify the downstream miRNAs of RP11284F219 the first five miRNAs in the output list were tested Among the predicted potential targets it was found that miR6273p had the most significant upregulation in NCIH1299 cells transfected with siRP11284F219 Fig S2Using sequence alignment it was identified that miR‘‘3p was partially complementary with the '‘UTR of RP11‘284F219 Fig 3A Subsequently 293T cells were transfected with the pmirGLORP11284F219wt or mut vector containing the wt or mut sequence of RP11284F219 '‘UTR with or without miR‘‘3p mimics Results from the luciferase reporter assay suggested that miR6273p mimics significantly decrease the signal of RP11‘284F219‘wt transfected cells but not the RP11‘284F219‘mut transfected cells indicating a direct interaction between the two non‘coding RNAs Fig 3A Furthermore transfection of siRP11284F219 into NCIH1299 and NCIH460 cells resulted in the suppression of endogenous RP11‘284F219 leading to a significant increase in miR‘‘3p expression Fig 3B Thus these findings suggested an inhibitory effect of RP11‘284F219 on the expression of miR‘‘3p in lung carcinoma cellsThe expression of miR‘‘3p was detected in both lung carcinoma tissues and cell lines It was demonstrated that miR‘‘3p was significantly downregulated in carcinoma tissues Fig 3C and NCIH460 NCIH1299 and A549 cells Fig 3D compared with healthy tissues and cells Collectively these data suggested a direct interaction between RP11284F219 and miR6273p in which RP11284F219 suppresses the expression of miR‘‘3pRP11‘284F219 regulates the proliferation and invasiveness of lung carcinoma cells via miR‘‘3p To rescue the antitumor effects of siRP11284F219 in lung carcinoma cells the miR‘‘3p inhibitor which specifically downregulates the expression of miR‘‘3p was transfected into NCI‘H1299 and NCI‘H460 cells Fig 4A The results from the CCK‘ and EdU assays demonstrated that treatment with si‘RP11‘284F219 0cONCOLOGY REPORTS Figure RP11‘284F219 knockdown inhibits lung carcinoma cell proliferation and invasion and promotes cell apoptosis A RP11‘284F219 knockdown was achieved via RP11‘284F219 siRNA and the knockdown efficiency was verified using reverse transcription‘quantitative PCR n3 Cell Counting Kit‘ assay was performed to measure the proliferation of B NCIH1299 and C NCIH460 cells after transfection with siRP11284F219 compared with the si‘NC group n5 D An EdU assay was performed to measure the proliferation of NCI‘H1299 and NCI‘H460 cells after transfection with si‘NC and si‘RP11‘284F219 Magnification x200 E Flow cytometry analysis was performed to determine the effects of RP11‘284F219 knockdown on apoptotic rates in NCI‘H1299 and NCI‘H460 cells n3 F Transwell assay was performed to determine the effects of RP11‘284F219 knockdown on NCI‘H1299 and NCI‘H460 cell invasion n3 Magnification x200 P005 P001 vs control group NC negative control siRNA small interfering RNA OD optical density and miR‘NC significantly decrease the proliferation of both NCIH1299 and NCIH460 cells Fig 4BD However the administration of miR‘‘3p inhibitor partially reversed the antiproliferative effect of siRP11284F219 indicating that RP11284F219 regulates the proliferation of lung carcinoma cells partially via miR6273p Fig 4BD In addition the 0cLI RP11‘284F219 PROMOTES LUNG CARCINOMA PROLIFERATION AND INVASIONFigure RP11‘284F219 directly interacts with miR‘‘3p A Binding site between RP11‘284F219 and miR‘‘3p that was identified using the DIANA tools and a luciferase reporter assay was conducted in pmirGLORP11284F219wt or mut treated cells in the presence of miR6273p mimics or miR‘NC n3 P005 vs miR‘NC B Expression of miR‘‘3p in NCI‘H1299 and NCI‘H460 cells transfected with si‘RP11‘284F219 was analyzed using RTqPCR P001 vs si‘NC n3 miR‘‘3p expression in C LC tissues and D NCI‘H460 NCI‘H1299 and A549 cells compared with adjacent healthy tissues and normal lung epithelial cells was analyzed using RT‘qPCR n3 P005 P001 vs adjacent tissue or BEAS‘2B cells NC negative control siRNA small interfering RNA wt wild‘type mut mutant miR microRNA LC lung carcinoma miR‘‘3p inhibitor restored the reduction in the number of NCIH1299 and NCIH460 cells that migrated through the Transwell membrane induced by si‘RP11‘284F219 treatment Fig 4F These data indicated the participation of miR6273p in the RP11284F219mediated invasive effectThe qPCR assay results identified that both MMP2 and MMP9 expression levels were restored in RP11284F219downregulated cells when miR6273p was inhibited compared with the miR‘NC group Fig S3 In addition transfection with miR‘‘3p inhibitor also diminished the pro‘apoptosis effect of si‘RP11‘284F219 in both NCIH1299 and NCIH460 cells Fig 4E Therefore it was suggested that RP11284F219 promoted the proliferation and invasion as well as suppressed the apoptosis of lung carcinoma cells by inhibiting the expression of miR‘‘3pRP11‘284F219 regulates CCAR1 via targeting miR‘‘3p To further evaluate how RP11‘284F219 exerts an oncogenic role via miR‘‘3p the publicly available algorithms of TargetScan httpwwwtargetscanorg and miRDB were used which identified CCAR1 as a potential target for miR6273p Fig 5A In order to validate this prediction miR6273p mimic was transfected into cells and the transfection efficiency was assessed The results demonstrated that transfection of miR6273p mimic increased the expression of miR‘‘3p by times compared with cells transfected with miRNC Fig S4After validating the upregulation of miR6273p mimic a CCAR1wt vector was constructed which contained the wt binding site between miR‘‘3p and the CCAR1 '‘UTR and CCAR1mut vector containing the mut sequence Fig 5A The results from luciferase reporter assays indicated that compared with the miRNC group the miR6273p mimic significantly decreased the luciferase activity of CCAR1‘wt treated cells but not the CCAR1‘mut treated cells suggesting a direct binding of miR‘‘3p to the '‘UTR of CCAR1 Fig 5B Increased expression levels of CCAR1 were present in the lung carcinoma tissues compared with the adjacent healthy tissues Fig 5C Moreover a significant decrease in both mRNA and protein expression levels of CCAR1 was detected upon transfecting NCIH1299 and NCIH460 cells with miR6273p mimics Fig 5D and E Thus CCAR1 may be a direct target of miR‘‘3p in lung carcinoma cells and tissuesRP11‘284F219 knockdown inhibits tumor growth and the expression of CCAR1 in vivo In order to investigate the effect of RP11284F219 on in vivo tumorigenicity NCIH1299 cells were transfected with siNC or siRP11284F219 and injected into the nude mice After weeks a significantly 0cONCOLOGY REPORTS Figure RP11‘284F219 regulates proliferation and invasiveness in lung carcinoma cells via miR‘‘3p A Expression of miR‘‘3p in NCI‘H1299 and NCI‘H460 cells transfected with miR‘NC or miR‘‘3p inhibitor was detected using RT‘qPCR analysis n3 P005 vs miR‘NC Cell Counting Kit‘ assay was performed in B NCI‘H1299 and C NCI‘H460 cells stably transfected with si‘RP11‘284F219 in the presence of miR‘NC or miR‘‘3p inhibitor n5 D EdU assay was performed in NCI‘H1299 and NCI‘H460 cells stably transfected with si‘RP11‘284F219 in the presence of miR‘NC or miR‘‘3p inhibitor Magnification x200 E Flow cytometry analysis was performed in NCI‘H1299 and NCI‘H460 cells stably transfected with si‘RP11‘284F219 in the presence of miR‘NC or miR‘‘3p inhibitor n3 F Transwell assay was performed in NCI‘H1299 and NCI‘H460 cells stably transfected with si‘RP11‘284F219 in the presence of miR‘NC or miR‘‘3p inhibitor Magnification x200 n3 P005 vs si‘NC NC negative control siRNA small interfering RNA OD optical density miR microRNA 0cLI RP11‘284F219 PROMOTES LUNG CARCINOMA PROLIFERATION AND INVASIONFigure miR‘‘3p directly targets CCAR1 A Bioinformatic analysis of the predicted binding sites between the CCAR1 '‘untranslated region and miR‘‘3p B Luciferase reporter assay in CCAR1‘wt or CCAR1‘mut treated cells in the presence of miR‘NC or miR‘‘3p mimic n3 P005 vs miR‘NC C CCAR1 expression in LC tissues compared with adjacent healthy tissues was analyzed using RT‘qPCR n13 P001 vs adjacent tissue Expression of CCAR1 in NCI‘H1299 and NCI‘H460 cells transfected with miR‘NC or miR‘‘3p mimics was detected using D RT‘qPCR and E western blotting n3 P005 vs miR‘NC miR microRNA NC negative control wt wild‘type mut mutant RT‘qPCR reverse transcription‘quantitative PCR CCAR1 cell division cycle and apoptosis regulator LC lung carcinoma slower proliferative rate of the tumors was observed in the siRP11284F219 group compared with the siNC group Fig 6A and B Furthermore the tumor volume and weight were significantly decreased in the si‘RP11‘284F219 group compared with the control group Fig 6A and B RTqPCR analysis also demonstrated that compared with the siNC group the tumors in the si‘RP11‘284F219 group expressed higher levels of miR6273p Fig 6C and lower levels of CCAR1 Fig 6D providing further evidence to the existence of the RP11‘284F219miR‘‘3pCCAR1 regulatory axis in lung carcinoma tumor tissuesDiscussionThe present study investigated the function of RP11284F219 in lung carcinoma It was initially found that RP11284F219 was significantly upregulated in both lung cancer tissues and cell lines Following the deduction of a potential oncogenic role of this lncRNA siRP11284F219 was transfected into NCIH460 and NCIH1299 cells and it was demonstrated that knockdown of RP11‘284F219 inhibited the proliferation and invasion while promoting apoptosis of lung carcinoma cells In the mechanistic studies using online prediction tools and in vitro assays the results indicated that miR‘‘3p directly interacts with RP11‘284F219 by binding to its '‘UTRThe function of miR627 was initially reported in colorectal cancer CRC Padi found that when upregulated by calcitriol miR627 targets the histone demethylase Jumonji domain containing 1A to increase methylation of histone H3K9 and suppresses the proliferative factors of CRC cells thus inhibiting the proliferation of CRC both in vitro and in vivo Moreover in CRC Sun discovered the role of miR‘ in vitamin D‘enhanced efficacy of irinotecan via inhibition of the cytochrome P450 enzyme‘mediated intratumoral drug metabolism miR‘ is also reported to be a potential non‘invasive diagnostic marker in gastric and breast cancer types In pulmonary diseases miR627 is downregulated in patients with chronic obstructive pulmonary disease and targets the high‘mobility group box protein to inhibit its expression thus improving transforming growth factorβ1‘induced pulmonary fibrosis The present results demonstrated the inhibitory effect of RP11‘284F219 on the expression of miR‘‘3p In addition it was identified that the miR‘‘3p inhibitor can neutralize the anti‘tumor effects of RP11‘284F219 knockdown indicating that RP11‘284F219 promotes the proliferation and invasiveness of lung carcinoma cells partially by regulating miR‘‘3p This anti‘tumor role of miR6273p under the regulation of RP11284F219 in lung carcinoma tissues and cells is in accordance with the previous aforementioned findings on human CRC gastric and breast cancer types\x0cONCOLOGY REPORTS Figure RP11‘284F219 knockdown inhibits tumor growth in vivo A Macroscopic image of xenografted tumors B Tumor volume in nude mice injected with NCI‘H1299 cells transfected with si‘NC or si‘RP11‘284F219 measured over weeks n5 C Weight of tumors in nude mice at weeks after injection of NCI‘H1299 cells transfected with si‘NC or si‘RP11‘284F219 n5 Expression levels of D miR‘‘3p and E CCAR1 in the tumor tissues from nude mice injected with NCI‘H1299 cells transfected with si‘NC or si‘RP11‘284F219 for weeks were detected using reverse transcription‘quantitative PCR n5 P005 P001 P0001 vs si‘NC miR microRNA NC negative control sh short hairpin RNA CCAR1 cell division cycle and apoptosis regulator Using the publicly available RNA interaction prediction algorithms the current study identified that CCAR1 which was initially shown as the target gene of miR6273p is also regulated by RP11‘284F219 Furthermore the regulatory axis of RP11‘284F219miR‘‘3pCCAR1 exists in the lung carcinoma cells both in vitro and in vivo in the tumor growth model The interaction between RP11‘284F219 and miR‘‘3p and the interaction between miR‘‘3p and CCAR1 were demonstrated by the dual‘luciferase assay Although this method has been used to validate RNA‘RNA interactions in previous studies other assays such as RNA pulldown and RNA binding protein immunoprecipitation that would provide more direct evidence for the RNARNA and RNA‘protein interactions should be performedCCAR1 was initially reported as a protein essential for cancer cell apoptosis induced by retinoids or chemotherapeutics such as Adriamycin and etoposide Subsequently Kim et al revealed that this protein functions as a transcriptional coactivator of nuclear receptors In human breast cancer cells as CCAR1 interacts and cooperates with the coactivators of estrogen receptor signaling it promotes the estrogendependent proliferation of cancer cells In CRC cells Ou reported that CCAR1 can be recruited by βcatenin to act as a coactivator for the transcriptional activation of lymphoid enhancer binding factor CCAR1 is essential for the expression of Wnt target genes as well as the neoplastic transformation of CRC cells In gastric cancer cells researchers have revealed the cooperation between CCAR1 and βcatenin which leads to the promotion of the proliferation and migration of cancer cells In lung cancer CCAR1 was reported to be an effector of Doxorubicin‘induced apoptosis Moreover Muthu demonstrated that certain chemical compounds that bind with CCAR1 can increase the expression of CCAR1 and induce apoptosis However a contradictory conclusion was reported in a recent study which observed that CCAR1 was promoted by serine and arginine rich splicing factor which is activated by glucose intake and further enhanced tumorigenesis by increasing the glucose consumption rate Corroborating this finding in the current study via the targeting of miR‘‘3p the expression of CCA

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You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: "Lung carcinoma is a prominent cause of mortality among patients with cancer Previous studies have reported the vital role of long noncoding RNAs lncRNAs in the malignant progression of lung cancer lncRNA RP11284F219 was originally identified to be expressed in lung carcinoma but its specific function remains unknown Therefore the present study aimed to elucidate the role of lncRNA RP11284F219 in lung carcinoma progression The expression of RP11‘284F219 in lung cell lines and tissues was measured using reverse transcription‘quantitative PCR The endogenous expression of RP11284F219 was silenced using RNA interference and cell viabilities were measured with a Cell Counting Kit‘ assay The invasion and apoptosis of cells were determined via Transwell assays and flow cytometry respectively The protein expression levels were measured by western blotting An increased expression of RP11‘284F219 was identified in both lung carcinoma tissues and cells Knockdown of RP11‘284F219 in lung carcinoma cells inhibited cell proliferation and invasion but promoted cell apoptosis The present study identified the existence of a direct interaction between RP11‘284F219 and microRNA miRNAmiR6273p Mechanistically it was demonstrated that RP11284F219 promoted the proliferation and invasiveness of lung carcinoma cells in part via the regulation of miR6273p Furthermore cell division cycle and apoptosis regulator CCAR1 was identified as a target gene of miR6273p The in vivo tumor growth assay also demonstrated that the knockdown of RP11‘284F219 suppressed tumor growth upregulated miR6273p and downregulated Correspondence to Dr Yuan Wang Department of Medical Imaging The First Affiliated Hospital of Xi'an Jiaotong University West Yanta Road Xi'an Shaanxi PR ChinaEmail wangyuan8003126comAbbreviations CCAR1 cell division cycle and apoptosis regulator NSCLC non‘small cell lung cancer SCLC small cell lung cancerKey words RP11284F219 lung carcinoma proliferation invasion microRNA6273p CCARCCAR1 in the xenograft model of nude mice Thus the present findings indicated the tumor promoting functions of RP11284F219 in the progression of lung carcinoma and provided a novel lncRNAmiRNA axis as a target for the management of lung cancerIntroductionPulmonary malignancies including lung and bronchus cancer rank first and second among different cancer types in terms of mortality and morbidity respectively in both men and women Furthermore of lung cancer cases are categorized as nonsmall cell lung cancer NSCLC while the remaining are classified as SCLC Although diagnostic methods and therapeutic strategies based on traditional surgical excision chemotherapy and chest radiotherapy have continuously improved the prognosis of lung carcinoma remains at for an overall 5year survival Therefore an increased understanding of the malignant progression and studies on novel therapeutic targets for the improved management of this disease are essentialLong noncoding RNAs lncRNAs are nucleotides in length and have little or no protein coding capacity The mechanisms via which lncRNAs regulate gene expression are diverse and include regulating the transcription of target genes functioning as transcriptional precursors of small RNAs generating different splice variants via regulating mRNA splicing patterns modulating protein activity and subcellular localization and scaffolding for the assembly of multiple component complexes In recent years previous studies have reported that various human cancer types exhibit lncRNAs dysfunction and these lncRNAs are involved in different aspects of pathogenesis such as the proliferation metastasis and apoptosis of tumor cells In lung cancer lncRNA metastasisassociated lung adenocarcinoma transcript is found to be upregulated in patients with advanced lung adenocarcinoma and may serve as a prognostic marker to predict the survival outcome of patients with cancer lncRNA HOX transcript antisense RNA is also highly expressed in lung cancer and it enhances the aggressiveness of lymph node metastasis and indicates a short diseasefree survival in patients with NSCLC Furthermore studies have shown that the expression of lncRNA Urothelial carcinoma‘associated 0cLI RP11‘284F219 PROMOTES LUNG CARCINOMA PROLIFERATION AND INVASIONis significantly upregulated in NSCLC and may induce resistance to treatment of EGFR‘tyrosine kinase inhibitors by activating the AKTmTOR pathway lncRNA RP11284F219 was primarily discovered in a Pancancer transcriptomic analysis lncRNA RP11‘284F219 exists as a cluster of three annotated lncRNAs RP11284F219107 antisense to brevican which is a proteoglycan linked to invasiveness in glioma but lacks expression in squamous cell lung carcinomas However the specific function and the underlying mechanism of RP11284F219 in lung carcinoma remain unknownTo the best of our knowledge the present study demonstrated for the first time that lncRNA RP11284F219 was significantly upregulated in lung carcinoma tissues and cell lines and was involved in the carcinogenesis of lung cancer Together with microRNA miRNAmiR6273p and cell division cycle and apoptosis regulator CCAR1 the regulatory axis of RP11‘284F219miR‘‘3pCCAR1 exists both in the lung carcinoma cells in vitro and in the tumor growth model in vivo The present study aimed to investigate RP11284F219 function in lung carcinoma and demonstrate the molecular mechanism underlying the regulation process via the RP11‘284F219miR‘‘3pCCAR1 axisMaterials and methodsTissue samples and cell lines Between May and Jan paired tumor and adjacent healthy tissues were isolated from patients with lung carcinoma age range ‘ years nine male patients four female patients who were diagnosed and treated in First Affiliated Hospital of Xi'an Jiaotong University The samples were dissected during the surgery and immediately flash‘frozen in liquid nitrogen and transferred to ‘ËšC storage for further extraction of both RNA and protein All the tissue samples were obtained with written informed consent from the patients The protocol was approved by The First Affiliated Hospital of Xi'an Jiaotong University approval no A normal lung epithelial cell line BEAS2B and lung carcinoma cell lines NCIH460 NCIH1299 and A549 were purchased from American Type Culture Collection ATCC and cultured according to the ATCC guidelines 293T cells were purchased from Procell Life ScienceTechnology Co Ltd and cultured in DMEM supplemented with FBS cat no ‘ ATCC and 1X Penicillin‘streptomycin Thermo Fisher Scientific Inc BEAS2B cells were cultured in bronchial epithelial growth medium BEGM cat no CC‘ Clonetics Corporation according to the manufacturer's instructions NCI‘H460 and NCI‘H1299 cells were cultured in RPMI‘ medium cat no ‘ ATCC and A549 cells in F12K medium cat no ‘ ATCC supplemented with FBS cat no ATCC and 1X Penicillin‘streptomycin Thermo Fisher Scientific Inc All cells were culture at ˚C with CO2RNA extraction and reverse transcription‘quantitative PCR RT‘qPCR Total RNA from both tissue samples and cell lines were extracted using TRIzol® reagent Invitrogen Thermo Fisher Scientific Inc For each sample ng total RNA was reverse transcribed to synthesize the first‘strand cDNA using the PrimeScript RT reagent kit Takara Bio InccDNA samples were diluted times to perform the RT‘qPCR using SYBR Premix Ex Taq Takara Bio Inc on a CFX96 realtime PCR detection system BioRad Laboratories Inc Expression levels of mRNAs lncRNAs and miRNAs were normalized to GAPDH The primers used for RTqPCR analyses were as follows GAPDH forward '‘AAC GAC CCC TTC ATT GAC C‘' and reverse '‘TCC ACG ACA TAC TCA GCA CC‘' RP11‘284F219 forward '‘AGG ATT GGC ACT CAC TTC GG‘' and reverse '‘TCT CTC ACC ACG TCT GGT CT‘' and CCAR1 forward '‘CTG ATG GCT AGC CCT AGT ATG GA‘' and reverse '‘TGC CTT TCA TGC CCA CTA AAA ‘' The temperature protocol used to perform RT was ˚C for h followed by ˚C for min Thermal conditions of PCR reactions were Initial denaturation at ˚C for min followed by cycles for sec at ˚C and sec at ˚C The mRNA expression levels were determined using the 2ΔΔCq method Oligonucleotides and cell transfection The small interfering RNA siRNA synthetic negative control siNC RP11284F219 siRNAs siRP11284F219 miRNC miR‘‘3p mimics and miR‘‘3p inhibitor were purchased from Shanghai GenePharma Co LtdAll primer sequence information is presented in Table I At a density of 2x105 cellswell the cells were plated in 6well plates h before transfection and were transfected at confluency All of the oligonucleotides were transfected at a final concentration of nM using Lipofectamine® reagent Invitrogen Thermo Fisher Scientific Inc according to the manufacturer's instruction Cells were collected at h posttransfection for subsequent experimentsCell Counting Kit CCK‘ assay and EdU labeling of prolif‘erating cells A CCK‘ was used for cell proliferation assay the cells were seeded into ‘well plates 2x103 cellswell and observed for and days or indicated time points following the manufacturer's instructions Dojindo Molecular Technologies Inc The optical density was measured at nm using a spectrophotometer Thermo Fisher Scientific IncFor the EdU assay cells were incubated with µM EdU cat no ab219801 Abcam for h at ˚C and fixed with formaldehyde at room temperature for min After a brief washing with PBS click reagent was added into each well and incubated in the dark for min at room temperature Followed by PBS washing the cells were stained with µgml DAPI at room temperature for min Images were captured using a fluorescence microscope Nikon Corporation and measured using Adobe Photoshop software Adobe Systems Inc The EdU labeled cells were analyzed with MoFlo Astrios Beckman‘Coulter Inc Magnification x200Transwell assay and flow cytometry measurement of cell apoptosis Transwell assays were performed with a coating of Matrigel BD Biosciences mixed with culture medium mixed at ratio at ˚C for h A total of 1x105 cells in µl serum‘free medium were added to the upper layer of the Transwell chambers µm pore size Corning Inc and cultured for h The lower chamber contained the culture medium with FBS The migrated cells were fixed with 0cONCOLOGY REPORTS Table I Sequence of siRNAs and miRNA mimics and inhibitorsOligonucleotides si‘NC si‘RP11‘284F219 miR‘NC miR‘‘3p mimics miR‘‘3p inhibitor miR microRNA siRNA small interfering RNA NC negative controlSequence '†’'UUCUCCGAACGUGUCACGUTTUAUUGGCACCAAGGAUAGCUCGUUAAUCGGCUAUAAUACGCUCUUUUCUUUGAGACUCACUUCUUUUCUUUGAGACUCACU paraformaldehyde for min at room temperature stained with crystal violet for min at room temperature and images of six randomly selected fields in each well were captured under a light microscope Magnification x200Cellular apoptosis was detected using the Apoptosis Detection kit cat no KGF001 Nanjing KeyGen Biotech Co Ltd according to the manufacturer's instructions Cells were stained with fluorescein isothiocyanateconjugated annexin V and PI After incubated for min at ˚C in the dark µl 1X Binding Buffer was added to each tube and stained cells were analyzed using BD FACS Canto II flow cytometry FACS Calibur BD Biosciences Data were analyzed using FlowJo software version Tree Star IncLuciferase reporter assay The RP11284F219 wildtype wt or mutant mut '‘untranslated region '‘UTR and CCAR1 wt or mut '‘UTR sequences were cloned into the pmirGLO plasmid Youbio httpwwwyoubiocn cat no VT1439 The vectors µgml were co‘transfected with miR‘NC or miR6273p mimic nM and Renilla plasmids ngwell used as an internal control into cells seeded in a 48well plate 1x104well using Lipofectamine® reagent Invitrogen Thermo Fisher Scientific Inc Cell lysates were collected at h after transfection and the luciferase activities were detected with the DualLuciferase Reporter Assay system Promega Corporation according to the manufacturer's instructionsWestern blotting Cell were lysed using RIPA lysis buffer Sigma‘Aldrich Merck KGaA and protein concentrations were assessed with the BCA Protein Assay kit according to the manufacturer's instructions Beyotime Institute of Biotechnology Shanghai China Equal amounts µg of cell protein lysates were loaded and separated by SDS‘PAGE transferred to a PVDF membrane and blocked with non‘fat milk at room temperature for h The membranes were then incubated with CCAR1 primary antibody cat no ab70243 Abcam overnight at ˚C followed by incubation with goat anti‘mouse or goat anti‘rabbit IgG‘horseradish peroxidase conjugate secondary antibodies cat no ab205718 Abcam at room temperature for h GAPDH cat no ab181602 Abcam was used as loading control The signals were detected using the ECL system Protein Simple according to the manufacturer's instructionsIn vivo tumorigenicity analysis in mice Male BALBc nude mice age weeks weight ‘ g were obtained from Beijing Vital River Laboratory Animal Technology Co Ltd and housed at a room temperature of ˚C with a h lightdark cycle The mice were maintained in an individually ventilated cage system under specific pathogen‘free conditions temperature ˚C humidity and fed with sterile food and water free access To evaluate the effect of RP11‘284F219 knockdown on the growth of lung carcinoma in vivo 5x106 siNC or siRP11284F219 treated NCI‘H1299 cells in µl serum‘free medium were subcutaneously injected into each mouse n5 per group under anesthesia which was induced by isoflurane and maintained by isoflurane flow rate 1lmin The animals were monitored daily and the following criteria for humane endpoint was used Severe tumor burden mm in diameter difficulty breathing significant body‘weight loss and clinical signs such as prostration hypothermia and significant abdominal distension Tumors were measured on days and and the volumes were calculated using the formula a x b22 [the largest diameter a and the smallest diameter b] Then weeks after inoculation the mice were euthanized by CO2 inhalation CO2 flow rate of cage volume and the death of animals were confirmed by cessation of heartbeat The xenografts were imaged and weighedThe total RNA was then extracted from the xenografts as aforementioned Animal care and study were approved by the Institutional Animal Care and Use Committee of The First Affiliated Hospital of Xi'an Jiaotong University approval no Target prediction Potential target miRNAs of RP11284F219 were predicted using LncBase V2 httpcarolinaimisathena‘ innovationgrdiana_toolswebindexphprlncbasev2index The target genes of miR‘‘3p were predicted using three bioinformatics algorithms TargetScanV72 httpwwwtargetscanorgvert_72 and miRDB httpwwwmirdborgmininghtmlStatistics analysis Data were analyzed using the GraphPad Prism software GraphPad Software Inc and presented as the mean ± SD from ‰¥ independent experiments A two‘tailed unpaired Student's t‘test or one‘way ANOVA with Tukey's post‘hoc analysis were performed to evaluate the statistical significance P005 was considered to indicate a statistically significant difference\x0cLI RP11‘284F219 PROMOTES LUNG CARCINOMA PROLIFERATION AND INVASIONFigure RP11‘284F219 expression is upregulated in LC tissues and cell lines A Expression of RP11‘284F219 in LC tissues in comparison with adjacent healthy tissues was analyzed using RTqPCR P0001 vs adjacent tissues n13 B Expression of RP11‘284F219 in human lung carcinoma cell lines NCIH460 NCIH1299 and A549 compared with normal human lung epithelial cell line BEAS2B was analyzed using RTqPCR P005 P0001 vs BEAS‘2B n3 LC lung carcinoma RT‘qPCR reverse transcription‘quantitative PCRResultsExpression of RP11‘284F219 is upregulated in lung carci‘noma To investigate the potential role of RP11284F219 in lung carcinoma its expression was analyzed in tissue samples and matched adjacent healthy tissues from patients with lung carcinoma The results demonstrated that the expression of RP11‘284F219 was significantly upregulated in tumor tissues compared with healthy tissues Fig 1A The expression of RP11‘284F219 was also analyzed in human lung carcinoma cell lines NCIH460 NCIH1299 and A549 and normal human lung epithelial cell line BEAS2B Consistent with the findings in the tissue samples the expression of RP11‘284F219 was significantly increased in carcinoma cell lines compared with the normal epithelial cell line Fig 1B These results indicated that RP11284F219 may serve an oncogenic role in lung carcinomaKnockdown of RP11‘284F219 exerts anti‘oncogenic effects in lung carcinoma cells To study the specific role of RP11284F219 in lung carcinoma cells RP11284F219 siRNA was transfected into NCIH1299 and NCIH460 cells Fig 2A After transfection the proliferation of these cells was measured using CCK‘ and EdU assays Fig 2B‘D The results suggested that knocking down RP11‘284F219 significantly reduced the proliferation of lung carcinoma cells compared with the NC group Fig 2BD The invasiveness of si‘RP11‘284F219 transfected cells also significantly decreased as indicated by the data from the Transwell assay Fig 2F To further validate the invasive capability a RT‘qPCR assay was performed to detect the expression levels of invasion‘related genes and the results identified that both MMP2 and MMP9 were significantly decreased when RP11‘284F219 was downregulated Fig S1The results of flow cytometry measurement based apoptosis assay suggested that cells transfected with siRP11284F219 had a higher apoptotic rate compared with the siNC transfected group Fig 2E These data demonstrated the antitumor effects of RP11‘284F219 knockdown in lung carcinoma cells indicating an oncogenic role of RP11284F219RP11‘284F219 directly interacts with miR‘‘3p Based on the prediction of the online tool lncBase v2 from DIANA Prediction module httpcarolinaimisathena‘innovationgrdiana_toolswebindexphprlncbasev2index which was used to identify the downstream miRNAs of RP11284F219 the first five miRNAs in the output list were tested Among the predicted potential targets it was found that miR6273p had the most significant upregulation in NCIH1299 cells transfected with siRP11284F219 Fig S2Using sequence alignment it was identified that miR‘‘3p was partially complementary with the '‘UTR of RP11‘284F219 Fig 3A Subsequently 293T cells were transfected with the pmirGLORP11284F219wt or mut vector containing the wt or mut sequence of RP11284F219 '‘UTR with or without miR‘‘3p mimics Results from the luciferase reporter assay suggested that miR6273p mimics significantly decrease the signal of RP11‘284F219‘wt transfected cells but not the RP11‘284F219‘mut transfected cells indicating a direct interaction between the two non‘coding RNAs Fig 3A Furthermore transfection of siRP11284F219 into NCIH1299 and NCIH460 cells resulted in the suppression of endogenous RP11‘284F219 leading to a significant increase in miR‘‘3p expression Fig 3B Thus these findings suggested an inhibitory effect of RP11‘284F219 on the expression of miR‘‘3p in lung carcinoma cellsThe expression of miR‘‘3p was detected in both lung carcinoma tissues and cell lines It was demonstrated that miR‘‘3p was significantly downregulated in carcinoma tissues Fig 3C and NCIH460 NCIH1299 and A549 cells Fig 3D compared with healthy tissues and cells Collectively these data suggested a direct interaction between RP11284F219 and miR6273p in which RP11284F219 suppresses the expression of miR‘‘3pRP11‘284F219 regulates the proliferation and invasiveness of lung carcinoma cells via miR‘‘3p To rescue the antitumor effects of siRP11284F219 in lung carcinoma cells the miR‘‘3p inhibitor which specifically downregulates the expression of miR‘‘3p was transfected into NCI‘H1299 and NCI‘H460 cells Fig 4A The results from the CCK‘ and EdU assays demonstrated that treatment with si‘RP11‘284F219 0cONCOLOGY REPORTS Figure RP11‘284F219 knockdown inhibits lung carcinoma cell proliferation and invasion and promotes cell apoptosis A RP11‘284F219 knockdown was achieved via RP11‘284F219 siRNA and the knockdown efficiency was verified using reverse transcription‘quantitative PCR n3 Cell Counting Kit‘ assay was performed to measure the proliferation of B NCIH1299 and C NCIH460 cells after transfection with siRP11284F219 compared with the si‘NC group n5 D An EdU assay was performed to measure the proliferation of NCI‘H1299 and NCI‘H460 cells after transfection with si‘NC and si‘RP11‘284F219 Magnification x200 E Flow cytometry analysis was performed to determine the effects of RP11‘284F219 knockdown on apoptotic rates in NCI‘H1299 and NCI‘H460 cells n3 F Transwell assay was performed to determine the effects of RP11‘284F219 knockdown on NCI‘H1299 and NCI‘H460 cell invasion n3 Magnification x200 P005 P001 vs control group NC negative control siRNA small interfering RNA OD optical density and miR‘NC significantly decrease the proliferation of both NCIH1299 and NCIH460 cells Fig 4BD However the administration of miR‘‘3p inhibitor partially reversed the antiproliferative effect of siRP11284F219 indicating that RP11284F219 regulates the proliferation of lung carcinoma cells partially via miR6273p Fig 4BD In addition the 0cLI RP11‘284F219 PROMOTES LUNG CARCINOMA PROLIFERATION AND INVASIONFigure RP11‘284F219 directly interacts with miR‘‘3p A Binding site between RP11‘284F219 and miR‘‘3p that was identified using the DIANA tools and a luciferase reporter assay was conducted in pmirGLORP11284F219wt or mut treated cells in the presence of miR6273p mimics or miR‘NC n3 P005 vs miR‘NC B Expression of miR‘‘3p in NCI‘H1299 and NCI‘H460 cells transfected with si‘RP11‘284F219 was analyzed using RTqPCR P001 vs si‘NC n3 miR‘‘3p expression in C LC tissues and D NCI‘H460 NCI‘H1299 and A549 cells compared with adjacent healthy tissues and normal lung epithelial cells was analyzed using RT‘qPCR n3 P005 P001 vs adjacent tissue or BEAS‘2B cells NC negative control siRNA small interfering RNA wt wild‘type mut mutant miR microRNA LC lung carcinoma miR‘‘3p inhibitor restored the reduction in the number of NCIH1299 and NCIH460 cells that migrated through the Transwell membrane induced by si‘RP11‘284F219 treatment Fig 4F These data indicated the participation of miR6273p in the RP11284F219mediated invasive effectThe qPCR assay results identified that both MMP2 and MMP9 expression levels were restored in RP11284F219downregulated cells when miR6273p was inhibited compared with the miR‘NC group Fig S3 In addition transfection with miR‘‘3p inhibitor also diminished the pro‘apoptosis effect of si‘RP11‘284F219 in both NCIH1299 and NCIH460 cells Fig 4E Therefore it was suggested that RP11284F219 promoted the proliferation and invasion as well as suppressed the apoptosis of lung carcinoma cells by inhibiting the expression of miR‘‘3pRP11‘284F219 regulates CCAR1 via targeting miR‘‘3p To further evaluate how RP11‘284F219 exerts an oncogenic role via miR‘‘3p the publicly available algorithms of TargetScan httpwwwtargetscanorg and miRDB were used which identified CCAR1 as a potential target for miR6273p Fig 5A In order to validate this prediction miR6273p mimic was transfected into cells and the transfection efficiency was assessed The results demonstrated that transfection of miR6273p mimic increased the expression of miR‘‘3p by times compared with cells transfected with miRNC Fig S4After validating the upregulation of miR6273p mimic a CCAR1wt vector was constructed which contained the wt binding site between miR‘‘3p and the CCAR1 '‘UTR and CCAR1mut vector containing the mut sequence Fig 5A The results from luciferase reporter assays indicated that compared with the miRNC group the miR6273p mimic significantly decreased the luciferase activity of CCAR1‘wt treated cells but not the CCAR1‘mut treated cells suggesting a direct binding of miR‘‘3p to the '‘UTR of CCAR1 Fig 5B Increased expression levels of CCAR1 were present in the lung carcinoma tissues compared with the adjacent healthy tissues Fig 5C Moreover a significant decrease in both mRNA and protein expression levels of CCAR1 was detected upon transfecting NCIH1299 and NCIH460 cells with miR6273p mimics Fig 5D and E Thus CCAR1 may be a direct target of miR‘‘3p in lung carcinoma cells and tissuesRP11‘284F219 knockdown inhibits tumor growth and the expression of CCAR1 in vivo In order to investigate the effect of RP11284F219 on in vivo tumorigenicity NCIH1299 cells were transfected with siNC or siRP11284F219 and injected into the nude mice After weeks a significantly 0cONCOLOGY REPORTS Figure RP11‘284F219 regulates proliferation and invasiveness in lung carcinoma cells via miR‘‘3p A Expression of miR‘‘3p in NCI‘H1299 and NCI‘H460 cells transfected with miR‘NC or miR‘‘3p inhibitor was detected using RT‘qPCR analysis n3 P005 vs miR‘NC Cell Counting Kit‘ assay was performed in B NCI‘H1299 and C NCI‘H460 cells stably transfected with si‘RP11‘284F219 in the presence of miR‘NC or miR‘‘3p inhibitor n5 D EdU assay was performed in NCI‘H1299 and NCI‘H460 cells stably transfected with si‘RP11‘284F219 in the presence of miR‘NC or miR‘‘3p inhibitor Magnification x200 E Flow cytometry analysis was performed in NCI‘H1299 and NCI‘H460 cells stably transfected with si‘RP11‘284F219 in the presence of miR‘NC or miR‘‘3p inhibitor n3 F Transwell assay was performed in NCI‘H1299 and NCI‘H460 cells stably transfected with si‘RP11‘284F219 in the presence of miR‘NC or miR‘‘3p inhibitor Magnification x200 n3 P005 vs si‘NC NC negative control siRNA small interfering RNA OD optical density miR microRNA 0cLI RP11‘284F219 PROMOTES LUNG CARCINOMA PROLIFERATION AND INVASIONFigure miR‘‘3p directly targets CCAR1 A Bioinformatic analysis of the predicted binding sites between the CCAR1 '‘untranslated region and miR‘‘3p B Luciferase reporter assay in CCAR1‘wt or CCAR1‘mut treated cells in the presence of miR‘NC or miR‘‘3p mimic n3 P005 vs miR‘NC C CCAR1 expression in LC tissues compared with adjacent healthy tissues was analyzed using RT‘qPCR n13 P001 vs adjacent tissue Expression of CCAR1 in NCI‘H1299 and NCI‘H460 cells transfected with miR‘NC or miR‘‘3p mimics was detected using D RT‘qPCR and E western blotting n3 P005 vs miR‘NC miR microRNA NC negative control wt wild‘type mut mutant RT‘qPCR reverse transcription‘quantitative PCR CCAR1 cell division cycle and apoptosis regulator LC lung carcinoma slower proliferative rate of the tumors was observed in the siRP11284F219 group compared with the siNC group Fig 6A and B Furthermore the tumor volume and weight were significantly decreased in the si‘RP11‘284F219 group compared with the control group Fig 6A and B RTqPCR analysis also demonstrated that compared with the siNC group the tumors in the si‘RP11‘284F219 group expressed higher levels of miR6273p Fig 6C and lower levels of CCAR1 Fig 6D providing further evidence to the existence of the RP11‘284F219miR‘‘3pCCAR1 regulatory axis in lung carcinoma tumor tissuesDiscussionThe present study investigated the function of RP11284F219 in lung carcinoma It was initially found that RP11284F219 was significantly upregulated in both lung cancer tissues and cell lines Following the deduction of a potential oncogenic role of this lncRNA siRP11284F219 was transfected into NCIH460 and NCIH1299 cells and it was demonstrated that knockdown of RP11‘284F219 inhibited the proliferation and invasion while promoting apoptosis of lung carcinoma cells In the mechanistic studies using online prediction tools and in vitro assays the results indicated that miR‘‘3p directly interacts with RP11‘284F219 by binding to its '‘UTRThe function of miR627 was initially reported in colorectal cancer CRC Padi found that when upregulated by calcitriol miR627 targets the histone demethylase Jumonji domain containing 1A to increase methylation of histone H3K9 and suppresses the proliferative factors of CRC cells thus inhibiting the proliferation of CRC both in vitro and in vivo Moreover in CRC Sun discovered the role of miR‘ in vitamin D‘enhanced efficacy of irinotecan via inhibition of the cytochrome P450 enzyme‘mediated intratumoral drug metabolism miR‘ is also reported to be a potential non‘invasive diagnostic marker in gastric and breast cancer types In pulmonary diseases miR627 is downregulated in patients with chronic obstructive pulmonary disease and targets the high‘mobility group box protein to inhibit its expression thus improving transforming growth factorβ1‘induced pulmonary fibrosis The present results demonstrated the inhibitory effect of RP11‘284F219 on the expression of miR‘‘3p In addition it was identified that the miR‘‘3p inhibitor can neutralize the anti‘tumor effects of RP11‘284F219 knockdown indicating that RP11‘284F219 promotes the proliferation and invasiveness of lung carcinoma cells partially by regulating miR‘‘3p This anti‘tumor role of miR6273p under the regulation of RP11284F219 in lung carcinoma tissues and cells is in accordance with the previous aforementioned findings on human CRC gastric and breast cancer types\x0cONCOLOGY REPORTS Figure RP11‘284F219 knockdown inhibits tumor growth in vivo A Macroscopic image of xenografted tumors B Tumor volume in nude mice injected with NCI‘H1299 cells transfected with si‘NC or si‘RP11‘284F219 measured over weeks n5 C Weight of tumors in nude mice at weeks after injection of NCI‘H1299 cells transfected with si‘NC or si‘RP11‘284F219 n5 Expression levels of D miR‘‘3p and E CCAR1 in the tumor tissues from nude mice injected with NCI‘H1299 cells transfected with si‘NC or si‘RP11‘284F219 for weeks were detected using reverse transcription‘quantitative PCR n5 P005 P001 P0001 vs si‘NC miR microRNA NC negative control sh short hairpin RNA CCAR1 cell division cycle and apoptosis regulator Using the publicly available RNA interaction prediction algorithms the current study identified that CCAR1 which was initially shown as the target gene of miR6273p is also regulated by RP11‘284F219 Furthermore the regulatory axis of RP11‘284F219miR‘‘3pCCAR1 exists in the lung carcinoma cells both in vitro and in vivo in the tumor growth model The interaction between RP11‘284F219 and miR‘‘3p and the interaction between miR‘‘3p and CCAR1 were demonstrated by the dual‘luciferase assay Although this method has been used to validate RNA‘RNA interactions in previous studies other assays such as RNA pulldown and RNA binding protein immunoprecipitation that would provide more direct evidence for the RNARNA and RNA‘protein interactions should be performedCCAR1 was initially reported as a protein essential for cancer cell apoptosis induced by retinoids or chemotherapeutics such as Adriamycin and etoposide Subsequently Kim et al revealed that this protein functions as a transcriptional coactivator of nuclear receptors In human breast cancer cells as CCAR1 interacts and cooperates with the coactivators of estrogen receptor signaling it promotes the estrogendependent proliferation of cancer cells In CRC cells Ou reported that CCAR1 can be recruited by βcatenin to act as a coactivator for the transcriptional activation of lymphoid enhancer binding factor CCAR1 is essential for the expression of Wnt target genes as well as the neoplastic transformation of CRC cells In gastric cancer cells researchers have revealed the cooperation between CCAR1 and βcatenin which leads to the promotion of the proliferation and migration of cancer cells In lung cancer CCAR1 was reported to be an effector of Doxorubicin‘induced apoptosis Moreover Muthu demonstrated that certain chemical compounds that bind with CCAR1 can increase the expression of CCAR1 and induce apoptosis However a contradictory conclusion was reported in a recent study which observed that CCAR1 was promoted by serine and arginine rich splicing factor which is activated by glucose intake and further enhanced tumorigenesis by increasing the glucose consumption rate Corroborating this finding in the current study via the targeting of miR‘‘3p the expression of CCA Answer:

Thyroid_Cancer

Drug repurposing identifying novel indications for drugs bypasses common drug development pitfalls to ultimately deliver therapies to patients faster However most repurposingdiscoveries have been led by anecdotal observations eg Viagra or experimentalbasedrepurposing screens which are costly timeconsuming and imprecise Recently more systematic computational approaches have been proposed however these rely on utilizing theinformation from the diseases a drug is already approved to treat This inherently limits thealgorithms making them unusable for investigational molecules Here we present a computational approach to drug repurposing CATNIP that requires only biological and chemicalinformation of a molecule CATNIP is trained with diverse small molecules and uses different drug similarity features such as structural target or pathway based similarityThis model obtains significant predictive power AUC Using our model we createda repurposing network to identify broad scale repurposing opportunities between drugtypes By exploiting this network we identified literaturesupported repurposing candidatessuch as the use of systemic hormonal preparations for the treatment of respiratory illnessesFurthermore we demonstrated that we can use our approach to identify novel uses fordefined drug classes We found that adrenergic uptake inhibitors specifically amitriptylineand trimipramine could be potential therapies for Parkinson™s disease Additionally usingCATNIP we predicted the kinase inhibitor vandetanib as a possible treatment for Type Diabetes Overall this systematic approach to drug repurposing lays the groundwork tostreamline future drug development effortsa1111111111a1111111111a1111111111a1111111111a1111111111 ACCESSCitation Gilvary C Elkhader J Madhukar NHenchcliffe C Goncalves MD Elemento O Amachine learning and network framework todiscover new indications for small moleculesPLoS Comput Biol e1008098 101371journalpcbi1008098Editor Avner Schlessinger Icahn School ofMedicine at Mount Sinai UNITED STATESReceived September Accepted June Published August Copyright Gilvary This is an access distributed under the terms of theCreative Commons Attribution License whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are creditedData Availability Statement Data is available atthe following URL wwwgithubcomcoryandarCATNIPFunding JE is supported by NLM of the NationalInstitutes of Health under award numberF31LM013058 The content is solely theresponsibility of the authors and does notnecessarily represent the official views of theNational Institutes of Health OE and his laboratoryare supported by NIH grants 1R01CA1945471U24CA210989 P50CA211024 UL1TR002384PLOS Computational Biology 101371journalpcbi1008098 August PLOS COMPUTATIONAL BIOLOGY 0cMachine learning approach to drug repurposingThe funders had no role in study design datacollection and analysis decision to publish orpreparation of the manuscriptCompeting interests OE is cofounder and equityholder in OneThree Biotech and Volastra twocompanies that use data science and machinelearning to develop novel therapies In addition OEis an advisor and equity holder in Freenome andOwkin NM is cofounder and equity holder inOneThree Biotech a company that use datascience and machine learning to develop noveltherapies CG is cofounder and equity holder inOneThree Biotech a company that use datascience and machine learning to develop noveltherapies This does not alter our adherence to allPLOS Computational Biology policies on sharingdata and materialsAuthor summaryCurrently clinical approval of a drug is an arduous process that results in an overwhelming number of compounds failing due to safety or efficacy concerns which leaves patientswithout novel lifesaving treatments The idea of drug repurposing is to take approveddrugs or compounds that were shelved due to reasons other than safety and identify newdiseases for them to treat This would allow drugs if they are sufficiently effective toquickly go through the FDA approval process and be available to patients quicker whichalso cuts the ever growing cost of novel compound research and development Here weintroduce CATNIP a computational model that can predict novel indications for specificdrugs or entire drug classes This approach analyzes drug similarity across a wide range ofbiological chemical and clinical features giving a complete picture of each drug™s mechanism and possible indications Interestingly CATNIP can be used for drugs that not onlyare previously approved but also shelved compounds which are often overlooked in previous repurposing analyses Most importantly CATNIP successfully identified noveltreatments for both Parkinson™s disease and Type Diabetes which are currently undergoing preclinical validationIntroductionWith over million spent bringing a single drug to market over the course of yearsdrug development has remained a costly and timeconsuming affair[] In response there hasbeen an increase in interest in drug repurposing the identification of novel indications forknown safe drugs Successes in this area have been seen in the past most notably in sildenafileg Viagra which was originally intended to treat hypertension and angina pectoris but waslater repurposed to treat erectile dysfunction Other examples of compounds repurposed fornew therapeutic applications include minoxidil[] and raloxifene[] which are now used totreat androgenic alopecia and osteoporosis respectively However most of these repurposingopportunities were discovered through inefficient approaches including anecdotal observations or hypothesisdriven investigations and a more efficient approach could lead to manymore repurposing opportunitiesComputational approaches for repurposing drugs are appealing in that they can be systematically and quickly applied to many drugs at a low cost compared to their experimental counterparts One computational approach that has proven to be invaluable in other areas of thedrug development pipeline is machine learning Machine learning is the use of computationalalgorithms to learn from available data to make novel predictions and gain new insight Usingthis technique one can create unbiased algorithms to match seemingly disparate drugs bycomparing their common features[] such as clinical indication toxicity profile[] or therapeutic target[ ] Previously our lab used a ˜similarity™ approach leveraging the principlethat similar drugs tend to have similar characteristics to predict a drug™s target by investigatingthe known targets of other drugs that were predicted to be œsimilar to the investigated drugbased on shared features[] We found that DRD2 a dopamine receptor was the predicted target for the compound ONC201 After identifying and experimentally validating this targetclinical trials were shifted to focus on gliomas which are now successfully completing phasetwo trials at the time of this publication[] The approach of leveraging drug similarity couldimmensely aid drug repurposing efforts with the appropriate dataPLOS Computational Biology 101371journalpcbi1008098 August PLOS COMPUTATIONAL BIOLOGY 0cMachine learning approach to drug repurposingOthers have successfully used this ˜similarity™ approach to repurpose drugs and demonstrated high predictive power when tested against FDA approved drugdiseases[] However these methods have primarily linked drugs together using a diseasecentric approachinstead of using features related to the drug itself ie drugcentric These repurposingopportunities are identified by predicting diseases similar to the diseases a drug is alreadyknown to treat Disease similarities can be based on semantic pathophysiological or clinicalsimilarities related to the drug™s clinical indication For example PREDICT a repurposingmethod developed by Gottlieb et al[] exploits the semantic similarity of disease terms asa form of diseasedisease similarity Such approaches while reliable limit the scope of therepositioning effort in several ways First the vast majority of small molecules never reachclinical approval and would be overlooked in this type of analysis Second the use of a diseasecentric approach biases repurposing predictions toward exclusively similar clinical diseases ie cancer drugs to other cancer types [] We postulated that using solely druginformation such as chemical and biological features would be a more effective andbroader approach to drug repurposingHere we propose a novel approach to drug repurposing which operates by a platform wecall Creating A Translational Network for Indication Prediction CATNIP CATNIP is amachinelearning algorithm that learns to predict whether two molecules share an indicationbased solely on the drug™s chemical and biological features using unique drugs The systematic application of CATNIP to molecule pairs creates a network with million nodesthat can then be used to identify potential drug repurposing opportunities By identifying feature importance through the use of chemical structure and target information to make broadscale predictions CATNIP is able to effectively bridge between different therapeutic indications to advance methods of drug repurposing In this report we have identified various candidate drug classes that are predicted to have therapeutic activity outside of their intendedindication in diseases such as Parkinson™s disease and Type DiabetesResultsVariance in drug indication nomenclature can be standardizedWe collected a wide variety of drugs N including both approved and investigationalmolecules with a diverse set of indications to ensure that our drug network covered a largeportion of the known chemical space A subset of these drugs FDA approved drugs and indications taken from DrugBank [] were used as a goldstandard of drugindicationassociations in the training set for the model Disease names are often not standardized whichcan lead to many diverse names for the same disease This problem leads to many drug pairsappearing to not have shared indications when they are associated with two different namesfor the same disease To address inconsistencies in nomenclature for drug indications such asœprostate carcinoma and œcarcinoma of the prostate the MetaMap tool [] was applied tomap disease names to UMLS concepts Methods This standardization of medical terminologies allowed us to reconcile various variations in the database allowing us to confirm thatdrugs did in fact treat the same disease Examples of these variations and their mappingsmay be seen in Table Using MetaMap we clustered the DrugBank indications into standardized indications A multitude of indication types were included in this standardization including but not limited to oncological mental health and neurological diseasesS1A Fig Our rigorous standardization of drug indications ensured an accurate training setallowing for the discovery and modeling of drugindication relationshipsPLOS Computational Biology 101371journalpcbi1008098 August PLOS COMPUTATIONAL BIOLOGY 0cTable Indication nomenclatures and their mappingsMetamap MappedIndicationIndicationDrugBankIndication IDDrugBankNumber of unique drugsassociated with IndicationUnique drugs associated with Indication IDMachine learning approach to drug repurposingProstateCarcinomaAcne VulgarisAdvanced ProstateCarcinomaAdvanced carcinomaof the prostateSevere AcneAcneDBCOND0070333DBCOND0020265DBCOND0077433DBCOND0019842DementiaVascularIdiopathicPulmonaryFibrosisPaget DiseaseModerate AcnevulgarisMild VascularDementiaDementia VascularDementiasIdiopathic PulmonaryFibrosis IPFMild IdiopathicPulmonary FibrosisPaget™s DiseasePaget™s Disease ofBoneDBCOND0022329DBCOND0022662DBCOND0029264DBCOND0060453DBCOND0031843DBCOND0093824DBCOND0038793DBCOND0030189101371journalpcbi1008098t001IDCyproterone acetate Esterified estrogensGoserelinCyproterone acetate Doxycycline TetracyclineAloe Vera Leaf Benzoyl peroxide Chloramphenicol ClioquinolGlycolic acid Linoleic acid Octasulfur Salicylic acid SilverSpironolactoneEthinylestradiol Minocycline Nestimate TazaroteneMemantineDonepezilGalantamine Trazodone TrifluoperazineNintedanib PrednisolonePirfenidoneAlendronic acid Pamidronic acid Risedronic acid ZoledronicacidEtidronic acidDrug pairs sharing indications have other similar characteristicsWe hypothesized that pairs of drugs that shared at least one indication would have other similar drug characteristics S1 Table To test this hypothesis we integrated the similarity of twodrugs across chemical and biological drug properties and created a computational model topredict if two drugs will share an indication Fig All of the drug similarity features S1Table collected could significantly distinguish between drug pairs known to share an indication and those not known to share an indication S2“S5 Figs For example we found thatdrug pairs with a shared clinical indication according to their listed DrugBank indicationstended to have significant overlap in targets Dstatistic pvalue S2A FigThe feature which best discriminated between drug pairs that shared a clinical indication versus drug pairs that do not was the similarity between the KEGG pathways that each drug™s targets are involved in Dstatistic p S4C Fig Pathway similarity was calculatedas the Jaccard Index between the KEGG pathways that contain each drug™s gene targets Methods The difference in effect size between the target similarity and the pathway similarity Dstatistic vs respectively indicates that the drugs do not necessarily have to targetthe same exact genes but rather the same biological pathway in order to share a clinical indication Additionally we found that drug pairs that share an indication had a more similarchemical structure than drug pairs that did not share an indication Dstatistic pvalue S5A Fig A biological network containing both physical and nonphysicalinteractions was curated containing proteincoding genes drugs and TFsThis curated network provided another feature for our model allowing us to utilize previouslyestablished interactions between proteins to aid with distinguishing drug pairs that share anindication Overall these features seem to indicate sufficient power in differentiating drugsPLOS Computational Biology 101371journalpcbi1008098 August PLOS COMPUTATIONAL BIOLOGY 0cMachine learning approach to drug repurposingFig Schematic of CATNIP repurposing approach A The use of drug similarity properties to predict if two drugs will share an indication using agradient boosting model the model is referred to as CATNIP B Schematic showing the use of CATNIP output scores to create a network with the scoresused as edge weights The colors of each drug represent the known disease and this demonstrates how one could identify novel indications for drugsthrough the network101371journalpcbi1008098g001PLOS Computational Biology 101371journalpcbi1008098 August PLOS COMPUTATIONAL BIOLOGY 0cMachine learning approach to drug repurposingFig CATNIP model accurately predicts drugs that share an indication and can be used for repurposing A Receiveroperating characteristic curve for CATNIP the performance for drug pairs with high and low structural similarity is alsoshown B A network of all drug pairs with a CATNIP score higher than Nodes drugs are colored based on ATCclassification and a specific example of repurposing between ATC classifications is highlighted C A graph of all ATCclassification and the median CATNIP score between the drugs belonging to each of them only including drug pairswith CATNIP score The edges between ATC Classifications with the highest median CATNIP scores are colored red101371journalpcbi1008098g002that share and do not share indications which we hypothesized can then be leveraged to createa predictive modelDrug pairs that share indications can be predicted by modelUsing these diverse drug properties as features we trained a Gradient Boosting model to predict if two drugs share a clinical indication A Gradient Boosting model showed superiorresults when compared with other algorithms Methods S2 Table The model output is adrug similarity score hereby referred to as a œCATNIP score which allows us to classifydrug pairs that share clinical indications We performed a 5fold crossvalidation analysis andachieved significant predictive performance with an areaunderthereceiveroperator curveAUC of Fig 2A We confirmed the statistical significance of our model with aPLOS Computational Biology 101371journalpcbi1008098 August PLOS COMPUTATIONAL BIOLOGY 0cMachine learning approach to drug repurposingprecisionrecall curve PRC because of the class imbalance in our dataset between drug pairsthat share indications against those that do not Shared Not Shared Whencompared to random predictions our model showed significant improvement vs areaunder PRC S6 Fig We retained a low percent of false positive predictions at various cutoffs false positives and false positives at a model prediction probability oftwo drugs sharing an indication of and respectively providing extra confidencethat our predictions can lead to strong repurposing candidatesWe found that the predictive model greatly benefited from the addition of diverse datatypes While structure similarity showed the highest feature importance of any single featureS11 Fig when used as a single feature within a gradient boosting model it only achieved anAUC of S12 Fig Interestingly when only supplying the model with ontology featuresa Jaccard index for the GO terms of the known targets of each drug within a drug pairachieved an AUC of However even at the highest AUC of any single feature typeit is significantly below the performance when combining all feature typesIn certain cases a high predictive performance is expected such as when two drugs arestructurally similar or share targets It has been shown before that structurally similar drugshave a high probability of treating the same indication[] However there continue to bedrug pairs known to treat the same indication that are not structurally similar For exampletamoxifen[] and anastrozole[] are structurally dissimilar compounds Dice similarity that treat the same indication Metathesaurus term Cancer Breast We recalculated our performance metrics to evaluate how our model performed in classifying drug pairsthat shared indications when only exposed to drug pairs with low structure similarityDice High performance was retained under with an AUC Fig 2A Additionally we found that our model performed similarly well when only exposed to drug pairs thatdid not have any known shared targets AUC Fig 2A These performance metricsconfirm that our model is robust enough to predict if a drug pair will share an indication evenfor more difficult prediction tasksNetwork clusters identify drugs with similar clinical characteristicsWe constructed a repurposing network by calculating a CATNIP score for all possible drugpairs found within DrugBank and assigning the drugs as nodes and the CATNIP score as theedge weight We pruned the network using a cutoff value of for the CATNIP scoresFig 2B which included different drug pairs This cutoff is equivalent to a predictedprobability of to share an indication and allowed for a balance between confidencewithin our predictions and drug diversity and availabilityWe hypothesized that drugs sharing at least one indication would cluster together in ournetwork To confirm this theory we classified each drug per its 1st order Anatomical Therapeutic Chemical ATC classification This identification is a method of distinguishing theclinical use of a drug that is widely used in European and North American chemoinformaticsdatabases[] Using ATC we observed clearly defined clusters within the repurposing network Fig 2B Many clusters featured multiple ATC classifications suggesting potential repurposing opportunities For example one cluster included the thiazolidinediones rosiglitazoneand pioglitazone ATC classification ˜Alimentary Tract and Metabolism™ and the fibratesfenofibrate and bezafibrate ATC classification ˜Cardiovascular system™ These two clusteredATC classifications were connected by a high CATNIP score between bezafibrate andpioglitazone an antidiabetic drug a relationship driven by the shared targeting of PPARa andPPARg resulting in the improvement of lipid and glucose metabolism Bezafibrate has shownefficacy in the treatment of Type Diabetes in numerous retrospective and preclinical studiesPLOS Computational Biology 101371journalpcbi1008098 August PLOS COMPUTATIONAL BIOLOGY 0cMachine learning approach to drug repurposingATC Code ReferenceRespiratory SystemRespiratory SystemRespiratory System[“][“][“][ ]Table Literature Support for ATC Repurposing PredictionsATC Code DermatologicalsAlimentary Tract and MetabolismSensory ansSystemic Hormonal Preparations Excluding Sex Hormones AndRespiratory SystemInsulinsSensory ans101371journalpcbi1008098t002Alimentary Tract and[“]Metabolismincluding Phase trials[“] however is still not an approved antidiabetic The identification of bezafibrate as a potential diabetes treatment is a key example of how CATNIP can beused to identify repurposing opportunitiesWe reasoned that the connections between ATC classifications across all the drug clusterscould provide additional aid for drug repurposing purposes Using the pruned network CATNIP Score we collected all the scores between drugs of differing ATC classificationsFrom this collection we were able to determine the median score associated between each pairof ATC classifications The ATC classifications with the highest median CATNIP scores hadliterature support for numerous repurposing efforts between them Table For exampledrugs with the ATC classifications of œRespiratory System and œSystemic Hormonal Preparations excluding sex hormones and insulins were strongly connected to each other median CATNIP score This connection was driven by highly scored pairs of drugs includingrimexolone to mometasone CATNIP score and prednisone to triamcinolone CATNIP score These connections are supported by the fact that hormonal agents like glucocorticoids and beta adrenergic agonists have been used for decades to relax the airway musculature in patients with reactive airways disease and chronic obstructive pulmonary disease[]Interestingly our analysis identified glucagon a peptide hormone that increases blood glucoselevels as a candidate for œRespiratory System repurposing and this use already has clinicalsupport[][] Additionally drugs classified as œRespiratory System and œDermatologicalwere also observed to be highly associated because of interactions such as the one betweenciclesonide and hydrocortisone CATNIP score Ciclesonide and hydrocortisone do infact share a clinical indication œAsthma Bronchial giving added confidence to our findingsThese types of network observations are important in laying the groundwork for suggestingnovel clinical repurposing strategies for FDAapproved drugsCATNIP identifies novel disease areas for drug classesWe investigated the ability to leverage CATNIP scores to identify repurposing opportunitiesby evaluating specific drug classes Drug classes are predefined in DrugBank In order to identify actionable repurposing possibilities we narrowed this list down to classes containinginhibitors antagonists or agonists of specific gene or protein families We focused our attention on specific disease areas that are attractive for drug repurposing opportunities due to alack of current treatments or high rates of acquired resistance The specific disease areas wereœmental disorders œneurological diseases œdiabetes and œcancer cancer was furtherdivided into specific cancer types due to the large variance in disease pathology between typesMethodsPLOS Computational Biology 101371journalpcbi1008098 August PLOS COMPUTATIONAL BIOLOGY 0cMachine learning approach to drug repurposingWe hypothesized that CATNIP scores could be used to identify specific drug classes thatwould be efficacious for a new disease area For each drug class and disease area we foundthe statistical difference in the CATNIP score distribution between two sets of drug pairsThe first set included pairs that had one drug within the drug class and the other drugapproved for the disease in question while the other set included drug pairs that had onedrug within the drug class and the other drug not approved for the disease in questionMethods We compared the effect size estimated by the Wilcoxon location shift for alldrug classdisease pairs that had a significant difference in distribution compared to drugclassnondisease pairs FDR Supplementary Data By using CATNIP scores wefound that many wellknown drug classdiseases associations could be recovered For example œmuscarinic antagonists were highly ranked for œneurological diseases and many suchagents are FDAapproved for this indication[] In addition we found that œkinase inhibitors were closely associated with the treatment of cancer and œdopamine antagonists forthe treatment of œmental disorders[ ] Wilcoxon Location Shift “ forœkinase inhibitors and select cancer types Location Shift for œdopamine antagonists and œmental disorders pvalue S7 Fig In fact almost all drug classdiseaseassociations contained at least one FDAapproved drug for the respective disease giving usadded confidence in our model Of note each drug was allowed to be categorized intonumerous drug classes leading to unexpected yet easily explained results for exampleœdopamine antagonists appearing as a top drug class for œneurological diseases This isdue to risperidone a drug traditionally used for schizophrenia and mood disorders alsohaving a secondary indication of Alzheimer™s type severe dementiaOur method reached significant levels of predictive power for predicting both drug class”disease associations and individual drugdisease association When predicting drug classdisease associations under our most lenient conditions calling cases where at least one drugwithin the class was known to treat the disease a true positives our method achieved a sensitivity of greater than However this improved to a sensitivity of when we implementedstricter cutoffs ie only calling drug classdisease associations true positives if of drugswithin the class treated that disease S10 Fig We additionally compared our method™s abilityto predict individual predictions to that of a previously highlighted method Gottlieb et al™sPREDICT[] We found our method had a slightly higher AUPRC vs andhigher sensitivity vs S4 Table S1 Methods While these results indicate modest improvements over PREDICT it is important to note that unlike in PREDICT diseaseinformation is not a required feature in CATNIP™s machine learning approach This meansthat CATNIP can be applied towards investigational molecules with no previously knownindications Additionally by not using disease information as a feature repositioning of drugswith known indications using CATNIP is not directly biased by the associated disease indication and instead uses mechanistic features chemical structure and properties targets etc aspart of the repositioning strategyNext we further interrogated the drug classes associated with œneurological diseases andœdiabetes specifically CATNIP scores could correctly identify drug classes known to treatthese diseases Table To identify possible repurposing candidates we focused our attentionon drug classes shown to have a large positive effect size with this CATNIP analysis but are notcurrently approved for treatment For œneurological diseases the use of adrenergic uptakeinhibitors traditionally used as antidepressants was the top repurposing candidate for œdiabetes alpha antagonists and kinase inhibitors were identified as possible novel treatmentsTable We believe further investigation into these drug classes and diseases could lead tosuccessful clinical applicationsPLOS Computational Biology 101371journalpcbi1008098 August PLOS COMPUTATIONAL BIOLOGY 0cMachine learning approach to drug repurposingTable Top Predictions of Drug Class Repurposing OpportunitiesClassDiabetesDiseasePrediction RankAlpha1 AntagonistsKinase InhibitorProtein Kinase InhibitorsProtein Synthesis InhibitorsCytochrome P450 CYP2E1 InhibitorsMonoamine Oxidase InhibitorsNeurologicalAdrenergic Uptake InhibitorsAdrenergic alpha AgonistsProtease Inhibitors101371journalpcbi1008098t003CATNIP interpretability reveals reasoning for repurposing candidatesFrom our list of repurposing candidates we chose two novel drug classdisease associations tofurther investigateAdrenergic uptake inhibitors applied to Parkinson™s disease First we evaluated therelationship between œneurological diseases and œadrenergic uptake inhibitors We focusedon the drug pairs with the highest CATNIP scores ie those predicted with the highest confidence to share at least one indication Fig 3A Of all the adrenergic uptake inhibitors wefound that amitriptyline and trimipramine two antidepressants had the highest CATNIPscores with the œneurological diseases drugs The drugs that shared the strongest connectionswith amitriptyline and trimipramine were drugs approved for Parkinson™s disease PD Specifically metixene atropine pergolide and benzatropine were associated with amitriptylineaccording to CATNIP and trimipramine was associated to benzatropine and rotigotine Trimipramine was also strongly connected with orphenadrine which is sometimes used off labelin PD but will not be included in the following analysesUsing the CATNIP model we evaluated which features contributed towards the predictionof amitriptyline and trimipramine to share an indication with PD drugs We found that targetgene ontology and pathway similarity all strongly contributed to the predictions for both amitriptyline and trimipramine Fig 3B S8 Fig Since target similarity and distance between targets in a proteinprotein interaction network were among the top contributing features weinvestigated which gene targets were shared amongst these drug pairs We found that amitriptyline targets three specific gene classes that are also targeted by at least one of the PD drugsmuscarinic acetylcholine receptors Gcoupled protein receptors GPCRs and alpha adrenergic receptor Trimipramine also targets muscarinic acetylcholine receptors alphaadrenergicreceptors and dopamine transporters which is similar to benzatropine a PD drug All thesereceptors have welldefined relationships with PD and other neurological diseases[ ]which adds support for repurposing amitriptyline andor trimipramineAmitriptyline may be an ideal candidate for use in PD patients We evaluated the sharedmolecular function gene ontology terms shared between amitriptyline and all four PD drugsGPCR activity was once again identified S1“S4 Files We then interrogated the biologicalpathways these drug targets are involved in and found many broad GPCR pathways overlapping between amitriptyline and the PD drugs S9 Fig including the Reactome pathway œGASTRIN_CREB_SIGNALLING PATHWAY VIA PKC AND MAPK Several recent studiessupport the link between gastrinreleasing peptide signaling to brain function[] ThroughCATNIP we have identified œadrenergic uptake inhibitor

cancer257

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: Drug repurposing identifying novel indications for drugs bypasses common drug development pitfalls to ultimately deliver therapies to patients faster However most repurposingdiscoveries have been led by anecdotal observations eg Viagra or experimentalbasedrepurposing screens which are costly timeconsuming and imprecise Recently more systematic computational approaches have been proposed however these rely on utilizing theinformation from the diseases a drug is already approved to treat This inherently limits thealgorithms making them unusable for investigational molecules Here we present a computational approach to drug repurposing CATNIP that requires only biological and chemicalinformation of a molecule CATNIP is trained with diverse small molecules and uses different drug similarity features such as structural target or pathway based similarityThis model obtains significant predictive power AUC Using our model we createda repurposing network to identify broad scale repurposing opportunities between drugtypes By exploiting this network we identified literaturesupported repurposing candidatessuch as the use of systemic hormonal preparations for the treatment of respiratory illnessesFurthermore we demonstrated that we can use our approach to identify novel uses fordefined drug classes We found that adrenergic uptake inhibitors specifically amitriptylineand trimipramine could be potential therapies for Parkinson™s disease Additionally usingCATNIP we predicted the kinase inhibitor vandetanib as a possible treatment for Type Diabetes Overall this systematic approach to drug repurposing lays the groundwork tostreamline future drug development effortsa1111111111a1111111111a1111111111a1111111111a1111111111 ACCESSCitation Gilvary C Elkhader J Madhukar NHenchcliffe C Goncalves MD Elemento O Amachine learning and network framework todiscover new indications for small moleculesPLoS Comput Biol e1008098 101371journalpcbi1008098Editor Avner Schlessinger Icahn School ofMedicine at Mount Sinai UNITED STATESReceived September Accepted June Published August Copyright Gilvary This is an access distributed under the terms of theCreative Commons Attribution License whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are creditedData Availability Statement Data is available atthe following URL wwwgithubcomcoryandarCATNIPFunding JE is supported by NLM of the NationalInstitutes of Health under award numberF31LM013058 The content is solely theresponsibility of the authors and does notnecessarily represent the official views of theNational Institutes of Health OE and his laboratoryare supported by NIH grants 1R01CA1945471U24CA210989 P50CA211024 UL1TR002384PLOS Computational Biology 101371journalpcbi1008098 August PLOS COMPUTATIONAL BIOLOGY 0cMachine learning approach to drug repurposingThe funders had no role in study design datacollection and analysis decision to publish orpreparation of the manuscriptCompeting interests OE is cofounder and equityholder in OneThree Biotech and Volastra twocompanies that use data science and machinelearning to develop novel therapies In addition OEis an advisor and equity holder in Freenome andOwkin NM is cofounder and equity holder inOneThree Biotech a company that use datascience and machine learning to develop noveltherapies CG is cofounder and equity holder inOneThree Biotech a company that use datascience and machine learning to develop noveltherapies This does not alter our adherence to allPLOS Computational Biology policies on sharingdata and materialsAuthor summaryCurrently clinical approval of a drug is an arduous process that results in an overwhelming number of compounds failing due to safety or efficacy concerns which leaves patientswithout novel lifesaving treatments The idea of drug repurposing is to take approveddrugs or compounds that were shelved due to reasons other than safety and identify newdiseases for them to treat This would allow drugs if they are sufficiently effective toquickly go through the FDA approval process and be available to patients quicker whichalso cuts the ever growing cost of novel compound research and development Here weintroduce CATNIP a computational model that can predict novel indications for specificdrugs or entire drug classes This approach analyzes drug similarity across a wide range ofbiological chemical and clinical features giving a complete picture of each drug™s mechanism and possible indications Interestingly CATNIP can be used for drugs that not onlyare previously approved but also shelved compounds which are often overlooked in previous repurposing analyses Most importantly CATNIP successfully identified noveltreatments for both Parkinson™s disease and Type Diabetes which are currently undergoing preclinical validationIntroductionWith over million spent bringing a single drug to market over the course of yearsdrug development has remained a costly and timeconsuming affair[] In response there hasbeen an increase in interest in drug repurposing the identification of novel indications forknown safe drugs Successes in this area have been seen in the past most notably in sildenafileg Viagra which was originally intended to treat hypertension and angina pectoris but waslater repurposed to treat erectile dysfunction Other examples of compounds repurposed fornew therapeutic applications include minoxidil[] and raloxifene[] which are now used totreat androgenic alopecia and osteoporosis respectively However most of these repurposingopportunities were discovered through inefficient approaches including anecdotal observations or hypothesisdriven investigations and a more efficient approach could lead to manymore repurposing opportunitiesComputational approaches for repurposing drugs are appealing in that they can be systematically and quickly applied to many drugs at a low cost compared to their experimental counterparts One computational approach that has proven to be invaluable in other areas of thedrug development pipeline is machine learning Machine learning is the use of computationalalgorithms to learn from available data to make novel predictions and gain new insight Usingthis technique one can create unbiased algorithms to match seemingly disparate drugs bycomparing their common features[] such as clinical indication toxicity profile[] or therapeutic target[ ] Previously our lab used a ˜similarity™ approach leveraging the principlethat similar drugs tend to have similar characteristics to predict a drug™s target by investigatingthe known targets of other drugs that were predicted to be œsimilar to the investigated drugbased on shared features[] We found that DRD2 a dopamine receptor was the predicted target for the compound ONC201 After identifying and experimentally validating this targetclinical trials were shifted to focus on gliomas which are now successfully completing phasetwo trials at the time of this publication[] The approach of leveraging drug similarity couldimmensely aid drug repurposing efforts with the appropriate dataPLOS Computational Biology 101371journalpcbi1008098 August PLOS COMPUTATIONAL BIOLOGY 0cMachine learning approach to drug repurposingOthers have successfully used this ˜similarity™ approach to repurpose drugs and demonstrated high predictive power when tested against FDA approved drugdiseases[] However these methods have primarily linked drugs together using a diseasecentric approachinstead of using features related to the drug itself ie drugcentric These repurposingopportunities are identified by predicting diseases similar to the diseases a drug is alreadyknown to treat Disease similarities can be based on semantic pathophysiological or clinicalsimilarities related to the drug™s clinical indication For example PREDICT a repurposingmethod developed by Gottlieb et al[] exploits the semantic similarity of disease terms asa form of diseasedisease similarity Such approaches while reliable limit the scope of therepositioning effort in several ways First the vast majority of small molecules never reachclinical approval and would be overlooked in this type of analysis Second the use of a diseasecentric approach biases repurposing predictions toward exclusively similar clinical diseases ie cancer drugs to other cancer types [] We postulated that using solely druginformation such as chemical and biological features would be a more effective andbroader approach to drug repurposingHere we propose a novel approach to drug repurposing which operates by a platform wecall Creating A Translational Network for Indication Prediction CATNIP CATNIP is amachinelearning algorithm that learns to predict whether two molecules share an indicationbased solely on the drug™s chemical and biological features using unique drugs The systematic application of CATNIP to molecule pairs creates a network with million nodesthat can then be used to identify potential drug repurposing opportunities By identifying feature importance through the use of chemical structure and target information to make broadscale predictions CATNIP is able to effectively bridge between different therapeutic indications to advance methods of drug repurposing In this report we have identified various candidate drug classes that are predicted to have therapeutic activity outside of their intendedindication in diseases such as Parkinson™s disease and Type DiabetesResultsVariance in drug indication nomenclature can be standardizedWe collected a wide variety of drugs N including both approved and investigationalmolecules with a diverse set of indications to ensure that our drug network covered a largeportion of the known chemical space A subset of these drugs FDA approved drugs and indications taken from DrugBank [] were used as a goldstandard of drugindicationassociations in the training set for the model Disease names are often not standardized whichcan lead to many diverse names for the same disease This problem leads to many drug pairsappearing to not have shared indications when they are associated with two different namesfor the same disease To address inconsistencies in nomenclature for drug indications such asœprostate carcinoma and œcarcinoma of the prostate the MetaMap tool [] was applied tomap disease names to UMLS concepts Methods This standardization of medical terminologies allowed us to reconcile various variations in the database allowing us to confirm thatdrugs did in fact treat the same disease Examples of these variations and their mappingsmay be seen in Table Using MetaMap we clustered the DrugBank indications into standardized indications A multitude of indication types were included in this standardization including but not limited to oncological mental health and neurological diseasesS1A Fig Our rigorous standardization of drug indications ensured an accurate training setallowing for the discovery and modeling of drugindication relationshipsPLOS Computational Biology 101371journalpcbi1008098 August PLOS COMPUTATIONAL BIOLOGY 0cTable Indication nomenclatures and their mappingsMetamap MappedIndicationIndicationDrugBankIndication IDDrugBankNumber of unique drugsassociated with IndicationUnique drugs associated with Indication IDMachine learning approach to drug repurposingProstateCarcinomaAcne VulgarisAdvanced ProstateCarcinomaAdvanced carcinomaof the prostateSevere AcneAcneDBCOND0070333DBCOND0020265DBCOND0077433DBCOND0019842DementiaVascularIdiopathicPulmonaryFibrosisPaget DiseaseModerate AcnevulgarisMild VascularDementiaDementia VascularDementiasIdiopathic PulmonaryFibrosis IPFMild IdiopathicPulmonary FibrosisPaget™s DiseasePaget™s Disease ofBoneDBCOND0022329DBCOND0022662DBCOND0029264DBCOND0060453DBCOND0031843DBCOND0093824DBCOND0038793DBCOND0030189101371journalpcbi1008098t001IDCyproterone acetate Esterified estrogensGoserelinCyproterone acetate Doxycycline TetracyclineAloe Vera Leaf Benzoyl peroxide Chloramphenicol ClioquinolGlycolic acid Linoleic acid Octasulfur Salicylic acid SilverSpironolactoneEthinylestradiol Minocycline Nestimate TazaroteneMemantineDonepezilGalantamine Trazodone TrifluoperazineNintedanib PrednisolonePirfenidoneAlendronic acid Pamidronic acid Risedronic acid ZoledronicacidEtidronic acidDrug pairs sharing indications have other similar characteristicsWe hypothesized that pairs of drugs that shared at least one indication would have other similar drug characteristics S1 Table To test this hypothesis we integrated the similarity of twodrugs across chemical and biological drug properties and created a computational model topredict if two drugs will share an indication Fig All of the drug similarity features S1Table collected could significantly distinguish between drug pairs known to share an indication and those not known to share an indication S2“S5 Figs For example we found thatdrug pairs with a shared clinical indication according to their listed DrugBank indicationstended to have significant overlap in targets Dstatistic pvalue S2A FigThe feature which best discriminated between drug pairs that shared a clinical indication versus drug pairs that do not was the similarity between the KEGG pathways that each drug™s targets are involved in Dstatistic p S4C Fig Pathway similarity was calculatedas the Jaccard Index between the KEGG pathways that contain each drug™s gene targets Methods The difference in effect size between the target similarity and the pathway similarity Dstatistic vs respectively indicates that the drugs do not necessarily have to targetthe same exact genes but rather the same biological pathway in order to share a clinical indication Additionally we found that drug pairs that share an indication had a more similarchemical structure than drug pairs that did not share an indication Dstatistic pvalue S5A Fig A biological network containing both physical and nonphysicalinteractions was curated containing proteincoding genes drugs and TFsThis curated network provided another feature for our model allowing us to utilize previouslyestablished interactions between proteins to aid with distinguishing drug pairs that share anindication Overall these features seem to indicate sufficient power in differentiating drugsPLOS Computational Biology 101371journalpcbi1008098 August PLOS COMPUTATIONAL BIOLOGY 0cMachine learning approach to drug repurposingFig Schematic of CATNIP repurposing approach A The use of drug similarity properties to predict if two drugs will share an indication using agradient boosting model the model is referred to as CATNIP B Schematic showing the use of CATNIP output scores to create a network with the scoresused as edge weights The colors of each drug represent the known disease and this demonstrates how one could identify novel indications for drugsthrough the network101371journalpcbi1008098g001PLOS Computational Biology 101371journalpcbi1008098 August PLOS COMPUTATIONAL BIOLOGY 0cMachine learning approach to drug repurposingFig CATNIP model accurately predicts drugs that share an indication and can be used for repurposing A Receiveroperating characteristic curve for CATNIP the performance for drug pairs with high and low structural similarity is alsoshown B A network of all drug pairs with a CATNIP score higher than Nodes drugs are colored based on ATCclassification and a specific example of repurposing between ATC classifications is highlighted C A graph of all ATCclassification and the median CATNIP score between the drugs belonging to each of them only including drug pairswith CATNIP score The edges between ATC Classifications with the highest median CATNIP scores are colored red101371journalpcbi1008098g002that share and do not share indications which we hypothesized can then be leveraged to createa predictive modelDrug pairs that share indications can be predicted by modelUsing these diverse drug properties as features we trained a Gradient Boosting model to predict if two drugs share a clinical indication A Gradient Boosting model showed superiorresults when compared with other algorithms Methods S2 Table The model output is adrug similarity score hereby referred to as a œCATNIP score which allows us to classifydrug pairs that share clinical indications We performed a 5fold crossvalidation analysis andachieved significant predictive performance with an areaunderthereceiveroperator curveAUC of Fig 2A We confirmed the statistical significance of our model with aPLOS Computational Biology 101371journalpcbi1008098 August PLOS COMPUTATIONAL BIOLOGY 0cMachine learning approach to drug repurposingprecisionrecall curve PRC because of the class imbalance in our dataset between drug pairsthat share indications against those that do not Shared Not Shared Whencompared to random predictions our model showed significant improvement vs areaunder PRC S6 Fig We retained a low percent of false positive predictions at various cutoffs false positives and false positives at a model prediction probability oftwo drugs sharing an indication of and respectively providing extra confidencethat our predictions can lead to strong repurposing candidatesWe found that the predictive model greatly benefited from the addition of diverse datatypes While structure similarity showed the highest feature importance of any single featureS11 Fig when used as a single feature within a gradient boosting model it only achieved anAUC of S12 Fig Interestingly when only supplying the model with ontology featuresa Jaccard index for the GO terms of the known targets of each drug within a drug pairachieved an AUC of However even at the highest AUC of any single feature typeit is significantly below the performance when combining all feature typesIn certain cases a high predictive performance is expected such as when two drugs arestructurally similar or share targets It has been shown before that structurally similar drugshave a high probability of treating the same indication[] However there continue to bedrug pairs known to treat the same indication that are not structurally similar For exampletamoxifen[] and anastrozole[] are structurally dissimilar compounds Dice similarity that treat the same indication Metathesaurus term Cancer Breast We recalculated our performance metrics to evaluate how our model performed in classifying drug pairsthat shared indications when only exposed to drug pairs with low structure similarityDice High performance was retained under with an AUC Fig 2A Additionally we found that our model performed similarly well when only exposed to drug pairs thatdid not have any known shared targets AUC Fig 2A These performance metricsconfirm that our model is robust enough to predict if a drug pair will share an indication evenfor more difficult prediction tasksNetwork clusters identify drugs with similar clinical characteristicsWe constructed a repurposing network by calculating a CATNIP score for all possible drugpairs found within DrugBank and assigning the drugs as nodes and the CATNIP score as theedge weight We pruned the network using a cutoff value of for the CATNIP scoresFig 2B which included different drug pairs This cutoff is equivalent to a predictedprobability of to share an indication and allowed for a balance between confidencewithin our predictions and drug diversity and availabilityWe hypothesized that drugs sharing at least one indication would cluster together in ournetwork To confirm this theory we classified each drug per its 1st order Anatomical Therapeutic Chemical ATC classification This identification is a method of distinguishing theclinical use of a drug that is widely used in European and North American chemoinformaticsdatabases[] Using ATC we observed clearly defined clusters within the repurposing network Fig 2B Many clusters featured multiple ATC classifications suggesting potential repurposing opportunities For example one cluster included the thiazolidinediones rosiglitazoneand pioglitazone ATC classification ˜Alimentary Tract and Metabolism™ and the fibratesfenofibrate and bezafibrate ATC classification ˜Cardiovascular system™ These two clusteredATC classifications were connected by a high CATNIP score between bezafibrate andpioglitazone an antidiabetic drug a relationship driven by the shared targeting of PPARa andPPARg resulting in the improvement of lipid and glucose metabolism Bezafibrate has shownefficacy in the treatment of Type Diabetes in numerous retrospective and preclinical studiesPLOS Computational Biology 101371journalpcbi1008098 August PLOS COMPUTATIONAL BIOLOGY 0cMachine learning approach to drug repurposingATC Code ReferenceRespiratory SystemRespiratory SystemRespiratory System[“][“][“][ ]Table Literature Support for ATC Repurposing PredictionsATC Code DermatologicalsAlimentary Tract and MetabolismSensory ansSystemic Hormonal Preparations Excluding Sex Hormones AndRespiratory SystemInsulinsSensory ans101371journalpcbi1008098t002Alimentary Tract and[“]Metabolismincluding Phase trials[“] however is still not an approved antidiabetic The identification of bezafibrate as a potential diabetes treatment is a key example of how CATNIP can beused to identify repurposing opportunitiesWe reasoned that the connections between ATC classifications across all the drug clusterscould provide additional aid for drug repurposing purposes Using the pruned network CATNIP Score we collected all the scores between drugs of differing ATC classificationsFrom this collection we were able to determine the median score associated between each pairof ATC classifications The ATC classifications with the highest median CATNIP scores hadliterature support for numerous repurposing efforts between them Table For exampledrugs with the ATC classifications of œRespiratory System and œSystemic Hormonal Preparations excluding sex hormones and insulins were strongly connected to each other median CATNIP score This connection was driven by highly scored pairs of drugs includingrimexolone to mometasone CATNIP score and prednisone to triamcinolone CATNIP score These connections are supported by the fact that hormonal agents like glucocorticoids and beta adrenergic agonists have been used for decades to relax the airway musculature in patients with reactive airways disease and chronic obstructive pulmonary disease[]Interestingly our analysis identified glucagon a peptide hormone that increases blood glucoselevels as a candidate for œRespiratory System repurposing and this use already has clinicalsupport[][] Additionally drugs classified as œRespiratory System and œDermatologicalwere also observed to be highly associated because of interactions such as the one betweenciclesonide and hydrocortisone CATNIP score Ciclesonide and hydrocortisone do infact share a clinical indication œAsthma Bronchial giving added confidence to our findingsThese types of network observations are important in laying the groundwork for suggestingnovel clinical repurposing strategies for FDAapproved drugsCATNIP identifies novel disease areas for drug classesWe investigated the ability to leverage CATNIP scores to identify repurposing opportunitiesby evaluating specific drug classes Drug classes are predefined in DrugBank In order to identify actionable repurposing possibilities we narrowed this list down to classes containinginhibitors antagonists or agonists of specific gene or protein families We focused our attention on specific disease areas that are attractive for drug repurposing opportunities due to alack of current treatments or high rates of acquired resistance The specific disease areas wereœmental disorders œneurological diseases œdiabetes and œcancer cancer was furtherdivided into specific cancer types due to the large variance in disease pathology between typesMethodsPLOS Computational Biology 101371journalpcbi1008098 August PLOS COMPUTATIONAL BIOLOGY 0cMachine learning approach to drug repurposingWe hypothesized that CATNIP scores could be used to identify specific drug classes thatwould be efficacious for a new disease area For each drug class and disease area we foundthe statistical difference in the CATNIP score distribution between two sets of drug pairsThe first set included pairs that had one drug within the drug class and the other drugapproved for the disease in question while the other set included drug pairs that had onedrug within the drug class and the other drug not approved for the disease in questionMethods We compared the effect size estimated by the Wilcoxon location shift for alldrug classdisease pairs that had a significant difference in distribution compared to drugclassnondisease pairs FDR Supplementary Data By using CATNIP scores wefound that many wellknown drug classdiseases associations could be recovered For example œmuscarinic antagonists were highly ranked for œneurological diseases and many suchagents are FDAapproved for this indication[] In addition we found that œkinase inhibitors were closely associated with the treatment of cancer and œdopamine antagonists forthe treatment of œmental disorders[ ] Wilcoxon Location Shift “ forœkinase inhibitors and select cancer types Location Shift for œdopamine antagonists and œmental disorders pvalue S7 Fig In fact almost all drug classdiseaseassociations contained at least one FDAapproved drug for the respective disease giving usadded confidence in our model Of note each drug was allowed to be categorized intonumerous drug classes leading to unexpected yet easily explained results for exampleœdopamine antagonists appearing as a top drug class for œneurological diseases This isdue to risperidone a drug traditionally used for schizophrenia and mood disorders alsohaving a secondary indication of Alzheimer™s type severe dementiaOur method reached significant levels of predictive power for predicting both drug class”disease associations and individual drugdisease association When predicting drug classdisease associations under our most lenient conditions calling cases where at least one drugwithin the class was known to treat the disease a true positives our method achieved a sensitivity of greater than However this improved to a sensitivity of when we implementedstricter cutoffs ie only calling drug classdisease associations true positives if of drugswithin the class treated that disease S10 Fig We additionally compared our method™s abilityto predict individual predictions to that of a previously highlighted method Gottlieb et al™sPREDICT[] We found our method had a slightly higher AUPRC vs andhigher sensitivity vs S4 Table S1 Methods While these results indicate modest improvements over PREDICT it is important to note that unlike in PREDICT diseaseinformation is not a required feature in CATNIP™s machine learning approach This meansthat CATNIP can be applied towards investigational molecules with no previously knownindications Additionally by not using disease information as a feature repositioning of drugswith known indications using CATNIP is not directly biased by the associated disease indication and instead uses mechanistic features chemical structure and properties targets etc aspart of the repositioning strategyNext we further interrogated the drug classes associated with œneurological diseases andœdiabetes specifically CATNIP scores could correctly identify drug classes known to treatthese diseases Table To identify possible repurposing candidates we focused our attentionon drug classes shown to have a large positive effect size with this CATNIP analysis but are notcurrently approved for treatment For œneurological diseases the use of adrenergic uptakeinhibitors traditionally used as antidepressants was the top repurposing candidate for œdiabetes alpha antagonists and kinase inhibitors were identified as possible novel treatmentsTable We believe further investigation into these drug classes and diseases could lead tosuccessful clinical applicationsPLOS Computational Biology 101371journalpcbi1008098 August PLOS COMPUTATIONAL BIOLOGY 0cMachine learning approach to drug repurposingTable Top Predictions of Drug Class Repurposing OpportunitiesClassDiabetesDiseasePrediction RankAlpha1 AntagonistsKinase InhibitorProtein Kinase InhibitorsProtein Synthesis InhibitorsCytochrome P450 CYP2E1 InhibitorsMonoamine Oxidase InhibitorsNeurologicalAdrenergic Uptake InhibitorsAdrenergic alpha AgonistsProtease Inhibitors101371journalpcbi1008098t003CATNIP interpretability reveals reasoning for repurposing candidatesFrom our list of repurposing candidates we chose two novel drug classdisease associations tofurther investigateAdrenergic uptake inhibitors applied to Parkinson™s disease First we evaluated therelationship between œneurological diseases and œadrenergic uptake inhibitors We focusedon the drug pairs with the highest CATNIP scores ie those predicted with the highest confidence to share at least one indication Fig 3A Of all the adrenergic uptake inhibitors wefound that amitriptyline and trimipramine two antidepressants had the highest CATNIPscores with the œneurological diseases drugs The drugs that shared the strongest connectionswith amitriptyline and trimipramine were drugs approved for Parkinson™s disease PD Specifically metixene atropine pergolide and benzatropine were associated with amitriptylineaccording to CATNIP and trimipramine was associated to benzatropine and rotigotine Trimipramine was also strongly connected with orphenadrine which is sometimes used off labelin PD but will not be included in the following analysesUsing the CATNIP model we evaluated which features contributed towards the predictionof amitriptyline and trimipramine to share an indication with PD drugs We found that targetgene ontology and pathway similarity all strongly contributed to the predictions for both amitriptyline and trimipramine Fig 3B S8 Fig Since target similarity and distance between targets in a proteinprotein interaction network were among the top contributing features weinvestigated which gene targets were shared amongst these drug pairs We found that amitriptyline targets three specific gene classes that are also targeted by at least one of the PD drugsmuscarinic acetylcholine receptors Gcoupled protein receptors GPCRs and alpha adrenergic receptor Trimipramine also targets muscarinic acetylcholine receptors alphaadrenergicreceptors and dopamine transporters which is similar to benzatropine a PD drug All thesereceptors have welldefined relationships with PD and other neurological diseases[ ]which adds support for repurposing amitriptyline andor trimipramineAmitriptyline may be an ideal candidate for use in PD patients We evaluated the sharedmolecular function gene ontology terms shared between amitriptyline and all four PD drugsGPCR activity was once again identified S1“S4 Files We then interrogated the biologicalpathways these drug targets are involved in and found many broad GPCR pathways overlapping between amitriptyline and the PD drugs S9 Fig including the Reactome pathway œGASTRIN_CREB_SIGNALLING PATHWAY VIA PKC AND MAPK Several recent studiessupport the link between gastrinreleasing peptide signaling to brain function[] ThroughCATNIP we have identified œadrenergic uptake inhibitor Answer:

Thyroid_Cancer

Signaling pathway analysis methods are commonly used to explainbiological behaviors of disease cells Effector genes typically decide functionalattributes associated with biological behaviors of disease cells by abnormal signalsthey received The signals that the effector genes receive can be quite different innormal vs disease conditions However most of current signaling pathway analysismethods do not take these signal variations into considerationMethods In this study we developed a novel signaling pathway analysis methodcalled signaling pathway functional attributes analysis SPFA method This methodanalyzes the signal variations that effector genes received between two conditionsnormal and disease in different signaling pathwaysResults We compared the SPFA method to seven other methods across GeneExpression Omnibus datasets using three measurements the median rank of targetpathways the median pvalue of target pathways and the percentages of significantpathways The results confirmed that SPFA was the topranking method in termsof median rank of target pathways and the fourth best method in terms of medianpvalue of target pathways SPFA™s percentage of significant pathways was modestindicating a good false positive rate and false negative rate Overall SPFA wascomparable to the other methods Our results also suggested that the signal variationscalculated by SPFA could help identify abnormal functional attributes and parts ofpathways The SPFA R code and functions can be accessed at githubcomZhenshenBaoSPFASubjects Bioinformatics Cell Biology Computational Biology Computational ScienceKeywords Signaling pathway analysis Functional attributes Cell behaviors SPFA Effector genesINTRODUCTIONRecently developed highthroughput functional genomics technologies have generatedlarge amounts of experimental disease data and detected new biological informationChallenge for biologists is understanding the biological behaviors of disease cells usingboth newly generated disease data and existing biological knowledge Signaling pathwayanalysis methods are used to better understand the biological behaviors of disease cellsHow to cite this Bao Z Zhang B Li L Ge Q Gu W Bai Y Identifying diseaseassociated signaling pathways through a noveleffector gene analysis PeerJ 8e9695 107717peerj9695Submitted February Accepted July Published August Corresponding authorYunfei Bai whitecfseueducnAcademic editorJun ChenAdditional Information andDeclarations can be found onpage 107717peerj9695Copyright Bao et alDistributed underCreative Commons CCBY 0cThe understanding of biological behaviors of disease cells benefits to understand thepathological scenery and treatment Overrepresentation analysis ORA based methodswere initially presented as signaling pathway analysis methods to help biologists identifyoverrepresented pathways from a list of relevant genes produced from experimentaldata ORAbased methods merely count the number of differentially expressed genes inspecific functional category gene sets such as the Gene Ontology GO Blake the Kyoto Encyclopedia of Genes and Genomes KEGG Kanehisa BioCarta Nishimura and Reactome Joshitope Then they determinesignificance of the overlaps via statistical tests such as Fisher™s exact test Many tools arebased on this method including OntoExpress Draghici Khatri Fisher Khatri Sirota Butte and the Gene Ontology Enrichment AnalysisSoftware Toolkit GOEAST Zheng Wang However ORAbased methods onlytake into account large changes in individual genes that significantly affect pathwaysand they do not account for smaller changes in sets of functionallyrelated genesie pathways capable of significant effects Functional class scoring FCS basedmethods have been used to avoid this limitation of ORAbased methods FCSbasedmethods take into account the coordinated gene expression changes in pathways such asgene set enrichment analysis GSEA Subramanian gene set analysis GSAEfron Tibshirani and meanrank gene set enrichment tests MRGSE Liu However ORAbased and FCSbased methods are both limited because theydo not take into account the complex interactions between genes or the complextopology of pathways To overcome this limitation pathwaytopologybased methodswere proposed Pathwaytopologybased methods integrate the complex interactionsbetween genes using pathway topology information specifically KEGG signalingpathway informationSignaling pathway impact analysis SPIA one of the most widelyused pathwaytopologybased methods considers both the number of differentially expressed genesDEGs in a given pathway and the topology information of that pathway Tarca Many improved methods based on SPIA have been proposed Li developed a method called subSPIA which used a minimum spanning tree way toprune signaling pathways and used the SPIA method to identify significant signalingsubpathways Li Bao developed two SPIAbased methodscalled PSPIA and MSPIA These two methods replaced or ˆ’ interaction strength inSPIA with the interaction strength of the Pearson correlation coefficients and mutualinformation respectively Bao There are different pathwaytopology methodsthat make use of the topological information of signaling pathways For instance GeneGraph Enrichment Analysis GGEA uses prior knowledge derived from directed generegulatory networks Geistlinger Liu Xu Bao used a subgraphmethod to take advantage of pathway topological information Liu Xu Bao ROntoTools introduced a term of perturbation factor by considering the type ofinteractions to take the pathway topology into consideration Tarca Bao PeerJ 107717peerj9695 0cVoichita Donato Draghici SebastianLeon developed a method usingtopology to detect liner subpathways in a signaling pathway SebastianLeon These methods still have their disadvantages Pathwaytopologybased methods donot consider the importance of genes in pathways Geneweightbased methods havebeen proposed to overcome this limitation Pathway analysis with downweighting ofoverlapping genes PADOG uses the frequency of a present gene in the analyzedpathways to improve gene set analysis Tarca Functional link enrichment ofgene ontology or gene sets LEGO measures gene weights in a gene set according toits relative association with genes inside and outside the gene set in a functional associationnetwork Dong Fang proposed an improved SPIA method calledSPIAIS that measured and assigned the importance as the average output degree ofthe gene in the pathwayA signaling pathway is a cascade of molecular reactions that bring out the functionalattributes eg cell proliferation apoptosis associated with the biological behaviors ofdisease cells using effector genes Effector genes receive signals without outputting signalsto other genes in an individual signaling pathway SebastianLeon Diseasesare always related to the abnormal signal that the effector genes receive Thereforethe signal that the effector genes receive can be very different under disease andnormal conditions The limitation of the previously mentioned methods includinggeneweightbased methods is that they do not consider the signal variations betweendisease and normal conditionsAdditionally the functional attributes in the same signaling pathway may be verydifferent from one another and can sometimes be opposites For example there are twoopposite functional attributes on the axon guidance pathway axon repulsion and axonattraction see the hsa04360 pathway in the KEGG dataset We cannot determinewhich functional attributes contribute more to the disease using most current pathwayanalysis methods Furthermore some pathways consist of several parts each with verydifferent contributions For example the Wnt signaling pathway is significant acrossdifferent diseases and can be divided into three parts Most existing pathway analysismethods cannot determine which part of the Wnt signaling pathway most significantlycontributes to a specific diseaseWe propose a new method that considers the signal variations between normal anddisease conditions that effector genes received in pathways the signaling pathwayfunctional attributes analysis SPFA method SPFA calculates the gene expression changesin a given pathway using an ORA method and then combines the ORA method resultswith the signal variation results under two conditions normal vs disease The signalvariations can help identify functional attributes and abnormal pathways We tested thecapabilities of the proposed signaling pathway analysis method on a series of real datasetsusing three parameters We also showed that the two types of probabilities consideredin this method were indeed independent Ultimately we verified the usefulness of thesignal variations the effector genes received under two different conditions using theSPFA methodBao PeerJ 107717peerj9695 0cMATERIALS AND METHODSData sources and preprocessingSignaling pathway analysis methods require two types of input a collection of pathwaysand a list of genes or gene products with accompanying expression values across differentsamples between the compared phenotypes We used the KEGG signaling pathway asit is the most common manuallycurated signaling pathway used for pathway analysisWe downloaded signaling pathways from the KEGG PATHWAY datasetWe acquired disease gene expression datasets from the KEGGdzPathwaysGEORpackage and KEGGandMetacoreDzPathwaysGEO Rpackages Table TarcaBhatti Romero Tarca Each disease gene expression dataset wasmatched with a corresponding disease KEGG pathway For example a colorectal cancerdataset was associated with the colorectal cancer pathway Tarca The corresponding disease KEGG pathways were called target pathways Three rules wereused to select the gene expression datasets The dataset™s DEGs were available If no DEGs were selected other comparable methodswould return null results The results of these datasets could be analyzed Pathway analysis result pvalues of could not be analyzed The target pathways of these datasets were KEGG pathways since we used KEGGpathways as examplesDEGs were selected if they contained more than genes with FDR adjustedpvalues Otherwise we selected more than genes with original pvalues and absolute log fold change If DEGs still less than genes we selected the top of genes ranked by pvalues as DEGs 0c 0cdse 0c 0cSPFA algorithm designTo assess the signal variations between two conditions normal vs disease that the effectorgenes received from upstream genes we calculated the sum of signal variations from allupstream genes to effector genes Given an effector gene ge and an upstream gene gsthe signal variation from the gene gs to the effector gene ge can be defined asese ¼ cordiseaseðgsgeÞ 00 cornormalðgsgeÞwhere cordiseaseðgsgeÞ and cornormalðgsgeÞ refer to the Pearson correlation coefficientbetween the gene expression data of gene gs and gene ge in the disease and normal statesrespectively dse is the network distance between gene gs and gene ge The Pearsoncorrelation coefficient is always used in gene coexpression networks to represent thestrength of interactions between two genes The Pearson correlation coefficient can also beused to represent the strength of an interaction between two gene expression valuesStudies have shown that the genetic regulatory patterns in signaling pathways betweenBao PeerJ 107717peerj9695 0cTable Data sets used for assessing the proposed method and compared methodsIDTarget pathwayGEO IDReferencesColorectal cancerColorectal cancerColorectal cancerColorectal cancerColorectal cancerNonsmall cell lung cancerPancreatic cancerPancreatic cancerPancreatic cancerThyroid cancerThyroid cancerAlzheimer™s diseaseAlzheimer™s diseaseAlzheimer™s diseaseAlzheimer™s diseaseAlzheimer™s diseaseChronic myeloid leukemiaChronic myeloid leukemiaAcute myeloid leukemiaAcute myeloid leukemiaAcute myeloid leukemiaDilated cardiomyopathyDilated cardiomyopathyEndometrial cancerGliomaGliomaHuntington™s diseaseParkinson™s diseaseParkinson™s diseaseProstate cancerProstate cancerRenal cell carcinomaRenal cell carcinomaGSE4107GSE4183GSE8671GSE9348GSE23878GSE18842GSE15471GSE16515GSE32676GSE3467GSE3678GSE5281_HIPGSE5281_ECGSE5281_VCXGSE1297GSE16759GSE24739_G0GSE24739_G1GSE14924_CD4GSE14924_CD8GSE9476GSE1145GSE3585GSE7305GSE19728GSE21354GSE8762GSE20291GSE20164GSE6956AAGSE6956CGSE781GSE14762Hong Galamb and Gyorffy SabatesBellver Hong Uddin SanchezPalencia Badea Pei Donahue He “Liang Liang Liang Blalock Juan Affer Affer Le Dieu Le Dieu Stirewalt “Barth Hever Liu Liu Runne Zhang Zheng Wallace Wallace Lenburg Wang genes are different under normal and disease conditions Jung If the geneticregulatory pattern between the two genes changes the signal transmitted between thetwo genes will be very different Thus we used the Pearson correlation coefficient tocalculate the signal variations that the effector genes received from their upstream genesHowever if an upstream gene does not directly transmit a signal the signal may beattenuated Therefore we used the network distance dse between gene gs and gene ge as apenalty coefficientBao PeerJ 107717peerj9695 0cFor each effector gene gi in a given pathway the accumulated signal variations betweeneijXsj¼1normal and disease conditions that the upstream genes received total s genes in theupstream of the gene gi were calculated using the formulaASVðgiÞ ¼The accumulated signal variation ASVðgiÞ of the effector gene gi in a pathway can helpus distinguish among the functional disease attributes Effector genes with high ASVðgiÞdemonstrate that these functional attributes significantly contribute to their correspondingdiseasesFor a given signaling pathway the total accumulated signal variation ASV can bedefined asASV ¼Xki¼1ASVðgiÞwhere k is the total number of effector genes in the given pathwayUltimately the probability Psd used to measure the signal variations between twoconditions normal vs disease that those effector genes received from genes upstream in agiven signaling pathway Px is based on the pathway™s ASVðPxÞ The same number of genesas the one observed on the given signaling pathway are randomly selected from allgenes random gene IDs and have any possible expression data in all samples in the rangeof the experimenter Therefore the observed signal variations were obtained by permutingthe gene IDs times ASVperðPxÞwas the total accumulated signal variation of thegiven pathway Px obtained in the perth time The probability PsdðPxÞ of the given pathwaywas calculated asPsdðPxÞ ¼IðASVperðPxÞ 15 ASVðPxÞÞPwhere I is a function that returns when the argument is true and when the argument isfalseThe probability Psd does not measure the gene differential expression in a givenpathway Thus we had to combine the probability Psd with the probability Pde which canmeasure the total gene differential expression in a given signaling pathway The probabilityPde of a given pathway Px can be calculated through the following hypergeometric test 13 12 13tr 12m 00 t 12 13n 00 rmnPdeðPxÞ ¼ 00where the whole genome contains a total of m genes n genes are the number of DEGs inthe m genes and the given pathway contains t genes and r DEGsBao PeerJ 107717peerj9695 0cThe probability Psd uses the Pearson correlation coefficient of the two genes™ expressiondata but the probability Pde uses the number of DEGs in a pathway Thus the twoprobabilities are independent of each other The significance of the given pathway wascalculated following the SPIA method which combines the probabilities Psd and Pde Tarca The formulas areP ¼ c 00 c 01 lnðcÞc ¼ Psd 02 Pdewhere c is a product of Pde and Psd P is the combined probability of the signalingpathwaySignificantly enriched pathway analysis using SPFAThe SPFA procedure identifies significantly enriched pathways in two steps Fig The first step measures the total gene differential expression in the signaling pathwaysDEGs need to first be identified from the gene expression datasets Then the DEGs aremapped onto the signaling pathways Finally the signaling pathway pvalues are calculatedusing a hypergeometric testThe second step is to measure the signal variations between the two conditions normalvs disease that effector genes received from upstream genes in the signaling pathwaysThis is completed by Finding all effector genes in each signaling pathway Ascertaining all paths from the upstream genes to the effector genes in each signalingpathway If a path exists between the upstream genes and effector genes an interactionmust exist between them The path™s network distances are used to weight thecorresponding interactions Using the Pearson correlation coefficient absolute difference values between the diseaseand normal samples to calculate the signal variations of the corresponding interactions Using the network distance of each interaction to decrease their signal variations Calculating the accumulation of the signal variations between the effector genes andupstream genes for each effector gene Calculating the sum of the accumulations of all effector genes in each signaling pathway Evaluating the statistical significance of each pathway based on their scoreUltimately the results of the two steps are combined into one pvalue We used the FDRadjust method on the combined pvalue to determine the significance of each signalingpathway The pathways with the adjusted combined pvalues smaller than a thresholdvalue were considered as significant pathwaysThe distribution of effector genes in the signaling pathwaysStudying the signal variations between two conditions normal vs disease that the effectorgenes received leads to a deeper understanding of the biological behaviors of disease cellsEffector genes are widely scattered throughout the signaling pathways If a gene has noBao PeerJ 107717peerj9695 0cFigure The workflow of SPFA method The step by step to identify significant signaling pathways using SPFAFullsize \ue90d 107717peerj9695fig1signal inputs in an individual signaling pathway the gene is not considered an effectorgene The distribution of effector genes in each signaling pathway can be seen in Fig One hundred and ninetyfive of the signaling pathways contained effector genesComparison methods and measuresWe compared seven methods to SPFA including Fisher Khatri Sirota Butte GSA Efron Tibshirani GSEA Subramanian MRGSE Liu SPIA Ullah ROnoTools Tarca Voichita Donato Draghici and PADOG Tarca We selected these methods for their stability andprevalence they can be compared using the same R environment as SPFAThere is no universally accepted technique for the validation of the results of pathwayanalysis methods Current pathway analysis methods use the results of a very smallnumber of datasets based on searching corresponding published life literature Thisapproach has its limitations First the number of datasets used is small Second authorsoften search their own leading to biased results Third complex biological phenomenaalways directly or indirectly correspond to multiple signaling pathwaysTarca compiled an objective and reproducible approach based on multipledatasets Tarca This approach avoided a biased literature search and requiredtesting on a large number of different datasets at least In this work we followedBao PeerJ 107717peerj9695 0cseneg rotceffe fo rebmuNPathwaysFigure The distribution of the effector genes™ number in each signaling pathway A total of ofFullsize \ue90d 107717peerj9695fig2 signaling pathways contain the effector genesTarca validation approach Two measurements were compared in thisvalidation approach The first measurement was the median pvalue of the targetpathways of the disease datasets Smaller median pvalues meant higher sensitivityThe second measurement was the median rank of the disease target pathwaysThe higher ranked methods were more accurate To further validate the different pathwayanalysis method results we used a third measurement the ratio of significant pathwaysusing a significance threshold of of the adjusted pvalue in the datasets Thismeasured the method™s ability to control false positive and false negative ratesRESULTSThe independence between the two probabilitiesThe two probabilities Pde and Psd are theoretically independent under the null hypothesisWe verified their independence by calculating the squared correlation coefficient betweenthe two probabilities using the gene expression datasets Table Our results showed thatthe average squared correlation coefficient of the datasets was R2 ¼ Only four ofthe squared correlation coefficients were slightly higher than R2 ¼ These resultsindicated essentially no correlation between the two probabilitiesSPFA method performanceWe compared SPFA with the other seven methods using three measurements the medianpvalue of the target pathways the median rank of the target pathways and the ratioof significant pathways The signaling pathways with adjusted pvalues ‰¤ weresignificantWhen comparing the median rank of the target pathways SPFA ranked first Fig When comparing the median pvalue of the target pathways SPFA ranked fourthBao PeerJ 107717peerj9695 0cTable The squared correlation coefficients between the two probabilities using the geneexpression datasets The four squared correlation coefficients which are slightly more than areshown in boldGEO IDGSE4107GSE4183GSE8671GSE9348GSE23878GSE18842GSE15471GSE16515GSE32676GSE3467GSE3678GSE5281_HIPGSE5281_ECGSE5281_VCXGSE1297GSE16759GSE24739_G0GSE24739_G1GSE14924_CD4GSE14924_CD8GSE9476GSE1145GSE3585GSE7305GSE19728GSE21354GSE8762GSE20291GSE20164GSE6956AAGSE6956CGSE781GSE14762AverageSquared correlation between the probabilities Pde and Psd661523Eˆ’748134E”Fig Notably the methods with the highest ranking in one measurement did notnecessarily rank the highest in the others This is because different measurements analyzedifferent abilities For example MRGSE was first in median pvalue but was sixth inmedian rank Fisher was second in median pvalue but ranked fourth in median rankTo better compare SPFA™s performance against the other methods we added the ranks ofBao PeerJ 107717peerj9695 0csyawhtap tegrat fo sknaRSPFAPADOGSPIAFisherGSAMRGSEGSEA ROntoToolsFigure The distribution of the target pathways ranks of the eight methods using datasets SPFAperforms the 1st among eight methods in terms of the median ranks of the target pathwaysFullsize \ue90d 107717peerj9695fig3the median pvalue and median rank values from each method together We found that thecombined value of SPFA and PADOG was the smallest Table To further assess the performance of the eight methods we collected the results fromother general pathways typically associated with cancer using the out of datasetswith a form of cancer in Table Apoptosis and Pathways in cancer When using theApoptosis pathway and Pathway in cancer pathway instead of target pathways SPFA™smedian ranks were both first and the median pvalues of MRGSE were also both rankedfirst These results were in alignment with the target pathway results However when usingBao PeerJ 107717peerj9695 0ctsyawhap tegrat fo seuavplMRGSEFisherPADOGSPFASPIAGSEAGSA ROntoToolsFigure The distribution of the target pathways pvalues of the eight methods using datasetsSPFA performs the 4th among eight methods in terms of the median pvalues of detecting the tarFullsize \ue90d 107717peerj9695fig4get pathwaysthe Apoptosis pathway and Pathway in cancer pathway instead of the target pathwaysPADOG™s median pvalues were both ranked fifth When using the Apoptosis pathwaySPFA™s median pvalue ranked third When using the Pathway in cancer pathway SPFA™smedian pvalue ranked fourth All these results suggest that SPFA had the best accuracyand a good sensitivity when compared with the other seven methodsAdditionally our results showed that SPFA™s ratio of significant pathways wasmoderate Fig compared to the others MRGSE™s ratio of significant pathways wasalmost and it could be questioned whether a such number of pathways was realisticBao PeerJ 107717peerj9695 0cTable The combined rank values of the ranks in terms of the median pvalues and the medianranks of target pathways of eight methodsMethodsSPFAPADOGFisherMRGSESPIAGSAGSEAROnoToolsRanks of the median pvaluesRanks of the median ranksSumTable The results of other general pathways apoptosis and pathway in cancer typically associatedwith cancer using the out of datasets with a form of cancer For each pathway the values for thetype of methods with the smallest median pvalues and ranks strongest association with the phenotypeare shown in boldPathway statisticApoptosisSPFAFisherSPIAGSAGSEAMRGSERontoToolsPADOGpValues medianRanks medianPathway in cancerpValues median794Eˆ’225Eˆ’162Eˆ’27Eˆ’Ranks medianGSA™s ratio of significant pathways was lower than and it reflected that the GSAmethod had a high false negative rate The methods had a modest ratio of significantpathways indicated that the method had a modest false positive rate and a modest falsenegative rate Thus the discriminative ability of SPFA was good when compared with theother seven methods In conclusion our results strongly supported that SPFA waswellsuited for signaling pathway analysis and confirmed previously reported results inDong Sources of improvement for SPFAThe main source of improvement in SPFA is that it uses signal variations that effectorgenes received under normal and disease conditions SPFA is compared to the simplerORAbased method used to calculate the probability Pde without accounting for signalvariations Fig As shown in Fig the ORAbased method has a higher worse rankand pvalue than SPFA for the target pathwaysBao PeerJ 107717peerj9695 0csyawhtap tnacifings tion dna tnacfings fio oitar ehTsignificancenot significantsignificantSPFAFisherSPIAROntoToolsGSAMRGSEGSEAPADOGmethodsFigure Average percentage of the pathways detected as significant and not significant by eachmethod using the threshold of pvalues ‰¤ Fullsize \ue90d 107717peerj9695fig5Validating the correlation between diseases and the signal variationsthat effector genes received under two different conditionsTo validate the correlation between diseases and the signal variations that effector genesreceived under two different conditions normal vs disease we analyzed a colorectalcancer dataset GSE4183 and an Alzheimer™s disease dataset GSE16759 The colorectalcancer microarray GSE4183 Affymetrix array HGU133 Plus20 included colorectalcancer samples and normal samples Galamb Gyorffy Bao PeerJ 107717peerj9695 0cABttttssssyyyyaaaawwwwhhhhaaaappppeeeeggggrrrraaaa ttt tttt tfff foooo sss seeekuuunaaaavvvRppplllttttssssyyyyaaaawwwwhhhhaaaappppeeeeggggrrrraaaa t tttt tttf fffoooo s sssekkkunnnaaaavRRRplSPFASPFASPFASPFAORAORAORAORASPFASPFASPFASPFAORAORAORAORAFigure Determining the contribution of signal variations received by effector genes between twodifferent conditions normal vs disease in SPFA performance The boxplots show the distribution ofFullsize \ue90d 107717peerj9695fig6the target pathways ranks A and pvalues BThe Alzheimer™s disease dataset GSE16759 included four disease samples and four normalsamples Juan The Wnt signaling pathway was altered in of the colorectal cancer samplesGalamb We assessed the signal variations that effector genes received in theWnt signaling pathway using the GSE4183 dataset Fig The results of Galamb coincided with our signal variation results Galamb reported thatoverexpression of TNS1 could induce the activation of JNK ENTREZID and The signal variation that the effector gene ENTREZID received ranked first inour results Galamb detected that RBMS1 is another overexpressed geneand modulator of cmyc ENTREZID cmyc can regulate cell cycles and cause cellsto transform pathways The signal variation that the effector gene ENTREZID received ranked second in our results Galamb also identified that TCF4 is anoverexpressed gene that can participate in the transcriptional regulation of genesassociated with colon carcinogenesis These colon carcinogenesis associated genes includecmyc ENTREZID cyclin D1 ENTREZID PPARδ ENTREZID and MMP7 ENTREZID The signal variations that these effector genes receivedranked second fourth fifth and sixth respectivelyBao PeerJ 107717peerj9695 0cseneg rotceffe eht yb devecer snoitairav liangSiEffector genesFigure The signal variations received by effector genes from the upstream genes in the Wntsignaling pathway using colorectal cancer datasets GSE4183Fullsize \ue90d 107717peerj9695fig7Many pathways can be studied in colorectal cancer datasets For example the PI3KAktsignaling pathway plays a critical role in the growth and progression of colorectal cancerJohnson The effector genes ENTREZID596 ENTREZID842 andENTREZID1027 have the highest signal variations and are linked to cell cycle progressionand cell survival Fig The GSE4183 dataset results further confirmed the role of thispathway in colorectal cancer developmentThe Wnt signaling pathway is also closely related to the occurrence and development ofAlzheimer™s disease Inestrosa The signal variations that different effectorgenes received calculating based on the Alzheimer™s disease dataset GSE16759 in the Wntsignaling pathway were shown in Fig The signal variations that the effector genesENTREZID and received were considerably higher than the other effector genesin the Wnt signaling pathway This result validated evidence of crosstalk between theAlzheimer™s disease signaling pathway and the two effector genes™ upstream genes in theWnt signaling pathwayAll these results indicated the high correlation between diseases and the signalvariations calculated using the SPFA methodBao PeerJ 107717peerj9695 0cseneg rotceffe eht yb devecer snoiitairav langSiEffector genesFigure The signal variations received by effector genes from the upstream genes in the PI3KAktsignaling pathway using colorectal cancer datasets GSE4183Fullsize \ue90d 107717peerj9695fig8The other usages of the signal variations that effector genes receivedunder two different conditionsThe signal variations that effector genes received under two different conditions canshow the different contributions of different functional attributes contributed to theircorresponding diseases We can also identify which parts of the pathway contribute to theircorresponding diseases through the signal variations that effector genes receivedWhen looking at the Wnt signaling pathway results of GSE4183 Fig first we knowthe functional attributes participating in the cell cycle have abnormal signal variationsbecause most effector genes with high signal variations participate in the pathway cell cycleincluding cmyc ENTREZID cyclin D1 ENTREZID and PPARδENTREZID and MMP7 ENTREZID Second we can know that theabnormal state of the first and second parts of the Wnt signaling pathway may contributemore to colorectal cancer because that the effector genes with high signal variations areall in the two parts If we were only to observe DEG distribution in the Wnt signalingpathway using the GSE4183 dataset we would not know which abnormal part contributedto the disease Fig Through the result of the Wnt signaling pathway in GSE16759Fig on one hand according to this result we can know that the functional attributeslinked with the effector genes ENTREZID and which had the highest signalBao PeerJ 107717peerj9695 0cseneg rotceff eehi t ybdevecer snoitairav langSiEffector genesFigure The signal variations received by effector genes from the upstream genes in the Wntsignaling pathway using Alzheimer™s disease datasets GSE16759Fullsize \ue90d 107717peerj9695fig9variations were abnormal in Alzheimer™s disease On the other hand this may dominatethat the first part of the Wnt signaling pathway may be more related to t

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You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: Signaling pathway analysis methods are commonly used to explainbiological behaviors of disease cells Effector genes typically decide functionalattributes associated with biological behaviors of disease cells by abnormal signalsthey received The signals that the effector genes receive can be quite different innormal vs disease conditions However most of current signaling pathway analysismethods do not take these signal variations into considerationMethods In this study we developed a novel signaling pathway analysis methodcalled signaling pathway functional attributes analysis SPFA method This methodanalyzes the signal variations that effector genes received between two conditionsnormal and disease in different signaling pathwaysResults We compared the SPFA method to seven other methods across GeneExpression Omnibus datasets using three measurements the median rank of targetpathways the median pvalue of target pathways and the percentages of significantpathways The results confirmed that SPFA was the topranking method in termsof median rank of target pathways and the fourth best method in terms of medianpvalue of target pathways SPFA™s percentage of significant pathways was modestindicating a good false positive rate and false negative rate Overall SPFA wascomparable to the other methods Our results also suggested that the signal variationscalculated by SPFA could help identify abnormal functional attributes and parts ofpathways The SPFA R code and functions can be accessed at githubcomZhenshenBaoSPFASubjects Bioinformatics Cell Biology Computational Biology Computational ScienceKeywords Signaling pathway analysis Functional attributes Cell behaviors SPFA Effector genesINTRODUCTIONRecently developed highthroughput functional genomics technologies have generatedlarge amounts of experimental disease data and detected new biological informationChallenge for biologists is understanding the biological behaviors of disease cells usingboth newly generated disease data and existing biological knowledge Signaling pathwayanalysis methods are used to better understand the biological behaviors of disease cellsHow to cite this Bao Z Zhang B Li L Ge Q Gu W Bai Y Identifying diseaseassociated signaling pathways through a noveleffector gene analysis PeerJ 8e9695 107717peerj9695Submitted February Accepted July Published August Corresponding authorYunfei Bai whitecfseueducnAcademic editorJun ChenAdditional Information andDeclarations can be found onpage 107717peerj9695Copyright Bao et alDistributed underCreative Commons CCBY 0cThe understanding of biological behaviors of disease cells benefits to understand thepathological scenery and treatment Overrepresentation analysis ORA based methodswere initially presented as signaling pathway analysis methods to help biologists identifyoverrepresented pathways from a list of relevant genes produced from experimentaldata ORAbased methods merely count the number of differentially expressed genes inspecific functional category gene sets such as the Gene Ontology GO Blake the Kyoto Encyclopedia of Genes and Genomes KEGG Kanehisa BioCarta Nishimura and Reactome Joshitope Then they determinesignificance of the overlaps via statistical tests such as Fisher™s exact test Many tools arebased on this method including OntoExpress Draghici Khatri Fisher Khatri Sirota Butte and the Gene Ontology Enrichment AnalysisSoftware Toolkit GOEAST Zheng Wang However ORAbased methods onlytake into account large changes in individual genes that significantly affect pathwaysand they do not account for smaller changes in sets of functionallyrelated genesie pathways capable of significant effects Functional class scoring FCS basedmethods have been used to avoid this limitation of ORAbased methods FCSbasedmethods take into account the coordinated gene expression changes in pathways such asgene set enrichment analysis GSEA Subramanian gene set analysis GSAEfron Tibshirani and meanrank gene set enrichment tests MRGSE Liu However ORAbased and FCSbased methods are both limited because theydo not take into account the complex interactions between genes or the complextopology of pathways To overcome this limitation pathwaytopologybased methodswere proposed Pathwaytopologybased methods integrate the complex interactionsbetween genes using pathway topology information specifically KEGG signalingpathway informationSignaling pathway impact analysis SPIA one of the most widelyused pathwaytopologybased methods considers both the number of differentially expressed genesDEGs in a given pathway and the topology information of that pathway Tarca Many improved methods based on SPIA have been proposed Li developed a method called subSPIA which used a minimum spanning tree way toprune signaling pathways and used the SPIA method to identify significant signalingsubpathways Li Bao developed two SPIAbased methodscalled PSPIA and MSPIA These two methods replaced or ˆ’ interaction strength inSPIA with the interaction strength of the Pearson correlation coefficients and mutualinformation respectively Bao There are different pathwaytopology methodsthat make use of the topological information of signaling pathways For instance GeneGraph Enrichment Analysis GGEA uses prior knowledge derived from directed generegulatory networks Geistlinger Liu Xu Bao used a subgraphmethod to take advantage of pathway topological information Liu Xu Bao ROntoTools introduced a term of perturbation factor by considering the type ofinteractions to take the pathway topology into consideration Tarca Bao PeerJ 107717peerj9695 0cVoichita Donato Draghici SebastianLeon developed a method usingtopology to detect liner subpathways in a signaling pathway SebastianLeon These methods still have their disadvantages Pathwaytopologybased methods donot consider the importance of genes in pathways Geneweightbased methods havebeen proposed to overcome this limitation Pathway analysis with downweighting ofoverlapping genes PADOG uses the frequency of a present gene in the analyzedpathways to improve gene set analysis Tarca Functional link enrichment ofgene ontology or gene sets LEGO measures gene weights in a gene set according toits relative association with genes inside and outside the gene set in a functional associationnetwork Dong Fang proposed an improved SPIA method calledSPIAIS that measured and assigned the importance as the average output degree ofthe gene in the pathwayA signaling pathway is a cascade of molecular reactions that bring out the functionalattributes eg cell proliferation apoptosis associated with the biological behaviors ofdisease cells using effector genes Effector genes receive signals without outputting signalsto other genes in an individual signaling pathway SebastianLeon Diseasesare always related to the abnormal signal that the effector genes receive Thereforethe signal that the effector genes receive can be very different under disease andnormal conditions The limitation of the previously mentioned methods includinggeneweightbased methods is that they do not consider the signal variations betweendisease and normal conditionsAdditionally the functional attributes in the same signaling pathway may be verydifferent from one another and can sometimes be opposites For example there are twoopposite functional attributes on the axon guidance pathway axon repulsion and axonattraction see the hsa04360 pathway in the KEGG dataset We cannot determinewhich functional attributes contribute more to the disease using most current pathwayanalysis methods Furthermore some pathways consist of several parts each with verydifferent contributions For example the Wnt signaling pathway is significant acrossdifferent diseases and can be divided into three parts Most existing pathway analysismethods cannot determine which part of the Wnt signaling pathway most significantlycontributes to a specific diseaseWe propose a new method that considers the signal variations between normal anddisease conditions that effector genes received in pathways the signaling pathwayfunctional attributes analysis SPFA method SPFA calculates the gene expression changesin a given pathway using an ORA method and then combines the ORA method resultswith the signal variation results under two conditions normal vs disease The signalvariations can help identify functional attributes and abnormal pathways We tested thecapabilities of the proposed signaling pathway analysis method on a series of real datasetsusing three parameters We also showed that the two types of probabilities consideredin this method were indeed independent Ultimately we verified the usefulness of thesignal variations the effector genes received under two different conditions using theSPFA methodBao PeerJ 107717peerj9695 0cMATERIALS AND METHODSData sources and preprocessingSignaling pathway analysis methods require two types of input a collection of pathwaysand a list of genes or gene products with accompanying expression values across differentsamples between the compared phenotypes We used the KEGG signaling pathway asit is the most common manuallycurated signaling pathway used for pathway analysisWe downloaded signaling pathways from the KEGG PATHWAY datasetWe acquired disease gene expression datasets from the KEGGdzPathwaysGEORpackage and KEGGandMetacoreDzPathwaysGEO Rpackages Table TarcaBhatti Romero Tarca Each disease gene expression dataset wasmatched with a corresponding disease KEGG pathway For example a colorectal cancerdataset was associated with the colorectal cancer pathway Tarca The corresponding disease KEGG pathways were called target pathways Three rules wereused to select the gene expression datasets The dataset™s DEGs were available If no DEGs were selected other comparable methodswould return null results The results of these datasets could be analyzed Pathway analysis result pvalues of could not be analyzed The target pathways of these datasets were KEGG pathways since we used KEGGpathways as examplesDEGs were selected if they contained more than genes with FDR adjustedpvalues Otherwise we selected more than genes with original pvalues and absolute log fold change If DEGs still less than genes we selected the top of genes ranked by pvalues as DEGs 0c 0cdse 0c 0cSPFA algorithm designTo assess the signal variations between two conditions normal vs disease that the effectorgenes received from upstream genes we calculated the sum of signal variations from allupstream genes to effector genes Given an effector gene ge and an upstream gene gsthe signal variation from the gene gs to the effector gene ge can be defined asese ¼ cordiseaseðgsgeÞ 00 cornormalðgsgeÞwhere cordiseaseðgsgeÞ and cornormalðgsgeÞ refer to the Pearson correlation coefficientbetween the gene expression data of gene gs and gene ge in the disease and normal statesrespectively dse is the network distance between gene gs and gene ge The Pearsoncorrelation coefficient is always used in gene coexpression networks to represent thestrength of interactions between two genes The Pearson correlation coefficient can also beused to represent the strength of an interaction between two gene expression valuesStudies have shown that the genetic regulatory patterns in signaling pathways betweenBao PeerJ 107717peerj9695 0cTable Data sets used for assessing the proposed method and compared methodsIDTarget pathwayGEO IDReferencesColorectal cancerColorectal cancerColorectal cancerColorectal cancerColorectal cancerNonsmall cell lung cancerPancreatic cancerPancreatic cancerPancreatic cancerThyroid cancerThyroid cancerAlzheimer™s diseaseAlzheimer™s diseaseAlzheimer™s diseaseAlzheimer™s diseaseAlzheimer™s diseaseChronic myeloid leukemiaChronic myeloid leukemiaAcute myeloid leukemiaAcute myeloid leukemiaAcute myeloid leukemiaDilated cardiomyopathyDilated cardiomyopathyEndometrial cancerGliomaGliomaHuntington™s diseaseParkinson™s diseaseParkinson™s diseaseProstate cancerProstate cancerRenal cell carcinomaRenal cell carcinomaGSE4107GSE4183GSE8671GSE9348GSE23878GSE18842GSE15471GSE16515GSE32676GSE3467GSE3678GSE5281_HIPGSE5281_ECGSE5281_VCXGSE1297GSE16759GSE24739_G0GSE24739_G1GSE14924_CD4GSE14924_CD8GSE9476GSE1145GSE3585GSE7305GSE19728GSE21354GSE8762GSE20291GSE20164GSE6956AAGSE6956CGSE781GSE14762Hong Galamb and Gyorffy SabatesBellver Hong Uddin SanchezPalencia Badea Pei Donahue He “Liang Liang Liang Blalock Juan Affer Affer Le Dieu Le Dieu Stirewalt “Barth Hever Liu Liu Runne Zhang Zheng Wallace Wallace Lenburg Wang genes are different under normal and disease conditions Jung If the geneticregulatory pattern between the two genes changes the signal transmitted between thetwo genes will be very different Thus we used the Pearson correlation coefficient tocalculate the signal variations that the effector genes received from their upstream genesHowever if an upstream gene does not directly transmit a signal the signal may beattenuated Therefore we used the network distance dse between gene gs and gene ge as apenalty coefficientBao PeerJ 107717peerj9695 0cFor each effector gene gi in a given pathway the accumulated signal variations betweeneijXsj¼1normal and disease conditions that the upstream genes received total s genes in theupstream of the gene gi were calculated using the formulaASVðgiÞ ¼The accumulated signal variation ASVðgiÞ of the effector gene gi in a pathway can helpus distinguish among the functional disease attributes Effector genes with high ASVðgiÞdemonstrate that these functional attributes significantly contribute to their correspondingdiseasesFor a given signaling pathway the total accumulated signal variation ASV can bedefined asASV ¼Xki¼1ASVðgiÞwhere k is the total number of effector genes in the given pathwayUltimately the probability Psd used to measure the signal variations between twoconditions normal vs disease that those effector genes received from genes upstream in agiven signaling pathway Px is based on the pathway™s ASVðPxÞ The same number of genesas the one observed on the given signaling pathway are randomly selected from allgenes random gene IDs and have any possible expression data in all samples in the rangeof the experimenter Therefore the observed signal variations were obtained by permutingthe gene IDs times ASVperðPxÞwas the total accumulated signal variation of thegiven pathway Px obtained in the perth time The probability PsdðPxÞ of the given pathwaywas calculated asPsdðPxÞ ¼IðASVperðPxÞ 15 ASVðPxÞÞPwhere I is a function that returns when the argument is true and when the argument isfalseThe probability Psd does not measure the gene differential expression in a givenpathway Thus we had to combine the probability Psd with the probability Pde which canmeasure the total gene differential expression in a given signaling pathway The probabilityPde of a given pathway Px can be calculated through the following hypergeometric test 13 12 13tr 12m 00 t 12 13n 00 rmnPdeðPxÞ ¼ 00where the whole genome contains a total of m genes n genes are the number of DEGs inthe m genes and the given pathway contains t genes and r DEGsBao PeerJ 107717peerj9695 0cThe probability Psd uses the Pearson correlation coefficient of the two genes™ expressiondata but the probability Pde uses the number of DEGs in a pathway Thus the twoprobabilities are independent of each other The significance of the given pathway wascalculated following the SPIA method which combines the probabilities Psd and Pde Tarca The formulas areP ¼ c 00 c 01 lnðcÞc ¼ Psd 02 Pdewhere c is a product of Pde and Psd P is the combined probability of the signalingpathwaySignificantly enriched pathway analysis using SPFAThe SPFA procedure identifies significantly enriched pathways in two steps Fig The first step measures the total gene differential expression in the signaling pathwaysDEGs need to first be identified from the gene expression datasets Then the DEGs aremapped onto the signaling pathways Finally the signaling pathway pvalues are calculatedusing a hypergeometric testThe second step is to measure the signal variations between the two conditions normalvs disease that effector genes received from upstream genes in the signaling pathwaysThis is completed by Finding all effector genes in each signaling pathway Ascertaining all paths from the upstream genes to the effector genes in each signalingpathway If a path exists between the upstream genes and effector genes an interactionmust exist between them The path™s network distances are used to weight thecorresponding interactions Using the Pearson correlation coefficient absolute difference values between the diseaseand normal samples to calculate the signal variations of the corresponding interactions Using the network distance of each interaction to decrease their signal variations Calculating the accumulation of the signal variations between the effector genes andupstream genes for each effector gene Calculating the sum of the accumulations of all effector genes in each signaling pathway Evaluating the statistical significance of each pathway based on their scoreUltimately the results of the two steps are combined into one pvalue We used the FDRadjust method on the combined pvalue to determine the significance of each signalingpathway The pathways with the adjusted combined pvalues smaller than a thresholdvalue were considered as significant pathwaysThe distribution of effector genes in the signaling pathwaysStudying the signal variations between two conditions normal vs disease that the effectorgenes received leads to a deeper understanding of the biological behaviors of disease cellsEffector genes are widely scattered throughout the signaling pathways If a gene has noBao PeerJ 107717peerj9695 0cFigure The workflow of SPFA method The step by step to identify significant signaling pathways using SPFAFullsize \ue90d 107717peerj9695fig1signal inputs in an individual signaling pathway the gene is not considered an effectorgene The distribution of effector genes in each signaling pathway can be seen in Fig One hundred and ninetyfive of the signaling pathways contained effector genesComparison methods and measuresWe compared seven methods to SPFA including Fisher Khatri Sirota Butte GSA Efron Tibshirani GSEA Subramanian MRGSE Liu SPIA Ullah ROnoTools Tarca Voichita Donato Draghici and PADOG Tarca We selected these methods for their stability andprevalence they can be compared using the same R environment as SPFAThere is no universally accepted technique for the validation of the results of pathwayanalysis methods Current pathway analysis methods use the results of a very smallnumber of datasets based on searching corresponding published life literature Thisapproach has its limitations First the number of datasets used is small Second authorsoften search their own leading to biased results Third complex biological phenomenaalways directly or indirectly correspond to multiple signaling pathwaysTarca compiled an objective and reproducible approach based on multipledatasets Tarca This approach avoided a biased literature search and requiredtesting on a large number of different datasets at least In this work we followedBao PeerJ 107717peerj9695 0cseneg rotceffe fo rebmuNPathwaysFigure The distribution of the effector genes™ number in each signaling pathway A total of ofFullsize \ue90d 107717peerj9695fig2 signaling pathways contain the effector genesTarca validation approach Two measurements were compared in thisvalidation approach The first measurement was the median pvalue of the targetpathways of the disease datasets Smaller median pvalues meant higher sensitivityThe second measurement was the median rank of the disease target pathwaysThe higher ranked methods were more accurate To further validate the different pathwayanalysis method results we used a third measurement the ratio of significant pathwaysusing a significance threshold of of the adjusted pvalue in the datasets Thismeasured the method™s ability to control false positive and false negative ratesRESULTSThe independence between the two probabilitiesThe two probabilities Pde and Psd are theoretically independent under the null hypothesisWe verified their independence by calculating the squared correlation coefficient betweenthe two probabilities using the gene expression datasets Table Our results showed thatthe average squared correlation coefficient of the datasets was R2 ¼ Only four ofthe squared correlation coefficients were slightly higher than R2 ¼ These resultsindicated essentially no correlation between the two probabilitiesSPFA method performanceWe compared SPFA with the other seven methods using three measurements the medianpvalue of the target pathways the median rank of the target pathways and the ratioof significant pathways The signaling pathways with adjusted pvalues ‰¤ weresignificantWhen comparing the median rank of the target pathways SPFA ranked first Fig When comparing the median pvalue of the target pathways SPFA ranked fourthBao PeerJ 107717peerj9695 0cTable The squared correlation coefficients between the two probabilities using the geneexpression datasets The four squared correlation coefficients which are slightly more than areshown in boldGEO IDGSE4107GSE4183GSE8671GSE9348GSE23878GSE18842GSE15471GSE16515GSE32676GSE3467GSE3678GSE5281_HIPGSE5281_ECGSE5281_VCXGSE1297GSE16759GSE24739_G0GSE24739_G1GSE14924_CD4GSE14924_CD8GSE9476GSE1145GSE3585GSE7305GSE19728GSE21354GSE8762GSE20291GSE20164GSE6956AAGSE6956CGSE781GSE14762AverageSquared correlation between the probabilities Pde and Psd661523Eˆ’748134E”Fig Notably the methods with the highest ranking in one measurement did notnecessarily rank the highest in the others This is because different measurements analyzedifferent abilities For example MRGSE was first in median pvalue but was sixth inmedian rank Fisher was second in median pvalue but ranked fourth in median rankTo better compare SPFA™s performance against the other methods we added the ranks ofBao PeerJ 107717peerj9695 0csyawhtap tegrat fo sknaRSPFAPADOGSPIAFisherGSAMRGSEGSEA ROntoToolsFigure The distribution of the target pathways ranks of the eight methods using datasets SPFAperforms the 1st among eight methods in terms of the median ranks of the target pathwaysFullsize \ue90d 107717peerj9695fig3the median pvalue and median rank values from each method together We found that thecombined value of SPFA and PADOG was the smallest Table To further assess the performance of the eight methods we collected the results fromother general pathways typically associated with cancer using the out of datasetswith a form of cancer in Table Apoptosis and Pathways in cancer When using theApoptosis pathway and Pathway in cancer pathway instead of target pathways SPFA™smedian ranks were both first and the median pvalues of MRGSE were also both rankedfirst These results were in alignment with the target pathway results However when usingBao PeerJ 107717peerj9695 0ctsyawhap tegrat fo seuavplMRGSEFisherPADOGSPFASPIAGSEAGSA ROntoToolsFigure The distribution of the target pathways pvalues of the eight methods using datasetsSPFA performs the 4th among eight methods in terms of the median pvalues of detecting the tarFullsize \ue90d 107717peerj9695fig4get pathwaysthe Apoptosis pathway and Pathway in cancer pathway instead of the target pathwaysPADOG™s median pvalues were both ranked fifth When using the Apoptosis pathwaySPFA™s median pvalue ranked third When using the Pathway in cancer pathway SPFA™smedian pvalue ranked fourth All these results suggest that SPFA had the best accuracyand a good sensitivity when compared with the other seven methodsAdditionally our results showed that SPFA™s ratio of significant pathways wasmoderate Fig compared to the others MRGSE™s ratio of significant pathways wasalmost and it could be questioned whether a such number of pathways was realisticBao PeerJ 107717peerj9695 0cTable The combined rank values of the ranks in terms of the median pvalues and the medianranks of target pathways of eight methodsMethodsSPFAPADOGFisherMRGSESPIAGSAGSEAROnoToolsRanks of the median pvaluesRanks of the median ranksSumTable The results of other general pathways apoptosis and pathway in cancer typically associatedwith cancer using the out of datasets with a form of cancer For each pathway the values for thetype of methods with the smallest median pvalues and ranks strongest association with the phenotypeare shown in boldPathway statisticApoptosisSPFAFisherSPIAGSAGSEAMRGSERontoToolsPADOGpValues medianRanks medianPathway in cancerpValues median794Eˆ’225Eˆ’162Eˆ’27Eˆ’Ranks medianGSA™s ratio of significant pathways was lower than and it reflected that the GSAmethod had a high false negative rate The methods had a modest ratio of significantpathways indicated that the method had a modest false positive rate and a modest falsenegative rate Thus the discriminative ability of SPFA was good when compared with theother seven methods In conclusion our results strongly supported that SPFA waswellsuited for signaling pathway analysis and confirmed previously reported results inDong Sources of improvement for SPFAThe main source of improvement in SPFA is that it uses signal variations that effectorgenes received under normal and disease conditions SPFA is compared to the simplerORAbased method used to calculate the probability Pde without accounting for signalvariations Fig As shown in Fig the ORAbased method has a higher worse rankand pvalue than SPFA for the target pathwaysBao PeerJ 107717peerj9695 0csyawhtap tnacifings tion dna tnacfings fio oitar ehTsignificancenot significantsignificantSPFAFisherSPIAROntoToolsGSAMRGSEGSEAPADOGmethodsFigure Average percentage of the pathways detected as significant and not significant by eachmethod using the threshold of pvalues ‰¤ Fullsize \ue90d 107717peerj9695fig5Validating the correlation between diseases and the signal variationsthat effector genes received under two different conditionsTo validate the correlation between diseases and the signal variations that effector genesreceived under two different conditions normal vs disease we analyzed a colorectalcancer dataset GSE4183 and an Alzheimer™s disease dataset GSE16759 The colorectalcancer microarray GSE4183 Affymetrix array HGU133 Plus20 included colorectalcancer samples and normal samples Galamb Gyorffy Bao PeerJ 107717peerj9695 0cABttttssssyyyyaaaawwwwhhhhaaaappppeeeeggggrrrraaaa ttt tttt tfff foooo sss seeekuuunaaaavvvRppplllttttssssyyyyaaaawwwwhhhhaaaappppeeeeggggrrrraaaa t tttt tttf fffoooo s sssekkkunnnaaaavRRRplSPFASPFASPFASPFAORAORAORAORASPFASPFASPFASPFAORAORAORAORAFigure Determining the contribution of signal variations received by effector genes between twodifferent conditions normal vs disease in SPFA performance The boxplots show the distribution ofFullsize \ue90d 107717peerj9695fig6the target pathways ranks A and pvalues BThe Alzheimer™s disease dataset GSE16759 included four disease samples and four normalsamples Juan The Wnt signaling pathway was altered in of the colorectal cancer samplesGalamb We assessed the signal variations that effector genes received in theWnt signaling pathway using the GSE4183 dataset Fig The results of Galamb coincided with our signal variation results Galamb reported thatoverexpression of TNS1 could induce the activation of JNK ENTREZID and The signal variation that the effector gene ENTREZID received ranked first inour results Galamb detected that RBMS1 is another overexpressed geneand modulator of cmyc ENTREZID cmyc can regulate cell cycles and cause cellsto transform pathways The signal variation that the effector gene ENTREZID received ranked second in our results Galamb also identified that TCF4 is anoverexpressed gene that can participate in the transcriptional regulation of genesassociated with colon carcinogenesis These colon carcinogenesis associated genes includecmyc ENTREZID cyclin D1 ENTREZID PPARδ ENTREZID and MMP7 ENTREZID The signal variations that these effector genes receivedranked second fourth fifth and sixth respectivelyBao PeerJ 107717peerj9695 0cseneg rotceffe eht yb devecer snoitairav liangSiEffector genesFigure The signal variations received by effector genes from the upstream genes in the Wntsignaling pathway using colorectal cancer datasets GSE4183Fullsize \ue90d 107717peerj9695fig7Many pathways can be studied in colorectal cancer datasets For example the PI3KAktsignaling pathway plays a critical role in the growth and progression of colorectal cancerJohnson The effector genes ENTREZID596 ENTREZID842 andENTREZID1027 have the highest signal variations and are linked to cell cycle progressionand cell survival Fig The GSE4183 dataset results further confirmed the role of thispathway in colorectal cancer developmentThe Wnt signaling pathway is also closely related to the occurrence and development ofAlzheimer™s disease Inestrosa The signal variations that different effectorgenes received calculating based on the Alzheimer™s disease dataset GSE16759 in the Wntsignaling pathway were shown in Fig The signal variations that the effector genesENTREZID and received were considerably higher than the other effector genesin the Wnt signaling pathway This result validated evidence of crosstalk between theAlzheimer™s disease signaling pathway and the two effector genes™ upstream genes in theWnt signaling pathwayAll these results indicated the high correlation between diseases and the signalvariations calculated using the SPFA methodBao PeerJ 107717peerj9695 0cseneg rotceffe eht yb devecer snoiitairav langSiEffector genesFigure The signal variations received by effector genes from the upstream genes in the PI3KAktsignaling pathway using colorectal cancer datasets GSE4183Fullsize \ue90d 107717peerj9695fig8The other usages of the signal variations that effector genes receivedunder two different conditionsThe signal variations that effector genes received under two different conditions canshow the different contributions of different functional attributes contributed to theircorresponding diseases We can also identify which parts of the pathway contribute to theircorresponding diseases through the signal variations that effector genes receivedWhen looking at the Wnt signaling pathway results of GSE4183 Fig first we knowthe functional attributes participating in the cell cycle have abnormal signal variationsbecause most effector genes with high signal variations participate in the pathway cell cycleincluding cmyc ENTREZID cyclin D1 ENTREZID and PPARδENTREZID and MMP7 ENTREZID Second we can know that theabnormal state of the first and second parts of the Wnt signaling pathway may contributemore to colorectal cancer because that the effector genes with high signal variations areall in the two parts If we were only to observe DEG distribution in the Wnt signalingpathway using the GSE4183 dataset we would not know which abnormal part contributedto the disease Fig Through the result of the Wnt signaling pathway in GSE16759Fig on one hand according to this result we can know that the functional attributeslinked with the effector genes ENTREZID and which had the highest signalBao PeerJ 107717peerj9695 0cseneg rotceff eehi t ybdevecer snoitairav langSiEffector genesFigure The signal variations received by effector genes from the upstream genes in the Wntsignaling pathway using Alzheimer™s disease datasets GSE16759Fullsize \ue90d 107717peerj9695fig9variations were abnormal in Alzheimer™s disease On the other hand this may dominatethat the first part of the Wnt signaling pathway may be more related to t Answer:

Thyroid_Cancer

Dysregulation of ribosome production can lead to a number of developmental disorderscalled ribosomopathies Despite the ubiquitous requirement for these cellular machinesused in protein synthesis ribosomopathies manifest in a tissuespecific manner with manyaffecting the development of the face Here we reveal yet another connection between craniofacial development and making ribosomes through the protein Paired Box PAX9PAX9 functions as an RNA Polymerase II transcription factor to regulate the expression ofproteins required for craniofacial and tooth development in humans We now expand thisfunction of PAX9 by demonstrating that PAX9 acts outside of the cell nucleolus to regulatethe levels of proteins critical for building the small subunit of the ribosome This function ofPAX9 is conserved to the anism Xenopus tropicalis an established model for humanribosomopathies Depletion of pax9 leads to craniofacial defects due to abnormalities inneural crest development a result consistent with that found for depletion of other ribosomebiogenesis factors This work highlights an unexpected layer of how the making of ribosomes is regulated in human cells and during embryonic developmentAuthor summaryWe are only beginning to understand the complex process of making human ribosomesthe cellular machines critical for all protein synthesis In humans making a ribosomerequires hundreds of regulatory factors to ensure proper cellular growth and development Dysregulation of this process can lead to a number of tissue specific disorderstermed ribosomopathies Here we have discovered a new role for the protein PairedBox PAX9 in making human ribosomes While PAX9 has traditionally been known toa1111111111a1111111111a1111111111a1111111111a1111111111 ACCESSCitation FarleyBarnes KI Deniz E Overton MMKhokha MK Baserga SJ Paired Box PAX9 the RNA polymerase II transcription factorregulates human ribosome biogenesis andcraniofacial development PLoS Genet e1008967 101371journalpgen1008967Editor Paul A Trainor Stowers Institute forMedical Research UNITED STATESReceived February Accepted June Published August Copyright FarleyBarnes This is an access distributed under the terms ofthe Creative Commons Attribution License whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are creditedData Availability Statement All RNAseq andRNAPII ChIPseq files are available from the GeneExpression Omnibus GEO database and areaccessible through the GEO series accessionnumber GSE154764 wwwncbinlmnihgovgeoqueryacccgiaccGSE154764 Allnumerical data that underlies graphs or summarystatistics is available in the Supporting InformationS4 TablePLOS Genetics 101371journalpgen1008967 August PLOS GENETICS 0cFunding This work was supported by the NationalInstitutes of Health R01GM115710R01GM122926 and R35GM131687 to SJBR01HD081379 to MKK T32GM007223 to SJB andKIF F31DE026946 to KIF and a pilot grant from theYale Cancer Center to SJB The funders had no rolein study design data collection and analysisdecision to publish or preparation of themanuscriptCompeting interests The authors have declaredthat no competing interests existPaired Box PAX9 regulates human ribosome biogenesisplay a role in regulating the levels of proteins required for craniofacial and tooth development in humans we expand this function of PAX9 by showing that PAX9 acts outside ofthe cell nucleolus to regulate the levels of proteins critical for building the small subunit ofthe ribosome In addition we show that this function is conserved to the model anismXenopus tropicalis This link between PAX9™s role in craniofacial development and inribosome biogenesis may lead to new insights into the pathogenesis of these PAX9 mutations in humansIntroductionWhen ribosome biogenesis is genetically disrupted in humans a number of surprisingly tissuespecific disorders called ribosomopathies arise For example genetic disruption of theTCOF1 POLR1C or POLR1D genes in the ribosomopathy Treacher Collins syndrome TCSOMIM results in reduced preribosomal RNA prerRNA transcription [“] Interestingly while these mutations each affect the global process of prerRNA transcriptionpatients have specific defects in craniofacial development TCS patients present with hypoplasia of the facial bones micrognathia with or without cleft palate narrowing of the ear canaland bilateral conductive hearing loss [“] Modeling the disease in mice has shown that thistissue specificity arises from differential tissue susceptibility to p53 levels [] p53 levels are stabilized upon disruptions in ribosome biogenesis when free ribosomal proteins bind to MDM2the E3 ligase for p53 [ ] This stabilization of p53 leads to apoptosis of the developing neural crest cells ultimately resulting in the mandibulofacial dysostosis of TCSTCS is not the only ribosomopathy to affect craniofacial development For example Diamond Blackfan anemia DBA OMIM is characterized by anemia low reticulocytecount and elevated erythrocyte adenosine deaminase activity [ ] However DBA patientsoften also have craniofacial anomalies and cleft palate reviewed in [] Additionally theribosomopathy Acrofacial dysostosis Cincinnati type OMIM is caused by mutationsin POLR1A that inhibit prerRNA transcription resulting in craniofacial defects [] Amore precise understanding of the role of the factors involved in human ribosome biogenesiscan therefore shed light on the molecular mechanisms underlying aberrant craniofacialdevelopmentThe process of making the cellular machines required for protein synthesis called ribosomebiogenesis includes a large number of factors that work together to control cell growth anddevelopment In humans these proteins are still being defined Previous studies have shownthat ribosome biogenesis begins with the transcription of the tandemly repeated ribosomalDNA rDNA into the 47S polycistronic prerRNA Fig 1A The prerRNA is further modified and processed to create the mature 18S 58S and 28S rRNAs These rRNAs are incorporated along with the 5S rRNA and ribosomal proteins into the small SSU and large LSUsubunits of the ribosome The 47S prerRNA is transcribed by RNA polymerase I RNAPIwhile the 5S rRNA is transcribed by RNA polymerase III RNAPIII Various assembly factorsand the ribosomal proteins are transcribed by RNA polymerase II RNAPII In addition torequiring all RNA polymerases synthesis and assembly of functional ribosomes integrates anumber of cellular signaling pathways to ensure proper regulation of this essential process inresponse to stimuliThe complex development of the face is controlled by a number of proteins including several RNAPII transcription factors such as Paired Box PAX9 PAX9 belongs to a family oftranscription factors that play key roles in anogenesis and neural crest cell development byPLOS Genetics 101371journalpgen1008967 August PLOS GENETICS 0cPaired Box PAX9 regulates human ribosome biogenesisPLOS Genetics 101371journalpgen1008967 August PLOS GENETICS 0cPaired Box PAX9 regulates human ribosome biogenesisFig PAX9 is required for human ribosome biogenesis A Ribosome biogenesis at a glance The tandemly repeated ribosomal DNA rDNA istranscribed into the 47S polycistronic preribosomal RNA prerRNA by RNA Polymerase I RNAPI This 47S prerRNA is processed throughmultiple steps to form the mature 18S 58S and 28S rRNAs which are incorporated into the small and large subunits of the ribosome along with the5S rRNA and ribosomal proteins Ribosomes perform cytoplasmic cellular protein synthesis through the translation of mRNAs B PAX9 depletionreduces nucleolar number from “ to only in MCF10A cells Left panel Nuclei stained in Hoechst are shown in blue Nucleoli are shown in pinkand stained with antifibrillarin antibody as in [] siGFP top was used as a negative control “ nucleolinucleus and siUTP4 middle was usedas a positive control nucleolusnucleus siPAX9 is shown at the bottom Right panel Quantitation of the number of nucleoli per nucleus for siGFPtop siUTP4 middle or siPAX9 bottom C PAX9 is not required for RNAPI transcription in MCF10A cells A dualluciferase reporter assay wasused to quantify luminescence after siRNA depletion of PAX9 The plasmids are pHrDIRESLuc firefly to report RNAPI transcription and aRenilla transfection control as in [] The ratio of firefly to Renilla luciferase was normalized to the siNT control N siNOL11 was used as apositive control [] Data were analyzed by Student™s t test using GraphPad Prism ���� p � D PAX9 is required for pre18S rRNAprocessing in MCF10A cells Left Schematic of prerRNA processing steps in human cells Intermediates detected by probe P3 are indicated with ablack box below Center Northern blot with probe P3 A probe for the 7SL RNA was used as a loading control Intermediates detected by probe P3 areshown to the right of the northern blot Negative controls were mock no siRNA and siNT nontargeting siUTP4 was used as a positive control[] Right Quantitation by RAMP [] of probe P3 upper and 7SL lower northern blots Graph is mean ± SEM N Data were analyzed by2way ANOVA using GraphPad Prism ���� p � ��� p � �� p � and � p � PTP indicates the 47S 45S and 43S processingintermediates E PAX9 siRNA depletion in MCF10A cells results in an increased ratio of 28S18S by Agilent BioAnalyzer Significance was calculatedby Student™s t test in GraphPad Prism where �� p � F PAX9 siRNA depletion in MCF10A cells results in decreased global protein synthesis asassessed by the puromycin incorporation assay [] A representative western blot using an antipuromycin antibody with a β actin loading control isshown to the left Protein was harvested after knockdown for hours using the indicated siRNAs Mock indicates no siRNA and Mock μMindicates no siRNA and half the concentration of puromycin siNT nontargeting was used a positive control Quantitation of replicates using cellsof different passage numbers is shown to the right Significance was calculated by Oneway ANOVA in GraphPad Prism where ���� p � and��� p � G PAX9 depletion in MCF10A cells results in decreased 40S 60S and 80S ribosome subunit levels Representative polysome profile ofMCF10A cells depleted using siRNAs targeting either PAX9 red or a nontargeting siNT blue control Equal amounts of protein were loaded oneach gradient This experiment was performed times using cells of different passage numbers101371journalpgen1008967g001controlling gene expression [ ] In humans mutations in PAX9 cause tooth agenesis aswell as hair loss [“] and reviewed in [] Indeed PAX9 mutations are the most prevalent mutation in patients with nonsyndromic tooth agenesis including oligodontia []Additionally mice homozygous for a partial deletion of Pax9 likely null have craniofacialmalformations including cleft palate skeletal abnormalities and arrested tooth developmentand die a few hours after birth [] Pax9 mice also exhibit a range of cardiac malformations[] another commonly impacted tissue in ribosomopathies [] While some research hasbeen done to identify the signaling pathways regulated by PAX9 attempts to correct the developmental defects have been only partially successful [“] Therefore further studies areneeded to identify all factors regulated by PAX9 in order to understand PAX9™s role in craniofacial developmentOur work ties together PAX9 and ribosome biogenesis filling in some gaps in our knowledge of the many cell growth and signaling pathways influenced by PAX9 depletion We originally identified PAX9 as a potential regulator of ribosome biogenesis in an siRNA screen forproteins required to maintain nucleolar number [] Probing PAX9™s specific role in makingribosomes in human tissue culture cells we discovered that PAX9 is required both for the prerRNA processing that produces the small subunit 18S rRNA and for global protein synthesisWe employed the genomewide transcriptomics analysis RNAseq to define PAX9 dependentmRNAs necessary for making ribosomes Several of the differentially expressed mRNAsincluding several ribosomal proteins were further examined to pinpoint roles for these proteins in prerRNA processing and global protein synthesis Finally given an established rolefor PAX9 in human craniofacial development we sought to model this disease in Xenopus tropicalis X tropicalis embryos an established model of human ribosomopathies Depletion ofPax9 in X tropicalis did indeed alter craniofacial patterning as well as neural crest development a migratory cell population that plays a major role in establishing craniofacial structureX tropicalis embryos depleted of Pax9 also show defective prerRNA processing These resultsshed light on the plethora of factors whose expression is regulated by PAX9 and the doorto likely connections between PAX9™s role in craniofacial development and human ribosomebiogenesisPLOS Genetics 101371journalpgen1008967 August PLOS GENETICS 0cPaired Box PAX9 regulates human ribosome biogenesisResultsPAX9 depletion disrupts small subunit ribosome biogenesisPreviously we performed an siRNA screen for new regulators of nucleolar number in humanMCF10A cells [] We identified PAX9 by this screening approach as a protein that whendepleted reduced the number of nucleoli per nucleus from “ to only Fig 1B As in []cells were fixed after hours of knockdown using pools of siRNAs targeting siGFP as a negative control siUTP4 as a positive control or siPAX9 This transient knockdown approach successfully depleted PAX9 S1A and S1B Fig The MCF10A cells were then stained with anantibody to fibrillarin FBL a nucleolar protein to detect nucleoli and with HOECHST todetect nuclei A CellProfiler [] pipeline was used to quantify the number of nucleoli per cellnucleus which shifted from “ in siGFP control cells to only in siPAX9 and siUTP4treatedcells We had previously used oligonucleotide deconvolution to additionally confirm thatPAX9 depletion leads to reductions in nucleolar number [] Using this approach individualdepletion of PAX9 using of the siRNAs from the original PAX9 pool reduced the numberof nucleoli from “ to only per cell nucleus [ ] As the siRNA screen served as a phenotypic readout of nucleolar function [] we hypothesized that PAX9 plays a role in humanribosome biogenesis through its function as an RNAPII transcription factorWe sought to investigate the extent to which PAX9 depletion affects human ribosome biogenesis using a panel of assays The first assay probes PAX9™s role in RNAPI transcriptionusing a dualluciferase reporter system previously published by our laboratory and others [ ] As PAX9 is a known RNAPII transcription factor it was relevant to test it for a possibleadditional role in RNAPI transcription In this reporter assay the ratio of firefly luciferasewhich is under the control of the rDNA promoter and measures RNAPI transcription wasquantified relative to a Renilla luciferase transfection control Relative to a nontargeting control siRNA siNT siRNAs targeting PAX9 had no significant effect on RNAPI transcriptionlevels after hours of knockdown in MCF10A cells Fig 1C Mock no siRNA and siNOL11were used as negative and positive controls respectively PAX9 is therefore not required forRNAPI transcription in MCF10A cellsNorthern blotting was used to define PAX9™s role in prerRNA processing In humans prerRNA processing occurs via a number of different pathways Fig 1D left and requires a number of transacting factors We therefore employed different probes to pinpoint any prerRNA processing defects occurring after PAX9 depletion in MCF10A cells S2A Fig After hours of siRNA knockdown PAX9 depletion resulted in a significant increase in the 30S prerRNA intermediate as well as a decrease in the levels of its 21S processing product relative tothe nontargeting siRNA siNT Fig 1D middle and right and S2 Fig Additionally 41S levelswere decreased relative to the primary processing transcript 47S plus the 45S and 43S processing intermediates herein termed the primary transcript plus or PTP Fig 1D middle andright and S2 Fig Quantitation of the ratios of each intermediate relative to its precursor in theprocessing pathway by Ratio Analysis of Multiple Precursors RAMP [] confirmed the statistical significance of these results Fig 1D right and S2 Fig These effects were also significant relative to a 7SL loading control Fig 1D right and S2 Fig Because the 30S and 21Sintermediates are both precursors to the 18S rRNA PAX9 is required for SSU biogenesis Thesame prerRNA processing defect was also detected in human embryonic kidney HEK293FTand colon carcinoma RKO cells depleted of PAX9 indicating that PAX9™s role in ribosomebiogenesis is conserved among diverse human cell lines S1 FigBecause the prerRNA processing defects indicate aberrant SSU biogenesis we sought todetermine the extent to which PAX9 depletion affects the production of the mature 18S rRNAAgilent BioAnalyzer quantitation shows an increase in the ratio of 28S to 18S Fig 1EPLOS Genetics 101371journalpgen1008967 August PLOS GENETICS 0cPaired Box PAX9 regulates human ribosome biogenesisCombined with the northern blot results indicating defects in the processing of the precursorsto the 18S rRNA this result is consistent with a predicted reduction in 18S rRNA levels Takentogether these results argue that PAX9 is required likely indirectly for the biogenesis of thesmall subunit of the ribosome which contains the 18S rRNAWe also utilized a puromycin incorporation assay to test the extent to which PAX9 depletion alters the final product of ribosome biogenesis global cellular translation Fig 1F Cellswith or without PAX9 depletion are treated with a low dose μM of puromycin which isincorporated into all nascent peptides produced during a hour pulse [] Western blottingfor incorporated puromycin shows decreased protein synthesis after hours of PAX9 depletion relative to a nontargeting siRNA control siNT Fig 1F Mock μM puromycin withno siRNA and Mock at a halfdose of puromycin μM were used as negative controls Fig1F These results confirm that PAX9 depletion leads to reduced protein synthesis consistentwith a role for PAX9 in SSU biogenesisBecause the above assays indicated a role for PAX9 in small subunit prerRNA processingand global protein synthesis we also tested the extent to which PAX9 depletion is required forribosomal subunit biogenesis and assembly Using polysome profiling we determined thatPAX9 depletion does result in significantly decreased SSU 40S levels in MCF10A cells Fig1G consistent with the reduction in 18S rRNA levels seen in Fig 1E Additionally 60S and80S polysome fractions also showed significant decreases after PAX9 knockdown Fig 1GInterestingly both here and in previous experiments MCF10A cells do not demonstraterobust polysome fractions using this technique [] Regardless we conclude that PAX9 depletion results in decreased SSU biogenesis that impacts the assembly and function of theribosomeAs disruptions in ribosome biogenesis result in interruptions in the cell cycle [“] wealso examined the extent to which PAX9 depletion changed the distribution of MCF10A cellswithin the cell cycle using flow cytometry S3 Fig Relative to a siNT control depletion ofPAX9 for hours resulted in a minor increase in the proportion of cells in G1 phase of thecell cycle but this was not statistically significant S3 Fig siRNAs targeting the ribosome biogenesis factor NOL11 were used as a positive control and depletion of this protein resulted inan increase in the proportion of cells in G2 S3 Fig consistent with previous findings [] Inall these assays allowed us to conclude that PAX9 regulates human ribosome biogenesisRNAseq analysis upon PAX9 depletion reveals decreased levels ofnucleolar mRNAs responsible for small subunit maturationAs PAX9 is a known RNAPII transcription factor we hypothesized that PAX9 works indirectlyto modulate SSU biogenesis Fig 2A This is consistent with a nuclear but not nucleolar localization of PAX9 in existing databases [“] PAX9 may act directly as a transcription factorfor nucleolar proteins or indirectly for proteins that affect the expression or function of nucleolar proteins To test the hypothesis that PAX9 affects nucleolar protein expression through itsfunction as a RNAPII transcription factor we used RNAseq in MCF10A cells to define the setof mRNAs that were differentially expressed after PAX9 depletion Relative to a nontargetingcontrol siRNA siNT PAX9 depletion resulted in the differential expression of over mRNAs fold change � or � q � Fig 2B and S1 Table Approximately half of these were reduced in their levels consistent with the hypothesis that PAX9 acts as a transcription factor to drive their expressionWhen considered as a whole the RNAseq dataset is enriched for several pathways knownto be regulated by PAX9 S4A Fig Ingenuity Pathway Analysis [] of the differentiallyexpressed mRNAs reveals enrichment of both WntCa2 signaling differential expression ofPLOS Genetics 101371journalpgen1008967 August PLOS GENETICS 0cPaired Box PAX9 regulates human ribosome biogenesisPLOS Genetics 101371journalpgen1008967 August PLOS GENETICS 0cPaired Box PAX9 regulates human ribosome biogenesisFig RNAseq transcriptomics analysis in human tissue culture cells reveals changes in the expression levels of over nucleolar mRNAs afterPAX9 knockdown A Schematic of how PAX9 would act as a RNAPII transcription factor to drive the levels of mRNAs required for making the smallsubunit SSU of the ribosome In the cell nucleus PAX9 binds to DNA to affect the transcription of mRNAs that either encode nucleolar proteinsdirect solid arrow or to transcribe mRNAs that affect the levels of mRNAs encoding nucleolar proteins indirect dotted arrows The resulting mRNAsare translated in the cytoplasm into proteins that function in SSU prerRNA processing in the nucleolus B RNAseq analysis after PAX9 siRNAdepletion in MCF10A cells reveals decreased levels of mRNAs encoding nucleolar proteins Relative to a nontargeting siRNA control siNT PAX9depletion resulted in differential expression of mRNAs fold change � or and FDR � Of these mRNAs had a decreased foldchange � and of those mRNAs code for proteins designated as nucleolar in at least one of three databases [“] Of the mRNAs whoselevels were decreased and that also code for nucleolar proteins were chosen as candidates for followup studies C qRTPCR confirms reduced mRNAlevels of the RNAseq candidates after PAX9 siRNA knockdown in MCF10A cells After depletion using siRNAs targeting either PAX9 or a nontargeting control siRNA siNT the levels of the indicated mRNAs were quantified by qRTPCR using primers to each target gene relative to a 7SLcontrol and siNT Data are shown as mean ± SEM Three replicates using cells of different passage numbers with technical replicates each wereperformed Significance was calculated by Oneway ANOVA using GraphPad Prism where ���� p � D Depletion of of the candidatemRNAs RPS6eS6 RPS9uS4 RPS28eS28 and FBL individually results in the same prerRNA processing defect as PAX9 siRNA depletion in MCF10Acells Representative northern blot after knockdown of the indicated siRNAs using probe P3 A probe for the 7SL RNA was used as a loading control PrerRNA processing intermediates detected by probe P3 are shown to the right of the northern blot PTP indicates the 47S 45S and 43S prerRNAprocessing intermediates E Quantitation of northern blots using probe P3 as shown in Fig 2D using RAMP [] Graph is mean ± SEM N Datawere analyzed using 2way ANOVA in GraphPad Prism where ���� p � ��� p � and �� p � Quantitation relative to the 7SL loadingcontrol is shown in S5 Fig F siRNA depletion of RNAseq candidates in MCF10A cells results in decreased global protein synthesis After hoursof knockdown with the indicated siRNAs MCF10A cells were pulsed with puromycin for hour and protein was harvested Western blotting with anantipuromycin antibody as well as a β actin loading control was carried out representative western blots shown to the left Mock mock at half theconcentration of puromycin μM and siNT nontargeting siRNAs were used as negative controls G Quantitation of three replicates usingMCF10A cells of different passage numbers of the puromycin incorporation assays following depletion with the indicated siRNAs relative to the siNT andβ actin loading controls is shown as mean ± SEM N Significance was calculated by Student™s ttest using GraphPad Prism where ���� p � ���p � and � p � 101371journalpgen1008967g002 pathway members p x ˆ’ S4B Fig and Wntβcatenin signaling differentialexpression of pathway members p x ˆ’ S4C Fig In cells depleted of PAX9expression levels are increased for many of the mRNAs in the Wnt signaling pathway This isconsistent with previous results suggesting a role for PAX9 in the negative regulation of Wntsignaling [ ]As an additional validation of the RNAseq dataset we determined that the genomesequences kb upstream of the start sites of the differentially expressed mRNAs containmultiple potential PAX9 binding sites making these mRNAs candidates for direct transcriptional regulation by PAX9 Scanning for known PAX9 binding sequences [™SGTCACGCWTGANTGMA3™ ™CGCGTGACCG3™ [] CD192Ains ™GCGTGACCA3™ and e5 ™GCGGAACGG3™] in the kb upstream of the mRNAs using CentriMo analysis []reveals and potential PAX9 binding sites respectively in the kbupstream of the mRNAs This number of potential binding sites upstream of the mRNAs sites per gene is similar to that observed in PAX9 ChIP experiments in the vertebral column of E125 mice sites per gene [] Additionally CentriMo enrichmentanalysis [] of the sequence kb upstream of each of the differentially expressedmRNAs reveals significant enrichment of different DNA binding sequences including thePAX3 PAX5 PAX6 and PAX7 DNA binding domains As multiple PAX proteins can bindthe same DNA sequence [] this provides further evidence for PAX9 regulation of these mRNAs These analyses therefore support the hypothesis that PAX9 regulates the levels of themRNAs identified in our RNAseq datasetIn the RNAseq dataset many of the differentially expressed mRNAs have knownroles in nucleolar function For example have appeared in other genomewidesiRNA screens for nucleolar function S1 Table [ ] Additionally of the differentially expressed mRNAs code for proteins designated as nucleolar in at least of nucleolar databases S1 Table [“] This is a significant enrichment in the expected number of nucleolar proteins assuming that nucleolar proteins make up only of the proteins inhuman cells [] Surprisingly expression of most of the mRNAs encoding nucleolar proteinsPLOS Genetics 101371journalpgen1008967 August PLOS GENETICS 0cPaired Box PAX9 regulates human ribosome biogenesis was downregulated upon PAX9 depletion indicating that PAX9 is requiredto maintain normal levels of many mRNAs whose protein products are destined for functionin the nucleolus S1 TableTo determine the mechanism of PAX9™s function in SSU biogenesis we chose candidatesfrom the RNAseq dataset to follow up on in greater detail The mRNA levels for the candidates were all downregulated after PAX9 depletion and all code for nucleolar proteins Fig 2Band S1 Table [“] Four of the candidates RPS6eS6 RPS9uS4 RPS28eS28 and FBLwere chosen on the basis of literature suggesting a role for these proteins in SSU prerRNAprocessing in HeLa cells [ ] Additionally it was pertinent to analyze RPL5uL18 as it hasa known role in the p53dependent nucleolar stress response [] qRTPCR confirmed theRNAseq results with reduced levels of each mRNA when PAX9 is depleted in MCF10A cellsFig 2C Interestingly depletion of either RPS9uS4 or RPS28eS28 resulted in a decrease innucleolar number from “ to only in our original siRNA screen [] Therefore it is possible that the mechanism through which PAX9 depletion results in decreased nucleolar numberrelies upon reduced expression of RPS9uS4 andor RPS28eS28 Fig 2AWe were able to confirm that depletion of of the tested candidates RPS6eS6 RPS9uS4 RPS28eS28 and FBL in MCF10A cells resulted in prerRNA processing defects similarto that of PAX9 depletion Fig 2D and 2E S5 Fig Only RPL5uL18 did not give the 30Sincrease characteristic of PAX9 depletion although this was expected given its known role inLSU prerRNA processing [] Additionally depletion of several of the candidates individually resulted in significantly decreased global protein synthesis by the puromycin incorporation assay similar to the effect seen after PAX9 depletion Fig 2F and 2G Puromycinincorporation was also reduced after RPS6eS6 depletion although it was not statistically significant Fig 2F and 2G Therefore PAX9 may function as a transcription factor to directly orindirectly increase the expression of RPS6eS6 RPS9uS4 RPS28eS28 andor FBL Fig 2AAs the proteins encoded by these mRNAs are required for SSU ribosome biogenesis Fig their depletion after PAX9 knockdown is a plausible mechanism through which PAX9 regulates prerRNA processing and global protein synthesisRNAPII ChIPseq analysis reveals decreased transcription of mRNAsencoding nucleolar proteins after PAX9 depletionAs the RNAseq analysis confirmed that levels of nucleolar mRNAs were decreased afterPAX9 depletion Fig we sought to map how RNAPII distributes on genes using RNAPIIChIPseq as a readout of transcription Through this approach we aimed to untangle the effectsof PAX9 depletion on RNAPII transcription from its effects on mRNA stability since RNAPIIChIPseq is able to detect genomewide changes in RNAPII occupancy As PAX proteins areable to both activate and repress target protein expression [] we have included genes withboth increased and decreased RNAPII occupancy in this analysis Approximately mRNAswere differentially occupied by RNAPII upon PAX9 knockdown compared to the nontargeting control siRNA siNT in MCF10A cells fold change cutoff � or � and MaxTags � S2 Table Of these had decreased RNAPII occupancy consistent with PAX9 acting as a transcriptional driver of these mRNAsTo assess the validity of the RNAPII ChIPseq dataset we again used CentriMo to identifypotential PAX9 DNAbinding sites in the kb upstream of the genes with differentialRNAPII occupancy [] Searching for the ™ CGCGTGACCG ™ PAX9 binding motif definedin [] revealed possible sites in the bp upstream of the differentially occupiedgenes Also the known PAX9 DNA binding motifs CD192Ains ™GCGTGACCA3™ ande5 ™GCGGAACGG3™ had and binding sites in these sequences respectivelyPLOS Genetics 101371journalpgen1008967 August PLOS GENETICS 0cPaired Box PAX9 regulates human ribosome biogenesisAdditionally Analysis of Motif Enrichment AME[] identified the PAX5 and PAX6 DNAbinding motifs as being significantly enriched in the kb of sequence upstream of the genes p � Since multiple PAX proteins can bind the same motif [] this suggests thatthis dataset does contain mRNAs that are regulated by PAX9 Of the differentially occupied genes have also been shown to be differentially regulated by PAX9 directly in PAX9ChIPseq experiments on E125 WT vertebral column murine tissue Fig 3A and S2 Table[] These analyses confirm the ability of RNAPII ChIPseq to detect changes in RNAPIImediated transcription after PAX9 knockdownBased on the hypothesis that PAX9 acts as an RNAPII transcription factor for regulators ofnucleolar function Fig we expected to detect changes in the RNAPIImediated transcription of a number of mRNAs encoding nucleolar proteins after PAX9 depletion IndeedmRNAs coding for nucleolar proteins were enriched with of the genes with differentialRNAPII occupancy coding for nucleolar proteins in at least one of three databases S2Table [“] This is again higher than would be expected assuming that nucleolar proteins acc

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You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: Dysregulation of ribosome production can lead to a number of developmental disorderscalled ribosomopathies Despite the ubiquitous requirement for these cellular machinesused in protein synthesis ribosomopathies manifest in a tissuespecific manner with manyaffecting the development of the face Here we reveal yet another connection between craniofacial development and making ribosomes through the protein Paired Box PAX9PAX9 functions as an RNA Polymerase II transcription factor to regulate the expression ofproteins required for craniofacial and tooth development in humans We now expand thisfunction of PAX9 by demonstrating that PAX9 acts outside of the cell nucleolus to regulatethe levels of proteins critical for building the small subunit of the ribosome This function ofPAX9 is conserved to the anism Xenopus tropicalis an established model for humanribosomopathies Depletion of pax9 leads to craniofacial defects due to abnormalities inneural crest development a result consistent with that found for depletion of other ribosomebiogenesis factors This work highlights an unexpected layer of how the making of ribosomes is regulated in human cells and during embryonic developmentAuthor summaryWe are only beginning to understand the complex process of making human ribosomesthe cellular machines critical for all protein synthesis In humans making a ribosomerequires hundreds of regulatory factors to ensure proper cellular growth and development Dysregulation of this process can lead to a number of tissue specific disorderstermed ribosomopathies Here we have discovered a new role for the protein PairedBox PAX9 in making human ribosomes While PAX9 has traditionally been known toa1111111111a1111111111a1111111111a1111111111a1111111111 ACCESSCitation FarleyBarnes KI Deniz E Overton MMKhokha MK Baserga SJ Paired Box PAX9 the RNA polymerase II transcription factorregulates human ribosome biogenesis andcraniofacial development PLoS Genet e1008967 101371journalpgen1008967Editor Paul A Trainor Stowers Institute forMedical Research UNITED STATESReceived February Accepted June Published August Copyright FarleyBarnes This is an access distributed under the terms ofthe Creative Commons Attribution License whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are creditedData Availability Statement All RNAseq andRNAPII ChIPseq files are available from the GeneExpression Omnibus GEO database and areaccessible through the GEO series accessionnumber GSE154764 wwwncbinlmnihgovgeoqueryacccgiaccGSE154764 Allnumerical data that underlies graphs or summarystatistics is available in the Supporting InformationS4 TablePLOS Genetics 101371journalpgen1008967 August PLOS GENETICS 0cFunding This work was supported by the NationalInstitutes of Health R01GM115710R01GM122926 and R35GM131687 to SJBR01HD081379 to MKK T32GM007223 to SJB andKIF F31DE026946 to KIF and a pilot grant from theYale Cancer Center to SJB The funders had no rolein study design data collection and analysisdecision to publish or preparation of themanuscriptCompeting interests The authors have declaredthat no competing interests existPaired Box PAX9 regulates human ribosome biogenesisplay a role in regulating the levels of proteins required for craniofacial and tooth development in humans we expand this function of PAX9 by showing that PAX9 acts outside ofthe cell nucleolus to regulate the levels of proteins critical for building the small subunit ofthe ribosome In addition we show that this function is conserved to the model anismXenopus tropicalis This link between PAX9™s role in craniofacial development and inribosome biogenesis may lead to new insights into the pathogenesis of these PAX9 mutations in humansIntroductionWhen ribosome biogenesis is genetically disrupted in humans a number of surprisingly tissuespecific disorders called ribosomopathies arise For example genetic disruption of theTCOF1 POLR1C or POLR1D genes in the ribosomopathy Treacher Collins syndrome TCSOMIM results in reduced preribosomal RNA prerRNA transcription [“] Interestingly while these mutations each affect the global process of prerRNA transcriptionpatients have specific defects in craniofacial development TCS patients present with hypoplasia of the facial bones micrognathia with or without cleft palate narrowing of the ear canaland bilateral conductive hearing loss [“] Modeling the disease in mice has shown that thistissue specificity arises from differential tissue susceptibility to p53 levels [] p53 levels are stabilized upon disruptions in ribosome biogenesis when free ribosomal proteins bind to MDM2the E3 ligase for p53 [ ] This stabilization of p53 leads to apoptosis of the developing neural crest cells ultimately resulting in the mandibulofacial dysostosis of TCSTCS is not the only ribosomopathy to affect craniofacial development For example Diamond Blackfan anemia DBA OMIM is characterized by anemia low reticulocytecount and elevated erythrocyte adenosine deaminase activity [ ] However DBA patientsoften also have craniofacial anomalies and cleft palate reviewed in [] Additionally theribosomopathy Acrofacial dysostosis Cincinnati type OMIM is caused by mutationsin POLR1A that inhibit prerRNA transcription resulting in craniofacial defects [] Amore precise understanding of the role of the factors involved in human ribosome biogenesiscan therefore shed light on the molecular mechanisms underlying aberrant craniofacialdevelopmentThe process of making the cellular machines required for protein synthesis called ribosomebiogenesis includes a large number of factors that work together to control cell growth anddevelopment In humans these proteins are still being defined Previous studies have shownthat ribosome biogenesis begins with the transcription of the tandemly repeated ribosomalDNA rDNA into the 47S polycistronic prerRNA Fig 1A The prerRNA is further modified and processed to create the mature 18S 58S and 28S rRNAs These rRNAs are incorporated along with the 5S rRNA and ribosomal proteins into the small SSU and large LSUsubunits of the ribosome The 47S prerRNA is transcribed by RNA polymerase I RNAPIwhile the 5S rRNA is transcribed by RNA polymerase III RNAPIII Various assembly factorsand the ribosomal proteins are transcribed by RNA polymerase II RNAPII In addition torequiring all RNA polymerases synthesis and assembly of functional ribosomes integrates anumber of cellular signaling pathways to ensure proper regulation of this essential process inresponse to stimuliThe complex development of the face is controlled by a number of proteins including several RNAPII transcription factors such as Paired Box PAX9 PAX9 belongs to a family oftranscription factors that play key roles in anogenesis and neural crest cell development byPLOS Genetics 101371journalpgen1008967 August PLOS GENETICS 0cPaired Box PAX9 regulates human ribosome biogenesisPLOS Genetics 101371journalpgen1008967 August PLOS GENETICS 0cPaired Box PAX9 regulates human ribosome biogenesisFig PAX9 is required for human ribosome biogenesis A Ribosome biogenesis at a glance The tandemly repeated ribosomal DNA rDNA istranscribed into the 47S polycistronic preribosomal RNA prerRNA by RNA Polymerase I RNAPI This 47S prerRNA is processed throughmultiple steps to form the mature 18S 58S and 28S rRNAs which are incorporated into the small and large subunits of the ribosome along with the5S rRNA and ribosomal proteins Ribosomes perform cytoplasmic cellular protein synthesis through the translation of mRNAs B PAX9 depletionreduces nucleolar number from “ to only in MCF10A cells Left panel Nuclei stained in Hoechst are shown in blue Nucleoli are shown in pinkand stained with antifibrillarin antibody as in [] siGFP top was used as a negative control “ nucleolinucleus and siUTP4 middle was usedas a positive control nucleolusnucleus siPAX9 is shown at the bottom Right panel Quantitation of the number of nucleoli per nucleus for siGFPtop siUTP4 middle or siPAX9 bottom C PAX9 is not required for RNAPI transcription in MCF10A cells A dualluciferase reporter assay wasused to quantify luminescence after siRNA depletion of PAX9 The plasmids are pHrDIRESLuc firefly to report RNAPI transcription and aRenilla transfection control as in [] The ratio of firefly to Renilla luciferase was normalized to the siNT control N siNOL11 was used as apositive control [] Data were analyzed by Student™s t test using GraphPad Prism ���� p � D PAX9 is required for pre18S rRNAprocessing in MCF10A cells Left Schematic of prerRNA processing steps in human cells Intermediates detected by probe P3 are indicated with ablack box below Center Northern blot with probe P3 A probe for the 7SL RNA was used as a loading control Intermediates detected by probe P3 areshown to the right of the northern blot Negative controls were mock no siRNA and siNT nontargeting siUTP4 was used as a positive control[] Right Quantitation by RAMP [] of probe P3 upper and 7SL lower northern blots Graph is mean ± SEM N Data were analyzed by2way ANOVA using GraphPad Prism ���� p � ��� p � �� p � and � p � PTP indicates the 47S 45S and 43S processingintermediates E PAX9 siRNA depletion in MCF10A cells results in an increased ratio of 28S18S by Agilent BioAnalyzer Significance was calculatedby Student™s t test in GraphPad Prism where �� p � F PAX9 siRNA depletion in MCF10A cells results in decreased global protein synthesis asassessed by the puromycin incorporation assay [] A representative western blot using an antipuromycin antibody with a β actin loading control isshown to the left Protein was harvested after knockdown for hours using the indicated siRNAs Mock indicates no siRNA and Mock μMindicates no siRNA and half the concentration of puromycin siNT nontargeting was used a positive control Quantitation of replicates using cellsof different passage numbers is shown to the right Significance was calculated by Oneway ANOVA in GraphPad Prism where ���� p � and��� p � G PAX9 depletion in MCF10A cells results in decreased 40S 60S and 80S ribosome subunit levels Representative polysome profile ofMCF10A cells depleted using siRNAs targeting either PAX9 red or a nontargeting siNT blue control Equal amounts of protein were loaded oneach gradient This experiment was performed times using cells of different passage numbers101371journalpgen1008967g001controlling gene expression [ ] In humans mutations in PAX9 cause tooth agenesis aswell as hair loss [“] and reviewed in [] Indeed PAX9 mutations are the most prevalent mutation in patients with nonsyndromic tooth agenesis including oligodontia []Additionally mice homozygous for a partial deletion of Pax9 likely null have craniofacialmalformations including cleft palate skeletal abnormalities and arrested tooth developmentand die a few hours after birth [] Pax9 mice also exhibit a range of cardiac malformations[] another commonly impacted tissue in ribosomopathies [] While some research hasbeen done to identify the signaling pathways regulated by PAX9 attempts to correct the developmental defects have been only partially successful [“] Therefore further studies areneeded to identify all factors regulated by PAX9 in order to understand PAX9™s role in craniofacial developmentOur work ties together PAX9 and ribosome biogenesis filling in some gaps in our knowledge of the many cell growth and signaling pathways influenced by PAX9 depletion We originally identified PAX9 as a potential regulator of ribosome biogenesis in an siRNA screen forproteins required to maintain nucleolar number [] Probing PAX9™s specific role in makingribosomes in human tissue culture cells we discovered that PAX9 is required both for the prerRNA processing that produces the small subunit 18S rRNA and for global protein synthesisWe employed the genomewide transcriptomics analysis RNAseq to define PAX9 dependentmRNAs necessary for making ribosomes Several of the differentially expressed mRNAsincluding several ribosomal proteins were further examined to pinpoint roles for these proteins in prerRNA processing and global protein synthesis Finally given an established rolefor PAX9 in human craniofacial development we sought to model this disease in Xenopus tropicalis X tropicalis embryos an established model of human ribosomopathies Depletion ofPax9 in X tropicalis did indeed alter craniofacial patterning as well as neural crest development a migratory cell population that plays a major role in establishing craniofacial structureX tropicalis embryos depleted of Pax9 also show defective prerRNA processing These resultsshed light on the plethora of factors whose expression is regulated by PAX9 and the doorto likely connections between PAX9™s role in craniofacial development and human ribosomebiogenesisPLOS Genetics 101371journalpgen1008967 August PLOS GENETICS 0cPaired Box PAX9 regulates human ribosome biogenesisResultsPAX9 depletion disrupts small subunit ribosome biogenesisPreviously we performed an siRNA screen for new regulators of nucleolar number in humanMCF10A cells [] We identified PAX9 by this screening approach as a protein that whendepleted reduced the number of nucleoli per nucleus from “ to only Fig 1B As in []cells were fixed after hours of knockdown using pools of siRNAs targeting siGFP as a negative control siUTP4 as a positive control or siPAX9 This transient knockdown approach successfully depleted PAX9 S1A and S1B Fig The MCF10A cells were then stained with anantibody to fibrillarin FBL a nucleolar protein to detect nucleoli and with HOECHST todetect nuclei A CellProfiler [] pipeline was used to quantify the number of nucleoli per cellnucleus which shifted from “ in siGFP control cells to only in siPAX9 and siUTP4treatedcells We had previously used oligonucleotide deconvolution to additionally confirm thatPAX9 depletion leads to reductions in nucleolar number [] Using this approach individualdepletion of PAX9 using of the siRNAs from the original PAX9 pool reduced the numberof nucleoli from “ to only per cell nucleus [ ] As the siRNA screen served as a phenotypic readout of nucleolar function [] we hypothesized that PAX9 plays a role in humanribosome biogenesis through its function as an RNAPII transcription factorWe sought to investigate the extent to which PAX9 depletion affects human ribosome biogenesis using a panel of assays The first assay probes PAX9™s role in RNAPI transcriptionusing a dualluciferase reporter system previously published by our laboratory and others [ ] As PAX9 is a known RNAPII transcription factor it was relevant to test it for a possibleadditional role in RNAPI transcription In this reporter assay the ratio of firefly luciferasewhich is under the control of the rDNA promoter and measures RNAPI transcription wasquantified relative to a Renilla luciferase transfection control Relative to a nontargeting control siRNA siNT siRNAs targeting PAX9 had no significant effect on RNAPI transcriptionlevels after hours of knockdown in MCF10A cells Fig 1C Mock no siRNA and siNOL11were used as negative and positive controls respectively PAX9 is therefore not required forRNAPI transcription in MCF10A cellsNorthern blotting was used to define PAX9™s role in prerRNA processing In humans prerRNA processing occurs via a number of different pathways Fig 1D left and requires a number of transacting factors We therefore employed different probes to pinpoint any prerRNA processing defects occurring after PAX9 depletion in MCF10A cells S2A Fig After hours of siRNA knockdown PAX9 depletion resulted in a significant increase in the 30S prerRNA intermediate as well as a decrease in the levels of its 21S processing product relative tothe nontargeting siRNA siNT Fig 1D middle and right and S2 Fig Additionally 41S levelswere decreased relative to the primary processing transcript 47S plus the 45S and 43S processing intermediates herein termed the primary transcript plus or PTP Fig 1D middle andright and S2 Fig Quantitation of the ratios of each intermediate relative to its precursor in theprocessing pathway by Ratio Analysis of Multiple Precursors RAMP [] confirmed the statistical significance of these results Fig 1D right and S2 Fig These effects were also significant relative to a 7SL loading control Fig 1D right and S2 Fig Because the 30S and 21Sintermediates are both precursors to the 18S rRNA PAX9 is required for SSU biogenesis Thesame prerRNA processing defect was also detected in human embryonic kidney HEK293FTand colon carcinoma RKO cells depleted of PAX9 indicating that PAX9™s role in ribosomebiogenesis is conserved among diverse human cell lines S1 FigBecause the prerRNA processing defects indicate aberrant SSU biogenesis we sought todetermine the extent to which PAX9 depletion affects the production of the mature 18S rRNAAgilent BioAnalyzer quantitation shows an increase in the ratio of 28S to 18S Fig 1EPLOS Genetics 101371journalpgen1008967 August PLOS GENETICS 0cPaired Box PAX9 regulates human ribosome biogenesisCombined with the northern blot results indicating defects in the processing of the precursorsto the 18S rRNA this result is consistent with a predicted reduction in 18S rRNA levels Takentogether these results argue that PAX9 is required likely indirectly for the biogenesis of thesmall subunit of the ribosome which contains the 18S rRNAWe also utilized a puromycin incorporation assay to test the extent to which PAX9 depletion alters the final product of ribosome biogenesis global cellular translation Fig 1F Cellswith or without PAX9 depletion are treated with a low dose μM of puromycin which isincorporated into all nascent peptides produced during a hour pulse [] Western blottingfor incorporated puromycin shows decreased protein synthesis after hours of PAX9 depletion relative to a nontargeting siRNA control siNT Fig 1F Mock μM puromycin withno siRNA and Mock at a halfdose of puromycin μM were used as negative controls Fig1F These results confirm that PAX9 depletion leads to reduced protein synthesis consistentwith a role for PAX9 in SSU biogenesisBecause the above assays indicated a role for PAX9 in small subunit prerRNA processingand global protein synthesis we also tested the extent to which PAX9 depletion is required forribosomal subunit biogenesis and assembly Using polysome profiling we determined thatPAX9 depletion does result in significantly decreased SSU 40S levels in MCF10A cells Fig1G consistent with the reduction in 18S rRNA levels seen in Fig 1E Additionally 60S and80S polysome fractions also showed significant decreases after PAX9 knockdown Fig 1GInterestingly both here and in previous experiments MCF10A cells do not demonstraterobust polysome fractions using this technique [] Regardless we conclude that PAX9 depletion results in decreased SSU biogenesis that impacts the assembly and function of theribosomeAs disruptions in ribosome biogenesis result in interruptions in the cell cycle [“] wealso examined the extent to which PAX9 depletion changed the distribution of MCF10A cellswithin the cell cycle using flow cytometry S3 Fig Relative to a siNT control depletion ofPAX9 for hours resulted in a minor increase in the proportion of cells in G1 phase of thecell cycle but this was not statistically significant S3 Fig siRNAs targeting the ribosome biogenesis factor NOL11 were used as a positive control and depletion of this protein resulted inan increase in the proportion of cells in G2 S3 Fig consistent with previous findings [] Inall these assays allowed us to conclude that PAX9 regulates human ribosome biogenesisRNAseq analysis upon PAX9 depletion reveals decreased levels ofnucleolar mRNAs responsible for small subunit maturationAs PAX9 is a known RNAPII transcription factor we hypothesized that PAX9 works indirectlyto modulate SSU biogenesis Fig 2A This is consistent with a nuclear but not nucleolar localization of PAX9 in existing databases [“] PAX9 may act directly as a transcription factorfor nucleolar proteins or indirectly for proteins that affect the expression or function of nucleolar proteins To test the hypothesis that PAX9 affects nucleolar protein expression through itsfunction as a RNAPII transcription factor we used RNAseq in MCF10A cells to define the setof mRNAs that were differentially expressed after PAX9 depletion Relative to a nontargetingcontrol siRNA siNT PAX9 depletion resulted in the differential expression of over mRNAs fold change � or � q � Fig 2B and S1 Table Approximately half of these were reduced in their levels consistent with the hypothesis that PAX9 acts as a transcription factor to drive their expressionWhen considered as a whole the RNAseq dataset is enriched for several pathways knownto be regulated by PAX9 S4A Fig Ingenuity Pathway Analysis [] of the differentiallyexpressed mRNAs reveals enrichment of both WntCa2 signaling differential expression ofPLOS Genetics 101371journalpgen1008967 August PLOS GENETICS 0cPaired Box PAX9 regulates human ribosome biogenesisPLOS Genetics 101371journalpgen1008967 August PLOS GENETICS 0cPaired Box PAX9 regulates human ribosome biogenesisFig RNAseq transcriptomics analysis in human tissue culture cells reveals changes in the expression levels of over nucleolar mRNAs afterPAX9 knockdown A Schematic of how PAX9 would act as a RNAPII transcription factor to drive the levels of mRNAs required for making the smallsubunit SSU of the ribosome In the cell nucleus PAX9 binds to DNA to affect the transcription of mRNAs that either encode nucleolar proteinsdirect solid arrow or to transcribe mRNAs that affect the levels of mRNAs encoding nucleolar proteins indirect dotted arrows The resulting mRNAsare translated in the cytoplasm into proteins that function in SSU prerRNA processing in the nucleolus B RNAseq analysis after PAX9 siRNAdepletion in MCF10A cells reveals decreased levels of mRNAs encoding nucleolar proteins Relative to a nontargeting siRNA control siNT PAX9depletion resulted in differential expression of mRNAs fold change � or and FDR � Of these mRNAs had a decreased foldchange � and of those mRNAs code for proteins designated as nucleolar in at least one of three databases [“] Of the mRNAs whoselevels were decreased and that also code for nucleolar proteins were chosen as candidates for followup studies C qRTPCR confirms reduced mRNAlevels of the RNAseq candidates after PAX9 siRNA knockdown in MCF10A cells After depletion using siRNAs targeting either PAX9 or a nontargeting control siRNA siNT the levels of the indicated mRNAs were quantified by qRTPCR using primers to each target gene relative to a 7SLcontrol and siNT Data are shown as mean ± SEM Three replicates using cells of different passage numbers with technical replicates each wereperformed Significance was calculated by Oneway ANOVA using GraphPad Prism where ���� p � D Depletion of of the candidatemRNAs RPS6eS6 RPS9uS4 RPS28eS28 and FBL individually results in the same prerRNA processing defect as PAX9 siRNA depletion in MCF10Acells Representative northern blot after knockdown of the indicated siRNAs using probe P3 A probe for the 7SL RNA was used as a loading control PrerRNA processing intermediates detected by probe P3 are shown to the right of the northern blot PTP indicates the 47S 45S and 43S prerRNAprocessing intermediates E Quantitation of northern blots using probe P3 as shown in Fig 2D using RAMP [] Graph is mean ± SEM N Datawere analyzed using 2way ANOVA in GraphPad Prism where ���� p � ��� p � and �� p � Quantitation relative to the 7SL loadingcontrol is shown in S5 Fig F siRNA depletion of RNAseq candidates in MCF10A cells results in decreased global protein synthesis After hoursof knockdown with the indicated siRNAs MCF10A cells were pulsed with puromycin for hour and protein was harvested Western blotting with anantipuromycin antibody as well as a β actin loading control was carried out representative western blots shown to the left Mock mock at half theconcentration of puromycin μM and siNT nontargeting siRNAs were used as negative controls G Quantitation of three replicates usingMCF10A cells of different passage numbers of the puromycin incorporation assays following depletion with the indicated siRNAs relative to the siNT andβ actin loading controls is shown as mean ± SEM N Significance was calculated by Student™s ttest using GraphPad Prism where ���� p � ���p � and � p � 101371journalpgen1008967g002 pathway members p x ˆ’ S4B Fig and Wntβcatenin signaling differentialexpression of pathway members p x ˆ’ S4C Fig In cells depleted of PAX9expression levels are increased for many of the mRNAs in the Wnt signaling pathway This isconsistent with previous results suggesting a role for PAX9 in the negative regulation of Wntsignaling [ ]As an additional validation of the RNAseq dataset we determined that the genomesequences kb upstream of the start sites of the differentially expressed mRNAs containmultiple potential PAX9 binding sites making these mRNAs candidates for direct transcriptional regulation by PAX9 Scanning for known PAX9 binding sequences [™SGTCACGCWTGANTGMA3™ ™CGCGTGACCG3™ [] CD192Ains ™GCGTGACCA3™ and e5 ™GCGGAACGG3™] in the kb upstream of the mRNAs using CentriMo analysis []reveals and potential PAX9 binding sites respectively in the kbupstream of the mRNAs This number of potential binding sites upstream of the mRNAs sites per gene is similar to that observed in PAX9 ChIP experiments in the vertebral column of E125 mice sites per gene [] Additionally CentriMo enrichmentanalysis [] of the sequence kb upstream of each of the differentially expressedmRNAs reveals significant enrichment of different DNA binding sequences including thePAX3 PAX5 PAX6 and PAX7 DNA binding domains As multiple PAX proteins can bindthe same DNA sequence [] this provides further evidence for PAX9 regulation of these mRNAs These analyses therefore support the hypothesis that PAX9 regulates the levels of themRNAs identified in our RNAseq datasetIn the RNAseq dataset many of the differentially expressed mRNAs have knownroles in nucleolar function For example have appeared in other genomewidesiRNA screens for nucleolar function S1 Table [ ] Additionally of the differentially expressed mRNAs code for proteins designated as nucleolar in at least of nucleolar databases S1 Table [“] This is a significant enrichment in the expected number of nucleolar proteins assuming that nucleolar proteins make up only of the proteins inhuman cells [] Surprisingly expression of most of the mRNAs encoding nucleolar proteinsPLOS Genetics 101371journalpgen1008967 August PLOS GENETICS 0cPaired Box PAX9 regulates human ribosome biogenesis was downregulated upon PAX9 depletion indicating that PAX9 is requiredto maintain normal levels of many mRNAs whose protein products are destined for functionin the nucleolus S1 TableTo determine the mechanism of PAX9™s function in SSU biogenesis we chose candidatesfrom the RNAseq dataset to follow up on in greater detail The mRNA levels for the candidates were all downregulated after PAX9 depletion and all code for nucleolar proteins Fig 2Band S1 Table [“] Four of the candidates RPS6eS6 RPS9uS4 RPS28eS28 and FBLwere chosen on the basis of literature suggesting a role for these proteins in SSU prerRNAprocessing in HeLa cells [ ] Additionally it was pertinent to analyze RPL5uL18 as it hasa known role in the p53dependent nucleolar stress response [] qRTPCR confirmed theRNAseq results with reduced levels of each mRNA when PAX9 is depleted in MCF10A cellsFig 2C Interestingly depletion of either RPS9uS4 or RPS28eS28 resulted in a decrease innucleolar number from “ to only in our original siRNA screen [] Therefore it is possible that the mechanism through which PAX9 depletion results in decreased nucleolar numberrelies upon reduced expression of RPS9uS4 andor RPS28eS28 Fig 2AWe were able to confirm that depletion of of the tested candidates RPS6eS6 RPS9uS4 RPS28eS28 and FBL in MCF10A cells resulted in prerRNA processing defects similarto that of PAX9 depletion Fig 2D and 2E S5 Fig Only RPL5uL18 did not give the 30Sincrease characteristic of PAX9 depletion although this was expected given its known role inLSU prerRNA processing [] Additionally depletion of several of the candidates individually resulted in significantly decreased global protein synthesis by the puromycin incorporation assay similar to the effect seen after PAX9 depletion Fig 2F and 2G Puromycinincorporation was also reduced after RPS6eS6 depletion although it was not statistically significant Fig 2F and 2G Therefore PAX9 may function as a transcription factor to directly orindirectly increase the expression of RPS6eS6 RPS9uS4 RPS28eS28 andor FBL Fig 2AAs the proteins encoded by these mRNAs are required for SSU ribosome biogenesis Fig their depletion after PAX9 knockdown is a plausible mechanism through which PAX9 regulates prerRNA processing and global protein synthesisRNAPII ChIPseq analysis reveals decreased transcription of mRNAsencoding nucleolar proteins after PAX9 depletionAs the RNAseq analysis confirmed that levels of nucleolar mRNAs were decreased afterPAX9 depletion Fig we sought to map how RNAPII distributes on genes using RNAPIIChIPseq as a readout of transcription Through this approach we aimed to untangle the effectsof PAX9 depletion on RNAPII transcription from its effects on mRNA stability since RNAPIIChIPseq is able to detect genomewide changes in RNAPII occupancy As PAX proteins areable to both activate and repress target protein expression [] we have included genes withboth increased and decreased RNAPII occupancy in this analysis Approximately mRNAswere differentially occupied by RNAPII upon PAX9 knockdown compared to the nontargeting control siRNA siNT in MCF10A cells fold change cutoff � or � and MaxTags � S2 Table Of these had decreased RNAPII occupancy consistent with PAX9 acting as a transcriptional driver of these mRNAsTo assess the validity of the RNAPII ChIPseq dataset we again used CentriMo to identifypotential PAX9 DNAbinding sites in the kb upstream of the genes with differentialRNAPII occupancy [] Searching for the ™ CGCGTGACCG ™ PAX9 binding motif definedin [] revealed possible sites in the bp upstream of the differentially occupiedgenes Also the known PAX9 DNA binding motifs CD192Ains ™GCGTGACCA3™ ande5 ™GCGGAACGG3™ had and binding sites in these sequences respectivelyPLOS Genetics 101371journalpgen1008967 August PLOS GENETICS 0cPaired Box PAX9 regulates human ribosome biogenesisAdditionally Analysis of Motif Enrichment AME[] identified the PAX5 and PAX6 DNAbinding motifs as being significantly enriched in the kb of sequence upstream of the genes p � Since multiple PAX proteins can bind the same motif [] this suggests thatthis dataset does contain mRNAs that are regulated by PAX9 Of the differentially occupied genes have also been shown to be differentially regulated by PAX9 directly in PAX9ChIPseq experiments on E125 WT vertebral column murine tissue Fig 3A and S2 Table[] These analyses confirm the ability of RNAPII ChIPseq to detect changes in RNAPIImediated transcription after PAX9 knockdownBased on the hypothesis that PAX9 acts as an RNAPII transcription factor for regulators ofnucleolar function Fig we expected to detect changes in the RNAPIImediated transcription of a number of mRNAs encoding nucleolar proteins after PAX9 depletion IndeedmRNAs coding for nucleolar proteins were enriched with of the genes with differentialRNAPII occupancy coding for nucleolar proteins in at least one of three databases S2Table [“] This is again higher than would be expected assuming that nucleolar proteins acc Answer:

Thyroid_Cancer

The role of serum inflammatory cytokines andberberine in the insulin signaling pathwayamong women with polycystic ovarysyndromeHongying Kuang12˜¯ Yuwei Duan34˜¯ Dan LiID5a Yanwen Xu34b Wenxia Ai2 Wei Li1Ying Wang1 Sha Liu2 Mushan Li2 Xiaoqiu Liu2 Manqi Shao2 The First Affiliated Hospital of Heilongjiang University of Chinese Medicine Harbin China HeilongjiangUniversity of Chinese Medicine Harbin China The First Affiliated Hospital of Sun Yatsen UniversityGuangzhou China Guangdong Provincial Key Laboratory of Reproductive Medicine Guangzhou China Department of Acupuncture the Third Affiliated Hospital Beijing University Of Chinese Medicine BeijingChina˜¯ These authors contributed equally to this worka Current address The First Affiliated Hospital of Sun Yatsen University Guangzhou Chinab Current address Reproductive Medicine Center The First Affiliated Hospital Sun Yatsen UniversityGuangzhou Guangdong China 3509437qqcom DL Xuyanwenlivecn YXAbstractObjectiveTo study the role of selected serum inflammatory cytokines and berberine in the insulin signaling pathway among women with polycystic ovary syndrome PCOSMethodsSelected serum inflammatory cytokines were analyzed in the p cells which were interfered by berberine from infertile women who were to be treated with In Vitro FertilizationIVF Intracytoplasmic Sperm InjectionEmbryo Transfer icsiet Among them patientshad PCOS infertility and were nonPCOS patients whose infertility resulted from fallopian tube and male factors The elisa method was used to detect the changes in the expression levels of inflammatory factors in the cells The correlations between the seruminflammatory cytokine expression levels and the corresponding clinical hormones were analyzed The changes in the expression mRNA and protein levels of the serum inflammatorycytokines were studied by realtime quantitative PCR and protein printing Fluorescencemicroscope and flow cytometry were used to detect the glucose uptake capacity of ovariangranulosa cells in PCOS patients under the action of insulin after berberineResultsIn the PCOS group IL17a P IL1Ra P00001 and IL6 P were significantly higher than those in the nonPCOS group In the nonPCOS group AMH level wasnegatively correlated with inflammatory cytokines IL17a r 0819P IL1a r a1111111111a1111111111a1111111111a1111111111a1111111111 ACCESSCitation Kuang H Duan Y Li D Xu Y Ai W Li W The role of serum inflammatorycytokines and berberine in the insulin signalingpathway among women with polycystic ovarysyndrome e0235404 101371journalpone0235404Editor Meijia Zhang China Agricultural UniversityCHINAReceived November Accepted June Published August Copyright Kuang This is an access distributed under the terms of theCreative Commons Attribution License whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are creditedData Availability Statement All relevant data arewithin the manuscript and its SupportingInformation filesFunding This work was supported by theOutstanding talents funding project of HeilongjiangUniversity of Chinese Medicine China [grantnumber 2018JC05] the Foundation for youngacademic leaders of Heilongjiang University ofChinese Medicine China [grant number2018RCD08] and the Foundation for HeilongjiangProvince Postdoctoral Research Startup ChinaPLOS ONE 101371journalpone0235404 August PLOS ONE 0c[grant number LBHQ18119] the Project ofUndergraduate Colleges and Universities YouthInnovation Talents by Education Department ofHeilongjiang Province China [grant numberUNPYST2018227] the Natural ScienceFoundation of Heilongjiang Province China [grantnumber H2016083]Competing interests The authors have declaredthat no competing interests existThe role of serum inflammatory cytokines and berberine in the insulin signaling pathway0716P IL1b r 0678P IL2 r 0765P and IL8r 0705P However in the PCOS group AMH levels were not significantly correlated with the levels of the examined inflammatory cytokines Berberine significantlyreduced the expression level of mTOR mRNA P and increased the expressionlevel of IRS1 mRNA P in the PCOS granule cellsConclusionIn this study we find that the elevated levels of serum inflammatory factors IL17a IL1Raand IL6 cause women to be in a subclinical inflammatory state for a long time Abnormalchanges in inflammatory factors alter their original negative correlations with AMH levelsthereby weakening the metabolism of glycolipids promoting insulin resistance destroyingthe normal ovulation and fertilization system of women leading to polycystic ovary syndrome characterized by menstrual thinning and abnormal ovulation Berberine can improvethe sensitivity of insulin by regulating the signal pathway of insulin receptor substrate1IRS1 and mammalian target of rapamycin mTOR in PCOS patients and achieve a therapeutic effect of treating PCOS IntroductionPolycystic ovary syndrome PCOS is the most common endocrinopathy affecting reproductive aged women It affects reproduction infertility irregular menstruation hirsutism etcmetabolism insulin resistance impaired glucose tolerance etc and psychological characteristics anxiety depression and deterioration in quality of life [] Berberine BBR as a quaternary ammonium salt extracted from plants such as Coptis chinensis and Phellodendronchinensis is currently used in the treatment of diabetes hyperlipidemia and PCOS [] Recentstudies have found that berberine has good hypoglycemic and hypolipidemic effects and is aneffective insulin sensitizer Berberine reduces the synthesis of steroid hormones and theexpression of ovarian aromatase through the action on the hypothalamuspituitaryovarianaxis HPOA improves the insulin resistance status of PCOS patients reduces body weightinduces ovulation and regulates menstruation thereby increasing pregnancy rate and livebirth rate [“] Clinical observations have demonstrated that even with longterm use of berberine its side effects are transient and mild suggesting that BBR is safe to use in PCOSpatients and a very promising plantbased compound for treating PCOS patients []Patients with PCOS have been found to be under a chronic lowgrade inflammation statusincluding high levels of leukocytes and disorder of the proinflammatory cytokines [] Interleukin IL6 is a multipotent cytokine that mediates inflammatory response by controllingcell differentiation migration proliferation and apoptosis thus playing a role in the development of insulin resistance [] IL17a is the œsignature cytokine secreted by the Th17 CD4ve T cell subset Activation of Th17type responses is important not only for host immunecontrol of extracellular bacterial and fungal infections but also associated with chronic inflammation and autoimmunity [] The IL1RA protein is a naturally occurring antagonist of proinflammatory cytokines These proinflammatory cytokines are involved in the underlyingmechanism of various chronic inflammatory conditions [] Therefore we hypothesize thatinflammatory factors are one of the important factors influencing the formation of PCOS andberberine may be an important drug that regulates PCOS inflammatory factorsPLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathwayAntiMu¨llerian hormone AMH is an indicator of a patient™s ability to respond quicklyand efficiently to ovarian reserve In women AMH is produced by the granulosa cells of theovarian follicles and its secretion begins at puberty and lasts until menopause [] PCOS ischaracterized by hyperandrogenism and follicular blockade These two characteristics may bedue to an imbalance between AMH and follicle stimulating hormone [] Circulating insulinlevels in patients with PCOS increase thereby inducing follicular developmental disorderswhich in turn lead to ovarian polycystic ovary formation and higher than normal AMH HighAMH is one of the indicators of stubborn anovulation [] Research has shown that serumAMH level is relatively stable throughout and between menstrual cycles [] More and morestudies have used AMH as a biomarker for PCOS []In the present study we examined the effect and mechanism of serum inflammatory cytokines including IL6 IL17a IL1RA etc on insulin sensitivity of PCOS In addition weassessed whether berberine can improve insulin sensitivity of PCOS by antagonizing the proinflammatory effect of serum inflammatory cytokines Materials and methods SampleSerum samples and granulosa cells were collected from infertile women who were to betreated with In Vitro Fertilization IVF Intracytoplasmic Sperm InjectionEmbryo Transfericsiet Among them patients had PCOS infertility and were nonPCOS patientswhose infertility resulted from fallopian tube and male factors The nonPCOS women wererequired to have regular menstrual cycles The essays were analyzed by Guangdong ProvincialKey Laboratory of Reproductive Medicine at the first affiliated hospital of Sun YatSen University Data collection time “ ParticipantsInclusion criteria age Diagnosis of PCOS according to Rotterdam StandardRotterdam Diagnostic Criteria for PCOS Ovarian Ovulation Disorder Manifests Oligomenorrhea or Amenorrhea Clinically or biochemically determined androgen level increasesmore than 2nmolL or clinically manifested hirsute acne excluding Kaohsiung caused byother diseases Ovarian morphology showed polycystic changes Only of the above items can be met IVFET treatmentExclusion criteria Having orally taken drugs in the past three months that affect the results such as contraceptives or other hormone drugs insulin sensitizers and lipidlowering drugs Suffering from other androgen excess related diseases including congenital adrenal hyperplasia with hydroxylase deficiency androgen secreting tumor excessive use of androgenproducing drugs Cushing syndrome severe insulin resistance thyroid dysfunction andhyperprolactinemia Having a history of anic diseases of heart lung liver kidney and other important ansor patients with mental diseases and other reasons that may interfere the present studyoutcomesPLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathway Ethics statement The study was approved by the ICE for Clinical Research andAnimal Trials of the First Affiliated Hospital of Sun Yatsen University reference [] No and was conducted in accordance with the ethical standards of the Declaration ofHelsinki All participants have signed written informed consent and there were no minors ReagentBerberine was provided by Yuanye BioTechnology Co Ltd Shanghai China2NBDG N13195 Invitrogen DMEMF12 “ Gibco and Fetal bovine serumSV3008702 Hyclone were purchased from Thermo Fisher Scientific Waltham USA Thefollowing is a list of used reagents and their manufacturers™ informationRealtime quantitative PCR kits ROCHE CAT Reverse transcriptase PROMEGA CAT M1701RIPA pyrolysis fluid yantian Bi CAT P0013BBcl2 abcam CAT ab196495BAD abcam CAT ab90435BAX abcam CAT ab182733ACTINβ abcam CAT sc70319 Methods Granulosa cell acquisition and grouping The follicular fluid was centrifuged at rpm for minutes at room temperature The supernatant was discarded and the precipitate after centrifugation was resuspended by phosphate buffer saline PBS The cell suspension was slowly centrifuged to percoll level at room temperature with 1800rpm for 10minAfter centrifugation the white floc was sucked out and washed with PBS three times at1200rpm for 5min at room temperature We discarded the supernatant and added typeIV collagenase and blew it evenly Digestion was performed at ˚C for 1520min We addedthe same amount of culture solution to stop digestion and blew it evenly The filtrate was filtered by micron cell filter and centrifuged at room temperature with 1000rpm for 4minThe supernatant was discarded the cells were suspended again with PBS 3ml erythrocytelysate was added to mix well and the mixture was allowed to stand at ˚C for 10min It wasthen centrifuged at 1000rpm for 4min at room temperature Some white precipitate whichwas verified as granulocytes were observed after centrifugation After the seed plate of PCOSwomen granulosa cells was seeded the fluid was changed once hours After hours solvent or berberine was added for continuous intervention for hours which led to the PCOScontrol group and PCOS berberine intervention group Detection of serum inflammatory cytokines The Merck luminex testing platformwas used to detect serum cytokines according to the manufacturer™s instructions Realtime quantitative PCR At the end of berberine treatment Trizol method wasused to extract total RNA in the cells which was then used to reverse transcription of mRNATotal RNA was isolated from cells using Trizol regent Takara BioInc Tokyo Japan and I mgmRNA was reverse transcribed to cDNA using a reverse transcription Takara Bio Inc TokyoJapan and subjected to quantitative PCR which was performed with the BioRad CFX96Touch TM RealTime PCR Detection System BioRad CA using iQ TM SYBR Green Supermix BioRad CA and threshold cycle numbers were obtained using BioRad CFX ManagerSoftware Real time PCR was carried out using the following conditions min at ˚C min at ˚C and cycles of s at ˚C min at ˚C using 1μl of cDNA reverse transcribed as mentioned above Quantifast SYBR green PCR kit Qiagen Cat No and500nM of forward and reverse primers The following primers were usedPLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathwayβactin GCCGTTCCGAAAGTTGCCTT GAGCGCGGCGATATCATCAAMPK ACAGCCGAGAAGCAGAAACA TTGCCAACCTTCACTTTGCCmTOR CTTAGAGGACAGCGGGGAAG TCCAAGCATCTTGCCCTGAGSTAT3 CTGAAACGGGCTTCAGGTCA TCCAGGGAGAAAGGGAGTCASOCS3 TCTGTCGGAAGACCGTCAAC CCTTAAAGCGGGGCATCGTAIRS1 TCTCTTCCCACGGCGATCTA TGACACTGCGGAAGGAACTCWe used the 2ΔΔCT method to calculate the relative expression level of target genes Western blot experiment After the end of each cell culture the cells were lysedusing RIPA lysate in an ice bath and the protein lysate was collected After the total proteinconcentration was determined by BCA method polyacrylamide gel electrophoresis was carried out according to the standard of μg of total protein per group After the electrophoresisthe protein in the gel was transferred to the polyvinylidene fluoride PVDF membraneblocked with skim milk powder and then incubated with the corresponding dilutedprimary antibody at ˚C overnight and finally labeled with the corresponding horseradish peroxidase After incubation for one hour at room temperature the ECL luminescentsolution was added and detected by autoradiography using a Biored gel imaging systemImmunoreactive protein bands were visualized with enhanced chemiluminescence ECL on aChemiDoc MP Imaging System Blots were scanned and quantified with the Image analysissoftware BioRad Image Lab All specific protein band densities were normalized to βactin amounts Detection of glucose uptake capacity in granulosa cells Granulosa cells of bothnonPCOS and PCOS patients were obtained in vitro for in vitro culture PCOS granulosacells were randomly divided into two groups one group was cultured normally and the othergroup was added with berberine with the final concentration of 100μM After 24h the serumfree culture solution containing 2nbdg with a final concentration of 50μM and insulin of100nM were replaced at ˚C After 1h incubation the difference in green fluorescence intensity of 530nm was compared under fluorescence microscope and the difference in fluorescence density between different groups was compared by using flowjov10 Statistical methodsData were represented by mean±standard deviation SD and SPSS statistical softwarewas used for analysis Analysis of variance ANOVA was used to test betweengroup differences and the pvalues were corrected by Bonferroni p value less than was considered statistically significant Results Analysis of clinical baseline indicators of PCOS group and nonPCOSgroupAs shown in Table compared with the nonPCOS group the PCOS group had significantlyhigher BMIP LHP00001 LHFSHP00001 FPGP AMHP00001 the number of follicles obtainedP00001 ICSI mature eggsP and thenumber of normal fertilization P FSH levels were significantly lower in the PCOSgroup than those in the nonPCOS group P Detection of serum inflammatory cytokinesAs shown in Table IL17a P IL1Ra P00001 and IL6 P in the PCOSgroup were significantly higher than those in the nonPCOS group IL10 P IL13PLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathwayTable Comparison of clinical indicators between PCOS and nonPCOS groupsnonPCOS n PCOS n PvalueAge years oldMenarche ageSBP mmHgDBPmmHgPulse sminBMI kgm2FSH mIUmlLH mIUmlLHFSHE2 pgmlPRL ngmlT nmolLFPG mmollAMHngmleggs obtainedICSI mature eggsNormal fertilized eggsMeanSDMedianMeanSDMedian101371journalpone0235404t001Table Comparison of serum inflammatory cytokines between the PCOS and nonPCOS groupsIL10 pgmlIL13 pgmlIL17a pgmlIL1Ra pgmlIL1α pgmlIL1β pgmlIL2 pgmlIL6 pgmlIL8 pgmlTNFα pgml101371journalpone0235404t002nonPCOSn ±±±±±±±±±±±PCOSn ±±±±±±±±±PvalueP IL1α P IL1β P IL8 P and TNFα P inthe two groups was not statistically significantly different Correlation analysis between serum inflammatory cytokines anddiagnostic clinical indicatorsWe examined the correlation between serum inflammatory cytokines and clinical serum hormones and other levels in PCOS and nonPCOS patients In the nonPCOS group AMH levelwas negatively correlated with inflammatory cytokines IL17ar 0819P IL1ar 0716P IL1br 0678P IL2r 0765P and IL8r 0705P However in the PCOS group AMH levels were not significantlyPLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathwaycorrelated with the levels of these inflammatory cytokines Among the diagnostic clinical indicators except AMH level there was no significant correlation between other clinical indicatorsand the expression level of inflammatory cytokines in the nonPCOS group and the PCOSgroup Tables “Table Correlation analysis of BMI FSH and other inflammatory cytokines in the nonPCOS groupnonPCOSBMIIL10IL13IL17aIL1raIL1aIL1bIL2IL6IL8TNFarP101371journalpone0235404t003FSHPrLHLHFSHrPrTable Correlation analysis of E2 PRL and other inflammatory cytokines in the nonPCOS groupnonPCOSE2PRLTFPGIL10IL13IL17aIL1raIL1aIL1bIL2IL6IL8TNFarPrPrPr101371journalpone0235404t004Table Correlation analysis of AMH the number of follicles obtained and other inflammatory cytokines in the nonPCOS groupnonPCOSIL10IL13IL17aIL1raIL1aIL1bIL2IL6IL8TNFarAMHPeggs obtainedICSI mature eggsNormal fertilized eggsrPrPrP101371journalpone0235404t005PPPLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathwayTable Correlation analysis of BMI FSH and other inflammatory cytokines in the PCOS groupPCOSIL10IL13IL17aIL1raIL1aIL1bIL2IL6IL8TNFαBMIFSHLHLHFSHrPrPrPr101371journalpone0235404t006Table Correlation analysis of E2 PRL and other inflammatory cytokines in the PCOS groupPCOSE2PRLTFPGIL10IL13IL17aIL1raIL1aIL1bIL2IL6IL8TNFαrPrPrPr101371journalpone0235404t007PPTable Correlation analysis of AMH the number of follicles obtained and other inflammatory cytokines in the PCOS groupPCOSIL10IL13IL17aIL1raIL1aIL1bIL2IL6IL8TNFαrAMHPeggs obtainedICSI mature eggsNormal fertilized eggsrPrPrP101371journalpone0235404t008 Detection of protein imprinting in granulosa cellsIt can be seen from Fig 1A that there was no significant difference in the expression levels ofAMPK P PAMPK P and ACC P protein in PCOS groupPLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathwayFig A Granule cell protein expression level B Gray expression analysis of protein expression A Representativewestern blot images and B Summary of expression changes among relative genes Data were represented by mean±standard deviation SD and SPSS statistical software was used for analysis Pvalue less than wasconsidered statistically significant101371journalpone0235404g001PLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathwayFig A Fluorescence density map of sugar uptake capacity of granular cells B Fluorescence density analysis Granulosa cells ofboth nonPCOS and PCOS patients were obtained in vitro for in vitro culture PCOS granule cells were randomly divided into twogroups one group was cultured normally and the other group was added with berberine with final concentration of 100μM After24h the serumfree culture solution containing 2nbdg with a final concentration of 50μM and insulin of 100nM was replaced at ˚CAfter 1h incubation the difference in green fluorescence intensity of 530nm was compared under fluorescence microscope and thedifference in fluorescence density between different groups was compared by using flowjov10 Data were represented by mean±standard deviation SD and SPSS statistical software was used for analysis Pvalue less than was considered statisticallysignificant101371journalpone0235404g002compared with the nonPCOS group while the expression levels of mTORP00001 PmTORP00001 and STAT3P00001 were significantly increased At the same time theprotein expression level of IRS1P was significantly decreased Detection of granulated cell sugar uptake capacityAs shown in Fig 2A and 2B compared with granulosa cells obtained from the nonPCOSgroup the fluorescence intensity of granulosa cells in PCOS group was significantly reducedP00001 and the fluorescence density in the cells increased significantly after berberineP00001 Detection of expression of key factors of glucose metabolism ingranulosa cellsAs shown in Fig 1B the increased expression level of AMPK mRNA after the addition of berberine was significantly different from that in the PCOS group P Compared with thenonPCOS group the level of mTOR in PCOS granule cells was significantly increasedP and the expression level of IRS1 mRNA was significantly reduced P Berberine significantly reduced the expression level of mTOR mRNA in PCOS granule cellsP and increased the expression level of IRS1 mRNA in PCOS granule cellsP Discussion The correlation between selected serum inflammatory cytokines andPCOSThis study finds that IL17a P IL1Ra P00001 and IL6 P in the PCOSgroup were significantly higher than those in the nonPCOS group Studies have shown thatPLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathwayPCOS associated with lowgrade chronic inflammation interleukin17A IL17A is one of themajor members of proinflammatory cytokines and is mainly involved in the development ofinflammatory and autoimmune diseases Within the immune process the genetic factors ofIL17A play a major role in the susceptibility of PCOS [] The level of IL1Ra is significantlyincreased in patients with PCOS which can lead to decreased insulin resistance and blood glucose metabolism causing obesity and metabolic syndrome in PCOS patients [] Tarkun [] studied the serum levels of IL6 in patients with PCOS higher than the normal control group and IL6 was significantly associated with insulin resistance and fasting blood glucose The increase of IL6 level is an important inflammatory factor inducing the occurrenceof PCOS AMH levels and inflammatory cytokines in PCOS patientsThis study finds that in the nonPCOS group AMH level was negatively correlated withinflammatory cytokines such asIL17a r 0819P 0004IL1a r 0716P 0002IL1b r 0678P 0031IL2 r 0765P 001IL8 r 0705P However in thePCOS group AMH levels were not significantly correlated with the levels of various inflammatory cytokineStudies have found that AMH plays a key role in the anovulatory mechanism of PCOS []AMH can promote the growth of preantral follicles to the small sinus stage in vitro whileincreasing the production of steroid hormones and paracrine factors as well as oocyte maturation AMH is a key follicular paracrineautocrine factor that has a positive effect on preantralfollicle survival and growth [] The alienation of AMH function affects the regulation of follicles and promotes the increase of small follicles in the body Due to the abnormal expressionof the number and function of follicles the regulation of the ovary on the matrix is weakenedand the follicular irregular growth is induced without follicular atresia The expression of normal levels of inflammatory cytokines may promote normal cell apoptosis Elevated inflammatory cytokines IL17aIL1aIL1bIL2IL8 may disrupt the ovarian follicle atresiaweakening the ability of apoptosis and inhibiting the maturation of oocytes In turn the AMHfunction is out of control and the correlation between AMH and inflammatory factor levels isinducedIt can be concluded that AMH levels under normal conditions can effectively regulate thelevel of inflammatory factors and promote the body™s own metabolism and reproductive function The expression of high AMH levels in PCOS patients causes a loss of correlation withinflammatory factors The abnormal level of AMH increases the level of inflammatory factorsresulting in a continuous low concentration of systemic inflammatory state in the humanbody leading to metabolic diseases such as insulin resistance and glycolipid metabolism andreproduction disfunction The effects of Berberine on granulosa cells™ insulin resistanceOur study finds that berberine can inhibit inflammatory factor levels increase AMPK mRNAand IRS1 mRNA levels and reduce the level of mTOR mRNA in granulosa cells of PCOSpatients Berberine can increase insulin sensitivity in patients reduce blood sugar improveinsulin resistance and achieve the therapeutic effect of treating PCOS Berberine is the mainactive ingredient of Chinese herbal medicine Coptis Cork and Mink and has been used totreat diarrhea metabolic disorders and infertility []Berberine regulates glucose metabolism through a variety of mechanisms and signalingpathways such as increased insulin sensitivity activation of the adenosine monophosphateactivated protein kinase AMPK pathway regulation of the intestinal microbiota andPLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathwayinhibition of liver sugar Exogenous stimulates peripheral cell glycolysis promotes intestinalglucagon like protein1 GLP1 secretion upregulates liver lowdensity lipoprotein receptormRNA expression and increases glucose transporter [“]PCOS is a multifactorial endocrine disease that affects reproduction and metabolism Studies in dheainduced PCOS mouse models have found that mTOR and pmTOR serine2448are highly expressed in the ovary In normal mice in another study mTOR protein levels inthe corpus luteum of PCOS patients were the same as in healthy women However comparedwith healthy patients receiving insulin stimulation the expression of mTOR protein in the corpus luteum of PCOS patients is less so another link between PCOS and mTOR is metabolicdisorders during PCOS [] The combination of activation of the mTOR pathway and oxidative damage to DNA that causes replication stress is a particularly effective factor in promoting aging and aging [] Berberine inhibits the level of DNA damage signals [“] It canreduce the expression and activation of γH2AX including tumor A and TK6 cells as wellas normal WI38 cells or mitogenic human lymphocytes [] It can also reduce intracellularreactive oxygen species and mitochondrial transmembrane potential which is a sign of mitochondrial activation [“] However these drugs also significantly reduce phosphorylationlevels of Ser235 of ribosomal S6 protein RpS6 Ser2448 of mTOR and Ser65 of 4EBP1These data indicate that the reduction of mTOR S6K signal which in turn reduces the translation rate and is accompanied by a reduction in oxidative phosphorylation which leads to areduction in ROS and a reduction in oxidative DNA damage [] The mechanism by whichBRB exerts these effects may be by targeting mitochondriaThe protective effect of berberine on islet function may involve two pathways On the onehand berberine can improve insulin sensitivity in patients with PCOS with IR as the core []On the other hand berberine can promote insulin secretion from islet cells of PCOS patientsand protect islet cells through antioxidant activity [] Kong [] found that themolecular mechanism of berberine on insulin resistance by upregulating the expression ofinsulin receptors confirmed that berberine can reduce fasting blood glucose and fasting seruminsulin At the same time they also found that berberine activated its promoter through protein kinase CPKCdependent increasing insulin receptor mRNA and protein expressionIn fat and muscle cells berberine may stimulate cells by upregulating glucose transporter type GLUT1 expression and inhibiting retinol binding protein4 RBP4 At the same timeberberine also has a certain effect on the phosphorylation of IRS1 which can finally alleviateinsulin resistance []In other words berberine can inhibit inflammatory factor levels increase AMPK mRNAand IRS1 mRNA levels and reduce the level of mTOR mRNA in granulosa cells of PCOSpatients The mechanism by which berberine regulates the metabolism of glycolipids is not tostimulate the patient to increase insulin secretion but to increase the glucose consumption ofthe patient™s cells and improve glucose tolerance At the same time by increasing insulin sensitivity in patients further lowering blood sugar and improving insulin resistance and achievingthe effect of treating PCOS ConclusionIn this study we found that the elevated levels of serum inflammatory factors IL17a IL1Raand IL6 caused women to be in a subclinical inflammatory state for a long time Abnormalchanges in inflammatory factors altered their original negative correlations with AMH levelsthereby weakening the metabolism of glycolipids promoting insulin resistance destroying thenormal ovulation and fertilization system of women and leading to polycystic ovary syndromecharacterized by menstrual thinning and abnormal ovulation Berberine can improve thePLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathwaysensitivity of insulin by regulating the signal pathway of mTOR mRNA and IRS1 mRNA inPCOS patients and achieve the therapeutic effect of treating PCOSSupporting informationS1 Raw ImagesPDFAuthor ContributionsConceptualization Yuwei Duan Wei LiData curation Wei LiFormal analysis Wei LiFunding acquisition Hongying KuangSoftware Manqi ShaoWriting “ original draft Hongying Kuang Yuwei Duan Wenxia AiWriting “ review editing Dan Li Yanwen Xu Wenxia Ai Wei Li Ying Wang Sha LiuMushan Li Xiaoqiu Liu Manqi ShaoReferencesTeede H Deeks A Moran L Polycystic Ovary Syndrome A complex Condition with PsychologicalReproductive and Metabolic Manifestations That Impacts on Health Across the Lifespan BMC Med ZHANG F MA T CUI P Diversity of the Gut Microbiota in DihydrotestosteroneInduced PCOSRats and the Pharmacologic Effects of Diane35 Probiotics and Berberine [J]Frontiers in microbiology IMENSHAHIDI M HOSSEINZADEH H Berberine and barberry Berberis vulgaris A clinical review [J] “LI M F ZHOU X M The Effect of Berberine on Polycystic Ovary Syndrome Patients with Insulin Resistance PCOSIR A MetaAnalysis and Systematic Review [J] LI W LI D KUANG H Berberine increases glucose uptake and intracellular ROS levels by promoting Sirtuin ubiquitination [J] Biomedicine pharmacotherapy Biomedecine pharmacotherapie RONDANELLI M INFANTINO V Polycystic ovary syndrome management a review of the possibleamazing role of berberine [J] “ Benson S Obesity Depression and Chronic Lowgrade Inflammation in Women with PolycysticOvary Syndrome Brain Behav Immun “ 101016jbbi200707003PMID Rehman K Akash MSH Liaqat A Role of Interleukin6 in Development of Insulin Resistance andType Diabetes

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You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: The role of serum inflammatory cytokines andberberine in the insulin signaling pathwayamong women with polycystic ovarysyndromeHongying Kuang12˜¯ Yuwei Duan34˜¯ Dan LiID5a Yanwen Xu34b Wenxia Ai2 Wei Li1Ying Wang1 Sha Liu2 Mushan Li2 Xiaoqiu Liu2 Manqi Shao2 The First Affiliated Hospital of Heilongjiang University of Chinese Medicine Harbin China HeilongjiangUniversity of Chinese Medicine Harbin China The First Affiliated Hospital of Sun Yatsen UniversityGuangzhou China Guangdong Provincial Key Laboratory of Reproductive Medicine Guangzhou China Department of Acupuncture the Third Affiliated Hospital Beijing University Of Chinese Medicine BeijingChina˜¯ These authors contributed equally to this worka Current address The First Affiliated Hospital of Sun Yatsen University Guangzhou Chinab Current address Reproductive Medicine Center The First Affiliated Hospital Sun Yatsen UniversityGuangzhou Guangdong China 3509437qqcom DL Xuyanwenlivecn YXAbstractObjectiveTo study the role of selected serum inflammatory cytokines and berberine in the insulin signaling pathway among women with polycystic ovary syndrome PCOSMethodsSelected serum inflammatory cytokines were analyzed in the p cells which were interfered by berberine from infertile women who were to be treated with In Vitro FertilizationIVF Intracytoplasmic Sperm InjectionEmbryo Transfer icsiet Among them patientshad PCOS infertility and were nonPCOS patients whose infertility resulted from fallopian tube and male factors The elisa method was used to detect the changes in the expression levels of inflammatory factors in the cells The correlations between the seruminflammatory cytokine expression levels and the corresponding clinical hormones were analyzed The changes in the expression mRNA and protein levels of the serum inflammatorycytokines were studied by realtime quantitative PCR and protein printing Fluorescencemicroscope and flow cytometry were used to detect the glucose uptake capacity of ovariangranulosa cells in PCOS patients under the action of insulin after berberineResultsIn the PCOS group IL17a P IL1Ra P00001 and IL6 P were significantly higher than those in the nonPCOS group In the nonPCOS group AMH level wasnegatively correlated with inflammatory cytokines IL17a r 0819P IL1a r a1111111111a1111111111a1111111111a1111111111a1111111111 ACCESSCitation Kuang H Duan Y Li D Xu Y Ai W Li W The role of serum inflammatorycytokines and berberine in the insulin signalingpathway among women with polycystic ovarysyndrome e0235404 101371journalpone0235404Editor Meijia Zhang China Agricultural UniversityCHINAReceived November Accepted June Published August Copyright Kuang This is an access distributed under the terms of theCreative Commons Attribution License whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are creditedData Availability Statement All relevant data arewithin the manuscript and its SupportingInformation filesFunding This work was supported by theOutstanding talents funding project of HeilongjiangUniversity of Chinese Medicine China [grantnumber 2018JC05] the Foundation for youngacademic leaders of Heilongjiang University ofChinese Medicine China [grant number2018RCD08] and the Foundation for HeilongjiangProvince Postdoctoral Research Startup ChinaPLOS ONE 101371journalpone0235404 August PLOS ONE 0c[grant number LBHQ18119] the Project ofUndergraduate Colleges and Universities YouthInnovation Talents by Education Department ofHeilongjiang Province China [grant numberUNPYST2018227] the Natural ScienceFoundation of Heilongjiang Province China [grantnumber H2016083]Competing interests The authors have declaredthat no competing interests existThe role of serum inflammatory cytokines and berberine in the insulin signaling pathway0716P IL1b r 0678P IL2 r 0765P and IL8r 0705P However in the PCOS group AMH levels were not significantly correlated with the levels of the examined inflammatory cytokines Berberine significantlyreduced the expression level of mTOR mRNA P and increased the expressionlevel of IRS1 mRNA P in the PCOS granule cellsConclusionIn this study we find that the elevated levels of serum inflammatory factors IL17a IL1Raand IL6 cause women to be in a subclinical inflammatory state for a long time Abnormalchanges in inflammatory factors alter their original negative correlations with AMH levelsthereby weakening the metabolism of glycolipids promoting insulin resistance destroyingthe normal ovulation and fertilization system of women leading to polycystic ovary syndrome characterized by menstrual thinning and abnormal ovulation Berberine can improvethe sensitivity of insulin by regulating the signal pathway of insulin receptor substrate1IRS1 and mammalian target of rapamycin mTOR in PCOS patients and achieve a therapeutic effect of treating PCOS IntroductionPolycystic ovary syndrome PCOS is the most common endocrinopathy affecting reproductive aged women It affects reproduction infertility irregular menstruation hirsutism etcmetabolism insulin resistance impaired glucose tolerance etc and psychological characteristics anxiety depression and deterioration in quality of life [] Berberine BBR as a quaternary ammonium salt extracted from plants such as Coptis chinensis and Phellodendronchinensis is currently used in the treatment of diabetes hyperlipidemia and PCOS [] Recentstudies have found that berberine has good hypoglycemic and hypolipidemic effects and is aneffective insulin sensitizer Berberine reduces the synthesis of steroid hormones and theexpression of ovarian aromatase through the action on the hypothalamuspituitaryovarianaxis HPOA improves the insulin resistance status of PCOS patients reduces body weightinduces ovulation and regulates menstruation thereby increasing pregnancy rate and livebirth rate [“] Clinical observations have demonstrated that even with longterm use of berberine its side effects are transient and mild suggesting that BBR is safe to use in PCOSpatients and a very promising plantbased compound for treating PCOS patients []Patients with PCOS have been found to be under a chronic lowgrade inflammation statusincluding high levels of leukocytes and disorder of the proinflammatory cytokines [] Interleukin IL6 is a multipotent cytokine that mediates inflammatory response by controllingcell differentiation migration proliferation and apoptosis thus playing a role in the development of insulin resistance [] IL17a is the œsignature cytokine secreted by the Th17 CD4ve T cell subset Activation of Th17type responses is important not only for host immunecontrol of extracellular bacterial and fungal infections but also associated with chronic inflammation and autoimmunity [] The IL1RA protein is a naturally occurring antagonist of proinflammatory cytokines These proinflammatory cytokines are involved in the underlyingmechanism of various chronic inflammatory conditions [] Therefore we hypothesize thatinflammatory factors are one of the important factors influencing the formation of PCOS andberberine may be an important drug that regulates PCOS inflammatory factorsPLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathwayAntiMu¨llerian hormone AMH is an indicator of a patient™s ability to respond quicklyand efficiently to ovarian reserve In women AMH is produced by the granulosa cells of theovarian follicles and its secretion begins at puberty and lasts until menopause [] PCOS ischaracterized by hyperandrogenism and follicular blockade These two characteristics may bedue to an imbalance between AMH and follicle stimulating hormone [] Circulating insulinlevels in patients with PCOS increase thereby inducing follicular developmental disorderswhich in turn lead to ovarian polycystic ovary formation and higher than normal AMH HighAMH is one of the indicators of stubborn anovulation [] Research has shown that serumAMH level is relatively stable throughout and between menstrual cycles [] More and morestudies have used AMH as a biomarker for PCOS []In the present study we examined the effect and mechanism of serum inflammatory cytokines including IL6 IL17a IL1RA etc on insulin sensitivity of PCOS In addition weassessed whether berberine can improve insulin sensitivity of PCOS by antagonizing the proinflammatory effect of serum inflammatory cytokines Materials and methods SampleSerum samples and granulosa cells were collected from infertile women who were to betreated with In Vitro Fertilization IVF Intracytoplasmic Sperm InjectionEmbryo Transfericsiet Among them patients had PCOS infertility and were nonPCOS patientswhose infertility resulted from fallopian tube and male factors The nonPCOS women wererequired to have regular menstrual cycles The essays were analyzed by Guangdong ProvincialKey Laboratory of Reproductive Medicine at the first affiliated hospital of Sun YatSen University Data collection time “ ParticipantsInclusion criteria age Diagnosis of PCOS according to Rotterdam StandardRotterdam Diagnostic Criteria for PCOS Ovarian Ovulation Disorder Manifests Oligomenorrhea or Amenorrhea Clinically or biochemically determined androgen level increasesmore than 2nmolL or clinically manifested hirsute acne excluding Kaohsiung caused byother diseases Ovarian morphology showed polycystic changes Only of the above items can be met IVFET treatmentExclusion criteria Having orally taken drugs in the past three months that affect the results such as contraceptives or other hormone drugs insulin sensitizers and lipidlowering drugs Suffering from other androgen excess related diseases including congenital adrenal hyperplasia with hydroxylase deficiency androgen secreting tumor excessive use of androgenproducing drugs Cushing syndrome severe insulin resistance thyroid dysfunction andhyperprolactinemia Having a history of anic diseases of heart lung liver kidney and other important ansor patients with mental diseases and other reasons that may interfere the present studyoutcomesPLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathway Ethics statement The study was approved by the ICE for Clinical Research andAnimal Trials of the First Affiliated Hospital of Sun Yatsen University reference [] No and was conducted in accordance with the ethical standards of the Declaration ofHelsinki All participants have signed written informed consent and there were no minors ReagentBerberine was provided by Yuanye BioTechnology Co Ltd Shanghai China2NBDG N13195 Invitrogen DMEMF12 “ Gibco and Fetal bovine serumSV3008702 Hyclone were purchased from Thermo Fisher Scientific Waltham USA Thefollowing is a list of used reagents and their manufacturers™ informationRealtime quantitative PCR kits ROCHE CAT Reverse transcriptase PROMEGA CAT M1701RIPA pyrolysis fluid yantian Bi CAT P0013BBcl2 abcam CAT ab196495BAD abcam CAT ab90435BAX abcam CAT ab182733ACTINβ abcam CAT sc70319 Methods Granulosa cell acquisition and grouping The follicular fluid was centrifuged at rpm for minutes at room temperature The supernatant was discarded and the precipitate after centrifugation was resuspended by phosphate buffer saline PBS The cell suspension was slowly centrifuged to percoll level at room temperature with 1800rpm for 10minAfter centrifugation the white floc was sucked out and washed with PBS three times at1200rpm for 5min at room temperature We discarded the supernatant and added typeIV collagenase and blew it evenly Digestion was performed at ˚C for 1520min We addedthe same amount of culture solution to stop digestion and blew it evenly The filtrate was filtered by micron cell filter and centrifuged at room temperature with 1000rpm for 4minThe supernatant was discarded the cells were suspended again with PBS 3ml erythrocytelysate was added to mix well and the mixture was allowed to stand at ˚C for 10min It wasthen centrifuged at 1000rpm for 4min at room temperature Some white precipitate whichwas verified as granulocytes were observed after centrifugation After the seed plate of PCOSwomen granulosa cells was seeded the fluid was changed once hours After hours solvent or berberine was added for continuous intervention for hours which led to the PCOScontrol group and PCOS berberine intervention group Detection of serum inflammatory cytokines The Merck luminex testing platformwas used to detect serum cytokines according to the manufacturer™s instructions Realtime quantitative PCR At the end of berberine treatment Trizol method wasused to extract total RNA in the cells which was then used to reverse transcription of mRNATotal RNA was isolated from cells using Trizol regent Takara BioInc Tokyo Japan and I mgmRNA was reverse transcribed to cDNA using a reverse transcription Takara Bio Inc TokyoJapan and subjected to quantitative PCR which was performed with the BioRad CFX96Touch TM RealTime PCR Detection System BioRad CA using iQ TM SYBR Green Supermix BioRad CA and threshold cycle numbers were obtained using BioRad CFX ManagerSoftware Real time PCR was carried out using the following conditions min at ˚C min at ˚C and cycles of s at ˚C min at ˚C using 1μl of cDNA reverse transcribed as mentioned above Quantifast SYBR green PCR kit Qiagen Cat No and500nM of forward and reverse primers The following primers were usedPLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathwayβactin GCCGTTCCGAAAGTTGCCTT GAGCGCGGCGATATCATCAAMPK ACAGCCGAGAAGCAGAAACA TTGCCAACCTTCACTTTGCCmTOR CTTAGAGGACAGCGGGGAAG TCCAAGCATCTTGCCCTGAGSTAT3 CTGAAACGGGCTTCAGGTCA TCCAGGGAGAAAGGGAGTCASOCS3 TCTGTCGGAAGACCGTCAAC CCTTAAAGCGGGGCATCGTAIRS1 TCTCTTCCCACGGCGATCTA TGACACTGCGGAAGGAACTCWe used the 2ΔΔCT method to calculate the relative expression level of target genes Western blot experiment After the end of each cell culture the cells were lysedusing RIPA lysate in an ice bath and the protein lysate was collected After the total proteinconcentration was determined by BCA method polyacrylamide gel electrophoresis was carried out according to the standard of μg of total protein per group After the electrophoresisthe protein in the gel was transferred to the polyvinylidene fluoride PVDF membraneblocked with skim milk powder and then incubated with the corresponding dilutedprimary antibody at ˚C overnight and finally labeled with the corresponding horseradish peroxidase After incubation for one hour at room temperature the ECL luminescentsolution was added and detected by autoradiography using a Biored gel imaging systemImmunoreactive protein bands were visualized with enhanced chemiluminescence ECL on aChemiDoc MP Imaging System Blots were scanned and quantified with the Image analysissoftware BioRad Image Lab All specific protein band densities were normalized to βactin amounts Detection of glucose uptake capacity in granulosa cells Granulosa cells of bothnonPCOS and PCOS patients were obtained in vitro for in vitro culture PCOS granulosacells were randomly divided into two groups one group was cultured normally and the othergroup was added with berberine with the final concentration of 100μM After 24h the serumfree culture solution containing 2nbdg with a final concentration of 50μM and insulin of100nM were replaced at ˚C After 1h incubation the difference in green fluorescence intensity of 530nm was compared under fluorescence microscope and the difference in fluorescence density between different groups was compared by using flowjov10 Statistical methodsData were represented by mean±standard deviation SD and SPSS statistical softwarewas used for analysis Analysis of variance ANOVA was used to test betweengroup differences and the pvalues were corrected by Bonferroni p value less than was considered statistically significant Results Analysis of clinical baseline indicators of PCOS group and nonPCOSgroupAs shown in Table compared with the nonPCOS group the PCOS group had significantlyhigher BMIP LHP00001 LHFSHP00001 FPGP AMHP00001 the number of follicles obtainedP00001 ICSI mature eggsP and thenumber of normal fertilization P FSH levels were significantly lower in the PCOSgroup than those in the nonPCOS group P Detection of serum inflammatory cytokinesAs shown in Table IL17a P IL1Ra P00001 and IL6 P in the PCOSgroup were significantly higher than those in the nonPCOS group IL10 P IL13PLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathwayTable Comparison of clinical indicators between PCOS and nonPCOS groupsnonPCOS n PCOS n PvalueAge years oldMenarche ageSBP mmHgDBPmmHgPulse sminBMI kgm2FSH mIUmlLH mIUmlLHFSHE2 pgmlPRL ngmlT nmolLFPG mmollAMHngmleggs obtainedICSI mature eggsNormal fertilized eggsMeanSDMedianMeanSDMedian101371journalpone0235404t001Table Comparison of serum inflammatory cytokines between the PCOS and nonPCOS groupsIL10 pgmlIL13 pgmlIL17a pgmlIL1Ra pgmlIL1α pgmlIL1β pgmlIL2 pgmlIL6 pgmlIL8 pgmlTNFα pgml101371journalpone0235404t002nonPCOSn ±±±±±±±±±±±PCOSn ±±±±±±±±±PvalueP IL1α P IL1β P IL8 P and TNFα P inthe two groups was not statistically significantly different Correlation analysis between serum inflammatory cytokines anddiagnostic clinical indicatorsWe examined the correlation between serum inflammatory cytokines and clinical serum hormones and other levels in PCOS and nonPCOS patients In the nonPCOS group AMH levelwas negatively correlated with inflammatory cytokines IL17ar 0819P IL1ar 0716P IL1br 0678P IL2r 0765P and IL8r 0705P However in the PCOS group AMH levels were not significantlyPLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathwaycorrelated with the levels of these inflammatory cytokines Among the diagnostic clinical indicators except AMH level there was no significant correlation between other clinical indicatorsand the expression level of inflammatory cytokines in the nonPCOS group and the PCOSgroup Tables “Table Correlation analysis of BMI FSH and other inflammatory cytokines in the nonPCOS groupnonPCOSBMIIL10IL13IL17aIL1raIL1aIL1bIL2IL6IL8TNFarP101371journalpone0235404t003FSHPrLHLHFSHrPrTable Correlation analysis of E2 PRL and other inflammatory cytokines in the nonPCOS groupnonPCOSE2PRLTFPGIL10IL13IL17aIL1raIL1aIL1bIL2IL6IL8TNFarPrPrPr101371journalpone0235404t004Table Correlation analysis of AMH the number of follicles obtained and other inflammatory cytokines in the nonPCOS groupnonPCOSIL10IL13IL17aIL1raIL1aIL1bIL2IL6IL8TNFarAMHPeggs obtainedICSI mature eggsNormal fertilized eggsrPrPrP101371journalpone0235404t005PPPLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathwayTable Correlation analysis of BMI FSH and other inflammatory cytokines in the PCOS groupPCOSIL10IL13IL17aIL1raIL1aIL1bIL2IL6IL8TNFαBMIFSHLHLHFSHrPrPrPr101371journalpone0235404t006Table Correlation analysis of E2 PRL and other inflammatory cytokines in the PCOS groupPCOSE2PRLTFPGIL10IL13IL17aIL1raIL1aIL1bIL2IL6IL8TNFαrPrPrPr101371journalpone0235404t007PPTable Correlation analysis of AMH the number of follicles obtained and other inflammatory cytokines in the PCOS groupPCOSIL10IL13IL17aIL1raIL1aIL1bIL2IL6IL8TNFαrAMHPeggs obtainedICSI mature eggsNormal fertilized eggsrPrPrP101371journalpone0235404t008 Detection of protein imprinting in granulosa cellsIt can be seen from Fig 1A that there was no significant difference in the expression levels ofAMPK P PAMPK P and ACC P protein in PCOS groupPLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathwayFig A Granule cell protein expression level B Gray expression analysis of protein expression A Representativewestern blot images and B Summary of expression changes among relative genes Data were represented by mean±standard deviation SD and SPSS statistical software was used for analysis Pvalue less than wasconsidered statistically significant101371journalpone0235404g001PLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathwayFig A Fluorescence density map of sugar uptake capacity of granular cells B Fluorescence density analysis Granulosa cells ofboth nonPCOS and PCOS patients were obtained in vitro for in vitro culture PCOS granule cells were randomly divided into twogroups one group was cultured normally and the other group was added with berberine with final concentration of 100μM After24h the serumfree culture solution containing 2nbdg with a final concentration of 50μM and insulin of 100nM was replaced at ˚CAfter 1h incubation the difference in green fluorescence intensity of 530nm was compared under fluorescence microscope and thedifference in fluorescence density between different groups was compared by using flowjov10 Data were represented by mean±standard deviation SD and SPSS statistical software was used for analysis Pvalue less than was considered statisticallysignificant101371journalpone0235404g002compared with the nonPCOS group while the expression levels of mTORP00001 PmTORP00001 and STAT3P00001 were significantly increased At the same time theprotein expression level of IRS1P was significantly decreased Detection of granulated cell sugar uptake capacityAs shown in Fig 2A and 2B compared with granulosa cells obtained from the nonPCOSgroup the fluorescence intensity of granulosa cells in PCOS group was significantly reducedP00001 and the fluorescence density in the cells increased significantly after berberineP00001 Detection of expression of key factors of glucose metabolism ingranulosa cellsAs shown in Fig 1B the increased expression level of AMPK mRNA after the addition of berberine was significantly different from that in the PCOS group P Compared with thenonPCOS group the level of mTOR in PCOS granule cells was significantly increasedP and the expression level of IRS1 mRNA was significantly reduced P Berberine significantly reduced the expression level of mTOR mRNA in PCOS granule cellsP and increased the expression level of IRS1 mRNA in PCOS granule cellsP Discussion The correlation between selected serum inflammatory cytokines andPCOSThis study finds that IL17a P IL1Ra P00001 and IL6 P in the PCOSgroup were significantly higher than those in the nonPCOS group Studies have shown thatPLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathwayPCOS associated with lowgrade chronic inflammation interleukin17A IL17A is one of themajor members of proinflammatory cytokines and is mainly involved in the development ofinflammatory and autoimmune diseases Within the immune process the genetic factors ofIL17A play a major role in the susceptibility of PCOS [] The level of IL1Ra is significantlyincreased in patients with PCOS which can lead to decreased insulin resistance and blood glucose metabolism causing obesity and metabolic syndrome in PCOS patients [] Tarkun [] studied the serum levels of IL6 in patients with PCOS higher than the normal control group and IL6 was significantly associated with insulin resistance and fasting blood glucose The increase of IL6 level is an important inflammatory factor inducing the occurrenceof PCOS AMH levels and inflammatory cytokines in PCOS patientsThis study finds that in the nonPCOS group AMH level was negatively correlated withinflammatory cytokines such asIL17a r 0819P 0004IL1a r 0716P 0002IL1b r 0678P 0031IL2 r 0765P 001IL8 r 0705P However in thePCOS group AMH levels were not significantly correlated with the levels of various inflammatory cytokineStudies have found that AMH plays a key role in the anovulatory mechanism of PCOS []AMH can promote the growth of preantral follicles to the small sinus stage in vitro whileincreasing the production of steroid hormones and paracrine factors as well as oocyte maturation AMH is a key follicular paracrineautocrine factor that has a positive effect on preantralfollicle survival and growth [] The alienation of AMH function affects the regulation of follicles and promotes the increase of small follicles in the body Due to the abnormal expressionof the number and function of follicles the regulation of the ovary on the matrix is weakenedand the follicular irregular growth is induced without follicular atresia The expression of normal levels of inflammatory cytokines may promote normal cell apoptosis Elevated inflammatory cytokines IL17aIL1aIL1bIL2IL8 may disrupt the ovarian follicle atresiaweakening the ability of apoptosis and inhibiting the maturation of oocytes In turn the AMHfunction is out of control and the correlation between AMH and inflammatory factor levels isinducedIt can be concluded that AMH levels under normal conditions can effectively regulate thelevel of inflammatory factors and promote the body™s own metabolism and reproductive function The expression of high AMH levels in PCOS patients causes a loss of correlation withinflammatory factors The abnormal level of AMH increases the level of inflammatory factorsresulting in a continuous low concentration of systemic inflammatory state in the humanbody leading to metabolic diseases such as insulin resistance and glycolipid metabolism andreproduction disfunction The effects of Berberine on granulosa cells™ insulin resistanceOur study finds that berberine can inhibit inflammatory factor levels increase AMPK mRNAand IRS1 mRNA levels and reduce the level of mTOR mRNA in granulosa cells of PCOSpatients Berberine can increase insulin sensitivity in patients reduce blood sugar improveinsulin resistance and achieve the therapeutic effect of treating PCOS Berberine is the mainactive ingredient of Chinese herbal medicine Coptis Cork and Mink and has been used totreat diarrhea metabolic disorders and infertility []Berberine regulates glucose metabolism through a variety of mechanisms and signalingpathways such as increased insulin sensitivity activation of the adenosine monophosphateactivated protein kinase AMPK pathway regulation of the intestinal microbiota andPLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathwayinhibition of liver sugar Exogenous stimulates peripheral cell glycolysis promotes intestinalglucagon like protein1 GLP1 secretion upregulates liver lowdensity lipoprotein receptormRNA expression and increases glucose transporter [“]PCOS is a multifactorial endocrine disease that affects reproduction and metabolism Studies in dheainduced PCOS mouse models have found that mTOR and pmTOR serine2448are highly expressed in the ovary In normal mice in another study mTOR protein levels inthe corpus luteum of PCOS patients were the same as in healthy women However comparedwith healthy patients receiving insulin stimulation the expression of mTOR protein in the corpus luteum of PCOS patients is less so another link between PCOS and mTOR is metabolicdisorders during PCOS [] The combination of activation of the mTOR pathway and oxidative damage to DNA that causes replication stress is a particularly effective factor in promoting aging and aging [] Berberine inhibits the level of DNA damage signals [“] It canreduce the expression and activation of γH2AX including tumor A and TK6 cells as wellas normal WI38 cells or mitogenic human lymphocytes [] It can also reduce intracellularreactive oxygen species and mitochondrial transmembrane potential which is a sign of mitochondrial activation [“] However these drugs also significantly reduce phosphorylationlevels of Ser235 of ribosomal S6 protein RpS6 Ser2448 of mTOR and Ser65 of 4EBP1These data indicate that the reduction of mTOR S6K signal which in turn reduces the translation rate and is accompanied by a reduction in oxidative phosphorylation which leads to areduction in ROS and a reduction in oxidative DNA damage [] The mechanism by whichBRB exerts these effects may be by targeting mitochondriaThe protective effect of berberine on islet function may involve two pathways On the onehand berberine can improve insulin sensitivity in patients with PCOS with IR as the core []On the other hand berberine can promote insulin secretion from islet cells of PCOS patientsand protect islet cells through antioxidant activity [] Kong [] found that themolecular mechanism of berberine on insulin resistance by upregulating the expression ofinsulin receptors confirmed that berberine can reduce fasting blood glucose and fasting seruminsulin At the same time they also found that berberine activated its promoter through protein kinase CPKCdependent increasing insulin receptor mRNA and protein expressionIn fat and muscle cells berberine may stimulate cells by upregulating glucose transporter type GLUT1 expression and inhibiting retinol binding protein4 RBP4 At the same timeberberine also has a certain effect on the phosphorylation of IRS1 which can finally alleviateinsulin resistance []In other words berberine can inhibit inflammatory factor levels increase AMPK mRNAand IRS1 mRNA levels and reduce the level of mTOR mRNA in granulosa cells of PCOSpatients The mechanism by which berberine regulates the metabolism of glycolipids is not tostimulate the patient to increase insulin secretion but to increase the glucose consumption ofthe patient™s cells and improve glucose tolerance At the same time by increasing insulin sensitivity in patients further lowering blood sugar and improving insulin resistance and achievingthe effect of treating PCOS ConclusionIn this study we found that the elevated levels of serum inflammatory factors IL17a IL1Raand IL6 caused women to be in a subclinical inflammatory state for a long time Abnormalchanges in inflammatory factors altered their original negative correlations with AMH levelsthereby weakening the metabolism of glycolipids promoting insulin resistance destroying thenormal ovulation and fertilization system of women and leading to polycystic ovary syndromecharacterized by menstrual thinning and abnormal ovulation Berberine can improve thePLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathwaysensitivity of insulin by regulating the signal pathway of mTOR mRNA and IRS1 mRNA inPCOS patients and achieve the therapeutic effect of treating PCOSSupporting informationS1 Raw ImagesPDFAuthor ContributionsConceptualization Yuwei Duan Wei LiData curation Wei LiFormal analysis Wei LiFunding acquisition Hongying KuangSoftware Manqi ShaoWriting “ original draft Hongying Kuang Yuwei Duan Wenxia AiWriting “ review editing Dan Li Yanwen Xu Wenxia Ai Wei Li Ying Wang Sha LiuMushan Li Xiaoqiu Liu Manqi ShaoReferencesTeede H Deeks A Moran L Polycystic Ovary Syndrome A complex Condition with PsychologicalReproductive and Metabolic Manifestations That Impacts on Health Across the Lifespan BMC Med ZHANG F MA T CUI P Diversity of the Gut Microbiota in DihydrotestosteroneInduced PCOSRats and the Pharmacologic Effects of Diane35 Probiotics and Berberine [J]Frontiers in microbiology IMENSHAHIDI M HOSSEINZADEH H Berberine and barberry Berberis vulgaris A clinical review [J] “LI M F ZHOU X M The Effect of Berberine on Polycystic Ovary Syndrome Patients with Insulin Resistance PCOSIR A MetaAnalysis and Systematic Review [J] LI W LI D KUANG H Berberine increases glucose uptake and intracellular ROS levels by promoting Sirtuin ubiquitination [J] Biomedicine pharmacotherapy Biomedecine pharmacotherapie RONDANELLI M INFANTINO V Polycystic ovary syndrome management a review of the possibleamazing role of berberine [J] “ Benson S Obesity Depression and Chronic Lowgrade Inflammation in Women with PolycysticOvary Syndrome Brain Behav Immun “ 101016jbbi200707003PMID Rehman K Akash MSH Liaqat A Role of Interleukin6 in Development of Insulin Resistance andType Diabetes Answer:

Thyroid_Cancer

The effects of tissue fixation on sequencingand transcript abundance of nucleic acidsfrom microdissected liver samples ofsmallmouth bass Micropterus dolomieuHeather L WalshID Adam J Sperry Vicki S BlazerUS Geological Survey National Fish Health Research Laboratory Leetown Science Center KearneysvilleWest Virginia United States of Americaa1111111111a1111111111a1111111111a1111111111a1111111111 hwalshusgsgovAbstractThere is an increasing emphasis on effectsbased monitoring to document responses associated with exposure to complex mixtures of chemicals climate change pathogens parasitesand other environmental stressors in fish populations For decades aquatic monitoring programs have included the collection of tissues preserved for microscopic pathology Consequently formalinfixed paraffinembedded FFPE tissue can be an important reservoir ofnucleic acids as technologies emerge that utilize molecular endpoints Despite the crosslinking effects of formalin its impact on nucleic acid quality and concentration amplification andsequencing are not well described While freshfrozen tissue is optimal for working withnucleic acids FFPE samples have been shown to be conducive for molecular studies Lasercapture microdissection LCM is one technology which allows for collection of specificregions or cell populations from fresh or preserved specimens with pathological alterationspathogens or parasites In this study smallmouth bass Micropterus dolomieu liver was preserved in three different fixatives including neutral buffered formalin NBF ZFix®ZF and PAXgene® PG for four time periods hr hr seven days and days Controls consisted of pieces of liver preserved in RNALater® or ethanol Smallmouth basswere chosen as they are an economically important sportfish and have been utilized as indicators of exposure to endocrine disruptors and other environmental stressors Small liversections were cut out with laser microdissection and DNA and RNA were purified and analyzed for nucleic acid concentration and quality Sanger sequencing and the NanoStringnCounter® technology were used to assess the suitability of these samples in downstreammolecular techniques The results revealed that of the formalin fixatives NBF samples fixedfor and hr were superior to ZF samples for both Sanger sequencing and the NanostringnCounter® The nonformalin PAXgene® samples were equally successful and they showedgreater stability in nucleic acid quality and concentration over longer fixation times This studydemonstrated that small quantities of preserved tissue from smallmouth bass can be utilizedin downstream molecular techniques however future studies will need to optimize the methods presented here for different tissue types fish species and pathological conditions ACCESSCitation Walsh HL Sperry AJ Blazer VS The effects of tissue fixation on sequencing andtranscript abundance of nucleic acids frommicrodissected liver samples of smallmouth bassMicropterus dolomieu e0236104 101371journalpone0236104Editor Rajakumar Anbazhagan National Instituteof Child Health and Human Development NICHDNIH UNITED STATESReceived April Accepted June Published August Copyright This is an access free of allcopyright and may be freely reproduceddistributed transmitted modified built upon orotherwise used by anyone for any lawful purposeThe work is made available under the CreativeCommons CC0 public domain dedicationData Availability Statement All relevant data arewithin the paper and its Supporting InformationfilesFunding This was fully funded internally by theUSGS Environmental Health Contaminant BiologyProgram and the USGS EcosystemsEnvironments and Fisheries Program MissionAreas There are no actual grant numbersPLOS ONE 101371journalpone0236104 August PLOS ONE 0cCompeting interests The authors have declaredthat no competing interests existEffects of tissue fixation on nucleic acids from microdissected tissue samples of a teleostIntroductionGlobally environmental monitoring programs are increasingly used to identify adverse effectsof human activities on aquatic resources [“] The recognition that there are numerouschemical contaminants environmental stressors as well as new and emerging pathogensparasites cooccurring has led to an emphasis on biological environmental effectsbased assessments utilizing resident indicator fish species or caged model species [“] Histopathologyhas been used for many years to assess the health of wild fishes both for specific studies as wellas part of effectsbased monitoring programs [“] More recently genomic endpoints arealso being incorporated into environmental monitoring and risk assessment [“] Whenboth histopathology and molecular analyses are part of an assessment pieces of a tissue arecommonly preserved in buffered formalin or a similar preservative and adjacent separatepieces are preserved in RNAlater1 ethanol or frozen for molecular analyses [“]However for alterations not visible the tissue piece chosen for gene expression may not contain the same cellular components or alterations as those within the histology sectionThe use of formalin fixed paraffinembedded FFPE tissue has been regarded as a valuablereservoir of preserved nucleic acids in mammalian studies [“] Although FFPE tissuesprovide a vast source of pathologically diverse types of genetic material there are drawbackscompared to other tissue preservation methods Formalin fixation causes nucleic acids to fragment degrade and crosslink [] Frozen tissues or tissues specifically preserved for downstream nucleic acid applications do not experience the type of degradation observed fromformalin fixation Despite these setbacks nucleic acids extracted from FFPE tissue have provento be suitable for use in endpoint PCR [] realtime qPCR [ ] and Nextgenerationsequencing [ ] Optimization of FFPE tissues for downstream nucleic acid applicationshas been attempted in multiple studies by evaluation of different fixation methods [ ] tissue handling and processing times [ ] and extraction methods [ ]Laser capture microdissection LCM utilizes a microscope equipped with a laser to targetand isolate specific cells from a heterogeneous population of cells [] Single cells foci of cellpopulations within a tissue or pathogens and parasites can be microdissected Hence nucleicacids from specific cell populations of interest can be analyzed for gene expression studiestranscriptome development or molecular identification of pathogens and parasites Thisallows for a more direct connection between the histopathology and molecular analyses LCMhas been previously utilized in fishrelated studies [ “] with frozen sections Snapfrozen tissue is optimal for use with LCM for the downstream recovery of nucleic acids However the use of snapfrozen tissue is not always feasible particularly in wild fish studies whereremoval and fixation of the ans occurs in the field and it can be days before tissues arereturned to the laboratory and processed LCM of FFPE tissue can bridge the gap betweenmicroscopy and molecular analyses [] As with other species there is a vast amount of archival FFPE or similarly preserved fish tissue that could be useful for molecular analysesThe aim of this study was to determine how fixative type and fixation time affects nucleicacids in FFPE smallmouth bass liver tissue dissected with LCM Smallmouth bass Micropterusdolomieu are utilized in ongoing monitoring and assessment studies as an indicator species ofexposure to endocrinedisrupting and other contaminants Additionally they are a nonmodel but economically important species To address the utility of paraffinembedded fishtissue for molecular studies smallmouth bass liver was sampled and preserved for four timeperiods hr hr seven days and days in neutral buffered formalin NBFZFix1 ZF and the nonformalin fixative PAXgene1 PG The PAXgene1 Tissue Systemwas designed to improve tissue quality for parallel molecular and morphological analyses []Similarly ZF a zincbased formalin solution was chosen as it has been shown to producePLOS ONE 101371journalpone0236104 August PLOS ONE 0cEffects of tissue fixation on nucleic acids from microdissected tissue samples of a teleosthigher yields of DNA and RNA when compared to samples fixed in NBF [] In addition toDNA and RNA quantification downstream molecular techniques including Sanger sequencing and the Nanostring nCounter1 digital multiplexed gene expression assay [] were usedto determine if nucleic acids extracted from LCM tissue sections would have utility in futurestudies To the best of our knowledge this study provides novel research on the optimizationof fixative type and fixation time for the use of fish tissue extracts with Nanostring nCounter1technologyMaterials and methodsEthics statement and smallmouth bass sample collectionAll procedures including the handling and euthanasia of fish were approved by the US Geological Survey™s Leetown Science Center™s Institutional Animal Care and Use CommitteeIACUC protocol Five smallmouth bass approximately years old were sampledfrom a flowthrough tank at the US Geological Survey Fish Health Laboratory in Kearneysville West Virginia Fish were placed in a lethal dosage mgL of tricaine methanesulfonate TricaineS Syndel Ferndale WA for euthanasia An incision from the anus tooperculum was made the liver was excised dissected into five equal pieces and placed into fixatives consisting of NBF ZF Product Anatech Ltd Battle Creek MI and PG Product QIAGEN Valencia CA Pieces of liver from each fish were also placed into RNALater1 Product AM7021 Thermo Fisher Scientific Waltham MA and ethyl alcoholETOH to serve as controls Samples in RNALater1 were stored at ˚C for hr prior tostorage at ˚C and samples in ETOH were stored at room temperature RT until extractionswere completedHistological preparation and laser capture microdissectionSamples were fixed for hrs hrs seven days and days at RT for NBF and ZF Tissues preserved in PG were removed from the PAXgene1 Tissue FIX Product QIAGEN after hrs at RT placed in the PAXgene1 Tissue STABILIZER solution Product QIAGEN and stored at ˚C for hrs hrs seven and days Tissue processing was performed on a Shandon CitadelTM Tissue Processor Thermo FisherScientific as follows hrs in alcohol hr in alcohol hr in alcohol 2x hr in alcohol 3x hr in a solution of alcohol and histoclear 2x Product HS200 National Diagnostics Atlanta GA hr in histoclear 2x and hr in paraffin 2x at ˚C Upon completion tissues were embedded into paraffin wax and cooled tohardenTissues were cut at a thickness of μm using a new sterile razor for each sample and sections placed onto Leica Microsystems UVsterilized polyethylene napthalate PEN membraneslides Product NC0496333 Thermo Fisher Scientific Sterilized diethyl pyrocarbonateDEPC Product Millipore Sigma Burlington MA water was used in the water bathand slides were allowed to air dry after sections were placed on the PEN membrane slideUnstained tissue sections were deparaffinized with Anatech Ltd ProPar Clearant Product NC9537734 Thermo Fisher Scientific for min 2x and allowed to air dry prior to lasermicrodissection Liver sections were cut at 5x magnification with a Leica LMD6500 microscope Leica Microsystems at a pulse rate of “ nm Sections x mm2were cut and dropped into the cap of a sterile microcentrifuge tube by gravity Fig and subsequently extracted for RNA or DNAPLOS ONE 101371journalpone0236104 August PLOS ONE 0cEffects of tissue fixation on nucleic acids from microdissected tissue samples of a teleostFig Laser capture microdissection of a smallmouth liver section A Liver section prior to microdissection BLiver section after microdissection C Liver section floating in buffer in the cap of a microcentrifuge tub prior tonucleic acid extraction101371journalpone0236104g001Nucleic acid extractions and downstream analysesFor RNA purification the EZNA1 FFPE RNA Kit Product R695401 Omega BioTekNorcross GA was used according to manufacturer™s protocols for the xylene extractionmethod Extraction began with the addition of GPL Buffer skipping the beginning of the protocol since the tissues were already deparaffinized Samples were digested with proteinase Kfor min and eluted in μl DEPC water As part of the assay protocol DNA contaminationwas removed with a step involving DNA Clearance Columns that binds genomic DNA andallows RNA to pass through the spin column For the controls preserved in RNALater1approximately “ mg liver was extracted with an EZNA1 Total RNA Kit I Product R683402 Omega BioTek according to manufacturer™s protocols and eluted in μl DEPCwater DNA contamination was also removed from these samples with the use of HiBind1RNA Mini Columns and RNasefree DNase Product E109102 Omega BioTek All samples were quantified with a Qubit1 Fluorometer Invitrogen Carlsbad CA using theQubit1 RNA HS Assay Kit Product Q32852 Thermo Fisher Scientific To analyze degradation RIN values were obtained with the Agilent RNA Pico Kit Product “Agilent Technologies Santa Clara CA on an Agilent Bioanalyzer Agilent Technologies Following quantification samples were stored at ˚C prior to use on the NanostringnCounter1DNA was extracted with a proteinase K digestion buffer 50mM TrisHCl pH mMEDTA Tween mgml proteinase K as described in Lehmann and Kreipe []Each sample was extracted in μl of proteinase K digestion buffer and incubated overnight at˚C The tubes were vortexed centrifuged and incubated at ˚C for min to deactivateproteinase K and stored at ˚C Approximately “ mg of control liver samples preservedin ETOH were extracted with a Qiagen DNeasy Blood Tissue Kit Product QIAGEN according to manufacturer™s protocols It is worth mentioning that in initial trialsfor this study the Qiagen DNeasy Blood Tissue Kit was also used to extract DNA from LCMsamples however no quantifiable DNA could be obtained which was why a single tube extraction method was subsequently utilized DNA was quantified with the Qubit1 dsDNA HSAssay Kit Product Q32851 Thermo Fisher Scientific Samples were analyzed for mean fragment size and distribution on an Agilent Bioanalyzer with the Agilent High SensitivityDNA Kit Product “ Agilent TechnologiesFor all LCM samples the final concentration of purified RNA and DNA was standardizedby dividing the total concentration by the total amount of tissue collected μgmm3 Since agreater amount of tissue was extracted from control samples the concentration of purifiedRNA and DNA was standardized to the amount of tissue collected for LCMTo assess the suitability of LCM samples for downstream molecular analyses Sangersequencing and the NanoString nCounter1 Technology were used For endpoint PCRPLOS ONE 101371journalpone0236104 August PLOS ONE 0cEffects of tissue fixation on nucleic acids from microdissected tissue samples of a teleostprimers EF1α5F ™GAG CCC CCT TAC AGC CAG AAG3™ and EF1α5R ™TTCACC TCA GTG GTC AGG CA3™ were designed with NCBI Primer BLAST [] to amplifya bp amplicon of the smallmouth bass elongation factor alpha EF1α accession HQ4248721 gene This housekeeping transcript was chosen since it has been used in othersmallmouth bass studies [ ] and sequence data was available for both smallmouth bassand the closely related largemouth bass accession KT8277941 PCR amplification was conducted under the following conditions denaturation at ˚C for min followed by cyclesof ˚C for s ˚C for s and ˚C for min s with a final extension at ˚C for min Each reaction consisted of μl clear Go Taq Master Mix Product M7132 PromegaMadison WI μl of each primer at 10μM and μl template for LCM samples approximately “ ng and μl template for ETOH samples approximately “ ng Uponcompletion samples were analyzed on an agarose gel with a bp ladder Amplicons werecleaned with a QIAquick1 PCR purification kit Product QIAGEN and eluted in μl of Buffer EB Purified amplicons were used as template in cycle sequencing reactionswith the Applied Biosystems BigDye Terminator v31 Cycle Sequencing Kit Product Thermo Fisher Scientific for cycles of ˚C for min ˚C for s ˚C for sand ˚C for min Cycle sequencing reactions were purified with an Agencourt CleanSEQKit Product A29151 Beckman Coulter Brea CA and sequenced on an ABI 3130xl GeneticAnalyzer Applied Biosystems Foster City CA Sequences were analyzed with Geneious wwwgeneiouscom and quality was assessed by the percentage of bases with aquality score of or higher Q40 NCBI BLASTn was used to determine sequence similarityto the Micropterus spp EF1α gene HQ4248721 or KT8277941NanoString nCounter1 Technology was used with a Custom CodeSet that targeted transcripts expressed in the liver of smallmouth bass as described in Hahn et al [] The previous establishment and availability of this CodeSet was the reason liver was chosen as the tissue of focus in this study The liver is also the principal an for many chemical detoxificationpathways metabolic pathways and the production of vitellogenin In brief the nCounter1platform provides the capability to quantify up to RNA DNA or protein targets called aCodeSet in a multiplex fashion providing results similar to quantitative PCR qPCR [] Itis a costeffective method to analyze specific mRNA targets unlike RNAsequencing whichproduces a vast amount of data and captures all mRNA in a sample Sample setup for hybridizations was carried out according to manufacturer™s protocols with ng of total RNA forevery sample The limit of detection LOD was calculated as the mean of the negative controls � the standard deviation of the negative controls and was transcriptsStatisticsSignificant differences in nucleic acid concentrations and transcript abundance between fixatives for each fixation time were determined with a nonparametric KruskalWallis onewayANOVA followed by a Dunn™s multiple comparison posthoc analysis with a Bonferroni correction in the statistical program R [] Normalized transcript abundance data was used forthe analysis Transcript abundance data was normalized in nSolver Analysis Software Nanostring Technologies Seattle WA where the geometric mean of the negative controlswas subtracted to estimate background and the normalization factor was computed from thegeometric mean of the positive controls and the housekeeping transcripts Housekeeping transcripts were log2 transformed and analyzed for stability with NormFinder [] in R A KruskalWallis test was also used to identify differences amongst each fixative for each fixationtime and the template concentration used for PCR the Q40 score and sequence lengthobtained with Sanger sequencing Finally Spearman™s rank correlation analyses werePLOS ONE 101371journalpone0236104 August PLOS ONE 0cEffects of tissue fixation on nucleic acids from microdissected tissue samples of a teleostconducted in R to determine if the concentration of DNA samples used for PCR sequencelength and the Q40 score were associated the number of sequences with similarity to theMicropterus spp EF1α gene with Sanger sequencing Pvalues � were considered statistically significantResultsNucleic acid concentrationsBoth RNA and DNA were recovered from samples of all fixatives and fixation times Fig Liver samples preserved in RNALater1 had more than times greater RNA concentrationsthan samples preserved in NBF ZF or PG with a mean concentration of ± ngmm3 mean ± standard error The highest concentration of RNA from LCM samples wasobtained from PG samples at hr ± ngmm3 Fig 2A The lowest concentrationsFig Nucleic acid auantification A Mean standard error of RNA and B DNA concentrations μgmm3 ofmicrodissected smallmouth bass liver samples fixed in neutral buffered formalin NBF ZFix1 ZF andPAXgene1 PG for hours hours and days Samples preserved in alcohol ETOH and RNALater1were included as controls101371journalpone0236104g002PLOS ONE 101371journalpone0236104 August PLOS ONE 0cEffects of tissue fixation on nucleic acids from microdissected tissue samples of a teleostwere from NBF preserved tissues at two weeks ± ngmm3 The concentration ofRNA was significantly greater in RNALater1 samples than in NBF Pvalue and PGPvalue at hr NBF Pvalue and ZF Pvalue at hr NBF Pvalue and ZF Pvalue at seven days and NBF Pvalue � ZF Pvalue and PG Pvalue at days Mean concentrations of RNA in samplesfixed in NBF and ZF decreased at seven and days while those fixed in PG remained stablethroughout the time course Fig 2AThe amount of DNA recovered was less than RNA with all mean concentrations of LCMsamples below μgmm3 Samples fixed in ETOH had more than times greater concentrations of DNA than samples fixed for LCM with a mean concentration of ± ngmm3 The concentration of DNA was significantly greater in ETOH samples than in PG at hr Pvalue NBF Pvalue and PG Pvalue at hr NBF Pvalue and PG Pvalue at seven days and NBF Pvalue and PG Pvalue at days There was little variation in DNA concentrations over time for anyof the fixatives although for all fixatives the lowest concentration was at days Fig 2BThe quality of RNA varied among fixatives Mean RIN values of samples fixed in RNALater1 were at least twice as great as samples fixed in NBF ZF and PG The highest RIN valuesfor LCM samples were observed in NBF fixed tissue at hrs seven and days Fig 3A RINvalues were significantly greater in RNALater1 samples than in PG Pvalue and ZFPvalue samples at hr PG Pvalue and ZF Pvalue samples atFig Nucleic acid quality A Mean RIN values of RNA and B fragment size bp of DNA from samples fixed in neutral buffered formalin NBF ZFix ZF and PAXgene PG for hours hours and days Samplespreserved in alcohol ETOH and RNAlater were included as controls101371journalpone0236104g003PLOS ONE 101371journalpone0236104 August PLOS ONE 0cEffects of tissue fixation on nucleic acids from microdissected tissue samples of a teleost hr PG Pvalue and ZF Pvalue samples at seven days and PG Pvalue � and ZF Pvalue samples at days There were no significant differences in RIN values between LCM samplesMean fragment size of DNA varied over time within each fixative group but was not significantly different than samples fixed in ETOH Fig 3B Fragment size was only significantlygreater in NBF samples than in PG samples P value at hrDownstream analysesEndpoint PCR and Sanger sequencing were successful for the amplification and sequencingof the smallmouth bass EF1α gene although differences in sequencing success were apparentWithout trimming the ™ or ™ ends samples preserved in ETOH produced sequences with amean percentage of bases with a Q40 score or greater of while samples fixed for LCMproduced lower quality sequences Fig Of the samples fixed for LCM PG preserved samplesproduced the highest quality sequences At days there were no samples fixed in NBF or ZFthat were successful for sequencing the EF1α gene For NBF and ZF the best quality sequenceswere generated by samples fixed for hr conversely PG had the lowest quality sequencesfrom samples fixed for hr Fig It should be noted that of the five PG samples sequencedat hr two samples had much lower quality sequences than the other three samples whichmay have contributed to the decrease in the mean percentage of high quality sequences at hr Additionally multiple samples failed to sequence These included one of the ETOH samples forward and reverse sequences three NBF day samples forward sequences oneNBF seven day sample forward and reverse sequences two PG seven day samples forwardsequences one PG day sample forward sequence one PG hr sample reversesequence one ZF seven day sample reverse sequence four ZF day samples three forwardand one reverse sequence and one ZF hr sample forward sequence In order to calculatethe percentage of sequences with similarity to the Micropterus spp EF1α gene failed sequenceswere not included ie of sequences with similarity to Micropterus spp EF1α ofsequences with similarity to Micropterus spp EF1α total of sequences that were successfullysequenced � For LCM fixed samples NBF samples fixed for and hr produced theFig Sanger sequencing quality Mean SEM percentage of bases with a Q40 score or above indicative of highquality sequencing Samples preserved in ethanol ETOH were included as controls routinely used for DNApreservation101371journalpone0236104g004PLOS ONE 101371journalpone0236104 August PLOS ONE 0cEffects of tissue fixation on nucleic acids from microdissected tissue samples of a teleostgreatest percentage of sequences with similarity to the EF1α gene In ZF samplesthe greatest percentage of samples with similarity to the EF1α gene was at hr and in PG samples it was at seven days Although PG samples produced the leastamount of sequences with similarity to the EF1α gene at and hr it produced the greatestnumber of sequences at seven and daysA Spearman™s rank correlation analysis with all samples revealed that sequence length pvalue � rho and template concentration pvalue rho were significantly associated with the number of sequences with similarity to the EF1α gene Fig Although PCR primers were estimated to produce an amplicon size around bp manysequences were longer than bp This could be due to the high degree of fragmentation inthe samples which may have resulted in the annealing of small fragments to the original template molecules in overlapping regions [] Fragment length and the percentage of bases witha Q40 score or greater were not significantly correlated with the number of sequences withsimilarity to the EF1α gene The correlations were also examined excluding the ETOH controlssince the DNA concentration of the controls was significantly greater than those of many fixedsamples and to examine the differences amongst the fixatives only Sequence length remainedsignificant pvalue � rho however template concentration was not significantpvalue rho Fragment length and the percentage of bases with a Q40 scoreFig Sanger sequencing and NCBI blastn results A Spearman™s rank correlation analysis of sequence length bpand the number of NCBI blastn matches to the Micropterus spp elongation factor alpha EF1α gene B Meansequence length bp of the EF1α gene obtained with Sanger sequencing and the percentage of sequences withsimilarity to the Micropterus spp EF1α gene101371journalpone0236104g005PLOS ONE 101371journalpone0236104 August PLOS ONE 0cEffects of tissue fixation on nucleic acids from microdissected tissue samples of a teleostTable KruskalWallis nonparametric ANOVA results Significant differences in template concentration usedin PCR and Sanger sequencing results for each fixative between fixation timesFixativeNBFZFPGFixativeNBFNBFZFPGFixativeNBFNBFZFZFPGTemplate ConcentrationpvalueZ scoreFixation TimeNo significant differencesNo significant differences hr vs days of Bases with � Q40 ScorepvaluepvalueZ scoreFixation Time hr vs days hr vs hrNo significant differencesNo significant differencesSequence LengthZ scoreFixation Time days vs hr days vs hr days vs hr days vs hrNo significant differencesSignificant differences pvalue � in template concentration of bases with a Q40 score or greater andsequence length between samples fixed in neutral buffered formalin NBF ZFix1 ZF and PAXgene PG101371journalpone0236104t001or greater remained not significantly correlated A KruskalWallis test was used to identify significant differences between fixation times and template concentration percentage of baseswith a Q40 score or greater and sequence length for each fixative Table The Nanostring nCounter1 results revealed multiple occurrences of samples fixed forLCM with similar or greater transcript abundance compared to RNALater1 samples S1Table In samples fixed for LCM there was significantly greater transcript abundance in PGsamples than in NBF andor ZF samples for one transcript at seven days and eight transcriptsat days Interestingly there were multiple significant differences in housekeeping transcriptabundances between samples preserved in RNALater1 and samples fixed for LCM MeanEF1α transcript abundance was significantly greater in RNALater1 samples than in samplesfixed for LCM at all fixation times Conversely at seven and days 40S ribosomal protein S12transcripts were higher in NBF and ZF samples when compared to RNALater1 and ribosomalprotein L8 was higher in the PG samples Table Significant differences were also identified between fixation times for each fixative typeused to preserve LCM samples Table All significant differences were between fixationtimes hr and seven or days and hr and seven or days In some instances sampleswith longer fixation times had transcripts with significantly greater transcript abundance thansamples fixed for or hr Once again significant differences were identified amongsthousekeeping transcripts There were no significant differences in PG samples over timeNormFinder results ranked the housekeeping transcripts according to stability For all fixatives and all fixation times including RNALater1 samples the most stable housekeepingtranscript was Ribosomal Protein L8 stability followed by EF1α stability Eukaryotic Translation Initiation Factor 3D stability and 40S ribosomal protein S12stability PLOS ONE 101371journalpone0236104 August PLOS ONE 0cEffects of tissue fixation on nucleic acids from microdissected tissue samples of a teleostTable KruskalWallis nonparametric ANOVA results Significant differences in transcript abundance betweenfixatives for each fixation timeTranscript NameElongation Factor Alpha�Heat Shock Protein Elongation Factor Alpha�Thyroid Hormone Receptor BetaTranscript NameElongation Factor Alpha�Heat Shock Protein Elongation Factor Alpha�Heat Shock Protein Transcript Name40S Ribosomal Protein S12�ArginaseBetacateninElongation Factor Alpha�Heat Shock Protein Transforming Growth Factor Beta40S ribosomal protein S12�C Reactive ProteinlikeElongation Factor Alpha�Eukaryotic Translation Initiation Factor 3D�Transforming Growth Factor BetaApolipoprotein Elongation Factor Alpha�Ribosomal Protein L8�Superoxide DismutaseThyroid Hormone Receptor BetaThyroid Hormone Receptor BetaTranscript Name40S ribosomal protein S12�ArginaseElongation Factor Alpha�40S ribosomal protein S12�ArginaseC Reactive ProteinlikeElongation Factor Alpha�Ribosomal Protein L8�Transforming Growth Factor BetaApolipoprotein Aryl Hydrocarbon ReceptorElongation Factor Alpha�Heat Shock Protein Ribosomal Protein L8� Hrpvalue Hrpvalue Dayspvalue DayspvalueZ scoreZ scoreZ scoreZ scorePLOS ONE 101371journalpone0236104 August FixativesRNALater vs NBFRNALater vs NBFRNALater vs PGRNALater vs PGFixativesRNALater vs PGRNALater vs NBFRNALater vs ZFRNALater vs ZFFixativesRNALater vs NBFRNALater vs NBFRNALater vs NBFRNALater vs NBFRNALater vs NBFRNALater vs NBFRNALater vs ZFRNALater vs ZFRNALater vs ZFRNALater vs ZFRNALater vs ZFRNALater vs PGRNALater vs PGRNALater vs PGRNALater vs PGRNALater vs PGPG vs NBFFixativesRNALater vs NBFRNALater vs NBFRNALater vs NBFRNALater vs ZFRNALater vs ZFRNALater vs ZFRNALater vs ZFRNALater vs ZFRNALater vs ZFRNALater vs PGRNALater vs PGRNALater vs PGRNALater vs PGRNALater vs PGContinued PLOS ONE 0cEffects of tissue fixation on nucleic acids from microdissected tissue samples of a teleostTable ContinuedAryl Hydrocarbon ReceptorEstrogen Receptor Beta Hepcidin Thyroid Hormone Receptor BetaType II Iodothyronine DeiodinaseAryl Hydrocarbon ReceptorHepcidin Thyroid Hormone Receptor BetaType II Iodothyronine DeiodinasePG vs NBFPG vs NBFPG vs NBFPG vs NBFPG vs NBFPG vs ZFPG vs ZFPG vs ZFPG vs ZFSignificant differences pvalue � in transcript abundance between RNALater1 samples and samples fixed in neutral buffered formalin NBF ZFix1 ZF and PAXgene PG� Indicates housekeeping transcripts101371journalpone0236104t002Table KruskalWallis nonparametric ANOVA results Significant differences in transcript abundance betweenfixation times for each fixativeTranscript Name40S ribosomal protein S12�ZFixpvalueCytochrome P450 family subfamily AElongation Factor Alpha�Eukaryotic Translation Initiation Factor 3D�Ribosomal Protein L8�TataBox Binding ProteinThyroid Hormone Receptor BetaTransforming Growth Factor BetaTranscript Name40S ribosomal protein S12�Cytochrome P450 family subfamily ABeta

cancer261

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: The effects of tissue fixation on sequencingand transcript abundance of nucleic acidsfrom microdissected liver samples ofsmallmouth bass Micropterus dolomieuHeather L WalshID Adam J Sperry Vicki S BlazerUS Geological Survey National Fish Health Research Laboratory Leetown Science Center KearneysvilleWest Virginia United States of Americaa1111111111a1111111111a1111111111a1111111111a1111111111 hwalshusgsgovAbstractThere is an increasing emphasis on effectsbased monitoring to document responses associated with exposure to complex mixtures of chemicals climate change pathogens parasitesand other environmental stressors in fish populations For decades aquatic monitoring programs have included the collection of tissues preserved for microscopic pathology Consequently formalinfixed paraffinembedded FFPE tissue can be an important reservoir ofnucleic acids as technologies emerge that utilize molecular endpoints Despite the crosslinking effects of formalin its impact on nucleic acid quality and concentration amplification andsequencing are not well described While freshfrozen tissue is optimal for working withnucleic acids FFPE samples have been shown to be conducive for molecular studies Lasercapture microdissection LCM is one technology which allows for collection of specificregions or cell populations from fresh or preserved specimens with pathological alterationspathogens or parasites In this study smallmouth bass Micropterus dolomieu liver was preserved in three different fixatives including neutral buffered formalin NBF ZFix®ZF and PAXgene® PG for four time periods hr hr seven days and days Controls consisted of pieces of liver preserved in RNALater® or ethanol Smallmouth basswere chosen as they are an economically important sportfish and have been utilized as indicators of exposure to endocrine disruptors and other environmental stressors Small liversections were cut out with laser microdissection and DNA and RNA were purified and analyzed for nucleic acid concentration and quality Sanger sequencing and the NanoStringnCounter® technology were used to assess the suitability of these samples in downstreammolecular techniques The results revealed that of the formalin fixatives NBF samples fixedfor and hr were superior to ZF samples for both Sanger sequencing and the NanostringnCounter® The nonformalin PAXgene® samples were equally successful and they showedgreater stability in nucleic acid quality and concentration over longer fixation times This studydemonstrated that small quantities of preserved tissue from smallmouth bass can be utilizedin downstream molecular techniques however future studies will need to optimize the methods presented here for different tissue types fish species and pathological conditions ACCESSCitation Walsh HL Sperry AJ Blazer VS The effects of tissue fixation on sequencing andtranscript abundance of nucleic acids frommicrodissected liver samples of smallmouth bassMicropterus dolomieu e0236104 101371journalpone0236104Editor Rajakumar Anbazhagan National Instituteof Child Health and Human Development NICHDNIH UNITED STATESReceived April Accepted June Published August Copyright This is an access free of allcopyright and may be freely reproduceddistributed transmitted modified built upon orotherwise used by anyone for any lawful purposeThe work is made available under the CreativeCommons CC0 public domain dedicationData Availability Statement All relevant data arewithin the paper and its Supporting InformationfilesFunding This was fully funded internally by theUSGS Environmental Health Contaminant BiologyProgram and the USGS EcosystemsEnvironments and Fisheries Program MissionAreas There are no actual grant numbersPLOS ONE 101371journalpone0236104 August PLOS ONE 0cCompeting interests The authors have declaredthat no competing interests existEffects of tissue fixation on nucleic acids from microdissected tissue samples of a teleostIntroductionGlobally environmental monitoring programs are increasingly used to identify adverse effectsof human activities on aquatic resources [“] The recognition that there are numerouschemical contaminants environmental stressors as well as new and emerging pathogensparasites cooccurring has led to an emphasis on biological environmental effectsbased assessments utilizing resident indicator fish species or caged model species [“] Histopathologyhas been used for many years to assess the health of wild fishes both for specific studies as wellas part of effectsbased monitoring programs [“] More recently genomic endpoints arealso being incorporated into environmental monitoring and risk assessment [“] Whenboth histopathology and molecular analyses are part of an assessment pieces of a tissue arecommonly preserved in buffered formalin or a similar preservative and adjacent separatepieces are preserved in RNAlater1 ethanol or frozen for molecular analyses [“]However for alterations not visible the tissue piece chosen for gene expression may not contain the same cellular components or alterations as those within the histology sectionThe use of formalin fixed paraffinembedded FFPE tissue has been regarded as a valuablereservoir of preserved nucleic acids in mammalian studies [“] Although FFPE tissuesprovide a vast source of pathologically diverse types of genetic material there are drawbackscompared to other tissue preservation methods Formalin fixation causes nucleic acids to fragment degrade and crosslink [] Frozen tissues or tissues specifically preserved for downstream nucleic acid applications do not experience the type of degradation observed fromformalin fixation Despite these setbacks nucleic acids extracted from FFPE tissue have provento be suitable for use in endpoint PCR [] realtime qPCR [ ] and Nextgenerationsequencing [ ] Optimization of FFPE tissues for downstream nucleic acid applicationshas been attempted in multiple studies by evaluation of different fixation methods [ ] tissue handling and processing times [ ] and extraction methods [ ]Laser capture microdissection LCM utilizes a microscope equipped with a laser to targetand isolate specific cells from a heterogeneous population of cells [] Single cells foci of cellpopulations within a tissue or pathogens and parasites can be microdissected Hence nucleicacids from specific cell populations of interest can be analyzed for gene expression studiestranscriptome development or molecular identification of pathogens and parasites Thisallows for a more direct connection between the histopathology and molecular analyses LCMhas been previously utilized in fishrelated studies [ “] with frozen sections Snapfrozen tissue is optimal for use with LCM for the downstream recovery of nucleic acids However the use of snapfrozen tissue is not always feasible particularly in wild fish studies whereremoval and fixation of the ans occurs in the field and it can be days before tissues arereturned to the laboratory and processed LCM of FFPE tissue can bridge the gap betweenmicroscopy and molecular analyses [] As with other species there is a vast amount of archival FFPE or similarly preserved fish tissue that could be useful for molecular analysesThe aim of this study was to determine how fixative type and fixation time affects nucleicacids in FFPE smallmouth bass liver tissue dissected with LCM Smallmouth bass Micropterusdolomieu are utilized in ongoing monitoring and assessment studies as an indicator species ofexposure to endocrinedisrupting and other contaminants Additionally they are a nonmodel but economically important species To address the utility of paraffinembedded fishtissue for molecular studies smallmouth bass liver was sampled and preserved for four timeperiods hr hr seven days and days in neutral buffered formalin NBFZFix1 ZF and the nonformalin fixative PAXgene1 PG The PAXgene1 Tissue Systemwas designed to improve tissue quality for parallel molecular and morphological analyses []Similarly ZF a zincbased formalin solution was chosen as it has been shown to producePLOS ONE 101371journalpone0236104 August PLOS ONE 0cEffects of tissue fixation on nucleic acids from microdissected tissue samples of a teleosthigher yields of DNA and RNA when compared to samples fixed in NBF [] In addition toDNA and RNA quantification downstream molecular techniques including Sanger sequencing and the Nanostring nCounter1 digital multiplexed gene expression assay [] were usedto determine if nucleic acids extracted from LCM tissue sections would have utility in futurestudies To the best of our knowledge this study provides novel research on the optimizationof fixative type and fixation time for the use of fish tissue extracts with Nanostring nCounter1technologyMaterials and methodsEthics statement and smallmouth bass sample collectionAll procedures including the handling and euthanasia of fish were approved by the US Geological Survey™s Leetown Science Center™s Institutional Animal Care and Use CommitteeIACUC protocol Five smallmouth bass approximately years old were sampledfrom a flowthrough tank at the US Geological Survey Fish Health Laboratory in Kearneysville West Virginia Fish were placed in a lethal dosage mgL of tricaine methanesulfonate TricaineS Syndel Ferndale WA for euthanasia An incision from the anus tooperculum was made the liver was excised dissected into five equal pieces and placed into fixatives consisting of NBF ZF Product Anatech Ltd Battle Creek MI and PG Product QIAGEN Valencia CA Pieces of liver from each fish were also placed into RNALater1 Product AM7021 Thermo Fisher Scientific Waltham MA and ethyl alcoholETOH to serve as controls Samples in RNALater1 were stored at ˚C for hr prior tostorage at ˚C and samples in ETOH were stored at room temperature RT until extractionswere completedHistological preparation and laser capture microdissectionSamples were fixed for hrs hrs seven days and days at RT for NBF and ZF Tissues preserved in PG were removed from the PAXgene1 Tissue FIX Product QIAGEN after hrs at RT placed in the PAXgene1 Tissue STABILIZER solution Product QIAGEN and stored at ˚C for hrs hrs seven and days Tissue processing was performed on a Shandon CitadelTM Tissue Processor Thermo FisherScientific as follows hrs in alcohol hr in alcohol hr in alcohol 2x hr in alcohol 3x hr in a solution of alcohol and histoclear 2x Product HS200 National Diagnostics Atlanta GA hr in histoclear 2x and hr in paraffin 2x at ˚C Upon completion tissues were embedded into paraffin wax and cooled tohardenTissues were cut at a thickness of μm using a new sterile razor for each sample and sections placed onto Leica Microsystems UVsterilized polyethylene napthalate PEN membraneslides Product NC0496333 Thermo Fisher Scientific Sterilized diethyl pyrocarbonateDEPC Product Millipore Sigma Burlington MA water was used in the water bathand slides were allowed to air dry after sections were placed on the PEN membrane slideUnstained tissue sections were deparaffinized with Anatech Ltd ProPar Clearant Product NC9537734 Thermo Fisher Scientific for min 2x and allowed to air dry prior to lasermicrodissection Liver sections were cut at 5x magnification with a Leica LMD6500 microscope Leica Microsystems at a pulse rate of “ nm Sections x mm2were cut and dropped into the cap of a sterile microcentrifuge tube by gravity Fig and subsequently extracted for RNA or DNAPLOS ONE 101371journalpone0236104 August PLOS ONE 0cEffects of tissue fixation on nucleic acids from microdissected tissue samples of a teleostFig Laser capture microdissection of a smallmouth liver section A Liver section prior to microdissection BLiver section after microdissection C Liver section floating in buffer in the cap of a microcentrifuge tub prior tonucleic acid extraction101371journalpone0236104g001Nucleic acid extractions and downstream analysesFor RNA purification the EZNA1 FFPE RNA Kit Product R695401 Omega BioTekNorcross GA was used according to manufacturer™s protocols for the xylene extractionmethod Extraction began with the addition of GPL Buffer skipping the beginning of the protocol since the tissues were already deparaffinized Samples were digested with proteinase Kfor min and eluted in μl DEPC water As part of the assay protocol DNA contaminationwas removed with a step involving DNA Clearance Columns that binds genomic DNA andallows RNA to pass through the spin column For the controls preserved in RNALater1approximately “ mg liver was extracted with an EZNA1 Total RNA Kit I Product R683402 Omega BioTek according to manufacturer™s protocols and eluted in μl DEPCwater DNA contamination was also removed from these samples with the use of HiBind1RNA Mini Columns and RNasefree DNase Product E109102 Omega BioTek All samples were quantified with a Qubit1 Fluorometer Invitrogen Carlsbad CA using theQubit1 RNA HS Assay Kit Product Q32852 Thermo Fisher Scientific To analyze degradation RIN values were obtained with the Agilent RNA Pico Kit Product “Agilent Technologies Santa Clara CA on an Agilent Bioanalyzer Agilent Technologies Following quantification samples were stored at ˚C prior to use on the NanostringnCounter1DNA was extracted with a proteinase K digestion buffer 50mM TrisHCl pH mMEDTA Tween mgml proteinase K as described in Lehmann and Kreipe []Each sample was extracted in μl of proteinase K digestion buffer and incubated overnight at˚C The tubes were vortexed centrifuged and incubated at ˚C for min to deactivateproteinase K and stored at ˚C Approximately “ mg of control liver samples preservedin ETOH were extracted with a Qiagen DNeasy Blood Tissue Kit Product QIAGEN according to manufacturer™s protocols It is worth mentioning that in initial trialsfor this study the Qiagen DNeasy Blood Tissue Kit was also used to extract DNA from LCMsamples however no quantifiable DNA could be obtained which was why a single tube extraction method was subsequently utilized DNA was quantified with the Qubit1 dsDNA HSAssay Kit Product Q32851 Thermo Fisher Scientific Samples were analyzed for mean fragment size and distribution on an Agilent Bioanalyzer with the Agilent High SensitivityDNA Kit Product “ Agilent TechnologiesFor all LCM samples the final concentration of purified RNA and DNA was standardizedby dividing the total concentration by the total amount of tissue collected μgmm3 Since agreater amount of tissue was extracted from control samples the concentration of purifiedRNA and DNA was standardized to the amount of tissue collected for LCMTo assess the suitability of LCM samples for downstream molecular analyses Sangersequencing and the NanoString nCounter1 Technology were used For endpoint PCRPLOS ONE 101371journalpone0236104 August PLOS ONE 0cEffects of tissue fixation on nucleic acids from microdissected tissue samples of a teleostprimers EF1α5F ™GAG CCC CCT TAC AGC CAG AAG3™ and EF1α5R ™TTCACC TCA GTG GTC AGG CA3™ were designed with NCBI Primer BLAST [] to amplifya bp amplicon of the smallmouth bass elongation factor alpha EF1α accession HQ4248721 gene This housekeeping transcript was chosen since it has been used in othersmallmouth bass studies [ ] and sequence data was available for both smallmouth bassand the closely related largemouth bass accession KT8277941 PCR amplification was conducted under the following conditions denaturation at ˚C for min followed by cyclesof ˚C for s ˚C for s and ˚C for min s with a final extension at ˚C for min Each reaction consisted of μl clear Go Taq Master Mix Product M7132 PromegaMadison WI μl of each primer at 10μM and μl template for LCM samples approximately “ ng and μl template for ETOH samples approximately “ ng Uponcompletion samples were analyzed on an agarose gel with a bp ladder Amplicons werecleaned with a QIAquick1 PCR purification kit Product QIAGEN and eluted in μl of Buffer EB Purified amplicons were used as template in cycle sequencing reactionswith the Applied Biosystems BigDye Terminator v31 Cycle Sequencing Kit Product Thermo Fisher Scientific for cycles of ˚C for min ˚C for s ˚C for sand ˚C for min Cycle sequencing reactions were purified with an Agencourt CleanSEQKit Product A29151 Beckman Coulter Brea CA and sequenced on an ABI 3130xl GeneticAnalyzer Applied Biosystems Foster City CA Sequences were analyzed with Geneious wwwgeneiouscom and quality was assessed by the percentage of bases with aquality score of or higher Q40 NCBI BLASTn was used to determine sequence similarityto the Micropterus spp EF1α gene HQ4248721 or KT8277941NanoString nCounter1 Technology was used with a Custom CodeSet that targeted transcripts expressed in the liver of smallmouth bass as described in Hahn et al [] The previous establishment and availability of this CodeSet was the reason liver was chosen as the tissue of focus in this study The liver is also the principal an for many chemical detoxificationpathways metabolic pathways and the production of vitellogenin In brief the nCounter1platform provides the capability to quantify up to RNA DNA or protein targets called aCodeSet in a multiplex fashion providing results similar to quantitative PCR qPCR [] Itis a costeffective method to analyze specific mRNA targets unlike RNAsequencing whichproduces a vast amount of data and captures all mRNA in a sample Sample setup for hybridizations was carried out according to manufacturer™s protocols with ng of total RNA forevery sample The limit of detection LOD was calculated as the mean of the negative controls � the standard deviation of the negative controls and was transcriptsStatisticsSignificant differences in nucleic acid concentrations and transcript abundance between fixatives for each fixation time were determined with a nonparametric KruskalWallis onewayANOVA followed by a Dunn™s multiple comparison posthoc analysis with a Bonferroni correction in the statistical program R [] Normalized transcript abundance data was used forthe analysis Transcript abundance data was normalized in nSolver Analysis Software Nanostring Technologies Seattle WA where the geometric mean of the negative controlswas subtracted to estimate background and the normalization factor was computed from thegeometric mean of the positive controls and the housekeeping transcripts Housekeeping transcripts were log2 transformed and analyzed for stability with NormFinder [] in R A KruskalWallis test was also used to identify differences amongst each fixative for each fixationtime and the template concentration used for PCR the Q40 score and sequence lengthobtained with Sanger sequencing Finally Spearman™s rank correlation analyses werePLOS ONE 101371journalpone0236104 August PLOS ONE 0cEffects of tissue fixation on nucleic acids from microdissected tissue samples of a teleostconducted in R to determine if the concentration of DNA samples used for PCR sequencelength and the Q40 score were associated the number of sequences with similarity to theMicropterus spp EF1α gene with Sanger sequencing Pvalues � were considered statistically significantResultsNucleic acid concentrationsBoth RNA and DNA were recovered from samples of all fixatives and fixation times Fig Liver samples preserved in RNALater1 had more than times greater RNA concentrationsthan samples preserved in NBF ZF or PG with a mean concentration of ± ngmm3 mean ± standard error The highest concentration of RNA from LCM samples wasobtained from PG samples at hr ± ngmm3 Fig 2A The lowest concentrationsFig Nucleic acid auantification A Mean standard error of RNA and B DNA concentrations μgmm3 ofmicrodissected smallmouth bass liver samples fixed in neutral buffered formalin NBF ZFix1 ZF andPAXgene1 PG for hours hours and days Samples preserved in alcohol ETOH and RNALater1were included as controls101371journalpone0236104g002PLOS ONE 101371journalpone0236104 August PLOS ONE 0cEffects of tissue fixation on nucleic acids from microdissected tissue samples of a teleostwere from NBF preserved tissues at two weeks ± ngmm3 The concentration ofRNA was significantly greater in RNALater1 samples than in NBF Pvalue and PGPvalue at hr NBF Pvalue and ZF Pvalue at hr NBF Pvalue and ZF Pvalue at seven days and NBF Pvalue � ZF Pvalue and PG Pvalue at days Mean concentrations of RNA in samplesfixed in NBF and ZF decreased at seven and days while those fixed in PG remained stablethroughout the time course Fig 2AThe amount of DNA recovered was less than RNA with all mean concentrations of LCMsamples below μgmm3 Samples fixed in ETOH had more than times greater concentrations of DNA than samples fixed for LCM with a mean concentration of ± ngmm3 The concentration of DNA was significantly greater in ETOH samples than in PG at hr Pvalue NBF Pvalue and PG Pvalue at hr NBF Pvalue and PG Pvalue at seven days and NBF Pvalue and PG Pvalue at days There was little variation in DNA concentrations over time for anyof the fixatives although for all fixatives the lowest concentration was at days Fig 2BThe quality of RNA varied among fixatives Mean RIN values of samples fixed in RNALater1 were at least twice as great as samples fixed in NBF ZF and PG The highest RIN valuesfor LCM samples were observed in NBF fixed tissue at hrs seven and days Fig 3A RINvalues were significantly greater in RNALater1 samples than in PG Pvalue and ZFPvalue samples at hr PG Pvalue and ZF Pvalue samples atFig Nucleic acid quality A Mean RIN values of RNA and B fragment size bp of DNA from samples fixed in neutral buffered formalin NBF ZFix ZF and PAXgene PG for hours hours and days Samplespreserved in alcohol ETOH and RNAlater were included as controls101371journalpone0236104g003PLOS ONE 101371journalpone0236104 August PLOS ONE 0cEffects of tissue fixation on nucleic acids from microdissected tissue samples of a teleost hr PG Pvalue and ZF Pvalue samples at seven days and PG Pvalue � and ZF Pvalue samples at days There were no significant differences in RIN values between LCM samplesMean fragment size of DNA varied over time within each fixative group but was not significantly different than samples fixed in ETOH Fig 3B Fragment size was only significantlygreater in NBF samples than in PG samples P value at hrDownstream analysesEndpoint PCR and Sanger sequencing were successful for the amplification and sequencingof the smallmouth bass EF1α gene although differences in sequencing success were apparentWithout trimming the ™ or ™ ends samples preserved in ETOH produced sequences with amean percentage of bases with a Q40 score or greater of while samples fixed for LCMproduced lower quality sequences Fig Of the samples fixed for LCM PG preserved samplesproduced the highest quality sequences At days there were no samples fixed in NBF or ZFthat were successful for sequencing the EF1α gene For NBF and ZF the best quality sequenceswere generated by samples fixed for hr conversely PG had the lowest quality sequencesfrom samples fixed for hr Fig It should be noted that of the five PG samples sequencedat hr two samples had much lower quality sequences than the other three samples whichmay have contributed to the decrease in the mean percentage of high quality sequences at hr Additionally multiple samples failed to sequence These included one of the ETOH samples forward and reverse sequences three NBF day samples forward sequences oneNBF seven day sample forward and reverse sequences two PG seven day samples forwardsequences one PG day sample forward sequence one PG hr sample reversesequence one ZF seven day sample reverse sequence four ZF day samples three forwardand one reverse sequence and one ZF hr sample forward sequence In order to calculatethe percentage of sequences with similarity to the Micropterus spp EF1α gene failed sequenceswere not included ie of sequences with similarity to Micropterus spp EF1α ofsequences with similarity to Micropterus spp EF1α total of sequences that were successfullysequenced � For LCM fixed samples NBF samples fixed for and hr produced theFig Sanger sequencing quality Mean SEM percentage of bases with a Q40 score or above indicative of highquality sequencing Samples preserved in ethanol ETOH were included as controls routinely used for DNApreservation101371journalpone0236104g004PLOS ONE 101371journalpone0236104 August PLOS ONE 0cEffects of tissue fixation on nucleic acids from microdissected tissue samples of a teleostgreatest percentage of sequences with similarity to the EF1α gene In ZF samplesthe greatest percentage of samples with similarity to the EF1α gene was at hr and in PG samples it was at seven days Although PG samples produced the leastamount of sequences with similarity to the EF1α gene at and hr it produced the greatestnumber of sequences at seven and daysA Spearman™s rank correlation analysis with all samples revealed that sequence length pvalue � rho and template concentration pvalue rho were significantly associated with the number of sequences with similarity to the EF1α gene Fig Although PCR primers were estimated to produce an amplicon size around bp manysequences were longer than bp This could be due to the high degree of fragmentation inthe samples which may have resulted in the annealing of small fragments to the original template molecules in overlapping regions [] Fragment length and the percentage of bases witha Q40 score or greater were not significantly correlated with the number of sequences withsimilarity to the EF1α gene The correlations were also examined excluding the ETOH controlssince the DNA concentration of the controls was significantly greater than those of many fixedsamples and to examine the differences amongst the fixatives only Sequence length remainedsignificant pvalue � rho however template concentration was not significantpvalue rho Fragment length and the percentage of bases with a Q40 scoreFig Sanger sequencing and NCBI blastn results A Spearman™s rank correlation analysis of sequence length bpand the number of NCBI blastn matches to the Micropterus spp elongation factor alpha EF1α gene B Meansequence length bp of the EF1α gene obtained with Sanger sequencing and the percentage of sequences withsimilarity to the Micropterus spp EF1α gene101371journalpone0236104g005PLOS ONE 101371journalpone0236104 August PLOS ONE 0cEffects of tissue fixation on nucleic acids from microdissected tissue samples of a teleostTable KruskalWallis nonparametric ANOVA results Significant differences in template concentration usedin PCR and Sanger sequencing results for each fixative between fixation timesFixativeNBFZFPGFixativeNBFNBFZFPGFixativeNBFNBFZFZFPGTemplate ConcentrationpvalueZ scoreFixation TimeNo significant differencesNo significant differences hr vs days of Bases with � Q40 ScorepvaluepvalueZ scoreFixation Time hr vs days hr vs hrNo significant differencesNo significant differencesSequence LengthZ scoreFixation Time days vs hr days vs hr days vs hr days vs hrNo significant differencesSignificant differences pvalue � in template concentration of bases with a Q40 score or greater andsequence length between samples fixed in neutral buffered formalin NBF ZFix1 ZF and PAXgene PG101371journalpone0236104t001or greater remained not significantly correlated A KruskalWallis test was used to identify significant differences between fixation times and template concentration percentage of baseswith a Q40 score or greater and sequence length for each fixative Table The Nanostring nCounter1 results revealed multiple occurrences of samples fixed forLCM with similar or greater transcript abundance compared to RNALater1 samples S1Table In samples fixed for LCM there was significantly greater transcript abundance in PGsamples than in NBF andor ZF samples for one transcript at seven days and eight transcriptsat days Interestingly there were multiple significant differences in housekeeping transcriptabundances between samples preserved in RNALater1 and samples fixed for LCM MeanEF1α transcript abundance was significantly greater in RNALater1 samples than in samplesfixed for LCM at all fixation times Conversely at seven and days 40S ribosomal protein S12transcripts were higher in NBF and ZF samples when compared to RNALater1 and ribosomalprotein L8 was higher in the PG samples Table Significant differences were also identified between fixation times for each fixative typeused to preserve LCM samples Table All significant differences were between fixationtimes hr and seven or days and hr and seven or days In some instances sampleswith longer fixation times had transcripts with significantly greater transcript abundance thansamples fixed for or hr Once again significant differences were identified amongsthousekeeping transcripts There were no significant differences in PG samples over timeNormFinder results ranked the housekeeping transcripts according to stability For all fixatives and all fixation times including RNALater1 samples the most stable housekeepingtranscript was Ribosomal Protein L8 stability followed by EF1α stability Eukaryotic Translation Initiation Factor 3D stability and 40S ribosomal protein S12stability PLOS ONE 101371journalpone0236104 August PLOS ONE 0cEffects of tissue fixation on nucleic acids from microdissected tissue samples of a teleostTable KruskalWallis nonparametric ANOVA results Significant differences in transcript abundance betweenfixatives for each fixation timeTranscript NameElongation Factor Alpha�Heat Shock Protein Elongation Factor Alpha�Thyroid Hormone Receptor BetaTranscript NameElongation Factor Alpha�Heat Shock Protein Elongation Factor Alpha�Heat Shock Protein Transcript Name40S Ribosomal Protein S12�ArginaseBetacateninElongation Factor Alpha�Heat Shock Protein Transforming Growth Factor Beta40S ribosomal protein S12�C Reactive ProteinlikeElongation Factor Alpha�Eukaryotic Translation Initiation Factor 3D�Transforming Growth Factor BetaApolipoprotein Elongation Factor Alpha�Ribosomal Protein L8�Superoxide DismutaseThyroid Hormone Receptor BetaThyroid Hormone Receptor BetaTranscript Name40S ribosomal protein S12�ArginaseElongation Factor Alpha�40S ribosomal protein S12�ArginaseC Reactive ProteinlikeElongation Factor Alpha�Ribosomal Protein L8�Transforming Growth Factor BetaApolipoprotein Aryl Hydrocarbon ReceptorElongation Factor Alpha�Heat Shock Protein Ribosomal Protein L8� Hrpvalue Hrpvalue Dayspvalue DayspvalueZ scoreZ scoreZ scoreZ scorePLOS ONE 101371journalpone0236104 August FixativesRNALater vs NBFRNALater vs NBFRNALater vs PGRNALater vs PGFixativesRNALater vs PGRNALater vs NBFRNALater vs ZFRNALater vs ZFFixativesRNALater vs NBFRNALater vs NBFRNALater vs NBFRNALater vs NBFRNALater vs NBFRNALater vs NBFRNALater vs ZFRNALater vs ZFRNALater vs ZFRNALater vs ZFRNALater vs ZFRNALater vs PGRNALater vs PGRNALater vs PGRNALater vs PGRNALater vs PGPG vs NBFFixativesRNALater vs NBFRNALater vs NBFRNALater vs NBFRNALater vs ZFRNALater vs ZFRNALater vs ZFRNALater vs ZFRNALater vs ZFRNALater vs ZFRNALater vs PGRNALater vs PGRNALater vs PGRNALater vs PGRNALater vs PGContinued PLOS ONE 0cEffects of tissue fixation on nucleic acids from microdissected tissue samples of a teleostTable ContinuedAryl Hydrocarbon ReceptorEstrogen Receptor Beta Hepcidin Thyroid Hormone Receptor BetaType II Iodothyronine DeiodinaseAryl Hydrocarbon ReceptorHepcidin Thyroid Hormone Receptor BetaType II Iodothyronine DeiodinasePG vs NBFPG vs NBFPG vs NBFPG vs NBFPG vs NBFPG vs ZFPG vs ZFPG vs ZFPG vs ZFSignificant differences pvalue � in transcript abundance between RNALater1 samples and samples fixed in neutral buffered formalin NBF ZFix1 ZF and PAXgene PG� Indicates housekeeping transcripts101371journalpone0236104t002Table KruskalWallis nonparametric ANOVA results Significant differences in transcript abundance betweenfixation times for each fixativeTranscript Name40S ribosomal protein S12�ZFixpvalueCytochrome P450 family subfamily AElongation Factor Alpha�Eukaryotic Translation Initiation Factor 3D�Ribosomal Protein L8�TataBox Binding ProteinThyroid Hormone Receptor BetaTransforming Growth Factor BetaTranscript Name40S ribosomal protein S12�Cytochrome P450 family subfamily ABeta Answer:

Thyroid_Cancer

Hydrophobicity drives receptormediateduptake of heatprocessed proteins by THP1macrophages and dendritic cells but notcytokine responsesYing Deng12 Coen Govers1 Malgorzata Teodorowicz3 Ieva Liobyte12 Ilaria DeSimone13 Kasper Hettinga4 Harry J WichersID12 Food and Biobased Research Wageningen University and Research Wageningen The Netherlands Laboratory of Food Chemistry Wageningen University and Research Wageningen The Netherlands Cell Biology and Immunology Wageningen University and Research Wageningen The Netherlands Food Quality and Design Wageningen University and Research Wageningen The Netherlands harrywicherswurnlAbstractAlthough an impact of processing on immunogenicity of food proteins has clearly been demonstrated the underlying mechanisms are still unclear We applied different processingmethods wet heating ˚C and low or hightemperature ˚C or ˚C respectivelydryheating in absence or presence of reducing sugars to lactoglobulin BLG lysozymeand thyroglobulin which represent dietary proteins with different pI or molecular weightUptake of the soluble fraction of the samples was tested in two types of genetically homogeneous antigenpresenting cells macrophages and dendritic cells derived from THP1monocytes This revealed a strong correlation between the uptake of the different proteinsamples by macrophages and dendritic cells and confirmed the key role of hydrophobicityover aggregation in determining the uptake Several uptake routes were shown to contribute to the uptake of BLG by macrophages However cytokine responses following exposureof macrophages to BLG samples were not related to the levels of uptake Together ourresults demonstrate that heattreatmentinduced increased hydrophobicity is the prime driving factor in uptake but not in cytokine production by THP1 macrophagesIntroductionDietary proteins play an important physiological role not only in providing amino acids andenergy but also for the maturation and regulation of the immune system Proteins are knownto be involved in the regulation of chronic inflammation and be the cause of allergies [] Forexample a higher intake of animal protein was found to enhance the proinflammatoryresponse of macrophages in mice [] Most of the proteins that humans ingest have beenthrough a heating process as part of the food processing as a result of which structural modifications such as glycation with reducing sugar andor aggregation of proteins may occura1111111111a1111111111a1111111111a1111111111a1111111111 ACCESSCitation Deng Y Govers C Teodorowicz MLiobyte I De Simone I Hettinga K Hydrophobicity drives receptormediated uptake ofheatprocessed proteins by THP1 macrophagesand dendritic cells but not cytokine responses e0236212 101371journalpone0236212Editor Yi Cao Xiangtan University CHINAReceived June Accepted August Published August Copyright Deng This is an access distributed under the terms of theCreative Commons Attribution License whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are creditedData Availability Statement All relevant data arewithin the paper and its Supporting InformationfilesFunding This work was financial supported by theChina Scholarship Council No awarded to YDCompeting interests The authors have declaredthat no competing interests existPLOS ONE 101371journalpone0236212 August PLOS ONE 0cUptake of heatprocessed proteins by THP1 macrophages and dendritic cells and subsequent cytokine responseDifferent processing methods would alter the immunogenicity of food protein in differentmanner For example the antigenicity of prawn allergen was found to be significantlyincreased after frying but decreased after boiling acid treatment or highpressure processing[] The allergenicity of milk protein was reported to decrease after glycation []In an earlier study on lactoglobulin BLG the major whey protein of milk [] weobserved that heat processing under different conditions altered the physicochemical properties and its uptake by THP1 macrophages In particular heating in solution at ˚C for days introduced changes in hydrophobicity and molecular weight which were shown to be thekey determinants for uptake This event may be relevant for a possible subsequent immuneresponse [ ] and has been linked to heattreatmentinduced changes in protein™s pr sityto induce an allergic reaction [“] Despite the new findings in that study there are obviouslimitations relate to the use of a single protein cell phenotype and immunological readoutHere we extend previous findings by investigating other proteins lysozyme and thyroglobulin another cell phenotype THP1 dendritic cells and different immunologicalresponses to yield novel insight Lysozyme and thyroglobulin were subjected to the same heattreatments as used for BLG Lysozyme from chicken egg white is a single chain polypeptidewith four disulphide bridges and is a rather inflexible protein with stable structure and properties [] It has a molecular weight of kDa which is similar to kDa of BLG and an isoelectric point pI of [] which is clearly different from BLG pI [] resulting in apositive net charge of this protein at the physiological pH Moreover it is also known as anallergen as exemplified by the high incidence of sensitization and allergenicity to this protein[] On the other hand bovine thyroglobulin which originates from follicular cells of the thyroid gland is a protein that is considered as less allergenic It has a molecular weight of kDa as a monomer making it more than times larger than the mass of the other two proteins under investigation It has a complex quaternary structure composed of four subunitsthat are disulphide bonded [ ] Its pI is close to that of BLG conferring it a similarcharge as BLG at the pH of the exposure medium We analysed uptake of heattreated proteinsby macrophages and dendritic cells and correlated this to physicochemical characteristics Furthermore we investigated the mechanism and receptors that are involved in uptake and identified downstream immunological responses by quantifying cytokine responseTaken together our findings provide insights into the role of protein size and pI towardsphysicochemical changes upon foodprocessing related heattreatments This appears to yieldgeneralisable correlations between a protein™s physicochemical characteristics and its uptakeby antigenpresenting cells APCs Finally we identified routes of uptake and downstreamimmunological responses which will help to clarify the interaction of protein ps withmacrophages and the potential immunoregulatory effect The results of this study could helpto better understand how the immunogenicity of food proteins can be modulated throughprocessingMaterials and methodsChemicalsAll chemicals were purchased from Sigma Aldrich St Louis Missouri USA unless otherwisestatedSample preparation and fluorescent labellingLactoglobulin BLG from cow™s milk lysozyme from chicken egg white and thyroglobulinfrom bovine thyroid were dissolved in sodium phosphate buffer mM pH same belowto a protein concentration of mgmL Besides this solution without saccharides DglucosePLOS ONE 101371journalpone0236212 August PLOS ONE 0cUptake of heatprocessed proteins by THP1 macrophages and dendritic cells and subsequent cytokine responseglu Dlactose lac and galactooligosaccharide GOS Royal FrieslandCampina Wageningen the Netherlands were added to reach a final molar ratio of total free amino groups tosaccharide reducing ends Identical heating methods being hightemperature dryheating Hwetheating W and lowtemperature dryheating L sample preparation and naming wereapplied to the proteins as indicated in a previous study [] Fluorescein isothiocyanate isomer IFITC labelling was performed as described previously []THP1 cell culture and differentiationThe human monocytic leukaemia cell line THP1 ATCC Manassas Virginia USA was differentiated into cells characteristic for resting macrophages M0 as described elsewhere []at a concentration of — cellsmL using ngmL phorbol12myristate13acetate PMAfor a hours stimulation followed by washing two times with medium and another hoursof rest Using a cell concentration of — cellsmL and incubation with ngmL of IL4and ngmL of PMA for days as described [] immature dendritic cells were generatedfrom THP1 monocytesPhysicochemical analysisThe analysis of solubility protein mass remaining in solution loss of amino group OPAmethod AGE formation fluorescent measurement exposure of hydrophobicity regionANS method surface charge zetapotential measurement secondary structure circulardichroism measurement and aggregation size exclusion chromatography were performedusing the protocols as described previously []Uptake and blocking of different uptake routesUptake experiments were performed as described previously [] No significant difference ofabsolute uptake value has been found for native BLG thyroglobulin or lysozyme in macrophages or dendritic cells S1 Fig As there is no bias for the basic uptake capability for the different proteins all the uptake results were presented relative to their corresponding nativeprotein to be able to compare among different proteins To determine which route of uptakewas involved in a protein™s uptake by THP1derived cells the cells were preincubated for minutes with DMSO control or different inhibitors μgmL nystatin caveolaedependentuptake inhibitor μgmL chlorpromazine clathrindependent uptake inhibitor μgmLcytochalasin B microphagocytosis inhibitor or all inhibitors combined dissolved in DMSOFollowing incubation the cells were washed with PBS and incubated for hours with FITClabelled protein samples Subsequently the cells were harvested and measured using flowcytometry as described previously [] For all uptake experiments μl trypan blue was addedper μl of cell suspension to quench extracellular FITC signal before flow cytometry analysis Uptake was calculated as either the fold change in mean fluorescence intensity MFI relative to control after correcting for FITC labelling efficiency control or as percentage tocontrol control Soluble Receptor for Advanced Glycation End Products sRAGE CD36and galectin3 inhibition ELISAThe experiment was performed according to the method of Liu [] As a positive control soy protein Bulk Powder Colchester UK was mixed with Dglucose in an equal wwratio in mM PBS buffer pH to a protein concentration of mgmL and heated for minutes at ˚C The positive control was diluted in mM of sodium carbonate buffer pHPLOS ONE 101371journalpone0236212 August PLOS ONE 0cUptake of heatprocessed proteins by THP1 macrophages and dendritic cells and subsequent cytokine response to μgml and used for plate coating by adding μL per well to a Nunc MaxiSorp„¢ flatbottom plate Thermo Fisher Waltham Massachusetts USA and incubating overnight at ˚C Then the plate was washed times with PBS buffer pH containing vv Tween After blocking with bovine serum albumin BSA in PBS for hour at room temperature the wells were washed times and incubated with μL protein samples μgmL at ˚C for hour The samples were preheated at ˚C for minutes with recombinanthuman sRAGE CD36 or galectin3 at a concentration of or μgmL RD SystemsMinneapolis Minnesota United States in PBS buffer with BSA and Tween20For detection the plate was washed times and incubated with μL per well of monoclonalmouse IgG2B human sRAGE or galectin3 antibody RD Systems Minneapolis MinnesotaUnited States at a concentration of μgml for minutes shaking at room temperatureAfter washing times the plate was incubated for minutes while shaking at room temperature with μL per well polyclonal goat antimouse HRPconjugated DAKO Glostrup Denmark at a concentration μgmL For galectin3 an additional incubation step with μgml Streptavidin SDT Baesweiler Germany μL per well for minutes was followed For CD36 only incubation with goat antihuman IgGHRP SouthernBiotech Birmingham Alabama United States at a concentration of μgml for minutes shaking atroom temperature was used for the detection step For all receptor inhibition ELISAs the platewas then washed times and μL per well TMB substrate was added and incubated for minutes before stopping the reaction by adding uL of HCl per well The absorbancemeasured at nm by an Infinite1 PRO NanoQuant Tecan Ma¨nnedorf Switzerlandwas subtracted from the measured absorbance at nm Each sample was measured in triplicate and values were averaged Absorbance of buffer was used as a control and was subtractedfrom every sample The absorbance of the control solution without any inhibition was codedby Abscontrol The percentage of inhibition for samples was equal to AbscontrolAbssampleAbscontrol�Cytokine production measurementTHP1 macrophages were incubated with μgmL of protein sample or medium as controlfor hours after which the supernatant was collected The cytokine production was measured using the ELISA Deluxe Set Human IL8CCL20IL1 Biolegend San Diego California United States following the manufacturer™s protocolStatisticsThe statistical analysis was performed using Prism software GraphPad Software San DiegoCalifornia United States with p considered to be significant The correlation analysiswas done by calculating the Pearson correlation coefficient r and the twotailed p value ThePCA plot was generated with the dataset of variables after being mean centred and weightedby 1standard deviation using Unscrambler software CAMO Oslo NorwayResultsWetheating significantly increased uptake of thyroglobulin by THP1derived macrophages whereas heattreatment did not increase uptake oflysozymeThyroglobulin and lysozyme were wetheated W hightemperature dryheated H or lowtemperature dryheated L in the absence or presence of saccharides with different lengthsmonosaccharide glucose disaccharide lactose or oligosaccharide GOS Soluble proteinPLOS ONE 101371journalpone0236212 August PLOS ONE 0cUptake of heatprocessed proteins by THP1 macrophages and dendritic cells and subsequent cytokine responsefrom each sample was concentration calibrated fluorescently labelled and incubated with resting macrophages that were derived from THP1 monocytes Hightemperature dryheating ofthyroglobulin in the presence of glucose and lactose and lysozyme in the presence of glucoseled to complete insolubility S2A and S4A Figs and therefore these samples were excludedfrom further analysis Prior to the experiment the lipopolysaccharide LPS contaminationwas measured S1 Table and found to be below the threshold level that significantly affecteduptake capacity of THP1 macrophages ie ngmL as reported in an earlier study []For uptake of thyroglobulin samples by macrophages Fig 1A the type of heat treatmentappeared to play a more determining role than the presence or absence of saccharides Notably all wetheated samples regardless of the presence of saccharides showed significantlyincreased uptake compared to untreated protein Although not significantly differing fromuntreated protein high and lowtemperature dryheating increased the uptake of thyroglobulin as well On the contrary no significant differences in uptake of lysozyme after differenttreatments and saccharide exposures was noticeable Fig 1BUptake of heattreated protein samples by THP1derived immaturedendritic cells is qualitatively similar to that of THP1derivedmacrophagesThe uptake of processed thyroglobulin and lysozyme as well as BLG which was previouslyreported [] was only tested for macrophages To expand the understanding of the proteins™uptake beyond only macrophages THP1derived immature dendritic cells iDC were alsotested with identical methodology as for macrophages The uptake of wetheated thyroglobulinin the absence or presence of saccharides by these iDC was significantly increased compared tothe native protein Fig 2A For lysozyme the relative uptake remained the same regardless ofthe treatment Fig 2B The uptake of wetheated BLG in the absence or presence of saccharidesby iDCs was also increased compared to the native form although only reaching significanceFig Uptake of thyroglobulin by macrophages was significantly increased upon wetheating of the sample Thyroglobulin THY A and lysozymeLYS B were untreated or heated W H or L in the absence or presence of saccharides glu lac or GOS THP1derived macrophages Mϕ wereincubated with FITClabelled soluble fraction of protein samples for hours after which uptake was determined using flow cytometry The resultsrepresent mean values ± SD of n measurement of independent experiments based on independent sample sets Statistical differences comparedto native proteins were calculated with Dunnett™s Test �p ��p 101371journalpone0236212g001PLOS ONE 101371journalpone0236212 August PLOS ONE 0cUptake of heatprocessed proteins by THP1 macrophages and dendritic cells and subsequent cytokine responseFig Uptake of thyroglobulin lysozyme and BLG samples by THP1derived iDC was strongly and significantly correlated with uptake bymacrophages AC Thyroglobulin THY lysozyme LYS and BLG were untreated or heated W H or L in the absence or presence of saccharidesglu lac or GOS THP1derived iDC were incubated with FITClabelled soluble fraction of protein samples for hours after which uptake wasdetermined using flow cytometry The results represent the mean values ± SD of n measurement of independent experiments based on independent sample sets Statistical differences compared to native proteins were calculated with Dunnett™s Test ��p ���p D Therelative uptake values of all protein samples by iDC was plotted against macrophages Mϕ and the correlation analysis was done by calculating thePearson correlation coefficient r and twotailed p value101371journalpone0236212g002when heattreated in the presence of glucose Fig 2C When compared to the results obtainedwith macrophages the increase in uptake upon protein wetheating for iDC was less pronounced BLGmacrophage data from previous study [] However the uptake pattern of allsamples correlated strongly r p between the two cell types Fig 2DIncreased uptake of thyroglobulin by THP1derived macrophages wascorrelated to hydrophobicity and aggregationDue to the strongly correlated responses of iDC and macrophages in uptake with a higher relative uptake by macrophages the latter was used for further testing To explain thePLOS ONE 101371journalpone0236212 August PLOS ONE 0cUptake of heatprocessed proteins by THP1 macrophages and dendritic cells and subsequent cytokine responsephysicochemical mechanism behind the differing uptake capability of macrophages for differently treated thyroglobulin and lysozyme a set of measurements was performed on the solublefraction after treatmentFor thyroglobulin dryheating at hightemperature significantly decreased its solubilityresulting in the absence of a soluble fraction when heating was performed in the presence ofglucose and lactose S2A Fig The soluble fraction of samples in the presence of GOS or without saccharide revealed a strong decrease of free amino groups and increases in AGErelatedfluorescence and formation of polymers in the presence of GOS S2B S2C and S3 Figs Wetheating did not result in loss of solubility but also led to formation of polymers and albeit lesspronounced as for hightemperature dryheating significant losses of amino groups andincreases in AGErelated fluorescence Moreover wetheating induced a significant increasein the exposure of hydrophobic regions as measured by ANSbinding S2D Fig Dryheatingat lowtemperature did not have much influence on the measured parameters for thyroglobulin except for a significant loss of amino groups and a slight increase in oligomer and polymerformation S2B and S3 Figs There was no significant change in the secondary structure forany of the treated thyroglobulin samples S2E and S2F FigApplying identical heating and glycation conditions as for thyroglobulin did not generateany soluble aggregates for lysozyme checked by size exclusion chromatography Hightemperature dryheating led to a significant decrease of solubility with complete insolubility forthe samples heated in the presence of glucose S4A Fig Among the soluble fraction of thehightemperature dryheated samples there was a significant decrease of free amino groupsincreases of AGErelated fluorescence and exposure of hydrophobic regions in the presence ofsaccharides S4B“S4D Fig Moreover results showed a significant loss of sheet structure forall samples except when heated in the presence of GOS S4F Fig Similarly lowtemperaturedryheating resulted in a significant loss of sheets in all lysozyme samples For wetheatedlysozyme only the exposure of hydrophobic regions in the presence of saccharides was significantly increased S4D FigTo identify the most important physicochemical properties of heatprocessed thyroglobulinthat are related to its uptake by macrophages we performed a principle component analysisPCA and a correlation analysis In the PCA plot Fig all lowtemperature dryheated samples clustered together with the untreated thyroglobulin indicating that the physicochemicalproperties of these samples were similar Uptake was strongly related with wetheating andpositively related to hydrophobicity ANS and polymer proportion and inversely to monomer and oligomer proportion These relations were further confirmed by correlation analysesAs shown in Fig there was a strong correlation between uptake and hydrophobicity and amoderate correlation between uptake and aggregation as indicated by the fraction of monomers polymers and oligomers Of note is that the samples were strongly clustering into twogroups at the extremes of the data scale for both the uptake and monomer or polymer contentcorrelation plots Fig 4B and 4C the correlation between uptake and oligomer content wasless profound Fig 4D p Contrastingly to thyroglobulin no clear correlation could befound between treatment and physicochemical properties for lysozyme in the PCA plotS5 FigEDCmediated increase in hydrophobicity but not crosslinking is themain factor driving uptake by macrophagesEDC links the carboxyl and amino group of amino acids and thereby offers the possibility tocrosslink proteins without heating This allows for a discrimination between aggregation andother heatingrelated effects like hydrophobicity changes with regard to their impact onPLOS ONE 101371journalpone0236212 August PLOS ONE 0cUptake of heatprocessed proteins by THP1 macrophages and dendritic cells and subsequent cytokine responseFig Uptake of thyroglobulin by macrophages is related to wetheating hydrophobicity and aggregation Thyroglobulin THYwas untreated or heated W H or L in the absence or presence of saccharides glu lac or GOS and tested for solubility uptake ofsoluble fraction by THP1 macrophages uptake AGE formation AGE glycation OPA percentage of αhelix helix or sheetsheet aggregation monomer and smaller mono oligomers oligo polymers poly and exposure of hydrophobic regions ANSPCA scores of the samples were given in blue and the parameter loadings of the principal components were given in red The clusteringof uptake with wetheated samples the formation of hydrophobic regions and polymer proportion is indicated in the red circle101371journalpone0236212g003uptake As shown in Fig 5A and 5B the proportion of polymer and monomer significantlyincreased resp decreased in the EDC crosslinked BLG and thyroglobulin On the contraryonly limited aggregation of lysozyme was observed upon EDCtreatment EDC crosslinkedBLG and thyroglobulin did not demonstrate an increase in uptake Surprisingly EDCmediated crosslinking significantly increased the hydrophobicity of lysozyme Fig 5C and also itsuptake by macrophages Fig 5DUptake of heatedtreated BLG was mediated via several routesTo better understand the mechanism of uptake we set out to identify which routes of uptakewere utilized for the various proteins that differed in physicochemical characteristics andtherefore in their recognition motifs for cells Native and heattreated BLG in the presence ofglucose was used as a subset because these samples covered the whole range of levels of physicochemical modifications and macrophages were pretreated with inhibitors targeting phagocytosis caveolae or clathrinmediated uptake or a combination thereof Preincubation ofmacrophages with nystatin caveolae inhibitor and the combination of inhibitors prior toBLG samples exposure significantly inhibited the uptake Fig The usage of cytochalasinmicrophagocytosis inhibitor significantly decreased the uptake of both hightemperaturedryheated and wetheated BLG Fig 6B and 6C Moreover chlorpromazine significantlyPLOS ONE 101371journalpone0236212 August PLOS ONE 0cUptake of heatprocessed proteins by THP1 macrophages and dendritic cells and subsequent cytokine responseFig Correlation analysis of uptake of heattreated thyroglobulin with physicochemical parameters Uptake of soluble fraction ofthyroglobulin THY samples by macrophages was correlated to hydrophobicity r07 and proportion of monomer polymer and oligomer07r05 The correlation analysis was done by calculating the Pearson correlation coefficient r and twotailed p value101371journalpone0236212g004reduced uptake of wetheated BLG indicating that clathrin was involved in this processFig 6CInhibition ELISA showed high binding of hightemperature dryheated andwetheated BLG by sRAGE CD36 and galectin3We investigated several receptors that might be involved in binding and subsequent uptake ofthe BLG samples that were heattreated in the presence of glucose Using an inhibition ELISAthe binding of the BLG samples to the soluble receptor for advanced glycation end productssRAGE CD36scavenger receptor and galectin3 was analysed Fig Native BLG did notshow any binding to sRAGE galectin3 or CD36 In contrast hightemperature dryheating ofBLG induced significant receptor binding for all three tested receptors Wetheating of BLGsimilarly resulted in binding to all three receptors albeit to a lesser extent and only significantly for sRAGE and galectin3 Finally lowtemperature dryheating did not induce anyreceptorbindingPLOS ONE 101371journalpone0236212 August PLOS ONE 0cUptake of heatprocessed proteins by THP1 macrophages and dendritic cells and subsequent cytokine responseFig EDCmediated hydrophobicity but not aggregation resulted in increased uptake of proteins by THP1 macrophagesThyroglobulin THY lysozyme LYS and BLG were nontreated or treated with EDC and subsequently analysed for the proportion ofpolymer A the proportion of monomerdimer B and protein hydrophobicity C EDCtreated and nontreated proteins were fluorescentlylabelled and their uptake by macrophage Mϕ was measured D The results represent the mean values ± SD of “ independent experimentswith independent sample sets Statistical differences were calculated with twotailed unpaired Ttest with Welch™s correction �p ��p ���p 101371journalpone0236212g005Cytokine responses of macrophages following exposure to BLG werereduced upon heattreatment of BLGReceptor binding and uptake of protein samples can lead to immune activity The immunological response could be estimated by the expression of secreted cytokines or chemokines Uponexposure to medium or native or heattreated BLG in the presence of glucose the response ofmacrophages was analysed by measuring the levels of secreted IL8 CCL20 and IL1 Asshown in Fig nonprocessed native BLG and lowtemperature dryheated BLG induced significant levels of IL8 CCL20 and IL1 secretion in macrophages when compared to mediumPLOS ONE 101371journalpone0236212 August PLOS ONE 0cUptake of heatprocessed proteins by THP1 macrophages and dendritic cells and subsequent cytokine responseFig Several routes appeared to be involved in the uptake of wetheated BLG BLG was untreated or heated W H or L in the presence of glucose gluand the soluble fraction was FITClabelled Macrophages were pretreated with DMSO control or inhibitors dissolved in DMSO of clathrindependentuptake chlorpromazine chlor caveolaedependent uptake nystatin nys microphagocytosis cytochalasin cyto or a combination of them combinedfor min before a 2hour incubation with protein samples Protein uptake was subsequently measured using flow cytometry The relative MFI wascalculated by dividing the MFI of a sample by the MFI of the corresponding control The results represent mean values ± SD of multiple independent cellexperiments N “ with independent sample sets The statistical differences were calculated with onetailed unpaired Ttest compared to control�p ��p ���p 101371journalpone0236212g006Hightemperature dryheating of BLG significantly reduced the secretion of all three analysedcytokines when compared to native BLG A similar reduction was observed in wetheated BLGfor IL8 and IL1 but less strongly and not significantly for CCL20DiscussionIn an earlier study we observed that hydrophobicity and the aggregation state of heatprocessed BLG were important parameters that were related to the protein uptake by THP1PLOS ONE 101371journalpone0236212 August PLOS ONE 0cUptake of heatprocessed proteins by THP1 macrophages and dendritic cells and subsequent cytokine responseFig Heattreatment in the presence of glucose confers receptor binding capacity to BLG BLG was untreated orheated W H or L in the presence of glucose glu and the soluble fraction was used to inhibit the binding of receptorto a positive control in the inhibition ELISA Data shown are the mean values ± SD of n based on measurement of parallel prepared samples Statistically significant differences between native and processed BLG for each receptorwas determined using unpaired twotailed tTest �p ��p ���p 101371journalpone0236212g007macrophages [] Here we extended these findings by analysing whether similar relationswould apply for a protein with similar pI but higher molecular weight thyroglobulin aprotein with similar molecular weight but higher pI lysozyme EDCaggregated proteinsand another cell type by using THP1 dendritic cells The comparison between THP1derived macrophages and dendritic cells regarding uptake revealed similar patterns for thethree tested proteins and the different heattreatments Macrophages and DCs are both able tocapture exogenous ps and induce an immunogenic response playing an essential role inFig Cytokine and chemokine responses of macrophages upon exposure to native or heattreated BLG varies BLG was untreated or heated W Hor L in the presence of glucose and the soluble fraction was incubated with macrophages The cytokine secretion of IL8 A CCL20 B and IL1 Cwas analysed in the supernatant with ELISA The results represent mean values ± SD of “ independent experimental measurements The statisticaldifferences were calculated with Tukey™s Test with different letters a“c in the bars indicated significantly different p 101371journalpone0236212g008PLOS ONE 101371journalpone0236212 August PLOS ONE 0cUptake of heatprocessed proteins by THP1 macrophages and dendritic cells and subsequent cytokine responseimmunological defence [] We did observe however a quantitative difference in uptake asthe uptake values for wetheated thyroglobulin and BLG were relatively higher in macrophagesthan in DCs This suggests that macrophages are more efficient in their phagocytic role thanDCs which is in line with literature stating that uptake by macrophages is part of their corerole in eliminating harmful substances whereas uptake by DCs is a means to initiating anadaptive immune response [] Upon comparing the three tested proteins we found that thephysicochemical alterations upon heattreatment to thyroglobulin and BLG were similar butdifferent from lysozyme These proteins differ in size thyroglobulin kDa BLG kDa lysozyme kDa pI lysozyme BLG thyroglobulin anddenaturation temperature lysozyme ˚C BLG ˚C thyroglobulin ˚C [“]suggesting that both pI and heat stability may be important for heatinduced structural proteinchanges The high pI may have conferred a positive charge to lysozyme during wetheatingpH thereby hindering aggregation under this condition due to electrostatic repulsionThe strong internal coherence and stability of lysozyme™s structure may also have contributedto its stability during heat processing [] It is reported

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You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: Hydrophobicity drives receptormediateduptake of heatprocessed proteins by THP1macrophages and dendritic cells but notcytokine responsesYing Deng12 Coen Govers1 Malgorzata Teodorowicz3 Ieva Liobyte12 Ilaria DeSimone13 Kasper Hettinga4 Harry J WichersID12 Food and Biobased Research Wageningen University and Research Wageningen The Netherlands Laboratory of Food Chemistry Wageningen University and Research Wageningen The Netherlands Cell Biology and Immunology Wageningen University and Research Wageningen The Netherlands Food Quality and Design Wageningen University and Research Wageningen The Netherlands harrywicherswurnlAbstractAlthough an impact of processing on immunogenicity of food proteins has clearly been demonstrated the underlying mechanisms are still unclear We applied different processingmethods wet heating ˚C and low or hightemperature ˚C or ˚C respectivelydryheating in absence or presence of reducing sugars to lactoglobulin BLG lysozymeand thyroglobulin which represent dietary proteins with different pI or molecular weightUptake of the soluble fraction of the samples was tested in two types of genetically homogeneous antigenpresenting cells macrophages and dendritic cells derived from THP1monocytes This revealed a strong correlation between the uptake of the different proteinsamples by macrophages and dendritic cells and confirmed the key role of hydrophobicityover aggregation in determining the uptake Several uptake routes were shown to contribute to the uptake of BLG by macrophages However cytokine responses following exposureof macrophages to BLG samples were not related to the levels of uptake Together ourresults demonstrate that heattreatmentinduced increased hydrophobicity is the prime driving factor in uptake but not in cytokine production by THP1 macrophagesIntroductionDietary proteins play an important physiological role not only in providing amino acids andenergy but also for the maturation and regulation of the immune system Proteins are knownto be involved in the regulation of chronic inflammation and be the cause of allergies [] Forexample a higher intake of animal protein was found to enhance the proinflammatoryresponse of macrophages in mice [] Most of the proteins that humans ingest have beenthrough a heating process as part of the food processing as a result of which structural modifications such as glycation with reducing sugar andor aggregation of proteins may occura1111111111a1111111111a1111111111a1111111111a1111111111 ACCESSCitation Deng Y Govers C Teodorowicz MLiobyte I De Simone I Hettinga K Hydrophobicity drives receptormediated uptake ofheatprocessed proteins by THP1 macrophagesand dendritic cells but not cytokine responses e0236212 101371journalpone0236212Editor Yi Cao Xiangtan University CHINAReceived June Accepted August Published August Copyright Deng This is an access distributed under the terms of theCreative Commons Attribution License whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are creditedData Availability Statement All relevant data arewithin the paper and its Supporting InformationfilesFunding This work was financial supported by theChina Scholarship Council No awarded to YDCompeting interests The authors have declaredthat no competing interests existPLOS ONE 101371journalpone0236212 August PLOS ONE 0cUptake of heatprocessed proteins by THP1 macrophages and dendritic cells and subsequent cytokine responseDifferent processing methods would alter the immunogenicity of food protein in differentmanner For example the antigenicity of prawn allergen was found to be significantlyincreased after frying but decreased after boiling acid treatment or highpressure processing[] The allergenicity of milk protein was reported to decrease after glycation []In an earlier study on lactoglobulin BLG the major whey protein of milk [] weobserved that heat processing under different conditions altered the physicochemical properties and its uptake by THP1 macrophages In particular heating in solution at ˚C for days introduced changes in hydrophobicity and molecular weight which were shown to be thekey determinants for uptake This event may be relevant for a possible subsequent immuneresponse [ ] and has been linked to heattreatmentinduced changes in protein™s pr sityto induce an allergic reaction [“] Despite the new findings in that study there are obviouslimitations relate to the use of a single protein cell phenotype and immunological readoutHere we extend previous findings by investigating other proteins lysozyme and thyroglobulin another cell phenotype THP1 dendritic cells and different immunologicalresponses to yield novel insight Lysozyme and thyroglobulin were subjected to the same heattreatments as used for BLG Lysozyme from chicken egg white is a single chain polypeptidewith four disulphide bridges and is a rather inflexible protein with stable structure and properties [] It has a molecular weight of kDa which is similar to kDa of BLG and an isoelectric point pI of [] which is clearly different from BLG pI [] resulting in apositive net charge of this protein at the physiological pH Moreover it is also known as anallergen as exemplified by the high incidence of sensitization and allergenicity to this protein[] On the other hand bovine thyroglobulin which originates from follicular cells of the thyroid gland is a protein that is considered as less allergenic It has a molecular weight of kDa as a monomer making it more than times larger than the mass of the other two proteins under investigation It has a complex quaternary structure composed of four subunitsthat are disulphide bonded [ ] Its pI is close to that of BLG conferring it a similarcharge as BLG at the pH of the exposure medium We analysed uptake of heattreated proteinsby macrophages and dendritic cells and correlated this to physicochemical characteristics Furthermore we investigated the mechanism and receptors that are involved in uptake and identified downstream immunological responses by quantifying cytokine responseTaken together our findings provide insights into the role of protein size and pI towardsphysicochemical changes upon foodprocessing related heattreatments This appears to yieldgeneralisable correlations between a protein™s physicochemical characteristics and its uptakeby antigenpresenting cells APCs Finally we identified routes of uptake and downstreamimmunological responses which will help to clarify the interaction of protein ps withmacrophages and the potential immunoregulatory effect The results of this study could helpto better understand how the immunogenicity of food proteins can be modulated throughprocessingMaterials and methodsChemicalsAll chemicals were purchased from Sigma Aldrich St Louis Missouri USA unless otherwisestatedSample preparation and fluorescent labellingLactoglobulin BLG from cow™s milk lysozyme from chicken egg white and thyroglobulinfrom bovine thyroid were dissolved in sodium phosphate buffer mM pH same belowto a protein concentration of mgmL Besides this solution without saccharides DglucosePLOS ONE 101371journalpone0236212 August PLOS ONE 0cUptake of heatprocessed proteins by THP1 macrophages and dendritic cells and subsequent cytokine responseglu Dlactose lac and galactooligosaccharide GOS Royal FrieslandCampina Wageningen the Netherlands were added to reach a final molar ratio of total free amino groups tosaccharide reducing ends Identical heating methods being hightemperature dryheating Hwetheating W and lowtemperature dryheating L sample preparation and naming wereapplied to the proteins as indicated in a previous study [] Fluorescein isothiocyanate isomer IFITC labelling was performed as described previously []THP1 cell culture and differentiationThe human monocytic leukaemia cell line THP1 ATCC Manassas Virginia USA was differentiated into cells characteristic for resting macrophages M0 as described elsewhere []at a concentration of — cellsmL using ngmL phorbol12myristate13acetate PMAfor a hours stimulation followed by washing two times with medium and another hoursof rest Using a cell concentration of — cellsmL and incubation with ngmL of IL4and ngmL of PMA for days as described [] immature dendritic cells were generatedfrom THP1 monocytesPhysicochemical analysisThe analysis of solubility protein mass remaining in solution loss of amino group OPAmethod AGE formation fluorescent measurement exposure of hydrophobicity regionANS method surface charge zetapotential measurement secondary structure circulardichroism measurement and aggregation size exclusion chromatography were performedusing the protocols as described previously []Uptake and blocking of different uptake routesUptake experiments were performed as described previously [] No significant difference ofabsolute uptake value has been found for native BLG thyroglobulin or lysozyme in macrophages or dendritic cells S1 Fig As there is no bias for the basic uptake capability for the different proteins all the uptake results were presented relative to their corresponding nativeprotein to be able to compare among different proteins To determine which route of uptakewas involved in a protein™s uptake by THP1derived cells the cells were preincubated for minutes with DMSO control or different inhibitors μgmL nystatin caveolaedependentuptake inhibitor μgmL chlorpromazine clathrindependent uptake inhibitor μgmLcytochalasin B microphagocytosis inhibitor or all inhibitors combined dissolved in DMSOFollowing incubation the cells were washed with PBS and incubated for hours with FITClabelled protein samples Subsequently the cells were harvested and measured using flowcytometry as described previously [] For all uptake experiments μl trypan blue was addedper μl of cell suspension to quench extracellular FITC signal before flow cytometry analysis Uptake was calculated as either the fold change in mean fluorescence intensity MFI relative to control after correcting for FITC labelling efficiency control or as percentage tocontrol control Soluble Receptor for Advanced Glycation End Products sRAGE CD36and galectin3 inhibition ELISAThe experiment was performed according to the method of Liu [] As a positive control soy protein Bulk Powder Colchester UK was mixed with Dglucose in an equal wwratio in mM PBS buffer pH to a protein concentration of mgmL and heated for minutes at ˚C The positive control was diluted in mM of sodium carbonate buffer pHPLOS ONE 101371journalpone0236212 August PLOS ONE 0cUptake of heatprocessed proteins by THP1 macrophages and dendritic cells and subsequent cytokine response to μgml and used for plate coating by adding μL per well to a Nunc MaxiSorp„¢ flatbottom plate Thermo Fisher Waltham Massachusetts USA and incubating overnight at ˚C Then the plate was washed times with PBS buffer pH containing vv Tween After blocking with bovine serum albumin BSA in PBS for hour at room temperature the wells were washed times and incubated with μL protein samples μgmL at ˚C for hour The samples were preheated at ˚C for minutes with recombinanthuman sRAGE CD36 or galectin3 at a concentration of or μgmL RD SystemsMinneapolis Minnesota United States in PBS buffer with BSA and Tween20For detection the plate was washed times and incubated with μL per well of monoclonalmouse IgG2B human sRAGE or galectin3 antibody RD Systems Minneapolis MinnesotaUnited States at a concentration of μgml for minutes shaking at room temperatureAfter washing times the plate was incubated for minutes while shaking at room temperature with μL per well polyclonal goat antimouse HRPconjugated DAKO Glostrup Denmark at a concentration μgmL For galectin3 an additional incubation step with μgml Streptavidin SDT Baesweiler Germany μL per well for minutes was followed For CD36 only incubation with goat antihuman IgGHRP SouthernBiotech Birmingham Alabama United States at a concentration of μgml for minutes shaking atroom temperature was used for the detection step For all receptor inhibition ELISAs the platewas then washed times and μL per well TMB substrate was added and incubated for minutes before stopping the reaction by adding uL of HCl per well The absorbancemeasured at nm by an Infinite1 PRO NanoQuant Tecan Ma¨nnedorf Switzerlandwas subtracted from the measured absorbance at nm Each sample was measured in triplicate and values were averaged Absorbance of buffer was used as a control and was subtractedfrom every sample The absorbance of the control solution without any inhibition was codedby Abscontrol The percentage of inhibition for samples was equal to AbscontrolAbssampleAbscontrol�Cytokine production measurementTHP1 macrophages were incubated with μgmL of protein sample or medium as controlfor hours after which the supernatant was collected The cytokine production was measured using the ELISA Deluxe Set Human IL8CCL20IL1 Biolegend San Diego California United States following the manufacturer™s protocolStatisticsThe statistical analysis was performed using Prism software GraphPad Software San DiegoCalifornia United States with p considered to be significant The correlation analysiswas done by calculating the Pearson correlation coefficient r and the twotailed p value ThePCA plot was generated with the dataset of variables after being mean centred and weightedby 1standard deviation using Unscrambler software CAMO Oslo NorwayResultsWetheating significantly increased uptake of thyroglobulin by THP1derived macrophages whereas heattreatment did not increase uptake oflysozymeThyroglobulin and lysozyme were wetheated W hightemperature dryheated H or lowtemperature dryheated L in the absence or presence of saccharides with different lengthsmonosaccharide glucose disaccharide lactose or oligosaccharide GOS Soluble proteinPLOS ONE 101371journalpone0236212 August PLOS ONE 0cUptake of heatprocessed proteins by THP1 macrophages and dendritic cells and subsequent cytokine responsefrom each sample was concentration calibrated fluorescently labelled and incubated with resting macrophages that were derived from THP1 monocytes Hightemperature dryheating ofthyroglobulin in the presence of glucose and lactose and lysozyme in the presence of glucoseled to complete insolubility S2A and S4A Figs and therefore these samples were excludedfrom further analysis Prior to the experiment the lipopolysaccharide LPS contaminationwas measured S1 Table and found to be below the threshold level that significantly affecteduptake capacity of THP1 macrophages ie ngmL as reported in an earlier study []For uptake of thyroglobulin samples by macrophages Fig 1A the type of heat treatmentappeared to play a more determining role than the presence or absence of saccharides Notably all wetheated samples regardless of the presence of saccharides showed significantlyincreased uptake compared to untreated protein Although not significantly differing fromuntreated protein high and lowtemperature dryheating increased the uptake of thyroglobulin as well On the contrary no significant differences in uptake of lysozyme after differenttreatments and saccharide exposures was noticeable Fig 1BUptake of heattreated protein samples by THP1derived immaturedendritic cells is qualitatively similar to that of THP1derivedmacrophagesThe uptake of processed thyroglobulin and lysozyme as well as BLG which was previouslyreported [] was only tested for macrophages To expand the understanding of the proteins™uptake beyond only macrophages THP1derived immature dendritic cells iDC were alsotested with identical methodology as for macrophages The uptake of wetheated thyroglobulinin the absence or presence of saccharides by these iDC was significantly increased compared tothe native protein Fig 2A For lysozyme the relative uptake remained the same regardless ofthe treatment Fig 2B The uptake of wetheated BLG in the absence or presence of saccharidesby iDCs was also increased compared to the native form although only reaching significanceFig Uptake of thyroglobulin by macrophages was significantly increased upon wetheating of the sample Thyroglobulin THY A and lysozymeLYS B were untreated or heated W H or L in the absence or presence of saccharides glu lac or GOS THP1derived macrophages Mϕ wereincubated with FITClabelled soluble fraction of protein samples for hours after which uptake was determined using flow cytometry The resultsrepresent mean values ± SD of n measurement of independent experiments based on independent sample sets Statistical differences comparedto native proteins were calculated with Dunnett™s Test �p ��p 101371journalpone0236212g001PLOS ONE 101371journalpone0236212 August PLOS ONE 0cUptake of heatprocessed proteins by THP1 macrophages and dendritic cells and subsequent cytokine responseFig Uptake of thyroglobulin lysozyme and BLG samples by THP1derived iDC was strongly and significantly correlated with uptake bymacrophages AC Thyroglobulin THY lysozyme LYS and BLG were untreated or heated W H or L in the absence or presence of saccharidesglu lac or GOS THP1derived iDC were incubated with FITClabelled soluble fraction of protein samples for hours after which uptake wasdetermined using flow cytometry The results represent the mean values ± SD of n measurement of independent experiments based on independent sample sets Statistical differences compared to native proteins were calculated with Dunnett™s Test ��p ���p D Therelative uptake values of all protein samples by iDC was plotted against macrophages Mϕ and the correlation analysis was done by calculating thePearson correlation coefficient r and twotailed p value101371journalpone0236212g002when heattreated in the presence of glucose Fig 2C When compared to the results obtainedwith macrophages the increase in uptake upon protein wetheating for iDC was less pronounced BLGmacrophage data from previous study [] However the uptake pattern of allsamples correlated strongly r p between the two cell types Fig 2DIncreased uptake of thyroglobulin by THP1derived macrophages wascorrelated to hydrophobicity and aggregationDue to the strongly correlated responses of iDC and macrophages in uptake with a higher relative uptake by macrophages the latter was used for further testing To explain thePLOS ONE 101371journalpone0236212 August PLOS ONE 0cUptake of heatprocessed proteins by THP1 macrophages and dendritic cells and subsequent cytokine responsephysicochemical mechanism behind the differing uptake capability of macrophages for differently treated thyroglobulin and lysozyme a set of measurements was performed on the solublefraction after treatmentFor thyroglobulin dryheating at hightemperature significantly decreased its solubilityresulting in the absence of a soluble fraction when heating was performed in the presence ofglucose and lactose S2A Fig The soluble fraction of samples in the presence of GOS or without saccharide revealed a strong decrease of free amino groups and increases in AGErelatedfluorescence and formation of polymers in the presence of GOS S2B S2C and S3 Figs Wetheating did not result in loss of solubility but also led to formation of polymers and albeit lesspronounced as for hightemperature dryheating significant losses of amino groups andincreases in AGErelated fluorescence Moreover wetheating induced a significant increasein the exposure of hydrophobic regions as measured by ANSbinding S2D Fig Dryheatingat lowtemperature did not have much influence on the measured parameters for thyroglobulin except for a significant loss of amino groups and a slight increase in oligomer and polymerformation S2B and S3 Figs There was no significant change in the secondary structure forany of the treated thyroglobulin samples S2E and S2F FigApplying identical heating and glycation conditions as for thyroglobulin did not generateany soluble aggregates for lysozyme checked by size exclusion chromatography Hightemperature dryheating led to a significant decrease of solubility with complete insolubility forthe samples heated in the presence of glucose S4A Fig Among the soluble fraction of thehightemperature dryheated samples there was a significant decrease of free amino groupsincreases of AGErelated fluorescence and exposure of hydrophobic regions in the presence ofsaccharides S4B“S4D Fig Moreover results showed a significant loss of sheet structure forall samples except when heated in the presence of GOS S4F Fig Similarly lowtemperaturedryheating resulted in a significant loss of sheets in all lysozyme samples For wetheatedlysozyme only the exposure of hydrophobic regions in the presence of saccharides was significantly increased S4D FigTo identify the most important physicochemical properties of heatprocessed thyroglobulinthat are related to its uptake by macrophages we performed a principle component analysisPCA and a correlation analysis In the PCA plot Fig all lowtemperature dryheated samples clustered together with the untreated thyroglobulin indicating that the physicochemicalproperties of these samples were similar Uptake was strongly related with wetheating andpositively related to hydrophobicity ANS and polymer proportion and inversely to monomer and oligomer proportion These relations were further confirmed by correlation analysesAs shown in Fig there was a strong correlation between uptake and hydrophobicity and amoderate correlation between uptake and aggregation as indicated by the fraction of monomers polymers and oligomers Of note is that the samples were strongly clustering into twogroups at the extremes of the data scale for both the uptake and monomer or polymer contentcorrelation plots Fig 4B and 4C the correlation between uptake and oligomer content wasless profound Fig 4D p Contrastingly to thyroglobulin no clear correlation could befound between treatment and physicochemical properties for lysozyme in the PCA plotS5 FigEDCmediated increase in hydrophobicity but not crosslinking is themain factor driving uptake by macrophagesEDC links the carboxyl and amino group of amino acids and thereby offers the possibility tocrosslink proteins without heating This allows for a discrimination between aggregation andother heatingrelated effects like hydrophobicity changes with regard to their impact onPLOS ONE 101371journalpone0236212 August PLOS ONE 0cUptake of heatprocessed proteins by THP1 macrophages and dendritic cells and subsequent cytokine responseFig Uptake of thyroglobulin by macrophages is related to wetheating hydrophobicity and aggregation Thyroglobulin THYwas untreated or heated W H or L in the absence or presence of saccharides glu lac or GOS and tested for solubility uptake ofsoluble fraction by THP1 macrophages uptake AGE formation AGE glycation OPA percentage of αhelix helix or sheetsheet aggregation monomer and smaller mono oligomers oligo polymers poly and exposure of hydrophobic regions ANSPCA scores of the samples were given in blue and the parameter loadings of the principal components were given in red The clusteringof uptake with wetheated samples the formation of hydrophobic regions and polymer proportion is indicated in the red circle101371journalpone0236212g003uptake As shown in Fig 5A and 5B the proportion of polymer and monomer significantlyincreased resp decreased in the EDC crosslinked BLG and thyroglobulin On the contraryonly limited aggregation of lysozyme was observed upon EDCtreatment EDC crosslinkedBLG and thyroglobulin did not demonstrate an increase in uptake Surprisingly EDCmediated crosslinking significantly increased the hydrophobicity of lysozyme Fig 5C and also itsuptake by macrophages Fig 5DUptake of heatedtreated BLG was mediated via several routesTo better understand the mechanism of uptake we set out to identify which routes of uptakewere utilized for the various proteins that differed in physicochemical characteristics andtherefore in their recognition motifs for cells Native and heattreated BLG in the presence ofglucose was used as a subset because these samples covered the whole range of levels of physicochemical modifications and macrophages were pretreated with inhibitors targeting phagocytosis caveolae or clathrinmediated uptake or a combination thereof Preincubation ofmacrophages with nystatin caveolae inhibitor and the combination of inhibitors prior toBLG samples exposure significantly inhibited the uptake Fig The usage of cytochalasinmicrophagocytosis inhibitor significantly decreased the uptake of both hightemperaturedryheated and wetheated BLG Fig 6B and 6C Moreover chlorpromazine significantlyPLOS ONE 101371journalpone0236212 August PLOS ONE 0cUptake of heatprocessed proteins by THP1 macrophages and dendritic cells and subsequent cytokine responseFig Correlation analysis of uptake of heattreated thyroglobulin with physicochemical parameters Uptake of soluble fraction ofthyroglobulin THY samples by macrophages was correlated to hydrophobicity r07 and proportion of monomer polymer and oligomer07r05 The correlation analysis was done by calculating the Pearson correlation coefficient r and twotailed p value101371journalpone0236212g004reduced uptake of wetheated BLG indicating that clathrin was involved in this processFig 6CInhibition ELISA showed high binding of hightemperature dryheated andwetheated BLG by sRAGE CD36 and galectin3We investigated several receptors that might be involved in binding and subsequent uptake ofthe BLG samples that were heattreated in the presence of glucose Using an inhibition ELISAthe binding of the BLG samples to the soluble receptor for advanced glycation end productssRAGE CD36scavenger receptor and galectin3 was analysed Fig Native BLG did notshow any binding to sRAGE galectin3 or CD36 In contrast hightemperature dryheating ofBLG induced significant receptor binding for all three tested receptors Wetheating of BLGsimilarly resulted in binding to all three receptors albeit to a lesser extent and only significantly for sRAGE and galectin3 Finally lowtemperature dryheating did not induce anyreceptorbindingPLOS ONE 101371journalpone0236212 August PLOS ONE 0cUptake of heatprocessed proteins by THP1 macrophages and dendritic cells and subsequent cytokine responseFig EDCmediated hydrophobicity but not aggregation resulted in increased uptake of proteins by THP1 macrophagesThyroglobulin THY lysozyme LYS and BLG were nontreated or treated with EDC and subsequently analysed for the proportion ofpolymer A the proportion of monomerdimer B and protein hydrophobicity C EDCtreated and nontreated proteins were fluorescentlylabelled and their uptake by macrophage Mϕ was measured D The results represent the mean values ± SD of “ independent experimentswith independent sample sets Statistical differences were calculated with twotailed unpaired Ttest with Welch™s correction �p ��p ���p 101371journalpone0236212g005Cytokine responses of macrophages following exposure to BLG werereduced upon heattreatment of BLGReceptor binding and uptake of protein samples can lead to immune activity The immunological response could be estimated by the expression of secreted cytokines or chemokines Uponexposure to medium or native or heattreated BLG in the presence of glucose the response ofmacrophages was analysed by measuring the levels of secreted IL8 CCL20 and IL1 Asshown in Fig nonprocessed native BLG and lowtemperature dryheated BLG induced significant levels of IL8 CCL20 and IL1 secretion in macrophages when compared to mediumPLOS ONE 101371journalpone0236212 August PLOS ONE 0cUptake of heatprocessed proteins by THP1 macrophages and dendritic cells and subsequent cytokine responseFig Several routes appeared to be involved in the uptake of wetheated BLG BLG was untreated or heated W H or L in the presence of glucose gluand the soluble fraction was FITClabelled Macrophages were pretreated with DMSO control or inhibitors dissolved in DMSO of clathrindependentuptake chlorpromazine chlor caveolaedependent uptake nystatin nys microphagocytosis cytochalasin cyto or a combination of them combinedfor min before a 2hour incubation with protein samples Protein uptake was subsequently measured using flow cytometry The relative MFI wascalculated by dividing the MFI of a sample by the MFI of the corresponding control The results represent mean values ± SD of multiple independent cellexperiments N “ with independent sample sets The statistical differences were calculated with onetailed unpaired Ttest compared to control�p ��p ���p 101371journalpone0236212g006Hightemperature dryheating of BLG significantly reduced the secretion of all three analysedcytokines when compared to native BLG A similar reduction was observed in wetheated BLGfor IL8 and IL1 but less strongly and not significantly for CCL20DiscussionIn an earlier study we observed that hydrophobicity and the aggregation state of heatprocessed BLG were important parameters that were related to the protein uptake by THP1PLOS ONE 101371journalpone0236212 August PLOS ONE 0cUptake of heatprocessed proteins by THP1 macrophages and dendritic cells and subsequent cytokine responseFig Heattreatment in the presence of glucose confers receptor binding capacity to BLG BLG was untreated orheated W H or L in the presence of glucose glu and the soluble fraction was used to inhibit the binding of receptorto a positive control in the inhibition ELISA Data shown are the mean values ± SD of n based on measurement of parallel prepared samples Statistically significant differences between native and processed BLG for each receptorwas determined using unpaired twotailed tTest �p ��p ���p 101371journalpone0236212g007macrophages [] Here we extended these findings by analysing whether similar relationswould apply for a protein with similar pI but higher molecular weight thyroglobulin aprotein with similar molecular weight but higher pI lysozyme EDCaggregated proteinsand another cell type by using THP1 dendritic cells The comparison between THP1derived macrophages and dendritic cells regarding uptake revealed similar patterns for thethree tested proteins and the different heattreatments Macrophages and DCs are both able tocapture exogenous ps and induce an immunogenic response playing an essential role inFig Cytokine and chemokine responses of macrophages upon exposure to native or heattreated BLG varies BLG was untreated or heated W Hor L in the presence of glucose and the soluble fraction was incubated with macrophages The cytokine secretion of IL8 A CCL20 B and IL1 Cwas analysed in the supernatant with ELISA The results represent mean values ± SD of “ independent experimental measurements The statisticaldifferences were calculated with Tukey™s Test with different letters a“c in the bars indicated significantly different p 101371journalpone0236212g008PLOS ONE 101371journalpone0236212 August PLOS ONE 0cUptake of heatprocessed proteins by THP1 macrophages and dendritic cells and subsequent cytokine responseimmunological defence [] We did observe however a quantitative difference in uptake asthe uptake values for wetheated thyroglobulin and BLG were relatively higher in macrophagesthan in DCs This suggests that macrophages are more efficient in their phagocytic role thanDCs which is in line with literature stating that uptake by macrophages is part of their corerole in eliminating harmful substances whereas uptake by DCs is a means to initiating anadaptive immune response [] Upon comparing the three tested proteins we found that thephysicochemical alterations upon heattreatment to thyroglobulin and BLG were similar butdifferent from lysozyme These proteins differ in size thyroglobulin kDa BLG kDa lysozyme kDa pI lysozyme BLG thyroglobulin anddenaturation temperature lysozyme ˚C BLG ˚C thyroglobulin ˚C [“]suggesting that both pI and heat stability may be important for heatinduced structural proteinchanges The high pI may have conferred a positive charge to lysozyme during wetheatingpH thereby hindering aggregation under this condition due to electrostatic repulsionThe strong internal coherence and stability of lysozyme™s structure may also have contributedto its stability during heat processing [] It is reported Answer:

Thyroid_Cancer

Comparison of different calculationtechniques for absorbed dose assessment inpatient specific peptide receptor radionuclidetherapyDomenico Finocchiaro12 Salvatore Berenato3 Valentina Bertolini1 Gastone Castellani2Nico Lanconelli2 Annibale Versari4 Emiliano Spezi35 Mauro Iori1 Federica FioroniID1Elisa Grassi1 Azienda Unit  Sanitaria Locale di Reggio Emilia”IRCCS Medical Physics Unit Reggio Emilia Italy Department of Physics and Astronomy University of Bologna Bologna Italy Department of MedicalPhysics Velindre Cancer Centre Cardiff United Kingdom Azienda Unit  Sanitaria Locale di Reggio Emilia”IRCCS Nuclear Medicine Unit Reggio Emilia Italy School of Engineering Cardiff University CardiffUnited Kingdom federicafioroniauslreita1111111111a1111111111a1111111111a1111111111a1111111111 ACCESSAbstractCitation Finocchiaro D Berenato S Bertolini VCastellani G Lanconelli N Versari A Comparison of different calculation techniques forabsorbed dose assessment in patient specificpeptide receptor radionuclide therapy e0236466 101371journalpone0236466Editor Choonsik Lee National Institute of HealthUNITED STATESReceived July Accepted July Published August Copyright Finocchiaro This is an access distributed under the terms ofthe Creative Commons Attribution License whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are creditedData Availability Statement All relevant data arewithin the manuscriptFunding This work was supported by theEuropean Metrology Programme For InnovationAnd Research EMPIR joint research project15HLT06 Metrology for clinical implementation ofdosimetry in molecular radiotherapyMRTDosimetry which has received funding fromthe European Union The EMPIR initiative is cofunded by the European Union™s Horizon AimThe present work concerns the comparison of the performances of three systems for dosimetry in RPT that use different techniques for absorbed dose calculation anlevel dosimetry voxellevel dose kernel convolution and Monte Carlo simulations The aim was toassess the importance of the choice of the most adequate calculation modality providingrecommendations about the choice of the computation toolMethodsThe performances were evaluated both on phantoms and patients in a multilevel approachDifferent phantoms filled with a 177Luradioactive solution were used a homogeneous cylindrical phantom a phantom with anshaped inserts and two cylindrical phantoms withinserts different for shape and volume A total of patients with NETs treated by PRRTwith 177LuDOTATOC were retrospectively analysedResultsThe comparisons were performed mainly between the mean values of the absorbed dose inthe regions of interest A general better agreement was obtained between Dose kernel convolution and Monte Carlo simulations results rather than between either of these two andanlevel dosimetry both for phantoms and patients Phantoms measurements alsoshowed the discrepancies mainly depend on the geometry of the inserts eg shape and volume For patients differences were more pronounced than phantoms and higher interintrapatient variability was observedPLOS ONE 101371journalpone0236466 August PLOS ONE 0cComparison of different absorbed dose calculation methods in MRTresearch and innovation programme and theEMPIR Participating States SB acknowledgesfunding from Cancer Research Wales throughgrant No Competing interests The authors have declaredthat no competing interests existConclusionThis study suggests that voxellevel techniques for dosimetry calculation are potentiallymore accurate and personalized than anlevel methods In particular a voxelconvolution method provides good results in a short time of calculation while Monte Carlo basedcomputation should be conducted with very fast calculation systems for a possible use inclinics despite its intrinsic higher accuracy Attention to the calculation modality is recommended in case of clinical regions of interest with irregular shape and far from sphericalgeometry in which Monte Carlo seems to be more accurate than voxelconvolutionmethodsIntroductionRadiopharmaceutical therapy RPT as defined in ICRP [] is based on the use of specificpharmaceuticals labelled with radionuclides to deliver a lethal dose of radiation to tumourareas Radiopharmaceuticals are specifically designed to have high affinity with given tumoursites so that ionizing radiations such as ps and photons emitted by the isotopes maydeposit energy inside or close to unhealthy tissues saving surrounding healthy tissues Thisapproach produced very encouraging results in the treatment of neuroendocrine tumoursNET in the last decades in particular in therapies which make use of somatostatin analogueslabelled with 90Y or 177Lu [] such as the recently registered Lutathera [] Different responserates and a large interpatient variability of the outcome were however reported by someauthors eg Campana D and Vinjamuri S [ ]The wellestablished experience with external beam radiation therapy EBRT has providedstrong evidence that tumour response and normal an toxicity is related to absorbed dosesFor this reason it was supposed that the treatment outcome correlates with the absorbed dosedelivered to tumours even in RPT [ ] Yet a dosimetry as more accurate and personalized aspossible is needed to this purpose to provide clinicians with reliable resultsDespite the general demand for a more individualized treatment based on pretherapeuticdosimetry study in NET dosimetry is not conducted always in the clinical routine This ismostly because dosimetry is often considered time consuming a lot of time required for imaging expensive costs for every image scan and every measurement and sometimes inaccuratefor the lack of standardization and harmonization mainly At present a standard procedurefor calculating the absorbed dose is not well defined for every kind of radionuclide therapy Inrelation to NET the evidence of prolonged survival has been demonstrated only recently [] ina subgroup of NETDifferent methods have been developed to perform dosimetry since its beginnings Techniques based on standardized reference models were first developed thanks to their simplicityof implementation and have been used for many years These models assume uniform activityie homogeneous uptake in the source regions However evidence indicates that deterministic biological effects including tumour response and normal tissue toxicity may not be wellpredicted by the mean absorbed dose in the region and may be significantly influenced bynonuniform doses [] To take into account this aspect voxelbased techniques were considered similarly to those which have been also used for decades as standard of care in EBRT [] Contrary to what happens for EBRT however in which plenty of software for therapyplanning are available on the market in RPT only few systems which are adequate toPLOS ONE 101371journalpone0236466 August PLOS ONE 0cComparison of different absorbed dose calculation methods in MRTdosimetry for Peptide Receptor Radionuclide Therapy PRRT and which can work with multiple 3D imaging have been officially released in the last few years [“] For this reasonmany dosimetry software and tools are in use worldwide but only some of them are commercially available Several of them are homemade tools which were developed before the commercial software were finalized [“] and have been fully customized by clinical users in themeantimeAt present standardization and harmonization of the calculation systems are importantTherefore it is essential to compare the various results obtained with the most advanced existing homemadecommercial software and other less advanced still used worldwide methodsYet both categories should be tested on a larger sample of cases than ever done before Thesetests should provide an example of the most accurate methodology for 3D dosimetry in RPTthanks to the gained experience in the last decades giving recommendations about the appropriate use and the limitations of each methodA few studies presenting some comparisons have already been published [“] Howeverthese works either did not report dosimetry studies performed completely at the voxel level[] or considered a limited number of clinical cases [] or showed a comparison based onthe dose factors and not based on absorbed doses [] Therefore more studies are needed tofully evaluate calculation performances in clinically relevant conditions considering a highnumber of casesIn this context the main objective of the present work is to compare different modalitiesfor absorbed dose calculation to point out the pros and the cons in each modality and to provide recommendations about the choice of the most adequate computation technique for thesingle clinical or research centres approaching the methodology The modalities here considered include the most used techniques in this field worldwide ranging from the less advancedand less personalised to the most accurate and patient specificThe considered modalities listed in growing complexity are an level dosimetry based onstandardized reference models such as OLINDA version [] which has been used fordecades before the recent release of the new updated commercial version OLINDA version [] voxellevel dosimetry based on dose kernel convolution VoxelMed20 [] and voxellevel dosimetry based on Monte Carlo MC simulations RAYDOSE [] OLINDA11 waschosen because it is still widely used for RPT dosimetry VoxelMed20 was chosen because itwas designed to achieve a good compromise between calculation accuracy and easy applicability in clinical practice RAYDOSE was considered because MC techniques are considered toprovide the most accurate approach to dose estimate []The comparison was performed on 3D images of specifically designed phantoms and onmultiple 3D dataset of images of a high number of clinical cases This multiapproach methodbased both on phantoms and patients allowed to investigate the differences of performancebetween the calculation modalities depending on the shape and the volume of the activity distribution and to provide a valuable comparison based on a conspicuous number of clinicalcasesMaterials and methodsThis study involves human participants All participants were enrolled in a clinical trialEUDRACT at Azienda USLIRCCS of Reggio Emilia Italy The study wasapproved by the ethics committee of Azienda USLIRCCS of Reggio Emilia Italy and eachpatient gave written informed consent for the study conductionThe following sections describe in detail the specific phantoms the image set the softwareand the data elaboration approachPLOS ONE 101371journalpone0236466 August PLOS ONE 0cComparison of different absorbed dose calculation methods in MRTPreparation of phantomsThree different phantoms filled with 177Lu radiolabelled peptides leftover from the clinicalapplication were used¢ a ˜Cylindrical phantom™ filled with a homogeneous radioactive solution Jaszczak Data Spectrum Corporation USA shown in Fig 1A Details are included in Table ¢ a cylindrical phantom and a set of fillable plastic inserts arranged in two different configurations to originate a couple of ˜Geometrical phantoms™ The inserts different for shapetoroidal pearshaped tubular and ellipsoidal and volume are depicted in Fig 1C Insertstake the name from the shape and the equivalent diameter ie the diameter for a spherewith the same volume as shown in Table Each insert was filled with the same activityconcentration and placed in a nonradioactive water background Details of volume andactivity concentration are shown in Table ¢ an ˜Anthropomorphic phantom™ with an shaped inserts LiquiPhill The Phantom Laboratory Greenwich NY shown in Fig 1B Details are included in Table Every insert wasfilled with an activity concentration typical of real ans in clinical cases and placed in aradioactive water backgroundTo accurately measure the volumes the weight of the phantoms and of the insertsbefore and after refilling was taken with a calibrated scale The density of the waterbasedsolution was of 1gml HCl M was used as a carrier solution to prevent radioactive177Lu deposition on the phantom walls and to guarantee a homogenous radionuclidesolutionEvery phantom was scanned once and the timeactivity curve was generated using the physical decay of the isotope All specific data regarding the volumes of inserts and phantoms andthe activity used are reported in Table Fig CT scans of phantoms used in this study a Cylindrical phantom b Anthropomorphic phantom c Insertswith different shapes placed in the Geometrical phantom101371journalpone0236466g001PLOS ONE 101371journalpone0236466 August PLOS ONE 0cTable Description of phantoms used to test the dosimetry toolsPhantomPhantom volumeInsert nameInsert volumeInsert activity concentration MBqBackground activity concentration MBqComparison of different absorbed dose calculation methods in MRTCylindricalGeometricalmlAnthropomorphicNATo17aTo26E20E30E38To17bP38P39aP39bTu38aTu38bLesionPancreasLeft kidneyRightkidneySpleenLivermlNA101371journalpone0236466t001Clinical trialmlmlNANAThe clinical cases considered in the present work were all extracted from a preexisting clinicalPRRT trial including patients and conducted by Azienda USLIRCCS of Reggio EmiliaItalyAll considered patients were previously enrolled in the trial EUDRACT between and The clinical trial design established that every patient had to besequentially administered with either 177Lu labelled radiopeptides 177LuDOTATOC or 90Ylabelled radiopeptides 90YDOTATOC up to a maximum of infusions or cycles Dosimetry was mandatory in the clinical trial and was to schedule during the first cycle of therapyafter a therapeutic administration of 177LuDOTATOC Each patient underwent SPECTCT scans at h post injection According to the trial design clinical absorbedTable Legend of the insert acronyms for the Geometrical phantomInsert geometryEquivalent diameter mmInsert nameTorusTorusTorusEllipsoidEllipsoidEllipsoidPearPearPearTubeTube101371journalpone0236466t002To17aTo17bTo26E20E30E38P38P39aP39bTu38aTu39bPLOS ONE 101371journalpone0236466 August PLOS ONE 0cComparison of different absorbed dose calculation methods in MRTdoses for 177Lu and 90Y labelled radiopeptides for liver spleen and kidneys were calculatedEach an was manually contoured and absorbed doses were calculated in compliance withthe MIRD scheme [] at anlevel from images The number of cycles the isotope and theactivity chosen for every injection were planned by an expert physician on the basis of thedosimetry results The activity prescription had to be determined based on the BiologicalEffective Dose BED delivered to kidneys Kidneys are regarded as the principal ans at riskin PRRT [ ] As suggested by different works [“] in this clinical trial the cumulativedose limit to kidneys was set to Gy of BED for patients with no risk factors hypertensiondiabetes renal failure are considered risk factors for this therapy and at 28Gy for patients withrisk factorsIn the present work absorbed doses to kidneys spleen and liver were calculated to comparethe three dosimetric methodsImage acquisition and reconstructionAll activity measurements were performed with an accurate activity calibrator for 177Lu Aktivimeter Isomed Nuklear Medizintechnik Germany and all image acquisitions were performed through a SPECTCT scanner Symbia T2 Siemens Medical Germany  NaITldetector previously calibrated [] The standard clinical protocol for body studies was usedboth for phantoms and patients with the following SPECT settings MEHR collimators matrix x zoom views x timeview s step and shoot mode degree of rotation ˚ noncircular orbit detector configuration ˚The first CT acquisition per patient was performed with the following parameters 130kVand max mAs using tube current modulation The subsequent CT images were acquiredwith kV and 40mAs for radiation protection safety of patients The CT reconstructed slicethickness was mm and a smooth reconstruction kernel was used B08s Siemens MedicalSolution Germany The higher image quality of the first CT scan is necessary for contouringvolumes of interest more accuratelyThe SPECT projections were reconstructed by an iterative algorithm including CT attenuation correction scatter correction and full collimatordetector response in Siemens ESoftworkstation Syngo MI Application version 32B Siemens Medical Solution Germany withFlash 3D iterative algorithm iterations subsets Gaussian filter cutoff mm mmcubic voxel []All cases of Sample A were rigidly registered to the first CT image of the sequence in Siemens Esoft workstation Images of patients included in Sample B were registered using adeformable multipass algorithm with the Velocity Advanced Imaging workstation Varian Medical Systems Palo Alto USA [] The registration procedures rescaled the originalvoxel size to 39x39x35 mm3The Volumes Of Interest VOI for each phantom and each patient were manually drawnon the reference CT image as recommended by Uribe [] using the VelocityworkstationSoftware for image processing and dosimetry calculationsOLINDA11 OLINDA version [] is an an level dosimetry software based on theMIRD methodology [] for internal dose estimation This is the method adopted in the clinical trial the clinical cases of this work are extracted from Absorbed doses to ans and tolesions can be calculated by using different models in the software human phantom modelsie mathematical representations of the human body to represent ans and whole body andsphere models ie mathematical representations of spheres to represent lesions [ ]PLOS ONE 101371journalpone0236466 August PLOS ONE 0cComparison of different absorbed dose calculation methods in MRTUnlike VoxelMed and RAYDOSE OLINDA needs timeintegrated activity A values ofVOIs as input parameters [] which were calculated with VoxelMed20 which will bedescribed in the next section and then inserted in OLINDAOLINDA sphere model commonly used to calculate doses to lesions was used to generatethe results for the inserts placed in the Geometrical phantom and for the dummy lesion housedin the anthropomorphic phantom while OLINDA an model adult male was used for thedummy ans placed in the anthropomorphic phantom Real insert volumes were used forcalculationsThe human models adult male or adult female were used to calculate dosimetry of thecohort of patients Doses were scaled using the true patient weight and the true an massesVoxelMed20 VoxelMed is a homemade software for dose calculation developed atAzienda USLIRCCS research hospital Reggio Emilia Italy It was developed in the MatlabThe Mathworks Natick MA programming environment and designed on the CERR platform wwwcerrinfo It performs voxellevel dosimetry based on the MIRD guidelines []The first version of the software along with the S value matrices for voxel dosimetry used incalculations were described in detail elsewhere []VoxelMed version includes a graphical user interface the possibility to export the resultsof calculations to Microsoft Excel file the visualization of the fitting curves both mono andbiexponential a module for renal BED calculation following the model suggested by StrigariL [] and the possibility to correct activity for partial volume effect PVE as presentedin [] Moreover VoxelMed20 provides the user with the timeintegrated activity A at VOIlevel which can be used for dosimetry with OLINDA version both for ans and lesionsTo calculate the number of disintegrations VoxelMed integrates the timeactivity curvewith the trapezoidal method in the time interval between the first and the last acquisitionBeyond this timeinterval the integration is performed analytically and the timeactivity curveis extrapolated using the effective halflife or the physical halflife it is chosen by the userThe effective halflife of the an or lesion is derived with a biexponential fit of the activitiesin the VOI the physical halflife is known from the selected isotope Timeintegrated activityis calculated in each voxel or in the whole an depending on the modality of dose calculationselected ie voxel level or an levelRAYDOSE RAYDOSE is a software package developed at Cardiff University School ofEngineering Cardiff University UK and designed to carry out 3D patientspecific imagebased dosimetry for RPT RAYDOSE provides personalized 3D dose map performing MonteCarlo simulations on radiation transport based on the Geant4 MC toolkit CERN Switzerland Geant4 is the stateoftheart package for the simulation of the transport of psthrough matter [] RAYDOSE generates voxellevel dose maps using anatomical and physiological data taken from morphologic and functional images []In order to obtain the area under the timeactivity curve RAYDOSE allows to use differentfitting modalities monoexponential decay linear uptake plus monoexponential decay or thetrapezoidal method In this study for the dose calculation of the clinical cases we used thetrapezoidal method at the voxel level up to the last time acquisition point while the timeactivity curve beyond the last scan time was extrapolated from the monoexponential curve fittingof the whole an activities in the VOI For dose calculation in phantoms we used the physical halflife of the isotope to extrapolate the activity from the scan time upwardsData and statistical analysisTwo groups of patients were considered for the purpose of this workPLOS ONE 101371journalpone0236466 August PLOS ONE 0cComparison of different absorbed dose calculation methods in MRTTable Demographic and baselines clinical characteristics of all patients�CharacteristicSample A N Sample B N Gender NoMaleFemaleAge yHeight cmWeight kgPrimary tumour site NoIleumPancreasLungThyroidRectumOthers177Lu activity for dosimetry MBq   ± ± ± ± ± ± ± N\\AN\\AN\\A ± � Plusminus values are means ± standard deviation  Injected activity at the first cycle of therapy Dosimetry was performed after the first injection101371journalpone0236466t003A first subgroup of cases named as œSample A was extracted by random samplingfrom the original clinical trial to adequately represent the whole population The number ofcases was calculated safely adopting a margin of error of and a standard deviation of A second independent subgroup of patients named as Sample B was extracted toofrom the original clinical trial similarly to Sample A A sample of cases was considered adequate in relation to the aim of the experiment conducted on Sample BPatient baseline characteristics for Sample A and Sample B are reported in Table The study type the image registration and the software used for the dose calculations of theclinical cases in sample A and sample B are summarised in Table Absorbed doses were calculated separately with OLINDA11 VoxelMed20 and RAYDOSEusing the same set of imagesKidney liver and spleen absorbed doses were calculated for each patient Two differentcomparison studies were performed The first study involved only comparison between VoxelMed and OLINDA based on absorbed dose calculations of patients in Sample A The secondstudy involved comparison between all the three software VoxelMed OLINDA and RAYDOSE based on absorbed dose calculations of patients in Sample B Furthermore in order toreduce the contribution of the fitting of the activitytime curves in the comparison of the software the VoxelMed dosimetry calculations for Sample B were repeated using the same effective halflife applied in RAYDOSE RAYDOSE estimates the effective halflife by fitting theTable Summary of phantom and patient studies performedStudy typePhantomObject of studyImage registrationSoftwareHomogeneous phantomNo registration only scanOLINDA11”VoxelMed”RAYDOSEGeometrical phantomNo registration only scanOLINDA11”VoxelMed”RAYDOSEAnthropomorphic phantomNo registration only scanOLINDA11”VoxelMed”RAYDOSEClinicalSample A patientsRigid registrationOLINDA11”VoxelMedSample B patientsDeformable registrationOLINDA11”VoxelMed VoxelMedλ RD”RAYDOSE101371journalpone0236466t004PLOS ONE 101371journalpone0236466 August PLOS ONE 0cComparison of different absorbed dose calculation methods in MRTFig Clinical comparison study workflow Procedure flow of absorbed dose calculation for each patient of sample A top of the image and sample B lowerpart of the image101371journalpone0236466g002an activities against time as previously described Flow chart in Fig illustrates methodology in clinical studyDosevolume histograms DVH were evaluated to compare spatial dose distribution atvoxellevelMean values of absorbed dose were used to compare an level and voxel level techniquesComparison between the different dosimetry methods was statistically evaluated using theLin™s concordance correlation coefficient CCC and the BlandAltman plot [] The CCCsymbolized by ρc allows to evaluate the degree of concordance between two measures whilethe BlandAltman plot is used to analyse the agreement between two quantities The CCC wascalculated using SAS SAS Institute Cary NC USA A value of ρc equal to denotes perfect concordance a value equal to perfect discordance while a value of no correlationResultsPhysical phantom studyThe values of mean absorbed dose for the physical phantoms calculated with OLINDA11VoxelMed and RAYDOSE are reported in Table Visual representation of the same data isprovided in Fig Similar DVH curves were generated with VoxelMed and RAYDOSE both for the Cylindrical phantom Fig and for the other two phantoms Fig Fig shows DHVs only for theinserts in the Geometrical and the Anthropomorphic phantoms with the smallest and the largest relative differences of mean absorbed dose respectivelyClinical studyAbsorbed dose for kidneys liver and spleen of the sample A of patients calculated withOLINDA11 and VoxelMed are shown in Table The absorbed doses to liver and to spleenwere found to be highly correlated while lower correlation was found for kidneys The CCCPLOS ONE 101371journalpone0236466 August PLOS ONE 0cComparison of different absorbed dose calculation methods in MRTTable Mean absorbed dose Gy calculated with OLINDA11 VoxelMed and RAYDOSE for all of the three phantoms The absorbed dose calculated withOLINDA11 was performed using either the an model and the Sphere model Absorbed doses to Pancreas Kidneys Spleen and Liver were calculated using the anmodel otherwise the Sphere model was usedPhantomCylindricalGeometricalAnthropomorphicInsert nameOLINDA11VoxelMedRAYDOSENATo17aTo17bTo26E20E30E38P38P39aP39bTu38aTu39bLesionPancreasKidneysSpleenLiver101371journalpone0236466t005[ confidence interval] values were ρc liver [ ] ρc spleen [ ]ρc kidneys [ ] The BlandAltman plot is shown in Fig OLINDA11 VoxelMed VoxelMedλ RD and RAYDOSE calculated mean absorbed dosefor patients of Sample B are shown in Table while the BlandAltman plot is shown in Fig The absorbed doses calculated with VoxelMed and RAYDOSE were highly correlated withρc kidneys [ ] ρc liver [ ] and ρc spleen [ ] andalmost complete agreement were found between VoxelMedλ RD and RAYDOSE withρc kidneys [ ] ρc liver [ ] and ρc spleen [ ]DiscussionIn this study we compared the performances of three tools for dosimetry calculationsOLINDA11 VoxelMed20 and RAYDOSE with the primary aim to evaluate the influence ofthe calculation modality on absorbed dose assessment anlevel based voxellevel dose kernel convolution based and Monte Carlo simulations based respectively The secondary aimwas to give some recommendations about the choice of the adequate technique for dosimetrycalculation to be implemented in a hospital in a research centre or in an academic instituteclinical or research purpose small or large number of patients clinical trials only or standardprocedures This analysis was performed in standard conditions by acquisition and processing of radioactive phantoms provided with inserts of specific volume and geometry and inclinical conditions over a large cohort of patients a selection of clinical cases taken from a clinical trial in which dosimetry had already been calculated Clinical conditions are indeed quitedistant from and more complicated than the standard conditions achievable in a phantom forseveral reasons biological kinetics in place of only physical decay of activity serial acquisitionsof functional images and associated issues related to image registration [] motion of thepatient that creates artefacts in images irregular shape of volumes of interest inhomogeneousactivity distributionPLOS ONE 101371journalpone0236466 August PLOS ONE 0cComparison of different absorbed dose calculation methods in MRTFig Comparison of mean absorbed dose Gy calculated using OLINDA11 VoxelMed and RAYDOSE a Homogeneous phantom b Anthropomorphicphantom and cf Geometrical phantom Note the Lesion insert in the Anthropomorphic phantom is missing because beyond the range of dose visualized in thegraph101371journalpone0236466g003Therefore to consider a large sample of clinical cases was of great interest since many studies about methods for dosimetry calculation are based on smaller groups of patients [“]and in a small group the inter patient variability cannot be properly investigatedThe quantitative intercomparison between all the three software was performed betweenthe mean values of absorbed doses In fact OLINDA provides only mean values while RAYDOSE and VoxelMed20 voxelbased tools provide the dose distribution that can be represented with DVHs from which the mean dose values can be derived To compare thetechniques of calculation the relative differences and the correlation between data pairs wereevaluatedFor standard conditions we evaluated discrepancies of calculated absorbed dose in a cylindrical phantom and in differently shaped inserts filled with a homogeneous radioactive solution Table shows the values of absorbed dose obtained with OLINDA11 VoxelMed andRAYDOSE in each of the phantoms These values are also plotted in Fig Lower values ofabsorbed dose were generally calculated using OLINDA in comparison with the dose calculated with other voxel modalities In the case of the cylindrical phantom a good agreementwas obtained between VoxelMed20 and RAYDOSE discrepancy equal to while largerdifference was observed between VoxelMed20 and OLINDA Absorbed dose map provided by VoxelMed and RAYDOSE showed similar spatial distribution close values of standard deviation across voxels around and analogues slope in DVHs Fig PLOS ONE 101371journalpone0236466 August PLOS ONE 0cComparison of different absorbed dose calculation methods in MRTFig Comparison of DVHs calculated using VoxelMed continuous line and RAYDOSE dotted line for the Cylindrical phantom101371journalpone0236466g004To compare the calculation techniques in different conditions of volume and geometry theGeometrical phantoms were acquired Relative differences in absorbed dose depend on theshape and on the volume of the inserts smaller is the volume and further from a regular sphereis the shape more the relative difference is higher In the Geometrical phantom the toroidalinserts provided the greatest discordance relative difference with VoxelMed dose rangingfrom to for OLINDA and from to for RAYDOSE while in the otherinserts differences ranged between [ ] for OLINDA11 and [ ] for RAYDOSEOn one hand the insert dose calculations in OLINDA were performed using the spheremodel since OLINDA only allows to perform dosimetry calculation for specified models ieans or spheres This approximation might explain the huge discrepancies obtained withthe voxelbased methods On the other hand a reason for the difference between RAYDOSEand VoxelMed is that the latter applies a mask before the convolution while RAYDOSE doesnot This contribution affects calculations in so far as the geometry and the volume of theinsert may influence the activity distribution and leave empty spaces around or inside theobjects This effect is especially pronounced for example in the case of the toroid The application of a mask also implies the lack of photon cross irradiation contribution between insertswhich has an impact on dose calculation contribution around [] Discrepancies ofPLOS ONE 101371journalpone0236466 August PLOS ONE 0cComparison of different absorbed dose calc

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You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: Comparison of different calculationtechniques for absorbed dose assessment inpatient specific peptide receptor radionuclidetherapyDomenico Finocchiaro12 Salvatore Berenato3 Valentina Bertolini1 Gastone Castellani2Nico Lanconelli2 Annibale Versari4 Emiliano Spezi35 Mauro Iori1 Federica FioroniID1Elisa Grassi1 Azienda Unit  Sanitaria Locale di Reggio Emilia”IRCCS Medical Physics Unit Reggio Emilia Italy Department of Physics and Astronomy University of Bologna Bologna Italy Department of MedicalPhysics Velindre Cancer Centre Cardiff United Kingdom Azienda Unit  Sanitaria Locale di Reggio Emilia”IRCCS Nuclear Medicine Unit Reggio Emilia Italy School of Engineering Cardiff University CardiffUnited Kingdom federicafioroniauslreita1111111111a1111111111a1111111111a1111111111a1111111111 ACCESSAbstractCitation Finocchiaro D Berenato S Bertolini VCastellani G Lanconelli N Versari A Comparison of different calculation techniques forabsorbed dose assessment in patient specificpeptide receptor radionuclide therapy e0236466 101371journalpone0236466Editor Choonsik Lee National Institute of HealthUNITED STATESReceived July Accepted July Published August Copyright Finocchiaro This is an access distributed under the terms ofthe Creative Commons Attribution License whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are creditedData Availability Statement All relevant data arewithin the manuscriptFunding This work was supported by theEuropean Metrology Programme For InnovationAnd Research EMPIR joint research project15HLT06 Metrology for clinical implementation ofdosimetry in molecular radiotherapyMRTDosimetry which has received funding fromthe European Union The EMPIR initiative is cofunded by the European Union™s Horizon AimThe present work concerns the comparison of the performances of three systems for dosimetry in RPT that use different techniques for absorbed dose calculation anlevel dosimetry voxellevel dose kernel convolution and Monte Carlo simulations The aim was toassess the importance of the choice of the most adequate calculation modality providingrecommendations about the choice of the computation toolMethodsThe performances were evaluated both on phantoms and patients in a multilevel approachDifferent phantoms filled with a 177Luradioactive solution were used a homogeneous cylindrical phantom a phantom with anshaped inserts and two cylindrical phantoms withinserts different for shape and volume A total of patients with NETs treated by PRRTwith 177LuDOTATOC were retrospectively analysedResultsThe comparisons were performed mainly between the mean values of the absorbed dose inthe regions of interest A general better agreement was obtained between Dose kernel convolution and Monte Carlo simulations results rather than between either of these two andanlevel dosimetry both for phantoms and patients Phantoms measurements alsoshowed the discrepancies mainly depend on the geometry of the inserts eg shape and volume For patients differences were more pronounced than phantoms and higher interintrapatient variability was observedPLOS ONE 101371journalpone0236466 August PLOS ONE 0cComparison of different absorbed dose calculation methods in MRTresearch and innovation programme and theEMPIR Participating States SB acknowledgesfunding from Cancer Research Wales throughgrant No Competing interests The authors have declaredthat no competing interests existConclusionThis study suggests that voxellevel techniques for dosimetry calculation are potentiallymore accurate and personalized than anlevel methods In particular a voxelconvolution method provides good results in a short time of calculation while Monte Carlo basedcomputation should be conducted with very fast calculation systems for a possible use inclinics despite its intrinsic higher accuracy Attention to the calculation modality is recommended in case of clinical regions of interest with irregular shape and far from sphericalgeometry in which Monte Carlo seems to be more accurate than voxelconvolutionmethodsIntroductionRadiopharmaceutical therapy RPT as defined in ICRP [] is based on the use of specificpharmaceuticals labelled with radionuclides to deliver a lethal dose of radiation to tumourareas Radiopharmaceuticals are specifically designed to have high affinity with given tumoursites so that ionizing radiations such as ps and photons emitted by the isotopes maydeposit energy inside or close to unhealthy tissues saving surrounding healthy tissues Thisapproach produced very encouraging results in the treatment of neuroendocrine tumoursNET in the last decades in particular in therapies which make use of somatostatin analogueslabelled with 90Y or 177Lu [] such as the recently registered Lutathera [] Different responserates and a large interpatient variability of the outcome were however reported by someauthors eg Campana D and Vinjamuri S [ ]The wellestablished experience with external beam radiation therapy EBRT has providedstrong evidence that tumour response and normal an toxicity is related to absorbed dosesFor this reason it was supposed that the treatment outcome correlates with the absorbed dosedelivered to tumours even in RPT [ ] Yet a dosimetry as more accurate and personalized aspossible is needed to this purpose to provide clinicians with reliable resultsDespite the general demand for a more individualized treatment based on pretherapeuticdosimetry study in NET dosimetry is not conducted always in the clinical routine This ismostly because dosimetry is often considered time consuming a lot of time required for imaging expensive costs for every image scan and every measurement and sometimes inaccuratefor the lack of standardization and harmonization mainly At present a standard procedurefor calculating the absorbed dose is not well defined for every kind of radionuclide therapy Inrelation to NET the evidence of prolonged survival has been demonstrated only recently [] ina subgroup of NETDifferent methods have been developed to perform dosimetry since its beginnings Techniques based on standardized reference models were first developed thanks to their simplicityof implementation and have been used for many years These models assume uniform activityie homogeneous uptake in the source regions However evidence indicates that deterministic biological effects including tumour response and normal tissue toxicity may not be wellpredicted by the mean absorbed dose in the region and may be significantly influenced bynonuniform doses [] To take into account this aspect voxelbased techniques were considered similarly to those which have been also used for decades as standard of care in EBRT [] Contrary to what happens for EBRT however in which plenty of software for therapyplanning are available on the market in RPT only few systems which are adequate toPLOS ONE 101371journalpone0236466 August PLOS ONE 0cComparison of different absorbed dose calculation methods in MRTdosimetry for Peptide Receptor Radionuclide Therapy PRRT and which can work with multiple 3D imaging have been officially released in the last few years [“] For this reasonmany dosimetry software and tools are in use worldwide but only some of them are commercially available Several of them are homemade tools which were developed before the commercial software were finalized [“] and have been fully customized by clinical users in themeantimeAt present standardization and harmonization of the calculation systems are importantTherefore it is essential to compare the various results obtained with the most advanced existing homemadecommercial software and other less advanced still used worldwide methodsYet both categories should be tested on a larger sample of cases than ever done before Thesetests should provide an example of the most accurate methodology for 3D dosimetry in RPTthanks to the gained experience in the last decades giving recommendations about the appropriate use and the limitations of each methodA few studies presenting some comparisons have already been published [“] Howeverthese works either did not report dosimetry studies performed completely at the voxel level[] or considered a limited number of clinical cases [] or showed a comparison based onthe dose factors and not based on absorbed doses [] Therefore more studies are needed tofully evaluate calculation performances in clinically relevant conditions considering a highnumber of casesIn this context the main objective of the present work is to compare different modalitiesfor absorbed dose calculation to point out the pros and the cons in each modality and to provide recommendations about the choice of the most adequate computation technique for thesingle clinical or research centres approaching the methodology The modalities here considered include the most used techniques in this field worldwide ranging from the less advancedand less personalised to the most accurate and patient specificThe considered modalities listed in growing complexity are an level dosimetry based onstandardized reference models such as OLINDA version [] which has been used fordecades before the recent release of the new updated commercial version OLINDA version [] voxellevel dosimetry based on dose kernel convolution VoxelMed20 [] and voxellevel dosimetry based on Monte Carlo MC simulations RAYDOSE [] OLINDA11 waschosen because it is still widely used for RPT dosimetry VoxelMed20 was chosen because itwas designed to achieve a good compromise between calculation accuracy and easy applicability in clinical practice RAYDOSE was considered because MC techniques are considered toprovide the most accurate approach to dose estimate []The comparison was performed on 3D images of specifically designed phantoms and onmultiple 3D dataset of images of a high number of clinical cases This multiapproach methodbased both on phantoms and patients allowed to investigate the differences of performancebetween the calculation modalities depending on the shape and the volume of the activity distribution and to provide a valuable comparison based on a conspicuous number of clinicalcasesMaterials and methodsThis study involves human participants All participants were enrolled in a clinical trialEUDRACT at Azienda USLIRCCS of Reggio Emilia Italy The study wasapproved by the ethics committee of Azienda USLIRCCS of Reggio Emilia Italy and eachpatient gave written informed consent for the study conductionThe following sections describe in detail the specific phantoms the image set the softwareand the data elaboration approachPLOS ONE 101371journalpone0236466 August PLOS ONE 0cComparison of different absorbed dose calculation methods in MRTPreparation of phantomsThree different phantoms filled with 177Lu radiolabelled peptides leftover from the clinicalapplication were used¢ a ˜Cylindrical phantom™ filled with a homogeneous radioactive solution Jaszczak Data Spectrum Corporation USA shown in Fig 1A Details are included in Table ¢ a cylindrical phantom and a set of fillable plastic inserts arranged in two different configurations to originate a couple of ˜Geometrical phantoms™ The inserts different for shapetoroidal pearshaped tubular and ellipsoidal and volume are depicted in Fig 1C Insertstake the name from the shape and the equivalent diameter ie the diameter for a spherewith the same volume as shown in Table Each insert was filled with the same activityconcentration and placed in a nonradioactive water background Details of volume andactivity concentration are shown in Table ¢ an ˜Anthropomorphic phantom™ with an shaped inserts LiquiPhill The Phantom Laboratory Greenwich NY shown in Fig 1B Details are included in Table Every insert wasfilled with an activity concentration typical of real ans in clinical cases and placed in aradioactive water backgroundTo accurately measure the volumes the weight of the phantoms and of the insertsbefore and after refilling was taken with a calibrated scale The density of the waterbasedsolution was of 1gml HCl M was used as a carrier solution to prevent radioactive177Lu deposition on the phantom walls and to guarantee a homogenous radionuclidesolutionEvery phantom was scanned once and the timeactivity curve was generated using the physical decay of the isotope All specific data regarding the volumes of inserts and phantoms andthe activity used are reported in Table Fig CT scans of phantoms used in this study a Cylindrical phantom b Anthropomorphic phantom c Insertswith different shapes placed in the Geometrical phantom101371journalpone0236466g001PLOS ONE 101371journalpone0236466 August PLOS ONE 0cTable Description of phantoms used to test the dosimetry toolsPhantomPhantom volumeInsert nameInsert volumeInsert activity concentration MBqBackground activity concentration MBqComparison of different absorbed dose calculation methods in MRTCylindricalGeometricalmlAnthropomorphicNATo17aTo26E20E30E38To17bP38P39aP39bTu38aTu38bLesionPancreasLeft kidneyRightkidneySpleenLivermlNA101371journalpone0236466t001Clinical trialmlmlNANAThe clinical cases considered in the present work were all extracted from a preexisting clinicalPRRT trial including patients and conducted by Azienda USLIRCCS of Reggio EmiliaItalyAll considered patients were previously enrolled in the trial EUDRACT between and The clinical trial design established that every patient had to besequentially administered with either 177Lu labelled radiopeptides 177LuDOTATOC or 90Ylabelled radiopeptides 90YDOTATOC up to a maximum of infusions or cycles Dosimetry was mandatory in the clinical trial and was to schedule during the first cycle of therapyafter a therapeutic administration of 177LuDOTATOC Each patient underwent SPECTCT scans at h post injection According to the trial design clinical absorbedTable Legend of the insert acronyms for the Geometrical phantomInsert geometryEquivalent diameter mmInsert nameTorusTorusTorusEllipsoidEllipsoidEllipsoidPearPearPearTubeTube101371journalpone0236466t002To17aTo17bTo26E20E30E38P38P39aP39bTu38aTu39bPLOS ONE 101371journalpone0236466 August PLOS ONE 0cComparison of different absorbed dose calculation methods in MRTdoses for 177Lu and 90Y labelled radiopeptides for liver spleen and kidneys were calculatedEach an was manually contoured and absorbed doses were calculated in compliance withthe MIRD scheme [] at anlevel from images The number of cycles the isotope and theactivity chosen for every injection were planned by an expert physician on the basis of thedosimetry results The activity prescription had to be determined based on the BiologicalEffective Dose BED delivered to kidneys Kidneys are regarded as the principal ans at riskin PRRT [ ] As suggested by different works [“] in this clinical trial the cumulativedose limit to kidneys was set to Gy of BED for patients with no risk factors hypertensiondiabetes renal failure are considered risk factors for this therapy and at 28Gy for patients withrisk factorsIn the present work absorbed doses to kidneys spleen and liver were calculated to comparethe three dosimetric methodsImage acquisition and reconstructionAll activity measurements were performed with an accurate activity calibrator for 177Lu Aktivimeter Isomed Nuklear Medizintechnik Germany and all image acquisitions were performed through a SPECTCT scanner Symbia T2 Siemens Medical Germany  NaITldetector previously calibrated [] The standard clinical protocol for body studies was usedboth for phantoms and patients with the following SPECT settings MEHR collimators matrix x zoom views x timeview s step and shoot mode degree of rotation ˚ noncircular orbit detector configuration ˚The first CT acquisition per patient was performed with the following parameters 130kVand max mAs using tube current modulation The subsequent CT images were acquiredwith kV and 40mAs for radiation protection safety of patients The CT reconstructed slicethickness was mm and a smooth reconstruction kernel was used B08s Siemens MedicalSolution Germany The higher image quality of the first CT scan is necessary for contouringvolumes of interest more accuratelyThe SPECT projections were reconstructed by an iterative algorithm including CT attenuation correction scatter correction and full collimatordetector response in Siemens ESoftworkstation Syngo MI Application version 32B Siemens Medical Solution Germany withFlash 3D iterative algorithm iterations subsets Gaussian filter cutoff mm mmcubic voxel []All cases of Sample A were rigidly registered to the first CT image of the sequence in Siemens Esoft workstation Images of patients included in Sample B were registered using adeformable multipass algorithm with the Velocity Advanced Imaging workstation Varian Medical Systems Palo Alto USA [] The registration procedures rescaled the originalvoxel size to 39x39x35 mm3The Volumes Of Interest VOI for each phantom and each patient were manually drawnon the reference CT image as recommended by Uribe [] using the VelocityworkstationSoftware for image processing and dosimetry calculationsOLINDA11 OLINDA version [] is an an level dosimetry software based on theMIRD methodology [] for internal dose estimation This is the method adopted in the clinical trial the clinical cases of this work are extracted from Absorbed doses to ans and tolesions can be calculated by using different models in the software human phantom modelsie mathematical representations of the human body to represent ans and whole body andsphere models ie mathematical representations of spheres to represent lesions [ ]PLOS ONE 101371journalpone0236466 August PLOS ONE 0cComparison of different absorbed dose calculation methods in MRTUnlike VoxelMed and RAYDOSE OLINDA needs timeintegrated activity A values ofVOIs as input parameters [] which were calculated with VoxelMed20 which will bedescribed in the next section and then inserted in OLINDAOLINDA sphere model commonly used to calculate doses to lesions was used to generatethe results for the inserts placed in the Geometrical phantom and for the dummy lesion housedin the anthropomorphic phantom while OLINDA an model adult male was used for thedummy ans placed in the anthropomorphic phantom Real insert volumes were used forcalculationsThe human models adult male or adult female were used to calculate dosimetry of thecohort of patients Doses were scaled using the true patient weight and the true an massesVoxelMed20 VoxelMed is a homemade software for dose calculation developed atAzienda USLIRCCS research hospital Reggio Emilia Italy It was developed in the MatlabThe Mathworks Natick MA programming environment and designed on the CERR platform wwwcerrinfo It performs voxellevel dosimetry based on the MIRD guidelines []The first version of the software along with the S value matrices for voxel dosimetry used incalculations were described in detail elsewhere []VoxelMed version includes a graphical user interface the possibility to export the resultsof calculations to Microsoft Excel file the visualization of the fitting curves both mono andbiexponential a module for renal BED calculation following the model suggested by StrigariL [] and the possibility to correct activity for partial volume effect PVE as presentedin [] Moreover VoxelMed20 provides the user with the timeintegrated activity A at VOIlevel which can be used for dosimetry with OLINDA version both for ans and lesionsTo calculate the number of disintegrations VoxelMed integrates the timeactivity curvewith the trapezoidal method in the time interval between the first and the last acquisitionBeyond this timeinterval the integration is performed analytically and the timeactivity curveis extrapolated using the effective halflife or the physical halflife it is chosen by the userThe effective halflife of the an or lesion is derived with a biexponential fit of the activitiesin the VOI the physical halflife is known from the selected isotope Timeintegrated activityis calculated in each voxel or in the whole an depending on the modality of dose calculationselected ie voxel level or an levelRAYDOSE RAYDOSE is a software package developed at Cardiff University School ofEngineering Cardiff University UK and designed to carry out 3D patientspecific imagebased dosimetry for RPT RAYDOSE provides personalized 3D dose map performing MonteCarlo simulations on radiation transport based on the Geant4 MC toolkit CERN Switzerland Geant4 is the stateoftheart package for the simulation of the transport of psthrough matter [] RAYDOSE generates voxellevel dose maps using anatomical and physiological data taken from morphologic and functional images []In order to obtain the area under the timeactivity curve RAYDOSE allows to use differentfitting modalities monoexponential decay linear uptake plus monoexponential decay or thetrapezoidal method In this study for the dose calculation of the clinical cases we used thetrapezoidal method at the voxel level up to the last time acquisition point while the timeactivity curve beyond the last scan time was extrapolated from the monoexponential curve fittingof the whole an activities in the VOI For dose calculation in phantoms we used the physical halflife of the isotope to extrapolate the activity from the scan time upwardsData and statistical analysisTwo groups of patients were considered for the purpose of this workPLOS ONE 101371journalpone0236466 August PLOS ONE 0cComparison of different absorbed dose calculation methods in MRTTable Demographic and baselines clinical characteristics of all patients�CharacteristicSample A N Sample B N Gender NoMaleFemaleAge yHeight cmWeight kgPrimary tumour site NoIleumPancreasLungThyroidRectumOthers177Lu activity for dosimetry MBq   ± ± ± ± ± ± ± N\\AN\\AN\\A ± � Plusminus values are means ± standard deviation  Injected activity at the first cycle of therapy Dosimetry was performed after the first injection101371journalpone0236466t003A first subgroup of cases named as œSample A was extracted by random samplingfrom the original clinical trial to adequately represent the whole population The number ofcases was calculated safely adopting a margin of error of and a standard deviation of A second independent subgroup of patients named as Sample B was extracted toofrom the original clinical trial similarly to Sample A A sample of cases was considered adequate in relation to the aim of the experiment conducted on Sample BPatient baseline characteristics for Sample A and Sample B are reported in Table The study type the image registration and the software used for the dose calculations of theclinical cases in sample A and sample B are summarised in Table Absorbed doses were calculated separately with OLINDA11 VoxelMed20 and RAYDOSEusing the same set of imagesKidney liver and spleen absorbed doses were calculated for each patient Two differentcomparison studies were performed The first study involved only comparison between VoxelMed and OLINDA based on absorbed dose calculations of patients in Sample A The secondstudy involved comparison between all the three software VoxelMed OLINDA and RAYDOSE based on absorbed dose calculations of patients in Sample B Furthermore in order toreduce the contribution of the fitting of the activitytime curves in the comparison of the software the VoxelMed dosimetry calculations for Sample B were repeated using the same effective halflife applied in RAYDOSE RAYDOSE estimates the effective halflife by fitting theTable Summary of phantom and patient studies performedStudy typePhantomObject of studyImage registrationSoftwareHomogeneous phantomNo registration only scanOLINDA11”VoxelMed”RAYDOSEGeometrical phantomNo registration only scanOLINDA11”VoxelMed”RAYDOSEAnthropomorphic phantomNo registration only scanOLINDA11”VoxelMed”RAYDOSEClinicalSample A patientsRigid registrationOLINDA11”VoxelMedSample B patientsDeformable registrationOLINDA11”VoxelMed VoxelMedλ RD”RAYDOSE101371journalpone0236466t004PLOS ONE 101371journalpone0236466 August PLOS ONE 0cComparison of different absorbed dose calculation methods in MRTFig Clinical comparison study workflow Procedure flow of absorbed dose calculation for each patient of sample A top of the image and sample B lowerpart of the image101371journalpone0236466g002an activities against time as previously described Flow chart in Fig illustrates methodology in clinical studyDosevolume histograms DVH were evaluated to compare spatial dose distribution atvoxellevelMean values of absorbed dose were used to compare an level and voxel level techniquesComparison between the different dosimetry methods was statistically evaluated using theLin™s concordance correlation coefficient CCC and the BlandAltman plot [] The CCCsymbolized by ρc allows to evaluate the degree of concordance between two measures whilethe BlandAltman plot is used to analyse the agreement between two quantities The CCC wascalculated using SAS SAS Institute Cary NC USA A value of ρc equal to denotes perfect concordance a value equal to perfect discordance while a value of no correlationResultsPhysical phantom studyThe values of mean absorbed dose for the physical phantoms calculated with OLINDA11VoxelMed and RAYDOSE are reported in Table Visual representation of the same data isprovided in Fig Similar DVH curves were generated with VoxelMed and RAYDOSE both for the Cylindrical phantom Fig and for the other two phantoms Fig Fig shows DHVs only for theinserts in the Geometrical and the Anthropomorphic phantoms with the smallest and the largest relative differences of mean absorbed dose respectivelyClinical studyAbsorbed dose for kidneys liver and spleen of the sample A of patients calculated withOLINDA11 and VoxelMed are shown in Table The absorbed doses to liver and to spleenwere found to be highly correlated while lower correlation was found for kidneys The CCCPLOS ONE 101371journalpone0236466 August PLOS ONE 0cComparison of different absorbed dose calculation methods in MRTTable Mean absorbed dose Gy calculated with OLINDA11 VoxelMed and RAYDOSE for all of the three phantoms The absorbed dose calculated withOLINDA11 was performed using either the an model and the Sphere model Absorbed doses to Pancreas Kidneys Spleen and Liver were calculated using the anmodel otherwise the Sphere model was usedPhantomCylindricalGeometricalAnthropomorphicInsert nameOLINDA11VoxelMedRAYDOSENATo17aTo17bTo26E20E30E38P38P39aP39bTu38aTu39bLesionPancreasKidneysSpleenLiver101371journalpone0236466t005[ confidence interval] values were ρc liver [ ] ρc spleen [ ]ρc kidneys [ ] The BlandAltman plot is shown in Fig OLINDA11 VoxelMed VoxelMedλ RD and RAYDOSE calculated mean absorbed dosefor patients of Sample B are shown in Table while the BlandAltman plot is shown in Fig The absorbed doses calculated with VoxelMed and RAYDOSE were highly correlated withρc kidneys [ ] ρc liver [ ] and ρc spleen [ ] andalmost complete agreement were found between VoxelMedλ RD and RAYDOSE withρc kidneys [ ] ρc liver [ ] and ρc spleen [ ]DiscussionIn this study we compared the performances of three tools for dosimetry calculationsOLINDA11 VoxelMed20 and RAYDOSE with the primary aim to evaluate the influence ofthe calculation modality on absorbed dose assessment anlevel based voxellevel dose kernel convolution based and Monte Carlo simulations based respectively The secondary aimwas to give some recommendations about the choice of the adequate technique for dosimetrycalculation to be implemented in a hospital in a research centre or in an academic instituteclinical or research purpose small or large number of patients clinical trials only or standardprocedures This analysis was performed in standard conditions by acquisition and processing of radioactive phantoms provided with inserts of specific volume and geometry and inclinical conditions over a large cohort of patients a selection of clinical cases taken from a clinical trial in which dosimetry had already been calculated Clinical conditions are indeed quitedistant from and more complicated than the standard conditions achievable in a phantom forseveral reasons biological kinetics in place of only physical decay of activity serial acquisitionsof functional images and associated issues related to image registration [] motion of thepatient that creates artefacts in images irregular shape of volumes of interest inhomogeneousactivity distributionPLOS ONE 101371journalpone0236466 August PLOS ONE 0cComparison of different absorbed dose calculation methods in MRTFig Comparison of mean absorbed dose Gy calculated using OLINDA11 VoxelMed and RAYDOSE a Homogeneous phantom b Anthropomorphicphantom and cf Geometrical phantom Note the Lesion insert in the Anthropomorphic phantom is missing because beyond the range of dose visualized in thegraph101371journalpone0236466g003Therefore to consider a large sample of clinical cases was of great interest since many studies about methods for dosimetry calculation are based on smaller groups of patients [“]and in a small group the inter patient variability cannot be properly investigatedThe quantitative intercomparison between all the three software was performed betweenthe mean values of absorbed doses In fact OLINDA provides only mean values while RAYDOSE and VoxelMed20 voxelbased tools provide the dose distribution that can be represented with DVHs from which the mean dose values can be derived To compare thetechniques of calculation the relative differences and the correlation between data pairs wereevaluatedFor standard conditions we evaluated discrepancies of calculated absorbed dose in a cylindrical phantom and in differently shaped inserts filled with a homogeneous radioactive solution Table shows the values of absorbed dose obtained with OLINDA11 VoxelMed andRAYDOSE in each of the phantoms These values are also plotted in Fig Lower values ofabsorbed dose were generally calculated using OLINDA in comparison with the dose calculated with other voxel modalities In the case of the cylindrical phantom a good agreementwas obtained between VoxelMed20 and RAYDOSE discrepancy equal to while largerdifference was observed between VoxelMed20 and OLINDA Absorbed dose map provided by VoxelMed and RAYDOSE showed similar spatial distribution close values of standard deviation across voxels around and analogues slope in DVHs Fig PLOS ONE 101371journalpone0236466 August PLOS ONE 0cComparison of different absorbed dose calculation methods in MRTFig Comparison of DVHs calculated using VoxelMed continuous line and RAYDOSE dotted line for the Cylindrical phantom101371journalpone0236466g004To compare the calculation techniques in different conditions of volume and geometry theGeometrical phantoms were acquired Relative differences in absorbed dose depend on theshape and on the volume of the inserts smaller is the volume and further from a regular sphereis the shape more the relative difference is higher In the Geometrical phantom the toroidalinserts provided the greatest discordance relative difference with VoxelMed dose rangingfrom to for OLINDA and from to for RAYDOSE while in the otherinserts differences ranged between [ ] for OLINDA11 and [ ] for RAYDOSEOn one hand the insert dose calculations in OLINDA were performed using the spheremodel since OLINDA only allows to perform dosimetry calculation for specified models ieans or spheres This approximation might explain the huge discrepancies obtained withthe voxelbased methods On the other hand a reason for the difference between RAYDOSEand VoxelMed is that the latter applies a mask before the convolution while RAYDOSE doesnot This contribution affects calculations in so far as the geometry and the volume of theinsert may influence the activity distribution and leave empty spaces around or inside theobjects This effect is especially pronounced for example in the case of the toroid The application of a mask also implies the lack of photon cross irradiation contribution between insertswhich has an impact on dose calculation contribution around [] Discrepancies ofPLOS ONE 101371journalpone0236466 August PLOS ONE 0cComparison of different absorbed dose calc Answer:

Thyroid_Cancer

The Adler grade by Doppler ultrasound isassociated with clinical pathology of cervicalcancer Implication for clinical managementDehong Che1 Zhirong Yang2 Hong Wei3 Xuedong Wang4 Jiayin GaoID1 Department of Obstetrics and Gynecology The Second Affiliated Hospital of Harbin Medical UniversityHarbin China Department of Ultrasound Harbin Red Cross Center Hospital Harbin China Departmentof Ultrasound The Second Affiliated Hospital of Harbin Medical University Harbin China Department ofObstetrics and Gynecology Longnan Hospital Daqing General Hospital Daqing Chinaa1111111111a1111111111a1111111111a1111111111a1111111111 dsp082901163comAbstract ACCESSCitation Che D Yang Z Wei H Wang X Gao J The Adler grade by Doppler ultrasound isassociated with clinical pathology of cervicalcancer Implication for clinical management PLoSONE e0236725 101371journalpone0236725Editor Linus Chuang University of Vermont LarnerCollege of Medicine UNITED STATESReceived February Accepted July Published August Copyright Che This is an access distributed under the terms of the CreativeCommons Attribution License which permitsunrestricted use distribution and reproduction inany medium provided the original author andsource are creditedData Availability Statement All relevant data arewithin the paper and its Supporting InformationfilesFunding The authors received no specific fundingfor this workCompeting interests The authors have declaredthat no competing interests existAbbreviations FIGO International Federation ofObstetrics and Gynecology TVCDFI transvaginalObjectiveTo analyze the relationship of Adler grade by transvaginal color Doppler flow imaging TVCDFI and the clinical pathological parameters of patients with cervical cancer and to identify the value of Adler grade in the diagnosis and treatment of cervical cancerMethodsPatients with cervical cancer diagnosed pathologically in our hospital from January to December were included All patients underwent TVCDFI examination and theimages were divided into to III grades according to the Adler grades and the correlationsbetween the Adler classification and clinical pathological parameters clinical stage masssize pathological type squamous cell carcinoma subtype CA125 CA199 were analyzedResultsA total of patients with cervical cancer were included With the increase of Adler severity the clinical stage of cervical cancer increased accordingly the cancer size differed significantly in patients with different Adler grade p There were significant differences inthe level of CA125 CA199 between the squamous cell carcinoma and adenocarcinoma allp005 the Adler grade was positively related with the clinical stage pathological type andsquamous cell carcinoma subtypes of cervical cancer all p005 no correlations werefound among the Adler grade and the cancer size CA125 CA199 all p005 The areaunder ROC curve of the cervical squamous cell carcinoma predicted by Adler grade basedon FIGO results and pathological results was 0811and respectively all p005ConclusionsAdler grades are closely associated with the clinical pathology of cervical cancer which maybe a convenient and effective approach for the assisting assessment of cervical cancerPLOS ONE 101371journalpone0236725 August PLOS ONE 0ccolor Doppler ultrasound WHO World HealthanizationUltrasound cervical cancerIntroductionCervical cancer is one of the most common malignant tumors in gynecology it has beenreported that the morbidity and mortality of cervical cancer ranks second among gynecological malignancies second only to breast cancer [ ] The number of new cases of cervical cancer has been increased every year around the world and the onset age of cervical cancer hasbecome much younger [] Cervical cancer is a malignant tumor which occurs at the junctionof squamous epithelial cells in the cervical vagina or the transitional zone and columnar epithelial cells in the endometrium of the cervical canal [] The etiology is not totally clear and itmay be related to sexual behavior frequency of deliveries and the infection of human papillomavirus [ ] The early detection and treatment of cervical cancer is very essential to theprognosis of patients with cervical cancerIn recent years with the popularization of many effective screening methods the overallmortality rate of cervical cancer patients has declined [] The clinical diagnosis mainlydepends on clinical manifestations and gynecological examinations which requires the combination of multiple auxiliary diagnostic methods including cervical cytology film cervicalmultipoint biopsy The International Federation of Obstetrics and Gynecology FIGO hasdeveloped a clinical staging standard for cervical cancer which referred to the colposcopypathological biopsy [] However when comparing the early diagnosed stage with the patient™spostoperative medical examination results the stage of cervical cancer has been much underestimated [“] More sophisticated radiological methods such as ultrasound computertomography and magnetic resonance imaging hasn™t been included in the stage of FIGO butthey have been the routine examination methods in clinical practice [] Many scholars [] have studied the characteristics of those examinations but so far there is no asto which is the best examination method for assessing cervical cancer staging It™s necessary toconduct more studies to identify the role of those tools for early diagnosis of cervical cancerPrevious studies [“] have reported that the transvaginal color Doppler ultrasoundTVCDFI can accurately reflect the size of the lesion the extent of infiltration and the bloodsupply and it is widely used in the detection of cancers in breast and thyroid but not in thecervical parts Besides it is well known that vascular patterns such as density of vessels is different for squamous and adenocarcinomas [] Therefore it™s necessary to show the difference to provide insights into clinical treatment Therefore in order to make clinicians moreclear about the blood supply of cervical cancer and facilitate communication between ultrasound doctors and clinicians we attempted to conduct a preliminary investigation on the correlation between the Adler grade and clinical pathology of cervical cancer to provide insightsinto the diagnosis and treatment of cervical cancer The study design was participantspatients with cervical cancer 2intervention TVCDFI detection and Adler grading 3comparison no applicable 4outcomes Adler secores and antigens CA125 and CA199MethodsEthical considerationsThe study was approved by the Medical Research Ethics Committee of our hospitalNo20180038 and written informed consents were obtained from all the patientsPatientsPatients with cervical cancer diagnosed pathologically in our hospital from January toDecember were selected as study objects in this present study The inclusion criteriawere as follows there were surgical pathology or biopsy detections verifying small cellPLOS ONE 101371journalpone0236725 August PLOS ONE 0cUltrasound cervical cancerneuroendocrine tumors by our experienced clinicans and the FIGO cervical cancer stagingwere used for severity differentiation [] no history of other malignant tumors or historyof radiotherapy and chemotherapy complete clinical data no history of cervical surgerysuch as we excluded patients undergone microwave freezing laser electrocautery surgeryThe exclusion criteria were as follows patients with congenital malformations of theuterus patients with coagulopathy patients with severe heart liver and kidney dysfunction patients with inability to cooperate with research or couldn™t followupTVCDFI detectionThe color doppler ultrasound system Philips E80 equipped with ultrasonographic probeDC58D were selected for ultrasound detection Before the detection all patients were askedto empty the rectum and bladder When performing the doppler ultrasound detection thepatients took the lithotomy positions Firstly we performed a transvaginal ultrasound examination of the uterus and bilateral accessory areas to determine the location number size shapeboundary echo of the cervical area and its relationship with surrounding tissues Secondly wechose the color Doppler blood flow imaging mode to observe the blood flow inside and aroundthe cervical area related parameters such the blood flow cancer size have been calculated andcollected The staging and TVCDFI done in the same setting and in different datesAdler grade classificationAdler grade classifications were conducted based on the detected blood flow [ ] We classified the blood flow signal of the lesion into four levels Adler refers to that no obvious bloodflow signal Adler I refers to or small blood vessels with a diameter of mm are detectedAdler II refers to that or small blood vessels are detected Adler III refers to that more than blood vessels or the blood vessels are intertwined into a network are detected The Adlergrade classifications were made by one radiologist and one gynecologist coherently and ifthere were any disparity further discussions were conducted for consensusThe detection of carbohydrate antigens CA125 and CA199Before any drug treatment ml venous blood without anticoagulation in the early morningwere collected from patients the blood specimens were left at room temperature for 15minthen centrifuged it and separated the serum then we stored it at environment with ˚C for further examination The levels of serum CA125 and CA199 were detected by professional stallwith electrochemical luminescence method The electrochemical luminometer and detectionkit used in the test were produced by Roche and the operation process was strictly performedaccording to the instructionsStatistical analysisSPSS statistical software were used for data analysis The measurement data for normal distribution was expressed as mean± standard deviation and the measurement data for skeweddistribution was expressed as median P25 P75 The tumor size CA125 CA199 were compared using the KruskalWallis H rank test of multiple sample comparisons of which Wilcoxon rank test was further used for pairwise comparisons And t tests were conducted in thecancer size between pathological types or squamous cell carcinoma subtypes Spearman correlation analysis was conducted for correlation analysis And receiver operating characteristicROC curves were drawn for evaluating the diagnosis value of Adler grade for cervical cancer[] p005 was considered as being statistically different in this present studyPLOS ONE 101371journalpone0236725 August PLOS ONE 0cUltrasound cervical cancerResultsThe ultrasound characteristics of cervical cancerAs Fig presented the ultrasound characteristics differed hugely among the ultrasoundimages with different Alder grades For Alder the cervical morphology was normal andno obvious blood flow signal were found in the images of color Doppler For Alder I thecervical morphology was slightly thickened and more enhanced intracervical echo couldbe collected one or two small spotlike blood flow signals could be detected For Alder IIuneven or thickened cervical echo distribution could be detected and strip blood flow signals at two to four locations could be seen For Alder III parauterine and extrauterineinvasion and blood metastasis could be found and reticular blood flow signals could bedetectedThe distribution of Adler classification and clinical stage of cervical cancerWe identified potential candidates firstly and patients were excluded As Table presented a total of patients were included for data analysis With the increase of Adler severity classification the clinical stage of cervical cancer increased accordinglyFig The Doppler ultrasound images on cervical cancer with different Adler grade a Adler b Adler I c Adler II d Adler III101371journalpone0236725g001PLOS ONE 101371journalpone0236725 August PLOS ONE 0cUltrasound cervical cancerTable The distribution of Adler classification and clinical stage of cervical cancerAdler gradeFIGO gradeGrade IGrade IIGrade IIIIVAdler Adler IAdler IIAdler IIIIn total101371journalpone0236725t001In totalThe comparison on the cancer size CA125 CA199 with Adler grade andpathological parametersAs Table showed the cancer size differed significantly among the different Adler gradesp but there were no significantly differences in the level of CA125 CA199 amongthe different Adler grades all p005 There were significant differences in the level ofCA125 CA199 between the squamous cell carcinoma and adenocarcinoma all p005 nosignificant difference was found in the cancer size between the squamous cell carcinoma andadenocarcinoma p No significant differences were found in the cancer size CA125and CA199 between the keratinized and nokeratinized squamous cell carcinoma all p005Furthermore as Table showed the Adler grade was positively related with the FIGO stagingpathological type and squamous cell carcinoma subtypes of cervical cancer all p005 nocorrelations were found among the Adler grade and the cancer size CA125 CA199 allp005The diagnosis value of Adler grade for cervical cancerAs Fig showed based on FIGO results the area under the ROC curve of the cervical squamous cell carcinoma predicted by Adler grade was p005 and its sensitivityTable Relationship analysis on the cancer size CA125 CA199 with Adler grade and pathological parametersItemsAdler gradeAdler Adler IAdler IIAdler IIItzpPathological typeSquamous cell carcinomaAdenocarcinomatzpsquamous cell carcinoma subtypesKeratinizedNonkeratinizedtzpCasesCancer sizemmCA125[MP25 P75 UmL]CA199[MP25 P75 UmL]±±± ± ± ± ± 101371journalpone0236725t002PLOS ONE 101371journalpone0236725 August PLOS ONE 0cUltrasound cervical cancerTable Correlation analysis between Adler grade and clinicopathological parametersClinicopathological parametersFIGO gradeCancer sizeCA125CA199Pathological typesquamous cell carcinoma subtypes101371journalpone0236725t003rpspecificity positive predictive value negative predictive value and Youden index were respectively And based on pathological results the area underthe ROC curve of the cervical squamous cell carcinoma subtype predicted by Adler grade was p005 and its sensitivity specificity positive predictive value negative predictivevalue and Youden index were respectively Thoseresults indicated that Adler grade could provide valuable reference for the diagnosis of cervicalcancerDiscussionsTVCDFI has the advantages of being noninvasive cheap with convenient operation [ ]It can not only clearly display the structure of each layer of the uterus accurately locate andqualitatively diagnose the lesion but also can observe the blood flow of detected area [ ]Dynamic ultrasound is currently one of the most widely used and mature imaging methods inthe diagnosis and treatment of obstetrics and gynecology diseases [] In this present studythe changes of cervical morphology and echo in Adler grade to I were small and Dopplerblood flow signals were less displayed but the blood flow signals in Adler grade II and IIIincreased significantly and the flakelike low echoes were seen inside With the increase ofAdler classification cervical masses continue to increase and color Doppler blood flow signalsare also displayed The results of this present study have indicated that Adler grade is expectedto be useful to reflect the richness of blood flow in cervical cancer which can provide important insights into the diagnosis and treatment of cervical cancerFig The ROC curve on the Adler grade for cervical cancer101371journalpone0236725g002PLOS ONE 101371journalpone0236725 August PLOS ONE 0cUltrasound cervical cancerThe occurrence proliferation invasion and metastasis of malignant tumors may largelydepend on tumor neovascularization [ ] Cervical cancer has characteristics of rapid proliferation and active cell division and growth which is closely related to the proliferation oftumor vessels in it [] On color Doppler ultrasound images there are often abundant different types of blood flow signals in tumor tissues which correspond to its rich vascular networkand are related to the special structure and blood flow characteristics of tumor blood vessels[ ] The Alder grades are mainly concentrated on the blood flow signals which to someextent can reflect the proliferation of vessels and the characteristics of microvessel densityaround the cervical cancer Previous studies [“] have reported that the blood signals maypredict the ability of tumor invasion and metastasis to a certain extent which is consistentwith the results of our studySquamous carcinoma is more common in the pathological and histological types of cervicalcancer [] We have found that there was correlation between Adler grade and the histopathological type The World Health anization WHO classifies cervical squamous cell carcinoma into two subtypes keratinized and nonkeratinized according to the presence ofkeratinized morphological features in the tissue [] Currently the studies on the relationshipof keratinized cervical squamous cell carcinoma and patient prognosis has been controversialIt™s been reported [“] that compared with nonkeratinized cervical squamous cell carcinoma the survival rate of keratinized cervical squamous cell carcinoma is significantlyreduced and it is not sensitive to radiotherapy and the prognosis is very poor for untreatedpatients or advanced patients Therefore differentiate the various subtypes of squamous cellcarcinoma of cervix is very helpful in clinical managementIn this study Spearman rank correlation analysis showed that Adler grade was positivelycorrelated with squamous cell carcinoma subtypes Therefore this technique is expected to bea reliable indicator for predicting the prognosis of patients with cervical cancerWith the continuous progress of cancer marker research the role of cancer related markerin the diagnosis and treatment of cancers has become increasingly important [ ] Severalstudies have shown that cervical cancer is also closely related to the marker CA125 and CA199It has been reported [“] that the carbohydrate antigens CA125 and CA199 are expressedat high levels in a variety of malignancies In this study the levels of CA125 and CA199 inpatients with adenocarcinoma were significantly higher than those in patients with squamouscell carcinoma The results were consistent with the findings of previous studies [ ] Eventhrough the Spearman correlation analysis shows that there is no correlation between Adlergrade and CA125 and CA199 it may be explained that the samples in this present study is notlarge enough to powerfully detect the difference future studies with larger population samplesare warranted to identify the role of CA125 and CA199 in cervical cancerSeveral limitations must be concerned in this present study Firstly the sample size of thisstudy is not large enough and the clinical staging is relatively rough biases may be existed inthis present study Secondly we did not use the cervical cancer specific markers such as squamous cell carcinoma tumor marker for reference it should be further elucidated in the futurestudies Thirdly it must bring to our attentions that the subjective factors in the Adler classification different operators and different parameter settings may bias the test results Howeverwe have performed related trainings on the TVCDFI before this study to avoid unnecessaryerrors in the process of ultrasound detection and analysisIn the results of this study have favored the value of Adler grade in the diagnosisand treatment of cervical cancer With the continuous development of ultrasound technologyand popularization of clinical ultrasound applications Adler grade should be promoted in theapplication of color Doppler ultrasound for the diagnosis and treatment of cervical cancerPLOS ONE 101371journalpone0236725 August PLOS ONE 0cUltrasound cervical cancerSupporting informationS1 ChecklistDOCXS2 ChecklistDOCXAuthor ContributionsConceptualization Dehong Che Zhirong Yang Jiayin GaoData curation Dehong Che Xuedong Wang Jiayin GaoFormal analysis Dehong Che Zhirong Yang Hong Wei Jiayin GaoInvestigation Dehong Che Zhirong Yang Hong Wei Xuedong Wang Jiayin GaoMethodology Xuedong WangResources Xuedong Wang Jiayin GaoSoftware Dehong CheValidation Dehong Che Jiayin GaoWriting “ original draft Dehong CheReferences Shrestha AD Neupane D Vedsted P Kallestrup P Cervical Cancer Prevalence Incidence and Mortality in Low and Middle Income Countries A Systematic Review Asian Pac J Cancer Prev “ Epub 1022034APJCP2018192319 PMID PubMed Central PMCID PMC5980914 Kalliala I Athanasiou A Veroniki AA Salanti G Efthimiou O Raftis N Incidence and mortalityfrom cervical cancer and other malignancies after treatment of cervical intraepithelial neoplasia a systematic review and metaanalysis of the literature Ann Oncol “ Epub 101016jannonc201911004 PMID Murfin J Irvine F MeechanRogers R Swift A Education income and occupation and their influenceon the uptake of cervical cancer prevention strategies A systematic review J 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disease a review of the literature Arch Gynecol Obstet “Epub 101007s0040401330916 PMID PLOS ONE 101371journalpone0236725 August PLOS ONE 0cUltrasound cervical cancerTariq M Zhang J Liang G Ding L He Q Yang B Macrophage Polarization AntiCancer Strategies toTarget TumorAssociated Macrophage in Breast Cancer J Cell Biochem “Epub 101002jcb25895 PMID Huang Z Zhao B Qin Z Li Y Wang T Zhou W Novel dual inhibitors targeting CDK4 andVEGFR2 synergistically suppressed cancer progression and angiogenesis Eur J Med Chem Epub 101016jejmech201907044 PMID Cohen PA Jhingran A Oaknin A Denny L Cervical cancer Lancet “ Epub 101016S014067361832470X PMID Ebina Y Mikami M Nagase S Tabata T Kaneuchi M Tashiro H Japan Society of GynecologicOncology guidelines for the treatment of uterine cervical cancer Int J Clin Oncol “ Epub 101007s101470181351y PMID The L Cervical cancer unequal progress Lancet Epub 101016S0140673619300030 PMID Ma G Zhang J Jiang H Zhang N Zhu Y Deng Y Microvessel density as a prognostic factor inesophageal squamous cell cancer patients A metaanalysis Medicine Baltimore e7600 Epub 101097MD0000000000007600 PMID PubMedCentral PMCID PMC5521944 Ruscito I Cacsire CastilloTong D Vergote I Ignat I Stanske M Vanderstichele A Characterisation of tumour microvessel density during progression of highgrade serous ovarian cancer clinicopathological impact an OCTIPS Consortium study Br J Cancer “ Epub 101038s414160180157z PMID PubMed Central PMCID PMC6070919 Hu X Liu H Ye M Zhu X Prognostic value of microvessel density in cervical cancer Cancer Cell Int Epub 101186s1293501806473 PMID PubMed Central PMCID PMC6169003 Yang Y He L Liu Y Xia S Fang A Xie Y Promising Nanocarriers for PEDF Gene TargetingDelivery to Cervical Cancer Cells Mediated by the Overexpressing FRalpha Sci Rep Epub 101038srep32427 PMID PubMed Central PMCIDPMC5006243Lekskul N Charakorn C Lertkhachonsuk AA Rattanasiri S Israngura Na Ayudhya N The Level ofSquamous Cell Carcinoma Antigen and Lymph Node Metastasis in Locally Advanced Cervical CancerAsian Pac J Cancer Prev “ Epub 107314apjcp PMID Xie X Song K Cui B Jiang J Yang X Kong B A comparison of the prognosis between adenocarcinoma and squamous cell carcinoma in stage IBIIA cervical cancer Int J Clin Oncol “ Epub 101007s1014701712258 PMID Grjibovski AM Dubovichenko D Saduakassova S Zhatkanbayeva G Omarova G Shalgumbayeva G Incidence mortality and determinants of survival from cervical cancer in Northwest Russia a registrybased cohort study Int Health “ Epub 101093inthealthihx068 PMID Charakorn C Thadanipon K Chaijindaratana S Rattanasiri S Numthavaj P Thakkinstian A The association between serum squamous cell carcinoma antigen and recurrence and survival of patients withcervical squamous cell carcinoma A systematic review and metaanalysis Gynecol Oncol “ Epub 101016jygyno201803056 PMID Kori M Yalcin Arga K Potential biomarkers and therapeutic targets in cervical cancer Insights from themetaanalysis of transcriptomics data within network biomedicine perspective PLoS One e0200717 Epub 101371journalpone0200717 PMID PubMedCentral PMCID PMC6051662 Wang W Xu X Tian B Wang Y Du L Sun T The diagnostic value of serum tumor markers CEACA199 CA125 CA153 and TPS in metastatic breast cancer Clin Chim Acta “ Epub 101016jcca201704023 PMID Laengsri V Kerdpin U Plabplueng C Treeratanapiboon L Nuchnoi P Cervical Cancer Markers Epigenetics and microRNAs Lab Med “ Epub 101093labmedlmx080 PMID Zhou X Wang H Wang X Preoperative CA125 and fibrinogen in patients with endometrial cancer arisk model for predicting lymphovascular space invasion J Gynecol Oncol 282e11 Epub 103802jgo201728e11 PMID PubMed Central PMCIDPMC5323282 Yu J Zheng Q Ding X Zheng B Chen X Chen B Systematic reanalysis strategy of serum indices identifies alkaline phosphatase as a potential predictive factor for cervical cancer Oncol Lett PLOS ONE 101371journalpone0236725 August PLOS ONE 0cUltrasound cervical cancer“ Epub 103892ol201910527 PMID PubMedCentral PMCID PMC6676666Fujii S Konishi I Nanbu Y Nonogaki H Kobayashi F Sagawa N Analysis of the levels of CA125carcinoembryonic antigen and CA199 in the cervical mucus for a detection of cervical adenocarcinoma Cancer “ Epub 1010021097014219880801623541aidcncr282062031730co24 PMID 24555900co2“ Mitsuhashi A Matsui H Usui H Nagai Y Tate S Unno Y Serum YKL40 as a marker for cervicaladenocarcinoma Ann Oncol “ Epub 101093annoncmdn552 PMID PLOS ONE 101371journalpone0236725 August PLOS ONE 0c'

cancer264

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: The Adler grade by Doppler ultrasound isassociated with clinical pathology of cervicalcancer Implication for clinical managementDehong Che1 Zhirong Yang2 Hong Wei3 Xuedong Wang4 Jiayin GaoID1 Department of Obstetrics and Gynecology The Second Affiliated Hospital of Harbin Medical UniversityHarbin China Department of Ultrasound Harbin Red Cross Center Hospital Harbin China Departmentof Ultrasound The Second Affiliated Hospital of Harbin Medical University Harbin China Department ofObstetrics and Gynecology Longnan Hospital Daqing General Hospital Daqing Chinaa1111111111a1111111111a1111111111a1111111111a1111111111 dsp082901163comAbstract ACCESSCitation Che D Yang Z Wei H Wang X Gao J The Adler grade by Doppler ultrasound isassociated with clinical pathology of cervicalcancer Implication for clinical management PLoSONE e0236725 101371journalpone0236725Editor Linus Chuang University of Vermont LarnerCollege of Medicine UNITED STATESReceived February Accepted July Published August Copyright Che This is an access distributed under the terms of the CreativeCommons Attribution License which permitsunrestricted use distribution and reproduction inany medium provided the original author andsource are creditedData Availability Statement All relevant data arewithin the paper and its Supporting InformationfilesFunding The authors received no specific fundingfor this workCompeting interests The authors have declaredthat no competing interests existAbbreviations FIGO International Federation ofObstetrics and Gynecology TVCDFI transvaginalObjectiveTo analyze the relationship of Adler grade by transvaginal color Doppler flow imaging TVCDFI and the clinical pathological parameters of patients with cervical cancer and to identify the value of Adler grade in the diagnosis and treatment of cervical cancerMethodsPatients with cervical cancer diagnosed pathologically in our hospital from January to December were included All patients underwent TVCDFI examination and theimages were divided into to III grades according to the Adler grades and the correlationsbetween the Adler classification and clinical pathological parameters clinical stage masssize pathological type squamous cell carcinoma subtype CA125 CA199 were analyzedResultsA total of patients with cervical cancer were included With the increase of Adler severity the clinical stage of cervical cancer increased accordingly the cancer size differed significantly in patients with different Adler grade p There were significant differences inthe level of CA125 CA199 between the squamous cell carcinoma and adenocarcinoma allp005 the Adler grade was positively related with the clinical stage pathological type andsquamous cell carcinoma subtypes of cervical cancer all p005 no correlations werefound among the Adler grade and the cancer size CA125 CA199 all p005 The areaunder ROC curve of the cervical squamous cell carcinoma predicted by Adler grade basedon FIGO results and pathological results was 0811and respectively all p005ConclusionsAdler grades are closely associated with the clinical pathology of cervical cancer which maybe a convenient and effective approach for the assisting assessment of cervical cancerPLOS ONE 101371journalpone0236725 August PLOS ONE 0ccolor Doppler ultrasound WHO World HealthanizationUltrasound cervical cancerIntroductionCervical cancer is one of the most common malignant tumors in gynecology it has beenreported that the morbidity and mortality of cervical cancer ranks second among gynecological malignancies second only to breast cancer [ ] The number of new cases of cervical cancer has been increased every year around the world and the onset age of cervical cancer hasbecome much younger [] Cervical cancer is a malignant tumor which occurs at the junctionof squamous epithelial cells in the cervical vagina or the transitional zone and columnar epithelial cells in the endometrium of the cervical canal [] The etiology is not totally clear and itmay be related to sexual behavior frequency of deliveries and the infection of human papillomavirus [ ] The early detection and treatment of cervical cancer is very essential to theprognosis of patients with cervical cancerIn recent years with the popularization of many effective screening methods the overallmortality rate of cervical cancer patients has declined [] The clinical diagnosis mainlydepends on clinical manifestations and gynecological examinations which requires the combination of multiple auxiliary diagnostic methods including cervical cytology film cervicalmultipoint biopsy The International Federation of Obstetrics and Gynecology FIGO hasdeveloped a clinical staging standard for cervical cancer which referred to the colposcopypathological biopsy [] However when comparing the early diagnosed stage with the patient™spostoperative medical examination results the stage of cervical cancer has been much underestimated [“] More sophisticated radiological methods such as ultrasound computertomography and magnetic resonance imaging hasn™t been included in the stage of FIGO butthey have been the routine examination methods in clinical practice [] Many scholars [] have studied the characteristics of those examinations but so far there is no asto which is the best examination method for assessing cervical cancer staging It™s necessary toconduct more studies to identify the role of those tools for early diagnosis of cervical cancerPrevious studies [“] have reported that the transvaginal color Doppler ultrasoundTVCDFI can accurately reflect the size of the lesion the extent of infiltration and the bloodsupply and it is widely used in the detection of cancers in breast and thyroid but not in thecervical parts Besides it is well known that vascular patterns such as density of vessels is different for squamous and adenocarcinomas [] Therefore it™s necessary to show the difference to provide insights into clinical treatment Therefore in order to make clinicians moreclear about the blood supply of cervical cancer and facilitate communication between ultrasound doctors and clinicians we attempted to conduct a preliminary investigation on the correlation between the Adler grade and clinical pathology of cervical cancer to provide insightsinto the diagnosis and treatment of cervical cancer The study design was participantspatients with cervical cancer 2intervention TVCDFI detection and Adler grading 3comparison no applicable 4outcomes Adler secores and antigens CA125 and CA199MethodsEthical considerationsThe study was approved by the Medical Research Ethics Committee of our hospitalNo20180038 and written informed consents were obtained from all the patientsPatientsPatients with cervical cancer diagnosed pathologically in our hospital from January toDecember were selected as study objects in this present study The inclusion criteriawere as follows there were surgical pathology or biopsy detections verifying small cellPLOS ONE 101371journalpone0236725 August PLOS ONE 0cUltrasound cervical cancerneuroendocrine tumors by our experienced clinicans and the FIGO cervical cancer stagingwere used for severity differentiation [] no history of other malignant tumors or historyof radiotherapy and chemotherapy complete clinical data no history of cervical surgerysuch as we excluded patients undergone microwave freezing laser electrocautery surgeryThe exclusion criteria were as follows patients with congenital malformations of theuterus patients with coagulopathy patients with severe heart liver and kidney dysfunction patients with inability to cooperate with research or couldn™t followupTVCDFI detectionThe color doppler ultrasound system Philips E80 equipped with ultrasonographic probeDC58D were selected for ultrasound detection Before the detection all patients were askedto empty the rectum and bladder When performing the doppler ultrasound detection thepatients took the lithotomy positions Firstly we performed a transvaginal ultrasound examination of the uterus and bilateral accessory areas to determine the location number size shapeboundary echo of the cervical area and its relationship with surrounding tissues Secondly wechose the color Doppler blood flow imaging mode to observe the blood flow inside and aroundthe cervical area related parameters such the blood flow cancer size have been calculated andcollected The staging and TVCDFI done in the same setting and in different datesAdler grade classificationAdler grade classifications were conducted based on the detected blood flow [ ] We classified the blood flow signal of the lesion into four levels Adler refers to that no obvious bloodflow signal Adler I refers to or small blood vessels with a diameter of mm are detectedAdler II refers to that or small blood vessels are detected Adler III refers to that more than blood vessels or the blood vessels are intertwined into a network are detected The Adlergrade classifications were made by one radiologist and one gynecologist coherently and ifthere were any disparity further discussions were conducted for consensusThe detection of carbohydrate antigens CA125 and CA199Before any drug treatment ml venous blood without anticoagulation in the early morningwere collected from patients the blood specimens were left at room temperature for 15minthen centrifuged it and separated the serum then we stored it at environment with ˚C for further examination The levels of serum CA125 and CA199 were detected by professional stallwith electrochemical luminescence method The electrochemical luminometer and detectionkit used in the test were produced by Roche and the operation process was strictly performedaccording to the instructionsStatistical analysisSPSS statistical software were used for data analysis The measurement data for normal distribution was expressed as mean± standard deviation and the measurement data for skeweddistribution was expressed as median P25 P75 The tumor size CA125 CA199 were compared using the KruskalWallis H rank test of multiple sample comparisons of which Wilcoxon rank test was further used for pairwise comparisons And t tests were conducted in thecancer size between pathological types or squamous cell carcinoma subtypes Spearman correlation analysis was conducted for correlation analysis And receiver operating characteristicROC curves were drawn for evaluating the diagnosis value of Adler grade for cervical cancer[] p005 was considered as being statistically different in this present studyPLOS ONE 101371journalpone0236725 August PLOS ONE 0cUltrasound cervical cancerResultsThe ultrasound characteristics of cervical cancerAs Fig presented the ultrasound characteristics differed hugely among the ultrasoundimages with different Alder grades For Alder the cervical morphology was normal andno obvious blood flow signal were found in the images of color Doppler For Alder I thecervical morphology was slightly thickened and more enhanced intracervical echo couldbe collected one or two small spotlike blood flow signals could be detected For Alder IIuneven or thickened cervical echo distribution could be detected and strip blood flow signals at two to four locations could be seen For Alder III parauterine and extrauterineinvasion and blood metastasis could be found and reticular blood flow signals could bedetectedThe distribution of Adler classification and clinical stage of cervical cancerWe identified potential candidates firstly and patients were excluded As Table presented a total of patients were included for data analysis With the increase of Adler severity classification the clinical stage of cervical cancer increased accordinglyFig The Doppler ultrasound images on cervical cancer with different Adler grade a Adler b Adler I c Adler II d Adler III101371journalpone0236725g001PLOS ONE 101371journalpone0236725 August PLOS ONE 0cUltrasound cervical cancerTable The distribution of Adler classification and clinical stage of cervical cancerAdler gradeFIGO gradeGrade IGrade IIGrade IIIIVAdler Adler IAdler IIAdler IIIIn total101371journalpone0236725t001In totalThe comparison on the cancer size CA125 CA199 with Adler grade andpathological parametersAs Table showed the cancer size differed significantly among the different Adler gradesp but there were no significantly differences in the level of CA125 CA199 amongthe different Adler grades all p005 There were significant differences in the level ofCA125 CA199 between the squamous cell carcinoma and adenocarcinoma all p005 nosignificant difference was found in the cancer size between the squamous cell carcinoma andadenocarcinoma p No significant differences were found in the cancer size CA125and CA199 between the keratinized and nokeratinized squamous cell carcinoma all p005Furthermore as Table showed the Adler grade was positively related with the FIGO stagingpathological type and squamous cell carcinoma subtypes of cervical cancer all p005 nocorrelations were found among the Adler grade and the cancer size CA125 CA199 allp005The diagnosis value of Adler grade for cervical cancerAs Fig showed based on FIGO results the area under the ROC curve of the cervical squamous cell carcinoma predicted by Adler grade was p005 and its sensitivityTable Relationship analysis on the cancer size CA125 CA199 with Adler grade and pathological parametersItemsAdler gradeAdler Adler IAdler IIAdler IIItzpPathological typeSquamous cell carcinomaAdenocarcinomatzpsquamous cell carcinoma subtypesKeratinizedNonkeratinizedtzpCasesCancer sizemmCA125[MP25 P75 UmL]CA199[MP25 P75 UmL]±±± ± ± ± ± 101371journalpone0236725t002PLOS ONE 101371journalpone0236725 August PLOS ONE 0cUltrasound cervical cancerTable Correlation analysis between Adler grade and clinicopathological parametersClinicopathological parametersFIGO gradeCancer sizeCA125CA199Pathological typesquamous cell carcinoma subtypes101371journalpone0236725t003rpspecificity positive predictive value negative predictive value and Youden index were respectively And based on pathological results the area underthe ROC curve of the cervical squamous cell carcinoma subtype predicted by Adler grade was p005 and its sensitivity specificity positive predictive value negative predictivevalue and Youden index were respectively Thoseresults indicated that Adler grade could provide valuable reference for the diagnosis of cervicalcancerDiscussionsTVCDFI has the advantages of being noninvasive cheap with convenient operation [ ]It can not only clearly display the structure of each layer of the uterus accurately locate andqualitatively diagnose the lesion but also can observe the blood flow of detected area [ ]Dynamic ultrasound is currently one of the most widely used and mature imaging methods inthe diagnosis and treatment of obstetrics and gynecology diseases [] In this present studythe changes of cervical morphology and echo in Adler grade to I were small and Dopplerblood flow signals were less displayed but the blood flow signals in Adler grade II and IIIincreased significantly and the flakelike low echoes were seen inside With the increase ofAdler classification cervical masses continue to increase and color Doppler blood flow signalsare also displayed The results of this present study have indicated that Adler grade is expectedto be useful to reflect the richness of blood flow in cervical cancer which can provide important insights into the diagnosis and treatment of cervical cancerFig The ROC curve on the Adler grade for cervical cancer101371journalpone0236725g002PLOS ONE 101371journalpone0236725 August PLOS ONE 0cUltrasound cervical cancerThe occurrence proliferation invasion and metastasis of malignant tumors may largelydepend on tumor neovascularization [ ] Cervical cancer has characteristics of rapid proliferation and active cell division and growth which is closely related to the proliferation oftumor vessels in it [] On color Doppler ultrasound images there are often abundant different types of blood flow signals in tumor tissues which correspond to its rich vascular networkand are related to the special structure and blood flow characteristics of tumor blood vessels[ ] The Alder grades are mainly concentrated on the blood flow signals which to someextent can reflect the proliferation of vessels and the characteristics of microvessel densityaround the cervical cancer Previous studies [“] have reported that the blood signals maypredict the ability of tumor invasion and metastasis to a certain extent which is consistentwith the results of our studySquamous carcinoma is more common in the pathological and histological types of cervicalcancer [] We have found that there was correlation between Adler grade and the histopathological type The World Health anization WHO classifies cervical squamous cell carcinoma into two subtypes keratinized and nonkeratinized according to the presence ofkeratinized morphological features in the tissue [] Currently the studies on the relationshipof keratinized cervical squamous cell carcinoma and patient prognosis has been controversialIt™s been reported [“] that compared with nonkeratinized cervical squamous cell carcinoma the survival rate of keratinized cervical squamous cell carcinoma is significantlyreduced and it is not sensitive to radiotherapy and the prognosis is very poor for untreatedpatients or advanced patients Therefore differentiate the various subtypes of squamous cellcarcinoma of cervix is very helpful in clinical managementIn this study Spearman rank correlation analysis showed that Adler grade was positivelycorrelated with squamous cell carcinoma subtypes Therefore this technique is expected to bea reliable indicator for predicting the prognosis of patients with cervical cancerWith the continuous progress of cancer marker research the role of cancer related markerin the diagnosis and treatment of cancers has become increasingly important [ ] Severalstudies have shown that cervical cancer is also closely related to the marker CA125 and CA199It has been reported [“] that the carbohydrate antigens CA125 and CA199 are expressedat high levels in a variety of malignancies In this study the levels of CA125 and CA199 inpatients with adenocarcinoma were significantly higher than those in patients with squamouscell carcinoma The results were consistent with the findings of previous studies [ ] Eventhrough the Spearman correlation analysis shows that there is no correlation between Adlergrade and CA125 and CA199 it may be explained that the samples in this present study is notlarge enough to powerfully detect the difference future studies with larger population samplesare warranted to identify the role of CA125 and CA199 in cervical cancerSeveral limitations must be concerned in this present study Firstly the sample size of thisstudy is not large enough and the clinical staging is relatively rough biases may be existed inthis present study Secondly we did not use the cervical cancer specific markers such as squamous cell carcinoma tumor marker for reference it should be further elucidated in the futurestudies Thirdly it must bring to our attentions that the subjective factors in the Adler classification different operators and different parameter settings may bias the test results Howeverwe have performed related trainings on the TVCDFI before this study to avoid unnecessaryerrors in the process of ultrasound detection and analysisIn the results of this study have favored the value of Adler grade in the diagnosisand treatment of cervical cancer With the continuous development of ultrasound technologyand popularization of clinical ultrasound applications Adler grade should be promoted in theapplication of color Doppler ultrasound for the diagnosis and treatment of cervical cancerPLOS ONE 101371journalpone0236725 August PLOS ONE 0cUltrasound cervical cancerSupporting informationS1 ChecklistDOCXS2 ChecklistDOCXAuthor ContributionsConceptualization Dehong Che Zhirong Yang Jiayin GaoData curation Dehong Che Xuedong Wang Jiayin GaoFormal analysis Dehong Che Zhirong Yang Hong Wei Jiayin GaoInvestigation Dehong Che Zhirong Yang Hong Wei Xuedong Wang Jiayin GaoMethodology Xuedong WangResources Xuedong Wang Jiayin GaoSoftware Dehong CheValidation Dehong Che Jiayin GaoWriting “ original draft Dehong CheReferences Shrestha AD Neupane D Vedsted P Kallestrup P Cervical Cancer Prevalence Incidence and Mortality in Low and Middle Income Countries A Systematic Review Asian Pac J Cancer Prev “ Epub 1022034APJCP2018192319 PMID PubMed Central PMCID PMC5980914 Kalliala I Athanasiou A Veroniki AA Salanti G Efthimiou O Raftis N Incidence and mortalityfrom cervical cancer and other malignancies after treatment of cervical intraepithelial neoplasia a systematic review and metaanalysis of the literature Ann Oncol “ Epub 101016jannonc201911004 PMID Murfin J Irvine F MeechanRogers R Swift A Education income and occupation and their influenceon the uptake of cervical cancer prevention 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of Gynecology and Obstetrics Cervical Cancer Staging GuidelinesObstet Gynecol “ Epub 101097AOG PMID Xiao C Zhou J Yang C Li J Pan Y Dai Y The Comparison of Vascular Characteristics of ThyroidMicrocarcinoma By Conventional Colour Doppler Ultrasonography and Superb Microvascular ImagingChinese Journal of Ultrasound in Medicine “PLOS ONE 101371journalpone0236725 August PLOS ONE 0cUltrasound cervical cancerInfante F Espada Vaquero M Bignardi T Lu C Testa AC Fauchon D Prediction of Tubal EctopicPregnancy Using Offline Analysis of 3Dimensional Transvaginal Ultrasonographic Data Sets An Interobserver and Diagnostic Accuracy Study J Ultrasound Med “ Epub 101002jum14489 PMID Haldorsen IS Lura N Blaakaer J Fischerova D Werner HMJ What Is the Role of Imaging at PrimaryDiagnostic WorkUp in Uterine Cervical Cancer Curr Oncol Rep Epub 101007s1191201908240 PMID PubMed Central PMCID PMC6663927 Ngan HYS Seckl MJ Berkowitz RS Xiang Y Golfier F Sekharan PK Update on the diagnosisand management of 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Epstein E Preoperative prediction of lymphnode metastasis and deep stromal invasion in women with invasive cervical cancer prospective multicenter study using 2D and 3D ultrasound Ultrasound Obstet Gynecol “ Epub 101002uog14643 PMID Wang S Yang W Fu JJ Sun Y Zhang H Bai J Microflow imaging of contrastenhanced ultrasound for evaluation of neovascularization in peripheral lung cancer Medicine Baltimore 32e4361 Epub 101097MD0000000000004361 PMID PubMed Central PMCID PMC4985302 Bhatla N Berek JS Cuello Fredes M Denny LA Grenman S Karunaratne K Revised FIGO staging for carcinoma of the cervix uteri Int J Gynaecol Obstet “ Epub 101002ijgo12749 PMID Adler DD Carson PL Rubin JM QuinnReid D Doppler ultrasound color flow imaging in the study ofbreast cancer preliminary findings Ultrasound Med Biol “ Epub 101016030156299090020d PMID Yang WT Tse GM Lam PK Metreweli C Chang J Correlation between color power Doppler sonographic measurement of breast tumor vasculature and immunohistochemical analysis of microvesseldensity for the quantitation of angiogenesis J Ultrasound Med “ Epub 107863jum200221111227 PMID Hickey GL Dunning J Seifert B Sodeck G Carr MJ Burger HU Statistical and data reportingguidelines for the European Journal of CardioThoracic Surgery and the Interactive CardioVascular andThoracic Surgery Eur J Cardiothorac Surg “ Epub 101093ejctsezv168 PMID Jurado M Galvan R MartinezMonge R Mazaira J Alcazar JL Neoangiogenesis in early cervical cancer correlation between color Doppler findings and risk factors A prospective observational studyWorld J Surg Oncol Epub 101186147778196126 PMID PubMed Central PMCID PMC2611993 Exacoustos C Zupi E Piccione E Ultrasound Imaging for Ovarian and Deep Infiltrating EndometriosisSemin Reprod Med “ Epub 101055s00361597127PMID Arya S Kupesic Plavsic S Preimplantation 3D ultrasound current uses and challenges J Perinat Med “ Epub 101515jpm20160361 PMID Romosan G Valentin L The sensitivity and specificity of transvaginal ultrasound with regard to acutepelvic inflammatory disease a review of the literature Arch Gynecol Obstet “Epub 101007s0040401330916 PMID PLOS ONE 101371journalpone0236725 August PLOS ONE 0cUltrasound cervical cancerTariq M Zhang J Liang G Ding L He Q Yang B Macrophage Polarization AntiCancer Strategies toTarget TumorAssociated Macrophage in Breast Cancer J Cell Biochem “Epub 101002jcb25895 PMID Huang Z Zhao B Qin Z Li Y Wang T Zhou W Novel dual inhibitors targeting CDK4 andVEGFR2 synergistically suppressed cancer progression and angiogenesis Eur J Med Chem Epub 101016jejmech201907044 PMID Cohen PA Jhingran A Oaknin A Denny L Cervical cancer Lancet “ Epub 101016S014067361832470X PMID Ebina Y Mikami M Nagase S Tabata T Kaneuchi M Tashiro H Japan Society of GynecologicOncology guidelines for the treatment of uterine cervical cancer Int J Clin Oncol “ Epub 101007s101470181351y PMID The L Cervical cancer unequal progress Lancet Epub 101016S0140673619300030 PMID Ma G Zhang J Jiang H Zhang N Zhu Y Deng Y Microvessel density as a prognostic factor inesophageal squamous cell cancer patients A metaanalysis Medicine Baltimore e7600 Epub 101097MD0000000000007600 PMID PubMedCentral PMCID PMC5521944 Ruscito I Cacsire CastilloTong D Vergote I Ignat I Stanske M Vanderstichele A Characterisation of tumour microvessel density during progression of highgrade serous ovarian cancer clinicopathological impact an OCTIPS Consortium study Br J Cancer “ Epub 101038s414160180157z PMID PubMed Central PMCID PMC6070919 Hu X Liu H Ye M Zhu X Prognostic value of microvessel density in cervical cancer Cancer Cell Int Epub 101186s1293501806473 PMID PubMed Central PMCID PMC6169003 Yang Y He L Liu Y Xia S Fang A Xie Y Promising Nanocarriers for PEDF Gene TargetingDelivery to Cervical Cancer Cells Mediated by the Overexpressing FRalpha Sci Rep Epub 101038srep32427 PMID PubMed Central PMCIDPMC5006243Lekskul N Charakorn C Lertkhachonsuk AA Rattanasiri S Israngura Na Ayudhya N The Level ofSquamous Cell Carcinoma Antigen and Lymph Node Metastasis in Locally Advanced Cervical CancerAsian Pac J Cancer Prev “ Epub 107314apjcp PMID Xie X Song K Cui B Jiang J Yang X Kong B A comparison of the prognosis between adenocarcinoma and squamous cell carcinoma in stage IBIIA cervical cancer Int J Clin Oncol “ Epub 101007s1014701712258 PMID Grjibovski AM Dubovichenko D Saduakassova S Zhatkanbayeva G Omarova G Shalgumbayeva G Incidence mortality and determinants of survival from cervical cancer in Northwest Russia a registrybased cohort study Int Health “ Epub 101093inthealthihx068 PMID Charakorn C Thadanipon K Chaijindaratana S Rattanasiri S Numthavaj P Thakkinstian A The association between serum squamous cell carcinoma antigen and recurrence and survival of patients withcervical squamous cell carcinoma A systematic review and metaanalysis Gynecol Oncol “ Epub 101016jygyno201803056 PMID Kori M Yalcin Arga K Potential biomarkers and therapeutic targets in cervical cancer Insights from themetaanalysis of transcriptomics data within network biomedicine perspective PLoS One e0200717 Epub 101371journalpone0200717 PMID PubMedCentral PMCID PMC6051662 Wang W Xu X Tian B Wang Y Du L Sun T The diagnostic value of serum tumor markers CEACA199 CA125 CA153 and TPS in metastatic breast cancer Clin Chim Acta “ Epub 101016jcca201704023 PMID Laengsri V Kerdpin U Plabplueng C Treeratanapiboon L Nuchnoi P Cervical Cancer Markers Epigenetics and microRNAs Lab Med “ Epub 101093labmedlmx080 PMID Zhou X Wang H Wang X Preoperative CA125 and fibrinogen in patients with endometrial cancer arisk model for predicting lymphovascular space invasion J Gynecol Oncol 282e11 Epub 103802jgo201728e11 PMID PubMed Central PMCIDPMC5323282 Yu J Zheng Q Ding X Zheng B Chen X Chen B Systematic reanalysis strategy of serum indices identifies alkaline phosphatase as a potential predictive factor for cervical cancer Oncol Lett PLOS ONE 101371journalpone0236725 August PLOS ONE 0cUltrasound cervical cancer“ Epub 103892ol201910527 PMID PubMedCentral PMCID PMC6676666Fujii S Konishi I Nanbu Y Nonogaki H Kobayashi F Sagawa N Analysis of the levels of CA125carcinoembryonic antigen and CA199 in the cervical mucus for a detection of cervical adenocarcinoma Cancer “ Epub 1010021097014219880801623541aidcncr282062031730co24 PMID 24555900co2“ Mitsuhashi A Matsui H Usui H Nagai Y Tate S Unno Y Serum YKL40 as a marker for cervicaladenocarcinoma Ann Oncol “ Epub 101093annoncmdn552 PMID PLOS ONE 101371journalpone0236725 August PLOS ONE 0c' Answer:

Thyroid_Cancer

Bone metastasis classification using wholebody images from prostate cancer patientsbased on convolutional neural networksapplicationNikolaos Papandrianos1 Elpiniki PapageiouID23 Athanasios Anagnostis34Konstantinos Papageiou4 General Department University of Thessaly Lamia Greece Faculty of Technology Dept of EnergySystems University of Thessaly Geopolis Campus Larisa Greece Institute for Bioeconomy and Agritechnology Center for Research and Technology Hellas Greece Department of Computer Science andTelecommunications University of Thessaly Lamia Greece npapandrianosuthgrAbstractBone metastasis is one of the most frequent diseases in prostate cancer scintigraphy imaging is particularly important for the clinical diagnosis of bone metastasis Up to date minimalresearch has been conducted regarding the application of machine learning with emphasison modern efficient convolutional neural networks CNNs algorithms for the diagnosis ofprostate cancer metastasis from bone scintigraphy images The advantageous and outstanding capabilities of deep learning machine learning™s groundbreaking technologicaladvancement have not yet been fully investigated regarding their application in computeraided diagnosis systems in the field of medical image analysis such as the problem of bonemetastasis classification in wholebody scans In particular CNNs are gaining great attention due to their ability to recognize complex visual patterns in the same way as human perception operates Considering all these new enhancements in the field of deep learning aset of simpler faster and more accurate CNN architectures designed for classification ofmetastatic prostate cancer in bones is explored This research study has a twofold goal tocreate and also demonstrate a set of simple but robust CNN models for automatic classification of wholebody scans in two categories malignant bone metastasis or healthy usingsolely the scans at the input level Through a meticulous exploration of CNN hyperparameter selection and finetuning the best architecture is selected with respect to classificationaccuracy Thus a CNN model with improved classification capabilities for bone metastasisdiagnosis is produced using bone scans from prostate cancer patients The achieved classification testing accuracy is whereas the average sensitivity is approximately Finally the bestperforming CNN method is compared to other popular and wellknown CNN architectures used for medical imaging like VGG16 ResNet50 GoogleNetand MobileNet The classification results show that the proposed CNNbased approach outperforms the popular CNN methods in nuclear medicine for metastatic prostate cancer diagnosis in bonesa1111111111a1111111111a1111111111a1111111111a1111111111 ACCESSCitation Papandrianos N Papageiou EAnagnostis A Papageiou K Bonemetastasis classification using whole body imagesfrom prostate cancer patients based onconvolutional neural networks application PLoSONE e0237213 101371journalpone0237213Editor Jeonghwan Gwak Korea NationalUniversity of Transportation REPUBLIC OF KOREAReceived November Accepted July Published August Copyright Papandrianos This is an access distributed under the terms ofthe Creative Commons Attribution License whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are creditedData Availability Statement The data which areanonymized bone scintigraphy images without anyfurther information are available only after requestfor research purposes Data availability Thedataset from Diagnostic Medical CenterœDiagnosticoIatriki AE was used under thelicense of the Board Committee Director of theDiagnostic Medical Center œDiagnosticoIatriki AE Dr Vasilios Parafestas for the current studyand is not publicly available The dataset may beavailable only under request from the Director ofPLOS ONE 101371journalpone0237213 August PLOS ONE 0cthe Diagnostic Center vparafestasyahoogr andthe relevant Doctor of Nuclear Medicine DrNikolaos Papandrianos npapandrianosuthgrnikpapandrgmailcomFunding The authors received no specificfunding for this workCompeting interests The authors have declaredthat no competing interests existBone metastasis classification using convolutional neural networks IntroductionMost common tumors such as those of the breast lung and prostate frequently metastasize tothe bone tissue and so the skeleton seems to be a site with the most significant tumor burdenin cancer patients with advanced disease Statistical analysis results have shown that of allbone metastases originate from the breast in women and from the prostate in men Theremaining emanates from thyroid lung and kidney cancers [] In the case of metastaticprostate cancer diagnosis has a significant impact on the quality of patient™s life [] In mostmen the metastatic prostate cancer mainly sites on the bones of the axial skeleton causingsevere lesions that can cause pain debility andor functional impairment [] As this type ofcancer has great avidity for bone and could cause painful and untreatable effects an early diagnosis is crucial for the patient Reviews on clinical evidences and diagnostic assessments ofbone metastases in men with prostate cancer can be found in []The implementation of a properly selected diagnostic imaging can reveal the number ofmetastatic foci in the skeletal system [ ] Rapid diagnosis of bone metastases can be achievedusing modern imaging techniques such as scintigraphy Positron Emission TomographyPET and wholebody Magnetic Resonance Imaging MRIThe primary imaging method in the diagnosis of metastases that offers the highest sensitivity among all imaging methods is Bone Scintigraphy BS [“] Through the depictionof the entire skeleton in one medical examination nuclear doctor is able to detect bone abnormalities in areas where intensive radionuclide activity is present However low specificityseems to be the main drawback of this method as it cannot tell whether the causes of boneturnovers are different than those of metastatic origin leukaemia healing fracture etc Atthe same time PET has been recognized as an efficient method for detecting cancer cellsbased on recent technological advancements in medical imaging PET and Computed Tomography CT combination can produce highresolution images [“]Although PET and PETCT are the most efficient screening techniques for bone metastasisBS remains the most common imaging procedure in nuclear medicine [ ] [] Asreported in European Association of Nuclear Medicine EANM guidelines [] BS is particularly important for clinical diagnosis of metastatic cancer both in men and women At presentwhen other imaging or examination methods are unable to provide a reliable diagnosis BSimaging becomes the proper modality for making a final diagnosis of bone metastasis []To address the considerable problem of bone metastasis diagnosis artificial intelligentmethods for medical image analysis implemented with deep learning algorithms have beenadequately investigated In this direction a recent survey reveals the entire penetration of deeplearning techniques into the field of medical image analysis detection segmentation classification retrieval image generation and enhancement registration and successful application ofdeep learning to medical imaging tasks are thoroughly examined [“]Implementation of deep learning in medical imaging is mainly conducted by ConvolutionalNeural Networks CNNs [ ] a relatively new and powerful way to learn useful representations of images and other structured data Before the application of CNNs these featurestypically had to be created by less powerful machine learning models or even handcraftedWith the introduction of CNNs such features could be learned directly from the provideddata since they include certain preferences in their structure that make them powerful deeplearning models for image analysis [ ] Typical CNNs have a similar structure withArtificial Neural Networks ANN and consist of one or more filters ie convolutional layers followed by aggregationpooling layers in order to extract features for classification tasks[] Gradient descent and backpropagation are both used as learning algorithms the sameway they are used in a standard ANN Their main difference lies in the fact that CNNs havePLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networkslayers of convolutions along with pooling layers in the beginning of their architecture Thefinal outputs are computed via fully connected layers located at the end of the network architecture []In recent years CNNs have gained wider recognition in medical image analysis domain aswell as in vision systems [“ ] Due to their enormous popularity several applications ofCNNs were investigated in the field of medical image analysis Two recent review studies []and [] gather all the important and most interesting applications of deep learningThe application of CNNs in medical imaging ranges from plain radiograph CT MRI andmicroscopy images to clinical photos dermatology capsule endoscopy and visual recognition[“] In addition a CNN was investigated in [] that regards automatic detection of tuberculosis on chest radiographs while in [] a brain tumor segmentation in magnetic resonanceimages was made possible with the use of a CNN More examples of CNNs™ successful application in medical domain include automated cardiac diagnosis [] detection of lesions and prediction of treatment response by PET [ ] as well as dynamic contrast agentenhancedcomputed tomography where CNN showed high diagnostic performance in the differentiation of liver masses [] Furthermore CNNs have shown outstanding performance in radiology and molecular imaging []Some models with major impact in the context of deep learning and medical image processing were introduced in several s the Unet model for biomedical data semanticsegmentation [] the GoogLeNet model introducing the inception module [] the ResNetmodel introducing the residual learning building block for extremely deep convolutional networks [] and also Deeplab which deals with the inclusion of many convolutional layersatrous for semantic segmentation of images in deep convolutional neural networks []In medical image analysis the most widely used CNN methods are the following i AlexNet [] This network has a quite deep architecture similar to GoogLeNet by YannLeCun [] incorporates more filters per layer and includes stacked convolutional layersIt attaches ReLU activations after every convolutional and fullyconnected layer ii ZFNet [] Being a rather slight modification of AlexNet this network won the ILSVRCcompetition iii VGGNet16 [ ] It consists of convolutional layers having avery uniform architecture similar to AlexNet iv GoogleNet [] It is a convolutional neuralnetwork with a standard stacked convolutional layer having one or more fully connected layers called inception modules able to extract various levels of features on the same time vResNet [] It is another efficient CNN architecture that introduced the œidentityshortcut connection to solve the notorious problem of the vanishing gradients of the deepnetworks vi DenseNet [] Being another important CNN architecture DenseNetoffers the main advantage of alleviating the gradient vanishment problem with the direct connection of all the layers Related work in nuclear medical imaging for metastatic prostate cancerdiagnosis in bonesReviewing the relevant literature for diagnosis of bone metastasis using bone scintigraphyscans the authors notice that only a couple of previous works have been adequately conductedfor metastatic prostate cancer classification using CNNs while the others are devoted to ANNsand their application in ComputerAided Diagnosis CAD These works have investigated theuse of Bone Scan Index BSI which was introduced to assess the bone scanning process andestimate the extent of bone metastasis [ ] Specifically it serves as a clinical quantitativeand reproducible parameter that can measure metastatic prostate cancer bone involvement[] The software developed for the BSIbased ANN approach was EXINI bone EXINIPLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networksDiagnostics AB Lund Sweden and afterwards a revised version of this software calledBONENAVI FUJIFILM Toyama Chemical Co Ltd Tokyo Japan was engineered using alarge number of Japanese multicenter training databases []The first of the reported studies was devoted to the development of a classification algorithm based on CNNs for bone scintigraphy image analysis [] It was carried out as a masterthesis in Lund University and is focused mainly on classification problems without considering any identification and segmentation tasks The used dataset was provided by Exini Diagnostics AB in the form of image patches of already found hotspots The process in whichhotspots were segmented cropped and collected from bone scans was implemented using asoftware developed at Exini BSI was calculated for wholebody bone scans by segmenting theentire skeleton from the background in both the anterior and posterior views Due to timeframe restrictions only the hotspots found in the spine have been used to train the CNN sincethey were considered to be the easiest to classify A shape model based on a mean shape of several normal wholebody scans was fitted to the skeleton using an image analysis algorithmcalled Morphon registration The outcomes of the aforementioned thesis [] have shown thatthe calculated accuracy of the validation set was whereas the calculated accuracy of thetesting set was The second study explored CNNs for classification of prostate cancer metastases usingbone scan images [] The tasks of this master project appeared to have a significant potentialon classifying bone scan images obtained by Exini Diagnostics AB too including BSI The twotasks were defined as i classifying anterior posterior pose and ii classifying metastatic nonmetastatic hotspots The outcome of this study is that the trained models produce highlyaccurate results in both tasks and they outperform other methods for all tested body regions inthe case of metastatic nonmetastatic hotspots classification The evaluation indicator of thearea under Receiver Operating Characteristic ROC score was equal to which is significantly higher than the respective ROC of obtained by methods reported in the literature for the same test setThe remaining research that concerns the same imaging modality BS is devoted to theintroduction of CAD systems with the use of ANN and other Machine Learning ML methods for bone metastasis detection in bone scintigraphy images Sadik were the first todevelop an automated CAD system as a clinical quality assurance tool for the interpretation ofbone scans [“] This bone scan CAD software was trained to interpret bone scans usingtraining databases that consist of bone scans from European patients who have the desiredimage interpretation metastatic disease or not The results showed a sensitivity of at aspecificity of These works result in certain outcomes that refer to the development of atotally automated computerassisted diagnosis system that can identify metastases after examining bone scans applying multilayer perceptron ANN techniques involving a small databaseof wholebody bone scans patients The highest sensitivity that was achieved from all thestudies and accomplished during this thesis was approximately []Horikoshi compared the diagnostic accuracy of two CAD systems one based on aEuropean and another on a Japanese training database in a group of bone scans from Japanesepatients [] The Japanese CAD software showed a higher specificity and accuracy comparedto the European Comparing the sensitivities the Japanese CAD software achieved whereas the European CAD software reached []In another study conducted by Tokuda the diagnostic capability of a completely automated CAD system which detects metastases in the images of bone scans by focusing on twodifferent patterns was investigated [] The first pattern was devoted to the detection ofmetastases per region the second one detects metastases per patient The investigated systemwas called œBONENAVI version  The produced results have shown that the new CADPLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networkssystem is able to decrease the number of false positive findings which depends on the primarylesion of cancerIn Aslantas proposed œCADBOSS as a fully automated diagnosis system forbone metastases detections using wholebody images [] The proposed CAD system combines an active contour segmentation algorithm for hotspots detection an advanced methodof image gridding to extract certain characteristics of metastatic regions as well as an ANNclassifier for identifying possible metastases The calculated accuracy sensitivity and specificity of CADBOSS were and respectively outperforming other state of theart CAD systemsAdditionally ML methods have been exploited and applied in CAD systems for bonemetastasis detection in bone scintigraphy images A parallelepiped classification method wasspecifically deployed in [] to assist physicians in bone metastases detection of cancer Decision Trees DT and Support Vector Machines SVM were exploited for predicting skeletalrelated events in cancer patients with bone metastases achieving higher accuracies with asmaller number of variables than the number of variables used in Linear Regression LR MLtechniques can be also used to build accurate models to predict skeletalrelated events in cancer patients with bone metastasis providing an overall classification accuracy of ± []As far as PET and PETCT imaging techniques in nuclear medicine are concerned thereare some recent and prominent studies that apply the advantageous features of CNNs In []deep learning has been applied for classification of benign and malignant bone lesions in [F]NaF PETCT images The authors in this work followed the VGG19 architecture for theirnetwork by employing × convolutional layers followed by fully connected layers and asoftmax layer as final activation The ImageNet database of natural images was further used topretrain the network™s weights In this way the network first is trained on general image features and later is tuned using the lesion images and the physician™s scores Taking a closer lookat the results it can be concluded that network™s performance was improved when it wastrained to differentiate between definitely benign score and definitely malignantscore lesions The values of prediction metrics were and concerningthe accuracy sensitivity specificity and positive predictive value respectivelyAlso a CNNbased system was examined in a recent retrospective study which included sequential patients who underwent wholebody FDG PETCT [] The main purpose ofthe study was to detect malignant findings in FDG PETCT examinations while a neural network model equivalent to ResNet24 was built Additionally GradCAM was employed toidentify the part of the image on which the neural network used the largest information Thefindings of the study showed that GradCAM reasonably highlighted the area of malignantuptake allowing physicians to make a diagnosis The same research team recently developed a CNNbased system that predicts the location of malignant uptake and furtherevaluated predictions accuracy [] A network model with configuration equivalent toResNet24 was used to classify wholebody FDG PET imagesIn the research work [] a simple CNNbased system that predicts patient sex from FDGPETCT images was proposed Specifically consecutive patients have participated in thestudy and underwent wholebody FDG PETCT The CNN system was used for classifyingthese patients by sex Another CNNbased diagnosis system for wholebody FDG PETCT wasdeveloped in [] that predicts whether physician™s further diagnosis is required or not Athorough analysis of the results shows that the accuracy considering images of patients presenting malignant uptake and images of equivocal was ± and ± respectivelyThe task of segmentation with the use of deep learning models in skeletal scintigraphyimages has been discussed in more research studies For example in [] the authors followedPLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networksdifferent approaches to convert convolutional neural networks designed for classificationtasks into powerful pixelwise predictors Moreover in [] a deeplearning based segmentation method was developed using prostatespecific membrane antigen PSMA PET imagesand showed significant promise towards automated delineation and quantification of prostatecancer lesions However this research domain that involves bone scintigraphy segmentationwith the inclusion of advanced deep neural networks has not been yet well establishedAlthough bone scintigraphy is extremely important for the diagnosis of metastatic cancer itis clear that a small number of research works have been carried out which presented onlysome preliminary results while the advantageous and outstanding capabilities of CNNs havenot been fully investigated Reviewing the relevant literature it appears that CNNs have notbeen sufficiently applied for the diagnosis of prostate cancer metastasis from whole bodyimages Aim and contribution of this research workCNN is an efficient deep learning network architecture that has recently found great applicability in the medical domain It has shown excellent performance on medical image applications including bone scintigraphy and nuclear medical imaging and can offer a positiveimpact on diagnosis tasksNowadays the main challenge in bone scintigraphy as being one of the most sensitiveimaging methods in nuclear medicine is to build an algorithm that automatically identifieswhether a patient is suffering from bone metastasis or not based on patient™s whole bodyscans It is of utmost importance that the algorithm needs to be extremely accurate due to thefact that patients™ lives could be at stake Deep learning algorithms whose potential lies in thefact that they can improve the accuracy of cancer screening have been recently investigatedfor nuclear medical imaging analysis Recent studies in BS and PET have shown that a deeplearningbased system can perform as well as nuclear physicians do in standalone mode andimprove physicians™ performance in support mode Even though BS is extremely importantfor the diagnosis of metastatic cancer there is currently no research paper regarding the diagnosis of prostate cancer metastasis from whole body scan images that applies robust and moreaccurate deep CNNsThis research study investigates the application of a deep learning CNN to classify bonemetastasis using whole body images of men who were initially diagnosed with prostate cancerThe proposed method employs different CNNbased architectures with data normalizationdata augmentation and shuffling in the preprocessing phase In the training phase the backpropagation technique has been used for updating the weights as part of the optimization process Finally the network architecture is finetuned and the configuration that offers the bestperformance is selected to train the CNN modelThe scope of the current work entails the following two main components First this studyintroduces the CNN method into the diagnosis of bone metastasis disease based on wholebody images In the second phase the paper deals with the improvement of the existing CNNmethod in terms of both network architecture and hyperparameter optimization Thedeployed process seemingly improves the diagnostic effect of the deep learning method making it more efficient compared to other benchmark and wellknown CNN methods such asResNet50 VGG16 GoogleNET Xception and MobileNet [ ]The innovations and contributions of this paper are well summarized as follows¢ The development and demonstration of a simple fast robust CNNbased classification toolfor the identification of bone metastasis in prostate cancer patients from wholebody scansPLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networks¢ The rigorous CNN hyperparameter exploration for determining the most appropriatearchitecture for an enhanced classification performance¢ A comparative experimental analysis utilizing popular image classification CNN architectures like ResNet50 VGG16 GoogleNET Xception and MobileNetThis paper is structured in the following fashion Section presents the material and methods related to this research study Section presents the proposed solution based on CNNmethod for bone metastasis diagnosis for prostate cancer patients using whole body imagesSection shows the results of exploration analysis of CNNs in Red Green and Blue RGBmode for different parameters and configurations thus providing the best CNN model forthis case study Furthermore all the performed experiments with representative results aregathered in section whereas section provides a thorough discussion on analysis of resultsSection concludes the paper and outlines future steps Materials and methods Patients and imagesA retrospective review of consecutive whole body scintigraphy images from differentmale patients who visited Nuclear Medicine Department of Diagnostic Medical Center œDiagnostico AE in Larisa Greece from June to June was performed The selection criterion was prostate cancer patients who had undergone wholebody scintigraphy because ofsuspected bone metastatic diseaseDue to the fact that whole body scan images contain some artifacts and other nonrelatedto bone uptake such as urine contamination and medical accessories ie urinary catheters[] as well as the frequent visible site of radiopharmaceutical injection [] a preprocessingapproach was accomplished to remove these artifacts and nonosseous uptake from the original images This preprocessing method was accomplished by a nuclear medicine physicianbefore the use of the dataset in the proposed classification approachThe initial dataset of images contained not only bone metastasis presence and absencepatient™s cases suffering from prostate cancer but also degenerative lesions [] Due to thisfact as well as aiming to cope with a twoclass classification problem in this study a preselection process concerning images of healthy and malignant patients was accomplished In specific out of consecutive wholebody scintigraphy images of men from differentpatients were selected and diagnosed accordingly by a nuclear medicine specialist with years of experience in bone scan interpretation Out of bone scan images bone scansconcern male patients with bone metastasis and male patients without bone metastasis Anuclear medicine physician classified all the patient cases into categories metastasis absentand metastasis present which was used as a gold standard see Fig The metastatic imageswere confirmed by further examinations performed by CTMRI Wholebody scintigraphy Bone scansA Siemens gamma camera Symbia S series SPECT System by dedicated workstation and software Syngo VE32B with two heads and low energy highresolution collimators was used forpatients scanning The speed of scanning was cmmin with no pixel zooming Two types ofradionuclide were used for bone scintigraphy 99mTcHDP TechneScan1 and 99TcMDPPoltechMDP 5mg Whole body scintigraphy was acquired approximately hours after intravenous injection of “ MBq of radiopharmaceutical agent depending on the patientbody type The common intravenous injection was MBq of radiopharmaceutical agentPLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networksFig Image samples in the dataset Label a Metastasis is present or b absent101371journalpone0237213g001In total planar bone scan images from patients with known prostate cancer werereviewed retrospectively The wholebody field was used to record anterior and posteriorviews digitally with resolution × pixels Images represent counts of detected gammadecays in each spatial unit with 16bit grayscale depthThe image data acquired were originally in DICOM files a commonly used protocol forstorage and communication in hospitals These image data were extracted from these DICOMfiles to create new images in JPEGformat instead A novel dataset of bone scintigraphyimages containing men patients suffering from prostate cancer with metastasis present andmetastasis absent two distinct classes of healthy and malignant cases was prepared for experimentation This dataset consisting of wholebody scans is available for research use afterrequestThis study was approved by the Board Committee Director of the Diagnostic Medical Center œDiagnosticoIatriki AE and the requirement to obtain informed consent was waived bythe Director of the Diagnostic Center due to its retrospective nature All procedures in thisstudy were in accordance with the Declaration of Helsinki MethodologyThe problem of classifying bone metastasis images is a complex procedure and so effectivemachine learning methods need to be exploited to cope with this diagnosis task Deep learningmethods such as CNNs are applied in order to train a classifier to distinguish images of prostate cancer patients with bone metastasis and metastasis absent on healthy patients The effective CNN method for bone metastasis classification proposed in this paper includes threeprocessing steps data preprocessing for the collected scan data normalization a trainingphase for CNN learning and validation and testing which includes the evaluation of the classification results as illustrated in Fig The proposed methodology is thoroughly presented inthe following sections Data preprocessing Step Load images to RGB The original images were saved inRGB mode All images are stored in a respective folder before loaded into the computer memory for CNN training In each image a suitable prefix was defined according to the patient™scategory for example œmalignant_ and œhealthy_ Next a small script was set up in order toPLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networksFig Flowchart of the proposed CNN methodology101371journalpone0237213g002assign a numerical value as label to each image according to its prefix In our case the value ˜™was assigned for œmalignant_ prefixes whereas the value ˜™ for œhealthy_ prefixesStep Data normalization It is common to follow a normalization process feature scalingin most machine learning algorithms [] The minmax normalization processnormalizes thedataset values within the to ensure that all feature data are in the same scale for trainingand testing The data normalization process also assists the convergence of the backpropagation algorithmStep Data shuffle To avoid or eliminate unbiased sampling in machine learning anappropriate shuffling method is needed to be defined More specifically a randomnumbergenerator is used to reorder the images An image sample chosen randomly is meant to be animpartial representation of the total images An unbiased random sample is important formachine learning to provide reliable conclusions In this study Python™s randomshufflemethod is used for dataset shufflingStep Data augmentation Data augmentation is used as a method to artificially increasethe diversity of training data by a large margin by manipulating the existing data instead ofcreating new Data augmentation techniques such as cropping padding and horizontal flipping are commonly used to train large neural networks [] In this research the number ofimages used for learning processes was followed by an image augmentation processing such asrotation enlargementreduction range and flip Note that the original images used for the testwere not subjected to such an augmentation processStep Data split The dataset was split into three sections a training portion a validationportion that would allow the training process to improve and a testing holdout portionwhich is part of the dataset that is completely hidden from the training process The first datasplit takes place by removing of the total dataset and saving for later use as testing Theremaining of the dataset is then split again into an ratio where the small po

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You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: Bone metastasis classification using wholebody images from prostate cancer patientsbased on convolutional neural networksapplicationNikolaos Papandrianos1 Elpiniki PapageiouID23 Athanasios Anagnostis34Konstantinos Papageiou4 General Department University of Thessaly Lamia Greece Faculty of Technology Dept of EnergySystems University of Thessaly Geopolis Campus Larisa Greece Institute for Bioeconomy and Agritechnology Center for Research and Technology Hellas Greece Department of Computer Science andTelecommunications University of Thessaly Lamia Greece npapandrianosuthgrAbstractBone metastasis is one of the most frequent diseases in prostate cancer scintigraphy imaging is particularly important for the clinical diagnosis of bone metastasis Up to date minimalresearch has been conducted regarding the application of machine learning with emphasison modern efficient convolutional neural networks CNNs algorithms for the diagnosis ofprostate cancer metastasis from bone scintigraphy images The advantageous and outstanding capabilities of deep learning machine learning™s groundbreaking technologicaladvancement have not yet been fully investigated regarding their application in computeraided diagnosis systems in the field of medical image analysis such as the problem of bonemetastasis classification in wholebody scans In particular CNNs are gaining great attention due to their ability to recognize complex visual patterns in the same way as human perception operates Considering all these new enhancements in the field of deep learning aset of simpler faster and more accurate CNN architectures designed for classification ofmetastatic prostate cancer in bones is explored This research study has a twofold goal tocreate and also demonstrate a set of simple but robust CNN models for automatic classification of wholebody scans in two categories malignant bone metastasis or healthy usingsolely the scans at the input level Through a meticulous exploration of CNN hyperparameter selection and finetuning the best architecture is selected with respect to classificationaccuracy Thus a CNN model with improved classification capabilities for bone metastasisdiagnosis is produced using bone scans from prostate cancer patients The achieved classification testing accuracy is whereas the average sensitivity is approximately Finally the bestperforming CNN method is compared to other popular and wellknown CNN architectures used for medical imaging like VGG16 ResNet50 GoogleNetand MobileNet The classification results show that the proposed CNNbased approach outperforms the popular CNN methods in nuclear medicine for metastatic prostate cancer diagnosis in bonesa1111111111a1111111111a1111111111a1111111111a1111111111 ACCESSCitation Papandrianos N Papageiou EAnagnostis A Papageiou K Bonemetastasis classification using whole body imagesfrom prostate cancer patients based onconvolutional neural networks application PLoSONE e0237213 101371journalpone0237213Editor Jeonghwan Gwak Korea NationalUniversity of Transportation REPUBLIC OF KOREAReceived November Accepted July Published August Copyright Papandrianos This is an access distributed under the terms ofthe Creative Commons Attribution License whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are creditedData Availability Statement The data which areanonymized bone scintigraphy images without anyfurther information are available only after requestfor research purposes Data availability Thedataset from Diagnostic Medical CenterœDiagnosticoIatriki AE was used under thelicense of the Board Committee Director of theDiagnostic Medical Center œDiagnosticoIatriki AE Dr Vasilios Parafestas for the current studyand is not publicly available The dataset may beavailable only under request from the Director ofPLOS ONE 101371journalpone0237213 August PLOS ONE 0cthe Diagnostic Center vparafestasyahoogr andthe relevant Doctor of Nuclear Medicine DrNikolaos Papandrianos npapandrianosuthgrnikpapandrgmailcomFunding The authors received no specificfunding for this workCompeting interests The authors have declaredthat no competing interests existBone metastasis classification using convolutional neural networks IntroductionMost common tumors such as those of the breast lung and prostate frequently metastasize tothe bone tissue and so the skeleton seems to be a site with the most significant tumor burdenin cancer patients with advanced disease Statistical analysis results have shown that of allbone metastases originate from the breast in women and from the prostate in men Theremaining emanates from thyroid lung and kidney cancers [] In the case of metastaticprostate cancer diagnosis has a significant impact on the quality of patient™s life [] In mostmen the metastatic prostate cancer mainly sites on the bones of the axial skeleton causingsevere lesions that can cause pain debility andor functional impairment [] As this type ofcancer has great avidity for bone and could cause painful and untreatable effects an early diagnosis is crucial for the patient Reviews on clinical evidences and diagnostic assessments ofbone metastases in men with prostate cancer can be found in []The implementation of a properly selected diagnostic imaging can reveal the number ofmetastatic foci in the skeletal system [ ] Rapid diagnosis of bone metastases can be achievedusing modern imaging techniques such as scintigraphy Positron Emission TomographyPET and wholebody Magnetic Resonance Imaging MRIThe primary imaging method in the diagnosis of metastases that offers the highest sensitivity among all imaging methods is Bone Scintigraphy BS [“] Through the depictionof the entire skeleton in one medical examination nuclear doctor is able to detect bone abnormalities in areas where intensive radionuclide activity is present However low specificityseems to be the main drawback of this method as it cannot tell whether the causes of boneturnovers are different than those of metastatic origin leukaemia healing fracture etc Atthe same time PET has been recognized as an efficient method for detecting cancer cellsbased on recent technological advancements in medical imaging PET and Computed Tomography CT combination can produce highresolution images [“]Although PET and PETCT are the most efficient screening techniques for bone metastasisBS remains the most common imaging procedure in nuclear medicine [ ] [] Asreported in European Association of Nuclear Medicine EANM guidelines [] BS is particularly important for clinical diagnosis of metastatic cancer both in men and women At presentwhen other imaging or examination methods are unable to provide a reliable diagnosis BSimaging becomes the proper modality for making a final diagnosis of bone metastasis []To address the considerable problem of bone metastasis diagnosis artificial intelligentmethods for medical image analysis implemented with deep learning algorithms have beenadequately investigated In this direction a recent survey reveals the entire penetration of deeplearning techniques into the field of medical image analysis detection segmentation classification retrieval image generation and enhancement registration and successful application ofdeep learning to medical imaging tasks are thoroughly examined [“]Implementation of deep learning in medical imaging is mainly conducted by ConvolutionalNeural Networks CNNs [ ] a relatively new and powerful way to learn useful representations of images and other structured data Before the application of CNNs these featurestypically had to be created by less powerful machine learning models or even handcraftedWith the introduction of CNNs such features could be learned directly from the provideddata since they include certain preferences in their structure that make them powerful deeplearning models for image analysis [ ] Typical CNNs have a similar structure withArtificial Neural Networks ANN and consist of one or more filters ie convolutional layers followed by aggregationpooling layers in order to extract features for classification tasks[] Gradient descent and backpropagation are both used as learning algorithms the sameway they are used in a standard ANN Their main difference lies in the fact that CNNs havePLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networkslayers of convolutions along with pooling layers in the beginning of their architecture Thefinal outputs are computed via fully connected layers located at the end of the network architecture []In recent years CNNs have gained wider recognition in medical image analysis domain aswell as in vision systems [“ ] Due to their enormous popularity several applications ofCNNs were investigated in the field of medical image analysis Two recent review studies []and [] gather all the important and most interesting applications of deep learningThe application of CNNs in medical imaging ranges from plain radiograph CT MRI andmicroscopy images to clinical photos dermatology capsule endoscopy and visual recognition[“] In addition a CNN was investigated in [] that regards automatic detection of tuberculosis on chest radiographs while in [] a brain tumor segmentation in magnetic resonanceimages was made possible with the use of a CNN More examples of CNNs™ successful application in medical domain include automated cardiac diagnosis [] detection of lesions and prediction of treatment response by PET [ ] as well as dynamic contrast agentenhancedcomputed tomography where CNN showed high diagnostic performance in the differentiation of liver masses [] Furthermore CNNs have shown outstanding performance in radiology and molecular imaging []Some models with major impact in the context of deep learning and medical image processing were introduced in several s the Unet model for biomedical data semanticsegmentation [] the GoogLeNet model introducing the inception module [] the ResNetmodel introducing the residual learning building block for extremely deep convolutional networks [] and also Deeplab which deals with the inclusion of many convolutional layersatrous for semantic segmentation of images in deep convolutional neural networks []In medical image analysis the most widely used CNN methods are the following i AlexNet [] This network has a quite deep architecture similar to GoogLeNet by YannLeCun [] incorporates more filters per layer and includes stacked convolutional layersIt attaches ReLU activations after every convolutional and fullyconnected layer ii ZFNet [] Being a rather slight modification of AlexNet this network won the ILSVRCcompetition iii VGGNet16 [ ] It consists of convolutional layers having avery uniform architecture similar to AlexNet iv GoogleNet [] It is a convolutional neuralnetwork with a standard stacked convolutional layer having one or more fully connected layers called inception modules able to extract various levels of features on the same time vResNet [] It is another efficient CNN architecture that introduced the œidentityshortcut connection to solve the notorious problem of the vanishing gradients of the deepnetworks vi DenseNet [] Being another important CNN architecture DenseNetoffers the main advantage of alleviating the gradient vanishment problem with the direct connection of all the layers Related work in nuclear medical imaging for metastatic prostate cancerdiagnosis in bonesReviewing the relevant literature for diagnosis of bone metastasis using bone scintigraphyscans the authors notice that only a couple of previous works have been adequately conductedfor metastatic prostate cancer classification using CNNs while the others are devoted to ANNsand their application in ComputerAided Diagnosis CAD These works have investigated theuse of Bone Scan Index BSI which was introduced to assess the bone scanning process andestimate the extent of bone metastasis [ ] Specifically it serves as a clinical quantitativeand reproducible parameter that can measure metastatic prostate cancer bone involvement[] The software developed for the BSIbased ANN approach was EXINI bone EXINIPLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networksDiagnostics AB Lund Sweden and afterwards a revised version of this software calledBONENAVI FUJIFILM Toyama Chemical Co Ltd Tokyo Japan was engineered using alarge number of Japanese multicenter training databases []The first of the reported studies was devoted to the development of a classification algorithm based on CNNs for bone scintigraphy image analysis [] It was carried out as a masterthesis in Lund University and is focused mainly on classification problems without considering any identification and segmentation tasks The used dataset was provided by Exini Diagnostics AB in the form of image patches of already found hotspots The process in whichhotspots were segmented cropped and collected from bone scans was implemented using asoftware developed at Exini BSI was calculated for wholebody bone scans by segmenting theentire skeleton from the background in both the anterior and posterior views Due to timeframe restrictions only the hotspots found in the spine have been used to train the CNN sincethey were considered to be the easiest to classify A shape model based on a mean shape of several normal wholebody scans was fitted to the skeleton using an image analysis algorithmcalled Morphon registration The outcomes of the aforementioned thesis [] have shown thatthe calculated accuracy of the validation set was whereas the calculated accuracy of thetesting set was The second study explored CNNs for classification of prostate cancer metastases usingbone scan images [] The tasks of this master project appeared to have a significant potentialon classifying bone scan images obtained by Exini Diagnostics AB too including BSI The twotasks were defined as i classifying anterior posterior pose and ii classifying metastatic nonmetastatic hotspots The outcome of this study is that the trained models produce highlyaccurate results in both tasks and they outperform other methods for all tested body regions inthe case of metastatic nonmetastatic hotspots classification The evaluation indicator of thearea under Receiver Operating Characteristic ROC score was equal to which is significantly higher than the respective ROC of obtained by methods reported in the literature for the same test setThe remaining research that concerns the same imaging modality BS is devoted to theintroduction of CAD systems with the use of ANN and other Machine Learning ML methods for bone metastasis detection in bone scintigraphy images Sadik were the first todevelop an automated CAD system as a clinical quality assurance tool for the interpretation ofbone scans [“] This bone scan CAD software was trained to interpret bone scans usingtraining databases that consist of bone scans from European patients who have the desiredimage interpretation metastatic disease or not The results showed a sensitivity of at aspecificity of These works result in certain outcomes that refer to the development of atotally automated computerassisted diagnosis system that can identify metastases after examining bone scans applying multilayer perceptron ANN techniques involving a small databaseof wholebody bone scans patients The highest sensitivity that was achieved from all thestudies and accomplished during this thesis was approximately []Horikoshi compared the diagnostic accuracy of two CAD systems one based on aEuropean and another on a Japanese training database in a group of bone scans from Japanesepatients [] The Japanese CAD software showed a higher specificity and accuracy comparedto the European Comparing the sensitivities the Japanese CAD software achieved whereas the European CAD software reached []In another study conducted by Tokuda the diagnostic capability of a completely automated CAD system which detects metastases in the images of bone scans by focusing on twodifferent patterns was investigated [] The first pattern was devoted to the detection ofmetastases per region the second one detects metastases per patient The investigated systemwas called œBONENAVI version  The produced results have shown that the new CADPLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networkssystem is able to decrease the number of false positive findings which depends on the primarylesion of cancerIn Aslantas proposed œCADBOSS as a fully automated diagnosis system forbone metastases detections using wholebody images [] The proposed CAD system combines an active contour segmentation algorithm for hotspots detection an advanced methodof image gridding to extract certain characteristics of metastatic regions as well as an ANNclassifier for identifying possible metastases The calculated accuracy sensitivity and specificity of CADBOSS were and respectively outperforming other state of theart CAD systemsAdditionally ML methods have been exploited and applied in CAD systems for bonemetastasis detection in bone scintigraphy images A parallelepiped classification method wasspecifically deployed in [] to assist physicians in bone metastases detection of cancer Decision Trees DT and Support Vector Machines SVM were exploited for predicting skeletalrelated events in cancer patients with bone metastases achieving higher accuracies with asmaller number of variables than the number of variables used in Linear Regression LR MLtechniques can be also used to build accurate models to predict skeletalrelated events in cancer patients with bone metastasis providing an overall classification accuracy of ± []As far as PET and PETCT imaging techniques in nuclear medicine are concerned thereare some recent and prominent studies that apply the advantageous features of CNNs In []deep learning has been applied for classification of benign and malignant bone lesions in [F]NaF PETCT images The authors in this work followed the VGG19 architecture for theirnetwork by employing × convolutional layers followed by fully connected layers and asoftmax layer as final activation The ImageNet database of natural images was further used topretrain the network™s weights In this way the network first is trained on general image features and later is tuned using the lesion images and the physician™s scores Taking a closer lookat the results it can be concluded that network™s performance was improved when it wastrained to differentiate between definitely benign score and definitely malignantscore lesions The values of prediction metrics were and concerningthe accuracy sensitivity specificity and positive predictive value respectivelyAlso a CNNbased system was examined in a recent retrospective study which included sequential patients who underwent wholebody FDG PETCT [] The main purpose ofthe study was to detect malignant findings in FDG PETCT examinations while a neural network model equivalent to ResNet24 was built Additionally GradCAM was employed toidentify the part of the image on which the neural network used the largest information Thefindings of the study showed that GradCAM reasonably highlighted the area of malignantuptake allowing physicians to make a diagnosis The same research team recently developed a CNNbased system that predicts the location of malignant uptake and furtherevaluated predictions accuracy [] A network model with configuration equivalent toResNet24 was used to classify wholebody FDG PET imagesIn the research work [] a simple CNNbased system that predicts patient sex from FDGPETCT images was proposed Specifically consecutive patients have participated in thestudy and underwent wholebody FDG PETCT The CNN system was used for classifyingthese patients by sex Another CNNbased diagnosis system for wholebody FDG PETCT wasdeveloped in [] that predicts whether physician™s further diagnosis is required or not Athorough analysis of the results shows that the accuracy considering images of patients presenting malignant uptake and images of equivocal was ± and ± respectivelyThe task of segmentation with the use of deep learning models in skeletal scintigraphyimages has been discussed in more research studies For example in [] the authors followedPLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networksdifferent approaches to convert convolutional neural networks designed for classificationtasks into powerful pixelwise predictors Moreover in [] a deeplearning based segmentation method was developed using prostatespecific membrane antigen PSMA PET imagesand showed significant promise towards automated delineation and quantification of prostatecancer lesions However this research domain that involves bone scintigraphy segmentationwith the inclusion of advanced deep neural networks has not been yet well establishedAlthough bone scintigraphy is extremely important for the diagnosis of metastatic cancer itis clear that a small number of research works have been carried out which presented onlysome preliminary results while the advantageous and outstanding capabilities of CNNs havenot been fully investigated Reviewing the relevant literature it appears that CNNs have notbeen sufficiently applied for the diagnosis of prostate cancer metastasis from whole bodyimages Aim and contribution of this research workCNN is an efficient deep learning network architecture that has recently found great applicability in the medical domain It has shown excellent performance on medical image applications including bone scintigraphy and nuclear medical imaging and can offer a positiveimpact on diagnosis tasksNowadays the main challenge in bone scintigraphy as being one of the most sensitiveimaging methods in nuclear medicine is to build an algorithm that automatically identifieswhether a patient is suffering from bone metastasis or not based on patient™s whole bodyscans It is of utmost importance that the algorithm needs to be extremely accurate due to thefact that patients™ lives could be at stake Deep learning algorithms whose potential lies in thefact that they can improve the accuracy of cancer screening have been recently investigatedfor nuclear medical imaging analysis Recent studies in BS and PET have shown that a deeplearningbased system can perform as well as nuclear physicians do in standalone mode andimprove physicians™ performance in support mode Even though BS is extremely importantfor the diagnosis of metastatic cancer there is currently no research paper regarding the diagnosis of prostate cancer metastasis from whole body scan images that applies robust and moreaccurate deep CNNsThis research study investigates the application of a deep learning CNN to classify bonemetastasis using whole body images of men who were initially diagnosed with prostate cancerThe proposed method employs different CNNbased architectures with data normalizationdata augmentation and shuffling in the preprocessing phase In the training phase the backpropagation technique has been used for updating the weights as part of the optimization process Finally the network architecture is finetuned and the configuration that offers the bestperformance is selected to train the CNN modelThe scope of the current work entails the following two main components First this studyintroduces the CNN method into the diagnosis of bone metastasis disease based on wholebody images In the second phase the paper deals with the improvement of the existing CNNmethod in terms of both network architecture and hyperparameter optimization Thedeployed process seemingly improves the diagnostic effect of the deep learning method making it more efficient compared to other benchmark and wellknown CNN methods such asResNet50 VGG16 GoogleNET Xception and MobileNet [ ]The innovations and contributions of this paper are well summarized as follows¢ The development and demonstration of a simple fast robust CNNbased classification toolfor the identification of bone metastasis in prostate cancer patients from wholebody scansPLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networks¢ The rigorous CNN hyperparameter exploration for determining the most appropriatearchitecture for an enhanced classification performance¢ A comparative experimental analysis utilizing popular image classification CNN architectures like ResNet50 VGG16 GoogleNET Xception and MobileNetThis paper is structured in the following fashion Section presents the material and methods related to this research study Section presents the proposed solution based on CNNmethod for bone metastasis diagnosis for prostate cancer patients using whole body imagesSection shows the results of exploration analysis of CNNs in Red Green and Blue RGBmode for different parameters and configurations thus providing the best CNN model forthis case study Furthermore all the performed experiments with representative results aregathered in section whereas section provides a thorough discussion on analysis of resultsSection concludes the paper and outlines future steps Materials and methods Patients and imagesA retrospective review of consecutive whole body scintigraphy images from differentmale patients who visited Nuclear Medicine Department of Diagnostic Medical Center œDiagnostico AE in Larisa Greece from June to June was performed The selection criterion was prostate cancer patients who had undergone wholebody scintigraphy because ofsuspected bone metastatic diseaseDue to the fact that whole body scan images contain some artifacts and other nonrelatedto bone uptake such as urine contamination and medical accessories ie urinary catheters[] as well as the frequent visible site of radiopharmaceutical injection [] a preprocessingapproach was accomplished to remove these artifacts and nonosseous uptake from the original images This preprocessing method was accomplished by a nuclear medicine physicianbefore the use of the dataset in the proposed classification approachThe initial dataset of images contained not only bone metastasis presence and absencepatient™s cases suffering from prostate cancer but also degenerative lesions [] Due to thisfact as well as aiming to cope with a twoclass classification problem in this study a preselection process concerning images of healthy and malignant patients was accomplished In specific out of consecutive wholebody scintigraphy images of men from differentpatients were selected and diagnosed accordingly by a nuclear medicine specialist with years of experience in bone scan interpretation Out of bone scan images bone scansconcern male patients with bone metastasis and male patients without bone metastasis Anuclear medicine physician classified all the patient cases into categories metastasis absentand metastasis present which was used as a gold standard see Fig The metastatic imageswere confirmed by further examinations performed by CTMRI Wholebody scintigraphy Bone scansA Siemens gamma camera Symbia S series SPECT System by dedicated workstation and software Syngo VE32B with two heads and low energy highresolution collimators was used forpatients scanning The speed of scanning was cmmin with no pixel zooming Two types ofradionuclide were used for bone scintigraphy 99mTcHDP TechneScan1 and 99TcMDPPoltechMDP 5mg Whole body scintigraphy was acquired approximately hours after intravenous injection of “ MBq of radiopharmaceutical agent depending on the patientbody type The common intravenous injection was MBq of radiopharmaceutical agentPLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networksFig Image samples in the dataset Label a Metastasis is present or b absent101371journalpone0237213g001In total planar bone scan images from patients with known prostate cancer werereviewed retrospectively The wholebody field was used to record anterior and posteriorviews digitally with resolution × pixels Images represent counts of detected gammadecays in each spatial unit with 16bit grayscale depthThe image data acquired were originally in DICOM files a commonly used protocol forstorage and communication in hospitals These image data were extracted from these DICOMfiles to create new images in JPEGformat instead A novel dataset of bone scintigraphyimages containing men patients suffering from prostate cancer with metastasis present andmetastasis absent two distinct classes of healthy and malignant cases was prepared for experimentation This dataset consisting of wholebody scans is available for research use afterrequestThis study was approved by the Board Committee Director of the Diagnostic Medical Center œDiagnosticoIatriki AE and the requirement to obtain informed consent was waived bythe Director of the Diagnostic Center due to its retrospective nature All procedures in thisstudy were in accordance with the Declaration of Helsinki MethodologyThe problem of classifying bone metastasis images is a complex procedure and so effectivemachine learning methods need to be exploited to cope with this diagnosis task Deep learningmethods such as CNNs are applied in order to train a classifier to distinguish images of prostate cancer patients with bone metastasis and metastasis absent on healthy patients The effective CNN method for bone metastasis classification proposed in this paper includes threeprocessing steps data preprocessing for the collected scan data normalization a trainingphase for CNN learning and validation and testing which includes the evaluation of the classification results as illustrated in Fig The proposed methodology is thoroughly presented inthe following sections Data preprocessing Step Load images to RGB The original images were saved inRGB mode All images are stored in a respective folder before loaded into the computer memory for CNN training In each image a suitable prefix was defined according to the patient™scategory for example œmalignant_ and œhealthy_ Next a small script was set up in order toPLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networksFig Flowchart of the proposed CNN methodology101371journalpone0237213g002assign a numerical value as label to each image according to its prefix In our case the value ˜™was assigned for œmalignant_ prefixes whereas the value ˜™ for œhealthy_ prefixesStep Data normalization It is common to follow a normalization process feature scalingin most machine learning algorithms [] The minmax normalization processnormalizes thedataset values within the to ensure that all feature data are in the same scale for trainingand testing The data normalization process also assists the convergence of the backpropagation algorithmStep Data shuffle To avoid or eliminate unbiased sampling in machine learning anappropriate shuffling method is needed to be defined More specifically a randomnumbergenerator is used to reorder the images An image sample chosen randomly is meant to be animpartial representation of the total images An unbiased random sample is important formachine learning to provide reliable conclusions In this study Python™s randomshufflemethod is used for dataset shufflingStep Data augmentation Data augmentation is used as a method to artificially increasethe diversity of training data by a large margin by manipulating the existing data instead ofcreating new Data augmentation techniques such as cropping padding and horizontal flipping are commonly used to train large neural networks [] In this research the number ofimages used for learning processes was followed by an image augmentation processing such asrotation enlargementreduction range and flip Note that the original images used for the testwere not subjected to such an augmentation processStep Data split The dataset was split into three sections a training portion a validationportion that would allow the training process to improve and a testing holdout portionwhich is part of the dataset that is completely hidden from the training process The first datasplit takes place by removing of the total dataset and saving for later use as testing Theremaining of the dataset is then split again into an ratio where the small po Answer:

Thyroid_Cancer

Is preoperative heart rate variability aprognostic indicator for overall survival andcancer recurrence in patients with primarycolorectal cancerM T A StrousID1 A M Daniels1 F M Zimmermann2 F N van Erning3 Y Gidron4 FJ Vogelaar1 Department of Surgery VieCuri Medical Centre Venlo The Netherlands Department of CardiologyCatharina Hospital Eindhoven The Netherlands Department of Research Netherlands ComprehensiveCancer anisation Utrecht The Netherlands Faculty of Welfare and Health University of Haifa HaifaIsrael maudstrousviecurinlAbstracta1111111111a1111111111a1111111111a1111111111a1111111111 ACCESSCitation Strous MTA Daniels AM ZimmermannFM van Erning FN Gidron Y Vogelaar FJ Ispreoperative heart rate variability a prognosticindicator for overall survival and cancer recurrencein patients with primary colorectal cancer PLoSONE e0237244 101371journalpone0237244Editor Louise Emilsson University of OsloNORWAYReceived February Accepted July Published August Copyright Strous This is an access distributed under the terms of theCreative Commons Attribution License whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are creditedData Availability Statement Data cannot beshared publicly because of ethical concernsPatients were included on a no objection base toconduct retrospective data studies and publishfindings but were not asked for permission topublish full encrypted data Data are available fromthe VieCuri Institutional Data Access contact viawetenschapsbureauviecurinl for researcherswho meet the criteria for access to confidentialdataBackgroundHeart Rate Variability HRV represents efferent vagus nerve activity which is suggested tobe inversely related to fundamental mechanisms of tumorigenesis and to be a predictor ofprognosis in various types of cancer HRV is also believed to predict the occurrence andseverity of postoperative complications We aimed to determine the role of preoperativeHRV as a prognostic factor in overall and cancer free survival in patients with colorectalcancerMethodsRetrospective analysis was performed in a detailed dataset of patients diagnosed with primary colorectal cancer between January and December who underwent curative surgical treatment HRV was measured as timedomain parameters SDNN StandardDeviation of NNintervals and RMSSD Root Mean Square of Successive Differencesbased on preoperative second ECGs Groups were created by baseline HRV Low HRVSDNN 20ms or RMSSD 19ms and normal HRV SDNN �20ms or RMSSD �19msPrimary endpoints were overall and cancer free survivalResultsA total of patients were included in this study HRV was not significantly associated withoverall survival SDNN 20ms vs SDNN �20ms244 vs adjusted HR “ p RMSSD 19ms vs RMSSD �19ms270 vs adjustedHR “ p or cancer recurrence SDNN 20ms vs�20ms201 vs adjusted HR “ p RMSSD 19ms vs�19ms vs adjusted HR “ p There was noPLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancerFunding The authors received no specific fundingfor this workCompeting interests The authors have declaredthat no competing interests existsignificant association between HRV and CEAlevel at one year followup or between HRVand occurrence of a postoperative complication or the severity of postoperativecomplicationsConclusionsHeart rate variability was not associated with overall or cancer free survival in patients withprimary colorectal cancer who underwent curative surgical treatment These results do notalign with results found in studies including only patients with advanced cancer which suggests that there is only an association in the other direction cancer causing low HRVIntroductionIn there were over million newly diagnosed colorectal cancer patients worldwide andover in the Netherlands alone It is the fourth most common cause of death worldwide[] To improve survival it is of importance to get a better insight into modifiable prognosticfactors Emerging evidence suggests that vagal nerve activity indexed by heart rate variabilityHRV could be one of these prognostic factors [“]HRV is the physiological phenomenon of the fluctuation in time intervals between adjacentheartbeats and represents efferent vagus nerve activity to the heart [“] It has been suggestedthat efferent vagal activity is inversely related with fundamental mechanisms of tumorigenesisas inflammation oxidative stress and excessive sympathetic activity [] These mechanisms arebelieved to be controlled by the vagus nerve via a bidirectional braintoimmune pathwaysthrough the release of neurotransmitters via the cholinergic antiinflammatory pathway[] A higher vagal tone may reflect a more flexible topdown regulation of the immunesystem and physiological activity moderated by the brain [] Absence of vagus activity due tovagotomy has been shown to increase the risk of developing colorectal cancer []In addition to influencing development of cancer vagus nerve activity seems to be a predictor of prognosis in various types of cancer Recent studies show an association betweendecreased activity of the vagus nerve and worse survival in patients with cancer of the gastrointestinal tract liver pancreas lung prostate and breast among others [] Also patientswith normal HRV seem to live longer in different sorts of metastatic cancer independent ofconfounders [] In patients with colorectal cancer a low HRV at baseline has shown to beassociated with higher CEA levels at months after diagnosis which predicts a poorer prognosis []In patients undergoing curative treatment for colorectal cancer HRV does not only seemto influence cancer prognosis A recent study showed that patients with lower HRV have moreintraoperative blood loss and more and more severe postoperative complications []Identifying patients with low HRV is easy and noninvasive When its predictive value forthe prognosis of cancer patients is of satisfactory significance vagus nerve activation prior toor during cancer treatment could theoretically be beneficial in improving prognosis [] Alsoif we could predict the occurrence and severity of postoperative complications based on HRVimproving HRV before surgery could possibly accelerate postoperative recovery and indirectlyaffect patients™ prognosis Recent studies focussing on improving HRV by improving physicalfitness by means of physical exercise show promising results in both older men and woman[] However the only previous study on colon cancer and HRV including patients receiving curative treatment included a small sample and did not examine whether HRV predictsPLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancersurvival in these patients [] To clarify the predictive value of HRV in prognosis of patientswith colorectal cancer further exploration is needed Current studies identifying HRV as aprognostic factor did not specifically focus on colorectal cancer have small study populationsdid not correct for confounders and mainly focused on metastatic disease [“]The aim of this study was to determine the role of preoperative HRV as a prognostic factorin overall and cancer free survival in patients with primary colorectal cancer who underwentcurative surgical treatmentMethodsData collectionData from the Netherlands Cancer Registry NCR were used The NCR collects data on allnewly diagnosed cancer patients in the Netherlands Information on patient and tumour characteristics diagnosis and treatment is routinely collected from the medical records by trainedadministrators of the cancer registry Anatomical site of the tumour is registered according tothe International Classification of Diseases Oncology The tumournodemetastasis TNMclassification is used for stage notification of the primary tumour according to the editionvalid at time of cancer diagnosis Quality of the data is high due to thorough training of theregistration team and consistency checks []For the study population additional data were collected from the medical records of thepatients This encompassed information on HRV CEAlevels ASA classification comorbidities identified at admission divided into groups cardiac disease hypertension diabetes mellitus thyroid disease pulmonary disease vascular disease neurological disease and otheroccurrence and severity of postoperative complications and cancer recurrence Groups ofcomorbidities were chosen based on matching features within these groups and their potentialinfluence on HRV or the endpoint being analysed Severity of the postoperative complicationsaccording to the ClavienDindo classification was also documented Medical records wereassessed between January and July and reevaluated for revision of this between the 20th and 25th of April This study was approved by the research committee and the Board of Directors of VieCuriMedical Centre Data was obtained under the law ˜scientific research and statistics in the interest of public health where asking for permission is not possible or appropriate for several reasons™ in the Netherlands unless patients objected to use of their personal medical record forscientific research Data was encrypted with an encryption key provided by the NCR Encryption was shortly lifted to access the patients™ number for accessing hisher medical record Following extraction data were encrypted againStudy populationThe study population included all consecutive patients diagnosed with primary colorectal adenocarcinoma between January and January at VieCuri Medical Centre who underwent curative surgical treatment Patients with metastatic disease at time of surgery orcarcinoma in situ were excluded as their treatment and prognosis differs from those receivingcurative treatment for colorectal cancer Metastasis found within months after surgery wereconsidered present at time of surgery and therefore excluded Other excluded patients werepatients with neuroendocrine tumours because of different tumour characteristics and prognosis patients with cardiac arrhythmias including atrial and ventricular extrasystole pacemakers patients taking betablockers as this enhances HRV indexes or patients withbradycardia heart rate bpm or tachycardia heart rate bpm as this precluded reliable calculation of HRV [] We did not exclude patients taking alphablockers calciumPLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancerinhibitors diuretics amiodarone ACEinhibitors or ARB™s as these types of medicationreduce central sympathetic functioning rather than peripheral and their influence on HRV istherefore less univocal and sometimes completely absent []Heart rate variabilityHeart rate variability was analysed using a 12lead 10second ECG 150Hz used for preoperative screening In case of multiple ECG™s per patient the most recent ECG before date of surgery was used for HRVanalysis In case of multiple ECG™s per patient on the same date theECG with the best quality was chosen meaning an ECG without motion artefacts In case ofmotion artefacts there was always an ECG without motion artefacts available recorded on thesame date Time between two consecutive Rpeaks was measured in lead II with an accuracy of02ms using MUSEECG HRV was presented using the timedomain HRV parameters SDNNStandard Deviation of NNintervals and RMSSD Root Mean Square of Successive Differences in milliseconds calculated using the following calculations []rSDNN ¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffii¼1 ðRRi 00 RRmeanÞ2Pnn 00 RMSSD ¼rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffii¼1 ðRRiþ1 00 RRiÞ2Pn 00 n 00 ð1Þð2ÞSDNN and RMSSD obtained from 10s ECGs were found to correlate with results of ECGsof longer durations Power spectral analysis HRV parameters as LF and HF can only beobtained in longer recording ECGs and were therefore not implementable in this study[]SDNN and RMSSD were both analysed as continuous variables as well as binary variablesusing cutoffs of 20ms versus �20ms and 19ms versus �19ms respectively In case of anSDNN 20ms or RMSSD 19ms HRV was classified as low and in case of SDNN �20ms orRMSSD �19ms as normal These cutoff values were based on cutoff values used in otherstudies showing an association between lowHRV as SDNN 20ms and RMSSD 19ms andcolorectal cancer as there is no standardised definition of low and normal HRV []Endpoints and definitionsThe primary endpoints of this study were overall and cancer free survival Overall survival wasdefined as the time between the date of surgery to the date of death or last followup date inmonths Cancer free survival was defined as the time in months from the date of surgery untilthe date of cancer recurrence defined as the first date of either radiologic or pathologic diagnosis of metastases or tumour recurrence of colorectal cancer Patients dying without cancerrecurrence were censored on day of death Secondary endpoints were elevated CEAlevel ugl at oneyear followup occurrence of postoperative complications within daysafter surgery and severity of postoperative complications according to the ClavienDindoclassificationStatistical analysisIn this retrospective observational cohort study we utilized descriptive statistics to provide anoverview of control variables of the study population patient characteristics as age sex BMIcomorbidities and ASAclassification heart rate and tumour characteristics as TNMstagetumour localisation and tumour differentiation and their association with HRV andPLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancerprognosis Normal distribution of the continuous variables heart rate age and BMI as well asSDNN and RMSSD were tested with a KolmogorovSmirnov test Because of normal distribution heart rate age and BMI were compared between HRVgroups using unpaired ttest Allother variables were categorical and were compared between HRVgroups using Chisquarestatistics as groups were all of sufficient powerDifferences in overall survival and cancer free survival in months according to SDNN andRMSSD were visualized by means of KaplanMeier curves and statistically tested using the logrank test Multivariate coxregression analyses were conducted to calculate the prognosticassociation between HRV and overall and cancer free survival while adjusting for other prognostic variables Multivariate logistic regression was used to assess the independent effect ofSDNN and RMSSD on CEAlevels and the occurrence and severity of postoperative complications Variables included for adjustment were chosen by means of forward stepwise selectionbased on clinical judgment differences at baseline eg differences on any predictor betweenpatients who later died or not and database availability and depended on the analysed endpoint Those included patient demographics age sex bodymassindex comorbidities identified at admission divided into groups cardiac disease hypertension diabetes mellitus thyroiddisease pulmonary disease vascular disease neurological disease other including Crohn™sdisease hepatitis kidney failure disorders anaemia depression arthritis tumour characteristics localisation stage differentiation and the occurrence of postoperative complicationswhen the later was not an outcome Differences in CEAlevel at baseline and one year checkup between and within groups of low HRV and normal HRV were assessed with a repeatedmeasures linear model and tested using the tukey test To test the implication of a longer timebetween ECG and treatment all analyses were repeated after excluding patients with an ECGolder than months A twotailed pvalue � was considered significant in all analysesData were analysed using IBM SPSS Statistics version IBM Corp NY Armonk USAResultsOf colorectal cancer patients that underwent a surgical resection a total of patientswere included in this study Reasons for exclusion are presented in Fig Median SDNN andRMSSD were 204ms interquartile range IQR 115ms351ms and 175ms IQR 99ms299ms respectively Table shows descriptive data of the included patients by HRV groupsBaseline heart rate and age were negatively associated with HRV The group of patients withlow HRV contains more patients with a history of cardiac disease regardless of the HRV defining parameter When defining low HRV by RMSSD 19ms more patients in this group havehypertension as comorbidity This group also contains more patients with an ASA classification greater than oneDuring a median followup of months IQR “ all causedeath occurred in patients Cancer recurrence occurred in patients during a median followup of months IQR “To rule out any distort in results caused by a delay between ECG and treatment all analyseswere repeated after exclusion of ECG™s older than months This did not lead to any new significant results Therefore these results were not displayed in detail in this paperSurvivalIn low HRV groups slightly more patients died compared to normal HRV groups SDNN20ms versus �20ms versus respectively RMSSD 19ms versus�19ms versus respectively These observed differences between HRVgroups in overall survival were not significant Fig A SDNN p B RMSSDPLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancerFig Flowchart of the study101371journalpone0237244g001p After adjustment for some potential confounders these associations remained notsignificant SDNN 20ms versus �20ms HR “ p and RMSSD19ms versus �19ms HR “ p Tables and Age cardiac disease tumour stage and postoperative complications had a significant influence on overall survival Age also differed at baseline and was identified as a confounder When defining low andnormal HRVgroups by SDNN cardiac disease was identified as confounder When conducting sensitivity analyses with SDNN and RMSS as continuous variables results remained thesame There was no association between HRV and overall survival SDNN HR “ p and RMSSD HR “ p In low HRV groups slightly more patients had recurrence of cancer compared to normalHRV groups SDNN 20ms versus �20ms versus respectivelyRMSSD 19ms versus �19ms versus respectively These observed differences between HRV groups in cancer free survival were not significant Fig A SDNNp B RMSSD p As in overall survival after adjustment for some potentialconfounders these associations remained not significant SDNN 20ms versus �20msHR “ p and RMSSD 19ms versus �19ms HR “ p Tables and In SDNNbased groups baseline heart rate was identified asa confounding variable Sensitivity analyses with SDNN and RMSSD as continuous variablesPLOS ONE 101371journalpone0237244 August PLOS ONE 0cTable Descriptive data of included patients according to prespecified HRV groupsSDNN 20ms n SDNN �20ms n pRMSSD 19ms n RMSSD �19ms n pHRV and prognosis in colorectal cancerHeart Rate�Age�Age n year� yearSex nMaleFemale [“] [“] [“] [“] [“] [“] [“] [“] Mean BMI SD [“] [“] [“] [“]Comorbidities nCardiac diseaseHypertensionDiabetes MellitusThyroid diseasePulmonary diseaseVascular diseaseNeurological diseaseOtherASA nASA1ASA2ASA34Localisation tumour nRight colonLeft colonRectumTumour stage nIIIIII Differentiation grade nWellmoderate Poorundifferentiated � Non normaldistributed data presented as median with interquartile range101371journalpone0237244t001did not alter these results There was no association between HRV and cancer free survivalSDNN HR “ p and RMSSD HR “p CEAlevelCEAlevel at baseline and one year checkup was registered in patients and elevated in of these patients This was elevated at one year checkup in only patients Low HRV was notsignificantly associated with elevated CEAlevels at one year checkup as shown in Table Sensitivity analyses with SDNN and RMSSD as continuous variables did not alter these resultsSDNN HR “ p and RMSSD HR “PLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancerFig KaplanMeier curves for overall survival in groups of low HRV and normal HRV presented as A SDNN andB RMSSD101371journalpone0237244g002PLOS ONE 101371journalpone0237244 August PLOS ONE 0cTable Multivariate analyses of associations of SDNN and covariates with overall and cancer free survivalHRV and prognosis in colorectal cancerSDNN20ms�20msHeart RateAgeBMICardiac diseaseNoYesHypertensionNoYesASAclassificationASA1ASA2ASA34LocalisationRight colonLeft colonRectumTumour stageIIIIIIOverall survivalHR CIpCancer free survivalHR CI “ “ reference “ “ “ reference “ “ “p reference reference “ “ reference reference “ “ reference “ “ reference “ “ reference “ “ “ “ reference “ “ reference “ “Postoperative complicationNoYes reference reference “ “101371journalpone0237244t002p Adjusting for covariates was not possible because of the small number of patientswith an elevated CEAlevelDifferences between CEAlevel at baseline and one year checkup were compared betweenand within HRVgroups Results were displayed in Fig There were no significant differencesin CEAlevel at baseline and one year checkup between the low HRV group and normal HRVgroup defined by SDNN and RMSSD p and p respectively Also there were nosignificant differences in CEAlevel at baseline and at one year checkup within the low HRVgroup and normal HRV group defined by SDNN and RMSSD p and p respectivelyPostoperative complicationsIn patients one or more postoperative complications occurred within days after surgeryThe occurrence of a postoperative complication was not significantly associated with lowHRV defined as SDNN 20ms or RMSSD 19ms even after adjustment for some potentialconfounders Table Heart rate age cardiac disease and hypertension were identified asconfounding covariates When conducting sensitivity analyses with SDNN and RMSS as continuous variables the association between occurrence of a postoperative complication withPLOS ONE 101371journalpone0237244 August PLOS ONE 0cTable Multivariate analyses of associations of RMSSD and covariates with overall and cancer free survivalHRV and prognosis in colorectal cancerRMSSD19ms�19msHeart RateAgeBMICardiac diseaseNoYesHypertensionNoYesASAclassificationASA1ASA2ASA34LocalisationRight colonLeft colonRectumTumour stageIIIIIIOverall survivalHR CIpCancer free survivalHR CI “ “ reference “ “ “ reference “ “ “p reference reference “ “ reference reference “ “ reference “ “ reference “ “ reference “ “ reference “ “ reference “ “ reference “ “Postoperative complicationNoYes reference reference “ “101371journalpone0237244t003baseline HRV remained not significant SDNN HR “ p andRMSSD “ p The same applied when postoperative complicationswere graded according to the ClavienDindo classification and the complication that is gradedthe highest for each patient is compared to the absence of postoperative complicationsTable DiscussionIn this observational cohortstudy we determined the Heart Rate Variability in preoperativeECGs of patients with primary colorectal cancer who underwent curative surgical treatment HRV refers to physiological variations in beattobeat intervals It was presented intimedomain parameters SDNN and RMSSD HRV was not significantly associated with overall survival or cancer free survival independent of some risk factors Also this study showedno significant differences in CEAlevels at one year checkup between patients with low HRVand patients with normal HRV Patients with low HRV did not have more or more severepostoperative complications compared to patients with normal HRVTumorigenesis has three fundamental mechanisms inflammation promoting oxidativestress and stimulating tumour growth oxidative stress causing DNAdamage andPLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancerFig KaplanMeier curves for cancer free survival in groups of low HRV and normal HRV presented as A SDNNand B RMSSD101371journalpone0237244g003PLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancerTable Univariate analyses of low HRV and risk of elevated CEAlevel at one year checkupCEA μglOR CIpCEA μglOR CIIndependent of baseline CEAIndependent of baseline CEASDNN 20ms n “RMSSD 19ms n “SDNN �20ms n Normal baseline CEA � μglSDNN 20ms n SDNN �20ms n Elevated baseline CEA μglSDNN 20ms n reference “ reference “RMSSD �19ms n Normal baseline CEA � μglRMSSD 19ms n RMSSD �19ms n Elevated baseline CEA μglRMSSD 19ms n reference “ reference “SDNN �20ms n referenceRMSSD �19ms n referencep101371journalpone0237244t004interfering with subsequent repair mechanisms and sympathetic neurotransmitters stimulating tumour metastasis and progression [] It has been suggested that afferent fibres of thevagus nerve inform the brain about peripheral inflammation This is followed by a braintoimmune response via the efferent route of the vagus nerve that modulates the function ofimmuneregulatory cells through the release of neurotransmitters via the cholinergic antiinflammatory pathway Malfunctioning of this route may play part in the onset of cancer []This theory has been supported by studies of patients with gastric ulcers who underwent avagotomy who then showed an increased risk of developing lung and colorectal cancer [“] The vagus nerve is also believed to modulate tumour progression An active vagus nervecan inhibit inflammation oxidative stress and the release of sympathetic neurotransmittersthat stimulate tumour metastasis and progression [“] Absence of this inhibitory effect inturn results in metastasis and tumour progression as shown in a study of Erin showingthat mice who underwent chemical vagotomy developed more metastasis of breastcancer cellsthan controls [] Epidemiologically low HRV has been associated with worse overall survivalover time in patients with recurrent or metastatic cancer of various types even after correctionfor confounders [“] However the results of the present study do not support thesefindingsTo our knowledge this is the first study including only patients with colorectal cancer whoare eligible for curative treatment by partial bowel resection and not those receiving palliativetreatment De Couck studied the relationship between HRV and tumour burden in bothcurative and palliative patients with prostate cancer or nonsmall cell lung cancer Independent of confounders the hypothesised inverse relationship of HRV and the tumour markerPSA at and months after diagnosis was only significant in patients with stage IV prostatecancer not stage II and III [] In colorectal cancer Mouton found that low HRV definedas SDNN ms predicted significantly higher levels of the tumour marker CEA at months after diagnosis Again these results were only found in patients receiving palliativetreatment not curative [] Only one previous study showed a significant inverse relationshipbetween HRV and mortality in cancer in general independent of stage [] This study of Guo had a large study population of patients with various types of cancer Low HRV wasdefined as SDNN 70ms and was significantly associated with shorter survival times Thissuggests that HRV is a predictive indicator of survival time not only in palliative but also incurative patients However results were not controlled for cancer type which could affect bothHRV and survival and should therefore be interpreted with caution [] The fact that thepatients recruited in the present sample were only with less advanced cancer may partlyexplain the lack of prognostic role in HRV in this samplePLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancerFig Bar chart for CEAlevel at baseline and one year checkup in groups of low HRV and normal HRV presented as ASDNN and B RMSSD101371journalpone0237244g004PLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancerTable Low HRV and risk of occurrence of a postoperative complication within daysPostoperative complicationOR CIUnadjustedSDNN 20ms n “SDNN �20ms n referenceAdjusted�SDNN 20ms n “SDNN �20ms n referenceUnadjustedRMSSD 19ms n “RMSSD �19ms n referenceAdjusted�RMSSD 19ms n “RMSSD �19ms n reference�adjusted for heart rate age cardiac disease and hypertension101371journalpone0237244t005pSome of these previous studies suggest a bidirectional relationship between vagus nerveactivity and cancer [] However based on current evidence on this subject we cannot supportthis hypothesis The positive association between low HRV and worse prognosis found inpatients with colorectal cancer receiving palliative treatment but not in patients receivingcurative treatment suggest that this relation is not bidirectional but that advanced cancer isassociated with low HRV Midlatestage tumours are often accompanied by damage to autonomic nerves resulting in decreased HRV [] A study of De Couck showed that cancerpatients in general have a significantly lower HRV than healthy people [] The same resultswere found in studies of Bijoor where RMSSD was found to be significantly lower inpatients with early and advanced stage cancer compared to a healthy control group []When comparing patients with advanced stage cancer TNM III and IV to patients with anearly stage of cancer TNM I and II RMSSD was found to be significantly lower in patientswith advanced stages of cancer [] Thus though experimental studies in animals showthat vagal nerve functioning can causally slow tumorigenesis the human data suggests that themalignant tumour causes vagal nerve dysfunction and therewith decreased HRV []Besides the proposed influence of low HRV on survival of colorectal cancer patientsthrough development and increased progression of cancer Reimer suggested that lowHRV could influence survival of those undergoing surgical treatment due to more and moresevere postoperative complications [] However the results found in this study were notconcurrent with those of Reimer who included patients with ASA undergoingelective surgery Their analysis of HRV was through powerspectrum parameters based on longer ECGrecordings instead of the timedomain parameters based on 10s ECGs used in thisstudy But the difference in used parameters used in both studies is probably not the explanation for the differences in results between both studies since it has been demonstrated thatRMSSD and SDNN based on ECG recordings of 10s are in substantial agreement with those of45min and a 10s ECG therefore suffices to determine time domain HRV parameters HoweverReimer did not correct for possible confounders In their study patients with low HRVwere more likely to have diabetes a known risk factor for postoperative ileus and wound infections both found to be common postoperative complications in their low HRV group Correcting for this comorbidity may change the significance of their findings [] A study ofPLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancerTable Low HRV and severity of postoperative complications according to ClavienDindo classificationUnadjustedMinor grade I and IIOR CIpMajor grade III IV VOR CISDNN 20msn “n “SDNN �20msn referencen referenceAdjusted�SDNN 20msn “SDNN �20msn referenceUnadjustedRMSSD 19msn “RMSSD �19msn referenceAdjusted�n “n referencen “n referenceRMSSD 19msn “n “RMSSD �19msn referencen referencep�adjusted for heart rate age categories vs � and hypertension101371journalpone0237244t006Scheffler did not show any significant preoperative differences in HRV between patientswith or without postoperative complications or between patients with postoperative complications of different severity levels [] Thus also in relation to predicting postoperative complications results are not uniform

cancer266

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: Is preoperative heart rate variability aprognostic indicator for overall survival andcancer recurrence in patients with primarycolorectal cancerM T A StrousID1 A M Daniels1 F M Zimmermann2 F N van Erning3 Y Gidron4 FJ Vogelaar1 Department of Surgery VieCuri Medical Centre Venlo The Netherlands Department of CardiologyCatharina Hospital Eindhoven The Netherlands Department of Research Netherlands ComprehensiveCancer anisation Utrecht The Netherlands Faculty of Welfare and Health University of Haifa HaifaIsrael maudstrousviecurinlAbstracta1111111111a1111111111a1111111111a1111111111a1111111111 ACCESSCitation Strous MTA Daniels AM ZimmermannFM van Erning FN Gidron Y Vogelaar FJ Ispreoperative heart rate variability a prognosticindicator for overall survival and cancer recurrencein patients with primary colorectal cancer PLoSONE e0237244 101371journalpone0237244Editor Louise Emilsson University of OsloNORWAYReceived February Accepted July Published August Copyright Strous This is an access distributed under the terms of theCreative Commons Attribution License whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are creditedData Availability Statement Data cannot beshared publicly because of ethical concernsPatients were included on a no objection base toconduct retrospective data studies and publishfindings but were not asked for permission topublish full encrypted data Data are available fromthe VieCuri Institutional Data Access contact viawetenschapsbureauviecurinl for researcherswho meet the criteria for access to confidentialdataBackgroundHeart Rate Variability HRV represents efferent vagus nerve activity which is suggested tobe inversely related to fundamental mechanisms of tumorigenesis and to be a predictor ofprognosis in various types of cancer HRV is also believed to predict the occurrence andseverity of postoperative complications We aimed to determine the role of preoperativeHRV as a prognostic factor in overall and cancer free survival in patients with colorectalcancerMethodsRetrospective analysis was performed in a detailed dataset of patients diagnosed with primary colorectal cancer between January and December who underwent curative surgical treatment HRV was measured as timedomain parameters SDNN StandardDeviation of NNintervals and RMSSD Root Mean Square of Successive Differencesbased on preoperative second ECGs Groups were created by baseline HRV Low HRVSDNN 20ms or RMSSD 19ms and normal HRV SDNN �20ms or RMSSD �19msPrimary endpoints were overall and cancer free survivalResultsA total of patients were included in this study HRV was not significantly associated withoverall survival SDNN 20ms vs SDNN �20ms244 vs adjusted HR “ p RMSSD 19ms vs RMSSD �19ms270 vs adjustedHR “ p or cancer recurrence SDNN 20ms vs�20ms201 vs adjusted HR “ p RMSSD 19ms vs�19ms vs adjusted HR “ p There was noPLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancerFunding The authors received no specific fundingfor this workCompeting interests The authors have declaredthat no competing interests existsignificant association between HRV and CEAlevel at one year followup or between HRVand occurrence of a postoperative complication or the severity of postoperativecomplicationsConclusionsHeart rate variability was not associated with overall or cancer free survival in patients withprimary colorectal cancer who underwent curative surgical treatment These results do notalign with results found in studies including only patients with advanced cancer which suggests that there is only an association in the other direction cancer causing low HRVIntroductionIn there were over million newly diagnosed colorectal cancer patients worldwide andover in the Netherlands alone It is the fourth most common cause of death worldwide[] To improve survival it is of importance to get a better insight into modifiable prognosticfactors Emerging evidence suggests that vagal nerve activity indexed by heart rate variabilityHRV could be one of these prognostic factors [“]HRV is the physiological phenomenon of the fluctuation in time intervals between adjacentheartbeats and represents efferent vagus nerve activity to the heart [“] It has been suggestedthat efferent vagal activity is inversely related with fundamental mechanisms of tumorigenesisas inflammation oxidative stress and excessive sympathetic activity [] These mechanisms arebelieved to be controlled by the vagus nerve via a bidirectional braintoimmune pathwaysthrough the release of neurotransmitters via the cholinergic antiinflammatory pathway[] A higher vagal tone may reflect a more flexible topdown regulation of the immunesystem and physiological activity moderated by the brain [] Absence of vagus activity due tovagotomy has been shown to increase the risk of developing colorectal cancer []In addition to influencing development of cancer vagus nerve activity seems to be a predictor of prognosis in various types of cancer Recent studies show an association betweendecreased activity of the vagus nerve and worse survival in patients with cancer of the gastrointestinal tract liver pancreas lung prostate and breast among others [] Also patientswith normal HRV seem to live longer in different sorts of metastatic cancer independent ofconfounders [] In patients with colorectal cancer a low HRV at baseline has shown to beassociated with higher CEA levels at months after diagnosis which predicts a poorer prognosis []In patients undergoing curative treatment for colorectal cancer HRV does not only seemto influence cancer prognosis A recent study showed that patients with lower HRV have moreintraoperative blood loss and more and more severe postoperative complications []Identifying patients with low HRV is easy and noninvasive When its predictive value forthe prognosis of cancer patients is of satisfactory significance vagus nerve activation prior toor during cancer treatment could theoretically be beneficial in improving prognosis [] Alsoif we could predict the occurrence and severity of postoperative complications based on HRVimproving HRV before surgery could possibly accelerate postoperative recovery and indirectlyaffect patients™ prognosis Recent studies focussing on improving HRV by improving physicalfitness by means of physical exercise show promising results in both older men and woman[] However the only previous study on colon cancer and HRV including patients receiving curative treatment included a small sample and did not examine whether HRV predictsPLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancersurvival in these patients [] To clarify the predictive value of HRV in prognosis of patientswith colorectal cancer further exploration is needed Current studies identifying HRV as aprognostic factor did not specifically focus on colorectal cancer have small study populationsdid not correct for confounders and mainly focused on metastatic disease [“]The aim of this study was to determine the role of preoperative HRV as a prognostic factorin overall and cancer free survival in patients with primary colorectal cancer who underwentcurative surgical treatmentMethodsData collectionData from the Netherlands Cancer Registry NCR were used The NCR collects data on allnewly diagnosed cancer patients in the Netherlands Information on patient and tumour characteristics diagnosis and treatment is routinely collected from the medical records by trainedadministrators of the cancer registry Anatomical site of the tumour is registered according tothe International Classification of Diseases Oncology The tumournodemetastasis TNMclassification is used for stage notification of the primary tumour according to the editionvalid at time of cancer diagnosis Quality of the data is high due to thorough training of theregistration team and consistency checks []For the study population additional data were collected from the medical records of thepatients This encompassed information on HRV CEAlevels ASA classification comorbidities identified at admission divided into groups cardiac disease hypertension diabetes mellitus thyroid disease pulmonary disease vascular disease neurological disease and otheroccurrence and severity of postoperative complications and cancer recurrence Groups ofcomorbidities were chosen based on matching features within these groups and their potentialinfluence on HRV or the endpoint being analysed Severity of the postoperative complicationsaccording to the ClavienDindo classification was also documented Medical records wereassessed between January and July and reevaluated for revision of this between the 20th and 25th of April This study was approved by the research committee and the Board of Directors of VieCuriMedical Centre Data was obtained under the law ˜scientific research and statistics in the interest of public health where asking for permission is not possible or appropriate for several reasons™ in the Netherlands unless patients objected to use of their personal medical record forscientific research Data was encrypted with an encryption key provided by the NCR Encryption was shortly lifted to access the patients™ number for accessing hisher medical record Following extraction data were encrypted againStudy populationThe study population included all consecutive patients diagnosed with primary colorectal adenocarcinoma between January and January at VieCuri Medical Centre who underwent curative surgical treatment Patients with metastatic disease at time of surgery orcarcinoma in situ were excluded as their treatment and prognosis differs from those receivingcurative treatment for colorectal cancer Metastasis found within months after surgery wereconsidered present at time of surgery and therefore excluded Other excluded patients werepatients with neuroendocrine tumours because of different tumour characteristics and prognosis patients with cardiac arrhythmias including atrial and ventricular extrasystole pacemakers patients taking betablockers as this enhances HRV indexes or patients withbradycardia heart rate bpm or tachycardia heart rate bpm as this precluded reliable calculation of HRV [] We did not exclude patients taking alphablockers calciumPLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancerinhibitors diuretics amiodarone ACEinhibitors or ARB™s as these types of medicationreduce central sympathetic functioning rather than peripheral and their influence on HRV istherefore less univocal and sometimes completely absent []Heart rate variabilityHeart rate variability was analysed using a 12lead 10second ECG 150Hz used for preoperative screening In case of multiple ECG™s per patient the most recent ECG before date of surgery was used for HRVanalysis In case of multiple ECG™s per patient on the same date theECG with the best quality was chosen meaning an ECG without motion artefacts In case ofmotion artefacts there was always an ECG without motion artefacts available recorded on thesame date Time between two consecutive Rpeaks was measured in lead II with an accuracy of02ms using MUSEECG HRV was presented using the timedomain HRV parameters SDNNStandard Deviation of NNintervals and RMSSD Root Mean Square of Successive Differences in milliseconds calculated using the following calculations []rSDNN ¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffii¼1 ðRRi 00 RRmeanÞ2Pnn 00 RMSSD ¼rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffii¼1 ðRRiþ1 00 RRiÞ2Pn 00 n 00 ð1Þð2ÞSDNN and RMSSD obtained from 10s ECGs were found to correlate with results of ECGsof longer durations Power spectral analysis HRV parameters as LF and HF can only beobtained in longer recording ECGs and were therefore not implementable in this study[]SDNN and RMSSD were both analysed as continuous variables as well as binary variablesusing cutoffs of 20ms versus �20ms and 19ms versus �19ms respectively In case of anSDNN 20ms or RMSSD 19ms HRV was classified as low and in case of SDNN �20ms orRMSSD �19ms as normal These cutoff values were based on cutoff values used in otherstudies showing an association between lowHRV as SDNN 20ms and RMSSD 19ms andcolorectal cancer as there is no standardised definition of low and normal HRV []Endpoints and definitionsThe primary endpoints of this study were overall and cancer free survival Overall survival wasdefined as the time between the date of surgery to the date of death or last followup date inmonths Cancer free survival was defined as the time in months from the date of surgery untilthe date of cancer recurrence defined as the first date of either radiologic or pathologic diagnosis of metastases or tumour recurrence of colorectal cancer Patients dying without cancerrecurrence were censored on day of death Secondary endpoints were elevated CEAlevel ugl at oneyear followup occurrence of postoperative complications within daysafter surgery and severity of postoperative complications according to the ClavienDindoclassificationStatistical analysisIn this retrospective observational cohort study we utilized descriptive statistics to provide anoverview of control variables of the study population patient characteristics as age sex BMIcomorbidities and ASAclassification heart rate and tumour characteristics as TNMstagetumour localisation and tumour differentiation and their association with HRV andPLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancerprognosis Normal distribution of the continuous variables heart rate age and BMI as well asSDNN and RMSSD were tested with a KolmogorovSmirnov test Because of normal distribution heart rate age and BMI were compared between HRVgroups using unpaired ttest Allother variables were categorical and were compared between HRVgroups using Chisquarestatistics as groups were all of sufficient powerDifferences in overall survival and cancer free survival in months according to SDNN andRMSSD were visualized by means of KaplanMeier curves and statistically tested using the logrank test Multivariate coxregression analyses were conducted to calculate the prognosticassociation between HRV and overall and cancer free survival while adjusting for other prognostic variables Multivariate logistic regression was used to assess the independent effect ofSDNN and RMSSD on CEAlevels and the occurrence and severity of postoperative complications Variables included for adjustment were chosen by means of forward stepwise selectionbased on clinical judgment differences at baseline eg differences on any predictor betweenpatients who later died or not and database availability and depended on the analysed endpoint Those included patient demographics age sex bodymassindex comorbidities identified at admission divided into groups cardiac disease hypertension diabetes mellitus thyroiddisease pulmonary disease vascular disease neurological disease other including Crohn™sdisease hepatitis kidney failure disorders anaemia depression arthritis tumour characteristics localisation stage differentiation and the occurrence of postoperative complicationswhen the later was not an outcome Differences in CEAlevel at baseline and one year checkup between and within groups of low HRV and normal HRV were assessed with a repeatedmeasures linear model and tested using the tukey test To test the implication of a longer timebetween ECG and treatment all analyses were repeated after excluding patients with an ECGolder than months A twotailed pvalue � was considered significant in all analysesData were analysed using IBM SPSS Statistics version IBM Corp NY Armonk USAResultsOf colorectal cancer patients that underwent a surgical resection a total of patientswere included in this study Reasons for exclusion are presented in Fig Median SDNN andRMSSD were 204ms interquartile range IQR 115ms351ms and 175ms IQR 99ms299ms respectively Table shows descriptive data of the included patients by HRV groupsBaseline heart rate and age were negatively associated with HRV The group of patients withlow HRV contains more patients with a history of cardiac disease regardless of the HRV defining parameter When defining low HRV by RMSSD 19ms more patients in this group havehypertension as comorbidity This group also contains more patients with an ASA classification greater than oneDuring a median followup of months IQR “ all causedeath occurred in patients Cancer recurrence occurred in patients during a median followup of months IQR “To rule out any distort in results caused by a delay between ECG and treatment all analyseswere repeated after exclusion of ECG™s older than months This did not lead to any new significant results Therefore these results were not displayed in detail in this paperSurvivalIn low HRV groups slightly more patients died compared to normal HRV groups SDNN20ms versus �20ms versus respectively RMSSD 19ms versus�19ms versus respectively These observed differences between HRVgroups in overall survival were not significant Fig A SDNN p B RMSSDPLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancerFig Flowchart of the study101371journalpone0237244g001p After adjustment for some potential confounders these associations remained notsignificant SDNN 20ms versus �20ms HR “ p and RMSSD19ms versus �19ms HR “ p Tables and Age cardiac disease tumour stage and postoperative complications had a significant influence on overall survival Age also differed at baseline and was identified as a confounder When defining low andnormal HRVgroups by SDNN cardiac disease was identified as confounder When conducting sensitivity analyses with SDNN and RMSS as continuous variables results remained thesame There was no association between HRV and overall survival SDNN HR “ p and RMSSD HR “ p In low HRV groups slightly more patients had recurrence of cancer compared to normalHRV groups SDNN 20ms versus �20ms versus respectivelyRMSSD 19ms versus �19ms versus respectively These observed differences between HRV groups in cancer free survival were not significant Fig A SDNNp B RMSSD p As in overall survival after adjustment for some potentialconfounders these associations remained not significant SDNN 20ms versus �20msHR “ p and RMSSD 19ms versus �19ms HR “ p Tables and In SDNNbased groups baseline heart rate was identified asa confounding variable Sensitivity analyses with SDNN and RMSSD as continuous variablesPLOS ONE 101371journalpone0237244 August PLOS ONE 0cTable Descriptive data of included patients according to prespecified HRV groupsSDNN 20ms n SDNN �20ms n pRMSSD 19ms n RMSSD �19ms n pHRV and prognosis in colorectal cancerHeart Rate�Age�Age n year� yearSex nMaleFemale [“] [“] [“] [“] [“] [“] [“] [“] Mean BMI SD [“] [“] [“] [“]Comorbidities nCardiac diseaseHypertensionDiabetes MellitusThyroid diseasePulmonary diseaseVascular diseaseNeurological diseaseOtherASA nASA1ASA2ASA34Localisation tumour nRight colonLeft colonRectumTumour stage nIIIIII Differentiation grade nWellmoderate Poorundifferentiated � Non normaldistributed data presented as median with interquartile range101371journalpone0237244t001did not alter these results There was no association between HRV and cancer free survivalSDNN HR “ p and RMSSD HR “p CEAlevelCEAlevel at baseline and one year checkup was registered in patients and elevated in of these patients This was elevated at one year checkup in only patients Low HRV was notsignificantly associated with elevated CEAlevels at one year checkup as shown in Table Sensitivity analyses with SDNN and RMSSD as continuous variables did not alter these resultsSDNN HR “ p and RMSSD HR “PLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancerFig KaplanMeier curves for overall survival in groups of low HRV and normal HRV presented as A SDNN andB RMSSD101371journalpone0237244g002PLOS ONE 101371journalpone0237244 August PLOS ONE 0cTable Multivariate analyses of associations of SDNN and covariates with overall and cancer free survivalHRV and prognosis in colorectal cancerSDNN20ms�20msHeart RateAgeBMICardiac diseaseNoYesHypertensionNoYesASAclassificationASA1ASA2ASA34LocalisationRight colonLeft colonRectumTumour stageIIIIIIOverall survivalHR CIpCancer free survivalHR CI “ “ reference “ “ “ reference “ “ “p reference reference “ “ reference reference “ “ reference “ “ reference “ “ reference “ “ “ “ reference “ “ reference “ “Postoperative complicationNoYes reference reference “ “101371journalpone0237244t002p Adjusting for covariates was not possible because of the small number of patientswith an elevated CEAlevelDifferences between CEAlevel at baseline and one year checkup were compared betweenand within HRVgroups Results were displayed in Fig There were no significant differencesin CEAlevel at baseline and one year checkup between the low HRV group and normal HRVgroup defined by SDNN and RMSSD p and p respectively Also there were nosignificant differences in CEAlevel at baseline and at one year checkup within the low HRVgroup and normal HRV group defined by SDNN and RMSSD p and p respectivelyPostoperative complicationsIn patients one or more postoperative complications occurred within days after surgeryThe occurrence of a postoperative complication was not significantly associated with lowHRV defined as SDNN 20ms or RMSSD 19ms even after adjustment for some potentialconfounders Table Heart rate age cardiac disease and hypertension were identified asconfounding covariates When conducting sensitivity analyses with SDNN and RMSS as continuous variables the association between occurrence of a postoperative complication withPLOS ONE 101371journalpone0237244 August PLOS ONE 0cTable Multivariate analyses of associations of RMSSD and covariates with overall and cancer free survivalHRV and prognosis in colorectal cancerRMSSD19ms�19msHeart RateAgeBMICardiac diseaseNoYesHypertensionNoYesASAclassificationASA1ASA2ASA34LocalisationRight colonLeft colonRectumTumour stageIIIIIIOverall survivalHR CIpCancer free survivalHR CI “ “ reference “ “ “ reference “ “ “p reference reference “ “ reference reference “ “ reference “ “ reference “ “ reference “ “ reference “ “ reference “ “ reference “ “Postoperative complicationNoYes reference reference “ “101371journalpone0237244t003baseline HRV remained not significant SDNN HR “ p andRMSSD “ p The same applied when postoperative complicationswere graded according to the ClavienDindo classification and the complication that is gradedthe highest for each patient is compared to the absence of postoperative complicationsTable DiscussionIn this observational cohortstudy we determined the Heart Rate Variability in preoperativeECGs of patients with primary colorectal cancer who underwent curative surgical treatment HRV refers to physiological variations in beattobeat intervals It was presented intimedomain parameters SDNN and RMSSD HRV was not significantly associated with overall survival or cancer free survival independent of some risk factors Also this study showedno significant differences in CEAlevels at one year checkup between patients with low HRVand patients with normal HRV Patients with low HRV did not have more or more severepostoperative complications compared to patients with normal HRVTumorigenesis has three fundamental mechanisms inflammation promoting oxidativestress and stimulating tumour growth oxidative stress causing DNAdamage andPLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancerFig KaplanMeier curves for cancer free survival in groups of low HRV and normal HRV presented as A SDNNand B RMSSD101371journalpone0237244g003PLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancerTable Univariate analyses of low HRV and risk of elevated CEAlevel at one year checkupCEA μglOR CIpCEA μglOR CIIndependent of baseline CEAIndependent of baseline CEASDNN 20ms n “RMSSD 19ms n “SDNN �20ms n Normal baseline CEA � μglSDNN 20ms n SDNN �20ms n Elevated baseline CEA μglSDNN 20ms n reference “ reference “RMSSD �19ms n Normal baseline CEA � μglRMSSD 19ms n RMSSD �19ms n Elevated baseline CEA μglRMSSD 19ms n reference “ reference “SDNN �20ms n referenceRMSSD �19ms n referencep101371journalpone0237244t004interfering with subsequent repair mechanisms and sympathetic neurotransmitters stimulating tumour metastasis and progression [] It has been suggested that afferent fibres of thevagus nerve inform the brain about peripheral inflammation This is followed by a braintoimmune response via the efferent route of the vagus nerve that modulates the function ofimmuneregulatory cells through the release of neurotransmitters via the cholinergic antiinflammatory pathway Malfunctioning of this route may play part in the onset of cancer []This theory has been supported by studies of patients with gastric ulcers who underwent avagotomy who then showed an increased risk of developing lung and colorectal cancer [“] The vagus nerve is also believed to modulate tumour progression An active vagus nervecan inhibit inflammation oxidative stress and the release of sympathetic neurotransmittersthat stimulate tumour metastasis and progression [“] Absence of this inhibitory effect inturn results in metastasis and tumour progression as shown in a study of Erin showingthat mice who underwent chemical vagotomy developed more metastasis of breastcancer cellsthan controls [] Epidemiologically low HRV has been associated with worse overall survivalover time in patients with recurrent or metastatic cancer of various types even after correctionfor confounders [“] However the results of the present study do not support thesefindingsTo our knowledge this is the first study including only patients with colorectal cancer whoare eligible for curative treatment by partial bowel resection and not those receiving palliativetreatment De Couck studied the relationship between HRV and tumour burden in bothcurative and palliative patients with prostate cancer or nonsmall cell lung cancer Independent of confounders the hypothesised inverse relationship of HRV and the tumour markerPSA at and months after diagnosis was only significant in patients with stage IV prostatecancer not stage II and III [] In colorectal cancer Mouton found that low HRV definedas SDNN ms predicted significantly higher levels of the tumour marker CEA at months after diagnosis Again these results were only found in patients receiving palliativetreatment not curative [] Only one previous study showed a significant inverse relationshipbetween HRV and mortality in cancer in general independent of stage [] This study of Guo had a large study population of patients with various types of cancer Low HRV wasdefined as SDNN 70ms and was significantly associated with shorter survival times Thissuggests that HRV is a predictive indicator of survival time not only in palliative but also incurative patients However results were not controlled for cancer type which could affect bothHRV and survival and should therefore be interpreted with caution [] The fact that thepatients recruited in the present sample were only with less advanced cancer may partlyexplain the lack of prognostic role in HRV in this samplePLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancerFig Bar chart for CEAlevel at baseline and one year checkup in groups of low HRV and normal HRV presented as ASDNN and B RMSSD101371journalpone0237244g004PLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancerTable Low HRV and risk of occurrence of a postoperative complication within daysPostoperative complicationOR CIUnadjustedSDNN 20ms n “SDNN �20ms n referenceAdjusted�SDNN 20ms n “SDNN �20ms n referenceUnadjustedRMSSD 19ms n “RMSSD �19ms n referenceAdjusted�RMSSD 19ms n “RMSSD �19ms n reference�adjusted for heart rate age cardiac disease and hypertension101371journalpone0237244t005pSome of these previous studies suggest a bidirectional relationship between vagus nerveactivity and cancer [] However based on current evidence on this subject we cannot supportthis hypothesis The positive association between low HRV and worse prognosis found inpatients with colorectal cancer receiving palliative treatment but not in patients receivingcurative treatment suggest that this relation is not bidirectional but that advanced cancer isassociated with low HRV Midlatestage tumours are often accompanied by damage to autonomic nerves resulting in decreased HRV [] A study of De Couck showed that cancerpatients in general have a significantly lower HRV than healthy people [] The same resultswere found in studies of Bijoor where RMSSD was found to be significantly lower inpatients with early and advanced stage cancer compared to a healthy control group []When comparing patients with advanced stage cancer TNM III and IV to patients with anearly stage of cancer TNM I and II RMSSD was found to be significantly lower in patientswith advanced stages of cancer [] Thus though experimental studies in animals showthat vagal nerve functioning can causally slow tumorigenesis the human data suggests that themalignant tumour causes vagal nerve dysfunction and therewith decreased HRV []Besides the proposed influence of low HRV on survival of colorectal cancer patientsthrough development and increased progression of cancer Reimer suggested that lowHRV could influence survival of those undergoing surgical treatment due to more and moresevere postoperative complications [] However the results found in this study were notconcurrent with those of Reimer who included patients with ASA undergoingelective surgery Their analysis of HRV was through powerspectrum parameters based on longer ECGrecordings instead of the timedomain parameters based on 10s ECGs used in thisstudy But the difference in used parameters used in both studies is probably not the explanation for the differences in results between both studies since it has been demonstrated thatRMSSD and SDNN based on ECG recordings of 10s are in substantial agreement with those of45min and a 10s ECG therefore suffices to determine time domain HRV parameters HoweverReimer did not correct for possible confounders In their study patients with low HRVwere more likely to have diabetes a known risk factor for postoperative ileus and wound infections both found to be common postoperative complications in their low HRV group Correcting for this comorbidity may change the significance of their findings [] A study ofPLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancerTable Low HRV and severity of postoperative complications according to ClavienDindo classificationUnadjustedMinor grade I and IIOR CIpMajor grade III IV VOR CISDNN 20msn “n “SDNN �20msn referencen referenceAdjusted�SDNN 20msn “SDNN �20msn referenceUnadjustedRMSSD 19msn “RMSSD �19msn referenceAdjusted�n “n referencen “n referenceRMSSD 19msn “n “RMSSD �19msn referencen referencep�adjusted for heart rate age categories vs � and hypertension101371journalpone0237244t006Scheffler did not show any significant preoperative differences in HRV between patientswith or without postoperative complications or between patients with postoperative complications of different severity levels [] Thus also in relation to predicting postoperative complications results are not uniform Answer:

Thyroid_Cancer

Identification and treatment of obstructive sleep apnea by a primarycare team with a subset focus on chronic painmanagement within the paper and its Supporting InformationfilesFunding The authors received no specific fundingfor this workCompeting interests The authors have declaredthat no competing interests existBackgroundPatients diagnosed with obstructive sleep apnea OSA who also consume prescription opioids have a greater likelihood of morbidity and mortality This study evaluated whether a primary care team focused on chronic pain care management could use a validatedquestionnaire STOPBang and motivational followup to increase identification and treatment of OSAMethodsThis study was a retrospective dual arm prepost controlled study Participants of thisstudy included the complete chronic pain management sub group treated by this primarycare team Participants were � years old and prescribed daily opioids for treatment ofchronic pain All participants had a multifaceted individualized educational meeting thatincluded completing a STOPBang questionnaire Participants who received a score �three were advised to follow up with their primary care physician Participants were seenquarterly throughout the studyResultsThe primary outcome of this study was that of participants with likely OSA were usingCPAP for a minimum of months range of “ months 18month average postintervention vs CPAPuse in the control group months of observation both groupswere chronic opioid users with OSA This was a relative improvement p Asecondary outcome was that of nonprior CPAP users obtained CPAP post intervention a prepost improvement p x2 with degree of freedom Alsoparticipants who were likely to have OSA STOPBang score � or had a positive polysomnography AHI with comorbidities compared to those unlikely to have OSA STOPPLOS ONE 101371journalpone0237359 August PLOS ONE 0cIdentification and treatment of obstructive sleep apnea by a primary care chronic pain management teamBang score or had a negative polysomnography AHI in this study were more likelyto be male have a higher BMI have hypertension have cardiovascular disease andorhave diabetes all typesConclusionTeam based care management for participants taking prescription opioids where STOPBang questionnaires were completed were associated with an increase in the identificationand treatment of OSAIntroductionStudy rationaleThe objective of this study was to evaluate whether a teambased care management intervention would increase identification and treatment of obstructive sleep apnea in participants taking medication for chronic painCurrent knowledgeObstructive sleep apnea OSA is a chronic sleep disorder characterized by episodes of apneasand hypopneas or the complete or partial collapse of the upper airway [] A diagnosis ismade when a patient has or more of these events in an hour or five of these events if otherpredicting symptoms for OSA are present [] Approximately million American adults havethis sleep disorder Patients with moderate to severe OSA are often treated with a continuouspositive airway pressure device or CPAP Treatment can reduce the risks of hypertension coronary artery disease heart failure arrhythmias sudden cardiac death and stroke [] A validated questionnaire STOPBang score can be used as a screening tool to identify patients atrisk for OSA A STOPBang sleep questionnaire is commonly distributed to patients whoshow signs of having potential risk factors for OSA œSTOP questions snoring tirednessobserved apnea and high blood pressure and œBang BMI kgm2 Age years Neckcircumference cm and Gender male [ “] A score of three or higher on STOPBangplaces patients at higher risk of OSA requiring further evaluation which includes an overnightpolysomnogram or sleep study to confirm diagnosis []Prescription opioid use for pain management is still a common practice in primary care settings though the rates of overdose due to these drugs in the United States are at an unprecedented high with deaths in [“] Patients using opiates for chronic painmanagement have also been found to have a greater likelihood of developing central sleepapnea which worsens the severity of OSA and ataxic breathing [] A relationship existsbetween pain and sleep disruption with over of patients who have chronic pain havingreported difficulty sleeping which increases hyperalgesia [ ] An analgesic effect may bepresent where pain creates sleep disorders and a shortage of sleep increases pain [ ]Patients with OSA who are then prescribed opioids have an increased risk of opioidinducedrespiratory depression OIRD [] Despite the potential morbidity of the combination ofOSA central sleep apnea and Opioid use studies have shown that those that have this combination only remain on definitive treatment for their OSA CPAP of the time [] It istherefore of the utmost importance that patients who chronically use opioids are evaluated forOSA and those diagnosed adhere to CPAPPLOS ONE 101371journalpone0237359 August PLOS ONE 0cIdentification and treatment of obstructive sleep apnea by a primary care chronic pain management teamIn order to decrease mortality it is necessary to educate patients on the risks and healthconcerns related to taking opioids Brief interventions can influence behavior of patients whoare at high risk for abuse of a substance [] Individualized nurseled management educationis a tool that can be used as a brief intervention within a teambased approach [] These individualized information gathering motivational meetings allow patients to experience empathy as their medical needs are met [ ] Individualized nurseled management educationis a tool that can be used as a brief intervention within a teambased approach [] Instructionand guidance can be coupled with an assessment of a patient™s potential risk factors for comorbidities such as OSA using a STOPBang questionnaireMaterials methodsDesignThis study was a retrospective dual arm prepost controlled study to evaluate whether anintervention STOPBang questionnaire education and motivational followup wouldimprove OSA diagnosis and treatment compliance The first investigation arm compared thecontrol group daily opioid use with OSA to the part of the intervention group with OSA onCPAP or at risk for OSA STOPBang � This arm™s purpose was to determine whether theintervention increased treatment compliance with CPAP The second investigation armcompared the study group to itself prepost intervention This arm evaluated whether applyingthe intervention increased diagnosis and treatment of OSA The single independent variablein this study was a participant™s admission to the chronic pain management subgroup withthe administration of a STOP Bang questionnaire and the dependent variable was CPAP useInclusion criteria consisted of ongoing participation in primary care pain management subgroup daily opioid use and an age � This study occurred between October and January which included monthsfor exposure and then at least months of follow up One of Intermountain Healthcare™s outpatient facilities in Utah served as the site for this study The total number of patients duringthe duration of the study who entered into the pain management sub group � years oldand were taking a daily dose of opioids was Out of the identified participants participants remained in the clinic during the entirety of this study Fig Ethics approvalIRB Number for this study was obtained from the Institutional Review Board ofIntermountain Healthcare Consent was waived for this retrospective studyControlsA Medline and Pubmed basic search from “ was used to identify a historical yet contemporary United States based control group that had Obstructive Sleep Apnea and met theinclusion criteria of being � years old and were taking a daily dose of opioids The controlthat was chosen was comparable to this study™s intervention group with regard to Race AgeGender Morphine Equivalent Daily Dose and Comorbidities Also the control used camefrom a study that lasted a similar duration []SubjectsEach participant received an information gathering and educational session with a registerednurse in the primary care facility at the time of their admittance into the chronic pain management program between “ During this session a full pain history was obtained fromparticipants All participants in the study underwent STOPBang screening All participantswho were at high risk for OSA STOPBang � were referred back to their primary carePLOS ONE 101371journalpone0237359 August PLOS ONE 0cIdentification and treatment of obstructive sleep apnea by a primary care chronic pain management teamFig Participant retention101371journalpone0237359g001physician Participants and physicians made joint decisions during a followup appointmentwhether to pursue polysomnography Reasons why participants did not complete polysomnography or attain treatment for OSA are that the participants refused workup there was ashared decision due to participant frailty had cancer end stage or were noncompliant toCPAP Fig Fig participants at high risk for sleep apnea STOPBang score � who did not undergo diagnostic evaluation orcomplete treatment101371journalpone0237359g002PLOS ONE 101371journalpone0237359 August PLOS ONE 0cIdentification and treatment of obstructive sleep apnea by a primary care chronic pain management teamEach participant received initial inperson education with the same registered nurse regarding nonpharmacological treatment of pain selfhelp groups risks and benefits of treatment ofpain management compliance with treatment of pain and its comorbidities and informationabout Naloxone an opioid reversal agent This education was provided in a hour sessionusing the FRAMES Feedback Responsibility Advise Menu for Change Empathy andEnhancing Self Efficacy motivational method [] A chronic pain management agreementwas executed Additional information obtained during this session included a Current OpioidMisuse Measure and a STOPBang questionnaire All participants who received a score greaterthan or equal to three on the STOPBang were told to follow up with their primary care physician All primary care providers were made aware they would receive electronic data onSTOPBang questionnaires and participants with scores � would make a follow up appointment with them Upon completion of this session information was recorded electronically andtransmitted to the primary care physician on record Each participant also had quarterly inperson followup appointments with their primary care physician In addition quarterly inperson or phone visits with the pain management nurse were continued Physician visits varied in length and scope Individualized nursing visits that continued to use the FRAMESmethod and lasted for a duration of minutes were focused on remaining compliant with allongoing therapies The nurse would document the interaction and any concerns about compliance with any therapy in the patient™s EMR Electronic Medical Record at the conclusionof the visits The information was based on the subjective input received from the participantQuarterly followup remained consistent throughout the study Attending all followupappointments both with the primary care physician and the pain management nurse was aprerequisite to remain in the pain management group Remaining in the pain managementwas group was the only avenue to receive controlled pain medication at this facilityData collectionThe baseline biometric parameters of Race Gender BMI Age Tobacco Use Alcohol Use andMorphine Equivalence Daily Dosage MEDD were extracted from each participant™s electronic medical record EMR Collected data also included information on Chronic DiseaseHypertension Cardiovascular Disease Pulmonary Disease Thyroid Disease Diabetes Gastroesophageal Reflux Disease and Chronic Kidney Disease and specific medication typesBenzodiazepines and Pregabalin STOPBang scores were gathered during the initial chronicpain management encounter when participants joined the study Subjective data on CPAPadherence was found in the EMR documentation of the pain management nurse and primarycare physician follow up reports postinclusion in the studyStatistical methodsMeans and standard deviations were calculated for demographic variables The DAgostinoPearson test was used to confirm normality [] Unequal variance ttesting was used in the statistical analysis of both arms of this study to compare Age BMI and Morphine EquivalentDaily Dosing Two proportion Ztesting was used to compare the control group daily opioiduse with OSA to the participants in the intervention group with OSA on CPAP or at highrisk for OSA STOPBang � with regards to CPAP use Race Gender Diabetes Cardiovascular Disease and Hypertension Two proportion Ztesting was also used to compare the participants not likely to have OSA STOPBang or had a negative polysomnography to thosewith OSA on CPAP or at high risk for OSA STOPBang � not completing polysomnography or not compliant with CPAP A x contingency table using McNemar™s test with correction for continuity was used to compare the participants™ CPAP use prepost intervention []PLOS ONE 101371journalpone0237359 August PLOS ONE 0cIdentification and treatment of obstructive sleep apnea by a primary care chronic pain management teamIRB approvalIRB Approval was granted from Intermountain Healthcare IRB IRB Number ResultsThe primary outcome of this study was that of participants with likely OSA were usingCPAP for a minimum of months range of “ months 18month average postintervention vs CPAP use in the control group months of observation which were chronic opioid users with OSA This was a relative improvement p A secondary outcomewas that of nonprior CPAP users obtained CPAP post intervention a prepostimprovement p x2 with degree of freedom Fig This resulted in ofintervention participants being treated with CPAP Comparing the average number of intervention participants with OSA on CPAP or at risk for OSA STOPBang � to the control dataset resulted in significant differences for BMI and Male Gender Otherdemographics between these two groups Age p MEDD p Race Caucasianp Hypertension p Cardiovascular Disease p and Diabetes all formsp showed no statistically significant differences between groups Comparing the averagenumber of intervention participants with OSA on CPAP or at risk for OSA STOPBang � to the average number of intervention participants not likely to have OSA STOPBang orhad a negative polysomnography resulted in the following statistically significant differencesBMI p Male Gender p00001 Hypertension p Cardiovascular Diseasep and Diabetes all forms p Other demographics between these two groupsAge p MEDD p Race Caucasian p Benzodiazepine Use p Pregabalin Use p and Active Chronic Disease States Gastroesophageal Reflux p Chronic Kidney Disease Thyroid Disease p and Pulmonary Diseasep showed no statistically significant differences between groups Table DiscussionMain findingsCurrent high death rates compared to healthy subjects of OSA and chronic pain managementwith daily opioid use warrant efforts to improve care [“] This study™s findings can be anFig CPAP use before and after intervention101371journalpone0237359g003PLOS ONE 101371journalpone0237359 August PLOS ONE 0cIdentification and treatment of obstructive sleep apnea by a primary care chronic pain management teamTable Comparison of clinical characteristics S1 AppendixDemographicsAverage Number of Participants withOSA on CPAP or at risk for OSASTOPBang � Control DailyOpioid Use withOSAAgeBMI ���Morphine Equivalent mgdat Conclusion of Study � months Pvalue Average Number of Participants Not Likelyto Have OSA STOPBang or NegPolysomnography PrePostPvalueDemographicsAverage Percentage of Participantswith OSA on CPAP or at risk for OSAControl DailyOpioid Use withPvalueAverage Percentage of Participants NotLikely to Have OSA STOPBang orPrePostPValueSTOPBang � OSANeg PolysomnographyRace CaucasianGender Male��Diabetes���Cardiovascular Disease��Hypertension�Gastroesophageal Reflux DiseaseThyroid Disease All FormsPulmonary Disease All Varieties Active CancerChronic Kidney DiseaseConcurrent Benzodiazepine UseConcurrent Pregabalin Use� p value of �� p value of ��� p value of 101371journalpone0237359t001NANANANANANANA NANANANANANANAimportant step towards improving mortality and morbidity when these two diseases are combined The results suggested that using a STOPBang questionnaire accompanied with motivational education initially as well as in followup improved diagnosis and treatment of OSAThese findings suggest that teambased care can positively impact the outcome of patientswith OSA when they are concurrently on chronic daily opioids and CPAP These findings warrant additional research Diagnosed sleep apnea participants who were using CPAP had a significantly higher rate of being male having hypertension having cardiovascular diseasehaving diabetes all types and having a higher BMI The fact that of participants at riskfor sleep apnea STOPBang � did not receive a full evaluation or end up on definitive treatment highlights the need for continued effort in this fieldLimitationsThe data found in this study regarding CPAP adherence was subject to limitations as wellSome participants that joined this teambased care management approach to treat chronicpain had already been prescribed CPAP and their adherence to the treatment was included inthis studies data set possibly overestimating the conclusions Adherence to CPAP in this studywas based on subjective treatment reconciliation at followup appointments with physiciansand nurses and since many participants received their sleep treatment from outside sourcesCPAP adherence data other than confirmation from the patient was not available If a participant indicated that they had not been using their CPAP � of the time between anyPLOS ONE 101371journalpone0237359 August PLOS ONE 0cIdentification and treatment of obstructive sleep apnea by a primary care chronic pain management teamfollowup appointments after the initiation of treatment they were considered to be nonadherent This type of data collection is subject to significant recall bias and could potentiallyinflate the percentage of participant adherenceThis preliminary research was done in the format of a prepost dual arm retrospectivestudy This type of study has inherent and significant limitations First due to the nature of theintervention team members nurses and physicians were not blinded and continued to evaluate participants throughout the course of this study and could have recommended evaluationand therapy of sleep apnea for alternative reasons This study could have been affected by aherd mentality since the primary care physicians worked together in one group and theircomposite style could have increased or decreased the identification and treatment of sleepapnea These limitations could be mitigated by performing a large prospective multicenterstudyThis study is also limited due to the nature of using a historical control [] Although the control used mimicked the experimental group in most ways daily opioid use MEDD race age andpercentages of diabetes hypertension and cardiovascular disease it varied in both BMI controlgroup had a higher average BMI and gender control group had a greater number of male participants It has been previously demonstrated that a higher BMI favors adherence to CPAP [] This does not detract from the finding that the intervention group in this study which had alower BMI had a higher adherence to CPAP Gender has also been evaluated in previous CPAPcompliance studies Two studies have shown no difference between genders whereas a differentstudy showed an increase in male noncompliance and another where there was an increase inmale compliance [“] As there is no definitive determination from these studies it is possiblethat the variance between the intervention and control groups were due to the gender differenceIt would seem from the above studies that there was no strong trend resulting from gender making gender unlikely to be the cause of the preponderance of the varianceThere was a loss of followup with primary care physicians after the administration of theSTOPBang questionnaire in this study Fig Patients may have fotten to or chosen notto schedule a followup appointment with their primary care physician after being determinedhigh risk for sleep apnea The participant received education on six other aspects of opioidmanaged pain during the one hour informational meeting Another limitation was that thedata from the intervention session was transmitted enblock to the primary care physicianelectronically These limitations could have been improved by having the nurse set up a specific appointment to consider sleep disorders in the highrisk group and to either verbally orelectronically deliver this information in an isolated approachComparisonsThis study reaffirmed previous studies that showed STOPBang was a tool that could identifyOSA [ “] Other similarities to previous investigations were found In those studies wheredaily opioid dosing for pain management was present “ of patients were found to sufferfrom OSA and of patients were found to be compliant to CPAP [ ] The data rangefor previous studies in regard to OSA incidence is consistent with this study in which ofparticipants were either diagnosed with OSA on CPAP or were at high risk for OSA STOPBang � not completing polysomnography or not compliant with CPAP This study was significantly higher with regards to the percentage of remaining on CPAP suggesting thatdiligence in repetitive diagnosis attempts and continued follow with motivational techniquesmay improve care in this type of patient Also this study like others suggested p thathypertension cardiovascular disease diabetes male gender and higher BMI are more common in patients with OSA []PLOS ONE 101371journalpone0237359 August PLOS ONE 0cIdentification and treatment of obstructive sleep apnea by a primary care chronic pain management teamConclusionsLeft untreated sleep apnea has a high rate of morbidity and mortality The use of prescribedopioids as a form of chronic pain management in a primary care setting can increase apatient™s likelihood of having sleep apnea A teambased intervention that included theadministration of a STOPBang questionnaire was associated with an increased diagnosis ofOSA prepost improvement level It was also associated with an increased adherenceto CPAP relative improvement The primary reasons CPAP treatment was not receivedincluded refusal of evaluation nonadherence to CPAP or shared physicianpatient decisionbased on agefrailtySupporting informationS1 Appendix Raw study data setXLSXAuthor ContributionsConceptualization Kathleen Whittington Leigh Simpson Dixie HarrisData curation Kathleen Whittington Michael ClayFormal analysis Kathleen Whittington Michael Clay Dixie HarrisInvestigation Joanna TierneyProject administration Joanna TierneySupervision Dixie HarrisWriting “ original draft Kathleen WhittingtonWriting “ review editing Kathleen Whittington Leigh Simpson Michael Clay JoannaTierney Dixie HarrisReferencesFarney Robert J œThe STOPBang equivalent model and prediction of severity of obstructivesleep apnea relation to polysomnographic measurements of the apneahypopnea index Journal ofclinical sleep medicine JCSM official publication of the American Academy of Sleep Medicine vol “65B 105664JCSM1306 PMID Manne Mahesh B œObstructive Sleep Apnea Who Should Be Tested and How ClevelandClinic Journal of Medicine Aug wwwmdedgecomccjmarticle105363cardiologyobstructivesleepapneawhoshouldbetestedandhow Abrishami A Khajehdehi A Chung F A systematic review of screening questionnaires for obstructivesleep apnea Can J Anaesth “ 101007s126300109280x PMID Vasu TS Doghramji K Cavallazzi R Obstructive sleep apnea syndrome and postoperative complications clinical use of the STOPBANG questionnaire Arch Otolaryngol Head Neck Surg “ 101001archoto20101020 PMID Kumar S Mcelligott D Goyal A Baugh M Ionita RN Risk of Obstructive Sleep Apnea OSA in Hospitalized Patients Chest 1384779A Ong TH Raudha S FookChong S Lew N Hsu AA Aal H Simplifying STOPBANG use of a simplequestionnaire to screen for OSA in an Asian population Sleep Breath “œDrug Overdose Deaths Centers for Disease Control and Prevention Centers for Disease Control andPrevention June wwwcdcgovdrugoverdosedatastatedeathshtml Substance Abuse and Mental Health Services Administration Drug Abuse Warning Network National Estimates of DrugRelated Emergency Department Visits HHS Publication No SMA “PLOS ONE 101371journalpone0237359 August PLOS ONE 0cIdentification and treatment of obstructive sleep apnea by a primary care chronic pain management team DAWN Series D39 Rockville MD Substance Abuse and Mental Health Services Administration Schneiderhan JMD Clauw DMD Schwenk TMD Primary care of patients with chronic pain J Am MedAssoc “ Walker James M œChronic Opioid Use Is a Risk Factor for the Development of Central SleepApnea and Ataxic Breathing Journal of Clinical Sleep Medicine JCSM Official Publication of theAmerican Academy of Sleep Medicine American Academy of Sleep Medicine Aug wwwncbinlmnihgovpmcarticlesPMC1978331 PMID Webster LR Choi Y Desai H Webster L Grant BJ Sleepdisordered breathing and chronic opioid therapy Pain Med “ 101111j15264637200700343x PMID Onen SH Onen F Courpron P Dubray C How pain and analgesics disturb sleep Clin J Pain “ 10109701ajp000012975731856f7 PMID Cozowicz Crispiana œOpioids for Acute Pain Management in Patients with Obstructive SleepApnea Anesthesia Analgesia vol no pp “ 101213aneJaoude Philippe Lal Ashima Vermont Leaj Porhomayon Jahan Ali A ElSolh Pain Intensity and Opioid Utilization in Response to CPAP Therapy in Veterans with Obstructive Sleep Apnea on Chronic Opioid Treatment Journal of Clinical Sleep Medicine Barbor TF HigginsBiddle JC World Health anization Dept of Mental Health and SubstanceDependence Brief Intervention of Hazardous and Harmful Drinking A Manual for Use in Primary CareGeneva World Health anization Group WBIS A crossnational trial of brief interventions with heavy drinkers Am J Public Health “ 102105ajph867948 PMID Miller WR Rollinick S Motivational Interviewing Preparing People to Change Addictive Behavior NewYork and London Guilford Press Saunders B Wilkinson C Phillips M The impact of a brief motivational intervention with opiate usersattending a methadone program Addiction “ 101046j13600443199590341510x PMID Hester RK Miller WR Handbook of Alcoholism Treatment Approaches Vol Boston MA Allyn andBacon D™Agostino R Belanger A A Suggestion for Using Powerful and Informative Tests of Normality The American Statistician “ 1023072684359 Edwards A Note on the ˜Correction for Continuity™ in testing the significance of differencebetween populations Psychometrika B “ Young T Finn L Sleep disordered breathing and mortality eighteenyear followup of the Wisconsin sleep cohort Sleep Aug “ PMID Marshall NS Wong KK Sleep apnea as an independent risk factor for allcause mortality the Busselton Health Study Sleep Aug “ PMID Liang Y Turner B Assesing Risk for Drug Overdose in a National Cohort Role for Both Daily and TotalOpioid Dose J Pain Apr “ 101016jjpain201411007 PMID Heejin Kim MinSukim Treatment Outcomes and Compliance According to Obesity in Patientswith OSA European Archives of OtoRhinoLaryngology “ PelletierFleury N Rakotonanahary D Fleury B The age and other factors in the evaluation of compliance with nasal continuous positive airway pressure for obstructive sleep apnea syndrome A Cox™sproportional hazard analysis Sleep Med “ 101016s1389945700 PMID Anttalainen U Saaresranta T Kalleinen N Aittokllio J Vahlbergy T Polo O CPAP adherence and partial upper airway obstruction during sleep Sleep Breath “ 101007s1132500701025 PMID Sin DD Mayers I Man GC Pawluk L Longterm compliance rates to continuous positive airway pressure in obstructive sleep apnea a populationbased study Chest “ 101378chest1212430 PMID Ye Lichun Pien Grace W Ratcliffe Sara J Weaver Terri E Gender Differences in Obstructive SleepApnea and Treatment Response to Continuous Positive Airway Pressure J Clin Sleep Med “ PMID PLOS ONE 101371journalpone0237359 August PLOS ONE 0cIdentification and treatment of obstructive sleep apnea by a primary care chronic pain management team Mador M Henderson J Effect of Opioids on Sleep and Breathing in Chronic Pain Patients Journal ofClinical Sleep Medicine Aug “ 105664jcsm3952 PMIDPLOS ONE 101371journalpone0237359 August PLOS ONE 0c'

cancer267

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: Identification and treatment of obstructive sleep apnea by a primarycare team with a subset focus on chronic painmanagement within the paper and its Supporting InformationfilesFunding The authors received no specific fundingfor this workCompeting interests The authors have declaredthat no competing interests existBackgroundPatients diagnosed with obstructive sleep apnea OSA who also consume prescription opioids have a greater likelihood of morbidity and mortality This study evaluated whether a primary care team focused on chronic pain care management could use a validatedquestionnaire STOPBang and motivational followup to increase identification and treatment of OSAMethodsThis study was a retrospective dual arm prepost controlled study Participants of thisstudy included the complete chronic pain management sub group treated by this primarycare team Participants were � years old and prescribed daily opioids for treatment ofchronic pain All participants had a multifaceted individualized educational meeting thatincluded completing a STOPBang questionnaire Participants who received a score �three were advised to follow up with their primary care physician Participants were seenquarterly throughout the studyResultsThe primary outcome of this study was that of participants with likely OSA were usingCPAP for a minimum of months range of “ months 18month average postintervention vs CPAPuse in the control group months of observation both groupswere chronic opioid users with OSA This was a relative improvement p Asecondary outcome was that of nonprior CPAP users obtained CPAP post intervention a prepost improvement p x2 with degree of freedom Alsoparticipants who were likely to have OSA STOPBang score � or had a positive polysomnography AHI with comorbidities compared to those unlikely to have OSA STOPPLOS ONE 101371journalpone0237359 August PLOS ONE 0cIdentification and treatment of obstructive sleep apnea by a primary care chronic pain management teamBang score or had a negative polysomnography AHI in this study were more likelyto be male have a higher BMI have hypertension have cardiovascular disease andorhave diabetes all typesConclusionTeam based care management for participants taking prescription opioids where STOPBang questionnaires were completed were associated with an increase in the identificationand treatment of OSAIntroductionStudy rationaleThe objective of this study was to evaluate whether a teambased care management intervention would increase identification and treatment of obstructive sleep apnea in participants taking medication for chronic painCurrent knowledgeObstructive sleep apnea OSA is a chronic sleep disorder characterized by episodes of apneasand hypopneas or the complete or partial collapse of the upper airway [] A diagnosis ismade when a patient has or more of these events in an hour or five of these events if otherpredicting symptoms for OSA are present [] Approximately million American adults havethis sleep disorder Patients with moderate to severe OSA are often treated with a continuouspositive airway pressure device or CPAP Treatment can reduce the risks of hypertension coronary artery disease heart failure arrhythmias sudden cardiac death and stroke [] A validated questionnaire STOPBang score can be used as a screening tool to identify patients atrisk for OSA A STOPBang sleep questionnaire is commonly distributed to patients whoshow signs of having potential risk factors for OSA œSTOP questions snoring tirednessobserved apnea and high blood pressure and œBang BMI kgm2 Age years Neckcircumference cm and Gender male [ “] A score of three or higher on STOPBangplaces patients at higher risk of OSA requiring further evaluation which includes an overnightpolysomnogram or sleep study to confirm diagnosis []Prescription opioid use for pain management is still a common practice in primary care settings though the rates of overdose due to these drugs in the United States are at an unprecedented high with deaths in [“] Patients using opiates for chronic painmanagement have also been found to have a greater likelihood of developing central sleepapnea which worsens the severity of OSA and ataxic breathing [] A relationship existsbetween pain and sleep disruption with over of patients who have chronic pain havingreported difficulty sleeping which increases hyperalgesia [ ] An analgesic effect may bepresent where pain creates sleep disorders and a shortage of sleep increases pain [ ]Patients with OSA who are then prescribed opioids have an increased risk of opioidinducedrespiratory depression OIRD [] Despite the potential morbidity of the combination ofOSA central sleep apnea and Opioid use studies have shown that those that have this combination only remain on definitive treatment for their OSA CPAP of the time [] It istherefore of the utmost importance that patients who chronically use opioids are evaluated forOSA and those diagnosed adhere to CPAPPLOS ONE 101371journalpone0237359 August PLOS ONE 0cIdentification and treatment of obstructive sleep apnea by a primary care chronic pain management teamIn order to decrease mortality it is necessary to educate patients on the risks and healthconcerns related to taking opioids Brief interventions can influence behavior of patients whoare at high risk for abuse of a substance [] Individualized nurseled management educationis a tool that can be used as a brief intervention within a teambased approach [] These individualized information gathering motivational meetings allow patients to experience empathy as their medical needs are met [ ] Individualized nurseled management educationis a tool that can be used as a brief intervention within a teambased approach [] Instructionand guidance can be coupled with an assessment of a patient™s potential risk factors for comorbidities such as OSA using a STOPBang questionnaireMaterials methodsDesignThis study was a retrospective dual arm prepost controlled study to evaluate whether anintervention STOPBang questionnaire education and motivational followup wouldimprove OSA diagnosis and treatment compliance The first investigation arm compared thecontrol group daily opioid use with OSA to the part of the intervention group with OSA onCPAP or at risk for OSA STOPBang � This arm™s purpose was to determine whether theintervention increased treatment compliance with CPAP The second investigation armcompared the study group to itself prepost intervention This arm evaluated whether applyingthe intervention increased diagnosis and treatment of OSA The single independent variablein this study was a participant™s admission to the chronic pain management subgroup withthe administration of a STOP Bang questionnaire and the dependent variable was CPAP useInclusion criteria consisted of ongoing participation in primary care pain management subgroup daily opioid use and an age � This study occurred between October and January which included monthsfor exposure and then at least months of follow up One of Intermountain Healthcare™s outpatient facilities in Utah served as the site for this study The total number of patients duringthe duration of the study who entered into the pain management sub group � years oldand were taking a daily dose of opioids was Out of the identified participants participants remained in the clinic during the entirety of this study Fig Ethics approvalIRB Number for this study was obtained from the Institutional Review Board ofIntermountain Healthcare Consent was waived for this retrospective studyControlsA Medline and Pubmed basic search from “ was used to identify a historical yet contemporary United States based control group that had Obstructive Sleep Apnea and met theinclusion criteria of being � years old and were taking a daily dose of opioids The controlthat was chosen was comparable to this study™s intervention group with regard to Race AgeGender Morphine Equivalent Daily Dose and Comorbidities Also the control used camefrom a study that lasted a similar duration []SubjectsEach participant received an information gathering and educational session with a registerednurse in the primary care facility at the time of their admittance into the chronic pain management program between “ During this session a full pain history was obtained fromparticipants All participants in the study underwent STOPBang screening All participantswho were at high risk for OSA STOPBang � were referred back to their primary carePLOS ONE 101371journalpone0237359 August PLOS ONE 0cIdentification and treatment of obstructive sleep apnea by a primary care chronic pain management teamFig Participant retention101371journalpone0237359g001physician Participants and physicians made joint decisions during a followup appointmentwhether to pursue polysomnography Reasons why participants did not complete polysomnography or attain treatment for OSA are that the participants refused workup there was ashared decision due to participant frailty had cancer end stage or were noncompliant toCPAP Fig Fig participants at high risk for sleep apnea STOPBang score � who did not undergo diagnostic evaluation orcomplete treatment101371journalpone0237359g002PLOS ONE 101371journalpone0237359 August PLOS ONE 0cIdentification and treatment of obstructive sleep apnea by a primary care chronic pain management teamEach participant received initial inperson education with the same registered nurse regarding nonpharmacological treatment of pain selfhelp groups risks and benefits of treatment ofpain management compliance with treatment of pain and its comorbidities and informationabout Naloxone an opioid reversal agent This education was provided in a hour sessionusing the FRAMES Feedback Responsibility Advise Menu for Change Empathy andEnhancing Self Efficacy motivational method [] A chronic pain management agreementwas executed Additional information obtained during this session included a Current OpioidMisuse Measure and a STOPBang questionnaire All participants who received a score greaterthan or equal to three on the STOPBang were told to follow up with their primary care physician All primary care providers were made aware they would receive electronic data onSTOPBang questionnaires and participants with scores � would make a follow up appointment with them Upon completion of this session information was recorded electronically andtransmitted to the primary care physician on record Each participant also had quarterly inperson followup appointments with their primary care physician In addition quarterly inperson or phone visits with the pain management nurse were continued Physician visits varied in length and scope Individualized nursing visits that continued to use the FRAMESmethod and lasted for a duration of minutes were focused on remaining compliant with allongoing therapies The nurse would document the interaction and any concerns about compliance with any therapy in the patient™s EMR Electronic Medical Record at the conclusionof the visits The information was based on the subjective input received from the participantQuarterly followup remained consistent throughout the study Attending all followupappointments both with the primary care physician and the pain management nurse was aprerequisite to remain in the pain management group Remaining in the pain managementwas group was the only avenue to receive controlled pain medication at this facilityData collectionThe baseline biometric parameters of Race Gender BMI Age Tobacco Use Alcohol Use andMorphine Equivalence Daily Dosage MEDD were extracted from each participant™s electronic medical record EMR Collected data also included information on Chronic DiseaseHypertension Cardiovascular Disease Pulmonary Disease Thyroid Disease Diabetes Gastroesophageal Reflux Disease and Chronic Kidney Disease and specific medication typesBenzodiazepines and Pregabalin STOPBang scores were gathered during the initial chronicpain management encounter when participants joined the study Subjective data on CPAPadherence was found in the EMR documentation of the pain management nurse and primarycare physician follow up reports postinclusion in the studyStatistical methodsMeans and standard deviations were calculated for demographic variables The DAgostinoPearson test was used to confirm normality [] Unequal variance ttesting was used in the statistical analysis of both arms of this study to compare Age BMI and Morphine EquivalentDaily Dosing Two proportion Ztesting was used to compare the control group daily opioiduse with OSA to the participants in the intervention group with OSA on CPAP or at highrisk for OSA STOPBang � with regards to CPAP use Race Gender Diabetes Cardiovascular Disease and Hypertension Two proportion Ztesting was also used to compare the participants not likely to have OSA STOPBang or had a negative polysomnography to thosewith OSA on CPAP or at high risk for OSA STOPBang � not completing polysomnography or not compliant with CPAP A x contingency table using McNemar™s test with correction for continuity was used to compare the participants™ CPAP use prepost intervention []PLOS ONE 101371journalpone0237359 August PLOS ONE 0cIdentification and treatment of obstructive sleep apnea by a primary care chronic pain management teamIRB approvalIRB Approval was granted from Intermountain Healthcare IRB IRB Number ResultsThe primary outcome of this study was that of participants with likely OSA were usingCPAP for a minimum of months range of “ months 18month average postintervention vs CPAP use in the control group months of observation which were chronic opioid users with OSA This was a relative improvement p A secondary outcomewas that of nonprior CPAP users obtained CPAP post intervention a prepostimprovement p x2 with degree of freedom Fig This resulted in ofintervention participants being treated with CPAP Comparing the average number of intervention participants with OSA on CPAP or at risk for OSA STOPBang � to the control dataset resulted in significant differences for BMI and Male Gender Otherdemographics between these two groups Age p MEDD p Race Caucasianp Hypertension p Cardiovascular Disease p and Diabetes all formsp showed no statistically significant differences between groups Comparing the averagenumber of intervention participants with OSA on CPAP or at risk for OSA STOPBang � to the average number of intervention participants not likely to have OSA STOPBang orhad a negative polysomnography resulted in the following statistically significant differencesBMI p Male Gender p00001 Hypertension p Cardiovascular Diseasep and Diabetes all forms p Other demographics between these two groupsAge p MEDD p Race Caucasian p Benzodiazepine Use p Pregabalin Use p and Active Chronic Disease States Gastroesophageal Reflux p Chronic Kidney Disease Thyroid Disease p and Pulmonary Diseasep showed no statistically significant differences between groups Table DiscussionMain findingsCurrent high death rates compared to healthy subjects of OSA and chronic pain managementwith daily opioid use warrant efforts to improve care [“] This study™s findings can be anFig CPAP use before and after intervention101371journalpone0237359g003PLOS ONE 101371journalpone0237359 August PLOS ONE 0cIdentification and treatment of obstructive sleep apnea by a primary care chronic pain management teamTable Comparison of clinical characteristics S1 AppendixDemographicsAverage Number of Participants withOSA on CPAP or at risk for OSASTOPBang � Control DailyOpioid Use withOSAAgeBMI ���Morphine Equivalent mgdat Conclusion of Study � months Pvalue Average Number of Participants Not Likelyto Have OSA STOPBang or NegPolysomnography PrePostPvalueDemographicsAverage Percentage of Participantswith OSA on CPAP or at risk for OSAControl DailyOpioid Use withPvalueAverage Percentage of Participants NotLikely to Have OSA STOPBang orPrePostPValueSTOPBang � OSANeg PolysomnographyRace CaucasianGender Male��Diabetes���Cardiovascular Disease��Hypertension�Gastroesophageal Reflux DiseaseThyroid Disease All FormsPulmonary Disease All Varieties Active CancerChronic Kidney DiseaseConcurrent Benzodiazepine UseConcurrent Pregabalin Use� p value of �� p value of ��� p value of 101371journalpone0237359t001NANANANANANANA NANANANANANANAimportant step towards improving mortality and morbidity when these two diseases are combined The results suggested that using a STOPBang questionnaire accompanied with motivational education initially as well as in followup improved diagnosis and treatment of OSAThese findings suggest that teambased care can positively impact the outcome of patientswith OSA when they are concurrently on chronic daily opioids and CPAP These findings warrant additional research Diagnosed sleep apnea participants who were using CPAP had a significantly higher rate of being male having hypertension having cardiovascular diseasehaving diabetes all types and having a higher BMI The fact that of participants at riskfor sleep apnea STOPBang � did not receive a full evaluation or end up on definitive treatment highlights the need for continued effort in this fieldLimitationsThe data found in this study regarding CPAP adherence was subject to limitations as wellSome participants that joined this teambased care management approach to treat chronicpain had already been prescribed CPAP and their adherence to the treatment was included inthis studies data set possibly overestimating the conclusions Adherence to CPAP in this studywas based on subjective treatment reconciliation at followup appointments with physiciansand nurses and since many participants received their sleep treatment from outside sourcesCPAP adherence data other than confirmation from the patient was not available If a participant indicated that they had not been using their CPAP � of the time between anyPLOS ONE 101371journalpone0237359 August PLOS ONE 0cIdentification and treatment of obstructive sleep apnea by a primary care chronic pain management teamfollowup appointments after the initiation of treatment they were considered to be nonadherent This type of data collection is subject to significant recall bias and could potentiallyinflate the percentage of participant adherenceThis preliminary research was done in the format of a prepost dual arm retrospectivestudy This type of study has inherent and significant limitations First due to the nature of theintervention team members nurses and physicians were not blinded and continued to evaluate participants throughout the course of this study and could have recommended evaluationand therapy of sleep apnea for alternative reasons This study could have been affected by aherd mentality since the primary care physicians worked together in one group and theircomposite style could have increased or decreased the identification and treatment of sleepapnea These limitations could be mitigated by performing a large prospective multicenterstudyThis study is also limited due to the nature of using a historical control [] Although the control used mimicked the experimental group in most ways daily opioid use MEDD race age andpercentages of diabetes hypertension and cardiovascular disease it varied in both BMI controlgroup had a higher average BMI and gender control group had a greater number of male participants It has been previously demonstrated that a higher BMI favors adherence to CPAP [] This does not detract from the finding that the intervention group in this study which had alower BMI had a higher adherence to CPAP Gender has also been evaluated in previous CPAPcompliance studies Two studies have shown no difference between genders whereas a differentstudy showed an increase in male noncompliance and another where there was an increase inmale compliance [“] As there is no definitive determination from these studies it is possiblethat the variance between the intervention and control groups were due to the gender differenceIt would seem from the above studies that there was no strong trend resulting from gender making gender unlikely to be the cause of the preponderance of the varianceThere was a loss of followup with primary care physicians after the administration of theSTOPBang questionnaire in this study Fig Patients may have fotten to or chosen notto schedule a followup appointment with their primary care physician after being determinedhigh risk for sleep apnea The participant received education on six other aspects of opioidmanaged pain during the one hour informational meeting Another limitation was that thedata from the intervention session was transmitted enblock to the primary care physicianelectronically These limitations could have been improved by having the nurse set up a specific appointment to consider sleep disorders in the highrisk group and to either verbally orelectronically deliver this information in an isolated approachComparisonsThis study reaffirmed previous studies that showed STOPBang was a tool that could identifyOSA [ “] Other similarities to previous investigations were found In those studies wheredaily opioid dosing for pain management was present “ of patients were found to sufferfrom OSA and of patients were found to be compliant to CPAP [ ] The data rangefor previous studies in regard to OSA incidence is consistent with this study in which ofparticipants were either diagnosed with OSA on CPAP or were at high risk for OSA STOPBang � not completing polysomnography or not compliant with CPAP This study was significantly higher with regards to the percentage of remaining on CPAP suggesting thatdiligence in repetitive diagnosis attempts and continued follow with motivational techniquesmay improve care in this type of patient Also this study like others suggested p thathypertension cardiovascular disease diabetes male gender and higher BMI are more common in patients with OSA []PLOS ONE 101371journalpone0237359 August PLOS ONE 0cIdentification and treatment of obstructive sleep apnea by a primary care chronic pain management teamConclusionsLeft untreated sleep apnea has a high rate of morbidity and mortality The use of prescribedopioids as a form of chronic pain management in a primary care setting can increase apatient™s likelihood of having sleep apnea A teambased intervention that included theadministration of a STOPBang questionnaire was associated with an increased diagnosis ofOSA prepost improvement level It was also associated with an increased adherenceto CPAP relative improvement The primary reasons CPAP treatment was not receivedincluded refusal of evaluation nonadherence to CPAP or shared physicianpatient decisionbased on agefrailtySupporting informationS1 Appendix Raw study data setXLSXAuthor ContributionsConceptualization Kathleen Whittington Leigh Simpson Dixie HarrisData curation Kathleen Whittington Michael ClayFormal analysis Kathleen Whittington Michael Clay Dixie HarrisInvestigation Joanna TierneyProject administration Joanna TierneySupervision Dixie HarrisWriting “ original draft Kathleen WhittingtonWriting “ review editing Kathleen Whittington Leigh Simpson Michael Clay JoannaTierney Dixie HarrisReferencesFarney Robert J œThe STOPBang equivalent model and prediction of severity of obstructivesleep apnea relation to polysomnographic measurements of the apneahypopnea index Journal ofclinical sleep medicine JCSM official publication of the American Academy of Sleep Medicine vol “65B 105664JCSM1306 PMID Manne Mahesh B œObstructive Sleep Apnea Who Should Be Tested and How ClevelandClinic Journal of Medicine Aug wwwmdedgecomccjmarticle105363cardiologyobstructivesleepapneawhoshouldbetestedandhow Abrishami A Khajehdehi A Chung F A systematic review of screening questionnaires for obstructivesleep apnea Can J Anaesth “ 101007s126300109280x PMID Vasu TS Doghramji K Cavallazzi R Obstructive sleep apnea syndrome and postoperative complications clinical use of the STOPBANG questionnaire Arch Otolaryngol Head Neck Surg “ 101001archoto20101020 PMID Kumar S Mcelligott D Goyal A Baugh M Ionita RN Risk of Obstructive Sleep Apnea OSA in Hospitalized Patients Chest 1384779A Ong TH Raudha S FookChong S Lew N Hsu AA Aal H Simplifying STOPBANG use of a simplequestionnaire to screen for OSA in an Asian population Sleep Breath “œDrug Overdose Deaths Centers for Disease Control and Prevention Centers for Disease Control andPrevention June wwwcdcgovdrugoverdosedatastatedeathshtml Substance Abuse and Mental Health Services Administration Drug Abuse Warning Network National Estimates of DrugRelated Emergency Department Visits HHS Publication No SMA “PLOS ONE 101371journalpone0237359 August PLOS ONE 0cIdentification and treatment of obstructive sleep apnea by a primary care chronic pain management team DAWN Series D39 Rockville MD Substance Abuse and Mental Health Services Administration Schneiderhan JMD Clauw DMD Schwenk TMD Primary care of patients with chronic pain J Am MedAssoc “ Walker James M œChronic Opioid Use Is a Risk Factor for the Development of Central SleepApnea and Ataxic Breathing Journal of Clinical Sleep Medicine JCSM Official Publication of theAmerican Academy of Sleep Medicine American Academy of Sleep Medicine Aug wwwncbinlmnihgovpmcarticlesPMC1978331 PMID Webster LR Choi Y Desai H Webster L Grant BJ Sleepdisordered breathing and chronic opioid therapy Pain Med “ 101111j15264637200700343x PMID Onen SH Onen F Courpron P Dubray C How pain and analgesics disturb sleep Clin J Pain “ 10109701ajp000012975731856f7 PMID Cozowicz Crispiana œOpioids for Acute Pain Management in Patients with Obstructive SleepApnea Anesthesia Analgesia vol no pp “ 101213aneJaoude Philippe Lal Ashima Vermont Leaj Porhomayon Jahan Ali A ElSolh Pain Intensity and Opioid Utilization in Response to CPAP Therapy in Veterans with Obstructive Sleep Apnea on Chronic Opioid Treatment Journal of Clinical Sleep Medicine Barbor TF HigginsBiddle JC World Health anization Dept of Mental Health and SubstanceDependence Brief Intervention of Hazardous and Harmful Drinking A Manual for Use in Primary CareGeneva World Health anization Group WBIS A crossnational trial of brief interventions with heavy drinkers Am J Public Health “ 102105ajph867948 PMID Miller WR Rollinick S Motivational Interviewing Preparing People to Change Addictive Behavior NewYork and London Guilford Press Saunders B Wilkinson C Phillips M The impact of a brief motivational intervention with opiate usersattending a methadone program Addiction “ 101046j13600443199590341510x PMID Hester RK Miller WR Handbook of Alcoholism Treatment Approaches Vol Boston MA Allyn andBacon D™Agostino R Belanger A A Suggestion for Using Powerful and Informative Tests of Normality The American Statistician “ 1023072684359 Edwards A Note on the ˜Correction for Continuity™ in testing the significance of differencebetween populations Psychometrika B “ Young T Finn L Sleep disordered breathing and mortality eighteenyear followup of the Wisconsin sleep cohort Sleep Aug “ PMID Marshall NS Wong KK Sleep apnea as an independent risk factor for allcause mortality the Busselton Health Study Sleep Aug “ PMID Liang Y Turner B Assesing Risk for Drug Overdose in a National Cohort Role for Both Daily and TotalOpioid Dose J Pain Apr “ 101016jjpain201411007 PMID Heejin Kim MinSukim Treatment Outcomes and Compliance According to Obesity in Patientswith OSA European Archives of OtoRhinoLaryngology “ PelletierFleury N Rakotonanahary D Fleury B The age and other factors in the evaluation of compliance with nasal continuous positive airway pressure for obstructive sleep apnea syndrome A Cox™sproportional hazard analysis Sleep Med “ 101016s1389945700 PMID Anttalainen U Saaresranta T Kalleinen N Aittokllio J Vahlbergy T Polo O CPAP adherence and partial upper airway obstruction during sleep Sleep Breath “ 101007s1132500701025 PMID Sin DD Mayers I Man GC Pawluk L Longterm compliance rates to continuous positive airway pressure in obstructive sleep apnea a populationbased study Chest “ 101378chest1212430 PMID Ye Lichun Pien Grace W Ratcliffe Sara J Weaver Terri E Gender Differences in Obstructive SleepApnea and Treatment Response to Continuous Positive Airway Pressure J Clin Sleep Med “ PMID PLOS ONE 101371journalpone0237359 August PLOS ONE 0cIdentification and treatment of obstructive sleep apnea by a primary care chronic pain management team Mador M Henderson J Effect of Opioids on Sleep and Breathing in Chronic Pain Patients Journal ofClinical Sleep Medicine Aug “ 105664jcsm3952 PMIDPLOS ONE 101371journalpone0237359 August PLOS ONE 0c' Answer:

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suffer from a high false positive rate because predicted expression of different genes may behighly correlated due to linkage disequilibrium between eQTL We propose a novel statistical method Gene Score Regression GSR to detect causal gene sets for complex traitswhile accounting for genetogene correlations We consider noncausal genes that arehighly correlated with the causal genes will also exhibit a high marginal association with thecomplex trait Consequently by regressing on the marginal associations of complex traitswith the sum of the genetogene correlations in each gene set we can assess the amountof variance of the complex traits explained by the predicted expression of the genes in eachgene set and identify plausible causal gene sets GSR can operate either on GWAS summary statistics or observed gene expression Therefore it may be widely applied to annotateGWAS results and identify the underlying biological pathways We demonstrate the highaccuracy and computational efficiency of GSR compared to stateoftheart methodsthrough simulations and real data applications GSR is ly available at githubcomlilabmcgillGSRIntroductionGenomewide association studies GWAS have been broadly successful in associating geneticvariants with complex traits and estimating trait heritabilities in large populations [“] Overthe past decade GWAS have quantified the effects of individual genetic variants on hundredsof polygenic phenotypes [ ] GWAS summary statistics have enabled various downstreamanalyses including partitioning heritability [] inferring causal single nucleotide polymorphisms SNPs using epigenomic annotations [] and gene sets enrichment analysis fora1111111111a1111111111a1111111111a1111111111a1111111111 ACCESSCitation Zhang W Li SY Liu T Li Y Partitioning genebased variance of complex traitsby gene score regression e0237657 101371journalpone0237657Editor F Alex Feltus Clemson University UNITEDSTATESReceived March Accepted July Published August Peer Review History PLOS recognizes thebenefits of transparency in the peer reviewprocess therefore we enable the publication ofall of the content of peer review and authorresponses alongside final published s Theeditorial history of this is available here101371journalpone0237657Copyright Zhang This is an access distributed under the terms of theCreative Commons Attribution License whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are creditedData Availability Statement All of the data used inthis paper are described under subsection Realdata application in the manuscript and pastedbelow as reference We applied our approach toinvestigate pathway enrichment for complexPLOS ONE 101371journalpone0237657 August PLOS ONE 0cGene score regressiontraits Fig 2b using publicly available summarystatistics and genotypeexpression weights basedon GTEx whole blood samples wwwgtexportalhomedatasets2 The GWASsummary statistics were downloaded from publicdatabase databroadinstitutealkesgroupsumstats_formatted We downloadedexpression weights and reference LD structureestimated in Genomes using Europeanindividuals from the TWASFUSION websitehttpgusevlabprojectsfusion Franke labcelltypespecific gene expression dataset wereobtained from databroadinstitutempgdepictdepict_downloadtissue_expression Inaddition we applied GSR to test for gene setenrichment in three wellpowered types of cancerbreast invasive carcinoma BRCA cases and controls thyroid carcinoma THCA casesand controls and prostate adenocarcinomaPRAD cases and controls using geneexpression datasets from The Cancer GenomeAtlas TCGA Uniformly processed normalizedand batcheffect corrected gene expressiondatasets from TCGA and GTEx were obtained fromfigsharecomsData_record_3 Gene expression and phenotype werestandardized before supplying to the GSR softwareStandard GSEA was also performed forcomparison Gene sets were downloaded from theMSigDb website oftwarebroadinstitutegseamsigdbindexjsp Here we combinedBIOCARTA KEGG and REACTOME to create a totalof gene sets We also downloaded the GO biological process terms as additional genesets as well as the gene sets pertaining tooncogenic signatures for the TCGA data analysisFunding The research is supported by CanadaFirst Research Excellence Fund CFREF HealthyBrains Healthy Life HBHL New Investigator fund at McGill University and Mon trealNeurologic Institute MNI and NSERC DiscoveryGrant RGPIN20190621 The funders had nocomplex traits [] However it remains challenging to link these genetic associations withknown biological mechanisms One main reason is that the majority of the GWAS loci are notlocated in known genic regions of the human genomeTranscriptomewide association studies TWAS [“] offer a systematic way to integrateGWAS and the reference genotypegene expression datasets such as the GenotypeTissueExpression project GTEx [] via expression quantitative loci eQTL In TWAS we couldfirst quantify the impact of each genetic variant on expression variability in a population andobtain predicted gene expression levels based on new genotypes Then we could correlate thepredicted gene expression with the phenotype of interest in order to identify pivotal genes[] Moreover when individuallevel genotypes and gene expression levels are not availablewe could still quantify genetophenotype association ie TWAS statistics using only themarginal effect sizes of SNPs on the phenotype and on gene expression respectively []These concepts and implementations have largely facilitated explanation of genetic associationfindings at the gene or the pathway levelHowever as depicted in Fig TWAS are often confounded by the genetogene correlationof the genetically predicted gene expression due to the SNPtoSNP correlation ie linkage disequilibrium LD [] Consequently relying on the TWAS statistics may lead to false positivediscoveries of causal genes and pathways One approach to address this problem is to finemapcausal genes by inferring the posterior probabilities of configurations of each gene being causalin a defined GWAS loci and then test gene set enrichment using the credible gene sets of prioritized genes [] However this approach is computationally expensive restricted to GWASloci and sensitive to the arbitrary thresholds used for determining the credible gene set andthe maximum number of causal genes per locusAnother method called PASCAL [] projects SNP signals onto genes while correcting forLD and then performs pathway enrichments as the aggregated transformed gene scoreswhich asymptotically follows a chisquare distribution However PASCAL does not leverage the eQTL information for each SNP thereby assuming that a priori all SNPs have thesame effect on the gene Stratified LD score regression LDSC offers a principle way to partition the SNP heritability into functional categories defined based on tissue or celltypespecific epigenomic regions [] or eQTL regions of the genes exhibiting a strong tissue specificity [] Although LDSC is able to obtain biologically meaningful tissuespecific enrichments it operates at the SNP level rendering it difficult to assess enrichment of gene setsMoreover neither PASCAL nor LDSC is able to integrate the observed gene expression datameasured in a disease cohort rather than the reference cohort that are broadly availableacross diverse studies of diseases including cancers such as The Cancer Genome AtlasTCGA []role in study design data collection and analysisAlthough expressionbased methods such as gene set enrichment analysis GSEA aredecision to publish or preparation of themanuscript No author received a salary from anyof the fundersCompeting interests The authors have declaredthat no competing interests existoften adopted in combination with the observed gene expression and phenotypes [] theygenerally do not account for the genetogene correlation While this type of correlation is usually caused by shared transcriptional regulatory mechanisms across genes GSEA still likelyproduces false positives in identifying causal pathwaysIn this study we present a novel and powerful genebased heritability partitioning methodthat jointly accounts for genetogene correlation and integrates information captured at eitherthe SNPtophenotype or the SNPtogene level We utilize this method to identify plausiblecausal gene sets or pathways for complex traits We showcase its high accuracy and computational efficiency in various simulated and real scenariosPLOS ONE 101371journalpone0237657 August PLOS ONE 0cGene score regressionFig Overview of confounding effects on pathway analysis a A hypothetical example illustrates the confounding issue when using the geneticallypredicted transcriptome to assess the pathway enrichments for a target phenotype The causal gene set includes a causal gene which is linked to noncausal gene via their respective causal SNPs and which are in strong linkage disequilibrium b A GWAS locus SNP associations with the targetphenotype are summarized The causal SNP for the causal gene is in red The SNPs that drive noncausal genes are in blue The rest of the SNPs are ingreen SNPs exhibit correlated signals due to the linkage disequilibrium LD as displayed by the upper triangle of the SNPSNP Pearson correlationmatrix c A TWAS locus The genetogene correlation is partly induced by the SNPtoSNP correlation and partly due to intrinsic coregulatoryexpression program d Pathway associations based on averaged gene associations101371journalpone0237657g001MethodsPartitioning genebased variance of complex traitsWe assume gene expression has linearly additive effects on a continuous polygenic trait yyi ¼XAijaj þ �ijð1Þwhere Aij denotes the expression of the jth gene in the ith individual for i N individuals and j G genes αj denotes the true effect size of the jth gene on the trait andj Þ �i denotes the residual for the ith individual in this linear model andaj � Nð0 s2�i � Nð0 s2� ÞPiyi ¼ N yy ¼ Here we further assume that both y and A are standardized such that NPiAij ¼ and j Aj ¼ for j GN ANPLOS ONE 101371journalpone0237657 August PLOS ONE 0cWe define the estimated marginal effect size of the jth gene on the trait as ajGene score regressionaj ¼NAj y¼NAj ðXAkak þ �ÞkX¼kNAj Akak þNAj �X¼krjkak þ �ð2Þð3Þð4Þð5Þwhere � ¼ N Aj � withVarð�0Þ ¼N Aj Varð�ÞAj ¼N s2�and rjk ¼ and the kth geneN Aj Ak is the estimated Pearson correlation in gene expression between the jth geneWe define w2j ¼ Na2j Then if we further assume α r and � are independent we haveE½w2j � ¼ E½Na2j �X¼ NE½Ã°rjkak þ �0Þ2�k¼ NE½r jk�E½a2k� þ s2�Xkð6Þð7Þð8ÞNow consider C gene sets Cc where c C and denote the proportion of total trait Here Cc denotes the numVarðajÞvariance explained by the cth gene set as τc with tc ¼ber of genes in the cth gene setPjCcjj2CcConsequentlyE½a2k� ¼ VarðakÞ ¼Xck2CctcBy approximating E½r N we have thatXjk� with r E½w2jk þ j � ¼ NE½r jk�E½a2k� þ s2�k¼ NXtcXr jk þXtc þ s2�ck2Ccc¼ NXtclðj cÞ þ cwhere we define gene score as lðj cÞ ¼tinuous trait is normalizedPr jk and VarðyÞ ¼k2Ccð9Þð10Þð11ÞPctc þ s2� ¼ since the conPLOS ONE 101371journalpone0237657 August PLOS ONE 0cGene score regressionTherefore if we are able to obtain estimates for w2j and C gene score lj c for j Gand c C we will be able to perform linear regression of the estimated w2j on lj cand derive regression coefficient that is an estimate for each τc c C respectivelyThese are available from GWAS summary statistics of SNPtotrait effect sizes eQTL summary statistics of SNPtogene expression effect sizes and a reference LD panel Specifically Suppose we have estimated effect sizes βp× of p SNPs based on a GWAS including Ngwassamples ieb ¼NgwasXywhere XNgwas×p is the standardized genotype Meanwhile we have the eQTL summary statistics W estimated usingAeQTL ¼ XeQTLWTherefore the predicted gene expression in GWAS is given bySinceA ¼ XWj ¼ Na2w2j¼ NðNAj yÞ2¼ NðNWj XyÞ2¼ NðW j bÞ2the required w2j can be estimated without accessing any individuallevel data Furthermore a reference LD panel Sp×p summarizing SNPtoSNP correlation in thematched population with the GWAS study can provide estimates for rjk asR ¼ ½rjk�¼NAA¼NW XXW¼NW SWPLOS ONE 101371journalpone0237657 August ð12Þð13Þð14Þð15Þð16Þð17Þð18Þð19Þ PLOS ONE 0cGene score regressionIt is noteworthy that with individuallevel gene expression data we can also easily obtainthe required w2j and R [rjk] by definitionIn practice many gene sets are not disjoint and share common genes with each otherTherefore we regress one gene set at a time along with a œdummy gene set that include theunion of all of the other genes The dummy gene set is used to account for unbalanced genesets and to stabilize estimates of τc We also include an intercept in the regression model toalleviate nongeneset biases for example positive correlation between gene scores and truegene effect sizes that could lead to intercept greater than and negative correlation betweengene scores and true gene effect sizes could lead to intercept smaller than Simulation designTo assess the accuracy of our GSR approach we simulated causal SNPs for gene expression aswell as causal gene sets for a continuous trait based on real genotypes and known gene setsfrom existing databases Our simulation included two stages At stage we first simulatedgene expression based on reference genotype panel We then estimated SNPgene effects W gfor each gene g based on the simulated gene expression and genotype which were then used topredict gene expression At stage separately we simulated the a continuous trait using simulated gene expression based on genotype and estimated the marginal SNPphenotype effectsSimulation step simulating gene expression To simulate individual genotype we first partitioned genotype data for individuals ofEuropean ancestry obtained from the Genomes Project [] into independent LD blocks as defined by LDetect [] We then randomly sampled LD blocks and used only those LD blocks for the subsequent simulation We used LD blocks as opposed to whole genome to reduce computational burden required for multiple simulation runs For LD block j j of an individual i i we randomly sampledfrom the available samples for block j and concatenated these sampled LD blocks for this individual We repeated this procedure to simulate genotype Xref forNref individuals as a reference population We standardized the simulated genotype Xref We randomly sampled k incis causal SNPs per gene within ± kb around the genewhere k default We also experimented different number of causal SNPs k allin cis SNPs We sampled SNPgene weights Wg � N ð0 h2g kÞ where gene expression heritability h2 default which is the variance of gene expression explained by genotype We alsoexperimented different gene heritability h2g ¼ f02 05gg ¼ We then simulated gene expression Agref Xref Wg � where � � N ð0 s2� Þ and� ¼ s2Nrefk XrefWgk2ð h2g 00 1ÞINrefto match the desired heritability 00 h2gh2g¼s2�kXref Wg k2Nref Finally we applied LASSO regression Agref � �XWg to get Wg for each geneSimulation step simulating phenotypePLOS ONE 101371journalpone0237657 August PLOS ONE 0cGene score regression We simulated another Ngwas GWAS individuals by the predefined LD blocksamong the Europeans in Genome data following the same procedures as decribedabove We then standardized the simulated genotype Xgwas We then sampled a causal pathway Cc from MSigDB such that all of the Gc � jCcj genes inCc were causal genes for the phenotype For each noncausal pathway we removed genes that were also present in the causal pathway We removed noncausal pathways containing fewer than five genes afterwardsdefault Alternatively in more realistic scenarios we allowed for sharing genes with causalpathways by noncausal pathways We sampled genephenotype effect a � N ð0 s2aGcIGcÞ where the phenotypic varianceexplained by gene expression s2s2a f01 05ga ¼ default We also experimented different We simulated gene expression Ac as in step for the Ngwas individuals and standardized itto obtain �Ac We simulated a continuous trait using causal gene expression y ¼ �Acα þ �y where�y � N ð0 s2Þ Here s2�yof variance explained 00 s2s2a�ya¼s2�yk �Acαk2Ngwas¼ Ngwask �Acαk2ð s2a 00 1ÞINgwasto match the predefined proportion Lastly we computed GWAS summary SNPtotrait effect size b ¼ N XgwasyWe repeated these simulation procedures times Unless otherwise stated while we wereexperimenting various settings we kept the other settings at their default values k causalSNP per gene gene expression variance explained per causal SNP h2ance explained per gene s2a ¼ one causal pathway Using these obtained summary statistics we were able to perform GSR PASCAL LDSC and FOCUS in each simulated scenarioApplying existing methods PASCAL PASCAL was downloaded from www2g ¼ 01k phenotypic variunilchcbgindexphptitlePascal [] We executed the software using default settings LDSCStratified LD score regression software was downloaded from githubcombulikldsc[] Because LDSC operates on SNP level we considered SNPs located within ± kbaround genes in each pathway to be involved in the corresponding pathway Then for eachpathway we computed the LD scores over all chromosomes We experimented the options ofrunning LDSC with and without the baseline annotations using our simulated data Wefound that LDSC running without the baseline worked better in our case One possible reason is that the baseline annotations cover genomewide SNPs whereas there are much fewerSNPs in the simulated pathways FOCUS We downloaded FOCUS [] from githubcombogdanlabfocus We used FOCUS to infer the posterior probability of each gene beingcausal for the phenotype across all of the LD blocks We then took the credible gene set asfollows We first summed all of the posteriors over all of the genes We then sorted the genesby the decreasing order of their FOCUSposteriors We kept adding the top ranked the geneinto the credible gene until the sum of their posteriors was greater than or equal to the of the total sum of posteriors We used the credible gene set for hypergeometric testfor each pathway to compute the pvalues We also tried other thresholds for credible setsranging from including the fewest genes to including the most genes GSEAGSEA software was obtained from oftwarebroadinstitutegsea [] We used thePLOS ONE 101371journalpone0237657 August PLOS ONE 0cGene score regressionFig Gene scores correlated with marginal TWAS summary statistics a Gene scores were correlated with marginal TWAS chisquare statistics ofSchizophrenia We grouped genes into bins by their gene scores to reduce noise For each bin we calculated the average gene scores and chisquarestatistics χ2 An unbinned version is supplied in S1 Fig in S1 File b Summary of Pearson correlation between marginal TWAS summary statisticsand gene scores for traits The negative correlation for T2D indicates that this trait possibly due to complicated genetic architecture andconfounding genetoenvironment interaction and drug effects is not suitable for using our approach101371journalpone0237657g002commandline version of GSEA to test for gene set enrichments using the observed geneexpression and phenotype dataReal data application We applied our approach to investigate pathway enrichment for complex traits Fig 2b using publicly available summary statistics and genotypeexpressionweights based on GTEx whole blood samples The GWAS summary statistics were downloaded from public database databroadinstitutealkesgroupsumstats_formatted[] We downloaded expression weights and reference LD structure estimated in Genomesusing European individuals from the TWASFUSION website httpgusevlabprojectsfusion [ ] Franke lab celltypespecific gene expression dataset were obtainedfrom databroadinstitutempgdepictdepict_downloadtissue_expressionIn addition we applied GSR to test for gene set enrichment in three wellpowered types ofcancer breast invasive carcinoma BRCA cases and controls thyroid carcinomaTHCA cases and controls and prostate adenocarcinoma PRAD cases and controls using gene expression datasets from The Cancer Genome Atlas TCGA Uniformlyprocessed normalized and batcheffect corrected gene expression datasets from TCGA andGTEx were obtained from figsharecomsData_record_35330593 [] Geneexpression and phenotype were standardized before supplying to the GSR software StandardGSEA was also performed for comparisonGene sets were downloaded from the MSigDb website oftwarebroadinstitutegseamsigdbindexjsp Here we combined BIOCARTA KEGG and REACTOME to create atotal of gene sets We also downloaded the GO biological process terms as additional gene sets as well as the gene sets pertaining to oncogenic signatures for the TCGAdata analysisResultsGene scores were correlated with TWAS statistics in polygenic complex traitsOur method GSR is built on the hypothesis that the marginal gene effect sizes on the phenotype should be positively correlated with the sum of correlation with other genes whichPLOS ONE 101371journalpone0237657 August PLOS ONE 0cGene score regressioninclude causal genes To validate this hypothesis we defined gene score for each gene as thesum of its squared Pearson correlation with all of the other genes derived from gene expression levels We calculated TWAS marginal statistics as the product of GWAS summary statistics β and eQTL weights W derived from the GTEx whole blood samples Eq To assessthe impact of genetogene correlation on TWAS statistic we correlated the gene scores withthe TWAS marginal statistics for complex traits Overall most traits had Pearson correlation between the gene score and the marginal TWAS statistic above For instance thecorrelation in schizophrenia was InterQuartile Range ” based on permutations Fig This implies a pervasive confounding impact on the downstream analysisincluding gene set or pathway enrichment analysis causal gene identification etc using theTWAS summary statistics while assuming independence of genes Fig GSR improved pathway enrichment powerIn simulated scenarios with default settings Methods compared to PASCAL and LDSC GSRdemonstrated hugely improved computational efficiency Table superior sensitivity indetecting causal pathways with an improved statistical power as well as competitive specificityin controlling for false positives Fig Specifically in simulations GSR achieved an overall area under the precisionrecall curve AUPRC of and identified the true causal pathway as the most significant one times compared to times by PASCAL which onlyachieved an overall AUPRC of Notably the FOCUSpredicted crediblegene sets were also significantly enriched for causal pathways Fig We then varied four different settings a the number of causal SNP per gene SNPgene heritabilities genephenotype variance explained overlapping causal pathway Wefocused our comparison with PASCAL because it directly tested for pathway enrichment andhas been demonstrated to outperform other relevant enrichment methods [] In all simulation settings GSR demonstrated an improved power in detecting the causal pathways S2 Figin S1 File as it was able to detect causal pathways when multiple SNPs influenced geneexpression when the proportion of variance explained by the gene expression was low orwhen the causal and noncausal pathways were allowed to overlap In contrast a lot of causalpathways were not deemed significant by PASCAL based on a pvalue threshold of which was equivalent to a Bonferronicorrected pvalue threshold of after correcting formultiple testing on approximately pathways tested per simulationImproved power in pathway enrichment leveraging observed geneexpressionOne unique feature of GSR is the ability to run not on only the summary statistics but also onobserved gene expression where the genegene expression correlation is directly estimatedTable Comparison of existing methods with GSRMethodPASCAL []LDSC []FOCUS []GSEA []GSRGWASTWASMeasured expressionRunning timesum stat �sum statsum statsum statsum statindividual expressionindividual expression m h   h m min� Summary statistics  For custom gene sets the main computation time for LDSC is calculating the LD score for all of the Genome SNPs101371journalpone0237657t001PLOS ONE 101371journalpone0237657 August PLOS ONE 0cGene score regressionFig Evaluation of power and robustness of GSR in detecting causal pathways a Precisionrecall curves for GSR and PASCAL summarizingresults from simulations b Summary of pvalues obtained by running GSR along with PASCAL LDSC and FOCUS times For each methodthe enrichment significance for causal pathways and noncausal pathways are displayed We experimented FOCUS with and crediblesets for the pathway enrichments For the ease of comparison we plotted the yaxis on a squareroot negative logarithmic scale Red line denotes pvalue threshold of Blue line denotes pvalue threshold of 101371journalpone0237657g003from the insample gene expression To evaluate the accuracy of this application we simulatedgene expression and phenotype for individuals which were provided as input to GSR forpathway enrichment analysis As a comparison we applied GSR to the summary statistics generated from the same datasetAs in the simulation above the SNPexpression weights were estimated from a separate setof reference individuals whereas the SNPphenotype associations were estimated fromonly individuals Notably the sample size for the GWAS cohort is much smaller than theprevious application to mimic the real data where usually fewer than individuals haveboth the RNAseq and phenotype available eg TCGA Additionally we applied standardGSEA [] to the same dataset with the observed gene expression We observed an improvedpower of GSR when using the observed gene expression over GSR using the summary statisticsFig whereas GSEA had a comparable performance as the latter Specifically all causal pathways in the simulated replicates had a pvalue below with the largest pvalue being × ˆ’ as determined by GSR using observed gene expression while no causal pathwayreached this level of significance with the smallest pvalue being × ˆ’ determined byGSEA We also compared the performances of GSR using observed gene expression to GSEAin various simulation settings and obtained consistent conclusions S3 Fig in S1 FileGene set enrichments in complex traitsApplying GSR to complex traits we revealed various pathways where the enriched gene setswere biologically meaningful For example the enriched gene sets for high density lipoproteinHDL predominantly involve lipid metabolism In contrast for Lupus gene sets wereenriched in interferon signalling pathways a known immunological hallmark We listed thetop enrichments over gene sets from MSigDB and Gene Ontology terms for HDL and theautoimmune trait Lupus in S1 Table in S1 FileAdditionally we applied GSR to test celltypespecific enrichments using cell types of which were derived from GTEx and cell types were derived from Franke lab datasets[] We observed biologically meaningful cell typespecific enrichment for the complexPLOS ONE 101371journalpone0237657 August PLOS ONE 0cGene score regressionFig Comparison of pathway enrichment determined by GSR using or not using observed gene expression information and by GSEA NominalNOM pvalues yielded by GSEA were summarized Red line denotes pvalue threshold of Blue line denotes pvalue threshold of 101371journalpone0237657g004traits Fig In particular central neural system cellspecific gene sets were enriched forschizophrenia immune cellspecific gene sets for lupus immune cellspecific and digestivetract cellspecific gene sets for Crohn™s disease and cardiac cellspecific gene sets for coronaryartery disease Lastly we correlated traits based on their gene set enrichments and observedmeaningful phenotypic clusters suggesting shared biological mechanisms by the related phenotypes S4 Fig in S1 File For example Crohn™s disease and ulcerative colitis two subtypes ofinflammatory bowel disease formed a cluster Neurological diseases schizophrenia and bipolardisorder formed a cluster Moreover lipid traits including LDL HDL and Triglyceridesformed their own clusterApplication on observed gene expressionLastly using expression profiles of BRCA THCA and PRAD from TCGA and GTEx [] wetested the enrichments of oncogenic gene sets as well as gene sets from BIOCARTAKEGG and REACTOME in each type of tumor Overall we observed a significantly strongerenrichments for the oncogenic signatures with higher p values compared to the more generalgene sets across all three tumour types ttest pvalue × ˆ’ × ˆ’ and × ˆ’for BRCA PRAD and THCA respectively S5 Fig in S1 File As a comparison we also ranstandard GSEA and observed qualitatively similar enrichments S5 Fig in S1 FilePLOS ONE 101371journalpone0237657 August PLOS ONE 0cGene score regressionFig Celltypespecific enrichment of gene sets for representative complex traits GSR was applied to each complex trait in order toidentify significantly enriched gene sets among predefined celltypespecific gene sets represented by nine different colors Gene setswere indicated by dots and were aligned in the same order on the xaxis Red lines indecate Bonferronicorrected pvalue threshold 101371journalpone0237657g005DiscussionIn this work we describe GSR an efficient method to test for gene set or pathway enrichmentsusing either GWAS summary statistics or observed gene expression and phenotype information We demonstrate robust and powerful detection of causal pathways in extensive simulation using our proposed method compared to several stateoftheart methods Whenapplying to the real data we also obtained biologically meaningful enrichments of relevantgene sets and pathways These features warrant GSR a widely applicable method in variousstudy settings with an aim to interpret association test results and capture the underlying biological mechanismsOur approach has superior computational efficiency In particular GSR took only minutes running on the full summary statistics and less than minutes on the full gene expressiondata with one million SNPs and genes to test for enrichments of over gene sets Inour simulations it is not surprising that FOCUS can accurately finemap causal genes as thesimulation designs followed similar assumptions adopted by FOCUS [] However FOCUSis at least times slower than GSR For the simulated data FOCUS took minutes to finemap all of the genes in GWAS loci whereas GSR took under three minutes to test for pathwayenrichments on the same machine Additionally the computational cost of FOCUS is exponential to the number of causal genes considered within each locus whereas GSR is not affectedPLOS ONE 101371journalpone0237657 August PLOS ONE 0cGene score regressionby the number of causal genes Also because GSR operates at genomewide level no thresholdis needed to decide which genes to be included whereas FOCUS needs userdefined thresholdfor constructing the credible gene set for the subsequent hypergeometric enrichment testGiven these advantages we envision that GSR will be a valuable tool for the bioinformaticcommunity and statistical genetic community as a fast way to investigate the functional implications of complex polygenic traitsIn different simulation settings GSR exhibits improved pathway enrichment power overPASCAL and LDSC two popular methods for partitioning heritability and identifying causalgene sets Since GSR leverages SNPtogene association summaried by eQTL weights whileeither PASCAL or LDSC operate

cancer268

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: suffer from a high false positive rate because predicted expression of different genes may behighly correlated due to linkage disequilibrium between eQTL We propose a novel statistical method Gene Score Regression GSR to detect causal gene sets for complex traitswhile accounting for genetogene correlations We consider noncausal genes that arehighly correlated with the causal genes will also exhibit a high marginal association with thecomplex trait Consequently by regressing on the marginal associations of complex traitswith the sum of the genetogene correlations in each gene set we can assess the amountof variance of the complex traits explained by the predicted expression of the genes in eachgene set and identify plausible causal gene sets GSR can operate either on GWAS summary statistics or observed gene expression Therefore it may be widely applied to annotateGWAS results and identify the underlying biological pathways We demonstrate the highaccuracy and computational efficiency of GSR compared to stateoftheart methodsthrough simulations and real data applications GSR is ly available at githubcomlilabmcgillGSRIntroductionGenomewide association studies GWAS have been broadly successful in associating geneticvariants with complex traits and estimating trait heritabilities in large populations [“] Overthe past decade GWAS have quantified the effects of individual genetic variants on hundredsof polygenic phenotypes [ ] GWAS summary statistics have enabled various downstreamanalyses including partitioning heritability [] inferring causal single nucleotide polymorphisms SNPs using epigenomic annotations [] and gene sets enrichment analysis fora1111111111a1111111111a1111111111a1111111111a1111111111 ACCESSCitation Zhang W Li SY Liu T Li Y Partitioning genebased variance of complex traitsby gene score regression e0237657 101371journalpone0237657Editor F Alex Feltus Clemson University UNITEDSTATESReceived March Accepted July Published August Peer Review History PLOS recognizes thebenefits of transparency in the peer reviewprocess therefore we enable the publication ofall of the content of peer review and authorresponses alongside final published s Theeditorial history of this is available here101371journalpone0237657Copyright Zhang This is an access distributed under the terms of theCreative Commons Attribution License whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are creditedData Availability Statement All of the data used inthis paper are described under subsection Realdata application in the manuscript and pastedbelow as reference We applied our approach toinvestigate pathway enrichment for complexPLOS ONE 101371journalpone0237657 August PLOS ONE 0cGene score regressiontraits Fig 2b using publicly available summarystatistics and genotypeexpression weights basedon GTEx whole blood samples wwwgtexportalhomedatasets2 The GWASsummary statistics were downloaded from publicdatabase databroadinstitutealkesgroupsumstats_formatted We downloadedexpression weights and reference LD structureestimated in Genomes using Europeanindividuals from the TWASFUSION websitehttpgusevlabprojectsfusion Franke labcelltypespecific gene expression dataset wereobtained from databroadinstitutempgdepictdepict_downloadtissue_expression Inaddition we applied GSR to test for gene setenrichment in three wellpowered types of cancerbreast invasive carcinoma BRCA cases and controls thyroid carcinoma THCA casesand controls and prostate adenocarcinomaPRAD cases and controls using geneexpression datasets from The Cancer GenomeAtlas TCGA Uniformly processed normalizedand batcheffect corrected gene expressiondatasets from TCGA and GTEx were obtained fromfigsharecomsData_record_3 Gene expression and phenotype werestandardized before supplying to the GSR softwareStandard GSEA was also performed forcomparison Gene sets were downloaded from theMSigDb website oftwarebroadinstitutegseamsigdbindexjsp Here we combinedBIOCARTA KEGG and REACTOME to create a totalof gene sets We also downloaded the GO biological process terms as additional genesets as well as the gene sets pertaining tooncogenic signatures for the TCGA data analysisFunding The research is supported by CanadaFirst Research Excellence Fund CFREF HealthyBrains Healthy Life HBHL New Investigator fund at McGill University and Mon trealNeurologic Institute MNI and NSERC DiscoveryGrant RGPIN20190621 The funders had nocomplex traits [] However it remains challenging to link these genetic associations withknown biological mechanisms One main reason is that the majority of the GWAS loci are notlocated in known genic regions of the human genomeTranscriptomewide association studies TWAS [“] offer a systematic way to integrateGWAS and the reference genotypegene expression datasets such as the GenotypeTissueExpression project GTEx [] via expression quantitative loci eQTL In TWAS we couldfirst quantify the impact of each genetic variant on expression variability in a population andobtain predicted gene expression levels based on new genotypes Then we could correlate thepredicted gene expression with the phenotype of interest in order to identify pivotal genes[] Moreover when individuallevel genotypes and gene expression levels are not availablewe could still quantify genetophenotype association ie TWAS statistics using only themarginal effect sizes of SNPs on the phenotype and on gene expression respectively []These concepts and implementations have largely facilitated explanation of genetic associationfindings at the gene or the pathway levelHowever as depicted in Fig TWAS are often confounded by the genetogene correlationof the genetically predicted gene expression due to the SNPtoSNP correlation ie linkage disequilibrium LD [] Consequently relying on the TWAS statistics may lead to false positivediscoveries of causal genes and pathways One approach to address this problem is to finemapcausal genes by inferring the posterior probabilities of configurations of each gene being causalin a defined GWAS loci and then test gene set enrichment using the credible gene sets of prioritized genes [] However this approach is computationally expensive restricted to GWASloci and sensitive to the arbitrary thresholds used for determining the credible gene set andthe maximum number of causal genes per locusAnother method called PASCAL [] projects SNP signals onto genes while correcting forLD and then performs pathway enrichments as the aggregated transformed gene scoreswhich asymptotically follows a chisquare distribution However PASCAL does not leverage the eQTL information for each SNP thereby assuming that a priori all SNPs have thesame effect on the gene Stratified LD score regression LDSC offers a principle way to partition the SNP heritability into functional categories defined based on tissue or celltypespecific epigenomic regions [] or eQTL regions of the genes exhibiting a strong tissue specificity [] Although LDSC is able to obtain biologically meaningful tissuespecific enrichments it operates at the SNP level rendering it difficult to assess enrichment of gene setsMoreover neither PASCAL nor LDSC is able to integrate the observed gene expression datameasured in a disease cohort rather than the reference cohort that are broadly availableacross diverse studies of diseases including cancers such as The Cancer Genome AtlasTCGA []role in study design data collection and analysisAlthough expressionbased methods such as gene set enrichment analysis GSEA aredecision to publish or preparation of themanuscript No author received a salary from anyof the fundersCompeting interests The authors have declaredthat no competing interests existoften adopted in combination with the observed gene expression and phenotypes [] theygenerally do not account for the genetogene correlation While this type of correlation is usually caused by shared transcriptional regulatory mechanisms across genes GSEA still likelyproduces false positives in identifying causal pathwaysIn this study we present a novel and powerful genebased heritability partitioning methodthat jointly accounts for genetogene correlation and integrates information captured at eitherthe SNPtophenotype or the SNPtogene level We utilize this method to identify plausiblecausal gene sets or pathways for complex traits We showcase its high accuracy and computational efficiency in various simulated and real scenariosPLOS ONE 101371journalpone0237657 August PLOS ONE 0cGene score regressionFig Overview of confounding effects on pathway analysis a A hypothetical example illustrates the confounding issue when using the geneticallypredicted transcriptome to assess the pathway enrichments for a target phenotype The causal gene set includes a causal gene which is linked to noncausal gene via their respective causal SNPs and which are in strong linkage disequilibrium b A GWAS locus SNP associations with the targetphenotype are summarized The causal SNP for the causal gene is in red The SNPs that drive noncausal genes are in blue The rest of the SNPs are ingreen SNPs exhibit correlated signals due to the linkage disequilibrium LD as displayed by the upper triangle of the SNPSNP Pearson correlationmatrix c A TWAS locus The genetogene correlation is partly induced by the SNPtoSNP correlation and partly due to intrinsic coregulatoryexpression program d Pathway associations based on averaged gene associations101371journalpone0237657g001MethodsPartitioning genebased variance of complex traitsWe assume gene expression has linearly additive effects on a continuous polygenic trait yyi ¼XAijaj þ �ijð1Þwhere Aij denotes the expression of the jth gene in the ith individual for i N individuals and j G genes αj denotes the true effect size of the jth gene on the trait andj Þ �i denotes the residual for the ith individual in this linear model andaj � Nð0 s2�i � Nð0 s2� ÞPiyi ¼ N yy ¼ Here we further assume that both y and A are standardized such that NPiAij ¼ and j Aj ¼ for j GN ANPLOS ONE 101371journalpone0237657 August PLOS ONE 0cWe define the estimated marginal effect size of the jth gene on the trait as ajGene score regressionaj ¼NAj y¼NAj ðXAkak þ �ÞkX¼kNAj Akak þNAj �X¼krjkak þ �ð2Þð3Þð4Þð5Þwhere � ¼ N Aj � withVarð�0Þ ¼N Aj Varð�ÞAj ¼N s2�and rjk ¼ and the kth geneN Aj Ak is the estimated Pearson correlation in gene expression between the jth geneWe define w2j ¼ Na2j Then if we further assume α r and � are independent we haveE½w2j � ¼ E½Na2j �X¼ NE½Ã°rjkak þ �0Þ2�k¼ NE½r jk�E½a2k� þ s2�Xkð6Þð7Þð8ÞNow consider C gene sets Cc where c C and denote the proportion of total trait Here Cc denotes the numVarðajÞvariance explained by the cth gene set as τc with tc ¼ber of genes in the cth gene setPjCcjj2CcConsequentlyE½a2k� ¼ VarðakÞ ¼Xck2CctcBy approximating E½r N we have thatXjk� with r E½w2jk þ j � ¼ NE½r jk�E½a2k� þ s2�k¼ NXtcXr jk þXtc þ s2�ck2Ccc¼ NXtclðj cÞ þ cwhere we define gene score as lðj cÞ ¼tinuous trait is normalizedPr jk and VarðyÞ ¼k2Ccð9Þð10Þð11ÞPctc þ s2� ¼ since the conPLOS ONE 101371journalpone0237657 August PLOS ONE 0cGene score regressionTherefore if we are able to obtain estimates for w2j and C gene score lj c for j Gand c C we will be able to perform linear regression of the estimated w2j on lj cand derive regression coefficient that is an estimate for each τc c C respectivelyThese are available from GWAS summary statistics of SNPtotrait effect sizes eQTL summary statistics of SNPtogene expression effect sizes and a reference LD panel Specifically Suppose we have estimated effect sizes βp× of p SNPs based on a GWAS including Ngwassamples ieb ¼NgwasXywhere XNgwas×p is the standardized genotype Meanwhile we have the eQTL summary statistics W estimated usingAeQTL ¼ XeQTLWTherefore the predicted gene expression in GWAS is given bySinceA ¼ XWj ¼ Na2w2j¼ NðNAj yÞ2¼ NðNWj XyÞ2¼ NðW j bÞ2the required w2j can be estimated without accessing any individuallevel data Furthermore a reference LD panel Sp×p summarizing SNPtoSNP correlation in thematched population with the GWAS study can provide estimates for rjk asR ¼ ½rjk�¼NAA¼NW XXW¼NW SWPLOS ONE 101371journalpone0237657 August ð12Þð13Þð14Þð15Þð16Þð17Þð18Þð19Þ PLOS ONE 0cGene score regressionIt is noteworthy that with individuallevel gene expression data we can also easily obtainthe required w2j and R [rjk] by definitionIn practice many gene sets are not disjoint and share common genes with each otherTherefore we regress one gene set at a time along with a œdummy gene set that include theunion of all of the other genes The dummy gene set is used to account for unbalanced genesets and to stabilize estimates of τc We also include an intercept in the regression model toalleviate nongeneset biases for example positive correlation between gene scores and truegene effect sizes that could lead to intercept greater than and negative correlation betweengene scores and true gene effect sizes could lead to intercept smaller than Simulation designTo assess the accuracy of our GSR approach we simulated causal SNPs for gene expression aswell as causal gene sets for a continuous trait based on real genotypes and known gene setsfrom existing databases Our simulation included two stages At stage we first simulatedgene expression based on reference genotype panel We then estimated SNPgene effects W gfor each gene g based on the simulated gene expression and genotype which were then used topredict gene expression At stage separately we simulated the a continuous trait using simulated gene expression based on genotype and estimated the marginal SNPphenotype effectsSimulation step simulating gene expression To simulate individual genotype we first partitioned genotype data for individuals ofEuropean ancestry obtained from the Genomes Project [] into independent LD blocks as defined by LDetect [] We then randomly sampled LD blocks and used only those LD blocks for the subsequent simulation We used LD blocks as opposed to whole genome to reduce computational burden required for multiple simulation runs For LD block j j of an individual i i we randomly sampledfrom the available samples for block j and concatenated these sampled LD blocks for this individual We repeated this procedure to simulate genotype Xref forNref individuals as a reference population We standardized the simulated genotype Xref We randomly sampled k incis causal SNPs per gene within ± kb around the genewhere k default We also experimented different number of causal SNPs k allin cis SNPs We sampled SNPgene weights Wg � N ð0 h2g kÞ where gene expression heritability h2 default which is the variance of gene expression explained by genotype We alsoexperimented different gene heritability h2g ¼ f02 05gg ¼ We then simulated gene expression Agref Xref Wg � where � � N ð0 s2� Þ and� ¼ s2Nrefk XrefWgk2ð h2g 00 1ÞINrefto match the desired heritability 00 h2gh2g¼s2�kXref Wg k2Nref Finally we applied LASSO regression Agref � �XWg to get Wg for each geneSimulation step simulating phenotypePLOS ONE 101371journalpone0237657 August PLOS ONE 0cGene score regression We simulated another Ngwas GWAS individuals by the predefined LD blocksamong the Europeans in Genome data following the same procedures as decribedabove We then standardized the simulated genotype Xgwas We then sampled a causal pathway Cc from MSigDB such that all of the Gc � jCcj genes inCc were causal genes for the phenotype For each noncausal pathway we removed genes that were also present in the causal pathway We removed noncausal pathways containing fewer than five genes afterwardsdefault Alternatively in more realistic scenarios we allowed for sharing genes with causalpathways by noncausal pathways We sampled genephenotype effect a � N ð0 s2aGcIGcÞ where the phenotypic varianceexplained by gene expression s2s2a f01 05ga ¼ default We also experimented different We simulated gene expression Ac as in step for the Ngwas individuals and standardized itto obtain �Ac We simulated a continuous trait using causal gene expression y ¼ �Acα þ �y where�y � N ð0 s2Þ Here s2�yof variance explained 00 s2s2a�ya¼s2�yk �Acαk2Ngwas¼ Ngwask �Acαk2ð s2a 00 1ÞINgwasto match the predefined proportion Lastly we computed GWAS summary SNPtotrait effect size b ¼ N XgwasyWe repeated these simulation procedures times Unless otherwise stated while we wereexperimenting various settings we kept the other settings at their default values k causalSNP per gene gene expression variance explained per causal SNP h2ance explained per gene s2a ¼ one causal pathway Using these obtained summary statistics we were able to perform GSR PASCAL LDSC and FOCUS in each simulated scenarioApplying existing methods PASCAL PASCAL was downloaded from www2g ¼ 01k phenotypic variunilchcbgindexphptitlePascal [] We executed the software using default settings LDSCStratified LD score regression software was downloaded from githubcombulikldsc[] Because LDSC operates on SNP level we considered SNPs located within ± kbaround genes in each pathway to be involved in the corresponding pathway Then for eachpathway we computed the LD scores over all chromosomes We experimented the options ofrunning LDSC with and without the baseline annotations using our simulated data Wefound that LDSC running without the baseline worked better in our case One possible reason is that the baseline annotations cover genomewide SNPs whereas there are much fewerSNPs in the simulated pathways FOCUS We downloaded FOCUS [] from githubcombogdanlabfocus We used FOCUS to infer the posterior probability of each gene beingcausal for the phenotype across all of the LD blocks We then took the credible gene set asfollows We first summed all of the posteriors over all of the genes We then sorted the genesby the decreasing order of their FOCUSposteriors We kept adding the top ranked the geneinto the credible gene until the sum of their posteriors was greater than or equal to the of the total sum of posteriors We used the credible gene set for hypergeometric testfor each pathway to compute the pvalues We also tried other thresholds for credible setsranging from including the fewest genes to including the most genes GSEAGSEA software was obtained from oftwarebroadinstitutegsea [] We used thePLOS ONE 101371journalpone0237657 August PLOS ONE 0cGene score regressionFig Gene scores correlated with marginal TWAS summary statistics a Gene scores were correlated with marginal TWAS chisquare statistics ofSchizophrenia We grouped genes into bins by their gene scores to reduce noise For each bin we calculated the average gene scores and chisquarestatistics χ2 An unbinned version is supplied in S1 Fig in S1 File b Summary of Pearson correlation between marginal TWAS summary statisticsand gene scores for traits The negative correlation for T2D indicates that this trait possibly due to complicated genetic architecture andconfounding genetoenvironment interaction and drug effects is not suitable for using our approach101371journalpone0237657g002commandline version of GSEA to test for gene set enrichments using the observed geneexpression and phenotype dataReal data application We applied our approach to investigate pathway enrichment for complex traits Fig 2b using publicly available summary statistics and genotypeexpressionweights based on GTEx whole blood samples The GWAS summary statistics were downloaded from public database databroadinstitutealkesgroupsumstats_formatted[] We downloaded expression weights and reference LD structure estimated in Genomesusing European individuals from the TWASFUSION website httpgusevlabprojectsfusion [ ] Franke lab celltypespecific gene expression dataset were obtainedfrom databroadinstitutempgdepictdepict_downloadtissue_expressionIn addition we applied GSR to test for gene set enrichment in three wellpowered types ofcancer breast invasive carcinoma BRCA cases and controls thyroid carcinomaTHCA cases and controls and prostate adenocarcinoma PRAD cases and controls using gene expression datasets from The Cancer Genome Atlas TCGA Uniformlyprocessed normalized and batcheffect corrected gene expression datasets from TCGA andGTEx were obtained from figsharecomsData_record_35330593 [] Geneexpression and phenotype were standardized before supplying to the GSR software StandardGSEA was also performed for comparisonGene sets were downloaded from the MSigDb website oftwarebroadinstitutegseamsigdbindexjsp Here we combined BIOCARTA KEGG and REACTOME to create atotal of gene sets We also downloaded the GO biological process terms as additional gene sets as well as the gene sets pertaining to oncogenic signatures for the TCGAdata analysisResultsGene scores were correlated with TWAS statistics in polygenic complex traitsOur method GSR is built on the hypothesis that the marginal gene effect sizes on the phenotype should be positively correlated with the sum of correlation with other genes whichPLOS ONE 101371journalpone0237657 August PLOS ONE 0cGene score regressioninclude causal genes To validate this hypothesis we defined gene score for each gene as thesum of its squared Pearson correlation with all of the other genes derived from gene expression levels We calculated TWAS marginal statistics as the product of GWAS summary statistics β and eQTL weights W derived from the GTEx whole blood samples Eq To assessthe impact of genetogene correlation on TWAS statistic we correlated the gene scores withthe TWAS marginal statistics for complex traits Overall most traits had Pearson correlation between the gene score and the marginal TWAS statistic above For instance thecorrelation in schizophrenia was InterQuartile Range ” based on permutations Fig This implies a pervasive confounding impact on the downstream analysisincluding gene set or pathway enrichment analysis causal gene identification etc using theTWAS summary statistics while assuming independence of genes Fig GSR improved pathway enrichment powerIn simulated scenarios with default settings Methods compared to PASCAL and LDSC GSRdemonstrated hugely improved computational efficiency Table superior sensitivity indetecting causal pathways with an improved statistical power as well as competitive specificityin controlling for false positives Fig Specifically in simulations GSR achieved an overall area under the precisionrecall curve AUPRC of and identified the true causal pathway as the most significant one times compared to times by PASCAL which onlyachieved an overall AUPRC of Notably the FOCUSpredicted crediblegene sets were also significantly enriched for causal pathways Fig We then varied four different settings a the number of causal SNP per gene SNPgene heritabilities genephenotype variance explained overlapping causal pathway Wefocused our comparison with PASCAL because it directly tested for pathway enrichment andhas been demonstrated to outperform other relevant enrichment methods [] In all simulation settings GSR demonstrated an improved power in detecting the causal pathways S2 Figin S1 File as it was able to detect causal pathways when multiple SNPs influenced geneexpression when the proportion of variance explained by the gene expression was low orwhen the causal and noncausal pathways were allowed to overlap In contrast a lot of causalpathways were not deemed significant by PASCAL based on a pvalue threshold of which was equivalent to a Bonferronicorrected pvalue threshold of after correcting formultiple testing on approximately pathways tested per simulationImproved power in pathway enrichment leveraging observed geneexpressionOne unique feature of GSR is the ability to run not on only the summary statistics but also onobserved gene expression where the genegene expression correlation is directly estimatedTable Comparison of existing methods with GSRMethodPASCAL []LDSC []FOCUS []GSEA []GSRGWASTWASMeasured expressionRunning timesum stat �sum statsum statsum statsum statindividual expressionindividual expression m h   h m min� Summary statistics  For custom gene sets the main computation time for LDSC is calculating the LD score for all of the Genome SNPs101371journalpone0237657t001PLOS ONE 101371journalpone0237657 August PLOS ONE 0cGene score regressionFig Evaluation of power and robustness of GSR in detecting causal pathways a Precisionrecall curves for GSR and PASCAL summarizingresults from simulations b Summary of pvalues obtained by running GSR along with PASCAL LDSC and FOCUS times For each methodthe enrichment significance for causal pathways and noncausal pathways are displayed We experimented FOCUS with and crediblesets for the pathway enrichments For the ease of comparison we plotted the yaxis on a squareroot negative logarithmic scale Red line denotes pvalue threshold of Blue line denotes pvalue threshold of 101371journalpone0237657g003from the insample gene expression To evaluate the accuracy of this application we simulatedgene expression and phenotype for individuals which were provided as input to GSR forpathway enrichment analysis As a comparison we applied GSR to the summary statistics generated from the same datasetAs in the simulation above the SNPexpression weights were estimated from a separate setof reference individuals whereas the SNPphenotype associations were estimated fromonly individuals Notably the sample size for the GWAS cohort is much smaller than theprevious application to mimic the real data where usually fewer than individuals haveboth the RNAseq and phenotype available eg TCGA Additionally we applied standardGSEA [] to the same dataset with the observed gene expression We observed an improvedpower of GSR when using the observed gene expression over GSR using the summary statisticsFig whereas GSEA had a comparable performance as the latter Specifically all causal pathways in the simulated replicates had a pvalue below with the largest pvalue being × ˆ’ as determined by GSR using observed gene expression while no causal pathwayreached this level of significance with the smallest pvalue being × ˆ’ determined byGSEA We also compared the performances of GSR using observed gene expression to GSEAin various simulation settings and obtained consistent conclusions S3 Fig in S1 FileGene set enrichments in complex traitsApplying GSR to complex traits we revealed various pathways where the enriched gene setswere biologically meaningful For example the enriched gene sets for high density lipoproteinHDL predominantly involve lipid metabolism In contrast for Lupus gene sets wereenriched in interferon signalling pathways a known immunological hallmark We listed thetop enrichments over gene sets from MSigDB and Gene Ontology terms for HDL and theautoimmune trait Lupus in S1 Table in S1 FileAdditionally we applied GSR to test celltypespecific enrichments using cell types of which were derived from GTEx and cell types were derived from Franke lab datasets[] We observed biologically meaningful cell typespecific enrichment for the complexPLOS ONE 101371journalpone0237657 August PLOS ONE 0cGene score regressionFig Comparison of pathway enrichment determined by GSR using or not using observed gene expression information and by GSEA NominalNOM pvalues yielded by GSEA were summarized Red line denotes pvalue threshold of Blue line denotes pvalue threshold of 101371journalpone0237657g004traits Fig In particular central neural system cellspecific gene sets were enriched forschizophrenia immune cellspecific gene sets for lupus immune cellspecific and digestivetract cellspecific gene sets for Crohn™s disease and cardiac cellspecific gene sets for coronaryartery disease Lastly we correlated traits based on their gene set enrichments and observedmeaningful phenotypic clusters suggesting shared biological mechanisms by the related phenotypes S4 Fig in S1 File For example Crohn™s disease and ulcerative colitis two subtypes ofinflammatory bowel disease formed a cluster Neurological diseases schizophrenia and bipolardisorder formed a cluster Moreover lipid traits including LDL HDL and Triglyceridesformed their own clusterApplication on observed gene expressionLastly using expression profiles of BRCA THCA and PRAD from TCGA and GTEx [] wetested the enrichments of oncogenic gene sets as well as gene sets from BIOCARTAKEGG and REACTOME in each type of tumor Overall we observed a significantly strongerenrichments for the oncogenic signatures with higher p values compared to the more generalgene sets across all three tumour types ttest pvalue × ˆ’ × ˆ’ and × ˆ’for BRCA PRAD and THCA respectively S5 Fig in S1 File As a comparison we also ranstandard GSEA and observed qualitatively similar enrichments S5 Fig in S1 FilePLOS ONE 101371journalpone0237657 August PLOS ONE 0cGene score regressionFig Celltypespecific enrichment of gene sets for representative complex traits GSR was applied to each complex trait in order toidentify significantly enriched gene sets among predefined celltypespecific gene sets represented by nine different colors Gene setswere indicated by dots and were aligned in the same order on the xaxis Red lines indecate Bonferronicorrected pvalue threshold 101371journalpone0237657g005DiscussionIn this work we describe GSR an efficient method to test for gene set or pathway enrichmentsusing either GWAS summary statistics or observed gene expression and phenotype information We demonstrate robust and powerful detection of causal pathways in extensive simulation using our proposed method compared to several stateoftheart methods Whenapplying to the real data we also obtained biologically meaningful enrichments of relevantgene sets and pathways These features warrant GSR a widely applicable method in variousstudy settings with an aim to interpret association test results and capture the underlying biological mechanismsOur approach has superior computational efficiency In particular GSR took only minutes running on the full summary statistics and less than minutes on the full gene expressiondata with one million SNPs and genes to test for enrichments of over gene sets Inour simulations it is not surprising that FOCUS can accurately finemap causal genes as thesimulation designs followed similar assumptions adopted by FOCUS [] However FOCUSis at least times slower than GSR For the simulated data FOCUS took minutes to finemap all of the genes in GWAS loci whereas GSR took under three minutes to test for pathwayenrichments on the same machine Additionally the computational cost of FOCUS is exponential to the number of causal genes considered within each locus whereas GSR is not affectedPLOS ONE 101371journalpone0237657 August PLOS ONE 0cGene score regressionby the number of causal genes Also because GSR operates at genomewide level no thresholdis needed to decide which genes to be included whereas FOCUS needs userdefined thresholdfor constructing the credible gene set for the subsequent hypergeometric enrichment testGiven these advantages we envision that GSR will be a valuable tool for the bioinformaticcommunity and statistical genetic community as a fast way to investigate the functional implications of complex polygenic traitsIn different simulation settings GSR exhibits improved pathway enrichment power overPASCAL and LDSC two popular methods for partitioning heritability and identifying causalgene sets Since GSR leverages SNPtogene association summaried by eQTL weights whileeither PASCAL or LDSC operate Answer:

Thyroid_Cancer

Exercise influences the impact ofpolychlorinated biphenyl exposure onimmune functionMahesh R PillaiID1 K Todd Keylock2 Howard C Cromwell3 Lee A Meserve1 Dept of Biological Sciences Bowling Green State University Bowling Green Ohio United States ofAmerica Dept of Exercise Science Bowling Green State University Bowling Green Ohio United States ofAmerica Dept of Psychology and JP Scott Center for Neuroscience Mind and Behavior Bowling GreenState University Bowling Green Ohio United States of America Current address Human Research Protection Program The University of Toledo Toledo Ohio UnitedStates of America lmeservbgsuedua1111111111a1111111111a1111111111a1111111111a1111111111Abstract ACCESSCitation Pillai MR Keylock KT Cromwell HCMeserve LA Exercise influences the impactof polychlorinated biphenyl exposure on immunefunction e0237705 101371journalpone0237705Editor HansJoachim Lehmler University of IowaUNITED STATESReceived April Accepted July Published August Peer Review History PLOS recognizes thebenefits of transparency in the peer reviewprocess therefore we enable the publication ofall of the content of peer review and authorresponses alongside final published s Theeditorial history of this is available here101371journalpone0237705Copyright Pillai This is an access distributed under the terms of theCreative Commons Attribution License whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are creditedData Availability Statement The data is availableon figshare 106084m9figshare12768332Funding Department of Biological SciencesBowling Green State University Bowling GreenOhio The funders had no role in study design dataPolychlorinated biphenyls PCBs are environmental pollutants and endocrine disruptorsharmfully affecting reproductive endocrine neurological and immunological systems Thisbroad influence has implications for processes such as wound healing which is modulatedby the immunological response of the body Conversely while PCBs can be linked to diminished wound healing outside of PCB pollution systems exercise has been shown to accelerate wound healing However the potential for moderate intensity exercise to modulateor offset the harmful effects of a toxin like PCB are yet unknown A key aim of the presentstudy was to examine how PCB exposure at different doses ppm ipaltered wound healing in exercised versus nonexercised subgroups of mice We examinedPCB effects on immune function in more depth by analyzing the concentrations of cytokinesinterleukin1 IL1 tumor necrosis factorα TNFα Interleukin6 IL6 and granulocytemacrophage colony stimulating factor GMCSF in these wounds inflicted by punch biopsyMice were euthanized at Day or Day after PCB injection n “ and skin excised fromthe wound area was homogenized and analyzed for cytokine content Results revealed thatwound healing was not signficantly impacted by either PCB exposure or exercise but therewere patterns of delays in healing that depended on PCB dose Changes in cytokines werealso observed and depended on PCB dose and exercise experience For example IL1concentrations in Day mice without PCB administration were less in exercisedmice than mice not exercised However IL1 concentrations in Day mice administered ppm were greater in exercised mice than not exercisedmice Changes in theother measured cytokines varied with mainly depressions at lesser PCB doses and elevations at higher doses Exercise had diverse effects on cytokine levels but increased cytokine levels in the two greater doses Explanations for these diverse effects include the useof young animals with more rapid wound healing rates less affected by toxin exposure aswell as PCBmediated compensatory effects at specific doses which could actually enhanceimmune function Future work should examine these interactions in more detail across adevelopmental time span Understanding how manipulating the effects of exposure toPLOS ONE 101371journalpone0237705 August PLOS ONE 0cExercise influences the impact of polychlorinated biphenyl exposure on immune functioncollection and analysis decision to publish orpreparation of the manuscriptCompeting interests The authors have declaredthat no competing interests existenvironemntal contaminants using behavioral modification could be very useful in certainhigh risk populations or exposed individualsIntroductionPolychlorinated biphenyls PCB are environmental contaminants that were manufacturedand used in large quantities for over years because of their wide range of applications However eventually the harmful effects of these chemicals were observed and their commercialproduction and use has been prohibited in the US since the 1970s [] Aroclor whichwas administered to mice in the present study is a mixture of PCB congeners with chlorination and was widely manufactured and released into the environment [] Because of theirvurtually indestructible nature PCBs remain in the environment and are known endocrinedisruptors [] PCB exposure has been shown to cause carcinogenecity genotoxicity reproductive toxicity and immunological effects [“] The majority of studies indicate that PCBexposure leads to immunosuppression in mice and humans however some studies haveshown an increase in immunological response [“] Exposure to polychlorinated biphenylsPCBs has been shown to have immunotoxic effect and to impair the functioning of severalimmune responsive cells [] Previous studies have revealed that PCB can hamper woundhealing [] However these earlier studies were done using an invertebrate model egearthworms with the PCB absorbance through the skin and the wound sizes were measuredonly at hours after wound creation We expanded the present study by examining a wellestablished mouse model and extending the time duration of the wound healing analysisThe present study expanded in another novel way by exploring the impact of exercise onimmune function in animal subjects exposed to PCB Researchers are slowly coming to a consensus that physical activity produces a Ushaped response curve with both no and extremephysical activity having deleterious effects whereas moderate physical activity has beneficialeffects on both overall health and immune system function [] Moderate intensity exercisehas been shown to provide beneficial effects retarding development of various chronic conditions like cardiovascular disease atherosclerosis and chronic inflammation and it also reducing the progression of these diseases and certain neoplasms if they are already present []The production of greater concentrations of antiinflammatory cytokines as a result of exerciseand this production has been demonstrated and this is essential for the positive effects of exercise [] The effect of exercise has also been studied in older mice in which the immune function is suboptimal resulting in greater susceptibility to and delayed recovery from infectionOlder animals also have also been found to have delayed wound healing Moderate intensityexercise in these animals improved wound healing rates It also reduced the concentrations ofproinflammatory cytokines in the wound tissue of these animals []The first aim of the present study was to determine whether PCB exposure impairs woundhealing and if exercise can reduce some of the negative PCB effects and improve wound healing To examine the relationship between PCB exposure and exercise in more detail we measured the concentrations of various cytokines in wound tissue of mice moderately exercised ornot exercised with or without PCB exposure Various immune responsive cells and the cytokines released by them play an important part in the inflammatory phase of wound healingby not only recruiting other immune cells but also in the reepithelization process [] Previous studies have shown that excessive inflammation with the presence of increased proinflammatory cytokines can delay wound healing especially in aged animals [] and thatPLOS ONE 101371journalpone0237705 August PLOS ONE 0cExercise influences the impact of polychlorinated biphenyl exposure on immune functionimmunologically suppressed mice lacking macrophages and neutrophils experience greaterrates of wound healing [] Furthermore it has been shown that exercise improves woundhealing rate in obese and aged mice [] The concentrations of proinflammatory cytokineslike TNFα KC and MCP1 have been observed to decrease in aged mice after exercise andthese findings correlated with more rapid wound healing rates in these animals Howeverthis study did not find significant positive effects of exercise on wound healing in youngermice [] In an evolutionarily less complex species PCB administration in earthworms hasimpaired wound healing and increased allograft rejection [] Additionally in vitro studieshave found PCB to cause enhanced stimulation of neutrophils to produce reactive oxygen species [] However the function of human and mouse macrophages and mouse splenocyteshas been shown to be impaired by PCB administration [“] No previous studies havedetermined the amounts of cytokines in wound tissue of animals administered PCB The cytokine data from the present study will give us a novel insight into whether exercise can modulate PCB induced modification of cytokine concentrations and thus negating some of theimmunotoxic effects of PCBMaterials and methodsAnimal careFemale C57BL6 mice weeks of age were obtained from Harlan Laboratories IndianapolisIN and housed in the Bowling Green State University BGSU animal facility All the animalstudies were conducted as approved by the BGSU Institutional Animal Care and Use Committee IACUC under protocol no “ The mice were individually housed in shoe box cagesand maintained on a reverse lightdark cycle They were provided food Teklad Mouse BreederDiet Envigo Madison WI and water ad libitum and their body weights were recordeddaily After PCB exposure exercise regimen and wound generation the animals were frequently monitored for evidence of distress poor grooming frantic appearance poor coatcondition aversion to handling and wound infection No signs of distress were observed inanimals in this study At the end of testing animals were euthanized by rapid CO2 inhalationfollowed by decapitation with a guillotinePCB exposureAll the animals were given weeks to acclimatize to the animal facility before the study beganAll the animals in the present study both not exercised exercised groups were administeredPCB Arochlor Accustandard Inc New Haven CT USA dissolved in corn oil via intraperitoneal injection at a volume of μlg body weight PCB doses administered were PCB PCB PCB or PCB μgg Previous studies have used similarPCB dosage [] and intraperitoneal injections [] Following acclimatization the animals inthe group not exercised were housed in the animal facility for weeks without exercise andPCB injection was administered days after this 3week period without exercise was overFollowing acclimatization the animals in the exercised group began moderate exercise for a3week period days after completion of the exercise period they were administered PCBExercise regimenThe animals not exercised remained in cages without exercise during the 3week period Thecarts holding the cages of these animals were tethered to the table containing the motorizedtreadmill using large metal clamps so that these no exercise animals not exercised wereexposed to the same sounds and vibrations as the animals in the exercise group The animalsPLOS ONE 101371journalpone0237705 August PLOS ONE 0cExercise influences the impact of polychlorinated biphenyl exposure on immune functionin the exercised group were exercised by running on a motorized treadmill JogADog modelDC6 1HP which was adapted with individual lanes for mice This running was done for minutes daily five days a week for weeks and consisted of moderate intensity running at “ mmin with a incline These exercise regimens were carried out at the beginning of theactive period of these animals “ as they are on a reverse lightdark cycle Previousstudies have shown that this intensity of exercise effectively alters immune function []Wound creation and wound size measurementThis procedure was carried out in the animals used in Aim I Table of the study Woundswere made two days following PCB exposure Mice were anesthetized with isoflurane Isoflo1 through a cone mask in oxygen at a flow rate of “ Lmin during wound creationMice were then administered μgg of analgesic Buprenex Buprenorphine hydrochloride mgml Reckitt Benckiser Healthcare UK Ltd Hull England A similar dose of Buprenex was administered twice daily for days post wounding A Wahl Peanut Hair TrimmerSterling IL was used without guides to remove the hair over an area on the upper back alocation inaccessible to the animal for the creation of wounds The shaved area was thencleaned with Betadine and ethanol Wounds were made using a mm sterile disposablepunch biopsy instrument Robbins Instruments Chatham NJ to create one full thicknessdermal punch resulting in two wounds A Canon EOS Rebel XTi camera was used to photograph the wound at the same time daily until the wounds were reduced to of original sizeor for two weeks after the wounds were made whichever came first Image J software version141o NIH was used to analyze the wound and compare it to the reference wound thus allowing comparison between rates of wound healingEuthanasiaThe animals were euthanized by rapid CO2 asphyxiation followed by decapitation with a guillotine after the wound size reached of the original wound or two weeks post woundingwhichever came first A mm punch biopsy instrument was used to harvest the wound andthe surrounding tissueWound cytokine analysisIL1 IL6 keratinocyte chemoattractant KC monocyte chemoattractant protein1 MCP1and tumor necrosis factor α TNFα protein concentrations in wound tissue Table weredetermined using a BioPlex Pro Mouse Cytokine 23plex Assay kit BioRad LaboratoriesInc Philadelphia PATissue extraction procedureThe wound and the surrounding tissue from animals that were euthanized by CO2 asphyxiation followed by decapitation with a guillotine at Day and Day after wound creation wereTable Detailed distribution of the mice used for wound healing studyWound HealingPCB doses μggTotal101371journalpone0237705t001Number of AnimalsNo ExerciseExercisePLOS ONE 101371journalpone0237705 August PLOS ONE 0cExercise influences the impact of polychlorinated biphenyl exposure on immune functionTable Detailed distribution of the mice for cytokine analysis studyPCB doses μggTotalDay Day Number of AnimalsNo ExerciseExerciseNo ExerciseExercise101371journalpone0237705t002harvested using a mm punch biopsy instrument Robbins Instruments Chatham NJ Previous study has measured cytokine levels in wound tissue at and days after wound creation[] The tissue was flash frozen in liquid nitrogen and then stored in ˚C freezer This tissuewas homogenized using a protocol developed by Frank and Kampfer [] The wound tissuewas homogenized in an extraction solution containing sterile 1X PBS and an antiproteasebuffer cOmplete EDTAfree Protease Inhibitor Cocktail Roche Diagnostics GmbH Mannheim Germany One tablet of cOmplete was added to ml of 1X PBS to prepare the extraction solution A single wound tissue sample was added to ml of the extraction solution andhomogenized using a PowerGen Generator PowerGen Fisher Scientific Pittsburg PAand a saw tooth blades 7X95mm The homogenate was centrifuged at rpm for min at˚C The supernatant thus obtained was aspirated into ml syringes BD Biosciences Mexicoand passed through a μm Sartorius Minisart Syringe filter Supelco Bellfonte PA The filtrate approx μl was aliquoted into tubes and stored at ˚C A μl aliquot was usedfor protein assayProtein assaysProtein concentrations of tissue extracts were determined by using BioRad Protein Assay“Dye Reagent Concentrate following the protocol of BioRad Laboratories Richmond CAProtein assays were done in order to express cytokine concentrations in the samples per milligram of protein Stock protein standard solution of 1mgml was prepared by using bovinealbumin purchased from Sigma Chemical Co St Louis MO dissolved in distilled waterBlank and serial dilutions of standard mgml mgml mgml mgml and mgml were used to generate the standard curve The μl aliquot of tissue extract was diluted in μl distilled water total volume μl and μl of this dilution was used to load a single wellof a Costar well plate All protein assays were done in triplicate The dye reagent concentratewas diluted with distilled water and μl of this diluted dye was added to each of thewells The plates were incubated on a shaker for min and then absorbance was measured at nm on a Clariostar plate reader BMG Labtech GmbH Ottenberg Germany and analyzed using Mars Data Analysis Software Version 301R2 BMG Labtech A standardcurve was plotted and used for calculating protein concentrations of tissue extracts The protein concentrations were expressed as mgmlCytokine analysisA BioPlex Pro Mouse Cytokine 23plex Assay kit BioRad Laboratories Inc PhiladelphiaPA was used for measuring cytokine content using a BioPlex instrument lab of Dr Stanislaw Stepkowski The University of Toledo Toledo OH and BioPlex Manager softwareBioRad Laboratories Inc Philadelphia PA was used for machine operation and dataPLOS ONE 101371journalpone0237705 August PLOS ONE 0cExercise influences the impact of polychlorinated biphenyl exposure on immune functioncollection A BioPlex handheld magnetic washer BioRad Laboratories Inc PhiladelphiaPA was used for all washing steps The protocol accompanying the kit was followed The following cytokines were measured Eotaxin GCSF GMCSF IFNγ Interleukins IL™s IL1αIL1 IL2 IL3 IL4 IL5 IL6 IL9 IL10 IL12p40 IL12p70 IL13 IL17A KCkeratinocyte MCP1MCAF monocyte chemotactic and activating factor MIP1α MIP1 macrophage inflammatory protein RANTES regulated on activation normal T cellexpressed and secreted and TNFα tumor necrosis factor alphaData analysisStatistical analysis for all data was completed using SPSS software IBM version Forthe wound size data general linear model Repeated Measures ANOVA along with post hoctests Bonferroni and Tukey was performed for PCB Exercise and Day Wound size on Day day of wound creation over all the groups was normalized to and all the values for thefolowing days were sizes compared to Day Error bars are standard error of the meanSEM Significance was ascribed to p � For the cytokine concentration data general linear model Univariate ANOVA along withpost hoc tests Bonferroni and Tukey was performed for PCB Exercise and Day Based on thesignificance of these data independent Ttests were run between groups within PCB Exerciseand Day Error bars are standard error of the mean SEM Significance was ascribed top � Note Before data were selected for running the statistical analysis in each of thegroups the numbers above or below the mean standard deviation SD were omittedsince there were a few outliers that were drastically skewing the group meansResultsWound healing PCB and exercise effectsDay effects Wounds showed signficant size reductions across days There was a significant main effect of time F6 p0001 There were significant differences for the pairwise comparisons from Day to Day of the analysis see Fig with the wounds decreasingin size in all groupsExercise effects Exercise had no signficant impact on wound healing in the controlgroup PCB However there was a general pattern in which wound sizes decreased at agreater rate in exercised mice administered no PCB Fig 2APCB effects Animals that were not exercised did not experience any significant changesor trends in wound healing rates with varying PCB doses There was a pattern where the ratesof wound healing are greater in PCB administered animals as compared to PCB Fig3C PCB Fig 3E and PCB Fig 3F Comparison of the other PCB doses did not yieldany obvious pattern Fig 3A 3B and 3DExercise and PCB interactions Exercise and PCB exposure appeared to interact indiverse ways In the low dose group exposed to PCB mice Fig 2B those not exercisedhealed at a greater rate than the exercised group A nonsignificant pattern was revealed wherewound sizes decrease at a greater rate in exercised mice administered PCB Fig 2C Additionally wound healing rates appear very similar in exercised and not exercised mice administered PCB Fig 2DNo significant changes or trends in wound healing rates with varying PCB doses wereobserved in animals that were exercised However the figures reveal a pattern where the ratesof wound healing are less in PCB administered animals as compared to PCB Fig 4APCB Fig 4D and PCB Fig 4E No particular pattern was observed with other PCBdoses Fig 4B 4C and 4FPLOS ONE 101371journalpone0237705 August PLOS ONE 0cExercise influences the impact of polychlorinated biphenyl exposure on immune functionFig Comparison of the wound size in animals from day day of wound creation to day n “ Animalsfrom all the treatment groups were combined and used to study the day effect The data have been normalized with theinitial wound size on the day of production Day being As can be expected the wound size decreased consistentlyover the period reduced to less than in days Wound size is significantly less on all the previous days except forthe immediate day before For example wound size on Day is significantly less than Day and Day but not Day All the other days followed a similar pattern101371journalpone0237705g001Fig Effect of exercise on wound size in mice administered varying doses of PCB n “ Animals that wereadmininstered PCB in varying doses did not show any significant differences or trends in wound size on different daysafter wound production irrespective of whether they were exercised or not However observation of the graph showsthat there seems to be a difference in the mean wound sizes Day through Day between the exercise and no exercisegroups with all doses of PCB except PCB 101371journalpone0237705g002PLOS ONE 101371journalpone0237705 August PLOS ONE 0cExercise influences the impact of polychlorinated biphenyl exposure on immune functionFig Comparison of wound sizes on different days in animals not exercised and administered varying doses ofPCB n “ C E and F The mean wound size is less on Day through Day in PCB treated animals ascompared to PCB PCB and PCB administered mice though no significant differences or trends wereobserved A B and D No significant differences or trends and no difference in means were observed when the otherdoses of PCB were compared101371journalpone0237705g003Cytokine concentrationsThe cytokine concentrations mentioned in the following sections are concentrations in thewound tissue and not circulating concentrations or whole animal concentrationsEffect of exercise and PCB on IL1 levelsIL1 concentrations were determined at Day and Day postwounding Fig UnivariateANOVA revealed following significant interactions PCB X exercise F351 p PCB X day F351 p and exercise X day F151 p Independentttests were performed to further explore the within group interactionsExercise and day interactions The effects of exercise depended on the day that levels ofcytokines were analyzed Concentrations of IL1 on Day in mice not administered PCBwere significantly less in exercised as compared to not exercised mice t5 p PLOS ONE 101371journalpone0237705 August PLOS ONE 0cExercise influences the impact of polychlorinated biphenyl exposure on immune functionFig Comparison of wound sizes on different days in exercised animals administered varying doses of PCBn “ A D and E The mean wound size is greater on Day through Day in PCB treated animals ascompared to PCB PCB and PCB administered mice though no significant differences or trends wereobserved B C and F No significant differences or trends and no difference in means were observed when the otherdoses of PCB were compared101371journalpone0237705g004Exercised mice not administered PCB revealed significantly less IL1 on Day as comparedto Day �t6 p Fig 5C and 5DPCB and day interactions On Day wound tissue from animals not exercised containedsignificantly less of IL1 in mice given μgg PCB as compared to the animals receiving noPCB t6 p Fig 5A In mice not exercised administered μgg PCB significantly greater concentrations of IL1 were present postwounding on Day as comparedto Day t6 p Fig 5A and 5B On postwounding Day not exercisedmice revealed significantly greater concentrations of IL1 in animals administered μggPCB as compared to mice not administered PCB t4 p Fig 5B Micethat were not exercised on postwounding Day revealed significantly greater concentrationsof IL1 in μgg PCB administered animals as compared to the ones administered μgg PCB t5 p Fig 5B A betweengroup comparison showedPLOS ONE 101371journalpone0237705 August PLOS ONE 0cExercise influences the impact of polychlorinated biphenyl exposure on immune functionFig Effect of exercise and PCB on levels of IL1 in wound tissue at day and day postwoundingComparison of IL1 levels days postwounding in animals not exercised reveals a pattern of reduced concentrationswith different doses of PCB as compared to no PCB administration with PCB demonstrating the most reductionSimilar pattern is observed at Day in exercised animals but with no change in PCB administration However onDay IL1 concentrations in not exercised animals reveals a pattern of dose dependent increase In exercisedanimals on Day concentrations of IL1 are higher in PCB and doses but remain the same with PCB administration101371journalpone0237705g005that postwounding Day mice that were not exercised revealed significantly greater concentrations of IL1 in μgg PCB treated animals as compared to animals administered μgg PCB §t5 p Fig 5B Between group comparison revealed thatmice that were not exercised and administered μgg PCB exhibited significantly greaterconcentrations of IL1 on postwounding Day as compared to Day t6 p Fig 5A and 5BPCB and exercise interactions Tissue from mice administered μgg PCB contained significantly greater concentrations of IL1 on Day in exercised animals as compared to animals not exercised £t6 p Fig 5A and 5C PostwoundingDay animals that were exercised demonstrated significantly lesser concentrations of IL1in μgg PCB administered animals as compared to the ones administered μgg PCB1t7 p Fig 5D On postwounding Day exercised animals demonstrated significantly greater concentrations of IL1 in mice administered μgg PCB ascompared to the animals not administered PCB ¶t6 p Fig 5D On Day mice administered μgg PCB displayed a trend towards less IL1 in exercised as compared to not exercised animals t7 p Fig 5B and 5D Concentrations of IL1 on Day in mice administered μgg PCB were significantly less in exercised mice ascompared to mice not exercised ¥t5 p Fig 5B and 5D Mice that wereexercised on Day postwounding revealed significantly greater concentrations of IL1 in μgg PCB administered animals as compared to the ones not administered PCB Δt6 p Fig 5DPLOS ONE 101371journalpone0237705 August PLOS ONE 0cExercise influences the impact of polychlorinated biphenyl exposure on immune functionFig Effect of exercise and PCB on levels of IL6 in wound tissue at day and day postwounding In notexercised animals on Day there appears to be no difference in the means of IL6 concentrations across PCB dosesHowever Day exercised animals demonstrate a pattern of reduced IL6 concentrations with PCB and PCB but increased with PCB administration as compared to PCB On Day in not exercised mice IL6concentrations appear to be greater with all PCB doses when compared to PCB with PCB being the highestWhereas Day exercised animals reveal pattern of slight decrease with all PCB doses as compared to no PCBadministration101371journalpone0237705g006Effect of exercise and PCB on IL6 levelsIL6 levels were measured at Day and Day postwounding Fig Univariate ANOVArevealed significant PCB effect F356 p It also revealed significant interactionsof the following PCB X exercise F356 p PCB X day F356 p and exercise X day F156 p There was a trend towards PCB X exercise X dayinteraction F356 p Independent ttests were performed to further explorethe within group interactionsExercise and day interactions There were no significant exercise and day interactionsPCB and day interactions On postwounding Day mice not exercised revealed significantly greater concentrations of IL6 in animals administered μgg PCB as compared tomice not administered PCB ¥t7 p Fig 6B Postwounding Day animalsthat were not exercised revealed significantly greater concentrations of IL6 in μgg PCBadministered animals as compared to the ones administered μgg PCB ¶t7 p Fig 6B Mice that were not exercised on postwounding Day demonstratedlower concentrations of IL6 in μGG PCB administered animals as compared to μgg PCB administered mice t7 p Fig 6B In mice that were not exercisedand administered μgg PCB there were significantly greater concentrations of IL6 onpostwounding Day as compared to Day t6 p Fig 6A and 6BPCB and exercise interactions Exercised animals on Day postwounding revealed significantly greater concentrations of IL6 in mice administered μgg PCB as compared tothe animals not administered PCB ‚¬t7 p Fig 6C Mice that were exercisedPLOS ONE 101371journalpone0237705 August PLOS ONE 0cExercise influences the impact of polychlorinated biphenyl exposure on immune functionon Day postwounding demonstrated significantly lesser concentrations of IL6 in μggPCB administered animals as compared to the ones not administered PCB t8 p Fig 6C Mice that were exercised on Day postwounding revealed significantlylesser concentrations of IL6 in μgg PCB administered animals as compared to μggPCB administered mice Δt8 p Fig 6C Postwounding Day mice thatwere exercised revealed significantly lesser concentrations of IL6 in μgg PCB treatedanimals as compared to animals administered μgg PCB 1t9 p Fig6C Concentrations of IL6 on Day postwounding in mice administered μgg PCBwere significantly greater in exercised mice as compared to mice not exercised t8 p Fig 6A and 6C In mice that were exercised and administered μgg PCB therewere significantly lesser concentrations of IL6 on Day postwounding as compared to Day �t8 p Fig 6C and 6D In mice administered μgg PCB on postwounding Day revealed significantly lower concentrations of IL6 in exercised animals as comparedto animals not exercised t7 p Fig 6B and 6D On Day postwoundingmice administered μgg PCB revealed significantly lower concentrations of IL6 in exercised animals as compared to animals not exercised £t8 p Fig 6A and 6CEffect of exercise and PCB on KC levelsKC levels were measured at Day and Day postwounding Fig Univariate ANOVA didnot reveal any significant individual or group interactions Independent ttests were performed to explore any within group interactionsFig Effect of exercise and PCB on levels of KC in wound tissue at day and day postwounding KCconcentrations on Day in not exercised animals reveal pattern of reduction with increasing PCB doses except PCB where there is an increase Similar pattern of reduction in KC concentrations with increasing dose is seen on Day exercised animals except for PCB which demonstrates an increase On Day in not exercised animals there is apattern of greater KC with all PCB does as compared to PCB with PCB revealing the highest concentrationsHowever exercised animals on Day demonstrate a pattern of dose dependent reduction in KC concentrations101371journalpone0237705g007PLOS ONE 101371journalpone0237705 August PLOS ONE 0cExercise influences the impact of polychlorinated biphenyl exposure on immune functionExercise and day interactions There were no significant exercise and day interactionsPCB and day interactions Animals not exercised on Day postwounding demonstrated significantly greater concentrations of KC in mice administered μg

cancer269

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: Exercise influences the impact ofpolychlorinated biphenyl exposure onimmune functionMahesh R PillaiID1 K Todd Keylock2 Howard C Cromwell3 Lee A Meserve1 Dept of Biological Sciences Bowling Green State University Bowling Green Ohio United States ofAmerica Dept of Exercise Science Bowling Green State University Bowling Green Ohio United States ofAmerica Dept of Psychology and JP Scott Center for Neuroscience Mind and Behavior Bowling GreenState University Bowling Green Ohio United States of America Current address Human Research Protection Program The University of Toledo Toledo Ohio UnitedStates of America lmeservbgsuedua1111111111a1111111111a1111111111a1111111111a1111111111Abstract ACCESSCitation Pillai MR Keylock KT Cromwell HCMeserve LA Exercise influences the impactof polychlorinated biphenyl exposure on immunefunction e0237705 101371journalpone0237705Editor HansJoachim Lehmler University of IowaUNITED STATESReceived April Accepted July Published August Peer Review History PLOS recognizes thebenefits of transparency in the peer reviewprocess therefore we enable the publication ofall of the content of peer review and authorresponses alongside final published s Theeditorial history of this is available here101371journalpone0237705Copyright Pillai This is an access distributed under the terms of theCreative Commons Attribution License whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are creditedData Availability Statement The data is availableon figshare 106084m9figshare12768332Funding Department of Biological SciencesBowling Green State University Bowling GreenOhio The funders had no role in study design dataPolychlorinated biphenyls PCBs are environmental pollutants and endocrine disruptorsharmfully affecting reproductive endocrine neurological and immunological systems Thisbroad influence has implications for processes such as wound healing which is modulatedby the immunological response of the body Conversely while PCBs can be linked to diminished wound healing outside of PCB pollution systems exercise has been shown to accelerate wound healing However the potential for moderate intensity exercise to modulateor offset the harmful effects of a toxin like PCB are yet unknown A key aim of the presentstudy was to examine how PCB exposure at different doses ppm ipaltered wound healing in exercised versus nonexercised subgroups of mice We examinedPCB effects on immune function in more depth by analyzing the concentrations of cytokinesinterleukin1 IL1 tumor necrosis factorα TNFα Interleukin6 IL6 and granulocytemacrophage colony stimulating factor GMCSF in these wounds inflicted by punch biopsyMice were euthanized at Day or Day after PCB injection n “ and skin excised fromthe wound area was homogenized and analyzed for cytokine content Results revealed thatwound healing was not signficantly impacted by either PCB exposure or exercise but therewere patterns of delays in healing that depended on PCB dose Changes in cytokines werealso observed and depended on PCB dose and exercise experience For example IL1concentrations in Day mice without PCB administration were less in exercisedmice than mice not exercised However IL1 concentrations in Day mice administered ppm were greater in exercised mice than not exercisedmice Changes in theother measured cytokines varied with mainly depressions at lesser PCB doses and elevations at higher doses Exercise had diverse effects on cytokine levels but increased cytokine levels in the two greater doses Explanations for these diverse effects include the useof young animals with more rapid wound healing rates less affected by toxin exposure aswell as PCBmediated compensatory effects at specific doses which could actually enhanceimmune function Future work should examine these interactions in more detail across adevelopmental time span Understanding how manipulating the effects of exposure toPLOS ONE 101371journalpone0237705 August PLOS ONE 0cExercise influences the impact of polychlorinated biphenyl exposure on immune functioncollection and analysis decision to publish orpreparation of the manuscriptCompeting interests The authors have declaredthat no competing interests existenvironemntal contaminants using behavioral modification could be very useful in certainhigh risk populations or exposed individualsIntroductionPolychlorinated biphenyls PCB are environmental contaminants that were manufacturedand used in large quantities for over years because of their wide range of applications However eventually the harmful effects of these chemicals were observed and their commercialproduction and use has been prohibited in the US since the 1970s [] Aroclor whichwas administered to mice in the present study is a mixture of PCB congeners with chlorination and was widely manufactured and released into the environment [] Because of theirvurtually indestructible nature PCBs remain in the environment and are known endocrinedisruptors [] PCB exposure has been shown to cause carcinogenecity genotoxicity reproductive toxicity and immunological effects [“] The majority of studies indicate that PCBexposure leads to immunosuppression in mice and humans however some studies haveshown an increase in immunological response [“] Exposure to polychlorinated biphenylsPCBs has been shown to have immunotoxic effect and to impair the functioning of severalimmune responsive cells [] Previous studies have revealed that PCB can hamper woundhealing [] However these earlier studies were done using an invertebrate model egearthworms with the PCB absorbance through the skin and the wound sizes were measuredonly at hours after wound creation We expanded the present study by examining a wellestablished mouse model and extending the time duration of the wound healing analysisThe present study expanded in another novel way by exploring the impact of exercise onimmune function in animal subjects exposed to PCB Researchers are slowly coming to a consensus that physical activity produces a Ushaped response curve with both no and extremephysical activity having deleterious effects whereas moderate physical activity has beneficialeffects on both overall health and immune system function [] Moderate intensity exercisehas been shown to provide beneficial effects retarding development of various chronic conditions like cardiovascular disease atherosclerosis and chronic inflammation and it also reducing the progression of these diseases and certain neoplasms if they are already present []The production of greater concentrations of antiinflammatory cytokines as a result of exerciseand this production has been demonstrated and this is essential for the positive effects of exercise [] The effect of exercise has also been studied in older mice in which the immune function is suboptimal resulting in greater susceptibility to and delayed recovery from infectionOlder animals also have also been found to have delayed wound healing Moderate intensityexercise in these animals improved wound healing rates It also reduced the concentrations ofproinflammatory cytokines in the wound tissue of these animals []The first aim of the present study was to determine whether PCB exposure impairs woundhealing and if exercise can reduce some of the negative PCB effects and improve wound healing To examine the relationship between PCB exposure and exercise in more detail we measured the concentrations of various cytokines in wound tissue of mice moderately exercised ornot exercised with or without PCB exposure Various immune responsive cells and the cytokines released by them play an important part in the inflammatory phase of wound healingby not only recruiting other immune cells but also in the reepithelization process [] Previous studies have shown that excessive inflammation with the presence of increased proinflammatory cytokines can delay wound healing especially in aged animals [] and thatPLOS ONE 101371journalpone0237705 August PLOS ONE 0cExercise influences the impact of polychlorinated biphenyl exposure on immune functionimmunologically suppressed mice lacking macrophages and neutrophils experience greaterrates of wound healing [] Furthermore it has been shown that exercise improves woundhealing rate in obese and aged mice [] The concentrations of proinflammatory cytokineslike TNFα KC and MCP1 have been observed to decrease in aged mice after exercise andthese findings correlated with more rapid wound healing rates in these animals Howeverthis study did not find significant positive effects of exercise on wound healing in youngermice [] In an evolutionarily less complex species PCB administration in earthworms hasimpaired wound healing and increased allograft rejection [] Additionally in vitro studieshave found PCB to cause enhanced stimulation of neutrophils to produce reactive oxygen species [] However the function of human and mouse macrophages and mouse splenocyteshas been shown to be impaired by PCB administration [“] No previous studies havedetermined the amounts of cytokines in wound tissue of animals administered PCB The cytokine data from the present study will give us a novel insight into whether exercise can modulate PCB induced modification of cytokine concentrations and thus negating some of theimmunotoxic effects of PCBMaterials and methodsAnimal careFemale C57BL6 mice weeks of age were obtained from Harlan Laboratories IndianapolisIN and housed in the Bowling Green State University BGSU animal facility All the animalstudies were conducted as approved by the BGSU Institutional Animal Care and Use Committee IACUC under protocol no “ The mice were individually housed in shoe box cagesand maintained on a reverse lightdark cycle They were provided food Teklad Mouse BreederDiet Envigo Madison WI and water ad libitum and their body weights were recordeddaily After PCB exposure exercise regimen and wound generation the animals were frequently monitored for evidence of distress poor grooming frantic appearance poor coatcondition aversion to handling and wound infection No signs of distress were observed inanimals in this study At the end of testing animals were euthanized by rapid CO2 inhalationfollowed by decapitation with a guillotinePCB exposureAll the animals were given weeks to acclimatize to the animal facility before the study beganAll the animals in the present study both not exercised exercised groups were administeredPCB Arochlor Accustandard Inc New Haven CT USA dissolved in corn oil via intraperitoneal injection at a volume of μlg body weight PCB doses administered were PCB PCB PCB or PCB μgg Previous studies have used similarPCB dosage [] and intraperitoneal injections [] Following acclimatization the animals inthe group not exercised were housed in the animal facility for weeks without exercise andPCB injection was administered days after this 3week period without exercise was overFollowing acclimatization the animals in the exercised group began moderate exercise for a3week period days after completion of the exercise period they were administered PCBExercise regimenThe animals not exercised remained in cages without exercise during the 3week period Thecarts holding the cages of these animals were tethered to the table containing the motorizedtreadmill using large metal clamps so that these no exercise animals not exercised wereexposed to the same sounds and vibrations as the animals in the exercise group The animalsPLOS ONE 101371journalpone0237705 August PLOS ONE 0cExercise influences the impact of polychlorinated biphenyl exposure on immune functionin the exercised group were exercised by running on a motorized treadmill JogADog modelDC6 1HP which was adapted with individual lanes for mice This running was done for minutes daily five days a week for weeks and consisted of moderate intensity running at “ mmin with a incline These exercise regimens were carried out at the beginning of theactive period of these animals “ as they are on a reverse lightdark cycle Previousstudies have shown that this intensity of exercise effectively alters immune function []Wound creation and wound size measurementThis procedure was carried out in the animals used in Aim I Table of the study Woundswere made two days following PCB exposure Mice were anesthetized with isoflurane Isoflo1 through a cone mask in oxygen at a flow rate of “ Lmin during wound creationMice were then administered μgg of analgesic Buprenex Buprenorphine hydrochloride mgml Reckitt Benckiser Healthcare UK Ltd Hull England A similar dose of Buprenex was administered twice daily for days post wounding A Wahl Peanut Hair TrimmerSterling IL was used without guides to remove the hair over an area on the upper back alocation inaccessible to the animal for the creation of wounds The shaved area was thencleaned with Betadine and ethanol Wounds were made using a mm sterile disposablepunch biopsy instrument Robbins Instruments Chatham NJ to create one full thicknessdermal punch resulting in two wounds A Canon EOS Rebel XTi camera was used to photograph the wound at the same time daily until the wounds were reduced to of original sizeor for two weeks after the wounds were made whichever came first Image J software version141o NIH was used to analyze the wound and compare it to the reference wound thus allowing comparison between rates of wound healingEuthanasiaThe animals were euthanized by rapid CO2 asphyxiation followed by decapitation with a guillotine after the wound size reached of the original wound or two weeks post woundingwhichever came first A mm punch biopsy instrument was used to harvest the wound andthe surrounding tissueWound cytokine analysisIL1 IL6 keratinocyte chemoattractant KC monocyte chemoattractant protein1 MCP1and tumor necrosis factor α TNFα protein concentrations in wound tissue Table weredetermined using a BioPlex Pro Mouse Cytokine 23plex Assay kit BioRad LaboratoriesInc Philadelphia PATissue extraction procedureThe wound and the surrounding tissue from animals that were euthanized by CO2 asphyxiation followed by decapitation with a guillotine at Day and Day after wound creation wereTable Detailed distribution of the mice used for wound healing studyWound HealingPCB doses μggTotal101371journalpone0237705t001Number of AnimalsNo ExerciseExercisePLOS ONE 101371journalpone0237705 August PLOS ONE 0cExercise influences the impact of polychlorinated biphenyl exposure on immune functionTable Detailed distribution of the mice for cytokine analysis studyPCB doses μggTotalDay Day Number of AnimalsNo ExerciseExerciseNo ExerciseExercise101371journalpone0237705t002harvested using a mm punch biopsy instrument Robbins Instruments Chatham NJ Previous study has measured cytokine levels in wound tissue at and days after wound creation[] The tissue was flash frozen in liquid nitrogen and then stored in ˚C freezer This tissuewas homogenized using a protocol developed by Frank and Kampfer [] The wound tissuewas homogenized in an extraction solution containing sterile 1X PBS and an antiproteasebuffer cOmplete EDTAfree Protease Inhibitor Cocktail Roche Diagnostics GmbH Mannheim Germany One tablet of cOmplete was added to ml of 1X PBS to prepare the extraction solution A single wound tissue sample was added to ml of the extraction solution andhomogenized using a PowerGen Generator PowerGen Fisher Scientific Pittsburg PAand a saw tooth blades 7X95mm The homogenate was centrifuged at rpm for min at˚C The supernatant thus obtained was aspirated into ml syringes BD Biosciences Mexicoand passed through a μm Sartorius Minisart Syringe filter Supelco Bellfonte PA The filtrate approx μl was aliquoted into tubes and stored at ˚C A μl aliquot was usedfor protein assayProtein assaysProtein concentrations of tissue extracts were determined by using BioRad Protein Assay“Dye Reagent Concentrate following the protocol of BioRad Laboratories Richmond CAProtein assays were done in order to express cytokine concentrations in the samples per milligram of protein Stock protein standard solution of 1mgml was prepared by using bovinealbumin purchased from Sigma Chemical Co St Louis MO dissolved in distilled waterBlank and serial dilutions of standard mgml mgml mgml mgml and mgml were used to generate the standard curve The μl aliquot of tissue extract was diluted in μl distilled water total volume μl and μl of this dilution was used to load a single wellof a Costar well plate All protein assays were done in triplicate The dye reagent concentratewas diluted with distilled water and μl of this diluted dye was added to each of thewells The plates were incubated on a shaker for min and then absorbance was measured at nm on a Clariostar plate reader BMG Labtech GmbH Ottenberg Germany and analyzed using Mars Data Analysis Software Version 301R2 BMG Labtech A standardcurve was plotted and used for calculating protein concentrations of tissue extracts The protein concentrations were expressed as mgmlCytokine analysisA BioPlex Pro Mouse Cytokine 23plex Assay kit BioRad Laboratories Inc PhiladelphiaPA was used for measuring cytokine content using a BioPlex instrument lab of Dr Stanislaw Stepkowski The University of Toledo Toledo OH and BioPlex Manager softwareBioRad Laboratories Inc Philadelphia PA was used for machine operation and dataPLOS ONE 101371journalpone0237705 August PLOS ONE 0cExercise influences the impact of polychlorinated biphenyl exposure on immune functioncollection A BioPlex handheld magnetic washer BioRad Laboratories Inc PhiladelphiaPA was used for all washing steps The protocol accompanying the kit was followed The following cytokines were measured Eotaxin GCSF GMCSF IFNγ Interleukins IL™s IL1αIL1 IL2 IL3 IL4 IL5 IL6 IL9 IL10 IL12p40 IL12p70 IL13 IL17A KCkeratinocyte MCP1MCAF monocyte chemotactic and activating factor MIP1α MIP1 macrophage inflammatory protein RANTES regulated on activation normal T cellexpressed and secreted and TNFα tumor necrosis factor alphaData analysisStatistical analysis for all data was completed using SPSS software IBM version Forthe wound size data general linear model Repeated Measures ANOVA along with post hoctests Bonferroni and Tukey was performed for PCB Exercise and Day Wound size on Day day of wound creation over all the groups was normalized to and all the values for thefolowing days were sizes compared to Day Error bars are standard error of the meanSEM Significance was ascribed to p � For the cytokine concentration data general linear model Univariate ANOVA along withpost hoc tests Bonferroni and Tukey was performed for PCB Exercise and Day Based on thesignificance of these data independent Ttests were run between groups within PCB Exerciseand Day Error bars are standard error of the mean SEM Significance was ascribed top � Note Before data were selected for running the statistical analysis in each of thegroups the numbers above or below the mean standard deviation SD were omittedsince there were a few outliers that were drastically skewing the group meansResultsWound healing PCB and exercise effectsDay effects Wounds showed signficant size reductions across days There was a significant main effect of time F6 p0001 There were significant differences for the pairwise comparisons from Day to Day of the analysis see Fig with the wounds decreasingin size in all groupsExercise effects Exercise had no signficant impact on wound healing in the controlgroup PCB However there was a general pattern in which wound sizes decreased at agreater rate in exercised mice administered no PCB Fig 2APCB effects Animals that were not exercised did not experience any significant changesor trends in wound healing rates with varying PCB doses There was a pattern where the ratesof wound healing are greater in PCB administered animals as compared to PCB Fig3C PCB Fig 3E and PCB Fig 3F Comparison of the other PCB doses did not yieldany obvious pattern Fig 3A 3B and 3DExercise and PCB interactions Exercise and PCB exposure appeared to interact indiverse ways In the low dose group exposed to PCB mice Fig 2B those not exercisedhealed at a greater rate than the exercised group A nonsignificant pattern was revealed wherewound sizes decrease at a greater rate in exercised mice administered PCB Fig 2C Additionally wound healing rates appear very similar in exercised and not exercised mice administered PCB Fig 2DNo significant changes or trends in wound healing rates with varying PCB doses wereobserved in animals that were exercised However the figures reveal a pattern where the ratesof wound healing are less in PCB administered animals as compared to PCB Fig 4APCB Fig 4D and PCB Fig 4E No particular pattern was observed with other PCBdoses Fig 4B 4C and 4FPLOS ONE 101371journalpone0237705 August PLOS ONE 0cExercise influences the impact of polychlorinated biphenyl exposure on immune functionFig Comparison of the wound size in animals from day day of wound creation to day n “ Animalsfrom all the treatment groups were combined and used to study the day effect The data have been normalized with theinitial wound size on the day of production Day being As can be expected the wound size decreased consistentlyover the period reduced to less than in days Wound size is significantly less on all the previous days except forthe immediate day before For example wound size on Day is significantly less than Day and Day but not Day All the other days followed a similar pattern101371journalpone0237705g001Fig Effect of exercise on wound size in mice administered varying doses of PCB n “ Animals that wereadmininstered PCB in varying doses did not show any significant differences or trends in wound size on different daysafter wound production irrespective of whether they were exercised or not However observation of the graph showsthat there seems to be a difference in the mean wound sizes Day through Day between the exercise and no exercisegroups with all doses of PCB except PCB 101371journalpone0237705g002PLOS ONE 101371journalpone0237705 August PLOS ONE 0cExercise influences the impact of polychlorinated biphenyl exposure on immune functionFig Comparison of wound sizes on different days in animals not exercised and administered varying doses ofPCB n “ C E and F The mean wound size is less on Day through Day in PCB treated animals ascompared to PCB PCB and PCB administered mice though no significant differences or trends wereobserved A B and D No significant differences or trends and no difference in means were observed when the otherdoses of PCB were compared101371journalpone0237705g003Cytokine concentrationsThe cytokine concentrations mentioned in the following sections are concentrations in thewound tissue and not circulating concentrations or whole animal concentrationsEffect of exercise and PCB on IL1 levelsIL1 concentrations were determined at Day and Day postwounding Fig UnivariateANOVA revealed following significant interactions PCB X exercise F351 p PCB X day F351 p and exercise X day F151 p Independentttests were performed to further explore the within group interactionsExercise and day interactions The effects of exercise depended on the day that levels ofcytokines were analyzed Concentrations of IL1 on Day in mice not administered PCBwere significantly less in exercised as compared to not exercised mice t5 p PLOS ONE 101371journalpone0237705 August PLOS ONE 0cExercise influences the impact of polychlorinated biphenyl exposure on immune functionFig Comparison of wound sizes on different days in exercised animals administered varying doses of PCBn “ A D and E The mean wound size is greater on Day through Day in PCB treated animals ascompared to PCB PCB and PCB administered mice though no significant differences or trends wereobserved B C and F No significant differences or trends and no difference in means were observed when the otherdoses of PCB were compared101371journalpone0237705g004Exercised mice not administered PCB revealed significantly less IL1 on Day as comparedto Day �t6 p Fig 5C and 5DPCB and day interactions On Day wound tissue from animals not exercised containedsignificantly less of IL1 in mice given μgg PCB as compared to the animals receiving noPCB t6 p Fig 5A In mice not exercised administered μgg PCB significantly greater concentrations of IL1 were present postwounding on Day as comparedto Day t6 p Fig 5A and 5B On postwounding Day not exercisedmice revealed significantly greater concentrations of IL1 in animals administered μggPCB as compared to mice not administered PCB t4 p Fig 5B Micethat were not exercised on postwounding Day revealed significantly greater concentrationsof IL1 in μgg PCB administered animals as compared to the ones administered μgg PCB t5 p Fig 5B A betweengroup comparison showedPLOS ONE 101371journalpone0237705 August PLOS ONE 0cExercise influences the impact of polychlorinated biphenyl exposure on immune functionFig Effect of exercise and PCB on levels of IL1 in wound tissue at day and day postwoundingComparison of IL1 levels days postwounding in animals not exercised reveals a pattern of reduced concentrationswith different doses of PCB as compared to no PCB administration with PCB demonstrating the most reductionSimilar pattern is observed at Day in exercised animals but with no change in PCB administration However onDay IL1 concentrations in not exercised animals reveals a pattern of dose dependent increase In exercisedanimals on Day concentrations of IL1 are higher in PCB and doses but remain the same with PCB administration101371journalpone0237705g005that postwounding Day mice that were not exercised revealed significantly greater concentrations of IL1 in μgg PCB treated animals as compared to animals administered μgg PCB §t5 p Fig 5B Between group comparison revealed thatmice that were not exercised and administered μgg PCB exhibited significantly greaterconcentrations of IL1 on postwounding Day as compared to Day t6 p Fig 5A and 5BPCB and exercise interactions Tissue from mice administered μgg PCB contained significantly greater concentrations of IL1 on Day in exercised animals as compared to animals not exercised £t6 p Fig 5A and 5C PostwoundingDay animals that were exercised demonstrated significantly lesser concentrations of IL1in μgg PCB administered animals as compared to the ones administered μgg PCB1t7 p Fig 5D On postwounding Day exercised animals demonstrated significantly greater concentrations of IL1 in mice administered μgg PCB ascompared to the animals not administered PCB ¶t6 p Fig 5D On Day mice administered μgg PCB displayed a trend towards less IL1 in exercised as compared to not exercised animals t7 p Fig 5B and 5D Concentrations of IL1 on Day in mice administered μgg PCB were significantly less in exercised mice ascompared to mice not exercised ¥t5 p Fig 5B and 5D Mice that wereexercised on Day postwounding revealed significantly greater concentrations of IL1 in μgg PCB administered animals as compared to the ones not administered PCB Δt6 p Fig 5DPLOS ONE 101371journalpone0237705 August PLOS ONE 0cExercise influences the impact of polychlorinated biphenyl exposure on immune functionFig Effect of exercise and PCB on levels of IL6 in wound tissue at day and day postwounding In notexercised animals on Day there appears to be no difference in the means of IL6 concentrations across PCB dosesHowever Day exercised animals demonstrate a pattern of reduced IL6 concentrations with PCB and PCB but increased with PCB administration as compared to PCB On Day in not exercised mice IL6concentrations appear to be greater with all PCB doses when compared to PCB with PCB being the highestWhereas Day exercised animals reveal pattern of slight decrease with all PCB doses as compared to no PCBadministration101371journalpone0237705g006Effect of exercise and PCB on IL6 levelsIL6 levels were measured at Day and Day postwounding Fig Univariate ANOVArevealed significant PCB effect F356 p It also revealed significant interactionsof the following PCB X exercise F356 p PCB X day F356 p and exercise X day F156 p There was a trend towards PCB X exercise X dayinteraction F356 p Independent ttests were performed to further explorethe within group interactionsExercise and day interactions There were no significant exercise and day interactionsPCB and day interactions On postwounding Day mice not exercised revealed significantly greater concentrations of IL6 in animals administered μgg PCB as compared tomice not administered PCB ¥t7 p Fig 6B Postwounding Day animalsthat were not exercised revealed significantly greater concentrations of IL6 in μgg PCBadministered animals as compared to the ones administered μgg PCB ¶t7 p Fig 6B Mice that were not exercised on postwounding Day demonstratedlower concentrations of IL6 in μGG PCB administered animals as compared to μgg PCB administered mice t7 p Fig 6B In mice that were not exercisedand administered μgg PCB there were significantly greater concentrations of IL6 onpostwounding Day as compared to Day t6 p Fig 6A and 6BPCB and exercise interactions Exercised animals on Day postwounding revealed significantly greater concentrations of IL6 in mice administered μgg PCB as compared tothe animals not administered PCB ‚¬t7 p Fig 6C Mice that were exercisedPLOS ONE 101371journalpone0237705 August PLOS ONE 0cExercise influences the impact of polychlorinated biphenyl exposure on immune functionon Day postwounding demonstrated significantly lesser concentrations of IL6 in μggPCB administered animals as compared to the ones not administered PCB t8 p Fig 6C Mice that were exercised on Day postwounding revealed significantlylesser concentrations of IL6 in μgg PCB administered animals as compared to μggPCB administered mice Δt8 p Fig 6C Postwounding Day mice thatwere exercised revealed significantly lesser concentrations of IL6 in μgg PCB treatedanimals as compared to animals administered μgg PCB 1t9 p Fig6C Concentrations of IL6 on Day postwounding in mice administered μgg PCBwere significantly greater in exercised mice as compared to mice not exercised t8 p Fig 6A and 6C In mice that were exercised and administered μgg PCB therewere significantly lesser concentrations of IL6 on Day postwounding as compared to Day �t8 p Fig 6C and 6D In mice administered μgg PCB on postwounding Day revealed significantly lower concentrations of IL6 in exercised animals as comparedto animals not exercised t7 p Fig 6B and 6D On Day postwoundingmice administered μgg PCB revealed significantly lower concentrations of IL6 in exercised animals as compared to animals not exercised £t8 p Fig 6A and 6CEffect of exercise and PCB on KC levelsKC levels were measured at Day and Day postwounding Fig Univariate ANOVA didnot reveal any significant individual or group interactions Independent ttests were performed to explore any within group interactionsFig Effect of exercise and PCB on levels of KC in wound tissue at day and day postwounding KCconcentrations on Day in not exercised animals reveal pattern of reduction with increasing PCB doses except PCB where there is an increase Similar pattern of reduction in KC concentrations with increasing dose is seen on Day exercised animals except for PCB which demonstrates an increase On Day in not exercised animals there is apattern of greater KC with all PCB does as compared to PCB with PCB revealing the highest concentrationsHowever exercised animals on Day demonstrate a pattern of dose dependent reduction in KC concentrations101371journalpone0237705g007PLOS ONE 101371journalpone0237705 August PLOS ONE 0cExercise influences the impact of polychlorinated biphenyl exposure on immune functionExercise and day interactions There were no significant exercise and day interactionsPCB and day interactions Animals not exercised on Day postwounding demonstrated significantly greater concentrations of KC in mice administered μg Answer:

Thyroid_Cancer

Integrinmediated adhesive properties ofneutrophils are reduced by hyperbaric oxygentherapy in patients with chronic nonhealingwoundMonica BaiulaID1 Roberto Greco2 Lucia Ferrazzano2 Alberto Caligiana1Klarida Hoxha3 Daniele Bandini3 Pasquale LongobardiID3 Santi Spampinato1˜¯Alessandra TolomelliID2˜¯ Department of Pharmacy and Biotechnology Alma Mater Studiorum University of Bologna Bologna Italy Department of Chemistry œGiacomo Ciamician Alma Mater Studiorum University of Bologna BolognaItaly Hyperbaric Centre Ravenna Italy˜¯ These authors contributed equally to this work Current address MediNeos Observational Research Modena Italy alessandratolomelliuniboit AT santispampinatouniboit SSa1111111111a1111111111a1111111111a1111111111a1111111111 ACCESSAbstractCitation Baiula M Greco R Ferrazzano L CaligianaA Hoxha K Bandini D Integrinmediated adhesive properties of neutrophils arereduced by hyperbaric oxygen therapy in patientswith chronic nonhealing wound e0237746 101371journalpone0237746Editor Nukhet AykinBurns University of Arkansasfor Medical Sciences College of Pharmacy UNITEDSTATESReceived May Accepted July Published August Copyright Baiula This is an access distributed under the terms of theCreative Commons Attribution License whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are creditedData Availability Statement All relevant data arewithin the manuscript and its SupportingInformation filesFunding Financial Disclosure This researchstudy was supported by Fondazione delMonte di Bologna e Ravenna wwwfondazionedelmonteit through a grant to ATFdM3980 prot N˚ bis2015 The authorswere also supported by the University of BolognaIn recent years several studies suggested that the ability of hyperbaric oxygen therapyHBOT to promote healing in patients with diabetic ulcers and chronic wounds is due to thereduction of inflammatory cytokines and to a significant decrease in neutrophils recruitmentto the damaged area α4 and 2 integrins are receptors mediating the neutrophil adhesionto the endothelium and the comprehension of the effects of hyperbaric oxygenation on theirexpression and functions in neutrophils could be of great importance for the design of noveltherapeutic protocols focused on antiinflammatory agents In this study the α4 and 2 integrins™ expression and functions have been evaluated in human primary neutrophils obtainedfrom patients with chronic nonhealing wounds and undergoing a prolonged HBOT kPaper minutes The effect of a peptidomimetic α41 integrin antagonist has been also analyzed under these conditions A statistically significant decrease in 2 integrin expression on neutrophils was observed during the treatment with HBO and maintained one monthafter the last treatment while α4 integrin levels remained unchanged However cell adhesion function of both neutrophilic integrins α41 and 2 was significantly reduced and respectively but α41 integrin was still sensitive to antagonist inhibition in the presence of fibronectin suggesting that a combined therapy between HBOT and integrin antagonists could have greater antinflammatory efficacyIntroductionHyperbaric oxygen HBO therapy has emerged in the last years as an innovative approachand an effective adjunctive therapy for the treatment of different pathologies The oxygenPLOS ONE 101371journalpone0237746 August PLOS ONE 0cRFO17 RFO18 RFO19 and by Italian Ministryof Education University and Research with aspecific research project PRIN project20157WW5EH and a grant to the Department ofChemistry œGiacomo Ciamician of the Universityof Bologna under the initiative Department ofExcellence L232 del The fundershad no role in study design data collection andanalysis decision to publish or preparation of themanuscriptCompeting interests The authors have declaredthat no competing interests existIntegrins™ role in HBOTpromoted wound healingpressure applied in the chamber is usually from to kiloPascal kPa“ to absoluteatmospheres ATA and the first effect of pressurizing the human body is the increase of partialpressure of gases and the decrease of volume of gasfilled spaces according to Boyle™s law [ ]The additionally available oxygen has the ability to restore oxygenation in areas where hypoxia or hypoperfusion occur and it can help damaged tissue to heal [] Moreover increasedoxygen levels that lead to changes in reactive oxygen species ROS and nitrogen speciesRNS production during HBO therapy HBOT are essential to stimulate specific repair functions of macrophages neutrophils and fibroblasts in the healing process [“] In additionHBOT regulates the inflammatory response reduction of NLRP3 inflammasome proinflammatory cytokines including IL1 IL6 e IL18 TNFα [“]HBOT has been successfully employed to control nonhealing diabetic ulcers and chronicwounds significantly minimizing the number of amputations relative to standard wound carealone in diabetic population []Wound healing is a complex process that involves growth factors components of the extracellular matrix and several cell types Inflammatory cytokines such as tumor necrosis factorαTNFα and interleukin1 IL1 are often present at high levels in the site of inflammationlike in chronic wounds [] The immune system is involved in all the steps of tissue repair[] Inflammatory response evolves in the leukocyteadhesion cascade primarily mediated bytwo major adhesion receptor families selectins and integrins []Neutrophils play a crucial role in wound healing process by sensing their environment andresponding to the extracellular signals by adhesion migration and other effector functions[] After ending their role at the site of inflammation neutrophils undergo apoptosis and areremoved by macrophages this latter event is considered a strong signal for inflammation resolution Although fighting infection neutrophils can also have harmful effects inducing damagein the inflamed tissue and leading to a delay in healing process [] and chronic inflammationNeutrophils express integrins on their surface significantly contributing to the recruitmentphase Among them 2 integrin family members including αL2 and αM2 bind to endothelial intercellular adhesion molecule1 ICAM1 and α41 integrin recognizes vascular celladhesion molecule1 VCAM1 expressed on endothelial cells [] Moreover α41 integrinand 2 integrins are involved in the onset and resolution of inflammatory process mediatingthe adhesion of monocytes lymphocytes and neutrophils to the blood vessels []Several studies carried out on animal models have evidenced that neutrophil recruitment issignificantly reduced by treatment with HBO in case of damage induced by ischemia andreperfusion [“] in other studies HBOT induces the reduction of tissue necrosis [ ]and lipid peroxidation []Moreover it has been shown that HBO treatment inhibits ischemia reperfusioninducedneutrophil adhesion to endothelium by blocking 2 integrin polarization [ ] and may alsoreduce leukocytes recruitment as it impaired adhesion molecule function by Snitrosation[] Conversely the role played by α41 integrin in neutrophilmediated adhesion to endothelium has been poorly investigated but an important role of α41 in 2 integrinindependentmigration of neutrophils across heart endothelium has been demonstrated in vitro suggestinga similar in vivo situation in neutrophil trafficking in reperfused myocardium []HBOT represents an effective therapy for chronic wounds as it reduces inflammation andaccelerates healing [] probably involving integrins In the present study we investigatedwhether HBOT could exert its effects by modulating functions of α41 and 2 integrinsexpressed on neutrophils obtained from patients with chronic nonhealing ulcers In a previous study Thom [] isolated polymorphonuclear leukocytes PMN from young healthyvolunteers exposed to only one session of HBO They observed a reduction in cell adhesionmediated by 2 integrins without any variation of its expression Our aim is to evaluate the rolePLOS ONE 101371journalpone0237746 August PLOS ONE 0cIntegrins™ role in HBOTpromoted wound healingof integrinmediated adhesion by characterizing the expression of integrin receptors on neutrophils during the HBOT and by analyzing the effect of a peptidomimetic α41 integrin antagonist under these conditions If integrins are a target for both HBOT and synthetic antagonistthat blockade their activation a joint therapy could be hypothesized leading to a faster andstronger decrease of inflammation To this purpose expression and function of α41 and 2integrins were investigated for the first time in human primary neutrophils isolated from theblood of patients with chronic nonhealing ulcer undergoing HBOT or standard wound therapy alone Expression of these integrins was monitored in patients before the beginning ofHBOT exposure and during the therapy using specific antibodies towards α4 integrin or 2integrin family Patient wound area size was measured and proinflammatory cytokine levelswere evaluated both in neutrophils and in plasma Furthermore in vitro cell adhesion assayswere performed in the presence of a peptidomimetic integrin antagonist previously developedby our group [] to investigate if the HBO treatment may influence neutrophil recruitmentand adhesion mediated by α41 integrinMaterials and methodsPatients™ recruitmentThe study protocol was conformed to the ethical guidelines of the Declaration ofHelsinkiThe patients followed exclusively the therapy prescribed by their medical doctor followingthe indications of the Italian public health system No additional treatments were done for thepurposes of the study which did not change any therapeutic protocol nor interfere with thehealing progress The patients of the HBOT group were submitted to the prescribed HBOTand to blood sampling following the standard protocols approved by the Institutional EthicsReview Board of the Comitato Etico della Romagna CEROM Version of June approved by CEROM Ethical Committee Reg Sperimentazioni Prot N45332018I5150 which has full access to the data in order to check conformity of the study to thecurrent regulationThirty patients from Hyperbaric Centre Ravenna Italy were enrolled in the study betweenJuly and January in two different groups control group patients n receivedstandard wound care alone as prescribed by their medical doctor whereas HBOT grouppatients n received HBOT in addition to conventional wound treatment The inclusioncriteria were adults years or older and chronic wounds that fail to demonstrate improvement wound area reduction after a minimum of weeks of standard wound therapyThe exclusion criteria were symptoms of bacterial infection malignancy pregnancy medications that can adversely affect healing including anticonvulsants steroids antibiotics angiogenesis inhibitors and NSAIDs such as drugs known to promote healing including vitaminsthyroid hormone and iron Contraindication for HBOT were claustrophobia uncontrolleddiabetes middle ear problems glaucoma heart failure pacemaker untreated pneumothoraxchronic obstructive pulmonary disease pulmonary emphysema with retention of CO2 prothesis and seizures Patients in the control group were matched by gender age and ethnicityExclusion criteria for control group patients were the same for patients in HBOT groupThe patients allowed donating blood samples and were informed of the aim of the studyand completed the written informed consent process before enrolling in the study At the endof the study all individuals received personal information about the results relative to theirown samplesThe following demographic information was collected for all recruited patients at baselinegender age smokernonsmoker and other medical conditions Photographs of the ulcer werePLOS ONE 101371journalpone0237746 August PLOS ONE 0cIntegrins™ role in HBOTpromoted wound healingtaken at various times After sharp wound debridement with a sterile scalpel the wound™s surface area was calculated by VISITRAK„¢ Digital System Smith Nephew For each patientthe ulcer was graded and staged by a clinician as described belowSample sizeSample size was based on a power analysis using G�Power [] The power was set at tolimit the risk of committing a type II error to the α level was set at The effect sizewas set at and the number of samples for each group was calculated to be to detect statistically significant differences P value of between groups In this study patientswere selected and allocated to study groups control and HBOT groupHyperbaric oxygen therapy protocolHBOT was conducted at the Hyperbaric Centre in Ravenna Italy The protocol procedureconsisted of HBO exposures in a multiplace hyperbaric chamber daily session five perweek from Monday to Friday HBOT group patients breathed oxygen at kPa per minutes in cycles for minutes separated by minutes medical air breathing intervalsClassification of woundsIn this study ulcers were graded using Falanga Wound Bed Preparation Score Staging copyrighted [] that provides staging of varying degrees based upon descriptions and characteristics of ulcers Table [] Staging of the wounds was done by combining the score of thewound bed appearance with that of the wound exudate Table Clinical assessment ofwound conditions was conducted for HBOT group before HBO treatment T0 immediatelyafter the fifteenth HBOT session T15 and one month after ending HBOT T1M for controlgroup during the first evaluation by a medical doctor T0 and after fifteen days of conventional wound therapy T15Neutrophil isolation from peripheral bloodVenous blood samples were obtained from the antecubital vein of participants in EDTAcontaining vacutainers Neutrophil isolation and further experiments were performed at theDepartment of Pharmacy and Biotechnology University of Bologna Bologna Italy Bloodsamples were obtained for patients in control group during the first evaluation by a clinicianTable Cutaneous ulcers were graded using Falanga wound classification systemWound Bed CharacteristicsWound appearanceGranulation tissueFibrinous tissueEscharABCDWound Exudate ScoreExtent of ControlFullyPartiallyUncontrolledAdapted from []101371journalpone0237746t001“ Any amountExudate AmountNoneminimal““““Dressing requirementNo absorptive dressing required If clinically feasibledressings could stay on for up to a weekModerate amountDressing changes required every “ daysVery exudative woundAbsorptive dressing changes required at least dailyPLOS ONE 101371journalpone0237746 August PLOS ONE 0cIntegrins™ role in HBOTpromoted wound healingT0 and after fifteen days of conventional wound therapy T15 while for HBOT grouppatients before T0 and immediately after the fourth T4 the eighth T8 the twelfth T12and the fifteenth T15 HBOT sessions In addition one month after ending HBOT T1Manother blood sample was collected from HBOT group patients For neutrophil isolationblood was carefully layered on top of an equal volume of Lympholyte1poly Cedarlaneand centrifuged at g for min at “ËšC as previously described [] Plasma wascarefully removed and store at ˚C for further analysis Neutrophils were transferred in aclean tube and were resuspended in mL of HBSS Hanks™ Balanced Salt Solution LifeTechnologies Italia without Ca2Mg2 and centrifuged at g for minutes To lyse theresidual red blood cells RBCs mL Red Cell Lysis Buffer Roche were added and the cellswere resuspended vortexing at low speed to avoid neutrophils activation Neutrophils werecentrifuged at g for min resuspended in HBSS without Ca2Mg2 and adjusted todesired concentrationCell viability was determined using Annexin V7AAD assay Guava Nexin Reagent Millipore as previously described [] and was Differential analysis of cells retrieved usingthis procedure showed granulocytes of which were neutrophils Neutrophilswere stored at room temperature and used for functional tests cell adhesion assay and flowcytometry analysis within h of collection An aliquot of the purified neutrophils was immediately stored at ˚C and used for mRNA extractionNeutrophil adhesion assayAdhesion assays on purified neutrophils were performed as previously described [ ]Briefly black 96well plates were coated overnight at ˚C with fibronectin FN or fibrinogenFg both μgmL to study respectively adhesion mediated by α41 and 2 integrins Neutrophils were counted and stained with CellTracker green CMFDA μM min at ˚CLife Technologies Italia Thereafter cells were plated 50000well on coated wells and incubated for min at ˚C After three washes adhered cells were lysed with Triton X100in BSA bovine serum albumin in HBSS min at ˚C and fluorescence was measuredEx485 nmEm535 nm To evaluate the ability of ligand [] named here RG66 to inhibitneutrophils™ adhesion cells were preincubated with various concentrations ˆ’“ˆ’ M ofRG66 or vehicle methanol for min at ˚C before plating cells into coated wells Neutrophil adhesion assays were also carried out in the presence of an antihuman 2 or α4 integrinantibody both μgmL purified mouse antihuman CD18 and purified mouse antihumanCD49d antibody BD Pharmingen Experiments were carried out in triplicate Data analysisand IC50 values were calculated using GraphPad Prism GraphPad Software San DiegoCA USAFlow cytometry analysisPurified neutrophils were suspended in BSA in HBSS at the concentration of cellsmL μLsample and incubated with FITClabeled antiα4 integrin antibody μLsampleFITC Mouse antihuman CD49d BD Pharmingen or FITClabeled anti2 integrin antibody μLsample FITC Mouse antihuman CD18 BD Pharmingen for min at ˚C as previously described [] After two washes with BSA in HBSS cells were resuspended in PBSand analyzed in a Guava EasyCyte Flow Cytometer Millipore and cellssample wereanalyzed Data were normalized with the relative fluorescence for nonspecific binding evaluated by exposing the cells to an isotype control monoclonal antibody FITC mouse IgG BectonDickinson Italia and set to PLOS ONE 101371journalpone0237746 August PLOS ONE 0cIntegrins™ role in HBOTpromoted wound healingIn another set of experiments purified neutrophils were suspended in BSA in HBSS atthe concentration of cellsmL μLsample and incubated with the conformational sensitive phycoerythrin PElabeled HUTS21 monoclonal antibody μLsample PE mouseantihuman CD29 antibody BD Pharmingen for min at room temperature Neutrophilswere washed twice with BSA in HBSS resuspended in PBS and analyzed at the flow cytometry cellssample were analyzed Data were normalized to nonspecific binding relativefluorescence evaluated by exposing the cells to an isotype control mAb monoclonal antibodyand set to Quantitative real time PCRTotal RNA was extracted from purified neutrophils with TRI Reagent SigmaAldrich andquantified using a NanoDrop spectrophotometer ThermoFisher Scientific For each sample“ μg of total RNA was treated with RNasefree DNase as previously described [] TheRNA samples were then converted into cDNA using HighCapacity cDNA Reverse Transcription Kits Life Technologies Italia according to the manufacturer™s instructions RealtimePCR was performed using GoTaq1 qPCR Master Mix Promega Corporation Madison WIUSA The protocol consisted of i for L19 and TNFα denaturation at ˚C for minutesfollowed by cycles of ˚C denaturation seconds and ˚C annealingextension minute ii for α4 integrin denaturation at ˚C for minutes followed by cycles of ˚Cdenaturation seconds ˚C annealing seconds and ˚C seconds iii for IL1denaturation at ˚C for minutes followed by cycles of ˚C denaturation seconds˚C annealing seconds and ˚C seconds Notemplate controls and DNA meltingcurve analysis were used as controls to ensure the lack of contaminating DNA in the RNApreparations and to rule out primerdimer formation respectively To amplify integrin andcytokine targets the following primers were used α4 integrin sense primer 50GTCGCATCCCGTGCAACTTTG30 and antisense primer 50GCTGTGCAGCACGACCGAGT30 amplifying a bp fragment TNFα sense primer 50CTTCTCCTTCCTGATCGTGG30 and antisense primer 50TCTCAGCTCCACGCCATT30 amplifying a bp fragment [] IL1sense primer 50CAAGGGCTTCAGGCAGGCCG30 and antisense primer 50TGAGTCCCGGAGCGTGCAGT30 amplifying a bp fragment [] To amplify 2 integrin cDNA primersequences were from PrimerBank [] a sense primer 50TGCGTCCTCTCTCAGGAGTG30and an antisense primer 50GGTCCATGATGTCGTCAGCC30 amplifying a bp fragmentwere usedAs reference control a bp fragment of the L19 ribosomal protein was amplified using asense primer 50CTAGTGTCCTCCGCTGTGG30 and an antisense primer 50AAGGTGTTTTTCCGGCATC30 [] For data analysis relative expression of RTPCR products wasdetermined using the ΔΔCT method [] as previously described [] the threshold cycle Ctvalues were normalized both on the basis of L19 content and on the values derived from T0sample Each sample was tested in triplicate Primers were synthesized by SigmaAldrichCytokine quantification in plasma by ELISACytokine protein levels TNFα and IL1 were determined in plasma of patients usingELISA kits Invitrogen LifeTechnologies Italia Monza Italy according to the manufacturer™sinstructions Briefly 50μLwell of sample were added to a 96well plate together with μL ofbiotin conjugated primary antibody the plate was then incubated for hours at room temperature After washing four times μL of streptavidinHRP solution were added and the platewas incubated for min at room temperature The wells were washed times and afterwards μL of stabilized chromogen were added and incubated for min at room temperaturePLOS ONE 101371journalpone0237746 August PLOS ONE 0cIntegrins™ role in HBOTpromoted wound healingAfter the addition of μL of stop solution absorbance was read at nm using an EnSpireMultimode Plate Reader PerkinElmer Waltham MA USA The calculated overall intraassay coefficient of variation was for TNFα and for IL1 the interassay coefficient of variation was calculated to be for TNFα and for IL1Synthesis and bioactivity of α41 synthetic ligandThe synthesis of RRG66 has been previously reported [] via a multistep synthesis startingfrom an enantiopure 3RZtertbutyl 3allylamino2ethylidene4methylpentanoate Thedetailed description of the synthetic protocol has been reported in S1 Fig The biological evaluation showed a strong dependence of the bioactivity on the ring stereochemistry could bedetected since S1 turned out to be completely inactive []Data and statistical analysisAll assays were carried out in triplicate for individual sample at each time pointperson and nrefers to the number of individuals Continuous variables are presented as mean ± standarddeviation when normally distributed data were tested using oneway ANOVA followed byNewmanKeuls posttest or using standard Student t test In addition data are presented asmedian and range and analyzed using MannWhitney™s test when non normally distributedCategorical data were analyzed using χ2square test Data analysis and IC50 values referring toadhesion assays in the presence of RG66 compound were fitted using sigmoidal doseresponseequation using GraphPad Prism software Statistical analyses were performed using GraphPadPrism version GraphPad Software Inc La Jolla CA USA P was consideredsignificantResultsDemographic and clinical data patients men and women age ± years presenting a chronic nonhealingwound condition were recruited and volunteered to participate in the study All the patientscompleted the study and no patient was excluded from the data analysis Demographic parameters such as age gender and clinical data are summarized in Table Age distributions demographic and clinical characteristics of patients were compatiblebetween HBOT and control groups P Overall the wounds of the patients enrolled in the study were caused by different etiologies caused by diabetes by venous insufficiency by critical limb ischemia bytrauma and by vasculitisAs regards comorbidities seven of the subjects suffered from diabetes of type1 and oftype2 twelve of them of hypertension six of obesity seven of venous insufficiency eight ofcardiomyopathy and six of hypothyroidism not requiring thyroid hormones Three subjectswere smokers and four previous smokers Common sites of wound were leg patients foot patients great toe patients and other sites patients Before beginning the HBOTall participants passed a standard medical and physical revision at the Hyperbaric Centre inRavenna All the patients both in control and HBOT groups received standard wound careie wounds were cleaned gently minimizing chemical or mechanical trauma at low pressure“ psi with saline solution and daily sterile sharp debridement with scalpel curette or scissors was performed for the necrotic tissue removal with caution to avoid excess tissue damagewhich may delay healing to get a wellbleeding granulating basePLOS ONE 101371journalpone0237746 August PLOS ONE 0cIntegrins™ role in HBOTpromoted wound healingPTable Demographic and clinical data of patients enrolled in the studyDemographic dataNumber of subjectsAge yearsaFemaleMalebSmokersComorbiditiesbDiabetesHypertensionObesityVenous insufficiencyGlaucomaCardiomyopathyHypothyroidismHBVHCV positiveRheumatoid arthritisControl groupHBOT group “type1 type2 “ extype1 type2 aAge is expressed as median rangeb Gender and comorbidities are expressed as number of individuals101371journalpone0237746t002Integrin expression on neutrophils deriving from patients undergoingHBOTIn order to study the effect of HBOT on neutrophil integrin expression these cells were isolated from blood samples deriving from patients with a chronic nonhealing wound receiving standard wound care alone control group or undergoing HBOT HBOT group Forcontrol group patients blood samples were collected during the first wound evaluation T0and after fifteen days of conventional wound therapy T15 for patients in the HBOT groupblood samples were obtained before T0 and immediately after the fourth T4 the eighthT8 the twelfth T12 and the fifteenth T15 HBOT treatment at the end of three weeks fiveexposuresweek moreover the last blood sample was drawn one month after ending HBOTT1MAs shown in Fig HBOT did not alter α4 integrin expression in primary human neutrophils both at the mRNA and protein levels Fig panels a and b nor induced any significantvariation in α4 integrin expression throughout the duration of HBO treatment No significantchange in α4 integrin expressed on neutrophils was observed in both control and HBOT grouppatientsOn the contrary 2 integrins were significantly reduced by HBOT both at mRNA andprotein levels this decrement was maintained up to the end of HBO treatment and also onemonth after the last HBOT session Fig panels c and d Neutrophils deriving from patientsreceiving conventional wound care alone control group did not show any changes in 2integrins expressionEffects of HBOT on integrinmediated adhesive properties of neutrophilsDuring an inflammatory process neutrophils adhere and transmigrate through bloodvesselwalls α41 and 2 integrins expressed on neutrophil cell membrane are required and stronglymediate rolling and firm adhesion crawling and transmigration steps of adhesion cascade [] as their activation is an essential step of this complex process To understand whetherPLOS ONE 101371journalpone0237746 August PLOS ONE 0cIntegrins™ role in HBOTpromoted wound healingFig HBOT does not modify α4 integrin levels panels a and b but reduces 2 expression panels c and d in neutrophilsderiving from patients receiving standard wound care alone control group and patients undergoing HBOT for sessionsHBOT group The decrement of 2 integrins was maintained up to one month after ending HBOT The effects of HBOT onintegrin expression were evaluated both by qPCR mRNA levels panel a c and by flow cytometry measuring integrin expressed oncell surface panel b d Results from qPCR are expressed as mean ± standard deviation of individual samples carried out intriplicate at each time point control group n HBOT group n Data from flow cytometry analysis are expressed as meanfluorescence intensity MFI ± standard deviation of individual samples carried out in triplicate at each time point control groupn HBOT group n MFI values for respective isotype control monoclonal antibody were set to ��� p versus T0both control and HBOT group101371journalpone0237746g001HBOT could influence integrinmediated neutrophil adhesion we performed cell adhesionassays to fibronectin FN or fibrinogen Fg ligands for α41 and 2 integrins respectivelyon neutrophils isolated from patients with chronic nonhealing wound receiving standardwound care alone control group or undergoing HBOT HBOT group Adhesion of neutrophils obtained from patients belonging to both control and HBOT groups was significantlyreduced by the addition of integrin specific antibodies able to block integrin functions Fig demonstrating that neutrophil adhesion to fibronectin or fibrinogen was mainly mediated byα41 or 2 integrin respectively Moreover as shown in Fig exposure to HBO induces a significant reduction of neutrophil adhesion mediated by either 2 or α41 integrins Fig panela and b respectively Interestingly this reduction of neutrophil adhesive properties is retainedthroughout the duration of HBO treatment and up to one month after the last HBOT sessionIn addition these data showed that adhesive properties of α41 integrin expressed on neutrophils were impaired during HBOT although its expression was not modified both at mRNAand protein levels as shown in Fig To better understand the involvement of α41 integrinin neutrophil adhesion we used a conformationspecific antibody that recognizes a specificepitope on integrin 1 subunit exposed only in a defined structural conformation [] Integrins exist in three major conformations a bent or inactive an intermediateactive and an highactivity conformation [] To monitor conformational changes in integrin subunits it is therefore possible to use conformationspecific antibodies [] We employed thePLOS ONE 101371journalpone0237746 August PLOS ONE 0cIntegrins™ role in HBOTpromoted wound healingFig HBOT reduces significantly integrinmediated neutrophil adhesion to fibrinogen Fg or fibronectin FN Integrinmediated adhesion wasdecreased in neutrophils obtained from patients belonging to HBOT group no changes were observed in control group patients The effects of HBOTon neutrophil adhesion mediated by 2 panel a or α41 panel b integrins were evaluated by adhesion assay to Fg or FN respectively as described inmethod section Adhesion mediated by 2 or α41 integrin is significantly prevented in neutrophils treated with a monoclonal antibody anti2 or antiα4 respectively HBOT exposure significantly reduced anti1 HUTS21 mAb binding to neutrophils namely changing α41 integrin conformationpanel c Mean fluorescence intensity MFI due to the anti1 integrin mAb PE conjugated HUTS21 binding in the presence of fibronectin μgmL was measured Nonspecific binding of an isotype control PE conjugated mAb added to neutrophils produced an MFI of ± that was subtractedfrom all samples Data are expressed as mean ± standard deviation of individual samples carried out in triplicate at each time point control groupn HBOT group n �� p � �� p ���� p versus T0 both control and HBOT group101371journalpone0237746g002PEconjugated HUTS21 mAb to determine whether the reduced adhesive properties of neutrophils mediated by α41 integrin during HBOT are due to a conformational change of 1subunit indicative of its activation status HUTS21 antibody recognizes a ligandinducedbinding site that is hidden in the inactive conformation but it is exposed when the agonistbinds or upon partial integrin activation namely when the integrin is in a high affinity conformation The epitope recognized by HUTS21 antibody is located in the hybrid domain of 1integrin subunit [] PEconjugated anti1 HUTS21 mAb was added to neutrophils in thepresence of fibronectin μgmL and fluorescence was measured by flow cytometry Exposure to HBOT induced a significant reduction of HUTS21 mAb binding to neutrophilsthroughout the duration of the treatment and up to one month after the last HBOT sessionFig panel c meaning th

cancer270

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: Integrinmediated adhesive properties ofneutrophils are reduced by hyperbaric oxygentherapy in patients with chronic nonhealingwoundMonica BaiulaID1 Roberto Greco2 Lucia Ferrazzano2 Alberto Caligiana1Klarida Hoxha3 Daniele Bandini3 Pasquale LongobardiID3 Santi Spampinato1˜¯Alessandra TolomelliID2˜¯ Department of Pharmacy and Biotechnology Alma Mater Studiorum University of Bologna Bologna Italy Department of Chemistry œGiacomo Ciamician Alma Mater Studiorum University of Bologna BolognaItaly Hyperbaric Centre Ravenna Italy˜¯ These authors contributed equally to this work Current address MediNeos Observational Research Modena Italy alessandratolomelliuniboit AT santispampinatouniboit SSa1111111111a1111111111a1111111111a1111111111a1111111111 ACCESSAbstractCitation Baiula M Greco R Ferrazzano L CaligianaA Hoxha K Bandini D Integrinmediated adhesive properties of neutrophils arereduced by hyperbaric oxygen therapy in patientswith chronic nonhealing wound e0237746 101371journalpone0237746Editor Nukhet AykinBurns University of Arkansasfor Medical Sciences College of Pharmacy UNITEDSTATESReceived May Accepted July Published August Copyright Baiula This is an access distributed under the terms of theCreative Commons Attribution License whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are creditedData Availability Statement All relevant data arewithin the manuscript and its SupportingInformation filesFunding Financial Disclosure This researchstudy was supported by Fondazione delMonte di Bologna e Ravenna wwwfondazionedelmonteit through a grant to ATFdM3980 prot N˚ bis2015 The authorswere also supported by the University of BolognaIn recent years several studies suggested that the ability of hyperbaric oxygen therapyHBOT to promote healing in patients with diabetic ulcers and chronic wounds is due to thereduction of inflammatory cytokines and to a significant decrease in neutrophils recruitmentto the damaged area α4 and 2 integrins are receptors mediating the neutrophil adhesionto the endothelium and the comprehension of the effects of hyperbaric oxygenation on theirexpression and functions in neutrophils could be of great importance for the design of noveltherapeutic protocols focused on antiinflammatory agents In this study the α4 and 2 integrins™ expression and functions have been evaluated in human primary neutrophils obtainedfrom patients with chronic nonhealing wounds and undergoing a prolonged HBOT kPaper minutes The effect of a peptidomimetic α41 integrin antagonist has been also analyzed under these conditions A statistically significant decrease in 2 integrin expression on neutrophils was observed during the treatment with HBO and maintained one monthafter the last treatment while α4 integrin levels remained unchanged However cell adhesion function of both neutrophilic integrins α41 and 2 was significantly reduced and respectively but α41 integrin was still sensitive to antagonist inhibition in the presence of fibronectin suggesting that a combined therapy between HBOT and integrin antagonists could have greater antinflammatory efficacyIntroductionHyperbaric oxygen HBO therapy has emerged in the last years as an innovative approachand an effective adjunctive therapy for the treatment of different pathologies The oxygenPLOS ONE 101371journalpone0237746 August PLOS ONE 0cRFO17 RFO18 RFO19 and by Italian Ministryof Education University and Research with aspecific research project PRIN project20157WW5EH and a grant to the Department ofChemistry œGiacomo Ciamician of the Universityof Bologna under the initiative Department ofExcellence L232 del The fundershad no role in study design data collection andanalysis decision to publish or preparation of themanuscriptCompeting interests The authors have declaredthat no competing interests existIntegrins™ role in HBOTpromoted wound healingpressure applied in the chamber is usually from to kiloPascal kPa“ to absoluteatmospheres ATA and the first effect of pressurizing the human body is the increase of partialpressure of gases and the decrease of volume of gasfilled spaces according to Boyle™s law [ ]The additionally available oxygen has the ability to restore oxygenation in areas where hypoxia or hypoperfusion occur and it can help damaged tissue to heal [] Moreover increasedoxygen levels that lead to changes in reactive oxygen species ROS and nitrogen speciesRNS production during HBO therapy HBOT are essential to stimulate specific repair functions of macrophages neutrophils and fibroblasts in the healing process [“] In additionHBOT regulates the inflammatory response reduction of NLRP3 inflammasome proinflammatory cytokines including IL1 IL6 e IL18 TNFα [“]HBOT has been successfully employed to control nonhealing diabetic ulcers and chronicwounds significantly minimizing the number of amputations relative to standard wound carealone in diabetic population []Wound healing is a complex process that involves growth factors components of the extracellular matrix and several cell types Inflammatory cytokines such as tumor necrosis factorαTNFα and interleukin1 IL1 are often present at high levels in the site of inflammationlike in chronic wounds [] The immune system is involved in all the steps of tissue repair[] Inflammatory response evolves in the leukocyteadhesion cascade primarily mediated bytwo major adhesion receptor families selectins and integrins []Neutrophils play a crucial role in wound healing process by sensing their environment andresponding to the extracellular signals by adhesion migration and other effector functions[] After ending their role at the site of inflammation neutrophils undergo apoptosis and areremoved by macrophages this latter event is considered a strong signal for inflammation resolution Although fighting infection neutrophils can also have harmful effects inducing damagein the inflamed tissue and leading to a delay in healing process [] and chronic inflammationNeutrophils express integrins on their surface significantly contributing to the recruitmentphase Among them 2 integrin family members including αL2 and αM2 bind to endothelial intercellular adhesion molecule1 ICAM1 and α41 integrin recognizes vascular celladhesion molecule1 VCAM1 expressed on endothelial cells [] Moreover α41 integrinand 2 integrins are involved in the onset and resolution of inflammatory process mediatingthe adhesion of monocytes lymphocytes and neutrophils to the blood vessels []Several studies carried out on animal models have evidenced that neutrophil recruitment issignificantly reduced by treatment with HBO in case of damage induced by ischemia andreperfusion [“] in other studies HBOT induces the reduction of tissue necrosis [ ]and lipid peroxidation []Moreover it has been shown that HBO treatment inhibits ischemia reperfusioninducedneutrophil adhesion to endothelium by blocking 2 integrin polarization [ ] and may alsoreduce leukocytes recruitment as it impaired adhesion molecule function by Snitrosation[] Conversely the role played by α41 integrin in neutrophilmediated adhesion to endothelium has been poorly investigated but an important role of α41 in 2 integrinindependentmigration of neutrophils across heart endothelium has been demonstrated in vitro suggestinga similar in vivo situation in neutrophil trafficking in reperfused myocardium []HBOT represents an effective therapy for chronic wounds as it reduces inflammation andaccelerates healing [] probably involving integrins In the present study we investigatedwhether HBOT could exert its effects by modulating functions of α41 and 2 integrinsexpressed on neutrophils obtained from patients with chronic nonhealing ulcers In a previous study Thom [] isolated polymorphonuclear leukocytes PMN from young healthyvolunteers exposed to only one session of HBO They observed a reduction in cell adhesionmediated by 2 integrins without any variation of its expression Our aim is to evaluate the rolePLOS ONE 101371journalpone0237746 August PLOS ONE 0cIntegrins™ role in HBOTpromoted wound healingof integrinmediated adhesion by characterizing the expression of integrin receptors on neutrophils during the HBOT and by analyzing the effect of a peptidomimetic α41 integrin antagonist under these conditions If integrins are a target for both HBOT and synthetic antagonistthat blockade their activation a joint therapy could be hypothesized leading to a faster andstronger decrease of inflammation To this purpose expression and function of α41 and 2integrins were investigated for the first time in human primary neutrophils isolated from theblood of patients with chronic nonhealing ulcer undergoing HBOT or standard wound therapy alone Expression of these integrins was monitored in patients before the beginning ofHBOT exposure and during the therapy using specific antibodies towards α4 integrin or 2integrin family Patient wound area size was measured and proinflammatory cytokine levelswere evaluated both in neutrophils and in plasma Furthermore in vitro cell adhesion assayswere performed in the presence of a peptidomimetic integrin antagonist previously developedby our group [] to investigate if the HBO treatment may influence neutrophil recruitmentand adhesion mediated by α41 integrinMaterials and methodsPatients™ recruitmentThe study protocol was conformed to the ethical guidelines of the Declaration ofHelsinkiThe patients followed exclusively the therapy prescribed by their medical doctor followingthe indications of the Italian public health system No additional treatments were done for thepurposes of the study which did not change any therapeutic protocol nor interfere with thehealing progress The patients of the HBOT group were submitted to the prescribed HBOTand to blood sampling following the standard protocols approved by the Institutional EthicsReview Board of the Comitato Etico della Romagna CEROM Version of June approved by CEROM Ethical Committee Reg Sperimentazioni Prot N45332018I5150 which has full access to the data in order to check conformity of the study to thecurrent regulationThirty patients from Hyperbaric Centre Ravenna Italy were enrolled in the study betweenJuly and January in two different groups control group patients n receivedstandard wound care alone as prescribed by their medical doctor whereas HBOT grouppatients n received HBOT in addition to conventional wound treatment The inclusioncriteria were adults years or older and chronic wounds that fail to demonstrate improvement wound area reduction after a minimum of weeks of standard wound therapyThe exclusion criteria were symptoms of bacterial infection malignancy pregnancy medications that can adversely affect healing including anticonvulsants steroids antibiotics angiogenesis inhibitors and NSAIDs such as drugs known to promote healing including vitaminsthyroid hormone and iron Contraindication for HBOT were claustrophobia uncontrolleddiabetes middle ear problems glaucoma heart failure pacemaker untreated pneumothoraxchronic obstructive pulmonary disease pulmonary emphysema with retention of CO2 prothesis and seizures Patients in the control group were matched by gender age and ethnicityExclusion criteria for control group patients were the same for patients in HBOT groupThe patients allowed donating blood samples and were informed of the aim of the studyand completed the written informed consent process before enrolling in the study At the endof the study all individuals received personal information about the results relative to theirown samplesThe following demographic information was collected for all recruited patients at baselinegender age smokernonsmoker and other medical conditions Photographs of the ulcer werePLOS ONE 101371journalpone0237746 August PLOS ONE 0cIntegrins™ role in HBOTpromoted wound healingtaken at various times After sharp wound debridement with a sterile scalpel the wound™s surface area was calculated by VISITRAK„¢ Digital System Smith Nephew For each patientthe ulcer was graded and staged by a clinician as described belowSample sizeSample size was based on a power analysis using G�Power [] The power was set at tolimit the risk of committing a type II error to the α level was set at The effect sizewas set at and the number of samples for each group was calculated to be to detect statistically significant differences P value of between groups In this study patientswere selected and allocated to study groups control and HBOT groupHyperbaric oxygen therapy protocolHBOT was conducted at the Hyperbaric Centre in Ravenna Italy The protocol procedureconsisted of HBO exposures in a multiplace hyperbaric chamber daily session five perweek from Monday to Friday HBOT group patients breathed oxygen at kPa per minutes in cycles for minutes separated by minutes medical air breathing intervalsClassification of woundsIn this study ulcers were graded using Falanga Wound Bed Preparation Score Staging copyrighted [] that provides staging of varying degrees based upon descriptions and characteristics of ulcers Table [] Staging of the wounds was done by combining the score of thewound bed appearance with that of the wound exudate Table Clinical assessment ofwound conditions was conducted for HBOT group before HBO treatment T0 immediatelyafter the fifteenth HBOT session T15 and one month after ending HBOT T1M for controlgroup during the first evaluation by a medical doctor T0 and after fifteen days of conventional wound therapy T15Neutrophil isolation from peripheral bloodVenous blood samples were obtained from the antecubital vein of participants in EDTAcontaining vacutainers Neutrophil isolation and further experiments were performed at theDepartment of Pharmacy and Biotechnology University of Bologna Bologna Italy Bloodsamples were obtained for patients in control group during the first evaluation by a clinicianTable Cutaneous ulcers were graded using Falanga wound classification systemWound Bed CharacteristicsWound appearanceGranulation tissueFibrinous tissueEscharABCDWound Exudate ScoreExtent of ControlFullyPartiallyUncontrolledAdapted from []101371journalpone0237746t001“ Any amountExudate AmountNoneminimal““““Dressing requirementNo absorptive dressing required If clinically feasibledressings could stay on for up to a weekModerate amountDressing changes required every “ daysVery exudative woundAbsorptive dressing changes required at least dailyPLOS ONE 101371journalpone0237746 August PLOS ONE 0cIntegrins™ role in HBOTpromoted wound healingT0 and after fifteen days of conventional wound therapy T15 while for HBOT grouppatients before T0 and immediately after the fourth T4 the eighth T8 the twelfth T12and the fifteenth T15 HBOT sessions In addition one month after ending HBOT T1Manother blood sample was collected from HBOT group patients For neutrophil isolationblood was carefully layered on top of an equal volume of Lympholyte1poly Cedarlaneand centrifuged at g for min at “ËšC as previously described [] Plasma wascarefully removed and store at ˚C for further analysis Neutrophils were transferred in aclean tube and were resuspended in mL of HBSS Hanks™ Balanced Salt Solution LifeTechnologies Italia without Ca2Mg2 and centrifuged at g for minutes To lyse theresidual red blood cells RBCs mL Red Cell Lysis Buffer Roche were added and the cellswere resuspended vortexing at low speed to avoid neutrophils activation Neutrophils werecentrifuged at g for min resuspended in HBSS without Ca2Mg2 and adjusted todesired concentrationCell viability was determined using Annexin V7AAD assay Guava Nexin Reagent Millipore as previously described [] and was Differential analysis of cells retrieved usingthis procedure showed granulocytes of which were neutrophils Neutrophilswere stored at room temperature and used for functional tests cell adhesion assay and flowcytometry analysis within h of collection An aliquot of the purified neutrophils was immediately stored at ˚C and used for mRNA extractionNeutrophil adhesion assayAdhesion assays on purified neutrophils were performed as previously described [ ]Briefly black 96well plates were coated overnight at ˚C with fibronectin FN or fibrinogenFg both μgmL to study respectively adhesion mediated by α41 and 2 integrins Neutrophils were counted and stained with CellTracker green CMFDA μM min at ˚CLife Technologies Italia Thereafter cells were plated 50000well on coated wells and incubated for min at ˚C After three washes adhered cells were lysed with Triton X100in BSA bovine serum albumin in HBSS min at ˚C and fluorescence was measuredEx485 nmEm535 nm To evaluate the ability of ligand [] named here RG66 to inhibitneutrophils™ adhesion cells were preincubated with various concentrations ˆ’“ˆ’ M ofRG66 or vehicle methanol for min at ˚C before plating cells into coated wells Neutrophil adhesion assays were also carried out in the presence of an antihuman 2 or α4 integrinantibody both μgmL purified mouse antihuman CD18 and purified mouse antihumanCD49d antibody BD Pharmingen Experiments were carried out in triplicate Data analysisand IC50 values were calculated using GraphPad Prism GraphPad Software San DiegoCA USAFlow cytometry analysisPurified neutrophils were suspended in BSA in HBSS at the concentration of cellsmL μLsample and incubated with FITClabeled antiα4 integrin antibody μLsampleFITC Mouse antihuman CD49d BD Pharmingen or FITClabeled anti2 integrin antibody μLsample FITC Mouse antihuman CD18 BD Pharmingen for min at ˚C as previously described [] After two washes with BSA in HBSS cells were resuspended in PBSand analyzed in a Guava EasyCyte Flow Cytometer Millipore and cellssample wereanalyzed Data were normalized with the relative fluorescence for nonspecific binding evaluated by exposing the cells to an isotype control monoclonal antibody FITC mouse IgG BectonDickinson Italia and set to PLOS ONE 101371journalpone0237746 August PLOS ONE 0cIntegrins™ role in HBOTpromoted wound healingIn another set of experiments purified neutrophils were suspended in BSA in HBSS atthe concentration of cellsmL μLsample and incubated with the conformational sensitive phycoerythrin PElabeled HUTS21 monoclonal antibody μLsample PE mouseantihuman CD29 antibody BD Pharmingen for min at room temperature Neutrophilswere washed twice with BSA in HBSS resuspended in PBS and analyzed at the flow cytometry cellssample were analyzed Data were normalized to nonspecific binding relativefluorescence evaluated by exposing the cells to an isotype control mAb monoclonal antibodyand set to Quantitative real time PCRTotal RNA was extracted from purified neutrophils with TRI Reagent SigmaAldrich andquantified using a NanoDrop spectrophotometer ThermoFisher Scientific For each sample“ μg of total RNA was treated with RNasefree DNase as previously described [] TheRNA samples were then converted into cDNA using HighCapacity cDNA Reverse Transcription Kits Life Technologies Italia according to the manufacturer™s instructions RealtimePCR was performed using GoTaq1 qPCR Master Mix Promega Corporation Madison WIUSA The protocol consisted of i for L19 and TNFα denaturation at ˚C for minutesfollowed by cycles of ˚C denaturation seconds and ˚C annealingextension minute ii for α4 integrin denaturation at ˚C for minutes followed by cycles of ˚Cdenaturation seconds ˚C annealing seconds and ˚C seconds iii for IL1denaturation at ˚C for minutes followed by cycles of ˚C denaturation seconds˚C annealing seconds and ˚C seconds Notemplate controls and DNA meltingcurve analysis were used as controls to ensure the lack of contaminating DNA in the RNApreparations and to rule out primerdimer formation respectively To amplify integrin andcytokine targets the following primers were used α4 integrin sense primer 50GTCGCATCCCGTGCAACTTTG30 and antisense primer 50GCTGTGCAGCACGACCGAGT30 amplifying a bp fragment TNFα sense primer 50CTTCTCCTTCCTGATCGTGG30 and antisense primer 50TCTCAGCTCCACGCCATT30 amplifying a bp fragment [] IL1sense primer 50CAAGGGCTTCAGGCAGGCCG30 and antisense primer 50TGAGTCCCGGAGCGTGCAGT30 amplifying a bp fragment [] To amplify 2 integrin cDNA primersequences were from PrimerBank [] a sense primer 50TGCGTCCTCTCTCAGGAGTG30and an antisense primer 50GGTCCATGATGTCGTCAGCC30 amplifying a bp fragmentwere usedAs reference control a bp fragment of the L19 ribosomal protein was amplified using asense primer 50CTAGTGTCCTCCGCTGTGG30 and an antisense primer 50AAGGTGTTTTTCCGGCATC30 [] For data analysis relative expression of RTPCR products wasdetermined using the ΔΔCT method [] as previously described [] the threshold cycle Ctvalues were normalized both on the basis of L19 content and on the values derived from T0sample Each sample was tested in triplicate Primers were synthesized by SigmaAldrichCytokine quantification in plasma by ELISACytokine protein levels TNFα and IL1 were determined in plasma of patients usingELISA kits Invitrogen LifeTechnologies Italia Monza Italy according to the manufacturer™sinstructions Briefly 50μLwell of sample were added to a 96well plate together with μL ofbiotin conjugated primary antibody the plate was then incubated for hours at room temperature After washing four times μL of streptavidinHRP solution were added and the platewas incubated for min at room temperature The wells were washed times and afterwards μL of stabilized chromogen were added and incubated for min at room temperaturePLOS ONE 101371journalpone0237746 August PLOS ONE 0cIntegrins™ role in HBOTpromoted wound healingAfter the addition of μL of stop solution absorbance was read at nm using an EnSpireMultimode Plate Reader PerkinElmer Waltham MA USA The calculated overall intraassay coefficient of variation was for TNFα and for IL1 the interassay coefficient of variation was calculated to be for TNFα and for IL1Synthesis and bioactivity of α41 synthetic ligandThe synthesis of RRG66 has been previously reported [] via a multistep synthesis startingfrom an enantiopure 3RZtertbutyl 3allylamino2ethylidene4methylpentanoate Thedetailed description of the synthetic protocol has been reported in S1 Fig The biological evaluation showed a strong dependence of the bioactivity on the ring stereochemistry could bedetected since S1 turned out to be completely inactive []Data and statistical analysisAll assays were carried out in triplicate for individual sample at each time pointperson and nrefers to the number of individuals Continuous variables are presented as mean ± standarddeviation when normally distributed data were tested using oneway ANOVA followed byNewmanKeuls posttest or using standard Student t test In addition data are presented asmedian and range and analyzed using MannWhitney™s test when non normally distributedCategorical data were analyzed using χ2square test Data analysis and IC50 values referring toadhesion assays in the presence of RG66 compound were fitted using sigmoidal doseresponseequation using GraphPad Prism software Statistical analyses were performed using GraphPadPrism version GraphPad Software Inc La Jolla CA USA P was consideredsignificantResultsDemographic and clinical data patients men and women age ± years presenting a chronic nonhealingwound condition were recruited and volunteered to participate in the study All the patientscompleted the study and no patient was excluded from the data analysis Demographic parameters such as age gender and clinical data are summarized in Table Age distributions demographic and clinical characteristics of patients were compatiblebetween HBOT and control groups P Overall the wounds of the patients enrolled in the study were caused by different etiologies caused by diabetes by venous insufficiency by critical limb ischemia bytrauma and by vasculitisAs regards comorbidities seven of the subjects suffered from diabetes of type1 and oftype2 twelve of them of hypertension six of obesity seven of venous insufficiency eight ofcardiomyopathy and six of hypothyroidism not requiring thyroid hormones Three subjectswere smokers and four previous smokers Common sites of wound were leg patients foot patients great toe patients and other sites patients Before beginning the HBOTall participants passed a standard medical and physical revision at the Hyperbaric Centre inRavenna All the patients both in control and HBOT groups received standard wound careie wounds were cleaned gently minimizing chemical or mechanical trauma at low pressure“ psi with saline solution and daily sterile sharp debridement with scalpel curette or scissors was performed for the necrotic tissue removal with caution to avoid excess tissue damagewhich may delay healing to get a wellbleeding granulating basePLOS ONE 101371journalpone0237746 August PLOS ONE 0cIntegrins™ role in HBOTpromoted wound healingPTable Demographic and clinical data of patients enrolled in the studyDemographic dataNumber of subjectsAge yearsaFemaleMalebSmokersComorbiditiesbDiabetesHypertensionObesityVenous insufficiencyGlaucomaCardiomyopathyHypothyroidismHBVHCV positiveRheumatoid arthritisControl groupHBOT group “type1 type2 “ extype1 type2 aAge is expressed as median rangeb Gender and comorbidities are expressed as number of individuals101371journalpone0237746t002Integrin expression on neutrophils deriving from patients undergoingHBOTIn order to study the effect of HBOT on neutrophil integrin expression these cells were isolated from blood samples deriving from patients with a chronic nonhealing wound receiving standard wound care alone control group or undergoing HBOT HBOT group Forcontrol group patients blood samples were collected during the first wound evaluation T0and after fifteen days of conventional wound therapy T15 for patients in the HBOT groupblood samples were obtained before T0 and immediately after the fourth T4 the eighthT8 the twelfth T12 and the fifteenth T15 HBOT treatment at the end of three weeks fiveexposuresweek moreover the last blood sample was drawn one month after ending HBOTT1MAs shown in Fig HBOT did not alter α4 integrin expression in primary human neutrophils both at the mRNA and protein levels Fig panels a and b nor induced any significantvariation in α4 integrin expression throughout the duration of HBO treatment No significantchange in α4 integrin expressed on neutrophils was observed in both control and HBOT grouppatientsOn the contrary 2 integrins were significantly reduced by HBOT both at mRNA andprotein levels this decrement was maintained up to the end of HBO treatment and also onemonth after the last HBOT session Fig panels c and d Neutrophils deriving from patientsreceiving conventional wound care alone control group did not show any changes in 2integrins expressionEffects of HBOT on integrinmediated adhesive properties of neutrophilsDuring an inflammatory process neutrophils adhere and transmigrate through bloodvesselwalls α41 and 2 integrins expressed on neutrophil cell membrane are required and stronglymediate rolling and firm adhesion crawling and transmigration steps of adhesion cascade [] as their activation is an essential step of this complex process To understand whetherPLOS ONE 101371journalpone0237746 August PLOS ONE 0cIntegrins™ role in HBOTpromoted wound healingFig HBOT does not modify α4 integrin levels panels a and b but reduces 2 expression panels c and d in neutrophilsderiving from patients receiving standard wound care alone control group and patients undergoing HBOT for sessionsHBOT group The decrement of 2 integrins was maintained up to one month after ending HBOT The effects of HBOT onintegrin expression were evaluated both by qPCR mRNA levels panel a c and by flow cytometry measuring integrin expressed oncell surface panel b d Results from qPCR are expressed as mean ± standard deviation of individual samples carried out intriplicate at each time point control group n HBOT group n Data from flow cytometry analysis are expressed as meanfluorescence intensity MFI ± standard deviation of individual samples carried out in triplicate at each time point control groupn HBOT group n MFI values for respective isotype control monoclonal antibody were set to ��� p versus T0both control and HBOT group101371journalpone0237746g001HBOT could influence integrinmediated neutrophil adhesion we performed cell adhesionassays to fibronectin FN or fibrinogen Fg ligands for α41 and 2 integrins respectivelyon neutrophils isolated from patients with chronic nonhealing wound receiving standardwound care alone control group or undergoing HBOT HBOT group Adhesion of neutrophils obtained from patients belonging to both control and HBOT groups was significantlyreduced by the addition of integrin specific antibodies able to block integrin functions Fig demonstrating that neutrophil adhesion to fibronectin or fibrinogen was mainly mediated byα41 or 2 integrin respectively Moreover as shown in Fig exposure to HBO induces a significant reduction of neutrophil adhesion mediated by either 2 or α41 integrins Fig panela and b respectively Interestingly this reduction of neutrophil adhesive properties is retainedthroughout the duration of HBO treatment and up to one month after the last HBOT sessionIn addition these data showed that adhesive properties of α41 integrin expressed on neutrophils were impaired during HBOT although its expression was not modified both at mRNAand protein levels as shown in Fig To better understand the involvement of α41 integrinin neutrophil adhesion we used a conformationspecific antibody that recognizes a specificepitope on integrin 1 subunit exposed only in a defined structural conformation [] Integrins exist in three major conformations a bent or inactive an intermediateactive and an highactivity conformation [] To monitor conformational changes in integrin subunits it is therefore possible to use conformationspecific antibodies [] We employed thePLOS ONE 101371journalpone0237746 August PLOS ONE 0cIntegrins™ role in HBOTpromoted wound healingFig HBOT reduces significantly integrinmediated neutrophil adhesion to fibrinogen Fg or fibronectin FN Integrinmediated adhesion wasdecreased in neutrophils obtained from patients belonging to HBOT group no changes were observed in control group patients The effects of HBOTon neutrophil adhesion mediated by 2 panel a or α41 panel b integrins were evaluated by adhesion assay to Fg or FN respectively as described inmethod section Adhesion mediated by 2 or α41 integrin is significantly prevented in neutrophils treated with a monoclonal antibody anti2 or antiα4 respectively HBOT exposure significantly reduced anti1 HUTS21 mAb binding to neutrophils namely changing α41 integrin conformationpanel c Mean fluorescence intensity MFI due to the anti1 integrin mAb PE conjugated HUTS21 binding in the presence of fibronectin μgmL was measured Nonspecific binding of an isotype control PE conjugated mAb added to neutrophils produced an MFI of ± that was subtractedfrom all samples Data are expressed as mean ± standard deviation of individual samples carried out in triplicate at each time point control groupn HBOT group n �� p � �� p ���� p versus T0 both control and HBOT group101371journalpone0237746g002PEconjugated HUTS21 mAb to determine whether the reduced adhesive properties of neutrophils mediated by α41 integrin during HBOT are due to a conformational change of 1subunit indicative of its activation status HUTS21 antibody recognizes a ligandinducedbinding site that is hidden in the inactive conformation but it is exposed when the agonistbinds or upon partial integrin activation namely when the integrin is in a high affinity conformation The epitope recognized by HUTS21 antibody is located in the hybrid domain of 1integrin subunit [] PEconjugated anti1 HUTS21 mAb was added to neutrophils in thepresence of fibronectin μgmL and fluorescence was measured by flow cytometry Exposure to HBOT induced a significant reduction of HUTS21 mAb binding to neutrophilsthroughout the duration of the treatment and up to one month after the last HBOT sessionFig panel c meaning th Answer:

Thyroid_Cancer

High burden of depression among cancerpatients on chemotherapy in University ofGondar comprehensive hospital and FelegeHiwot referral hospital Northwest EthiopiaAdhanom Gebreegziabher BarakiID1 Getahun Mengistu Tessema2 EyayawAdisu Demeke3 Department of Epidemiology and Biostatistics College of Medicine and Health Sciences Institute of PublicHealth University of Gondar Gondar Ethiopia Department of Internal Medicine College of Medicine andHealth Sciences School of Medicine University of Gondar Gondar Ethiopia Department ofPhysiotherapy Bahirdar University Bahirdar Ethiopia adsh04gmailcomAbstracta1111111111a1111111111a1111111111a1111111111a1111111111 ACCESSCitation Baraki AG Tessema GM Demeke EA High burden of depression among cancerpatients on chemotherapy in University of Gondarcomprehensive hospital and Felege Hiwot referralhospital Northwest Ethiopia e0237837 101371journalpone0237837Editor Nu¨lu¨fer Erbil Ordu University TURKEYReceived September Accepted August Published August Copyright Baraki This is an access distributed under the terms of theCreative Commons Attribution License whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are creditedData Availability Statement Data containspotentially identifying characteristics as well assensitive patient information eg HIV statusTherefore please send all data requests to theDirector of School of Medicine Dr MezgebuSilamsew at msilamsawgmailcom orPostgraduate committee Mr Getasew Amare atgetasewa23gmailcomFunding The authors received no specificfunding for this workIntroductionCancer the most stressful event a person may experience often triggers depressionDepression among these groups of people in turn affects chemotherapy adherence lengthof hospitalization quality of life and cancer treatment outcome Even though the problem isenormous studies that address it are limited Therefore this study was conducted to determine the prevalence of depression and associated factors among cancer patients on chemotherapy in FelegeHiwot referral hospital and University of Gondar referral hospitalNorthwest EthiopiaMethodsAn institutionbased crosssectional study was conducted from April to May A total of cancer patients on chemotherapy were included Depression was assessed using thepatient health questionnaire PHQ9 Binary logistic regression was used to select variablesand determine Crude Odds Ratio COR Variables with P value were entered into multivariable logistic regression Adjusted Odds Ratio AOR with confidence intervals forvariables with Pvalue was estimated to show factors affecting depression amongcancer patients The fitness of the model was checked by using the HosmerLemeshowgoodnessoffit testResultsThe prevalence of depression among cancer patients on chemotherapy was CI Educational status of college and above AOR CI Jobless AOR CI UnderweightAOR CI PLOS ONE 101371journalpone0237837 August PLOS ONE 0cHigh burden of depression among cancer patients on chemotherapy in Northwest EthiopiaCompeting interests The authors have declaredthat no competing interests existAbbreviations AOR Adjusted Odds Ratio CICrude Odds Ratio COR Crude Odds Ratio PHQPatient Health Questionnaire SD StandardDeviation UoGCSH University of GondarComprehensive Specialized Hospitalchemotherapy duration � months or more AOR CI were notablyassociated with depressionConclusionThe burden of depression among cancer patients in this study was high We recommendconcerned bodies working to curve the problem to intervene based on the identified risk factors Improving educational status reducing work stress and maintaining normal weightwould reduce depressionIntroductionThe global burden of cancer has risen to million new cases and million deaths in Worldwide the total number of people who are alive within years of a cancer diagnosis isestimated to be million []Depression is a common mental disorder characterized by persistent sadness and a loss ofinterest in activities that one normally enjoys accompanied by an inability to carry out dailyactivities for at least two weeks More than million people are now living with depressionan increase of more than between and [] The national prevalence of depression among the general population in Ethiopia was []Cancer the most stressful event that a person may experience often triggers depression [] The prevalence of depression among cancer exceeds that observed in the general population [] and it ranges from to [ “] Depression among cancer patientsaffects treatment since they have to take medications for both cancer and depression [] affectacceptance of adjuvant cancer treatment [] adherence [] extend hospitalization reducesthe quality of life [ ] and increases the risk of suicide [] Depression also predicts cancerprogression and mortality [ ]Several factors affect depression among cancer patients these include age sex marital status educational status occupation pain type of cancer phase of treatment [ ] andsocial support [ ]Even though routine screening of distress is recommended internationally for good cancercare [] less emphasis is given in the study area and most of the care focuses on cancer Studies on the magnitude and the contributing factors are also limited Therefore this study wasconducted to fill this information gap by determining the prevalence of depression among cancer patients and factors affecting itMethodsStudy design and periodAn institutionbased crosssectional study was conducted among cancer patients from Aprilto May Study areaThis study was conducted on cancer patients who are getting treatment and have followed upat the oncology unit of the University of Gondar comprehensive specialized hospitalUoGCSH and FelegeHiwot referral hospital FHRH The two hospitals are found in theAmhara region northwest Ethiopia km and km away from the capital Addis AbabaPLOS ONE 101371journalpone0237837 August PLOS ONE 0cHigh burden of depression among cancer patients on chemotherapy in Northwest Ethiopiarespectively The oncology unit of UoGCSH currently has beds for the management of cancer patients whereas the oncology unit of FHRH has currently beds for inpatient treatmentof cancer patientsParticipantsThe source populations were all adult cancer patients visiting the oncology unit and treatedwith chemotherapy in these hospitals All adults with any type of cancer patients under chemotherapy treatment and follow up during the study period were included in the studySample size and sampling procedureA final sample size of was found by using single population proportion formula with population correction for total cancer patients of in the two hospitals using the followingassumptions the prevalence of depression Za2 for confidence interval andmargin of error of The final sample size was proportionally allocated to the two hospitals for UoGCSHand 176FHRH A systematic random sampling method was employed to select every 3rdpatients who were coming to the oncology unit during the data collection period and full fillthe inclusion criteria We had a plan to randomly select and replace the nonresponders butno study participant refused to participateVariablesThe dependent variable of depression was measured using the widely used Patient HealthQuestionnaire PHQ9 The Amharic Local language version of the scale has been validatedin Ethiopia sensitivity and specificity [] We have used a cutoff point of toclassify patients as having depression or notIndependent variables like Age sex marital status average monthly income educationallevel smoking habit alcoholic habit physical activity were collected by intervieweradministered questionnaire whereas variables like Body Mass Index Type of cancer clinicalstagetype of chemotherapy Duration of chemotherapy and comorbidities like Hypertension DMHIV and Anemia were collected from patient charts The smoking and Alcohol use habitswere assessed by asking the patients if they ever smoke cigarette or drink alcohol and socialsupport was evaluated using the Oslo item social support scale with scores ranging from to poor “ moderate “ and strong “ []Data collection procedure and quality assuranceThe data was collected by interviewing the participants using a structured pretested questionnaire and chart review The data was collected by three nurses working in each oncology unitData collectors were trained for one day about the objective of the study and ethical considerations Data collectors were supervised by the principal investigator Data was reviewed andchecked for completeness accuracy and consistency after each day of data collectionData processing and analysisData were entered into Epiinfo version and STATA version was used for analysis Frequencies and percentages were computed for all variables Data were presented in tables andgraphs Binary logistic regression was used to select variables and to determine Crude OddsRatio COR Variables with P value were entered into multivariable logistic regressionAdjusted Odds Ratio AOR with confidence intervals for variables with Pvalue PLOS ONE 101371journalpone0237837 August PLOS ONE 0cHigh burden of depression among cancer patients on chemotherapy in Northwest Ethiopiawas estimated to show factors affecting depression among cancer patients The fitness of themodel was checked by using the HosmerLemeshow goodnessoffit testEthics statementEthical approval to conduct the study was received from the University of Gondar ethicalreview board School of Medicine Reference number SOM12372019 A permission letterwas received from the two hospitals To keep the privacy of participants™ name and other personal identifiers were not collected Consent to participate in the study was also orally takenfrom patients Patients with depression were also linked to the psychiatry clinicResultsSociodemographic characteristic of study participantsA total of patients participated in the study From the total respondents the majority ofthem were females were married and were housewivesRegarding to age distribution the mean and standard deviation of participant™s age was SD Table Behavioral and comorbidity characteristicsFour participants were declared that they were smoking cigarettes daily and were alcohol consumers Most of the participants were physically active Most ofthe participants were in normal weight category based on their BMI whereasTable Baseline characteristics of cancer patients in UoGCSH and FHRH FrequencyPercentageVariablesSexMarital statusMaleFemaleSingleMarriedDivorcedWidowedEducational levelOccupationNo educationPrimary educationSecondary educationCollege and aboveGovernment employeeMonthly Income ETBFarmerMerchantUnemployed ““ 101371journalpone0237837t001PLOS ONE 101371journalpone0237837 August PLOS ONE 0cHigh burden of depression among cancer patients on chemotherapy in Northwest EthiopiaTable Behavioral factors and comorbidities among study participant at UoGCSH and FHRH VariablesSmoking statusNoYesAlcohol drinkingNoYesPhysical activityNoYesBody mass index “ComorbidityNoYesDiabetesNoYesHypertensionNoYesAnemiaNoYesHIVAIDSNoYesFrequencyPercentage101371journalpone0237837t002 and were underweight and overweight respectively Ninetytwo participants had additional comorbidity Table Type of cancer and treatmentrelated characteristicsBreast cancer was the commonest cancer Whereas cervical cancer colorectal cancer35 and lung cancer are ranked second to fourth Most of thepatients were diagnosed with the disease in the past six months prior to the studyRegarding the clinical stage of the disease the third stage accounts for patients Atotal of participants have taken chemotherapy for less than three months Table Prevalence of depression among cancer patientsIn this study patients had depression making the prevalence CI The prevalence of depression among male cancer patients was CI whereas it was CI among female patients From the totalpatients with depression and had moderate moderatelysevere and severe depression respectively The magnitude of depression has also shown thedifference among different types of cancer Fig PLOS ONE 101371journalpone0237837 August PLOS ONE 0cHigh burden of depression among cancer patients on chemotherapy in Northwest EthiopiaTable Type of cancer and treatmentrelated characteristics of study participant at UoGCSH and FHRH VariablesFrequencyPercentageType of cancerBreast cancerLung cancerColorectal cancerGastric cancerCervical cancerHead and Neck cancerEsophageal cancerBlood cancerSkin cancerThyroid cancerBladder cancerLymphomaLiver cancerSarcomaTesticular cancerClinical stageStage Stage Stage Stage UnknownDuration since diagnosis months“ months monthsDuration since start of chemotherapy“ months“ months months101371journalpone0237837t003Factors associated with depression among cancer patientsIn bivariable logistic regression age sex marital status educational status occupation BMISocial support and duration of chemotherapy were found to have Pvalue 02subsequentlythese variables were subjected to multivariable analysis and educational level occupational status BMI status and duration of chemotherapy were statistically associated with depressionamong cancer patientsThe odds of depression among patients who attended college and above was significantlyreduced when compared to those with no education AOR CI Whencompared to government employees patients who are unemployed had less risk of depressionAOR CI Underweight patients had 239AOR CI times higher odds of depression as compared to those with normal body mass indexPatients who took chemotherapy for six months or more had AOR CI times higher odds of depression as compared to their counterparts Table PLOS ONE 101371journalpone0237837 August PLOS ONE 0cHigh burden of depression among cancer patients on chemotherapy in Northwest EthiopiaFig Prevalence of depression among cancer patients on chemotherapy in UoGCSH and FHRH northwest Ethiopia101371journalpone0237837g001DiscussionIn this study we have assessed the magnitude of depression among cancer patients and the factors affecting it We have found prevalence and occupation educational status bodymass index and duration of chemotherapy were found to be independent predictors ofdepressionThe magnitude of depression in this study was consistent with other studies conductedamong Chinese cancer patients [] whereas this figure was higher than a study conducted in Addis Ababa [] Iran [] and metaanalysis done by Krebber [] This discrepancy could be attributable to the difference in the study populations in terms of types ofcancer the tool used for screening or other sociodemographic variations and severity ofdepression consideredThe odds of depression was significantly reduced in patients who are unemployed whencompared to government employees This piece of evidence is supported by another multicenter study [] This could be related to workrelated stress which worsens feelings of inadequate control over one™s work frustrated hopes and expectations leading to depression []The odds of depression among patient who attended college and above was reduced whencompared to those who have no education This finding is supported by a study from China[] Atlanta [] and Greece [] The possible reason could be these patients may have a betterunderstanding of the disease and have early screening which increases their recovery A higherproportion of educated people are in the first or second clinical stage of cancer ascompared to of patients without educationUnderweight cancer patients had more than double odds of depression as compared tothose who have a normal body mass index This finding is supported by several single studies[ ] and systematic review and metaanalysis [] showing underweight people at higherrisk of depression This shows malnutrition has a significant role in the mental health of peopleand maintaining a healthy weight is essential to improve health in general and mental healthin particularEven though the chemotherapy duration has shown no significant association with depression in few pieces of literature [ ] The odds of depression among cancer patients whoPLOS ONE 101371journalpone0237837 August PLOS ONE 0cHigh burden of depression among cancer patients on chemotherapy in Northwest EthiopiaTable Factors affecting depression among cancer patientsat UoGCSH and FHRH VariablesDepressionCOR CIAOR95 CIYesNoAge mean sd SexMarital statusMale Female Single Married Divorced Widowed Educational levelNo education Primary Secondary College OccupationGovernment Farmer Merchant Unemployed Body mass indexUnderweightNormalOver weight Social supportPoorModerateStrongDuration of chemotherapy� months months� Pvalue � � � �101371journalpone0237837t004took chemotherapy for more than six months was higher than their counterparts in this studyThis could be a side effect of chemotherapy [] or it could be also associated with the staggering cost of chemotherapy which makes these patients stress to buy it for an extended durationThis study assessed the frequently ignored aspect of cancer comorbidity depression Butthe study has some limitations as it was a crosssectional study The causeeffect relationshipsare not guaranteed in these studies therefore we recommend a prospective study Even thoughwe have used a validated tool some of the symptoms used in PHQ“ like weight loss andtiredness might be related to cancer itself and may overestimate depressionConclusionThe burden of depression among cancer patients in this study was high Occupation educational status body mass index and duration of chemotherapy were found to be independentlyassociated to depression We recommend concerned bodies working to curve the problem toPLOS ONE 101371journalpone0237837 August PLOS ONE 0cHigh burden of depression among cancer patients on chemotherapy in Northwest Ethiopiaintervene based on the identified risk factors Improving educational status reducing workstress and maintaining normal weight would reduce depression Clinicians shall also provideintegrated care of mental health and cancer treatmentAcknowledgmentsWe would like to thank the University of Gondar Oncology unit staff of FelegeHiwot referralhospital and University of Gondar comprehensive specialized hospitals and all data collectorsAuthor ContributionsConceptualization Adhanom Gebreegziabher Baraki Getahun Mengistu Tessema EyayawAdisu DemekeData curation Eyayaw Adisu DemekeFormal analysis Adhanom Gebreegziabher Baraki Eyayaw Adisu DemekeInvestigation Adhanom Gebreegziabher BarakiMethodology Adhanom Gebreegziabher Baraki Getahun Mengistu TessemaProject administration Eyayaw Adisu DemekeSoftware Adhanom Gebreegziabher BarakiValidation Getahun Mengistu TessemaVisualization Getahun Mengistu TessemaWriting “ original draft Adhanom Gebreegziabher BarakiWriting “ review editing Adhanom Gebreegziabher Baraki Getahun Mengistu TessemaEyayaw Adisu DemekeReferences Bray F Global cancer statistics GLOBOCAN estimates of incidence and mortality worldwide for cancers in countries CA a cancer journal for clinicians p “ World Health anization Mental Health [cited August ] wwwwhointmental_healthmanagementdepressionen Hailemariam S The prevalence of depression and associated factors in Ethiopia findings fromthe National Health Survey International journal of mental health systems p 10118617524458623 PMID Nikbakhsh N Prevalence of depression and anxiety among cancer patients Caspian journal ofinternal medicine p “ PMID AlShakhli H Harcourt D and Kenealy J Psychological distress surrounding diagnosis of malignantand nonmalignant skin lesions at a pigmented lesion clinic Journal of Plastic Reconstructive Aesthetic Surgery p “ Sotelo JL Musselman D and Nemeroff C The biology of depression in cancer and the relationshipbetween depression and cancer progression Int Rev Psychiatry p “ PMID Krebber A Prevalence of depression in cancer patients a metaanalysis of diagnostic interviewsand selfreport instruments PsychoOncology p “ 101002pon PMID Hong JS and Tian J Prevalence of anxiety and depression and their risk factors in Chinese cancerpatients Supportive care in cancer p “ 101007s005200131997y PMID Mitchell AJ Prevalence of depression anxiety and adjustment disorder in oncological haematological and palliativecare settings a metaanalysis of interviewbased studies The lancet oncology p “ 101016S147020451170002X PMID PLOS ONE 101371journalpone0237837 August PLOS ONE 0cHigh burden of depression among cancer patients on chemotherapy in Northwest EthiopiaLinden W Anxiety and depression after cancer diagnosis prevalence rates by cancer type gender and age J Affect Disord “ p “ 101016jjad201203025PMID JimenezFonseca P Factors associated with anxiety and depression in cancer patients prior toinitiating adjuvant therapy Clin Transl Oncol p “ 101007s1209401818739 PMID Alemayehu M Deyessa N Medihin G Fekadu A A descriptive analysis of depression and pain complaints among patients with cancer in a low income country PloS one Colleoni M Depression and degree of acceptance of adjuvant cytotoxic drugs Lancet p “ 101016S014067360002821X PMID Pitman A Depression and anxiety in patients with cancer Bmj p k1415 101136bmjk1415 PMID Prieto JM Psychiatric morbidity and impact on hospital length of stay among hematologic cancerpatients receiving stemcell transplantation J Clin Oncol p “ 101200JCO200207101 PMID Tian J Chen ZC and Hang LF The effects of psychological status of the patients with digestive systemcancers on prognosis of the disease Europe PMC p “ Yousaf U Suicides among Danish cancer patients “ British journal of cancer p “ 101038sjbjc6602424 PMID Spiegel D and GieseDavis J Depression and cancer mechanisms and disease progression Biologicalpsychiatry p “ 101016s0006322303005663 PMID Ng CG Anxiety depression perceived social support and quality of life in Malaysian breast cancer patients a 1year prospective study Health and quality of life outcomes p Burgess C Depression and anxiety in women with early breast cancer five year observationalcohort study Bmj p 101136bmj38343670868D3 PMID Grassi L Screening for distress in cancer patients a multicenter nationwide study in Italy Cancer p “ 101002cncr27902 PMID Gelaye B Williams MA Lemma S Deyessa N Bahretibeb Y Shibre T Validity of the patienthealth questionnaire9 for depression screening and diagnosis in East Africa Psychiatry research Dec “ 101016jpsychres201307015 PMID Dalgard OS Dowrick C Lehtinen V VazquezBarquero JL Casey P Wilkinson G Negative lifeevents social support and gender difference in depression Social psychiatry and psychiatric epidemiology Jun “ 101007s0012700600515 PMID Iacovides A The relationship between job stress burnout and clinical depression Journal ofaffective disorders p “ 101016s0165032702001015 PMIDTorres MA Predictors of depression in breast cancer patients treated with radiation role of priorchemotherapy and nuclear factor kappa B Cancer p “ 101002cncr28003 PMID Polikandrioti M EVALUATION OF DEPRESSION IN PATIENTS UNDERGOING CHEMOTHERAPY Health Science Journal De Wit LM Depression and body mass index a ushaped association BMC public health p MartinRodriguez E Relationship between body mass index and depression in women a 7yearprospective cohort study The APNA study European Psychiatry p “ 101016jeurpsy201511003 PMID Jung SJ Association between body size weight change and depression systematic review andmetaanalysis The British Journal of Psychiatry p “ 101192bjpbp116186726 PMID Ciaramella A and Poli P Assessment of depression among cancer patients the role of pain cancertype and treatment PsychoOncology Journal of the Psychological Social and Behavioral Dimensionsof Cancer p “ Atag E Prevalence of depressive symptoms in elderly cancer patients receiving chemotherapyand influencing factors Psychogeriatrics p “ 101111psyg12329PMID Thornton LM Delayed emotional recovery after taxanebased chemotherapy Cancer Interdisciplinary International Journal of the American Cancer Society p “PLOS ONE 101371journalpone0237837 August PLOS ONE 0c'

cancer271

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: High burden of depression among cancerpatients on chemotherapy in University ofGondar comprehensive hospital and FelegeHiwot referral hospital Northwest EthiopiaAdhanom Gebreegziabher BarakiID1 Getahun Mengistu Tessema2 EyayawAdisu Demeke3 Department of Epidemiology and Biostatistics College of Medicine and Health Sciences Institute of PublicHealth University of Gondar Gondar Ethiopia Department of Internal Medicine College of Medicine andHealth Sciences School of Medicine University of Gondar Gondar Ethiopia Department ofPhysiotherapy Bahirdar University Bahirdar Ethiopia adsh04gmailcomAbstracta1111111111a1111111111a1111111111a1111111111a1111111111 ACCESSCitation Baraki AG Tessema GM Demeke EA High burden of depression among cancerpatients on chemotherapy in University of Gondarcomprehensive hospital and Felege Hiwot referralhospital Northwest Ethiopia e0237837 101371journalpone0237837Editor Nu¨lu¨fer Erbil Ordu University TURKEYReceived September Accepted August Published August Copyright Baraki This is an access distributed under the terms of theCreative Commons Attribution License whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are creditedData Availability Statement Data containspotentially identifying characteristics as well assensitive patient information eg HIV statusTherefore please send all data requests to theDirector of School of Medicine Dr MezgebuSilamsew at msilamsawgmailcom orPostgraduate committee Mr Getasew Amare atgetasewa23gmailcomFunding The authors received no specificfunding for this workIntroductionCancer the most stressful event a person may experience often triggers depressionDepression among these groups of people in turn affects chemotherapy adherence lengthof hospitalization quality of life and cancer treatment outcome Even though the problem isenormous studies that address it are limited Therefore this study was conducted to determine the prevalence of depression and associated factors among cancer patients on chemotherapy in FelegeHiwot referral hospital and University of Gondar referral hospitalNorthwest EthiopiaMethodsAn institutionbased crosssectional study was conducted from April to May A total of cancer patients on chemotherapy were included Depression was assessed using thepatient health questionnaire PHQ9 Binary logistic regression was used to select variablesand determine Crude Odds Ratio COR Variables with P value were entered into multivariable logistic regression Adjusted Odds Ratio AOR with confidence intervals forvariables with Pvalue was estimated to show factors affecting depression amongcancer patients The fitness of the model was checked by using the HosmerLemeshowgoodnessoffit testResultsThe prevalence of depression among cancer patients on chemotherapy was CI Educational status of college and above AOR CI Jobless AOR CI UnderweightAOR CI PLOS ONE 101371journalpone0237837 August PLOS ONE 0cHigh burden of depression among cancer patients on chemotherapy in Northwest EthiopiaCompeting interests The authors have declaredthat no competing interests existAbbreviations AOR Adjusted Odds Ratio CICrude Odds Ratio COR Crude Odds Ratio PHQPatient Health Questionnaire SD StandardDeviation UoGCSH University of GondarComprehensive Specialized Hospitalchemotherapy duration � months or more AOR CI were notablyassociated with depressionConclusionThe burden of depression among cancer patients in this study was high We recommendconcerned bodies working to curve the problem to intervene based on the identified risk factors Improving educational status reducing work stress and maintaining normal weightwould reduce depressionIntroductionThe global burden of cancer has risen to million new cases and million deaths in Worldwide the total number of people who are alive within years of a cancer diagnosis isestimated to be million []Depression is a common mental disorder characterized by persistent sadness and a loss ofinterest in activities that one normally enjoys accompanied by an inability to carry out dailyactivities for at least two weeks More than million people are now living with depressionan increase of more than between and [] The national prevalence of depression among the general population in Ethiopia was []Cancer the most stressful event that a person may experience often triggers depression [] The prevalence of depression among cancer exceeds that observed in the general population [] and it ranges from to [ “] Depression among cancer patientsaffects treatment since they have to take medications for both cancer and depression [] affectacceptance of adjuvant cancer treatment [] adherence [] extend hospitalization reducesthe quality of life [ ] and increases the risk of suicide [] Depression also predicts cancerprogression and mortality [ ]Several factors affect depression among cancer patients these include age sex marital status educational status occupation pain type of cancer phase of treatment [ ] andsocial support [ ]Even though routine screening of distress is recommended internationally for good cancercare [] less emphasis is given in the study area and most of the care focuses on cancer Studies on the magnitude and the contributing factors are also limited Therefore this study wasconducted to fill this information gap by determining the prevalence of depression among cancer patients and factors affecting itMethodsStudy design and periodAn institutionbased crosssectional study was conducted among cancer patients from Aprilto May Study areaThis study was conducted on cancer patients who are getting treatment and have followed upat the oncology unit of the University of Gondar comprehensive specialized hospitalUoGCSH and FelegeHiwot referral hospital FHRH The two hospitals are found in theAmhara region northwest Ethiopia km and km away from the capital Addis AbabaPLOS ONE 101371journalpone0237837 August PLOS ONE 0cHigh burden of depression among cancer patients on chemotherapy in Northwest Ethiopiarespectively The oncology unit of UoGCSH currently has beds for the management of cancer patients whereas the oncology unit of FHRH has currently beds for inpatient treatmentof cancer patientsParticipantsThe source populations were all adult cancer patients visiting the oncology unit and treatedwith chemotherapy in these hospitals All adults with any type of cancer patients under chemotherapy treatment and follow up during the study period were included in the studySample size and sampling procedureA final sample size of was found by using single population proportion formula with population correction for total cancer patients of in the two hospitals using the followingassumptions the prevalence of depression Za2 for confidence interval andmargin of error of The final sample size was proportionally allocated to the two hospitals for UoGCSHand 176FHRH A systematic random sampling method was employed to select every 3rdpatients who were coming to the oncology unit during the data collection period and full fillthe inclusion criteria We had a plan to randomly select and replace the nonresponders butno study participant refused to participateVariablesThe dependent variable of depression was measured using the widely used Patient HealthQuestionnaire PHQ9 The Amharic Local language version of the scale has been validatedin Ethiopia sensitivity and specificity [] We have used a cutoff point of toclassify patients as having depression or notIndependent variables like Age sex marital status average monthly income educationallevel smoking habit alcoholic habit physical activity were collected by intervieweradministered questionnaire whereas variables like Body Mass Index Type of cancer clinicalstagetype of chemotherapy Duration of chemotherapy and comorbidities like Hypertension DMHIV and Anemia were collected from patient charts The smoking and Alcohol use habitswere assessed by asking the patients if they ever smoke cigarette or drink alcohol and socialsupport was evaluated using the Oslo item social support scale with scores ranging from to poor “ moderate “ and strong “ []Data collection procedure and quality assuranceThe data was collected by interviewing the participants using a structured pretested questionnaire and chart review The data was collected by three nurses working in each oncology unitData collectors were trained for one day about the objective of the study and ethical considerations Data collectors were supervised by the principal investigator Data was reviewed andchecked for completeness accuracy and consistency after each day of data collectionData processing and analysisData were entered into Epiinfo version and STATA version was used for analysis Frequencies and percentages were computed for all variables Data were presented in tables andgraphs Binary logistic regression was used to select variables and to determine Crude OddsRatio COR Variables with P value were entered into multivariable logistic regressionAdjusted Odds Ratio AOR with confidence intervals for variables with Pvalue PLOS ONE 101371journalpone0237837 August PLOS ONE 0cHigh burden of depression among cancer patients on chemotherapy in Northwest Ethiopiawas estimated to show factors affecting depression among cancer patients The fitness of themodel was checked by using the HosmerLemeshow goodnessoffit testEthics statementEthical approval to conduct the study was received from the University of Gondar ethicalreview board School of Medicine Reference number SOM12372019 A permission letterwas received from the two hospitals To keep the privacy of participants™ name and other personal identifiers were not collected Consent to participate in the study was also orally takenfrom patients Patients with depression were also linked to the psychiatry clinicResultsSociodemographic characteristic of study participantsA total of patients participated in the study From the total respondents the majority ofthem were females were married and were housewivesRegarding to age distribution the mean and standard deviation of participant™s age was SD Table Behavioral and comorbidity characteristicsFour participants were declared that they were smoking cigarettes daily and were alcohol consumers Most of the participants were physically active Most ofthe participants were in normal weight category based on their BMI whereasTable Baseline characteristics of cancer patients in UoGCSH and FHRH FrequencyPercentageVariablesSexMarital statusMaleFemaleSingleMarriedDivorcedWidowedEducational levelOccupationNo educationPrimary educationSecondary educationCollege and aboveGovernment employeeMonthly Income ETBFarmerMerchantUnemployed ““ 101371journalpone0237837t001PLOS ONE 101371journalpone0237837 August PLOS ONE 0cHigh burden of depression among cancer patients on chemotherapy in Northwest EthiopiaTable Behavioral factors and comorbidities among study participant at UoGCSH and FHRH VariablesSmoking statusNoYesAlcohol drinkingNoYesPhysical activityNoYesBody mass index “ComorbidityNoYesDiabetesNoYesHypertensionNoYesAnemiaNoYesHIVAIDSNoYesFrequencyPercentage101371journalpone0237837t002 and were underweight and overweight respectively Ninetytwo participants had additional comorbidity Table Type of cancer and treatmentrelated characteristicsBreast cancer was the commonest cancer Whereas cervical cancer colorectal cancer35 and lung cancer are ranked second to fourth Most of thepatients were diagnosed with the disease in the past six months prior to the studyRegarding the clinical stage of the disease the third stage accounts for patients Atotal of participants have taken chemotherapy for less than three months Table Prevalence of depression among cancer patientsIn this study patients had depression making the prevalence CI The prevalence of depression among male cancer patients was CI whereas it was CI among female patients From the totalpatients with depression and had moderate moderatelysevere and severe depression respectively The magnitude of depression has also shown thedifference among different types of cancer Fig PLOS ONE 101371journalpone0237837 August PLOS ONE 0cHigh burden of depression among cancer patients on chemotherapy in Northwest EthiopiaTable Type of cancer and treatmentrelated characteristics of study participant at UoGCSH and FHRH VariablesFrequencyPercentageType of cancerBreast cancerLung cancerColorectal cancerGastric cancerCervical cancerHead and Neck cancerEsophageal cancerBlood cancerSkin cancerThyroid cancerBladder cancerLymphomaLiver cancerSarcomaTesticular cancerClinical stageStage Stage Stage Stage UnknownDuration since diagnosis months“ months monthsDuration since start of chemotherapy“ months“ months months101371journalpone0237837t003Factors associated with depression among cancer patientsIn bivariable logistic regression age sex marital status educational status occupation BMISocial support and duration of chemotherapy were found to have Pvalue 02subsequentlythese variables were subjected to multivariable analysis and educational level occupational status BMI status and duration of chemotherapy were statistically associated with depressionamong cancer patientsThe odds of depression among patients who attended college and above was significantlyreduced when compared to those with no education AOR CI Whencompared to government employees patients who are unemployed had less risk of depressionAOR CI Underweight patients had 239AOR CI times higher odds of depression as compared to those with normal body mass indexPatients who took chemotherapy for six months or more had AOR CI times higher odds of depression as compared to their counterparts Table PLOS ONE 101371journalpone0237837 August PLOS ONE 0cHigh burden of depression among cancer patients on chemotherapy in Northwest EthiopiaFig Prevalence of depression among cancer patients on chemotherapy in UoGCSH and FHRH northwest Ethiopia101371journalpone0237837g001DiscussionIn this study we have assessed the magnitude of depression among cancer patients and the factors affecting it We have found prevalence and occupation educational status bodymass index and duration of chemotherapy were found to be independent predictors ofdepressionThe magnitude of depression in this study was consistent with other studies conductedamong Chinese cancer patients [] whereas this figure was higher than a study conducted in Addis Ababa [] Iran [] and metaanalysis done by Krebber [] This discrepancy could be attributable to the difference in the study populations in terms of types ofcancer the tool used for screening or other sociodemographic variations and severity ofdepression consideredThe odds of depression was significantly reduced in patients who are unemployed whencompared to government employees This piece of evidence is supported by another multicenter study [] This could be related to workrelated stress which worsens feelings of inadequate control over one™s work frustrated hopes and expectations leading to depression []The odds of depression among patient who attended college and above was reduced whencompared to those who have no education This finding is supported by a study from China[] Atlanta [] and Greece [] The possible reason could be these patients may have a betterunderstanding of the disease and have early screening which increases their recovery A higherproportion of educated people are in the first or second clinical stage of cancer ascompared to of patients without educationUnderweight cancer patients had more than double odds of depression as compared tothose who have a normal body mass index This finding is supported by several single studies[ ] and systematic review and metaanalysis [] showing underweight people at higherrisk of depression This shows malnutrition has a significant role in the mental health of peopleand maintaining a healthy weight is essential to improve health in general and mental healthin particularEven though the chemotherapy duration has shown no significant association with depression in few pieces of literature [ ] The odds of depression among cancer patients whoPLOS ONE 101371journalpone0237837 August PLOS ONE 0cHigh burden of depression among cancer patients on chemotherapy in Northwest EthiopiaTable Factors affecting depression among cancer patientsat UoGCSH and FHRH VariablesDepressionCOR CIAOR95 CIYesNoAge mean sd SexMarital statusMale Female Single Married Divorced Widowed Educational levelNo education Primary Secondary College OccupationGovernment Farmer Merchant Unemployed Body mass indexUnderweightNormalOver weight Social supportPoorModerateStrongDuration of chemotherapy� months months� Pvalue � � � �101371journalpone0237837t004took chemotherapy for more than six months was higher than their counterparts in this studyThis could be a side effect of chemotherapy [] or it could be also associated with the staggering cost of chemotherapy which makes these patients stress to buy it for an extended durationThis study assessed the frequently ignored aspect of cancer comorbidity depression Butthe study has some limitations as it was a crosssectional study The causeeffect relationshipsare not guaranteed in these studies therefore we recommend a prospective study Even thoughwe have used a validated tool some of the symptoms used in PHQ“ like weight loss andtiredness might be related to cancer itself and may overestimate depressionConclusionThe burden of depression among cancer patients in this study was high Occupation educational status body mass index and duration of chemotherapy were found to be independentlyassociated to depression We recommend concerned bodies working to curve the problem toPLOS ONE 101371journalpone0237837 August PLOS ONE 0cHigh burden of depression among cancer patients on chemotherapy in Northwest Ethiopiaintervene based on the identified risk factors Improving educational status reducing workstress and maintaining normal weight would reduce depression Clinicians shall also provideintegrated care of mental health and cancer treatmentAcknowledgmentsWe would like to thank the University of Gondar Oncology unit staff of FelegeHiwot referralhospital and University of Gondar comprehensive specialized hospitals and all data collectorsAuthor ContributionsConceptualization Adhanom Gebreegziabher Baraki Getahun Mengistu Tessema EyayawAdisu DemekeData curation Eyayaw Adisu DemekeFormal analysis Adhanom Gebreegziabher Baraki Eyayaw Adisu DemekeInvestigation Adhanom Gebreegziabher BarakiMethodology Adhanom Gebreegziabher Baraki Getahun Mengistu TessemaProject administration Eyayaw Adisu DemekeSoftware Adhanom Gebreegziabher BarakiValidation Getahun Mengistu TessemaVisualization Getahun Mengistu TessemaWriting “ original draft Adhanom Gebreegziabher BarakiWriting “ review editing Adhanom Gebreegziabher Baraki Getahun Mengistu TessemaEyayaw Adisu DemekeReferences Bray F Global cancer statistics GLOBOCAN estimates of incidence and mortality worldwide for cancers in countries CA a cancer journal for clinicians p “ World Health anization Mental Health [cited August ] wwwwhointmental_healthmanagementdepressionen Hailemariam S The prevalence of depression and associated factors in Ethiopia findings fromthe National Health Survey International journal of mental health systems p 10118617524458623 PMID Nikbakhsh N Prevalence of depression and anxiety among cancer patients Caspian journal ofinternal medicine p “ PMID AlShakhli H Harcourt D and Kenealy J Psychological distress surrounding diagnosis of malignantand nonmalignant skin lesions at a pigmented lesion clinic Journal of Plastic Reconstructive Aesthetic Surgery p “ Sotelo JL Musselman D and Nemeroff C The biology of depression in cancer and the relationshipbetween depression and cancer progression Int Rev Psychiatry p “ PMID Krebber A Prevalence of depression in cancer patients a metaanalysis of diagnostic interviewsand selfreport instruments PsychoOncology p “ 101002pon PMID Hong JS and Tian J Prevalence of anxiety and depression and their risk factors in Chinese cancerpatients Supportive care in cancer p “ 101007s005200131997y PMID Mitchell AJ Prevalence of depression anxiety and adjustment disorder in oncological haematological and palliativecare settings a metaanalysis of interviewbased studies The lancet oncology p “ 101016S147020451170002X PMID PLOS ONE 101371journalpone0237837 August PLOS ONE 0cHigh burden of depression among cancer patients on chemotherapy in Northwest EthiopiaLinden W Anxiety and depression after cancer diagnosis prevalence rates by cancer type gender and age J Affect Disord “ p “ 101016jjad201203025PMID JimenezFonseca P Factors associated with anxiety and depression in cancer patients prior toinitiating adjuvant therapy Clin Transl Oncol p “ 101007s1209401818739 PMID Alemayehu M Deyessa N Medihin G Fekadu A A descriptive analysis of depression and pain complaints among patients with cancer in a low income country PloS one Colleoni M Depression and degree of acceptance of adjuvant cytotoxic drugs Lancet p “ 101016S014067360002821X PMID Pitman A Depression and anxiety in patients with cancer Bmj p k1415 101136bmjk1415 PMID Prieto JM Psychiatric morbidity and impact on hospital length of stay among hematologic cancerpatients receiving stemcell transplantation J Clin Oncol p “ 101200JCO200207101 PMID Tian J Chen ZC and Hang LF The effects of psychological status of the patients with digestive systemcancers on prognosis of the disease Europe PMC p “ Yousaf U Suicides among Danish cancer patients “ British journal of cancer p “ 101038sjbjc6602424 PMID Spiegel D and GieseDavis J Depression and cancer mechanisms and disease progression Biologicalpsychiatry p “ 101016s0006322303005663 PMID Ng CG Anxiety depression perceived social support and quality of life in Malaysian breast cancer patients a 1year prospective study Health and quality of life outcomes p Burgess C Depression and anxiety in women with early breast cancer five year observationalcohort study Bmj p 101136bmj38343670868D3 PMID Grassi L Screening for distress in cancer patients a multicenter nationwide study in Italy Cancer p “ 101002cncr27902 PMID Gelaye B Williams MA Lemma S Deyessa N Bahretibeb Y Shibre T Validity of the patienthealth questionnaire9 for depression screening and diagnosis in East Africa Psychiatry research Dec “ 101016jpsychres201307015 PMID Dalgard OS Dowrick C Lehtinen V VazquezBarquero JL Casey P Wilkinson G Negative lifeevents social support and gender difference in depression Social psychiatry and psychiatric epidemiology Jun “ 101007s0012700600515 PMID Iacovides A The relationship between job stress burnout and clinical depression Journal ofaffective disorders p “ 101016s0165032702001015 PMIDTorres MA Predictors of depression in breast cancer patients treated with radiation role of priorchemotherapy and nuclear factor kappa B Cancer p “ 101002cncr28003 PMID Polikandrioti M EVALUATION OF DEPRESSION IN PATIENTS UNDERGOING CHEMOTHERAPY Health Science Journal De Wit LM Depression and body mass index a ushaped association BMC public health p MartinRodriguez E Relationship between body mass index and depression in women a 7yearprospective cohort study The APNA study European Psychiatry p “ 101016jeurpsy201511003 PMID Jung SJ Association between body size weight change and depression systematic review andmetaanalysis The British Journal of Psychiatry p “ 101192bjpbp116186726 PMID Ciaramella A and Poli P Assessment of depression among cancer patients the role of pain cancertype and treatment PsychoOncology Journal of the Psychological Social and Behavioral Dimensionsof Cancer p “ Atag E Prevalence of depressive symptoms in elderly cancer patients receiving chemotherapyand influencing factors Psychogeriatrics p “ 101111psyg12329PMID Thornton LM Delayed emotional recovery after taxanebased chemotherapy Cancer Interdisciplinary International Journal of the American Cancer Society p “PLOS ONE 101371journalpone0237837 August PLOS ONE 0c' Answer:

Thyroid_Cancer

Electronic health records EHRs contain rich documentation regarding disease symptomsand progression but EHR data is challenging to use for diagnosis prediction due to its highdimensionality relative scarcity and substantial level of noise We investigated how to bestrepresent EHR data for predicting cervical cancer a serious disease where early detectionis beneficial for the outcome of treatment A case group of patients with cervical cancerwere matched to ten times as many controls and for both groups several types of eventswere extracted from their EHRs These events included clinical codes lab results and contents of free text notes retrieved using a LSTM neural network Clinical events are describedwith great variation in EHR texts leading to a very large feature space Therefore an eventhierarchy inferred from the textual events was created to represent the clinical texts Overallthe events extracted from free text notes contributed the most to the final prediction and thehierarchy of textual events further improved performance Four classifiers were evaluatedfor predicting a future cancer diagnosis where Random Forest achieved the best resultswith an AUC of from a year before diagnosis up to one day before diagnosis Weconclude that our approach is sound and had excellent discrimination at diagnosis but onlymodest discrimination capacity before this point Since our study objective was earlier disease prediction than such we propose further work should consider extending patient histories through eg the integration of primary health records preceding referral to hospitalIntroductionInformation on disease progression documented in electronic health records EHRs is apotential source of valuable new knowledge which could lead to improved health care [“]Since EHR information is derived directly from health care there is a great interest on how tobest use this source for reallife applications by way of advanced medical informatics Applications that can benefit from EHR mining include clinical decision support adverse eventa1111111111a1111111111a1111111111a1111111111a1111111111 ACCESSCitation Weegar R Sundstro¨m K Usingmachine learning for predicting cervical cancerfrom Swedish electronic health records by mininghierarchical representations e0237911 101371journalpone0237911Editor Sreeram V Ramagopalan University ofOxford UNITED KINGDOMReceived January Accepted August Published August Copyright Weegar Sundstro¨m This is an access distributed under the terms ofthe Creative Commons Attribution License whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are creditedData Availability Statement The data set consistsof second party data and is part of Health Bank Swedish Health Record Research Bank which ismanaged by the Department of Computer andSystems Sciences at Stockholm University dsvsusehealthbank The ethical permissionsassociated with this data set does not allow for access to the data For interestedresearchers inquiries can be made to the directorof Health Bank herculesdsvsuse or to thecorresponding author to access the data in thesame way as the authors ie on site at DepartmentPLOS ONE 101371journalpone0237911 August PLOS ONE 0cof Computer and Systems Sciences at StockholmUniversityFunding Both authors RW and KS were fundedby the Nordic Information for Action eScienceCenter of Excellence in HealthRelated eSciencesNIASC project number wwwnordicehealthse The funders had no role in studydesign data collection and analysis decision topublish or preparation of the manuscriptCompeting interests The authors have declaredthat no competing interests existUsing machine learning for predicting cervical cancer from Swedish electronic health recordsdetection and risk prediction [] One important potential of mining of EHRs is to generatenew clinical hypotheses and since EHRs document large populations observed over time theyallow for investigations regarding the relationship between clinical events and outcomes []Compared to cohort studies where specific information about individuals is typically collected at predefined time intervals the use of EHR data has several distinct strengths but alsolimitations Some of the advantages of using EHR data are that data are collected continuouslythat a richer set of information types is included and that less resources may be required toacquire the data However since EHRs are not primarily used to collect data for research purposes EHR data are typically sparse and can contain a high level of noise making this type ofdata challenging to analyze []In this work the focus is prediction of a major human cancer ie cervical cancer usingEHRs as input Globally cervical cancer is one of the dominating cancer forms in womenwith half a million cases each year In Sweden due to prevention through anized cervicalscreening the disease is rarer but there are still about cases of cervical cancer per year andthe incidence has risen in the past two years The median age of women getting the diagnosisis years and thus it is a disease which strikes relatively early in life [] Improved predictionof risk could lead to interventions at an earlier stage where the illness may still exist as a precancerous lesions amenable to surgical removal For cancer diseases earlier diagnosis couldalso lead to improvements in the outcomes of treatment and quality of life as well as for survival [] and for cervical cancer relative survival is higher when the cancer is detected at anearly stage [] The latter improvement is termed downstaging and would be of value especially in a disease such as cervical cancer where higher stages are associated with very highmortality Machine learning models created from EHRs could if sufficiently accurate potentially lead to an earlier diagnosis and increased knowledge regarding the events preceding adiagnosis In this work the aim is therefore to apply machine learning to EHRs and to explorehow well classifiers can identify future cervical cancer cases To this end both the informativeness of different event types found in EHRs diagnosis codes drug codes free text proceduresand lab results was evaluated together with the issue of how to best represent such events fordiagnosis predictionMachine learning methods have been applied to EHRs for predicting a number of differentoutcomes both for specific diseases and also for the risk of mortality and hospitalization andthe number of studies using EHR data for creating risk prediction models is increasing []Zhao and Weng [] used variables known to be related to pancreatic cancer andweighted them using PubMed s The variables included symptoms labresults andcomorbidities and were extracted from EHRs for cases and controls Each selected variable was assigned a weight according to if the association to pancreatic cancer mined from thes was positive or negative and a Bayesian Network Inference model using the assignedweights gave a better predictive performance compared to not using the weights derived fromPubMedIn their study on using machine learning to develop risk prediction models from healthrecord data Mani [] aimed to identify patients at risk of type diabetes They extracted variables representing demographic information clinical findings and laboratory valuesfor over patients where of were patients with a diabetes type diagnosis and theremaining were controls Three data sets representing different time intervals were createdIn the first one all data up to the diagnosis date were included while in the second and thirdones data were included from over days and over days before the diagnosis A numberof classifiers were evaluated for predicting which patients would develop type diabetes andan AUC of was achieved for each of the three time intervalsPLOS ONE 101371journalpone0237911 August PLOS ONE 0cUsing machine learning for predicting cervical cancer from Swedish electronic health recordsHuang [] aimed to predict future cases of depression the severity of the depressionand the response to treatment In brief patients with a future depression diagnosis andat least years of data before the diagnosis were matched to six times as many controlsDiagnosis codes medication codes demographics and free text from EHRs belonging to thecases and controls were used as input and their model could predict depression diagnoses months in advance with an AUC area under the curve of and when including all available data up to the diagnosis data the corresponding result was an AUC of Kop [] used structured data from EHRs to predict future cases of colorectal cancerCRC Six months of data for over patients whereof CRC cases were extractedfrom primary care health records The data included ICPC codes International Classificationof Primary Care ATC codes Anatomical Therapeutic Chemical Classification System andlaboratory values Temporal and cooccurrence patterns were mined from the data set andused as input for three different classifiers CART decision trees logistic regression and Random Forest An AUC of was achieved and the input features were ranked according tothe importance factor provided by the logistic regression model Most of the features with highranks corresponded to events known to be linked to colorectal cancer providing validation oftheir modelFurther promising examples of studies have used neural network models such denoisingautoencoders in combination with Random Forest convolutional networks and recurrentnetworks for risk prediction of subsequent clinically relevant diseases through the use of EPRdata [“]Materials and methodsEthics statementThe use of health record data in this project was approved by the Regional Ethical ReviewBoard in Stockholm Sweden which determined that informed consent from the study participants was not required The data was anonymized before being accessed by the researchersEthical permission number Feature extractionWhen health records are mined a first necessary step is feature extraction during which dataof interest are selected and extracted from the records There are two possible ways of selectingwhich features to include either a topdown approach is used where domain or expert knowledge guides the feature selection or a bottomup approach is applied with an datadriven feature selection [] A benefit of using the bottom up approach is that it allows fordetecting new previously unknown links between events and outcomes since no a prioriassumptions are made regarding which features are relevant to include in the model []EHRs contain several information types requiring different levels of preprocessing beforethey can be included in a machine learning model Often a distinction is made between structured and unstructured information where diagnosis codes drug codes and demographicinformation such as age or gender is considered as structured and therefore easier to representfor machine learning purposes Free text on the other hand is regarded as unstructured information as it is not possible to directly determine the value or meaning of an EHR free textnote This free text makes up a substantial part of the documentation in EHRs and since itdescribes a patient™s health status symptoms and treatments it is potentially valuable toinclude free text in risk prediction models Free text notes require a higher level of preprocessing and therefore Natural Language Processing methods may be applied to structure the freetext and extract relevant information from it []PLOS ONE 101371journalpone0237911 August PLOS ONE 0cUsing machine learning for predicting cervical cancer from Swedish electronic health recordsThe level or degree of structure can vary also for the structured data for example even ifthe type of a lab test is coded the result of the test might be a in free text with different unitsused for the same test Such information can be regarded as semistructured and thereforeadditional preprocessing is required also for these types of eventsDataThe health records collected for this study comes from Karolinska University Hospital inStockholm from the years [] These records contain coded information such asdiagnosis codes drug codes and procedural codes semistructured information such as labtest results and free text From this data set the patients with an ICD10 diagnosis coderepresenting cervical cancer ie a code starting with C53 were selected as the case groupNext a control group was created where each patient in the C53 case group was matched onage to ten other control women who did not have any C53 diagnosis on record in the dataSince one aim of this work was to investigate the events leading up to a first diagnosis of cervical cancer only cases with EHR data before the diagnosis date of cancer were included andall patients with the code Z854C previous cervical cancer were also filtered out leaving cases and controls Five basic event types were extracted from the ERHs for both casesand controls clinical entities found in the free text notes diagnosis codes drug codes labresults and procedure codes The extracted data were then divided into intervals for the firstof which all data up to the day before diagnosis were included For the next interval only dataregistered at least days before diagnosis were included and so on in intervals of days upto a year before diagnosis The available data was divided into two parts a development setcontaining of the data and a test set with the remaining The development set was setaside for hyperparameter tuning of the classifiers and for feature selection The test set wasused to evaluate the selected classifiers through a process of 10fold crossvalidation wherebyten rounds of training and testing was performed with of the data used for training and for testing for each round The development set was not included in the final evaluationof the classifiersICD codesThe health records contain diagnosis codes from the Swedish version of the ICD10 codesInternational Statistical Classification of Diseases and Related Health Problems”Tenth Revision ICD10 codes are hierarchically arranged with chapters at the highest level thesechapters are further divided into sections subsections and full codes This makes it possible toinclude both very finegrained information in form of the full diagnosis codes as well as higherlevel information such as chapters and subsections Table gives an example of the ICD10Table Example from the ICD hierarchyLevelChapterSectionExampleC00D48 NeoplasmsC51C58 Malignant neoplasms of female genital ansSubsectionC53 Malignant neoplasm of cervix uteriCodeC530 Malignant neoplasm endocervixSizeIn dataThe top of the hierarchy consists of different chapters where chapter II comprises codes for neoplasms Furtherdown in the hierarchy more detailed information is included in the codes The column titled Size gives the numberof different codes for each level in the complete code hierarchy and the last column In data shows the number ofdifferent codes included in the current data sets101371journalpone0237911t001PLOS ONE 101371journalpone0237911 August PLOS ONE 0cUsing machine learning for predicting cervical cancer from Swedish electronic health recordsFig ICD chapters and sections for the case group and the control group The left part corresponds to the case group and the right part the control group The innercircle represents the ICD chapters and the outer circle the ICD sections included in the chapter the labels show the sections with the most frequent codes101371journalpone0237911g001hierarchy for the code C530 Fig provides a visualization of the distribution of ICD10 codesfor the control group and the C53 case group Comparing cases and controls it can be notedthat many of the patients in the case groups have experienced other types of cancers ofcases and of controls had a diagnosis code from chapter II of ICD10 which is the chaptercontaining codes for tumorsneoplasms Additionally a large part of these codes came fromthe section representing codes for malignant neoplasms of the female genital ans C51C58other than cervical cancerATC codesThe next feature type are the ATC codes from the Anatomical Therapeutic Chemical Classification System representing drugs As with ICDcodes these codes are hierarchical and therefore it was possible to represent them with different levels of detail Table shows an exampleof the included ATC levelsClinical entities extracted from textMost of the data extracted from the EHRs were in the form of free text notes and differentapproaches have previously been used to create representations of the free text in EHRs OneTable Example from the ATC code hierarchyLevelMain group3rd level subgroup4th level subgroupFull codeExampleN Nervous systemN02B Other analgesics and antipyreticsN02BE AnilidesN02BE01 ParacetamolIn dataThe last column of the table shows how many different codes appear in the current data set for each code level101371journalpone0237911t002PLOS ONE 101371journalpone0237911 August PLOS ONE 0cUsing machine learning for predicting cervical cancer from Swedish electronic health recordsFig Example of patient record text This example contains one multiword Disorder œsquamous cell carcinoma insitu101371journalpone0237911g002possibility is to map the text to some existing ontology or terminology Roque []matched free text to ICD codes to enrich coded information extracted from EHRs This hasthe advantage of getting a coded representation practical for interpretation and machinelearning purposes however mapping directly from clinical text to standard terminologies canlead to low recall [] as the language used in clinical text differs from the standardised language in terminologies Tools for matching text to terminologies such as MetaMap [] arenot available for Swedish Another approach which also reduces the size of the feature spacewas applied by Miotto [] They included free text in their model by extracting entitiesfrom clinical notes using the Biomedical Annotator and topic modeling this approachwas effective for predicting diagnoses but has the drawback of reduced interpretabilityHere named entity recognition NER was applied to the free text notes belonging to casesand controls Using NER allowed for extracting the most relevant parts of the text and compared to including the full texts in a bagofwords model named entities makes it possible torepresent multiword expressions such as diabetes typ diabetes type and cancer in situ Toextract these entities from the free text a bidirectional long shortterm memory biLSTMnetwork was trained on notes annotated by two medical experts The notes used as trainingdata for the network were annotated for the entity types Body part annotations Disorder annotations and Finding annotations For this work findings and disorderswere included as events and following the SNOMED definitions a œFinding represent bothnormal and abnormal observations regarding a patient while a œDisorder always is the resultof an underlying pathological process see Fig A large corpus of clinical texts was used to generate word2vec [] embeddings of the textsas input representation for the network This corpus contained GB of text from SwedishEHRs with a complete vocabulary of about words The properties of the LSTM network are described in detail in Weegar [] This network was applied to all clinical notesfor the cases and the controls to extract all mentions of findings and disorders from these textswhich contained on average tokens for each patient Next negation detection was used toidentify negated entities using a rulebased module NegEx adapted specifically to the domainof Swedish clinical text Swedish [] This was an important step because clinical notes oftencontain documentation of discussions and reasoning around a potential condition in text andit is thus particularly common that findings are negated Indeed in this material up to ten percent of findings and six percent of the extracted disorders were actually in the negated formSimilarly to mitigate the risk of including information regarding individuals other than theactual patient to whom the record belonged deriving from eg discussions on family historyof disease clinical notes with headings related to family members were excluded The use ofNER for event extraction resulted in events for the controls on average For the cases onlyevents occurring before the C53 diagnosis were included on average eventsThe extracted events were lemmatized which reduced the scarcity of the free text data aslemmatization maps inflected versions of a word to the same basic representation By usinglemmatization words such as hudirritationskin irritation singular and hudirritationerskinirritations plural will be considered as the same event Similarly the event hostadecoughedPLOS ONE 101371journalpone0237911 August PLOS ONE 0cUsing machine learning for predicting cervical cancer from Swedish electronic health recordspast tense will be joined with hostarcoughs present tense But even after lemmatizationthe number of different events extracted from the texts is very large There were about different events appearing at least two times This is partially due to the possibility of describing the same event in many different ways in writing for example œfractured patella andœpatella fracture refers to the same type of event but will be considered as two different eventsas their surface forms are different Therefore to further reduce the feature space two additional preprocessing steps were applied to the textual events Firstly a spelling normalizationmodule was used to group different spelling variations of the same concept This grouping wasachieved through calculating the edit distance between each pair of events A Levenshtein editdistance of two strings is a measurement of how different the two strings are and is calculatedby counting the number of insertions deletions and substitutions of characters that is requiredto make two strings equal [] Groups of events were formed where each member in thegroup had at most an edit distance of one from some other member of the group and the samefirst letter as all other members in the group as it was found that allowing larger distancesintroduced errors For multiword expressions the distance was calculated per word and itwas also required that each included word had the same first letter The spelling differences inthe text data were mainly the result of misspellings but also of inflections or instances of writing the same concept as a either a compound word or a as two separate words After groupingthe events using edit distance all variations were exchanged with the most frequent surfaceform One example of such a group is the spelling variations for thyroid cancerthyreoideacancer tyreoideacancer thyroidecancer thyreoidacancer thyreoidcancer tyroideacancer tyreoidecancer thyroidea cancer thyroideacancer thyreoidecancerwhere each variation was mapped to the most frequently occurring form œthyreoideacancerThis normalization mapped about different surface forms of events into about different groupsNext a hierarchical representation of the events extracted from the free texts was createdThe hierarchy was constructed by first sorting the events extracted from the texts according totheir length and placing oneword entities at the top of the hierarchy Next for each level anyevent that contained all words of an event on the higher level was added as a child node of thatevent In this hierarchy the oneword event œleukaemia at the top level had the child nodesœacute leukaemia and œchronic leukaemia where œchronic leukaemia in turn had the childnodes œchronic myeloid leukaemia and œchronic lymphocytic leukaemia Events furtherdown in the hierarchy became gradually more detailed as they often contained some type ofmodifier to its parent node such as œnormal œserious or œmalignant capturing many relevant relationships between eventsA hierarchy of four levels was created with oneword entities at level one and events containing four or more words at the fourth level It is worth noting that when representing thetextual events for a single patient the longer lowlevel events had influence over higher levelrepresentations in the same way as for the ICD or ATC code hierarchies Using the ICD codehierarchy a diagnosis can be represented by the corresponding full lowlevel code or by thesection or chapter it belongs to In a similar way the lower level event œchronic myeloid leukaemia can be represented by the higher level event œleukaemiaThis hierarchy of textual events was however different from the ICD and ATC code hierarchies in two ways Firstly each child node could have more than one parent node an œinfectedwound had the parent œwound and the parent œinfected The second difference was thatwhile the top levels are the smallest in the code hierarchies the opposite is true for the textualevents There was a higher number of short textual events and the short events also make up alarger part of the total set of textual events The top level of the hierarchy contained PLOS ONE 101371journalpone0237911 August PLOS ONE 0cUsing machine learning for predicting cervical cancer from Swedish electronic health recordsdifferent events followed by events at level two events at level three and events at level fourLab resultsThe included data for lab results consists of the type of test and the result of the test Includingthe value of test result can be too finegrained for the task of prediction since many tests arerare [] Therefore the result is represented as being either inside or outside the referencerange for a test value For example the result of a Hemoglobin test can either be in the normalrange for Hemoglobin above the range or below it Therefore each lab result in the includedhealth records will be represented by one of three different features depending on the outcomeof the testProcedure codesProcedure codes classify procedures including surgical procedures medical investigationspreventive measures and treatments for example the code AK044 corresponding to an ultrasound of the kidneys The codes are used in health records for statistical and administrativepurposes and have been included as an event type in this workA number of the events that were extracted from the EHRs were unique meaning that theyonly occurred for a single patient Since such events cannot contribute to the diagnosis prediction but only increase the scarcity of the data they were removed at this stage leaving in total different events in the data set Each case had on average different events and events in total before the diagnosis For the controls the corresponding numbers were on average different events and an average total of The reason for the larger number of eventsfor the control group is that only data up to a cervical cancer diagnosis is included for the casegroup and all events after the diagnosis were discarded For the controls on the other hand alldocumented events were kept even if they occurred after the time of their matched case™s diagnosis Since the objective of our study was correct classification based on EHR events withpotentially low expected contrast between cases and controls we opted for this choice to maximize the control information for the classifiers to be trained on minimize the risk ofmisclassification of disease status by ensuring to the best of our knowledge that no hospitalbased female controls were diagnosed with cervical cancer later during the study period and to increase generalizability to a reallife clinical situation where EHRs are available but diseasestatus of the individuals is not already known Fig gives an overview of the available data forthe different time intervals However some events will appear outside of the study periodleading to data censoring [ ] Important and informative events could have occurred beforethe start of data collection and this also means as in most casecontrol or other studies thatwe do not know whether some members of the control group develop cervical cancer after thestudy end point when no more EHR data was available to us However this is a wellknownfact resulting from this type of study design and does not invalidate the comparison made during the actual study period definedClassification experimentsFour different classifiers were used to evaluate the appropriateness of the different featuretypes for classification of future C53 cases These classifiers were Random Forest ComplementNaive Bayes Bernoulli Naive Bayes and Support Vector Machines all implemented in Scikitlearn [] The first classifier Random Forest is an ensemble classifier robust to noise andlarge feature spaces [] Complement Naive Bayes [] is capable in cases of data sets withclass imbalance Bernoulli Naive Bayes additionally takes absence of features into account forPLOS ONE 101371journalpone0237911 August PLOS ONE 0cUsing machine learning for predicting cervical cancer from Swedish electronic health recordsFig Available data for the case group The xaxis denotes the number of days before a diagnosis and events only appearing onetime in the data set have been excluded At days before a diagnosis events were available for patients101371journalpone0237911g003classification [] and Support Vector Machines SVM is a classifier well suited for highdimensional spaces []This classification task can be understood as a text classification problem where each eventextracted from a patient record corresponds to a word and each patient is represented by a vector with the same dimension as the complete vocabulary of events For Random Forest andComplement Naive Bayes the input vectors consisted of the raw counts of events For Bernoulli Naive Bayes binary input vectors were used and for the SVM classifier normalized vector counts were used as input Binary vectors correspond to if a patient ever experienced anevent and count vectors also represent how many times each event occurred Another possibility is to use tfidf term frequencyinverse document frequency weights The idea behindtfidf is to give more weight to the events that are representative for individual patients However using tfidf did not improve classification resultsResultsEvent typesEach type of event was evaluated individually for its ability to correctly classify the patients Fig shows the average AUC using the four classifiers over time for each event type ICD codesATC codes Procedure codes Clinical entities and Lab results The average AUC was calculated as the sum of the scores AUCi for the individual classifiers divided by the number of classifiers AUCavg ¼ the classifiers were the clinical entities extracted from the text as the highest AUC scores wereachieved using only the text entities ðAUC1 þ AUC2 þ AUC3 þ AUC4Þ The most informative event type forPLOS ONE 101371journalpone0237911 August PLOS ONE 0cUsing machine learning for predicting cervical cancer from Swedish electronic health recordsFig Average AUC for the different event types over time101371journalpone0237911g004Event levelsSince the data in EHRs typically are noisy high dimensional and sparse it is necessary toinvestigate data representation how the information included in the records should be presented to the model [] In this work both which types of feature to include and also the detailwith which those features should be represented was evaluatedIt has previously been found that including features derived from several levels of hierarchical clinical codes improves the performance for predicting adverse drug events from EHR data[] and as the input events can be represented with different levels of detail the next set ofexperiments aimed to evaluate the most suitable representation of the events in this regardUsing the most detailed levels such as a full ICD10 code gives very detailed information buta very sparse data set By including the higherlevel representations the sparsity of the data canbe reduced Fig shows the average AUC o

cancer272

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: Electronic health records EHRs contain rich documentation regarding disease symptomsand progression but EHR data is challenging to use for diagnosis prediction due to its highdimensionality relative scarcity and substantial level of noise We investigated how to bestrepresent EHR data for predicting cervical cancer a serious disease where early detectionis beneficial for the outcome of treatment A case group of patients with cervical cancerwere matched to ten times as many controls and for both groups several types of eventswere extracted from their EHRs These events included clinical codes lab results and contents of free text notes retrieved using a LSTM neural network Clinical events are describedwith great variation in EHR texts leading to a very large feature space Therefore an eventhierarchy inferred from the textual events was created to represent the clinical texts Overallthe events extracted from free text notes contributed the most to the final prediction and thehierarchy of textual events further improved performance Four classifiers were evaluatedfor predicting a future cancer diagnosis where Random Forest achieved the best resultswith an AUC of from a year before diagnosis up to one day before diagnosis Weconclude that our approach is sound and had excellent discrimination at diagnosis but onlymodest discrimination capacity before this point Since our study objective was earlier disease prediction than such we propose further work should consider extending patient histories through eg the integration of primary health records preceding referral to hospitalIntroductionInformation on disease progression documented in electronic health records EHRs is apotential source of valuable new knowledge which could lead to improved health care [“]Since EHR information is derived directly from health care there is a great interest on how tobest use this source for reallife applications by way of advanced medical informatics Applications that can benefit from EHR mining include clinical decision support adverse eventa1111111111a1111111111a1111111111a1111111111a1111111111 ACCESSCitation Weegar R Sundstro¨m K Usingmachine learning for predicting cervical cancerfrom Swedish electronic health records by mininghierarchical representations e0237911 101371journalpone0237911Editor Sreeram V Ramagopalan University ofOxford UNITED KINGDOMReceived January Accepted August Published August Copyright Weegar Sundstro¨m This is an access distributed under the terms ofthe Creative Commons Attribution License whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are creditedData Availability Statement The data set consistsof second party data and is part of Health Bank Swedish Health Record Research Bank which ismanaged by the Department of Computer andSystems Sciences at Stockholm University dsvsusehealthbank The ethical permissionsassociated with this data set does not allow for access to the data For interestedresearchers inquiries can be made to the directorof Health Bank herculesdsvsuse or to thecorresponding author to access the data in thesame way as the authors ie on site at DepartmentPLOS ONE 101371journalpone0237911 August PLOS ONE 0cof Computer and Systems Sciences at StockholmUniversityFunding Both authors RW and KS were fundedby the Nordic Information for Action eScienceCenter of Excellence in HealthRelated eSciencesNIASC project number wwwnordicehealthse The funders had no role in studydesign data collection and analysis decision topublish or preparation of the manuscriptCompeting interests The authors have declaredthat no competing interests existUsing machine learning for predicting cervical cancer from Swedish electronic health recordsdetection and risk prediction [] One important potential of mining of EHRs is to generatenew clinical hypotheses and since EHRs document large populations observed over time theyallow for investigations regarding the relationship between clinical events and outcomes []Compared to cohort studies where specific information about individuals is typically collected at predefined time intervals the use of EHR data has several distinct strengths but alsolimitations Some of the advantages of using EHR data are that data are collected continuouslythat a richer set of information types is included and that less resources may be required toacquire the data However since EHRs are not primarily used to collect data for research purposes EHR data are typically sparse and can contain a high level of noise making this type ofdata challenging to analyze []In this work the focus is prediction of a major human cancer ie cervical cancer usingEHRs as input Globally cervical cancer is one of the dominating cancer forms in womenwith half a million cases each year In Sweden due to prevention through anized cervicalscreening the disease is rarer but there are still about cases of cervical cancer per year andthe incidence has risen in the past two years The median age of women getting the diagnosisis years and thus it is a disease which strikes relatively early in life [] Improved predictionof risk could lead to interventions at an earlier stage where the illness may still exist as a precancerous lesions amenable to surgical removal For cancer diseases earlier diagnosis couldalso lead to improvements in the outcomes of treatment and quality of life as well as for survival [] and for cervical cancer relative survival is higher when the cancer is detected at anearly stage [] The latter improvement is termed downstaging and would be of value especially in a disease such as cervical cancer where higher stages are associated with very highmortality Machine learning models created from EHRs could if sufficiently accurate potentially lead to an earlier diagnosis and increased knowledge regarding the events preceding adiagnosis In this work the aim is therefore to apply machine learning to EHRs and to explorehow well classifiers can identify future cervical cancer cases To this end both the informativeness of different event types found in EHRs diagnosis codes drug codes free text proceduresand lab results was evaluated together with the issue of how to best represent such events fordiagnosis predictionMachine learning methods have been applied to EHRs for predicting a number of differentoutcomes both for specific diseases and also for the risk of mortality and hospitalization andthe number of studies using EHR data for creating risk prediction models is increasing []Zhao and Weng [] used variables known to be related to pancreatic cancer andweighted them using PubMed s The variables included symptoms labresults andcomorbidities and were extracted from EHRs for cases and controls Each selected variable was assigned a weight according to if the association to pancreatic cancer mined from thes was positive or negative and a Bayesian Network Inference model using the assignedweights gave a better predictive performance compared to not using the weights derived fromPubMedIn their study on using machine learning to develop risk prediction models from healthrecord data Mani [] aimed to identify patients at risk of type diabetes They extracted variables representing demographic information clinical findings and laboratory valuesfor over patients where of were patients with a diabetes type diagnosis and theremaining were controls Three data sets representing different time intervals were createdIn the first one all data up to the diagnosis date were included while in the second and thirdones data were included from over days and over days before the diagnosis A numberof classifiers were evaluated for predicting which patients would develop type diabetes andan AUC of was achieved for each of the three time intervalsPLOS ONE 101371journalpone0237911 August PLOS ONE 0cUsing machine learning for predicting cervical cancer from Swedish electronic health recordsHuang [] aimed to predict future cases of depression the severity of the depressionand the response to treatment In brief patients with a future depression diagnosis andat least years of data before the diagnosis were matched to six times as many controlsDiagnosis codes medication codes demographics and free text from EHRs belonging to thecases and controls were used as input and their model could predict depression diagnoses months in advance with an AUC area under the curve of and when including all available data up to the diagnosis data the corresponding result was an AUC of Kop [] used structured data from EHRs to predict future cases of colorectal cancerCRC Six months of data for over patients whereof CRC cases were extractedfrom primary care health records The data included ICPC codes International Classificationof Primary Care ATC codes Anatomical Therapeutic Chemical Classification System andlaboratory values Temporal and cooccurrence patterns were mined from the data set andused as input for three different classifiers CART decision trees logistic regression and Random Forest An AUC of was achieved and the input features were ranked according tothe importance factor provided by the logistic regression model Most of the features with highranks corresponded to events known to be linked to colorectal cancer providing validation oftheir modelFurther promising examples of studies have used neural network models such denoisingautoencoders in combination with Random Forest convolutional networks and recurrentnetworks for risk prediction of subsequent clinically relevant diseases through the use of EPRdata [“]Materials and methodsEthics statementThe use of health record data in this project was approved by the Regional Ethical ReviewBoard in Stockholm Sweden which determined that informed consent from the study participants was not required The data was anonymized before being accessed by the researchersEthical permission number Feature extractionWhen health records are mined a first necessary step is feature extraction during which dataof interest are selected and extracted from the records There are two possible ways of selectingwhich features to include either a topdown approach is used where domain or expert knowledge guides the feature selection or a bottomup approach is applied with an datadriven feature selection [] A benefit of using the bottom up approach is that it allows fordetecting new previously unknown links between events and outcomes since no a prioriassumptions are made regarding which features are relevant to include in the model []EHRs contain several information types requiring different levels of preprocessing beforethey can be included in a machine learning model Often a distinction is made between structured and unstructured information where diagnosis codes drug codes and demographicinformation such as age or gender is considered as structured and therefore easier to representfor machine learning purposes Free text on the other hand is regarded as unstructured information as it is not possible to directly determine the value or meaning of an EHR free textnote This free text makes up a substantial part of the documentation in EHRs and since itdescribes a patient™s health status symptoms and treatments it is potentially valuable toinclude free text in risk prediction models Free text notes require a higher level of preprocessing and therefore Natural Language Processing methods may be applied to structure the freetext and extract relevant information from it []PLOS ONE 101371journalpone0237911 August PLOS ONE 0cUsing machine learning for predicting cervical cancer from Swedish electronic health recordsThe level or degree of structure can vary also for the structured data for example even ifthe type of a lab test is coded the result of the test might be a in free text with different unitsused for the same test Such information can be regarded as semistructured and thereforeadditional preprocessing is required also for these types of eventsDataThe health records collected for this study comes from Karolinska University Hospital inStockholm from the years [] These records contain coded information such asdiagnosis codes drug codes and procedural codes semistructured information such as labtest results and free text From this data set the patients with an ICD10 diagnosis coderepresenting cervical cancer ie a code starting with C53 were selected as the case groupNext a control group was created where each patient in the C53 case group was matched onage to ten other control women who did not have any C53 diagnosis on record in the dataSince one aim of this work was to investigate the events leading up to a first diagnosis of cervical cancer only cases with EHR data before the diagnosis date of cancer were included andall patients with the code Z854C previous cervical cancer were also filtered out leaving cases and controls Five basic event types were extracted from the ERHs for both casesand controls clinical entities found in the free text notes diagnosis codes drug codes labresults and procedure codes The extracted data were then divided into intervals for the firstof which all data up to the day before diagnosis were included For the next interval only dataregistered at least days before diagnosis were included and so on in intervals of days upto a year before diagnosis The available data was divided into two parts a development setcontaining of the data and a test set with the remaining The development set was setaside for hyperparameter tuning of the classifiers and for feature selection The test set wasused to evaluate the selected classifiers through a process of 10fold crossvalidation wherebyten rounds of training and testing was performed with of the data used for training and for testing for each round The development set was not included in the final evaluationof the classifiersICD codesThe health records contain diagnosis codes from the Swedish version of the ICD10 codesInternational Statistical Classification of Diseases and Related Health Problems”Tenth Revision ICD10 codes are hierarchically arranged with chapters at the highest level thesechapters are further divided into sections subsections and full codes This makes it possible toinclude both very finegrained information in form of the full diagnosis codes as well as higherlevel information such as chapters and subsections Table gives an example of the ICD10Table Example from the ICD hierarchyLevelChapterSectionExampleC00D48 NeoplasmsC51C58 Malignant neoplasms of female genital ansSubsectionC53 Malignant neoplasm of cervix uteriCodeC530 Malignant neoplasm endocervixSizeIn dataThe top of the hierarchy consists of different chapters where chapter II comprises codes for neoplasms Furtherdown in the hierarchy more detailed information is included in the codes The column titled Size gives the numberof different codes for each level in the complete code hierarchy and the last column In data shows the number ofdifferent codes included in the current data sets101371journalpone0237911t001PLOS ONE 101371journalpone0237911 August PLOS ONE 0cUsing machine learning for predicting cervical cancer from Swedish electronic health recordsFig ICD chapters and sections for the case group and the control group The left part corresponds to the case group and the right part the control group The innercircle represents the ICD chapters and the outer circle the ICD sections included in the chapter the labels show the sections with the most frequent codes101371journalpone0237911g001hierarchy for the code C530 Fig provides a visualization of the distribution of ICD10 codesfor the control group and the C53 case group Comparing cases and controls it can be notedthat many of the patients in the case groups have experienced other types of cancers ofcases and of controls had a diagnosis code from chapter II of ICD10 which is the chaptercontaining codes for tumorsneoplasms Additionally a large part of these codes came fromthe section representing codes for malignant neoplasms of the female genital ans C51C58other than cervical cancerATC codesThe next feature type are the ATC codes from the Anatomical Therapeutic Chemical Classification System representing drugs As with ICDcodes these codes are hierarchical and therefore it was possible to represent them with different levels of detail Table shows an exampleof the included ATC levelsClinical entities extracted from textMost of the data extracted from the EHRs were in the form of free text notes and differentapproaches have previously been used to create representations of the free text in EHRs OneTable Example from the ATC code hierarchyLevelMain group3rd level subgroup4th level subgroupFull codeExampleN Nervous systemN02B Other analgesics and antipyreticsN02BE AnilidesN02BE01 ParacetamolIn dataThe last column of the table shows how many different codes appear in the current data set for each code level101371journalpone0237911t002PLOS ONE 101371journalpone0237911 August PLOS ONE 0cUsing machine learning for predicting cervical cancer from Swedish electronic health recordsFig Example of patient record text This example contains one multiword Disorder œsquamous cell carcinoma insitu101371journalpone0237911g002possibility is to map the text to some existing ontology or terminology Roque []matched free text to ICD codes to enrich coded information extracted from EHRs This hasthe advantage of getting a coded representation practical for interpretation and machinelearning purposes however mapping directly from clinical text to standard terminologies canlead to low recall [] as the language used in clinical text differs from the standardised language in terminologies Tools for matching text to terminologies such as MetaMap [] arenot available for Swedish Another approach which also reduces the size of the feature spacewas applied by Miotto [] They included free text in their model by extracting entitiesfrom clinical notes using the Biomedical Annotator and topic modeling this approachwas effective for predicting diagnoses but has the drawback of reduced interpretabilityHere named entity recognition NER was applied to the free text notes belonging to casesand controls Using NER allowed for extracting the most relevant parts of the text and compared to including the full texts in a bagofwords model named entities makes it possible torepresent multiword expressions such as diabetes typ diabetes type and cancer in situ Toextract these entities from the free text a bidirectional long shortterm memory biLSTMnetwork was trained on notes annotated by two medical experts The notes used as trainingdata for the network were annotated for the entity types Body part annotations Disorder annotations and Finding annotations For this work findings and disorderswere included as events and following the SNOMED definitions a œFinding represent bothnormal and abnormal observations regarding a patient while a œDisorder always is the resultof an underlying pathological process see Fig A large corpus of clinical texts was used to generate word2vec [] embeddings of the textsas input representation for the network This corpus contained GB of text from SwedishEHRs with a complete vocabulary of about words The properties of the LSTM network are described in detail in Weegar [] This network was applied to all clinical notesfor the cases and the controls to extract all mentions of findings and disorders from these textswhich contained on average tokens for each patient Next negation detection was used toidentify negated entities using a rulebased module NegEx adapted specifically to the domainof Swedish clinical text Swedish [] This was an important step because clinical notes oftencontain documentation of discussions and reasoning around a potential condition in text andit is thus particularly common that findings are negated Indeed in this material up to ten percent of findings and six percent of the extracted disorders were actually in the negated formSimilarly to mitigate the risk of including information regarding individuals other than theactual patient to whom the record belonged deriving from eg discussions on family historyof disease clinical notes with headings related to family members were excluded The use ofNER for event extraction resulted in events for the controls on average For the cases onlyevents occurring before the C53 diagnosis were included on average eventsThe extracted events were lemmatized which reduced the scarcity of the free text data aslemmatization maps inflected versions of a word to the same basic representation By usinglemmatization words such as hudirritationskin irritation singular and hudirritationerskinirritations plural will be considered as the same event Similarly the event hostadecoughedPLOS ONE 101371journalpone0237911 August PLOS ONE 0cUsing machine learning for predicting cervical cancer from Swedish electronic health recordspast tense will be joined with hostarcoughs present tense But even after lemmatizationthe number of different events extracted from the texts is very large There were about different events appearing at least two times This is partially due to the possibility of describing the same event in many different ways in writing for example œfractured patella andœpatella fracture refers to the same type of event but will be considered as two different eventsas their surface forms are different Therefore to further reduce the feature space two additional preprocessing steps were applied to the textual events Firstly a spelling normalizationmodule was used to group different spelling variations of the same concept This grouping wasachieved through calculating the edit distance between each pair of events A Levenshtein editdistance of two strings is a measurement of how different the two strings are and is calculatedby counting the number of insertions deletions and substitutions of characters that is requiredto make two strings equal [] Groups of events were formed where each member in thegroup had at most an edit distance of one from some other member of the group and the samefirst letter as all other members in the group as it was found that allowing larger distancesintroduced errors For multiword expressions the distance was calculated per word and itwas also required that each included word had the same first letter The spelling differences inthe text data were mainly the result of misspellings but also of inflections or instances of writing the same concept as a either a compound word or a as two separate words After groupingthe events using edit distance all variations were exchanged with the most frequent surfaceform One example of such a group is the spelling variations for thyroid cancerthyreoideacancer tyreoideacancer thyroidecancer thyreoidacancer thyreoidcancer tyroideacancer tyreoidecancer thyroidea cancer thyroideacancer thyreoidecancerwhere each variation was mapped to the most frequently occurring form œthyreoideacancerThis normalization mapped about different surface forms of events into about different groupsNext a hierarchical representation of the events extracted from the free texts was createdThe hierarchy was constructed by first sorting the events extracted from the texts according totheir length and placing oneword entities at the top of the hierarchy Next for each level anyevent that contained all words of an event on the higher level was added as a child node of thatevent In this hierarchy the oneword event œleukaemia at the top level had the child nodesœacute leukaemia and œchronic leukaemia where œchronic leukaemia in turn had the childnodes œchronic myeloid leukaemia and œchronic lymphocytic leukaemia Events furtherdown in the hierarchy became gradually more detailed as they often contained some type ofmodifier to its parent node such as œnormal œserious or œmalignant capturing many relevant relationships between eventsA hierarchy of four levels was created with oneword entities at level one and events containing four or more words at the fourth level It is worth noting that when representing thetextual events for a single patient the longer lowlevel events had influence over higher levelrepresentations in the same way as for the ICD or ATC code hierarchies Using the ICD codehierarchy a diagnosis can be represented by the corresponding full lowlevel code or by thesection or chapter it belongs to In a similar way the lower level event œchronic myeloid leukaemia can be represented by the higher level event œleukaemiaThis hierarchy of textual events was however different from the ICD and ATC code hierarchies in two ways Firstly each child node could have more than one parent node an œinfectedwound had the parent œwound and the parent œinfected The second difference was thatwhile the top levels are the smallest in the code hierarchies the opposite is true for the textualevents There was a higher number of short textual events and the short events also make up alarger part of the total set of textual events The top level of the hierarchy contained PLOS ONE 101371journalpone0237911 August PLOS ONE 0cUsing machine learning for predicting cervical cancer from Swedish electronic health recordsdifferent events followed by events at level two events at level three and events at level fourLab resultsThe included data for lab results consists of the type of test and the result of the test Includingthe value of test result can be too finegrained for the task of prediction since many tests arerare [] Therefore the result is represented as being either inside or outside the referencerange for a test value For example the result of a Hemoglobin test can either be in the normalrange for Hemoglobin above the range or below it Therefore each lab result in the includedhealth records will be represented by one of three different features depending on the outcomeof the testProcedure codesProcedure codes classify procedures including surgical procedures medical investigationspreventive measures and treatments for example the code AK044 corresponding to an ultrasound of the kidneys The codes are used in health records for statistical and administrativepurposes and have been included as an event type in this workA number of the events that were extracted from the EHRs were unique meaning that theyonly occurred for a single patient Since such events cannot contribute to the diagnosis prediction but only increase the scarcity of the data they were removed at this stage leaving in total different events in the data set Each case had on average different events and events in total before the diagnosis For the controls the corresponding numbers were on average different events and an average total of The reason for the larger number of eventsfor the control group is that only data up to a cervical cancer diagnosis is included for the casegroup and all events after the diagnosis were discarded For the controls on the other hand alldocumented events were kept even if they occurred after the time of their matched case™s diagnosis Since the objective of our study was correct classification based on EHR events withpotentially low expected contrast between cases and controls we opted for this choice to maximize the control information for the classifiers to be trained on minimize the risk ofmisclassification of disease status by ensuring to the best of our knowledge that no hospitalbased female controls were diagnosed with cervical cancer later during the study period and to increase generalizability to a reallife clinical situation where EHRs are available but diseasestatus of the individuals is not already known Fig gives an overview of the available data forthe different time intervals However some events will appear outside of the study periodleading to data censoring [ ] Important and informative events could have occurred beforethe start of data collection and this also means as in most casecontrol or other studies thatwe do not know whether some members of the control group develop cervical cancer after thestudy end point when no more EHR data was available to us However this is a wellknownfact resulting from this type of study design and does not invalidate the comparison made during the actual study period definedClassification experimentsFour different classifiers were used to evaluate the appropriateness of the different featuretypes for classification of future C53 cases These classifiers were Random Forest ComplementNaive Bayes Bernoulli Naive Bayes and Support Vector Machines all implemented in Scikitlearn [] The first classifier Random Forest is an ensemble classifier robust to noise andlarge feature spaces [] Complement Naive Bayes [] is capable in cases of data sets withclass imbalance Bernoulli Naive Bayes additionally takes absence of features into account forPLOS ONE 101371journalpone0237911 August PLOS ONE 0cUsing machine learning for predicting cervical cancer from Swedish electronic health recordsFig Available data for the case group The xaxis denotes the number of days before a diagnosis and events only appearing onetime in the data set have been excluded At days before a diagnosis events were available for patients101371journalpone0237911g003classification [] and Support Vector Machines SVM is a classifier well suited for highdimensional spaces []This classification task can be understood as a text classification problem where each eventextracted from a patient record corresponds to a word and each patient is represented by a vector with the same dimension as the complete vocabulary of events For Random Forest andComplement Naive Bayes the input vectors consisted of the raw counts of events For Bernoulli Naive Bayes binary input vectors were used and for the SVM classifier normalized vector counts were used as input Binary vectors correspond to if a patient ever experienced anevent and count vectors also represent how many times each event occurred Another possibility is to use tfidf term frequencyinverse document frequency weights The idea behindtfidf is to give more weight to the events that are representative for individual patients However using tfidf did not improve classification resultsResultsEvent typesEach type of event was evaluated individually for its ability to correctly classify the patients Fig shows the average AUC using the four classifiers over time for each event type ICD codesATC codes Procedure codes Clinical entities and Lab results The average AUC was calculated as the sum of the scores AUCi for the individual classifiers divided by the number of classifiers AUCavg ¼ the classifiers were the clinical entities extracted from the text as the highest AUC scores wereachieved using only the text entities ðAUC1 þ AUC2 þ AUC3 þ AUC4Þ The most informative event type forPLOS ONE 101371journalpone0237911 August PLOS ONE 0cUsing machine learning for predicting cervical cancer from Swedish electronic health recordsFig Average AUC for the different event types over time101371journalpone0237911g004Event levelsSince the data in EHRs typically are noisy high dimensional and sparse it is necessary toinvestigate data representation how the information included in the records should be presented to the model [] In this work both which types of feature to include and also the detailwith which those features should be represented was evaluatedIt has previously been found that including features derived from several levels of hierarchical clinical codes improves the performance for predicting adverse drug events from EHR data[] and as the input events can be represented with different levels of detail the next set ofexperiments aimed to evaluate the most suitable representation of the events in this regardUsing the most detailed levels such as a full ICD10 code gives very detailed information buta very sparse data set By including the higherlevel representations the sparsity of the data canbe reduced Fig shows the average AUC o Answer:

Thyroid_Cancer

lack of COVID19 diagnostic tests for the whole Spanish population thecurrent strategy is to identify the disease early to limit contagion in the communityAimTo determine clinical factors of a poor prognosis in patients with COVID19 infectionDesign and settingDescriptive observational retrospective study in three primary healthcare centres with anassigned population of MethodExamination of the medical records of patients with COVID19 infections confirmed by polymerase chain reaction Logistic multivariate regression models adjusted for age and sexwere constructed to analyse independent predictive factors associated with death ICUadmission and hospitalizationResultsWe included patients mean age years female aged � years were health workers doctors nurses auxiliaries Predictors of ICUadmission or death were greater age OR 95CI to male sex OR 95CI to autoimmune disease OR 95CI to bilateral pulmonary infiltrates OR 95CI to elevated lactatedehydrogenase OR 95CI to elevated Ddimer OR 95CI to and elevated Creactive protein OR 95CI to Myalgia or arthralgia OR 95CI to was protective factor against ICU admission andPLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientsdeath Predictors of hospitalization were chills OR 95CI to feverOR 95CI to dyspnoea OR 95CI to depressionOR 95CI to lymph ia OR 95CI to andelevated Creactive protein OR 95CI to Anosmia OR 95CI to was the only significant protective factor for hospitalization after adjusting forage and sexConclusionDetermining the clinical biological and radiological characteristics of patients with suspected COVID19 infection will be key to early treatment and isolation and the tracing ofcontactsIntroductionOn December the health authorities of Wuhan city Hubei Province China reporteda cluster of cases of pneumonia of unknown aetiology with onset of symptoms on December including severe cases with a common exposure identified in a city market [] whichwas closed on January On January the Chinese authorities identified a newCoronaviridae family virus initially named coronavirus 2019nCoV and later coronavirusSARSCoV2 as the causal agent [] The genetic sequence was shared by the Chinese authorities on January On January the first case was detected in the USA in Washingtonstate [] On January the World Health anization declared the SARSCoV2 outbreak in China a public health emergency of international concern [] Subsequently the outbreak has spread outside China with Europe especially affected []The first positive case diagnosed in Spain was confirmed on January on the islandof La Gomera while the first death occurred on February in Valencia city the date was confirmed twenty days later The first confirmed case in Barcelona was on February and fromthen until June there have been confirmed cases in Spain []The most common signs of infection are respiratory symptoms fever cough and shortnessof breath In more severe cases the infection may cause pneumonia severe acute respiratorysyndrome renal failure and death [] Transmission appears to be mainly persontopersonvia the airway through respiratory droplets measuring microns when the patient has respiratory symptoms cough and sneezing and contact with fomites [] Most estimates of theincubation period of COVID19 range from to days with most around five days Evidenceon the transmission of the virus before symptom onset is unclear There is currently no specifictreatment for COVID19 infections To date the most important scientific efforts have focusedon three areas strategies to contain the spread of the disease the initiation of clinical trialswith antivirals and multiple therapies and the design of a new vaccine which is still unclearThese strategies include some of a community nature where primary healthcare plays a centralrole in disease prevention and control [] Few studies have described the clinical characteristics of the disease fewer the predictive factors and virtually none have described the Mediterranean population compared with the rest of the world Therefore this study aimed todescribe the clinical biological and radiological manifestations the evolution treatments andmortality rate of patients with COVID19 infection in the population of Barcelona city anddetermine the most important predictors of a poor prognosisPLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientsMaterials and methodsA multicentre observational descriptive study was carried out in three urban primary healthcare centres serving an assigned population of with one reference hospital The studyincluded all consecutive adult patients with COVID19 confirmed by polymerase chain reaction PCR from nasal and pharyngeal samples during the study period of February to April Diagnostic confirmation was made in the hospital laboratories as PCR is not available in primary healthcare centres Signs and symptoms the main available haematologicaland biochemical data and the results of imaging tests were recorded as were comorbiditiesthe evolution the hospitalization rate intensive care unit ICU admission and the treatmentsreceived The study population was divided into four age groups “ years “ years“ years and � years Other variables recorded were the type of followup the need fortemporary work disability and the source of possible contacts The time to first medical visitwas defined as the difference in days between symptom onset and medical visit by a familyphysician The factors that determined a poor prognosis hospitalization ICU admissiondeath were collected The data were obtained from the electronic medical record Missingdata were collected by telephone interviews with patients when possible Patients from nursinghomes were excluded as the rate of infections and mortality has been shown to be muchhigher than in the noninstitutionalized population The study was approved by the EthicsCommittee of the Hospital Clinic of Barcelona registration number HCB20200525 Thestudy was conducted according to the Helsinki Declaration and Spanish legislation on biomedical studies data protection and respect for human rightsStatistical analysisCategorical variables are presented as absolute frequencies and percentages and continuous variables as means and standard deviations SD Predictors of death ICU admission andhospitalization were determined using the student™s t test for continuous variables and the chisquare test for categorical variables Logistic multivariate regression models adjusted for ageand sex were constructed to analyse independent predictive factors associated with death ICUadmission and hospitalization Odds ratios OR and their confidence intervals 95CIobtained in the adjusted regression analysis were calculated Forest plots were used to represent OR and 95CI Values of p005 were considered statistically significant The statisticalanalysis was performed using the R version for WindowsResultsClinical characteristics and comorbiditiesWe included patients mean age years female aged � yearsThe mean time from symptom onset to the medical visit was SD days Clinical characteristics are shown in Table Notably were health workers doctors nurses auxiliaries The most frequentclinical symptoms were cough fever general malaise fatigue myalgia or arthralgia dyspnoea diarrhoea headache anosmia and dysgeusia Physical examination in patients showed had auscultatory alterations tachypnoea and an oxygen saturation of � ICU admission and death were associated with a greater mean age years vs yearsp male sex vs p dyspnoea vs p fever vs p auscultatory alterations vs p and low oxygen saturation vs p Table Myalgia or arthralgia vs PLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientsTable Clinical and exploratory factors predicting hospitalization and ICU admissiondeathVariablesTotalDeath or ICU admissionHospitalizationAge”yearsDistribution”no “ years“ years“ years� yearsMale”no Occupation”no n No n YesPAdjusted ORNoYesPAdjusted OR ± ± n ± [“][ CI]n ± n ± [“][ CI] [“] [“]Other type of exposure Health professionalOther health workersSmoking exsmokersmoker”nototal no Temperature at admission” ˚C Patients with fever �˚C”nototal no Time from symptom onset tomedical visit”days¡Symptoms”no ¶ ± ± ± ± ± ± NANANA [“] [“] [“] [“] ± ± ± [“] [“] ± [“] NANANA [“]CoughGeneral malaiseFatigue [“] [“] [“] [“] [“] [“]Myalgia or arthralgia [“] [“]DyspnoeaDiarrhoeaHeadacheAnosmiaDysgeusiaSore throatBlocked noseNausea or vomitingSputum productionChillsAstheniaChest painAlterations in physical examination”nototal no §Auscultatory alterationsTachypnoeaTachycardiaPharyngitis [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] NA [“]Continued PLOS ONE 101371journalpone0237960 August PLOS ONE 0cTable ContinuedVariablesTotalDeath or ICU admissionHospitalizationPrognostic factors in Spanish COVID19 patientsOxygen saturation �”nototalno n n No n YesPAdjusted ORNoYes[ CI]n n [“] PAdjusted OR[ CI] [“]In bold statistically significant independent predictive factors associated with hospitalization death or ICU admission logistic multivariate regression adjusted for ageand sex  Temperature distribution was ˚C “ËšC “ËšC and ˚C ¡ In patients™ data on period between symptom onset and medical visit were lacking¶ Symptoms with a frequency of patients were disorientation n conjunctivitis n haemoptysis n and cutaneous lesions n § patients had a physical examination The alterations with a frequency of patients were cutaneous lesions n and tonsillopharyngitis n Ref reference NA not applicable101371journalpone0237960t001p headache vs p dysgeusia vs p andanosmia vs p were less frequent in patients admitted to the ICU or whodied than the remaining patients Age OR 95CI to and male sexOR 95CI to were independent predictors of ICU admission and deathMyalgia or arthralgia OR 95CI to was the only significant protective factor against ICU admission and death after adjusting for age and sex Fig The best clinicalpredictors of hospitalization were chills OR 95CI to fever OR 95CI to and dyspnoea OR 95CI to Anosmia OR 95CI to was the only significant protective factor for hospitalization after adjusting for age and sex Table and Fig Comorbidities were presented by patients the most common were hypertension in diabetes mellitus in and obesity in Table Heartdisease vs p autoimmune disease vs p diabetes vs p hypertension vs p and chronic kidney disease vs p were the comorbidities significantly associated with ICUadmission and death Table Autoimmune disease was the only significant predictivecomorbidity for ICU admission and death after adjusting for age and sex OR CI to Fig Depression was the best predictor of hospitalization among allcomorbidities OR 95CI to Fig Having � comorbidity was associated with ICU admission and death OR 95CI to and hospitalizationOR 95CI to independently of age and sexImaging and laboratory testsChest Xray was necessary in patients and showed lobar pulmonary infiltrates in bilateral pulmonary infiltrates in and an interstitial pattern in Table Chest CT was required in patients and pulmonary ultrasound in Biologically of patients had lymph ia mm3 Likewise had a lactate dehydrogenase LDH Uml and liver test alterations were commonelevated ASTGOT in and ALTGPT in In of cases Ddimer waselevated 500mgL The most important factors for ICU admission and death were bilateralpulmonary infiltrates OR 95CI to elevated lactatedehydrogenaseOR 95CI to elevated Ddimer OR 95CI to andelevated Creactive protein OR 95CI to Fig Significant predictivePLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientsFig Prognostic factors for death and ICU admission �The upper limits of the confidence intervals were restricted to in order not to mask the significant effects of other variables with smaller ranges101371journalpone0237960g001factors associated with hospitalization after adjusting for age and sex were lymph iaOR 95CI to and elevated Creactive protein OR 95CI to Fig Treatment complications and evolutionTreatment included hydroxychloroquine in patients azithromycin in lopinavirritonavir in glucocorticoids in and tocilizumab in among others Table and of patients required hospitalization Phone follow up was registered in patients patients were monitored at homePLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientsFig Prognostic factors for hospitalization �The upper limits of the confidence intervals were restricted to inorder not to mask the significant effects of other variables with smaller ranges101371journalpone0237960g002 of the patients of working age sought work disability due to COVID19 TheICU admission rate was The evolution included pneumonia in patientsadult respiratory distress syndrome in severe renal failure in pulmonarythromboembolism in and sepsis in patients Occupational contact with persons with confirmed or suspected COVID19 infection was reported by patientswhile reported that contact occurred in the family setting Occupational contactwas a protective factor against hospitalization OR 95CI to ICU admission and death OR 95CI to after adjusting for age and sex The mortalityrate to date was PLOS ONE 101371journalpone0237960 August PLOS ONE 0cTable Comorbidities associated with hospitalization and ICU admissiondeathVariablesTotalDeath or ICU admissionHospitalizationn NoYesPAdjusted OR [NoYesPAdjusted OR [n n CI]n n CI]Prognostic factors in Spanish COVID19 patientsComorbidities”no  Any comorbidityHypertensionDiabetesObesityDyslipidaemiaCancer [“] [“] [“] [“] [“] [“] [“] [“] Chronic kidney disease Heart disease Autoimmune disease Chronic obstructive pulmonary diseaseDepressionCardiac arrhythmiaThyroid alterationsAsthmaLiver diseaseCerebrovascular diseaseAlzheimer disease [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“]In bold statistically significant independent predictive factors associated with hospitalization death or ICU admission logistic multivariate regression adjusted for ageand sex  Comorbidities with a frequency of patients were bronchiectasis n fibromyalgia n anaemia n arthritis n HIV n syphilis n andtuberculosis n 101371journalpone0237960t002DiscussionThis study summarizes the clinical biological and radiological characteristics evolution andprognostic factors of patients with COVID19 disease in primary and community healthcareTo date we are aware of three published Spanish studies [“] The first reported data from patients on ICU admissions in a region where the pandemic was reported early [] Thestudy by Borobia [] describes the first adult patients with COVID19 consecutivelyadmitted to a University Hospital in Madrid The third focuses on the differences by agedependent categories in the clinical profile presentation management and shortterm outcomes [] Although there have been two systematic reviews and metaanalysis that analysethe clinical characteristics of COVID19 they are limited to Chinese cohorts or case series [] and a large USA cohort [] that did not analyse clinical predictors of a poor prognosisClinically the same main symptoms of cough and fever are reported in all series Howeverin Barcelona city we have observed diarrhoea anosmia and dysgeusia which is hardlyreported in the Chinese series [] which unlike ours comes principally from hospitals diarrhoea occurred in of cases very similar to the in New York [] and clearly higherthan the reported in China Nearly of patients had anosmia and dysgeusia similar tothe results obtained in French patients [] In contrast expectoration was found in only compared with in the Chinese seriesPLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientsTable Analytical and radiological predictors of hospitalization and ICU admissiondeathVariablesTotalDeath or ICU admission n Hospitalization n n NoYesPAdjusted ORNoYesn n [ CI]n n PAdjusted OR[ CI]Alterations in chest Xray”nototalno  Bilateral pulmonary infiltratesInterstitialground glass patternLobar pulmonary infiltrateAlterations in chest CAT scan”nototal no ¡ [“] [“] [“] [“] [“] [“]Bilateral pulmonary infiltrates Interstitialground glass pattern Laboratory parameters”nototalno [“] [“] [“] [“]Leukocytes mm3 [“] [“]Lymphocytes mm3Platelets mm3 [“] [“] [“] [“]Haemoglobin gdl [“] [“]Creactive protein mglitreProcalcitonin ngmlLactate dehydrogenase UlitreAminotransferase aspartate UlitreAlanine aminotransferase UlitreTotal bilirubin mgdL [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“]Creatine kinase Ulitre [“] [“]Creatinine 15mgdL Ddimer mglitreSodium mEqlitrePotassium mEqlitre [“] [“] [“] [“] [“] [“] [“] [“]In bold statistically significant independent predictive factors associated with hospitalization death or ICU admission logistic multivariate regression adjusted for ageand sex  patients had a chest Xray The alterations with a frequency patients were pneumothorax n and pleural effusion n Chest Xray resultswere not available in patients¡ patients had a chest CAT scan Alterations with a frequency of patients were pulmonary thromboembolism n emphysema n lobarpulmonary infiltrates n pneumonia n atelectasis n and pleural effusion n CAT scan results were not available in five patients101371journalpone0237960t003PLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientsTable Predictors of the evolution complications and treatment in patients hospitalized or with ICU admissiondeathVariablesTotalDeath or ICU admission n Hospitalization n n NoYesPAdjusted OR [NoYesPAdjusted OR [n n CI]n n CI]Complications”no  Any complicationPneumonia NA [“] [“] [“]Adult respiratory distress [“ [“]syndromeRenal failurePulmonary thromboembolismTreatments”no ¡HydroxychloroquineAzithromycinLopinavirRitonavir [“] [“] [“]NA] [“] [“] [“] [“] [“] [“]Oxygen therapy [“] [“]Intravenous antibiotics [“] [“]GlucocorticoidsTocilizumabCephalosporinsLow molecular weight heparin Remdesivir Covid19 infection”no [“] [“] [“] [“] [“] [“] [“] [“] [“] [“]Any cohabitant [“] [“]Any work colleague [“] [“]Any contact person in other [“] [“]settingsIn bold statistically significant independent predictive factors associated with hospitalization death or ICU admission logistic multivariate regression adjusted for ageand sex  Complications in patients were sepsis n multian failure n electrolyte alterations n hematologic alterations n and lung cancer n ¡ Treatments with a frequency of patients except remdesivir were amoxicillin n interferon n rituximab n darunavir n and entecavirn NA not applicable101371journalpone0237960t004Chinese patients had a mean age of years ten years lower than our series and ofour patients were aged � years compared with and in China Germany andthe USA respectively but in Italy [ “] Older age and male sex predisposed to ahigher mortality rate in our and all large series [ ] In our patients comorbidities werethree times higher than in the Chinese cohort [] and were similar to the findings of the NewYork study [] Any comorbidity was a risk factor for hospitalization ICU admission anddeath Depression was an independent risk factor for hospitalization which has not beenobserved in other cohorts studied Depression was often accompanied by a vulnerable socialsituation which may have justified hospitalization Likewise autoimmune diseases were independent risk factor for ICU admission and death Various hypotheses have been postulated onpossible autoimmune alterations in the pathogenic evolution of the disease With respect toPLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientstreatment no drug has proved effective against Covid19 until now Moreover many treatments were unavailable in the outpatient setting Currently we are only certain that treatmentwith tocilizumab showed better survival rates in retrospective cohorts [] although its efficacy has not been tested in randomized clinical trials Therefore the results on the outcomesassociated with treatment should be interpreted with cautionThe same comorbidities were identified with hypertension and diabetes being the twomost common while in the USA and Italy obesity seems to be higher Our results show thatobesity was close to being an independent risk factor for hospitalization OR CI to Strikingly of our patients were healthcare workers compared with in Wuhanand in Germany [ ] Although these studies recognized an important degree ofunderreporting of cases in health workers the difference remains important There are at leasttwo possible explanations first the lack of personal protective equipment in the initial phaseof the epidemic a constant revindication of health professionals who felt undersupplied Secondly many cases were health professionals from primary healthcare or the reference hospitalwho reside in the same area where they workIn all reported series bilateral pneumonia was the most common radiological finding waspresent in more than half the cases [] and was a factor of a poor prognosis and mortality Incontrast an interstitial radiological pattern did not confer an increased risk of mortality TheWuhan study reported a CAT scan use of compared with in Barcelona In contrast chest Xrays were carried out in and respectively the availability of diagnostic means was higher in China A recent international consensus states that radiologicalassessment is not necessary in asymptomatic patients or those with mild disease but is requiredin patients with moderate or severe disease regardless of whether a definite diagnosis ofCOVID19 has been made [] In addition simple chest Xrays are preferable in a resourceconstrained environment with difficulties in accessing CAT scans [] The possible use of pulmonary ultrasound for the pointofcare diagnosis of COVID19 pneumonia has not been sufficiently analysed but might be an efficient alternative due to its portability and reliability []In fact the regional Catalan government has recently acquired ultrasound machines toenable family physicians to make doctors can make pointofcare home or nursing homediagnoses of pneumonia [] Biologically lymph ia and increased CRP LDH and Ddimer were usually constant and similar in all series and were associated with an increased riskof mortality A differential variable in our series is a greater number of alterations in liver testswhich was present in “ of patients data similar to the USA and Italian cohorts but different from the Chinese cohort where it was [] We also found hypokalaemia in of patients a factor not reported in other studiesWe found a hospitalization rate of compared with “ in the USA and inChina and an ICU admission rate of which was similar to the Chinese USA “ and German results While the protocols of action and admission are similarand depend on the level of clinical involvement the therapeutic protocols differ between hospitals cities and countries There remain many unknowns in the treatment of COVID19The only truth is that we do not have a vaccine an etiological treatment or a treatment withsufficient scientific evidence to generalize its use Currently the systematic review of antiretroviral treatments has not offered conclusive results [] and despite in vitro results for hydroxychloroquine COVID19 infections are currently intractable [ ]The mortality rate in our study was compared with in New York in hospitalized patients in China in Germany and in Italy Different informationand recording systems the availability of diagnostic tests and above all the anization ofnational health systems may have contributed to the differences observedPLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientsThe study had some limitations due to the observational retrospective design However itis sufficiently representative of the population with confirmed COVID19 to permit betteridentification of the factors of a poor prognosis of the disease from a clinical perspective Wecannot rule out some heterogeneity in data codification due to observers™ interpretations of themedical records However this bias is minimal as most clinical factors included are clearlydefined in the electronic medical record Another limitation of this study is the percentage ofpatients without laboratory parameters more than Even though in real clinical practicethese percentages may be expected the results corresponding to laboratory parameters shouldbe interpreted with cautionFour months after the declaration of the pandemic there is not a sufficiently reliable available and generalizable diagnostic test that can analyse the seroprevalence of COVID19 evenin the most industrialized countries Given this lack determining the clinical biological andradiological characteristics of probable cases of COVID19 infection will be key to the initiation of early treatment and isolation and for contact tracing especially in primary healthcareSupporting informationS1 DatasetXLSXAcknowledgmentsThe authors thank David Buss for editorial assistanceAuthor ContributionsConceptualization Antoni Siso´Almirall Belchin Kostov Jaume BenaventÃreuData curation Antoni Siso´Almirall Belchin Kostov Minerva MasHeredia Sergi VilanovaRotllan Ethel SequeiraAymar Mireia SansCorrales Elisenda SantArderiu Laia CayuelasRedondo Angela Martı´nezPe´rez Noemı´ Garcı´aPlana August AnguitaGuimetJaume BenaventÃreuFormal analysis Antoni Siso´Almirall Belchin Kostov Jaume BenaventÃreuInvestigation Antoni Siso´Almirall Belchin Kostov Minerva MasHeredia Sergi VilanovaRotllan Ethel SequeiraAymar Mireia SansCorrales Elisenda SantArderiu Laia CayuelasRedondo Angela Martı´nezPe´rez Noemı´ Garcı´aPlana August AnguitaGuimetJaume BenaventÃreuMethodology Antoni Siso´Almirall Belchin Kostov Minerva MasHeredia Sergi VilanovaRotllan Ethel SequeiraAymar Mireia SansCorrales Elisenda SantArderiu Laia CayuelasRedondo Angela Martı´nezPe´rez Noemı´ Garcı´aPlana August AnguitaGuimetJaume BenaventÃreuProject administration Antoni Siso´Almirall Belchin Kostov Jaume BenaventÃreuResources Antoni Siso´Almirall Belchin Kostov Jaume BenaventÃreuSupervision Antoni Siso´Almirall Belchin Kostov Jaume BenaventÃreuValidation Antoni Siso´Almirall Belchin Kostov Minerva MasHeredia Sergi VilanovaRotllan Ethel SequeiraAymar Mireia SansCorrales Elisenda SantArderiu Laia CayuelasRedondo Angela Martı´nezPe´rez Noemı´ Garcı´aPlana August AnguitaGuimetJaume BenaventÃreuPLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientsVisualization Antoni Siso´Almirall Belchin Kostov Jaume BenaventÃreuWriting “ original draft Antoni Siso´Almirall Belchin Kostov Jaume BenaventÃreuWriting “ review editing Antoni Siso´Almirall Belchin Kostov Minerva MasHerediaSergi VilanovaRotllan Ethel SequeiraAymar Mireia SansCorrales Elisenda SantArderiu Laia CayuelasRedondo Angela Martı´nezPe´rez Noemı´ Garcı´aPlana AugustAnguitaGuimet Jaume BenaventÃreuReferences World Health anization Pneumonia of unknown cause”China wwwwhointcsrdon05january2020pneumoniaofunkowncausechinaen accessed April Centers for Disease Control and Prevention Novel Coronavirus 2019nCoV Situation Summarystackscdcgovviewcdc84806cdc_84806_DS1pdf accessed April Holshue ML DeBolt C Lindquist S Lofy KH Wiesman J Bruce H First Case of Novel Coronavirus in the United States N Engl J Med “ 101056NEJMoa2001191 PMID World Health anization Novel Coronavirus2019nCoV wwwwhointdocsdefaultsourcecoronavirusesituationreports20200130sitrep10ncovpdfsfvrsnd0b2e480_2 accessed AprilJohns Hopkins University CSSE COVID19 Dashboard by the Center for Systems Science and Engineering CSSE at Johns Hopkins University JHU gisanddatamapsarcgiscomappsopsdashboardindexhtmlbda7594740fd40299423467b48e9ecf6 accessed April Ministerio de Sanidad Gobierno de España Situacio´n del COVID19 en España covid19isciiies accessed April Guan WJ Ni ZY Hu Y Liang WH Ou CQ He JX Clinical Characteristics of Coronavirus Disease in China N Engl J Med 101056NEJMoa2002032 PMID Yeo C Kaushal S Yeo D Enteric involvement of coronaviruses is faecaloral transmission of SARSCoV2 possible Lancet Gastroenterol Hepatol “ 101016S2468 PMID Chang BB Chiu TY Ready for a long fight against the COVID19 outbreak an innovative model oftiered primary health care in Taiwan BJGP 103399bjgp 20X101068PMID Barrasa H Rello J Tejada S Martı´n A Balziskueta G Vinuesa C SARSCov2 in Spanish Intensive Care Early Experience with 15day Survival In Vitoria Anaesth Crit Care Pain Med 101016jaccpm202004001 PMID Borobia AM Carcas AJ Arnalich F A´ lvarezSala R MonserratVillatoro J Quintana M A Cohortof Patients with COVID19 in a Major Teaching Hospital in Europe J Clin Med Martı´nSa´nchez FJ Del Toro E Cardassay E Valls Carbo´ A Cuesta F Vigara M Clinical presentation and outcome across age categories among patients with COVID19 admitted to a Spanish Emergency Department Eur Geriatr Med 101007s41999020003592 PMIDFu L Wang B Yuan T Chen X Ao Y Fitzpatrick T Clinical characteristics of coronavirus disease COVID19 in China A systematic review and metaanalysis J Infect 101016jjinf202003041 PMID RodriguezMorales AJ CardonaOspina JA Gutie´rrezOcampo E VillamizarPeña R HolguinRiveraY EscaleraAntezana JP Clinical laboratory and imaging features of COVID19 A systematicreview and metaanalysis Travel Med Infect Dis 101016jtmaid2020101623PMID Richardson S Hirsch JS Narasimhan M Crawford JM McGinn T Davidson KW PresentingCharacteristics Comorbidities and Outcomes Among Patients Hospitalized With COVID19 inthe New York City Area JAMA 101001jama20206775 PMID Goyal P Choi JJ Pinh

cancer273

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: lack of COVID19 diagnostic tests for the whole Spanish population thecurrent strategy is to identify the disease early to limit contagion in the communityAimTo determine clinical factors of a poor prognosis in patients with COVID19 infectionDesign and settingDescriptive observational retrospective study in three primary healthcare centres with anassigned population of MethodExamination of the medical records of patients with COVID19 infections confirmed by polymerase chain reaction Logistic multivariate regression models adjusted for age and sexwere constructed to analyse independent predictive factors associated with death ICUadmission and hospitalizationResultsWe included patients mean age years female aged � years were health workers doctors nurses auxiliaries Predictors of ICUadmission or death were greater age OR 95CI to male sex OR 95CI to autoimmune disease OR 95CI to bilateral pulmonary infiltrates OR 95CI to elevated lactatedehydrogenase OR 95CI to elevated Ddimer OR 95CI to and elevated Creactive protein OR 95CI to Myalgia or arthralgia OR 95CI to was protective factor against ICU admission andPLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientsdeath Predictors of hospitalization were chills OR 95CI to feverOR 95CI to dyspnoea OR 95CI to depressionOR 95CI to lymph ia OR 95CI to andelevated Creactive protein OR 95CI to Anosmia OR 95CI to was the only significant protective factor for hospitalization after adjusting forage and sexConclusionDetermining the clinical biological and radiological characteristics of patients with suspected COVID19 infection will be key to early treatment and isolation and the tracing ofcontactsIntroductionOn December the health authorities of Wuhan city Hubei Province China reporteda cluster of cases of pneumonia of unknown aetiology with onset of symptoms on December including severe cases with a common exposure identified in a city market [] whichwas closed on January On January the Chinese authorities identified a newCoronaviridae family virus initially named coronavirus 2019nCoV and later coronavirusSARSCoV2 as the causal agent [] The genetic sequence was shared by the Chinese authorities on January On January the first case was detected in the USA in Washingtonstate [] On January the World Health anization declared the SARSCoV2 outbreak in China a public health emergency of international concern [] Subsequently the outbreak has spread outside China with Europe especially affected []The first positive case diagnosed in Spain was confirmed on January on the islandof La Gomera while the first death occurred on February in Valencia city the date was confirmed twenty days later The first confirmed case in Barcelona was on February and fromthen until June there have been confirmed cases in Spain []The most common signs of infection are respiratory symptoms fever cough and shortnessof breath In more severe cases the infection may cause pneumonia severe acute respiratorysyndrome renal failure and death [] Transmission appears to be mainly persontopersonvia the airway through respiratory droplets measuring microns when the patient has respiratory symptoms cough and sneezing and contact with fomites [] Most estimates of theincubation period of COVID19 range from to days with most around five days Evidenceon the transmission of the virus before symptom onset is unclear There is currently no specifictreatment for COVID19 infections To date the most important scientific efforts have focusedon three areas strategies to contain the spread of the disease the initiation of clinical trialswith antivirals and multiple therapies and the design of a new vaccine which is still unclearThese strategies include some of a community nature where primary healthcare plays a centralrole in disease prevention and control [] Few studies have described the clinical characteristics of the disease fewer the predictive factors and virtually none have described the Mediterranean population compared with the rest of the world Therefore this study aimed todescribe the clinical biological and radiological manifestations the evolution treatments andmortality rate of patients with COVID19 infection in the population of Barcelona city anddetermine the most important predictors of a poor prognosisPLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientsMaterials and methodsA multicentre observational descriptive study was carried out in three urban primary healthcare centres serving an assigned population of with one reference hospital The studyincluded all consecutive adult patients with COVID19 confirmed by polymerase chain reaction PCR from nasal and pharyngeal samples during the study period of February to April Diagnostic confirmation was made in the hospital laboratories as PCR is not available in primary healthcare centres Signs and symptoms the main available haematologicaland biochemical data and the results of imaging tests were recorded as were comorbiditiesthe evolution the hospitalization rate intensive care unit ICU admission and the treatmentsreceived The study population was divided into four age groups “ years “ years“ years and � years Other variables recorded were the type of followup the need fortemporary work disability and the source of possible contacts The time to first medical visitwas defined as the difference in days between symptom onset and medical visit by a familyphysician The factors that determined a poor prognosis hospitalization ICU admissiondeath were collected The data were obtained from the electronic medical record Missingdata were collected by telephone interviews with patients when possible Patients from nursinghomes were excluded as the rate of infections and mortality has been shown to be muchhigher than in the noninstitutionalized population The study was approved by the EthicsCommittee of the Hospital Clinic of Barcelona registration number HCB20200525 Thestudy was conducted according to the Helsinki Declaration and Spanish legislation on biomedical studies data protection and respect for human rightsStatistical analysisCategorical variables are presented as absolute frequencies and percentages and continuous variables as means and standard deviations SD Predictors of death ICU admission andhospitalization were determined using the student™s t test for continuous variables and the chisquare test for categorical variables Logistic multivariate regression models adjusted for ageand sex were constructed to analyse independent predictive factors associated with death ICUadmission and hospitalization Odds ratios OR and their confidence intervals 95CIobtained in the adjusted regression analysis were calculated Forest plots were used to represent OR and 95CI Values of p005 were considered statistically significant The statisticalanalysis was performed using the R version for WindowsResultsClinical characteristics and comorbiditiesWe included patients mean age years female aged � yearsThe mean time from symptom onset to the medical visit was SD days Clinical characteristics are shown in Table Notably were health workers doctors nurses auxiliaries The most frequentclinical symptoms were cough fever general malaise fatigue myalgia or arthralgia dyspnoea diarrhoea headache anosmia and dysgeusia Physical examination in patients showed had auscultatory alterations tachypnoea and an oxygen saturation of � ICU admission and death were associated with a greater mean age years vs yearsp male sex vs p dyspnoea vs p fever vs p auscultatory alterations vs p and low oxygen saturation vs p Table Myalgia or arthralgia vs PLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientsTable Clinical and exploratory factors predicting hospitalization and ICU admissiondeathVariablesTotalDeath or ICU admissionHospitalizationAge”yearsDistribution”no “ years“ years“ years� yearsMale”no Occupation”no n No n YesPAdjusted ORNoYesPAdjusted OR ± ± n ± [“][ CI]n ± n ± [“][ CI] [“] [“]Other type of exposure Health professionalOther health workersSmoking exsmokersmoker”nototal no Temperature at admission” ˚C Patients with fever �˚C”nototal no Time from symptom onset tomedical visit”days¡Symptoms”no ¶ ± ± ± ± ± ± NANANA [“] [“] [“] [“] ± ± ± [“] [“] ± [“] NANANA [“]CoughGeneral malaiseFatigue [“] [“] [“] [“] [“] [“]Myalgia or arthralgia [“] [“]DyspnoeaDiarrhoeaHeadacheAnosmiaDysgeusiaSore throatBlocked noseNausea or vomitingSputum productionChillsAstheniaChest painAlterations in physical examination”nototal no §Auscultatory alterationsTachypnoeaTachycardiaPharyngitis [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] NA [“]Continued PLOS ONE 101371journalpone0237960 August PLOS ONE 0cTable ContinuedVariablesTotalDeath or ICU admissionHospitalizationPrognostic factors in Spanish COVID19 patientsOxygen saturation �”nototalno n n No n YesPAdjusted ORNoYes[ CI]n n [“] PAdjusted OR[ CI] [“]In bold statistically significant independent predictive factors associated with hospitalization death or ICU admission logistic multivariate regression adjusted for ageand sex  Temperature distribution was ˚C “ËšC “ËšC and ˚C ¡ In patients™ data on period between symptom onset and medical visit were lacking¶ Symptoms with a frequency of patients were disorientation n conjunctivitis n haemoptysis n and cutaneous lesions n § patients had a physical examination The alterations with a frequency of patients were cutaneous lesions n and tonsillopharyngitis n Ref reference NA not applicable101371journalpone0237960t001p headache vs p dysgeusia vs p andanosmia vs p were less frequent in patients admitted to the ICU or whodied than the remaining patients Age OR 95CI to and male sexOR 95CI to were independent predictors of ICU admission and deathMyalgia or arthralgia OR 95CI to was the only significant protective factor against ICU admission and death after adjusting for age and sex Fig The best clinicalpredictors of hospitalization were chills OR 95CI to fever OR 95CI to and dyspnoea OR 95CI to Anosmia OR 95CI to was the only significant protective factor for hospitalization after adjusting for age and sex Table and Fig Comorbidities were presented by patients the most common were hypertension in diabetes mellitus in and obesity in Table Heartdisease vs p autoimmune disease vs p diabetes vs p hypertension vs p and chronic kidney disease vs p were the comorbidities significantly associated with ICUadmission and death Table Autoimmune disease was the only significant predictivecomorbidity for ICU admission and death after adjusting for age and sex OR CI to Fig Depression was the best predictor of hospitalization among allcomorbidities OR 95CI to Fig Having � comorbidity was associated with ICU admission and death OR 95CI to and hospitalizationOR 95CI to independently of age and sexImaging and laboratory testsChest Xray was necessary in patients and showed lobar pulmonary infiltrates in bilateral pulmonary infiltrates in and an interstitial pattern in Table Chest CT was required in patients and pulmonary ultrasound in Biologically of patients had lymph ia mm3 Likewise had a lactate dehydrogenase LDH Uml and liver test alterations were commonelevated ASTGOT in and ALTGPT in In of cases Ddimer waselevated 500mgL The most important factors for ICU admission and death were bilateralpulmonary infiltrates OR 95CI to elevated lactatedehydrogenaseOR 95CI to elevated Ddimer OR 95CI to andelevated Creactive protein OR 95CI to Fig Significant predictivePLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientsFig Prognostic factors for death and ICU admission �The upper limits of the confidence intervals were restricted to in order not to mask the significant effects of other variables with smaller ranges101371journalpone0237960g001factors associated with hospitalization after adjusting for age and sex were lymph iaOR 95CI to and elevated Creactive protein OR 95CI to Fig Treatment complications and evolutionTreatment included hydroxychloroquine in patients azithromycin in lopinavirritonavir in glucocorticoids in and tocilizumab in among others Table and of patients required hospitalization Phone follow up was registered in patients patients were monitored at homePLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientsFig Prognostic factors for hospitalization �The upper limits of the confidence intervals were restricted to inorder not to mask the significant effects of other variables with smaller ranges101371journalpone0237960g002 of the patients of working age sought work disability due to COVID19 TheICU admission rate was The evolution included pneumonia in patientsadult respiratory distress syndrome in severe renal failure in pulmonarythromboembolism in and sepsis in patients Occupational contact with persons with confirmed or suspected COVID19 infection was reported by patientswhile reported that contact occurred in the family setting Occupational contactwas a protective factor against hospitalization OR 95CI to ICU admission and death OR 95CI to after adjusting for age and sex The mortalityrate to date was PLOS ONE 101371journalpone0237960 August PLOS ONE 0cTable Comorbidities associated with hospitalization and ICU admissiondeathVariablesTotalDeath or ICU admissionHospitalizationn NoYesPAdjusted OR [NoYesPAdjusted OR [n n CI]n n CI]Prognostic factors in Spanish COVID19 patientsComorbidities”no  Any comorbidityHypertensionDiabetesObesityDyslipidaemiaCancer [“] [“] [“] [“] [“] [“] [“] [“] Chronic kidney disease Heart disease Autoimmune disease Chronic obstructive pulmonary diseaseDepressionCardiac arrhythmiaThyroid alterationsAsthmaLiver diseaseCerebrovascular diseaseAlzheimer disease [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“]In bold statistically significant independent predictive factors associated with hospitalization death or ICU admission logistic multivariate regression adjusted for ageand sex  Comorbidities with a frequency of patients were bronchiectasis n fibromyalgia n anaemia n arthritis n HIV n syphilis n andtuberculosis n 101371journalpone0237960t002DiscussionThis study summarizes the clinical biological and radiological characteristics evolution andprognostic factors of patients with COVID19 disease in primary and community healthcareTo date we are aware of three published Spanish studies [“] The first reported data from patients on ICU admissions in a region where the pandemic was reported early [] Thestudy by Borobia [] describes the first adult patients with COVID19 consecutivelyadmitted to a University Hospital in Madrid The third focuses on the differences by agedependent categories in the clinical profile presentation management and shortterm outcomes [] Although there have been two systematic reviews and metaanalysis that analysethe clinical characteristics of COVID19 they are limited to Chinese cohorts or case series [] and a large USA cohort [] that did not analyse clinical predictors of a poor prognosisClinically the same main symptoms of cough and fever are reported in all series Howeverin Barcelona city we have observed diarrhoea anosmia and dysgeusia which is hardlyreported in the Chinese series [] which unlike ours comes principally from hospitals diarrhoea occurred in of cases very similar to the in New York [] and clearly higherthan the reported in China Nearly of patients had anosmia and dysgeusia similar tothe results obtained in French patients [] In contrast expectoration was found in only compared with in the Chinese seriesPLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientsTable Analytical and radiological predictors of hospitalization and ICU admissiondeathVariablesTotalDeath or ICU admission n Hospitalization n n NoYesPAdjusted ORNoYesn n [ CI]n n PAdjusted OR[ CI]Alterations in chest Xray”nototalno  Bilateral pulmonary infiltratesInterstitialground glass patternLobar pulmonary infiltrateAlterations in chest CAT scan”nototal no ¡ [“] [“] [“] [“] [“] [“]Bilateral pulmonary infiltrates Interstitialground glass pattern Laboratory parameters”nototalno [“] [“] [“] [“]Leukocytes mm3 [“] [“]Lymphocytes mm3Platelets mm3 [“] [“] [“] [“]Haemoglobin gdl [“] [“]Creactive protein mglitreProcalcitonin ngmlLactate dehydrogenase UlitreAminotransferase aspartate UlitreAlanine aminotransferase UlitreTotal bilirubin mgdL [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“]Creatine kinase Ulitre [“] [“]Creatinine 15mgdL Ddimer mglitreSodium mEqlitrePotassium mEqlitre [“] [“] [“] [“] [“] [“] [“] [“]In bold statistically significant independent predictive factors associated with hospitalization death or ICU admission logistic multivariate regression adjusted for ageand sex  patients had a chest Xray The alterations with a frequency patients were pneumothorax n and pleural effusion n Chest Xray resultswere not available in patients¡ patients had a chest CAT scan Alterations with a frequency of patients were pulmonary thromboembolism n emphysema n lobarpulmonary infiltrates n pneumonia n atelectasis n and pleural effusion n CAT scan results were not available in five patients101371journalpone0237960t003PLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientsTable Predictors of the evolution complications and treatment in patients hospitalized or with ICU admissiondeathVariablesTotalDeath or ICU admission n Hospitalization n n NoYesPAdjusted OR [NoYesPAdjusted OR [n n CI]n n CI]Complications”no  Any complicationPneumonia NA [“] [“] [“]Adult respiratory distress [“ [“]syndromeRenal failurePulmonary thromboembolismTreatments”no ¡HydroxychloroquineAzithromycinLopinavirRitonavir [“] [“] [“]NA] [“] [“] [“] [“] [“] [“]Oxygen therapy [“] [“]Intravenous antibiotics [“] [“]GlucocorticoidsTocilizumabCephalosporinsLow molecular weight heparin Remdesivir Covid19 infection”no [“] [“] [“] [“] [“] [“] [“] [“] [“] [“]Any cohabitant [“] [“]Any work colleague [“] [“]Any contact person in other [“] [“]settingsIn bold statistically significant independent predictive factors associated with hospitalization death or ICU admission logistic multivariate regression adjusted for ageand sex  Complications in patients were sepsis n multian failure n electrolyte alterations n hematologic alterations n and lung cancer n ¡ Treatments with a frequency of patients except remdesivir were amoxicillin n interferon n rituximab n darunavir n and entecavirn NA not applicable101371journalpone0237960t004Chinese patients had a mean age of years ten years lower than our series and ofour patients were aged � years compared with and in China Germany andthe USA respectively but in Italy [ “] Older age and male sex predisposed to ahigher mortality rate in our and all large series [ ] In our patients comorbidities werethree times higher than in the Chinese cohort [] and were similar to the findings of the NewYork study [] Any comorbidity was a risk factor for hospitalization ICU admission anddeath Depression was an independent risk factor for hospitalization which has not beenobserved in other cohorts studied Depression was often accompanied by a vulnerable socialsituation which may have justified hospitalization Likewise autoimmune diseases were independent risk factor for ICU admission and death Various hypotheses have been postulated onpossible autoimmune alterations in the pathogenic evolution of the disease With respect toPLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientstreatment no drug has proved effective against Covid19 until now Moreover many treatments were unavailable in the outpatient setting Currently we are only certain that treatmentwith tocilizumab showed better survival rates in retrospective cohorts [] although its efficacy has not been tested in randomized clinical trials Therefore the results on the outcomesassociated with treatment should be interpreted with cautionThe same comorbidities were identified with hypertension and diabetes being the twomost common while in the USA and Italy obesity seems to be higher Our results show thatobesity was close to being an independent risk factor for hospitalization OR CI to Strikingly of our patients were healthcare workers compared with in Wuhanand in Germany [ ] Although these studies recognized an important degree ofunderreporting of cases in health workers the difference remains important There are at leasttwo possible explanations first the lack of personal protective equipment in the initial phaseof the epidemic a constant revindication of health professionals who felt undersupplied Secondly many cases were health professionals from primary healthcare or the reference hospitalwho reside in the same area where they workIn all reported series bilateral pneumonia was the most common radiological finding waspresent in more than half the cases [] and was a factor of a poor prognosis and mortality Incontrast an interstitial radiological pattern did not confer an increased risk of mortality TheWuhan study reported a CAT scan use of compared with in Barcelona In contrast chest Xrays were carried out in and respectively the availability of diagnostic means was higher in China A recent international consensus states that radiologicalassessment is not necessary in asymptomatic patients or those with mild disease but is requiredin patients with moderate or severe disease regardless of whether a definite diagnosis ofCOVID19 has been made [] In addition simple chest Xrays are preferable in a resourceconstrained environment with difficulties in accessing CAT scans [] The possible use of pulmonary ultrasound for the pointofcare diagnosis of COVID19 pneumonia has not been sufficiently analysed but might be an efficient alternative due to its portability and reliability []In fact the regional Catalan government has recently acquired ultrasound machines toenable family physicians to make doctors can make pointofcare home or nursing homediagnoses of pneumonia [] Biologically lymph ia and increased CRP LDH and Ddimer were usually constant and similar in all series and were associated with an increased riskof mortality A differential variable in our series is a greater number of alterations in liver testswhich was present in “ of patients data similar to the USA and Italian cohorts but different from the Chinese cohort where it was [] We also found hypokalaemia in of patients a factor not reported in other studiesWe found a hospitalization rate of compared with “ in the USA and inChina and an ICU admission rate of which was similar to the Chinese USA “ and German results While the protocols of action and admission are similarand depend on the level of clinical involvement the therapeutic protocols differ between hospitals cities and countries There remain many unknowns in the treatment of COVID19The only truth is that we do not have a vaccine an etiological treatment or a treatment withsufficient scientific evidence to generalize its use Currently the systematic review of antiretroviral treatments has not offered conclusive results [] and despite in vitro results for hydroxychloroquine COVID19 infections are currently intractable [ ]The mortality rate in our study was compared with in New York in hospitalized patients in China in Germany and in Italy Different informationand recording systems the availability of diagnostic tests and above all the anization ofnational health systems may have contributed to the differences observedPLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientsThe study had some limitations due to the observational retrospective design However itis sufficiently representative of the population with confirmed COVID19 to permit betteridentification of the factors of a poor prognosis of the disease from a clinical perspective Wecannot rule out some heterogeneity in data codification due to observers™ interpretations of themedical records However this bias is minimal as most clinical factors included are clearlydefined in the electronic medical record Another limitation of this study is the percentage ofpatients without laboratory parameters more than Even though in real clinical practicethese percentages may be expected the results corresponding to laboratory parameters shouldbe interpreted with cautionFour months after the declaration of the pandemic there is not a sufficiently reliable available and generalizable diagnostic test that can analyse the seroprevalence of COVID19 evenin the most industrialized countries Given this lack determining the clinical biological andradiological characteristics of probable cases of COVID19 infection will be key to the initiation of early treatment and isolation and for contact tracing especially in primary healthcareSupporting informationS1 DatasetXLSXAcknowledgmentsThe authors thank David Buss for editorial assistanceAuthor ContributionsConceptualization Antoni Siso´Almirall Belchin Kostov Jaume BenaventÃreuData curation Antoni Siso´Almirall Belchin Kostov Minerva MasHeredia Sergi VilanovaRotllan Ethel SequeiraAymar Mireia SansCorrales Elisenda SantArderiu Laia CayuelasRedondo Angela Martı´nezPe´rez Noemı´ Garcı´aPlana August AnguitaGuimetJaume BenaventÃreuFormal analysis Antoni Siso´Almirall Belchin Kostov Jaume BenaventÃreuInvestigation Antoni Siso´Almirall Belchin Kostov Minerva MasHeredia Sergi VilanovaRotllan Ethel SequeiraAymar Mireia SansCorrales Elisenda SantArderiu Laia CayuelasRedondo Angela Martı´nezPe´rez Noemı´ Garcı´aPlana August AnguitaGuimetJaume BenaventÃreuMethodology Antoni Siso´Almirall Belchin Kostov Minerva MasHeredia Sergi VilanovaRotllan Ethel SequeiraAymar Mireia SansCorrales Elisenda SantArderiu Laia CayuelasRedondo Angela Martı´nezPe´rez Noemı´ Garcı´aPlana August AnguitaGuimetJaume BenaventÃreuProject administration Antoni Siso´Almirall Belchin Kostov Jaume BenaventÃreuResources Antoni Siso´Almirall Belchin Kostov Jaume BenaventÃreuSupervision Antoni Siso´Almirall Belchin Kostov Jaume BenaventÃreuValidation Antoni Siso´Almirall Belchin Kostov Minerva MasHeredia Sergi VilanovaRotllan Ethel SequeiraAymar Mireia SansCorrales Elisenda SantArderiu Laia CayuelasRedondo Angela Martı´nezPe´rez Noemı´ Garcı´aPlana August AnguitaGuimetJaume BenaventÃreuPLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientsVisualization Antoni Siso´Almirall Belchin Kostov Jaume BenaventÃreuWriting “ original draft Antoni Siso´Almirall Belchin Kostov Jaume BenaventÃreuWriting “ review editing Antoni Siso´Almirall Belchin Kostov Minerva MasHerediaSergi VilanovaRotllan Ethel SequeiraAymar Mireia SansCorrales Elisenda SantArderiu Laia CayuelasRedondo Angela Martı´nezPe´rez Noemı´ Garcı´aPlana AugustAnguitaGuimet Jaume BenaventÃreuReferences World Health anization Pneumonia of unknown cause”China wwwwhointcsrdon05january2020pneumoniaofunkowncausechinaen accessed April Centers for Disease Control and Prevention Novel Coronavirus 2019nCoV Situation Summarystackscdcgovviewcdc84806cdc_84806_DS1pdf accessed April Holshue ML DeBolt C Lindquist S Lofy KH Wiesman J Bruce H First Case of Novel Coronavirus in the United States N Engl J Med “ 101056NEJMoa2001191 PMID World Health anization Novel Coronavirus2019nCoV wwwwhointdocsdefaultsourcecoronavirusesituationreports20200130sitrep10ncovpdfsfvrsnd0b2e480_2 accessed AprilJohns Hopkins University CSSE COVID19 Dashboard by the Center for Systems Science and Engineering CSSE at Johns Hopkins University JHU gisanddatamapsarcgiscomappsopsdashboardindexhtmlbda7594740fd40299423467b48e9ecf6 accessed April Ministerio de Sanidad Gobierno de España Situacio´n del COVID19 en España covid19isciiies accessed April Guan WJ Ni ZY Hu Y Liang WH Ou CQ He JX Clinical Characteristics of Coronavirus Disease in China N Engl J Med 101056NEJMoa2002032 PMID Yeo C Kaushal S Yeo D Enteric involvement of coronaviruses is faecaloral transmission of SARSCoV2 possible Lancet Gastroenterol Hepatol “ 101016S2468 PMID Chang BB Chiu TY Ready for a long fight against the COVID19 outbreak an innovative model oftiered primary health care in Taiwan BJGP 103399bjgp 20X101068PMID Barrasa H Rello J Tejada S Martı´n A Balziskueta G Vinuesa C SARSCov2 in Spanish Intensive Care Early Experience with 15day Survival In Vitoria Anaesth Crit Care Pain Med 101016jaccpm202004001 PMID Borobia AM Carcas AJ Arnalich F A´ lvarezSala R MonserratVillatoro J Quintana M A Cohortof Patients with COVID19 in a Major Teaching Hospital in Europe J Clin Med Martı´nSa´nchez FJ Del Toro E Cardassay E Valls Carbo´ A Cuesta F Vigara M Clinical presentation and outcome across age categories among patients with COVID19 admitted to a Spanish Emergency Department Eur Geriatr Med 101007s41999020003592 PMIDFu L Wang B Yuan T Chen X Ao Y Fitzpatrick T Clinical characteristics of coronavirus disease COVID19 in China A systematic review and metaanalysis J Infect 101016jjinf202003041 PMID RodriguezMorales AJ CardonaOspina JA Gutie´rrezOcampo E VillamizarPeña R HolguinRiveraY EscaleraAntezana JP Clinical laboratory and imaging features of COVID19 A systematicreview and metaanalysis Travel Med Infect Dis 101016jtmaid2020101623PMID Richardson S Hirsch JS Narasimhan M Crawford JM McGinn T Davidson KW PresentingCharacteristics Comorbidities and Outcomes Among Patients Hospitalized With COVID19 inthe New York City Area JAMA 101001jama20206775 PMID Goyal P Choi JJ Pinh Answer:

Thyroid_Cancer

edible fungus with a high nutritional value and medicinal effect theBachu mushroom has a broad market To distinguish among Bachu mushrooms with highvalue and other fungi effectively and accurately as well as to explore a universal identification method this study proposed a method to identify Bachu mushrooms by Fourier Transform Infrared Spectroscopy FTIR combined with machine learning In this experimenttwo kinds of common edible mushrooms Lentinus edodes and club fungi were selectedand classified with Bachu mushrooms Due to the different distribution of nutrients in thecaps and stalks the caps and stalks were studied in this experiment By comparing the average normalized infrared spectra of the caps and stalks of the three types of fungi we founddifferences in their infrared spectra indicating that the latter can be used to classify andidentify the three types of fungi We also used machine learning to process the spectraldata The overall steps of data processing are as follows use partial least squares PLS toextract spectral features select the appropriate characteristic number use different classification algorithms for classification and finally determine the best algorithm according to theclassification results Among them the basis of selecting the characteristic number was thecumulative variance interpretation rate To improve the reliability of the experimental resultsthis study also used the classification results to verify the feasibility The classification algorithms used in this study were the support vector machine SVM backpropagation neuralnetwork BPNN and knearest neighbors KNN algorithm The results showed that thethree algorithms achieved good results in the multivariate classification of the caps andstalks data Moreover the cumulative variance explanation rate could be used to select thecharacteristic number Finally by comparing the classification results of the three algorithms the classification effect of KNN was found to be the best Additionally the classification results were as follows according to the caps data classification the accuracy was according to the stalks data classification the accuracy was This studyshowed that infrared spectroscopy combined with a machine learning algorithm has thepotential to be applied to identify Bachu mushrooms and the cumulative variancea1111111111a1111111111a1111111111a1111111111a1111111111 ACCESSCitation Gao R Chen C Wang H Chen C Yan ZHan H Classification of multicategoryedible fungi based on the infrared spectra of capsand stalks e0238149 101371journalpone0238149Editor Jie Zhang Newcastle University UNITEDKINGDOMReceived April Accepted August Published August Peer Review History PLOS recognizes thebenefits of transparency in the peer reviewprocess therefore we enable the publication ofall of the content of peer review and authorresponses alongside final published s Theeditorial history of this is available here101371journalpone0238149Copyright Gao This is an access distributed under the terms of theCreative Commons Attribution License whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are creditedData Availability Statement All relevant data arewithin the manuscript and its SupportingInformation filesFunding This work has been supported by theScience and Technology Project on aid to XinjiangPLOS ONE 101371journalpone0238149 August PLOS ONE 0cUygur Autonomous Region No2018E02058 theNational Science Foundation of ChinaNo61765014 Xinjiang Uygur AutonomousRegion Graduate Innovation Project of ChinaNational Innovation Program for College studentsNo201910755039 the Urumqi Science andTechnology Project No P161310002 theReserve Talents Project of the National HighlevelPersonnel of Special Support ProgramQN2016YX0324Competing interests The authors have declaredthat no competing interests existClassification of multicategory edible fungi based on the infrared spectra of caps and stalksexplanation rate can be used to select the characteristic number This method can also beused to identify other types of edible fungi and has a broad application prospect IntroductionThe Bachu mushroom is a characteristic edible fungus in Xinjiang China It belongs to the Saddle fungus genus and is produced in the natural Populus euphratica forest region of the Yeerqiang River Basin in Xinjiang [] The Bachu mushroom not only has high nutritional valuerich in various amino acids and proteins but also has high medicinal value [] Studies haveshown that the Bachu mushroom has antitumor antioxidation and cholesterollowering effectsand is used to treat gastric cancer cerebral arteriosclerosis cardiovascular disease hypertensionand other diseases [] Its nutritional value is much higher than that of general edible fungithus it is of great research value However because the Bachu mushroom cannot be cultivatedartificially the market has been in short supply increasing its price Presently the processingtechnology of the Bachu mushroom is developing and relatively mature such as polysaccharideextraction of the Bachu mushroom and preparation of compound beverages and these processing technologies have a wide application prospect [“] In the future after the derivatives ofthe Bachu mushroom are mass produced and the treatment process is widely used controllingthe quality of raw materials will be very significant to ensure product quality Therefore to prevent businesses from choosing other lowpriced mushrooms as raw materials for high profits itis necessary to identify a simple and rapid way to distinguish Bachu mushrooms from othertypes of mushrooms However current methods to identify Bachu mushrooms and other ediblefungi depend on appearance This method can distinguish mushroom species to a large extentbut it also has great limitations”that is it is limited to individual intact edible bacteria”but liquid extract and powdered mushroom powder cannot be distinguished Thus to overcome thelimitations of conventional methods and explore a more universal mushroom classificationmethod the spectral data of Bachu mushroom powder and two other types of mushroom powder were measured and the classification of Bachu mushroom powder and other types of mushroom powder was identified by a machine learning algorithm in this studyDue to the differences in the types and contents of nutrients in the stalks and caps of ediblefungi the caps and stalks were analyzed in this study [] Previous studies have shown somedifferences in the contents of proteins and amino acids between caps and stalks in edible fungi[] Additionally the difference in the distribution of nutrients is related to the species of edible fungi [] Therefore to make the experimental results more accurate and persuasive aswell as to avoid the uneven distribution of the substance content in the samples this studyadopted the classification analysis method of grouping according to the attribute index tostudy the caps and stalks []The research method used in this experiment classified the Bachu mushroom and two othertypes of edible fungi by infrared spectra combined with a machine learning algorithm Infraredspectra have the characteristics of wide applicability high efficiency convenience repeatabilityand high sensitivity thus it has been widely used in physics remote sensing biology foodmedical and other research fields [ ] Infrared spectroscopy has great application value infood research [ ] Additionally infrared spectroscopy combined with a machine learningalgorithm has been applied to the classification of mushroom as food the effect of the producing area on the nutritional value of fungi and the fine classification of rare edible fungi [“] The purpose of this study was to explore a universal identification method to identifyBachu mushroom using infrared spectra combined with a machine learning algorithm andPLOS ONE 101371journalpone0238149 August PLOS ONE 0cClassification of multicategory edible fungi based on the infrared spectra of caps and stalksverify the feasibility of the application of infrared spectra combined with an algorithm in theidentification of edible mushroom species Experimental methods Sample preparationIn this study Lentinus edodes and club fungi were selected and classified with Bachu mushrooms Among them Lentinus edodes are produced in Fujian Province of China and purchased from Fuchang Food Limited Company Fujian Province of China club fungi areproduced in Yunnan Province of China purchased from Wuweijin Store and Bachu mushrooms are produced in Bachu County Xinjiang Province and purchased from the mostfamous wholesale market in Urumqi”Six Markets The three kinds of mushrooms purchasedare all dried The caps of the three kinds of mushrooms are umbrellashaped and dark brownThe stalks of club fungi are longer and those of Lentinus edodes and Bachu mushrooms areshorter The three kinds of mushrooms are similar in appearance The samples of the threetypes of edible fungi were purchased from the market After incubating the prepared sample inan ˚C electric steam oven for dehydration treatment for hour the stalks were separatedfrom the caps Next every three complete caps were crushed together and the powder waspassed through a 200mesh sieve as a sample and named according to the mushroom speciesThe stalks were treated in the same way Finally samples of Lentinus edodes powder samples of club fungi powder and samples of Bachu mushroom powder were obtained Measurement of NIF spectraThe sample powder was placed into a 4ml sample tube and its infrared spectrum was measured The spectrum acquisition instrument was a VERTEX infrared spectrometer fromBRUKER Germany Before each measurement of the FTIR spectrum the atmospheric background data were measured using OPUS65 software The selected resolution was cm1 thenumber of scans was the scan range was “ cm1 and the atmospheric compensation parameter was CO2 To reduce the influence of human error and other factors each sample was scanned times Finally the data obtained for the caps were for Lentinus edodes for club fungi for Bachu mushrooms and the same number of stalks data Statistical algorithm analysisStatistical algorithms have been widely used to manage infrared spectral data [] In thisstudy PLS SVM KNN and BPNN were used to process and analyze the spectral data Thecaps data were reduced by PLS to extract features and then the appropriate characteristicnumber was selected as the input of the three classification algorithms namely SVM KNNand BPNN and then the accuracy was obtained Additionally the stalk data were processed inthe same way All algorithms in this study are implemented on MATLAB 2018aThe partial least squares PLS method is a mathematical optimization supervised learningmethod that can identify the best matching function for a set of data by minimizing the sum ofthe squares of errors Based on the advantages of the PLS model which is easy to identify noiseand allows regression modeling with a small sample number the PLS algorithm is widely usedin various research fields [ ] In food research PLS has been used in food nutrition testingfood quality research and food industry research [“] PLS is often used in combinationwith spectra for feature extraction and further spectral data analysis [] In this study toimprove the classification efficiency and filter out the worthless spectral information PLS wasused to reduce the dimensionality of the original spectral dataPLOS ONE 101371journalpone0238149 August PLOS ONE 0cClassification of multicategory edible fungi based on the infrared spectra of caps and stalksAfter dimensionality reduction of the original data an appropriate characteristic numbershould be selected as the basis for classification In this study the characteristic number wasselected based on the cumulative variance interpretation rate of the characteristic number ThePLS program used in this study is the plsregress function The variance explanation rates of thefactors extracted from the first and second columns of the PCTVAR matrix correspond to thevariances of x and y respectively this study chose the variance explanation rate of y [] Thevariance interpretation rate is the degree of interpretation of the data characteristics of thedependent variables by a single factor and the cumulative interpretation rate of n factors is thedegree of interpretation of the data characteristics of the dependent variables by n factors”that is the influence of n factors on the dependent variables Therefore in theory we can selectthe appropriate number of factors according to the cumulative variance interpretation rateand select as few factors as possible to improve the classification efficiency to ensure the integrity of the extracted features To explore the applicability of the theory this study will furtheruse the classification results to verify the theory After selecting an appropriate characteristicnumber it can be used as the input of the classification algorithms SVM KNN and BPNNSupport vector machine SVM is a commonly used generalized linear classifier in whichthe core idea is to apply the principle of risk minimization to the field of classification Regarding pattern classification it has good generalization performance robustness versatility andsimple calculation [] Therefore in food science SVM is widely applied to food classificationand food quality testing [ ] Based on the advantages of SVM and characteristics that canbe used for multiple classifications in this study SVM was used to classify the spectral data ofmushroom powder directly after three features were extractedThe knearest neighbors KNN classification algorithm is one of the most practical algorithms in data mining classification technology It is easy to understand and powerful at thesame time [] Different from other classification algorithms KNN does not need training Itdirectly finds the k samples nearest to the sample and divides them into categories with thelargest number of samples among the k samples thus KNN is suitable for multivariate classification and has high classification accuracy when the category boundary is obvious [] Additionally KNN has been widely used in food classification and quality inspection [“]Therefore we chose the KNN algorithm as the second algorithm of multivariate classificationThe backpropagation neural network BPNN is a multilayer feedforward neural networktrained according to the error backpropagation algorithm The BP neural network has strongnonlinear mapping ability parallel information processing ability and excellent selflearningability thus it has been widely used in food research biomedical fields and other researchfields [“] Additionally the BP neural network can achieve good classification resultswhen it is used in multivariate classification [] In summary we chose the BPNN as the thirdclassification algorithm Results and discussion Spectral analysisAfter the obtained spectral data were averaged normalized and smoothed the obtained spectrogram is shown in Fig Fig shows the FTIR spectra of the Bachu mushroom and Lentinusedodes stalks were similar both with characteristic peaks at cm1 and cm1 and thespectral intensity of Lentinus edodes was higher than that of the Bachu mushroom Additionally the spectrum of the Lentinus edodes stalk has a characteristic peak at cm1 and thespectral intensity in the range of cm1 was significantly lower than that of Lentinusedodes and the Bachu mushroom Comparing the spectra of the three caps the spectra of clubfungi are quite different from those of the other two types of fungi Fig shows that the averagePLOS ONE 101371journalpone0238149 August PLOS ONE 0cClassification of multicategory edible fungi based on the infrared spectra of caps and stalksFig The average normalized spectrum of the caps of the three types of edible fungi101371journalpone0238149g001normalized spectra of Lentinus edodes club fungi and Bachu mushroom stalks all had peaks at cm1 and cm1 and the spectral intensity of Lentinus edodes was the highest in thesetwo places At cm1 the peak intensity of club fungi was higher than that of the Bachumushroom however at cm1 the peak intensity of club fungi was slightly lower than thatof the Bachu mushroom Although the three spectral lines changed roughly the same howeverin the range of cm1 the spectral intensity of the Bachu mushroom was significantlylower than that of the other two kinds of fungi and the spectral intensity of the Bachu mushroom was significantly lower than that of the other two kinds of fungiThrough comparative analysis of the infrared spectra of the caps and stalks of the threetypes of fungi their infrared spectral images showed the same trend but many peak intensitieswere different Therefore we can classify them according to the spectral data based on thePLOS ONE 101371journalpone0238149 August PLOS ONE 0cClassification of multicategory edible fungi based on the infrared spectra of caps and stalksFig The average normalized spectrum of the stalks of the three types of edible fungi101371journalpone0238149g002infrared spectral differences among the three types However it is difficult to distinguishamong the three types of edible fungi directly and accurately only by spectroscopy Thus toclassify them efficiently and accurately we used the combined infrared spectrum analysis withmachine learning Data analysis Dimensionality reduction with PLSIn the PLS algorithm features wereselected to obtain the cumulative variance explanation rate curve Figs and The cumulative variance explanation rate of the first five features of the caps reached while the variance explanation rate of the first five features of the stalks also reached more than and thePLOS ONE 101371journalpone0238149 August PLOS ONE 0cClassification of multicategory edible fungi based on the infrared spectra of caps and stalksFig The principal component cumulative variance explanation rate curve of the caps101371journalpone0238149g003cumulative variance explanation rate of the first features of both was close to Figs and Thus the extracted features can fully express the features of the original data [] Thestalks data and caps data were classified by the SVM BPNN and KNN algorithms Classification by algorithmIn this experiment three classification algorithmswere used”SVM BPNN and KNN”to classify the caps data according to different characteristic numbers Additionally the stalk data were processed in the same way The parameter setting and classification results of the algorithm were as followsThe main ideas of the SVM model in this experiment were as follows Select the test set andtraining set preprocess the data select the best C and g parameters and then use the bestparameters for network training and prediction and obtain the accuracy Among them thetraining set and test set are randomly selected according to the proportion Preprocessingwas used to normalize all the sample data [] In the SVM algorithm the selection of C and gPLOS ONE 101371journalpone0238149 August PLOS ONE 0cClassification of multicategory edible fungi based on the infrared spectra of caps and stalksFig The principal component cumulative variance explanation rate curve of the stalks101371journalpone0238149g004parameters will directly affect the classification results thus it is necessary to select the best Cand g parameters to achieve the best classification results In this study the variation range ofparameter C was [“] the range of parameter g was [“] and the method of parameteroptimization was grid optimization [] To classify the caps data and features were selected The stalks data were processed in the same way The multipleclassification results of the stalks and caps were then obtained as shown in Table In the KNN algorithm the k value was the proportion of the random selection of the testset was and the method to calculate the distance between data was the cosine distanceCosine KNN [] To classify the caps data and featureswere selected The stalks data were processed in the same way Each result was expressed as theaverage of five computational results The multiple classification results of the stalks and capswere then obtained as shown in Table PLOS ONE 101371journalpone0238149 August PLOS ONE 0cClassification of multicategory edible fungi based on the infrared spectra of caps and stalksTable Classification results of the SVM algorithmNumber of PLS componentsClassification accuracy of CapsClassification accuracy of Stalks101371journalpone0238149t001In the BPNN algorithm of this experiment the transfer function of the hidden layer wastamsig the output layer was purelin the learning training function was trainlm and the weightlearning function was learngdm The network parameters were set to training times thenetwork performance goal was and the learning rate was [] Thirty percent of allsamples were selected randomly as the test set To classify the caps data and features were selected The stalks data were processed in the same way Eachresult was expressed as the average of five computational results The multiple classificationresults of the stalks and caps were then obtained as shown in Table Verification of the feasibility of selecting the characteristic number with thecumulative variance explanation rate Figs and shows the line chart of the classificationresults of the three algorithms when selecting different characteristic numbers The accuracyof SVM in the classification of caps was when selecting and features decreasedslightly with the increase in the characteristic number and then stabilized at Accordingto the fungal stalks data the accuracy of SVM classification increased gradually when selecting features fluctuated slightly with the increase in the characteristic number andthen stabilized at Using KNN to classify the caps data the accuracy was stable between and but fluctuated slightly When the characteristic number tended to theaccuracy was stable at According to the classification of stalks data by KNN the accuracy varied greatly when selecting “ features fluctuated slightly with the increase in thecharacteristic number and then stabilized at For the classification of caps by BPNNthe accuracy varied greatly when the characteristic number was and the classificationTable Classification results of the KNN algorithmNumber of PLS componentsClassification accuracy of CapsClassification accuracy of Stalks101371journalpone0238149t002PLOS ONE 101371journalpone0238149 August PLOS ONE 0cClassification of multicategory edible fungi based on the infrared spectra of caps and stalksTable Classification results of the BPNN algorithmNumber of PLS componentsClassification accuracy of CapsClassification accuracy of Stalks 101371journalpone0238149t003Fig Accuracy of the three algorithms to select different characteristic numbers according to the data of the caps101371journalpone0238149g005PLOS ONE 101371journalpone0238149 August PLOS ONE 0cClassification of multicategory edible fungi based on the infrared spectra of caps and stalksFig Accuracy of the three algorithms to select different characteristic numbers according to the data of the stalks101371journalpone0238149g006effect was unstable When the characteristic number was more than the accuracy decreasedslightly and finally stabilized at When BPNN classified stalks its accuracy variedgreatly when the characteristic number was less than when the characteristic number wasmore than the accuracy was stabilized at Combined with the cumulative variance explanation rate with the increase in the characteristic number the extraction degree of the extracted features to the original data informationgradually increased Thus the classification results become more reliable and the accuracywill gradually tend to a certain value”that is in the ideal state the accuracy of all featuresextracted for classification However in this process not all information is conducive toimproving accuracy and some information will interfere with the classification results thusthe accuracy will fluctuate slightly [] Therefore when we select the characteristic numberaccording to the variance interpretation rate we should consider the degree of featurePLOS ONE 101371journalpone0238149 August PLOS ONE 0cClassification of multicategory edible fungi based on the infrared spectra of caps and stalksextraction accuracy and classification efficiency comprehensively In summary when thecumulative variance interpretation rate of features reaches the classification results aresufficiently reliable and the accuracy is high confirming that it is feasible to select the characteristic number according to the cumulative variance interpretation rate Selection of the best algorithm According to the caps data classification thePLSSVM algorithm has the best classification effect when selecting features with anaccuracy of The PLSKNN algorithm has the best classification effect when selecting features with an accuracy of When selecting features the PLSBPNN algorithmhas the best classification effect with an accuracy of Fig According to the stalksdata classification the classification effect of the PLSSVM algorithm was the best when selecting features with an accuracy of The classification effect of the PLSKNN algorithm was the best when selecting features with an accuracy of When featureswere selected the classification effect of the PLSBPNN algorithm was the best reaching Fig Combined with the selection of the characteristic number to analyze the three algorithmsthe PLSKNN algorithm has a better classification effect on stalks and caps and the accuracy ismore stable when selecting different characteristic numbers Thus the PLSKNN algorithmwas chosen as the optimal algorithm Using comprehensive analysis of the accuracy ofPLSKNN classification when selecting different characteristic numbers a characteristic number of reveals a higher accuracy for both caps and stalks and a high classification efficiencyTherefore in this experiment the PLSKNN algorithm was finally selected and the characteristic number was The final classification accuracy was for the caps and forthe stalks ConclusionsThis study verified the feasibility of infrared spectroscopy combined with the PLSSVMPLSKNN and PLSBPNN algorithms in the classification of the Bachu mushroom and otheredible mushrooms We compared the classification results and selected the optimal algorithmand best feature number to reveal an efficient rapid and universal method to identify theBachu mushroom overcoming the limitation that the current identification of Bachu mushroom only depends on its appearance Moreover the method is universal and can be applied tothe classification and identification of other types of food Additionally this study proposed toselect the characteristic number according to the cumulative variance interpretation rate andused the classification results of the three algorithms to verify its feasibility This method ofselecting the characteristic number can also be extended to other research fields of factor analysis and the appropriate characteristic number can be selected intuitively and quicklySupporting informationS1 DataZIPAuthor ContributionsConceptualization Cheng Chen Zhiao WangData curation Rui Gao Cheng Chen Hang Wang Chen Chen Ziwei Yan Yan WuFormal analysis Hang WangFunding acquisition Cheng Chen Chen Chen Fangfang Chen Zhiao WangPLOS ONE 101371journalpone0238149 August PLOS ONE 0cClassification of multicategory edible fungi based on the infrared spectra of caps and stalksInvestigation Rui Gao Fangfang ChenMethodology Rui Gao Cheng Chen Chen Chen Yuxiu ZhouProject administration Rui Gao Cheng Chen Rumeng Si Xiaoyi LvResources Rui Gao Ziwei Yan Fangfang Chen Yan WuSoftware Ziwei YanSupervision Cheng ChenValidation Rui Gao Rumeng SiWriting “ original draft Rui Gao Hang Wang Zhiao Wang Yuxiu ZhouWriting “ review editing Rui Gao Cheng Chen Huijie Han Xiaoyi LvReferencesZhao Q Species clarification of the culinary Bachu mushroom in western China Mycologia p “ 10385216002 PMID Chen X Wang J and Li H Basic Ingredientsand Nutritional Assessment Of Baehu Mushroom FarmProducts Processing p “Zeng D and Zhu S Purification characterization antioxidant and anticancer activities of novel polysaccharides extracted from Bachu mushroom International journal of biological macromolecules p “ 101016jijbiomac201709088 PMID XuJie H Extraction of BaChu mushroom polysaccharides and preparation of a compound beverage Carbohydrate Polymers p “ XuJie H and Wei C Optimization of extraction process of crude polysaccharides from wild edibleBaChu mushroom by response surface methodology Carbohydrate Polymers p “ Wang CY A Review on the Potential Reuse of Functional Polysaccharides Extracted from the ByProducts of Mushroom Processing Food and Bioprocess Technology p “ Akindahunsi AA and Oyetayo FL Nutrient and antinutrient distribution of edible mushroom Pleurotustuberregium fries singer LWT”Food Science and Technology p “Zhu Y and Tan ATL Chemometric Feature Selection and Classification of ltigtGanoderma lucidumltigt Spores and Fruiting Body Using ATRFTIR Spectroscopy American Journal of AnalyticalChemistry p “ Oboh G and Shodehinde S Distribution of nutrients polyphenols and antioxidant activities in the pileiand stipes of some commonly consumed edible mushrooms in Nigeria Bulletin of the Chemical Societyof Ethiopia Ball GH Classification Analysis Ferrari M and Quaresima V A brief review on the history of human functional nearinfrared spectroscopy fNIRS development and fields of application Neuroimage p “ 101016jneuroimage201203049 PMID Chen C Exploration research on the fusion of multimodal spectrum technology to improve performance of rapid diagnosis scheme for Thyroid Dysfunction Journal of biophotonics pe201900099 101002jbio201900099 PMID Van de Voort F Fourier transform infrared spectroscopy applied to food analysis Food Research International p “ Chen C Application of near infrared spectroscopy combined with SVR algorithm in rapid detection of cAMP content in red jujube Optik p Meenu M and Xu B Application of vibrational spectroscopy for classification authentication and quality analysis of mushroom A concise review Food chemistry Li Y Geographical traceability of wild Boletus edulis based on data fusion of FTMIR and ICPAES coupled with data mining methods SVM Spectrochim Acta A Mol Biomol Spectrosc p “ 101016jsaa201701029 PMID Fu H A comprehensive quality evaluation method by FTNIR spectroscopy and chemometricFine classification and untargeted authentication against multiple frauds for Chinese Ganoderma lucidum Spectrochimica Acta Part A Molecular and Biomolecular Spectroscopy p “PLOS ONE 101371journalpone0238149 August PLOS ONE 0cClassification of multicategory edible fungi based on the infrared spectra of caps and stalks Geladi P and DÃ¥bakk E An overview of chemometrics applications in near infrared spectrometryJournal of Near Infrared Spectroscopy p “ Rosipal R and Kra¨mer N Overview and recent advances in partial least squares in International Statistical and Optimization Perspectives Workshop Subspace Latent Structure and Feature Selection Springer Xie L Quantification of glucose fructose and sucrose in bayberry juice by NIR and PLS FoodChemistry p “Ivorra E Detection of expired vacuumpacked smoked salmon based on PLSDA method usinghyperspectral images Journal of food engineering p “ Heyder M Theuvsen L and HollmannHespos T Investments in tracking and tracing systems in thefood industry a PLS analysis Food Policy p “ Mehmood T et a

cancer274

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: edible fungus with a high nutritional value and medicinal effect theBachu mushroom has a broad market To distinguish among Bachu mushrooms with highvalue and other fungi effectively and accurately as well as to explore a universal identification method this study proposed a method to identify Bachu mushrooms by Fourier Transform Infrared Spectroscopy FTIR combined with machine learning In this experimenttwo kinds of common edible mushrooms Lentinus edodes and club fungi were selectedand classified with Bachu mushrooms Due to the different distribution of nutrients in thecaps and stalks the caps and stalks were studied in this experiment By comparing the average normalized infrared spectra of the caps and stalks of the three types of fungi we founddifferences in their infrared spectra indicating that the latter can be used to classify andidentify the three types of fungi We also used machine learning to process the spectraldata The overall steps of data processing are as follows use partial least squares PLS toextract spectral features select the appropriate characteristic number use different classification algorithms for classification and finally determine the best algorithm according to theclassification results Among them the basis of selecting the characteristic number was thecumulative variance interpretation rate To improve the reliability of the experimental resultsthis study also used the classification results to verify the feasibility The classification algorithms used in this study were the support vector machine SVM backpropagation neuralnetwork BPNN and knearest neighbors KNN algorithm The results showed that thethree algorithms achieved good results in the multivariate classification of the caps andstalks data Moreover the cumulative variance explanation rate could be used to select thecharacteristic number Finally by comparing the classification results of the three algorithms the classification effect of KNN was found to be the best Additionally the classification results were as follows according to the caps data classification the accuracy was according to the stalks data classification the accuracy was This studyshowed that infrared spectroscopy combined with a machine learning algorithm has thepotential to be applied to identify Bachu mushrooms and the cumulative variancea1111111111a1111111111a1111111111a1111111111a1111111111 ACCESSCitation Gao R Chen C Wang H Chen C Yan ZHan H Classification of multicategoryedible fungi based on the infrared spectra of capsand stalks e0238149 101371journalpone0238149Editor Jie Zhang Newcastle University UNITEDKINGDOMReceived April Accepted August Published August Peer Review History PLOS recognizes thebenefits of transparency in the peer reviewprocess therefore we enable the publication ofall of the content of peer review and authorresponses alongside final published s Theeditorial history of this is available here101371journalpone0238149Copyright Gao This is an access distributed under the terms of theCreative Commons Attribution License whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are creditedData Availability Statement All relevant data arewithin the manuscript and its SupportingInformation filesFunding This work has been supported by theScience and Technology Project on aid to XinjiangPLOS ONE 101371journalpone0238149 August PLOS ONE 0cUygur Autonomous Region No2018E02058 theNational Science Foundation of ChinaNo61765014 Xinjiang Uygur AutonomousRegion Graduate Innovation Project of ChinaNational Innovation Program for College studentsNo201910755039 the Urumqi Science andTechnology Project No P161310002 theReserve Talents Project of the National HighlevelPersonnel of Special Support ProgramQN2016YX0324Competing interests The authors have declaredthat no competing interests existClassification of multicategory edible fungi based on the infrared spectra of caps and stalksexplanation rate can be used to select the characteristic number This method can also beused to identify other types of edible fungi and has a broad application prospect IntroductionThe Bachu mushroom is a characteristic edible fungus in Xinjiang China It belongs to the Saddle fungus genus and is produced in the natural Populus euphratica forest region of the Yeerqiang River Basin in Xinjiang [] The Bachu mushroom not only has high nutritional valuerich in various amino acids and proteins but also has high medicinal value [] Studies haveshown that the Bachu mushroom has antitumor antioxidation and cholesterollowering effectsand is used to treat gastric cancer cerebral arteriosclerosis cardiovascular disease hypertensionand other diseases [] Its nutritional value is much higher than that of general edible fungithus it is of great research value However because the Bachu mushroom cannot be cultivatedartificially the market has been in short supply increasing its price Presently the processingtechnology of the Bachu mushroom is developing and relatively mature such as polysaccharideextraction of the Bachu mushroom and preparation of compound beverages and these processing technologies have a wide application prospect [“] In the future after the derivatives ofthe Bachu mushroom are mass produced and the treatment process is widely used controllingthe quality of raw materials will be very significant to ensure product quality Therefore to prevent businesses from choosing other lowpriced mushrooms as raw materials for high profits itis necessary to identify a simple and rapid way to distinguish Bachu mushrooms from othertypes of mushrooms However current methods to identify Bachu mushrooms and other ediblefungi depend on appearance This method can distinguish mushroom species to a large extentbut it also has great limitations”that is it is limited to individual intact edible bacteria”but liquid extract and powdered mushroom powder cannot be distinguished Thus to overcome thelimitations of conventional methods and explore a more universal mushroom classificationmethod the spectral data of Bachu mushroom powder and two other types of mushroom powder were measured and the classification of Bachu mushroom powder and other types of mushroom powder was identified by a machine learning algorithm in this studyDue to the differences in the types and contents of nutrients in the stalks and caps of ediblefungi the caps and stalks were analyzed in this study [] Previous studies have shown somedifferences in the contents of proteins and amino acids between caps and stalks in edible fungi[] Additionally the difference in the distribution of nutrients is related to the species of edible fungi [] Therefore to make the experimental results more accurate and persuasive aswell as to avoid the uneven distribution of the substance content in the samples this studyadopted the classification analysis method of grouping according to the attribute index tostudy the caps and stalks []The research method used in this experiment classified the Bachu mushroom and two othertypes of edible fungi by infrared spectra combined with a machine learning algorithm Infraredspectra have the characteristics of wide applicability high efficiency convenience repeatabilityand high sensitivity thus it has been widely used in physics remote sensing biology foodmedical and other research fields [ ] Infrared spectroscopy has great application value infood research [ ] Additionally infrared spectroscopy combined with a machine learningalgorithm has been applied to the classification of mushroom as food the effect of the producing area on the nutritional value of fungi and the fine classification of rare edible fungi [“] The purpose of this study was to explore a universal identification method to identifyBachu mushroom using infrared spectra combined with a machine learning algorithm andPLOS ONE 101371journalpone0238149 August PLOS ONE 0cClassification of multicategory edible fungi based on the infrared spectra of caps and stalksverify the feasibility of the application of infrared spectra combined with an algorithm in theidentification of edible mushroom species Experimental methods Sample preparationIn this study Lentinus edodes and club fungi were selected and classified with Bachu mushrooms Among them Lentinus edodes are produced in Fujian Province of China and purchased from Fuchang Food Limited Company Fujian Province of China club fungi areproduced in Yunnan Province of China purchased from Wuweijin Store and Bachu mushrooms are produced in Bachu County Xinjiang Province and purchased from the mostfamous wholesale market in Urumqi”Six Markets The three kinds of mushrooms purchasedare all dried The caps of the three kinds of mushrooms are umbrellashaped and dark brownThe stalks of club fungi are longer and those of Lentinus edodes and Bachu mushrooms areshorter The three kinds of mushrooms are similar in appearance The samples of the threetypes of edible fungi were purchased from the market After incubating the prepared sample inan ˚C electric steam oven for dehydration treatment for hour the stalks were separatedfrom the caps Next every three complete caps were crushed together and the powder waspassed through a 200mesh sieve as a sample and named according to the mushroom speciesThe stalks were treated in the same way Finally samples of Lentinus edodes powder samples of club fungi powder and samples of Bachu mushroom powder were obtained Measurement of NIF spectraThe sample powder was placed into a 4ml sample tube and its infrared spectrum was measured The spectrum acquisition instrument was a VERTEX infrared spectrometer fromBRUKER Germany Before each measurement of the FTIR spectrum the atmospheric background data were measured using OPUS65 software The selected resolution was cm1 thenumber of scans was the scan range was “ cm1 and the atmospheric compensation parameter was CO2 To reduce the influence of human error and other factors each sample was scanned times Finally the data obtained for the caps were for Lentinus edodes for club fungi for Bachu mushrooms and the same number of stalks data Statistical algorithm analysisStatistical algorithms have been widely used to manage infrared spectral data [] In thisstudy PLS SVM KNN and BPNN were used to process and analyze the spectral data Thecaps data were reduced by PLS to extract features and then the appropriate characteristicnumber was selected as the input of the three classification algorithms namely SVM KNNand BPNN and then the accuracy was obtained Additionally the stalk data were processed inthe same way All algorithms in this study are implemented on MATLAB 2018aThe partial least squares PLS method is a mathematical optimization supervised learningmethod that can identify the best matching function for a set of data by minimizing the sum ofthe squares of errors Based on the advantages of the PLS model which is easy to identify noiseand allows regression modeling with a small sample number the PLS algorithm is widely usedin various research fields [ ] In food research PLS has been used in food nutrition testingfood quality research and food industry research [“] PLS is often used in combinationwith spectra for feature extraction and further spectral data analysis [] In this study toimprove the classification efficiency and filter out the worthless spectral information PLS wasused to reduce the dimensionality of the original spectral dataPLOS ONE 101371journalpone0238149 August PLOS ONE 0cClassification of multicategory edible fungi based on the infrared spectra of caps and stalksAfter dimensionality reduction of the original data an appropriate characteristic numbershould be selected as the basis for classification In this study the characteristic number wasselected based on the cumulative variance interpretation rate of the characteristic number ThePLS program used in this study is the plsregress function The variance explanation rates of thefactors extracted from the first and second columns of the PCTVAR matrix correspond to thevariances of x and y respectively this study chose the variance explanation rate of y [] Thevariance interpretation rate is the degree of interpretation of the data characteristics of thedependent variables by a single factor and the cumulative interpretation rate of n factors is thedegree of interpretation of the data characteristics of the dependent variables by n factors”that is the influence of n factors on the dependent variables Therefore in theory we can selectthe appropriate number of factors according to the cumulative variance interpretation rateand select as few factors as possible to improve the classification efficiency to ensure the integrity of the extracted features To explore the applicability of the theory this study will furtheruse the classification results to verify the theory After selecting an appropriate characteristicnumber it can be used as the input of the classification algorithms SVM KNN and BPNNSupport vector machine SVM is a commonly used generalized linear classifier in whichthe core idea is to apply the principle of risk minimization to the field of classification Regarding pattern classification it has good generalization performance robustness versatility andsimple calculation [] Therefore in food science SVM is widely applied to food classificationand food quality testing [ ] Based on the advantages of SVM and characteristics that canbe used for multiple classifications in this study SVM was used to classify the spectral data ofmushroom powder directly after three features were extractedThe knearest neighbors KNN classification algorithm is one of the most practical algorithms in data mining classification technology It is easy to understand and powerful at thesame time [] Different from other classification algorithms KNN does not need training Itdirectly finds the k samples nearest to the sample and divides them into categories with thelargest number of samples among the k samples thus KNN is suitable for multivariate classification and has high classification accuracy when the category boundary is obvious [] Additionally KNN has been widely used in food classification and quality inspection [“]Therefore we chose the KNN algorithm as the second algorithm of multivariate classificationThe backpropagation neural network BPNN is a multilayer feedforward neural networktrained according to the error backpropagation algorithm The BP neural network has strongnonlinear mapping ability parallel information processing ability and excellent selflearningability thus it has been widely used in food research biomedical fields and other researchfields [“] Additionally the BP neural network can achieve good classification resultswhen it is used in multivariate classification [] In summary we chose the BPNN as the thirdclassification algorithm Results and discussion Spectral analysisAfter the obtained spectral data were averaged normalized and smoothed the obtained spectrogram is shown in Fig Fig shows the FTIR spectra of the Bachu mushroom and Lentinusedodes stalks were similar both with characteristic peaks at cm1 and cm1 and thespectral intensity of Lentinus edodes was higher than that of the Bachu mushroom Additionally the spectrum of the Lentinus edodes stalk has a characteristic peak at cm1 and thespectral intensity in the range of cm1 was significantly lower than that of Lentinusedodes and the Bachu mushroom Comparing the spectra of the three caps the spectra of clubfungi are quite different from those of the other two types of fungi Fig shows that the averagePLOS ONE 101371journalpone0238149 August PLOS ONE 0cClassification of multicategory edible fungi based on the infrared spectra of caps and stalksFig The average normalized spectrum of the caps of the three types of edible fungi101371journalpone0238149g001normalized spectra of Lentinus edodes club fungi and Bachu mushroom stalks all had peaks at cm1 and cm1 and the spectral intensity of Lentinus edodes was the highest in thesetwo places At cm1 the peak intensity of club fungi was higher than that of the Bachumushroom however at cm1 the peak intensity of club fungi was slightly lower than thatof the Bachu mushroom Although the three spectral lines changed roughly the same howeverin the range of cm1 the spectral intensity of the Bachu mushroom was significantlylower than that of the other two kinds of fungi and the spectral intensity of the Bachu mushroom was significantly lower than that of the other two kinds of fungiThrough comparative analysis of the infrared spectra of the caps and stalks of the threetypes of fungi their infrared spectral images showed the same trend but many peak intensitieswere different Therefore we can classify them according to the spectral data based on thePLOS ONE 101371journalpone0238149 August PLOS ONE 0cClassification of multicategory edible fungi based on the infrared spectra of caps and stalksFig The average normalized spectrum of the stalks of the three types of edible fungi101371journalpone0238149g002infrared spectral differences among the three types However it is difficult to distinguishamong the three types of edible fungi directly and accurately only by spectroscopy Thus toclassify them efficiently and accurately we used the combined infrared spectrum analysis withmachine learning Data analysis Dimensionality reduction with PLSIn the PLS algorithm features wereselected to obtain the cumulative variance explanation rate curve Figs and The cumulative variance explanation rate of the first five features of the caps reached while the variance explanation rate of the first five features of the stalks also reached more than and thePLOS ONE 101371journalpone0238149 August PLOS ONE 0cClassification of multicategory edible fungi based on the infrared spectra of caps and stalksFig The principal component cumulative variance explanation rate curve of the caps101371journalpone0238149g003cumulative variance explanation rate of the first features of both was close to Figs and Thus the extracted features can fully express the features of the original data [] Thestalks data and caps data were classified by the SVM BPNN and KNN algorithms Classification by algorithmIn this experiment three classification algorithmswere used”SVM BPNN and KNN”to classify the caps data according to different characteristic numbers Additionally the stalk data were processed in the same way The parameter setting and classification results of the algorithm were as followsThe main ideas of the SVM model in this experiment were as follows Select the test set andtraining set preprocess the data select the best C and g parameters and then use the bestparameters for network training and prediction and obtain the accuracy Among them thetraining set and test set are randomly selected according to the proportion Preprocessingwas used to normalize all the sample data [] In the SVM algorithm the selection of C and gPLOS ONE 101371journalpone0238149 August PLOS ONE 0cClassification of multicategory edible fungi based on the infrared spectra of caps and stalksFig The principal component cumulative variance explanation rate curve of the stalks101371journalpone0238149g004parameters will directly affect the classification results thus it is necessary to select the best Cand g parameters to achieve the best classification results In this study the variation range ofparameter C was [“] the range of parameter g was [“] and the method of parameteroptimization was grid optimization [] To classify the caps data and features were selected The stalks data were processed in the same way The multipleclassification results of the stalks and caps were then obtained as shown in Table In the KNN algorithm the k value was the proportion of the random selection of the testset was and the method to calculate the distance between data was the cosine distanceCosine KNN [] To classify the caps data and featureswere selected The stalks data were processed in the same way Each result was expressed as theaverage of five computational results The multiple classification results of the stalks and capswere then obtained as shown in Table PLOS ONE 101371journalpone0238149 August PLOS ONE 0cClassification of multicategory edible fungi based on the infrared spectra of caps and stalksTable Classification results of the SVM algorithmNumber of PLS componentsClassification accuracy of CapsClassification accuracy of Stalks101371journalpone0238149t001In the BPNN algorithm of this experiment the transfer function of the hidden layer wastamsig the output layer was purelin the learning training function was trainlm and the weightlearning function was learngdm The network parameters were set to training times thenetwork performance goal was and the learning rate was [] Thirty percent of allsamples were selected randomly as the test set To classify the caps data and features were selected The stalks data were processed in the same way Eachresult was expressed as the average of five computational results The multiple classificationresults of the stalks and caps were then obtained as shown in Table Verification of the feasibility of selecting the characteristic number with thecumulative variance explanation rate Figs and shows the line chart of the classificationresults of the three algorithms when selecting different characteristic numbers The accuracyof SVM in the classification of caps was when selecting and features decreasedslightly with the increase in the characteristic number and then stabilized at Accordingto the fungal stalks data the accuracy of SVM classification increased gradually when selecting features fluctuated slightly with the increase in the characteristic number andthen stabilized at Using KNN to classify the caps data the accuracy was stable between and but fluctuated slightly When the characteristic number tended to theaccuracy was stable at According to the classification of stalks data by KNN the accuracy varied greatly when selecting “ features fluctuated slightly with the increase in thecharacteristic number and then stabilized at For the classification of caps by BPNNthe accuracy varied greatly when the characteristic number was and the classificationTable Classification results of the KNN algorithmNumber of PLS componentsClassification accuracy of CapsClassification accuracy of Stalks101371journalpone0238149t002PLOS ONE 101371journalpone0238149 August PLOS ONE 0cClassification of multicategory edible fungi based on the infrared spectra of caps and stalksTable Classification results of the BPNN algorithmNumber of PLS componentsClassification accuracy of CapsClassification accuracy of Stalks 101371journalpone0238149t003Fig Accuracy of the three algorithms to select different characteristic numbers according to the data of the caps101371journalpone0238149g005PLOS ONE 101371journalpone0238149 August PLOS ONE 0cClassification of multicategory edible fungi based on the infrared spectra of caps and stalksFig Accuracy of the three algorithms to select different characteristic numbers according to the data of the stalks101371journalpone0238149g006effect was unstable When the characteristic number was more than the accuracy decreasedslightly and finally stabilized at When BPNN classified stalks its accuracy variedgreatly when the characteristic number was less than when the characteristic number wasmore than the accuracy was stabilized at Combined with the cumulative variance explanation rate with the increase in the characteristic number the extraction degree of the extracted features to the original data informationgradually increased Thus the classification results become more reliable and the accuracywill gradually tend to a certain value”that is in the ideal state the accuracy of all featuresextracted for classification However in this process not all information is conducive toimproving accuracy and some information will interfere with the classification results thusthe accuracy will fluctuate slightly [] Therefore when we select the characteristic numberaccording to the variance interpretation rate we should consider the degree of featurePLOS ONE 101371journalpone0238149 August PLOS ONE 0cClassification of multicategory edible fungi based on the infrared spectra of caps and stalksextraction accuracy and classification efficiency comprehensively In summary when thecumulative variance interpretation rate of features reaches the classification results aresufficiently reliable and the accuracy is high confirming that it is feasible to select the characteristic number according to the cumulative variance interpretation rate Selection of the best algorithm According to the caps data classification thePLSSVM algorithm has the best classification effect when selecting features with anaccuracy of The PLSKNN algorithm has the best classification effect when selecting features with an accuracy of When selecting features the PLSBPNN algorithmhas the best classification effect with an accuracy of Fig According to the stalksdata classification the classification effect of the PLSSVM algorithm was the best when selecting features with an accuracy of The classification effect of the PLSKNN algorithm was the best when selecting features with an accuracy of When featureswere selected the classification effect of the PLSBPNN algorithm was the best reaching Fig Combined with the selection of the characteristic number to analyze the three algorithmsthe PLSKNN algorithm has a better classification effect on stalks and caps and the accuracy ismore stable when selecting different characteristic numbers Thus the PLSKNN algorithmwas chosen as the optimal algorithm Using comprehensive analysis of the accuracy ofPLSKNN classification when selecting different characteristic numbers a characteristic number of reveals a higher accuracy for both caps and stalks and a high classification efficiencyTherefore in this experiment the PLSKNN algorithm was finally selected and the characteristic number was The final classification accuracy was for the caps and forthe stalks ConclusionsThis study verified the feasibility of infrared spectroscopy combined with the PLSSVMPLSKNN and PLSBPNN algorithms in the classification of the Bachu mushroom and otheredible mushrooms We compared the classification results and selected the optimal algorithmand best feature number to reveal an efficient rapid and universal method to identify theBachu mushroom overcoming the limitation that the current identification of Bachu mushroom only depends on its appearance Moreover the method is universal and can be applied tothe classification and identification of other types of food Additionally this study proposed toselect the characteristic number according to the cumulative variance interpretation rate andused the classification results of the three algorithms to verify its feasibility This method ofselecting the characteristic number can also be extended to other research fields of factor analysis and the appropriate characteristic number can be selected intuitively and quicklySupporting informationS1 DataZIPAuthor ContributionsConceptualization Cheng Chen Zhiao WangData curation Rui Gao Cheng Chen Hang Wang Chen Chen Ziwei Yan Yan WuFormal analysis Hang WangFunding acquisition Cheng Chen Chen Chen Fangfang Chen Zhiao WangPLOS ONE 101371journalpone0238149 August PLOS ONE 0cClassification of multicategory edible fungi based on the infrared spectra of caps and stalksInvestigation Rui Gao Fangfang ChenMethodology Rui Gao Cheng Chen Chen Chen Yuxiu ZhouProject administration Rui Gao Cheng Chen Rumeng Si Xiaoyi LvResources Rui Gao Ziwei Yan Fangfang Chen Yan WuSoftware Ziwei YanSupervision Cheng ChenValidation Rui Gao Rumeng SiWriting “ original draft Rui Gao Hang Wang Zhiao Wang Yuxiu ZhouWriting “ review editing Rui Gao Cheng Chen Huijie Han Xiaoyi LvReferencesZhao Q Species clarification of the culinary Bachu mushroom in western China Mycologia p “ 10385216002 PMID Chen X Wang J and Li H Basic Ingredientsand Nutritional Assessment Of Baehu Mushroom FarmProducts Processing p “Zeng D and Zhu S Purification characterization antioxidant and anticancer activities of novel polysaccharides extracted from Bachu mushroom International journal of biological macromolecules p “ 101016jijbiomac201709088 PMID XuJie H Extraction of BaChu mushroom polysaccharides and preparation of a compound beverage Carbohydrate Polymers p “ XuJie H and Wei C Optimization of extraction process of crude polysaccharides from wild edibleBaChu mushroom by response surface methodology Carbohydrate Polymers p “ Wang CY A Review on the Potential Reuse of Functional Polysaccharides Extracted from the ByProducts of Mushroom Processing Food and Bioprocess Technology p “ Akindahunsi AA and Oyetayo FL Nutrient and antinutrient distribution of edible mushroom Pleurotustuberregium fries singer LWT”Food Science and Technology p “Zhu Y and Tan ATL Chemometric Feature Selection and Classification of ltigtGanoderma lucidumltigt Spores and Fruiting Body Using ATRFTIR Spectroscopy American Journal of AnalyticalChemistry p “ Oboh G and Shodehinde S Distribution of nutrients polyphenols and antioxidant activities in the pileiand stipes of some commonly consumed edible mushrooms in Nigeria Bulletin of the Chemical Societyof Ethiopia Ball GH Classification Analysis Ferrari M and Quaresima V A brief review on the history of human functional nearinfrared spectroscopy fNIRS development and fields of application Neuroimage p “ 101016jneuroimage201203049 PMID Chen C Exploration research on the fusion of multimodal spectrum technology to improve performance of rapid diagnosis scheme for Thyroid Dysfunction Journal of biophotonics pe201900099 101002jbio201900099 PMID Van de Voort F Fourier transform infrared spectroscopy applied to food analysis Food Research International p “ Chen C Application of near infrared spectroscopy combined with SVR algorithm in rapid detection of cAMP content in red jujube Optik p Meenu M and Xu B Application of vibrational spectroscopy for classification authentication and quality analysis of mushroom A concise review Food chemistry Li Y Geographical traceability of wild Boletus edulis based on data fusion of FTMIR and ICPAES coupled with data mining methods SVM Spectrochim Acta A Mol Biomol Spectrosc p “ 101016jsaa201701029 PMID Fu H A comprehensive quality evaluation method by FTNIR spectroscopy and chemometricFine classification and untargeted authentication against multiple frauds for Chinese Ganoderma lucidum Spectrochimica Acta Part A Molecular and Biomolecular Spectroscopy p “PLOS ONE 101371journalpone0238149 August PLOS ONE 0cClassification of multicategory edible fungi based on the infrared spectra of caps and stalks Geladi P and DÃ¥bakk E An overview of chemometrics applications in near infrared spectrometryJournal of Near Infrared Spectroscopy p “ Rosipal R and Kra¨mer N Overview and recent advances in partial least squares in International Statistical and Optimization Perspectives Workshop Subspace Latent Structure and Feature Selection Springer Xie L Quantification of glucose fructose and sucrose in bayberry juice by NIR and PLS FoodChemistry p “Ivorra E Detection of expired vacuumpacked smoked salmon based on PLSDA method usinghyperspectral images Journal of food engineering p “ Heyder M Theuvsen L and HollmannHespos T Investments in tracking and tracing systems in thefood industry a PLS analysis Food Policy p “ Mehmood T et a Answer:

Thyroid_Cancer

"ligandactivated transcriptional factors that belong to the nuclear receptor superfamily Among them PPAR alpha andPPAR gamma are prone to exert an antiangiogenic eï¬ect whereas PPAR betadelta has an opposite eï¬ect in physiological andpathological conditions Angiogenesis has been known as a hallmark of cancer and our recent works also demonstrate thatvascularspecific PPAR betadelta overexpression promotes tumor angiogenesis and progression in vivo In this review we willmainly focus on the role of PPAR betadelta in tumor angiogenesis linked to the tumor microenvironment to further facilitatetumor progression and metastasis Moreover the crosstalk between PPAR betadelta and its downstream key signal moleculesinvolved in tumor angiogenesis will also be discussed and the network of interplay between them will further be established inthe review IntroductionPeroxisome proliferatoractivated receptorsPPARs asligandactivated transcription factors belong to the steroidreceptor superfamily which includes three isoforms PPARalpha PPAR betadelta and PPAR gamma [] PPARs formheterodimers with retinoic X receptors and regulate theexpression of various genes upon ligand binding PPARs alsointeract with corepressors or coactivators to modulate thetranscription of its downstream target genes PPARs asimportant transcriptional regulators have been suggested tobe involved in lipid metabolism and multiple cellular functions For instance PPAR alpha also functions in fatty acidbetaoxidation and vascular ‚ammation [] PPAR gammaacts as a regulator in adipocyte diï¬erentiation and type diabetes [] PPAR betadelta is a key player in cardiac energyproduction angiogenesis and particularly in cancer progression []PPAR alpha and PPAR gamma exert predominantly anantiangiogenic eï¬ect [“] but there still exist conflictingstudies showing opposite results [ ] On the contraryPPAR betadelta produces more obviously proangiogeniceï¬ects [“] In this review we will focus on the promotingrole of PPAR betadelta in angiogenesis especially in tumorangiogenesis The network of interplay between PPAR betadelta and its various downstream signal molecules and alsobetween those key molecules will be further discussed andestablished Remarkably diverse important signal moleculesinvolved in tumor angiogenesis and progression and cancercell metabolism have been identified as direct PPAR betadelta target genes AngiogenesisAngiogenesis is the physiological process through which anew capillary network forms from the preexisting vasculature[ ] whereas vasculogenesis denotes de novo bloodvessel formation mostly during embryogenesis in whichendothelial progenitor cells EPC migrate to sites of vascularization then diï¬erentiate into endothelial cells EC andcoalesce into the initial vascular plexus [ ] Besides theinteraction between proangiogenic factors and antiangiogenic factors angiogenesis is also a multiple step biologicalprocess during which a variety of molecules cooperateincluding cell adhesion molecules matrix metalloproteinases 0cPPAR ResearchMMPs extracellular matrix ECM and basement membrane componentsAngiogenesis is a physiological and vital process in development and growth An imbalance of proangiogenic andantiangiogenic factors causes angiogenesis in pathologicalconditions such as diabetic retinopathy and tumor growthThus when the imbalance comes to a point at which angiogenesis is triggered by tumor cells then an œangiogenicswitch of tumor cells is turned on during tumor progression the œangiogenic switch is often activated and remainson [“] Inducing angiogenesis is known as a hallmarkof cancer [] and angiogenesis is also a fundamental stepby which most benign tumors transition into malignant ones Tumor Angiogenesis Tumor needs to sprout new vesselsand further develop a vascular network in order to supplynutrients and oxygen remove waste products support a continually high proliferative rate and ultimately expand neoplastic growth [ ] Hence angiogenesis is essential forhelping sustain tumor growth and facilitate tumor progression Besides being a requirement for angiogenesis an abnormal vasculature also helps to promote tumor progression andmetastasis The tumor vascular wall is imperfect and prone toleakage so it is much easier for tumor cells to directly penetrate into the blood vessels or lymphatic vessels and then proliferate at another distant site to form metastasis []Due to intensive abnormal neovascularization in tumortissues most malignant tumors grow rapidly and acquirethe ability to spread to adjacent and distant ans whichmakes them more malignant and even life threateningTherefore angiogenesis indeed plays an important role intumor progression and metastasis and to intervene with thisprocess would obviously prevent tumor development andspread Thus this has been regarded as a critical target forantitumor therapy PPAR Alpha and AngiogenesisIt was reported firstly that a selective PPAR alpha agonistWY14643 did not show any eï¬ect on angiogenesis or EC proliferation [] But some subsequent studies showed that theactivation of PPAR alpha inhibited angiogenesis in vitro byusing fenofibrate a clinically used PPAR alpha agonist []Moreover fenofibrate suppressed EC proliferation migration and tube formation through inhibition of protein kinaseB Akt and disruption of the cytoskeleton [] Furthermore PPAR alpha activation was shown to inhibit vascularendothelial growth factor VEGF induced EC migrationand basic fibroblast growth factor bFGFFGF2 inducedcorneal angiogenesis in vitro and in vivo [] Especiallyin vivo reduced tumor growth and microvessel numberswere observed in mice implanted with melanoma Lewis lungcarcinoma LLC fibrosarcoma and glioblastoma due to asystemic treatment of PPAR alpha ligand and the antiangiogenic state induced through activation of PPAR alpha withelevated thrombospondin1 TSP1 and endostatin expression []Howeverit was demonstrated inanother observation that activation of PPAR alpha stimuin that same yearlated neovascularization in vivo with increased phosphorylation of endothelial nitric oxide synthase eNOS and Akt via aVEGFdependent manner [] Furthermore Zhang andWard also suggested that PPAR alpha activation inducedproangiogenic responses in human ocular cells [] Inanother study it was shown that a new PPAR alpha agonistRK13675 had no eï¬ect on angiogenesis [] RecentlyPPAR alpha activation is further shown to have antineovascularization eï¬ects with downregulation of VEGF and angiopoietin expression in a rat alkali burn model []In summary the role of PPAR alpha in angiogenesis isstill controversial Some observations showed that ligandactivation of PPAR alpha had antiangiogenic eï¬ects mediated either through upregulation of antiangiogenic factorssuch as TSP1 and endostatin or downregulation of proangiogenic factors including VEGF FGF2 AKT and angiopoietins Others also reported opposite results showing aproangiogenic role upon PPAR alpha activation Thus thespecific molecular mechanism is still unclear and needs tobe further studied PPAR Gamma and AngiogenesisLigand activation of PPAR gamma was previously shown toinhibit human umbilical vein endothelial cell HUVEC tubeformation in collagen gels [] and VEGFinduced choroidalneovascularization in vitro and in vivo [] Another studyalso demonstrated that EC apoptosis was induced throughtreatment with the PPAR gamma ligand 15dPGJ2 [] Furthermore rosiglitazone a potent PPAR gamma agonist wasshown to inhibit primary tumor growth and metastasisthrough both direct and indirect antiangiogenic eï¬ectsin vitro and bFGFinduced corneal neovascularizationin vivo [] Moreover a similar observation also displayedthe inhibition of VEGFinduced angiogenesis in a chickchorioallantonic membrane model [] In a mouse modelwith ischemiainduced retinopathy pioglitazone a PPARgamma agonist also showed a protective eï¬ect against pathological neoangiogenesis through upregulation of anti‚ammatory adipokine adiponectin [] Additionally thePPAR gamma antagonist GW9662 was shown to reverseOmega3 polyunsaturated fatty acidinduced reduction ofESelectin angiopoietin2 vascular cell adhesion molecule and intracellular adhesion molecule1 [] implicatingan antiangiogenic potential of PPAR gamma itself Howeveropposite results also showed that pioglitazone enhanced neovascularization and inhibited apoptosis of EPC in vitro andin vivo via a Phosphoinositide3Kinase PI3K dependentmanner []Nadra observed that PPAR gammanull embryosdisplayed a vascular structural defect at E95 Moreover disanized placental layers and an altered placental microvasculature were observed in pregnant wildtype mice treatedwith the PPAR gamma agonist rosiglitazone as well asreduced expression of proangiogenic factorsincludingVEGF proliferin and plateletendothelial cell adhesionmolecule1 PECAM1CD31 [] suggesting a crucial roleof PPAR gamma in placental vascular development The 0cPPAR Researchmajor antiangiogenic properties on PPAR gamma activationwere also reviewed here []Notablyin most cancersthe canonical Wntbetacatenin pathway is upregulated while on the contrary PPARgamma is downregulated Interestingly in numerous tissuesthe activation of PPAR gamma inhibits the betacateninpathway whereascanonicalWntbetacatenin signal cascade also inactivates PPARgamma [] implicating a negative regulatory role of PPARgamma in carcinogenesis where tumor angiogenesis mightbe a fundamental stepstimulation ofthetheIn summary PPAR gamma predominantly displays anantiangiogenic eï¬ect that may be mediated through the inhibition of VEGF or bFGFinduced neovascularization andreduction of the expression level of some proangiogenicfactors PPAR BetaDelta and AngiogenesisUnlike PPAR alpha and PPAR gamma on the contrarymany studies have explicitly shown the proangiogenic eï¬ectsof PPAR betadelta on physiological and pathological angiogenesis The first evidence provided in a study is that activation of PPAR betadelta with GW501516 a highly selectivePPAR betadelta agonistinduces HUVEC proliferationand an increased expression of VEGF and its receptorVEGFR1 FLT1 [] Besides inducing EC proliferationPPAR betadelta activation by itsligand prostacyclinPGI2 also stimulates upregulation of alpha expression an antiapoptotic and anti‚ammatory protein whichthereby protects ECs from H2O2induced apoptosis and oxidant injury [] Moreover a subsequent study further provides evidence that activation of PPAR betadelta withGW501516 induces angiogenesis during which VEGFrelease is considered as a major trigger factor [] firstlysuggesting the promotion for angiogenesis upon PPARbetadelta activationMüllerBrüsselbach show that PPAR betadelta mice implanted with LLC and B16 melanoma exhibit diminished blood flow and immature microvascular structurescompared with wildtype mice Moreover reexpression ofPPAR betadelta into the matrigelinvading cells triggersmicrovessel maturation and restores normal vascularization[] indicating a crucial role of PPAR betadelta in tumorvascularization Additionally another study also observedreduced levels of calcium intracellular channel protein CLIC4 but it observed enhanced expression of cellular retinol binding protein CRBP1 in migrating ECs from PPARbetadeltanull mice [] both of which play a role in tumorvascularization [ ] It was reported that PPAR betadeltawas required for placentation [] and most of the PPARbetadeltanull mutant embryos died at E95 to E105 due toabnormal celltocell communication atthe placentaldecidual interface [] However in these studies [“] adefect in angiogenesis was not observed during normal development in PPAR betadeltaknockout miceSome observations also show the important role of PPARbetadelta in physiological angiogenesis For instance skeletal musclespecific PPAR betadelta overexpression leads toPPAR betadeltaan increase in the number of oxidative muscle fibers andrunning endurance in adult mice [“] Moreover PPARbetadelta activation promotes a rapid muscle remodelingvia a calcineurindependent manner and induces muscleangiogenesis in highly selective PPAR betadelta agonistGW0742treated animals [] Furthermore in the heartpharmacologicalstimulation withGW0742 induces rapid cardiac growth and cardiac angiogenesis through direct transcriptional activation of calcineurin [] Interestingly the same cardiac phenotype wasalso observed after treatment with the PPAR betadelta agonist GW501516implicating a response specificity forPPAR betadelta stimulation [] Calcineurin activationfurther leads to the stimulation of nuclear factoractivatedT cell c3 NFATc3 and an enhanced expression of hypoxiainducible factor alpha HIF1alpha and cyclindependentkinase CDK9 [] Overall the remodeling in skeletalmuscle and heart is perfectly the same as the phenotypeobserved with exercise and both of them are mediatedthrough activation of calcineurinPPAR betadelta may act as a key regulator in mediatingpathological angiogenesis For instance PPAR betadelta wasshown to regulate retinal angiogenesis in vitro and in vivoand its inhibition reduced preretinal neovascularization possibly via an Angiopoietinlike protein Angptl4 dependent manner []implicating the potential of PPARbetadelta in modulating pathological ocular angiogenesisRecently an observation reported that PPAR betadeltaknockdown in both retinal pigment epithelial and choroidalendothelial cells caused an antiangiogenic phenotype andPPAR betadelta promoted laserinduced choroidal neovascular CNV lesions in PPAR betadelta mice [] Moreover pharmacological inhibition of PPAR betadelta with theantagonist GSK0660 also resulted in a significantly decreasedCNV lesion size in vivo suggesting a functional role of PPARbetadelta in the development of CNV lesions [] This indicates that PPAR betadelta has an important association withpathological angiogenesisAngiotensin II Ang II the biologically active peptide ofthe reninangiotensin system RAS is a major blood pressure and cardiovascular homeostasis regulator and is alsorecognized as a potent mitogen Angiotensinconvertingenzyme inhibitors were introduced approximately yearsago as antihypertensive agents and have since become asuccessful therapeutic approach for high blood pressurecongestive heart failure and postmyocardial infarction Inexperimental systemsthe antitumor eï¬ects of diverseACE inhibitors show that these inhibit cell proliferationand possessand anti‚ammatory eï¬ects [“] It has been shown recentlythat activation of PPAR betadelta inhibits Ang IIstimulated protein synthesis in a concentrationdependentmanner and suppresses Ang IIinduced generation of reactive oxygen species ROS in vascular smooth muscle cells[] PPAR betadelta was further shown to inhibit AngIImediated atherosclerosis [] However it is not clearuntil now if PPAR betadelta activation can be consideredis foras an ACE inhibitormimicking approach as itexample the case for PPAR gamma activators[]antiangiogenicantimetastatic 0cPPAR Researchthe relevance ofFurthermorethis hypothetical PPARbetadelta feature might be limited for tumor angiogenesiswhere vascular smooth muscle hypertrophy and atherosclerosis do not contribute to the major pathologyBesides inducing angiogenesis it has been demonstratedthat PPAR betadelta directly acts on early EPC through activation of the AKT pathway and induces an enhanced vasculogenesis [] Similarlythe PPAR betadeltamediatedprovasculogenic eï¬ects are also observed on late EPC []He showed that PPAR betadelta activation withGW501516 induced EPC proliferation and tube formationwhereas EPC treated with an inhibitor of cyclooxygenaseCOX or PGI2 synthase or with PPAR betadeltaspecificsiRNA also displayed an opposite eï¬ect [] Furthermoreit has been demonstrated that PPAR betadelta inducesangiogenesis and skeletal muscle regeneration throughmatrix metalloproteinase MMP 9mediated insulinlikegrowth factor1 paracrine networks upon EPC activation[] Han also observed that PPAR betadelta activationpromoted a rapid wound healing with enhanced angiogenesis in a mouse model with skin punch wound [] Overallin addition to EC PPAR betadelta is also a key regulator ofEPC or even may act as an initiator of activation of EPC tofurther induce vasculogenesis PPAR BetaDelta and Tumor AngiogenesisLinked to Tumor MicroenvironmentPPAR betadelta expression is often upregulated and promotes cancer progression in many major human cancerslung breast and gastric cancers [“]such as colonwhich suggests a crucial role of PPAR betadelta in cancercells even though there exist some conflicting studies indicating that the functional role of PPAR betadelta in tumorigenesis or carcinogenesis still remains highly controversial [“] and dependent on specific tumor or cancer cell typesThus here we discuss the promotion of PPAR betadelta intumor progression through facilitating tumor angiogenesisPPAR betadelta has been suggested as a critical œhubnode transcriptional factor which governs a tumor œangiogenic switch [ “] In the transcriptional networkanalysis it was reported that tumor growth and tumor angiogenesis were markedly inhibited in PPAR betadeltanullmice in comparison with wildtype mice [] Moreoverthe elevated PPAR betadelta expression level was also considered to be highly correlated to pathologically advancedtumor stage and increased cancer risk for recurrence and distant metastasis in patients with pancreatic cancer [] indicating the crucial association of PPAR betadelta withtumor angiogenesis progression and cancer invasivenessPPAR betadelta may indirectly facilitate tumor angiogenesis and progression through its function on the tumormicroenvironment TME where tumor angiogenesis is fostered Moreover a tumor also releases some extracellular signals to closely communicate and constantly collaborate withTME to facilitate tumor angiogenesis in order to furtherenable tumor growth and progression For instance it wasshown that colon cancer cells with PPAR betadelta knockoutfailed to stimulate EC vascularization in response to hypoxicstress whereas wildtype cells exposed to hypoxia were ableto induce angiogenesis [ ] suggesting that PPAR betadelta is required for the promotion of angiogenesis in hypoxic stressmediated TME Moreover in the TME tumorltrating myeloid cells are considered as the most important cells for fostering tumor angiogenesis among the multiple diï¬erent kinds of stromal cells [] Besides stimulatingtumor angiogenesis tumor myeloid cells also support tumrowth by suppressing tumor immunity and promotingtumor metastasis to distinct sites [] Interestingly it hasbeen demonstrated that PPAR betadelta activation intumorltrating myeloid cells stimulates cancer cell invasion and facilitates tumor angiogenesis via an Interleukin IL10 dependent manner [] Moreover impairedtumor growth and angiogenesis were observed in PPARbetadelta KO BMT mice due to PPAR betadelta deficiencyin tumor myeloid cells [] suggesting that PPAR betadeltaplays a key role in tumor angiogenesis and progression intumor myeloid cells of TMEFurthermore the endoplasmic reticulum ER an essential anelle involved in many cellular functions is implicated in TME In cancer stressors like hypoxia nutrientdeprivation and acidosis disrupt ER function and lead toaccumulation of unfolded proteins in ER a condition knownas ER stress Cells adapt to ER stress by activating an integrated signal transduction pathway called the unfolded protein response UPR UPR represents a survival response bythe cells to restore ER homeostasis and has both survivaland cell death eï¬ects The mechanisms that determine cellfate during ER stress are not well understood For instanceshort exposure to ER stress initially increases AKT signalingbut longterm ER stress suppresses AKT signaling []PPAR betadelta activation has been shown to reduce endoplasmic reticulum ER stressassociated ‚ammation inskeletal muscle through an AMPKdependent mechanism[] and to reduce ‚ammation in response to chronic ERstress in cardiac cells [] Furthermore it has been nicelyshown that PPAR betadelta can repress RASoncogeneinduced ER stress to promote senescence in tumors [] Thisis mediated through the decrease of pAKT activity promoting cellular senescence through upregulation of p53 and p27expression [] It would be interesting to investigate thedirect eï¬ects of PPAR betadelta on senescence of tumorendothelial cells in an in vivo setting We recently showedthat senescent endothelial cells are indispensable for ahealthy lifespan and that removal of senescent endotheliumdisrupts vascular function leading to diminished vessel densities and fibrotic lesions [] If PPAR betadelta mediatessenescence of tumor endothelium thereby protecting vesselintegrity this might explain the enhanced tumor growthand vascularization upon PPAR betadelta activationobserved by us and others [ ]Most recently Zuo demonstrated that PPARbetadelta in cancer cells regulates tumor angiogenesisin vivo and in vitro by promoting the secretion of proangiogenic factors including VEGF and Interleukin IL8[] Most importantly in our recent works it has beenshown that conditionalinducible vascular endotheliumspecific PPAR betadelta overexpression in vivo leads to 0cPPAR Researchenhanced tumor angiogenesis tumor growth and metastasis formationfurther indicating a vascular ECspecificPPAR betadelta action mechanism in tumor progressionindependent of some controversial observations of PPARbetadelta in specific tumor or cancer cell types [] Wagner also firstly reported the mouse model in whichrapid induction of cardiac angiogenesis and cardiac hypertrophy were observed [ ] Crosstalk between PPAR BetaDelta and SignalMolecules PPAR betadelta activation or overexpressionmay upregulate the expression of its various downstream signal molecules involved in tumor angiogenesis includingproangiogenic factors such as VEGF PDGF and FGFproinvasive matrixdegrading enzymes such as MMP9pro‚ammatory mediators such as COX2 and cytokinesand chemokines such as IL1 and CXCL8 even some ofwhich have been further identified as PPAR betadelta directtarget genes Besides a leading role of PPAR betadelta amongthe signal molecules PPAR betadelta may function in TMElinked to diverse kinds of cells through direct or indirectmodulation of its downstream molecules Interplay between PPAR BetaDelta and InflammatoryAngiogenesis Inflammatory angiogenesis is a crucial processin tumor progression For instance the pro‚ammatorymediator cyclooxygenase2 COX2 is considered as a keyregulator of angiogenesis and tumor growth through multiple downstream proangiogenic mechanisms such as production of VEGF and induction of MMPs Moreover selectiveinhibition of COX2 has also been shown to suppress angiogenesis in vivo and in vitro [] It is well known that VEGFAplays a critical role in both angiogenesis and vasculogenesis[] and it leads the directional migration of tip cells andstalk cell proliferation in microtubule branches [ ] Ithas also been demonstrated that MMP9 triggers the œangiogenic switch during carcinogenesis and enhances the availability of VEGF to its receptors [] Furthermore it hasbeen reported that ‚ammatory cell MMP9 initiates theonset of tumor neovascularization during which there existsfunctionalincludingMMP9 [] LEPTIN is shown to mediate angiogenesisin vivo and in vitro through induction of EC proliferationand expression of MMP2 and MMP9 [] and to furtherpromote EC diï¬erentiation and directional migrationthrough enhancement of COX2 activity [] LEPTIN couldalso induce angiogenesis via transactivation of VEGFR inECs [] Additionally besides inducing angiogenesisPPAR betadelta also functions in chronic ‚ammationfacilitating tumorigenesis through induction of COX2 andits product prostaglandin E2 PGE2 in vivo [ ]Interestingly COX2 VEGF MMP9 and LEPTIN have beenidentified as PPAR betadelta target genes via a direct transcriptional activation mechanism in hepatocellular carcinoma cells [] colorectal cancer cells [ ] EPCs[ ] and liposarcoma cells [] respectivelylinks between VEGF and MMPsIn TME tumorltrating ‚ammatory cells also helpto induce and sustain tumor angiogenesis and further tofacilitate tissue invasion and tumor metastatic spread byreleasing some signal molecules such as proinvasive MMP9and ‚ammatory chemokines [“] Chemotaxis is alsoa crucial process for inducing angiogenesis in tumors eitherdirectly by attracting ECs towards tumor cells to form newvessels or indirectly by mediating immune ‚ammatorycells to ltrate eventually promoting tumor angiogenesis[] Chemotaxis of tumor cells and stromal cells in TMEis also required for tumor dissemination during tumor progression and metastasis [ ]CXC chemokines such as CXCL8 encoding IL8 andCXCL5 are also involved in COX2associated angiogenesisto contribute to nonsmallcelllung cancer progression[ ] It is further shown that IL8 directly regulatesangiogenesis via recruitment of neutrophils [] whichfurther drives VEGF activation [] MoreoverIL8responding neutrophils are considered as the major sourceof angiogenesisinducing MMP9 [ ] Chemokine CC motif ligand CCL2 in addition to the promotionof angiogenesis [ ] also enhances tumor metastasis[] Furthermore myeloid monocytic cellssuch asmyeloidderivedtumorMDSCsassociated macrophages TAMs and dendritic cells arerecruited to the tumor site mainly by CCL2 and producemany proangiogenic factorssuch as VEGF CXCL8plateletderived growth factor PDGF and transforminggrowth factor beta TGF beta [“] In fact bothTGF beta and hypoxia are potentinducers of VEGFexpression in tumor cells and collaborate with TME toprovide the foundation of tumor angiogenesis and cancercell invasion [] Importantly IL8 has been reported asa key target gene of PPAR betadelta to promote angiogenesis in vivo and in vitro [] and CCL2 expression isalso significantly upregulated upon vascular PPAR betadelta overexpression in vivo []suppressorcellsCOX2 also mediates IL1 betainduced angiogenesisin vitro and in vivo [ ] IL1 beta supports neovascularization through the regulation of the expression of VEGFand its receptor VEGFR2 FLK1KDR on ECs [] IL1 actsas an upstream pro‚ammatory mediator that initiates anddisseminates the ‚ammatory state by inducing a localinteractive network and increasing adhesion moleculeexpression on ECs and leukocytes which facilitates tumorassociated angiogenesis [] In TME ‚ammatory IL1beta recruits myeloid cells from bone marrow and activatesthem to produce proangiogenic factors such as VEGF VEGFfurther activates ECs and myeloid cells promoting tumorinvasiveness and fostering tumor angiogenesis [] In addition IL6 also stimulates angiogenesis and vasculogenesis[ ] However Gopinathan observed an IL6induced newly forming vascular structure with defectivepericyte PC coverage ex vivo [] thus facilitating cancercell ltration and tumor metastasis through vascular leakage Interestingly IL1 and IL6 expression levels are significantly upregulated in the PPAR betadelta overexpressionmouse model reported recently []In summary PPAR betadelta seems to act as a key leaderin ‚ammatory mediatordriven tumor angiogenesis linkedto TME in which many pro‚ammatory mediators chemokines and proangiogenic factors closely communicate with 0cPPAR Researcheach other and also associate with tumorltrating myeloid cells such as neutrophils TAMs and MDSCs Other Key PPAR BetaDeltaMediated ProangiogenicFactors It has been demonstrated that Wilms™ tumor suppressor WT1 is a major regulator of tumor neovascularization andtumor progression [] E26 avian leukemia oncogene ETS1 also plays a key role in regulating vascular development and haemopoiesis particularly in angiogenesis []In addition ETS1 promotes cancer cell invasion throughupregulation of MMPs [] Consistent with this silencingof ETS1 in highly invasive breast cancer cells also reducesthe expression of MMP9 and MMP1 []ETS1 also acts as a key regulator of MMPs such asMMP1 MMP3 and MMP9 in human cancerassociatedfibroblasts CAFs [ ] CAFs support tumor growthby secreting growth factors such as VEGF FGF PDGF andchemokines to stimulate angiogenesis and thereby promotecancer cell invasion and metastasis formation [ ]CAFs as metastatic tumor stroma are a crucial componentin tumor progression through the remodeling of the ECMstructure thus helping a tumor to acquire an aggressive phenotype [ ] PPAR betadelta in CAFs also exhibits aprotumorigenic eï¬ect It was reported that ablation of PPARbetadelta in CAFs attenuated tumor growth by altering theredox balance in TME [] suggesting that PPAR betadeltain CAFs is also an important player in tumor developmentETS1 induces the expression of VEGF VEGFR1 andVEGFR2 in ECs [“] In turn VEGF is also a majorinducer of ETS1 in ECs through the activation of either thePI3KAKT pathway or the MEKERK12 signal cascade[ ] WT1 is also reported to regulate tumor angiogenesis via direct transactivation of ETS1 []SRYrelated HMGbox SOX18 has also beenreported previously to induce angiogenesis during tissuerepair and wound healing [] and cancer progression[] And most recently it was further shown that specificECderived endovascular progenitors initiated a vasculogenic process and diï¬erentiated into more mature endothelial phenotypes within the core of the growing tumorsthrough reactivation of SOX18 [] Interestingly theseimportant proangiogenic molecules including WT1 ETS1and SOX18 are also significantly upregulated in the vascularPPAR betadelta overexpression model in vivo [] AndWT1 is also identified as a target gene of PPAR betadeltain melanoma cells [] PPAR BetaDelta May Facilitate Cancer Progression atDiverse Cellular Levels in TME PPAR betadelta activationis shown to induce colonic cancer stem cell CSC expansionand to promote the liver metastasis of colorectal cancerin vivo via direct transactivation of the Nanog gene []NANOG as a key transcriptional factor governs the selfrenewal and pluripotency of stem cells [] and cancer cellsexpressing NANOG also often exhibit stem cell properties[] Protooncogene cKITCD117 is known as the maststem cell factor receptor and receptor tyrosine kinase andits activation in CSCs may regulate the stemness to controltumor progression and drug resistance to tyrosine kinaseinhibitors Moreover cKIT has been identified as a potentialmarker of the cancer stemlike cells [] In addition cKITnot only functions on ECs [ ] but also belongs to thetumor angiogenesispromoting molecule [“] Studiesalso suggested that activation of cKIT enhances the expression of VEGF that can be suppressed by imatinib an inhibitor of cKIT in gastrointestinal stromal tumor cells whichthereby has an impact on tumor angiogenesis [ ] cKIT is also involved in pathological ocular neovascularization [] and is regulated transcriptionally by WT1 []and PPAR betadelta []PDGFB and its receptor PDGFR beta also known asangiogenic factors are suggested to enhance angiogenesisand vasculogenesis via their function in ECs [“] andEPCs [] and to regulate vascular permeability and vesselmaturation through recruitment of pericytes PCs [] and smooth muscle cells SMCs [] in newly formingvessels Moreover PDGFB and PDGFR beta also interactwith other proangiogenic factors such as FGF2 [ ]VEGFA and its receptor VEGFR2 [] FurthermorePDGFB and PDGFR beta may also aï¬ect cancer growthand progression by directly acting on TME Besides thecrosstalk with CAFs [“] PDGFR beta in stromalfibroblasts may mediate PDGFBinduced TAM recruitment[] thus implicating a role of PDGFR beta in tumorstroma to facilitate tumor progression Most recently it wasfurther shown that specific targeting of PDGFR beta kinaseactivity in TME inhibited cancer growth and vascularizationin cancers with high PDGFB expression such as LLC []Therefore this indicates the diverse role of PDGFB andPDGFR beta in facilitating tumor angiogenesis and progression at diï¬erent cellular levels in TME PDGFR beta is demonstrated as a target of telomeric repeat binding factor TRF2 that is further activated transcriptionally by WT1[] PDGFB and PDGFR beta have further been identifiedas critical targets of PPAR betadelta via a direct transactivation mechanism

cancer275

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: "ligandactivated transcriptional factors that belong to the nuclear receptor superfamily Among them PPAR alpha andPPAR gamma are prone to exert an antiangiogenic eï¬ect whereas PPAR betadelta has an opposite eï¬ect in physiological andpathological conditions Angiogenesis has been known as a hallmark of cancer and our recent works also demonstrate thatvascularspecific PPAR betadelta overexpression promotes tumor angiogenesis and progression in vivo In this review we willmainly focus on the role of PPAR betadelta in tumor angiogenesis linked to the tumor microenvironment to further facilitatetumor progression and metastasis Moreover the crosstalk between PPAR betadelta and its downstream key signal moleculesinvolved in tumor angiogenesis will also be discussed and the network of interplay between them will further be established inthe review IntroductionPeroxisome proliferatoractivated receptorsPPARs asligandactivated transcription factors belong to the steroidreceptor superfamily which includes three isoforms PPARalpha PPAR betadelta and PPAR gamma [] PPARs formheterodimers with retinoic X receptors and regulate theexpression of various genes upon ligand binding PPARs alsointeract with corepressors or coactivators to modulate thetranscription of its downstream target genes PPARs asimportant transcriptional regulators have been suggested tobe involved in lipid metabolism and multiple cellular functions For instance PPAR alpha also functions in fatty acidbetaoxidation and vascular ‚ammation [] PPAR gammaacts as a regulator in adipocyte diï¬erentiation and type diabetes [] PPAR betadelta is a key player in cardiac energyproduction angiogenesis and particularly in cancer progression []PPAR alpha and PPAR gamma exert predominantly anantiangiogenic eï¬ect [“] but there still exist conflictingstudies showing opposite results [ ] On the contraryPPAR betadelta produces more obviously proangiogeniceï¬ects [“] In this review we will focus on the promotingrole of PPAR betadelta in angiogenesis especially in tumorangiogenesis The network of interplay between PPAR betadelta and its various downstream signal molecules and alsobetween those key molecules will be further discussed andestablished Remarkably diverse important signal moleculesinvolved in tumor angiogenesis and progression and cancercell metabolism have been identified as direct PPAR betadelta target genes AngiogenesisAngiogenesis is the physiological process through which anew capillary network forms from the preexisting vasculature[ ] whereas vasculogenesis denotes de novo bloodvessel formation mostly during embryogenesis in whichendothelial progenitor cells EPC migrate to sites of vascularization then diï¬erentiate into endothelial cells EC andcoalesce into the initial vascular plexus [ ] Besides theinteraction between proangiogenic factors and antiangiogenic factors angiogenesis is also a multiple step biologicalprocess during which a variety of molecules cooperateincluding cell adhesion molecules matrix metalloproteinases 0cPPAR ResearchMMPs extracellular matrix ECM and basement membrane componentsAngiogenesis is a physiological and vital process in development and growth An imbalance of proangiogenic andantiangiogenic factors causes angiogenesis in pathologicalconditions such as diabetic retinopathy and tumor growthThus when the imbalance comes to a point at which angiogenesis is triggered by tumor cells then an œangiogenicswitch of tumor cells is turned on during tumor progression the œangiogenic switch is often activated and remainson [“] Inducing angiogenesis is known as a hallmarkof cancer [] and angiogenesis is also a fundamental stepby which most benign tumors transition into malignant ones Tumor Angiogenesis Tumor needs to sprout new vesselsand further develop a vascular network in order to supplynutrients and oxygen remove waste products support a continually high proliferative rate and ultimately expand neoplastic growth [ ] Hence angiogenesis is essential forhelping sustain tumor growth and facilitate tumor progression Besides being a requirement for angiogenesis an abnormal vasculature also helps to promote tumor progression andmetastasis The tumor vascular wall is imperfect and prone toleakage so it is much easier for tumor cells to directly penetrate into the blood vessels or lymphatic vessels and then proliferate at another distant site to form metastasis []Due to intensive abnormal neovascularization in tumortissues most malignant tumors grow rapidly and acquirethe ability to spread to adjacent and distant ans whichmakes them more malignant and even life threateningTherefore angiogenesis indeed plays an important role intumor progression and metastasis and to intervene with thisprocess would obviously prevent tumor development andspread Thus this has been regarded as a critical target forantitumor therapy PPAR Alpha and AngiogenesisIt was reported firstly that a selective PPAR alpha agonistWY14643 did not show any eï¬ect on angiogenesis or EC proliferation [] But some subsequent studies showed that theactivation of PPAR alpha inhibited angiogenesis in vitro byusing fenofibrate a clinically used PPAR alpha agonist []Moreover fenofibrate suppressed EC proliferation migration and tube formation through inhibition of protein kinaseB Akt and disruption of the cytoskeleton [] Furthermore PPAR alpha activation was shown to inhibit vascularendothelial growth factor VEGF induced EC migrationand basic fibroblast growth factor bFGFFGF2 inducedcorneal angiogenesis in vitro and in vivo [] Especiallyin vivo reduced tumor growth and microvessel numberswere observed in mice implanted with melanoma Lewis lungcarcinoma LLC fibrosarcoma and glioblastoma due to asystemic treatment of PPAR alpha ligand and the antiangiogenic state induced through activation of PPAR alpha withelevated thrombospondin1 TSP1 and endostatin expression []Howeverit was demonstrated inanother observation that activation of PPAR alpha stimuin that same yearlated neovascularization in vivo with increased phosphorylation of endothelial nitric oxide synthase eNOS and Akt via aVEGFdependent manner [] Furthermore Zhang andWard also suggested that PPAR alpha activation inducedproangiogenic responses in human ocular cells [] Inanother study it was shown that a new PPAR alpha agonistRK13675 had no eï¬ect on angiogenesis [] RecentlyPPAR alpha activation is further shown to have antineovascularization eï¬ects with downregulation of VEGF and angiopoietin expression in a rat alkali burn model []In summary the role of PPAR alpha in angiogenesis isstill controversial Some observations showed that ligandactivation of PPAR alpha had antiangiogenic eï¬ects mediated either through upregulation of antiangiogenic factorssuch as TSP1 and endostatin or downregulation of proangiogenic factors including VEGF FGF2 AKT and angiopoietins Others also reported opposite results showing aproangiogenic role upon PPAR alpha activation Thus thespecific molecular mechanism is still unclear and needs tobe further studied PPAR Gamma and AngiogenesisLigand activation of PPAR gamma was previously shown toinhibit human umbilical vein endothelial cell HUVEC tubeformation in collagen gels [] and VEGFinduced choroidalneovascularization in vitro and in vivo [] Another studyalso demonstrated that EC apoptosis was induced throughtreatment with the PPAR gamma ligand 15dPGJ2 [] Furthermore rosiglitazone a potent PPAR gamma agonist wasshown to inhibit primary tumor growth and metastasisthrough both direct and indirect antiangiogenic eï¬ectsin vitro and bFGFinduced corneal neovascularizationin vivo [] Moreover a similar observation also displayedthe inhibition of VEGFinduced angiogenesis in a chickchorioallantonic membrane model [] In a mouse modelwith ischemiainduced retinopathy pioglitazone a PPARgamma agonist also showed a protective eï¬ect against pathological neoangiogenesis through upregulation of anti‚ammatory adipokine adiponectin [] Additionally thePPAR gamma antagonist GW9662 was shown to reverseOmega3 polyunsaturated fatty acidinduced reduction ofESelectin angiopoietin2 vascular cell adhesion molecule and intracellular adhesion molecule1 [] implicatingan antiangiogenic potential of PPAR gamma itself Howeveropposite results also showed that pioglitazone enhanced neovascularization and inhibited apoptosis of EPC in vitro andin vivo via a Phosphoinositide3Kinase PI3K dependentmanner []Nadra observed that PPAR gammanull embryosdisplayed a vascular structural defect at E95 Moreover disanized placental layers and an altered placental microvasculature were observed in pregnant wildtype mice treatedwith the PPAR gamma agonist rosiglitazone as well asreduced expression of proangiogenic factorsincludingVEGF proliferin and plateletendothelial cell adhesionmolecule1 PECAM1CD31 [] suggesting a crucial roleof PPAR gamma in placental vascular development The 0cPPAR Researchmajor antiangiogenic properties on PPAR gamma activationwere also reviewed here []Notablyin most cancersthe canonical Wntbetacatenin pathway is upregulated while on the contrary PPARgamma is downregulated Interestingly in numerous tissuesthe activation of PPAR gamma inhibits the betacateninpathway whereascanonicalWntbetacatenin signal cascade also inactivates PPARgamma [] implicating a negative regulatory role of PPARgamma in carcinogenesis where tumor angiogenesis mightbe a fundamental stepstimulation ofthetheIn summary PPAR gamma predominantly displays anantiangiogenic eï¬ect that may be mediated through the inhibition of VEGF or bFGFinduced neovascularization andreduction of the expression level of some proangiogenicfactors PPAR BetaDelta and AngiogenesisUnlike PPAR alpha and PPAR gamma on the contrarymany studies have explicitly shown the proangiogenic eï¬ectsof PPAR betadelta on physiological and pathological angiogenesis The first evidence provided in a study is that activation of PPAR betadelta with GW501516 a highly selectivePPAR betadelta agonistinduces HUVEC proliferationand an increased expression of VEGF and its receptorVEGFR1 FLT1 [] Besides inducing EC proliferationPPAR betadelta activation by itsligand prostacyclinPGI2 also stimulates upregulation of alpha expression an antiapoptotic and anti‚ammatory protein whichthereby protects ECs from H2O2induced apoptosis and oxidant injury [] Moreover a subsequent study further provides evidence that activation of PPAR betadelta withGW501516 induces angiogenesis during which VEGFrelease is considered as a major trigger factor [] firstlysuggesting the promotion for angiogenesis upon PPARbetadelta activationMüllerBrüsselbach show that PPAR betadelta mice implanted with LLC and B16 melanoma exhibit diminished blood flow and immature microvascular structurescompared with wildtype mice Moreover reexpression ofPPAR betadelta into the matrigelinvading cells triggersmicrovessel maturation and restores normal vascularization[] indicating a crucial role of PPAR betadelta in tumorvascularization Additionally another study also observedreduced levels of calcium intracellular channel protein CLIC4 but it observed enhanced expression of cellular retinol binding protein CRBP1 in migrating ECs from PPARbetadeltanull mice [] both of which play a role in tumorvascularization [ ] It was reported that PPAR betadeltawas required for placentation [] and most of the PPARbetadeltanull mutant embryos died at E95 to E105 due toabnormal celltocell communication atthe placentaldecidual interface [] However in these studies [“] adefect in angiogenesis was not observed during normal development in PPAR betadeltaknockout miceSome observations also show the important role of PPARbetadelta in physiological angiogenesis For instance skeletal musclespecific PPAR betadelta overexpression leads toPPAR betadeltaan increase in the number of oxidative muscle fibers andrunning endurance in adult mice [“] Moreover PPARbetadelta activation promotes a rapid muscle remodelingvia a calcineurindependent manner and induces muscleangiogenesis in highly selective PPAR betadelta agonistGW0742treated animals [] Furthermore in the heartpharmacologicalstimulation withGW0742 induces rapid cardiac growth and cardiac angiogenesis through direct transcriptional activation of calcineurin [] Interestingly the same cardiac phenotype wasalso observed after treatment with the PPAR betadelta agonist GW501516implicating a response specificity forPPAR betadelta stimulation [] Calcineurin activationfurther leads to the stimulation of nuclear factoractivatedT cell c3 NFATc3 and an enhanced expression of hypoxiainducible factor alpha HIF1alpha and cyclindependentkinase CDK9 [] Overall the remodeling in skeletalmuscle and heart is perfectly the same as the phenotypeobserved with exercise and both of them are mediatedthrough activation of calcineurinPPAR betadelta may act as a key regulator in mediatingpathological angiogenesis For instance PPAR betadelta wasshown to regulate retinal angiogenesis in vitro and in vivoand its inhibition reduced preretinal neovascularization possibly via an Angiopoietinlike protein Angptl4 dependent manner []implicating the potential of PPARbetadelta in modulating pathological ocular angiogenesisRecently an observation reported that PPAR betadeltaknockdown in both retinal pigment epithelial and choroidalendothelial cells caused an antiangiogenic phenotype andPPAR betadelta promoted laserinduced choroidal neovascular CNV lesions in PPAR betadelta mice [] Moreover pharmacological inhibition of PPAR betadelta with theantagonist GSK0660 also resulted in a significantly decreasedCNV lesion size in vivo suggesting a functional role of PPARbetadelta in the development of CNV lesions [] This indicates that PPAR betadelta has an important association withpathological angiogenesisAngiotensin II Ang II the biologically active peptide ofthe reninangiotensin system RAS is a major blood pressure and cardiovascular homeostasis regulator and is alsorecognized as a potent mitogen Angiotensinconvertingenzyme inhibitors were introduced approximately yearsago as antihypertensive agents and have since become asuccessful therapeutic approach for high blood pressurecongestive heart failure and postmyocardial infarction Inexperimental systemsthe antitumor eï¬ects of diverseACE inhibitors show that these inhibit cell proliferationand possessand anti‚ammatory eï¬ects [“] It has been shown recentlythat activation of PPAR betadelta inhibits Ang IIstimulated protein synthesis in a concentrationdependentmanner and suppresses Ang IIinduced generation of reactive oxygen species ROS in vascular smooth muscle cells[] PPAR betadelta was further shown to inhibit AngIImediated atherosclerosis [] However it is not clearuntil now if PPAR betadelta activation can be consideredis foras an ACE inhibitormimicking approach as itexample the case for PPAR gamma activators[]antiangiogenicantimetastatic 0cPPAR Researchthe relevance ofFurthermorethis hypothetical PPARbetadelta feature might be limited for tumor angiogenesiswhere vascular smooth muscle hypertrophy and atherosclerosis do not contribute to the major pathologyBesides inducing angiogenesis it has been demonstratedthat PPAR betadelta directly acts on early EPC through activation of the AKT pathway and induces an enhanced vasculogenesis [] Similarlythe PPAR betadeltamediatedprovasculogenic eï¬ects are also observed on late EPC []He showed that PPAR betadelta activation withGW501516 induced EPC proliferation and tube formationwhereas EPC treated with an inhibitor of cyclooxygenaseCOX or PGI2 synthase or with PPAR betadeltaspecificsiRNA also displayed an opposite eï¬ect [] Furthermoreit has been demonstrated that PPAR betadelta inducesangiogenesis and skeletal muscle regeneration throughmatrix metalloproteinase MMP 9mediated insulinlikegrowth factor1 paracrine networks upon EPC activation[] Han also observed that PPAR betadelta activationpromoted a rapid wound healing with enhanced angiogenesis in a mouse model with skin punch wound [] Overallin addition to EC PPAR betadelta is also a key regulator ofEPC or even may act as an initiator of activation of EPC tofurther induce vasculogenesis PPAR BetaDelta and Tumor AngiogenesisLinked to Tumor MicroenvironmentPPAR betadelta expression is often upregulated and promotes cancer progression in many major human cancerslung breast and gastric cancers [“]such as colonwhich suggests a crucial role of PPAR betadelta in cancercells even though there exist some conflicting studies indicating that the functional role of PPAR betadelta in tumorigenesis or carcinogenesis still remains highly controversial [“] and dependent on specific tumor or cancer cell typesThus here we discuss the promotion of PPAR betadelta intumor progression through facilitating tumor angiogenesisPPAR betadelta has been suggested as a critical œhubnode transcriptional factor which governs a tumor œangiogenic switch [ “] In the transcriptional networkanalysis it was reported that tumor growth and tumor angiogenesis were markedly inhibited in PPAR betadeltanullmice in comparison with wildtype mice [] Moreoverthe elevated PPAR betadelta expression level was also considered to be highly correlated to pathologically advancedtumor stage and increased cancer risk for recurrence and distant metastasis in patients with pancreatic cancer [] indicating the crucial association of PPAR betadelta withtumor angiogenesis progression and cancer invasivenessPPAR betadelta may indirectly facilitate tumor angiogenesis and progression through its function on the tumormicroenvironment TME where tumor angiogenesis is fostered Moreover a tumor also releases some extracellular signals to closely communicate and constantly collaborate withTME to facilitate tumor angiogenesis in order to furtherenable tumor growth and progression For instance it wasshown that colon cancer cells with PPAR betadelta knockoutfailed to stimulate EC vascularization in response to hypoxicstress whereas wildtype cells exposed to hypoxia were ableto induce angiogenesis [ ] suggesting that PPAR betadelta is required for the promotion of angiogenesis in hypoxic stressmediated TME Moreover in the TME tumorltrating myeloid cells are considered as the most important cells for fostering tumor angiogenesis among the multiple diï¬erent kinds of stromal cells [] Besides stimulatingtumor angiogenesis tumor myeloid cells also support tumrowth by suppressing tumor immunity and promotingtumor metastasis to distinct sites [] Interestingly it hasbeen demonstrated that PPAR betadelta activation intumorltrating myeloid cells stimulates cancer cell invasion and facilitates tumor angiogenesis via an Interleukin IL10 dependent manner [] Moreover impairedtumor growth and angiogenesis were observed in PPARbetadelta KO BMT mice due to PPAR betadelta deficiencyin tumor myeloid cells [] suggesting that PPAR betadeltaplays a key role in tumor angiogenesis and progression intumor myeloid cells of TMEFurthermore the endoplasmic reticulum ER an essential anelle involved in many cellular functions is implicated in TME In cancer stressors like hypoxia nutrientdeprivation and acidosis disrupt ER function and lead toaccumulation of unfolded proteins in ER a condition knownas ER stress Cells adapt to ER stress by activating an integrated signal transduction pathway called the unfolded protein response UPR UPR represents a survival response bythe cells to restore ER homeostasis and has both survivaland cell death eï¬ects The mechanisms that determine cellfate during ER stress are not well understood For instanceshort exposure to ER stress initially increases AKT signalingbut longterm ER stress suppresses AKT signaling []PPAR betadelta activation has been shown to reduce endoplasmic reticulum ER stressassociated ‚ammation inskeletal muscle through an AMPKdependent mechanism[] and to reduce ‚ammation in response to chronic ERstress in cardiac cells [] Furthermore it has been nicelyshown that PPAR betadelta can repress RASoncogeneinduced ER stress to promote senescence in tumors [] Thisis mediated through the decrease of pAKT activity promoting cellular senescence through upregulation of p53 and p27expression [] It would be interesting to investigate thedirect eï¬ects of PPAR betadelta on senescence of tumorendothelial cells in an in vivo setting We recently showedthat senescent endothelial cells are indispensable for ahealthy lifespan and that removal of senescent endotheliumdisrupts vascular function leading to diminished vessel densities and fibrotic lesions [] If PPAR betadelta mediatessenescence of tumor endothelium thereby protecting vesselintegrity this might explain the enhanced tumor growthand vascularization upon PPAR betadelta activationobserved by us and others [ ]Most recently Zuo demonstrated that PPARbetadelta in cancer cells regulates tumor angiogenesisin vivo and in vitro by promoting the secretion of proangiogenic factors including VEGF and Interleukin IL8[] Most importantly in our recent works it has beenshown that conditionalinducible vascular endotheliumspecific PPAR betadelta overexpression in vivo leads to 0cPPAR Researchenhanced tumor angiogenesis tumor growth and metastasis formationfurther indicating a vascular ECspecificPPAR betadelta action mechanism in tumor progressionindependent of some controversial observations of PPARbetadelta in specific tumor or cancer cell types [] Wagner also firstly reported the mouse model in whichrapid induction of cardiac angiogenesis and cardiac hypertrophy were observed [ ] Crosstalk between PPAR BetaDelta and SignalMolecules PPAR betadelta activation or overexpressionmay upregulate the expression of its various downstream signal molecules involved in tumor angiogenesis includingproangiogenic factors such as VEGF PDGF and FGFproinvasive matrixdegrading enzymes such as MMP9pro‚ammatory mediators such as COX2 and cytokinesand chemokines such as IL1 and CXCL8 even some ofwhich have been further identified as PPAR betadelta directtarget genes Besides a leading role of PPAR betadelta amongthe signal molecules PPAR betadelta may function in TMElinked to diverse kinds of cells through direct or indirectmodulation of its downstream molecules Interplay between PPAR BetaDelta and InflammatoryAngiogenesis Inflammatory angiogenesis is a crucial processin tumor progression For instance the pro‚ammatorymediator cyclooxygenase2 COX2 is considered as a keyregulator of angiogenesis and tumor growth through multiple downstream proangiogenic mechanisms such as production of VEGF and induction of MMPs Moreover selectiveinhibition of COX2 has also been shown to suppress angiogenesis in vivo and in vitro [] It is well known that VEGFAplays a critical role in both angiogenesis and vasculogenesis[] and it leads the directional migration of tip cells andstalk cell proliferation in microtubule branches [ ] Ithas also been demonstrated that MMP9 triggers the œangiogenic switch during carcinogenesis and enhances the availability of VEGF to its receptors [] Furthermore it hasbeen reported that ‚ammatory cell MMP9 initiates theonset of tumor neovascularization during which there existsfunctionalincludingMMP9 [] LEPTIN is shown to mediate angiogenesisin vivo and in vitro through induction of EC proliferationand expression of MMP2 and MMP9 [] and to furtherpromote EC diï¬erentiation and directional migrationthrough enhancement of COX2 activity [] LEPTIN couldalso induce angiogenesis via transactivation of VEGFR inECs [] Additionally besides inducing angiogenesisPPAR betadelta also functions in chronic ‚ammationfacilitating tumorigenesis through induction of COX2 andits product prostaglandin E2 PGE2 in vivo [ ]Interestingly COX2 VEGF MMP9 and LEPTIN have beenidentified as PPAR betadelta target genes via a direct transcriptional activation mechanism in hepatocellular carcinoma cells [] colorectal cancer cells [ ] EPCs[ ] and liposarcoma cells [] respectivelylinks between VEGF and MMPsIn TME tumorltrating ‚ammatory cells also helpto induce and sustain tumor angiogenesis and further tofacilitate tissue invasion and tumor metastatic spread byreleasing some signal molecules such as proinvasive MMP9and ‚ammatory chemokines [“] Chemotaxis is alsoa crucial process for inducing angiogenesis in tumors eitherdirectly by attracting ECs towards tumor cells to form newvessels or indirectly by mediating immune ‚ammatorycells to ltrate eventually promoting tumor angiogenesis[] Chemotaxis of tumor cells and stromal cells in TMEis also required for tumor dissemination during tumor progression and metastasis [ ]CXC chemokines such as CXCL8 encoding IL8 andCXCL5 are also involved in COX2associated angiogenesisto contribute to nonsmallcelllung cancer progression[ ] It is further shown that IL8 directly regulatesangiogenesis via recruitment of neutrophils [] whichfurther drives VEGF activation [] MoreoverIL8responding neutrophils are considered as the major sourceof angiogenesisinducing MMP9 [ ] Chemokine CC motif ligand CCL2 in addition to the promotionof angiogenesis [ ] also enhances tumor metastasis[] Furthermore myeloid monocytic cellssuch asmyeloidderivedtumorMDSCsassociated macrophages TAMs and dendritic cells arerecruited to the tumor site mainly by CCL2 and producemany proangiogenic factorssuch as VEGF CXCL8plateletderived growth factor PDGF and transforminggrowth factor beta TGF beta [“] In fact bothTGF beta and hypoxia are potentinducers of VEGFexpression in tumor cells and collaborate with TME toprovide the foundation of tumor angiogenesis and cancercell invasion [] Importantly IL8 has been reported asa key target gene of PPAR betadelta to promote angiogenesis in vivo and in vitro [] and CCL2 expression isalso significantly upregulated upon vascular PPAR betadelta overexpression in vivo []suppressorcellsCOX2 also mediates IL1 betainduced angiogenesisin vitro and in vivo [ ] IL1 beta supports neovascularization through the regulation of the expression of VEGFand its receptor VEGFR2 FLK1KDR on ECs [] IL1 actsas an upstream pro‚ammatory mediator that initiates anddisseminates the ‚ammatory state by inducing a localinteractive network and increasing adhesion moleculeexpression on ECs and leukocytes which facilitates tumorassociated angiogenesis [] In TME ‚ammatory IL1beta recruits myeloid cells from bone marrow and activatesthem to produce proangiogenic factors such as VEGF VEGFfurther activates ECs and myeloid cells promoting tumorinvasiveness and fostering tumor angiogenesis [] In addition IL6 also stimulates angiogenesis and vasculogenesis[ ] However Gopinathan observed an IL6induced newly forming vascular structure with defectivepericyte PC coverage ex vivo [] thus facilitating cancercell ltration and tumor metastasis through vascular leakage Interestingly IL1 and IL6 expression levels are significantly upregulated in the PPAR betadelta overexpressionmouse model reported recently []In summary PPAR betadelta seems to act as a key leaderin ‚ammatory mediatordriven tumor angiogenesis linkedto TME in which many pro‚ammatory mediators chemokines and proangiogenic factors closely communicate with 0cPPAR Researcheach other and also associate with tumorltrating myeloid cells such as neutrophils TAMs and MDSCs Other Key PPAR BetaDeltaMediated ProangiogenicFactors It has been demonstrated that Wilms™ tumor suppressor WT1 is a major regulator of tumor neovascularization andtumor progression [] E26 avian leukemia oncogene ETS1 also plays a key role in regulating vascular development and haemopoiesis particularly in angiogenesis []In addition ETS1 promotes cancer cell invasion throughupregulation of MMPs [] Consistent with this silencingof ETS1 in highly invasive breast cancer cells also reducesthe expression of MMP9 and MMP1 []ETS1 also acts as a key regulator of MMPs such asMMP1 MMP3 and MMP9 in human cancerassociatedfibroblasts CAFs [ ] CAFs support tumor growthby secreting growth factors such as VEGF FGF PDGF andchemokines to stimulate angiogenesis and thereby promotecancer cell invasion and metastasis formation [ ]CAFs as metastatic tumor stroma are a crucial componentin tumor progression through the remodeling of the ECMstructure thus helping a tumor to acquire an aggressive phenotype [ ] PPAR betadelta in CAFs also exhibits aprotumorigenic eï¬ect It was reported that ablation of PPARbetadelta in CAFs attenuated tumor growth by altering theredox balance in TME [] suggesting that PPAR betadeltain CAFs is also an important player in tumor developmentETS1 induces the expression of VEGF VEGFR1 andVEGFR2 in ECs [“] In turn VEGF is also a majorinducer of ETS1 in ECs through the activation of either thePI3KAKT pathway or the MEKERK12 signal cascade[ ] WT1 is also reported to regulate tumor angiogenesis via direct transactivation of ETS1 []SRYrelated HMGbox SOX18 has also beenreported previously to induce angiogenesis during tissuerepair and wound healing [] and cancer progression[] And most recently it was further shown that specificECderived endovascular progenitors initiated a vasculogenic process and diï¬erentiated into more mature endothelial phenotypes within the core of the growing tumorsthrough reactivation of SOX18 [] Interestingly theseimportant proangiogenic molecules including WT1 ETS1and SOX18 are also significantly upregulated in the vascularPPAR betadelta overexpression model in vivo [] AndWT1 is also identified as a target gene of PPAR betadeltain melanoma cells [] PPAR BetaDelta May Facilitate Cancer Progression atDiverse Cellular Levels in TME PPAR betadelta activationis shown to induce colonic cancer stem cell CSC expansionand to promote the liver metastasis of colorectal cancerin vivo via direct transactivation of the Nanog gene []NANOG as a key transcriptional factor governs the selfrenewal and pluripotency of stem cells [] and cancer cellsexpressing NANOG also often exhibit stem cell properties[] Protooncogene cKITCD117 is known as the maststem cell factor receptor and receptor tyrosine kinase andits activation in CSCs may regulate the stemness to controltumor progression and drug resistance to tyrosine kinaseinhibitors Moreover cKIT has been identified as a potentialmarker of the cancer stemlike cells [] In addition cKITnot only functions on ECs [ ] but also belongs to thetumor angiogenesispromoting molecule [“] Studiesalso suggested that activation of cKIT enhances the expression of VEGF that can be suppressed by imatinib an inhibitor of cKIT in gastrointestinal stromal tumor cells whichthereby has an impact on tumor angiogenesis [ ] cKIT is also involved in pathological ocular neovascularization [] and is regulated transcriptionally by WT1 []and PPAR betadelta []PDGFB and its receptor PDGFR beta also known asangiogenic factors are suggested to enhance angiogenesisand vasculogenesis via their function in ECs [“] andEPCs [] and to regulate vascular permeability and vesselmaturation through recruitment of pericytes PCs [] and smooth muscle cells SMCs [] in newly formingvessels Moreover PDGFB and PDGFR beta also interactwith other proangiogenic factors such as FGF2 [ ]VEGFA and its receptor VEGFR2 [] FurthermorePDGFB and PDGFR beta may also aï¬ect cancer growthand progression by directly acting on TME Besides thecrosstalk with CAFs [“] PDGFR beta in stromalfibroblasts may mediate PDGFBinduced TAM recruitment[] thus implicating a role of PDGFR beta in tumorstroma to facilitate tumor progression Most recently it wasfurther shown that specific targeting of PDGFR beta kinaseactivity in TME inhibited cancer growth and vascularizationin cancers with high PDGFB expression such as LLC []Therefore this indicates the diverse role of PDGFB andPDGFR beta in facilitating tumor angiogenesis and progression at diï¬erent cellular levels in TME PDGFR beta is demonstrated as a target of telomeric repeat binding factor TRF2 that is further activated transcriptionally by WT1[] PDGFB and PDGFR beta have further been identifiedas critical targets of PPAR betadelta via a direct transactivation mechanism Answer:

Thyroid_Cancer

Official Case Reports Journal of the Asian Pacific Society of RespirologyRespirology Case ReportsEndobronchial metastases from a primary embryonalcarcinomaChiKang Teng1ChihYen Tu121Division of Pulmonary and Critical Care Medicine Department of Internal Medicine China Medical University Hospital Taichung Taiwan2School of Medicine China Medical University Taichung Taiwan3Division of Pathology China Medical University Hospital Taichung Taiwan WenChien Cheng12 ChiehLung Chen1 TingHan Chen1 YunShan Lin3 AbstractWe report the case of a 24yearold man who presented with chief complaintsof shortness of breath and haemoptysis chest radiography revealed completecollapse of the left lung Bronchoscopy revealed an endobronchial tumourwith complete obstruction of the left main bronchus Cryosurgical excisionwas performed tissue pathology confirmed the diagnosis of metastatic embryonal carcinoma The patient underwent a right orchiectomy followed by ableomycin etoposide cisplatin BEP chemotherapy regimenKeywordsCryosurgery embryonal carcinoma endobronchialmetastases endobronchial tumourCorrespondenceWenChien Cheng Division of Pulmonary and Critical Care Medicine Department of Internal MedicineChina Medical University Hospital No YudeRoad North Dis Taichung City TaiwanEmail wcchengdrgmailcomReceived July Revised July Accepted July Associate Editor James HoRespirology Case Reports e00644 101002rcr2644IntroductionLung metastases from extrapulmonary malignancies areobserved frequently in clinical practice by contrastendobronchial metastases EBMs from extrapulmonarymalignancies are rare and may have distinct histopathological etiologies [“] Primary lung cancer is the most common cause of endobronchialtumours Extrapulmonarymalignancies that are frequently associated with metastasesto the central airways include tumours of breast colorectalthyroid origin [“] Although mediastinalrenal orlymphadenopathy is frequently observed in associationwith testicular seminoma EBMs from embryonal carcinomas are extremely rare [] In this report we present acase of EBM from a primary embryonal carcinomaCase ReportA 24yearold man with no relevant past medical historypresented to our hospital with a chief complaint of shortness of breath lasting for several days Upon questioningthecoughexperiencedrevealedpatientthatheanendobronchialrevealedleft main bronchushaemoptysis shortness of breath and occasional chest painfor the past several days He reported no fever chills coldsweats weight loss or decreased appetite A chest radiograph at admission revealed complete collapse of the leftlung Fig 1A Computed tomography CT was notable forleft pleural masses an endobronchial tumour obstructingthe left main bronchus and complete collapse of the leftlung and a soft tissue mass lesion in the right scrotumBronchoscopytumourobstructing theFig 1B Theendobronchialtumour was excised with bronchoscopiccryosurgery a followup chest radiograph revealed someimprovement in the status of the left lung Immunohistochemical staining of the tumour tissue revealed that the cellswere thyroid transcription factor1 TTF1negative sallike protein SALL4positive and cluster of differentiation CD30positive These findings were consistentwith a final diagnosis of metastatic embryonal carcinomaFig We checked the levels of tumour markers in thepatient including those of alphafetoprotein AFP betahuman chorionic gonadotropin BhCG and lactate dehydrogenase LDH Each tumour marker was found to be The Authors Respirology Case Reports published by John Wiley Sons Australia Ltdon behalf of The Asian Pacific Society of RespirologyThis is an access under the terms of the Creative Commons AttributionNonCommercialNoDerivs License which permits use and distribution in any mediumprovided the original work is properly cited the use is noncommercial and no modifications or adaptations are made Vol Iss e00644Page 0cEBM from embryonal carcinomaCK Teng et alFigure Chestradiograph and bronchoscopic view of the endobronchial metastasesEBM A Complete collapse of the left lungon chest radiograph B Bronchoscopic viewof the endobronchial tumour within the leftmain bronchusFigure Tumour pathology of metastatic embryonal carcinoma A Embryonal carcinoma with a complex glandular growth pattern The characteristic large cohesive and highly pleomorphic tumour cells with moderate amounts of amphophilic cytoplasm overlapping nuclei vesicular chromatinand frequent mitoses are shown as indicated by the arrows original magnification — B Immunohistochemical staining with antithyroid transcription factor1 TTF1 highlighting cells in the alveolar space original magnification — C Immunohistochemical staining with antisallikeprotein SALL4 revealed diffuse nuclear staining original magnification — D Immunohistochemical staining with anticluster of differentiation CD30 highlighting diffuse membranous staining original magnification —presentin high levels AFP ngmL BhCG mIUmL and LDH IUL The patientunderwent a right orchiectomy followed by a BEPbleomycin etoposide cisplatin chemotherapy regimenDiscussionWe report here the case of a young man with an EBMfrom a primary embryonal carcinoma who presentedshortness of breath and haemoptysis chest radiography atpresentation revealed complete collapse of the left lungtumoursLikewise manykindsLung metastases from extrapulmonary malignancies arereported relatively frequently in clinical practice howeverEBMs from extrapulmonary malignancies are rare [“] Primary lung cancer is the most common cause ofendobronchialofextrapulmonary primary tumours have been associatedwith EBM primarily breast colon and renal carcinomas[“] EBMs from testicular seminomas are also extremelyrare The majority of testicular tumours arise from testicular germ cells and are frequently composed of multiple celltypesaretumours most ofie mixedtypethese The Authors Respirology Case Reports published by John Wiley Sons Australia Ltdon behalf of The Asian Pacific Society of Respirology 0cCK Teng et alEBM from embryonal carcinomaTable Reports of previous cases of EBMsLocationDiagnostic methodPathology–zt¼rk []MoreiraMeyer []Case Case The orifice of right upper lobeRight main bronchusLeft main bronchus main carina andright main bronchus Fibreoptic bronchoscopy Mixed GCTFibreoptic bronchoscopy Mixed GCTVideobronchoscopyEmbryonal carcinoma–zsu []The orifice of the right upper lobeFibreoptic bronchoscopyTesticular seminomaTuran []Varkey []Our caseand right intermediary lobeRight intermediate bronchusLeft main bronchusLeft main bronchusEBM endobronchial metastases GCT germ cell tumourembryonic carcinomas or seminomas “There are only a few published reports of primary testicularembryonic carcinomas resulting in EBMs [“]The mostofcommon symptomsendobronchialtumours are haemoptysis and coughing with shortness ofbreath and wheezing reported less frequently Howeversome patients are asymptomatic [] In our patient symptoms on presentation included haemoptysis and shortnessof breathResults from chest radiography in patients with EBMcan be quite variable and may include mediastinal lymphadenopathy hilar masses atelectasis and multiple pulmonary nodules chest radiographs may also be normal onpresentation [] Our patient presented with complete collapse of the left lung that was revealed initially by chestradiographyHowever the full diagnosis cannot be made based onlyon symptoms and chest radiographs it can be difficult todistinguish between primary lung cancer and tumours ofextrapulmonary origin based on these findings alone Toconfirm the diagnosis we obtained a bronchoscopic biopsyspecimen to examine the tumour tissue The flexible bronchoscopy fibreoptic bronchoscopy can be performedunder sedation without general anaesthesia as comparedwith rigid bronchoscopy The former is a simple techniquewhich is well tolerated and most commonly performed asan outpatient day procedure [] The patient underwentbronchoscopic cryosurgery under sedation in our bronchoscopy room to excise the tumour the final pathology reportconfirmed the diagnosis of metastatic embryonal carcinomaWe had evaluated the presence of AFP BhCG andLDH tumour markers Elevated AFP levels can be secretedby germ cell tumours GCTs including embryonal carcinoma yolk sac tumour or teratoma In GCTs detectableRigid bronchoscopyBronchoscopyFibreoptic bronchoscopyand cryosurgerySomatictype GCTEmbryonal carcinomaEmbryonal carcinomaBhCG elevation is observed in both seminomas and nonseminomas The serum level of LDH was directly correlated with tumour burden in nonseminomatous GCTswhich is also useful for the surveillance of patients withadvanced seminoma [] The tumour markers in ourpatient showed elevated levels of AFP BhCG and LDHThis was compatible with the diagnosis of embryonal carcinoma MoreiraMeyer also evaluated the patienttumour markers and found elevated levels of AFP ngmL and BhCG mIUmL The elevatedlevels of both tumour markers contribute to the diagnosisof metastatic embryonal carcinoma [] –zsu onlyevaluated the patient™s BhCG level which was found to beelevated mIUmL and the final diagnosis wasmetastatic testicular seminoma []On comparison with previous case reports Table ours was the first case in which the tissue was obtainedusing cryosurgery Other reports obtained tissue samplesusing forceps biopsy alone or forceps biopsy combinedwith argon plasma coagulation APC to control bleedingCryobiopsy provided us with enough sample to performmore extensive immunohistochemical staining Cryobiopsyhas more successful diagnostic results than forceps biopsiesdue to larger and highquality artefactfree samplesHaemorrhage was observed to be similar during both procedures [] Further study on this topic will be needed toevaluate which of the diagnostic methods result in superioroutcomesIn conclusion EBMs from primary GCTs notably thoseassociated with total or partial collapse are extremely rareWe have presented this case to emphasize the importanceof distinguishing EBM from primary lung carcinoma andto report the first case in which metastatic embryonalcarcinoma was diagnosed by bronchoscopic cryosurgery The Authors Respirology Case Reports published by John Wiley Sons Australia Ltdon behalf of The Asian Pacific Society of Respirology 0cEBM from embryonal carcinomaDisclosure StatementAppropriate written informed consent was obtained forpublication ofand accompanyingimagesreportcasethisReferences –zt¼rk A Aktas¸ Z and Yılmaz A Endobronchialmetastasis of mixed germ cell tumors two cases TuberkToraks “ Lee SH Jung JY Kim DH Endobronchialmetastases from extrathoracic malignancy Yonsei Med J“ Ikemura K Lin DM Martyn CP Endobronchialmetastasis from extrapulmonary neoplasms analysis ofclinicopathologic features and cytological evaluation bybronchial brushing Lung “ MoreiraMeyer A BautistaHerrera D Hern¡ndezembryonal EndobronchialGonz¡lez MCK Teng et alcarcinoma“J BronchologyInterv Pulmonol –zsu S Erol MM Oztuna F Endobronchial metastasis from testicular seminoma Med Princ Pract “tumoraltesticular germ cell Turan D Akif –zg¼l M Kirkil GEndobronchial metastasis ofEurasian J Pulmonol “et Varkey B and Heckman MG Diagnosis of a case ofembryonal carcinoma by bronchial biopsy Chest “ Paradis TJ Dixon J and Tieu BH The role of bronchoscopy in the diagnosis of airway disease J Thorac Dis“ Aktas Z Gunay E Hoca NT Endobronchialcryobiopsy or forceps biopsy for lung cancer diagnosisAnn Thorac Med “ Barlow LJ Badalato GM and McKiernan JM Serumtumor markers in the evaluation of male germ cell tumorsNat Rev Urol “ The Authors Respirology Case Reports published by John Wiley Sons Australia Ltdon behalf of The Asian Pacific Society of Respirology 0c'

cancer276

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: Official Case Reports Journal of the Asian Pacific Society of RespirologyRespirology Case ReportsEndobronchial metastases from a primary embryonalcarcinomaChiKang Teng1ChihYen Tu121Division of Pulmonary and Critical Care Medicine Department of Internal Medicine China Medical University Hospital Taichung Taiwan2School of Medicine China Medical University Taichung Taiwan3Division of Pathology China Medical University Hospital Taichung Taiwan WenChien Cheng12 ChiehLung Chen1 TingHan Chen1 YunShan Lin3 AbstractWe report the case of a 24yearold man who presented with chief complaintsof shortness of breath and haemoptysis chest radiography revealed completecollapse of the left lung Bronchoscopy revealed an endobronchial tumourwith complete obstruction of the left main bronchus Cryosurgical excisionwas performed tissue pathology confirmed the diagnosis of metastatic embryonal carcinoma The patient underwent a right orchiectomy followed by ableomycin etoposide cisplatin BEP chemotherapy regimenKeywordsCryosurgery embryonal carcinoma endobronchialmetastases endobronchial tumourCorrespondenceWenChien Cheng Division of Pulmonary and Critical Care Medicine Department of Internal MedicineChina Medical University Hospital No YudeRoad North Dis Taichung City TaiwanEmail wcchengdrgmailcomReceived July Revised July Accepted July Associate Editor James HoRespirology Case Reports e00644 101002rcr2644IntroductionLung metastases from extrapulmonary malignancies areobserved frequently in clinical practice by contrastendobronchial metastases EBMs from extrapulmonarymalignancies are rare and may have distinct histopathological etiologies [“] Primary lung cancer is the most common cause of endobronchialtumours Extrapulmonarymalignancies that are frequently associated with metastasesto the central airways include tumours of breast colorectalthyroid origin [“] Although mediastinalrenal orlymphadenopathy is frequently observed in associationwith testicular seminoma EBMs from embryonal carcinomas are extremely rare [] In this report we present acase of EBM from a primary embryonal carcinomaCase ReportA 24yearold man with no relevant past medical historypresented to our hospital with a chief complaint of shortness of breath lasting for several days Upon questioningthecoughexperiencedrevealedpatientthatheanendobronchialrevealedleft main bronchushaemoptysis shortness of breath and occasional chest painfor the past several days He reported no fever chills coldsweats weight loss or decreased appetite A chest radiograph at admission revealed complete collapse of the leftlung Fig 1A Computed tomography CT was notable forleft pleural masses an endobronchial tumour obstructingthe left main bronchus and complete collapse of the leftlung and a soft tissue mass lesion in the right scrotumBronchoscopytumourobstructing theFig 1B Theendobronchialtumour was excised with bronchoscopiccryosurgery a followup chest radiograph revealed someimprovement in the status of the left lung Immunohistochemical staining of the tumour tissue revealed that the cellswere thyroid transcription factor1 TTF1negative sallike protein SALL4positive and cluster of differentiation CD30positive These findings were consistentwith a final diagnosis of metastatic embryonal carcinomaFig We checked the levels of tumour markers in thepatient including those of alphafetoprotein AFP betahuman chorionic gonadotropin BhCG and lactate dehydrogenase LDH Each tumour marker was found to be The Authors Respirology Case Reports published by John Wiley Sons Australia Ltdon behalf of The Asian Pacific Society of RespirologyThis is an access under the terms of the Creative Commons AttributionNonCommercialNoDerivs License which permits use and distribution in any mediumprovided the original work is properly cited the use is noncommercial and no modifications or adaptations are made Vol Iss e00644Page 0cEBM from embryonal carcinomaCK Teng et alFigure Chestradiograph and bronchoscopic view of the endobronchial metastasesEBM A Complete collapse of the left lungon chest radiograph B Bronchoscopic viewof the endobronchial tumour within the leftmain bronchusFigure Tumour pathology of metastatic embryonal carcinoma A Embryonal carcinoma with a complex glandular growth pattern The characteristic large cohesive and highly pleomorphic tumour cells with moderate amounts of amphophilic cytoplasm overlapping nuclei vesicular chromatinand frequent mitoses are shown as indicated by the arrows original magnification — B Immunohistochemical staining with antithyroid transcription factor1 TTF1 highlighting cells in the alveolar space original magnification — C Immunohistochemical staining with antisallikeprotein SALL4 revealed diffuse nuclear staining original magnification — D Immunohistochemical staining with anticluster of differentiation CD30 highlighting diffuse membranous staining original magnification —presentin high levels AFP ngmL BhCG mIUmL and LDH IUL The patientunderwent a right orchiectomy followed by a BEPbleomycin etoposide cisplatin chemotherapy regimenDiscussionWe report here the case of a young man with an EBMfrom a primary embryonal carcinoma who presentedshortness of breath and haemoptysis chest radiography atpresentation revealed complete collapse of the left lungtumoursLikewise manykindsLung metastases from extrapulmonary malignancies arereported relatively frequently in clinical practice howeverEBMs from extrapulmonary malignancies are rare [“] Primary lung cancer is the most common cause ofendobronchialofextrapulmonary primary tumours have been associatedwith EBM primarily breast colon and renal carcinomas[“] EBMs from testicular seminomas are also extremelyrare The majority of testicular tumours arise from testicular germ cells and are frequently composed of multiple celltypesaretumours most ofie mixedtypethese The Authors Respirology Case Reports published by John Wiley Sons Australia Ltdon behalf of The Asian Pacific Society of Respirology 0cCK Teng et alEBM from embryonal carcinomaTable Reports of previous cases of EBMsLocationDiagnostic methodPathology–zt¼rk []MoreiraMeyer []Case Case The orifice of right upper lobeRight main bronchusLeft main bronchus main carina andright main bronchus Fibreoptic bronchoscopy Mixed GCTFibreoptic bronchoscopy Mixed GCTVideobronchoscopyEmbryonal carcinoma–zsu []The orifice of the right upper lobeFibreoptic bronchoscopyTesticular seminomaTuran []Varkey []Our caseand right intermediary lobeRight intermediate bronchusLeft main bronchusLeft main bronchusEBM endobronchial metastases GCT germ cell tumourembryonic carcinomas or seminomas “There are only a few published reports of primary testicularembryonic carcinomas resulting in EBMs [“]The mostofcommon symptomsendobronchialtumours are haemoptysis and coughing with shortness ofbreath and wheezing reported less frequently Howeversome patients are asymptomatic [] In our patient symptoms on presentation included haemoptysis and shortnessof breathResults from chest radiography in patients with EBMcan be quite variable and may include mediastinal lymphadenopathy hilar masses atelectasis and multiple pulmonary nodules chest radiographs may also be normal onpresentation [] Our patient presented with complete collapse of the left lung that was revealed initially by chestradiographyHowever the full diagnosis cannot be made based onlyon symptoms and chest radiographs it can be difficult todistinguish between primary lung cancer and tumours ofextrapulmonary origin based on these findings alone Toconfirm the diagnosis we obtained a bronchoscopic biopsyspecimen to examine the tumour tissue The flexible bronchoscopy fibreoptic bronchoscopy can be performedunder sedation without general anaesthesia as comparedwith rigid bronchoscopy The former is a simple techniquewhich is well tolerated and most commonly performed asan outpatient day procedure [] The patient underwentbronchoscopic cryosurgery under sedation in our bronchoscopy room to excise the tumour the final pathology reportconfirmed the diagnosis of metastatic embryonal carcinomaWe had evaluated the presence of AFP BhCG andLDH tumour markers Elevated AFP levels can be secretedby germ cell tumours GCTs including embryonal carcinoma yolk sac tumour or teratoma In GCTs detectableRigid bronchoscopyBronchoscopyFibreoptic bronchoscopyand cryosurgerySomatictype GCTEmbryonal carcinomaEmbryonal carcinomaBhCG elevation is observed in both seminomas and nonseminomas The serum level of LDH was directly correlated with tumour burden in nonseminomatous GCTswhich is also useful for the surveillance of patients withadvanced seminoma [] The tumour markers in ourpatient showed elevated levels of AFP BhCG and LDHThis was compatible with the diagnosis of embryonal carcinoma MoreiraMeyer also evaluated the patienttumour markers and found elevated levels of AFP ngmL and BhCG mIUmL The elevatedlevels of both tumour markers contribute to the diagnosisof metastatic embryonal carcinoma [] –zsu onlyevaluated the patient™s BhCG level which was found to beelevated mIUmL and the final diagnosis wasmetastatic testicular seminoma []On comparison with previous case reports Table ours was the first case in which the tissue was obtainedusing cryosurgery Other reports obtained tissue samplesusing forceps biopsy alone or forceps biopsy combinedwith argon plasma coagulation APC to control bleedingCryobiopsy provided us with enough sample to performmore extensive immunohistochemical staining Cryobiopsyhas more successful diagnostic results than forceps biopsiesdue to larger and highquality artefactfree samplesHaemorrhage was observed to be similar during both procedures [] Further study on this topic will be needed toevaluate which of the diagnostic methods result in superioroutcomesIn conclusion EBMs from primary GCTs notably thoseassociated with total or partial collapse are extremely rareWe have presented this case to emphasize the importanceof distinguishing EBM from primary lung carcinoma andto report the first case in which metastatic embryonalcarcinoma was diagnosed by bronchoscopic cryosurgery The Authors Respirology Case Reports published by John Wiley Sons Australia Ltdon behalf of The Asian Pacific Society of Respirology 0cEBM from embryonal carcinomaDisclosure StatementAppropriate written informed consent was obtained forpublication ofand accompanyingimagesreportcasethisReferences –zt¼rk A Aktas¸ Z and Yılmaz A Endobronchialmetastasis of mixed germ cell tumors two cases TuberkToraks “ Lee SH Jung JY Kim DH Endobronchialmetastases from extrathoracic malignancy Yonsei Med J“ Ikemura K Lin DM Martyn CP Endobronchialmetastasis from extrapulmonary neoplasms analysis ofclinicopathologic features and cytological evaluation bybronchial brushing Lung “ MoreiraMeyer A BautistaHerrera D Hern¡ndezembryonal EndobronchialGonz¡lez MCK Teng et alcarcinoma“J BronchologyInterv Pulmonol –zsu S Erol MM Oztuna F Endobronchial metastasis from testicular seminoma Med Princ Pract “tumoraltesticular germ cell Turan D Akif –zg¼l M Kirkil GEndobronchial metastasis ofEurasian J Pulmonol “et Varkey B and Heckman MG Diagnosis of a case ofembryonal carcinoma by bronchial biopsy Chest “ Paradis TJ Dixon J and Tieu BH The role of bronchoscopy in the diagnosis of airway disease J Thorac Dis“ Aktas Z Gunay E Hoca NT Endobronchialcryobiopsy or forceps biopsy for lung cancer diagnosisAnn Thorac Med “ Barlow LJ Badalato GM and McKiernan JM Serumtumor markers in the evaluation of male germ cell tumorsNat Rev Urol “ The Authors Respirology Case Reports published by John Wiley Sons Australia Ltdon behalf of The Asian Pacific Society of Respirology 0c' Answer:

Thyroid_Cancer

"preadipocytes diï¬erentiate into adipocytes During this process the preadipocytes ceaseto proliferate begin to accumulate lipid droplets and develop morphologic and biochemical characteristics of mature adipocytesMesenchymal stem cells MSCs are a type of adult stem cells known for their high plasticity and capacity to generate mesodermaland nonmesodermal tissues Many mature cell types can be generated from MSCs including adipocyte osteocyte and chondrocyteThe diï¬erentiation of stem cells into multiple mature phenotypes is at the basis for tissue regeneration and repair Cancer stem cellsCSCs play a very important role in tumor development and have the potential to diï¬erentiate into multiple cell lineagesAccumulating evidence has shown that cancer cells can be induced to diï¬erentiate into various benign cells such as adipocytesfibrocytes osteoblast by a variety of small molecular compounds which may provide new strategies for cancer treatmentRecent studies have reported that tumor cells undergoing epithelialtomesenchymal transition can be induced to diï¬erentiateinto adipocytes In this review molecular mechanisms signal pathways and the roles of various biological processes in adiposediï¬erentiation are summarized Understanding the molecular mechanism of adipogenesis and adipose diï¬erentiation of cancercells may contribute to cancer treatments that involve inducing diï¬erentiation into benign cells IntroductionAdipogenesis is the process through which mesenchymalstem cells MSCs commit to the adipose lineage and diï¬erentiate into adipocytes During this process preadipocytescease to proliferate begin to accumulate lipid droplets anddevelop morphologic and biochemical characteristics ofmature adipocytes such as hormoneresponsive lipogenesisand lipolytic programs Currently there are mainly twomodels of benign adipocyte diï¬erentiation in vitro One isfibroid pluripotent stem cells which can diï¬erentiate intonot only adipocytes but also muscle cartilage and othercells There are two kinds of fibroid pluripotent stem cellsbone marrow and adipose mesenchymal stem cells Anothergroup is fibroblastic preadipocytes which have a single direction of diï¬erentiation namely lipid diï¬erentiation including3T3L1 and 3T3F422A cells [] Cancer cells with tumorinitiation ability designated as cancer stem cells CSCshave the characteristics of tumorigenesis and the expressionof specific stem cell markers as well as the longterm selfrenewal proliferation capacity and adipose diï¬erentiationpotential [] In addition to CSCs [] cancer cells undergoing epithelialmesenchymaltransformation EMT havebeen reported to be induced to diï¬erentiate into adipocytes[“] Lung cancer NCIH446 cells can be induced to differentiate into neurons adipocytes and bone cells in vitro[] The adipogenesis diï¬erentiation treatment is promisingin the p53 gene deletion type of fibroblastderived cancer[] Cancer cells with homologous recombination defectssuch as ovarian and breast cancer cells with breast cancersusceptibility genes BRCA mutations can be inducedto diï¬erentiate by poly ADPribose polymerase PARP 0cStem Cells Internationalinhibitors [] The nuclear receptor peroxisome proliferatoractivated receptor Î PPARÎ agonist antidiabetic thiazolidinedione drug can induce growth arrest and adipogenicdiï¬erentiation in human mouse and dog osteosarcoma cells[] Thyroid cancer cells expressing the PPARÎ fusion proteinPPFP can be induced to diï¬erentiate into adipocytes bypioglitazone [] Adipogenesis can be induced in welldiï¬erentiated liposarcoma WDLPS and dediï¬erentiatedliposarcoma DDLPS cells by dexamethasone indomethacininsulin and 3isobutyl1methyl xanthine IBMX []In this review we highlight some of the crucial transcription factors that induce adipogenesis both in MSCs and inincluding the wellstudied PPARÎ and CCAATCSCsenhancerbinding proteins CEBPs [] as well as othercell factors that have been recently shown to have an important role in adipocyte diï¬erentiation We focus on understanding the complex regulatory mechanism of adipocytediï¬erentiation that can contribute to the clinical treatmentof human diseases including those caused by obesity andadipocytes dysfunction especially for the malignant tumorwhich can be transdiï¬erentiated into mature adipocytes Adipocyte DifferentiationCell proliferation and diï¬erentiation are two opposingprocesses and there is a transition between these two processes in the early stages of adipocyte diï¬erentiation Theinteraction of cell cycle regulators and diï¬erentiation factors produces a cascade of events which ultimately resultsin the expression of adipocyte phenotype [] Adipogenesishas diï¬erent stages Each stage has a specific gene expression pattern [] In general adipocyte diï¬erentiation ofpluripotent stem cells is divided into two phases The firstphase known as determination involves the commitment ofpluripotent stem cells to preadipocytes The preadipocytescannot be distinguished morphologically from their precursor cells but also have lost the potential to diï¬erentiate intoother cell types In the second phase which is known asterminal diï¬erentiation the preadipocytes gradually acquirethe characteristics of mature adipocytes and acquire physiological functions including lipid transport and synthesisinsulin sensitivity and the secretion of adipocytespecificproteins []The diï¬erentiation of precursor adipocytes is also dividedinto four stages proliferation mitotic cloning early diï¬erentiation and terminal diï¬erentiation [] After the precursors are inoculated into the cell culture plates the cellsgrow exponentially until they converge After reaching contact inhibition the growth rate slows and gradually stagnatesand the proliferation of precursor adipocytes stops which isvery necessary for initiating the diï¬erentiation of precursoradipocytes Adipocyte precursors exhibit transient mitosiscalled œclonal expansion a process that relies on the actionof induced diï¬erentiation factors Some preadipocyte cellsmouse cell lines 3T3L1 3T3F442A undergo one or tworounds of cell division prior to diï¬erentiation [] whereasother cell lines mouse C3H10T12 diï¬erentiating into adipocyte do not undergo mitosis clonal expansion []Whether œmitotic clonal expansion is required for adiposediï¬erentiation remains controversial However it is certainthat some of the checkpoint proteins for mitosis regulateaspects of adipogenesis [ ] When cells enter the terminaldiï¬erentiation stage the de novo synthesis of fatty acidsincreases significantly the transcription factors and adipocyterelated genes work cooperatively to maintain precursor adipocyte diï¬erentiation into mature adipocytes containing largelipid droplets [] Regulatory Pathways inPreadipocytes CommitmentAdipocyte diï¬erentiation is a complex process in which geneexpression is finely regulated The most basic regulatory network of adipose diï¬erentiation has not been updated inrecent years but some factors and signaling pathways thatdo aï¬ect adipose diï¬erentiation have been continuouslyreported Adipocyte diï¬erentiation is the result of the geneexpression that determines the phenotype of adipocyteswhich is a complex and delicate regulatory process Figure Wnt Signal Pathway in Adipogenesis Wnt signaling isimportant for adipocytes proliferation and diï¬erentiationboth in vitro and in vivo [] The Wnt family of secretedglycoproteins functions through paracrine and autocrinemechanisms to ‚uence cell fate and development Wntprotein binding to frizzled receptors initiates signalingthrough catenindependent and independent pathways[] Wnt signaling inhibits adipocyte diï¬erentiation in vitroby blocking the expression of PPARÎ and CEBPα [] Constitutive Wnt10b expression inhibits adipogenesis Wnt10b isexpressed in preadipocytes and stromal vascular cells butnot in adipocytes In vivo transgenic expression of Wnt10bin adipocytes results in a reduction in white adipose tissuemass and absent brown adipose tissue development []Wnt10a and Wnt6 have also been identified as determinantsof brown adipocyte development [ ] Wnt5b is transiently induced during adipogenesis and promotes diï¬erentiation [] indicating that preadipocytes integrate inputs fromseveral competing Wnt signals The Hedgehog HH Signaling Pathway MechanismThree vertebrate HH ligands including sonic hedgehogSHHIndian hedgehog IHH and desert hedgehogDHH have been identified and initiated a signaling cascademediated by patched Ptch1 and Ptch2 receptors [ ]HH signaling had an inhibitory eï¬ect on adipogenesis inmurine cells such as C3H10T12 KS483 calvaria MSCslines and mouse adiposederived stromal cells [] Thesecells were visualized by decreased cytoplasmic fat accumulation and the expression of adipocyte marker genes after HHsignaling was inhibited [] Although it is generally agreedthat HH expression has an inhibitory eï¬ect on preadipocytediï¬erentiation the mechanisms linking HH signaling andadipogenesis remain poorly defined [] ERKMAPKPPAR Signal Pathway Extracellularregulated protein kinase ERK is required in the proliferativephase of diï¬erentiation ERK activity blockade in 3T3L1 0cStem Cells InternationalDEX insulin DEMXWNT 10band othersSHHPBC SMOTGF𝛽P SMAD3 SMAD3Testosterone𝛽catentinARIRSPI3KAKTCREBPKAPCREBFOXO1A2TCFLEF GATA23CEBP𝛽MAPKG3K3𝛽P2CEBP𝛽CEBPαPPARá¿«BMPsSMAD1SREBPAdipocytegenesFigure Regulation pathways in preadipocytes commitment BMP and Wnt families are mediators of MSCs commitment to producepreadipocytes Exposure of growtharrested preadipocytes to diï¬erentiation inducers IGF1 glucocorticoid and cAMP triggers DNAreplication leading to adipocyte gene expression due to a transcription factor cascade The dotted line indicates an uncertain molecularregulatory mechanismcells and embryonic stem cells can inhibit adipogenesis Inthe terminal diï¬erentiation phase ERK1 activity leads toPPARÎ phosphorylation which inhibits adipocyte diï¬erentiation This implies that ERK1 activity must be reduced afteradipocyte proliferation so that diï¬erentiation can proceedThis reduction is mediated in part by mitogenactivatedprotein kinase MAPK phosphatase1 MKP1 [ ]These extracellular and intracellular regulation factors causeadipocytespecific gene expression and eventually lead toadipocyte formation Adipocyte DifferentiationRegulatory Proteins PPARÎ and Adipocyte Diï¬erentiation PPARÎ is a member of the nuclearreceptor superfamily and is both necessaryand sufficient for adipogenesis [] Forced expression ofPPARÎ is sufficient to induce adipocyte diï¬erentiation broblasts [] Indeedthe proadipogenic CEBPs andKrüppellike factors KLFs have all been shown to induceat least one of the two PPARÎ promoters In contrast antiadipogenic transcription factor GATA functioned in part byrepressing PPARÎ expression [] PPARÎ itself has twoisomers The relative roles of PPARÎ1 and PPARÎ2 in adipogenesis remain an question PPARÎ2 is mainlyexpressed in adipose tissue while PPARÎ1 is expressed inmany other tissues Although both can promote adipocytediï¬erentiation PPARÎ2 could do so eï¬ectively at very lowligand concentration compared with PPARÎ1 [] The twoprotein isoforms are generated by alternative splicing andpromoter usage and both are induced during adipogenesisPPARÎ1 can also be expressed in cell types other than adipocytes Ren [] used engineered zincfinger proteins tothe expression ofthe endogenous PPARÎ1 andinhibitPPARÎ2 promoters in 3T3L1 cells Ectopic expression ofPPARÎ2 promotes adipogenesis whereas that of PPARÎ1does not Zhang reported that PPARÎ2 deficiencyimpairs the development of adipose tissue and insulin sensitivity []There are transcriptional cascades between adipocytesgenes including PPARÎ and CEBPα which are the coreadipocyte diï¬erentiation regulators In the early stage of adipocyte diï¬erentiation the expression of CEBP and CEBPδincrease which upregulates CEBPα expressionfurtheractivate PPARÎ PPARÎ activating CEBPα in turn resultsin a positive feedback PPARÎ binding with retinoic acid Xreceptor RXR forms diï¬erent heterodimers The variousdimmers can combine with the PPARÎ response elementPPRE and initiate the transcription of downstream genesfor diï¬erentiation into adipocytes []CEBPs participate in adipogenesis and several CEBPfamily members are expressed in adipocytesincludingCEBPα CEBP CEBPÎ CEBPδ and CEBPhomologous protein CHOP The temporal expression of thesefactors during adipocyte diï¬erentiation triggers a cascadewhereby early induction of CEBP and CEBPδ leads toCEBPα expression This notion is further supported by thesequential binding of these transcription factors to severaladipocyte promoters duringadipocyte diï¬erentiationCEBP is crucial for adipogenesis in immortalized preadipocyte lines CEBP and CEBPδ promote adipogenesis atleast in part by inducing CEBPα and PPARÎ CEBPαinduces many adipocyte genes directly and plays an important role in adipose tissue development Once CEBPα isexpressed its expression is maintained through autoactivation [] Despite the importance of CEBPs in adipogenesis 0cStem Cells Internationalthese transcription factors clearly cannot function efficientlyin the absence of PPARÎ CEBP cannot induce CEBPαexpression in the absence of PPARÎ which is required torelease histone deacetylase1 HDAC1 from the CEBPαpromoter [] Furthermore ectopic CEBPα expressioncannot induce adipogenesis in PPARΓ“ï¬broblasts []However CEBPα also plays an important role in diï¬erentiated adipocytes Overexpression of exogenous PPARÎ inCEBPαdeficient cells showed that although CEBPα isnot required for lipid accumulation and the expression ofmany adipocyte genes it is necessary for the acquisition ofinsulin sensitivity [ ] Figure Human fibroblasts withthe ability to diï¬erentiate into adipocytes also do not undergomitotic cloning amplification However PPARÎ exogenousligands need to be added to promote adipocyte diï¬erentiation Therefore it can be inferred that mitotic cloning expansion can produce endogenous ligands of PPARÎ [] BMP and Transforming Growth Factor TGF inAdipocyte Diï¬erentiation A variety of extracellular factorsaï¬ect the preadipocyte commitment of stem cells includingbone morphogenetic protein BMP []transforminggrowth factor TGF [] insulininsulinlike growthfactor IGF1 [] tumor necrosis factor α and interleukin [] matrix metalloproteinase [] fibroblast growthfactor FGF and FGF2 [] BMP and TGF have variedeï¬ects on the diï¬erentiation fate of mesenchymal cells []The TGF superfamily members BMPs and myostatinregulate the diï¬erentiation of many cell types includingadipocytes [] TGF inhibitor can promote adipose diï¬erentiation of cancer cells with a mesenchymal phenotypein vitro and transgenic overexpression of TGF impairsadipocyte development [] Inhibition of adipogenesis couldbe obtained through blocking of endogenous TGF with adominantnegative TGF receptor or drosophila mothersagainst decapentaplegic protein SMAD inhibitionSMAD3 binds to CEBPs and inhibits their transcriptionalactivity including their ability to transactivate the PPARÎ2promoter [ ] Exposure of multipotent mesenchymalcells to BMP4 commits these cells to the adipocyte lineageallowing them to undergo adipose conversion [] Theeï¬ects of BMP2 are more complex and depend on the presence of other signaling molecules BMP2 alone has little eï¬ecton adipogenesis and it interacts with other factors such asTGF and insulin to stimulate adipogenesis of embryonicstem cells [] BMP2 stimulates adipogenesis of multipotentC3H10T12 cells at low concentrations and can contribute tochondrocyte and osteoblast development at higher concentrations [] KLFs in Adipocyte Diï¬erentiation During adipocyte differentiation some KLF family members are overexpressedsuch as KLF4 KLF5 KLF9 and KLF15 while KLF16 expression is reduced [ ] KLF15 is the first KLF family members which were identified to be involved in adipocytediï¬erentiation Its expression increased significantly on thesixth day of 3T3L1 adipocyte diï¬erentiation and peakedon the second day of adipocyte induction in MSCs andmouse embryonic fibroblasts Inhibition of KLF15 by siRNAor mutation led to a decrease in PPARÎ CEBPα fatty acidbinding protein FABP4 and glucose transporter GLUT4 However overexpression of KLF15 in NIH3T3cells was found to be associated with lipid accumulation aswell as increases in PPARÎ and FABP4 [] Mice with complete absence of KLF5 showed embryonal lethality and micewith singlechromosome KLF5 knockout showed a significant reduction in white fat in adulthood suggesting thatKLF5 plays an important role in adipocyte diï¬erentiationKLF5 can be activated by CEBP or CEBPδ which isinvolved in early adipocyte diï¬erentiation KLF5 can beactivated by CEBP or CEBPδ which is involved in earlyadipocyte diï¬erentiation Direct binding of KLF5 to thePPARÎ2 promoter in combination with CEBPs inducesPPARÎ2 expression [] Transfection of KLF5 dominantnegative mutants in 3T3L1 cells reduced lipid droplet accumulation and inhibited PPARÎ and CEBPα expressionwhereas overexpression of wild KLF5 significantly promotedadipocyte diï¬erentiation even without exogenous hormonestimulation Similar to KLF5 KLF9 knockdown can inhibitthe expression of a series of adipocyte diï¬erentiation genessuch as PPARÎ CEBPα and FABP4 hence inhibitingadipocyte diï¬erentiation However KLF9 overexpressiondid not upregulate the expression of PPARÎ and CEBPα[] In addition KLF4 can transactivate CEBP by bindingto the region of KB upstream of the CEBP promoter and promote lipid diï¬erentiation [] KLF6 can forma complex with histone deacetylase3 HDAC3 inhibitingpreadipocyte factor1 Pref1 expression and promotinglipid diï¬erentiation [] KLF2 is highly expressed in adiposeprogenitors and its expression decreases during the processof lipid diï¬erentiation Overexpressed KLF2 can bind to theCACCC region of PPARÎ2 proximal promoter and inhibitlipid diï¬erentiation as well as the expression of PPARÎCEBPα and sterolregulated elementbinding proteinsSREBP by inhibiting the promoter activity [] RNAsequence analysisshowed that KLFl6 expression wasdecreased on the first day of adipocyte diï¬erentiation of3T3L1 cells Adipocyte diï¬erentiation was promoted byKLF16 knockdown but was inhibited by KLF16 overexpression via inhibition of PPARÎ promoter activity [] In addition KLF3 and KLF7 were also found to play a negativeregulatory role in adipocyte diï¬erentiation [ ] Signal Transducers and Activators of TranscriptionSTATs and Adipocyte Diï¬erentiation The activated STATprotein enters the nucleus as a dimer and binds to the targetgene to regulate gene transcription In the adipocyte diï¬erentiation of mouse 3T3L1 cells the expression of STAT1 andSTAT5 was significantly increased while that of STAT3and STAT6 was not significantly changed [] In the adipocyte diï¬erentiation of human subcutaneous adipose precursor cells STAT1 expression was significantly decreased[] while the expression of STAT3 and STAT5 wasincreased and STAT6 expression was unchanged [] Therole of STAT1 in adipocyte diï¬erentiation is not clearbecause its expression trend in humans and mice diï¬ersduring the adipocyte diï¬erentiation process Early adipocytediï¬erentiation of 3T3L1 cells was inhibited by STAT1 0cStem Cells InternationalKLF5SREBP1cKLF15KLF2CHOPCEBPá¿«KROX20LigandCEBP𝛽CEBP𝛿GATA23PPARá¿«CEBP𝛼ProadipogenicAntiadipogenicGenes of terminaladipocytedifferentiationFigure A cascade of transcription factors that regulate adipogenesis PPARÎ is one of the key transcription factors in adipogenesis and thecore of the transcriptional cascade that regulates adipogenesis PPARÎ expression is regulated by several proadipogenic blue andantiadipogenic red factors CEBPα is regulated through a series of inhibitory protein“protein interactions Some transcription factorfamilies include several members that participate in adipogenesis such as the KLFs Black lines indicate eï¬ects on gene expression violetlines represent eï¬ects on protein activityagonist interferon Î Loss of STAT1 in 3T3L1 cells can rescue the inhibition of adipocyte diï¬erentiation caused byprostaglandin factor 2α [] Other studies have found thatSTAT1 is required for adipose diï¬erentiation and STAT1overexpression in C3H10T12 cells can prevent the inhibition of lipid diï¬erentiation caused by Bcell lymphoma6knockdown [] There was no abnormal adipose tissuein STAT1 knockout mice [] STAT3 not only aï¬ectsthe proliferation of 3T3L1 cells but also coregulates theiradipocyte diï¬erentiation with high mobility group protein [] The FABP4 promoter was used to specificallyknock out STAT3 in the adipose tissue of mice and theresults showed that mice weight significantly increasedand the adipocyte quantity increased compared with thewildtype mice [] STAT5A and STAT5B have diï¬erenteï¬ects on adipocyte diï¬erentiation Abnormal adipose tissuewas found in the mice with STAT5A or STAT5B knockout ordouble knockout and the amount of adipose tissue was onlyonefifth of the original adipose tissue in mice withoutknockdown [] Histone Modification in Adipocyte Diï¬erentiation Histone deacetylase sirtuin SIRT plays an important rolein biological processes such as stress tolerance energymetabolism and cell diï¬erentiation [] During the adipocyte diï¬erentiation of C3H1012 cells SIRT1 expressiondecreased [] Overexpression of SIRT1 activated theWnt signal which caused the deacetylation of cateninThe accumulation of catenin in the nucleus could inhibitadipocyte diï¬erentiation SIRT1 knockdown resulted inincreased acetylation of the histones H3K9 and H4K16 inthe secreted frizzledrelated protein sFRP and sFRP2 promoters thereby promoting transcription of these genes andpromoting lipid diï¬erentiation [] Forkhead box proteinO FOXO is a member of the transcription factor FOXOfamily It can recruit cyclic AMP response elementbindingprotein CBPhistone acetyltransferase p300 to initiate anacetylation The acetylated FOXO1 can be phosphorylatedby phosphorylated protein kinase B PKBAKT The phosphorylation of FOXO1 by AKT inhibits the transcriptionalactivation of FOXO1 The acetylation of FOXO1 lost the ability of DNAbinding affinity and promoted its shuttling fromnuclei to cytoplasm [] SIRT1 and SIRT2 can deacetylateand active FOXO1 Activated FOXO1 nonphosphorylatednuclear FOXO1 in the nucleus binds to the promoters of target genes encoding p21 p27 and PPARÎ and initiates subsequent transcriptions [] SIRT2 inhibits the acetylation andphosphorylation of FOXO1 thereby induces the accumulation of activated FOXO1 in the nucleus Activated FOXO1could inhibit adipogenesis via PPARÎ [“] Lysinespecific histone demethylase LSD1 expression increasedduring the adipocyte diï¬erentiation of 3T3L1 cells LSD1could reduce the dimethylation levels of histone H3K9 andH3K4 in the CEBPα promoter region thereby promotingadipocyte diï¬erentiation [] SET domaincontaining SETD8 catalyzed the monomethylation of H4K20 andpromoted PPARÎ expression The activation of PPARÎ transcriptional activity leads to the induction of monomethylatedH4K20 and modification of PPARÎ and its targets therebypromoting adipogenesis [] Enhancer of zeste homolog EZH2 is a methyltransferase and can bind methyl groupsto histone H3K27 which is also necessary for lipid diï¬erentiation The absence of EZH2 in brown fat precursors results inreduced levels of the Wnt promoter histone H3K27me3which is also saved by the ectopic EZH2 expression or theuse of a Wntcatenin signal inhibitor [] In addition histone demethylases such as lysinespecific histone demethylase LSDKDM KDM6 and histone lysine demethylasePHF2 are also involved in adipose diï¬erentiation andKDM2B inhibits transcription factor activator protein 2αpromoter via H3K4me3 and H3K36me2 [] Role of microRNA and Long NoncodingRNA in AdipogenesismicroRNA miR can bind and cut target genes or inhibittarget gene translation Endogenous siRNA can be producedby the action of Dicer enzyme and bind to a specific proteinto change its cellular location [] Many kinds of miRsare involved in regulating adipocyte diï¬erentiation The 0cStem Cells Internationalexpression of miR143 increased during the diï¬erentiationof adipose progenitor cells Overexpression of miR143promoted gene expression involved in adipose diï¬erentiationand triglyceride accumulation Inhibition of miR143 prevented the adipose diï¬erentiation of human fat progenitorcells [ ] Additionally miR8 promotes adipocyte diï¬erentiation by inhibiting Wnt signaling [] Moreover miR miR103 miR21 miR519d miR210 miR30miR204211 and miR375 also play a certain role in promoting adipocyte diï¬erentiation while miR130 miR448and let7y inhibit lipid diï¬erentiation [ ] In additionto miRs long noncoding RNA LncRNA is a type of noncoding RNA and is important during epigenetic regulationand can form a doublestranded RNA complex with mRNAcauses protein transcription Lncu90926 inhibits adipocytediï¬erentiation by inhibiting the transactivation of PPARÎ2[] As a novel LncRNA HOXAAS3 expression increasedduring the adipose diï¬erentiation of MSCs and HOXAAS3 silencing reduced the marker gene of adipose diï¬erentiation and inhibited the adipose diï¬erentiation [] Zhu et al[] reported that HOXAAS3 interacted with EZH2 toregulate lineage commitment of MSCs HOXA AS3 canregulate the trimethylation level of H3K27 in the Runx2promoter region by binding to EZH2 Therefore HOXAAS3 is considered to be an epigenetic switch regulating MSCslineage specificity [] Adipocyte diï¬erentiationassociatedLncRNA can act as a competitive endogenous RNA of miR in the process of lipid diï¬erentiation thereby promotingthe expression of SIRT1 the target gene of miR204 and thusinhibiting lipid diï¬erentiation [] The LncRNA NEAT1can also regulate adipocyte diï¬erentiation under the ‚uence of miRNA140 [] Other LncRNA including LncRNABlnc1 and Plnc are also involved in regulating adipocytediï¬erentiation [ ] Other Biochemical Response Involved inAdipocyte Differentiation Unfolded Protein Responses in Adipocyte Diï¬erentiationIn the endoplasmic reticulum of eukaryotes unfolded protein response involves three proteinsinositolrequiringenzyme 1α doublestranded RNAdependent proteinkinaselike ER kinase and activating transcription factorATF 6α [] Knockdown of ATF6α aï¬ects the expressionof adipocytes genes and inhibits C3H10T12 adipocyte differentiation [] The inhibitory eï¬ect of berberine on adipocyte diï¬erentiation of 3T3L1 cells is also due to inducedCHOP and decorin expressions and this inhibitory eï¬ectis ameliorated by CHOP knockout [] In the adipocytediï¬erentiation process of 3T3L1 cells increases in PPARÎand CEBPα as markers of adipocyte diï¬erentiation wereaccompanied by an increase in the corresponding proteinexpressions of phosphorylated Eukaryotic translation initiaEIF 2α phosphorylated endoribonucleasetion factorIRE1α ATF4 CHOP and other unfolded protein responsesEndoplasmic reticulum stress inducer or hypoxic endoplasmic reticulum stress can inhibit adipocyte diï¬erentiationAdditionally EIF2α mutation results in continuous activation or overexpression of CHOP which also inhibits adipocyte diï¬erentiation [] After the initiation of adiposediï¬erentiation numerous diï¬erentiationassociated proteinsare synthesized Exogenous endoplasmic reticulum stressinducers can lead to excessive endoplasmic reticulumresponse which in turn aï¬ects the synthesis of proteinsrelated to diï¬erentiation and inhibits adipocyte formationFigure Role of Oxidative Stress in Adipogenesis During thedirectional diï¬erentiation of MSCs mitochondrial complexI and III and NADPH oxidase NOX4 are the main sourcesof oxygen species ROS production Currently it is believedthat ROS aï¬ects not only the cell cycle and apoptosis but alsodiï¬erentiation through ‚uencing the signaling pathwaysincluding the Wnt HH and FOXO signaling cascade duringMSCs diï¬erentiation [] The diï¬erentiation ability ofstem cells is determined by the arrangement of perinuclearmitochondria which specifically manifests as low ATPcellcontents and a high rate of oxygen consumption The lackof these characteristics indicates stem cell diï¬erentiation[] Adipocyte diï¬erentiation is a highly dependent ROSactivation factor related to mitosis and cell maturation[] Schroder found that exogenous H2O2 could stimulate adipocyte diï¬erentiation of mouse 3T3L1 cells andhuman adipocyte progenitor cells in the absence of insulinH2O2 regulates adipocyte diï¬erentiation of 3T3L1 cells ina dosedependent manner High doses of H2O2 and μM promote adipocyte diï¬erentiation [ ] Tormos found that ROS synthesis increased in humanMSCs at the early stage of adipose diï¬erentiation and targeted antioxidants could inhibit lipid diï¬erentiation Byknocking down Rieske ironsulfur protein and ubiquinonebinding protein ROS produced by mitochondrial complexIII was found to be necessary in initiating adipose diï¬erentiation [] However other studies have shown that theexpression levels of adiponectin and PPARÎ were decreasedby using H2O2 “ mM in 3T3L1 cells [] Free radical nitric oxide NO also promotes lipid diï¬erentiationbecause treatment with NO inducer hydroxylamine or NOsynthase NOS substrate arginine can significantly induceadipose diï¬erentiation of rat adipose progenitor cells NOSinduced adipose diï¬erentiation mainly via eNOS rather thaniNOS [] ROS can induce adipose diï¬erentiation primarily by inhibiting Wnt FOXO and HH signaling pathwaysthat inhibit lipid diï¬erentiation Autophagy in Adipocyte Diï¬erentiation The increase inautophagosomes during lipid diï¬erentiation indicates thatautophagy may play an important role in lipid diï¬erentiation[] Baerga confirmed that the adipocyte diï¬erentiation efficiency was significantly inhibited in mouse embryonic fibroblasts lacking autophagyrelated gene Atg agene encoding an essential protein required for autophagy[] Knockdown of Atg5 in 3T3L1 cells promotesproteasomedependent degradation of PPARÎ2therebyinhibiting adipocyte diï¬erentiation [] Zhang reportedthat autophagyrelated gene 7Atg7 is also crucial for adipose development Atg7deficient mice were slim and onlyhad of white fat compared to wildtype mice and the 0cStem Cells InternationalCEBP𝛽 geneEBF1 geneKLF4EGR2CEBP𝛽CytosolCEBP𝛿 geneCEBP𝛿KLF5genePPARá¿« geneKLF5NR2F2NFKB11433RELASREBF1A2RXRAPPARá¿«PPARá¿«RXRA heterodimerPPARá¿«RXRAcorepressor complexFABP4Ligands of PPARá¿«FAM120BTHRAP3EP300NCOA2NCOA3HELZ2NCOA1CREBBPEBF1ADIPOQ geneAIDRFCEBP𝛼 geneZNF638ZNF467CEBP𝛼NCOR1HDAC3NCOR2 SLC2A4 geneGLUT4 geneLEP geneFABP4 geneCDK4CCND3PLIN1 genePCK1 geneFABP4CD36 genePPARARXRAcoactivator complexPPARá¿«fatty acidRXRAmediatorcoactivator complexANGPTLgenePPARGC1AMediator complex consensusLPL geneNucleoplasmProteins bind to gene promotersTranscription of genes into proteinsActing on proteins compoundingTGF𝛽1WNT1WNT10BTNF77233ADIPOQGLUT4SLC2A4 tetramerLEPFABP4lipid dropletPLIN1PCK1PaPa Pa4xPalmCCD36PaANGPTL4LPLFigure Regulation of adipocyte diï¬erentiation A regulatory loop exists between PPARÎ and CEBP activation Transcription factor CoeEBF activates CEBPα CEBPα activates EBF1 and EBF1 activates PPARÎ CEBP and CEBPδ act directly on the PPARÎ gene bybinding its promoter and activating transcription CEBPα CEBP and CEBPδ can activate the EBF1 gene and KLF5 The EBF1 and KLF5proteins in turn bind the promoter of PPARÎ which becomes activated Other hormones such as insulin can aï¬ect the expression ofPPARÎ and other transcription factors such as SREBP1c PPARÎ can form a heterodimer with the RXRα In the absence of activatingligands the PPARÎRXRα complex recruits transcription repressors such as nuclear receptor corepressor NCoR NCoR1 andHDAC3 Upon binding with activating ligands PPARÎ causes a rearrangement of adjacent factors Corepressors such as NCoR2 are lostand coactivators such as Transcription intermediary factor TIF2 CBP and p300 are recruited which can result in the expression of CyclicAMPresponsive elementbinding protein CREB followed by PPA

cancer277

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: "preadipocytes diï¬erentiate into adipocytes During this process the preadipocytes ceaseto proliferate begin to accumulate lipid droplets and develop morphologic and biochemical characteristics of mature adipocytesMesenchymal stem cells MSCs are a type of adult stem cells known for their high plasticity and capacity to generate mesodermaland nonmesodermal tissues Many mature cell types can be generated from MSCs including adipocyte osteocyte and chondrocyteThe diï¬erentiation of stem cells into multiple mature phenotypes is at the basis for tissue regeneration and repair Cancer stem cellsCSCs play a very important role in tumor development and have the potential to diï¬erentiate into multiple cell lineagesAccumulating evidence has shown that cancer cells can be induced to diï¬erentiate into various benign cells such as adipocytesfibrocytes osteoblast by a variety of small molecular compounds which may provide new strategies for cancer treatmentRecent studies have reported that tumor cells undergoing epithelialtomesenchymal transition can be induced to diï¬erentiateinto adipocytes In this review molecular mechanisms signal pathways and the roles of various biological processes in adiposediï¬erentiation are summarized Understanding the molecular mechanism of adipogenesis and adipose diï¬erentiation of cancercells may contribute to cancer treatments that involve inducing diï¬erentiation into benign cells IntroductionAdipogenesis is the process through which mesenchymalstem cells MSCs commit to the adipose lineage and diï¬erentiate into adipocytes During this process preadipocytescease to proliferate begin to accumulate lipid droplets anddevelop morphologic and biochemical characteristics ofmature adipocytes such as hormoneresponsive lipogenesisand lipolytic programs Currently there are mainly twomodels of benign adipocyte diï¬erentiation in vitro One isfibroid pluripotent stem cells which can diï¬erentiate intonot only adipocytes but also muscle cartilage and othercells There are two kinds of fibroid pluripotent stem cellsbone marrow and adipose mesenchymal stem cells Anothergroup is fibroblastic preadipocytes which have a single direction of diï¬erentiation namely lipid diï¬erentiation including3T3L1 and 3T3F422A cells [] Cancer cells with tumorinitiation ability designated as cancer stem cells CSCshave the characteristics of tumorigenesis and the expressionof specific stem cell markers as well as the longterm selfrenewal proliferation capacity and adipose diï¬erentiationpotential [] In addition to CSCs [] cancer cells undergoing epithelialmesenchymaltransformation EMT havebeen reported to be induced to diï¬erentiate into adipocytes[“] Lung cancer NCIH446 cells can be induced to differentiate into neurons adipocytes and bone cells in vitro[] The adipogenesis diï¬erentiation treatment is promisingin the p53 gene deletion type of fibroblastderived cancer[] Cancer cells with homologous recombination defectssuch as ovarian and breast cancer cells with breast cancersusceptibility genes BRCA mutations can be inducedto diï¬erentiate by poly ADPribose polymerase PARP 0cStem Cells Internationalinhibitors [] The nuclear receptor peroxisome proliferatoractivated receptor Î PPARÎ agonist antidiabetic thiazolidinedione drug can induce growth arrest and adipogenicdiï¬erentiation in human mouse and dog osteosarcoma cells[] Thyroid cancer cells expressing the PPARÎ fusion proteinPPFP can be induced to diï¬erentiate into adipocytes bypioglitazone [] Adipogenesis can be induced in welldiï¬erentiated liposarcoma WDLPS and dediï¬erentiatedliposarcoma DDLPS cells by dexamethasone indomethacininsulin and 3isobutyl1methyl xanthine IBMX []In this review we highlight some of the crucial transcription factors that induce adipogenesis both in MSCs and inincluding the wellstudied PPARÎ and CCAATCSCsenhancerbinding proteins CEBPs [] as well as othercell factors that have been recently shown to have an important role in adipocyte diï¬erentiation We focus on understanding the complex regulatory mechanism of adipocytediï¬erentiation that can contribute to the clinical treatmentof human diseases including those caused by obesity andadipocytes dysfunction especially for the malignant tumorwhich can be transdiï¬erentiated into mature adipocytes Adipocyte DifferentiationCell proliferation and diï¬erentiation are two opposingprocesses and there is a transition between these two processes in the early stages of adipocyte diï¬erentiation Theinteraction of cell cycle regulators and diï¬erentiation factors produces a cascade of events which ultimately resultsin the expression of adipocyte phenotype [] Adipogenesishas diï¬erent stages Each stage has a specific gene expression pattern [] In general adipocyte diï¬erentiation ofpluripotent stem cells is divided into two phases The firstphase known as determination involves the commitment ofpluripotent stem cells to preadipocytes The preadipocytescannot be distinguished morphologically from their precursor cells but also have lost the potential to diï¬erentiate intoother cell types In the second phase which is known asterminal diï¬erentiation the preadipocytes gradually acquirethe characteristics of mature adipocytes and acquire physiological functions including lipid transport and synthesisinsulin sensitivity and the secretion of adipocytespecificproteins []The diï¬erentiation of precursor adipocytes is also dividedinto four stages proliferation mitotic cloning early diï¬erentiation and terminal diï¬erentiation [] After the precursors are inoculated into the cell culture plates the cellsgrow exponentially until they converge After reaching contact inhibition the growth rate slows and gradually stagnatesand the proliferation of precursor adipocytes stops which isvery necessary for initiating the diï¬erentiation of precursoradipocytes Adipocyte precursors exhibit transient mitosiscalled œclonal expansion a process that relies on the actionof induced diï¬erentiation factors Some preadipocyte cellsmouse cell lines 3T3L1 3T3F442A undergo one or tworounds of cell division prior to diï¬erentiation [] whereasother cell lines mouse C3H10T12 diï¬erentiating into adipocyte do not undergo mitosis clonal expansion []Whether œmitotic clonal expansion is required for adiposediï¬erentiation remains controversial However it is certainthat some of the checkpoint proteins for mitosis regulateaspects of adipogenesis [ ] When cells enter the terminaldiï¬erentiation stage the de novo synthesis of fatty acidsincreases significantly the transcription factors and adipocyterelated genes work cooperatively to maintain precursor adipocyte diï¬erentiation into mature adipocytes containing largelipid droplets [] Regulatory Pathways inPreadipocytes CommitmentAdipocyte diï¬erentiation is a complex process in which geneexpression is finely regulated The most basic regulatory network of adipose diï¬erentiation has not been updated inrecent years but some factors and signaling pathways thatdo aï¬ect adipose diï¬erentiation have been continuouslyreported Adipocyte diï¬erentiation is the result of the geneexpression that determines the phenotype of adipocyteswhich is a complex and delicate regulatory process Figure Wnt Signal Pathway in Adipogenesis Wnt signaling isimportant for adipocytes proliferation and diï¬erentiationboth in vitro and in vivo [] The Wnt family of secretedglycoproteins functions through paracrine and autocrinemechanisms to ‚uence cell fate and development Wntprotein binding to frizzled receptors initiates signalingthrough catenindependent and independent pathways[] Wnt signaling inhibits adipocyte diï¬erentiation in vitroby blocking the expression of PPARÎ and CEBPα [] Constitutive Wnt10b expression inhibits adipogenesis Wnt10b isexpressed in preadipocytes and stromal vascular cells butnot in adipocytes In vivo transgenic expression of Wnt10bin adipocytes results in a reduction in white adipose tissuemass and absent brown adipose tissue development []Wnt10a and Wnt6 have also been identified as determinantsof brown adipocyte development [ ] Wnt5b is transiently induced during adipogenesis and promotes diï¬erentiation [] indicating that preadipocytes integrate inputs fromseveral competing Wnt signals The Hedgehog HH Signaling Pathway MechanismThree vertebrate HH ligands including sonic hedgehogSHHIndian hedgehog IHH and desert hedgehogDHH have been identified and initiated a signaling cascademediated by patched Ptch1 and Ptch2 receptors [ ]HH signaling had an inhibitory eï¬ect on adipogenesis inmurine cells such as C3H10T12 KS483 calvaria MSCslines and mouse adiposederived stromal cells [] Thesecells were visualized by decreased cytoplasmic fat accumulation and the expression of adipocyte marker genes after HHsignaling was inhibited [] Although it is generally agreedthat HH expression has an inhibitory eï¬ect on preadipocytediï¬erentiation the mechanisms linking HH signaling andadipogenesis remain poorly defined [] ERKMAPKPPAR Signal Pathway Extracellularregulated protein kinase ERK is required in the proliferativephase of diï¬erentiation ERK activity blockade in 3T3L1 0cStem Cells InternationalDEX insulin DEMXWNT 10band othersSHHPBC SMOTGF𝛽P SMAD3 SMAD3Testosterone𝛽catentinARIRSPI3KAKTCREBPKAPCREBFOXO1A2TCFLEF GATA23CEBP𝛽MAPKG3K3𝛽P2CEBP𝛽CEBPαPPARá¿«BMPsSMAD1SREBPAdipocytegenesFigure Regulation pathways in preadipocytes commitment BMP and Wnt families are mediators of MSCs commitment to producepreadipocytes Exposure of growtharrested preadipocytes to diï¬erentiation inducers IGF1 glucocorticoid and cAMP triggers DNAreplication leading to adipocyte gene expression due to a transcription factor cascade The dotted line indicates an uncertain molecularregulatory mechanismcells and embryonic stem cells can inhibit adipogenesis Inthe terminal diï¬erentiation phase ERK1 activity leads toPPARÎ phosphorylation which inhibits adipocyte diï¬erentiation This implies that ERK1 activity must be reduced afteradipocyte proliferation so that diï¬erentiation can proceedThis reduction is mediated in part by mitogenactivatedprotein kinase MAPK phosphatase1 MKP1 [ ]These extracellular and intracellular regulation factors causeadipocytespecific gene expression and eventually lead toadipocyte formation Adipocyte DifferentiationRegulatory Proteins PPARÎ and Adipocyte Diï¬erentiation PPARÎ is a member of the nuclearreceptor superfamily and is both necessaryand sufficient for adipogenesis [] Forced expression ofPPARÎ is sufficient to induce adipocyte diï¬erentiation broblasts [] Indeedthe proadipogenic CEBPs andKrüppellike factors KLFs have all been shown to induceat least one of the two PPARÎ promoters In contrast antiadipogenic transcription factor GATA functioned in part byrepressing PPARÎ expression [] PPARÎ itself has twoisomers The relative roles of PPARÎ1 and PPARÎ2 in adipogenesis remain an question PPARÎ2 is mainlyexpressed in adipose tissue while PPARÎ1 is expressed inmany other tissues Although both can promote adipocytediï¬erentiation PPARÎ2 could do so eï¬ectively at very lowligand concentration compared with PPARÎ1 [] The twoprotein isoforms are generated by alternative splicing andpromoter usage and both are induced during adipogenesisPPARÎ1 can also be expressed in cell types other than adipocytes Ren [] used engineered zincfinger proteins tothe expression ofthe endogenous PPARÎ1 andinhibitPPARÎ2 promoters in 3T3L1 cells Ectopic expression ofPPARÎ2 promotes adipogenesis whereas that of PPARÎ1does not Zhang reported that PPARÎ2 deficiencyimpairs the development of adipose tissue and insulin sensitivity []There are transcriptional cascades between adipocytesgenes including PPARÎ and CEBPα which are the coreadipocyte diï¬erentiation regulators In the early stage of adipocyte diï¬erentiation the expression of CEBP and CEBPδincrease which upregulates CEBPα expressionfurtheractivate PPARÎ PPARÎ activating CEBPα in turn resultsin a positive feedback PPARÎ binding with retinoic acid Xreceptor RXR forms diï¬erent heterodimers The variousdimmers can combine with the PPARÎ response elementPPRE and initiate the transcription of downstream genesfor diï¬erentiation into adipocytes []CEBPs participate in adipogenesis and several CEBPfamily members are expressed in adipocytesincludingCEBPα CEBP CEBPÎ CEBPδ and CEBPhomologous protein CHOP The temporal expression of thesefactors during adipocyte diï¬erentiation triggers a cascadewhereby early induction of CEBP and CEBPδ leads toCEBPα expression This notion is further supported by thesequential binding of these transcription factors to severaladipocyte promoters duringadipocyte diï¬erentiationCEBP is crucial for adipogenesis in immortalized preadipocyte lines CEBP and CEBPδ promote adipogenesis atleast in part by inducing CEBPα and PPARÎ CEBPαinduces many adipocyte genes directly and plays an important role in adipose tissue development Once CEBPα isexpressed its expression is maintained through autoactivation [] Despite the importance of CEBPs in adipogenesis 0cStem Cells Internationalthese transcription factors clearly cannot function efficientlyin the absence of PPARÎ CEBP cannot induce CEBPαexpression in the absence of PPARÎ which is required torelease histone deacetylase1 HDAC1 from the CEBPαpromoter [] Furthermore ectopic CEBPα expressioncannot induce adipogenesis in PPARΓ“ï¬broblasts []However CEBPα also plays an important role in diï¬erentiated adipocytes Overexpression of exogenous PPARÎ inCEBPαdeficient cells showed that although CEBPα isnot required for lipid accumulation and the expression ofmany adipocyte genes it is necessary for the acquisition ofinsulin sensitivity [ ] Figure Human fibroblasts withthe ability to diï¬erentiate into adipocytes also do not undergomitotic cloning amplification However PPARÎ exogenousligands need to be added to promote adipocyte diï¬erentiation Therefore it can be inferred that mitotic cloning expansion can produce endogenous ligands of PPARÎ [] BMP and Transforming Growth Factor TGF inAdipocyte Diï¬erentiation A variety of extracellular factorsaï¬ect the preadipocyte commitment of stem cells includingbone morphogenetic protein BMP []transforminggrowth factor TGF [] insulininsulinlike growthfactor IGF1 [] tumor necrosis factor α and interleukin [] matrix metalloproteinase [] fibroblast growthfactor FGF and FGF2 [] BMP and TGF have variedeï¬ects on the diï¬erentiation fate of mesenchymal cells []The TGF superfamily members BMPs and myostatinregulate the diï¬erentiation of many cell types includingadipocytes [] TGF inhibitor can promote adipose diï¬erentiation of cancer cells with a mesenchymal phenotypein vitro and transgenic overexpression of TGF impairsadipocyte development [] Inhibition of adipogenesis couldbe obtained through blocking of endogenous TGF with adominantnegative TGF receptor or drosophila mothersagainst decapentaplegic protein SMAD inhibitionSMAD3 binds to CEBPs and inhibits their transcriptionalactivity including their ability to transactivate the PPARÎ2promoter [ ] Exposure of multipotent mesenchymalcells to BMP4 commits these cells to the adipocyte lineageallowing them to undergo adipose conversion [] Theeï¬ects of BMP2 are more complex and depend on the presence of other signaling molecules BMP2 alone has little eï¬ecton adipogenesis and it interacts with other factors such asTGF and insulin to stimulate adipogenesis of embryonicstem cells [] BMP2 stimulates adipogenesis of multipotentC3H10T12 cells at low concentrations and can contribute tochondrocyte and osteoblast development at higher concentrations [] KLFs in Adipocyte Diï¬erentiation During adipocyte differentiation some KLF family members are overexpressedsuch as KLF4 KLF5 KLF9 and KLF15 while KLF16 expression is reduced [ ] KLF15 is the first KLF family members which were identified to be involved in adipocytediï¬erentiation Its expression increased significantly on thesixth day of 3T3L1 adipocyte diï¬erentiation and peakedon the second day of adipocyte induction in MSCs andmouse embryonic fibroblasts Inhibition of KLF15 by siRNAor mutation led to a decrease in PPARÎ CEBPα fatty acidbinding protein FABP4 and glucose transporter GLUT4 However overexpression of KLF15 in NIH3T3cells was found to be associated with lipid accumulation aswell as increases in PPARÎ and FABP4 [] Mice with complete absence of KLF5 showed embryonal lethality and micewith singlechromosome KLF5 knockout showed a significant reduction in white fat in adulthood suggesting thatKLF5 plays an important role in adipocyte diï¬erentiationKLF5 can be activated by CEBP or CEBPδ which isinvolved in early adipocyte diï¬erentiation KLF5 can beactivated by CEBP or CEBPδ which is involved in earlyadipocyte diï¬erentiation Direct binding of KLF5 to thePPARÎ2 promoter in combination with CEBPs inducesPPARÎ2 expression [] Transfection of KLF5 dominantnegative mutants in 3T3L1 cells reduced lipid droplet accumulation and inhibited PPARÎ and CEBPα expressionwhereas overexpression of wild KLF5 significantly promotedadipocyte diï¬erentiation even without exogenous hormonestimulation Similar to KLF5 KLF9 knockdown can inhibitthe expression of a series of adipocyte diï¬erentiation genessuch as PPARÎ CEBPα and FABP4 hence inhibitingadipocyte diï¬erentiation However KLF9 overexpressiondid not upregulate the expression of PPARÎ and CEBPα[] In addition KLF4 can transactivate CEBP by bindingto the region of KB upstream of the CEBP promoter and promote lipid diï¬erentiation [] KLF6 can forma complex with histone deacetylase3 HDAC3 inhibitingpreadipocyte factor1 Pref1 expression and promotinglipid diï¬erentiation [] KLF2 is highly expressed in adiposeprogenitors and its expression decreases during the processof lipid diï¬erentiation Overexpressed KLF2 can bind to theCACCC region of PPARÎ2 proximal promoter and inhibitlipid diï¬erentiation as well as the expression of PPARÎCEBPα and sterolregulated elementbinding proteinsSREBP by inhibiting the promoter activity [] RNAsequence analysisshowed that KLFl6 expression wasdecreased on the first day of adipocyte diï¬erentiation of3T3L1 cells Adipocyte diï¬erentiation was promoted byKLF16 knockdown but was inhibited by KLF16 overexpression via inhibition of PPARÎ promoter activity [] In addition KLF3 and KLF7 were also found to play a negativeregulatory role in adipocyte diï¬erentiation [ ] Signal Transducers and Activators of TranscriptionSTATs and Adipocyte Diï¬erentiation The activated STATprotein enters the nucleus as a dimer and binds to the targetgene to regulate gene transcription In the adipocyte diï¬erentiation of mouse 3T3L1 cells the expression of STAT1 andSTAT5 was significantly increased while that of STAT3and STAT6 was not significantly changed [] In the adipocyte diï¬erentiation of human subcutaneous adipose precursor cells STAT1 expression was significantly decreased[] while the expression of STAT3 and STAT5 wasincreased and STAT6 expression was unchanged [] Therole of STAT1 in adipocyte diï¬erentiation is not clearbecause its expression trend in humans and mice diï¬ersduring the adipocyte diï¬erentiation process Early adipocytediï¬erentiation of 3T3L1 cells was inhibited by STAT1 0cStem Cells InternationalKLF5SREBP1cKLF15KLF2CHOPCEBPá¿«KROX20LigandCEBP𝛽CEBP𝛿GATA23PPARá¿«CEBP𝛼ProadipogenicAntiadipogenicGenes of terminaladipocytedifferentiationFigure A cascade of transcription factors that regulate adipogenesis PPARÎ is one of the key transcription factors in adipogenesis and thecore of the transcriptional cascade that regulates adipogenesis PPARÎ expression is regulated by several proadipogenic blue andantiadipogenic red factors CEBPα is regulated through a series of inhibitory protein“protein interactions Some transcription factorfamilies include several members that participate in adipogenesis such as the KLFs Black lines indicate eï¬ects on gene expression violetlines represent eï¬ects on protein activityagonist interferon Î Loss of STAT1 in 3T3L1 cells can rescue the inhibition of adipocyte diï¬erentiation caused byprostaglandin factor 2α [] Other studies have found thatSTAT1 is required for adipose diï¬erentiation and STAT1overexpression in C3H10T12 cells can prevent the inhibition of lipid diï¬erentiation caused by Bcell lymphoma6knockdown [] There was no abnormal adipose tissuein STAT1 knockout mice [] STAT3 not only aï¬ectsthe proliferation of 3T3L1 cells but also coregulates theiradipocyte diï¬erentiation with high mobility group protein [] The FABP4 promoter was used to specificallyknock out STAT3 in the adipose tissue of mice and theresults showed that mice weight significantly increasedand the adipocyte quantity increased compared with thewildtype mice [] STAT5A and STAT5B have diï¬erenteï¬ects on adipocyte diï¬erentiation Abnormal adipose tissuewas found in the mice with STAT5A or STAT5B knockout ordouble knockout and the amount of adipose tissue was onlyonefifth of the original adipose tissue in mice withoutknockdown [] Histone Modification in Adipocyte Diï¬erentiation Histone deacetylase sirtuin SIRT plays an important rolein biological processes such as stress tolerance energymetabolism and cell diï¬erentiation [] During the adipocyte diï¬erentiation of C3H1012 cells SIRT1 expressiondecreased [] Overexpression of SIRT1 activated theWnt signal which caused the deacetylation of cateninThe accumulation of catenin in the nucleus could inhibitadipocyte diï¬erentiation SIRT1 knockdown resulted inincreased acetylation of the histones H3K9 and H4K16 inthe secreted frizzledrelated protein sFRP and sFRP2 promoters thereby promoting transcription of these genes andpromoting lipid diï¬erentiation [] Forkhead box proteinO FOXO is a member of the transcription factor FOXOfamily It can recruit cyclic AMP response elementbindingprotein CBPhistone acetyltransferase p300 to initiate anacetylation The acetylated FOXO1 can be phosphorylatedby phosphorylated protein kinase B PKBAKT The phosphorylation of FOXO1 by AKT inhibits the transcriptionalactivation of FOXO1 The acetylation of FOXO1 lost the ability of DNAbinding affinity and promoted its shuttling fromnuclei to cytoplasm [] SIRT1 and SIRT2 can deacetylateand active FOXO1 Activated FOXO1 nonphosphorylatednuclear FOXO1 in the nucleus binds to the promoters of target genes encoding p21 p27 and PPARÎ and initiates subsequent transcriptions [] SIRT2 inhibits the acetylation andphosphorylation of FOXO1 thereby induces the accumulation of activated FOXO1 in the nucleus Activated FOXO1could inhibit adipogenesis via PPARÎ [“] Lysinespecific histone demethylase LSD1 expression increasedduring the adipocyte diï¬erentiation of 3T3L1 cells LSD1could reduce the dimethylation levels of histone H3K9 andH3K4 in the CEBPα promoter region thereby promotingadipocyte diï¬erentiation [] SET domaincontaining SETD8 catalyzed the monomethylation of H4K20 andpromoted PPARÎ expression The activation of PPARÎ transcriptional activity leads to the induction of monomethylatedH4K20 and modification of PPARÎ and its targets therebypromoting adipogenesis [] Enhancer of zeste homolog EZH2 is a methyltransferase and can bind methyl groupsto histone H3K27 which is also necessary for lipid diï¬erentiation The absence of EZH2 in brown fat precursors results inreduced levels of the Wnt promoter histone H3K27me3which is also saved by the ectopic EZH2 expression or theuse of a Wntcatenin signal inhibitor [] In addition histone demethylases such as lysinespecific histone demethylase LSDKDM KDM6 and histone lysine demethylasePHF2 are also involved in adipose diï¬erentiation andKDM2B inhibits transcription factor activator protein 2αpromoter via H3K4me3 and H3K36me2 [] Role of microRNA and Long NoncodingRNA in AdipogenesismicroRNA miR can bind and cut target genes or inhibittarget gene translation Endogenous siRNA can be producedby the action of Dicer enzyme and bind to a specific proteinto change its cellular location [] Many kinds of miRsare involved in regulating adipocyte diï¬erentiation The 0cStem Cells Internationalexpression of miR143 increased during the diï¬erentiationof adipose progenitor cells Overexpression of miR143promoted gene expression involved in adipose diï¬erentiationand triglyceride accumulation Inhibition of miR143 prevented the adipose diï¬erentiation of human fat progenitorcells [ ] Additionally miR8 promotes adipocyte diï¬erentiation by inhibiting Wnt signaling [] Moreover miR miR103 miR21 miR519d miR210 miR30miR204211 and miR375 also play a certain role in promoting adipocyte diï¬erentiation while miR130 miR448and let7y inhibit lipid diï¬erentiation [ ] In additionto miRs long noncoding RNA LncRNA is a type of noncoding RNA and is important during epigenetic regulationand can form a doublestranded RNA complex with mRNAcauses protein transcription Lncu90926 inhibits adipocytediï¬erentiation by inhibiting the transactivation of PPARÎ2[] As a novel LncRNA HOXAAS3 expression increasedduring the adipose diï¬erentiation of MSCs and HOXAAS3 silencing reduced the marker gene of adipose diï¬erentiation and inhibited the adipose diï¬erentiation [] Zhu et al[] reported that HOXAAS3 interacted with EZH2 toregulate lineage commitment of MSCs HOXA AS3 canregulate the trimethylation level of H3K27 in the Runx2promoter region by binding to EZH2 Therefore HOXAAS3 is considered to be an epigenetic switch regulating MSCslineage specificity [] Adipocyte diï¬erentiationassociatedLncRNA can act as a competitive endogenous RNA of miR in the process of lipid diï¬erentiation thereby promotingthe expression of SIRT1 the target gene of miR204 and thusinhibiting lipid diï¬erentiation [] The LncRNA NEAT1can also regulate adipocyte diï¬erentiation under the ‚uence of miRNA140 [] Other LncRNA including LncRNABlnc1 and Plnc are also involved in regulating adipocytediï¬erentiation [ ] Other Biochemical Response Involved inAdipocyte Differentiation Unfolded Protein Responses in Adipocyte Diï¬erentiationIn the endoplasmic reticulum of eukaryotes unfolded protein response involves three proteinsinositolrequiringenzyme 1α doublestranded RNAdependent proteinkinaselike ER kinase and activating transcription factorATF 6α [] Knockdown of ATF6α aï¬ects the expressionof adipocytes genes and inhibits C3H10T12 adipocyte differentiation [] The inhibitory eï¬ect of berberine on adipocyte diï¬erentiation of 3T3L1 cells is also due to inducedCHOP and decorin expressions and this inhibitory eï¬ectis ameliorated by CHOP knockout [] In the adipocytediï¬erentiation process of 3T3L1 cells increases in PPARÎand CEBPα as markers of adipocyte diï¬erentiation wereaccompanied by an increase in the corresponding proteinexpressions of phosphorylated Eukaryotic translation initiaEIF 2α phosphorylated endoribonucleasetion factorIRE1α ATF4 CHOP and other unfolded protein responsesEndoplasmic reticulum stress inducer or hypoxic endoplasmic reticulum stress can inhibit adipocyte diï¬erentiationAdditionally EIF2α mutation results in continuous activation or overexpression of CHOP which also inhibits adipocyte diï¬erentiation [] After the initiation of adiposediï¬erentiation numerous diï¬erentiationassociated proteinsare synthesized Exogenous endoplasmic reticulum stressinducers can lead to excessive endoplasmic reticulumresponse which in turn aï¬ects the synthesis of proteinsrelated to diï¬erentiation and inhibits adipocyte formationFigure Role of Oxidative Stress in Adipogenesis During thedirectional diï¬erentiation of MSCs mitochondrial complexI and III and NADPH oxidase NOX4 are the main sourcesof oxygen species ROS production Currently it is believedthat ROS aï¬ects not only the cell cycle and apoptosis but alsodiï¬erentiation through ‚uencing the signaling pathwaysincluding the Wnt HH and FOXO signaling cascade duringMSCs diï¬erentiation [] The diï¬erentiation ability ofstem cells is determined by the arrangement of perinuclearmitochondria which specifically manifests as low ATPcellcontents and a high rate of oxygen consumption The lackof these characteristics indicates stem cell diï¬erentiation[] Adipocyte diï¬erentiation is a highly dependent ROSactivation factor related to mitosis and cell maturation[] Schroder found that exogenous H2O2 could stimulate adipocyte diï¬erentiation of mouse 3T3L1 cells andhuman adipocyte progenitor cells in the absence of insulinH2O2 regulates adipocyte diï¬erentiation of 3T3L1 cells ina dosedependent manner High doses of H2O2 and μM promote adipocyte diï¬erentiation [ ] Tormos found that ROS synthesis increased in humanMSCs at the early stage of adipose diï¬erentiation and targeted antioxidants could inhibit lipid diï¬erentiation Byknocking down Rieske ironsulfur protein and ubiquinonebinding protein ROS produced by mitochondrial complexIII was found to be necessary in initiating adipose diï¬erentiation [] However other studies have shown that theexpression levels of adiponectin and PPARÎ were decreasedby using H2O2 “ mM in 3T3L1 cells [] Free radical nitric oxide NO also promotes lipid diï¬erentiationbecause treatment with NO inducer hydroxylamine or NOsynthase NOS substrate arginine can significantly induceadipose diï¬erentiation of rat adipose progenitor cells NOSinduced adipose diï¬erentiation mainly via eNOS rather thaniNOS [] ROS can induce adipose diï¬erentiation primarily by inhibiting Wnt FOXO and HH signaling pathwaysthat inhibit lipid diï¬erentiation Autophagy in Adipocyte Diï¬erentiation The increase inautophagosomes during lipid diï¬erentiation indicates thatautophagy may play an important role in lipid diï¬erentiation[] Baerga confirmed that the adipocyte diï¬erentiation efficiency was significantly inhibited in mouse embryonic fibroblasts lacking autophagyrelated gene Atg agene encoding an essential protein required for autophagy[] Knockdown of Atg5 in 3T3L1 cells promotesproteasomedependent degradation of PPARÎ2therebyinhibiting adipocyte diï¬erentiation [] Zhang reportedthat autophagyrelated gene 7Atg7 is also crucial for adipose development Atg7deficient mice were slim and onlyhad of white fat compared to wildtype mice and the 0cStem Cells InternationalCEBP𝛽 geneEBF1 geneKLF4EGR2CEBP𝛽CytosolCEBP𝛿 geneCEBP𝛿KLF5genePPARá¿« geneKLF5NR2F2NFKB11433RELASREBF1A2RXRAPPARá¿«PPARá¿«RXRA heterodimerPPARá¿«RXRAcorepressor complexFABP4Ligands of PPARá¿«FAM120BTHRAP3EP300NCOA2NCOA3HELZ2NCOA1CREBBPEBF1ADIPOQ geneAIDRFCEBP𝛼 geneZNF638ZNF467CEBP𝛼NCOR1HDAC3NCOR2 SLC2A4 geneGLUT4 geneLEP geneFABP4 geneCDK4CCND3PLIN1 genePCK1 geneFABP4CD36 genePPARARXRAcoactivator complexPPARá¿«fatty acidRXRAmediatorcoactivator complexANGPTLgenePPARGC1AMediator complex consensusLPL geneNucleoplasmProteins bind to gene promotersTranscription of genes into proteinsActing on proteins compoundingTGF𝛽1WNT1WNT10BTNF77233ADIPOQGLUT4SLC2A4 tetramerLEPFABP4lipid dropletPLIN1PCK1PaPa Pa4xPalmCCD36PaANGPTL4LPLFigure Regulation of adipocyte diï¬erentiation A regulatory loop exists between PPARÎ and CEBP activation Transcription factor CoeEBF activates CEBPα CEBPα activates EBF1 and EBF1 activates PPARÎ CEBP and CEBPδ act directly on the PPARÎ gene bybinding its promoter and activating transcription CEBPα CEBP and CEBPδ can activate the EBF1 gene and KLF5 The EBF1 and KLF5proteins in turn bind the promoter of PPARÎ which becomes activated Other hormones such as insulin can aï¬ect the expression ofPPARÎ and other transcription factors such as SREBP1c PPARÎ can form a heterodimer with the RXRα In the absence of activatingligands the PPARÎRXRα complex recruits transcription repressors such as nuclear receptor corepressor NCoR NCoR1 andHDAC3 Upon binding with activating ligands PPARÎ causes a rearrangement of adjacent factors Corepressors such as NCoR2 are lostand coactivators such as Transcription intermediary factor TIF2 CBP and p300 are recruited which can result in the expression of CyclicAMPresponsive elementbinding protein CREB followed by PPA Answer:

Thyroid_Cancer

THYROID development of thyroid hormone TH analogues was prompted by the attempt to exploit the effects of THon lipid metabolism avoiding cardiac thyrotoxicosis Analysis of the relative distribution of the a and bsubtypes of nuclear TH receptors TRa and TRb showed that TRa and TRb are responsible for cardiac andmetabolic responses respectively Therefore analogues with TRb selectivity were developed and four differentcompounds have been used in clinical trials GC1 sobetirome KB2115 eprotirome MB07344VK2809and MGL3196 resmetirom Each of these compounds was able to reduce lowdensity lipoprotein cholesterolbut a phase trial with eprotirome was interrupted because of a significant increase in liver enzymes and thecontemporary report of cartilage side effects in animals As a consequence the other projects were terminatedas well However in recent years TRb agonists have raised new interest for the treatment of nonalcoholic fattyliver disease NAFLD After obtaining excellent results in experimental models clinical trials have beenstarted with MGL3196 and VK2809 and the initial reports are encouraging Sobetirome turned out to beeffective also in experimental models of demyelinating disease Aside TRb agonists TH analogues includesome TH metabolites that are biologically active on their own and their synthetic analogues ¢triiodothyroacetic acid has already found clinical use in the treatment of some cases of TH resistance due toTRb mutations and interesting results have recently been reported in patients with the Allan“Herndon“Dudleysyndrome a rare disease caused by mutations in the TH transporter MCT8 35diiodothyronine T2 has beenused with success in rat models of dyslipidemia and NAFLD but the outcome of a clinical trial with a syntheticT2 analogue was disappointing 3iodothyronamine T1AM is the last entry in the group of active TH metabolites Promising results have been obtained in animal models of neurological injury induced by bamyloidor by convulsive agents but no clinical data are available so farKeywords TH analogues sobetirome eprotirome resmetirom triac 35diiodothyronine 3iodothyronamineIntroductionT he term thyroid hormone TH analogue is used withregard to compounds that have a similar molecularstructure as TH and can therefore interact with at least someof its molecular targetsIt should be kept in mind that TH signaling is particularlycomplex Canonical TH signaling is based on the interactionwith nuclear TH receptors TRs leading to either activation orrepression of the transcription of a large number of genes Twogene subtypes exist TRa and TRb and different isoformswhich are designated by numerical subscripts can be producedby alternative splicing TRa1 is the major TR isoform in theheart and it is also expressed in other tissues including skeletal muscle brain and bone TRa2 lacks part of the THbinding domain so it is unable to bind TH and is thought tomediate constitutive repression of transcription TRb1 iswidely expressed in most tissues while TRb2 expression ismore circumscribed and it is particularly relevant in the brainpituitary retina and inner ear TRb3 has been identified in ratadipose tissue but it does not appear to be present in humansWith regard to the topic of this review the main functionalconsequence of TR subtype distribution is that the metaboliceffects of TH are largely mediated by TRb1 while cardiacTH actions depend on TRa1In addition to canonical signaling TH may activate anumber of noncanonical signaling pathways The latterinvolves TRs that do not bind DNA directly extranuclearDepartment of Pathology University of Pisa Pisa Italyª Riccardo Zucchi Published by Mary Ann Liebert Inc This Access is distributed under the terms of the CreativeCommons Attribution Noncommercial License httpcreativecommonslicensesbync40 which permits any noncommercial usedistribution and reproduction in any medium provided the original authors and the source are cited 0cZUCCHITRs that activate the phosphatidylinositol 3kinase pathwayand membrane receptors belonging to the integrin family In addition some TH metabolites have been suggested tobe potentially active although their physiological role is stillunclear By definition these compounds should be regardedas TH analogues although they are endogenous moleculesThey have also been used as templates to develop novelclasses of synthetic analogues Active TH metabolites include35diiodothyronine T2 3iodothyronamine T1AM andseveral thyroacetic acids ¢¢tetraiodothyroacetic acid orTetrac ¢triiodothyroacetic acid or Triac 3iodothroaceticacid or TA1 They have been the object of several recentreviews “ and their properties are briefly recalled in thesubsequent paragraphs only in as much as this is relevant to theobject of the present succinct reviewTH analogues have been developed for clinical and therapeutic purposes namely to exploit some aspects of THsignaling to produce beneficial effects in the diseaseTherefore in the following paragraphs these analogues arediscussed with the perspective of their potential therapeuticuse Special emphasis is placed on clinical investigationsalthough some results obtained in experimental models ofdisease are mentioned when appropriateTH Analogues with Selective TRb Activityin DyslipidemiaThe development of selective TRb agonists was promptedby the aim of treating dyslipidemia particularly hypercholesterolemia avoiding cardiac thyrotoxicosis The ligandbinding pocket is more flexible in TRb than in TRa and one ofthe amino acids involved in triiodothyronine T3 binding isdifferent since TRa serine is replaced by asparagine in TRb These differences have been exploited to synthesizeselective TRb agonists The analogues that have reached the clinical field areshown in Figure In GC1 now called sobetirome the iodine atoms and the oxygen linking the two aromatic rings arereplaced by alkyl groups and the amino acid side chain isreplaced by an oxoacetic chain The ratio of TRb to TRaaffinity is 10fold higher for GC1 than for T3 and an additional useful property is represented by selective liver uptakeIn KB2115 now referred to as eprotirome iodine is replaced by bromine in the tyrosyl ring or isopropyl in thephenolic ring and an amidoacetic side chain is presentyielding nearly 20fold TRb selectivityA different strategy was followed by researchers at MetabasisTheir compound known as MB07811 and now renamedVK2809 is a prodrug selectively taken up by the liver where it isconverted into the active principle MB07344 which differsfrom GC1 only for the presence of a phosphoryl group in the sidechainAnother compound that has been used in human is MGL also called resmetirom whose more complex chemicalstructure a substituted pyridazinone ring replaces the phenolicring and a heterocyclic cyanoazauracil group is included in theside chain allows nearly 30fold TRb selectivityAll these compounds have been used in animal models ofhypercholesterolemia where they reduced total and lowdensity lipoprotein LDL cholesterol without significantchanges in heart rate Based on these preclinical findingsclinical trials were started In both sobetirome andeprotirome were reported to produce a significant reduction in total and LDL cholesterol after “ weeks oftreatment in small groups of patients affected by hypercholesterolemia Similar results were obtained with MGL3196which reduced serum triglycerides and LDL cholesterol after weeks of treatment in hypercholesterolemic patients In patients treated with statins the addition of eprotirome caused further reduction in serum LDL cholesterol andtriglycerides No significant side effects were reported in any of theseinvestigations and therefore a phase trial was undertakento compare eprotirome at the daily doses of and lgversus placebo in patients with familial hypercholesterolemia After weeks total cholesterol LDL cholesterolFIG Chemical structureof the synthetic TRb analogues that have been used inclinical trials In the leftlower panel please note thatMB07811 now known asVK2809 is a prodrug in theliver it is converted into theactive principle formerlyknown as MB07344 by thehydroxylase CYP3A in a reaction requiring glutathioneGSH See text for furtherdetails 0cTHYROID HORMONE ANALOGUESand triglycerides were significantly reduced in both treatmentgroups without any change in HDL cholesterol However the trial was interrupted it was originally planned to last“ weeks because parallel experimental investigationsfound that eprotirome caused cartilage damage in dogs Notably during the study period a significant increase in liverenzymes was detected which determined treatment interruption in four patientsResulting in a partial domino effect termination of theeprotirome project caused parallel projects to be terminatedas well In particular the sobetirome project was interruptedand Metabasis announced that a phase investigation withMB07344 was aborted after observing increased liver enzymes in some patients Additional reasons probably contributed to these decisions While selective TRb agonistswere devised and tested excellent clinical results were obtained with statins in the primary and secondary preventionsof major cardiac events At the same time investigationsperformed with other experimental drugs showed that LDLcholesterol reduction per se was not necessarily associatedwith reduced cardiovascular risk In aggregate selective TRbstimulation was no longer regarded as a promising strategy totreat hypercholesterolemiaTH Analogues with Selective TRb Activityin Liver DiseaseDespite the disappointing results obtained with TRb agonists in hypercholesterolemia in recent years new potentialuses have been proposed for these agents The most attractivefield is probably represented by nonalcoholic fatty liver disease NAFLDThis is a highly prevalent condition since it is estimated toaffect “ of the adult population in the United Statesand Europe Its clinical presentation is quite variableMost NAFLD patients show a simple increase in liver enzymes with a histological pattern of increased hepatocytetriglyceride steatosis Symptoms may be minimal or absentand the clinical picture may be stable over time However asignificant fraction of NAFLD patients up to in someseries develop histological evidence of lobular inflammationand hepatocyte ballooning defining a variant of the diseaseknown as nonalcoholic steatohepatitis NASH NASH is aserious condition since it is associated with a high risk about“ of evolution into cirrhosis that is derangement inliver architecture leading to hepatic insufficiency Patientswith NASH andor cirrhosis are also prone to develop hepatocellular carcinoma HCCNAFLD is frequently associated with insulin resistancehypercholesterolemia or other components of the socalledmetabolic syndrome and patients are usually treated forthese underlying conditions but no specific treatment toprevent liver damage has been approved so far In principleTH should be beneficial in NAFLD due to reduced fatty acidsynthesis increased triglyceride and fatty acid breakdownand enhanced hepatocyte regeneration The liver effects of TH are mediated by TRb so selective TRb agonistshave been tested in experimental models and several compounds including sobetirome eprotirome and MB07811were able to reduce liver steatosis “ Sobetirome wasalso reported to prevent the development of HCC induced byactivation of the catenin pathway The study of TRb agonists in NAFLD has just entered theclinical field and the results of two clinical trials have recently been reported In patients with biopsyconfirmedNASH treated with MGL3196 resmetirom mg dailyfor weeks liver fat as assessed by magnetic resonanceimaging was significantly reduced versus with placebo if expressed as percentage of absolute livermass versus if expressed as percentage ofbaseline liver fat The effect was retained after weeksand in a subgroup of patients biopsy revealed a reduction ofhistological markers of inflammationSimilarresults were reported with VK2809 aliasMB07811 in patients with NAFLD treated for weeksat the dosages of mg every other day or mg daily In this trial liver fat content assessed by magnetic resonanceimaging decreased by “ versus with placebo“ vs if expressed as percentage ofbaseline liver fat While these results have raised a greatinterest and discussions have already started aboutthecomparison of these two drugs it should be stressed that thefinal results of the latter trial have not been published yetTH Analogues with Selective TRb Activity in CentralNervous System DiseaseIn recent years theoretical arguments have been developed suggesting the potential usefulness of TH analogues indemyelinating disease since TH favors both oligodendrocytedifferentiation and myelin sheet synthesis AlthoughTRa1 is widely expressed in the central nervous systemefforts have been focused on TRb agonists because of thenecessity to avoid cardiac side effects To increase bioavailability an ethanolamine ester of sobetirome has beensynthesized which acts as a prodrug since it crosses thebloodbrain barrier and is converted into sobetirome withinthe central nervous system This compound has beenrecently tested in different experimental models of demyelination with good results biochemical evidence of remyelination was confirmed by morphological findingsobtained by nuclear magnetic imaging and it was associatedwith improved functional recovery Clinical tests may be imminent for a specific diseaseknown as Xlinked adrenoleukodystrophy ALD This is arare congenital disease due to mutations in the ALD protein atransporter for verylongchain fatty acids ie with ¡carbon atoms physiologically located in the peroxisomalmembrane and encoded by the ABCD1 gene The consequence is the accumulation of verylongchain fatty acids andtheir derivatives which are eventually incorporated in cellular membranes whose structure and function are derangedAffected males develop adrenal insufficiency in childhoodand progressive myelopathy occurs in adulthood Demyelinating lesions in cerebral white matter also appear since theage of years ALD patients may benefit from hematopoietic stem celltransplantation but this treatment is effective only if performed in the early stages of the disease and it carries asignificant risk of mortality “ No specific pharmacological therapy is available for ALD Notably TH inducesthe expression of ABCD2 coding for an additional peroxisomal transporter and in a transgenic mouse model of ALDsobetirome administration reduced the brain and adrenal 0cZUCCHIcontent of verylongchain fatty acids Based on theseobservations a clinical trial with sobetirome in XlinkedALD has been posted in the NIH database NCT01787578The trial is presently labeled as withdrawn and the allegedreason is the need for revisions to the original protocolTriac in Syndromes of Reduced Sensitivity to THReduced sensitivity to TH is diagnosed when symptoms ofhypothyroidism occur despite normal or increased serum THThis finding may be the consequence of mutations in TRstransporters or metabolizing enzymes The expression˜˜resistance to TH™™ is usually reserved for syndromes causedby TR mutations Most cases are associated with TRb mutations Since TRb is involved in the inhibition of thyrotropin TSH secretion by T3 and thyroxine T4 circulatinglevels of T4 and T3 are usually high TSH is normal or highand goiter may be present The clinical picture is quitevariable and includes signs and symptoms of both hypothyroidism in TRbdependent ans and thyrotoxicosisin TRbdependent ans The most common findings aredelayed growth delayed bone maturation cognitive impairment and tachycardia Treating TH resistance is not easy Exogenous T4 or T3administration may improve some symptoms but it mayworsen others and symptomatic therapy is often prescribedfor example betablockers to reduce heart rate The idealtreatment would be represented by a T3 analogue able toactivate the mutated receptorIn some patients this can occur with Triac Fig Thelack of the amine group does not prevent Triac from activating TRs and it has similar affinity as T3 for the wildtypereceptor Apparently the different shape and charge of theTriac molecule allow several classes of mutated TRb to beactivated as well In these patients chronic Triac administration at dosages on the order of “ lg per kg of bodyweight daily represents the best therapeutic regimen Ingeneral Triacsensitive cases of TH resistance are caused bymutations in the carboxyterminal region of the T3 bindingFIG Chemical structure of some active thyroid hormone metabolites that have been used in patients or in animal models of human disease See text for further detailsdomain while mutations located close to the hinge region donot respond to Triac No positive response to Triac has beenreported in TRa mutations so farA different cause of reduced sensitivity to TH is represented by mutations in MCT8 a T3T4 transporter that isthe major pathway mediating T4 and T3 uptake in the centralnervous system The clinical syndrome associated withMCT8 mutations is known as the Allan“Herndon“Dudleysyndrome The MCT8 gene is located on the X chromosome and therefore virtually all patients are male Diagnosis is suspected in the presence of high T3 with low tonormal T4 and low to normal TSH associated with congenital brain hypothyroidism The phenotype is variable depending on the location and type of the mutationSymptoms usually include cognitive impairment associated with congenital hypotonia and weakness which mayprogress to spasticity Paroxysmal dyskinesias and seizuresmay also occur Peripheral hyperthyroidism often causestachycardia muscle wasting and progressive body weightreduction “Since it has been observed that cellular Triac uptake doesnot depend on MCT8 a clinical trial has been undertakenwith this endogenous TH analogue in patients with amedian age of years The results obtained after monthsof treatment at the dosage of “ lg daily have recently been published The treatment was highly effective in reducing serum T3 and Triac dosage was actuallytitrated on T3 reduction Signs of peripheral hypothyroidismnotably tachycardia and body weight reduction were significantly attenuated On the contrary the effects on theneurological symptoms were limited and improvement inthe indices of motor function was limited to patients youngerthan years It seems therefore that once the neurologicalphenotype is fully developed it is hardly reversibleOn this basis another phase trialis underwayNCT02396459 in which Triac treatment will be started asearly as possible in postnatal lifeSynthetic TH analogues might also be useful Notably in asmall clinical study diiodothyropropionic acid DITPA administration “ mgkg per day was able to normalizeT3 in four children affected by MCT8 deficiency aged “months Heart rate decreased in three patients and weightgain occurred in two Although DITPA was introduced over years ago as a relatively weak and poorly selective TRagonist its mode of interaction with TRs has not been specifically investigated so farPotential Uses of T2 and T1AMWhile Triac is basically a thyromimetic other active THmetabolites Fig can interact with different moleculartargets T2 has direct mitochondrial actions allegedly onrespiratory chain complex IV subunit Va and it stimulatesmitochondrial respiration and fatty acid oxidation These metabolic responses are further supported by genomiceffects whose molecular basis is unclear since they appear tobe different from those elicited by T3 In any case in ratstreated with highfat diet exogenous T2 decreased serumtriglycerides and LDL cholesterol as well as liver fat andbiochemicalindices of liver injury suggesting potentialtherapeutic value for either dyslipidemia or NAFLD However it is still controversial whether these positive 0cTHYROID HORMONE ANALOGUESeffects may be achieved without inducing tachycardia orcardiac hypertrophy since cardiac thyrotoxicosis has beenreported in mice A single pilot investigation was performed in two humanvolunteers taking T2 lgkg for weeks A slightbut significant decrease in body weight was reported without any change in serum T3 T4 TSH and cardiac function More recently a synthetic T2 analogue TRC150094was used in patients with metabolic syndrome but theresults were rather disappointing since no significant effect on insulin sensitivity and plasma lipid profile was observed The last entry into the group of active TH metabolites isrepresented by T1AM It does not interact with canonical ornoncanonical TH targets and it was discovered as a highaffinity agonist of TAAR1 a membrane G proteincoupledreceptor GPCR that is expressed in the brain and manyother tissues T1AM is a biogenic amine and sharesseveral properties of this class of compounds includingthe ability to interact with multiple targets namely otherGPCRs eg a2A adrenergic receptor membrane ionicchannels eg TRPM8 and monoamine transporters In experimental animals the administration of exogenousT1AM induced many different functional effects Neurological and metabolic effects are particularly interesting sincethey appear to be elicited at relative low doses and mighthave physiological relevance The former includesmodulation of feeding behavior and sleepwake cycle reduction of pain threshold prolearning and antiamnestic responses The major metabolic effects consistin thestimulation of triglyceride and fatty acid catabolism but antiinsulin effects on glucose metabolism are also elicited atslightly higher dosesAlthough the administration of exogenous T1AM has notbeen tested in humans some experimental results obtainedin murine models of disease have raised interest aboutpotential therapeutic applications T1AM reduced serumcholesterol in spontaneously obese mice as well asliver triglycerides in a mouse model of polycystic ovarysyndrome Neuroprotective effects appear even more promising Intracerebral T1AM rescued longterm potentiation and behavioral evidence of cognitive dysfunction in an in vivomodel of bamyloid toxicity namely transgenic mice overexpressing humanmutated amyloid precursor protein In addition the intracerebral injection of T1AM metabolite iodothyroacetic acid protected from the convulsive effect ofpentylenetetrazole and from kainate toxicity while exogenous T1AM reduced apoptosis and functional injury in amouse model of spinal cord clamp Because of the multitude of T1AM effects an active research line is focused on the development of synthetic derivatives with more favorable biodistribution andor receptorselectivity In conclusion the development TH analogues was initially prompted by the attempt to exploit the effects of THon lipid metabolism while avoiding unwanted cardiac effects TRb agonists have provided good results in a fewsmall clinical trials performed in hypercholesterolemic patients but these projects have been terminated after the report of potential side effects In recent years TRb agonistshave raised new interest for the treatment of NAFLD and acouple of clinical trials have provided encouraging initialresults Triac has already found clinical use in the treatmentof selected cases of TH resistance due to TRb mutationsand interesting results have recently been reported in theAllan“Herndon“Dudley syndromeOther TH analogues are under consideration for neurological diseases although human results are not yetavailable In particular sobetirome derivatives have beensuccessfulin animal models of multiple sclerosis andT1AM has been beneficial in an animal model of bamyloidtoxicityOverall research on TH analogues is experiencing a shiftin its focus but it still appears to be an active and promisingfieldAuthor Disclosure StatementNo competing financial interests existFunding InformationThis work was supported by a grant from Pisa UniversityPRA to RZReferences OrtigaCarvalho TM Sidhaye AR Wondisford FE Thyroid hormone receptors and resistance to thyroid hormone disorders Nat Rev Endocrinol “ 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Therapeutic applications of thyroid hormone analogues inresistance to thyroid hormone RTH syndromes Mol CellEndocrinol “ Friesema ECH Visser WE Visser TJ Genetics andphenomics of thyroid hormone transport by MCT8 MolCell Endocrinol “ Schwartz CE Stevenson RE The MCT8 thyroidhormone transporter and AllanHerndonDudley Syndrome Best Pract Clin Endocrinol Metab “ Groeneweg S Peeters RP Moran C Stoupa A Aurial FTonduti D Dica A Paone L Rozenkova K Malikova Jvan der Walt A de Coo IFM McGowan A Lyons GAarsen FK Barca D van Beynum IM van der Knoop MMJansen J Manshande M Lunsing RJ Nowak S den UilCA Zillikens MC Visser FE Vrijmoeth P de Wit MCYWolf NI Zandstra A Ambegaonkar G Singh Y de RijkeYB Medici M Bertini ES Depoorter S Lebl J Cappa MDe Meirleir L Krude H Craiu D Zibordi F Oliver Petit IPolak M Chatterjee K Visser TJ Visser WE Effectiveness and safety of the triiodothyronine analogue Triacin children and adults with MCT8 deficiency an international single arm label phase trial Lancet Diabetes Endocrinol “ Verge CF Konrad D Cohen M Di Cosmo C DumitrescuAM Marcinkowski T Hameed S Hamilton J Weiss RERefetoff S Diiodothyropionic acid DITPA in thetreatment of MCT8 deficiency J Clin Endocrinol Metab“ Goglia F The effects of 35diiodothyronine on energybalance Front Physiol Jonas W Lietzow J Wohlgemuth F Hoefig CS WiedmerP Schweizer U Ko¨hrle J Schurmann A 35DiiodoLthyronine 35T2 exertsthyromimetic effects onhypothalamuspituitarythyroid axis body compositionand energy metabolism in male dietinduced obese miceEndocrinology “ Antonelli A Fallahi P Ferrari SM Di Domenicantonio AMoreno M Lanni A Goglia F 35diiodoLthyronineincreases resting metabolic rate and reduces body weightwithout undesirable side effects J Biol Regul HomeostAgents “ Van der Val F Hassing C Visser M Thakkar P MohananA Pathak K Dutt C Chauthaiwale V Ackermans MNederveen A Serlie M Nieuwdorp M Stroes E Theeffect of a diodothyronine mimetic on insulin sensitivity inmale cardiometabolic patients a doubleblind randomizedcontrolled trial PLoS One 9e86890 0cTHYROID HORMONE ANALOGUES Zucchi R Chiellini S Scanlan TS Grandy DK Traceamineassociated receptors and their ligands Br J Pharmacol “ Rutigliano G Accorroni A Zucchi R The case forTAAR1 as a modulator of central nervous system functionFront Pharmacol Zucchi R Accorroni A Chiellini G Update on iodothyronamine and its neurological and metabolic actions Front Physiol AssadiPorter FM Reiland H Sabatini M Lorenzini LCarnicelli V Rogowski M Selen Alpergin ES Tonelli MGhelardoni S Saba A Zucchi R Chiellini G Metabolic reprogramming by 3iodothyronamine T1AM anew perspective to reverse obesity through regulation ofsirtuin and expression Int J Mol Sci 19e1535 Selen Alpergin ES Bolandnazar Z Sabatini M RogowskiM Chiellini G Zucchi R AssadiPorter FM Metabolic profiling reveals reprogramming of lipid metabolicpathways in treatment of polycystic ovary syndrome with3iodothyronamine Physiol Rep 5e13097 Accorroni A Rutigliano G Sabatini M Frascarelli S BorsoM Novelli E Bandini L Ghelardoni L Saba A Zucchi ROriglia N Exogenous 3iodothyronamine rescues theentorhinal cortex from bamyloid toxicity Thyroid “ Laurino A Landucci E Resta F De Siena F PellegriniGiampietro DE Masi A Mannaioni G Raimondi L Anticonvulsant and neuroprotective effects of the thyroidhormone metabolite 3Iodothyroacetic Acid Thyroid “ Lv J Liao J Tan W Yang L Shi X Zhang H ChenL Wang S Li Q 3Iodothyronamine acting throughan antiapoptotic mechanism is neuroprotective againstspinal cord injury in rats Ann Clin Lab Sci “

cancer278

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: THYROID development of thyroid hormone TH analogues was prompted by the attempt to exploit the effects of THon lipid metabolism avoiding cardiac thyrotoxicosis Analysis of the relative distribution of the a and bsubtypes of nuclear TH receptors TRa and TRb showed that TRa and TRb are responsible for cardiac andmetabolic responses respectively Therefore analogues with TRb selectivity were developed and four differentcompounds have been used in clinical trials GC1 sobetirome KB2115 eprotirome MB07344VK2809and MGL3196 resmetirom Each of these compounds was able to reduce lowdensity lipoprotein cholesterolbut a phase trial with eprotirome was interrupted because of a significant increase in liver enzymes and thecontemporary report of cartilage side effects in animals As a consequence the other projects were terminatedas well However in recent years TRb agonists have raised new interest for the treatment of nonalcoholic fattyliver disease NAFLD After obtaining excellent results in experimental models clinical trials have beenstarted with MGL3196 and VK2809 and the initial reports are encouraging Sobetirome turned out to beeffective also in experimental models of demyelinating disease Aside TRb agonists TH analogues includesome TH metabolites that are biologically active on their own and their synthetic analogues ¢triiodothyroacetic acid has already found clinical use in the treatment of some cases of TH resistance due toTRb mutations and interesting results have recently been reported in patients with the Allan“Herndon“Dudleysyndrome a rare disease caused by mutations in the TH transporter MCT8 35diiodothyronine T2 has beenused with success in rat models of dyslipidemia and NAFLD but the outcome of a clinical trial with a syntheticT2 analogue was disappointing 3iodothyronamine T1AM is the last entry in the group of active TH metabolites Promising results have been obtained in animal models of neurological injury induced by bamyloidor by convulsive agents but no clinical data are available so farKeywords TH analogues sobetirome eprotirome resmetirom triac 35diiodothyronine 3iodothyronamineIntroductionT he term thyroid hormone TH analogue is used withregard to compounds that have a similar molecularstructure as TH and can therefore interact with at least someof its molecular targetsIt should be kept in mind that TH signaling is particularlycomplex Canonical TH signaling is based on the interactionwith nuclear TH receptors TRs leading to either activation orrepression of the transcription of a large number of genes Twogene subtypes exist TRa and TRb and different isoformswhich are designated by numerical subscripts can be producedby alternative splicing TRa1 is the major TR isoform in theheart and it is also expressed in other tissues including skeletal muscle brain and bone TRa2 lacks part of the THbinding domain so it is unable to bind TH and is thought tomediate constitutive repression of transcription TRb1 iswidely expressed in most tissues while TRb2 expression ismore circumscribed and it is particularly relevant in the brainpituitary retina and inner ear TRb3 has been identified in ratadipose tissue but it does not appear to be present in humansWith regard to the topic of this review the main functionalconsequence of TR subtype distribution is that the metaboliceffects of TH are largely mediated by TRb1 while cardiacTH actions depend on TRa1In addition to canonical signaling TH may activate anumber of noncanonical signaling pathways The latterinvolves TRs that do not bind DNA directly extranuclearDepartment of Pathology University of Pisa Pisa Italyª Riccardo Zucchi Published by Mary Ann Liebert Inc This Access is distributed under the terms of the CreativeCommons Attribution Noncommercial License httpcreativecommonslicensesbync40 which permits any noncommercial usedistribution and reproduction in any medium provided the original authors and the source are cited 0cZUCCHITRs that activate the phosphatidylinositol 3kinase pathwayand membrane receptors belonging to the integrin family In addition some TH metabolites have been suggested tobe potentially active although their physiological role is stillunclear By definition these compounds should be regardedas TH analogues although they are endogenous moleculesThey have also been used as templates to develop novelclasses of synthetic analogues Active TH metabolites include35diiodothyronine T2 3iodothyronamine T1AM andseveral thyroacetic acids ¢¢tetraiodothyroacetic acid orTetrac ¢triiodothyroacetic acid or Triac 3iodothroaceticacid or TA1 They have been the object of several recentreviews “ and their properties are briefly recalled in thesubsequent paragraphs only in as much as this is relevant to theobject of the present succinct reviewTH analogues have been developed for clinical and therapeutic purposes namely to exploit some aspects of THsignaling to produce beneficial effects in the diseaseTherefore in the following paragraphs these analogues arediscussed with the perspective of their potential therapeuticuse Special emphasis is placed on clinical investigationsalthough some results obtained in experimental models ofdisease are mentioned when appropriateTH Analogues with Selective TRb Activityin DyslipidemiaThe development of selective TRb agonists was promptedby the aim of treating dyslipidemia particularly hypercholesterolemia avoiding cardiac thyrotoxicosis The ligandbinding pocket is more flexible in TRb than in TRa and one ofthe amino acids involved in triiodothyronine T3 binding isdifferent since TRa serine is replaced by asparagine in TRb These differences have been exploited to synthesizeselective TRb agonists The analogues that have reached the clinical field areshown in Figure In GC1 now called sobetirome the iodine atoms and the oxygen linking the two aromatic rings arereplaced by alkyl groups and the amino acid side chain isreplaced by an oxoacetic chain The ratio of TRb to TRaaffinity is 10fold higher for GC1 than for T3 and an additional useful property is represented by selective liver uptakeIn KB2115 now referred to as eprotirome iodine is replaced by bromine in the tyrosyl ring or isopropyl in thephenolic ring and an amidoacetic side chain is presentyielding nearly 20fold TRb selectivityA different strategy was followed by researchers at MetabasisTheir compound known as MB07811 and now renamedVK2809 is a prodrug selectively taken up by the liver where it isconverted into the active principle MB07344 which differsfrom GC1 only for the presence of a phosphoryl group in the sidechainAnother compound that has been used in human is MGL also called resmetirom whose more complex chemicalstructure a substituted pyridazinone ring replaces the phenolicring and a heterocyclic cyanoazauracil group is included in theside chain allows nearly 30fold TRb selectivityAll these compounds have been used in animal models ofhypercholesterolemia where they reduced total and lowdensity lipoprotein LDL cholesterol without significantchanges in heart rate Based on these preclinical findingsclinical trials were started In both sobetirome andeprotirome were reported to produce a significant reduction in total and LDL cholesterol after “ weeks oftreatment in small groups of patients affected by hypercholesterolemia Similar results were obtained with MGL3196which reduced serum triglycerides and LDL cholesterol after weeks of treatment in hypercholesterolemic patients In patients treated with statins the addition of eprotirome caused further reduction in serum LDL cholesterol andtriglycerides No significant side effects were reported in any of theseinvestigations and therefore a phase trial was undertakento compare eprotirome at the daily doses of and lgversus placebo in patients with familial hypercholesterolemia After weeks total cholesterol LDL cholesterolFIG Chemical structureof the synthetic TRb analogues that have been used inclinical trials In the leftlower panel please note thatMB07811 now known asVK2809 is a prodrug in theliver it is converted into theactive principle formerlyknown as MB07344 by thehydroxylase CYP3A in a reaction requiring glutathioneGSH See text for furtherdetails 0cTHYROID HORMONE ANALOGUESand triglycerides were significantly reduced in both treatmentgroups without any change in HDL cholesterol However the trial was interrupted it was originally planned to last“ weeks because parallel experimental investigationsfound that eprotirome caused cartilage damage in dogs Notably during the study period a significant increase in liverenzymes was detected which determined treatment interruption in four patientsResulting in a partial domino effect termination of theeprotirome project caused parallel projects to be terminatedas well In particular the sobetirome project was interruptedand Metabasis announced that a phase investigation withMB07344 was aborted after observing increased liver enzymes in some patients Additional reasons probably contributed to these decisions While selective TRb agonistswere devised and tested excellent clinical results were obtained with statins in the primary and secondary preventionsof major cardiac events At the same time investigationsperformed with other experimental drugs showed that LDLcholesterol reduction per se was not necessarily associatedwith reduced cardiovascular risk In aggregate selective TRbstimulation was no longer regarded as a promising strategy totreat hypercholesterolemiaTH Analogues with Selective TRb Activityin Liver DiseaseDespite the disappointing results obtained with TRb agonists in hypercholesterolemia in recent years new potentialuses have been proposed for these agents The most attractivefield is probably represented by nonalcoholic fatty liver disease NAFLDThis is a highly prevalent condition since it is estimated toaffect “ of the adult population in the United Statesand Europe Its clinical presentation is quite variableMost NAFLD patients show a simple increase in liver enzymes with a histological pattern of increased hepatocytetriglyceride steatosis Symptoms may be minimal or absentand the clinical picture may be stable over time However asignificant fraction of NAFLD patients up to in someseries develop histological evidence of lobular inflammationand hepatocyte ballooning defining a variant of the diseaseknown as nonalcoholic steatohepatitis NASH NASH is aserious condition since it is associated with a high risk about“ of evolution into cirrhosis that is derangement inliver architecture leading to hepatic insufficiency Patientswith NASH andor cirrhosis are also prone to develop hepatocellular carcinoma HCCNAFLD is frequently associated with insulin resistancehypercholesterolemia or other components of the socalledmetabolic syndrome and patients are usually treated forthese underlying conditions but no specific treatment toprevent liver damage has been approved so far In principleTH should be beneficial in NAFLD due to reduced fatty acidsynthesis increased triglyceride and fatty acid breakdownand enhanced hepatocyte regeneration The liver effects of TH are mediated by TRb so selective TRb agonistshave been tested in experimental models and several compounds including sobetirome eprotirome and MB07811were able to reduce liver steatosis “ Sobetirome wasalso reported to prevent the development of HCC induced byactivation of the catenin pathway The study of TRb agonists in NAFLD has just entered theclinical field and the results of two clinical trials have recently been reported In patients with biopsyconfirmedNASH treated with MGL3196 resmetirom mg dailyfor weeks liver fat as assessed by magnetic resonanceimaging was significantly reduced versus with placebo if expressed as percentage of absolute livermass versus if expressed as percentage ofbaseline liver fat The effect was retained after weeksand in a subgroup of patients biopsy revealed a reduction ofhistological markers of inflammationSimilarresults were reported with VK2809 aliasMB07811 in patients with NAFLD treated for weeksat the dosages of mg every other day or mg daily In this trial liver fat content assessed by magnetic resonanceimaging decreased by “ versus with placebo“ vs if expressed as percentage ofbaseline liver fat While these results have raised a greatinterest and discussions have already started aboutthecomparison of these two drugs it should be stressed that thefinal results of the latter trial have not been published yetTH Analogues with Selective TRb Activity in CentralNervous System DiseaseIn recent years theoretical arguments have been developed suggesting the potential usefulness of TH analogues indemyelinating disease since TH favors both oligodendrocytedifferentiation and myelin sheet synthesis AlthoughTRa1 is widely expressed in the central nervous systemefforts have been focused on TRb agonists because of thenecessity to avoid cardiac side effects To increase bioavailability an ethanolamine ester of sobetirome has beensynthesized which acts as a prodrug since it crosses thebloodbrain barrier and is converted into sobetirome withinthe central nervous system This compound has beenrecently tested in different experimental models of demyelination with good results biochemical evidence of remyelination was confirmed by morphological findingsobtained by nuclear magnetic imaging and it was associatedwith improved functional recovery Clinical tests may be imminent for a specific diseaseknown as Xlinked adrenoleukodystrophy ALD This is arare congenital disease due to mutations in the ALD protein atransporter for verylongchain fatty acids ie with ¡carbon atoms physiologically located in the peroxisomalmembrane and encoded by the ABCD1 gene The consequence is the accumulation of verylongchain fatty acids andtheir derivatives which are eventually incorporated in cellular membranes whose structure and function are derangedAffected males develop adrenal insufficiency in childhoodand progressive myelopathy occurs in adulthood Demyelinating lesions in cerebral white matter also appear since theage of years ALD patients may benefit from hematopoietic stem celltransplantation but this treatment is effective only if performed in the early stages of the disease and it carries asignificant risk of mortality “ No specific pharmacological therapy is available for ALD Notably TH inducesthe expression of ABCD2 coding for an additional peroxisomal transporter and in a transgenic mouse model of ALDsobetirome administration reduced the brain and adrenal 0cZUCCHIcontent of verylongchain fatty acids Based on theseobservations a clinical trial with sobetirome in XlinkedALD has been posted in the NIH database NCT01787578The trial is presently labeled as withdrawn and the allegedreason is the need for revisions to the original protocolTriac in Syndromes of Reduced Sensitivity to THReduced sensitivity to TH is diagnosed when symptoms ofhypothyroidism occur despite normal or increased serum THThis finding may be the consequence of mutations in TRstransporters or metabolizing enzymes The expression˜˜resistance to TH™™ is usually reserved for syndromes causedby TR mutations Most cases are associated with TRb mutations Since TRb is involved in the inhibition of thyrotropin TSH secretion by T3 and thyroxine T4 circulatinglevels of T4 and T3 are usually high TSH is normal or highand goiter may be present The clinical picture is quitevariable and includes signs and symptoms of both hypothyroidism in TRbdependent ans and thyrotoxicosisin TRbdependent ans The most common findings aredelayed growth delayed bone maturation cognitive impairment and tachycardia Treating TH resistance is not easy Exogenous T4 or T3administration may improve some symptoms but it mayworsen others and symptomatic therapy is often prescribedfor example betablockers to reduce heart rate The idealtreatment would be represented by a T3 analogue able toactivate the mutated receptorIn some patients this can occur with Triac Fig Thelack of the amine group does not prevent Triac from activating TRs and it has similar affinity as T3 for the wildtypereceptor Apparently the different shape and charge of theTriac molecule allow several classes of mutated TRb to beactivated as well In these patients chronic Triac administration at dosages on the order of “ lg per kg of bodyweight daily represents the best therapeutic regimen Ingeneral Triacsensitive cases of TH resistance are caused bymutations in the carboxyterminal region of the T3 bindingFIG Chemical structure of some active thyroid hormone metabolites that have been used in patients or in animal models of human disease See text for further detailsdomain while mutations located close to the hinge region donot respond to Triac No positive response to Triac has beenreported in TRa mutations so farA different cause of reduced sensitivity to TH is represented by mutations in MCT8 a T3T4 transporter that isthe major pathway mediating T4 and T3 uptake in the centralnervous system The clinical syndrome associated withMCT8 mutations is known as the Allan“Herndon“Dudleysyndrome The MCT8 gene is located on the X chromosome and therefore virtually all patients are male Diagnosis is suspected in the presence of high T3 with low tonormal T4 and low to normal TSH associated with congenital brain hypothyroidism The phenotype is variable depending on the location and type of the mutationSymptoms usually include cognitive impairment associated with congenital hypotonia and weakness which mayprogress to spasticity Paroxysmal dyskinesias and seizuresmay also occur Peripheral hyperthyroidism often causestachycardia muscle wasting and progressive body weightreduction “Since it has been observed that cellular Triac uptake doesnot depend on MCT8 a clinical trial has been undertakenwith this endogenous TH analogue in patients with amedian age of years The results obtained after monthsof treatment at the dosage of “ lg daily have recently been published The treatment was highly effective in reducing serum T3 and Triac dosage was actuallytitrated on T3 reduction Signs of peripheral hypothyroidismnotably tachycardia and body weight reduction were significantly attenuated On the contrary the effects on theneurological symptoms were limited and improvement inthe indices of motor function was limited to patients youngerthan years It seems therefore that once the neurologicalphenotype is fully developed it is hardly reversibleOn this basis another phase trialis underwayNCT02396459 in which Triac treatment will be started asearly as possible in postnatal lifeSynthetic TH analogues might also be useful Notably in asmall clinical study diiodothyropropionic acid DITPA administration “ mgkg per day was able to normalizeT3 in four children affected by MCT8 deficiency aged “months Heart rate decreased in three patients and weightgain occurred in two Although DITPA was introduced over years ago as a relatively weak and poorly selective TRagonist its mode of interaction with TRs has not been specifically investigated so farPotential Uses of T2 and T1AMWhile Triac is basically a thyromimetic other active THmetabolites Fig can interact with different moleculartargets T2 has direct mitochondrial actions allegedly onrespiratory chain complex IV subunit Va and it stimulatesmitochondrial respiration and fatty acid oxidation These metabolic responses are further supported by genomiceffects whose molecular basis is unclear since they appear tobe different from those elicited by T3 In any case in ratstreated with highfat diet exogenous T2 decreased serumtriglycerides and LDL cholesterol as well as liver fat andbiochemicalindices of liver injury suggesting potentialtherapeutic value for either dyslipidemia or NAFLD However it is still controversial whether these positive 0cTHYROID HORMONE ANALOGUESeffects may be achieved without inducing tachycardia orcardiac hypertrophy since cardiac thyrotoxicosis has beenreported in mice A single pilot investigation was performed in two humanvolunteers taking T2 lgkg for weeks A slightbut significant decrease in body weight was reported without any change in serum T3 T4 TSH and cardiac function More recently a synthetic T2 analogue TRC150094was used in patients with metabolic syndrome but theresults were rather disappointing since no significant effect on insulin sensitivity and plasma lipid profile was observed The last entry into the group of active TH metabolites isrepresented by T1AM It does not interact with canonical ornoncanonical TH targets and it was discovered as a highaffinity agonist of TAAR1 a membrane G proteincoupledreceptor GPCR that is expressed in the brain and manyother tissues T1AM is a biogenic amine and sharesseveral properties of this class of compounds includingthe ability to interact with multiple targets namely otherGPCRs eg a2A adrenergic receptor membrane ionicchannels eg TRPM8 and monoamine transporters In experimental animals the administration of exogenousT1AM induced many different functional effects Neurological and metabolic effects are particularly interesting sincethey appear to be elicited at relative low doses and mighthave physiological relevance The former includesmodulation of feeding behavior and sleepwake cycle reduction of pain threshold prolearning and antiamnestic responses The major metabolic effects consistin thestimulation of triglyceride and fatty acid catabolism but antiinsulin effects on glucose metabolism are also elicited atslightly higher dosesAlthough the administration of exogenous T1AM has notbeen tested in humans some experimental results obtainedin murine models of disease have raised interest aboutpotential therapeutic applications T1AM reduced serumcholesterol in spontaneously obese mice as well asliver triglycerides in a mouse model of polycystic ovarysyndrome Neuroprotective effects appear even more promising Intracerebral T1AM rescued longterm potentiation and behavioral evidence of cognitive dysfunction in an in vivomodel of bamyloid toxicity namely transgenic mice overexpressing humanmutated amyloid precursor protein In addition the intracerebral injection of T1AM metabolite iodothyroacetic acid protected from the convulsive effect ofpentylenetetrazole and from kainate toxicity while exogenous T1AM reduced apoptosis and functional injury in amouse model of spinal cord clamp Because of the multitude of T1AM effects an active research line is focused on the development of synthetic derivatives with more favorable biodistribution andor receptorselectivity In conclusion the development TH analogues was initially prompted by the attempt to exploit the effects of THon lipid metabolism while avoiding unwanted cardiac effects TRb agonists have provided good results in a fewsmall clinical trials performed in hypercholesterolemic patients but these projects have been terminated after the report of potential side effects In recent years TRb agonistshave raised new interest for the treatment of NAFLD and acouple of clinical trials have provided encouraging initialresults Triac has already found clinical use in the treatmentof selected cases of TH resistance due to TRb mutationsand interesting results have recently been reported in theAllan“Herndon“Dudley syndromeOther TH analogues are under consideration for neurological diseases although human results are not yetavailable In particular sobetirome derivatives have beensuccessfulin animal models of multiple sclerosis andT1AM has been beneficial in an animal model of bamyloidtoxicityOverall research on TH analogues is experiencing a shiftin its focus but it still appears to be an active and promisingfieldAuthor Disclosure StatementNo competing financial interests existFunding InformationThis work was supported by a grant from Pisa UniversityPRA to RZReferences OrtigaCarvalho TM Sidhaye AR Wondisford FE Thyroid hormone receptors and resistance to thyroid hormone disorders Nat Rev Endocrinol “ Flamant F Cheng SY Hollenberg AN Moeller LC Samarut J Wondisford FE Yen PM Refetoff S Thyroidhormone signaling pathways time for a more precise nomenclature Endocrinology “ Davis PJ Goglia F Leonard JL Nongenomic actionsof thyrid hormone Nat Rev Endocrinol “ Senese R de Lange P Petito G Moreno M Goglia FLanni A 35diiodothyronine a novel thyroid hormone metabolite and a potent modulator of energy metabolism Front Endocrinol Lausanne Groeneweg S Peeters RP Visser TJ Visser WE Triiodothyroacetic acid in health and disease J Endocrinol234R99“R121 Hoefig CS Zucchi R Ko¨ hrle J Thyronaminesand derivatives physiological relevance pharmacological actions and future research directions Thyroid “ Ko¨hrle J Biebermann H 3iodohyronamine”a thyroid hormone metabolite with distinct target profiles andmodes of action Endocrine Rev “ Zucchi R Rutigliano G Saponaro F Novel thyroidhormones Endocrine “ Joharapurak AA Dhote VV Jain MR Selectivethyromimetics using receptor and tissue selectivity approaches prospects for dyslipidemia J Med Chem “ Mondal S Mugesh G Novel thyroid hormone analogues enzyme inhibitors and mimetics and their actionMol Cell Endocrinol “ Tancevski I Rudling M Eller P Thyromimetics ajourney from bench to bedside Pharmacol Ther “ Lin VW Klepp HM Hanley RM Sobetirome is aTRb and liverselective thyromimetic that can affectsubstantial LDLC lowering without significant changes inheart rate or the thyroid axis in euthyroid men [abstract] 0cZUCCHISan FranciscoENDO OR3633the Endocrine Society Annual Meeting Berkenstam A Kristensen J Mellstro¨m K Carlsson BMalmJ Rehnmark S Garg N Andersson CM Rudling MSjo¨berg F Angelin B Baxter JD The thyroid mimeticcompound KB2115 lowers plasma LDL cholesterol andstimulates bile acid synthesis without cardiac effects inhumans Proc Natl Acad Sci U S A “ Taub R Chiang E ChabotBlanchet M Kelly MJ ReevesRA Guertin MC Tardif JC Lipid lowering in healthyvolunteers treated with multiple doses of MGL3196 alivertargeted thyroid hormone receptorb agonist Atherosclerosis “ Ladenson PW Kristensen JD Ridgway EC Olsson AGCarlsson B Klein I Baxter JD Angelin B Use of thethyroid hormone analogue eprotirome in statintreateddyslipidemia N Engl J Med “ Sjouke B Langslet G Ceska R Nicholls SJ Nissen SEOhlander M Ladenson PW Olsson AG Hovingh GKKastelein JJ Eprotirome in patients with familial hypercholesterolaemia the AKKA trial a randomizeddoubleblind placebocontrolled phase study LancetDiabetes Endocrinol “ European Association for the Study of the Liver EASLEuropean Association for the Study of Diabetes EASDEuropean Association for the Study of Obesity EASO ESALEASDEASO clinical practice guidelines forthe management of nonalcoholic fatty liver diseaseJ Hepatol “ Coppola M Glinni D Moreno M Cioffi F Silvestri EGoglia F Thyroid hormone analogues and derivativesactions in fatty liver World J Hepatol “ Sinha RA Singh BK Yen PM Direct effects of thyroid hormones on hepatic lipid metabolism Nat Rev Endocrinol “ Martagon AJ Lin JZ Cimini SL Webb P Phillips KJ The amelioration of hepatic steatosis by thyroid hormonereceptor agonists is insufficient to restore insulin sensitivityin obob mice PLoS One 10e0122987 Puliga E Min Q Tao J Zhang R PradhanSundd TPoddar M Singh S Columbano A Yu J Momga SP Thyroid hormone receptorb agonist GC1 inhibits metbcatenindriven hepatocellular cancer Am J Pathol “ Harrison SA Bashir MR Guy CD Zhou L Moylan CAFrias JP Alkhouri N Bansal MB Baum S NeuschwanderTetri BA Taub R Moussa SE Resmetirom MGL for the treatment of nonalcoholic steatohepatitis amulticenter randomized doubleblind placebocontrolledphase trial Lancet “ Loomba R Neutel J Bernard D Severance R Mohseni RDao M Saini S Margaritescu C Homer K Tran B Mancini M Masamune H Lian B VK2809 a novel liverdirected thyroid receptor beta agonist significantly reducesliver fat in patients with nonalcoholic fatty liver disease aphase randomized placebocontrolled trial [abstract]Hepatology 681447A Zhang M Ma Z Qin H Yao Z Thyroid hormonepotentially benefits multiple sclerosis via facilitating remyelination Mol Neurobiol “ Placzek AT Ferrara SJ Hartley MD SanfordCrane HSMeining M Scanlan TS Sobetirome prodrug esterswith enhanced bloodbrain barrier permeability BioMed Chem “ Hartley MD Banerji T Tagge IJ Kirkemo LL ChaudharyP Calkins E Galipeau D Shokat MD DeBell MJ VanLeuven S Miller H Myelin repair stimulated by CNSselective thyroid hormone action JCI Insights 4e126329 Kemp S Huffnagel IC Linthorst GE Wanders RJ EngelenM Adrenoleukodystrophy”neuroendocrine pathogenesis and redefinition of natural history Nat Rev Endocrinol “ Hartley MD Kirkemo LL Banerji T Scanlan TS Athyroid hormonebased strategy for correcting the biochemical abnormality in Xlinked adrenoleukodystrophyEndocrinology “ Refetoff S Dumitrescu AM Syndromes of reducedsensitivity to thyroid hormone genetic defects in hormonereceptors cell transporters and deiodination Best PractClin Endocrinol Metabolism “ Groeneweg S Peeters RP Visser TJ Visser WE Therapeutic applications of thyroid hormone analogues inresistance to thyroid hormone RTH syndromes Mol CellEndocrinol “ Friesema ECH Visser WE Visser TJ Genetics andphenomics of thyroid hormone transport by MCT8 MolCell Endocrinol “ Schwartz CE Stevenson RE The MCT8 thyroidhormone transporter and AllanHerndonDudley Syndrome Best Pract Clin Endocrinol Metab “ Groeneweg S Peeters RP Moran C Stoupa A Aurial FTonduti D Dica A Paone L Rozenkova K Malikova Jvan der Walt A de Coo IFM McGowan A Lyons GAarsen FK Barca D van Beynum IM van der Knoop MMJansen J Manshande M Lunsing RJ Nowak S den UilCA Zillikens MC Visser FE Vrijmoeth P de Wit MCYWolf NI Zandstra A Ambegaonkar G Singh Y de RijkeYB Medici M Bertini ES Depoorter S Lebl J Cappa MDe Meirleir L Krude H Craiu D Zibordi F Oliver Petit IPolak M Chatterjee K Visser TJ Visser WE Effectiveness and safety of the triiodothyronine analogue Triacin children and adults with MCT8 deficiency an international single arm label phase trial Lancet Diabetes Endocrinol “ Verge CF Konrad D Cohen M Di Cosmo C DumitrescuAM Marcinkowski T Hameed S Hamilton J Weiss RERefetoff S Diiodothyropionic acid DITPA in thetreatment of MCT8 deficiency J Clin Endocrinol Metab“ Goglia F The effects of 35diiodothyronine on energybalance Front Physiol Jonas W Lietzow J Wohlgemuth F Hoefig CS WiedmerP Schweizer U Ko¨hrle J Schurmann A 35DiiodoLthyronine 35T2 exertsthyromimetic effects onhypothalamuspituitarythyroid axis body compositionand energy metabolism in male dietinduced obese miceEndocrinology “ Antonelli A Fallahi P Ferrari SM Di Domenicantonio AMoreno M Lanni A Goglia F 35diiodoLthyronineincreases resting metabolic rate and reduces body weightwithout undesirable side effects J Biol Regul HomeostAgents “ Van der Val F Hassing C Visser M Thakkar P MohananA Pathak K Dutt C Chauthaiwale V Ackermans MNederveen A Serlie M Nieuwdorp M Stroes E Theeffect of a diodothyronine mimetic on insulin sensitivity inmale cardiometabolic patients a doubleblind randomizedcontrolled trial PLoS One 9e86890 0cTHYROID HORMONE ANALOGUES Zucchi R Chiellini S Scanlan TS Grandy DK Traceamineassociated receptors and their ligands Br J Pharmacol “ Rutigliano G Accorroni A Zucchi R The case forTAAR1 as a modulator of central nervous system functionFront Pharmacol Zucchi R Accorroni A Chiellini G Update on iodothyronamine and its neurological and metabolic actions Front Physiol AssadiPorter FM Reiland H Sabatini M Lorenzini LCarnicelli V Rogowski M Selen Alpergin ES Tonelli MGhelardoni S Saba A Zucchi R Chiellini G Metabolic reprogramming by 3iodothyronamine T1AM anew perspective to reverse obesity through regulation ofsirtuin and expression Int J Mol Sci 19e1535 Selen Alpergin ES Bolandnazar Z Sabatini M RogowskiM Chiellini G Zucchi R AssadiPorter FM Metabolic profiling reveals reprogramming of lipid metabolicpathways in treatment of polycystic ovary syndrome with3iodothyronamine Physiol Rep 5e13097 Accorroni A Rutigliano G Sabatini M Frascarelli S BorsoM Novelli E Bandini L Ghelardoni L Saba A Zucchi ROriglia N Exogenous 3iodothyronamine rescues theentorhinal cortex from bamyloid toxicity Thyroid “ Laurino A Landucci E Resta F De Siena F PellegriniGiampietro DE Masi A Mannaioni G Raimondi L Anticonvulsant and neuroprotective effects of the thyroidhormone metabolite 3Iodothyroacetic Acid Thyroid “ Lv J Liao J Tan W Yang L Shi X Zhang H ChenL Wang S Li Q 3Iodothyronamine acting throughan antiapoptotic mechanism is neuroprotective againstspinal cord injury in rats Ann Clin Lab Sci “ Answer:

Thyroid_Cancer

"conceptualization data curation investigation project administration supervision validation visualization writing review and editing Goneim I and Ibraheim A performed data curation Kamal NM was involved in literature review writingoriginal draft writing review and editing Alsofiani F and Alawur A performed literature review and writingoriginal draft All authors had read and approved the final manuscriptInformed consent statement Written informed consent in the patient™s native language was obtained from her fatherConflictofinterest statement The authors declare that they have no conflict of interestCARE Checklist statement The authors have read the CARE Checklist and the manuscript was prepared and revised according to the CARE Laila M Sherief Department of Pediatric Hematology and Oncology Faculty of Medicine Zagazig University Zagazig EgyptLaila M Sherief Amr Ibraheim Department of Pediatrics Faculty of Medicine Zagazig University Zagazig EgyptEsmael Goneim Department of Pediatric Oncology Tanta Cancer Institute Tanta EgyptNaglaa M Kamal Department of Pediatrics and Pediatric Hepatology Faculty of Medicine Cairo University Cairo EgyptNaglaa M Kamal Fuad A alsofiani Abdulraouf H Alawur Department of Pediatrics Alhada Armed Forces Hospital Taif Saudi ArabiaCorresponding author Naglaa M Kamal MD Full Professor Department of Pediatrics and Pediatric Hepatology Faculty of Medicine Cairo University Kasralainy Cairo Egypt naglakamalkasralainyeduegAbstractBACKGROUND thalassemia intermedia TI is one of the hemoglobinopathies It constitutes of thalassemia cases yet being associated with a better quality of life than thalassemia major TMCASE SUMMARY We recently reported the first case of acute lymphoblastic leukemia ALL from Egypt in a child with TM and we herein report the first case of ALL from Egypt in a child with TI In this report literature was reviewed for cases of malignancies associated with TI and the possible factors underling the relationship between the two entities We stress that physicians should have a high index of suspicion of malignancies in thalassemia patients if they present with any suggestive symptoms or signsKey words Acute lymphoblastic leukemia Thalassemia intermedia Children Malignancies Iron overload Hydroxyurea Case reportThe Authors Published by Baishideng Publishing Group Inc All rights reservedWJCPwwwwjgnetcomAugust Volume Issue 0cSherief LM ALL in a child with TIChecklist Access This is an access that was selected by an inhouse editor and fully peerreviewed by external reviewers It is distributed in accordance with the Creative Commons Attribution NonCommercial CC BYNC license which permits others to distribute remix adapt build upon this work noncommercially and license their derivative works on different terms provided the original work is properly cited and the use is noncommercial See httpcreativecommonslicensesbync40Manuscript source Unsolicited manuscriptReceived January Peerreview started January First decision April Revised May Accepted June in press June Published online August PReviewer Fujioka K Moschovi MA SEditor Dou Y LEditor A EEditor Li JHCore tip Cases have been reported for malignancies in patients of thalassemia major However rare case reports have been reported for malignancies in patients of thalassemiaintermedia as it is a nontransfusion dependent anemia Physicians should have high index of suspicion to diagnose malignancies in patients with thalassemiaintermediaCitation Sherief LM Goneim E Kamal NM Ibraheim A Alsofiani F Alawur A Acute lymphoblastic leukemia in a thalassemia intermedia child A case report World J Clin Pediatr URL wwwwjgnetcom22192808fullv9i11htm dx105409wjcpv9i11INTRODUCTIONThalassemia represents the most common singlegene disorder worldwide The total annual incidence of symptomatic individuals with thalassemia is estimated at in throughout the world of whom nearly have thalassemia intermedia TI which is intermediate in severity between the milder thalassemiaminor and the more severe transfusiondependent thalassemiamajor TM[]We herein report the first case from Egypt with TI who developed acute lymphoblastic leukemia ALLCASE PRESENTATIONChief complaintsThe reported patient is a 15yearold girl with TI who presented at the age of years with pallor decreased growth rate and decreased activity She had severe microcytic hypochromic anemia with hemoglobin Hb of gdLHistory of present illnessPediatric hematologist workup proved the diagnosis of TI Her Hb electrophoresis showed HbA HbF and HbA2 Genetic molecular testing revealed compound heterozygosity for cd27 GT and cd39 CT mutations Hydroxyurea at a dose of mgkg per day was started in addition to folic acidShe was then followed at the pediatric hematology unit at regular intervals to monitor her tolerance to drug therapy with special attention to hematological toxicity There were no significant side effects during seven years of therapy and the patient showed good response with occasional need for blood transfusions She underwent splenectomy during her late teensHistory of past illnessAt the age of years she developed generalized bone aches abdominal pain persistent fever and dyspnea and so she was referred to our hospitalPhysical examinationOn physical examination there was severe pallor tachypnea tachycardia and hepatomegalyLaboratory examinationsInitial complete blood picture showed a Hb level gdL white blood cell count of — 109L and platelets count of — 109LSerum electrolytes cerebrospinal fluid analysis and kidney and liver function tests were normal expect for mild elevation of total serum bilirubin which was mgdLSerum ferritin was ngdL Serological studies including EpsteinBarr virus cytomegalovirus human immunodeficiency virus hepatitis C virus and hepatitis B virus were negative Lactate dehydrogenase was UL and serum uric acid was mgdLWJCPwwwwjgnetcomAugust Volume Issue 0cSherief LM ALL in a child with TIImaging examinationsHer chest Xray was normal Abdominal ultrasonography revealed hepatomegaly with calcular cholecystitis and bilateral diffuse renal enlargement Echocardiography showed mitral valve prolapse with trivial mitral regurgitationMULTIDISCIPLINARY EXPERT CONSULTATIONThe pediatric haematologistoncologist assessment requested bone marrow biopsy which was carried by the hematopathologistBone marrow examination revealed blast cells in a hypercellular marrow with depressed erythropoiesis and granulopoiesies and normal thrombopoiesis Immunophenotyping showed lymphoblasts that are CD10 positive CD19 positive CD34 positive TDT positive HLADR positive CD13 positive and CD33 positiveCytogenetic examination showed a normal karyotype with a DNA index of and negative t1221 t119 BCRABL or 11q23 translocationsmutationsFINAL DIAGNOSISA final diagnosis of Bacutelymphoblasticleukemia ALL with aberrant expression of CD13 and CD33 was achievedTREATMENTInduction chemotherapy of the total XV protocol with prednisone vincristine Lasparaginase doxorubicin cyclophosamide cytarabine 6mercaptopurine and intrathecal chemotherapy was commencedShe received multiple packed red cell transfusions which eventually led to elevation of serum ferritin to ngdL Thus she was started on oral chelation therapy with deferasirox with no complicationsThe patient eventually went into complete remission She then received consolidation chemotherapy of standard risk of the total XV protocol with times of high dose methotrexate HDMTX 6mercaptopurine and intrathecal chemotherapyShe received multiple packed red blood cell transfusions and other supportive measures during the periods of induction and consolidation The transfusions therapy was given according to the guidelines of pediatric oncologists who usually transfuse if Hb level is less than gdL and if associated with pulmonary or cardiac comorbidities or exposed to invasive procedure and hemorrhage and they transfuse with Hb less than gdL The transfusions were not associated with any complications Deferasirox was stopped in consolidation phase during infusion of high dose methotrexateThe main problem observed during the periods of induction and consolidation therapy was increased requirement of blood transfusions as well as repeated infections as during this period the child received intensive chemotherapy which caused bone marrow suppressionOUTCOME AND FOLLOWUPThe child is still in complete remission while being now in the continuation phase for standard risk week fortyDISCUSSIONWe have recently reported the first case from Egypt with thalassemia major TM who developed ALL[] herein we report similarly the first report from Egypt for a patient with TI who developed ALL to highlight that the coexistence of malignancy and beta thalassemia is not rareA thorough look in literature for previously reported cases of malignancies in WJCPwwwwjgnetcomAugust Volume Issue 0cSherief LM ALL in a child with TIpatients with TI revealed only one report in from Turkey on a years old boy with TI who developed ALL[] Other previous reports on malignancies associated with TI described nonHodjkinlymphoma[] chronic myeloid leukemia[] Hodjkin lymphoma[] hepatocellular carcinoma[] and thyroid malignancies[] Table To our knowledge our patient is the second worldwide and the first from EgyptAlthough reported cases of malignancies associated with TI are few but it raises the attention of physicians to have high index of suspicion of malignancies in this group of patients when they present with unexplained new symptoms or proposed symptoms and signs of malignancySpecial concern about management plans in these patients as they usually require more frequent blood transfusions as the chemotherapy causes suppression of the bone marrow which adds to the base line chronic hemolysisIn spite that reported cases of malignancies in TI are scarce which makes our trial to find causal relationship between TI and cancer development beyond the scope of our report but we tried to search literature foe possible contributing factors Those factors can™t rise to the level of conclusions and definitely need to be proved and validated by larger prospective cohort with large control groups multicenter worldwide studies addressing all possible hypothesesIndeed the most practical logical thinking about that underlying factors for the development of malignancy in TI is being multifactorial[]In a large multicenter study on thalassemia patients from Iran the proportion of patients with cancer was higher in those with TI than those with TM and respectively[] They explained it by the fact that bone marrow in TM patients is suppressed by the regular transfusions while it is very active with high turnover in those with TI[] They suggested that this can lead to a higher rate of DNA repair faults and mutations with subsequent higher rate of hematological malignancies[]Another potential factor is the prolonged use of hydroxyurea Conflicting data are there regarding its carcinogenic potential Hydroxyurea as an antimetabolite interferes with both DNA synthesis and repair mechanisms with later accumulation of mutations and subsequent chromosomal damage Although no studies have yet investigated the relationship between hydroxyurea and the development of cancers in thalassemia but clinically concerns have been raised regarding its potential leukemogenic potential[] Other authors were against this assumption[] The BABYHUG clinical trial which compared hydroxyurea with placebo treated controls refuted this assumption and did not suggest any increased risk of genotoxicity[]Overall there is no evidence to suggest an increased risk of carcinogenesis in patients with thalassemia with hydroxyurea and further studies will need to be designed to establish any potential relationship[]One more probable factor is that patients with TI being having milder disease than those with TM with fewer blood transfusions might lead to delayed diagnosis and even if diagnosed usually there is underestimation of their iron overload problem and sometimes the deceiving relatively mildly elevated ferritin as compared to TM which has been shown to underestimate the true iron burden in TI patient with ultimate fate that these patients accumulate iron but it usually goes unnoticed unchelated and unmonitored Anemia hypoxia and ineffective erythropoiesis suppress the expression of hepcidin by increasing expression of growth differentiation factor and hypoxiainducible transcription factors with the resultant increased intestinal iron absorption and in turn adds to the problem of iron overload[]The longstanding iron overload with its deposition in different body ans with the wellknown association between excess iron and cancer development can be a predisposing factor for all types of malignancies through direct and indirect effects[]Iron can directly damage DNA by nontransferrinbound iron with the consequent inactivation of tumorsuppressor genes such as p53 or their products The indirect effects include the formation of reactive oxygen species ironinduced lipid peroxidation and altered immune system with decreased immune surveillance suppression of tumoricidal action of macrophages and alteration of cytokine activities TI patients usually survive longer than TM patients with enough time for iron overload to develop[]Some authors suggested that improved management protocols of thalassemia patients have led to increased survival with most of them reaching adult age with the consequent occurrence of diseases associated with long life span like malignancies[] This assumption can partially explain other reports in elder patients but it can™t work in our patient and the Turkish one who are teenagersMany authors suggested that the occurrence of malignancies in thalassemia patients could be a pure coincidence or a combination of genetic and environmental factors[]WJCPwwwwjgnetcomAugust Volume Issue 0cTable Previously reported cases of thalassemia intermedia who developed malignanciesSherief LM ALL in a child with TINumber of patientsType of malignancyAcute lymphoblastic leukemiaNonHodgkin lymphoma NHLNHL Hodgkin lymphoma HLNHL HL chronic myeloid leukemia CMLCMLHLHepatocellular carcinoma HCCHCCHCCHCCHCCThyroid cancerSome patients have thalassemia major and others have thalassemia intermedia in references and Ref[][][][][][][][][][][][]We can sum up to a clear message that whatever the pathogenesis of malignancies in thalassemias the most important message is to alarm physicians to have high index of suspicion for malignancies if their thalassemia patients develop suggestive symptoms and signs Worsening anemia leukocytosis fever boneache lymphadenopathy and splenomegaly are alarming to look for leukemias and other hematological malignanciesREFERENCES Galanello R Origa R Betathalassemia Orphanet J Rare Dis [PMID ]Sherief LM Kamal NM Abdelrahman HM Hassan BA Zakaria MM First report of acute lymphoblastic leukemia in an Egyptian child with thalassemia major Hemoglobin [PMID ]Tuğcu D Karakaş Z G¶k§e M Ağaoğlu L Un¼var A Sarıbeyoğlu E Ak§ay A Devecioğlu O Thalassemia Intermedia and Acute Lymphoblastic Leukemia Is it a Coincidental Double Diagnosis Turk J Haematol [PMID 104274tjh20140068]Chehal A Loutfi R Taher A Betathalassemia intermedia and nonHodgkin's lymphoma Hemoglobin [PMID 101081hem120015025]Benetatos L Alymara V Vassou A Bourantas KL Malignancies in betathalassemia patients a singlecenter experience and a concise review of the literature Int J Lab Hematol [PMID 101111j1751553X200700929x]Karimi M Giti R Haghpanah S Azarkeivan A Hoofar H Eslami M Malignancies in patients with betathalassemia major and betathalassemia intermedia a multicenter study in Iran Pediatr Blood Cancer [PMID 101002pbc22144]Alavi S Safari A Sadeghi E Amiri S Hematological malignancies complicating thalassemia syndromes a single center experience Blood Res [PMID 105045br2013482149]Jabr FI Aoun E Yassine H Azar C Taher A Betathalassemia intermedia and Hodgkin lymphoma Am J Hematol [PMID 101002ajh20478]BnaPignatti C Vergine G Lombardo T Cappellini MD Cianciulli P Maggio A Renda D Lai ME Mandas A Forni G Piga A Bisconte MG Hepatocellular carcinoma in the thalassaemia syndromes Br J Haematol [PMID 101046j13652141200304732x]Mancuso A Sciarrino E Renda MC Maggio A A prospective study of hepatocellular carcinoma incidence in thalassemia Hemoglobin [PMID ]Restivo Pantalone G Renda D Valenza F D'Amato F Vitrano A Cassar  F Rigano P Di Salvo V Giangreco A Bevacqua E Maggio A Hepatocellular carcinoma in patients with thalassaemia syndromes clinical characteristics and outcome in a long term single centre experience Br J Haematol [PMID 101111j13652141201008180x]Fragatou S Tsourveloudis I Manesis G Incidence of hepatocellular carcinoma in a thalassemia unit Hemoglobin [PMID ] WJCPwwwwjgnetcomAugust Volume Issue 0cSherief LM ALL in a child with TI Maakaron JE Cappellini MD Graziadei G Ayache JB Taher AT Hepatocellular carcinoma in hepatitisnegative patients with thalassemia intermedia a closer look at the role of siderosis Ann Hepatol [PMID ]Poggi M Sorrentino F Pascucci C Monti S Lauri C Bisogni V Toscano V Cianciulli P Malignancies in thalassemia patients first description of two cases of thyroid cancer and review of the literature Hemoglobin [PMID ]Halawi R Cappellini MD Taher A A higher prevalence of hematologic malignancies in patients with thalassemia Background and culprits Am J Hematol [PMID 101002ajh24682]McGann PT Flanagan JM Howard TA Dertinger SD He J Kulharya AS Thompson BW Ware RE BABY HUG Investigators Genotoxicity associated with hydroxyurea exposure in infants with sickle cell anemia results from the BABYHUG Phase III Clinical Trial Pediatr Blood Cancer [PMID 101002pbc23365] WJCPwwwwjgnetcomAugust Volume Issue 0cPublished by Baishideng Publishing Group Inc Koll Center Parkway Suite Pleasanton CA USA Telephone Email bpgofficewjgnetcom Help Desk wwwf6publishingcomhelpdesk wwwwjgnetcom Baishideng Publishing Group Inc All rights reserved\x0c"

cancer279

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: "conceptualization data curation investigation project administration supervision validation visualization writing review and editing Goneim I and Ibraheim A performed data curation Kamal NM was involved in literature review writingoriginal draft writing review and editing Alsofiani F and Alawur A performed literature review and writingoriginal draft All authors had read and approved the final manuscriptInformed consent statement Written informed consent in the patient™s native language was obtained from her fatherConflictofinterest statement The authors declare that they have no conflict of interestCARE Checklist statement The authors have read the CARE Checklist and the manuscript was prepared and revised according to the CARE Laila M Sherief Department of Pediatric Hematology and Oncology Faculty of Medicine Zagazig University Zagazig EgyptLaila M Sherief Amr Ibraheim Department of Pediatrics Faculty of Medicine Zagazig University Zagazig EgyptEsmael Goneim Department of Pediatric Oncology Tanta Cancer Institute Tanta EgyptNaglaa M Kamal Department of Pediatrics and Pediatric Hepatology Faculty of Medicine Cairo University Cairo EgyptNaglaa M Kamal Fuad A alsofiani Abdulraouf H Alawur Department of Pediatrics Alhada Armed Forces Hospital Taif Saudi ArabiaCorresponding author Naglaa M Kamal MD Full Professor Department of Pediatrics and Pediatric Hepatology Faculty of Medicine Cairo University Kasralainy Cairo Egypt naglakamalkasralainyeduegAbstractBACKGROUND thalassemia intermedia TI is one of the hemoglobinopathies It constitutes of thalassemia cases yet being associated with a better quality of life than thalassemia major TMCASE SUMMARY We recently reported the first case of acute lymphoblastic leukemia ALL from Egypt in a child with TM and we herein report the first case of ALL from Egypt in a child with TI In this report literature was reviewed for cases of malignancies associated with TI and the possible factors underling the relationship between the two entities We stress that physicians should have a high index of suspicion of malignancies in thalassemia patients if they present with any suggestive symptoms or signsKey words Acute lymphoblastic leukemia Thalassemia intermedia Children Malignancies Iron overload Hydroxyurea Case reportThe Authors Published by Baishideng Publishing Group Inc All rights reservedWJCPwwwwjgnetcomAugust Volume Issue 0cSherief LM ALL in a child with TIChecklist Access This is an access that was selected by an inhouse editor and fully peerreviewed by external reviewers It is distributed in accordance with the Creative Commons Attribution NonCommercial CC BYNC license which permits others to distribute remix adapt build upon this work noncommercially and license their derivative works on different terms provided the original work is properly cited and the use is noncommercial See httpcreativecommonslicensesbync40Manuscript source Unsolicited manuscriptReceived January Peerreview started January First decision April Revised May Accepted June in press June Published online August PReviewer Fujioka K Moschovi MA SEditor Dou Y LEditor A EEditor Li JHCore tip Cases have been reported for malignancies in patients of thalassemia major However rare case reports have been reported for malignancies in patients of thalassemiaintermedia as it is a nontransfusion dependent anemia Physicians should have high index of suspicion to diagnose malignancies in patients with thalassemiaintermediaCitation Sherief LM Goneim E Kamal NM Ibraheim A Alsofiani F Alawur A Acute lymphoblastic leukemia in a thalassemia intermedia child A case report World J Clin Pediatr URL wwwwjgnetcom22192808fullv9i11htm dx105409wjcpv9i11INTRODUCTIONThalassemia represents the most common singlegene disorder worldwide The total annual incidence of symptomatic individuals with thalassemia is estimated at in throughout the world of whom nearly have thalassemia intermedia TI which is intermediate in severity between the milder thalassemiaminor and the more severe transfusiondependent thalassemiamajor TM[]We herein report the first case from Egypt with TI who developed acute lymphoblastic leukemia ALLCASE PRESENTATIONChief complaintsThe reported patient is a 15yearold girl with TI who presented at the age of years with pallor decreased growth rate and decreased activity She had severe microcytic hypochromic anemia with hemoglobin Hb of gdLHistory of present illnessPediatric hematologist workup proved the diagnosis of TI Her Hb electrophoresis showed HbA HbF and HbA2 Genetic molecular testing revealed compound heterozygosity for cd27 GT and cd39 CT mutations Hydroxyurea at a dose of mgkg per day was started in addition to folic acidShe was then followed at the pediatric hematology unit at regular intervals to monitor her tolerance to drug therapy with special attention to hematological toxicity There were no significant side effects during seven years of therapy and the patient showed good response with occasional need for blood transfusions She underwent splenectomy during her late teensHistory of past illnessAt the age of years she developed generalized bone aches abdominal pain persistent fever and dyspnea and so she was referred to our hospitalPhysical examinationOn physical examination there was severe pallor tachypnea tachycardia and hepatomegalyLaboratory examinationsInitial complete blood picture showed a Hb level gdL white blood cell count of — 109L and platelets count of — 109LSerum electrolytes cerebrospinal fluid analysis and kidney and liver function tests were normal expect for mild elevation of total serum bilirubin which was mgdLSerum ferritin was ngdL Serological studies including EpsteinBarr virus cytomegalovirus human immunodeficiency virus hepatitis C virus and hepatitis B virus were negative Lactate dehydrogenase was UL and serum uric acid was mgdLWJCPwwwwjgnetcomAugust Volume Issue 0cSherief LM ALL in a child with TIImaging examinationsHer chest Xray was normal Abdominal ultrasonography revealed hepatomegaly with calcular cholecystitis and bilateral diffuse renal enlargement Echocardiography showed mitral valve prolapse with trivial mitral regurgitationMULTIDISCIPLINARY EXPERT CONSULTATIONThe pediatric haematologistoncologist assessment requested bone marrow biopsy which was carried by the hematopathologistBone marrow examination revealed blast cells in a hypercellular marrow with depressed erythropoiesis and granulopoiesies and normal thrombopoiesis Immunophenotyping showed lymphoblasts that are CD10 positive CD19 positive CD34 positive TDT positive HLADR positive CD13 positive and CD33 positiveCytogenetic examination showed a normal karyotype with a DNA index of and negative t1221 t119 BCRABL or 11q23 translocationsmutationsFINAL DIAGNOSISA final diagnosis of Bacutelymphoblasticleukemia ALL with aberrant expression of CD13 and CD33 was achievedTREATMENTInduction chemotherapy of the total XV protocol with prednisone vincristine Lasparaginase doxorubicin cyclophosamide cytarabine 6mercaptopurine and intrathecal chemotherapy was commencedShe received multiple packed red cell transfusions which eventually led to elevation of serum ferritin to ngdL Thus she was started on oral chelation therapy with deferasirox with no complicationsThe patient eventually went into complete remission She then received consolidation chemotherapy of standard risk of the total XV protocol with times of high dose methotrexate HDMTX 6mercaptopurine and intrathecal chemotherapyShe received multiple packed red blood cell transfusions and other supportive measures during the periods of induction and consolidation The transfusions therapy was given according to the guidelines of pediatric oncologists who usually transfuse if Hb level is less than gdL and if associated with pulmonary or cardiac comorbidities or exposed to invasive procedure and hemorrhage and they transfuse with Hb less than gdL The transfusions were not associated with any complications Deferasirox was stopped in consolidation phase during infusion of high dose methotrexateThe main problem observed during the periods of induction and consolidation therapy was increased requirement of blood transfusions as well as repeated infections as during this period the child received intensive chemotherapy which caused bone marrow suppressionOUTCOME AND FOLLOWUPThe child is still in complete remission while being now in the continuation phase for standard risk week fortyDISCUSSIONWe have recently reported the first case from Egypt with thalassemia major TM who developed ALL[] herein we report similarly the first report from Egypt for a patient with TI who developed ALL to highlight that the coexistence of malignancy and beta thalassemia is not rareA thorough look in literature for previously reported cases of malignancies in WJCPwwwwjgnetcomAugust Volume Issue 0cSherief LM ALL in a child with TIpatients with TI revealed only one report in from Turkey on a years old boy with TI who developed ALL[] Other previous reports on malignancies associated with TI described nonHodjkinlymphoma[] chronic myeloid leukemia[] Hodjkin lymphoma[] hepatocellular carcinoma[] and thyroid malignancies[] Table To our knowledge our patient is the second worldwide and the first from EgyptAlthough reported cases of malignancies associated with TI are few but it raises the attention of physicians to have high index of suspicion of malignancies in this group of patients when they present with unexplained new symptoms or proposed symptoms and signs of malignancySpecial concern about management plans in these patients as they usually require more frequent blood transfusions as the chemotherapy causes suppression of the bone marrow which adds to the base line chronic hemolysisIn spite that reported cases of malignancies in TI are scarce which makes our trial to find causal relationship between TI and cancer development beyond the scope of our report but we tried to search literature foe possible contributing factors Those factors can™t rise to the level of conclusions and definitely need to be proved and validated by larger prospective cohort with large control groups multicenter worldwide studies addressing all possible hypothesesIndeed the most practical logical thinking about that underlying factors for the development of malignancy in TI is being multifactorial[]In a large multicenter study on thalassemia patients from Iran the proportion of patients with cancer was higher in those with TI than those with TM and respectively[] They explained it by the fact that bone marrow in TM patients is suppressed by the regular transfusions while it is very active with high turnover in those with TI[] They suggested that this can lead to a higher rate of DNA repair faults and mutations with subsequent higher rate of hematological malignancies[]Another potential factor is the prolonged use of hydroxyurea Conflicting data are there regarding its carcinogenic potential Hydroxyurea as an antimetabolite interferes with both DNA synthesis and repair mechanisms with later accumulation of mutations and subsequent chromosomal damage Although no studies have yet investigated the relationship between hydroxyurea and the development of cancers in thalassemia but clinically concerns have been raised regarding its potential leukemogenic potential[] Other authors were against this assumption[] The BABYHUG clinical trial which compared hydroxyurea with placebo treated controls refuted this assumption and did not suggest any increased risk of genotoxicity[]Overall there is no evidence to suggest an increased risk of carcinogenesis in patients with thalassemia with hydroxyurea and further studies will need to be designed to establish any potential relationship[]One more probable factor is that patients with TI being having milder disease than those with TM with fewer blood transfusions might lead to delayed diagnosis and even if diagnosed usually there is underestimation of their iron overload problem and sometimes the deceiving relatively mildly elevated ferritin as compared to TM which has been shown to underestimate the true iron burden in TI patient with ultimate fate that these patients accumulate iron but it usually goes unnoticed unchelated and unmonitored Anemia hypoxia and ineffective erythropoiesis suppress the expression of hepcidin by increasing expression of growth differentiation factor and hypoxiainducible transcription factors with the resultant increased intestinal iron absorption and in turn adds to the problem of iron overload[]The longstanding iron overload with its deposition in different body ans with the wellknown association between excess iron and cancer development can be a predisposing factor for all types of malignancies through direct and indirect effects[]Iron can directly damage DNA by nontransferrinbound iron with the consequent inactivation of tumorsuppressor genes such as p53 or their products The indirect effects include the formation of reactive oxygen species ironinduced lipid peroxidation and altered immune system with decreased immune surveillance suppression of tumoricidal action of macrophages and alteration of cytokine activities TI patients usually survive longer than TM patients with enough time for iron overload to develop[]Some authors suggested that improved management protocols of thalassemia patients have led to increased survival with most of them reaching adult age with the consequent occurrence of diseases associated with long life span like malignancies[] This assumption can partially explain other reports in elder patients but it can™t work in our patient and the Turkish one who are teenagersMany authors suggested that the occurrence of malignancies in thalassemia patients could be a pure coincidence or a combination of genetic and environmental factors[]WJCPwwwwjgnetcomAugust Volume Issue 0cTable Previously reported cases of thalassemia intermedia who developed malignanciesSherief LM ALL in a child with TINumber of patientsType of malignancyAcute lymphoblastic leukemiaNonHodgkin lymphoma NHLNHL Hodgkin lymphoma HLNHL HL chronic myeloid leukemia CMLCMLHLHepatocellular carcinoma HCCHCCHCCHCCHCCThyroid cancerSome patients have thalassemia major and others have thalassemia intermedia in references and Ref[][][][][][][][][][][][]We can sum up to a clear message that whatever the pathogenesis of malignancies in thalassemias the most important message is to alarm physicians to have high index of suspicion for malignancies if their thalassemia patients develop suggestive symptoms and signs Worsening anemia leukocytosis fever boneache lymphadenopathy and splenomegaly are alarming to look for leukemias and other hematological malignanciesREFERENCES Galanello R Origa R Betathalassemia Orphanet J Rare Dis [PMID ]Sherief LM Kamal NM Abdelrahman HM Hassan BA Zakaria MM First report of acute lymphoblastic leukemia in an Egyptian child with thalassemia major Hemoglobin [PMID ]Tuğcu D Karakaş Z G¶k§e M Ağaoğlu L Un¼var A Sarıbeyoğlu E Ak§ay A Devecioğlu O Thalassemia Intermedia and Acute Lymphoblastic Leukemia Is it a Coincidental Double Diagnosis Turk J Haematol [PMID 104274tjh20140068]Chehal A Loutfi R Taher A Betathalassemia intermedia and nonHodgkin's lymphoma Hemoglobin [PMID 101081hem120015025]Benetatos L Alymara V Vassou A Bourantas KL Malignancies in betathalassemia patients a singlecenter experience and a concise review of the literature Int J Lab Hematol [PMID 101111j1751553X200700929x]Karimi M Giti R Haghpanah S Azarkeivan A Hoofar H Eslami M Malignancies in patients with betathalassemia major and betathalassemia intermedia a multicenter study in Iran Pediatr Blood Cancer [PMID 101002pbc22144]Alavi S Safari A Sadeghi E Amiri S Hematological malignancies complicating thalassemia syndromes a single center experience Blood Res [PMID 105045br2013482149]Jabr FI Aoun E Yassine H Azar C Taher A Betathalassemia intermedia and Hodgkin lymphoma Am J Hematol [PMID 101002ajh20478]BnaPignatti C Vergine G Lombardo T Cappellini MD Cianciulli P Maggio A Renda D Lai ME Mandas A Forni G Piga A Bisconte MG Hepatocellular carcinoma in the thalassaemia syndromes Br J Haematol [PMID 101046j13652141200304732x]Mancuso A Sciarrino E Renda MC Maggio A A prospective study of hepatocellular carcinoma incidence in thalassemia Hemoglobin [PMID ]Restivo Pantalone G Renda D Valenza F D'Amato F Vitrano A Cassar  F Rigano P Di Salvo V Giangreco A Bevacqua E Maggio A Hepatocellular carcinoma in patients with thalassaemia syndromes clinical characteristics and outcome in a long term single centre experience Br J Haematol [PMID 101111j13652141201008180x]Fragatou S Tsourveloudis I Manesis G Incidence of hepatocellular carcinoma in a thalassemia unit Hemoglobin [PMID ] WJCPwwwwjgnetcomAugust Volume Issue 0cSherief LM ALL in a child with TI Maakaron JE Cappellini MD Graziadei G Ayache JB Taher AT Hepatocellular carcinoma in hepatitisnegative patients with thalassemia intermedia a closer look at the role of siderosis Ann Hepatol [PMID ]Poggi M Sorrentino F Pascucci C Monti S Lauri C Bisogni V Toscano V Cianciulli P Malignancies in thalassemia patients first description of two cases of thyroid cancer and review of the literature Hemoglobin [PMID ]Halawi R Cappellini MD Taher A A higher prevalence of hematologic malignancies in patients with thalassemia Background and culprits Am J Hematol [PMID 101002ajh24682]McGann PT Flanagan JM Howard TA Dertinger SD He J Kulharya AS Thompson BW Ware RE BABY HUG Investigators Genotoxicity associated with hydroxyurea exposure in infants with sickle cell anemia results from the BABYHUG Phase III Clinical Trial Pediatr Blood Cancer [PMID 101002pbc23365] WJCPwwwwjgnetcomAugust Volume Issue 0cPublished by Baishideng Publishing Group Inc Koll Center Parkway Suite Pleasanton CA USA Telephone Email bpgofficewjgnetcom Help Desk wwwf6publishingcomhelpdesk wwwwjgnetcom Baishideng Publishing Group Inc All rights reserved\x0c" Answer:

Thyroid_Cancer

"clinicopathological characteristics with risk factors of breast cancer patients in NigeriaMethods Newly diagnosed female patients with breast cancer were assessed over months Patients were reviewed using a predesigned proforma which focused on sociodemographic information clinical information risk factors and tumor biologyResults A total of women were identified their mean age was years More than half are premenopausal at presentation with Eastern Cooperative Oncology Group ECOG score of and right side as the most common primary site of disease Less than half of them are estrogen receptor ER positive are progesterone receptor PR positive and are hormone receptor positive and triple negative respectively Most patients presented at the latter stage of the disease stage III and stage IV Only are well differentiated and almost all had invasive ductal histological type Obesity and physical inactivity are the most common risk factors for the disease A significant relationship was found between immunohistochemistry status and family history of breast cancer tumor site previManuscript submitted June accepted July Published online August aOncology and Radiotherapy Department Lagos University Teaching Hospital Lagos NigeriabMolecular and Anatomical Pathology Department College of Medicine University of Lagos Lagos NigeriacRadiotherapy Radiobiology Radiodiagnosis and Radiography Department College of Medicine University of Lagos Lagos NigeriadWest Cancer Centre and Research Institute Memphis TN USAeArrive Alive Diagnostics and Imaging Services Ltd Lagos NigeriafCorresponding Author Adeoluwa Akeem Adeniji Oncology and Radiotherapy Department Lagos University Teaching Hospital Lagos Nigeria Email godscrownbestyahoocom 1014740wjon1303ous breast surgery previous lump and alcohol intakeConclusion Findings from this study showed that Nigerian breast cancer patients differ from their counterparts in the high human development index HHDI countries in terms of the patients and disease characteristics In view of this prevention and treatment options should consider this uniqueness to ensure better outcomeKeywords Breast cancer Subtypes Tumor biology Risk factors Correlation NigeriaIntroductionCancer is a major public health concern globally [] According to GLOBACAN cancer is the single most important factor impacting life expectancy worldwide [] In women worldwide breast cancer is the most common malignancy [] Every year about million new cases are diagnosed worldwide and this represents of the female population [] In alone there were million new cases of breast cancer worldwide and over deaths and this represents new cases of cancer and of all cancerrelated deaths []One of the indicators that reflect the development of each country the status and their living conditions is the human development index HDI The HDI is defined as the average achievement of three factors including life expectancy at birth gross national income per capita and mean and expected years of schooling Low HDI level includes countries that are the least developed and the very high HDI level includes the most developed countries [] Although low human development index countries LHDI like Nigeria have a lower incidence of breast cancer when compared to high human development index HHDI countries like the United States of America USA mortality rates are higher [] The incidence rate in the LHDI countries is rising likely because of westernization and its lifestyle choices []The high mortality rate is seen because of late stage presentation misdiagnosis and poor health seeking behavior s The authors Journal compilation World J Oncol and Elmer Press Inc„¢ wwwwjonThis is distributed under the terms of the Creative Commons Attribution NonCommercial International License which permits unrestricted noncommercial use distribution and reproduction in any medium provided the original work is properly cited 0cBreast Cancer Among Nigerian WomenWorld J Oncol among other factors of the African population in general The screening rates is still low ranging from to for reasons ranging from cost quality assurance and fear of radiation [] Studies have also shown that the genetic and histopathologic subtypes in African women are likely to be more aggressive than those seen in their Caucasian counterparts []Worldwide the treatment of breast cancer is now personalized dependent on the patient the stage and grade of disease histological type immunohistochemistry drug preference surgery and radiation impact and techniques for best outcomes When the pathology immunohistochemistry and tumor biology types are not factored into treatment modalities for these patients coupled with late stage at presentation poverty and lack of funding for treatment there are worse outcomes for patients and this accounts for the high incidence of morbidity and mortality that is seenAccording to the Central Intelligence Agency fact book there is prevalence rate of poverty in Nigeria [] There is paucity of studies detailing biology or genetics of breast cancer in SubSaharan Africa likely because of the difficulty involved in obtaining and processing tissue samples usually because of financial constraint [] and due to the lack of laboratory facilities to carry out these investigations []Currently the management of Nigerian women with breast cancer is dependent on protocols imported from developed countries like the USA even though the patient population and disease profile may differ Understanding the profile of Nigerian women with breast cancer helps to create prevention and treatment in a more personalized approach in management of the disease in NigeriaThis study therefore focuses on exploring those characteristics in Nigerian patients the differences seen when compared to their counterparts in HHDI countries and hopes that these findings could impact prevention and management of breast cancer patients in NigeriaMaterials and MethodsStudy designThis is a noninterventional prospective study among participants recruited from the Radiotherapy Unit of Lagos University Teaching Hospital IdiAraba Nigeria Participants were selected newly histologically diagnosed with tumor staging according to American Joint Committee on Cancer 8th edition breast cancer patients who attended the outpatient clinics for treatment for the first time from July to July Participants were all females aged years or more Patients who were acutely ill Eastern Cooperative Oncology Group ECOG score were excluded from the study A structured intervieweradministered proforma was used to obtain required data from all study participants during the study period The proforma collected data on sociodemographic and disease characteristics Neutr ia and febrile neutr ia was graded using the Common Terminology Criteria for Adverse Events CTCAE version The CTCAE is a set of criteria for the standardized classification of adverse effects of drugs used in cancer therapyMeasuresStudy proforma Sociodemographic and socioeconomic informationParticipants were administered questionnaires aimed at gathering information about their age marital status level of education occupation partner™s occupation and economic statusPatient™s occupation was categorized under three domains unemployed including student housewife minimally skilled artisan civil servant trader and skilledprofessional doctor lawyer accountantPatient™s marital status was defined into two categories married or unmarried divorced separated single and widowed Clinical information and risk factorsParticipants were asked questions about their past medical history including parity first symptom menopausal status and duration of illness Risk factors like alcohol use smoking family history use of contraceptive breastfeeding and previous history of benign breast lesions were also elicited Some clinical data were obtained by reviewing the patient™s hospital folder with a specific focus on cancer diagnosis staging surgery and ECOG performance of participantsBody mass index BMI of each patient was calculated using the height and weight recorded in their medical case files at first presentation to the hospital A BMI of kgm2 or more was defined as obesity and kgm2 or more was considered overweightPresence of comorbidities including hypertension diabetes human immunodeficiency virus and peptic ulcer disease was recorded Positive family history of breast cancer is defined as breast cancer both in first and second degree of patient™s family Physical inactivity is measured by inability to move around carry out day to day activities or at least min of moderate intensity physical activity per week as recommended by the World Health anization [] Early menarche is defined as first menstrual period in a female adolescent before the age of years [] while late first pregnancy is defined as above years [] Previous lump is the presence of a benign lump that was removed before the onset of the breast malignancy Pathology and immunohistochemistryParticipants™ hospital folders were reviewed for data on pathologic staging of disease pathologic information including histologic type tumor grade and immunohistochemistry classifis The authors Journal compilation World J Oncol and Elmer Press Inc„¢ wwwwjon 0cAdeniji et alWorld J Oncol Frequency ± Mean ± SD Range Living with a partner Not living with a partnerTable Characteristics of Breast Cancer PatientsCharacteristicsAge years Marital status Occupation Education level Unemployed Minimally skilled Skilled and professional None Primary Secondary Tertiary Table Immunohistochemistry Distribution Among Breast Cancer Patients Negative Positive Negative PositiveReceptor statusEstrogen receptor status Progesterone receptor status HER2 receptor status Hormonal receptor status Triple negativity Equivocal Negative Positive Negative PositiveFrequency SD standard deviationHER2 human epidermal growth factor receptor cation of disease Human epidermal growth factor receptor HER2 is defined by immunohistochemistry onlyData analysisData analysis was done using Statistical Package for Social Sciences software for Windows version SPSS Chicago IL Univariate analyses were presented in the forms of tables as descriptive frequency distribution of the sociodemographic and immunohistochemistry of the patients Correlation and association analyses were conducted using Chisquared and analysis of variance ANOVA with a precision index of ‰¤ Ethical considerationsEthical approval was sought from the ethics committee of the Lagos University Teaching Hospital and the study was conducted according to the principles of the Declaration of Helsinki Informed consent was sought from every participant before undertaking to participate in the studyResultsA total of patients were seen as outpatients with histologically diagnosed breast cancer The mean age of the patients studied was years with a range of years Table Majority live with a partner were unemployed and attained tertiary level of educationTable summarizes the immunohistochemical status of the patients Estrogen receptor ER and progesterone receptor PR positivity were cases and respectively About one in every five had HER2 positivity Almost half have triple negative subtypeThe majority of participants sampled suffered from right breast cancer Table The mean age at diagnosis and body mass index BMI at first presentation to the clinic were years and kgm2 A total of were premenopausal A total of had preexisting comorbidities while have had breast surgery before and more than half presented with ECOG performance score ‰¥ The most common primary site of tumor was the rightThe most frequent histological type was invasive ductal with cases Table Of these cancers were grade were grade and were grade Stages I II III and IV were and respectively with having confirmed cases of an metastasis and two cases did not have documented investigation of an metastasis in their medical case filesThe most common risk factors identified with the participants were overweightobesity and physical inactivity About of patients studied had a family history of breast or any other type of cancer Table A significant relationship was found between the HER status and history of breast surgery P tumor site P Table family history of breast cancer P and previous lump P Table There was also a significant relationship between HR status and alcohol intake P and family history of breast cancer P Table The only significant relationship seen in triple negative subtype was with family history of breast cancer P Table Immunohistochemistry status correlations with the age of the patients age at diagnosis menopausal status and the histologic type were not statistically significants The authors Journal compilation World J Oncol and Elmer Press Inc„¢ wwwwjon 0cBreast Cancer Among Nigerian WomenWorld J Oncol eulavP ± n ± eulavP lacoviuqE n evitageN n evitisoPn ± ± ± ± ± ± eulavP evitageNn ± ± ± ± ± ± yregrus tsaerb fo yrotsiHlasuaponemerPlasuaponemtsoP sutats lasuaponeMerocs GOCEmgk IMBseitidibromoCseY oN laretaliBthgiRtfeL etis romuT scitsiretcarahCsisongaid ta egA DS±nae M evitagen elpirTsutats REHsutats lanomroH evitisoP n ycneuqerFepytbuS romuT yb scitsiretcarahC lacniilC fo noitubirtsDi lebaTpu ygoocnO evitlarepooCnre tsaE GOCE xedni ssam ydob IMB noitiaved dradnats DS rotpecer rotcaf thw liamredpe namuh REHs The authors Journal compilation World J Oncol and Elmer Press Inc„¢ wwwwjon 0cAdeniji et alWorld J Oncol eulavPn eulavP lacoviuqE n evitageN n evitisoPn eulavP evitageNn evitisoPn ycneuqerF evitagen elpirTsutats REHsutats lanomroHepytbuS romuT yb scitsiretcarahC cgoohtaPli amonicrac latcud evisavnIamonicrac ralubol evisavnIasrehtO rotpecer rotcaf thw lamredpei namuh REH iamoncrac yrallipap dna suoncumi yralludem srehOat fo noitubirtsDi lebaTscitsiretcarahCezis romuT sutats ladoN sisatsatem romuTegats esaesiDedarg romuTIIIIII epyt ygolotsiHIIIIIIVI s The authors Journal compilation World J Oncol and Elmer Press Inc„¢ wwwwjon 0cBreast Cancer Among Nigerian WomenWorld J Oncol evitagen elpirTsutats REHsutats lanomroHeulavPn eulavP lacoviuqE n evitageN n evitisoPn eulavP evitageNn evitisoPn ycneuqerFepytbuS romuT yb srotcaF ksRi fo noitubirtsDi lebaTpmul tsaerb tnangilamngineb suoiverPdeeftsaerb ton diDehcranemylraE ycnangerp tsrfi etaLytisebothgiewrevOytirapilluNesu evitpecartnoc larOerusopxe noitaidaRytivitcani lacisyhPyrotsih ylimaFekatni lohoclAgnikomSscitsiretcarahCDiscussionAfricanAmericans in USA have more metastatic breast cancer when compared to other races and the same said for highgrade disease larger tumor size and hormone receptor negativity in the blacks [] These are significantly increased among the blacks living in Africa [ ]This study clearly itemizes the sociodemographic clinical histological and immunohistochemistry characteristics of the Nigerian breast cancer patients in a hospitalbased study In this study the mean age was years with majority of women aged between and years similar to the findings in previous studies in Nigeria but in contrast to western countries where most of the breast cancer patients are postmenopausal [ ] Some studies have postulated a decrease in levels of circulating estrogen levels as responsible for the decreasing age of breast cancer patients worldwide [ ] This finding emphasizes on the need for preventive health education and screening programs not only targeted at the elderly because of the assumption that they are the prime age group at risk while this might be true for western countries and the pattern of disease presentation in Nigerian patients clearly highlights the need to begin screening for the disease before yearsThe previous studies conducted in Africa and Nigeria are largely hospitalbased studies and with small sample sizes making it difficult to predict associations between patients and risk factors The finding of obesity and physical inactivity as the largest risk factors for breast cancer is new in the premenopausal group although this was always true for many western countries mostly for postmenopausal women [] This finding is new in LHDI countries like Nigeria and the predominance of the triple negative subset may account for this finding compared to the hormone receptor positive subset predominance in the western countries In a similar study carried out in Nigerian women in early menarche and not breast feeding were the risk factors associated with increased risk of development of breast cancer In this study early menarche was only seen in of breast cancer patients []Family history is not a common risk factor in our patients as compared to their counterparts in HHDI countries [ ] The same is true for early menarche and nulliparity Not surprisingly the profile of risk in Nigerian cancer patients has evolved to mirror their Caucasian counterparts in the areas of obesity and physical inactivity [ ] This finding helps to focus healthcare professionals during screening exercises not to rule out likely patients because of the absence of traditional risk factors like family history early menarche or nulliparityIn this study like many other studies conducted in SubSaharan Africa patients are seen in locally advanced and advanced stages of their diseases [ ] This finding is not true for women in developed countries as patients tend to present at earlier stages [] Majority of the respondents were of moderate economic status which suggests that funds may not be the reason for late stage presentation as seen in previous studies [ ] Finding the reason why these patients presented late despite the fairly stable economic status is beyond the scope of this study and is for further review Perhaps the reason P rotpecer rotcaf thw liamredpe namuh REHs The authors Journal compilation World J Oncol and Elmer Press Inc„¢ wwwwjon 0cAdeniji et alWorld J Oncol may be associated to the painless nature of their first symptom which may have affected their health seeking behaviorIn recent times targeted therapies based on grade histology and immunohistochemistry have resulted in better outcomes for patients [ ] Globally the invasive ductal carcinoma is the commonest histologic subtype of breast cancer [] This is true for breast cancer patients in this study representing of the breast cancer patients seenTriple negative was the commonest subtype seen in these patients and differed from the less aggressive subtypes seen in Caucasian women [ ] This subtype is associated with high rates of tumor invasion and metastases and is associated with a poorer prognosis [] This may explain the high mortality rates seen in the Nigerian breast cancer patients despite the comparatively lower incidence ratesConclusionNigerian breast cancer patient are likely to be premenopausal obese or overweight with no family history of higher tumor grade triple negative subtype late stage and hormone receptor negative These findings explain the high mortality rates seen in the Nigerian breast cancer patients and can be modified or useful in targeted treatment to ensure a better outcome Supplementary Material wwwwjonla Samuel Olalekan Keshinro Financial support Adeoluwa Adeniji Akeem Olayemi Olubunmi Dawodu Muhammad Yaqub Habeebu Ademola Oluwatosin Oyekan Bashir Mariam Adebola Samuel Olalekan Keshinro Administrative support Adeoluwa Adeniji Akeem Timilehin Gabriel Fagbenro Bashir Mariam Adebola Michael Martin Provision of study materials or patients Adeoluwa Adeniji Akeem Muhammad Yaqub Habeebu Ademola Oluwatosin Oyekan Collection and assembly of data Adeoluwa Adeniji Akeem Ademola Oluwatosin Oyekan Michael Martin Bashir Mariam Adebola Samuel Olalekan Keshinro Data analysis and interpretation Adeoluwa Adeniji Akeem Timilehin Gabriel Fagbenro Olayemi Olubunmi Dawodu Muhammad Yaqub Habeebu Manuscript writing all authors Final approval of manuscript all authors Accountable for all aspects of the work all authorsData AvailabilityThe data supporting the findings of this study have been deposited in OneDrive and can be accessed via the link at cuttlyadenijibreastcancerSupplementary MaterialReferencesSuppl Data of All Study Participants During the Study PeriodAcknowledgmentsWe acknowledge the entire staff of the department especially the medical record officers nurses and the resident doctorsFinancial DisclosureNone to declareConflict of InterestThe authors declare that they have no conflict of interest regarding this workInformed ConsentInformed consent was obtainedAuthor ContributionsConception and design Adeoluwa Adeniji Akeem Olayemi Olubunmi Dawodu Muhammad Yaqub Habeebu Ademola Oluwatosin Oyekan Michael Martin Bashir Mariam Adebo Ginsburg O Bray F Coleman MP Vanderpuye V Eniu A Kotha SR Sarker M et al The global burden of women's cancers a grand challenge in global health Lancet Bray F Ferlay J Soerjomataram I Siegel RL Torre LA Jemal A Global cancer statistics GLOBOCAN estimates of incidence and mortality worldwide for cancers in countries CA Cancer J Clin Ghoncheh M Momenimovahed Z Salehiniya H Epidemiology incidence and mortality of breast cancer in Asia Asian Pac J Cancer Prev 201617S34752 Shrivastava S Singh N Nigam AK et al Comparative study of hematological parameters along with effect of chemotherapy and radiotherapy in different stages of breast cancer Int J Res Med Sci Soheylizad M Khazaei S Jenabi E Delpisheh A Veisani Y The relationship between human development index and its components with thyroid cancer incidence and mortality using the decomposition approach Int J Endocrinol Metab 2018164e65078Igene H Global health inequalities and breast cancer an impending public health problem for developing countries Breast J Brinton LA Figueroa JD Awuah B Yarney J Wiafe S Wood SN Ansong D et al Breast cancer in SubSaharan Africa opportunities for prevention Breast Cancer Res Treat Akhigbe AO Omuemu VO Knowledge attitudes and practice of breast cancer screening among female health workers in a Nigerian urban city BMC Cancer Lawal O Murphy FJ Hogg P Irurhe N Nightingale J s The authors Journal compilation World J Oncol and Elmer Press Inc„¢ wwwwjon 0cBreast Cancer Among Nigerian WomenWorld J Oncol Mammography screening in Nigeria A critical comparison to other countries Radiography Akinola R Wright K Osunfidiya O Orogbemi O Akinola O Mammography and mammographic screening are female patients at a teaching hospital in Lagos Nigeria aware of these procedures Diagn Interv Radiol MO O Ayodele SO Umar AS Breast cancer and mammography Current knowledge attitudes and practices of female health workers in a tertiary health institution in Northern Nigeria Screening Agboola AJ Musa AA Wanangwa N AbdelFatah T Nolan CC Ayoade BA Oyebadejo TY et al Molecular characteristics and prognostic features of breast cancer in Nigerian compared with UK women Breast Cancer Res Treat Factbook CI The world factbook Available at wwwciagovlibrarypublicationstheworldfactbook Accessed on January 2nd Fregene A Newman LA Breast cancer in subSaharan Africa how does it relate to breast cancer in AfricanAmerican women Cancer AkaroloAnthony SN Ogundiran TO Adebamowo CA Emerging breast cancer epidemic evidence from Africa Breast Cancer Res 201012Suppl 4S8 Fuzeki E Banzer W Physical activity recommendations for health and beyond in currently inactive populations Int J Environ Res Public Health Ibitoye M Choi C Tai H Lee G Sommer M Early menarche A systematic review of its effect on sexual and reproductive health in low and middleincome countries PLoS One 2017126e0178884 Lampinen R VehvilainenJulkunen K Kankkunen P A review of pregnancy in women over years of age Nurs J DeSantis CE Ma J Gaudet MM Newman LA Miller KD Goding Sauer A Jemal A et al Breast cancer statistics CA Cancer J Clin Ntekim A Nufu FT Campbell OB Breast cancer in young women in Ibadan Nigeria Afr Health Sci Henderson BE Ross R Bernstein L Estrogens as a cause of human cancer the Richard and Hinda Rosenthal Foundation award lecture Cancer Res Bray F McCarron P Parkin DM The changing global patterns of female breast cancer incidence and mortality Breast Cancer Res Wolin KY Carson K Colditz GA Obesity and cancer Oncologist Huo D Adebamowo CA Ogundiran TO Akang EE Campbell O Adenipekun A Cummings S et al Parity and breastfeeding are protective against breast cancer in Nigerian women Br J Cancer Adesunkanmi AR Lawal OO Adelusola KA Durosimi MA The severity outcome and challenges of breast cancer in Nigeria Breast Adebamowo CA Adekunle OO Casecontrolled study of the epidemiological risk factors for breast cancer in Nigeria Br J Surg Porter P Westernizing women's risks Breast cancer in lowerincome countries N Engl J Med McPherson K Steel CM Dixon JM ABC of breast diseases Breast cancerepidemiology risk factors and genetics BMJ Awofeso O Roberts AA Salako O Balogun L Okediji P Prevalence and pattern of latestage presentation in women with breast and cervical cancers in Lagos University Teaching Hospital Nigeria Niger Med J JedyAgba E McCormack V Adebamowo C DosSantosSilva I Stage at diagnosis of breast cancer in subSaharan Africa a systematic review and metaanalysis Lancet Glob Health 2016412e923e935 Vanderpuye V Grover S Hammad N PoojaPrabhakar Simonds H Olopade F Stefan DC An update on the management of breast cancer in Africa Infect Agent Cancer Ibrahim NA Oludara MA Sociodemographic factors and reasons associated with delay in breast cancer presentation a study in Nigerian women Breast Lannin DR Mathews HF Mitchell J Swanson MS Swanson FH Edwards MS Influence of socioeconomic and cultural factors on racial differences in latestage presentation of breast cancer JAMA Sohn YM Han K Seo M Immunohistochemical subtypes of breast cancer correlation with clinicopathological and radiological factors Iran J Radiol 2016134e31386 Beral V Bull D Doll R Peto R Reeves G Collaborative Group on Hormonal Factors in Breast C Breast cancer and abortion collaborative reanalysis of data from epidemiological studies including women with breast cancer from countries Lancet Li CI Anderson BO Daling JR Moe RE Trends in incidence rates of invasive lobular and ductal breast carcinoma JAMA Wu X Baig A Kasymjanova G Kafi K Holcroft C Mekouar H Carbonneau A et al Pattern of Local recurrence and distant metastasis in breast cancer by molecular subtype Cureus 2016812e924s The authors Journal compilation World J Oncol and Elmer Press Inc„¢ wwwwjon 0c"

cancer280

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: "clinicopathological characteristics with risk factors of breast cancer patients in NigeriaMethods Newly diagnosed female patients with breast cancer were assessed over months Patients were reviewed using a predesigned proforma which focused on sociodemographic information clinical information risk factors and tumor biologyResults A total of women were identified their mean age was years More than half are premenopausal at presentation with Eastern Cooperative Oncology Group ECOG score of and right side as the most common primary site of disease Less than half of them are estrogen receptor ER positive are progesterone receptor PR positive and are hormone receptor positive and triple negative respectively Most patients presented at the latter stage of the disease stage III and stage IV Only are well differentiated and almost all had invasive ductal histological type Obesity and physical inactivity are the most common risk factors for the disease A significant relationship was found between immunohistochemistry status and family history of breast cancer tumor site previManuscript submitted June accepted July Published online August aOncology and Radiotherapy Department Lagos University Teaching Hospital Lagos NigeriabMolecular and Anatomical Pathology Department College of Medicine University of Lagos Lagos NigeriacRadiotherapy Radiobiology Radiodiagnosis and Radiography Department College of Medicine University of Lagos Lagos NigeriadWest Cancer Centre and Research Institute Memphis TN USAeArrive Alive Diagnostics and Imaging Services Ltd Lagos NigeriafCorresponding Author Adeoluwa Akeem Adeniji Oncology and Radiotherapy Department Lagos University Teaching Hospital Lagos Nigeria Email godscrownbestyahoocom 1014740wjon1303ous breast surgery previous lump and alcohol intakeConclusion Findings from this study showed that Nigerian breast cancer patients differ from their counterparts in the high human development index HHDI countries in terms of the patients and disease characteristics In view of this prevention and treatment options should consider this uniqueness to ensure better outcomeKeywords Breast cancer Subtypes Tumor biology Risk factors Correlation NigeriaIntroductionCancer is a major public health concern globally [] According to GLOBACAN cancer is the single most important factor impacting life expectancy worldwide [] In women worldwide breast cancer is the most common malignancy [] Every year about million new cases are diagnosed worldwide and this represents of the female population [] In alone there were million new cases of breast cancer worldwide and over deaths and this represents new cases of cancer and of all cancerrelated deaths []One of the indicators that reflect the development of each country the status and their living conditions is the human development index HDI The HDI is defined as the average achievement of three factors including life expectancy at birth gross national income per capita and mean and expected years of schooling Low HDI level includes countries that are the least developed and the very high HDI level includes the most developed countries [] Although low human development index countries LHDI like Nigeria have a lower incidence of breast cancer when compared to high human development index HHDI countries like the United States of America USA mortality rates are higher [] The incidence rate in the LHDI countries is rising likely because of westernization and its lifestyle choices []The high mortality rate is seen because of late stage presentation misdiagnosis and poor health seeking behavior s The authors Journal compilation World J Oncol and Elmer Press Inc„¢ wwwwjonThis is distributed under the terms of the Creative Commons Attribution NonCommercial International License which permits unrestricted noncommercial use distribution and reproduction in any medium provided the original work is properly cited 0cBreast Cancer Among Nigerian WomenWorld J Oncol among other factors of the African population in general The screening rates is still low ranging from to for reasons ranging from cost quality assurance and fear of radiation [] Studies have also shown that the genetic and histopathologic subtypes in African women are likely to be more aggressive than those seen in their Caucasian counterparts []Worldwide the treatment of breast cancer is now personalized dependent on the patient the stage and grade of disease histological type immunohistochemistry drug preference surgery and radiation impact and techniques for best outcomes When the pathology immunohistochemistry and tumor biology types are not factored into treatment modalities for these patients coupled with late stage at presentation poverty and lack of funding for treatment there are worse outcomes for patients and this accounts for the high incidence of morbidity and mortality that is seenAccording to the Central Intelligence Agency fact book there is prevalence rate of poverty in Nigeria [] There is paucity of studies detailing biology or genetics of breast cancer in SubSaharan Africa likely because of the difficulty involved in obtaining and processing tissue samples usually because of financial constraint [] and due to the lack of laboratory facilities to carry out these investigations []Currently the management of Nigerian women with breast cancer is dependent on protocols imported from developed countries like the USA even though the patient population and disease profile may differ Understanding the profile of Nigerian women with breast cancer helps to create prevention and treatment in a more personalized approach in management of the disease in NigeriaThis study therefore focuses on exploring those characteristics in Nigerian patients the differences seen when compared to their counterparts in HHDI countries and hopes that these findings could impact prevention and management of breast cancer patients in NigeriaMaterials and MethodsStudy designThis is a noninterventional prospective study among participants recruited from the Radiotherapy Unit of Lagos University Teaching Hospital IdiAraba Nigeria Participants were selected newly histologically diagnosed with tumor staging according to American Joint Committee on Cancer 8th edition breast cancer patients who attended the outpatient clinics for treatment for the first time from July to July Participants were all females aged years or more Patients who were acutely ill Eastern Cooperative Oncology Group ECOG score were excluded from the study A structured intervieweradministered proforma was used to obtain required data from all study participants during the study period The proforma collected data on sociodemographic and disease characteristics Neutr ia and febrile neutr ia was graded using the Common Terminology Criteria for Adverse Events CTCAE version The CTCAE is a set of criteria for the standardized classification of adverse effects of drugs used in cancer therapyMeasuresStudy proforma Sociodemographic and socioeconomic informationParticipants were administered questionnaires aimed at gathering information about their age marital status level of education occupation partner™s occupation and economic statusPatient™s occupation was categorized under three domains unemployed including student housewife minimally skilled artisan civil servant trader and skilledprofessional doctor lawyer accountantPatient™s marital status was defined into two categories married or unmarried divorced separated single and widowed Clinical information and risk factorsParticipants were asked questions about their past medical history including parity first symptom menopausal status and duration of illness Risk factors like alcohol use smoking family history use of contraceptive breastfeeding and previous history of benign breast lesions were also elicited Some clinical data were obtained by reviewing the patient™s hospital folder with a specific focus on cancer diagnosis staging surgery and ECOG performance of participantsBody mass index BMI of each patient was calculated using the height and weight recorded in their medical case files at first presentation to the hospital A BMI of kgm2 or more was defined as obesity and kgm2 or more was considered overweightPresence of comorbidities including hypertension diabetes human immunodeficiency virus and peptic ulcer disease was recorded Positive family history of breast cancer is defined as breast cancer both in first and second degree of patient™s family Physical inactivity is measured by inability to move around carry out day to day activities or at least min of moderate intensity physical activity per week as recommended by the World Health anization [] Early menarche is defined as first menstrual period in a female adolescent before the age of years [] while late first pregnancy is defined as above years [] Previous lump is the presence of a benign lump that was removed before the onset of the breast malignancy Pathology and immunohistochemistryParticipants™ hospital folders were reviewed for data on pathologic staging of disease pathologic information including histologic type tumor grade and immunohistochemistry classifis The authors Journal compilation World J Oncol and Elmer Press Inc„¢ wwwwjon 0cAdeniji et alWorld J Oncol Frequency ± Mean ± SD Range Living with a partner Not living with a partnerTable Characteristics of Breast Cancer PatientsCharacteristicsAge years Marital status Occupation Education level Unemployed Minimally skilled Skilled and professional None Primary Secondary Tertiary Table Immunohistochemistry Distribution Among Breast Cancer Patients Negative Positive Negative PositiveReceptor statusEstrogen receptor status Progesterone receptor status HER2 receptor status Hormonal receptor status Triple negativity Equivocal Negative Positive Negative PositiveFrequency SD standard deviationHER2 human epidermal growth factor receptor cation of disease Human epidermal growth factor receptor HER2 is defined by immunohistochemistry onlyData analysisData analysis was done using Statistical Package for Social Sciences software for Windows version SPSS Chicago IL Univariate analyses were presented in the forms of tables as descriptive frequency distribution of the sociodemographic and immunohistochemistry of the patients Correlation and association analyses were conducted using Chisquared and analysis of variance ANOVA with a precision index of ‰¤ Ethical considerationsEthical approval was sought from the ethics committee of the Lagos University Teaching Hospital and the study was conducted according to the principles of the Declaration of Helsinki Informed consent was sought from every participant before undertaking to participate in the studyResultsA total of patients were seen as outpatients with histologically diagnosed breast cancer The mean age of the patients studied was years with a range of years Table Majority live with a partner were unemployed and attained tertiary level of educationTable summarizes the immunohistochemical status of the patients Estrogen receptor ER and progesterone receptor PR positivity were cases and respectively About one in every five had HER2 positivity Almost half have triple negative subtypeThe majority of participants sampled suffered from right breast cancer Table The mean age at diagnosis and body mass index BMI at first presentation to the clinic were years and kgm2 A total of were premenopausal A total of had preexisting comorbidities while have had breast surgery before and more than half presented with ECOG performance score ‰¥ The most common primary site of tumor was the rightThe most frequent histological type was invasive ductal with cases Table Of these cancers were grade were grade and were grade Stages I II III and IV were and respectively with having confirmed cases of an metastasis and two cases did not have documented investigation of an metastasis in their medical case filesThe most common risk factors identified with the participants were overweightobesity and physical inactivity About of patients studied had a family history of breast or any other type of cancer Table A significant relationship was found between the HER status and history of breast surgery P tumor site P Table family history of breast cancer P and previous lump P Table There was also a significant relationship between HR status and alcohol intake P and family history of breast cancer P Table The only significant relationship seen in triple negative subtype was with family history of breast cancer P Table Immunohistochemistry status correlations with the age of the patients age at diagnosis menopausal status and the histologic type were not statistically significants The authors Journal compilation World J Oncol and Elmer Press Inc„¢ wwwwjon 0cBreast Cancer Among Nigerian WomenWorld J Oncol eulavP ± n ± eulavP lacoviuqE n evitageN n evitisoPn ± ± ± ± ± ± eulavP evitageNn ± ± ± ± ± ± yregrus tsaerb fo yrotsiHlasuaponemerPlasuaponemtsoP sutats lasuaponeMerocs GOCEmgk IMBseitidibromoCseY oN laretaliBthgiRtfeL etis romuT scitsiretcarahCsisongaid ta egA DS±nae M evitagen elpirTsutats REHsutats lanomroH evitisoP n ycneuqerFepytbuS romuT yb scitsiretcarahC lacniilC fo noitubirtsDi lebaTpu ygoocnO evitlarepooCnre tsaE GOCE xedni ssam ydob IMB noitiaved dradnats DS rotpecer rotcaf thw liamredpe namuh REHs The authors Journal compilation World J Oncol and Elmer Press Inc„¢ wwwwjon 0cAdeniji et alWorld J Oncol eulavPn eulavP lacoviuqE n evitageN n evitisoPn eulavP evitageNn evitisoPn ycneuqerF evitagen elpirTsutats REHsutats lanomroHepytbuS romuT yb scitsiretcarahC cgoohtaPli amonicrac latcud evisavnIamonicrac ralubol evisavnIasrehtO rotpecer rotcaf thw lamredpei namuh REH iamoncrac yrallipap dna suoncumi yralludem srehOat fo noitubirtsDi lebaTscitsiretcarahCezis romuT sutats ladoN sisatsatem romuTegats esaesiDedarg romuTIIIIII epyt ygolotsiHIIIIIIVI s The authors Journal compilation World J Oncol and Elmer Press Inc„¢ wwwwjon 0cBreast Cancer Among Nigerian WomenWorld J Oncol evitagen elpirTsutats REHsutats lanomroHeulavPn eulavP lacoviuqE n evitageN n evitisoPn eulavP evitageNn evitisoPn ycneuqerFepytbuS romuT yb srotcaF ksRi fo noitubirtsDi lebaTpmul tsaerb tnangilamngineb suoiverPdeeftsaerb ton diDehcranemylraE ycnangerp tsrfi etaLytisebothgiewrevOytirapilluNesu evitpecartnoc larOerusopxe noitaidaRytivitcani lacisyhPyrotsih ylimaFekatni lohoclAgnikomSscitsiretcarahCDiscussionAfricanAmericans in USA have more metastatic breast cancer when compared to other races and the same said for highgrade disease larger tumor size and hormone receptor negativity in the blacks [] These are significantly increased among the blacks living in Africa [ ]This study clearly itemizes the sociodemographic clinical histological and immunohistochemistry characteristics of the Nigerian breast cancer patients in a hospitalbased study In this study the mean age was years with majority of women aged between and years similar to the findings in previous studies in Nigeria but in contrast to western countries where most of the breast cancer patients are postmenopausal [ ] Some studies have postulated a decrease in levels of circulating estrogen levels as responsible for the decreasing age of breast cancer patients worldwide [ ] This finding emphasizes on the need for preventive health education and screening programs not only targeted at the elderly because of the assumption that they are the prime age group at risk while this might be true for western countries and the pattern of disease presentation in Nigerian patients clearly highlights the need to begin screening for the disease before yearsThe previous studies conducted in Africa and Nigeria are largely hospitalbased studies and with small sample sizes making it difficult to predict associations between patients and risk factors The finding of obesity and physical inactivity as the largest risk factors for breast cancer is new in the premenopausal group although this was always true for many western countries mostly for postmenopausal women [] This finding is new in LHDI countries like Nigeria and the predominance of the triple negative subset may account for this finding compared to the hormone receptor positive subset predominance in the western countries In a similar study carried out in Nigerian women in early menarche and not breast feeding were the risk factors associated with increased risk of development of breast cancer In this study early menarche was only seen in of breast cancer patients []Family history is not a common risk factor in our patients as compared to their counterparts in HHDI countries [ ] The same is true for early menarche and nulliparity Not surprisingly the profile of risk in Nigerian cancer patients has evolved to mirror their Caucasian counterparts in the areas of obesity and physical inactivity [ ] This finding helps to focus healthcare professionals during screening exercises not to rule out likely patients because of the absence of traditional risk factors like family history early menarche or nulliparityIn this study like many other studies conducted in SubSaharan Africa patients are seen in locally advanced and advanced stages of their diseases [ ] This finding is not true for women in developed countries as patients tend to present at earlier stages [] Majority of the respondents were of moderate economic status which suggests that funds may not be the reason for late stage presentation as seen in previous studies [ ] Finding the reason why these patients presented late despite the fairly stable economic status is beyond the scope of this study and is for further review Perhaps the reason P rotpecer rotcaf thw liamredpe namuh REHs The authors Journal compilation World J Oncol and Elmer Press Inc„¢ wwwwjon 0cAdeniji et alWorld J Oncol may be associated to the painless nature of their first symptom which may have affected their health seeking behaviorIn recent times targeted therapies based on grade histology and immunohistochemistry have resulted in better outcomes for patients [ ] Globally the invasive ductal carcinoma is the commonest histologic subtype of breast cancer [] This is true for breast cancer patients in this study representing of the breast cancer patients seenTriple negative was the commonest subtype seen in these patients and differed from the less aggressive subtypes seen in Caucasian women [ ] This subtype is associated with high rates of tumor invasion and metastases and is associated with a poorer prognosis [] This may explain the high mortality rates seen in the Nigerian breast cancer patients despite the comparatively lower incidence ratesConclusionNigerian breast cancer patient are likely to be premenopausal obese or overweight with no family history of higher tumor grade triple negative subtype late stage and hormone receptor negative These findings explain the high mortality rates seen in the Nigerian breast cancer patients and can be modified or useful in targeted treatment to ensure a better outcome Supplementary Material wwwwjonla Samuel Olalekan Keshinro Financial support Adeoluwa Adeniji Akeem Olayemi Olubunmi Dawodu Muhammad Yaqub Habeebu Ademola Oluwatosin Oyekan Bashir Mariam Adebola Samuel Olalekan Keshinro Administrative support Adeoluwa Adeniji Akeem Timilehin Gabriel Fagbenro Bashir Mariam Adebola Michael Martin Provision of study materials or patients Adeoluwa Adeniji Akeem Muhammad Yaqub Habeebu Ademola Oluwatosin Oyekan Collection and assembly of data Adeoluwa Adeniji Akeem Ademola Oluwatosin Oyekan Michael Martin Bashir Mariam Adebola Samuel Olalekan Keshinro Data analysis and interpretation Adeoluwa Adeniji Akeem Timilehin Gabriel Fagbenro Olayemi Olubunmi Dawodu Muhammad Yaqub Habeebu Manuscript writing all authors Final approval of manuscript all authors Accountable for all aspects of the work all authorsData AvailabilityThe data supporting the findings of this study have been deposited in OneDrive and can be accessed via the link at cuttlyadenijibreastcancerSupplementary MaterialReferencesSuppl Data of All Study Participants During the Study PeriodAcknowledgmentsWe acknowledge the entire staff of the department especially the medical record officers nurses and the resident doctorsFinancial DisclosureNone to declareConflict of InterestThe authors declare that they have no conflict of interest regarding this workInformed ConsentInformed consent was obtainedAuthor ContributionsConception and design Adeoluwa Adeniji Akeem Olayemi Olubunmi Dawodu Muhammad Yaqub Habeebu Ademola Oluwatosin Oyekan Michael Martin Bashir Mariam Adebo Ginsburg O Bray F Coleman MP Vanderpuye V Eniu A Kotha SR Sarker M et al The global burden of women's cancers a grand challenge in global health Lancet Bray F Ferlay J Soerjomataram I Siegel RL Torre LA Jemal A Global cancer statistics GLOBOCAN estimates of incidence and mortality worldwide for cancers in countries CA Cancer J Clin Ghoncheh M Momenimovahed Z Salehiniya H Epidemiology incidence and mortality of breast cancer in Asia Asian Pac J Cancer Prev 201617S34752 Shrivastava S Singh N Nigam AK et al Comparative study of hematological parameters along with effect of chemotherapy and radiotherapy in different stages of breast cancer Int J Res Med Sci Soheylizad M Khazaei S Jenabi E Delpisheh A Veisani Y The relationship between human development index and its components with thyroid cancer incidence and mortality using the decomposition approach Int J Endocrinol Metab 2018164e65078Igene H Global health inequalities and breast cancer an impending public health problem for developing countries Breast J Brinton LA Figueroa JD Awuah B Yarney J Wiafe S Wood SN Ansong D et al Breast cancer in SubSaharan Africa opportunities for prevention Breast Cancer Res Treat Akhigbe AO Omuemu VO Knowledge attitudes and practice of breast cancer screening among female health workers in a Nigerian urban city BMC Cancer Lawal O Murphy FJ Hogg P Irurhe N Nightingale J s The authors Journal compilation World J Oncol and Elmer Press Inc„¢ wwwwjon 0cBreast Cancer Among Nigerian WomenWorld J Oncol Mammography screening in Nigeria A critical comparison to other countries Radiography Akinola R Wright K Osunfidiya O Orogbemi O Akinola O Mammography and mammographic screening are female patients at a teaching hospital in Lagos Nigeria aware of these procedures Diagn Interv Radiol MO O Ayodele SO Umar AS Breast cancer and mammography Current knowledge attitudes and practices of female health workers in a tertiary health institution in Northern Nigeria Screening Agboola AJ Musa AA Wanangwa N AbdelFatah T Nolan CC Ayoade BA Oyebadejo TY et al Molecular characteristics and prognostic features of breast cancer in Nigerian compared with UK women Breast Cancer Res Treat Factbook CI The world factbook Available at wwwciagovlibrarypublicationstheworldfactbook Accessed on January 2nd Fregene A Newman LA Breast cancer in subSaharan Africa how does it relate to breast cancer in AfricanAmerican women Cancer AkaroloAnthony SN Ogundiran TO Adebamowo CA Emerging breast cancer epidemic evidence from Africa Breast Cancer Res 201012Suppl 4S8 Fuzeki E Banzer W Physical activity recommendations for health and beyond in currently inactive populations Int J Environ Res Public Health Ibitoye M Choi C Tai H Lee G Sommer M Early menarche A systematic review of its effect on sexual and reproductive health in low and middleincome countries PLoS One 2017126e0178884 Lampinen R VehvilainenJulkunen K Kankkunen P A review of pregnancy in women over years of age Nurs J DeSantis CE Ma J Gaudet MM Newman LA Miller KD Goding Sauer A Jemal A et al Breast cancer statistics CA Cancer J Clin Ntekim A Nufu FT Campbell OB Breast cancer in young women in Ibadan Nigeria Afr Health Sci Henderson BE Ross R Bernstein L Estrogens as a cause of human cancer the Richard and Hinda Rosenthal Foundation award lecture Cancer Res Bray F McCarron P Parkin DM The changing global patterns of female breast cancer incidence and mortality Breast Cancer Res Wolin KY Carson K Colditz GA Obesity and cancer Oncologist Huo D Adebamowo CA Ogundiran TO Akang EE Campbell O Adenipekun A Cummings S et al Parity and breastfeeding are protective against breast cancer in Nigerian women Br J Cancer Adesunkanmi AR Lawal OO Adelusola KA Durosimi MA The severity outcome and challenges of breast cancer in Nigeria Breast Adebamowo CA Adekunle OO Casecontrolled study of the epidemiological risk factors for breast cancer in Nigeria Br J Surg Porter P Westernizing women's risks Breast cancer in lowerincome countries N Engl J Med McPherson K Steel CM Dixon JM ABC of breast diseases Breast cancerepidemiology risk factors and genetics BMJ Awofeso O Roberts AA Salako O Balogun L Okediji P Prevalence and pattern of latestage presentation in women with breast and cervical cancers in Lagos University Teaching Hospital Nigeria Niger Med J JedyAgba E McCormack V Adebamowo C DosSantosSilva I Stage at diagnosis of breast cancer in subSaharan Africa a systematic review and metaanalysis Lancet Glob Health 2016412e923e935 Vanderpuye V Grover S Hammad N PoojaPrabhakar Simonds H Olopade F Stefan DC An update on the management of breast cancer in Africa Infect Agent Cancer Ibrahim NA Oludara MA Sociodemographic factors and reasons associated with delay in breast cancer presentation a study in Nigerian women Breast Lannin DR Mathews HF Mitchell J Swanson MS Swanson FH Edwards MS Influence of socioeconomic and cultural factors on racial differences in latestage presentation of breast cancer JAMA Sohn YM Han K Seo M Immunohistochemical subtypes of breast cancer correlation with clinicopathological and radiological factors Iran J Radiol 2016134e31386 Beral V Bull D Doll R Peto R Reeves G Collaborative Group on Hormonal Factors in Breast C Breast cancer and abortion collaborative reanalysis of data from epidemiological studies including women with breast cancer from countries Lancet Li CI Anderson BO Daling JR Moe RE Trends in incidence rates of invasive lobular and ductal breast carcinoma JAMA Wu X Baig A Kasymjanova G Kafi K Holcroft C Mekouar H Carbonneau A et al Pattern of Local recurrence and distant metastasis in breast cancer by molecular subtype Cureus 2016812e924s The authors Journal compilation World J Oncol and Elmer Press Inc„¢ wwwwjon 0c" Answer:

Colon_Cancer

the bacteroides fragilis b fragilis produce biofilm for colonisation in the intestinal tract can cause a series of inflammatory reactions due to b fragilis toxin bft which can lead to chronic intestinal inflammation and tissue injury and play a crucial role leading to colorectal cancer crc the enterotoxigenic b fragilis etbf forms biofilm and produce toxin and play a role in crc whereas the nontoxigenic b fragilis ntbf does not produce toxin the etbf triggers the expression of cyclooxygenase cox2 that releases pge2 for inducing inflammation and control cell proliferation from chronic intestinal inflammation to cancer development it involves signal transducers and activators of transcription stat3 activation stat3 activates by the interaction between epithelial cells and bft thus regulatory tcell tregs will activates and reduce interleukin il2 amount as the level of il2 drops thelper th17 cells are generated leading to increase in il17 levels il17 is implicated in early intestinal inflammation and promotes cancer cell survival and proliferation and consequently triggers il6 production that activate stat3 pathway additionally bft degrades ecadherin hence alteration of signalling pathways can upregulate spermine oxidase leading to cell morphology and promote carcinogenesis and irreversible dna damage patient with familial adenomatous polyposis fap disease displays a high level of tumour load in the colon this disease is caused by germline mutation of the adenomatous polyposis coli apc gene that increases bacterial adherence to the mucosa layer mutatedapc gene genotype with etbf increases the chances of crc development therefore the colonisation of the etbf in the intestinal tract depicts tumour aetiology can result in risk of hostility and effect on human health keywords bacteroides fragilis colon cancer stat3 pathway bacteroides fragilis toxin inflammationintroductionbacteroides species are nonspore forming anaerobe and gramnegative bacteria there are more than different species of bacteroides these bacteria act as normal flora in the intestine to maintain healthy intestinal microflora in humans bacteroides fragilis b fragilis has two classes nontoxigenic b fragilis ntbf and enterotoxigenic b fragilis etbf the differences between ntbf and etbf are the presence of b fragilis toxin bft gene and its ability to produce biofilm bft product is a kda zincdependent metalloprotease toxin also known as fragilysin or bft “ bft plays an important role in intestinal inflammation and tissue injury by damaging the tight junction and increasing intestinal permeability furthermore it has been proven that tissue inflammation and injury promote cancer formation simultaneously the biofilm produced by b fragilis induces carcinogenesis fortunately only etbf encompasses bft and can produce biofilms hence ntbf does not harm the intestinal tract malays j med sci jul“aug “wwwmjmsusmmy penerbit universiti sains malaysia this work is licensed under the terms of the creative commons attribution cc by httpcreativecommonslicensesby40 0cmalays j med sci jul“aug “in the united states colorectal cancer crc is the third most common cancer in both genders it is also the second most common cancerrelated death especially for older patients who are ‰¥ years old in the american cancer society stated that there were new cases of crcs that led to the death of people moreover crc is the fourth leading cancer resulting in deaths worldwide inflammatory bowel disease ibd and genetic mutations are factors predisposing an individual towards colon cancer this indicates that crc has a high mortality rate “microbes are capable in promoting cancer development through several routes such as activation of chronic inflammation alteration of tumour microenvironment and production of toxins that damage dna when there is chronic etbf colonisation in the intestine it stimulates chronic intestinal inflammation triggering signal transducers and activators of transcription stat3 activation which contributes to interleukin il17 production il17 is involved in colon inflammation bft produced by etbf causes the alteration of signalling pathways and production of reactive oxygen species ros that leads to dna damage and cleavage of ecadherin in the below review we have provided a general information regarding bft produced by etbf triggering crc developmentliterature reviewcolon cancer associated with microbespromote nutrition in the human gastrointestinal tract there are nearly trillion microbes out of which make up normal flora in the intestine meanwhile the normal flora is characterised into beneficial and harmful microbes beneficial microbes including production of vitamins in the intestine and prevent disease formation however harmful microbes produce carcinogenic toxin and substances in the intestine these harmful substances may cause cancer there are many types of bacteria that stimulate a variety of cancer formation through their respective site of inflammation eg bacteria such as enterococcus faecalis e faecalis colibactinproducing escherichia coli e coli and etbf are involved in crc development however the mechanisms between each bacterium in contributing to crc formation are different for instance e faecalis damages the dna through ros colibactinproducing e coli produces colibactin that damages the dna and etbf produces bft that contributes to inflammation and immunecell infiltration intestinal dysbiosis inflammation and colon cancergenetic normal flora is advantageous to a person as it maintains intestinal health and gut homeostasis however as the bacteria such as etbf in the gut undergoes dysbiosis it brings harmful effects to the person according to deng et al a correlation was observed between microbiota imbalance and cancer progression while liu et al claimed is associated with that crc development intestinal microecology disorder imbalance among microbiota leads to bacterial infection that can progress to chronic inflammation one of the main environmental risk factors contributing to crc development is chronic intestinal inflammation chronic inflammation alters cellular microenvironment enhances gene mutation inhibits apoptosis and induces neovascularisation and cell proliferation that causes precancerous conditions eventually leading cancer simultaneously chronic inflammation alterations that directly affect the stat3 pathway and promoting there are three stages involved in tumour development namely initiation promotion and progression during initiation and progression cancer cells and microbes interact both producing genetic inflammatory“immunological factors that are responsible for their survival and replication in tumour progression tumour cells interact with the inflammatory cells in the tumour microenvironment these tumour cells inflammatory“immunological factors to attract the inflammatory cells and the stromal cells simultaneously activate both stromal cells start to produce various soluble factors including growth factors and protease these soluble factors play an important role in facilitating the growth differentiation and survival of tumour cells hence it promotes tumour progression and promotion additionally cytokines or microbes promote cancer by changing genetic sequence during gene mutation epithelial cells inflammatory and activated carcinogenesis cytokines chemokines causes and secrete wwwmjmsusmmy 0creplicate rapidly and develop into a hyperplastic epithelium which progresses into adenomas and then towards adenocarcinomas both adenomas and adenocarcinomas affect the growth rate of colonic epithelial cells and improve the cells™ toleration towards apoptosis and abnormal cells escape from the immune cells furthermore these invade submucosa turning into cancer when the growth of malignant cells continues the tumour continues to spread in the colon thus carcinogenesis becomes more efficientadenocarcinomas begin to ibd is an example of chronic intestinal inflammation that is associated with etbf pathogenic bacteria are capable of stimulating infection inflammation and carcinogenesis whereas the relationship between ibd and crc is well established surprisingly patients with ibd show a high level of immunoglobulin ig g antibodies il6 vascular endothelial growth factor vegf and tumour necrosis factor tnf igg antibodies are responsible for killing bacteria moving into the intestinal lumen simultaneously il6 and vegf are responsible for stat3 activation ibd is also known as ulcerative colitis uc and crohn™s disease cd this chronic intestinal inflammation increases the risk of colitisassociated crc the probability of which depends on multiple casual factors including severity duration of inflammation in the intestine and gut microbiota imbalance “ patients with uc or cd have “ folds higher incidence of crc when compared to healthy individuals it is also stated that patients with uc and cd have and respectively higher risks of crc compared to a normal healthy person this indicates that patients with uc tend to be more susceptible to crc than those with cd furthermore it is evident that the large intestine tends to have a higher risk of crc compared to the small intestine which can be attributed to the higher amount of bacteria simultaneously people with ibd and crc have a higher quantity of etbf in the intestine or stool examination compared to healthy persons additionally etbf are biofilm producers they can reduce or redistribute ecadherin in the colonic epithelial cells trigger the production of il6 by epithelial cells activate stat3 pathway and enhance cells proliferation at the site of crypt epithelial in normal colon mucosa this shows that biofilms are associated with the risk of colon cancer development review article formation of colon cancercox enzymes involved in inflammation carcinogenesis and biomarkerchronic inflammation is a principal factor that contributes to carcinogenesis prostaglandin is a paracrine hormone that plays an important role in inflammation cyclooxygenase cox is the ratelimiting enzyme responsible for producing prostaglandins cox1 and cox2 are the isoforms of cox enzymes that break down arachidonic acid into prostaglandins cox2 plays an important role in maintaining environment for the development of cancer inflammation cox2 is normally expressed in epithelial and stromal cells and the expression level is increased in both inflammation and cancer due to the presence of proinflammatory cytokines additionally bft triggers colonic epithelial cells to express cox2 but not cox1 cox2 releases prostaglandin e2 pge2 that triggers pain and inflammation at the site of tissue injury simultaneously pge2 controls cell proliferation by binding at the cell receptor and activating oncogenic signalling pathways thus it is proven that cox2 plays an important role in carcinogenesis and cancer progression by promoting cell proliferation angiogenesis and cancer stem cell formation inhibiting cell apoptosis and heightening metastatic potential through producing pge2 “the in certain studies it is stated that aspirin and nonsteroidal antiinflammatory drugs have the ability to inhibit the activity of cox enzyme which reduces inflammatory response thus it delays crc occurrence fortunately cox1 and cox2 act as biomarkers for screening purposes the biomarker is defined as any substance structure or process that is measurable in the body to determine the incidence of a disease it is commonly detected in circulation and body fluids cox1 is present in most cells thus it is not a specific biomarker however cox2 is only detected when is stimulated by trauma release of cytokines and stimulation of arachidonate metabolism by a toxin such as bft thus cox2 acts as a useful biomarker to detect “ cox2 is also a useful biomarker for colorectal carcinogenesis screening the level of cox2 biomarker in the blood is dependent upon epithelial cell proliferation apoptosis inhibition and neoangiogenesis patients with crc have high levels of cox2 compared to normal individuals “ indicating more aggressive growth rate and higher mortality rate this inflammatory responses inflammation the wwwmjmsusmmy 0cmalays j med sci jul“aug “suggests that cox2 expression is correlated to the aggressiveness of growth rate and mortality rate etbf activates stat3immune is activated to eliminate regulation of etbf is associated with ibd due to the abnormal response to bacteria the systemic adaptive immune response foreign antigens in the body this action eventually reduces intestinal mucosal tolerance although immune cells kill foreign antigens neutrophils and th17 cells contribute to inflammation and tumourigenesis transcription factors are known as stat protein family comprising seven members each stat protein responds to its specific cytokines they play an important role in regulating immune responses by controlling th cell types generation for instance the activation and generation of th17 cells require transcription factor stat3 protein the roles of stat3 protein include promotion of cell proliferation cell survival inflammation cellular transformation metastasis of cancer blood vessel formation and tumourpromoting moreover stat3 intrinsic pathway for cancer inflammation it induces genes in tumour cells that are responsible for inflammation within a tumour cell it exhibits an overly expressed stat3 pathway inflammation is a major factors etbf has the ability to activate stat3 rapidly in both colonic epithelial cells and colonic mucosal immune cells through phosphorylation and nuclear translocation however stat3 activation first occurs in colonic mucosal immune cells followed by colonic epithelial cells to activate stat3 in immune cells epithelial cells should respond in the production of cytokines such as il6 il10 and il23 besides cytokines growth including vegf and fibroblast growth factor fgf2 are also involved in activating stat3 when etbf and bft first interact with colonic epithelial cells they stimulate early stat3 activation in colonic mucosal immune cells this stat3 activation continuously rises slowly until it reaches the peak level the peak indicates that etbf activates the immune system due to barrier dysfunction during etbfinduced colitis it activates both stat3 and th17 immune response in the colonic mucosa stat3 activation induces prooncogenic inflammatory pathways and increases the permeability of mucosa although stat3 activation is longterm and lasts for months it highly increases the chance of getting a tumour as a result of chronic inflammation additionally stat3 activation promotes the accumulation of tumour regulatory tcell tregs and blocks the generation of antitumour immune responses which give an adverse effect to the body this abnormal persistent stat3 activation increases the cancer cell tolerance prevents rejection of cancer by the immune system reduces the effectiveness of immunotherapy and enhances the effectiveness of oncogenesis activated stat3 predominantly detected in human cancers is constitutively activated and depicts its association with neoplasms patients with ibd tend to show stat3 activation and a high level of th17 cells and il17 the level of activated stat3 in patients with ibd and dysplasia is different from patients with ibd and without dysplasia patients with ibd and dysplasia show a higher level of activated stat3 compared to those without dysplasia simultaneously the level of activated stat3 increases together with the continuum of dysplasia to colitisassociated cancer it is clear that b fragilis can either be toxigenic or nontoxigenic the latter does not activate stat3 because it does not produce bft therefore ntbf does not contribute to colon cancer development but etbf does are tregs th17 and il17 good or badreduces intestinal il17 this process in a normal healthy condition tregs play an important role in inflammatory responses and immune homeostasis they express high levels of il2 receptor and produce endogenous il2 which inhibits the production of intestinal inflammation and prevents carcinogenesis however when etbf colonises a particular site of the colon it produces a large amount of bft damaging the intestinal mucosa to initiate etbftriggered colitis with the activation of the stat3 pathway this leads to direct contact between tregs and etbf and promotes tregs activation activated tregs lack the ability to produce endogenous il2 “ instead of producing endogenous il2 tregs consume exogenous il2 for their survival the consumption of exogenous il2 by tregs reduces the levels of exogenous il2 and produces an environment that favours the growth of th17 cells as the levels of il2 drop th17 cells are no longer inhibited and undergo expansion to produce a large quantity of naïve tcells this naïve subset of tcells then differentiates into th17 cells in excess wwwmjmsusmmy 0ccarcinogenesis this shows that colonisation of etbf promotes the accumulation of both tregs and th17 cells “ th17 cells start to produce large amounts of cytokines including tnf and il17 these cytokines promote cell survival and proliferation during injuries although th17 cells heal an injured site they turn into pathogenic th17 cells when deregulated these pathogenic th17 cells initiate chronic inflammatory condition il17 produced by pathogenic th17 cells are involved in an early inflammatory stage of the injuries it promotes tumour cell survival proliferation tumour neovascularisation and metastasis which allow “ additionally tumour cells and fibroblasts are stimulated by il17 to produce high amounts of angiogenic factors for angiogenesis il17 can activate stat3 pathway indirectly through il6 when il17 binds to il17 receptorbearing tumour cells it stimulates il6 production that is highly important for stat3 pathway activation as mentioned above this stat3 pathway activation contributes to several characteristics such as cancer proliferation antiapoptosis and angiogenesis that favour carcinogenesis in the colon this shows that there is a relationship between stat3 pathway and tregs in contributing to crc formation when etbf is accumulating in the intestinal tract as shown in figure to some extent stat5 and stat6 have been reported to be involved in inhibiting antitumour immunity when all stat3 and are activated together it highly enhances the tumourigenesis effect cleavage of ecadherin stimulate cell proliferationapart from inflammation bft alters the structure and function of colon epithelial cells by degrading ecadherin ecadherin is a 120kda glycoprotein that is the major structural protein in zonula adherens and is also known to be a tumour suppressor and zonula adherence protein this protein is responsible for the epithelial polarity in normal conditions the expression of ecadherin is linked to cellular functions including apoptosis and homotypic cell“cell “ unfortunately when ecadherin interacts with bft in the intestinal epithelial cells it degrades ecadherin rapidly in an atpindependent manner this cleavage promotes colonic injury inflammation and loss of membraneassociation resulting in morphological enhances cellular metastatic potential it is proven that changes and adhesion review article formation of colon canceretbfbftintestinal lumenepithelial cells†“ il2intestinal immune system†‘ th17tnfcell proliferation†‘ il17tregsinflammationcarcinogenesisil17 receptorbearing tumour cellsstat3 activationfigure the mechanism through abnormal systemof intestinal carcinogenesis immune factordependent the cleavage of ecadherin correlates with the changes of cell morphologies simultaneously degradation of ecadherin also promotes the binding of nuclear localisation of βcatenin and tcell transcriptional activator this binding promotes gene regulation and transcription additionally βcatenin plays an important role in wingless and int wnt signalling pathway promoting cell proliferation and epithelialmesenchymal transition and enhancing the expression of protooncogene in primary colorectal tumours cells in the centre of the tumour exhibit the presence of βcatenin and ecadherin however when the cells move away from the centre of the tumour they exhibit high amounts of nuclear βcatenin and the junction of ecadherin is lost important role ecadherin plays an in maintaining the morphology of cells there is a relationship with the ecadherin and the apical factin ring of the intestinal epithelial cells™ secretion when the loss of ecadherin increases the integrity of the apical factin is lost resulting in the increase in cell volume and chloride secretion and cell and epithelial barriers become wwwmjmsusmmy 0cmalays j med sci jul“aug “more permeable this contributes to intestinal inflammation diarrhoea and colon carcinogenesisalteration of the signalling pathway of colorectal cancerbft is involved in many colonic epithelial cell signal transductions when bft disturbs or activates the signalling pathway it brings adverse effects to the body and can lead to colorectal tumourigenesis figure the colonic epithelial cell signal transduction transpires through the nuclear factor kappalightchainenhancer of activated bcells nfκb wnt and mitogenactivated protein kinase mapk signalling pathways bft can stimulate nfκb pathway in the intestinal epithelial cells with the expression of heme oxygenase1 ho1 and cytokines to induce mucosal inflammation this pathway has the ability to enhance the survival of neoplastic cells by preventing them from undergoing apoptosis leading to tumour formation furthermore in figure it shows that when nfκb of intestinal epithelial cells is activated for a long time it induces the activity of nitric oxide synthase that breaks down larginine to produce nitric oxide which can damage cellular dna wnt signalling pathway is important to maintain the structures of the intestinal epithelium however wnt signalling pathway contributes negatively and affects cells which are extremely important for colorectal carcinogenesis and progression as wnt signalling pathway is activated it weakens tight junctions and reduces cellular adhesion this allows the cancer cells to undergo migration and metastasis hence cancerous cells can migrate to another ans spermine oxidase is a catabolic enzyme that increases ros which can be upregulated by bft in normal conditions figure ros acts as an important mediator in multiple cell signalling pathways and immune response that is produced naturally within biological systems it consists of superoxide hydroxyl radical and hydrogen peroxide however as the amount of ros becomes excessive it imparts negative effects in the disruption of redox homeostasis figure this excessive ros induces oxidative stress it oxidises cellular components including dna lipids and proteins within the cells etbfbftbftepithelial cellactivation of signaling pathwaynfκbwntnitric oxide synthase — reduce cell adhesion — weaken tight junction — cancer cell migratenitric oxidedamage cellular dnafigure the role of the signalling pathways when epithelial cells contact bftspermine oxidase smorelative oxygen species ros “ mediatorimmune responsemultiple cell signalling pathwaysfigure normal condition of the smo and ros that helps in immune response and cell signalling pathwayswwwmjmsusmmy 0conce the cellular components are oxidised it generates irreversible damage to host cells additionally ros plays an important role in the survival of cancer cells enhancing the effectiveness of carcinogenesis and aggravating cancer formed in the body “spermine oxidase smo “ upregulated†‘ relative oxygen species ros “ mediatordnalipidproteinirreversible damagecarcinogenesisfigure the adverse effect of smo contacted bftfamilial adenomatous polyposisfactors contributes the combination of both genetic and environmental to crc formation it is estimated that of crc development is due to genetic predisposition wherein nearly of all crcs are attributed to familial adenomatous polyposis fap fap is an autosomal dominant inherited disorder that describes the development of numerous colorectal adenomatous polyps these polyps are able to develop in the teenager™s colon meanwhile the number of polyps formed in the colon depends on the age of a person which means the number of polyps is directly proportional to the age of a person if these polyps are not removed from the colon they may transform from benign to malignant developing crc the source of fap disease is mainly due in the adenomatous to germline mutation polyposis coli apc gene “ this apc mutation occurs due to frameshifts insertions or deletions that may introduce a premature stop codon during the halfway through the transcription process these earlyintroduced premature stop codons in the gene sequence lead to incompletetruncated apc protein formation thus the normal function of apc protein is lost eventually facilitating carcinogenesis review article formation of colon canceradditionally germline mutations along with somatic mutations of the normal allele or loss of the normal allele lead to inactivation of apc once apc is inactivated it precisely commences carcinogenesis in normal conditions apc pathway acts as a gatekeeper controlling a part of wnt signalling pathway unfortunately when apc is mutated the function of apc pathway is lost or inactivated this inactivation of the apc pathway results in the activation of wnt signalling pathway this characteristic is mainly found in crc moreover apc mutation has the ability to alter bacteria“host epithelial interaction where it allows the bacteria to attach onto the mucosa if a person has the apcmutated gene and is exposed to etbf the chances of developing crc are high concurrently high amount of tumour load is displayed in the person™s colon conclusionthe human gastrointestinal tract contains its own bacterial flora that benefit humans daily b fragilis is one of them and consists of two classes namely ntbf and etbf the differences between both the classes is the presence of bft etbf is able to produce bft that can disrupt the intestinal environment and promotes inflammation simultaneously bft degrade ecadherin and causes inflammation ibd is a chronic intestinal inflammation associated with etbf and can induce crc however patients with cd have lower risk of developing crc as compared to those with uc patients with ibd exhibit stat3 activation due to the stimulation of immune response that favours th17 cell generation as the levels of th17 cell increase it brings a huge disadvantage to the intestinal tract due to the production of il17 furthermore il17 stimulates the production of il6 that is required to activate stat3 this indicates that the stat3 pathway activates for a long time longterm stat3 activation blocks antitumour immune response which supports the growth of cancer cells thus stat3 th17 and il17 are highly important in carcinogenesis concurrently the production of proinflammatory cytokines at the site of inflammation triggers the production of cox2 enzymes that release pge2 cox2 is also known for its carcinogenic abilities due to the production of pge2 that controls cell proliferation additionally bft affects signal transductions such as wnt wwwmjmsusmmy 0cmalays j med sci jul“aug “nfκb and mapk signalling pathways and induces tumourigenesis considering that bft induces inflammation activates stat3 and alters signalling pathways it can be concluded that bft produced by etbf plays an important role in colon carcinogenesis boleij a hechenbleikner em goodwin ac badani r stein em lazarev mg et al the bacteroides fragilis toxin gene is prevalent the colon mucosa of colorectal cancer in patients clin infect dis “ httpsdoi101093cidciu787acknowledgementsnoneconflict of interestnonefundsnoneauthors™ contributionsconception and design hkkdrafting of the article with supervision cwtcritical revision of the article for important intellectual content fdfinal approval of the article hkk fdcorrespondencedr fabian davamaniphd microbiology university of madrasfaculty of biomedical scienceschool of health sciences international medical university kuala lumpur malaysia tel fax email fabian_davamaniimuedumyreferences snezhkina av krasnov gs lipatova av sadritdinova af kardymon ol fedorova ms et al the dysregulation of polyamine metabolism in colorectal cancer is associated with overexpression of cmyc and cebpβ rather than enterotoxigenic bacteroides fragilis infection oxid med cell longev “ httpsdoi10115520162353560 sears cl geis al housseau f bacteroides fragilis from carcinogenesis j clin symbiont invest “ httpsdoi101172jci72334subverts mucosal to biology colon lv y ye t wang h zhao j chen w wang x et al suppression of colorectal tumorigenesis by recombinant bacteroides fragilis enterotoxin2 in vivo world j gastroenterol “ httpsdoi103748wjgv23i4603 pierce jv bernstein hd genomic diversity of enterotoxigenic strains of bacteroides fragilis plos one 2016116e0158171 httpsdoi 101371journalpone0158171 sun j kato i gut microbiota inflammation and colorectal cancer genes dis “ httpsdoi101016jgendis201603004 erdrich j zhang x giovannucci e willett w proportion of colon cancer attributable to lifestyle in a cohort of us women cancer causes control “ httpsdoi101007s105520150619z mughinigras l schaapveld m kramers j mooij s neefjesborst ea van pelt w et al increased colon cancer risk after severe salmonella infection plos one 2018131e0189721 httpsdoi101371journalpone0189721 francescone r hou v grivennikov si microbiome inflammation and cancer cancer j “ httpsdoi101097ppo0000000000000048 wick ec rabizadeh s albesiano e wu x wu s chan j et al stat3 activation in murine enterotoxigenic bacteroides inflamm bowel dis “ httpsdoi101097mib0000000000000019induced by fragili

cancer281

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: the bacteroides fragilis b fragilis produce biofilm for colonisation in the intestinal tract can cause a series of inflammatory reactions due to b fragilis toxin bft which can lead to chronic intestinal inflammation and tissue injury and play a crucial role leading to colorectal cancer crc the enterotoxigenic b fragilis etbf forms biofilm and produce toxin and play a role in crc whereas the nontoxigenic b fragilis ntbf does not produce toxin the etbf triggers the expression of cyclooxygenase cox2 that releases pge2 for inducing inflammation and control cell proliferation from chronic intestinal inflammation to cancer development it involves signal transducers and activators of transcription stat3 activation stat3 activates by the interaction between epithelial cells and bft thus regulatory tcell tregs will activates and reduce interleukin il2 amount as the level of il2 drops thelper th17 cells are generated leading to increase in il17 levels il17 is implicated in early intestinal inflammation and promotes cancer cell survival and proliferation and consequently triggers il6 production that activate stat3 pathway additionally bft degrades ecadherin hence alteration of signalling pathways can upregulate spermine oxidase leading to cell morphology and promote carcinogenesis and irreversible dna damage patient with familial adenomatous polyposis fap disease displays a high level of tumour load in the colon this disease is caused by germline mutation of the adenomatous polyposis coli apc gene that increases bacterial adherence to the mucosa layer mutatedapc gene genotype with etbf increases the chances of crc development therefore the colonisation of the etbf in the intestinal tract depicts tumour aetiology can result in risk of hostility and effect on human health keywords bacteroides fragilis colon cancer stat3 pathway bacteroides fragilis toxin inflammationintroductionbacteroides species are nonspore forming anaerobe and gramnegative bacteria there are more than different species of bacteroides these bacteria act as normal flora in the intestine to maintain healthy intestinal microflora in humans bacteroides fragilis b fragilis has two classes nontoxigenic b fragilis ntbf and enterotoxigenic b fragilis etbf the differences between ntbf and etbf are the presence of b fragilis toxin bft gene and its ability to produce biofilm bft product is a kda zincdependent metalloprotease toxin also known as fragilysin or bft “ bft plays an important role in intestinal inflammation and tissue injury by damaging the tight junction and increasing intestinal permeability furthermore it has been proven that tissue inflammation and injury promote cancer formation simultaneously the biofilm produced by b fragilis induces carcinogenesis fortunately only etbf encompasses bft and can produce biofilms hence ntbf does not harm the intestinal tract malays j med sci jul“aug “wwwmjmsusmmy penerbit universiti sains malaysia this work is licensed under the terms of the creative commons attribution cc by httpcreativecommonslicensesby40 0cmalays j med sci jul“aug “in the united states colorectal cancer crc is the third most common cancer in both genders it is also the second most common cancerrelated death especially for older patients who are ‰¥ years old in the american cancer society stated that there were new cases of crcs that led to the death of people moreover crc is the fourth leading cancer resulting in deaths worldwide inflammatory bowel disease ibd and genetic mutations are factors predisposing an individual towards colon cancer this indicates that crc has a high mortality rate “microbes are capable in promoting cancer development through several routes such as activation of chronic inflammation alteration of tumour microenvironment and production of toxins that damage dna when there is chronic etbf colonisation in the intestine it stimulates chronic intestinal inflammation triggering signal transducers and activators of transcription stat3 activation which contributes to interleukin il17 production il17 is involved in colon inflammation bft produced by etbf causes the alteration of signalling pathways and production of reactive oxygen species ros that leads to dna damage and cleavage of ecadherin in the below review we have provided a general information regarding bft produced by etbf triggering crc developmentliterature reviewcolon cancer associated with microbespromote nutrition in the human gastrointestinal tract there are nearly trillion microbes out of which make up normal flora in the intestine meanwhile the normal flora is characterised into beneficial and harmful microbes beneficial microbes including production of vitamins in the intestine and prevent disease formation however harmful microbes produce carcinogenic toxin and substances in the intestine these harmful substances may cause cancer there are many types of bacteria that stimulate a variety of cancer formation through their respective site of inflammation eg bacteria such as enterococcus faecalis e faecalis colibactinproducing escherichia coli e coli and etbf are involved in crc development however the mechanisms between each bacterium in contributing to crc formation are different for instance e faecalis damages the dna through ros colibactinproducing e coli produces colibactin that damages the dna and etbf produces bft that contributes to inflammation and immunecell infiltration intestinal dysbiosis inflammation and colon cancergenetic normal flora is advantageous to a person as it maintains intestinal health and gut homeostasis however as the bacteria such as etbf in the gut undergoes dysbiosis it brings harmful effects to the person according to deng et al a correlation was observed between microbiota imbalance and cancer progression while liu et al claimed is associated with that crc development intestinal microecology disorder imbalance among microbiota leads to bacterial infection that can progress to chronic inflammation one of the main environmental risk factors contributing to crc development is chronic intestinal inflammation chronic inflammation alters cellular microenvironment enhances gene mutation inhibits apoptosis and induces neovascularisation and cell proliferation that causes precancerous conditions eventually leading cancer simultaneously chronic inflammation alterations that directly affect the stat3 pathway and promoting there are three stages involved in tumour development namely initiation promotion and progression during initiation and progression cancer cells and microbes interact both producing genetic inflammatory“immunological factors that are responsible for their survival and replication in tumour progression tumour cells interact with the inflammatory cells in the tumour microenvironment these tumour cells inflammatory“immunological factors to attract the inflammatory cells and the stromal cells simultaneously activate both stromal cells start to produce various soluble factors including growth factors and protease these soluble factors play an important role in facilitating the growth differentiation and survival of tumour cells hence it promotes tumour progression and promotion additionally cytokines or microbes promote cancer by changing genetic sequence during gene mutation epithelial cells inflammatory and activated carcinogenesis cytokines chemokines causes and secrete wwwmjmsusmmy 0creplicate rapidly and develop into a hyperplastic epithelium which progresses into adenomas and then towards adenocarcinomas both adenomas and adenocarcinomas affect the growth rate of colonic epithelial cells and improve the cells™ toleration towards apoptosis and abnormal cells escape from the immune cells furthermore these invade submucosa turning into cancer when the growth of malignant cells continues the tumour continues to spread in the colon thus carcinogenesis becomes more efficientadenocarcinomas begin to ibd is an example of chronic intestinal inflammation that is associated with etbf pathogenic bacteria are capable of stimulating infection inflammation and carcinogenesis whereas the relationship between ibd and crc is well established surprisingly patients with ibd show a high level of immunoglobulin ig g antibodies il6 vascular endothelial growth factor vegf and tumour necrosis factor tnf igg antibodies are responsible for killing bacteria moving into the intestinal lumen simultaneously il6 and vegf are responsible for stat3 activation ibd is also known as ulcerative colitis uc and crohn™s disease cd this chronic intestinal inflammation increases the risk of colitisassociated crc the probability of which depends on multiple casual factors including severity duration of inflammation in the intestine and gut microbiota imbalance “ patients with uc or cd have “ folds higher incidence of crc when compared to healthy individuals it is also stated that patients with uc and cd have and respectively higher risks of crc compared to a normal healthy person this indicates that patients with uc tend to be more susceptible to crc than those with cd furthermore it is evident that the large intestine tends to have a higher risk of crc compared to the small intestine which can be attributed to the higher amount of bacteria simultaneously people with ibd and crc have a higher quantity of etbf in the intestine or stool examination compared to healthy persons additionally etbf are biofilm producers they can reduce or redistribute ecadherin in the colonic epithelial cells trigger the production of il6 by epithelial cells activate stat3 pathway and enhance cells proliferation at the site of crypt epithelial in normal colon mucosa this shows that biofilms are associated with the risk of colon cancer development review article formation of colon cancercox enzymes involved in inflammation carcinogenesis and biomarkerchronic inflammation is a principal factor that contributes to carcinogenesis prostaglandin is a paracrine hormone that plays an important role in inflammation cyclooxygenase cox is the ratelimiting enzyme responsible for producing prostaglandins cox1 and cox2 are the isoforms of cox enzymes that break down arachidonic acid into prostaglandins cox2 plays an important role in maintaining environment for the development of cancer inflammation cox2 is normally expressed in epithelial and stromal cells and the expression level is increased in both inflammation and cancer due to the presence of proinflammatory cytokines additionally bft triggers colonic epithelial cells to express cox2 but not cox1 cox2 releases prostaglandin e2 pge2 that triggers pain and inflammation at the site of tissue injury simultaneously pge2 controls cell proliferation by binding at the cell receptor and activating oncogenic signalling pathways thus it is proven that cox2 plays an important role in carcinogenesis and cancer progression by promoting cell proliferation angiogenesis and cancer stem cell formation inhibiting cell apoptosis and heightening metastatic potential through producing pge2 “the in certain studies it is stated that aspirin and nonsteroidal antiinflammatory drugs have the ability to inhibit the activity of cox enzyme which reduces inflammatory response thus it delays crc occurrence fortunately cox1 and cox2 act as biomarkers for screening purposes the biomarker is defined as any substance structure or process that is measurable in the body to determine the incidence of a disease it is commonly detected in circulation and body fluids cox1 is present in most cells thus it is not a specific biomarker however cox2 is only detected when is stimulated by trauma release of cytokines and stimulation of arachidonate metabolism by a toxin such as bft thus cox2 acts as a useful biomarker to detect “ cox2 is also a useful biomarker for colorectal carcinogenesis screening the level of cox2 biomarker in the blood is dependent upon epithelial cell proliferation apoptosis inhibition and neoangiogenesis patients with crc have high levels of cox2 compared to normal individuals “ indicating more aggressive growth rate and higher mortality rate this inflammatory responses inflammation the wwwmjmsusmmy 0cmalays j med sci jul“aug “suggests that cox2 expression is correlated to the aggressiveness of growth rate and mortality rate etbf activates stat3immune is activated to eliminate regulation of etbf is associated with ibd due to the abnormal response to bacteria the systemic adaptive immune response foreign antigens in the body this action eventually reduces intestinal mucosal tolerance although immune cells kill foreign antigens neutrophils and th17 cells contribute to inflammation and tumourigenesis transcription factors are known as stat protein family comprising seven members each stat protein responds to its specific cytokines they play an important role in regulating immune responses by controlling th cell types generation for instance the activation and generation of th17 cells require transcription factor stat3 protein the roles of stat3 protein include promotion of cell proliferation cell survival inflammation cellular transformation metastasis of cancer blood vessel formation and tumourpromoting moreover stat3 intrinsic pathway for cancer inflammation it induces genes in tumour cells that are responsible for inflammation within a tumour cell it exhibits an overly expressed stat3 pathway inflammation is a major factors etbf has the ability to activate stat3 rapidly in both colonic epithelial cells and colonic mucosal immune cells through phosphorylation and nuclear translocation however stat3 activation first occurs in colonic mucosal immune cells followed by colonic epithelial cells to activate stat3 in immune cells epithelial cells should respond in the production of cytokines such as il6 il10 and il23 besides cytokines growth including vegf and fibroblast growth factor fgf2 are also involved in activating stat3 when etbf and bft first interact with colonic epithelial cells they stimulate early stat3 activation in colonic mucosal immune cells this stat3 activation continuously rises slowly until it reaches the peak level the peak indicates that etbf activates the immune system due to barrier dysfunction during etbfinduced colitis it activates both stat3 and th17 immune response in the colonic mucosa stat3 activation induces prooncogenic inflammatory pathways and increases the permeability of mucosa although stat3 activation is longterm and lasts for months it highly increases the chance of getting a tumour as a result of chronic inflammation additionally stat3 activation promotes the accumulation of tumour regulatory tcell tregs and blocks the generation of antitumour immune responses which give an adverse effect to the body this abnormal persistent stat3 activation increases the cancer cell tolerance prevents rejection of cancer by the immune system reduces the effectiveness of immunotherapy and enhances the effectiveness of oncogenesis activated stat3 predominantly detected in human cancers is constitutively activated and depicts its association with neoplasms patients with ibd tend to show stat3 activation and a high level of th17 cells and il17 the level of activated stat3 in patients with ibd and dysplasia is different from patients with ibd and without dysplasia patients with ibd and dysplasia show a higher level of activated stat3 compared to those without dysplasia simultaneously the level of activated stat3 increases together with the continuum of dysplasia to colitisassociated cancer it is clear that b fragilis can either be toxigenic or nontoxigenic the latter does not activate stat3 because it does not produce bft therefore ntbf does not contribute to colon cancer development but etbf does are tregs th17 and il17 good or badreduces intestinal il17 this process in a normal healthy condition tregs play an important role in inflammatory responses and immune homeostasis they express high levels of il2 receptor and produce endogenous il2 which inhibits the production of intestinal inflammation and prevents carcinogenesis however when etbf colonises a particular site of the colon it produces a large amount of bft damaging the intestinal mucosa to initiate etbftriggered colitis with the activation of the stat3 pathway this leads to direct contact between tregs and etbf and promotes tregs activation activated tregs lack the ability to produce endogenous il2 “ instead of producing endogenous il2 tregs consume exogenous il2 for their survival the consumption of exogenous il2 by tregs reduces the levels of exogenous il2 and produces an environment that favours the growth of th17 cells as the levels of il2 drop th17 cells are no longer inhibited and undergo expansion to produce a large quantity of naïve tcells this naïve subset of tcells then differentiates into th17 cells in excess wwwmjmsusmmy 0ccarcinogenesis this shows that colonisation of etbf promotes the accumulation of both tregs and th17 cells “ th17 cells start to produce large amounts of cytokines including tnf and il17 these cytokines promote cell survival and proliferation during injuries although th17 cells heal an injured site they turn into pathogenic th17 cells when deregulated these pathogenic th17 cells initiate chronic inflammatory condition il17 produced by pathogenic th17 cells are involved in an early inflammatory stage of the injuries it promotes tumour cell survival proliferation tumour neovascularisation and metastasis which allow “ additionally tumour cells and fibroblasts are stimulated by il17 to produce high amounts of angiogenic factors for angiogenesis il17 can activate stat3 pathway indirectly through il6 when il17 binds to il17 receptorbearing tumour cells it stimulates il6 production that is highly important for stat3 pathway activation as mentioned above this stat3 pathway activation contributes to several characteristics such as cancer proliferation antiapoptosis and angiogenesis that favour carcinogenesis in the colon this shows that there is a relationship between stat3 pathway and tregs in contributing to crc formation when etbf is accumulating in the intestinal tract as shown in figure to some extent stat5 and stat6 have been reported to be involved in inhibiting antitumour immunity when all stat3 and are activated together it highly enhances the tumourigenesis effect cleavage of ecadherin stimulate cell proliferationapart from inflammation bft alters the structure and function of colon epithelial cells by degrading ecadherin ecadherin is a 120kda glycoprotein that is the major structural protein in zonula adherens and is also known to be a tumour suppressor and zonula adherence protein this protein is responsible for the epithelial polarity in normal conditions the expression of ecadherin is linked to cellular functions including apoptosis and homotypic cell“cell “ unfortunately when ecadherin interacts with bft in the intestinal epithelial cells it degrades ecadherin rapidly in an atpindependent manner this cleavage promotes colonic injury inflammation and loss of membraneassociation resulting in morphological enhances cellular metastatic potential it is proven that changes and adhesion review article formation of colon canceretbfbftintestinal lumenepithelial cells†“ il2intestinal immune system†‘ th17tnfcell proliferation†‘ il17tregsinflammationcarcinogenesisil17 receptorbearing tumour cellsstat3 activationfigure the mechanism through abnormal systemof intestinal carcinogenesis immune factordependent the cleavage of ecadherin correlates with the changes of cell morphologies simultaneously degradation of ecadherin also promotes the binding of nuclear localisation of βcatenin and tcell transcriptional activator this binding promotes gene regulation and transcription additionally βcatenin plays an important role in wingless and int wnt signalling pathway promoting cell proliferation and epithelialmesenchymal transition and enhancing the expression of protooncogene in primary colorectal tumours cells in the centre of the tumour exhibit the presence of βcatenin and ecadherin however when the cells move away from the centre of the tumour they exhibit high amounts of nuclear βcatenin and the junction of ecadherin is lost important role ecadherin plays an in maintaining the morphology of cells there is a relationship with the ecadherin and the apical factin ring of the intestinal epithelial cells™ secretion when the loss of ecadherin increases the integrity of the apical factin is lost resulting in the increase in cell volume and chloride secretion and cell and epithelial barriers become wwwmjmsusmmy 0cmalays j med sci jul“aug “more permeable this contributes to intestinal inflammation diarrhoea and colon carcinogenesisalteration of the signalling pathway of colorectal cancerbft is involved in many colonic epithelial cell signal transductions when bft disturbs or activates the signalling pathway it brings adverse effects to the body and can lead to colorectal tumourigenesis figure the colonic epithelial cell signal transduction transpires through the nuclear factor kappalightchainenhancer of activated bcells nfκb wnt and mitogenactivated protein kinase mapk signalling pathways bft can stimulate nfκb pathway in the intestinal epithelial cells with the expression of heme oxygenase1 ho1 and cytokines to induce mucosal inflammation this pathway has the ability to enhance the survival of neoplastic cells by preventing them from undergoing apoptosis leading to tumour formation furthermore in figure it shows that when nfκb of intestinal epithelial cells is activated for a long time it induces the activity of nitric oxide synthase that breaks down larginine to produce nitric oxide which can damage cellular dna wnt signalling pathway is important to maintain the structures of the intestinal epithelium however wnt signalling pathway contributes negatively and affects cells which are extremely important for colorectal carcinogenesis and progression as wnt signalling pathway is activated it weakens tight junctions and reduces cellular adhesion this allows the cancer cells to undergo migration and metastasis hence cancerous cells can migrate to another ans spermine oxidase is a catabolic enzyme that increases ros which can be upregulated by bft in normal conditions figure ros acts as an important mediator in multiple cell signalling pathways and immune response that is produced naturally within biological systems it consists of superoxide hydroxyl radical and hydrogen peroxide however as the amount of ros becomes excessive it imparts negative effects in the disruption of redox homeostasis figure this excessive ros induces oxidative stress it oxidises cellular components including dna lipids and proteins within the cells etbfbftbftepithelial cellactivation of signaling pathwaynfκbwntnitric oxide synthase — reduce cell adhesion — weaken tight junction — cancer cell migratenitric oxidedamage cellular dnafigure the role of the signalling pathways when epithelial cells contact bftspermine oxidase smorelative oxygen species ros “ mediatorimmune responsemultiple cell signalling pathwaysfigure normal condition of the smo and ros that helps in immune response and cell signalling pathwayswwwmjmsusmmy 0conce the cellular components are oxidised it generates irreversible damage to host cells additionally ros plays an important role in the survival of cancer cells enhancing the effectiveness of carcinogenesis and aggravating cancer formed in the body “spermine oxidase smo “ upregulated†‘ relative oxygen species ros “ mediatordnalipidproteinirreversible damagecarcinogenesisfigure the adverse effect of smo contacted bftfamilial adenomatous polyposisfactors contributes the combination of both genetic and environmental to crc formation it is estimated that of crc development is due to genetic predisposition wherein nearly of all crcs are attributed to familial adenomatous polyposis fap fap is an autosomal dominant inherited disorder that describes the development of numerous colorectal adenomatous polyps these polyps are able to develop in the teenager™s colon meanwhile the number of polyps formed in the colon depends on the age of a person which means the number of polyps is directly proportional to the age of a person if these polyps are not removed from the colon they may transform from benign to malignant developing crc the source of fap disease is mainly due in the adenomatous to germline mutation polyposis coli apc gene “ this apc mutation occurs due to frameshifts insertions or deletions that may introduce a premature stop codon during the halfway through the transcription process these earlyintroduced premature stop codons in the gene sequence lead to incompletetruncated apc protein formation thus the normal function of apc protein is lost eventually facilitating carcinogenesis review article formation of colon canceradditionally germline mutations along with somatic mutations of the normal allele or loss of the normal allele lead to inactivation of apc once apc is inactivated it precisely commences carcinogenesis in normal conditions apc pathway acts as a gatekeeper controlling a part of wnt signalling pathway unfortunately when apc is mutated the function of apc pathway is lost or inactivated this inactivation of the apc pathway results in the activation of wnt signalling pathway this characteristic is mainly found in crc moreover apc mutation has the ability to alter bacteria“host epithelial interaction where it allows the bacteria to attach onto the mucosa if a person has the apcmutated gene and is exposed to etbf the chances of developing crc are high concurrently high amount of tumour load is displayed in the person™s colon conclusionthe human gastrointestinal tract contains its own bacterial flora that benefit humans daily b fragilis is one of them and consists of two classes namely ntbf and etbf the differences between both the classes is the presence of bft etbf is able to produce bft that can disrupt the intestinal environment and promotes inflammation simultaneously bft degrade ecadherin and causes inflammation ibd is a chronic intestinal inflammation associated with etbf and can induce crc however patients with cd have lower risk of developing crc as compared to those with uc patients with ibd exhibit stat3 activation due to the stimulation of immune response that favours th17 cell generation as the levels of th17 cell increase it brings a huge disadvantage to the intestinal tract due to the production of il17 furthermore il17 stimulates the production of il6 that is required to activate stat3 this indicates that the stat3 pathway activates for a long time longterm stat3 activation blocks antitumour immune response which supports the growth of cancer cells thus stat3 th17 and il17 are highly important in carcinogenesis concurrently the production of proinflammatory cytokines at the site of inflammation triggers the production of cox2 enzymes that release pge2 cox2 is also known for its carcinogenic abilities due to the production of pge2 that controls cell proliferation additionally bft affects signal transductions such as wnt wwwmjmsusmmy 0cmalays j med sci jul“aug “nfκb and mapk signalling pathways and induces tumourigenesis considering that bft induces inflammation activates stat3 and alters signalling pathways it can be concluded that bft produced by etbf plays an important role in colon carcinogenesis boleij a hechenbleikner em goodwin ac badani r stein em lazarev mg et al the bacteroides fragilis toxin gene is prevalent the colon mucosa of colorectal cancer in patients clin infect dis “ httpsdoi101093cidciu787acknowledgementsnoneconflict of interestnonefundsnoneauthors™ contributionsconception and design hkkdrafting of the article with supervision cwtcritical revision of the article for important intellectual content fdfinal approval of the article hkk fdcorrespondencedr fabian davamaniphd microbiology university of madrasfaculty of biomedical scienceschool of health sciences international medical university kuala lumpur malaysia tel fax email fabian_davamaniimuedumyreferences snezhkina av krasnov gs lipatova av sadritdinova af kardymon ol fedorova ms et al the dysregulation of polyamine metabolism in colorectal cancer is associated with overexpression of cmyc and cebpβ rather than enterotoxigenic bacteroides fragilis infection oxid med cell longev “ httpsdoi10115520162353560 sears cl geis al housseau f bacteroides fragilis from carcinogenesis j clin symbiont invest “ httpsdoi101172jci72334subverts mucosal to biology colon lv y ye t wang h zhao j chen w wang x et al suppression of colorectal tumorigenesis by recombinant bacteroides fragilis enterotoxin2 in vivo world j gastroenterol “ httpsdoi103748wjgv23i4603 pierce jv bernstein hd genomic diversity of enterotoxigenic strains of bacteroides fragilis plos one 2016116e0158171 httpsdoi 101371journalpone0158171 sun j kato i gut microbiota inflammation and colorectal cancer genes dis “ httpsdoi101016jgendis201603004 erdrich j zhang x giovannucci e willett w proportion of colon cancer attributable to lifestyle in a cohort of us women cancer causes control “ httpsdoi101007s105520150619z mughinigras l schaapveld m kramers j mooij s neefjesborst ea van pelt w et al increased colon cancer risk after severe salmonella infection plos one 2018131e0189721 httpsdoi101371journalpone0189721 francescone r hou v grivennikov si microbiome inflammation and cancer cancer j “ httpsdoi101097ppo0000000000000048 wick ec rabizadeh s albesiano e wu x wu s chan j et al stat3 activation in murine enterotoxigenic bacteroides inflamm bowel dis “ httpsdoi101097mib0000000000000019induced by fragili Answer:

Colon_Cancer

advances in technology hardware and computing have created new opportunities to improve the quality of cancer care and research by leveraging informatics innovations digital health machine learning and precision oncology are key areas where there are noteworthy advances in the field of cancer informatics as the adoption of smartphones and wearable technologies increases patientgenerated health data such as physical activity and electronic patientreported outcomes pros are being leveraged for prevention and in interventions for remote monitoring during treatment and survivorship and to predict health outcomes moving beyond cancer clinical trials health systems are increasingly seeking to integrate pros into routine cancer care processes as part of the national cancer institute™s nci cancer moonshot initiative six healthcare systems will systematically integrate pros into clinical workflows and electronic health records ehrs additionally much attention surrounds the hype and challenges of the application of machine learning algorithms in clinical care including for a number of oncology use cases however there are concerns around biases and homogeneous training data sets thus machine learning has had limited implementation into clinical care considerable progress in genomics and computational medicine has ushered in a new era in precision oncology generating vast amounts of data much of which is publicly accessible for example the nci precision medicine initiativeoncology has had recent success defining genetically targeted therapies understanding tumor treatment resistance and developing a us cancer knowledge system while the opportunities within cancer informatics have much potential for positive impact if not applied thoughtfully these innovations risk propagating disparities and inequities operationalizing these informatics discoveries into routine cancer care also continues to present a diverse set of challenges objectivesgiven the rapid pace of cancer informatics research our objective was to conduct a survey of the literature for advancements over the past several years in three key areas of growth digital health machine learning and precision oncology we also highlight the ethical implications challenges and opportunities for each topic methodsa literature search was conducted in two electronic databases pubmed and google scholar to identify peerreviewed s and conference proceedings search terms included variations of the following neoplasms[mesh] informatics[mesh] cancer oncology clinical cancer informatics medical cancer informatics the search covered the period from october to october imia yearbook of medical informatics imia and ge thieme verlag kg 0c and returned too many s for practical review from pubmed and from google scholar thus we narrowed the search to key pubmedindexed informatics s and proceedings such as the of the american medical informatics association jamia jamia open applied clinical informatics of medical internet research bioinformatics of biomedical informatics jco clinical cancer informatics and nature digital medicine we also performed a manual review of cancer informatics s that were not yet indexed in pubmed additionally we searched the proceedings of the amia annual symposia and amia informatics summits given the sheer number of results we focused on manuscripts that demonstrated a clear focus on clinical or translational cancer informatics manuscripts were selected based on methodological rigor scientific impact innovation and contributions towards cancer informatics as a field or on impact on cancer care the reviewed manuscripts were grouped into three topics including digital health machine learning and precision oncology for each topic we describe the current state implications challenges and opportunities results digital healthdigital health is the intersection of technologies with health and wellness to enhance communication and delivery of health services it includes mobile health health information technologies wearable devices telehealth or virtual health and personalized medicine as health systems transition from traditional officebased care to more continuous interactions with patients and caregivers emerging technologies are key drivers of the capture of patientgenerated health data currently it is still challenging to bring together disparate data streams generated by patients from technologies such as smartphones social media platforms wearable activity trackers and internet of things iot devices such as smart home sensors however multiple studies have begun to demonstrate the value of employing digital health technologies to improve cancer prevention and care for example apps and webbased interventions have been used to support exercise and in digital outreach efforts focused on cancer screening in a webbased exercise intervention to mitigate the effects of chemotherapy breast cancer patients demonstrated improvements on the sixminute walk test and abdominal back lower body strength compared to the control group over the eightweek study one study demonstrated that google search and facebook advertisements could be used to promote cancer screening these advertising campaigns were associated with increased visits to institutional websites and scheduled screening exams compared to the week before and after the campaign these studies reveal the potential for digital health apps to engage patients reach broad populations and improve physical activity outcomes in addition to being a vehicle to deliver interventions social media can be used for behavioral surveillance several studies explored communications within patient communities with the most common posts or tweets on the topics of clinical trials treatment and support taylor found evidence of differing levels of emotional informational and social support for patients with lung cancer across platforms ie facebook twitter macmillanuk which may be due to differences in user characteristics or the nature of selfmoderating communities in terms of engagement cho examined the relationship between melanomarelated instagram posts and engagement outcomes ie likes comments social support they found that posts about physical consequences decreased the number of œlikes but increased comments and perceived emotional support and that the inclusion of images increased the number of comments made about the posts overall these highlighted studies contribute to an understanding of participative engagement and experiences on social media platforms for written and visual cancerrelated content that is usergenerated and suggest that patients may have varied experiences related to social support across platforms and communitiesthere is mounting evidence that using pros for cancer care symptom assessments can improve survival quality of life and help patients remain longer on treatment when compared to usual care while there is a wide range of pros that could be assessed during cancer treatment and in survivorship several efforts have aimed to identify key domains relevant to improving outcomes in cancer care for example a clinical advisory group identified priority domains and measures to enhance cancer care which included distress and symptoms and unmet needs girgis then developed clinical recommendations from these domains mapped them into wellbeing categories and demonstrated that these recommendations could algorithmically provide automated clinical feedback there is also concern that the use of structured questionnaires to assess pros may limit the ability to capture the broad range of symptoms experienced by patients during treatment chung et al examined whether patiententered freetext narratives of symptomatic adverse events could be mapped to established terminologies from the national cancer institute nci patientreported outcomes version of the common terminology criteria for adverse events proctcae among patients enrolled in three multicenter cancer trials over half provided unstructured freetext entries and most of these entries could be mapped post hoc to a proctcae or medical dictionary for regulatory activities meddra term this suggests that mapping to existing terminologies is not only feasible but also provides the opportunity to supplement trialspecific questionnaires to better capture diverse patient experiences during cancer treatmentsuse of wearable activity trackers and other iot sensors to capture additional patientgenerated health data is expanding several feasibility studies have had favorable results although sample size was generally small studies noted that higher daily steps recorded from activity trackers were correlated with pros such as increased performance status lower distress [ ] fatigue depressive symptoms [ ] adverse events hospitalizations and death despite high dropoff rates for imia yearbook of medical informatics 2020griffin 0cnetwork to classify topics within an online health community focused on breast cancer and found that patients with different cancer stages have different topics of conversations lee et al assessed conversations about living with cancer among sexual minority women using latent dirichl location which revealed that the most common topic was about coping and connecting to others with shared gender identities utilizing nlp techniques to analyze and understand topics in online communities could help to optimize interventions and deliver valuable supportrecognizing the ongoing challenges with concept extraction and the scarcity of annotated datasets researchers have employed open science efforts to foster interdisciplinary collaborations in several areas for example temporal history of your medical events thyme corpora [ ] informatics for integrating biology the bedside i2b2 challenge sets and medical information mart of intensive care mimiciii contain collections of deidentified clinical text for research dream challenges have also hosted several cancerfocused competitions that yielded a number of r packages and established benchmarks for prognostic models [ ]in medical imaging ml has been deployed for early detection improved diagnosis and better prognostic accuracy ardila used lowdose chest computed tomography images to perform endtoend lung cancer screening with high accuracy similarly predictive models to calculate breast cancer risk from mammograms demonstrated improved risk discrimination over current clinical standards in dermatology several deep learning methods were found to be superior to dermatologists in diagnosing and detecting skin cancers [ ] wei also found employing deep learning approaches to classify lung pathology images yielded more accurate results when compared to pathologists additionally advancements have been made in mlbased cds tools for classification of skin lesions estimates of colon cancer outcomes based on various therapies and management of uterine cervical abnormalities given the lack of large imaging datasets for training models investigators have also focused on producing realistic synthetic data [ ] research by senaras revealed that generative adversarial networks are a promising technique to generate labeled synthetic immunohistochemical stained breast tissue for precision cancer care genomics tools have also demonstrated the potential to advance the discovery of novel targeted cancer therapies deepmind™s alphafold tool has shown promise in predicting protein structure from its genetic sequence crisprcas9 screens have also facilitated tremendous progress in identifying essential genes across cancer cell lines several studies have used ml to identify cancer subtypes using gene expression data and pathological images for example coudray established that ml algorithms could predict six of the most commonly mutated genes within adenocarcinoma lung cancer ml could also be used to assist pathologists in detecting gene mutations or cancer subtypes given the variety of data sources necessary to compare personalized cancer care pathways incorporating genetic clinical imaging cost and quality of life data into multifaceted cds tools could aid in reducing the cognitive load of clinicians precision oncologyprecision medicine is an evolving set of diagnostic and treatment paradigms which seeks to optimize and tailor safe and effective care for patients based on their individual characteristics precision medicine has been advanced considerably by cancer genomics increasingly discoveries using genomic technologies that have fueled translational research are impacting clinical settings single cell level assays are proving transformational in the understanding of tumor heterogeneity disease biology and treatment resistance and will likely play an increasingly important role in clinical research and precision medicine applications several studies have demonstrated the feasibility of large cohorts prospectively assayed using genomic technologies with turnaround times short enough to impact wear time for activity trackers [ ] this research highlights that wearable devices could provide accurate assessments of performance status and physical function to inform treatment selection and could serve as a viable proxy for pros machine learningrecent advancements in machine learning ml reveal opportunities for ml to transform cancer care the field has seen progress in early detection of cancers and improved diagnostic accuracy personalized therapeutics and clinical workflows in the last two years the field has also experienced substantial growth in fda approvals for algorithms including oncology applications for the detection of suspicious lesions and clinical decision support cds recent reviews have highlighted a number of promising mlbased knowledge extractions from unstructured and semistructured oncology data from ehrs social media platforms and online health communities [ ] using ehr clinical documents savova created a natural language processing nlp system deepphe to generate cancer phenotypes which could help reduce time spent reviewing clinical documents guan used nlp techniques to extract genomic sequencing information from clinical progress notes and then classified the notes on whether sequencing led to a treatment change qiu also leveraged nlp to extract the primary site of tumor origin from pathology reports given that cancer stage and origin are rarely captured in structured format within ehrs many of the recent approaches have focused on nlp to extract these data however seneviratne used the observational medical outcomes partnership omop common data model to create a feature vector framework to classify patients with prostate cancer by stage using structured ehr data this approach may be more generalizable than nlp methods since clinical notes vary widely in content depending on the type and author there have also been a number of studies that apply nlp to online health community and social media datasets [ ] zhang used a convolutional neural imia yearbook of medical informatics 2020from patient engagement to precision oncology leveraging informatics to advance cancer care 0cclinical decisions [ ] for example in an institutional precision oncology initiative at memorial sloan kettering cancer center mskccimpact approximately patients sequenced at a rate of more than patients per month underwent paired tumor and germline dna sequencing of a panel of approximately known cancer genes turnaround time to final report was less than days on average most of the somatic variants were not present in the large public database of somatic variants called the catalogue of somatic mutations in cancer cosmic at the time of the study suggesting a gap between finding a variant and interpreting its relevance to a patient™s clinical context as with other similar studies researchers relied on publicly available data resources such as my cancer genome oncokb clinical interpretation of variants in cancer civic hemonc and communitybased resources to facilitate interpretation of results the clinical impact of largescale precision oncology sequencing efforts suggest mixed results an overview suggests that only a minority of sequenced patients have at least one actionable molecular finding specifically only and of patients respectively from the ncimatch approximately patients and the mskccimpact trials received targeted therapies [ ] increasing the number and effectiveness of available therapeutics remains a central challenge to broadening the impact of genomic precision medicine however it will also be critical to identify treatments that may yield low therapeutic benefitbiomarker definition is another growth area in precision oncology precision immunooncologics a class of relatively new therapeutics have shown remarkable efficacy for some cancers several defined prognostic and predictive indices suggest which patients are most likely to benefit from for example immune checkpoint inhibitors [ ] for chimeric antigen receptor tcell cart therapy there are over trials targeting at least different biomarkers in multiple cancer types public health projects are also beginning to adopt strategies to capitalize on biomarkers and genomic information to enhance precision medicine applications for instance the us centers for disease control and prevention maintains the public health genomics knowledge base phgkb which includes cancerspecific resources to œprovide systematically curated and updated information that bridges populationbased research on genomics with clinical and public health applications  the nci has also created an online resource that announces and coordinates œprecision cancer surveillance efforts including the addition of patientlevel genomic linkages into cancer registries discussioninnovations in cancer informatics are abundant in the areas of digital health machine learning and precision oncology and have great potential in improving cancer care while there are considerable challenges there are also many opportunities for future research to advance cancer informatics as a field opportunities and implications for digital health in cancer caregiven the ubiquitous adoption of smartphones and growing ownership of wearable and iot devices digital health technologies have the unique capability to gather longitudinal data outside of clinical settings that could yield deeper insights into a patient™s symptom experience or help identify modifiable risk factors for cancer interoperability standards and numerous us initiatives such as the 21st century cures act and the promoting interoperability program formerly named the meaningful use program are transforming the way individuals access medical services and manage their health thereby accelerating the meaningful application of digital health technologiesthe evidence base for digital health in cancer care is currently limited by small sample size short study duration and limited focus on digital accessibility underserved or underrepresented populations or the needs and psychosocial outcomes of caregivers of patients with cancer patients with cognitive or physical disabilities may not be able to use some technologies and different modalities are needed to reach patients based on their abilities preferences cultural norms level of training and lived environment caregivers may be an important bridge to reach patients who have technical physical or cognitive limitations but this is an understudied area many studies specifically exclude participants based on nonownership of a smartphone lack of internet access severity of medical conditions being on chemotherapy treatment or a recurrence of cancer yet these populations may be those who could benefit the most from digital health technologies and interventions as companies are increasingly engaged in acquiring and sharing individuals™ healthrelated information there are noteworthy concerns about data privacy security and the ethical use of digital health data given the growing number of partnerships between health systems pharmaceutical companies insurers and technology companies and the increased access by third parties to cloudbased patient data and associated analytical tools there is growing unease about data linkages and the potential for misuse and data breaches for example google recently sparked a federal investigation following their partnership with ascension due to concerns for sharing protected health information and a department of justice inquiry over their acquisition agreement for fitbit apple has several partnerships with insurers and pharmaceutical companies such as aetna and eli lilly and fitbit has partnerships with bristolmyers squibbpfizer alliance and humana recently it was revealed that the uk™s national health service has given amazon free use of all health data under an alexa advice deal consumers may be unaware or inadequately informed about how their data are being used and by whom suggesting the need for greater transparency of privacy and data sharing practices and policies to be consumable at the patient level although the fda regulates some digital health apps or technologies the majority of apps available in the marketplace are not validated through rigorous research additionally it remains challenging for patients and caregivers to determine which apps are trustworthy effective and usefulimia yearbook of medical informatics 2020griffin 0c opportunities and implications for ml in cancer caremachine learning research has had meaningful advances in cancer prevention diagnostics and prediction this progress has been driven by increased availability of data and scalable computing infrastructures cloudbased platforms such as microsoft azure databricks google cloud automl and amazon web services sagemaker have simplified building training and deploying models though developers should be cognizant of the computational and environmental costs associated with cloudbased platforms realtime nlp and ml innovations within ehr systems are also being explored by companies like nuance and epic which could lead to better capture of tumor staging information as structured data elements through ambient computing despite the promise of ml to improve cancer care there are substantial challenges that must be addressed before ml is implemented into routine cancer care for instance women missed screening mammograms in the uk due to algorithm failure the ibm watson for oncology project highlighted a number of issues in the ml and oncology communities related to lack of validation and benchmarking data quality and subjectivity in interpretation of results data quality and availability challenges in healthcare are an acknowledged barrier to research particularly the paucity of data for underrepresented populations this may lead to biases since training data does not reflect the attributes of these populations yet may be applied in clinical care there are also growing concerns about the œdeskilling of physicians that could occur when some or all of the tasks become automated such as a drop in a clinician™s diagnostic accuracy this has future implications for graduate medical education and how curricula may require transformation to address these emerging issuesgiven the large volume and diversity of training data needed for ml research federated learning approaches which do not require direct data sharing have strong potential but remain difficult to implement due to privacy preservation challenges furthermore the œblack box nature of many algorithms renders interpretation and benchmarking performance difficult to improve algorithm transparency price proposed a threestep framework for validating œblack box algorithms which involves having high quality training data and development procedures testing algorithm performance against independent test data and evaluating performance continuously even with benchmarking appropriate selection of realistic and biascontrolled test cases may still be an issue the œintrinsic uncertainty in medicine introduces variations in results interpretation which also suggests that model performance criteria should be use case specific rather than based on standard scoring metrics in the era of deepfake ethical issues are inevitable if there are no appropriate regulatory frameworks for the deployment of ml algorithms health disparities could be widened due to lack of representative training data and the possible consequences from algorithm failures limit the current utility of implementing ml algorithms in realworld cancer care settings governance and policies for deployment audits of performance and implementation of best practices will be critical for safe implementation but these are not yet widely used within health systems to mitigate some of the challenges facing the field of ml several anizations have proposed ethical and regulatory frameworks the fda has proposed policies for ensuring safe and effective use of mlbased software for medical purposes including regulatory frameworks for software as a medical device for cds as well as a precertification program in realworld deployment into clinical care it is critical to have rigorous change protocols for algorithm modifications to ensure safety and to provide transparency to users during updates of algorithms however these may not be routinely implemented while mlbased cds tools can assist in automated detection classification or reporting safeguards are essential to proactively mitigate errors that may arise from these complex systems professional societies such as the american college of radiology along with several other us and international radiology anizations have released an exemplar consensus and guidance document on the importance of developing ethical standards for ml toolkits such as the american college of radiology™s ailab framework that promote a vendorneutral approach to develop algorithms based on patient populations may also allow for extensibility of algorithms opportunities and implications for precision oncology precision medicine utilizes characteristics of individual patients but relies on populations of patients to actually guide treatment decisions even when highquality data are available experts may disagree about interpretation and actionability applied within a clinical setting perhaps more problematic is the bias that can arise when extrapolating findings to ethnic racial or age groups not wellrepresented in research cohorts [ ] despite the growing application of genomic testing in research and precision oncology use cases are still limited by lack of availability of codified data elements from molecular testing on tumor samples germline dna and serum biomarkers even for healthcare systems with advanced ehrs and cancer research efforts currently there are numerous opensource cancer genomics tools and ml training platforms that have the potential to accelerate cancer informatics research such as oncosim oncowiki and google collaboratory [ ] the nci informatics technology for cancer research program has a number of openaccess tools that support the analysis of genomic imaging and clinical data yet despite the momentum in precision oncology discoveries and actionability of results their application to the context of the treatment selection for individual patients remains an open challenge in accelerating the implementation of precision oncology conclusionsbringing informatics innovations within digital health machine learning and precision oncology into cancer care will require thoughtful approaches to operationalize the collection and meaningful summarization imia yearbook of medical informatics 2020from patient engagement to precision oncology leveraging informatics to advance cancer care 0cof disparate data sources to actualize the promise of cancer informatics moving from patient engagement in collecting and sharing health data with care teams and researchers to the delivery of precision cancer care necessitates leveraging informatics innovations the research highlighted in this survey paper reflects the fastpaced everevolving field and its challenges as we move discoveries into cancer careacknowledgementsashley griffin is supported by nlm training grant 5t15lm01250003 references simpro study team electronic symptom management implementation of patient reported outcomes in oncology research available from httpswwwesymcancermoonshot [accessed nov ] us national cancer institute nci and the precision medicine initiative available from httpswwwcancergovresearchareastreatmentpmioncology [accessed nov ] us food and drug administration digital health available from httpswwwfdagovmedicaldevicesdigitalhealth [accessed nov ] chung ae jensen re basch em leveraging emerging technologies and the œinternet of things to improve the quality of cancer care j oncol pract arizagarcia a lozanolozano m galianocastillo n postigomartin p arroyomorales m cantarerovillanueva i a webbased exercise system ecuidatechemo to counter the side effects of chemotherapy in patients with breast cancer randomized controlled trial j med internet res 2019217e14418 jessup dl glover iv m daye d banzi l jones p choy g implementation of digital awareness strategies to engage patients and providers in a lung cancer screening program retrospective study j med internet res 2018202e52 sedrak ms salgia mm decat bergerot c ashinggiwa k cotta bn adashek jj examining public communication about kidney cancer on twitter jco clin cancer inform chen l wang x peng tq nature and diffusion of gynecologic cancerrelated misinformation on social media analysis of tweets j med internet res 20182010e11515 taylor j pagliari c the social dynamics of lung cancer talk on twitter facebook and macmillanuk npj digit med cho h silver n na k adams d luong kt song c visual cancer communication on social media an examination of content and effects of melanomasucks j med internet res 2018209e10501 basch e deal am dueck ac scher hi kris mg hudis c overall survival results of a trial assessing patientreported outcomes for symptom monitoring during routine cancer treatment jama roth aj kornblith ab batelcopel l peabody e scher hi holland jc rapid screening for psychologic distress in men with prostate carcinoma a pilot study cancer national comprehensive cancer network nccn clinical practice guidelines in oncology distress management available from httpswwwnccnprofessionalsphysician_glsdefaultaspx [accessed nov ] richardson l jones g a review of the reliability and validity of the edmonton symptom assessment system curr oncol girgis a durcinoska i koh es ng w arnold a delaney gp development of health pathways to standardize cancer care pathways informed by patientreported outcomes and clinical practice guidelines jco clin cancer inform chung ae shoenbill k mitchell sa dueck ac schrag d bruner dw et al patient free text reporting of symptomatic adverse events in cancer clinical research using the national cancer institute™s patientreported outcomes version of the common terminology criteria for adverse events proctcae j am med inform assoc gresham g hendifar ae spiegel b neeman e tuli r rimel bj wearable activity monitors to assess performance status and predict clinical outcomes in advanced cancer patients npj digit med gupta a stewart t bhulani n dong y rahimi z crane k feasibility of wearable physical activity monitors in patients with cancer jco clin cancer inform chung iy jung m

cancer282

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: advances in technology hardware and computing have created new opportunities to improve the quality of cancer care and research by leveraging informatics innovations digital health machine learning and precision oncology are key areas where there are noteworthy advances in the field of cancer informatics as the adoption of smartphones and wearable technologies increases patientgenerated health data such as physical activity and electronic patientreported outcomes pros are being leveraged for prevention and in interventions for remote monitoring during treatment and survivorship and to predict health outcomes moving beyond cancer clinical trials health systems are increasingly seeking to integrate pros into routine cancer care processes as part of the national cancer institute™s nci cancer moonshot initiative six healthcare systems will systematically integrate pros into clinical workflows and electronic health records ehrs additionally much attention surrounds the hype and challenges of the application of machine learning algorithms in clinical care including for a number of oncology use cases however there are concerns around biases and homogeneous training data sets thus machine learning has had limited implementation into clinical care considerable progress in genomics and computational medicine has ushered in a new era in precision oncology generating vast amounts of data much of which is publicly accessible for example the nci precision medicine initiativeoncology has had recent success defining genetically targeted therapies understanding tumor treatment resistance and developing a us cancer knowledge system while the opportunities within cancer informatics have much potential for positive impact if not applied thoughtfully these innovations risk propagating disparities and inequities operationalizing these informatics discoveries into routine cancer care also continues to present a diverse set of challenges objectivesgiven the rapid pace of cancer informatics research our objective was to conduct a survey of the literature for advancements over the past several years in three key areas of growth digital health machine learning and precision oncology we also highlight the ethical implications challenges and opportunities for each topic methodsa literature search was conducted in two electronic databases pubmed and google scholar to identify peerreviewed s and conference proceedings search terms included variations of the following neoplasms[mesh] informatics[mesh] cancer oncology clinical cancer informatics medical cancer informatics the search covered the period from october to october imia yearbook of medical informatics imia and ge thieme verlag kg 0c and returned too many s for practical review from pubmed and from google scholar thus we narrowed the search to key pubmedindexed informatics s and proceedings such as the of the american medical informatics association jamia jamia open applied clinical informatics of medical internet research bioinformatics of biomedical informatics jco clinical cancer informatics and nature digital medicine we also performed a manual review of cancer informatics s that were not yet indexed in pubmed additionally we searched the proceedings of the amia annual symposia and amia informatics summits given the sheer number of results we focused on manuscripts that demonstrated a clear focus on clinical or translational cancer informatics manuscripts were selected based on methodological rigor scientific impact innovation and contributions towards cancer informatics as a field or on impact on cancer care the reviewed manuscripts were grouped into three topics including digital health machine learning and precision oncology for each topic we describe the current state implications challenges and opportunities results digital healthdigital health is the intersection of technologies with health and wellness to enhance communication and delivery of health services it includes mobile health health information technologies wearable devices telehealth or virtual health and personalized medicine as health systems transition from traditional officebased care to more continuous interactions with patients and caregivers emerging technologies are key drivers of the capture of patientgenerated health data currently it is still challenging to bring together disparate data streams generated by patients from technologies such as smartphones social media platforms wearable activity trackers and internet of things iot devices such as smart home sensors however multiple studies have begun to demonstrate the value of employing digital health technologies to improve cancer prevention and care for example apps and webbased interventions have been used to support exercise and in digital outreach efforts focused on cancer screening in a webbased exercise intervention to mitigate the effects of chemotherapy breast cancer patients demonstrated improvements on the sixminute walk test and abdominal back lower body strength compared to the control group over the eightweek study one study demonstrated that google search and facebook advertisements could be used to promote cancer screening these advertising campaigns were associated with increased visits to institutional websites and scheduled screening exams compared to the week before and after the campaign these studies reveal the potential for digital health apps to engage patients reach broad populations and improve physical activity outcomes in addition to being a vehicle to deliver interventions social media can be used for behavioral surveillance several studies explored communications within patient communities with the most common posts or tweets on the topics of clinical trials treatment and support taylor found evidence of differing levels of emotional informational and social support for patients with lung cancer across platforms ie facebook twitter macmillanuk which may be due to differences in user characteristics or the nature of selfmoderating communities in terms of engagement cho examined the relationship between melanomarelated instagram posts and engagement outcomes ie likes comments social support they found that posts about physical consequences decreased the number of œlikes but increased comments and perceived emotional support and that the inclusion of images increased the number of comments made about the posts overall these highlighted studies contribute to an understanding of participative engagement and experiences on social media platforms for written and visual cancerrelated content that is usergenerated and suggest that patients may have varied experiences related to social support across platforms and communitiesthere is mounting evidence that using pros for cancer care symptom assessments can improve survival quality of life and help patients remain longer on treatment when compared to usual care while there is a wide range of pros that could be assessed during cancer treatment and in survivorship several efforts have aimed to identify key domains relevant to improving outcomes in cancer care for example a clinical advisory group identified priority domains and measures to enhance cancer care which included distress and symptoms and unmet needs girgis then developed clinical recommendations from these domains mapped them into wellbeing categories and demonstrated that these recommendations could algorithmically provide automated clinical feedback there is also concern that the use of structured questionnaires to assess pros may limit the ability to capture the broad range of symptoms experienced by patients during treatment chung et al examined whether patiententered freetext narratives of symptomatic adverse events could be mapped to established terminologies from the national cancer institute nci patientreported outcomes version of the common terminology criteria for adverse events proctcae among patients enrolled in three multicenter cancer trials over half provided unstructured freetext entries and most of these entries could be mapped post hoc to a proctcae or medical dictionary for regulatory activities meddra term this suggests that mapping to existing terminologies is not only feasible but also provides the opportunity to supplement trialspecific questionnaires to better capture diverse patient experiences during cancer treatmentsuse of wearable activity trackers and other iot sensors to capture additional patientgenerated health data is expanding several feasibility studies have had favorable results although sample size was generally small studies noted that higher daily steps recorded from activity trackers were correlated with pros such as increased performance status lower distress [ ] fatigue depressive symptoms [ ] adverse events hospitalizations and death despite high dropoff rates for imia yearbook of medical informatics 2020griffin 0cnetwork to classify topics within an online health community focused on breast cancer and found that patients with different cancer stages have different topics of conversations lee et al assessed conversations about living with cancer among sexual minority women using latent dirichl location which revealed that the most common topic was about coping and connecting to others with shared gender identities utilizing nlp techniques to analyze and understand topics in online communities could help to optimize interventions and deliver valuable supportrecognizing the ongoing challenges with concept extraction and the scarcity of annotated datasets researchers have employed open science efforts to foster interdisciplinary collaborations in several areas for example temporal history of your medical events thyme corpora [ ] informatics for integrating biology the bedside i2b2 challenge sets and medical information mart of intensive care mimiciii contain collections of deidentified clinical text for research dream challenges have also hosted several cancerfocused competitions that yielded a number of r packages and established benchmarks for prognostic models [ ]in medical imaging ml has been deployed for early detection improved diagnosis and better prognostic accuracy ardila used lowdose chest computed tomography images to perform endtoend lung cancer screening with high accuracy similarly predictive models to calculate breast cancer risk from mammograms demonstrated improved risk discrimination over current clinical standards in dermatology several deep learning methods were found to be superior to dermatologists in diagnosing and detecting skin cancers [ ] wei also found employing deep learning approaches to classify lung pathology images yielded more accurate results when compared to pathologists additionally advancements have been made in mlbased cds tools for classification of skin lesions estimates of colon cancer outcomes based on various therapies and management of uterine cervical abnormalities given the lack of large imaging datasets for training models investigators have also focused on producing realistic synthetic data [ ] research by senaras revealed that generative adversarial networks are a promising technique to generate labeled synthetic immunohistochemical stained breast tissue for precision cancer care genomics tools have also demonstrated the potential to advance the discovery of novel targeted cancer therapies deepmind™s alphafold tool has shown promise in predicting protein structure from its genetic sequence crisprcas9 screens have also facilitated tremendous progress in identifying essential genes across cancer cell lines several studies have used ml to identify cancer subtypes using gene expression data and pathological images for example coudray established that ml algorithms could predict six of the most commonly mutated genes within adenocarcinoma lung cancer ml could also be used to assist pathologists in detecting gene mutations or cancer subtypes given the variety of data sources necessary to compare personalized cancer care pathways incorporating genetic clinical imaging cost and quality of life data into multifaceted cds tools could aid in reducing the cognitive load of clinicians precision oncologyprecision medicine is an evolving set of diagnostic and treatment paradigms which seeks to optimize and tailor safe and effective care for patients based on their individual characteristics precision medicine has been advanced considerably by cancer genomics increasingly discoveries using genomic technologies that have fueled translational research are impacting clinical settings single cell level assays are proving transformational in the understanding of tumor heterogeneity disease biology and treatment resistance and will likely play an increasingly important role in clinical research and precision medicine applications several studies have demonstrated the feasibility of large cohorts prospectively assayed using genomic technologies with turnaround times short enough to impact wear time for activity trackers [ ] this research highlights that wearable devices could provide accurate assessments of performance status and physical function to inform treatment selection and could serve as a viable proxy for pros machine learningrecent advancements in machine learning ml reveal opportunities for ml to transform cancer care the field has seen progress in early detection of cancers and improved diagnostic accuracy personalized therapeutics and clinical workflows in the last two years the field has also experienced substantial growth in fda approvals for algorithms including oncology applications for the detection of suspicious lesions and clinical decision support cds recent reviews have highlighted a number of promising mlbased knowledge extractions from unstructured and semistructured oncology data from ehrs social media platforms and online health communities [ ] using ehr clinical documents savova created a natural language processing nlp system deepphe to generate cancer phenotypes which could help reduce time spent reviewing clinical documents guan used nlp techniques to extract genomic sequencing information from clinical progress notes and then classified the notes on whether sequencing led to a treatment change qiu also leveraged nlp to extract the primary site of tumor origin from pathology reports given that cancer stage and origin are rarely captured in structured format within ehrs many of the recent approaches have focused on nlp to extract these data however seneviratne used the observational medical outcomes partnership omop common data model to create a feature vector framework to classify patients with prostate cancer by stage using structured ehr data this approach may be more generalizable than nlp methods since clinical notes vary widely in content depending on the type and author there have also been a number of studies that apply nlp to online health community and social media datasets [ ] zhang used a convolutional neural imia yearbook of medical informatics 2020from patient engagement to precision oncology leveraging informatics to advance cancer care 0cclinical decisions [ ] for example in an institutional precision oncology initiative at memorial sloan kettering cancer center mskccimpact approximately patients sequenced at a rate of more than patients per month underwent paired tumor and germline dna sequencing of a panel of approximately known cancer genes turnaround time to final report was less than days on average most of the somatic variants were not present in the large public database of somatic variants called the catalogue of somatic mutations in cancer cosmic at the time of the study suggesting a gap between finding a variant and interpreting its relevance to a patient™s clinical context as with other similar studies researchers relied on publicly available data resources such as my cancer genome oncokb clinical interpretation of variants in cancer civic hemonc and communitybased resources to facilitate interpretation of results the clinical impact of largescale precision oncology sequencing efforts suggest mixed results an overview suggests that only a minority of sequenced patients have at least one actionable molecular finding specifically only and of patients respectively from the ncimatch approximately patients and the mskccimpact trials received targeted therapies [ ] increasing the number and effectiveness of available therapeutics remains a central challenge to broadening the impact of genomic precision medicine however it will also be critical to identify treatments that may yield low therapeutic benefitbiomarker definition is another growth area in precision oncology precision immunooncologics a class of relatively new therapeutics have shown remarkable efficacy for some cancers several defined prognostic and predictive indices suggest which patients are most likely to benefit from for example immune checkpoint inhibitors [ ] for chimeric antigen receptor tcell cart therapy there are over trials targeting at least different biomarkers in multiple cancer types public health projects are also beginning to adopt strategies to capitalize on biomarkers and genomic information to enhance precision medicine applications for instance the us centers for disease control and prevention maintains the public health genomics knowledge base phgkb which includes cancerspecific resources to œprovide systematically curated and updated information that bridges populationbased research on genomics with clinical and public health applications  the nci has also created an online resource that announces and coordinates œprecision cancer surveillance efforts including the addition of patientlevel genomic linkages into cancer registries discussioninnovations in cancer informatics are abundant in the areas of digital health machine learning and precision oncology and have great potential in improving cancer care while there are considerable challenges there are also many opportunities for future research to advance cancer informatics as a field opportunities and implications for digital health in cancer caregiven the ubiquitous adoption of smartphones and growing ownership of wearable and iot devices digital health technologies have the unique capability to gather longitudinal data outside of clinical settings that could yield deeper insights into a patient™s symptom experience or help identify modifiable risk factors for cancer interoperability standards and numerous us initiatives such as the 21st century cures act and the promoting interoperability program formerly named the meaningful use program are transforming the way individuals access medical services and manage their health thereby accelerating the meaningful application of digital health technologiesthe evidence base for digital health in cancer care is currently limited by small sample size short study duration and limited focus on digital accessibility underserved or underrepresented populations or the needs and psychosocial outcomes of caregivers of patients with cancer patients with cognitive or physical disabilities may not be able to use some technologies and different modalities are needed to reach patients based on their abilities preferences cultural norms level of training and lived environment caregivers may be an important bridge to reach patients who have technical physical or cognitive limitations but this is an understudied area many studies specifically exclude participants based on nonownership of a smartphone lack of internet access severity of medical conditions being on chemotherapy treatment or a recurrence of cancer yet these populations may be those who could benefit the most from digital health technologies and interventions as companies are increasingly engaged in acquiring and sharing individuals™ healthrelated information there are noteworthy concerns about data privacy security and the ethical use of digital health data given the growing number of partnerships between health systems pharmaceutical companies insurers and technology companies and the increased access by third parties to cloudbased patient data and associated analytical tools there is growing unease about data linkages and the potential for misuse and data breaches for example google recently sparked a federal investigation following their partnership with ascension due to concerns for sharing protected health information and a department of justice inquiry over their acquisition agreement for fitbit apple has several partnerships with insurers and pharmaceutical companies such as aetna and eli lilly and fitbit has partnerships with bristolmyers squibbpfizer alliance and humana recently it was revealed that the uk™s national health service has given amazon free use of all health data under an alexa advice deal consumers may be unaware or inadequately informed about how their data are being used and by whom suggesting the need for greater transparency of privacy and data sharing practices and policies to be consumable at the patient level although the fda regulates some digital health apps or technologies the majority of apps available in the marketplace are not validated through rigorous research additionally it remains challenging for patients and caregivers to determine which apps are trustworthy effective and usefulimia yearbook of medical informatics 2020griffin 0c opportunities and implications for ml in cancer caremachine learning research has had meaningful advances in cancer prevention diagnostics and prediction this progress has been driven by increased availability of data and scalable computing infrastructures cloudbased platforms such as microsoft azure databricks google cloud automl and amazon web services sagemaker have simplified building training and deploying models though developers should be cognizant of the computational and environmental costs associated with cloudbased platforms realtime nlp and ml innovations within ehr systems are also being explored by companies like nuance and epic which could lead to better capture of tumor staging information as structured data elements through ambient computing despite the promise of ml to improve cancer care there are substantial challenges that must be addressed before ml is implemented into routine cancer care for instance women missed screening mammograms in the uk due to algorithm failure the ibm watson for oncology project highlighted a number of issues in the ml and oncology communities related to lack of validation and benchmarking data quality and subjectivity in interpretation of results data quality and availability challenges in healthcare are an acknowledged barrier to research particularly the paucity of data for underrepresented populations this may lead to biases since training data does not reflect the attributes of these populations yet may be applied in clinical care there are also growing concerns about the œdeskilling of physicians that could occur when some or all of the tasks become automated such as a drop in a clinician™s diagnostic accuracy this has future implications for graduate medical education and how curricula may require transformation to address these emerging issuesgiven the large volume and diversity of training data needed for ml research federated learning approaches which do not require direct data sharing have strong potential but remain difficult to implement due to privacy preservation challenges furthermore the œblack box nature of many algorithms renders interpretation and benchmarking performance difficult to improve algorithm transparency price proposed a threestep framework for validating œblack box algorithms which involves having high quality training data and development procedures testing algorithm performance against independent test data and evaluating performance continuously even with benchmarking appropriate selection of realistic and biascontrolled test cases may still be an issue the œintrinsic uncertainty in medicine introduces variations in results interpretation which also suggests that model performance criteria should be use case specific rather than based on standard scoring metrics in the era of deepfake ethical issues are inevitable if there are no appropriate regulatory frameworks for the deployment of ml algorithms health disparities could be widened due to lack of representative training data and the possible consequences from algorithm failures limit the current utility of implementing ml algorithms in realworld cancer care settings governance and policies for deployment audits of performance and implementation of best practices will be critical for safe implementation but these are not yet widely used within health systems to mitigate some of the challenges facing the field of ml several anizations have proposed ethical and regulatory frameworks the fda has proposed policies for ensuring safe and effective use of mlbased software for medical purposes including regulatory frameworks for software as a medical device for cds as well as a precertification program in realworld deployment into clinical care it is critical to have rigorous change protocols for algorithm modifications to ensure safety and to provide transparency to users during updates of algorithms however these may not be routinely implemented while mlbased cds tools can assist in automated detection classification or reporting safeguards are essential to proactively mitigate errors that may arise from these complex systems professional societies such as the american college of radiology along with several other us and international radiology anizations have released an exemplar consensus and guidance document on the importance of developing ethical standards for ml toolkits such as the american college of radiology™s ailab framework that promote a vendorneutral approach to develop algorithms based on patient populations may also allow for extensibility of algorithms opportunities and implications for precision oncology precision medicine utilizes characteristics of individual patients but relies on populations of patients to actually guide treatment decisions even when highquality data are available experts may disagree about interpretation and actionability applied within a clinical setting perhaps more problematic is the bias that can arise when extrapolating findings to ethnic racial or age groups not wellrepresented in research cohorts [ ] despite the growing application of genomic testing in research and precision oncology use cases are still limited by lack of availability of codified data elements from molecular testing on tumor samples germline dna and serum biomarkers even for healthcare systems with advanced ehrs and cancer research efforts currently there are numerous opensource cancer genomics tools and ml training platforms that have the potential to accelerate cancer informatics research such as oncosim oncowiki and google collaboratory [ ] the nci informatics technology for cancer research program has a number of openaccess tools that support the analysis of genomic imaging and clinical data yet despite the momentum in precision oncology discoveries and actionability of results their application to the context of the treatment selection for individual patients remains an open challenge in accelerating the implementation of precision oncology conclusionsbringing informatics innovations within digital health machine learning and precision oncology into cancer care will require thoughtful approaches to operationalize the collection and meaningful summarization imia yearbook of medical informatics 2020from patient engagement to precision oncology leveraging informatics to advance cancer care 0cof disparate data sources to actualize the promise of cancer informatics moving from patient engagement in collecting and sharing health data with care teams and researchers to the delivery of precision cancer care necessitates leveraging informatics innovations the research highlighted in this survey paper reflects the fastpaced everevolving field and its challenges as we move discoveries into cancer careacknowledgementsashley griffin is supported by nlm training grant 5t15lm01250003 references simpro study team electronic symptom management implementation of patient reported outcomes in oncology research available from httpswwwesymcancermoonshot [accessed nov ] us national cancer institute nci and the precision medicine initiative available from httpswwwcancergovresearchareastreatmentpmioncology [accessed nov ] us food and drug administration digital health available from httpswwwfdagovmedicaldevicesdigitalhealth [accessed nov ] chung ae jensen re basch em leveraging emerging technologies and the œinternet of things to improve the quality of cancer care j oncol pract arizagarcia a lozanolozano m galianocastillo n postigomartin p arroyomorales m cantarerovillanueva i a webbased exercise system ecuidatechemo to counter the side effects of chemotherapy in patients with breast cancer randomized controlled trial j med internet res 2019217e14418 jessup dl glover iv m daye d banzi l jones p choy g implementation of digital awareness strategies to engage patients and providers in a lung cancer screening program retrospective study j med internet res 2018202e52 sedrak ms salgia mm decat bergerot c ashinggiwa k cotta bn adashek jj examining public communication about kidney cancer on twitter jco clin cancer inform chen l wang x peng tq nature and diffusion of gynecologic cancerrelated misinformation on social media analysis of tweets j med internet res 20182010e11515 taylor j pagliari c the social dynamics of lung cancer talk on twitter facebook and macmillanuk npj digit med cho h silver n na k adams d luong kt song c visual cancer communication on social media an examination of content and effects of melanomasucks j med internet res 2018209e10501 basch e deal am dueck ac scher hi kris mg hudis c overall survival results of a trial assessing patientreported outcomes for symptom monitoring during routine cancer treatment jama roth aj kornblith ab batelcopel l peabody e scher hi holland jc rapid screening for psychologic distress in men with prostate carcinoma a pilot study cancer national comprehensive cancer network nccn clinical practice guidelines in oncology distress management available from httpswwwnccnprofessionalsphysician_glsdefaultaspx [accessed nov ] richardson l jones g a review of the reliability and validity of the edmonton symptom assessment system curr oncol girgis a durcinoska i koh es ng w arnold a delaney gp development of health pathways to standardize cancer care pathways informed by patientreported outcomes and clinical practice guidelines jco clin cancer inform chung ae shoenbill k mitchell sa dueck ac schrag d bruner dw et al patient free text reporting of symptomatic adverse events in cancer clinical research using the national cancer institute™s patientreported outcomes version of the common terminology criteria for adverse events proctcae j am med inform assoc gresham g hendifar ae spiegel b neeman e tuli r rimel bj wearable activity monitors to assess performance status and predict clinical outcomes in advanced cancer patients npj digit med gupta a stewart t bhulani n dong y rahimi z crane k feasibility of wearable physical activity monitors in patients with cancer jco clin cancer inform chung iy jung m Answer:

Colon_Cancer

bousmalis leveraged cgan with the contentsimilarity loss to generate realistic target images and jointly trained the gan discriminator with the task network unidirectional translation has been applied to remove dataset variations for example bentaieb et al designed a stain normalization approach using a task conditional gan to translate he images to a reference stain madani et al proposed a semisupervised approach for cardiac abnormality classification using using gan discriminator for abnormality classification in minimally labeled xray images and showed that the adversarial loss could reduce domain overfitting mahmood translated real endoscopy images to graphicallyrendered synthetic colon images with groundtruth annotations for depthestimation during surgical navigation unidirectional translation has also been applied to crossmodality scenario for instance zhao proposed a modified unet to translate paired brain ct to mri bidirectional translationbidirectional image translation also known as reconstructionbased dt leverages two gans constraining the mapping space by enforcing semanticconsistency between the original and reconstructed images cyclegan by zhu is one of the most popular architectures for bidirectional translation cyclegan utilizes cycleconsistency to constrain the translation mapping and improve the quality of generated images cyclegan has been expanded to handle larger domain shifts with semanticconsistency loss functions cycada multidomain translation stargan and translation between two domains with multimodal conditional distributions munit in supervised learning bidirectional translation expands the training data to make the segmentation task model imia yearbook of medical informatics 2020choudhary 0crobust the translation and segmentation network can be trained either independently two stages or jointly zhang et al presented a onestage framework with an additional shapeconsistency loss in cyclegan to achieve better segmentation masks and lower failures chartsias used a twostage framework to segment mri images using ct images cai combined segmentation loss on generated images as an additional shape constraint for 3d translation and leveraged mri for pancreas segmentation in ct images in the unsupervised setting image translation is used to create labeled data for the target domain huo proposed a joint optimization approach for the synthesis and segmentation of ct images using labeled mri their framework achieved comparable performance in comparison to the fully labeled case there are a few observations about gans a cyclegan does not guarantee consistent translation of minor anatomical structures and boundaries and thus needs additional constraints like gradient and shape consistency for instance jiang incorporated tumorshape and featurebased losses to preserve tumors while translating ct data to mri data b attention networks can account for varying transferability of different image regions for instance liu proposed a novel attentionbased unet as a gan generator to translate hardtogenerate textured regions from mri to ct for alternate scenarios such as 3d2d paired images or semisupervised dadt zhang segmented xray images by using synthetic xray images created from accessible 3d ct annotations nguyen et al used semisupervised da with paired ct images to constrain cyclegan to generate more realistic images pan leveraged mri to generate missing pet images for patients for alzheimer™s disease diagnosis chen proposed stateoftheart unsupervised segmentation method using bidirectional dadt between mri and ct combining cyclegan with shared feature encoder layers between domains their method resembled cycada and showed the efficacy of combining dt with featurebased alignment c dadt can be used for singlemodality medical imaging chen leveraged a cyclegan with semanticaware adversarial loss to perform lung segmentation across different chest xray datasets latent feature space transformation in domain adaptation unlike the imagetoimage translation in dtda the lfstda transforms the source domain and target domain images to a shared latent feature space to learn a domaininvariant feature representation the goal is to minimize domainspecific information while preserving the taskrelated information the lfstda can be trained in an unsupervised fashion to obtain a domaininvariant representation or in a concurrent manner where the representation network and the task network eg image classification network are trained simultaneously to improve the performance lfstda is used in three basic implementations divergence minimization adversarial training and crossdomain reconstruction compared to dtda lsftda is more computationally efficient because it focuses on translating relevant information only instead of the complete image also featurebased domain alignment outperforms dtda by preserving taskcritical features divergence minimizationa simple approach to learn domaininvariant features and remove distributionshift is to minimize some divergence criterion between source and target data distributions common choices include maximum mean discrepancy mmd correlation alignment coral [ ] contrastive domain discrepancy cdd and wasserstein distance mmd coral and wasserstein distances are classagnostic divergence metrics and do not discriminate class labels when aligning samples cddbased da aligns samples based on their labels by minimizing the intraclass discrepancy and maximizing the interclass discrepancy mmd and coral are two of the most utilized divergence metrics that match the firstorder moment mean and the secondorder moment covariance of distributions however the represented hidden features can be complicated in the real world and may not be fully characterized by mean and covariance wasserstein distance aligns feature distributions between domains via optimal transport theory compared to the adversarialbased approaches divergencebased da has not been as widely explored in medical imaging for crossmodality da zhu utilized maximum mean discrepancy to map mr and pet images to a common space to mitigate missing data several works have used samemodality da to mitigate dataset variations in xray retinal fundus and electron microscopy images adversarial traininginstead of minimizing a divergence metric adversarial methods train a discriminator typically a separate network in an adversarial fashion against the feature encoder network the goal of the feature network is to learn a latent representation such that the discriminator is unable to identify the input sample domain from the representation for medical imaging featurebased adversarial domain adaptation has been widely utilized for various applications for example in crossmodality adaptation zhang applied a domain discriminator to adapt models trained for pathology images to microscopy images lsftda is also used for singlemodality adaptation to overcome dataset variations in pathology images mr images and ultrasound images for example lafarge have utilized a domain discriminator to mitigate the color variations of histopathology images for mitosis detection in breast cancer kamnitsas have applied a domain discriminator to mr images from different scanners and imaging protocols to improve the brain lesion segmentation performance reconstructionbased adaptationthe reconstructionbased adaptation maximizes the interdomain similarity by encoding images from each domain to reconstruct images in the other domain the reconstruction network decoder performs feature alignment by recreating the feature extractor™s input while the feature extractor encoder transforms input image into latent representation ghifary proposed imia yearbook of medical informatics 2020advancing medical imaging informatics by deep learningbased domain adaptation 0cdrcn for object detection using only target domain data reconstruction while bousmalis et al proposed a domain separation network that extracts image representations in two subspaces the private domain features and the shareddomain features the latter being used to reconstruct input image for medical imaging reconstructionbased methods are less developed and are usually combined with adversarial learning for samemodality adaptation oliveira et al have combined imagetoimage translation with a featurebased discriminator to mitigate the variations in xray images and improve segmentation performance for crossmodality adaptation ouyang combined variational autoencoder vae with adversarial training to adapt mr to ct scans challenges and opportunities domain selection and direction of domain adaptationselecting related domains for effective knowledge transfer is an openresearch area in ml in medical imaging domains are often selected based on the type of imaging technique eg radiology anatomy availability of labeled data and whether the modalities are complementary for the underlying task regarding whether da could be performed symmetrically across domains the potential information loss in a particular direction is critical for assessing task performance for example for unsupervised da from ct to mri reverse da may sometimes be needed to preserve tumors for supervised da between multiple he stained images tellez showed that mitosisdetection and cancer tissue classification in a particular color space leads to higher accuracy typically to assess domain relationship and da direction it is necessary to use a largescale empirical studies such as exploring bidirectional da across multiple datasets b a representationshift metric to roughly quantify the risk of applying learnedrepresentations from a particular domain to a new domain or c multisource da which automatically explores latent source domains in multisource datasets and quantifies the membership of each target sample however such experimentation requires extensive benchmarking studies that are lacking in medical imaging transferability of individual samplesmost da studies for medical imaging assume that all samples are equally transferable across two domains thus they focus on globally aligning domain distributions however the ability to transfer or align varies across clinical samples because of a intradomain variations eg in multimodal da between mri and ct each modality can have contrast variations b noisy annotations due to human subjectivity c target label space being a subset of source label space and d varying transferability among different image regions eg tumors are difficult to translate and could be missed during ct to mri imagetranslation some samples in the source domain may be less useful and can lead to negative transferring which adversely impacts da selecting relevant samples or reducing the impact of outlier samples using transferability frameworks is a potential solution some strategies include weighting samples based on classifier discrepancy downweighting outlier classes using the classification probability for target data leveraging openset based optimization and leveraging an attention mechanism to focus on hardtotransfer samples or using a noise coadaption layer recent medical imaging studies have explored sample selection and transferability assessment using reverse classification accuracy attentionbased unet and transferable semantic representations limitations of domain adaptation in medical imagingfor medical imaging most dlbased da uses adversarial methods primarily gan for unsupervised da adversarial methods are prone to errors because the discriminator can be confused and there is no guarantee that the domain distributions are sufficiently similar moreover the generator in gan is prone to œhallucinating content to convince the discriminator that data belongs to the target distribution as such cyclegan could be trained to synthesize tumors in images of healthy patients beyond applying consistency constraints during image translation artifacts which are not directly visible in synthesized images are also important for consideration for example cyclegans incorporate highfrequency information in the intermediate representation used by the second generator to translate the image back to the source domain this high frequency information can interfere with downstream tasks dtda approaches require translating the entire image increasing the complexity of the models for largesized medical images like whole slide images few studies [ ] have compared adversarial da methods for mrict translation however a comprehensive comparison of various featurebased da approaches is lacking future studies could explore combining dtda and lfstda approaches moreover current frameworks typically focus on sourcetarget domain pair while many tasks such as stain normalization in histopathology images can be multidomain leveraging synthetic datada for medical imaging can be applied in relatively underexplored applications such as singleview 3d reconstruction or temporal disease analysis this could benefit imageguided surgery in which training data is very scarce and difficult to obtain one way is to leverage synthetic data with ground truth information adapting it to the real data this approach has been successfully applied in natural images reverse domain adaptation ie translating real data to synthetic data is also a promising solution mahmood generated synthetic endoscopy data with known depth information by using an anatomical colon model and a virtual endoscope this simulated data was used for 3d reconstruction of real endoscopic images pan translated mr data to generate synthetic pet images to infer missing patient scans for temporal analysis of alzheimer™s disease another area that could benefit from synthetic data is skin lesion detection imia yearbook of medical informatics 2020choudhary 0ctable summary of da studies in medical imaging categorized by da methodology task modality anatomy and learning scenarios s segmentation c classification 3dr 3d reconstructionda method framework task source target domains anatomy learning scenario publications single modality dt lfst unidirectional reconstruction s c s c c s s s s s s s divergence s reconstruction s adversarial crossmodality radiology xray different demographics xray different demographics synthetic data †’ mri xray different demographics ultrasound different sources xray different disease states ultrasound different sources mri different flair sequences mri moderate to severe tbi mri different disease types demographics mri different vendors mri crossinstitutional xray different disease types demographics contrast lung xray different datasets breast lung lung brain lung fetal head lung heart brain brain brain brain brain spine reconstruction ct †’ mri s ct †’ xray s mri †” ct s mri †’ ct s s ct †” mri s 3d ct †” mri s mri †’ ct mri †’ ct mri t1 †’ t2 s ct †’ mri s mri †’ pet c mri †’ ct s 3dr ct †’ 3d rendered depthmap mri †’ pet c ct†’ mri s s mri †’ ct reconstruction s 3d mri †’ ct unidirectional divergence adversarial dt lfst single modality dt lfst adversarial unidirectional c c c s s divergence wsi different he stains wsi different stains wsi crossinstitutional wsi different stains microscopy different specimens crossmodality dt lfst reconstruction s s unidirectional adversarial c wsi ck †’ pdl1 wsi he †” if wsi †’ microscopy lung lung heart heart heart heart breast pancreas abdomen abdomen brain hip thigh pelvis brain brain colon brain abdomen heart heart pathology breast colon ovary breast prostate colon brain lung breast bladder lung colon opthalmology single modality dt unidirectional adversarial divergence lfst s s s s retinal fundus multiple datasets retinal fundus multiple datasets retinal fundus multiple datasets retinal fundus multiple datasets retina retina retina retina unsupervised semisupervised unsupervised semisupervised unsupervised chen tang gholami et a madani yang dong degel orbesarteaga kamnitsas novosad yan shanis venkataramani oliveira unsupervised supervised unsupervised supervised unsupervised unsupervised supervised unsupervised unsupervised unsupervised jiang zhang chen chartsias zhang cai huo liu hiasa pan zhao mahmood zhu yang dou ouyang bentaieb lafarge ren hou chacon kapil brieu zhang zhao javanmardi wang zhuang imia yearbook of medical informatics 2020advancing medical imaging informatics by deep learningbased domain adaptation 0c conclusions and future directionsdeep learning has been widely applied to medical imaging data analysis however the lack of wellannotated images and the heterogeneity of multicenter medical imaging datasets are two key challenges for dl performance da has emerged as an effective approach for minimizing domainshift and leveraging labeled data from distinct but related domains fstda and dtda are two popular approaches to minimize the distribution divergence in multiple medical imaging studies exploring samemodality or crossmodality scenarios they have proven to achieve good performance particularly in unsupervised da settings and organ segmentation tasks current approaches are primarily adversarial with domains being selected based on certain heuristics and underlying tasks extensive benchmarking studies are needed to quantify the domain relationship for different imaging modalities and to compare adversarial and nonadversarial approaches varying sample transferability and multimodal domains for medical imaging are two other major issues one strategy is to explore downweighting or attentionbased networks also alternative multimodal frameworks such as munit can be explored finally for certain application areas in medical imaging such as 3d reconstruction and temporal disease analysis where da is relatively unexplored synthetic data can be usedacknowledgmentsthe work was supported in part by grants from the national science foundation eager award nsf1651360 children™s healthcare of atlanta and georgia tech partnership grant giglio breast cancer research fund petit institute faculty fellow award and carol ann and david d flanagan faculty fellow research fund for professor may d wang this work was also supported in part by the scholarship from china scholarship council csc under the grant csc no the content of this article is solely the responsibility of the authors and does not necessarily represent the official views of the nihreferences mendelson ds rubin dl imaging informatics essential tools for the delivery of imaging services acad radiol oct20101195“ litjens g kooi t bejnordi be setio aaa ciompi f ghafoorian m a survey on deep learning in medical image analysis med image anal dec4260“ adlermilstein j jha ak sharing clinical data electronically a critical challenge for fixing the health care system jama apr sharma p shamout fe clifton da preserving patient privacy while training a predictive model of inhospital mortality arxiv preprint arxiv191200354 dec zhang y wei y zhao p niu s wu q tan m huang j collaborative unsupervised 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asymmetrical maximum classifier discrepancy in proceedings of the acm turing celebration conferencechina p bermúdezchacón r altingövde o becker c salzmann m fua p visual correspondences for unsupervised domain adaptation on electron microscopy images ieee trans med imaging otteholler i homeyer a karssemeijer n stain specific standardization of wholeslide histopathological images ieee trans med imaging feb352404“ csurka g domain adaptation for visual applications a comprehensive survey arxiv preprint arxiv170205374 feb wilson g cook dj a survey of unsupervised deep domain adaptation arxiv preprint arxiv181202849 dec wang m deng w deep visual domain adaptation a survey neurocomputing oct312135“ hoffman j tzeng e park t zhu jy isola p saenko k cycada cycleconsistent adversarial domain adaptation in international conference on machine learning p dou q ouyang c chen c chen h glocker b zhuang x pnpadanet plugandplay adversarial domain adaptation network with a benchmark at crossmodality cardiac segmentation arxiv preprint arxiv181207907 dec isola p zhu jy zhou t efros aa imagetoimage translation with conditional adversarial networks in proceedings of the ieee conference on computer vision and pattern recognition p goodfellow i pougetabadie j mirza m xu b wardefarley d ozair s courville a generative adversarial nets in advances in neural information processing systems p mahmood f chen r durr nj unsupervised reverse domain adaptation for synthetic medical images via adversarial training ieee trans med imaging dec37122572“ bousmalis k silberman n dohan d erhan d krishnan d unsupervised pixellevel domain adaptation with generative adversarial networks in proceedings of the ieee conference on computer vision and pattern recognition p zhang z yang l zheng y translating and segmenting multimodal medical volumes with cycleand shapeconsistency generative adversarial network in proceedings of the ieee conference on computer vision and pattern recognition p de bel t hermsen m kers j van der laak j litjens g staintransforming cycleconsistent generative adversarial networks for improved segmentation of renal histopathology in proceedings of the 2nd international conference on medical imaging with deep learning proceedings of machine learning research may p mirza m osindero s conditional generative adversarial nets arxiv preprint arxiv14111784 nov yoo d kim n park s paek as kweon is pixellevel domain transfer in european conference on computer vision p liu my tuzel o coupled generative adversarial networks in advances in neural information processing systems p madani a moradi m karargyris a syedamahmood t semisupervised learning with generative adversarial networks for chest xray imia yearbook of medical informatics 2020advancing medical imaging informatics by deep learningbased domain adaptation 0c zhang y chen h wei y zhao p cao j fan x from whole slide imaging to microscopy deep microscopy adaptation network for histopathology cancer image classification in international conference on medical image computing and computerassisted intervention p lafarge mw pluim jp eppenhof ka moeskops p veta m domainadversarial neural networks to address the appearance

cancer283

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: bousmalis leveraged cgan with the contentsimilarity loss to generate realistic target images and jointly trained the gan discriminator with the task network unidirectional translation has been applied to remove dataset variations for example bentaieb et al designed a stain normalization approach using a task conditional gan to translate he images to a reference stain madani et al proposed a semisupervised approach for cardiac abnormality classification using using gan discriminator for abnormality classification in minimally labeled xray images and showed that the adversarial loss could reduce domain overfitting mahmood translated real endoscopy images to graphicallyrendered synthetic colon images with groundtruth annotations for depthestimation during surgical navigation unidirectional translation has also been applied to crossmodality scenario for instance zhao proposed a modified unet to translate paired brain ct to mri bidirectional translationbidirectional image translation also known as reconstructionbased dt leverages two gans constraining the mapping space by enforcing semanticconsistency between the original and reconstructed images cyclegan by zhu is one of the most popular architectures for bidirectional translation cyclegan utilizes cycleconsistency to constrain the translation mapping and improve the quality of generated images cyclegan has been expanded to handle larger domain shifts with semanticconsistency loss functions cycada multidomain translation stargan and translation between two domains with multimodal conditional distributions munit in supervised learning bidirectional translation expands the training data to make the segmentation task model imia yearbook of medical informatics 2020choudhary 0crobust the translation and segmentation network can be trained either independently two stages or jointly zhang et al presented a onestage framework with an additional shapeconsistency loss in cyclegan to achieve better segmentation masks and lower failures chartsias used a twostage framework to segment mri images using ct images cai combined segmentation loss on generated images as an additional shape constraint for 3d translation and leveraged mri for pancreas segmentation in ct images in the unsupervised setting image translation is used to create labeled data for the target domain huo proposed a joint optimization approach for the synthesis and segmentation of ct images using labeled mri their framework achieved comparable performance in comparison to the fully labeled case there are a few observations about gans a cyclegan does not guarantee consistent translation of minor anatomical structures and boundaries and thus needs additional constraints like gradient and shape consistency for instance jiang incorporated tumorshape and featurebased losses to preserve tumors while translating ct data to mri data b attention networks can account for varying transferability of different image regions for instance liu proposed a novel attentionbased unet as a gan generator to translate hardtogenerate textured regions from mri to ct for alternate scenarios such as 3d2d paired images or semisupervised dadt zhang segmented xray images by using synthetic xray images created from accessible 3d ct annotations nguyen et al used semisupervised da with paired ct images to constrain cyclegan to generate more realistic images pan leveraged mri to generate missing pet images for patients for alzheimer™s disease diagnosis chen proposed stateoftheart unsupervised segmentation method using bidirectional dadt between mri and ct combining cyclegan with shared feature encoder layers between domains their method resembled cycada and showed the efficacy of combining dt with featurebased alignment c dadt can be used for singlemodality medical imaging chen leveraged a cyclegan with semanticaware adversarial loss to perform lung segmentation across different chest xray datasets latent feature space transformation in domain adaptation unlike the imagetoimage translation in dtda the lfstda transforms the source domain and target domain images to a shared latent feature space to learn a domaininvariant feature representation the goal is to minimize domainspecific information while preserving the taskrelated information the lfstda can be trained in an unsupervised fashion to obtain a domaininvariant representation or in a concurrent manner where the representation network and the task network eg image classification network are trained simultaneously to improve the performance lfstda is used in three basic implementations divergence minimization adversarial training and crossdomain reconstruction compared to dtda lsftda is more computationally efficient because it focuses on translating relevant information only instead of the complete image also featurebased domain alignment outperforms dtda by preserving taskcritical features divergence minimizationa simple approach to learn domaininvariant features and remove distributionshift is to minimize some divergence criterion between source and target data distributions common choices include maximum mean discrepancy mmd correlation alignment coral [ ] contrastive domain discrepancy cdd and wasserstein distance mmd coral and wasserstein distances are classagnostic divergence metrics and do not discriminate class labels when aligning samples cddbased da aligns samples based on their labels by minimizing the intraclass discrepancy and maximizing the interclass discrepancy mmd and coral are two of the most utilized divergence metrics that match the firstorder moment mean and the secondorder moment covariance of distributions however the represented hidden features can be complicated in the real world and may not be fully characterized by mean and covariance wasserstein distance aligns feature distributions between domains via optimal transport theory compared to the adversarialbased approaches divergencebased da has not been as widely explored in medical imaging for crossmodality da zhu utilized maximum mean discrepancy to map mr and pet images to a common space to mitigate missing data several works have used samemodality da to mitigate dataset variations in xray retinal fundus and electron microscopy images adversarial traininginstead of minimizing a divergence metric adversarial methods train a discriminator typically a separate network in an adversarial fashion against the feature encoder network the goal of the feature network is to learn a latent representation such that the discriminator is unable to identify the input sample domain from the representation for medical imaging featurebased adversarial domain adaptation has been widely utilized for various applications for example in crossmodality adaptation zhang applied a domain discriminator to adapt models trained for pathology images to microscopy images lsftda is also used for singlemodality adaptation to overcome dataset variations in pathology images mr images and ultrasound images for example lafarge have utilized a domain discriminator to mitigate the color variations of histopathology images for mitosis detection in breast cancer kamnitsas have applied a domain discriminator to mr images from different scanners and imaging protocols to improve the brain lesion segmentation performance reconstructionbased adaptationthe reconstructionbased adaptation maximizes the interdomain similarity by encoding images from each domain to reconstruct images in the other domain the reconstruction network decoder performs feature alignment by recreating the feature extractor™s input while the feature extractor encoder transforms input image into latent representation ghifary proposed imia yearbook of medical informatics 2020advancing medical imaging informatics by deep learningbased domain adaptation 0cdrcn for object detection using only target domain data reconstruction while bousmalis et al proposed a domain separation network that extracts image representations in two subspaces the private domain features and the shareddomain features the latter being used to reconstruct input image for medical imaging reconstructionbased methods are less developed and are usually combined with adversarial learning for samemodality adaptation oliveira et al have combined imagetoimage translation with a featurebased discriminator to mitigate the variations in xray images and improve segmentation performance for crossmodality adaptation ouyang combined variational autoencoder vae with adversarial training to adapt mr to ct scans challenges and opportunities domain selection and direction of domain adaptationselecting related domains for effective knowledge transfer is an openresearch area in ml in medical imaging domains are often selected based on the type of imaging technique eg radiology anatomy availability of labeled data and whether the modalities are complementary for the underlying task regarding whether da could be performed symmetrically across domains the potential information loss in a particular direction is critical for assessing task performance for example for unsupervised da from ct to mri reverse da may sometimes be needed to preserve tumors for supervised da between multiple he stained images tellez showed that mitosisdetection and cancer tissue classification in a particular color space leads to higher accuracy typically to assess domain relationship and da direction it is necessary to use a largescale empirical studies such as exploring bidirectional da across multiple datasets b a representationshift metric to roughly quantify the risk of applying learnedrepresentations from a particular domain to a new domain or c multisource da which automatically explores latent source domains in multisource datasets and quantifies the membership of each target sample however such experimentation requires extensive benchmarking studies that are lacking in medical imaging transferability of individual samplesmost da studies for medical imaging assume that all samples are equally transferable across two domains thus they focus on globally aligning domain distributions however the ability to transfer or align varies across clinical samples because of a intradomain variations eg in multimodal da between mri and ct each modality can have contrast variations b noisy annotations due to human subjectivity c target label space being a subset of source label space and d varying transferability among different image regions eg tumors are difficult to translate and could be missed during ct to mri imagetranslation some samples in the source domain may be less useful and can lead to negative transferring which adversely impacts da selecting relevant samples or reducing the impact of outlier samples using transferability frameworks is a potential solution some strategies include weighting samples based on classifier discrepancy downweighting outlier classes using the classification probability for target data leveraging openset based optimization and leveraging an attention mechanism to focus on hardtotransfer samples or using a noise coadaption layer recent medical imaging studies have explored sample selection and transferability assessment using reverse classification accuracy attentionbased unet and transferable semantic representations limitations of domain adaptation in medical imagingfor medical imaging most dlbased da uses adversarial methods primarily gan for unsupervised da adversarial methods are prone to errors because the discriminator can be confused and there is no guarantee that the domain distributions are sufficiently similar moreover the generator in gan is prone to œhallucinating content to convince the discriminator that data belongs to the target distribution as such cyclegan could be trained to synthesize tumors in images of healthy patients beyond applying consistency constraints during image translation artifacts which are not directly visible in synthesized images are also important for consideration for example cyclegans incorporate highfrequency information in the intermediate representation used by the second generator to translate the image back to the source domain this high frequency information can interfere with downstream tasks dtda approaches require translating the entire image increasing the complexity of the models for largesized medical images like whole slide images few studies [ ] have compared adversarial da methods for mrict translation however a comprehensive comparison of various featurebased da approaches is lacking future studies could explore combining dtda and lfstda approaches moreover current frameworks typically focus on sourcetarget domain pair while many tasks such as stain normalization in histopathology images can be multidomain leveraging synthetic datada for medical imaging can be applied in relatively underexplored applications such as singleview 3d reconstruction or temporal disease analysis this could benefit imageguided surgery in which training data is very scarce and difficult to obtain one way is to leverage synthetic data with ground truth information adapting it to the real data this approach has been successfully applied in natural images reverse domain adaptation ie translating real data to synthetic data is also a promising solution mahmood generated synthetic endoscopy data with known depth information by using an anatomical colon model and a virtual endoscope this simulated data was used for 3d reconstruction of real endoscopic images pan translated mr data to generate synthetic pet images to infer missing patient scans for temporal analysis of alzheimer™s disease another area that could benefit from synthetic data is skin lesion detection imia yearbook of medical informatics 2020choudhary 0ctable summary of da studies in medical imaging categorized by da methodology task modality anatomy and learning scenarios s segmentation c classification 3dr 3d reconstructionda method framework task source target domains anatomy learning scenario publications single modality dt lfst unidirectional reconstruction s c s c c s s s s s s s divergence s reconstruction s adversarial crossmodality radiology xray different demographics xray different demographics synthetic data †’ mri xray different demographics ultrasound different sources xray different disease states ultrasound different sources mri different flair sequences mri moderate to severe tbi mri different disease types demographics mri different vendors mri crossinstitutional xray different disease types demographics contrast lung xray different datasets breast lung lung brain lung fetal head lung heart brain brain brain brain brain spine reconstruction ct †’ mri s ct †’ xray s mri †” ct s mri †’ ct s s ct †” mri s 3d ct †” mri s mri †’ ct mri †’ ct mri t1 †’ t2 s ct †’ mri s mri †’ pet c mri †’ ct s 3dr ct †’ 3d rendered depthmap mri †’ pet c ct†’ mri s s mri †’ ct reconstruction s 3d mri †’ ct unidirectional divergence adversarial dt lfst single modality dt lfst adversarial unidirectional c c c s s divergence wsi different he stains wsi different stains wsi crossinstitutional wsi different stains microscopy different specimens crossmodality dt lfst reconstruction s s unidirectional adversarial c wsi ck †’ pdl1 wsi he †” if wsi †’ microscopy lung lung heart heart heart heart breast pancreas abdomen abdomen brain hip thigh pelvis brain brain colon brain abdomen heart heart pathology breast colon ovary breast prostate colon brain lung breast bladder lung colon opthalmology single modality dt unidirectional adversarial divergence lfst s s s s retinal fundus multiple datasets retinal fundus multiple datasets retinal fundus multiple datasets retinal fundus multiple datasets retina retina retina retina unsupervised semisupervised unsupervised semisupervised unsupervised chen tang gholami et a madani yang dong degel orbesarteaga kamnitsas novosad yan shanis venkataramani oliveira unsupervised supervised unsupervised supervised unsupervised unsupervised supervised unsupervised unsupervised unsupervised jiang zhang chen chartsias zhang cai huo liu hiasa pan zhao mahmood zhu yang dou ouyang bentaieb lafarge ren hou chacon kapil brieu zhang zhao javanmardi wang zhuang imia yearbook of medical informatics 2020advancing medical imaging informatics by deep learningbased domain adaptation 0c conclusions and future directionsdeep learning has been widely applied to medical imaging data analysis however the lack of wellannotated images and the heterogeneity of multicenter medical imaging datasets are two key challenges for dl performance da has emerged as an effective approach for minimizing domainshift and leveraging labeled data from distinct but related domains fstda and dtda are two popular approaches to minimize the distribution divergence in multiple medical imaging studies exploring samemodality or crossmodality scenarios they have proven to achieve good performance particularly in unsupervised da settings and organ segmentation tasks current approaches are primarily adversarial with domains being selected based on certain heuristics and underlying tasks extensive benchmarking studies are needed to quantify the domain relationship for different imaging modalities and to compare adversarial and nonadversarial approaches varying sample transferability and multimodal domains for medical imaging are two other major issues one strategy is to explore downweighting or attentionbased networks also alternative multimodal frameworks such as munit can be explored finally for certain application areas in medical imaging such as 3d reconstruction and temporal disease analysis where da is relatively unexplored synthetic data can be usedacknowledgmentsthe work was supported in part by grants from the national science foundation eager award nsf1651360 children™s healthcare of atlanta and georgia tech partnership grant giglio breast cancer research fund petit institute faculty fellow award and carol ann and david d flanagan faculty fellow research fund for professor may d wang this work was also supported in part by the scholarship from china scholarship council csc under the grant csc no the content of this article is solely the responsibility of the authors and does not necessarily represent the official views of the nihreferences mendelson ds rubin dl imaging informatics essential tools for the delivery of imaging services acad radiol oct20101195“ litjens g kooi t bejnordi be setio aaa ciompi f ghafoorian m a survey on deep learning in medical image analysis med image anal dec4260“ adlermilstein j jha ak sharing clinical data electronically a critical challenge for fixing the health care system jama apr sharma p shamout fe clifton da preserving patient privacy while training a predictive model of inhospital mortality arxiv preprint arxiv191200354 dec zhang y wei y zhao p niu s wu q tan m huang j collaborative unsupervised domain adaptation for medical image diagnosis arxiv preprint arxiv191107293 nov yao l prosky j covington b lyman k a strong baseline for domain adaptation and generalization in medical imaging arxiv preprint arxiv190401638 apr pan sj yan q a survey on transfer learning ieee trans knowl data eng oct tajbakhsh n jeyaseelan l li q chiang jn wu z ding x embracing imperfect datasets a review of deep learning solutions for medical image segmentation med image anal apr dai c mo y angelini e guo y bai w transfer learning from partial annotations for whole brain segmentation in domain adaptation and representation transfer and medical image learning with less labels and imperfect data oct p razzak mi naz s zaib a deep learning for medical image processing overview challenges and the future in classification in bioapps p chartsias a joyce t dharmakumar r tsaftaris sa adversarial image synthesis for unpaired multimodal cardiac data in international workshop on simulation and synthesis in medical imaging sep p huo y xu z bao s assad a abramson rg landman ba adversarial synthesis learning enables segmentation without target modality ground truth in ieee 15th international symposium on biomedical imaging isbi p chen c dou q chen h qin j heng pa synergistic image and feature adaptation towards crossmodality domain adaptation for medical image segmentation in proceedings of the aaai conference on artificial intelligence lee b newberg a neuroimaging in traumatic brain imaging neurorx apr22372“ gurcan mn boucheron le can a madabhushi a rajpoot nm yener b histopathological image analysis a review ieee rev biomed eng oct brieu n meier a kapil a schoenmeyer r gavriel cg caie pd et al domain adaptationbased augmentation for weakly supervised nuclei detection arxiv preprint arxiv190704681 jul tomczak k czerwińska p wiznerowicz m the cancer genome atlas tcga an immeasurable source of knowledge contemp oncol 2015191aa68“ irvin j rajpurkar p ko m yu y ciureailcus s chute c et al chexpert a large chest radiograph dataset with uncertainty labels and expert comparison arxiv preprint arxiv jan ciompi f geessink o bejnordi be de souza gs baidoshvili a litjens g the importance of stain normalization in colorectal tissue classification with convolutional networks in ieee 14th international symposium on biomedical imaging isbi p kushibar k valverde s gonzálezvillà s bernal j cabezas m oliver a supervised domain adaptation for automatic subcortical brain structure segmentation with minimal user interaction sci rep dec916742 pooch ehp ballester pl barros rc can we trust deep learning models diagnosis the impact of domain shift in chest radiograph classification arxiv preprint arxiv sep lampert t merveille o schmitz j forestier g feuerhake f wemmert c strategies for training stain invariant cnns in ieee 16th international symposium on biomedical imaging isbi p albadawy ea saha a mazurowski ma deep learning for segmentation of brain tumors impact of crossinstitutional training and testing med phys mar4531150“ stacke k eilertsen g unger j lundström c a closer look at domain shift for deep learning in histopathology arxiv preprint arxiv sep yang x dou h li r wang x bian c li s generalizing deep models for ultrasound image segmentation in medical image computing and computer assisted intervention miccai p degel ma navab n albarqouni s domain and geometry agnostic cnns for left atrium segmentation in 3d ultrasound arxiv180500357 cs “ li h loehr t wiestler b zhang j menze b euda efficient unsupervised domain adaptation for crosssite medical image segmentation arxiv preprint arxiv jan chen c dou q chen h heng pa semanticaware generative adversarial nets for unsupervised domain adaptation in chest xray segmentation in international workshop on machine learning in medical imaging p bentaieb a hamarneh g adversarial stain transfer for histopathology image analysis ieee trans med imaging mar373792“ pan y liu m lian c zhou t xia y shen d synthesizing missing pet from mri with cycleconsistent generative adversarial networks for alzheimer™s disease diagnosis in international conference on medical image computing and computerassisted intervention p ehteshami bejnordi b litjens g timofeeva n imia yearbook of medical informatics 2020choudhary 0cclassification with ability of data domain adaptation in ieee 15th international symposium on biomedical imaging isbi p zhao c carass a lee j he y prince jl whole brain segmentation and labeling from ct using synthetic mr images in international workshop on machine learning in medical imaging p zhu jy park t isola p efros aa unpaired imagetoimage translation using cycleconsistent adversarial networks in proceedings of the ieee international conference on computer vision p choi y choi m kim m ha jw kim s choo j stargan unified generative adversarial networks for multidomain imagetoimage translation in proceedings of the ieee conference on computer vision and pattern recognition p huang x liu my belongie s kautz j multimodal unsupervised imagetoimage translation in proceedings of the european conference on computer vision eccv p zhang y miao s mansi t liao r task driven generative modeling for unsupervised domain adaptation application to xray image segmentation in international conference on medical image computing and computerassisted intervention p cai j zhang z cui l zheng y yang l towards crossmodal organ translation and segmentation a cycleand shapeconsistent generative adversarial network med image anal feb hiasa y otake y takao m matsuoka t takashima k carass a et al crossmodality image synthesis from unpaired data using cyclegan in international workshop on simulation and synthesis in medical imaging p jiang j hu yc tyagi n zhang p rimner a mageras gs et al tumoraware adversarial domain adaptation from ct to mri for lung cancer segmentation in international conference on medical image computing and computerassisted intervention pp wang x li l ye w long m wang j transferable attention for domain adaptation in proceedings of the aaai conference on artificial intelligence liu x wei x yu a pan z unpaired data based crossdomain synthesis and segmentation using attention neural network in asian conference on machine learning p oktay o schlemper j folgoc ll lee m heinrich m misawa k attention unet learning where to look for the pancreas arxiv preprint arxiv180403999 apr nguyen h luo s ramos f semisupervised learning approach to generate neuroimaging modalities with adversarial training in pacificasia conference on knowledge discovery and data mining p chen c dou q chen h qin j heng pa unsupervised bidirectional crossmodality adaptation via deeply synergistic image and feature alignment for medical image segmentation ieee trans med imaging rozantsev a salzmann m fua p beyond sharing weights for deep domain adaptation ieee transactions trans pattern anal mach intell sun b saenko k deep coral correlation alignment for deep domain adaptation in european conference on computer vision p bhushan damodaran b kellenberger b flamary r tuia d courty n deepjdot deep joint distribution optimal transport for unsupervised domain adaptation in proceedings of the european conference on computer vision eccv p sun b feng j saenko k return of frustratingly easy domain adaptation in thirtieth aaai conference on artificial intelligence mar kang g jiang l yang y hauptmann ag contrastive adaptation network for unsupervised domain adaptation in proceedings of the ieee conference on computer vision and pattern recognition p tzeng e hoffman j saenko k darrell t adversarial discriminative domain adaptation in proceedings of the ieee conference on computer vision and pattern recognition p ganin y ustinova e ajakan h germain p larochelle h laviolette f domainadversarial training of neural networks the journal of machine learning research jan tzeng e hoffman j zhang n saenko k darrell t deep domain confusion maximizing for domain invariance arxiv preprint arxiv14123474 dec tsai yh hung wc schulter s sohn k yang mh chandraker m learning to adapt structured output space for semantic segmentation in proceedings of the ieee conference on computer vision and pattern recognition p bousmalis k trigeorgis g silberman n krishnan d erhan d domain separation networks in advances in neural information processing systems p ghifary m kleijn wb zhang m balduzzi d li w deep reconstructionclassification networks for unsupervised domain adaptation in european conference on computer vision p zhu x thung kh adeli e zhang y shen d maximum mean discrepancy based multiple kernel learning for incomplete multimodality neuroimaging data in international conference on medical image computing and computerassisted intervention p venkataramani r ravishankar h anamandra s towards continuous domain adaptation for medical imaging in ieee 16th international symposium on biomedical imaging isbi p zhuang j chen z zhang j zhang d cai z domain adaptation for retinal vessel segmentation using asymmetrical maximum classifier discrepancy in proceedings of the acm turing celebration conferencechina p bermúdezchacón r altingövde o becker c salzmann m fua p visual correspondences for unsupervised domain adaptation on electron microscopy images ieee trans med imaging otteholler i homeyer a karssemeijer n stain specific standardization of wholeslide histopathological images ieee trans med imaging feb352404“ csurka g domain adaptation for visual applications a comprehensive survey arxiv preprint arxiv170205374 feb wilson g cook dj a survey of unsupervised deep domain adaptation arxiv preprint arxiv181202849 dec wang m deng w deep visual domain adaptation a survey neurocomputing oct312135“ hoffman j tzeng e park t zhu jy isola p saenko k cycada cycleconsistent adversarial domain adaptation in international conference on machine learning p dou q ouyang c chen c chen h glocker b zhuang x pnpadanet plugandplay adversarial domain adaptation network with a benchmark at crossmodality cardiac segmentation arxiv preprint arxiv181207907 dec isola p zhu jy zhou t efros aa imagetoimage translation with conditional adversarial networks in proceedings of the ieee conference on computer vision and pattern recognition p goodfellow i pougetabadie j mirza m xu b wardefarley d ozair s courville a generative adversarial nets in advances in neural information processing systems p mahmood f chen r durr nj unsupervised reverse domain adaptation for synthetic medical images via adversarial training ieee trans med imaging dec37122572“ bousmalis k silberman n dohan d erhan d krishnan d unsupervised pixellevel domain adaptation with generative adversarial networks in proceedings of the ieee conference on computer vision and pattern recognition p zhang z yang l zheng y translating and segmenting multimodal medical volumes with cycleand shapeconsistency generative adversarial network in proceedings of the ieee conference on computer vision and pattern recognition p de bel t hermsen m kers j van der laak j litjens g staintransforming cycleconsistent generative adversarial networks for improved segmentation of renal histopathology in proceedings of the 2nd international conference on medical imaging with deep learning proceedings of machine learning research may p mirza m osindero s conditional generative adversarial nets arxiv preprint arxiv14111784 nov yoo d kim n park s paek as kweon is pixellevel domain transfer in european conference on computer vision p liu my tuzel o coupled generative adversarial networks in advances in neural information processing systems p madani a moradi m karargyris a syedamahmood t semisupervised learning with generative adversarial networks for chest xray imia yearbook of medical informatics 2020advancing medical imaging informatics by deep learningbased domain adaptation 0c zhang y chen h wei y zhao p cao j fan x from whole slide imaging to microscopy deep microscopy adaptation network for histopathology cancer image classification in international conference on medical image computing and computerassisted intervention p lafarge mw pluim jp eppenhof ka moeskops p veta m domainadversarial neural networks to address the appearance Answer:

Colon_Cancer

surfacecontrolled waterdispersible form of curcumin that we called theracurmin® the area under the blood concentrationtime curve auc of theracurmin in humans was 27fold higher than that of curcumin powder previously we reported on the antiinflammatory effects of theracurmin for knee osteoarthritishypothesispurpose we determined the clinical effects of orally administered theracurmin in patients with knee osteoarthritis over a 6month periodstudy design open prospective studymethods fifty patients kellgrenlawrence grade ii iii or iv knee osteoarthritis who were above years old were enrolled in this clinical study theracurmin containing mgday of curcumin was administered orally every day for months to monitor for adverse events blood biochemistry analyses were performed before and after months of each intervention the patients™ knee symptoms were evaluated at and months based on the japanese knee osteoarthritis measure the knee pain visual analog scale and the knee scoring system of the japanese orthopedic associationresults five cases dropped out during the study but no cases dropped out because of major problems no major side effects were observed with theracurmin treatment including the blood biochemistry analysis results the effective group included cases while the noteffective group included cases this study demonstrates the safety and good efficacy of theracurmin for various types of knee osteoarthritis theracurmin shows great potential for the treatment of human knee osteoarthritiskeywords curcumin highly bioavailable knee osteoarthritis safety humanreceived june accepted june type original researchdeclaration of conflicting interests the authors declared no potential conflicts of interest with respect to the research authorship andor publication of this funding the authors disclosed receipt of the following financial support for the research authorship andor publication of this this research was financially supported by theravalues corporation tokyo japan corresponding author yasuaki nakagawa department of orthopedic surgery national hospital anization kyoto medical center fukakusa mukaihatacho fushimiku kyoto japan email yasu0202nakagawagmailcomintroductionhip or knee osteoarthritis which is also referred to as degenerative joint disease is a slow destructive process of the joints that affects millions of people worldwide although the exact biochemical cause of osteoarthritis remains unknown the process usually begins with abnormal joint structures or with unusually high stress placed on joint surfaces hip knee or other types of joint osteoarthritis are longterm conditions mostly treated with analgesics and nonsteroidal antiinflammatory drugs nsaids however these drugs sometimes cause serious gastrointestinal and cardiovascular adverse events particularly with longterm use12 thus there is a need for diseasemodifying agents that not only decrease joint pain but also slow the progression of the conditioncurcumin is a polyphenol extracted from turmeric which has been safely used in foods such as curry for a long time3 curcumin is a promising therapeutic food material because of its antiinflammatory and antioxidative functions it has long been used as an antiinflammatory treatment in traditional chinese and ayurvedic medicine3 curcumin regulates various biochemical and molecular pathways by modulating several molecular targets including transcription factors cytokines enzymes and genes that regulate cell proliferation or apoptosis4 the antiinflammatory effect of curcumin seems comparable with those of steroidal drugs and nsaids such as indomethacin and phenylbutazone5 some studies have shown that curcumin™s antiinflammatory properties were related to the suppression of prostaglandin synthesis by its effect on cyclooxygenase cox6 a key enzyme responsible for the conversion of arachidonic acid to prostaglandins curcumin has also been shown to inhibit proteasome activity and induce apoptosis in human colon cancer cells in vitro and in vivo7 moreover an important mechanism of creative commons non commercial cc bync this is distributed under the terms of the creative commons attributionnoncommercial license creativecommonslicensesbync40 which permits noncommercial use reproduction and distribution of the work without further permission provided the original work is attributed as specified on the sage and open access pages ussagepubcomenusnamopenaccessatsage 0c clinical medicine insights arthritis and musculoskeletal disorders curcumin is its inhibition of nuclear factor kappa b nfkb activation8 which is a key event in the longterm inflammatory process based on these findings curcumin is expected to be effective for a range of diseases related to longterm inflammation including cancer cardiovascular disease metabolic syndrome alzheimer™s disease osteoarthritis and other common diseases and aging conditions34910 furthermore curcumin can be a potent inhibitor of the production of inflammatory and catabolic mediators by chondrocytes9 as osteoarthritis and related osteoarticular conditions of the synovial joints are characterized by inflammation curcumin™s biological actions in joint tissues may facilitate the development of clinically safe orally administered therapeutic agents for treating joint diseaseshowever curcumin™s poor bioavailability has been an obstacle to realizing its beneficial health effects because only a small amount of curcumin is absorbed via oral administration11 to overcome the bioavailability problem we previously developed a surfacecontrolled waterdispersible curcumin that we named it theracurmin® theracurmin theravalues tokyo japan12 the absorption efficacy of theracurmin was investigated and compared with that of curcumin powder in rats the area under the blood concentrationtime curve auc after the oral administration of theracurmin was more than 40fold higher than that of curcumin powder in healthy human volunteers the auc of theracurmin was 27fold higher than that of curcumin powder these findings demonstrate theracurmin™s significantly higher bioavailability compared to currently available curcumin preparations thus theracurmin is believed to be useful for providing the clinical benefits of curcumin in humansin our previous study we conducted a randomized doubleblinded placebocontrolled prospective clinical study of the efficacy of theracurmin which is a highly bioavailable form of curcumin in patients with osteoarthritis13 the knee pain visual analog scale vas scores were significantly lower in the patients treated with theracurmin than in those treated with placebo at weeks among those who had initial vas scores the same tendency was displayed in the total japanese knee osteoarthritis measure jkom14 score and its subcategory scores including pain and stiffness in the knees conditions in daily life general activities and health conditions moreover theracurmin lowered celecoxib dependence compared to placebo these results suggest that theracurmin may decrease knee osteoarthritis pain and discomfort and may improve patients™ general condition and quality of daily life as osteoarthritis is a slowly progressive and longterm condition that decreases quality of life agents such as theracurmin which is also a common food ingredient that is mildly effective with no major side effects have modest potential for the treatment of human knee osteoarthritisthe purpose of this study was to determine the clinical efficacy and safety of orally administered theracurmin in patients with knee osteoarthritis over months of treatment we hypothesized that theracurmin ingestion for months would significantly improve the symptoms and functional abilities of patients with knee osteoarthritis with no major side effectsmaterials and methodsthis was an open prospective study a total of patients with knee osteoarthritis confirmed by radiographic analysis were selected and enrolled in this study written informed consent was obtained from all subjects before participation all procedures were reviewed and approved by the research ethics committee of our hospital and this study was performed in accordance with the world medical association™s declaration of helsinkithe inclusion criteria were knee osteoarthritis patients with primary medial or lateral type in the femorotibial joints or patellofemoral joints above years of age and with kellgrenlawrence kl grade ii iii or iv osteoarthritis with radiographic classification the other combined therapies allowed during the study were nsaids pain relief patches and hyaluronic acid knee injection treatment the exclusion criteria were knee surgeries during the study knee steroid injections within months before the study or other steroid administrations within weeks before the study we examined the previous treatments before enrolment of patients in this study but did not assess them except the above exclusion criteria the flow chart of this study was shown in figure theracurmin theracurmin theravalues tokyo japan was administered orally twice a day for months theracurmin consisted of curcumin undisclosed components curcumin derivatives citric acid dextrin gum ghatti and maltose15 subjects in the theracurmin group took capsules of theracurmin per day corresponding to a total daily dose of mg of curcumin its dose was same as nakagawa™s study13 the subjects were requested to report the number of remaining capsules at their and 6month visits at our outpatient clinic to assess complianceblood biochemistry analyses including highsensitivity creactive protein hscrp were performed before the study and after months of each intervention the patients™ knee symptoms were evaluated at and months according to the following criteria the jkom14 the knee pain vas included in the jkom and the knee scoring system of the japanese orthopedic association joa13 the vas has a minimum score of and a maximum score of the jkom consists of questions divided into subcategories pain and stiffness condition in daily life general activities and health conditions the jkom is used for patient selfassessment and is based on the world health anization™s international classification of functioning disability and health it is validated in the same manner as the western ontario and mcmaster universities™ arthritis index womac the joa scale evaluates items ability to walk points ability to climb up and down stairs points range of motion points and joint swelling points each knee joint can achieve a maximum score of points on the joa scale the 0cnakagawa et al cid44cid81cid70cid79cid88cid86cid76cid82cid81cid3cid38cid85cid76cid87cid72cid85cid76cid68cid3cid46cid81cid72cid72cid3cid82cid86cid87cid72cid82cid68cid85cid87cid75cid85cid76cid87cid76cid86cid3cid83cid68cid87cid76cid72cid81cid87cid86cid3cid90cid76cid87cid75cid3cid83cid85cid76cid80cid68cid85cid92cid3cid80cid72cid71cid76cid68cid79cid3cid82cid85cid3cid79cid68cid87cid72cid85cid68cid79cid3cid87cid92cid83cid72cid3cid76cid81cid3cid87cid75cid72cid3cid73cid72cid80cid82cid85cid82cid87cid76cid69cid76cid68cid79cid3cid77cid82cid76cid81cid87cid86cid3cid82cid85cid3cid83cid68cid87cid72cid79cid79cid82cid73cid72cid80cid82cid85cid68cid79cid3cid77cid82cid76cid81cid87cid86cid15cid50cid89cid72cid85cid3cid23cid19cid3cid92cid72cid68cid85cid86cid3cid82cid73cid3cid68cid74cid72cid15cid46cid72cid79cid79cid74cid85cid72cid81cid16cid47cid68cid90cid85cid72cid81cid70cid72cid3cid74cid85cid68cid71cid72cid3cid44cid44cid15cid3cid44cid44cid44cid3cid82cid85cid3cid44cid57cid3cid82cid86cid87cid72cid82cid68cid85cid87cid75cid85cid76cid87cid76cid86cid3cid90cid76cid87cid75cid3cid85cid68cid71cid76cid82cid74cid85cid68cid83cid75cid76cid70cid3cid70cid79cid68cid86cid86cid76cid73cid76cid70cid68cid87cid76cid82cid81cid17cid40cid91cid70cid79cid88cid86cid76cid82cid81cid3cid38cid85cid76cid87cid72cid85cid76cid68cid46cid81cid72cid72cid3cid86cid88cid85cid74cid72cid85cid76cid72cid86cid3cid71cid88cid85cid76cid81cid74cid3cid87cid75cid72cid3cid86cid87cid88cid71cid92cid15cid3cid3cid46cid81cid72cid72cid3cid86cid87cid72cid85cid82cid76cid71cid3cid76cid81cid77cid72cid70cid87cid76cid82cid81cid86cid3cid90cid76cid87cid75cid76cid81cid3cid87cid90cid82cid3cid80cid82cid81cid87cid75cid86cid3cid69cid72cid73cid82cid85cid72cid3cid87cid75cid72cid3cid86cid87cid88cid71cid92cid15cid3cid82cid85cid3cid82cid87cid75cid72cid85cid3cid86cid87cid72cid85cid82cid76cid71cid3cid68cid71cid80cid76cid81cid76cid86cid87cid85cid68cid87cid76cid82cid81cid86cid3cid90cid76cid87cid75cid76cid81cid3cid73cid82cid88cid85cid3cid90cid72cid72cid78cid86cid3cid69cid72cid73cid82cid85cid72cid3cid87cid75cid72cid3cid86cid87cid88cid71cid92cid17cid36cid79cid79cid82cid70cid68cid87cid76cid82cid81cid3cid11cid81cid32cid24cid19cid12cid47cid82cid86cid87cid3cid87cid82cid3cid73cid82cid79cid79cid82cid90cid3cid88cid83cid3cid11cid81cid32cid24cid12cid39cid72cid70cid85cid72cid68cid86cid72cid3cid82cid73cid3cid68cid83cid83cid72cid87cid76cid87cid72cid3cid21cid21cid3cid80cid82cid81cid87cid75theracurmincid17791rcid3cid76cid86cid3cid72cid91cid83cid72cid81cid86cid76cid89cid72cid3cid68cid87cid3cid20cid3cid80cid82cid81cid87cid75cid39cid76cid68cid85cid85cid75cid82cid72cid68cid3cid68cid87cid3cid25cid3cid71cid68cid92cid86cid45cid82cid76cid81cid87cid3cid83cid68cid76cid81cid3cid68cid87cid3cid21cid3cid80cid82cid81cid87cid75cid86cid54cid87cid82cid83cid83cid72cid71cid3cid89cid76cid86cid76cid87cid76cid81cid74cid3cid87cid75cid72cid3cid75cid82cid86cid83cid76cid87cid68cid79cid3cid68cid73cid87cid72cid85cid3cid73cid76cid89cid72cid3cid41cid82cid79cid79cid82cid90cid16cid88cid83cid3cid11cid81cid32cid23cid24cid12cid40cid73cid73cid72cid70cid87cid76cid89cid72cid3cid70cid68cid86cid72cid86cid3cid11cid81cid32cid22cid23cid12cid49cid82cid81cid16cid72cid73cid73cid72cid70cid87cid76cid89cid72cid3cid70cid68cid86cid72cid86cid3cid11cid81cid32cid20cid20cid12cid36cid81cid68cid79cid92cid86cid76figure flow chart in this studyjoa score was significantly correlated with validated patientrelated outcome measures jkom and the medical outcome study shortform 36item health survey indicating the concurrent validity of the joa16 we evaluated adverse events and the combined therapies that the subjects required during the 6month periodin this study we defined effective cases as follows patients in the effective group felt that the treatment was effective and the vas jkom or joa scores improved between and months at a minimum of assessment we examined the proportion of effective cases according to the kl classification and type of knee osteoarthritis thirteen patients were treated with only theracurmin without combined therapy because the patients desired this strategy we also examined the proportion of effective cases in the theracurmin only groupto determine the clinical and chronological effects of orally administered theracurmin in patients with knee osteoarthritis over the months treatment period we calculated the differences in vas jkom and joa scores between the assessment and pretreatment periods we then compared the data from the assessment periods with the pretreatment period we also compared the data between each assessment periodthe mannwhitney utest was used to statistically analyze the vas jkom and joa scores a paired ttest was used to statistically analyze the vas jkom and joa scores of each patient between the pretreatment period and months after the treatment a p value of was considered statistically significantresultsfive cases dropped out of this study figure and subject did not attend her month followup visit one subject dropped out at month because the patient felt that her personal costs for the theracurmin treatment were expensive yen per month the other patients discontinued participation in the study because of minor side effects one patient had a decreased appetite at weeks and another patient had diarrhea on day in addition one patient felt increased joint pain at months therefore we included patients men and women for further analysis the mean therapeutic age was range yearsusing a paired ttest the vas jkom and joa scores at months after the treatment were significantly better than those at the pretreatment period of all patients table 0c clinical medicine insights arthritis and musculoskeletal disorders table comparison of the clinical scores between the pretreatment period and months after the treatment of all patients using a paired ttestpretreatment monthsp valuevas point jkom point joa point abbreviations jkom japanese knee osteoarthritis measure joa japanese orthopedic association vas visual analog scalemean value standard deviationtable comparison of the clinical scores between the pretreatment period and months after the treatment of patients who were treated theracurmin only using a paired ttestpretreatment monthsp valuevas pointjkom pointjoa point abbreviations jkom japanese knee osteoarthritis measure joa japanese orthopedic association vas visual analog scalemean value standard deviationof the theracurmin only group the joa score at months after the treatment was significantly better than that at the pretreatment period table improved scores of vas jkom and joa between pretreatment period and months after treatment for all patients were significant figure and also improved score of joa between them for theracurmin only group was significant figure of the patients the were effective cases and were not effective cases the latter group included patients who did not feel the effectiveness of theracurmin the improvements in the vas figure 4a jkom figure 4b and joa scores figure 4c between 6month treatment and pretreatment periods are shown in figure the proportion of effective cases for each kl classification grade is shown in table the proportion of effectiveness ranged from to there were no significant differences in effectiveness between different grades of kl classification the proportions of effective cases for each type of knee osteoarthritis are shown in table the proportion of effectiveness ranged from to there were no significant differences in effectiveness between various types of knee osteoarthritisfive patients felt itchy there were a slight increase in neutral fat cases and hepatic enzymes cases in the blood analyses during the study however no major side effects were observed with theracurmin treatment including in the blood biochemistry analyses performed during this study there was no significant difference in the improvement of sensitive crp values between the effective group and the noteffective groupthe theracurmin only group included effective cases and noteffective cases the latter group included patients who did not feel the effectiveness of theracurminthe chronological changes of the clinical scores in the effective group are shown in tables to the p values for improvement in vas table jkom table and joa table scores during each assessment period are indicated in the effective group months of theracurmin treatment resulted in significant clinical effects these positive effects were maintained for months according to the vas and joa scores one month of theracurmin treatment also resulted significant clinical effects that lasted for months according to the jkom scoresdiscussionthis study showed that theracurmin was effective for treating knee osteoarthritis in of the total population patients and in of the theracurmin only patients patients there were no significant differences in effectiveness between different kl classification grades or various types of knee osteoarthritis we also evaluated the safety of theracurmin for knee osteoarthritis no major side effects were observed with theracurmin treatment including in the blood biochemistry analyses performed during this study however there was no significant difference in the improvement of sensitive crp levels between the effective group and the noteffective group in the effective group or months of theracurmin treatment resulted in significant clinical effects that could be maintained for monthsthere have been several reports on the effectiveness of curcumin to treat knee osteoarthritis nakagawa reported a trial involving knee oa patients with a kl grade ii or iii classification a significant reduction in pain was observed after an 8week administration of a surfacecontrolled waterdispersible form of curcumin in a randomized doubleblinded placebocontrolled study13 the authors also observed a more pronounced lowering of celecoxib dependence in the group of patients treated with curcumin than in the placebo group three other randomized doubleblinded placebocontrolled trials have been conducted treatment of knee oa with curcuminoids plus glycosaminoglycans combined with physical therapy improves vas scores at motion and lequesne index scores17 treatment with curcuminoids was associated with significantly greater reductions in womac vas and lequesne™s pain function index scores compared to placebo18 in addition the 12week use of curcumin complex or its combination with boswellic acid reduces painrelated symptoms in patients with oa19 in a multicenter study curcuma domestica extracts were shown to be effective as ibuprofen for the treatment of knee osteoarthritis20 this study showed that theracurmin were effective for treating knee osteoarthritis in of the total population of casesthe use of curcumin is limited by its bioavailability to overcome these obstacles nakagawa reported that the blood 0cnakagawa et al cid16092cid16090cid16096cid16097cid16092cid16090cid16096cid16092cid16090cid16095cid16097cid16092cid16090cid16095cid16092cid16090cid16094cid16097cid16092cid16090cid16094cid16092cid16090cid16093cid16097cid16092cid16090cid16093cid16092cid16090cid16092cid16097cid16092cid16117cid16153cid16156cid16158cid16155cid16162cid16145cid16144 cid16159cid16143cid16155cid16158cid16145cid16156cid16155cid16149cid16154cid16160cid16159cid16156cid16155cid16149cid16154cid16160cid16159cid16117cid16153cid16156cid16158cid16155cid16162cid16145cid16144 cid16159cid16143cid16155cid16158cid16145cid16095cid16092cid16094cid16097cid16094cid16092cid16093cid16097cid16093cid16092cid16097cid16092cid16156cid16158cid16145cid16160cid16158cid16145cid16141cid16160cid16153cid16145cid16154cid16160cid16098cid16076cid16153cid16155cid16154cid16160cid16148cid16159acid16156cid16158cid16145cid16160cid16158cid16145cid16141cid16160cid16153cid16145cid16154cid16160cid16098cid16076cid16153cid16155cid16154cid16160cid16148cid16159bcid16156cid16155cid16149cid16154cid16160cid16159cid16117cid16153cid16156cid16158cid16155cid16162cid16145cid16144 cid16159cid16143cid16155cid16158cid16145cid16095cid16092cid16094cid16097cid16094cid16092cid16093cid16097cid16093cid16092cid16097cid16092figure improved scores of vas jkom and joa between pretreatment period and months after treatment for all patients a vas p b jkom p and c joa p jkom indicates japanese knee osteoarthritis measure joa japanese orthopedic association vas visual analog scalecid16156cid16158cid16145cid16160cid16158cid16145cid16141cid16160cid16153cid16145cid16154cid16160cid16098cid16076cid16153cid16155cid16154cid16160cid16148cid16159ccid16117cid16153cid16156cid16158cid16155cid16162cid16145cid16144 cid16159cid16143cid16155cid16158cid16145cid16156cid16155cid16149cid16154cid16160cid16159cid16095cid16097cid16095cid16092cid16094cid16097cid16094cid16092cid16093cid16097cid16093cid16092cid16097cid16092cid16156cid16158cid16145cid16160cid16158cid16145cid16141cid16160cid16153cid16145cid16154cid16160cid16098cid16076cid16153cid16155cid16154cid16160cid16148cid16159figure improved score of joa between pretreatment period and months after treatment for theracurmin only group p joa indicates japanese orthopedic associationconcentrationtime curve of theracurmin was 27fold higher than that of curcumin powder with healthy human volunteers13 appelboom reported that the administration of mg of flexofytol was equivalent to the ingestion of g of native curcumin21 belcaro reported that meriva was a lecithin delivery form of curcumin22 and panahi reported that curcuminoids were coadministered with piperine to improve bioavailability23 in these studies curcumin was more effective for the treatment of knee osteoarthritis in this study the proportion of effective cases was in the theracurmin only groupclinical studies on the efficacy of curcumin for the treatment of knee osteoarthritis have reported on specific biomarkers there was significant elevation in serum superoxide dismutase activities a borderline significant elevation in glutathione concentrations and a significant reduction in malonedialdehyde concentrations in the curcuminoids compared with the placebo group22 curcumin significantly reduced the serum levels of coll21 and tended to decrease crp levels in flexofytol for months23 belcaro reported that the treatment group experienced a greater decrease in crp levels compared to the control group24 unfortunately in our study there was no significant difference in the improvement of sensitive crp levels between the effective group and the noteffective group similar to another study17 chin reported that the extensive clinical trials on the effects of curcumin in osteoarthritic 0c clinical medicine insights arthritis and musculoskeletal disorders cid16130cid16109cid16127 cid16149cid16153cid16156cid16158cid16155cid16162cid16145cid16153cid16145cid16154cid16160cid16118cid16119cid16123cid16121 cid16149cid16153cid16156cid16158cid16155cid16162cid16145cid16153cid16145cid16154cid16160cid16118cid16123cid16109 cid16149cid16153cid16156cid16158cid16155cid16162cid16145cid16153cid16145cid16154cid16160cid16092cid16090cid16097cid16092cid16090cid16096cid16092cid16090cid16095cid16092cid16090cid16094cid16092cid16090cid16093cid16092cid16095cid16092cid16094cid16097cid16094cid16092cid16093cid16097cid16093cid16092cid16097cid16092cid16089cid16097cid16145cid16146cid16146cid16145cid16143cid16160cid16149cid16162cid16145cid16154cid16155acid16145cid16146cid16146cid16145cid16143cid16160cid16149cid16162cid16145cid16154cid16155bcid16095cid16097cid16095cid16092cid16094cid16097cid16094cid16092cid16093cid16097cid16093cid16092cid16097cid16092cid16145cid16146cid16146cid16145cid16143cid16160cid16149cid16162cid16145cid16154cid16155cfigure improvements in the clinical scores a vas scores improved by points in the effective group and in the noteffective group this difference was significant p b jkom scores improved by points in the effective group and ˆ’ in the noteffective group this difference was significant p c joa scores improved by points in the effective group and in the noteffective group this difference was no significant p jkom indicates japanese knee osteoarthritis measure joa japanese orthopedic association vas visual analog scaletable the proportion of effective cases according to kl classificationkl classificationeffective groupnoteffective groupiiiiiiv cases cases cases cases cases caseabbreviation kl kellgrenlawrencetable the proportion of effective cases according to the various types of knee osteoarthritistype of knee oaft lateraleffective groupno effective group cases ft medial cases patello femoral joint cases abbreviations ft femorotibial joint oa osteoarthritis cases cases casepatients reported reduced pain and improved functionality of the patients treated with curcumin these effects could be achieved as early as weeks25 similarly or months of theracurmin treatment resulted in significant clinical effects that could be maintained for months in this studyseveral reports have discussed the effectiveness of curcumin for cartilage degeneration in animal models and cell cultures curcumin treatment led to reduced proteoglycan loss and cartilage erosion in a posttraumatic osteoarthritis mouse model26 curcumin treatment enhanced autophagy and reduced apoptosis and cartilage loss in a spontaneous and surgically induced osteoarthritis mice model27 curcumin prevented the deterioration of articular cartilage compared to the control group in estrogendeficient rats28 in cell culture studies noncytotoxic concentrations of curcumin exerted anticatabolic and antiinflammatory effects in cartilage explants29 curcumin was demonstrated to inhibit the interleukin il1binduced activation of nfkb by suppressing ikba phosphorylation30 curcumin inhibited chondrocyte hypertrophy through indian hedgehog homolog and notch signaling curcumin was a potential agent in modulating cartilage homeostasis and maintaining the chondrocyte phenotype31 however although this study was unable to demonstrate improvements in patient knee cartilage the above findings suggest that curcumin might improve the knee cartilage of such patientsin this study patients felt itchy there were a slight increase in neutral fat cases and hepatic enzymes cases in the blood analyses during the study however no major side effects were observed with theracurmin treatment including in the blood biochemistry analyses performed during this study likewise there were no considerable adverse effects in curcumin groups in other clinical studies17181921 curcuma domestica extracts were noninferior to ibuprofen for the treatment of knee osteoarthritis and the curcuma domestica extracts group had a similar incidence of adverse events but with gastrointestinal adverse events20 daily reported that turmeric preparations and curcumin were considered safe at doses not exceeding mgday for up to months32 a study on the safety of theracurmin revealed that a dose of up to mg for months was safe in cancer patients receiving chemotherapy33 in this study the dosage of curcumin was mgday similar to nakagawa study13 this was the smallest dose used in the published clinical studies34 the safety of curcumin was demonstrated in this study and the dosage of curcumin used in our study was safethere are main limitations in this study there was a short followup period months the sample size was small cases the lack of other treatment adjustment in the analysis the placebo effects could not be eliminated and this was an 0cnakagawa et al table the chronological changes of the clinical scores in vas scores month month month months months months months months months months months monthsabbreviation vas visual analog scalethe p values between the assessment periods for improvement in vas scoresbold numbers indicate a significant differencetable the chronological changes of the clinical scores in jkom scores month month month months months months months months months months months monthsabbreviation jkom japanese knee osteoarthritis measurethe p values between the assessment periods for improvement in jkom scoresbold numbers indicate a significant differencetable the chronological changes of the clinical scores in joa scores month months months months months months month month months months months monthsabbreviation joa japanese orthopedic associationthe p values between the assessment periods for improvement in joa scoresbold numbers indicate a significant differenceopen study also it is a potential selection bias that the patients are paying for their treatment from the startfifty patients with knee osteoarthritis took capsules of theracurmin per day corresponding to a total daily dose of mg of curcumin for months five cases dropped out because of minor side effects therefore we included patients men and women in the further analyses this study showed that theracurmin was effective for treating knee osteoarthritis in of all cases and in of cases in the theracurmin only group of the theracurminonly group the joa score at months after the treatment was significantly better than that at the pretreatment period using the paired ttest in our evaluation of the safety of theracurmin for knee osteoarthritis no major side effects were observed with theracurmin treatment including in the blood biochemistry analyses performed during this study theracurmin showed great potential for the treatment of human knee osteoarthritis based on efficacy and sa

cancer284

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: surfacecontrolled waterdispersible form of curcumin that we called theracurmin® the area under the blood concentrationtime curve auc of theracurmin in humans was 27fold higher than that of curcumin powder previously we reported on the antiinflammatory effects of theracurmin for knee osteoarthritishypothesispurpose we determined the clinical effects of orally administered theracurmin in patients with knee osteoarthritis over a 6month periodstudy design open prospective studymethods fifty patients kellgrenlawrence grade ii iii or iv knee osteoarthritis who were above years old were enrolled in this clinical study theracurmin containing mgday of curcumin was administered orally every day for months to monitor for adverse events blood biochemistry analyses were performed before and after months of each intervention the patients™ knee symptoms were evaluated at and months based on the japanese knee osteoarthritis measure the knee pain visual analog scale and the knee scoring system of the japanese orthopedic associationresults five cases dropped out during the study but no cases dropped out because of major problems no major side effects were observed with theracurmin treatment including the blood biochemistry analysis results the effective group included cases while the noteffective group included cases this study demonstrates the safety and good efficacy of theracurmin for various types of knee osteoarthritis theracurmin shows great potential for the treatment of human knee osteoarthritiskeywords curcumin highly bioavailable knee osteoarthritis safety humanreceived june accepted june type original researchdeclaration of conflicting interests the authors declared no potential conflicts of interest with respect to the research authorship andor publication of this funding the authors disclosed receipt of the following financial support for the research authorship andor publication of this this research was financially supported by theravalues corporation tokyo japan corresponding author yasuaki nakagawa department of orthopedic surgery national hospital anization kyoto medical center fukakusa mukaihatacho fushimiku kyoto japan email yasu0202nakagawagmailcomintroductionhip or knee osteoarthritis which is also referred to as degenerative joint disease is a slow destructive process of the joints that affects millions of people worldwide although the exact biochemical cause of osteoarthritis remains unknown the process usually begins with abnormal joint structures or with unusually high stress placed on joint surfaces hip knee or other types of joint osteoarthritis are longterm conditions mostly treated with analgesics and nonsteroidal antiinflammatory drugs nsaids however these drugs sometimes cause serious gastrointestinal and cardiovascular adverse events particularly with longterm use12 thus there is a need for diseasemodifying agents that not only decrease joint pain but also slow the progression of the conditioncurcumin is a polyphenol extracted from turmeric which has been safely used in foods such as curry for a long time3 curcumin is a promising therapeutic food material because of its antiinflammatory and antioxidative functions it has long been used as an antiinflammatory treatment in traditional chinese and ayurvedic medicine3 curcumin regulates various biochemical and molecular pathways by modulating several molecular targets including transcription factors cytokines enzymes and genes that regulate cell proliferation or apoptosis4 the antiinflammatory effect of curcumin seems comparable with those of steroidal drugs and nsaids such as indomethacin and phenylbutazone5 some studies have shown that curcumin™s antiinflammatory properties were related to the suppression of prostaglandin synthesis by its effect on cyclooxygenase cox6 a key enzyme responsible for the conversion of arachidonic acid to prostaglandins curcumin has also been shown to inhibit proteasome activity and induce apoptosis in human colon cancer cells in vitro and in vivo7 moreover an important mechanism of creative commons non commercial cc bync this is distributed under the terms of the creative commons attributionnoncommercial license creativecommonslicensesbync40 which permits noncommercial use reproduction and distribution of the work without further permission provided the original work is attributed as specified on the sage and open access pages ussagepubcomenusnamopenaccessatsage 0c clinical medicine insights arthritis and musculoskeletal disorders curcumin is its inhibition of nuclear factor kappa b nfkb activation8 which is a key event in the longterm inflammatory process based on these findings curcumin is expected to be effective for a range of diseases related to longterm inflammation including cancer cardiovascular disease metabolic syndrome alzheimer™s disease osteoarthritis and other common diseases and aging conditions34910 furthermore curcumin can be a potent inhibitor of the production of inflammatory and catabolic mediators by chondrocytes9 as osteoarthritis and related osteoarticular conditions of the synovial joints are characterized by inflammation curcumin™s biological actions in joint tissues may facilitate the development of clinically safe orally administered therapeutic agents for treating joint diseaseshowever curcumin™s poor bioavailability has been an obstacle to realizing its beneficial health effects because only a small amount of curcumin is absorbed via oral administration11 to overcome the bioavailability problem we previously developed a surfacecontrolled waterdispersible curcumin that we named it theracurmin® theracurmin theravalues tokyo japan12 the absorption efficacy of theracurmin was investigated and compared with that of curcumin powder in rats the area under the blood concentrationtime curve auc after the oral administration of theracurmin was more than 40fold higher than that of curcumin powder in healthy human volunteers the auc of theracurmin was 27fold higher than that of curcumin powder these findings demonstrate theracurmin™s significantly higher bioavailability compared to currently available curcumin preparations thus theracurmin is believed to be useful for providing the clinical benefits of curcumin in humansin our previous study we conducted a randomized doubleblinded placebocontrolled prospective clinical study of the efficacy of theracurmin which is a highly bioavailable form of curcumin in patients with osteoarthritis13 the knee pain visual analog scale vas scores were significantly lower in the patients treated with theracurmin than in those treated with placebo at weeks among those who had initial vas scores the same tendency was displayed in the total japanese knee osteoarthritis measure jkom14 score and its subcategory scores including pain and stiffness in the knees conditions in daily life general activities and health conditions moreover theracurmin lowered celecoxib dependence compared to placebo these results suggest that theracurmin may decrease knee osteoarthritis pain and discomfort and may improve patients™ general condition and quality of daily life as osteoarthritis is a slowly progressive and longterm condition that decreases quality of life agents such as theracurmin which is also a common food ingredient that is mildly effective with no major side effects have modest potential for the treatment of human knee osteoarthritisthe purpose of this study was to determine the clinical efficacy and safety of orally administered theracurmin in patients with knee osteoarthritis over months of treatment we hypothesized that theracurmin ingestion for months would significantly improve the symptoms and functional abilities of patients with knee osteoarthritis with no major side effectsmaterials and methodsthis was an open prospective study a total of patients with knee osteoarthritis confirmed by radiographic analysis were selected and enrolled in this study written informed consent was obtained from all subjects before participation all procedures were reviewed and approved by the research ethics committee of our hospital and this study was performed in accordance with the world medical association™s declaration of helsinkithe inclusion criteria were knee osteoarthritis patients with primary medial or lateral type in the femorotibial joints or patellofemoral joints above years of age and with kellgrenlawrence kl grade ii iii or iv osteoarthritis with radiographic classification the other combined therapies allowed during the study were nsaids pain relief patches and hyaluronic acid knee injection treatment the exclusion criteria were knee surgeries during the study knee steroid injections within months before the study or other steroid administrations within weeks before the study we examined the previous treatments before enrolment of patients in this study but did not assess them except the above exclusion criteria the flow chart of this study was shown in figure theracurmin theracurmin theravalues tokyo japan was administered orally twice a day for months theracurmin consisted of curcumin undisclosed components curcumin derivatives citric acid dextrin gum ghatti and maltose15 subjects in the theracurmin group took capsules of theracurmin per day corresponding to a total daily dose of mg of curcumin its dose was same as nakagawa™s study13 the subjects were requested to report the number of remaining capsules at their and 6month visits at our outpatient clinic to assess complianceblood biochemistry analyses including highsensitivity creactive protein hscrp were performed before the study and after months of each intervention the patients™ knee symptoms were evaluated at and months according to the following criteria the jkom14 the knee pain vas included in the jkom and the knee scoring system of the japanese orthopedic association joa13 the vas has a minimum score of and a maximum score of the jkom consists of questions divided into subcategories pain and stiffness condition in daily life general activities and health conditions the jkom is used for patient selfassessment and is based on the world health anization™s international classification of functioning disability and health it is validated in the same manner as the western ontario and mcmaster universities™ arthritis index womac the joa scale evaluates items ability to walk points ability to climb up and down stairs points range of motion points and joint swelling points each knee joint can achieve a maximum score of points on the joa scale the 0cnakagawa et al cid44cid81cid70cid79cid88cid86cid76cid82cid81cid3cid38cid85cid76cid87cid72cid85cid76cid68cid3cid46cid81cid72cid72cid3cid82cid86cid87cid72cid82cid68cid85cid87cid75cid85cid76cid87cid76cid86cid3cid83cid68cid87cid76cid72cid81cid87cid86cid3cid90cid76cid87cid75cid3cid83cid85cid76cid80cid68cid85cid92cid3cid80cid72cid71cid76cid68cid79cid3cid82cid85cid3cid79cid68cid87cid72cid85cid68cid79cid3cid87cid92cid83cid72cid3cid76cid81cid3cid87cid75cid72cid3cid73cid72cid80cid82cid85cid82cid87cid76cid69cid76cid68cid79cid3cid77cid82cid76cid81cid87cid86cid3cid82cid85cid3cid83cid68cid87cid72cid79cid79cid82cid73cid72cid80cid82cid85cid68cid79cid3cid77cid82cid76cid81cid87cid86cid15cid50cid89cid72cid85cid3cid23cid19cid3cid92cid72cid68cid85cid86cid3cid82cid73cid3cid68cid74cid72cid15cid46cid72cid79cid79cid74cid85cid72cid81cid16cid47cid68cid90cid85cid72cid81cid70cid72cid3cid74cid85cid68cid71cid72cid3cid44cid44cid15cid3cid44cid44cid44cid3cid82cid85cid3cid44cid57cid3cid82cid86cid87cid72cid82cid68cid85cid87cid75cid85cid76cid87cid76cid86cid3cid90cid76cid87cid75cid3cid85cid68cid71cid76cid82cid74cid85cid68cid83cid75cid76cid70cid3cid70cid79cid68cid86cid86cid76cid73cid76cid70cid68cid87cid76cid82cid81cid17cid40cid91cid70cid79cid88cid86cid76cid82cid81cid3cid38cid85cid76cid87cid72cid85cid76cid68cid46cid81cid72cid72cid3cid86cid88cid85cid74cid72cid85cid76cid72cid86cid3cid71cid88cid85cid76cid81cid74cid3cid87cid75cid72cid3cid86cid87cid88cid71cid92cid15cid3cid3cid46cid81cid72cid72cid3cid86cid87cid72cid85cid82cid76cid71cid3cid76cid81cid77cid72cid70cid87cid76cid82cid81cid86cid3cid90cid76cid87cid75cid76cid81cid3cid87cid90cid82cid3cid80cid82cid81cid87cid75cid86cid3cid69cid72cid73cid82cid85cid72cid3cid87cid75cid72cid3cid86cid87cid88cid71cid92cid15cid3cid82cid85cid3cid82cid87cid75cid72cid85cid3cid86cid87cid72cid85cid82cid76cid71cid3cid68cid71cid80cid76cid81cid76cid86cid87cid85cid68cid87cid76cid82cid81cid86cid3cid90cid76cid87cid75cid76cid81cid3cid73cid82cid88cid85cid3cid90cid72cid72cid78cid86cid3cid69cid72cid73cid82cid85cid72cid3cid87cid75cid72cid3cid86cid87cid88cid71cid92cid17cid36cid79cid79cid82cid70cid68cid87cid76cid82cid81cid3cid11cid81cid32cid24cid19cid12cid47cid82cid86cid87cid3cid87cid82cid3cid73cid82cid79cid79cid82cid90cid3cid88cid83cid3cid11cid81cid32cid24cid12cid39cid72cid70cid85cid72cid68cid86cid72cid3cid82cid73cid3cid68cid83cid83cid72cid87cid76cid87cid72cid3cid21cid21cid3cid80cid82cid81cid87cid75theracurmincid17791rcid3cid76cid86cid3cid72cid91cid83cid72cid81cid86cid76cid89cid72cid3cid68cid87cid3cid20cid3cid80cid82cid81cid87cid75cid39cid76cid68cid85cid85cid75cid82cid72cid68cid3cid68cid87cid3cid25cid3cid71cid68cid92cid86cid45cid82cid76cid81cid87cid3cid83cid68cid76cid81cid3cid68cid87cid3cid21cid3cid80cid82cid81cid87cid75cid86cid54cid87cid82cid83cid83cid72cid71cid3cid89cid76cid86cid76cid87cid76cid81cid74cid3cid87cid75cid72cid3cid75cid82cid86cid83cid76cid87cid68cid79cid3cid68cid73cid87cid72cid85cid3cid73cid76cid89cid72cid3cid41cid82cid79cid79cid82cid90cid16cid88cid83cid3cid11cid81cid32cid23cid24cid12cid40cid73cid73cid72cid70cid87cid76cid89cid72cid3cid70cid68cid86cid72cid86cid3cid11cid81cid32cid22cid23cid12cid49cid82cid81cid16cid72cid73cid73cid72cid70cid87cid76cid89cid72cid3cid70cid68cid86cid72cid86cid3cid11cid81cid32cid20cid20cid12cid36cid81cid68cid79cid92cid86cid76figure flow chart in this studyjoa score was significantly correlated with validated patientrelated outcome measures jkom and the medical outcome study shortform 36item health survey indicating the concurrent validity of the joa16 we evaluated adverse events and the combined therapies that the subjects required during the 6month periodin this study we defined effective cases as follows patients in the effective group felt that the treatment was effective and the vas jkom or joa scores improved between and months at a minimum of assessment we examined the proportion of effective cases according to the kl classification and type of knee osteoarthritis thirteen patients were treated with only theracurmin without combined therapy because the patients desired this strategy we also examined the proportion of effective cases in the theracurmin only groupto determine the clinical and chronological effects of orally administered theracurmin in patients with knee osteoarthritis over the months treatment period we calculated the differences in vas jkom and joa scores between the assessment and pretreatment periods we then compared the data from the assessment periods with the pretreatment period we also compared the data between each assessment periodthe mannwhitney utest was used to statistically analyze the vas jkom and joa scores a paired ttest was used to statistically analyze the vas jkom and joa scores of each patient between the pretreatment period and months after the treatment a p value of was considered statistically significantresultsfive cases dropped out of this study figure and subject did not attend her month followup visit one subject dropped out at month because the patient felt that her personal costs for the theracurmin treatment were expensive yen per month the other patients discontinued participation in the study because of minor side effects one patient had a decreased appetite at weeks and another patient had diarrhea on day in addition one patient felt increased joint pain at months therefore we included patients men and women for further analysis the mean therapeutic age was range yearsusing a paired ttest the vas jkom and joa scores at months after the treatment were significantly better than those at the pretreatment period of all patients table 0c clinical medicine insights arthritis and musculoskeletal disorders table comparison of the clinical scores between the pretreatment period and months after the treatment of all patients using a paired ttestpretreatment monthsp valuevas point jkom point joa point abbreviations jkom japanese knee osteoarthritis measure joa japanese orthopedic association vas visual analog scalemean value standard deviationtable comparison of the clinical scores between the pretreatment period and months after the treatment of patients who were treated theracurmin only using a paired ttestpretreatment monthsp valuevas pointjkom pointjoa point abbreviations jkom japanese knee osteoarthritis measure joa japanese orthopedic association vas visual analog scalemean value standard deviationof the theracurmin only group the joa score at months after the treatment was significantly better than that at the pretreatment period table improved scores of vas jkom and joa between pretreatment period and months after treatment for all patients were significant figure and also improved score of joa between them for theracurmin only group was significant figure of the patients the were effective cases and were not effective cases the latter group included patients who did not feel the effectiveness of theracurmin the improvements in the vas figure 4a jkom figure 4b and joa scores figure 4c between 6month treatment and pretreatment periods are shown in figure the proportion of effective cases for each kl classification grade is shown in table the proportion of effectiveness ranged from to there were no significant differences in effectiveness between different grades of kl classification the proportions of effective cases for each type of knee osteoarthritis are shown in table the proportion of effectiveness ranged from to there were no significant differences in effectiveness between various types of knee osteoarthritisfive patients felt itchy there were a slight increase in neutral fat cases and hepatic enzymes cases in the blood analyses during the study however no major side effects were observed with theracurmin treatment including in the blood biochemistry analyses performed during this study there was no significant difference in the improvement of sensitive crp values between the effective group and the noteffective groupthe theracurmin only group included effective cases and noteffective cases the latter group included patients who did not feel the effectiveness of theracurminthe chronological changes of the clinical scores in the effective group are shown in tables to the p values for improvement in vas table jkom table and joa table scores during each assessment period are indicated in the effective group months of theracurmin treatment resulted in significant clinical effects these positive effects were maintained for months according to the vas and joa scores one month of theracurmin treatment also resulted significant clinical effects that lasted for months according to the jkom scoresdiscussionthis study showed that theracurmin was effective for treating knee osteoarthritis in of the total population patients and in of the theracurmin only patients patients there were no significant differences in effectiveness between different kl classification grades or various types of knee osteoarthritis we also evaluated the safety of theracurmin for knee osteoarthritis no major side effects were observed with theracurmin treatment including in the blood biochemistry analyses performed during this study however there was no significant difference in the improvement of sensitive crp levels between the effective group and the noteffective group in the effective group or months of theracurmin treatment resulted in significant clinical effects that could be maintained for monthsthere have been several reports on the effectiveness of curcumin to treat knee osteoarthritis nakagawa reported a trial involving knee oa patients with a kl grade ii or iii classification a significant reduction in pain was observed after an 8week administration of a surfacecontrolled waterdispersible form of curcumin in a randomized doubleblinded placebocontrolled study13 the authors also observed a more pronounced lowering of celecoxib dependence in the group of patients treated with curcumin than in the placebo group three other randomized doubleblinded placebocontrolled trials have been conducted treatment of knee oa with curcuminoids plus glycosaminoglycans combined with physical therapy improves vas scores at motion and lequesne index scores17 treatment with curcuminoids was associated with significantly greater reductions in womac vas and lequesne™s pain function index scores compared to placebo18 in addition the 12week use of curcumin complex or its combination with boswellic acid reduces painrelated symptoms in patients with oa19 in a multicenter study curcuma domestica extracts were shown to be effective as ibuprofen for the treatment of knee osteoarthritis20 this study showed that theracurmin were effective for treating knee osteoarthritis in of the total population of casesthe use of curcumin is limited by its bioavailability to overcome these obstacles nakagawa reported that the blood 0cnakagawa et al cid16092cid16090cid16096cid16097cid16092cid16090cid16096cid16092cid16090cid16095cid16097cid16092cid16090cid16095cid16092cid16090cid16094cid16097cid16092cid16090cid16094cid16092cid16090cid16093cid16097cid16092cid16090cid16093cid16092cid16090cid16092cid16097cid16092cid16117cid16153cid16156cid16158cid16155cid16162cid16145cid16144 cid16159cid16143cid16155cid16158cid16145cid16156cid16155cid16149cid16154cid16160cid16159cid16156cid16155cid16149cid16154cid16160cid16159cid16117cid16153cid16156cid16158cid16155cid16162cid16145cid16144 cid16159cid16143cid16155cid16158cid16145cid16095cid16092cid16094cid16097cid16094cid16092cid16093cid16097cid16093cid16092cid16097cid16092cid16156cid16158cid16145cid16160cid16158cid16145cid16141cid16160cid16153cid16145cid16154cid16160cid16098cid16076cid16153cid16155cid16154cid16160cid16148cid16159acid16156cid16158cid16145cid16160cid16158cid16145cid16141cid16160cid16153cid16145cid16154cid16160cid16098cid16076cid16153cid16155cid16154cid16160cid16148cid16159bcid16156cid16155cid16149cid16154cid16160cid16159cid16117cid16153cid16156cid16158cid16155cid16162cid16145cid16144 cid16159cid16143cid16155cid16158cid16145cid16095cid16092cid16094cid16097cid16094cid16092cid16093cid16097cid16093cid16092cid16097cid16092figure improved scores of vas jkom and joa between pretreatment period and months after treatment for all patients a vas p b jkom p and c joa p jkom indicates japanese knee osteoarthritis measure joa japanese orthopedic association vas visual analog scalecid16156cid16158cid16145cid16160cid16158cid16145cid16141cid16160cid16153cid16145cid16154cid16160cid16098cid16076cid16153cid16155cid16154cid16160cid16148cid16159ccid16117cid16153cid16156cid16158cid16155cid16162cid16145cid16144 cid16159cid16143cid16155cid16158cid16145cid16156cid16155cid16149cid16154cid16160cid16159cid16095cid16097cid16095cid16092cid16094cid16097cid16094cid16092cid16093cid16097cid16093cid16092cid16097cid16092cid16156cid16158cid16145cid16160cid16158cid16145cid16141cid16160cid16153cid16145cid16154cid16160cid16098cid16076cid16153cid16155cid16154cid16160cid16148cid16159figure improved score of joa between pretreatment period and months after treatment for theracurmin only group p joa indicates japanese orthopedic associationconcentrationtime curve of theracurmin was 27fold higher than that of curcumin powder with healthy human volunteers13 appelboom reported that the administration of mg of flexofytol was equivalent to the ingestion of g of native curcumin21 belcaro reported that meriva was a lecithin delivery form of curcumin22 and panahi reported that curcuminoids were coadministered with piperine to improve bioavailability23 in these studies curcumin was more effective for the treatment of knee osteoarthritis in this study the proportion of effective cases was in the theracurmin only groupclinical studies on the efficacy of curcumin for the treatment of knee osteoarthritis have reported on specific biomarkers there was significant elevation in serum superoxide dismutase activities a borderline significant elevation in glutathione concentrations and a significant reduction in malonedialdehyde concentrations in the curcuminoids compared with the placebo group22 curcumin significantly reduced the serum levels of coll21 and tended to decrease crp levels in flexofytol for months23 belcaro reported that the treatment group experienced a greater decrease in crp levels compared to the control group24 unfortunately in our study there was no significant difference in the improvement of sensitive crp levels between the effective group and the noteffective group similar to another study17 chin reported that the extensive clinical trials on the effects of curcumin in osteoarthritic 0c clinical medicine insights arthritis and musculoskeletal disorders cid16130cid16109cid16127 cid16149cid16153cid16156cid16158cid16155cid16162cid16145cid16153cid16145cid16154cid16160cid16118cid16119cid16123cid16121 cid16149cid16153cid16156cid16158cid16155cid16162cid16145cid16153cid16145cid16154cid16160cid16118cid16123cid16109 cid16149cid16153cid16156cid16158cid16155cid16162cid16145cid16153cid16145cid16154cid16160cid16092cid16090cid16097cid16092cid16090cid16096cid16092cid16090cid16095cid16092cid16090cid16094cid16092cid16090cid16093cid16092cid16095cid16092cid16094cid16097cid16094cid16092cid16093cid16097cid16093cid16092cid16097cid16092cid16089cid16097cid16145cid16146cid16146cid16145cid16143cid16160cid16149cid16162cid16145cid16154cid16155acid16145cid16146cid16146cid16145cid16143cid16160cid16149cid16162cid16145cid16154cid16155bcid16095cid16097cid16095cid16092cid16094cid16097cid16094cid16092cid16093cid16097cid16093cid16092cid16097cid16092cid16145cid16146cid16146cid16145cid16143cid16160cid16149cid16162cid16145cid16154cid16155cfigure improvements in the clinical scores a vas scores improved by points in the effective group and in the noteffective group this difference was significant p b jkom scores improved by points in the effective group and ˆ’ in the noteffective group this difference was significant p c joa scores improved by points in the effective group and in the noteffective group this difference was no significant p jkom indicates japanese knee osteoarthritis measure joa japanese orthopedic association vas visual analog scaletable the proportion of effective cases according to kl classificationkl classificationeffective groupnoteffective groupiiiiiiv cases cases cases cases cases caseabbreviation kl kellgrenlawrencetable the proportion of effective cases according to the various types of knee osteoarthritistype of knee oaft lateraleffective groupno effective group cases ft medial cases patello femoral joint cases abbreviations ft femorotibial joint oa osteoarthritis cases cases casepatients reported reduced pain and improved functionality of the patients treated with curcumin these effects could be achieved as early as weeks25 similarly or months of theracurmin treatment resulted in significant clinical effects that could be maintained for months in this studyseveral reports have discussed the effectiveness of curcumin for cartilage degeneration in animal models and cell cultures curcumin treatment led to reduced proteoglycan loss and cartilage erosion in a posttraumatic osteoarthritis mouse model26 curcumin treatment enhanced autophagy and reduced apoptosis and cartilage loss in a spontaneous and surgically induced osteoarthritis mice model27 curcumin prevented the deterioration of articular cartilage compared to the control group in estrogendeficient rats28 in cell culture studies noncytotoxic concentrations of curcumin exerted anticatabolic and antiinflammatory effects in cartilage explants29 curcumin was demonstrated to inhibit the interleukin il1binduced activation of nfkb by suppressing ikba phosphorylation30 curcumin inhibited chondrocyte hypertrophy through indian hedgehog homolog and notch signaling curcumin was a potential agent in modulating cartilage homeostasis and maintaining the chondrocyte phenotype31 however although this study was unable to demonstrate improvements in patient knee cartilage the above findings suggest that curcumin might improve the knee cartilage of such patientsin this study patients felt itchy there were a slight increase in neutral fat cases and hepatic enzymes cases in the blood analyses during the study however no major side effects were observed with theracurmin treatment including in the blood biochemistry analyses performed during this study likewise there were no considerable adverse effects in curcumin groups in other clinical studies17181921 curcuma domestica extracts were noninferior to ibuprofen for the treatment of knee osteoarthritis and the curcuma domestica extracts group had a similar incidence of adverse events but with gastrointestinal adverse events20 daily reported that turmeric preparations and curcumin were considered safe at doses not exceeding mgday for up to months32 a study on the safety of theracurmin revealed that a dose of up to mg for months was safe in cancer patients receiving chemotherapy33 in this study the dosage of curcumin was mgday similar to nakagawa study13 this was the smallest dose used in the published clinical studies34 the safety of curcumin was demonstrated in this study and the dosage of curcumin used in our study was safethere are main limitations in this study there was a short followup period months the sample size was small cases the lack of other treatment adjustment in the analysis the placebo effects could not be eliminated and this was an 0cnakagawa et al table the chronological changes of the clinical scores in vas scores month month month months months months months months months months months monthsabbreviation vas visual analog scalethe p values between the assessment periods for improvement in vas scoresbold numbers indicate a significant differencetable the chronological changes of the clinical scores in jkom scores month month month months months months months months months months months monthsabbreviation jkom japanese knee osteoarthritis measurethe p values between the assessment periods for improvement in jkom scoresbold numbers indicate a significant differencetable the chronological changes of the clinical scores in joa scores month months months months months months month month months months months monthsabbreviation joa japanese orthopedic associationthe p values between the assessment periods for improvement in joa scoresbold numbers indicate a significant differenceopen study also it is a potential selection bias that the patients are paying for their treatment from the startfifty patients with knee osteoarthritis took capsules of theracurmin per day corresponding to a total daily dose of mg of curcumin for months five cases dropped out because of minor side effects therefore we included patients men and women in the further analyses this study showed that theracurmin was effective for treating knee osteoarthritis in of all cases and in of cases in the theracurmin only group of the theracurminonly group the joa score at months after the treatment was significantly better than that at the pretreatment period using the paired ttest in our evaluation of the safety of theracurmin for knee osteoarthritis no major side effects were observed with theracurmin treatment including in the blood biochemistry analyses performed during this study theracurmin showed great potential for the treatment of human knee osteoarthritis based on efficacy and sa Answer:

Colon_Cancer

during the covid19 pandemic emergency departments have noted a significant decrease in strokepatients we performed a timely analysis of the bavarian telestroke tempis œworking diagnosis databasemethods twelve hospitals from the tempis network were selected data collected for january through april in years through were extracted and analyzed for presumed and definite ischemic stroke is amongst otherdisorders in addition recommendations for intravenous thrombolysis rtpa and endovascular thrombectomy evtwere noted and mobility data of the region analyzed if statistically valid groupcomparison was tested with fisher™sexact test considering unpaired observations and apvalue was considered significantresults upon lockdown in midmarch we observed a significant reduction in recommendations for rtpa compared to the preceding three years [“] vs p¼ recommendations for evt werep¼ reflecting its increasing importance following the covid19 lockdown midmarch the number ofevt decreased back to levels in “ [“] vs p¼ absolute numbers of issignificantly higher in january to midmarch compared to “ [“] vs decreased in parallel to mobility datas the reduced stroke incidence during the covid19 pandemic may in part be explained by patientavoidance to seek emergency stroke care and may have an association to population mobility increasing mobilitymay induce a rebound effect and may conflict with a potential second covid19 wave telemedical networks maybe ideal databases to study such effects in nearreal timekeywordstelestroke covid19 lockdown stroke thrombolysisdate received may date accepted june introductionimplementation of social distancing to combat theimpact of corona virus pandemic sequelae has emergedas the major strategy to contain the spread of infectiongiven the lack of specific treatments for covid19 andlimited intensive care resources1 major concerns forstroke neurologistsin this extraordinary scenarioinclude the following a rapid specific managementof cases of acute stroke with possible covid19from initiation of the stroke call in the preclinical setting through the ambulance system emergency department and hospital stroke department and in the1department of neurology university of regensburg bezirksklinikumregensburg tempis telemedical stroke center regensburg germany2cts herdecke germany3department of neurology tempis telemedical stroke centeracademic teaching hospital of the university of munich mu¨nchen klinikharlaching munich germanycorresponding authorfelix schlachetzki md department of neurology university ofregensburg center for vascular neurology and intensive care tempistelemedical stroke center bezirksklinikum regensburguniversit‚¬atsstr84 regensburg germanyemails felixschlachetzkiklinikuniregensburgde 0c of telemedicine and telecare bthe factneuroradiological department when needed to aid instroke diagnosis and treatmentthatpatients with mild stroke symptoms or transient ischemic attacks tias may be reluctant to request hospitaladmission for acute stroke23 and c that covid19itself is associated with severe stroke syndromes this issuggested in a recent case series of covid19 patientsfrom wuhan china focusing on neurological symptoms that described cerebrovascular events in of cases especially in elderly patients and in thosewith more severe infections also authors of a secondcase series reported unusual cases of young covid19patients yrs with large vessel stroke and otherauthors reported three stroke patients with coagulopathy and antiphospholipid antibodies in the context ofsevere covid infections4“the number ofin contrast several stroke departments in germanyincluding our own the usa and china have noted asignificant drop in the number of stroke patient admissions during the corona pandemic7 data on this phenomenon are still scarce howeverin a descriptivereport by morelli from piacenza lombardyitaly covering the period february appearance ofthe first sarscov2 patient recorded in italy to march stroke admissionsdecreased from an average of with largevessel occlusions lvos to two tias one lvoand three lacunar strokes8 using a commercial neuroimaging database with the rapid software platform kasangra and hamilton observed a decrease in stroke imaging procedures with the nadirfollowing the first statewide stayathome order in theusa9 the decrease was observed in all age sex andstroke severity subgroups within all participatinghospitals which processed overall patientsbetween july and april cardiologistsin france observed a similar significant drop in admissions to nine intensive cardiac care units after initiationof social distancing and selfquarantine in midmarch overall there are scarce data available on theimpact of the covid19 infection itself on cardiovascular morbidity including cerebral stroke11aims and hypothesisthe primary aim of this study was to evaluate the effectof the covid19 pandemic lockdown on stroke consultations and treatment recommendations using theacute consultant database of the telestroke networktempis12 we focused on data collected during thefirst four months of which included the emergence of the corona virus pandemic in southeasternbavaria through the first two months of social distancingregion shutdown we compared these data withcomparable data collected during the same months inthe years “methodsdata from daily consultations at clinics withoutneurology departmentsin the telestroke networktempis form the basis of this study the consultationstook place between january and april in the years“ all data were pseudonymized weextracted the actual working diagnoses based on telemedical consultation and neuroimaging results mainlycerebral computed tomography two major databaseswere used to calculate the population within these districts wwwdestatisde and experiencearcgiscomexperience478220a4c454480e823b17327b2bf1d4pagepage_1 this retrospective study was approvedby the local ethics committee of the university ofregensburg and performed in accordance with guidelines of the declaration of helsinkimobility data available at wwwapplecomcovid19mobility were extracted these data were generated from the relative request volume for directionsin munich germany compared with a base volumeon january to observe the relationship ofmobility and the reported stroke decline in piacenzawe also extracted mobility data from milan close topiacenza italy8the major ˜working diagnostic groups™ were asfollows a ischemic stroke b tia c intracranialhaemorrhage d epileptic seizure e migraine andf other disorder including facial palsy headacheand brain tumour also included were cases in whichthere were recommendations for iv thrombolysis ivrtpa or endovascular therapy evt thrombectomyfor lvoexploratory descriptive summary statistics withmean values and standard deviations were appliedin an analysis of data covering january through aprilin years “ in comparison with data coveringthe same period in counts are presented as agraphic display showing incidences standardized to15day periods if statistically valid especially percentage of recommendations for iv thrombolysis andthrombectomy groupcomparison was tested withfisher™s exact test considering unpaired observationsa pvalue was considered significantresultsthere were telemedical consultations during thespecific time frames investigated and the population inthe geographical areas covered by these rural hospitals is most hospitals reside in areas with ahigh number of covid19 cases figure 1a the 0cschlachetzki number of covid19“positive cases in the whole ofbavaria rose from five at the end of february to cases on april the public lockdownwas initiated on march however the recommendation of personal quarantine for people who hadtravelled to northern italy was broadcast earlier on march in munich applevr mobility trends demonstrated a decrease in walking activity in midmarch to “ “ to “ of the baseline levelin milan lombardy italy on march walking activity began to decrease soon reaching “ of baselineactivity and remaining fairly constantthereafterfigure 1boverall consultations were analysed and excluded being nonacute consultations within thenetwork ie followup examinations statistically significant changes in the number of recommendations foriv thrombolysis were observed in figure 1cwhile in “ iv thrombolysis was recommendedin of consultations with suspected ischemicstroke of the frequency of this recommendation decreased to of in p¼ no differences in the number of ivthrombolysis recommendations were observed duringthe time period covering january to march in “ vs in notfigure a incidence of new covid19 infections in bavaria on april red dots indicate network hospitals and green andyellow squares depict the two academic stroke centres that alternate weekly for the tempis consult service modified with permission from the bavarian state office for health and food safety httpwwwlglbayerndegesundheitinfektionsschutzinfektionskrankheiten_a_zcoronaviruskarte_coronavirus b mobility data according to covid19 mobility trends reports apple thedata reflect requests for routing in apple maps for munich which resides in the centre of the tempis network and for milan nearpiacenza where the first decline in the number of strokes was reported morelli 8 horizontal dotted line indicates reportedreduced stroke activity in piacenza c recommendations absolute numbers for application of iv thrombolysis and thrombectomyvertical dashed line indicates the official beginning of lockdown in bavaria time and patient numbers on yaxis are standardized to15day periods xaxis in each month to compensate for shorter february and longer january and march months j1¼ january first half j2 “ january second half f¼ february m¼ march a¼ april d working diagnoses of the telestrokeconsultations vertical dashed line indicates the official beginning of lockdown in bavaria time and patient numbers on yaxis arestandardized to 15day periods xaxis in each month to compensate for shorter february and longer january and march months j1¼ january first half j2 “ january second half f¼ february m¼ march a¼ april 0c of telemedicine and telecare significant no trend in fewer recommendations forevt was observed between march and april in compared with the same time periods in “ of vs “ of however in the preceding time frame january to march significantly more recommendations for thrombectomy were made comparedwith “ of vs of in “ p¼ the data reflect the development of consultationsand treatment recommendations for lvo in the network from onward the number of recommendations for evt steadily rose with increasing evidencefor recanalization even in later time windows andincreasing employment of computed tomography angiography in the tempis network table shows thedevelopment of consultations in up to the end ofthe study including the lockdown period it shows adrop in the number of consultations and more importantly fewer recommendations for iv thrombolysisand evt which suggests fewer incidences of ischemicstroke severities table figure 1dalthough bavaria is the state with the highestnumber of covid19 cases in germany especially inour region we only performed five telestroke consultations for the network hospitals in which possiblecovid19 infection was discussed including a singlepatient with stroke symptoms and feverdiscussionthe tempis telestroke working data confirm thecurrent observation of a low stroke incidence insoutheastern bavaria with relative proportions of theworking diagnosis remaining similar the number ofcases of disabling stroke from intracranial haemorrhage and ischemic stroke requiring iv rtpa or evtalso diminished challenging the theory that onlypatient avoidance to call for emergency treatment isresponsible for this phenomenon this study also demonstrates the potential and importance of telestrokenetworks in the current covid19 pandemic313the observation of fewer stroke cases during thecovid19 pandemic seems to contradict two essentialassumptions with regard to stroke risk a sarcov is a strong risk factor for stroke and b physicalinactivity in a lockdown setting may increase the riskof stroke especially among elderly persons firstsarcov2 may induce hypercoagulability and highlevels of creactive protein ddimer and interleukin6placing patients at risk to develop thrombotic complications14 in a series of intensive care unit patientsin the netherlands reported by klok only threestrokes complicated the course of covid19 whereasthe majority of complications included pulmonarythrombosiscatheterassociatedembolism n¼ and peripheral venous thrombosisn¼ andobservations in case series that concurrent covid infection complicates or triggers unusual ischemicstroke may well prevail but case control studies focusing on this phenomenon are urgently needed to affirmor deny the assertion5 second physical inactivity has aprofound effect on atrial fibrillation obesity diabetesmellitus management and hypertension among othersand contradicts current recommendations on mid andlongterm stroke prevention16 a recent study in consecutive patients with nonstsegment elevationacute coronary syndromes acss and optical coherence tomography of the culprit lesion reported bykato found that the combination of greaterphysical activity outdoor acs onset and high bodymass index had a significant effect on the incidence ofcoronary plaque erosion17 interestingly mobility datasuch as those provided by the apple mobility databasevr demonstrated a parallel reduction in incidencesof stroke and acs in three published papers8“ inaddition to oursour data confirm the observation from morelli who termed the phrase ˜baffling case of ischemicstroke disappearance™ these authors also discuss thatthis effect cannot be totally explained merely by thereluctance of patients to call for help in a stroke emergency because the number of cases presenting withsevere stroke requiring evt and the number of generalconsultations in tempis also decreased an analysisbased on a large database associated with the application of rapid software in acute stroke by kansagra is in line with our observation that also severestroke patients diminished during the early lockdownphase9 the number of ischemic core volumes “ml and greater than ml were observed to decreaseby and respectively core volumes “ ml decreased by and and very smallcore infarct volumes measuring “ ml decreased the decrease in the number of very small infarctvolumes may well be explained by the generally proposed hesitation to seek emergency care while thereduction in large ischemic core volumes is morelikely due to fewer lvos as observed in our studywith a sharp decline in iv thrombolysis and thrombectomy recommendationsanother explanation may be a concurrent low infection rate with other viruses that can trigger atherosclerosis and plaque rupture resulting in neuro andcardiovascular morbidity18 the lockdown not onlyreduces physical activity strict social distancing anduse of facial masks should also lead to low rates ofexposure to and transmission of other common virusesand allergens that by themselves appear to triggerstroke19 additional studies with detailed analyses of 0cschlachetzki stekcarberauqsniatadhtnomehtfoshtgneltnereffidrofstnemtsudajtuohtiwtubsdoirepkeewotnidedvdiilirpa“yraunajrofsnoitatlusnocforebmunlatotlebatrpa“rpa“ram“ram“bef“bef“naj“naj“sopdvocilatotairavabnisesacnoitaiveddradnatsdnaseulavnaemni“morfatadwohssipmetkrowtenekortsnoitatlusnoceetl] 06 06 06 06 06 06 06 06 06 06 06 06 06 06 06 06[snp p¡ 8a 06[¡snp§p§§§§ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[  8a 06[§ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[lsisyobmorhtvi 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06 06[ 8a 06[ 8a 06 06[ytilibadaerrettebrofldoblynoerasrebmunldobskcattaicmehcsitneisnartatiamotamehlarudbushdsegahrromehidonhcarabushasegahrromeahlainarcartnihciigndeebllainarcartnsnsn¼¼¼¼bp§pcpp§§ ¡iids 06“naemsnoitatlusnocekortsicmehcsids 06“naemds 06“naemymotcebmorhtds 06“naemds 06“naema tids 06“naemh cids 06“naemhashdsbcieniargimeruziessrehto 0c of telemedicine and telecare symptom onsettodoor times stroke severity neuroimaging and inflammatory markers are needed tounderstand the reason for the reduced number of revascularization therapies requested during the covid19pandemiclimitations of the studyanalysis of daily working diagnoses in the tempistelestroke network has the advantage of being highlytimely yet it lacks specificity because the final diagnosismay differ from the initial one this may be compensated by the creation of a large common database fortelestroke networks that incorporates corrections forthe actual population covered analyses of otherstrokerelated databases such as the one associatedwith rapid software healthcare provider databasesand common stroke registries for quality control thedecrease in the number of thrombectomy recommendations in our cohort midmarch did not reach statistical significance when compared with the sameperiod in years through because rates forthis procedure increased according with levels of evidence2021 in agreement with this development thrombectomy recommendations by tempis neurologists in prior to the covid19 pandemic occurred morefrequently than in previous yearssour study using the tempis telestroke database confirms lower incidences of ischemic stroke and otheracute neurological disorders requiring consultationsuch as intracerebral haemorrhage seizure disorderand migraine next to a reluctance within the population to seek immediate medical assistance for acutestroke the covid19 lockdown which resulted inless physical activity and fewer other common infections may also be responsible for the fewer numberof patients with severe stroke especially those withintracranial haemorrhage and those eligible for recanalization therapies if lockdownassociated factors areindeed responsible for a lower stroke incidence we mayexpect a rebound effect following the lockdown periodwith an increased incidence of stroke as well as ofmyocardial infarcts and traumatic brain injuries aspatients™ frailty may have increased during the lockdown analyses of large stroke databases may revealfurther insights into this phenomenon however telestroke networks such as tempis may be ideal tools tomonitor stroke occurrence in real timeacknowledgmentsthe authors acknowledge all consulting neurologists intempis and colleaguesin badin partner hospitalsebersbergburglengenfeldreichenhalleggenfeldenerding freising kelheim mu¨ hldorf rotthalmu¨ nstervilsbiburg dingolfing and zwiesel the authors like tothank jo ann elison ma elsdfor editing thispaper for english grammar and languagedeclaration of conflicting intereststhe authors declared no potential conflicts of interest withrespect to the research authorship andor publication of thisarticleanonymized data are available on requestfundingthe authors received no financial support for the researchauthorship andor publication of this articleorcid idfelix schlachetzkiorcidorg0000000161672597references jawaid a protecting older adults during social distancing science khosravani h rajendram p notario l protectedcode stroke hyperacute stroke management during thecoronavirus disease covid19 pandemic stroke “ markus hs and brainin m covid19 and stroke a global world stroke organization perspective int jstroke “ mao l jin h wang m neurologic manifestationsof hospitalized patients with coronavirus disease inwuhan china jama neurol “ oxley tj mocco j majidi s largevessel stroke asa presenting feature of covid19 in the young n engl jmed e60 zhang y xiao m zhang s coagulopathy andantiphospholipid antibodies in patients with covid19n engl j med e38 zhao j rudd a and liu r challenges and potentialsolutions of stroke care during the coronavirus disease covid19 outbreak stroke “ morelli n rota e terracciano c the baffling caseofischemic stroke disappearance from the casualtydepartment in the covid19 era eur neurol “ kansagra ap goyal ms hamilton s collateraleffect of covid19 on stroke evaluation in the unitedstates n englnejmc2014816 online ahead of printj meddoi huet f prieur c schurtz g one train may hideanother acute cardiovascular diseases could be neglectedbecause of the covid19 pandemic arch cardiovasc dis “ bansal m cardiovascular disease and covid19diabetes metab syndr “ audebert hj schenkel j heuschmann pu effectsof the implementation of a telemedical stroke networkthe telemedic pilot project for integrative stroke care 0cschlachetzki tempis in bavaria germany lancet neurol “ patel uk malik p demasi m multidisciplinaryapproach and outcomes of teleneurology a reviewcureus e4410 terpos e ntanasisstathopoulos i elalamy i hematological findings and complications of covid am j hematol “ klok fa kruip m van der meer njm incidenceof thrombotic complications in critically ill icu patientswith covid19 thromb res “ kyu hh bachman vf alexander lt physicalactivity and risk of breast cancer colon cancer diabetesischemic heart disease and ischemic stroke eventsaystematic review and doseresponse metaanalysis forthe global burden of disease study bmj i3857 kato a minami y katsura a physical exertion asa trigger of acute coronary syndrome caused by plaqueerosion j thromb thrombolysis “ grau aj urbanek c and palm f common infectionsand the risk of stroke nat rev neurol “ pagliano p spera am ascione t infections causing stroke or strokelike syndromes infection “ campbell bcv donnan ga lees kr endovascular stent thrombectomy the new standardof care for large vessel ischaemic stroke lancet neurol “ vinny pw vishnu vy and padma srivastava mvthrombectomy to hours after stroke n engl jmed 0c'

cancer285

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: during the covid19 pandemic emergency departments have noted a significant decrease in strokepatients we performed a timely analysis of the bavarian telestroke tempis œworking diagnosis databasemethods twelve hospitals from the tempis network were selected data collected for january through april in years through were extracted and analyzed for presumed and definite ischemic stroke is amongst otherdisorders in addition recommendations for intravenous thrombolysis rtpa and endovascular thrombectomy evtwere noted and mobility data of the region analyzed if statistically valid groupcomparison was tested with fisher™sexact test considering unpaired observations and apvalue was considered significantresults upon lockdown in midmarch we observed a significant reduction in recommendations for rtpa compared to the preceding three years [“] vs p¼ recommendations for evt werep¼ reflecting its increasing importance following the covid19 lockdown midmarch the number ofevt decreased back to levels in “ [“] vs p¼ absolute numbers of issignificantly higher in january to midmarch compared to “ [“] vs decreased in parallel to mobility datas the reduced stroke incidence during the covid19 pandemic may in part be explained by patientavoidance to seek emergency stroke care and may have an association to population mobility increasing mobilitymay induce a rebound effect and may conflict with a potential second covid19 wave telemedical networks maybe ideal databases to study such effects in nearreal timekeywordstelestroke covid19 lockdown stroke thrombolysisdate received may date accepted june introductionimplementation of social distancing to combat theimpact of corona virus pandemic sequelae has emergedas the major strategy to contain the spread of infectiongiven the lack of specific treatments for covid19 andlimited intensive care resources1 major concerns forstroke neurologistsin this extraordinary scenarioinclude the following a rapid specific managementof cases of acute stroke with possible covid19from initiation of the stroke call in the preclinical setting through the ambulance system emergency department and hospital stroke department and in the1department of neurology university of regensburg bezirksklinikumregensburg tempis telemedical stroke center regensburg germany2cts herdecke germany3department of neurology tempis telemedical stroke centeracademic teaching hospital of the university of munich mu¨nchen klinikharlaching munich germanycorresponding authorfelix schlachetzki md department of neurology university ofregensburg center for vascular neurology and intensive care tempistelemedical stroke center bezirksklinikum regensburguniversit‚¬atsstr84 regensburg germanyemails felixschlachetzkiklinikuniregensburgde 0c of telemedicine and telecare bthe factneuroradiological department when needed to aid instroke diagnosis and treatmentthatpatients with mild stroke symptoms or transient ischemic attacks tias may be reluctant to request hospitaladmission for acute stroke23 and c that covid19itself is associated with severe stroke syndromes this issuggested in a recent case series of covid19 patientsfrom wuhan china focusing on neurological symptoms that described cerebrovascular events in of cases especially in elderly patients and in thosewith more severe infections also authors of a secondcase series reported unusual cases of young covid19patients yrs with large vessel stroke and otherauthors reported three stroke patients with coagulopathy and antiphospholipid antibodies in the context ofsevere covid infections4“the number ofin contrast several stroke departments in germanyincluding our own the usa and china have noted asignificant drop in the number of stroke patient admissions during the corona pandemic7 data on this phenomenon are still scarce howeverin a descriptivereport by morelli from piacenza lombardyitaly covering the period february appearance ofthe first sarscov2 patient recorded in italy to march stroke admissionsdecreased from an average of with largevessel occlusions lvos to two tias one lvoand three lacunar strokes8 using a commercial neuroimaging database with the rapid software platform kasangra and hamilton observed a decrease in stroke imaging procedures with the nadirfollowing the first statewide stayathome order in theusa9 the decrease was observed in all age sex andstroke severity subgroups within all participatinghospitals which processed overall patientsbetween july and april cardiologistsin france observed a similar significant drop in admissions to nine intensive cardiac care units after initiationof social distancing and selfquarantine in midmarch overall there are scarce data available on theimpact of the covid19 infection itself on cardiovascular morbidity including cerebral stroke11aims and hypothesisthe primary aim of this study was to evaluate the effectof the covid19 pandemic lockdown on stroke consultations and treatment recommendations using theacute consultant database of the telestroke networktempis12 we focused on data collected during thefirst four months of which included the emergence of the corona virus pandemic in southeasternbavaria through the first two months of social distancingregion shutdown we compared these data withcomparable data collected during the same months inthe years “methodsdata from daily consultations at clinics withoutneurology departmentsin the telestroke networktempis form the basis of this study the consultationstook place between january and april in the years“ all data were pseudonymized weextracted the actual working diagnoses based on telemedical consultation and neuroimaging results mainlycerebral computed tomography two major databaseswere used to calculate the population within these districts wwwdestatisde and experiencearcgiscomexperience478220a4c454480e823b17327b2bf1d4pagepage_1 this retrospective study was approvedby the local ethics committee of the university ofregensburg and performed in accordance with guidelines of the declaration of helsinkimobility data available at wwwapplecomcovid19mobility were extracted these data were generated from the relative request volume for directionsin munich germany compared with a base volumeon january to observe the relationship ofmobility and the reported stroke decline in piacenzawe also extracted mobility data from milan close topiacenza italy8the major ˜working diagnostic groups™ were asfollows a ischemic stroke b tia c intracranialhaemorrhage d epileptic seizure e migraine andf other disorder including facial palsy headacheand brain tumour also included were cases in whichthere were recommendations for iv thrombolysis ivrtpa or endovascular therapy evt thrombectomyfor lvoexploratory descriptive summary statistics withmean values and standard deviations were appliedin an analysis of data covering january through aprilin years “ in comparison with data coveringthe same period in counts are presented as agraphic display showing incidences standardized to15day periods if statistically valid especially percentage of recommendations for iv thrombolysis andthrombectomy groupcomparison was tested withfisher™s exact test considering unpaired observationsa pvalue was considered significantresultsthere were telemedical consultations during thespecific time frames investigated and the population inthe geographical areas covered by these rural hospitals is most hospitals reside in areas with ahigh number of covid19 cases figure 1a the 0cschlachetzki number of covid19“positive cases in the whole ofbavaria rose from five at the end of february to cases on april the public lockdownwas initiated on march however the recommendation of personal quarantine for people who hadtravelled to northern italy was broadcast earlier on march in munich applevr mobility trends demonstrated a decrease in walking activity in midmarch to “ “ to “ of the baseline levelin milan lombardy italy on march walking activity began to decrease soon reaching “ of baselineactivity and remaining fairly constantthereafterfigure 1boverall consultations were analysed and excluded being nonacute consultations within thenetwork ie followup examinations statistically significant changes in the number of recommendations foriv thrombolysis were observed in figure 1cwhile in “ iv thrombolysis was recommendedin of consultations with suspected ischemicstroke of the frequency of this recommendation decreased to of in p¼ no differences in the number of ivthrombolysis recommendations were observed duringthe time period covering january to march in “ vs in notfigure a incidence of new covid19 infections in bavaria on april red dots indicate network hospitals and green andyellow squares depict the two academic stroke centres that alternate weekly for the tempis consult service modified with permission from the bavarian state office for health and food safety httpwwwlglbayerndegesundheitinfektionsschutzinfektionskrankheiten_a_zcoronaviruskarte_coronavirus b mobility data according to covid19 mobility trends reports apple thedata reflect requests for routing in apple maps for munich which resides in the centre of the tempis network and for milan nearpiacenza where the first decline in the number of strokes was reported morelli 8 horizontal dotted line indicates reportedreduced stroke activity in piacenza c recommendations absolute numbers for application of iv thrombolysis and thrombectomyvertical dashed line indicates the official beginning of lockdown in bavaria time and patient numbers on yaxis are standardized to15day periods xaxis in each month to compensate for shorter february and longer january and march months j1¼ january first half j2 “ january second half f¼ february m¼ march a¼ april d working diagnoses of the telestrokeconsultations vertical dashed line indicates the official beginning of lockdown in bavaria time and patient numbers on yaxis arestandardized to 15day periods xaxis in each month to compensate for shorter february and longer january and march months j1¼ january first half j2 “ january second half f¼ february m¼ march a¼ april 0c of telemedicine and telecare significant no trend in fewer recommendations forevt was observed between march and april in compared with the same time periods in “ of vs “ of however in the preceding time frame january to march significantly more recommendations for thrombectomy were made comparedwith “ of vs of in “ p¼ the data reflect the development of consultationsand treatment recommendations for lvo in the network from onward the number of recommendations for evt steadily rose with increasing evidencefor recanalization even in later time windows andincreasing employment of computed tomography angiography in the tempis network table shows thedevelopment of consultations in up to the end ofthe study including the lockdown period it shows adrop in the number of consultations and more importantly fewer recommendations for iv thrombolysisand evt which suggests fewer incidences of ischemicstroke severities table figure 1dalthough bavaria is the state with the highestnumber of covid19 cases in germany especially inour region we only performed five telestroke consultations for the network hospitals in which possiblecovid19 infection was discussed including a singlepatient with stroke symptoms and feverdiscussionthe tempis telestroke working data confirm thecurrent observation of a low stroke incidence insoutheastern bavaria with relative proportions of theworking diagnosis remaining similar the number ofcases of disabling stroke from intracranial haemorrhage and ischemic stroke requiring iv rtpa or evtalso diminished challenging the theory that onlypatient avoidance to call for emergency treatment isresponsible for this phenomenon this study also demonstrates the potential and importance of telestrokenetworks in the current covid19 pandemic313the observation of fewer stroke cases during thecovid19 pandemic seems to contradict two essentialassumptions with regard to stroke risk a sarcov is a strong risk factor for stroke and b physicalinactivity in a lockdown setting may increase the riskof stroke especially among elderly persons firstsarcov2 may induce hypercoagulability and highlevels of creactive protein ddimer and interleukin6placing patients at risk to develop thrombotic complications14 in a series of intensive care unit patientsin the netherlands reported by klok only threestrokes complicated the course of covid19 whereasthe majority of complications included pulmonarythrombosiscatheterassociatedembolism n¼ and peripheral venous thrombosisn¼ andobservations in case series that concurrent covid infection complicates or triggers unusual ischemicstroke may well prevail but case control studies focusing on this phenomenon are urgently needed to affirmor deny the assertion5 second physical inactivity has aprofound effect on atrial fibrillation obesity diabetesmellitus management and hypertension among othersand contradicts current recommendations on mid andlongterm stroke prevention16 a recent study in consecutive patients with nonstsegment elevationacute coronary syndromes acss and optical coherence tomography of the culprit lesion reported bykato found that the combination of greaterphysical activity outdoor acs onset and high bodymass index had a significant effect on the incidence ofcoronary plaque erosion17 interestingly mobility datasuch as those provided by the apple mobility databasevr demonstrated a parallel reduction in incidencesof stroke and acs in three published papers8“ inaddition to oursour data confirm the observation from morelli who termed the phrase ˜baffling case of ischemicstroke disappearance™ these authors also discuss thatthis effect cannot be totally explained merely by thereluctance of patients to call for help in a stroke emergency because the number of cases presenting withsevere stroke requiring evt and the number of generalconsultations in tempis also decreased an analysisbased on a large database associated with the application of rapid software in acute stroke by kansagra is in line with our observation that also severestroke patients diminished during the early lockdownphase9 the number of ischemic core volumes “ml and greater than ml were observed to decreaseby and respectively core volumes “ ml decreased by and and very smallcore infarct volumes measuring “ ml decreased the decrease in the number of very small infarctvolumes may well be explained by the generally proposed hesitation to seek emergency care while thereduction in large ischemic core volumes is morelikely due to fewer lvos as observed in our studywith a sharp decline in iv thrombolysis and thrombectomy recommendationsanother explanation may be a concurrent low infection rate with other viruses that can trigger atherosclerosis and plaque rupture resulting in neuro andcardiovascular morbidity18 the lockdown not onlyreduces physical activity strict social distancing anduse of facial masks should also lead to low rates ofexposure to and transmission of other common virusesand allergens that by themselves appear to triggerstroke19 additional studies with detailed analyses of 0cschlachetzki stekcarberauqsniatadhtnomehtfoshtgneltnereffidrofstnemtsudajtuohtiwtubsdoirepkeewotnidedvdiilirpa“yraunajrofsnoitatlusnocforebmunlatotlebatrpa“rpa“ram“ram“bef“bef“naj“naj“sopdvocilatotairavabnisesacnoitaiveddradnatsdnaseulavnaemni“morfatadwohssipmetkrowtenekortsnoitatlusnoceetl] 06 06 06 06 06 06 06 06 06 06 06 06 06 06 06 06[snp p¡ 8a 06[¡snp§p§§§§ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[  8a 06[§ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[lsisyobmorhtvi 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06 06[ 8a 06[ 8a 06 06[ytilibadaerrettebrofldoblynoerasrebmunldobskcattaicmehcsitneisnartatiamotamehlarudbushdsegahrromehidonhcarabushasegahrromeahlainarcartnihciigndeebllainarcartnsnsn¼¼¼¼bp§pcpp§§ ¡iids 06“naemsnoitatlusnocekortsicmehcsids 06“naemds 06“naemymotcebmorhtds 06“naemds 06“naema tids 06“naemh cids 06“naemhashdsbcieniargimeruziessrehto 0c of telemedicine and telecare symptom onsettodoor times stroke severity neuroimaging and inflammatory markers are needed tounderstand the reason for the reduced number of revascularization therapies requested during the covid19pandemiclimitations of the studyanalysis of daily working diagnoses in the tempistelestroke network has the advantage of being highlytimely yet it lacks specificity because the final diagnosismay differ from the initial one this may be compensated by the creation of a large common database fortelestroke networks that incorporates corrections forthe actual population covered analyses of otherstrokerelated databases such as the one associatedwith rapid software healthcare provider databasesand common stroke registries for quality control thedecrease in the number of thrombectomy recommendations in our cohort midmarch did not reach statistical significance when compared with the sameperiod in years through because rates forthis procedure increased according with levels of evidence2021 in agreement with this development thrombectomy recommendations by tempis neurologists in prior to the covid19 pandemic occurred morefrequently than in previous yearssour study using the tempis telestroke database confirms lower incidences of ischemic stroke and otheracute neurological disorders requiring consultationsuch as intracerebral haemorrhage seizure disorderand migraine next to a reluctance within the population to seek immediate medical assistance for acutestroke the covid19 lockdown which resulted inless physical activity and fewer other common infections may also be responsible for the fewer numberof patients with severe stroke especially those withintracranial haemorrhage and those eligible for recanalization therapies if lockdownassociated factors areindeed responsible for a lower stroke incidence we mayexpect a rebound effect following the lockdown periodwith an increased incidence of stroke as well as ofmyocardial infarcts and traumatic brain injuries aspatients™ frailty may have increased during the lockdown analyses of large stroke databases may revealfurther insights into this phenomenon however telestroke networks such as tempis may be ideal tools tomonitor stroke occurrence in real timeacknowledgmentsthe authors acknowledge all consulting neurologists intempis and colleaguesin badin partner hospitalsebersbergburglengenfeldreichenhalleggenfeldenerding freising kelheim mu¨ hldorf rotthalmu¨ nstervilsbiburg dingolfing and zwiesel the authors like tothank jo ann elison ma elsdfor editing thispaper for english grammar and languagedeclaration of conflicting intereststhe authors declared no potential conflicts of interest withrespect to the research authorship andor publication of thisarticleanonymized data are available on requestfundingthe authors received no financial support for the researchauthorship andor publication of this articleorcid idfelix schlachetzkiorcidorg0000000161672597references jawaid a protecting older adults during social distancing science khosravani h rajendram p notario l protectedcode stroke hyperacute stroke management during thecoronavirus disease covid19 pandemic stroke “ markus hs and brainin m covid19 and stroke a global world stroke organization perspective int jstroke “ mao l jin h wang m neurologic manifestationsof hospitalized patients with coronavirus disease inwuhan china jama neurol “ oxley tj mocco j majidi s largevessel stroke asa presenting feature of covid19 in the young n engl jmed e60 zhang y xiao m zhang s coagulopathy andantiphospholipid antibodies in patients with covid19n engl j med e38 zhao j rudd a and liu r challenges and potentialsolutions of stroke care during the coronavirus disease covid19 outbreak stroke “ morelli n rota e terracciano c the baffling caseofischemic stroke disappearance from the casualtydepartment in the covid19 era eur neurol “ kansagra ap goyal ms hamilton s collateraleffect of covid19 on stroke evaluation in the unitedstates n englnejmc2014816 online ahead of printj meddoi huet f prieur c schurtz g one train may hideanother acute cardiovascular diseases could be neglectedbecause of the covid19 pandemic arch cardiovasc dis “ bansal m cardiovascular disease and covid19diabetes metab syndr “ audebert hj schenkel j heuschmann pu effectsof the implementation of a telemedical stroke networkthe telemedic pilot project for integrative stroke care 0cschlachetzki tempis in bavaria germany lancet neurol “ patel uk malik p demasi m multidisciplinaryapproach and outcomes of teleneurology a reviewcureus e4410 terpos e ntanasisstathopoulos i elalamy i hematological findings and complications of covid am j hematol “ klok fa kruip m van der meer njm incidenceof thrombotic complications in critically ill icu patientswith covid19 thromb res “ kyu hh bachman vf alexander lt physicalactivity and risk of breast cancer colon cancer diabetesischemic heart disease and ischemic stroke eventsaystematic review and doseresponse metaanalysis forthe global burden of disease study bmj i3857 kato a minami y katsura a physical exertion asa trigger of acute coronary syndrome caused by plaqueerosion j thromb thrombolysis “ grau aj urbanek c and palm f common infectionsand the risk of stroke nat rev neurol “ pagliano p spera am ascione t infections causing stroke or strokelike syndromes infection “ campbell bcv donnan ga lees kr endovascular stent thrombectomy the new standardof care for large vessel ischaemic stroke lancet neurol “ vinny pw vishnu vy and padma srivastava mvthrombectomy to hours after stroke n engl jmed 0c' Answer:

Colon_Cancer

purpose to explore a new therapeutic option for patients with hepatocellular carcinoma hcc the efficacy and safety of a group of traditional chinese medicines banxia xiexin recipe as monotherapy for patients with advanced hcc was studied materials and methods the study included patients with advanced hcc from august to august for analysis these eligible patients received treatment with banxia xiexin recipe for at least month the primary endpoints were progressionfree survival pfs and overall survival os the secondary efficacy endpoints included objective response rate orr and disease control rate dcr in addition safety was also assessed results the median treatment duration of these patients was months range months and followup is still ongoing the median pfs was months confidence interval [ci] months and the median os was months ci months the orr was and the dcr was in the subgroup analysis the median os in the transcatheter arterial chemoembolization tace group was not reached and the median os in the no tace group was months ci months in addition no drugrelated serious adverse events were observed during the study this is the first clinical analysis of traditional chinese medicine as a single treatment for advanced hcc the obtained results are encouraging as they suggest that this panel of chinese herbs is safe and it may be effective for patients with advanced hcc in a realworld clinical settingkeywordshepatocellular carcinoma herbs tace banxia xiexin recipe clinical researchsubmitted december revised june accepted june introductionliver cancer is one of the most frequent malignant tumors and the third leading cause of cancerrelated deaths worldwide both the incidence and mortality rates for liver cancer are increasing while the overall incidence and mortality for all cancers combined tend to decline12 hepatocellular carcinoma hcc is the most common and deadly form of liver cancer in china chronic hepatitis b virus hbv infection and subsequent liver fibrosis and cirrhosis are the 1affiliated hospital of chengdu university of traditional chinese medicine chengdu sichuan province chinathese authors contributed to the work equally and should be regarded as co“first authorscorresponding authorshaoquan xiong department of medical oncology affiliated hospital of chengdu university of traditional chinese medicine chengdu sichuan province china email xsquan106163comcreative commons non commercial cc bync this is distributed under the terms of the creative commons attributionnoncommercial license creativecommonslicensesbync40 which permits noncommercial use reproduction and distribution of the work without further permission provided the original work is attributed as specified on the sage and open access pages ussagepubcomenusnamopenaccessatsage 0c integrative cancer therapies leading causes of liver cancer due to the lack of effective treatment options as well as the high recurrence and metastasis rates hcc is associated with an extremely poor prognosis at present surgical resection and liver transplantation are effective treatments for liver cancer but only a few patients are surgical candidates due to tumor extension poor hepatic functional reserve or underlying liver cirrhosis34 even for the few lucky patients who have completed the surgery the recurrence rates at years following liver resection have been reported to exceed for patients with advanced liver cancer it is essential to improve general wellbeing promote quality of life and prolong survival time it is well known that chemotherapy effects in patients with advanced liver cancer are not outstanding67 some patients can consider local treatment such as transarterial chemoembolization tace the progressionfree survival pfs of patients receiving tace treatment is reported to be months8 molecular targeted drugs are also one of the options for patients with advanced liver cancer such as sorafenib and lenvatinib however according to reports on sorafenib the only approved molecular targeted therapy for advanced hcc the median pfs of patients treated with sorafenib was only months in the asiapacific region9 in recent years immunotherapy which has been attracting worldwide attention is also becoming an important treatment method for example nivolumab and pabolizumab which have been approved by the food and drug administration for secondline treatment of advanced hcc can bring certain benefits to patients with advanced liver cancer but are expensive and difficult to afford for many patientsin china traditional chinese medicine tcm therapies which can date back more than years are widely used in cancer treatment10 banxia xiexin decoction was created by the ancient doctor zhang zhongjing it is mainly used to treat digestive diseases such as bloating and abdominal pain it has a history of more than years in a previous retrospective study it was found that banxia xiexin decoction can bring survival benefits to patients with advanced liver cancer and one of them achieved complete response cr11 as a tcm hospital many patients with advanced liver cancer come to our hospital to undergo chinese medicine for treatment therefore we designed a prospective study of banxia xiexin decoction as monotherapy for advanced liver cancer and analyzed its treatment effect and safetymethodsstudy design and patient enrollmentin this prospective openlabel study patients from the oncology department of the affiliated hospital of the chengdu university of traditional chinese medicine chengdu china were recruitedaccording to the american association for the study of liver diseases criteria eligible patients were adults with advanced hcc confirmed by pathological assessment or noninvasive assessment12 these patients had not received any cancerrelated treatment within weeks before the start of study treatment and the disease had progressed after the prior treatment in addition these patients had to have at least one measurable lesion defined by modified response evaluation criteria in solid tumors for hcc mrecist and recist version inclusion criteria also included eastern cooperative oncology group ecog performance status of to an estimated life expectancy of months childpugh liver function class a or b adequate an function such as white blood cell count ‰¥ — cellsl neutrophils ‰¥ — cellsl and platelets ‰¥ — cellsl and the absence of any serious comorbidities such as significant cardiac cerebrovascular hepatic or nephritic abnormities or other important an dysfunctionskey exclusion criteria included patients with low compliance or use of other antitumor treatments at the same time diagnosis of other malignant tumors and hcc with brain metastasisthe protocol was approved by the ethics committee of the affiliated hospital of chengdu university of traditional chinese medicine no 2015bl003 all patients provided written informed consent before study entrystudy treatment and proceduresthe enrolled patients received treatment with banxia xiexin recipe in the form of decoction the herbal components and their doses in the banxia xiexin recipe decoction are listed in the appendix with the use of an automatic herbboiling machine the decoction volume of each prescription was about ml patients were instructed to take ml each time times dailytreatment was continued until disease progression death development of unacceptable toxicity patient™s refusal to continue or at the discretion of the investigator when the disease progressed treatment could also be continued for month to assess tumor response as long as the patient agreed to do sofollowing an initial baseline assessment within days of the start of study treatment the investigator assessed tumor response by using computed tomography ct scan after to months of treatment subsequently followup assessments were done every months until the patient died adverse events were recorded continuously from enrollment to the end of the final study visitoutcomesthe primary endpoints were pfs time from enrollment to radiological disease progression or death and overall survival os time from enrollment to death due to any cause the secondary efficacy endpoints included the proportion 0cwang et al of patients who had an objective response complete or partial response [pr] according to recist version namely objective response rate orr and disease control rate dcr the proportion of patients who had a cr pr or stable disease besides safety was also assessed by the incidence and nature of adverse eventsstatistical analysisthe pfs and os were estimated using the kaplanmeier method all statistical analyses were performed using graphpad prism software version graphpad software inc and logrank testresultsfrom august to august a total of patients with advanced hcc were enrolled from the oncology department of the affiliated hospital of chengdu university of traditional chinese medicine however only the results of the mediumterm analysis are presented here since patients were still on treatment and had not yet reached the assessment time point therefore patients were finally included in the current analysis meaningful results were found to report in this midterm analysis of course we will update the results according to the latest followup data in the future the baseline characteristics of the included patients are shown in table notably patients were without any treatment in this study all patients who were previously treated with surgery or tace had progressive disease which was similar to the clinical trials of sorafenib9 the number of patients receiving chemotherapy and molecular targeted therapy is and respectively patients who had received previous systemic therapy were excluded in the clinical trials of sorafenib but these patients had progressive disease after the prior treatment according to recist version in this studythe data cutoff date for the current analysis was august the median treatment duration of these patients was months range months and followup is still ongoing since if the disease progressed treatment could also be continued for month to assess tumor response as long as the patient agreed to do so or if thepatient would like to use it as an adjuvant treatment to other treatment such as tace moleculartargeted therapy and so onin the primary analysis the median pfs was months confidence interval [ci] figure and the median os was months ci figure thirtysix patients died all due to disease progression the 1year survival rate was notably most of these patients received prior treatments such as tace and targeted molecular therapy before enrollment in order to explore the potential effect of previous treatment we performed a subgroup analysis the median pfs months ci was longer in the subgroup of patients who had previously received tace compared with the median pfs months ci who had not previously received tace thus the treatment effect in this type of patient seems to be better p figure the median os of advanced hcc patients who had previously received tace was not reached and the median os of subgroups who had not received tace was months ci as shown in figure in terms of tumor response after treatment with banxia xiexin recipe patients achieved a pr had stable disease the orr was and the dcr was table particularly the imaging data of typical cases were presented as follows the first patient was a 58yearold male who was diagnosed with advanced hcc with metastasis to the right kidney on august at baseline the ct scan showed that the size of the target lesion in the liver was cm — cm — cm figure 5a blood tests revealed elevated αfetoprotein µgml and the ecog performance status score was this patient had not received any anticancer treatment previously after months treatment with banxia xiexin recipe the ct scan showed that the target lesion in the liver shrank to cm — cm figure 5b and reached a pr according to recist version the ecog performance status score improved from to the patient is still currently in treatment and has not yet had progressive diseaseone 54yearold male patient was first diagnosed to have hcc in december he was treated by surgical resection on january no antineoplastic treatment was done thereafter when a regular review was conducted on september it showed intrahepatic recurrence and right lung metastasis and then he received treatment with chinese herbs at baseline the ct scan showed that the size of the target lesion in the liver was cm — cm — cm figure 6a blood tests revealed elevated αfetoprotein µgml and the ecog performance status score was after a 32month treatment with modified xiexin recipe the ct scan showed that the target lesion in the liver shrank to — cm figure 6b and reached a pr according to recist version the duration of the pr was months he continued to insist on treatment after the disease progressed and he died in october concerning safety no adverse events were observed except for the occurrence of mild nausea three patients reported mild nausea after taking pills but the symptom was relieved spontaneously after week without any 0c integrative cancer therapies table the clinical characteristics of the patients with advanced hepatocellular carcinomavariablesnumber of patientspercentageage years ‰¥sex male femaleecogps childspugh class a bhepatitis b virus infection yes noliver cirrhosis yes noafp µgl ‰¥ tumor size cm ‰¥pvtt yes noextrahepatic spread yes noinvolved disease sites per patient ‰¥previous treatment no sur tace rf che mttajcc stage iiibivaivbabbreviations ecogps eastern cooperative oncology group“performance status pvtt portal vein tumor thrombus sur surgical resection tace transcatheter arterial chemoembolization rf radiofrequency che chemotherapy mtt molecular targeted therapy ajcc american joint committee on cancerfigure kaplanmeier analysis of progressionfree survivalfigure kaplanmeier analysis of overall survivalfigure kaplanmeier subgroup analysis of progressionfree survival stratified by prior tace treatmenttreatment in addition no serious adverse events such as liver or kidney damage were detected during the followup period thus the tcm treatment was safe and welltolerated in these patients with advanced hccaccording to the international tumor chemotherapy adverse drug evaluation system“ctcae v4014 patients included in this study were evaluated for safety during the followup the adverse reactions were nausea 0cwang et al control effect of tcm as monotherapy for patients with liver cancer banxia xiexin decoction has been used for treating digestive diseases for more than years in our previous retrospective analysis it was found that banxia xiexin decoction can relieve the symptoms of patients with advanced liver cancer inhibit tumor growth and prolong survival one of the patients™ tumors bulk was significantly reduced to reach cr status this state has been maintained for up to months11 therefore it is necessary to carry out further scientific researchto the authors™ best knowledge this is the first prospective study of chinese herbs alone for the treatment of advanced hcc that used the widely accepted endpoints of pfs os orr and dcr according to recist version in the present study the median pfs was months in clinical studies of sorafenib an oral multikinase inhibitor which is the first molecularly targeted drug approved by multiple countries around the world for firstline treatment of advanced hcc the median pfs was months in western countries19 and only months in the asiapacific region9 in current research work all patients were from china and in particular of patients had been diagnosed with liver cirrhosis when enrolled table the manifestations of liver decompensation such as splenomegaly and portal hypertension had appeared however the childpugh class was a or b it had been reported that hcc patients with liver cirrhosis showed significantly worse os than noncirrhotic hcc patients months vs months p even so the median pfs reached months and the median os reached months in a realworld clinical setting these findings suggest that the efficacy of the banxia xiexin recipe may be superior to that of sorafenib in patients with advanced hcc although this was not a headtohead comparison a further study directly comparing chinese herbs versus sorafenib for advanced hcc will be conducted in the futurein the reflect trial21 a subgroup analysis of the chinese population found that the os of the lenvatinib group was significantly longer than that of the sorafenib group by months with statistical differences in addition when combined with hbv infection the survival benefit was significantly different between the lenvatinib group and the sorafenib group os months vs months therefore it was approved by the china food and drug administration on september and approved for domestic listing it is important to note that the experimental conditions for entering the reflect trial were strict excluding ecog performance status patients with a score of or and a large liver mass however in patients enrolled in the present study had liver masses cm and had ecog scores of to nevertheless the median os of this study was figure kaplanmeier subgroup analysis of overall survival stratified by prior tace treatmenttable tumor response according to recist version variablesnumber of patientspercentagecrprsdpdorrdcrabbreviations cr complete response pr partial response sd stable disease pd progressive disease orr objective response rate dcr disease control ratevomiting diarrhea abdominal distension oral ulcers the painless mass of the scalp and so on the overall adverse reaction rate was all of which were grade i to ii adverse reactions and responded to symptomatic treatment there was no adverse reaction of grade iii or above and no drugrelated lethal events occurred specific adverse reaction events and their incidence rates are shown in table discussionit is wellknown that hcc is one of the leading causes of cancerrelated mortality worldwide the longterm survival rate remains unsatisfactory due to the high recurrence and metastasis rates after conventional treatment to improve the outcome of patients with hcc there is an urgent need for more effective therapies at present tcm is often used as an adjuvant therapy to conventional treatments such as tace in treating patients with liver cancer1518 to reduce the toxicities of other treatments relieving symptoms and improving quality of life tcm has only been considered as a complementary or alternative treatment for cancer patients and there is a paucity of data regarding the tumor 0c integrative cancer therapies figure a b the imaging data of one partial response patient diagnosed as advanced hepatocellular carcinoma with metastasis to the right kidneyfigure a b the imaging data of another partial response patient diagnosed as advanced hepatocellular carcinoma with intrahepatic recurrence and right lung metastasismonths therefore for patients with worse economic and physical conditions banxia xiexin decoction may be a better choice for many patients with advanced liver cancer in addition some other new drugs have been tried for the treatment of hcc unfortunately all phase trials assessing novel systemic drugs2224 have failed to improve outcomes over sorafenibin immunotherapy immune checkpoint inhibitors represented by pd1pdl1 monoclonal antibodies have made rapid progress the results of the checkmate040 study25 showed that nivolumab achieved an os of months in newly diagnosed patients and a 150month survival benefit in patients who had undergone sorafenib treatment pabrizumab which is also a pd1 immunosuppressant used in the keynote224 study26 for patients with advanced hcc who had previously progressed with sorafenib achieved a pfs of months median os of months compared with the enrollment conditions for keynote224 the condition of the patients enrolled in the present study are more complicated the largest diameter of the mass in of patients is ‰¥ cm of patients are in stage iv of patients had hbv infection and of patients had hepatic cirrhosis even so this study still achieved a pfs of months higher than the above chemotherapy and immune drugs it can be seen that the efficacy of banxia xiexin decoction for the treatment of advanced liver cancer may be better than the abovementioned immune drugs and it has the potential to become one of the important methods for liver cancer treatmentfurther subgroup analysis revealed that previous treatment might affect the efficacy of chinese herbs in the current analysis patients had been treated with tace previously whose outcome was slightly better compared with those who had not been treated with tace previously pfs vs months p in terms of os the difference is more obvious os undefined vs months p subsequent treatment with banxia xiexin decoction seems to have a greater benefit for patients with advanced liver cancer who have progressed with interventional therapyoverall the orr was and the dcr was in this study in comparison with the data reported in 0cwang et al table adverse reactionsgrade igrade iigrade iiigrade ivany grade grade iii nauseavomitingdiarrheabloatingulcersuperficial painless mass related literature the orr and dcr were respectively and in the orient trial of sorafenib919 it should be noted that the ecogps score of the included patients was to in our study but to in the orient trial indicating that tcm treatment may have a wider scope of application and may benefit more patients compared with targeted therapyin terms of safety no serious adverse events such as liver or kidney damage were observed during the followup period thus banxia xiexin recipe was generally safe and welltolerated in patients with advanced hccas for the antitumor mechanism of tcm it has been reported that tcm or its extracts have direct antitumor effects such as pinellia27 ginseng2829 coptis30 glycyrrhiza31 atractylodes macrocephala32 tangerine peel33 and scutellaria34 the effect of inducing apoptosis is also noted such as with berberine contained in coptis3537 the effect of regulating immunity is noted such as with pinellia27 ginseng3839 atractylodes macrocephala40 and milkvetch root41 antitumor vascular growth such as that of scutellarin4243 can inhibit protein kinase b akt phosphorylation downregulate vegf and inhibit tumor angiogenesis thus playing a role in inhibiting tumor growth and metastasis also it has been reported that the imbalance of intestinal flora not only affects the occurrence and development of intestinal cancer and irritable bowel syndrome but also is closely related to the occurrence of breast cancer lung cancer liver cancer and other malignant tumors4446 however atractylodes macrocephala scutellaria baicalensis and coptis4047 can indirectly inhibit tumors by regulating intestinal flora it is worth mentioning that much basic research has found that pinellia48 coptis4950 ginseng51 scutellaria52 atractylodes macrocephala53 and astragalus54 have antihepatoma effects for example berberine4950 a constituent of the extract of coptis in banxia xiexin decoction can inhibit the pi3kakt pathway of liver cancer downregulate mhcc97h and hepg2 cells phosphorylate the expression of akt and pi3k and inhibit the growth of liver cancer cells in a dosedependent manner besides it can induce cell cycle arrest and promote apoptosis to treat hccthere are only a few patients with tumor shrinkage in the present work it is believed that the direct antitumor effect of banxia xiexin decoction is not outstanding and there may be other mechanisms after analysis it was observed that the more prominent effect of banxia xiexin decoction on liver cancer is reflected in os which is similar to the efficacy characteristics of pd1 immune checkpoint inhibitors banxia xiexin decoction contains pinellia27 ginseng3839 atractylodes40 and astragalus41 medicinal herbs with immunomodulatory effects for example ginseng38 could relieve immunosuppression by increased viability of natural killer cells enhanced immune an index improved cellmediated immune response increased content of cd4 and ratio of cd4cd8 and recovery of macrophage function therefore it was speculated that liver cancer patients in this study may benefit from indirect antitumor mechanisms such as antivascular immunomodulation and others in addition some studies5556 confirmed that some ingredients of banxia xiexin decoction could improve liver function and regulate gastrointestinal function these comprehensive effects can also partially explain why liver cancer patients in this study seem to obtain longer pfs and osin china most patients with hcc have liver diseases such as hepatitis and cirrhosis among the patients enrolled in this study patients had a history of hepatitis b and patients had liver cirrhosis hcc and basal liver disease often affect each other and form a vicious circle the scutellaria baicalensis ginseng licorice tangerine peel and astragalus in banxia xiexin decoction all have liverprotective effects and licorice has a direct antihbv and hepatitis c virus effects5758 from this point of view this is also one of the reasons why banxia xiexin decoction may be effective in treating liver cancer although much basic research has confirmed the antitumor effects of single herbal extracts in banxia xiexin decoction a single herb may not have a good antitumor effect in clinical use even though the decoction affects liver cancer after being prescribed still each herb may play its respective role whether it causes a new antitumor effect or through compatibility with the effects of other herbs the antitumor mechanism is unclear and needs further studylimitationsthe report is limited by its singlearm research design and cannot be directly compared with standard chemotherapy 0c integrative cancer therapies targeted therapy or immunotherapy at the same time the small number of cases in this study is also one of the disadvantages but this study is still continuing and there will be more data analysis in the future due to the willingness of patients to choose tcm for treatment in tcm hospitals it is difficult to avoid certain limitations when we adopt the single treatment of tcm although oral chinese medicine decoction is difficult to take according to scientific equal dosage and concentration it is in line with the use habits of tcmsin this is the first prospective study of chinese herbs as monotherapy for the treatment of advanced hcc that used the internationally accepted endpoints of pfs os orr and dcr the findings are encouraging as they suggest that this panel of chinese herbs is safe and may be effective for patients with advanced hcc in a realworld clinical setting further studies are required to assess the comparative efficacy of tcm treatment versus other antitumor therapies in this patient populationappendixcomponents and their doses in the modified xiexin recipe decoction chinese latin and english nameschinese namelatin nameenglish nameplace of origin production batch dose gbanxiahuangqinganjiangrenshenhuangliandazaogancaobaizhuchenpihuangqirhizoma pinelliaeradix scutellariaerhizoma zingiberisradix ginsengrhizoma coptidisfructus jujubaeradix glycyrrhizaerhizoma atractylodis macrocephalaepericarpium citri reticulataeradix astragalipinellia tuberbaikal skullcap rootdried gingerginsengcoptisjujubae chinese datelicorice rootlargehead atractylodes rhizometangerine peelmilkvetch rootsichuanshanxisichuanjilinsichuanxinjiangsichuanzhejiangsichuangansuacknowledgmentsthe authors acknowledge the contributions of the other investigators in this trialauthor contributionslijuan wang jianlong ke and shaoquan xiong conceived and designed the study lijuan wang jianlong ke cui wang yaling li qiuyue luo rui cai qian ding panpan lv and tingting song collected the data lijuan wang jianlong ke and cui wang processed the data lijuan wang and jianlong ke wrote the manuscript all authors have participated in the drafting review and approval of the report and the decision to submit for publicationdeclaration of conflicting intereststhe authors declared no potential conflicts of interest with respect to the research authorship andor publication of this fundingthe authors disclosed receipt of the following financial support for the research authorship andor publication of this this research was supported by the national natural science foundation of china no and sichuan provincial science and technology department project no2017jy0327orcid idslijuan wang jianlong ke panpan lv orcid0000000317048847 orcid0000000150587581 orcid0000000271727659references torre la bray f siegel rl ferlay j lortettieulent j jemal a global cancer statistics ca cancer j clin doi103322caac21262 chen w zheng r baade pd et al cancer statistics in china ca cancer j clin doi103322caac21338 hung h treatment modalities for hepatocellular carcinoma curr cancer drug targets doi102174 vitale a trevisani f farinati f cillo u treatment of 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doi101089acm20080060 xu h deng y zhou z huang y chinese herbal medicine chaihuhuaji decoction alleviates postem

cancer286

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: purpose to explore a new therapeutic option for patients with hepatocellular carcinoma hcc the efficacy and safety of a group of traditional chinese medicines banxia xiexin recipe as monotherapy for patients with advanced hcc was studied materials and methods the study included patients with advanced hcc from august to august for analysis these eligible patients received treatment with banxia xiexin recipe for at least month the primary endpoints were progressionfree survival pfs and overall survival os the secondary efficacy endpoints included objective response rate orr and disease control rate dcr in addition safety was also assessed results the median treatment duration of these patients was months range months and followup is still ongoing the median pfs was months confidence interval [ci] months and the median os was months ci months the orr was and the dcr was in the subgroup analysis the median os in the transcatheter arterial chemoembolization tace group was not reached and the median os in the no tace group was months ci months in addition no drugrelated serious adverse events were observed during the study this is the first clinical analysis of traditional chinese medicine as a single treatment for advanced hcc the obtained results are encouraging as they suggest that this panel of chinese herbs is safe and it may be effective for patients with advanced hcc in a realworld clinical settingkeywordshepatocellular carcinoma herbs tace banxia xiexin recipe clinical researchsubmitted december revised june accepted june introductionliver cancer is one of the most frequent malignant tumors and the third leading cause of cancerrelated deaths worldwide both the incidence and mortality rates for liver cancer are increasing while the overall incidence and mortality for all cancers combined tend to decline12 hepatocellular carcinoma hcc is the most common and deadly form of liver cancer in china chronic hepatitis b virus hbv infection and subsequent liver fibrosis and cirrhosis are the 1affiliated hospital of chengdu university of traditional chinese medicine chengdu sichuan province chinathese authors contributed to the work equally and should be regarded as co“first authorscorresponding authorshaoquan xiong department of medical oncology affiliated hospital of chengdu university of traditional chinese medicine chengdu sichuan province china email xsquan106163comcreative commons non commercial cc bync this is distributed under the terms of the creative commons attributionnoncommercial license creativecommonslicensesbync40 which permits noncommercial use reproduction and distribution of the work without further permission provided the original work is attributed as specified on the sage and open access pages ussagepubcomenusnamopenaccessatsage 0c integrative cancer therapies leading causes of liver cancer due to the lack of effective treatment options as well as the high recurrence and metastasis rates hcc is associated with an extremely poor prognosis at present surgical resection and liver transplantation are effective treatments for liver cancer but only a few patients are surgical candidates due to tumor extension poor hepatic functional reserve or underlying liver cirrhosis34 even for the few lucky patients who have completed the surgery the recurrence rates at years following liver resection have been reported to exceed for patients with advanced liver cancer it is essential to improve general wellbeing promote quality of life and prolong survival time it is well known that chemotherapy effects in patients with advanced liver cancer are not outstanding67 some patients can consider local treatment such as transarterial chemoembolization tace the progressionfree survival pfs of patients receiving tace treatment is reported to be months8 molecular targeted drugs are also one of the options for patients with advanced liver cancer such as sorafenib and lenvatinib however according to reports on sorafenib the only approved molecular targeted therapy for advanced hcc the median pfs of patients treated with sorafenib was only months in the asiapacific region9 in recent years immunotherapy which has been attracting worldwide attention is also becoming an important treatment method for example nivolumab and pabolizumab which have been approved by the food and drug administration for secondline treatment of advanced hcc can bring certain benefits to patients with advanced liver cancer but are expensive and difficult to afford for many patientsin china traditional chinese medicine tcm therapies which can date back more than years are widely used in cancer treatment10 banxia xiexin decoction was created by the ancient doctor zhang zhongjing it is mainly used to treat digestive diseases such as bloating and abdominal pain it has a history of more than years in a previous retrospective study it was found that banxia xiexin decoction can bring survival benefits to patients with advanced liver cancer and one of them achieved complete response cr11 as a tcm hospital many patients with advanced liver cancer come to our hospital to undergo chinese medicine for treatment therefore we designed a prospective study of banxia xiexin decoction as monotherapy for advanced liver cancer and analyzed its treatment effect and safetymethodsstudy design and patient enrollmentin this prospective openlabel study patients from the oncology department of the affiliated hospital of the chengdu university of traditional chinese medicine chengdu china were recruitedaccording to the american association for the study of liver diseases criteria eligible patients were adults with advanced hcc confirmed by pathological assessment or noninvasive assessment12 these patients had not received any cancerrelated treatment within weeks before the start of study treatment and the disease had progressed after the prior treatment in addition these patients had to have at least one measurable lesion defined by modified response evaluation criteria in solid tumors for hcc mrecist and recist version inclusion criteria also included eastern cooperative oncology group ecog performance status of to an estimated life expectancy of months childpugh liver function class a or b adequate an function such as white blood cell count ‰¥ — cellsl neutrophils ‰¥ — cellsl and platelets ‰¥ — cellsl and the absence of any serious comorbidities such as significant cardiac cerebrovascular hepatic or nephritic abnormities or other important an dysfunctionskey exclusion criteria included patients with low compliance or use of other antitumor treatments at the same time diagnosis of other malignant tumors and hcc with brain metastasisthe protocol was approved by the ethics committee of the affiliated hospital of chengdu university of traditional chinese medicine no 2015bl003 all patients provided written informed consent before study entrystudy treatment and proceduresthe enrolled patients received treatment with banxia xiexin recipe in the form of decoction the herbal components and their doses in the banxia xiexin recipe decoction are listed in the appendix with the use of an automatic herbboiling machine the decoction volume of each prescription was about ml patients were instructed to take ml each time times dailytreatment was continued until disease progression death development of unacceptable toxicity patient™s refusal to continue or at the discretion of the investigator when the disease progressed treatment could also be continued for month to assess tumor response as long as the patient agreed to do sofollowing an initial baseline assessment within days of the start of study treatment the investigator assessed tumor response by using computed tomography ct scan after to months of treatment subsequently followup assessments were done every months until the patient died adverse events were recorded continuously from enrollment to the end of the final study visitoutcomesthe primary endpoints were pfs time from enrollment to radiological disease progression or death and overall survival os time from enrollment to death due to any cause the secondary efficacy endpoints included the proportion 0cwang et al of patients who had an objective response complete or partial response [pr] according to recist version namely objective response rate orr and disease control rate dcr the proportion of patients who had a cr pr or stable disease besides safety was also assessed by the incidence and nature of adverse eventsstatistical analysisthe pfs and os were estimated using the kaplanmeier method all statistical analyses were performed using graphpad prism software version graphpad software inc and logrank testresultsfrom august to august a total of patients with advanced hcc were enrolled from the oncology department of the affiliated hospital of chengdu university of traditional chinese medicine however only the results of the mediumterm analysis are presented here since patients were still on treatment and had not yet reached the assessment time point therefore patients were finally included in the current analysis meaningful results were found to report in this midterm analysis of course we will update the results according to the latest followup data in the future the baseline characteristics of the included patients are shown in table notably patients were without any treatment in this study all patients who were previously treated with surgery or tace had progressive disease which was similar to the clinical trials of sorafenib9 the number of patients receiving chemotherapy and molecular targeted therapy is and respectively patients who had received previous systemic therapy were excluded in the clinical trials of sorafenib but these patients had progressive disease after the prior treatment according to recist version in this studythe data cutoff date for the current analysis was august the median treatment duration of these patients was months range months and followup is still ongoing since if the disease progressed treatment could also be continued for month to assess tumor response as long as the patient agreed to do so or if thepatient would like to use it as an adjuvant treatment to other treatment such as tace moleculartargeted therapy and so onin the primary analysis the median pfs was months confidence interval [ci] figure and the median os was months ci figure thirtysix patients died all due to disease progression the 1year survival rate was notably most of these patients received prior treatments such as tace and targeted molecular therapy before enrollment in order to explore the potential effect of previous treatment we performed a subgroup analysis the median pfs months ci was longer in the subgroup of patients who had previously received tace compared with the median pfs months ci who had not previously received tace thus the treatment effect in this type of patient seems to be better p figure the median os of advanced hcc patients who had previously received tace was not reached and the median os of subgroups who had not received tace was months ci as shown in figure in terms of tumor response after treatment with banxia xiexin recipe patients achieved a pr had stable disease the orr was and the dcr was table particularly the imaging data of typical cases were presented as follows the first patient was a 58yearold male who was diagnosed with advanced hcc with metastasis to the right kidney on august at baseline the ct scan showed that the size of the target lesion in the liver was cm — cm — cm figure 5a blood tests revealed elevated αfetoprotein µgml and the ecog performance status score was this patient had not received any anticancer treatment previously after months treatment with banxia xiexin recipe the ct scan showed that the target lesion in the liver shrank to cm — cm figure 5b and reached a pr according to recist version the ecog performance status score improved from to the patient is still currently in treatment and has not yet had progressive diseaseone 54yearold male patient was first diagnosed to have hcc in december he was treated by surgical resection on january no antineoplastic treatment was done thereafter when a regular review was conducted on september it showed intrahepatic recurrence and right lung metastasis and then he received treatment with chinese herbs at baseline the ct scan showed that the size of the target lesion in the liver was cm — cm — cm figure 6a blood tests revealed elevated αfetoprotein µgml and the ecog performance status score was after a 32month treatment with modified xiexin recipe the ct scan showed that the target lesion in the liver shrank to — cm figure 6b and reached a pr according to recist version the duration of the pr was months he continued to insist on treatment after the disease progressed and he died in october concerning safety no adverse events were observed except for the occurrence of mild nausea three patients reported mild nausea after taking pills but the symptom was relieved spontaneously after week without any 0c integrative cancer therapies table the clinical characteristics of the patients with advanced hepatocellular carcinomavariablesnumber of patientspercentageage years ‰¥sex male femaleecogps childspugh class a bhepatitis b virus infection yes noliver cirrhosis yes noafp µgl ‰¥ tumor size cm ‰¥pvtt yes noextrahepatic spread yes noinvolved disease sites per patient ‰¥previous treatment no sur tace rf che mttajcc stage iiibivaivbabbreviations ecogps eastern cooperative oncology group“performance status pvtt portal vein tumor thrombus sur surgical resection tace transcatheter arterial chemoembolization rf radiofrequency che chemotherapy mtt molecular targeted therapy ajcc american joint committee on cancerfigure kaplanmeier analysis of progressionfree survivalfigure kaplanmeier analysis of overall survivalfigure kaplanmeier subgroup analysis of progressionfree survival stratified by prior tace treatmenttreatment in addition no serious adverse events such as liver or kidney damage were detected during the followup period thus the tcm treatment was safe and welltolerated in these patients with advanced hccaccording to the international tumor chemotherapy adverse drug evaluation system“ctcae v4014 patients included in this study were evaluated for safety during the followup the adverse reactions were nausea 0cwang et al control effect of tcm as monotherapy for patients with liver cancer banxia xiexin decoction has been used for treating digestive diseases for more than years in our previous retrospective analysis it was found that banxia xiexin decoction can relieve the symptoms of patients with advanced liver cancer inhibit tumor growth and prolong survival one of the patients™ tumors bulk was significantly reduced to reach cr status this state has been maintained for up to months11 therefore it is necessary to carry out further scientific researchto the authors™ best knowledge this is the first prospective study of chinese herbs alone for the treatment of advanced hcc that used the widely accepted endpoints of pfs os orr and dcr according to recist version in the present study the median pfs was months in clinical studies of sorafenib an oral multikinase inhibitor which is the first molecularly targeted drug approved by multiple countries around the world for firstline treatment of advanced hcc the median pfs was months in western countries19 and only months in the asiapacific region9 in current research work all patients were from china and in particular of patients had been diagnosed with liver cirrhosis when enrolled table the manifestations of liver decompensation such as splenomegaly and portal hypertension had appeared however the childpugh class was a or b it had been reported that hcc patients with liver cirrhosis showed significantly worse os than noncirrhotic hcc patients months vs months p even so the median pfs reached months and the median os reached months in a realworld clinical setting these findings suggest that the efficacy of the banxia xiexin recipe may be superior to that of sorafenib in patients with advanced hcc although this was not a headtohead comparison a further study directly comparing chinese herbs versus sorafenib for advanced hcc will be conducted in the futurein the reflect trial21 a subgroup analysis of the chinese population found that the os of the lenvatinib group was significantly longer than that of the sorafenib group by months with statistical differences in addition when combined with hbv infection the survival benefit was significantly different between the lenvatinib group and the sorafenib group os months vs months therefore it was approved by the china food and drug administration on september and approved for domestic listing it is important to note that the experimental conditions for entering the reflect trial were strict excluding ecog performance status patients with a score of or and a large liver mass however in patients enrolled in the present study had liver masses cm and had ecog scores of to nevertheless the median os of this study was figure kaplanmeier subgroup analysis of overall survival stratified by prior tace treatmenttable tumor response according to recist version variablesnumber of patientspercentagecrprsdpdorrdcrabbreviations cr complete response pr partial response sd stable disease pd progressive disease orr objective response rate dcr disease control ratevomiting diarrhea abdominal distension oral ulcers the painless mass of the scalp and so on the overall adverse reaction rate was all of which were grade i to ii adverse reactions and responded to symptomatic treatment there was no adverse reaction of grade iii or above and no drugrelated lethal events occurred specific adverse reaction events and their incidence rates are shown in table discussionit is wellknown that hcc is one of the leading causes of cancerrelated mortality worldwide the longterm survival rate remains unsatisfactory due to the high recurrence and metastasis rates after conventional treatment to improve the outcome of patients with hcc there is an urgent need for more effective therapies at present tcm is often used as an adjuvant therapy to conventional treatments such as tace in treating patients with liver cancer1518 to reduce the toxicities of other treatments relieving symptoms and improving quality of life tcm has only been considered as a complementary or alternative treatment for cancer patients and there is a paucity of data regarding the tumor 0c integrative cancer therapies figure a b the imaging data of one partial response patient diagnosed as advanced hepatocellular carcinoma with metastasis to the right kidneyfigure a b the imaging data of another partial response patient diagnosed as advanced hepatocellular carcinoma with intrahepatic recurrence and right lung metastasismonths therefore for patients with worse economic and physical conditions banxia xiexin decoction may be a better choice for many patients with advanced liver cancer in addition some other new drugs have been tried for the treatment of hcc unfortunately all phase trials assessing novel systemic drugs2224 have failed to improve outcomes over sorafenibin immunotherapy immune checkpoint inhibitors represented by pd1pdl1 monoclonal antibodies have made rapid progress the results of the checkmate040 study25 showed that nivolumab achieved an os of months in newly diagnosed patients and a 150month survival benefit in patients who had undergone sorafenib treatment pabrizumab which is also a pd1 immunosuppressant used in the keynote224 study26 for patients with advanced hcc who had previously progressed with sorafenib achieved a pfs of months median os of months compared with the enrollment conditions for keynote224 the condition of the patients enrolled in the present study are more complicated the largest diameter of the mass in of patients is ‰¥ cm of patients are in stage iv of patients had hbv infection and of patients had hepatic cirrhosis even so this study still achieved a pfs of months higher than the above chemotherapy and immune drugs it can be seen that the efficacy of banxia xiexin decoction for the treatment of advanced liver cancer may be better than the abovementioned immune drugs and it has the potential to become one of the important methods for liver cancer treatmentfurther subgroup analysis revealed that previous treatment might affect the efficacy of chinese herbs in the current analysis patients had been treated with tace previously whose outcome was slightly better compared with those who had not been treated with tace previously pfs vs months p in terms of os the difference is more obvious os undefined vs months p subsequent treatment with banxia xiexin decoction seems to have a greater benefit for patients with advanced liver cancer who have progressed with interventional therapyoverall the orr was and the dcr was in this study in comparison with the data reported in 0cwang et al table adverse reactionsgrade igrade iigrade iiigrade ivany grade grade iii nauseavomitingdiarrheabloatingulcersuperficial painless mass related literature the orr and dcr were respectively and in the orient trial of sorafenib919 it should be noted that the ecogps score of the included patients was to in our study but to in the orient trial indicating that tcm treatment may have a wider scope of application and may benefit more patients compared with targeted therapyin terms of safety no serious adverse events such as liver or kidney damage were observed during the followup period thus banxia xiexin recipe was generally safe and welltolerated in patients with advanced hccas for the antitumor mechanism of tcm it has been reported that tcm or its extracts have direct antitumor effects such as pinellia27 ginseng2829 coptis30 glycyrrhiza31 atractylodes macrocephala32 tangerine peel33 and scutellaria34 the effect of inducing apoptosis is also noted such as with berberine contained in coptis3537 the effect of regulating immunity is noted such as with pinellia27 ginseng3839 atractylodes macrocephala40 and milkvetch root41 antitumor vascular growth such as that of scutellarin4243 can inhibit protein kinase b akt phosphorylation downregulate vegf and inhibit tumor angiogenesis thus playing a role in inhibiting tumor growth and metastasis also it has been reported that the imbalance of intestinal flora not only affects the occurrence and development of intestinal cancer and irritable bowel syndrome but also is closely related to the occurrence of breast cancer lung cancer liver cancer and other malignant tumors4446 however atractylodes macrocephala scutellaria baicalensis and coptis4047 can indirectly inhibit tumors by regulating intestinal flora it is worth mentioning that much basic research has found that pinellia48 coptis4950 ginseng51 scutellaria52 atractylodes macrocephala53 and astragalus54 have antihepatoma effects for example berberine4950 a constituent of the extract of coptis in banxia xiexin decoction can inhibit the pi3kakt pathway of liver cancer downregulate mhcc97h and hepg2 cells phosphorylate the expression of akt and pi3k and inhibit the growth of liver cancer cells in a dosedependent manner besides it can induce cell cycle arrest and promote apoptosis to treat hccthere are only a few patients with tumor shrinkage in the present work it is believed that the direct antitumor effect of banxia xiexin decoction is not outstanding and there may be other mechanisms after analysis it was observed that the more prominent effect of banxia xiexin decoction on liver cancer is reflected in os which is similar to the efficacy characteristics of pd1 immune checkpoint inhibitors banxia xiexin decoction contains pinellia27 ginseng3839 atractylodes40 and astragalus41 medicinal herbs with immunomodulatory effects for example ginseng38 could relieve immunosuppression by increased viability of natural killer cells enhanced immune an index improved cellmediated immune response increased content of cd4 and ratio of cd4cd8 and recovery of macrophage function therefore it was speculated that liver cancer patients in this study may benefit from indirect antitumor mechanisms such as antivascular immunomodulation and others in addition some studies5556 confirmed that some ingredients of banxia xiexin decoction could improve liver function and regulate gastrointestinal function these comprehensive effects can also partially explain why liver cancer patients in this study seem to obtain longer pfs and osin china most patients with hcc have liver diseases such as hepatitis and cirrhosis among the patients enrolled in this study patients had a history of hepatitis b and patients had liver cirrhosis hcc and basal liver disease often affect each other and form a vicious circle the scutellaria baicalensis ginseng licorice tangerine peel and astragalus in banxia xiexin decoction all have liverprotective effects and licorice has a direct antihbv and hepatitis c virus effects5758 from this point of view this is also one of the reasons why banxia xiexin decoction may be effective in treating liver cancer although much basic research has confirmed the antitumor effects of single herbal extracts in banxia xiexin decoction a single herb may not have a good antitumor effect in clinical use even though the decoction affects liver cancer after being prescribed still each herb may play its respective role whether it causes a new antitumor effect or through compatibility with the effects of other herbs the antitumor mechanism is unclear and needs further studylimitationsthe report is limited by its singlearm research design and cannot be directly compared with standard chemotherapy 0c integrative cancer therapies targeted therapy or immunotherapy at the same time the small number of cases in this study is also one of the disadvantages but this study is still continuing and there will be more data analysis in the future due to the willingness of patients to choose tcm for treatment in tcm hospitals it is difficult to avoid certain limitations when we adopt the single treatment of tcm although oral chinese medicine decoction is difficult to take according to scientific equal dosage and concentration it is in line with the use habits of tcmsin this is the first prospective study of chinese herbs as monotherapy for the treatment of advanced hcc that used the internationally accepted endpoints of pfs os orr and dcr the findings are encouraging as they suggest that this panel of chinese herbs is safe and may be effective for patients with advanced hcc in a realworld clinical setting further studies are required to assess the comparative efficacy of tcm treatment versus other antitumor therapies in this patient populationappendixcomponents and their doses in the modified xiexin recipe decoction chinese latin and english nameschinese namelatin nameenglish nameplace of origin production batch dose gbanxiahuangqinganjiangrenshenhuangliandazaogancaobaizhuchenpihuangqirhizoma pinelliaeradix scutellariaerhizoma zingiberisradix ginsengrhizoma coptidisfructus jujubaeradix glycyrrhizaerhizoma atractylodis macrocephalaepericarpium citri reticulataeradix astragalipinellia tuberbaikal skullcap rootdried gingerginsengcoptisjujubae chinese datelicorice rootlargehead atractylodes rhizometangerine peelmilkvetch rootsichuanshanxisichuanjilinsichuanxinjiangsichuanzhejiangsichuangansuacknowledgmentsthe authors acknowledge the contributions of the other investigators in this trialauthor contributionslijuan wang jianlong ke and shaoquan xiong conceived and designed the study lijuan wang jianlong ke cui wang yaling li qiuyue luo rui cai qian ding panpan lv and tingting song collected the data lijuan wang jianlong ke and cui wang processed the data lijuan wang and jianlong ke wrote the manuscript all authors have participated in the drafting review and approval of the report and the decision to submit for publicationdeclaration of conflicting intereststhe authors declared no potential conflicts of interest with respect to the research authorship andor publication of this fundingthe authors disclosed receipt of the following financial support for the research authorship andor publication of this this research was supported by the national natural science foundation of china no and sichuan provincial science and technology department project no2017jy0327orcid idslijuan wang jianlong ke panpan lv orcid0000000317048847 orcid0000000150587581 orcid0000000271727659references torre la bray f siegel rl ferlay j lortettieulent j jemal a global cancer statistics ca cancer j clin doi103322caac21262 chen w zheng r baade pd et al cancer statistics in china ca cancer j clin doi103322caac21338 hung h treatment modalities for hepatocellular carcinoma curr cancer drug targets doi102174 vitale a trevisani f farinati f cillo u treatment of hepatocellular carcinoma in the precision medicine era from treatment stage migration to therapeutic hierarchy hepatology published online february doi101002hep31187 bruix j sherman m practice guidelines committee american association for the study of liver diseases management of hepatocellular carcinoma hepatology doi101002hep20933 lee dw lee kh kim hj et al a phase ii trial of s1 and oxaliplatin in patients with advanced hepatocellular carcinoma bmc cancer doi101186s1288501840399 0cwang et al zaanan a williet n hebbar m et al gemcitabine plus oxaliplatin in advanced hepatocellular carcinoma a large multicenter ageo study j hepatol doi101016jjhep201209006 wang y cui w raltitrexed based transcatheter arterial chemoembolization tace for unresectable hepatocellular carcinoma a singlecenter randomized controlled study chemotherapy doi104172216777001000192 cheng al kang yk chen z et al efficacy and safety of sorafenib in patients in the asiapacific region with advanced hepatocellular carcinoma a phase iii randomised doubleblind placebocontrolled trial lancet oncol doi101016s1470204508702857 anonymous miraculous pivot ling shu jing originally published during warring states period to bc people™s health press shaoquan x lijian w qiuyue l et al retrospective analysis of modified xiexin recipe combined retrospective analysis of modified xiexin recipe combined chin j integr tradit western med bruix j sherman m american association for the study of liver diseases management of hepatocellular carcinoma an update hepatology doi101002hep24199 lencioni r llovet jm modified recist mrecist assessment for hepatocellular carcinoma semin liver dis doi101055s00301247132 gao wj liu yy yuan cr international evaluation system for adverse events of chemotherapeutic drugs in cancer treatment ctcae v40 tumor meng mb cui yl guan ys et al traditional chinese medicine plus transcatheter arterial chemoembolization for unresectable hepatocellular carcinoma j altern complement med doi101089acm20080060 xu h deng y zhou z huang y chinese herbal medicine chaihuhuaji decoction alleviates postem Answer:

Colon_Cancer

this study hypothesizes that bromelain bl acts as radiosensitizer of tumor cells and that it protects normal cells from radiation effects in vitro and in vivo studies have been carried out to prove that assumption in vitro mtt cell proliferation assay has shown that the irradiated ehrlich ascites carcinoma eac cell line could be sensitized by bl pretreatment in vivo animals were randomly divided into groups group control pbs ip for days group ehrlich solid tumor est bearing mice group est Îradiation fractionated dose gy — group est bl mgkg ip daily for days group est bl for days followed by Îirradiation gy — the size and weight of tumors in gammairradiated est bearing mice treated with bl decreased significantly with a significant amelioration in the histopathological examination besides bl mitigated the effect of Îirradiation on the liver relative gene expression of poly adp ribose polymerase1 parp1 nuclear factor kappa activated b cells nfκb and peroxisome proliferatoractivated receptor α pparα and it restored liver function via amelioration of paraoxonase1 pon1 activity reactive oxygen species ros content lipid peroxidation lpo and serum aspartate transaminase ast alanine transaminase alt and albumin alb it is concluded that bl can be considered as a radiosensitizer and radioprotector suggesting a possible role in reducing radiation exposure dose during radiotherapykeywordsbromelain tumor Îradiation radiosensitizer radioprotectorsubmitted april revised july accepted july introductionradiotherapy has been used for a long time in treating cancer1 however from the clinical perspective radiotherapy provides inadequate success due to the radioresistance of many tumors as well as the high risk of recurrence and effects on normal cells may occur23 radioresistance occurs as the microenvironment of solid tumors is hypoxic compared with normal tissue4 in addition some tumors have either an intrinsic resistance to ionizing radiation or can attain this property through accumulation of genetic mutations causing an increased survival and proliferation5 thus strategies to improve radiation therapy could include increasing resistance of normal tissues to radiation andor increasing sensitivity of the tumor cells6radiosensitizing agents increase the sensitivity of tumor cells via enhancing the generation of reactive oxygen species ros increasing lipid peroxidation depletion of glutathione which leads to dna damage inhibition of dna repair inhibition of dna synthesis induction of cell cycle arrest induction of apoptosis and inhibition of proliferation7 numerous nutritive cancer chemopreventive compounds having antioxidant properties have been recognized to potentiate radiation therapyinduced cytotoxic 1drug radiation research department national centre for radiation research and technology egyptian atomic energy authority nasr city cairo egypt2biochemistry department alazhar university cairo egyptcorresponding authorhanan a fahmy drug radiation research department national centre for radiation research and technology atomic energy authority p o box nasr city cairo egypt email fahmyhananyahoocomcreative commons non commercial cc bync this is distributed under the terms of the creative commons attributionnoncommercial license creativecommonslicensesbync40 which permits noncommercial use reproduction and distribution of the work without further permission provided the original work is attributed as specified on the sage and open access pages ussagepubcomenusnamopenaccessatsage 0c integrative cancer therapies effects on cancer cells inversely decreasing its toxicity on normal adjacent tissues89 in this regard much research has aimed to develop numerous antioxidant drugs of both natural and synthetic origin tested in both in vitro and in vivo models and also human clinical trials to overcome injuries caused by ir exposure and to induce killing of cancer cells at the same time previous studies have reported that phytochemical soy isoflavones genistein daidzein and glycitein which exhibit anticarcinogenic properties through their antioxidant activities could be used as potent radiosensitizers to enhance the efficacy of radiotherapymediated suppression of the growth and metastatic ability of cancers1011 along parallel lines resveratrol and piperine which possess antitumor activity have been shown to augment ionizing radiation irinduced apoptosis and loss of mitochondrial membrane potential in murine colon carcinoma and melanoma cells via enhancing irinduced ros generation12 moreover pentoxifylline ptx a methylxanthine that possesses antioxidant properties is known for improving tumor tissue oxygenation in murine hypoxic tumors and inhibiting post radiation induced normal tissue injury in mice1314 consequently searching for a natural product possessing anticancer activity that increases radiosensitivity of tumor cells and radioresistance of normal cells may lead to a potential future drug in cancer therapyamong the natural products bromelain bl extract attracts interest due to its anticancer antioxidant as well as antiinflammatory effects1517 bl an extract from pineapple stem ananas comosus belongs to a group of protein digesting enzymes it is a mixture of diï¬erent thiol endopeptidases and other components like phosphatase glucosidase peroxidase cellulase escharase calcium and several protease inhibitors1819 the anticancer activity of bl has been examined in various types of gastrointestinal and breast cancers cell lines in in vivo models bl has shown antimetastatic effect and reduction in local tumor growth2023 it is also used for reducing the severity of such radiation therapy side effects as mucositis skin reactions and dysphagia in patients24 hence this study was aimed to evaluate the radiosensitizing and radioprotective effect of bl using in vivo and in vitro approachesmaterials and methodin vitro studiesmtt cell proliferation assay the growth and viability of ehrlich ascites carcinoma eac cell line were tested in vitro by 345dimethylthiazol2yl25diphenyltetrazolium bromide mtt assay according to freimoser and buch 2526 to verify the antitumor and radiosensitizing effect of bl two plates were designed for this study the first one contained eac cells maintained by serial subculturing at the national cancer institute egypt incubated for hour before irradiation irr gy alone and with different concentrations of bromelain bl in phosphate buffer saline pbs the second one contained eac cells serving as a control and eac with different concentrations of bl each test was seeded in triplicate into a plate at concentration of — cellswell containing rpmi media with fbs nahco3 uml penicillin and µgml streptomycin and each plate was incubated for hours at °c in co2 and humidity atmosphere then μl mtt reagent bio basic inc canada was added over the cells in each well and the plate was incubated in the dark for to hours until a purple precipitate was seen and the absorbance was measured at nm the amount of color produced was directly proportional to the number of viable cells viable cell a samples ˆ’ a blanka control ˆ’ a blank — the inhibitory concentration ic50 is the dose of a drug which reduces the viability to and was calculated using nonlinear regression analysisfree radical scavenging assay the antioxidant activity of bromelain was evaluated by 1diphenyl2picrylhydrazyl dpph radical assay and its scavenging power was compared with some antioxidants naringin polyphenolic antioxidant garlic oil and glutathione sulfur containing antioxidants about µl of samples mgml dissolved in dist water was added to µl of a solution of dpph g100 ml dissolved in vv methanol after minutes incubation at room temperature in the dark the absorbance was read at nm against a blank µl dist water µl dpphmethanol solution the experiments were done in triplicate according to the method of braca 27 glutathione mgml was used as a standard antioxidant the scavenging percentage of dpph was calculated according to the followscavengin ing equation where b was the absorbance of the blank and a was the absorbance of samples or standard ec50 is defined as concentration of sample that causes dpph loss there values were calculated using nonlinear regression analysisˆ’b ab\uf8ee\uf8ef\uf8f0\uf8f9\uf8fa —\uf8fbin vivo studiesradiation processing whole body Îirradiation of mice was carried out using gamma cell40 137cesium manufactured by the atomic energy of canada limited ontario canada installed in the national center for radiation research and technology ncrrt cairo egypt the dose rate was gymin during the experimental period daily correction for humidity barometric pressure and temperature were madeanimals adult female swiss albino mice weighing to g obtained from the breeding unit of ncrrt cairo egypt all animal procedures were performed in accordance with the committee of scientific ethics at faculty of 0cmekkawy table sequences of primers for realtime quantitative pcrgeneparp1 nm0074152nfκb nc0000696pparα nc0000816βactin nc0000716forward primerreverse primer²ccatcgacgtcaactacga3²²caatggctacacaggacca3²²actccacctgcagagcaacca3²²gcgtggggacagccgcatctt3²²gtgcgtggtagcatgagtgt3²²cactgtcacctggaaccaga3²²tagatctcctgcagtagcggg3²²atcggcagaaggggcggaga3²pharmacy alazhar university egypt following the guidelines for animal use the animals were housed in colony cages micecage under proper environmental conditions that is hours darklight cycle good ventilation condition and temperature to humidity at the ncrrt animal house fed with standard diet pellets and provided with water ad libitum animals were left week for acclimatization on the lab environment before starting the experimenttumor transplantation the eac cell line was supplied by serial subculturing at the national cancer institute cairo university egypt it was implanted in each donor female swiss albino mice by ip injection of — cells22 g b wt and allowed to multiply28 the ehrlich solid tumor est was obtained by the intramuscular inoculation of ml of — viable eac in the right lower limb of each mouse29 mice with a palpable solid tumor diameter mm3 that was maintained within to days after inoculation were used in the studyanimal grouping animals were randomly divided into groups mice each group control not bearing tumor received pbs ip for days group ehrlich solid tumor est bearing mice received pbs ip for days group est Îirradiation gy — fractionated doses starting days after tumor appearance mm3 and lasting for days group est bearing mice receiving freshly prepared bl dissolved in pbs mgkg ip daily for days according to pilot study starting once est becomes mm3 bl was purchased from merck kgaa co darmstadt germany group est bearing mice received bl as in group hours before Îirradiation as in group mice were anesthetized days after last irradiation dose using urethane mgkg30 blood samples were collected through cardiac puncture and divided into parts edta coated and plain vials at that time they were euthanized by cervical dislocation liver and tumor tissues were dissected out rinsed with icecold saline dried on a filter paper and weighed then homogenized in icecold pbs ph and stored at ˆ’°c until used for subsequent biochemical analysisestimation of total body tumor and liver weights animals in each group were checked daily for any adverse clinical symptoms and deaths after to days post inoculation with eac body weights were recorded so body weight change could be estimated tumor and liver weights were measured during sample collection and then the tumor inhibitory ratio was calculated by the following formula inhibition ratio aˆ’ba — where a is the tumor weight average of the control and b is that of the treated group also relative liver weight was calculated as liver weighttotal body weight — histopathological examination three tumors of each group were collected and fixed in neutral buffered formalin the specimens were dehydrated in ascending grades of ethyl alcohol cleared in xylene and embedded in paraffin wax four micron thick paraffin sections were mounted on clean slides stained with ehrlich™s hematoxylineosin he31 and examined using an olympus microscope bx41 hamburg germany histopathological evaluation was done by assessment of necrosis and calculation of tumor area percentage using image analysis software image j 146a nih usa through the following equation of tumor area area of tumortotal area of the field — molecular analyses the mrna levels of poly adpribose polymerase parp1 nuclear factor kappa b nfκb and peroxisome proliferatoractivated receptors pparα genes and of the housekeeping gene βactin were measured by real time polymerase chain reaction rtpcr total rna was isolated from liver tissues using qiagen tissue extraction kit qiagen usa in accordance with the manufacturer™s instructions the extracted rna μg was used for cdna conversion using high capacity cdna reverse transcription kit fermentas usa and μl reaction volume sybr chemistry in applied biosystems thermal cycler usa to amplify pcr under the following conditions °c for denaturation then °c to °c for annealing using primers mentioned in table and °c for elongationresults were expressed using the comparative ˆ†ˆ†ct method for relative mrna quantification of target genes normalized to an endogenous reference βactin and a relevant control equal to ˆ’ˆ†ˆ†ct ˆ†ˆ†ct is the difference between the mean ˆ†ctsample and mean ˆ†ctcontrol where ˆ†ctsample is the difference between the mean ctsample and the mean ctβactin and ˆ†ctcontrol is the difference between the mean ctcontrol and the mean ctβactin 0c integrative cancer therapies estimation of lipid peroxidation lpo reactive oxygen species ros and paraoxonase pon1 in liver homogenate liver lipid peroxidation was estimated by measurement of malondialdehyde mda formation using the thiobarbituric acid method of yoshioka 32 a modified technique of vrablic 33 was used to measure the generation of ros by the intracellular conversion of nitro blue tetrazolium nbt to formazan by the action of superoxide anion paraoxonase activity was estimated by using fluorometric assay enzchek® kit invitrogen uk for the anophosphatase activity of paraoxonase based on the hydrolysis of a fluorogenic anophosphate analog34hematological and biochemical analyses whole blood was immediately analyzed for complete blood count with platelet count using the fully automated analyzer abx cobas micros roche germany estimation of serum alanine aminotransferase alt aspartate aminotransferase ast and albumin alb assays follow the recommendations of the international federation of clinical chemistry ifcc but were optimized for performance and stability using the rochehitachi cobas c 311systemstatistical analysis the statistical analysis was performed using oneway analysis of variance anova and the groups were compared by tukeykramer test viability percentage at different concentrations and body weight change analyzed by twoway anova followed by bonferroni™s posttest graphs were sketched using graph pad prism isi® software usa version software data were presented as mean ± standard error se and p values considered significantresultsin vitro studieseffect of bromelain and gammairradiation blirr on tumor cell growth and viability the radiosensitizing effect of bl on eac cells was determined by performing mtt assay eac cells exposed to gy Îradiation showed high cell viability percentage reflecting a radioresistance of eac cell line while bl treatment showed in vitro cytotoxic activity with ic50 value of mgml however the maximum cytotoxic effect appeared when the eac cells were subjected to bl then Îradiation gy compared to control or irradiated group with ic50 mgml table effect of bromelain and some natural compounds as free radical scavengers the inhibitory percentage of each compound is shown in figure the ec50 value concentration of sample that causes dpph activity loss is a reliable way for estimation of the radical scavenging activity the ec50 value of glutathione referenced antioxidant is mgml while table cytotoxic activity of blirr against eac cell line bromelain concentration mgmleac bl mgmlic50 mgmlviability non irradiated eac irradiated eac48ab59ab60ab69a787a10ab158ab18ab27ab52ab ± ± each value indicates the mean of records statistical analysis carried out by twoway anova followed by bonferroni posttests a significant versus control ehrlich ascites carcinoma eac group where b significant versus irradiated eac group at p ic50 ± se values were calculated by using nonlinear regression analysisbromelain and garlic oil ec50 are almost equal and mgml respectively however the naringin phenolic antioxidant is the least potent one ec50 mgml in this comparisonin vivo studieseffect of bromelain and gammairradiation blirr on tumor weight and volume table shows a significant decrease in tumor weight in groups treated with bl andor Îirradiation as compared to the est nontreated group the more drastic decrease in the tumor weight ratio observed in the combined therapy group bl irr compared with the estirradiated group as well as est group indicates that combination therapy is more significantly effective than single agent therapy the photograph of est xenografts at the time of sacrifice shows the synergistic effect of bl and irr on tumor volume figure effect of bromelain and gammairradiation blirr on tumor histopathological features of est bearing mice histopathological examination of solid tumor sections revealed typical malignant features including sheets of malignant cells infiltrating adjacent muscular tissue the malignant cells show pleomorphism hyperchromatism and mitotic activity while the necrotic cells appear as nonviable homogenous structureless material with degenerated or karyorrhectic nuclei untreated est bearing group shows a deeply stained tumor cells arrow head and areas of necrosis arrow figure 3a — also it displays intact cancer cells arrow and giant cells arrow figure 3b and c — the estirradiated group shows muscle fibers invaded by deeply stained tumor cells arrow head and large areas of necrosis arrow figure 3d — also displays a notable necrosis of cancer cells n figure 3e — the bl treated group shows a 0cmekkawy figure dpph 1diphenyl2picrylhydrazyl reduction curve for glutathione bromelain naringin and garlic oil each value represents mean ± se all experiments were replicated timestable tumor weight and inhibition ratio of ehrlich solid tumor estbearing mice treated with gammairradiation irr gy — andor bromelain bl mgkggroupsestest irrest blest bl irrtumor weight g ± ± 004a ± 005a ± 005abtumor inhibitory ratio ” ± 24a ± 14ab ± 204ababbreviations est ehrlich solid tumor est irr ehrlich solid tumor irradiation est bl ehrlich solid tumor bromelain est bl irr ehrlich solid tumor bromelain irradiationthe values shown are mean ± se of data a significant versus est group where b significant versus estirradiated group at p figure a photograph of ehrlich solid tumor est xenografts at the time of scarification showing the effect of bromelain and gammairradation blirr on tumor volume e ehrlich solid tumor e ir ehrlich solid tumor irradiation e br ehrlich solid tumor bromelain e ir br ehrlich solid tumor bromelain irradiation 0c integrative cancer therapies figure photo micrograph of ehrlich solid tumor est xenografts in different animal groups est sections show the degree of tumorogenesis necrosis n regression of tumor by appearance of muscle fibers m — and — a b c est ehrlich solid tumor d e est irr ehrlich solid tumor irradiation f g est bl ehrlich solid tumor bromelain h i est bl irr ehrlich solid tumor bromelain irradiationwide area of necrosis arrow and n few groups of cancer cells arrow head and muscle fiber m figures 3f — and 4g — however combined treatment bl irr displays muscle fiber m significant regression of tumor or wide areas of necrotic cancer cells n and few groups of intact cancer cells arrow figure 3h — and 3i —the tumor area percentage per total tissue area could determine the degree of proliferation as seen in figure there is a great regression of tumor area in the group treated with bl alone or bl and irr compared with untreated est or estirradiated group indicating that combination therapy significantly more effective than single agent therapy 0cmekkawy bl irr shows nonsignificant group change additionally bl irr group significantly upregulated pparα expression compared with est and estirradiated groups indicating that bl might have a hepato as well as radioprotective effect figure effect of bromelain and gammairradiation blirr on the hepatic lipid peroxidation lpo level reactive oxygen species ros content and paraoxonase1 pon1 activity of ehrlich solid tumor est bearing mice lpo in liver tissues significantly increased in all est bearing groups compared to the control group except the combined treated group irr bl succeeded in returning mda lpo measured as mda malondialdehyde level to the normal level however liver ros significantly increased only in untreated and Îirradiated est bearing groups when compared to the control group while a significant decrease in liver ros showed in estirradiated mice treated with bl in comparison with both est untreated and estirradiated groups pon1 activity in liver homogenate was significantly decreased in est untreated and estirradiated groups when compared with the control group bl treated groups revealed significant increases in pon1 when compared with both est untreated and estirradiated groups showing that bl might have a hepato and radioprotective effect figure effect of bromelain and gammairradiation blirr on hematological measurements wbcs and plts were significantly elevated while hgb and hct significantly decreased in the untreated estbearing mice in comparison with control mice however Îirradiation resulted in a significant decrease in wbcs rbcs plt hgb and hct compared with the control mice treatment of the estbearing mice with bl shows a significant amelioration in wbcs plt and hct compared to est untreated mice combined treatment bl irr shows an enhancement in wbcs plt and hct compared to est untreated and gammairradiated est bearing mice table effect of bromelain and gammairradiation blirr on the serum alanine transaminase alt aspartate transaminase ast and albumin alb to investigate the cytoprotective effects of bl against irradiation the levels of serum alt ast and alb were measured figure it was found that alt and ast significantly increased and conversely alb significantly decreased in est untreated and estirradiated groups compared with the control group however estbearing mice treated with bl alone show nearly the same result of alt and alb as control values estbearing mice treated with bl in combination with irradiation initiated a significant decrease in ast and alt as compared with estirradiated group which may reflect a potential hepatic radioprotective effect of blfigure percentage of tumor areatotal tissue area of ehrlich solid tumor est bearing mice treated with gammairradiation irr gy — andor bl mgkg the values shown in the plotted area are mean of records from animals ± se significant versus est group where significant versus estirradiated group at p effect of bromelain and gammairradiation blirr on body weight change and relative liver weight regarding the day by day documented recording of body weight bwt illustrated in figure there is almost no change in bwt of bl treated group while it increases significantly in the untreated est group conversely estirradiated groups with or without bl treatment show a significant decrease in bwt when compared with the control group table relative liver weight was compared after normalization to mg body weight untreated estbearing group shows a significant increase in liver weight by hepatomegaly while nonsignificant changes were observed in bl treated groups compared to the normal group table effect of bromelain and gammairradiation blirr on the poly adpribose polymerase parp1 nuclear factor kappa b nfκb and peroxisome proliferatoractivated receptors pparα relative gene expression of ehrlich solid tumor est bearing mice to test the possibility that bl reduces radiation damage to the liver mrna gene expression of parp1 nfκb and pparα was measured in the liver homogenates of est bearing mice and compared to control pbs treated mice the results illustrated in figure show that irr causes significant increases in parp1 and nfκb expression compared to the control group however combined treatment bl irr shows a significant increase in parp1 and nfκb expression compared to control group and a significant attenuation compared to estirradiated groupmoreover all est bearing groups show significant decreases in hepatic pparα relative gene expression compared to the control group except the combined therapy 0c integrative cancer therapies figure effect of bromelain and gammairradiation blirr on body weight during experiment period each value represents the mean of records ± se significant versus control group where significant versus est group and significant versus estirradiated group at p est ehrlich solid tumor est irr ehrlich solid tumor irradiation est bl ehrlich solid tumor bromelain est bl irr ehrlich solid tumor bromelain irradiationtable change in body weight and relative liver weight of control mice and ehrlich solid tumor est bearing mice treated with gammairradiation irr gy — andor bromelain bl mgkggroupscontrolestest irrest blest bl irrbody weight change ± ± 101aˆ’ ± 217abˆ’ ± 201bˆ’ ± 341abrelative liver weight ± ± 026a ± 022a ± 026a ± 026ababbreviations est ehrlich solid tumor est irr ehrlich solid tumor irradiation est bl ehrlich solid tumor bromelain est bl irr ehrlich solid tumor bromelain irradiationeach value represents the mean ± se a significant versus control group where b significant versus est group at p body weight changes percent are related to the initial weight of animalsdiscussionresistance of tumor cells to chemoradiotherapy as well as the damaging effects to nearby normal tissues remains a major obstacle to successful cancer management therefore the current study has been conducted to estimate the effect of bromelain bl as a tumor radiosensetizer and to show to what extent it can protect normal tissue from radiation hazardsradiosensitizers are compounds that when combined with radiation therapy achieve greater cytotoxicity they can be determined in vitro by the mtt assay2635 the present study has found that the radioresistant eac cells could be sensitized when incubated with bl before irradiation it was known previously that in vitro treatment with bl on mouse tumor cell lines resulted in inhibition of cell growth and invasion capacities3637 the anticancer property of bl has been mainly attributed to the protease component through digestion and diffusion in tumor cells38 it may also be due to the bl enhancement of p53 expression as well as another activator of apoptosis eg bax39 in addition it decreases the activity of cell survival regulators such as akt and erk it also deactivates aktdependent proapoptotic regulator foxo3a thus promoting apoptotic cell death in tumors40it is well known that during cancer and radiotherapy excessive energy is used from the host41 ultimately contributing to mechanisms that promote loss of weight as shown in the present study which also showed that bl could return body weight to a normal level by decreasing tumor weight and volume currently the combined therapy bl irr has been shown to be more effective than single agent therapy in reducing tumor volume and weight indicating that bl could possess a radiosensitizing effect in addition the combined therapy has revealed a drastic decrease in tumor area percentage wide areas of necrotic cancer cells and presence of muscle fiber in the histopathological examination compared with the control est and estirradiated groups this seems to be in agreement with other findings of the role of bl in reducing metastasis and local tumor growth2342 in chemically induced mouse skin papillomas topical application of bl reduced tumor formation tumor volume and caused apoptotic cell death39 bl is a hydrolytic enzymatic complex which shows an efficient digestion and diffusion in tumor cells through attacking the glycosidic linkages and hence denatures glycoproteins thus it protects against tumor growth37 another study has demonstrated the use of controlled proteolytic activity on tumor as a successful strategy to increase therapeutic efficacy43 0cmekkawy figure effect of bromelain and gammairradiation blirr on relative gene expression of liver a parp1 b nfκb and c pparα each value represents the mean of records ± se significant versus control group where significant versus est group and significant versus estirradiated group at p est ehrlich solid tumor est irr ehrlich solid tumor irradiation est bl ehrlich solid tumor bromelain est bl irr ehrlich solid tumor bromelain irradiationthe aim of the radiotherapy protocols is to achieve the maximum curative effect on tumor cells with minimal damaging effect on normal cells hence antioxidants and other nutrients which do not interfere with therapeutic modalities for cancer may enhance the killing property decrease side effects and protect normal tissue44for estimation of the antioxidant ability of bl dpph assay was conducted in vitro and the free radical inhibitory action of bl was compared with some antioxidant compounds it was found that bl has a powerful free radical scavenging power bl belongs to thiol proteases in which the catalytic nucleophile is sulfhydryl groups of cysteine residues which in turn accounts for its antioxidant activity45the involvement of ros mda and pon1 are important mechanisms that play a vital role during radiation toxicity the use of antioxidants is an important preventive to decrease the toxic and pathological effects associated with oxidative stress caused by radiation46 the attained results show a hepatic impairment on the third day from exposure to Îradiation elevation of lpo and ros levels and inhibition of pon1 activity compared to normal mice however treatment with bl revealed an amelioration in hepatic damage caused by irradiation these results were in accordance with liu 47 who described the effect of radiation induced ros generation which in turn might attack cell membrane phospholipids and circulating lipids and thus increases production of mda48 lpo acts as a sensitive biomarker for oxidative stress that occurs as part of the pathogenesis of irradiation49 bl has sulfhydryl groups consequently accounting for its antioxidant activity45 thus it could act as ros scavengermeasurement of pon1 postradiotherapy could be an effective clinical biomarker of hepatic and systemic oxidative stress and may be used as an index of the usefulness of radiotherapy50 it has been demonstrated to catalyze hydrolysis of lipid hydroperoxides and lactones51 pon1 protects serum hdl and ldl ps against lipid peroxidation52 in the present study the decreased activity of pon1 upon radiation exposure might be due to its super saturation of lipid hydroperoxides and lactones upon treatment with bl the activity of pon1 was restored near to the normal level hence the pon1

cancer287

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: this study hypothesizes that bromelain bl acts as radiosensitizer of tumor cells and that it protects normal cells from radiation effects in vitro and in vivo studies have been carried out to prove that assumption in vitro mtt cell proliferation assay has shown that the irradiated ehrlich ascites carcinoma eac cell line could be sensitized by bl pretreatment in vivo animals were randomly divided into groups group control pbs ip for days group ehrlich solid tumor est bearing mice group est Îradiation fractionated dose gy — group est bl mgkg ip daily for days group est bl for days followed by Îirradiation gy — the size and weight of tumors in gammairradiated est bearing mice treated with bl decreased significantly with a significant amelioration in the histopathological examination besides bl mitigated the effect of Îirradiation on the liver relative gene expression of poly adp ribose polymerase1 parp1 nuclear factor kappa activated b cells nfκb and peroxisome proliferatoractivated receptor α pparα and it restored liver function via amelioration of paraoxonase1 pon1 activity reactive oxygen species ros content lipid peroxidation lpo and serum aspartate transaminase ast alanine transaminase alt and albumin alb it is concluded that bl can be considered as a radiosensitizer and radioprotector suggesting a possible role in reducing radiation exposure dose during radiotherapykeywordsbromelain tumor Îradiation radiosensitizer radioprotectorsubmitted april revised july accepted july introductionradiotherapy has been used for a long time in treating cancer1 however from the clinical perspective radiotherapy provides inadequate success due to the radioresistance of many tumors as well as the high risk of recurrence and effects on normal cells may occur23 radioresistance occurs as the microenvironment of solid tumors is hypoxic compared with normal tissue4 in addition some tumors have either an intrinsic resistance to ionizing radiation or can attain this property through accumulation of genetic mutations causing an increased survival and proliferation5 thus strategies to improve radiation therapy could include increasing resistance of normal tissues to radiation andor increasing sensitivity of the tumor cells6radiosensitizing agents increase the sensitivity of tumor cells via enhancing the generation of reactive oxygen species ros increasing lipid peroxidation depletion of glutathione which leads to dna damage inhibition of dna repair inhibition of dna synthesis induction of cell cycle arrest induction of apoptosis and inhibition of proliferation7 numerous nutritive cancer chemopreventive compounds having antioxidant properties have been recognized to potentiate radiation therapyinduced cytotoxic 1drug radiation research department national centre for radiation research and technology egyptian atomic energy authority nasr city cairo egypt2biochemistry department alazhar university cairo egyptcorresponding authorhanan a fahmy drug radiation research department national centre for radiation research and technology atomic energy authority p o box nasr city cairo egypt email fahmyhananyahoocomcreative commons non commercial cc bync this is distributed under the terms of the creative commons attributionnoncommercial license creativecommonslicensesbync40 which permits noncommercial use reproduction and distribution of the work without further permission provided the original work is attributed as specified on the sage and open access pages ussagepubcomenusnamopenaccessatsage 0c integrative cancer therapies effects on cancer cells inversely decreasing its toxicity on normal adjacent tissues89 in this regard much research has aimed to develop numerous antioxidant drugs of both natural and synthetic origin tested in both in vitro and in vivo models and also human clinical trials to overcome injuries caused by ir exposure and to induce killing of cancer cells at the same time previous studies have reported that phytochemical soy isoflavones genistein daidzein and glycitein which exhibit anticarcinogenic properties through their antioxidant activities could be used as potent radiosensitizers to enhance the efficacy of radiotherapymediated suppression of the growth and metastatic ability of cancers1011 along parallel lines resveratrol and piperine which possess antitumor activity have been shown to augment ionizing radiation irinduced apoptosis and loss of mitochondrial membrane potential in murine colon carcinoma and melanoma cells via enhancing irinduced ros generation12 moreover pentoxifylline ptx a methylxanthine that possesses antioxidant properties is known for improving tumor tissue oxygenation in murine hypoxic tumors and inhibiting post radiation induced normal tissue injury in mice1314 consequently searching for a natural product possessing anticancer activity that increases radiosensitivity of tumor cells and radioresistance of normal cells may lead to a potential future drug in cancer therapyamong the natural products bromelain bl extract attracts interest due to its anticancer antioxidant as well as antiinflammatory effects1517 bl an extract from pineapple stem ananas comosus belongs to a group of protein digesting enzymes it is a mixture of diï¬erent thiol endopeptidases and other components like phosphatase glucosidase peroxidase cellulase escharase calcium and several protease inhibitors1819 the anticancer activity of bl has been examined in various types of gastrointestinal and breast cancers cell lines in in vivo models bl has shown antimetastatic effect and reduction in local tumor growth2023 it is also used for reducing the severity of such radiation therapy side effects as mucositis skin reactions and dysphagia in patients24 hence this study was aimed to evaluate the radiosensitizing and radioprotective effect of bl using in vivo and in vitro approachesmaterials and methodin vitro studiesmtt cell proliferation assay the growth and viability of ehrlich ascites carcinoma eac cell line were tested in vitro by 345dimethylthiazol2yl25diphenyltetrazolium bromide mtt assay according to freimoser and buch 2526 to verify the antitumor and radiosensitizing effect of bl two plates were designed for this study the first one contained eac cells maintained by serial subculturing at the national cancer institute egypt incubated for hour before irradiation irr gy alone and with different concentrations of bromelain bl in phosphate buffer saline pbs the second one contained eac cells serving as a control and eac with different concentrations of bl each test was seeded in triplicate into a plate at concentration of — cellswell containing rpmi media with fbs nahco3 uml penicillin and µgml streptomycin and each plate was incubated for hours at °c in co2 and humidity atmosphere then μl mtt reagent bio basic inc canada was added over the cells in each well and the plate was incubated in the dark for to hours until a purple precipitate was seen and the absorbance was measured at nm the amount of color produced was directly proportional to the number of viable cells viable cell a samples ˆ’ a blanka control ˆ’ a blank — the inhibitory concentration ic50 is the dose of a drug which reduces the viability to and was calculated using nonlinear regression analysisfree radical scavenging assay the antioxidant activity of bromelain was evaluated by 1diphenyl2picrylhydrazyl dpph radical assay and its scavenging power was compared with some antioxidants naringin polyphenolic antioxidant garlic oil and glutathione sulfur containing antioxidants about µl of samples mgml dissolved in dist water was added to µl of a solution of dpph g100 ml dissolved in vv methanol after minutes incubation at room temperature in the dark the absorbance was read at nm against a blank µl dist water µl dpphmethanol solution the experiments were done in triplicate according to the method of braca 27 glutathione mgml was used as a standard antioxidant the scavenging percentage of dpph was calculated according to the followscavengin ing equation where b was the absorbance of the blank and a was the absorbance of samples or standard ec50 is defined as concentration of sample that causes dpph loss there values were calculated using nonlinear regression analysisˆ’b ab\uf8ee\uf8ef\uf8f0\uf8f9\uf8fa —\uf8fbin vivo studiesradiation processing whole body Îirradiation of mice was carried out using gamma cell40 137cesium manufactured by the atomic energy of canada limited ontario canada installed in the national center for radiation research and technology ncrrt cairo egypt the dose rate was gymin during the experimental period daily correction for humidity barometric pressure and temperature were madeanimals adult female swiss albino mice weighing to g obtained from the breeding unit of ncrrt cairo egypt all animal procedures were performed in accordance with the committee of scientific ethics at faculty of 0cmekkawy table sequences of primers for realtime quantitative pcrgeneparp1 nm0074152nfκb nc0000696pparα nc0000816βactin nc0000716forward primerreverse primer²ccatcgacgtcaactacga3²²caatggctacacaggacca3²²actccacctgcagagcaacca3²²gcgtggggacagccgcatctt3²²gtgcgtggtagcatgagtgt3²²cactgtcacctggaaccaga3²²tagatctcctgcagtagcggg3²²atcggcagaaggggcggaga3²pharmacy alazhar university egypt following the guidelines for animal use the animals were housed in colony cages micecage under proper environmental conditions that is hours darklight cycle good ventilation condition and temperature to humidity at the ncrrt animal house fed with standard diet pellets and provided with water ad libitum animals were left week for acclimatization on the lab environment before starting the experimenttumor transplantation the eac cell line was supplied by serial subculturing at the national cancer institute cairo university egypt it was implanted in each donor female swiss albino mice by ip injection of — cells22 g b wt and allowed to multiply28 the ehrlich solid tumor est was obtained by the intramuscular inoculation of ml of — viable eac in the right lower limb of each mouse29 mice with a palpable solid tumor diameter mm3 that was maintained within to days after inoculation were used in the studyanimal grouping animals were randomly divided into groups mice each group control not bearing tumor received pbs ip for days group ehrlich solid tumor est bearing mice received pbs ip for days group est Îirradiation gy — fractionated doses starting days after tumor appearance mm3 and lasting for days group est bearing mice receiving freshly prepared bl dissolved in pbs mgkg ip daily for days according to pilot study starting once est becomes mm3 bl was purchased from merck kgaa co darmstadt germany group est bearing mice received bl as in group hours before Îirradiation as in group mice were anesthetized days after last irradiation dose using urethane mgkg30 blood samples were collected through cardiac puncture and divided into parts edta coated and plain vials at that time they were euthanized by cervical dislocation liver and tumor tissues were dissected out rinsed with icecold saline dried on a filter paper and weighed then homogenized in icecold pbs ph and stored at ˆ’°c until used for subsequent biochemical analysisestimation of total body tumor and liver weights animals in each group were checked daily for any adverse clinical symptoms and deaths after to days post inoculation with eac body weights were recorded so body weight change could be estimated tumor and liver weights were measured during sample collection and then the tumor inhibitory ratio was calculated by the following formula inhibition ratio aˆ’ba — where a is the tumor weight average of the control and b is that of the treated group also relative liver weight was calculated as liver weighttotal body weight — histopathological examination three tumors of each group were collected and fixed in neutral buffered formalin the specimens were dehydrated in ascending grades of ethyl alcohol cleared in xylene and embedded in paraffin wax four micron thick paraffin sections were mounted on clean slides stained with ehrlich™s hematoxylineosin he31 and examined using an olympus microscope bx41 hamburg germany histopathological evaluation was done by assessment of necrosis and calculation of tumor area percentage using image analysis software image j 146a nih usa through the following equation of tumor area area of tumortotal area of the field — molecular analyses the mrna levels of poly adpribose polymerase parp1 nuclear factor kappa b nfκb and peroxisome proliferatoractivated receptors pparα genes and of the housekeeping gene βactin were measured by real time polymerase chain reaction rtpcr total rna was isolated from liver tissues using qiagen tissue extraction kit qiagen usa in accordance with the manufacturer™s instructions the extracted rna μg was used for cdna conversion using high capacity cdna reverse transcription kit fermentas usa and μl reaction volume sybr chemistry in applied biosystems thermal cycler usa to amplify pcr under the following conditions °c for denaturation then °c to °c for annealing using primers mentioned in table and °c for elongationresults were expressed using the comparative ˆ†ˆ†ct method for relative mrna quantification of target genes normalized to an endogenous reference βactin and a relevant control equal to ˆ’ˆ†ˆ†ct ˆ†ˆ†ct is the difference between the mean ˆ†ctsample and mean ˆ†ctcontrol where ˆ†ctsample is the difference between the mean ctsample and the mean ctβactin and ˆ†ctcontrol is the difference between the mean ctcontrol and the mean ctβactin 0c integrative cancer therapies estimation of lipid peroxidation lpo reactive oxygen species ros and paraoxonase pon1 in liver homogenate liver lipid peroxidation was estimated by measurement of malondialdehyde mda formation using the thiobarbituric acid method of yoshioka 32 a modified technique of vrablic 33 was used to measure the generation of ros by the intracellular conversion of nitro blue tetrazolium nbt to formazan by the action of superoxide anion paraoxonase activity was estimated by using fluorometric assay enzchek® kit invitrogen uk for the anophosphatase activity of paraoxonase based on the hydrolysis of a fluorogenic anophosphate analog34hematological and biochemical analyses whole blood was immediately analyzed for complete blood count with platelet count using the fully automated analyzer abx cobas micros roche germany estimation of serum alanine aminotransferase alt aspartate aminotransferase ast and albumin alb assays follow the recommendations of the international federation of clinical chemistry ifcc but were optimized for performance and stability using the rochehitachi cobas c 311systemstatistical analysis the statistical analysis was performed using oneway analysis of variance anova and the groups were compared by tukeykramer test viability percentage at different concentrations and body weight change analyzed by twoway anova followed by bonferroni™s posttest graphs were sketched using graph pad prism isi® software usa version software data were presented as mean ± standard error se and p values considered significantresultsin vitro studieseffect of bromelain and gammairradiation blirr on tumor cell growth and viability the radiosensitizing effect of bl on eac cells was determined by performing mtt assay eac cells exposed to gy Îradiation showed high cell viability percentage reflecting a radioresistance of eac cell line while bl treatment showed in vitro cytotoxic activity with ic50 value of mgml however the maximum cytotoxic effect appeared when the eac cells were subjected to bl then Îradiation gy compared to control or irradiated group with ic50 mgml table effect of bromelain and some natural compounds as free radical scavengers the inhibitory percentage of each compound is shown in figure the ec50 value concentration of sample that causes dpph activity loss is a reliable way for estimation of the radical scavenging activity the ec50 value of glutathione referenced antioxidant is mgml while table cytotoxic activity of blirr against eac cell line bromelain concentration mgmleac bl mgmlic50 mgmlviability non irradiated eac irradiated eac48ab59ab60ab69a787a10ab158ab18ab27ab52ab ± ± each value indicates the mean of records statistical analysis carried out by twoway anova followed by bonferroni posttests a significant versus control ehrlich ascites carcinoma eac group where b significant versus irradiated eac group at p ic50 ± se values were calculated by using nonlinear regression analysisbromelain and garlic oil ec50 are almost equal and mgml respectively however the naringin phenolic antioxidant is the least potent one ec50 mgml in this comparisonin vivo studieseffect of bromelain and gammairradiation blirr on tumor weight and volume table shows a significant decrease in tumor weight in groups treated with bl andor Îirradiation as compared to the est nontreated group the more drastic decrease in the tumor weight ratio observed in the combined therapy group bl irr compared with the estirradiated group as well as est group indicates that combination therapy is more significantly effective than single agent therapy the photograph of est xenografts at the time of sacrifice shows the synergistic effect of bl and irr on tumor volume figure effect of bromelain and gammairradiation blirr on tumor histopathological features of est bearing mice histopathological examination of solid tumor sections revealed typical malignant features including sheets of malignant cells infiltrating adjacent muscular tissue the malignant cells show pleomorphism hyperchromatism and mitotic activity while the necrotic cells appear as nonviable homogenous structureless material with degenerated or karyorrhectic nuclei untreated est bearing group shows a deeply stained tumor cells arrow head and areas of necrosis arrow figure 3a — also it displays intact cancer cells arrow and giant cells arrow figure 3b and c — the estirradiated group shows muscle fibers invaded by deeply stained tumor cells arrow head and large areas of necrosis arrow figure 3d — also displays a notable necrosis of cancer cells n figure 3e — the bl treated group shows a 0cmekkawy figure dpph 1diphenyl2picrylhydrazyl reduction curve for glutathione bromelain naringin and garlic oil each value represents mean ± se all experiments were replicated timestable tumor weight and inhibition ratio of ehrlich solid tumor estbearing mice treated with gammairradiation irr gy — andor bromelain bl mgkggroupsestest irrest blest bl irrtumor weight g ± ± 004a ± 005a ± 005abtumor inhibitory ratio ” ± 24a ± 14ab ± 204ababbreviations est ehrlich solid tumor est irr ehrlich solid tumor irradiation est bl ehrlich solid tumor bromelain est bl irr ehrlich solid tumor bromelain irradiationthe values shown are mean ± se of data a significant versus est group where b significant versus estirradiated group at p figure a photograph of ehrlich solid tumor est xenografts at the time of scarification showing the effect of bromelain and gammairradation blirr on tumor volume e ehrlich solid tumor e ir ehrlich solid tumor irradiation e br ehrlich solid tumor bromelain e ir br ehrlich solid tumor bromelain irradiation 0c integrative cancer therapies figure photo micrograph of ehrlich solid tumor est xenografts in different animal groups est sections show the degree of tumorogenesis necrosis n regression of tumor by appearance of muscle fibers m — and — a b c est ehrlich solid tumor d e est irr ehrlich solid tumor irradiation f g est bl ehrlich solid tumor bromelain h i est bl irr ehrlich solid tumor bromelain irradiationwide area of necrosis arrow and n few groups of cancer cells arrow head and muscle fiber m figures 3f — and 4g — however combined treatment bl irr displays muscle fiber m significant regression of tumor or wide areas of necrotic cancer cells n and few groups of intact cancer cells arrow figure 3h — and 3i —the tumor area percentage per total tissue area could determine the degree of proliferation as seen in figure there is a great regression of tumor area in the group treated with bl alone or bl and irr compared with untreated est or estirradiated group indicating that combination therapy significantly more effective than single agent therapy 0cmekkawy bl irr shows nonsignificant group change additionally bl irr group significantly upregulated pparα expression compared with est and estirradiated groups indicating that bl might have a hepato as well as radioprotective effect figure effect of bromelain and gammairradiation blirr on the hepatic lipid peroxidation lpo level reactive oxygen species ros content and paraoxonase1 pon1 activity of ehrlich solid tumor est bearing mice lpo in liver tissues significantly increased in all est bearing groups compared to the control group except the combined treated group irr bl succeeded in returning mda lpo measured as mda malondialdehyde level to the normal level however liver ros significantly increased only in untreated and Îirradiated est bearing groups when compared to the control group while a significant decrease in liver ros showed in estirradiated mice treated with bl in comparison with both est untreated and estirradiated groups pon1 activity in liver homogenate was significantly decreased in est untreated and estirradiated groups when compared with the control group bl treated groups revealed significant increases in pon1 when compared with both est untreated and estirradiated groups showing that bl might have a hepato and radioprotective effect figure effect of bromelain and gammairradiation blirr on hematological measurements wbcs and plts were significantly elevated while hgb and hct significantly decreased in the untreated estbearing mice in comparison with control mice however Îirradiation resulted in a significant decrease in wbcs rbcs plt hgb and hct compared with the control mice treatment of the estbearing mice with bl shows a significant amelioration in wbcs plt and hct compared to est untreated mice combined treatment bl irr shows an enhancement in wbcs plt and hct compared to est untreated and gammairradiated est bearing mice table effect of bromelain and gammairradiation blirr on the serum alanine transaminase alt aspartate transaminase ast and albumin alb to investigate the cytoprotective effects of bl against irradiation the levels of serum alt ast and alb were measured figure it was found that alt and ast significantly increased and conversely alb significantly decreased in est untreated and estirradiated groups compared with the control group however estbearing mice treated with bl alone show nearly the same result of alt and alb as control values estbearing mice treated with bl in combination with irradiation initiated a significant decrease in ast and alt as compared with estirradiated group which may reflect a potential hepatic radioprotective effect of blfigure percentage of tumor areatotal tissue area of ehrlich solid tumor est bearing mice treated with gammairradiation irr gy — andor bl mgkg the values shown in the plotted area are mean of records from animals ± se significant versus est group where significant versus estirradiated group at p effect of bromelain and gammairradiation blirr on body weight change and relative liver weight regarding the day by day documented recording of body weight bwt illustrated in figure there is almost no change in bwt of bl treated group while it increases significantly in the untreated est group conversely estirradiated groups with or without bl treatment show a significant decrease in bwt when compared with the control group table relative liver weight was compared after normalization to mg body weight untreated estbearing group shows a significant increase in liver weight by hepatomegaly while nonsignificant changes were observed in bl treated groups compared to the normal group table effect of bromelain and gammairradiation blirr on the poly adpribose polymerase parp1 nuclear factor kappa b nfκb and peroxisome proliferatoractivated receptors pparα relative gene expression of ehrlich solid tumor est bearing mice to test the possibility that bl reduces radiation damage to the liver mrna gene expression of parp1 nfκb and pparα was measured in the liver homogenates of est bearing mice and compared to control pbs treated mice the results illustrated in figure show that irr causes significant increases in parp1 and nfκb expression compared to the control group however combined treatment bl irr shows a significant increase in parp1 and nfκb expression compared to control group and a significant attenuation compared to estirradiated groupmoreover all est bearing groups show significant decreases in hepatic pparα relative gene expression compared to the control group except the combined therapy 0c integrative cancer therapies figure effect of bromelain and gammairradiation blirr on body weight during experiment period each value represents the mean of records ± se significant versus control group where significant versus est group and significant versus estirradiated group at p est ehrlich solid tumor est irr ehrlich solid tumor irradiation est bl ehrlich solid tumor bromelain est bl irr ehrlich solid tumor bromelain irradiationtable change in body weight and relative liver weight of control mice and ehrlich solid tumor est bearing mice treated with gammairradiation irr gy — andor bromelain bl mgkggroupscontrolestest irrest blest bl irrbody weight change ± ± 101aˆ’ ± 217abˆ’ ± 201bˆ’ ± 341abrelative liver weight ± ± 026a ± 022a ± 026a ± 026ababbreviations est ehrlich solid tumor est irr ehrlich solid tumor irradiation est bl ehrlich solid tumor bromelain est bl irr ehrlich solid tumor bromelain irradiationeach value represents the mean ± se a significant versus control group where b significant versus est group at p body weight changes percent are related to the initial weight of animalsdiscussionresistance of tumor cells to chemoradiotherapy as well as the damaging effects to nearby normal tissues remains a major obstacle to successful cancer management therefore the current study has been conducted to estimate the effect of bromelain bl as a tumor radiosensetizer and to show to what extent it can protect normal tissue from radiation hazardsradiosensitizers are compounds that when combined with radiation therapy achieve greater cytotoxicity they can be determined in vitro by the mtt assay2635 the present study has found that the radioresistant eac cells could be sensitized when incubated with bl before irradiation it was known previously that in vitro treatment with bl on mouse tumor cell lines resulted in inhibition of cell growth and invasion capacities3637 the anticancer property of bl has been mainly attributed to the protease component through digestion and diffusion in tumor cells38 it may also be due to the bl enhancement of p53 expression as well as another activator of apoptosis eg bax39 in addition it decreases the activity of cell survival regulators such as akt and erk it also deactivates aktdependent proapoptotic regulator foxo3a thus promoting apoptotic cell death in tumors40it is well known that during cancer and radiotherapy excessive energy is used from the host41 ultimately contributing to mechanisms that promote loss of weight as shown in the present study which also showed that bl could return body weight to a normal level by decreasing tumor weight and volume currently the combined therapy bl irr has been shown to be more effective than single agent therapy in reducing tumor volume and weight indicating that bl could possess a radiosensitizing effect in addition the combined therapy has revealed a drastic decrease in tumor area percentage wide areas of necrotic cancer cells and presence of muscle fiber in the histopathological examination compared with the control est and estirradiated groups this seems to be in agreement with other findings of the role of bl in reducing metastasis and local tumor growth2342 in chemically induced mouse skin papillomas topical application of bl reduced tumor formation tumor volume and caused apoptotic cell death39 bl is a hydrolytic enzymatic complex which shows an efficient digestion and diffusion in tumor cells through attacking the glycosidic linkages and hence denatures glycoproteins thus it protects against tumor growth37 another study has demonstrated the use of controlled proteolytic activity on tumor as a successful strategy to increase therapeutic efficacy43 0cmekkawy figure effect of bromelain and gammairradiation blirr on relative gene expression of liver a parp1 b nfκb and c pparα each value represents the mean of records ± se significant versus control group where significant versus est group and significant versus estirradiated group at p est ehrlich solid tumor est irr ehrlich solid tumor irradiation est bl ehrlich solid tumor bromelain est bl irr ehrlich solid tumor bromelain irradiationthe aim of the radiotherapy protocols is to achieve the maximum curative effect on tumor cells with minimal damaging effect on normal cells hence antioxidants and other nutrients which do not interfere with therapeutic modalities for cancer may enhance the killing property decrease side effects and protect normal tissue44for estimation of the antioxidant ability of bl dpph assay was conducted in vitro and the free radical inhibitory action of bl was compared with some antioxidant compounds it was found that bl has a powerful free radical scavenging power bl belongs to thiol proteases in which the catalytic nucleophile is sulfhydryl groups of cysteine residues which in turn accounts for its antioxidant activity45the involvement of ros mda and pon1 are important mechanisms that play a vital role during radiation toxicity the use of antioxidants is an important preventive to decrease the toxic and pathological effects associated with oxidative stress caused by radiation46 the attained results show a hepatic impairment on the third day from exposure to Îradiation elevation of lpo and ros levels and inhibition of pon1 activity compared to normal mice however treatment with bl revealed an amelioration in hepatic damage caused by irradiation these results were in accordance with liu 47 who described the effect of radiation induced ros generation which in turn might attack cell membrane phospholipids and circulating lipids and thus increases production of mda48 lpo acts as a sensitive biomarker for oxidative stress that occurs as part of the pathogenesis of irradiation49 bl has sulfhydryl groups consequently accounting for its antioxidant activity45 thus it could act as ros scavengermeasurement of pon1 postradiotherapy could be an effective clinical biomarker of hepatic and systemic oxidative stress and may be used as an index of the usefulness of radiotherapy50 it has been demonstrated to catalyze hydrolysis of lipid hydroperoxides and lactones51 pon1 protects serum hdl and ldl ps against lipid peroxidation52 in the present study the decreased activity of pon1 upon radiation exposure might be due to its super saturation of lipid hydroperoxides and lactones upon treatment with bl the activity of pon1 was restored near to the normal level hence the pon1 Answer:

Colon_Cancer

malignant mesothelioma is an aggressive cancer associated with asbestos exposure with median survival time of to months following diagnosis given that mesothelial cells also line the peritoneum and pericardium malignant mesothelioma can present in unusual sites and in patients with nonrespiratory complaints a 73yearold male presented to the emergency department for worsening intermittent diffuse abdominal pain for the past months with associated unintentional 40pound weight loss early satiety and diarrhea he denied exposure to asbestos computed tomography imaging revealed multiple masses concerning for malignancy including the primary retroperitoneal mass a mass involving the terminal ileum and a mass in the right upper lung esophagogastroduodenoscopy demonstrated significant mass effect within the stomach without signs of endoluminal infiltration computed tomography“guided biopsy of the retroperitoneal abdominal and intramuscular paraspinal masses was performed stage iv epithelioid mesothelioma was confirmed when hematoxylin and eosin staining revealed pleomorphic malignancy nuclei containing a vesicular chromatin pattern and prominent nucleoli and immunohistochemical staining was positive for ck oscar cytokeratin gata3 calretinin ema and ck56 he was started on cisplatin pemetrexed and bevacizumab but developed severe abdominal pain with pneumoperitoneum and bowel perforation month later and expired shortly thereafter to our knowledge this represents a highly atypical presentation of malignant mesothelioma considering the involvement of the retroperitoneum with diffuse lesions in the abdominopelvic cavity and thorax sparing the lung pleurae this case also calls attention to the occurrence of malignant mesothelioma in patients without known asbestos exposure and the crucial role of pathology in diagnosing atypical presentationskeywordsepithelioid mesothelioma neoplasm asbestos esophagogastroduodenoscopy retroperitonealintroductionmalignant mesothelioma mm is a rare cancer originating from mesothelial cells forming the linings of the pleura to of mm cases12 peritoneum to of cases23 and pericardium of cases456 typically the pathophysiology of mm is believed to occur in patients with asbestos exposure leading to chronic inflammation of the pleura67 subsequently mm presents in a patient with shortness of breath with findings on imaging of nodular thickening of the pleura pleural effusion or a mass7 in cases involving the peritoneum a more common initial presentation would be ascites with computed tomography ct findings including irregular thickening of the peritoneum lymph node enlargement and possible metastasis to the chest table regardless of location published guidelines detail the importance of first looking at the histology of a biopsy prior to immunohistochemical ihc and molecular testing to confirm suspicion of mm from the histology mm is classified into epithelioid sarcomatoid or biphasic mixed with epithelioid mm remaining the most common with its polygonal cells resembling reactive mesothelial cells other histologic features include scalloped cell borders with increased cytoplasm prominent and enlarged nucleoli and nuclear atypia10 the presence of a solid mass separate from the pleura and peritoneum with histologic features of mm eliminates the requirement for stromal invasion for the diagnosis910immunohistochemical testing should use markers of mesothelial origin such as cytokeratin markers and alternative tumor markers to narrow the differential diagnosis based on histology10 this will vary by histology and location 1mercyone des moines medical center des moines ia usa2des moines university des moines ia usareceived may revised june accepted july corresponding authordustin j uhlenhopp mercyone des moines medical center 6th avenue des moines ia usa email duhlenhoppmercydesmoinescreative commons non commercial cc bync this is distributed under the terms of the creative commons attributionnoncommercial license creativecommonslicensesbync40 which permits noncommercial use reproduction and distribution of the work without further permission provided the original work is attributed as specified on the sage and open access pages ussagepubcomenusnamopenaccessatsage 0c of investigative medicine high impact case reportstable typical characteristics of malignant abdominal mesothelioma1819characteristicspercentage of casespathological subtypes epithelial sarcomatous mixedasbestos exposure male femalepresenting symptoms ascitesa abdominal pain asthenia weight loss anorexia fevera diarrhea vomitingact scan findings ascites abdominal mass peritoneal thickening mesenterial thickeningadditional laboratory findings thrombocytosis 000mm3 anemia male gdl female gdlmaletofemale ratiomedian overall survivalaverage age of onset months yearsabbreviation ct computed tomographyamost significant symptoms on presentation associated with deathof the mm epithelioid markers are needed to rule out carcinomas and peritoneal markers are needed to rule out adenocarcinomas and cancers causing peritoneal carcinomatosis810 key markers for mesothelioma are positive ihc staining for calretinin d240 podoplanin and cytokeratin in this case report we present an unusual case of mm with metastatic disease in a patient with no known asbestos exposure and a history of abdominal pain and weight loss found to have multiple lesions and diffuse lymphadenopathy on imagingcase descriptiona 73yearold male presented to the emergency department with worsening intermittent diffuse abdominal pain and 40pound weight loss over the past months associated with increased flatulence early satiety and frequent exive nonbloody diarrhea the patient described the pain as cramping that typically worsened in severity at night he denied feverchills dyspnea cough epistaxis hematemesis hemoptysis hematochezia and melena the patient was a longtime science instructor at a local community college prior to transitioning to the swedish importexport business with no significant exposure history including asbestos he had a prior history of right middle cerebral artery stroke and was a former smoker with a 60packyear smoking historyinitial testing revealed normal electrolytes and liver function panel elevated lactic acid mmoll leukocytosis white blood cell 12100mm3 and anemia hematocrit abdominal ct with oral and intravenous iv contrast revealed a homogenous retroperitoneal mass measuring — — cm with displacement of the gastroesophageal junction and lesser curvature of stomach as well as a soft tissue mass involving the terminal ileum measuring — — cm both were concerning for neoplasm additional findings included diffuse lymphadenopathy in the retroperitoneum likely signifying metastatic disease no evidence of obstruction was noted see figure esophagogastroduodenoscopy confirmed concerns for extrinsic compression of gastroesophageal junction and fundus of stomach suggesting an underlying mass no endoluminal gastric masslesions were appreciated see figure colonoscopy was attempted but scope was not able to advance past the sigmoid colon safely due to colonic stricturecomputed tomography chest with iv contrast identified moderate diffuse emphysema a right upper lung lobe spiculated mass measuring cm and indeterminate small left upper and lower lobe nodules see figure diffuse retrocrural and gastroesophageal lymphadenopathy was noted in addition to the retroperitoneal lymphadenopathygiven the concern for lymphoma versus alternative neoplastic disease ctguided biopsy was performed on the retroperitoneal abdominal mass and an intramuscular paraspinal mass hematoxylin and eosin staining revealed pleomorphic malignancy nuclei containing a vesicular chromatin pattern and prominent nucleoli see figure cells were predominantly polygonal with occasional spindling noted ihc staining was positive for ck oscar cytokeratin gata3 calretinin ema and ck56 staining was negative for pax8 tte1 p40 p63 d240 wt1 moc31 cd34 mari cd45 dog desmin cd117 sox10 actin ca9 and myogenthese findings were suggestive of epithelioid mm with metastasis to the terminal ileum and lung a diagnosis that was confirmed by mayo clinic laboratories noting this is a particularly atypical presentation of mesotheliomathe patient opted for treatment of stage iv mesothelioma with plans to pursue a 21day cycle of cisplatin pemetrexed and bevacizumab for up to cycles however he presented to the emergency department days after starting the second cycle with severe abdominal pain of hours duration he was found to have a moderate pneumoperitoneum with bowel perforation worsening intraabdominal mesothelioma and several small bilateral acute renal parenchymal infarcts the patient and his family opted for comfort care and he passed the following day 0cuhlenhopp et al figure abdominal computed tomography with oral and intravenous contrast demonstrating a homogenous retroperitoneal neoplastic mass measuring — — cm green arrows on left image with displacement of the gastroesophageal junction and lesser curvature of stomach a soft tissue mass involving the terminal ileum measuring — — cm and a 17cm midmesentery mass are also noted green arrows on right imagefigure esophagogastroduodenoscopy revealed extrinsic compression of the fundus suggesting an underlying mass or lesion no endoluminal gastric masseslesions were appreciated computed tomography“guided biopsy later revealed this to be epithelioid mesothelioma originating from the retroperitoneal spacediscussionthe diagnosis of mm carries a poor prognosis and requires careful review by pathology our case details the workup necessary to diagnosis mm in a patient with an atypical presentation of intermittent abdominal pain found to have figure computed tomography chest with intravenous contrast identified moderate diffuse emphysema a right upper lung lobe spiculated mass measuring cm green arrows and indeterminate small left upper and lower lobe nodules as this did not involve the pleura as is traditionally seen this was felt to be a metastasis of the primary retroperitoneal mass with metastasis to lungs and ileumsignificant metastatic disease prior to identification of a primary malignancyreview of the case reveals the importance of tissue analysis and the continued public health concern of mesothelioma 0c of investigative medicine high impact case reportsongoing research is investigating whether these genetic changes have a role in disease development in cases without asbestos exposure610 in the case of malignant peritoneal mesothelioma there is a higher fraction of mm without an identifiable environmental exposure which raises the question whether the disease process and role of genetics vary by the location of mm14 in the future these will hopefully lead to the development of immunotherapies that are able to target these pathways and improved disease prognosis6known asbestos exposure was not a factor in our case and should not be used to rule out mesothelioma10 over of mm cases are attributable to asbestos exposure with of malignant peritoneal mesothelioma attributed to asbestos1516 recent studies detail that despite the restriction of asbestos products in the united states for over years the role of asbestos in chronic inflammation and dna damage will continue to cause malignancy615 in the united states alone there were approximately new mm cases in with current estimates anticipating a peak incidence of mm worldwide in given the continued use of asbestos worldwide and time to disease development6715 this case highlights the importance of considering mesothelioma in the differential diagnosis of abdominal pain regardless of asbestos exposure historydeclaration of conflicting intereststhe authors declared no potential conflicts of interest with respect to the research authorship andor publication of this fundingthe authors received no financial support for the research authorship andor publication of this ethics approvalour institution does not require ethical approval for reporting individual cases or case seriesinformed consentverbal informed consent was obtained from the patient for their anonymized information to be published in this orcid idsdustin j uhlenhopp orcid0000000315092203ann saliares orcid0000000281484435tagore sunkara orcid0000000195369027references robinson bm malignant pleural mesothelioma an epidemiological perspective ann cardiothorac surg doi103978jissn2225319x20121104 hui m harshavardhana kr uppin sg pericardial mesothelioma presenting as chronic constrictive pericarditis a series of three cases from a single institution indian j pathol microbiol doi104103ijpmijpm_711_17figure hematoxylin and eosin stain of the retroperitoneal mass — malignant mesothelioma that is somewhat pleomorphic but mostly epithelioid polygonal cells with some spindling are seen cells have pleomorphic nuclei occasionally multinucleated with moderate amount of eosinophilic cytoplasm nuclei have a vesicular chromatin pattern with distinct nucleoli there is no obvious glandular or squamous differentiationmm carries a median survival time of to months following diagnosis7 this poor prognosis is partially due to the fact that over of cases are not discovered until the distant metastasis stage11 while most cases of epithelioid mm have an improved prognosis compared with sarcomatoid mm it can possess pleomorphic features that indicate highly aggressive cancer and shortened expected survival time71012 treatment consists of chemotherapy radiation and surgery although no therapy is curative7in our case ihc staining had an important role in developing the diagnosis of epithelioid mm with pleomorphic features it distinguished the disease from reactive mesothelial hyperplasia and other malignancies with possibly better prognoses10 the right lung lobe mass is not the typical presentation for mm involving the thorax and complicates the clinical picture the presentation of the retroperitoneal mass coupled with diffuse metastatic lesions and atypical involvement of the thorax make this patient™s presentation particularly unique calretinin gata3 and ck56 were positive in the tissue obtained from the retroperitoneal and paraspinal muscle masses which implicated a mesothelial origin for the malignancy calretinin is a strong indicator of epithelioid mm and is useful in distinguishing mm from adenocarcinoma10 recent reports estimate a positive staining for gata3 in mesothelioma in of cases and note a presence in epithelioid mm in one third of cases1013 another important ihc markers for mm is d240 which was negative in our patientwhile not completed in our patient other markers present in mm include the deletion of p16 occurring in of epithelioid mm and the presence of a bap1 mutation101214 0cuhlenhopp et al lev½ m boubl­kov¡ l b¼chler t Å¡imÅ¡a j treatment of malignant peritoneal mesothelioma klin onkol doi1014735amko2019333 sardar mr kuntz c patel t et al primary pericardial mesothelioma unique case and literature review tex heart inst j mutsaers se the mesothelial cell int j biochem cell biol napolitano a carbone m malignant mesothelioma time to translate trends cancer bibby ac tsim s kanellakis n et al malignant pleural mesothelioma an update on investigation diagnosis and treatment eur respir rev doi10118316000617 liang yf zheng gq chen yf song h yin wj zhang l ct differentiation of diffuse malignant peritoneal mesothelioma and peritoneal carcinomatosis j gastroenterol hepatol gill rr imaging of mesothelioma in a tannapfel ed malignant mesothelioma recent results in cancer research vol springer husain an colby tv ordonez ng et al guidelines for pathologic diagnosis of malignant mesothelioma update of the consensus statement from the international mesothelioma interest group arch pathol lab med howlader n noone am krapcho m et al seer cancer statistics review accessed february seercancergovcsr1975_2016 arif q husain an malignant mesothelioma diagnosis arch pathol lab med miettinen m mccue pa sarlomorikala m et al gata3 a multispecific but potentially useful marker in surgical pathology a systematic analysis of epithelial and nonepithelial tumors am j surg pathol attanoos rl churg a galateausalle f gibbs ar roggli vl malignant mesothelioma and its nonasbestos causes arch pathol lab med craighead je epidemiology of mesothelioma and historical background in a tannapfel ed malignant mesothelioma recent results in cancer research vol springer spirtas r heineman ef bernstein l et al malignant mesothelioma attributable risk of asbestos exposure occup environ med bray f ferlay j soerjomataram i siegel rl torre la jemal a global cancer statistics globocan estimates of incidence and mortality worldwide for cancers in countries ca cancer j clin doi103322caac21492 manzini vde p recchia l cafferata m et al malignant peritoneal mesothelioma a multicenter study on cases ann oncol doi101093annoncmdp shih ca ho sp tsay fw lai kh hsu pi diffuse malignant peritoneal mesothelioma kaohsiung j med sci doi101016jkjms201305003 0c'

cancer288

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: malignant mesothelioma is an aggressive cancer associated with asbestos exposure with median survival time of to months following diagnosis given that mesothelial cells also line the peritoneum and pericardium malignant mesothelioma can present in unusual sites and in patients with nonrespiratory complaints a 73yearold male presented to the emergency department for worsening intermittent diffuse abdominal pain for the past months with associated unintentional 40pound weight loss early satiety and diarrhea he denied exposure to asbestos computed tomography imaging revealed multiple masses concerning for malignancy including the primary retroperitoneal mass a mass involving the terminal ileum and a mass in the right upper lung esophagogastroduodenoscopy demonstrated significant mass effect within the stomach without signs of endoluminal infiltration computed tomography“guided biopsy of the retroperitoneal abdominal and intramuscular paraspinal masses was performed stage iv epithelioid mesothelioma was confirmed when hematoxylin and eosin staining revealed pleomorphic malignancy nuclei containing a vesicular chromatin pattern and prominent nucleoli and immunohistochemical staining was positive for ck oscar cytokeratin gata3 calretinin ema and ck56 he was started on cisplatin pemetrexed and bevacizumab but developed severe abdominal pain with pneumoperitoneum and bowel perforation month later and expired shortly thereafter to our knowledge this represents a highly atypical presentation of malignant mesothelioma considering the involvement of the retroperitoneum with diffuse lesions in the abdominopelvic cavity and thorax sparing the lung pleurae this case also calls attention to the occurrence of malignant mesothelioma in patients without known asbestos exposure and the crucial role of pathology in diagnosing atypical presentationskeywordsepithelioid mesothelioma neoplasm asbestos esophagogastroduodenoscopy retroperitonealintroductionmalignant mesothelioma mm is a rare cancer originating from mesothelial cells forming the linings of the pleura to of mm cases12 peritoneum to of cases23 and pericardium of cases456 typically the pathophysiology of mm is believed to occur in patients with asbestos exposure leading to chronic inflammation of the pleura67 subsequently mm presents in a patient with shortness of breath with findings on imaging of nodular thickening of the pleura pleural effusion or a mass7 in cases involving the peritoneum a more common initial presentation would be ascites with computed tomography ct findings including irregular thickening of the peritoneum lymph node enlargement and possible metastasis to the chest table regardless of location published guidelines detail the importance of first looking at the histology of a biopsy prior to immunohistochemical ihc and molecular testing to confirm suspicion of mm from the histology mm is classified into epithelioid sarcomatoid or biphasic mixed with epithelioid mm remaining the most common with its polygonal cells resembling reactive mesothelial cells other histologic features include scalloped cell borders with increased cytoplasm prominent and enlarged nucleoli and nuclear atypia10 the presence of a solid mass separate from the pleura and peritoneum with histologic features of mm eliminates the requirement for stromal invasion for the diagnosis910immunohistochemical testing should use markers of mesothelial origin such as cytokeratin markers and alternative tumor markers to narrow the differential diagnosis based on histology10 this will vary by histology and location 1mercyone des moines medical center des moines ia usa2des moines university des moines ia usareceived may revised june accepted july corresponding authordustin j uhlenhopp mercyone des moines medical center 6th avenue des moines ia usa email duhlenhoppmercydesmoinescreative commons non commercial cc bync this is distributed under the terms of the creative commons attributionnoncommercial license creativecommonslicensesbync40 which permits noncommercial use reproduction and distribution of the work without further permission provided the original work is attributed as specified on the sage and open access pages ussagepubcomenusnamopenaccessatsage 0c of investigative medicine high impact case reportstable typical characteristics of malignant abdominal mesothelioma1819characteristicspercentage of casespathological subtypes epithelial sarcomatous mixedasbestos exposure male femalepresenting symptoms ascitesa abdominal pain asthenia weight loss anorexia fevera diarrhea vomitingact scan findings ascites abdominal mass peritoneal thickening mesenterial thickeningadditional laboratory findings thrombocytosis 000mm3 anemia male gdl female gdlmaletofemale ratiomedian overall survivalaverage age of onset months yearsabbreviation ct computed tomographyamost significant symptoms on presentation associated with deathof the mm epithelioid markers are needed to rule out carcinomas and peritoneal markers are needed to rule out adenocarcinomas and cancers causing peritoneal carcinomatosis810 key markers for mesothelioma are positive ihc staining for calretinin d240 podoplanin and cytokeratin in this case report we present an unusual case of mm with metastatic disease in a patient with no known asbestos exposure and a history of abdominal pain and weight loss found to have multiple lesions and diffuse lymphadenopathy on imagingcase descriptiona 73yearold male presented to the emergency department with worsening intermittent diffuse abdominal pain and 40pound weight loss over the past months associated with increased flatulence early satiety and frequent exive nonbloody diarrhea the patient described the pain as cramping that typically worsened in severity at night he denied feverchills dyspnea cough epistaxis hematemesis hemoptysis hematochezia and melena the patient was a longtime science instructor at a local community college prior to transitioning to the swedish importexport business with no significant exposure history including asbestos he had a prior history of right middle cerebral artery stroke and was a former smoker with a 60packyear smoking historyinitial testing revealed normal electrolytes and liver function panel elevated lactic acid mmoll leukocytosis white blood cell 12100mm3 and anemia hematocrit abdominal ct with oral and intravenous iv contrast revealed a homogenous retroperitoneal mass measuring — — cm with displacement of the gastroesophageal junction and lesser curvature of stomach as well as a soft tissue mass involving the terminal ileum measuring — — cm both were concerning for neoplasm additional findings included diffuse lymphadenopathy in the retroperitoneum likely signifying metastatic disease no evidence of obstruction was noted see figure esophagogastroduodenoscopy confirmed concerns for extrinsic compression of gastroesophageal junction and fundus of stomach suggesting an underlying mass no endoluminal gastric masslesions were appreciated see figure colonoscopy was attempted but scope was not able to advance past the sigmoid colon safely due to colonic stricturecomputed tomography chest with iv contrast identified moderate diffuse emphysema a right upper lung lobe spiculated mass measuring cm and indeterminate small left upper and lower lobe nodules see figure diffuse retrocrural and gastroesophageal lymphadenopathy was noted in addition to the retroperitoneal lymphadenopathygiven the concern for lymphoma versus alternative neoplastic disease ctguided biopsy was performed on the retroperitoneal abdominal mass and an intramuscular paraspinal mass hematoxylin and eosin staining revealed pleomorphic malignancy nuclei containing a vesicular chromatin pattern and prominent nucleoli see figure cells were predominantly polygonal with occasional spindling noted ihc staining was positive for ck oscar cytokeratin gata3 calretinin ema and ck56 staining was negative for pax8 tte1 p40 p63 d240 wt1 moc31 cd34 mari cd45 dog desmin cd117 sox10 actin ca9 and myogenthese findings were suggestive of epithelioid mm with metastasis to the terminal ileum and lung a diagnosis that was confirmed by mayo clinic laboratories noting this is a particularly atypical presentation of mesotheliomathe patient opted for treatment of stage iv mesothelioma with plans to pursue a 21day cycle of cisplatin pemetrexed and bevacizumab for up to cycles however he presented to the emergency department days after starting the second cycle with severe abdominal pain of hours duration he was found to have a moderate pneumoperitoneum with bowel perforation worsening intraabdominal mesothelioma and several small bilateral acute renal parenchymal infarcts the patient and his family opted for comfort care and he passed the following day 0cuhlenhopp et al figure abdominal computed tomography with oral and intravenous contrast demonstrating a homogenous retroperitoneal neoplastic mass measuring — — cm green arrows on left image with displacement of the gastroesophageal junction and lesser curvature of stomach a soft tissue mass involving the terminal ileum measuring — — cm and a 17cm midmesentery mass are also noted green arrows on right imagefigure esophagogastroduodenoscopy revealed extrinsic compression of the fundus suggesting an underlying mass or lesion no endoluminal gastric masseslesions were appreciated computed tomography“guided biopsy later revealed this to be epithelioid mesothelioma originating from the retroperitoneal spacediscussionthe diagnosis of mm carries a poor prognosis and requires careful review by pathology our case details the workup necessary to diagnosis mm in a patient with an atypical presentation of intermittent abdominal pain found to have figure computed tomography chest with intravenous contrast identified moderate diffuse emphysema a right upper lung lobe spiculated mass measuring cm green arrows and indeterminate small left upper and lower lobe nodules as this did not involve the pleura as is traditionally seen this was felt to be a metastasis of the primary retroperitoneal mass with metastasis to lungs and ileumsignificant metastatic disease prior to identification of a primary malignancyreview of the case reveals the importance of tissue analysis and the continued public health concern of mesothelioma 0c of investigative medicine high impact case reportsongoing research is investigating whether these genetic changes have a role in disease development in cases without asbestos exposure610 in the case of malignant peritoneal mesothelioma there is a higher fraction of mm without an identifiable environmental exposure which raises the question whether the disease process and role of genetics vary by the location of mm14 in the future these will hopefully lead to the development of immunotherapies that are able to target these pathways and improved disease prognosis6known asbestos exposure was not a factor in our case and should not be used to rule out mesothelioma10 over of mm cases are attributable to asbestos exposure with of malignant peritoneal mesothelioma attributed to asbestos1516 recent studies detail that despite the restriction of asbestos products in the united states for over years the role of asbestos in chronic inflammation and dna damage will continue to cause malignancy615 in the united states alone there were approximately new mm cases in with current estimates anticipating a peak incidence of mm worldwide in given the continued use of asbestos worldwide and time to disease development6715 this case highlights the importance of considering mesothelioma in the differential diagnosis of abdominal pain regardless of asbestos exposure historydeclaration of conflicting intereststhe authors declared no potential conflicts of interest with respect to the research authorship andor publication of this fundingthe authors received no financial support for the research authorship andor publication of this ethics approvalour institution does not require ethical approval for reporting individual cases or case seriesinformed consentverbal informed consent was obtained from the patient for their anonymized information to be published in this orcid idsdustin j uhlenhopp orcid0000000315092203ann saliares orcid0000000281484435tagore sunkara orcid0000000195369027references robinson bm malignant pleural mesothelioma an epidemiological perspective ann cardiothorac surg doi103978jissn2225319x20121104 hui m harshavardhana kr uppin sg pericardial mesothelioma presenting as chronic constrictive pericarditis a series of three cases from a single institution indian j pathol microbiol doi104103ijpmijpm_711_17figure hematoxylin and eosin stain of the retroperitoneal mass — malignant mesothelioma that is somewhat pleomorphic but mostly epithelioid polygonal cells with some spindling are seen cells have pleomorphic nuclei occasionally multinucleated with moderate amount of eosinophilic cytoplasm nuclei have a vesicular chromatin pattern with distinct nucleoli there is no obvious glandular or squamous differentiationmm carries a median survival time of to months following diagnosis7 this poor prognosis is partially due to the fact that over of cases are not discovered until the distant metastasis stage11 while most cases of epithelioid mm have an improved prognosis compared with sarcomatoid mm it can possess pleomorphic features that indicate highly aggressive cancer and shortened expected survival time71012 treatment consists of chemotherapy radiation and surgery although no therapy is curative7in our case ihc staining had an important role in developing the diagnosis of epithelioid mm with pleomorphic features it distinguished the disease from reactive mesothelial hyperplasia and other malignancies with possibly better prognoses10 the right lung lobe mass is not the typical presentation for mm involving the thorax and complicates the clinical picture the presentation of the retroperitoneal mass coupled with diffuse metastatic lesions and atypical involvement of the thorax make this patient™s presentation particularly unique calretinin gata3 and ck56 were positive in the tissue obtained from the retroperitoneal and paraspinal muscle masses which implicated a mesothelial origin for the malignancy calretinin is a strong indicator of epithelioid mm and is useful in distinguishing mm from adenocarcinoma10 recent reports estimate a positive staining for gata3 in mesothelioma in of cases and note a presence in epithelioid mm in one third of cases1013 another important ihc markers for mm is d240 which was negative in our patientwhile not completed in our patient other markers present in mm include the deletion of p16 occurring in of epithelioid mm and the presence of a bap1 mutation101214 0cuhlenhopp et al lev½ m boubl­kov¡ l b¼chler t Å¡imÅ¡a j treatment of malignant peritoneal mesothelioma klin onkol doi1014735amko2019333 sardar mr kuntz c patel t et al primary pericardial mesothelioma unique case and literature review tex heart inst j mutsaers se the mesothelial cell int j biochem cell biol napolitano a carbone m malignant mesothelioma time to translate trends cancer bibby ac tsim s kanellakis n et al malignant pleural mesothelioma an update on investigation diagnosis and treatment eur respir rev doi10118316000617 liang yf zheng gq chen yf song h yin wj zhang l ct differentiation of diffuse malignant peritoneal mesothelioma and peritoneal carcinomatosis j gastroenterol hepatol gill rr imaging of mesothelioma in a tannapfel ed malignant mesothelioma recent results in cancer research vol springer husain an colby tv ordonez ng et al guidelines for pathologic diagnosis of malignant mesothelioma update of the consensus statement from the international mesothelioma interest group arch pathol lab med howlader n noone am krapcho m et al seer cancer statistics review accessed february seercancergovcsr1975_2016 arif q husain an malignant mesothelioma diagnosis arch pathol lab med miettinen m mccue pa sarlomorikala m et al gata3 a multispecific but potentially useful marker in surgical pathology a systematic analysis of epithelial and nonepithelial tumors am j surg pathol attanoos rl churg a galateausalle f gibbs ar roggli vl malignant mesothelioma and its nonasbestos causes arch pathol lab med craighead je epidemiology of mesothelioma and historical background in a tannapfel ed malignant mesothelioma recent results in cancer research vol springer spirtas r heineman ef bernstein l et al malignant mesothelioma attributable risk of asbestos exposure occup environ med bray f ferlay j soerjomataram i siegel rl torre la jemal a global cancer statistics globocan estimates of incidence and mortality worldwide for cancers in countries ca cancer j clin doi103322caac21492 manzini vde p recchia l cafferata m et al malignant peritoneal mesothelioma a multicenter study on cases ann oncol doi101093annoncmdp shih ca ho sp tsay fw lai kh hsu pi diffuse malignant peritoneal mesothelioma kaohsiung j med sci doi101016jkjms201305003 0c' Answer:

Colon_Cancer

in a novel coronavirus sarscov2 was found to cause a highly contagious disease characterized by pneumonia the disease covid19 quickly spread around the globe escalating to a global pandemic in this review we discuss the virological immunological and imaging approaches harnessed for covid19 diagnosis and research covid19 shares many clinical characteristics with other respiratory illnessesaccurate and early detection of the infection is pivotal to controlling the outbreak as this enables case identification isolation and contact tracing we summarize the available literature on current laboratory and pointofcare diagnostics highlight their strengths and limitations and describe the emerging diagnostic approaches on the horizonwe also discuss the various research techniques that are being used to evaluate host immunity in laboratoryconfirmed patients additionally pathological imaging of tissue samples from affected patients has a critical role in guiding investigations on this disease conventional techniques such as immunohistochemistry and immunofluorescence have been frequently used to characterize the immune microenvironment in covid19 we also outline the emerging imaging techniques such as the rnascope which might also aid in our understanding of the significance of covid19specific biomarkers such as the angiotensinconverting enzyme ace2 cellular receptoroverall great progress has been made in covid19 research in a short period extensive global collation of our current knowledge of sarscov2 will provide insights into novel treatment modalities such as monoclonal antibodies and support the development of a sarscov2 vaccinekeywordscovid19 immunology pathology diagnostics specific t cellsintroductionin december a novel respiratory disease named coronavirus disease covid19 was detected by physicians in wuhan china the disease was found to be caused by the severe acute respiratory syndrome sars“cov2 rna virus12 within a matter of weeks covid19 had spread rapidly and escalated to a global pandemic at the time of writing june million cases had been reported and patients had succumbed to the disease worldwide3 indeed patients with covid19 are at high risk of developing a severe and critical disease4 therefore rapid and accurate diagnostic tests are urgently needed to effectively isolate identify and treat infected individuals and to contain the spread of the virus failure to do so will inevitably lead to spikes in cases and the resultant overcrowding and collapse of healthcare services5 moreover research into this novel virus is also critical to understand its pathogenesis and its interaction with the human immune system insights from such research will guide the design of public health policies and protocols to 1lee kong chian school of medicine nanyang technological university singapore singapore2yong loo lin school of medicine national university of singapore singapore singapore3institute of molecular cell biology imcb agency of science technology and research astar singapore singapore4department of anatomical pathology singapore general hospital singapore singaporethese authors contributed equallyreceived june and in revised form july accepted for publication july corresponding authorjoe poh sheng yeong institute of molecular cell biology imcb agency of science technology and research astar college road academia level diagnostics tower singapore singapore email yeongpsimcbastaredusg 0c slas technology identify susceptible individuals and diagnostic prognostic and treatment approaches for patientscurrent diagnostic approaches predominantly involve established virological procedures such as nucleic acid hybridization techniques reversetranscriptase pcr [rtpcr] and recombinase polymerase amplification rpa as well as immunologic approaches like antibody assays each approach boasts unique strengths and weaknesses for instance while rtpcr demonstrates high sensitivity and specificity its capabilities have been severely limited for practical reasons during this current pandemic due to global shortages of skilled personnel reagents and equipment and a processing time of up to days by contrast immunologic tests such as antibody assays are rapid and require minimal equipment but they have limited utility in the context of acute diagnosis of sarscov2 infections this is because it can take several days to weeks following symptom onset for a patient to mount a detectable antibody response6immunological tools in research include enzymelinked immunosorbent assays elisas flow cytometry and mass cytometry cytof imaging techniques for pathological analyses include conventional approaches such as hematoxylin“eosin he staining immunohistochemical ihc staining or transmission electron microscopy tem and rnascope each of these methods is used to examine the pathophysiology underlying covid19 from a different perspective each with their own advantages and disadvantages for example it has been established that the entry of sarscov2 intro cells depends on the binding of viral proteins with the human receptor angiotensinconverting enzyme ace2 receptors7 additionally evidence shows that the type ii transmembrane protease tmprss2 is also essential for viral entry by priming the viral spike protein for binding to ace28 therefore considerable research efforts employing different techniques have been directed at mapping the distribution of ace2 and tmprss2 in tissues and their relationship to the observed manifestations of disease together the combination of these approaches has advanced our understanding of covid19in this review we discuss the current approaches in covid19 diagnosis and research with a focus on findings from virological and pathological imaging methods we also discuss immunological methods which are increasingly recognized as an integral component of the disease processdiagnosticsthe most common symptoms of covid19 at initial presentation are nonspecific and include a high fever a new and persistent cough and fatigue910 due to similarities between the clinical characteristics of covid19 and many other respiratory illnesses the accurate and early detection of infection is pivotal for outbreak control any delays in diagnosis are increasingly measured in lives lostaccording to the world health anization who the immediate goal for research into covid19 diagnostics is the development of rna assays antibody and antigen assays and pointofcare detection11 the intermediateterm priority would be their integration into multiplex diagnostic platforms while the longterm goal would be the investigation of prognostic markersin this section we summarize the current and emerging diagnostic tools for sarscov2 through the lens of immunologylabbased testsrtpcr molecular testing the detection of viral nucleic acids by rtpcr is the primary method used to confirm a suspected case of covid19 rtpcr and other nucleic acid hybridization techniques are an integral part of virology and are applied in a broad range of settings including screening diagnosis informing medical and therapeutic decisions and assessing cure rates from therapy12 chinese officials released the genomic sequence of sarscov2 to public databases early in the course of the outbreak13 and the who has since published seven protocols for rtpcrbased diagnostics because of the high sensitivity and specificity of rtpcr it is regarded as the œgold standard for virus detection14 there are two essential steps in the process viral rna extraction and pcr amplification and probebased detection multiple largescale highthroughput instruments are available for automating both steps such as the roche cobas system which has an advertised throughput of tests per hours15however rtpcrbased testing is costly and timeconsuming requiring up to days using centralized laboratory equipment and skilled personnel furthermore global supply chain challenges have led to significant shortages of essential reagents lastly falsenegative results due to low sample volumes variable sampling techniques and sampling locations sample degradation during transportation andor improper nucleic acid extraction are a concern16“ in addition the differences in detectable viral material in different sampling locations eg nasopharyngeal vs bronchoalveolar lavage fluid [balf] vs rectal samples might also explain the falsepositive rtpcr results on repeat testing in œrecovered covid19 patients indeed one postmortem case study revealed residual virus in lung tissue despite consecutive negative results on pcr testing from nasopharyngeal swabs19 separately winichakoon et al outlined a case of repeatedly negative nasopharyngeal and oropharyngeal swabs in a clinically deteriorating patient where only a balf pcr test returned positive20given the high expression of ace2 on alveolar epithelial cells and negative expression on nasal oral and nasopharynx 0ctan et al cells21 it would be prudent to perform bronchoalveolar lavage on patients to rule out falsenegative results from swabs of upper respiratory tract samples20labbased immunological assays in contrast to rtpcr techniques that detect viral nucleic acids serological and immunological assays aim to detect antibodies against sarscov2 or antigenic proteins in infected individuals neutralization assays are considered the gold standard for assessing neutralizing protective antibodies22 however these assays require specialized biosafety level bsl3 facilities and still take several days to complete another type of labbased antibody assay is the traditional elisa which detects all binding antibodies the four principal types of elisa are direct indirect competitive and sandwich elisa the indirect elisa is the most common method for determining antibody concentrations elisas have good concordance with neutralization assays for the detection of antibody responses in sarscov223 unfortunately both methods require skilled operators and are limited by low throughput due to the absence of fully automated systemsserological diagnostics offer several advantages re quirements for specimen quality are comparatively less stringent than for nucleic acid tests as the antibodies are uniformly distributed in the serum24 consequently sampling location concerns do not apply here furthermore good correlation between igg elisas performed on both conventional serum samples and plasma samples have been reported25 of which the latter may be conveniently obtained from residual blood submitted for other routine laboratory testsone pitfall of antibody assays is their limited utility early in the course of any infection sparse data are available with regard to the antibody responses produced by patients with covid19 it seems that sarscov2 igm is detectable at a median of days after symptom onset while igg is detectable after days26 with the seroconversion rate approaching by day an italian research group noted that the performance of a commercial vivadiag covid19 igmigg test was very poor with a sensitivity of only and a negative predictive value of in a cohort of suspected covid19 patients in the emergency room setting27 as such we believe that for now rtpcr testing is likely more appropriate for diagnosing acute covid19notably a longitudinal study examining the iggigm profiles of patients found that seroconversion for igg and igm occurred in no specific chronological order with a median of days after symptom onset28 all patients achieved seroconversion by day consequently the detection of both igg and igm simultaneously rather than one antibody alone would be idealanother concern surrounding serologic diagnostics is the production of falsepositive results from crossreactivity due to the high prevalence of the four endemic human coronaviruses in the human population in sarscov2 the spike s protein which includes two regions s1 and s2 and the nucleocapsid n protein np are the major immunogens29 and therefore most diagnostics rely on the detection of antibodies specific for these antigens one work suggests that of the possible targets the s1 subunit antigen is more specific than either the whole s antigen or the n antigen for detecting sarscov2 antibodies with no crossreactivity to other coronaviruses except for sarscov23 given that only sarscov infections were recorded worldwide30 the risk of false positives from this crossreactivity is miniscule however np elisas are more sensitive than s1 in detecting antibodies in those with a mild infection23 importantly as in sarscov most of the neutralizing antibodies are directed against the s protein31 of which s1 contains a receptorbinding domain rbd responsible for making contact with ace2 to facilitate viral entry7 thus theoretically only diagnostics that detect s1specific antibodies are suitable to infer immunity to covid19 this fact is corroborated by evidence that antis rbd but not antinp igg levels correlated with neutralizing antibody titers in sera from a cohort of recovered patients32 the number of commercial antibody assays is growing detecting either antinp antibodies antis1s antibodies or both there is also large variation in their claimed sensitivities and specificities33 based on the available evidence an ideal serological assay would be a combined test that simultaneously detects both antibodies to np and s1 antigens assessment of antinp antibodies has good sensitivity and would be best suited for supporting the diagnosis of infection while the additional antis1 antibody assay would allow for the determination of immunityrapid testspointofcare rtpcr tests a small number of commercial pointofcare tests utilizing rtpcr have been developed these typically involve the same methodology as conventional rtpcr but implemented with automated and portable benchtopsized instruments that can be operated closer to patient care settings than a centralized laboratory a prominent example is cepheid™s xpert xpress sarscov2 run on the gene xpert platform this apparatus can provide a result within min others include the mesabiotech accula test and microsensdx rapiprep covid19 despite displaying good sensitivity and specificity these instruments are generally limited by a very low throughput of only one to four tests per run per machine34 and as such are only suited to small laboratories or clinics 0c slas technology figure loopmediated isothermal amplification lamp a lamp begins when the forward inner primer fip binds to the a2c region while the forward primer a1 binds to a1c which displaces the fip complementary strand b the backward inner primer bip binds b2c while the backward primer b3 binds b3c and displaces the bip complementary strand c a complementary sequence that initiates loop formation is produced d loop structures are formed that allow for lamp with the use of loop primersfigure crispr technique viral rna is converted to dsdna using rtrpa recombinase polymerase amplification a the cas12a nuclease enzyme is activated upon complex binding to the target sequence resulting in cleavage of the target sequence and the fluorescent rna reporter b t7 transcription converts dna to complementary rna cas13 nuclease enzyme activity is activated upon complex binding to the target sequence resulting in a similar cleavage of the target sequence and the fluorescent rna reporterimmunological assaysrapid antibody assays compared with labbased antibody assays rapid assays such as lateral flow immunoassays lfias fig and chemiluminescent immunoassays clias fig offer the benefits of rapid diagnostic testing at a low cost these assays do not require specialized equipment or expertise35 and are thus excellent candidates for pointofcare testing or deployment on a large scale this an area of intense interest with governments worldwide aiming to order millions of tests to inform policy makers about attack rates in their populations36 lfias are predominantly singleuse kits designed for pointofcare use while clias are fully automated analyzers that permit very high testing throughputunfortunately these tests do not quantify the antibody titers and the performance of lfias has been called into question one evaluation of nine commercial lfias reported a sensitivity ranging from only to versus rtpcr and to versus elisa37 meanwhile the performance of clias is superior with good sensitivity and specificity levels similar to those of elisa38 otherwise these tests share the same advantages and drawbacks as the 0ctan et al table summary of diagnostic approaches for covid19categorytype of testtypical test result timecharacteristicsexamplesvirologicmolecular rtpcrdaysgold standard high sensitivity who rtpcr protocolstests pointofcare rtpcr“ minlamp crispr himmunologic testslfia for antibodies“ minantigensand specificity high throughput but lab basedrapid good sensitivity and specificity pointofcare testing but low throughputrapid good sensitivity and specificity pointofcare testing but low throughputrapid pointofcare testing but not quantitative poor sensitivitycepheid xpert xpress sarscov2sherlock biosciences sherlockvivadiag covid19 igmigg rapid testcorisbio covid19 ag respistripepitope diagnostics kt1033 edi novel coronavirus covid19 elisa kitroche elecsys antisarscov2 traditional elisa“ hgood sensitivity and specificity but lab based not automatedclia minrapid good sensitivity and specificity high throughput but lab basedneutralization assaydaysgold standard high sensitivity not commercially and specificity able to quantify neutralizing antibodies but requires bsl3 lab facilityavailablelabbased antibody assays discussed above the characteristics and unique advantages and disadvantages of these different methodologies are outlined in table antigen assays an alternative approach to immunological assays is to directly detect sarscov2 viral antigens several commercial pointofcare antigen tests are available but their performance remains to be evaluated these tests may be suitable for making an early diagnosis and are deployable as pointofcare assays however they face the same sampling limitations as rtpcr and are hypothetically hampered by limited sensitivity due to the omission of an amplification process unlike nucleic acid testing for example one multicenter study evaluating the corisbio covid19 ag respistrip a lateral flow assay for the sarscov2 np reported a test sensitivity of only rapid nonpcr molecular testing nucleic acid testing using nonpcr methods is an emerging approach for rapid diagnostics and several assays have received food and drug administration fda emergency use authorization which facilitates the distribution of unapproved medical products or the offlabel use of approved medical products when certain criteria are met these methods share high sensitivity and specificity on par with rtpcr but with the principal advantages of more rapid testing at a lower cost40“lamp fig is one such novel isothermal nucleic acid amplification method that does not require a thermal cycler one example is the id now covid19 test from abbott diagnostics which can deliver results in just min43 and uses a lightweight portable instrument allowing onsite testing of swab samples however it has a limited throughput of only one sample per runthe crispr enzymes cas12 and cas13 have also been adapted for rapid nucleic acid sensing fig the detectr assay by mammoth biosciences45 as well as the sherlock assay by sherlock biosciences46 potentially offers sensitivity and specificity comparable to those of rtpcr but can be completed in h however these approaches are still in the early stages of commercialization and current applications are available only as test kits to be run in labs while pointofcare versions exist as proofofconcept demonstrations47 nonetheless their inherent characteristics hold great potential for diagnosis in the futureprognostication of diseaseprofiling of genetic susceptibility work is in progress to ascertain the possible genetic basis for the apparent variations in covid19 susceptibility and disease severity cao et al compared expression quantitative trait loci eqtl for ace2 in different populations finding significantly greater eqtl variants associated with higher ace2 expression in 0c slas technology figure lateral flow immunoassay lfia a serum sample deposited on the sample pad b antisarscov2 antibodies in the sample will bind to the target antigen with a labeled tag c immobilized antihuman igm antibodies will capture the sarscov2 antibody“antigen complex d control antibodies are captured by immobilized antibodies in the control lineserum prognostic markers another application of immunological methods would be to measure markers that enable prognostication in covid19 higher titers of antibodies against sarscov2 have been associated with more severe disease2350 similar to previous studies in middle east respiratory syndrome mers“cov51 elisa has been used to provide a quantitative measurement of serum and plasma igm and igg antibodies by monitoring the kinetics of igm and igg antibodies specific to the n and s proteins on sarscov2 it was found that intensive care unit icu patients had a significantly lower level of sigg within weeks of symptom onset but a higher level of nigg antibodies compared with nonicu patients52 this finding highlights the possible utility of sigg and nigg as a prognostic tool for covid19 patientsthe ddimer level which consists of crosslinked fibrin degradation products that reflect ongoing blood clot formation and breakdown activity in the body is another proposed prognostic marker modern commercial assays for ddimers are based on monoclonal antibodies employing either elisa or microlatex agglutination assays53 reports have emerged that elevated ddimer levels suggestive of a hypercoagulable state are associated with drastically worse outcomes a chinese group reported that ddimer levels of ‰¥ µgml on admission were associated with a times increased mortality relative to ddimer levels of µgml in a cohort of covid19 patients54 this finding of ddimer levels as a negative prognostic marker was also noted in other studies conducted in china455 and the netherlands56similarly interleukin il6 a key component of the cytokine release syndrome is another marker measured by elisa and has been described to independently predict adverse outcomes in covid195758 tumor necrosis factor alpha tnfα another important proinflammatory cytokine has also been found to be strongly correlated with figure chemiluminescence enzyme immunoassay clia sarscov2 antigens will capture igm and igg antibodies from the sample serum secondary antibodies that are conjugated with horseradish peroxidase hrp bind to the captured primary igm and igg antibodies and react with a chemiluminescent substrate to generate a strong chemiluminescent signal that is measured in terms of relative light units rlueast asian populations but reported no direct evidence supporting the existence of s proteinbindingresistant ace2 mutants48 out of identified protein altering variants separately stawiski et al analyzed ace2 polymorphisms within a much larger population dataset spanning more than population groups across the world and performed structural predictions to identify variants that might confer protection or rather increase susceptibility to sarscov2 s protein binding49 out of a total of identified proteinaltering ace2 variants variants were predicted to increase susceptibility while variants were speculated to confer protection however the degree of changes in receptor“virus binding interactions for each structural variant was not quantified these findings represent significant developments in our understanding of population risk profiles for covid19 and future coronavirus infections 0ctan et al endan damage and mortality even after adjusting for disease severity scores59 gao et al examined both il6 and ddimer levels they proposed a panel comprising tandem testing of these two markers which produced a sensitivity of and specificity of in early prediction of severe covid1958 elevated troponin levels a marker of myocardial injury measured with elisa immunoassays also strongly predict progression to death in patients with severe illness60 these results suggest that multiplex cytokine and serum marker profiling will be a powerful tool in stratifying patients that may guide clinical decisions and resource allocationsummary in sum rapid progress has been made in diagnostics for covid19 yet the race against time continues for researchers and biotechnology firms to develop rapid costeffective and reliable test kits that can be deployed on a large scale at the time of writing labbased rtpcr testing has been the dominant diagnostic approach but alternative molecular approaches like isothermal amplification and crispr which have clear advantages are on the horizon immunological tests such as clia and lfia will become increasingly important because of the urgent need for pointofcare diagnostics for mass testing of infected asymptomatic individuals and their close contacts and will be valuable in complementing molecular approaches for confirming infection furthermore immunological assays will be in great demand by policy makers worldwide for the assessment of immunity to covid19 however the performance of these serological tests varies significantly particularly their degree of sensitivity and specificity we believe that caution must be taken in the interpretation of these tests detailed evaluation of the reliability of serological tests will be a key area for future research lastly given the importance of techniques like elisa in prognosticating covid19 immunological methods will undoubtedly occupy a crucial role in achieving all levels of the who™s short medium and longterm diagnostic goalscovid19 research toolsimmunological approachescovid19 infection has a poor prognosis in individuals with comorbidities and abnormal immune functions although research surrounding covid19 is still in its infancy several studies have revealed lymphopenia and the cytokine storm as underlying mechanisms correlating to disease progression here we discuss the various immunological techniques involved in assessing host immunity in covid19 patientselisa as discussed elisa has also been used to detect the inflammatory cytokines implicated in the cytokine storm seen in patients with severe respiratory failure due to covid19 one study found that the immune dysregulation in patients with severe respiratory failure was due to a significantly increased production of il6 and defective lymphoid function because of an il6mediated decrease in hladr expression on cd14 monocytes interestingly interferongamma ifnγ levels were below the detection level in these patients suggesting that t helper th cells are unlikely to be major players in the overinflammatory response of severe patients61 a similar observation was made in a separate study whereby inflammatory cytokines that mediate major immune responses such as tnfα and il1β were not significantly elevated in icu patients62 these findings demonstrate that the immunophenotype of patients with covid19 can vary depending on presently unclear host immune factors and the severity of their condition this relationship between disease severity and cytokine storm has also been highlighted in other studies that found a significantly elevated plasma concentration of granulocyte colonystimulating factor gcsf ip10 ccl2 and ccl3 in icu patients compared with nonicu patients63elisa is also being used as a companion diagnostic tool for therapeutic purposes in a study that explored the use of convalescent plasma therapy from donors as a form of treatment in severe covid19 elisa was used to assess the neutralizing activity of the rbdspecific igm and igg antibodies found in the donor convalescent plasma64 after the transfusion was complete elisa was also used to detect igg igm and neutralizing antibody titers in the sera of patients to assess the response to treatment65enzymelinked immunosorbent spot enzymelinked immunosorbent spot elispot is a sensitive immunoassay that quantitatively measures cytokinesecreting cells at the singlecell level providing insight into immunerelated cellular activities66 hence it is a promising tool for characterizing specific tcell immunity in covid19 patients by ifnγ elispot analysis it was revealed that convalescent covid19 patients had significantly increased levels of ifnγsecreting t cells when compared with healthy donors a significant correlation between neutralizing antibody titers and npspecific t cells was identified in these patients suggesting that a combination of humoral and cellular immunity is integral to clearing sarscov2 interestingly it was noted that in convalescent patients weeks ifnγsecreting tcell numbers had postdischarge decreased suggesting that they may not be maintained for a prolonged period of time even in recovered patients67elispot is also serving a vital role in vaccine development through the detection of potential tcell epitopes in the s protein rbd of sarscov268 one study was able to harness elispot assays to identify three tcell epitopes that induced a strong adaptive immune response 0c slas technology postimmunization demonstrating the promise of elispot assays in the area of vaccine development32 recently elispot has also been applied to assess the immunogenicity of newly developed vaccines one such study successfully utilized an ifnγ elispot assay to evaluate tcell responses to a new sarscov2 vaccine in murine splenocytes and rhesus macaque peripheral blood mononuclear cells pbmcs the promising findings from this animal study informed the start of a phase i clinical trial with the same vaccine highlighting the usefulness of elispot in assessing immune responses to new vaccines and promoting vaccine development69flow cytometry unlike elisa and elispot flow cytometry determines the number of cytokinesecreting cells and has the capacity to immunophenotype based on surface and intracellular markers70 in relation to the current pandemic this technique enables the detection sorting and analysis of multiple subpopulations of immune cells specific to covid using flow cytometry researchers detected a cytotoxic immune environment in patient blood samples despite a reduction in the overall lymphocyte population71“ as part of the sarscov2 antiviral response peripheral lymphocytes retain the capacity to activate and differentiate into subpopulations such as antibodysecreting cells cd3“cd19cd27hicd38hi follicular t cells cd4cxcr5icospd1 cd4 th cells cd38hladrcd4 cytotoxic t tc cells cd38hladrcd8 and regulatory t treg cells cd3cd4cd25cd127“ these tc cells harbor large amounts of cytotoxic granules while cd4 th cells skewed toward a proinflammatory th1 and th17 phenotype727375 the overall hyperinflammation and cytotoxic environment supports the notion that a cytokine storm could be liable for the multisystemic insults in patients with severe covid19elicitation of antiviral tcell responses specific to sarscov2 is of utmost importance to establishing viral control many studies have demonstrated robust antiviral responses however there is no known set of markers reported to identify sarscov2specific t cells collectively most groups have characterized sarscov2specific t cells based on hladr cd38 cd69 cd25 cd44 and ki67 expression th

cancer289

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: in a novel coronavirus sarscov2 was found to cause a highly contagious disease characterized by pneumonia the disease covid19 quickly spread around the globe escalating to a global pandemic in this review we discuss the virological immunological and imaging approaches harnessed for covid19 diagnosis and research covid19 shares many clinical characteristics with other respiratory illnessesaccurate and early detection of the infection is pivotal to controlling the outbreak as this enables case identification isolation and contact tracing we summarize the available literature on current laboratory and pointofcare diagnostics highlight their strengths and limitations and describe the emerging diagnostic approaches on the horizonwe also discuss the various research techniques that are being used to evaluate host immunity in laboratoryconfirmed patients additionally pathological imaging of tissue samples from affected patients has a critical role in guiding investigations on this disease conventional techniques such as immunohistochemistry and immunofluorescence have been frequently used to characterize the immune microenvironment in covid19 we also outline the emerging imaging techniques such as the rnascope which might also aid in our understanding of the significance of covid19specific biomarkers such as the angiotensinconverting enzyme ace2 cellular receptoroverall great progress has been made in covid19 research in a short period extensive global collation of our current knowledge of sarscov2 will provide insights into novel treatment modalities such as monoclonal antibodies and support the development of a sarscov2 vaccinekeywordscovid19 immunology pathology diagnostics specific t cellsintroductionin december a novel respiratory disease named coronavirus disease covid19 was detected by physicians in wuhan china the disease was found to be caused by the severe acute respiratory syndrome sars“cov2 rna virus12 within a matter of weeks covid19 had spread rapidly and escalated to a global pandemic at the time of writing june million cases had been reported and patients had succumbed to the disease worldwide3 indeed patients with covid19 are at high risk of developing a severe and critical disease4 therefore rapid and accurate diagnostic tests are urgently needed to effectively isolate identify and treat infected individuals and to contain the spread of the virus failure to do so will inevitably lead to spikes in cases and the resultant overcrowding and collapse of healthcare services5 moreover research into this novel virus is also critical to understand its pathogenesis and its interaction with the human immune system insights from such research will guide the design of public health policies and protocols to 1lee kong chian school of medicine nanyang technological university singapore singapore2yong loo lin school of medicine national university of singapore singapore singapore3institute of molecular cell biology imcb agency of science technology and research astar singapore singapore4department of anatomical pathology singapore general hospital singapore singaporethese authors contributed equallyreceived june and in revised form july accepted for publication july corresponding authorjoe poh sheng yeong institute of molecular cell biology imcb agency of science technology and research astar college road academia level diagnostics tower singapore singapore email yeongpsimcbastaredusg 0c slas technology identify susceptible individuals and diagnostic prognostic and treatment approaches for patientscurrent diagnostic approaches predominantly involve established virological procedures such as nucleic acid hybridization techniques reversetranscriptase pcr [rtpcr] and recombinase polymerase amplification rpa as well as immunologic approaches like antibody assays each approach boasts unique strengths and weaknesses for instance while rtpcr demonstrates high sensitivity and specificity its capabilities have been severely limited for practical reasons during this current pandemic due to global shortages of skilled personnel reagents and equipment and a processing time of up to days by contrast immunologic tests such as antibody assays are rapid and require minimal equipment but they have limited utility in the context of acute diagnosis of sarscov2 infections this is because it can take several days to weeks following symptom onset for a patient to mount a detectable antibody response6immunological tools in research include enzymelinked immunosorbent assays elisas flow cytometry and mass cytometry cytof imaging techniques for pathological analyses include conventional approaches such as hematoxylin“eosin he staining immunohistochemical ihc staining or transmission electron microscopy tem and rnascope each of these methods is used to examine the pathophysiology underlying covid19 from a different perspective each with their own advantages and disadvantages for example it has been established that the entry of sarscov2 intro cells depends on the binding of viral proteins with the human receptor angiotensinconverting enzyme ace2 receptors7 additionally evidence shows that the type ii transmembrane protease tmprss2 is also essential for viral entry by priming the viral spike protein for binding to ace28 therefore considerable research efforts employing different techniques have been directed at mapping the distribution of ace2 and tmprss2 in tissues and their relationship to the observed manifestations of disease together the combination of these approaches has advanced our understanding of covid19in this review we discuss the current approaches in covid19 diagnosis and research with a focus on findings from virological and pathological imaging methods we also discuss immunological methods which are increasingly recognized as an integral component of the disease processdiagnosticsthe most common symptoms of covid19 at initial presentation are nonspecific and include a high fever a new and persistent cough and fatigue910 due to similarities between the clinical characteristics of covid19 and many other respiratory illnesses the accurate and early detection of infection is pivotal for outbreak control any delays in diagnosis are increasingly measured in lives lostaccording to the world health anization who the immediate goal for research into covid19 diagnostics is the development of rna assays antibody and antigen assays and pointofcare detection11 the intermediateterm priority would be their integration into multiplex diagnostic platforms while the longterm goal would be the investigation of prognostic markersin this section we summarize the current and emerging diagnostic tools for sarscov2 through the lens of immunologylabbased testsrtpcr molecular testing the detection of viral nucleic acids by rtpcr is the primary method used to confirm a suspected case of covid19 rtpcr and other nucleic acid hybridization techniques are an integral part of virology and are applied in a broad range of settings including screening diagnosis informing medical and therapeutic decisions and assessing cure rates from therapy12 chinese officials released the genomic sequence of sarscov2 to public databases early in the course of the outbreak13 and the who has since published seven protocols for rtpcrbased diagnostics because of the high sensitivity and specificity of rtpcr it is regarded as the œgold standard for virus detection14 there are two essential steps in the process viral rna extraction and pcr amplification and probebased detection multiple largescale highthroughput instruments are available for automating both steps such as the roche cobas system which has an advertised throughput of tests per hours15however rtpcrbased testing is costly and timeconsuming requiring up to days using centralized laboratory equipment and skilled personnel furthermore global supply chain challenges have led to significant shortages of essential reagents lastly falsenegative results due to low sample volumes variable sampling techniques and sampling locations sample degradation during transportation andor improper nucleic acid extraction are a concern16“ in addition the differences in detectable viral material in different sampling locations eg nasopharyngeal vs bronchoalveolar lavage fluid [balf] vs rectal samples might also explain the falsepositive rtpcr results on repeat testing in œrecovered covid19 patients indeed one postmortem case study revealed residual virus in lung tissue despite consecutive negative results on pcr testing from nasopharyngeal swabs19 separately winichakoon et al outlined a case of repeatedly negative nasopharyngeal and oropharyngeal swabs in a clinically deteriorating patient where only a balf pcr test returned positive20given the high expression of ace2 on alveolar epithelial cells and negative expression on nasal oral and nasopharynx 0ctan et al cells21 it would be prudent to perform bronchoalveolar lavage on patients to rule out falsenegative results from swabs of upper respiratory tract samples20labbased immunological assays in contrast to rtpcr techniques that detect viral nucleic acids serological and immunological assays aim to detect antibodies against sarscov2 or antigenic proteins in infected individuals neutralization assays are considered the gold standard for assessing neutralizing protective antibodies22 however these assays require specialized biosafety level bsl3 facilities and still take several days to complete another type of labbased antibody assay is the traditional elisa which detects all binding antibodies the four principal types of elisa are direct indirect competitive and sandwich elisa the indirect elisa is the most common method for determining antibody concentrations elisas have good concordance with neutralization assays for the detection of antibody responses in sarscov223 unfortunately both methods require skilled operators and are limited by low throughput due to the absence of fully automated systemsserological diagnostics offer several advantages re quirements for specimen quality are comparatively less stringent than for nucleic acid tests as the antibodies are uniformly distributed in the serum24 consequently sampling location concerns do not apply here furthermore good correlation between igg elisas performed on both conventional serum samples and plasma samples have been reported25 of which the latter may be conveniently obtained from residual blood submitted for other routine laboratory testsone pitfall of antibody assays is their limited utility early in the course of any infection sparse data are available with regard to the antibody responses produced by patients with covid19 it seems that sarscov2 igm is detectable at a median of days after symptom onset while igg is detectable after days26 with the seroconversion rate approaching by day an italian research group noted that the performance of a commercial vivadiag covid19 igmigg test was very poor with a sensitivity of only and a negative predictive value of in a cohort of suspected covid19 patients in the emergency room setting27 as such we believe that for now rtpcr testing is likely more appropriate for diagnosing acute covid19notably a longitudinal study examining the iggigm profiles of patients found that seroconversion for igg and igm occurred in no specific chronological order with a median of days after symptom onset28 all patients achieved seroconversion by day consequently the detection of both igg and igm simultaneously rather than one antibody alone would be idealanother concern surrounding serologic diagnostics is the production of falsepositive results from crossreactivity due to the high prevalence of the four endemic human coronaviruses in the human population in sarscov2 the spike s protein which includes two regions s1 and s2 and the nucleocapsid n protein np are the major immunogens29 and therefore most diagnostics rely on the detection of antibodies specific for these antigens one work suggests that of the possible targets the s1 subunit antigen is more specific than either the whole s antigen or the n antigen for detecting sarscov2 antibodies with no crossreactivity to other coronaviruses except for sarscov23 given that only sarscov infections were recorded worldwide30 the risk of false positives from this crossreactivity is miniscule however np elisas are more sensitive than s1 in detecting antibodies in those with a mild infection23 importantly as in sarscov most of the neutralizing antibodies are directed against the s protein31 of which s1 contains a receptorbinding domain rbd responsible for making contact with ace2 to facilitate viral entry7 thus theoretically only diagnostics that detect s1specific antibodies are suitable to infer immunity to covid19 this fact is corroborated by evidence that antis rbd but not antinp igg levels correlated with neutralizing antibody titers in sera from a cohort of recovered patients32 the number of commercial antibody assays is growing detecting either antinp antibodies antis1s antibodies or both there is also large variation in their claimed sensitivities and specificities33 based on the available evidence an ideal serological assay would be a combined test that simultaneously detects both antibodies to np and s1 antigens assessment of antinp antibodies has good sensitivity and would be best suited for supporting the diagnosis of infection while the additional antis1 antibody assay would allow for the determination of immunityrapid testspointofcare rtpcr tests a small number of commercial pointofcare tests utilizing rtpcr have been developed these typically involve the same methodology as conventional rtpcr but implemented with automated and portable benchtopsized instruments that can be operated closer to patient care settings than a centralized laboratory a prominent example is cepheid™s xpert xpress sarscov2 run on the gene xpert platform this apparatus can provide a result within min others include the mesabiotech accula test and microsensdx rapiprep covid19 despite displaying good sensitivity and specificity these instruments are generally limited by a very low throughput of only one to four tests per run per machine34 and as such are only suited to small laboratories or clinics 0c slas technology figure loopmediated isothermal amplification lamp a lamp begins when the forward inner primer fip binds to the a2c region while the forward primer a1 binds to a1c which displaces the fip complementary strand b the backward inner primer bip binds b2c while the backward primer b3 binds b3c and displaces the bip complementary strand c a complementary sequence that initiates loop formation is produced d loop structures are formed that allow for lamp with the use of loop primersfigure crispr technique viral rna is converted to dsdna using rtrpa recombinase polymerase amplification a the cas12a nuclease enzyme is activated upon complex binding to the target sequence resulting in cleavage of the target sequence and the fluorescent rna reporter b t7 transcription converts dna to complementary rna cas13 nuclease enzyme activity is activated upon complex binding to the target sequence resulting in a similar cleavage of the target sequence and the fluorescent rna reporterimmunological assaysrapid antibody assays compared with labbased antibody assays rapid assays such as lateral flow immunoassays lfias fig and chemiluminescent immunoassays clias fig offer the benefits of rapid diagnostic testing at a low cost these assays do not require specialized equipment or expertise35 and are thus excellent candidates for pointofcare testing or deployment on a large scale this an area of intense interest with governments worldwide aiming to order millions of tests to inform policy makers about attack rates in their populations36 lfias are predominantly singleuse kits designed for pointofcare use while clias are fully automated analyzers that permit very high testing throughputunfortunately these tests do not quantify the antibody titers and the performance of lfias has been called into question one evaluation of nine commercial lfias reported a sensitivity ranging from only to versus rtpcr and to versus elisa37 meanwhile the performance of clias is superior with good sensitivity and specificity levels similar to those of elisa38 otherwise these tests share the same advantages and drawbacks as the 0ctan et al table summary of diagnostic approaches for covid19categorytype of testtypical test result timecharacteristicsexamplesvirologicmolecular rtpcrdaysgold standard high sensitivity who rtpcr protocolstests pointofcare rtpcr“ minlamp crispr himmunologic testslfia for antibodies“ minantigensand specificity high throughput but lab basedrapid good sensitivity and specificity pointofcare testing but low throughputrapid good sensitivity and specificity pointofcare testing but low throughputrapid pointofcare testing but not quantitative poor sensitivitycepheid xpert xpress sarscov2sherlock biosciences sherlockvivadiag covid19 igmigg rapid testcorisbio covid19 ag respistripepitope diagnostics kt1033 edi novel coronavirus covid19 elisa kitroche elecsys antisarscov2 traditional elisa“ hgood sensitivity and specificity but lab based not automatedclia minrapid good sensitivity and specificity high throughput but lab basedneutralization assaydaysgold standard high sensitivity not commercially and specificity able to quantify neutralizing antibodies but requires bsl3 lab facilityavailablelabbased antibody assays discussed above the characteristics and unique advantages and disadvantages of these different methodologies are outlined in table antigen assays an alternative approach to immunological assays is to directly detect sarscov2 viral antigens several commercial pointofcare antigen tests are available but their performance remains to be evaluated these tests may be suitable for making an early diagnosis and are deployable as pointofcare assays however they face the same sampling limitations as rtpcr and are hypothetically hampered by limited sensitivity due to the omission of an amplification process unlike nucleic acid testing for example one multicenter study evaluating the corisbio covid19 ag respistrip a lateral flow assay for the sarscov2 np reported a test sensitivity of only rapid nonpcr molecular testing nucleic acid testing using nonpcr methods is an emerging approach for rapid diagnostics and several assays have received food and drug administration fda emergency use authorization which facilitates the distribution of unapproved medical products or the offlabel use of approved medical products when certain criteria are met these methods share high sensitivity and specificity on par with rtpcr but with the principal advantages of more rapid testing at a lower cost40“lamp fig is one such novel isothermal nucleic acid amplification method that does not require a thermal cycler one example is the id now covid19 test from abbott diagnostics which can deliver results in just min43 and uses a lightweight portable instrument allowing onsite testing of swab samples however it has a limited throughput of only one sample per runthe crispr enzymes cas12 and cas13 have also been adapted for rapid nucleic acid sensing fig the detectr assay by mammoth biosciences45 as well as the sherlock assay by sherlock biosciences46 potentially offers sensitivity and specificity comparable to those of rtpcr but can be completed in h however these approaches are still in the early stages of commercialization and current applications are available only as test kits to be run in labs while pointofcare versions exist as proofofconcept demonstrations47 nonetheless their inherent characteristics hold great potential for diagnosis in the futureprognostication of diseaseprofiling of genetic susceptibility work is in progress to ascertain the possible genetic basis for the apparent variations in covid19 susceptibility and disease severity cao et al compared expression quantitative trait loci eqtl for ace2 in different populations finding significantly greater eqtl variants associated with higher ace2 expression in 0c slas technology figure lateral flow immunoassay lfia a serum sample deposited on the sample pad b antisarscov2 antibodies in the sample will bind to the target antigen with a labeled tag c immobilized antihuman igm antibodies will capture the sarscov2 antibody“antigen complex d control antibodies are captured by immobilized antibodies in the control lineserum prognostic markers another application of immunological methods would be to measure markers that enable prognostication in covid19 higher titers of antibodies against sarscov2 have been associated with more severe disease2350 similar to previous studies in middle east respiratory syndrome mers“cov51 elisa has been used to provide a quantitative measurement of serum and plasma igm and igg antibodies by monitoring the kinetics of igm and igg antibodies specific to the n and s proteins on sarscov2 it was found that intensive care unit icu patients had a significantly lower level of sigg within weeks of symptom onset but a higher level of nigg antibodies compared with nonicu patients52 this finding highlights the possible utility of sigg and nigg as a prognostic tool for covid19 patientsthe ddimer level which consists of crosslinked fibrin degradation products that reflect ongoing blood clot formation and breakdown activity in the body is another proposed prognostic marker modern commercial assays for ddimers are based on monoclonal antibodies employing either elisa or microlatex agglutination assays53 reports have emerged that elevated ddimer levels suggestive of a hypercoagulable state are associated with drastically worse outcomes a chinese group reported that ddimer levels of ‰¥ µgml on admission were associated with a times increased mortality relative to ddimer levels of µgml in a cohort of covid19 patients54 this finding of ddimer levels as a negative prognostic marker was also noted in other studies conducted in china455 and the netherlands56similarly interleukin il6 a key component of the cytokine release syndrome is another marker measured by elisa and has been described to independently predict adverse outcomes in covid195758 tumor necrosis factor alpha tnfα another important proinflammatory cytokine has also been found to be strongly correlated with figure chemiluminescence enzyme immunoassay clia sarscov2 antigens will capture igm and igg antibodies from the sample serum secondary antibodies that are conjugated with horseradish peroxidase hrp bind to the captured primary igm and igg antibodies and react with a chemiluminescent substrate to generate a strong chemiluminescent signal that is measured in terms of relative light units rlueast asian populations but reported no direct evidence supporting the existence of s proteinbindingresistant ace2 mutants48 out of identified protein altering variants separately stawiski et al analyzed ace2 polymorphisms within a much larger population dataset spanning more than population groups across the world and performed structural predictions to identify variants that might confer protection or rather increase susceptibility to sarscov2 s protein binding49 out of a total of identified proteinaltering ace2 variants variants were predicted to increase susceptibility while variants were speculated to confer protection however the degree of changes in receptor“virus binding interactions for each structural variant was not quantified these findings represent significant developments in our understanding of population risk profiles for covid19 and future coronavirus infections 0ctan et al endan damage and mortality even after adjusting for disease severity scores59 gao et al examined both il6 and ddimer levels they proposed a panel comprising tandem testing of these two markers which produced a sensitivity of and specificity of in early prediction of severe covid1958 elevated troponin levels a marker of myocardial injury measured with elisa immunoassays also strongly predict progression to death in patients with severe illness60 these results suggest that multiplex cytokine and serum marker profiling will be a powerful tool in stratifying patients that may guide clinical decisions and resource allocationsummary in sum rapid progress has been made in diagnostics for covid19 yet the race against time continues for researchers and biotechnology firms to develop rapid costeffective and reliable test kits that can be deployed on a large scale at the time of writing labbased rtpcr testing has been the dominant diagnostic approach but alternative molecular approaches like isothermal amplification and crispr which have clear advantages are on the horizon immunological tests such as clia and lfia will become increasingly important because of the urgent need for pointofcare diagnostics for mass testing of infected asymptomatic individuals and their close contacts and will be valuable in complementing molecular approaches for confirming infection furthermore immunological assays will be in great demand by policy makers worldwide for the assessment of immunity to covid19 however the performance of these serological tests varies significantly particularly their degree of sensitivity and specificity we believe that caution must be taken in the interpretation of these tests detailed evaluation of the reliability of serological tests will be a key area for future research lastly given the importance of techniques like elisa in prognosticating covid19 immunological methods will undoubtedly occupy a crucial role in achieving all levels of the who™s short medium and longterm diagnostic goalscovid19 research toolsimmunological approachescovid19 infection has a poor prognosis in individuals with comorbidities and abnormal immune functions although research surrounding covid19 is still in its infancy several studies have revealed lymphopenia and the cytokine storm as underlying mechanisms correlating to disease progression here we discuss the various immunological techniques involved in assessing host immunity in covid19 patientselisa as discussed elisa has also been used to detect the inflammatory cytokines implicated in the cytokine storm seen in patients with severe respiratory failure due to covid19 one study found that the immune dysregulation in patients with severe respiratory failure was due to a significantly increased production of il6 and defective lymphoid function because of an il6mediated decrease in hladr expression on cd14 monocytes interestingly interferongamma ifnγ levels were below the detection level in these patients suggesting that t helper th cells are unlikely to be major players in the overinflammatory response of severe patients61 a similar observation was made in a separate study whereby inflammatory cytokines that mediate major immune responses such as tnfα and il1β were not significantly elevated in icu patients62 these findings demonstrate that the immunophenotype of patients with covid19 can vary depending on presently unclear host immune factors and the severity of their condition this relationship between disease severity and cytokine storm has also been highlighted in other studies that found a significantly elevated plasma concentration of granulocyte colonystimulating factor gcsf ip10 ccl2 and ccl3 in icu patients compared with nonicu patients63elisa is also being used as a companion diagnostic tool for therapeutic purposes in a study that explored the use of convalescent plasma therapy from donors as a form of treatment in severe covid19 elisa was used to assess the neutralizing activity of the rbdspecific igm and igg antibodies found in the donor convalescent plasma64 after the transfusion was complete elisa was also used to detect igg igm and neutralizing antibody titers in the sera of patients to assess the response to treatment65enzymelinked immunosorbent spot enzymelinked immunosorbent spot elispot is a sensitive immunoassay that quantitatively measures cytokinesecreting cells at the singlecell level providing insight into immunerelated cellular activities66 hence it is a promising tool for characterizing specific tcell immunity in covid19 patients by ifnγ elispot analysis it was revealed that convalescent covid19 patients had significantly increased levels of ifnγsecreting t cells when compared with healthy donors a significant correlation between neutralizing antibody titers and npspecific t cells was identified in these patients suggesting that a combination of humoral and cellular immunity is integral to clearing sarscov2 interestingly it was noted that in convalescent patients weeks ifnγsecreting tcell numbers had postdischarge decreased suggesting that they may not be maintained for a prolonged period of time even in recovered patients67elispot is also serving a vital role in vaccine development through the detection of potential tcell epitopes in the s protein rbd of sarscov268 one study was able to harness elispot assays to identify three tcell epitopes that induced a strong adaptive immune response 0c slas technology postimmunization demonstrating the promise of elispot assays in the area of vaccine development32 recently elispot has also been applied to assess the immunogenicity of newly developed vaccines one such study successfully utilized an ifnγ elispot assay to evaluate tcell responses to a new sarscov2 vaccine in murine splenocytes and rhesus macaque peripheral blood mononuclear cells pbmcs the promising findings from this animal study informed the start of a phase i clinical trial with the same vaccine highlighting the usefulness of elispot in assessing immune responses to new vaccines and promoting vaccine development69flow cytometry unlike elisa and elispot flow cytometry determines the number of cytokinesecreting cells and has the capacity to immunophenotype based on surface and intracellular markers70 in relation to the current pandemic this technique enables the detection sorting and analysis of multiple subpopulations of immune cells specific to covid using flow cytometry researchers detected a cytotoxic immune environment in patient blood samples despite a reduction in the overall lymphocyte population71“ as part of the sarscov2 antiviral response peripheral lymphocytes retain the capacity to activate and differentiate into subpopulations such as antibodysecreting cells cd3“cd19cd27hicd38hi follicular t cells cd4cxcr5icospd1 cd4 th cells cd38hladrcd4 cytotoxic t tc cells cd38hladrcd8 and regulatory t treg cells cd3cd4cd25cd127“ these tc cells harbor large amounts of cytotoxic granules while cd4 th cells skewed toward a proinflammatory th1 and th17 phenotype727375 the overall hyperinflammation and cytotoxic environment supports the notion that a cytokine storm could be liable for the multisystemic insults in patients with severe covid19elicitation of antiviral tcell responses specific to sarscov2 is of utmost importance to establishing viral control many studies have demonstrated robust antiviral responses however there is no known set of markers reported to identify sarscov2specific t cells collectively most groups have characterized sarscov2specific t cells based on hladr cd38 cd69 cd25 cd44 and ki67 expression th Answer:

Colon_Cancer

netosis is a type of regulated cell death dependent on the formation of neutrophil extracellular traps net where netlike structures of decondensed chromatin andproteases are produced by polymorphonuclear pmn granulocytes these structuresimmobilise pathogens and restrict them with antimicrobial molecules thus preventing theirspread whilst nets possess a fundamental antimicrobial function within the innate immunesystem under physiological circumstances increasing evidence also indicates that netosisoccurs in the pathogenic process of other disease type including but not limited to atherosclerosis airway inflammation alzheimer™s and stroke here we reviewed the role ofnetosis in the development of an injury including injury to the brain lung heart kidneymusculoskeletal system gut and reproductive system whilst therapeutic agents in blockinginjuries induced by netosis in its primitive stages were also discussed this review providesnovel insights into the involvement of netosis in different an injuries and whilstpotential therapeutic measures targeting netosis remain a largely unexplored area thesewarrant further investigationkey words netosis neutrophil an injury cell death inflammation cell death is commonly segregated into necrosisand apoptosis apoptosis being programmed cell death anaesthetics pain medicine and intensive care department of surgeryand cancer faculty of medicine imperial college london chelsea andwestminster hospital fulham road london sw10 9nh uk department of anesthesiology shanghai fengxian district central hospital shanghai jiao tong university affiliated sixth people™s hospitalsouth campus fengxian district shanghai china to whom correspondence should be addressed at anaesthetics painmedicine and intensive care department of surgery and cancer faculty of medicine imperial college london chelsea and westminsterhospital fulham road london sw10 9nh uk emaildmaimperialacukfor instance during development and physiological cellular turnover whilst necrosis predominantly takesplace in an unregulated manner netosis like necrosis is a mode of cell death that involves the loss ofmembrane integrity during netosis decondensationof chromatin is thought to be initiated by peptidyl arginine deiminase pad4 its subsequent releasetogether with granule contents is vital in the innateimmune response to infection and inflammation recentstudies suggest that net formation is of central topathogenesis of an injury this review will summarise the current understanding of the molecular mechanisms of netosis and the therapeutic approaches underdevelopment targeting netinduced an injury the authors this is an open access publication 0cmolecular mechanism of netformationalthough netosis is closely associated with netformation not all net formation requires the process ofcell death to take place beforehand according to nomenclature committee on cell death the term ˜netosis™should only be used in the context of cell death and notjust based on the presence of net formation two main pathways of net formation have beendescribed and categorised according to their dependenceon the activity of nicotinamide adenine dinucleotide phosphate nadph oxidase pathway fig nadph oxidasedependent net formationthe nadph oxidasedependent molecular pathwayof net formation begins with activation of neutrophilsurface receptors by stimuli derived from pathogenic ornonpathogenic sources such as cholesterol or urate andends with cellular lysis these stimuli trigger calciumrelease from the endoplasmic reticulum er resulting inthe activation of protein kinase c pkc and the assemblyof the nadph oxidase complex generating reactive oxygen species ros following this neutrophil elastasene a protease stored in the cytoplasmic granules migrates to the nucleus in a myeloperoxidase mpodependent manner and cleaves histones to initiate chromatindecondensation this is promoted by the citrullinationof histone arginine residues by peptidylarginine deiminaseiv pad4 finally mixing of the chromatin andgranule proteins takes place as cellular membranes arebroken down interestingly there have been reports ofmitochondrial dna mtdna instead of nuclear dnabeing the source of the dna fibres in nets with observations of mtdna being released from granulocytes inresponse to disease states such as trauma and systemiclupus erythematosus sle [ ] moreover it seemsthat histone citrullination is not always required for netformation as observed by kenny and colleagues in theirstudy of neutrophils activated by the pkc agonist phorbol12myristate 13acetate pma this highlights the diversity of pathways for net formation following their induction degradation of nets can take place through severalpathways for example by dnases or endocytosed bymacrophages factors that influence net formation include phco2 and hco3ˆ’ levels which modulate neutrophil activation this explains why nets form more readily in thecahilog zhao wu alam eguchi weng and maperiphery of an inflammatory site where the ph is morealkaline this may influence treatment efficacy asnets can seal off the affected area an acidic environmenthas been hypothesised to reduce nadph oxidasedependent net formation by reducing neutrophil glycolysis nadph oxidasedependent net formation also requires neutrophils to be in the cell cycle necessitating theactivation of cyclindependent kinases cdk phosphorylating the retinoblastoma proteinnadph oxidaseindependent net formationthis mechanism of net formation is more relevant inthe context of infection as inducers of netosis here arecalcium ionophore a23187 and the potassium ionophorenigericin which are products of streptomyceschartreusensis and streptomyces hygroscopicus respectively how this pathway leads to net release ispoorly understoodnetosis and inflammationnets under physiological conditions are central topathogen clearance when there is excessive formation orsuboptimal nets are able to initiate further destructivesignalling through interaction with other tissue constituentsand the immune system moreover the antimicrobial histones and peptides within the net structure impose adirect cytotoxic effect on tissues to date there havebeen numerous accounts of netosis being present indiseases of major ans understanding of netosis inpathophysiology may offer unique opportunities for clinical managementnetosis in an injurythere is an expanding body of research describingnetosis in infectious and noninfectious an injurysummarised in fig although it is valid that in thesescenarios nuclear dna released during necrotic cell deathcan contribute to tissue injury and exacerbate the extent ofan damage here we focus on the contribution by aberrant net formation and the implication of understandingits underlying pathogenesis for therapeutic interventions 0crole of neutrophil netosis in an injuryfig type of cell death for neutrophil in an injury during solid an injury neutrophils could be prompted to undergo caspasedependent apoptosiswhich results in controlled dissolution of cell into apoptotic bodies containing cellular debris to prevent immune and inflammatory responses neutrophilextracellular traps nets form via two pathways the first is through lytic netosis a cell death pathway characterised by nuclear delobulation anddisintegration of the nuclear envelope which precedes loss of cellular polarisation chromatin decondensation and plasma membrane rupture the secondmechanism involves the nonlytic form of netosis which is not associated with cellular death but prompts expulsion of nuclear chromatin together withrelease of granule proteins through degranulation these components can assemble in the extracellular space into nets leaving behind enucleated cytoplaststhat continue to ingest microanisms in addition neutrophils could undergo unregulated necrosis that does not involve specific molecular pathwayswith uncontrolled release of cellular debris acting as dangerassociated molecular patterns damps to trigger proinflammatory responsebrainalzheimer™s disease alzheimer™s disease ad is acommon disorder of neurodegeneration characterised bygradual loss of cognitive functions in ad patients neutrophils have been observed to invade the brain parenchyma and release nets causing destruction of neural cellsand the bloodbrain barrier stroke it is well known that the adaptive immunesystem is altered after a stroke predisposing patients toinfections [“] interestingly netosis has also beendescribed as significantly impaired up until on day inthose with an ischaemic stroke though netosis inhaemorrhagic stroke patients has yet to be documented ithas been noted that the generation of ros a keyrequirement for chromatin decondensation is suppressedin these patients for up to days lungcystic fibrosis it has been well established that chronic infections in cystic fibrosis cf patients are due to thehighly viscous mucus production harbouring microbialgrowth although impaired clearance of mucus has beenprincipally named responsible there is increasing evidencethat the high viscosity is also due vast amounts of freedna found in sputum samples which was in concordance with the high concentration of neutrophil and 0ccahilog zhao wu alam eguchi weng and mafig involvement of netosis in an injury accumulating evidences now point to an important role of netosis in infectious and noninfectious solidan injury neutrophil invasion into brain parenchyma and release of neutrophil extracellular traps nets have been established in the pathophysiology ofalzheimer™s disease to cause destruction to neural cells and bloodbrain barrier abnormal netosis activity and reactive oxygen species ros response akey element to netosis initiation were observed in stroke patients the degree of neutrophil infiltration net formationcomponent eg cellfreenucleosomes and netosis have been found to correlate with the severity of a range of lung diseases including cystic fibrosis acute lung injury aliacute respiratory distress syndrome ards and lung infection netosis was also shown to be involved in allergic asthma chronic obstructive pulmonarydisease and pulmonary hypertension wherein degree of net formation correlates with disease severity during liver ischaemiareperfusion tolllikereceptordependent net release has been suggested to mediate liver inflammation and injury conversely deficiency in net release was reported indecompensated cirrhotic liver disease and could explain susceptibility to bacterial peritonitis infection in those patients net formation and netosis havefurther been implicated in atherosclerosis and myocardial infarction wherein net was found in thrombi and infarct lesion and correlate with disease severityin rheumatoid arthritis enhanced net release and netosis are observed in synovial tissue rheumatoid nodules and skin whilst proinflammatory cytokinesand autoantibodies further aggravate neutrophil infiltration and netosis neutrophils could also be potently activated by monosodiumurate msu crystals ingout joints and point to a potential role of netnetosis in gout pathogenesis moreover neutrophil activation and net deposition were also observed incolon mucosa of ulcerative colitis excessive neutrophil activation net formation and netosis could also be responsible different pregnancyrelateddisorders including preeclampsia wherein net deposition and netosis in the intervillous space may damage maternal endothelium and impair foetaloxygen exchangenets found in cf lungs the source was believed to befrom necrotic neutrophils but is now considered to besecondary to netosis additionally net productionwas found to be promoted by bacterial infection in cfairways and was defective in clearance of the bacteriapseudomonas aeruginosa nets are also named as a facilitative factor for biofilm formation there is evidencethat surfactant protein d spd responsible foropsonising pathogens and dying cells for clearance byalveolar macrophages is essential for net clearancethrough binding directly to the chromatin within the netsspd levels are decreased in cf patients and the level ofdecrease is proportional to the degree of inflammationthrough accumulation of nets 0crole of neutrophil netosis in an injurylung infection neutrophils migrated into the affectedsite and initiate the cascade of antimicrobial mechanismsincluding net generation to combat microanismsthis happens more readily in the lungs compared with inother tissues with neutrophils found to exist in higherconcentrations in pulmonary vasculature compared withsystemic blood vessels a prominent pathway leadingto net formation in infection is through the interaction oflipopolysaccharide lps with tolllike receptor tlr4found on neutrophils in patients with communityacquired pneumoniacap increased levels of cellfree nucleosomes in serumsamples used as surrogate markers of netosis werefound this was associated with prolonged hospitalisationand a greater 30day allcause mortality this findingsuggests nets could function as a novel marker of prognosis in capacute lung injury and acute respiratory distresssyndrome the degree of neutrophil influx into the lungsand net release during ali and ards positively correlates with disease progression and severity with neutrophil depletion conferring protection in a transfusionrelated ali animal model netosis seems to be akey component of ventilatorinduced lung injury vili as evidenced by detection of citrullinated histone3suggesting that this was a mode of cell death independentfrom apoptosis and necrosis the authors suggestedthat this may be due to increased levels of il1β andhmgb1 which have been both shown to be able to inducenetosisallergic asthma patients with neutrophilic asthmahave a greater severity of disease and reduced response tocorticosteroid treatment compared with the eosinophilictype the increased expression of neutrophilchemoattractant il8 in airway smooth muscle cells couldbe contributing to disease severity through inducingnetosischronic obstructive pulmonary disease netosis hasbeen documented as an integral part of chronic obstructivepulmonary disease copd pathophysiology unlike asthma where neutrophils are important in certain subtypesnetosis has been directly linked to disease severity incopd [ ] tlr4 expression one of the main potentiators for net formation is increased during copd exacerbations pulmonary hypertension nets are also able to potentiate dysregulated angiogenesis as seen in patients withchronic thromboembolic pulmonary hypertension and idiopathic pulmonary hypertension as plasma levels of dnane and mpo levels are significantly elevated moreovernets also seem to destabilise intercellular junctions andincrease endothelial cell motility through direct contactwith endothelial cells nets were found to induce theactivity of the proinflammatory transcription factor nfκbby approximately 3fold moreover nets increase thesurface expression of von willebrand factor and plateletadhesion thereby producing a prothrombotic state kidneyglomerulonephritides nets have been visualised upon immunostaining renal biopsies from patients with sleand antineutrophil cytoplasmic antibodiesassociated vasculitis aav and may be at least partially responsiblefor activating complement pathways resulting in diseaseexacerbations these autoimmune conditions alsoseem to affect the patient™s ability to degrade nets amplifying their deleterious inflammatory effects increscentic glomerulonephritis neutrophilmediated glomerular damage is worsened by addition of extracellularmpo which could have been released during netosis netosis could also contribute to the loss of immunetolerance through further externalisation of crucialautoantigens during cell death haemolytic uraemic syndrome hus plasma from affected patients exhibited a greater capacity to undergonetosis compared with healthy patients the ensuingdamage has been linked to the proinflammatory cytokinesil6 and il8 released from glomerular epithelial cellsupon stimulation by nets this potentiates microvasculature inflammation and thrombosis precipitating renal failure liverdecompensated cirrhotic liver disease a deficiency innet release has been demonstrated to play a role in theonset of endstage liver disease as neutrophils incirrhotic patients are found to have defective ros production which commonly triggers net release thismay also partially explain why these have a predispositionto recurrent bacterial infections and increased rates ofdecompensatory complications such as spontaneous bacterial peritonitis sbp this is corroborated by defectivenet release from ascitic fluid neutrophils in cirrhoticpatients compared with controls in vitro cirrhoticpatients with sbp were also found to have an increase in 0cpro and antiinflammatory cytokines in peripheral bloodand ascitic fluid ischaemiareperfusion injury ischaemiareperfusioninjury iri is an inherent consequence of liver transplantation hypovolaemia or trauma and results in the release ofdamageassociated molecular patterns damps which inturn cause net formation in a tlrdependent mannerexacerbating inflammation treatment with apeptidylargininedeiminase pad4 inhibitor ordnase has been shown to be significantly hepatoprotectivefollowing liver iri cardiovascular systematherosclerosis nets are a wellknown constituent ofatherosclerotic lesions mpo has been strongly associated with diminishing the cardioprotective effects ofhighdensity lipoprotein cholesterol hdlc through oxidation reactions and an increased enzymatic activity islinked to increased plaque rupture other proteinsfound in nets such as cathelinrelated antimicrobial peptide cramp have also been shown to contribute todisease progression moreover in vitro studies showthat hypercholesterolemia triggers net formation alargescale study in patients with suspected coronary arterydisease revealed that the markers of netosis such asextracellular dna are independently associated with disease severity coronary specimens from patients afteran acute myocardial infarction mi showed the presenceof nets in both fresh and lytic thrombi therefore suggesting netosis happens in the early stages of thrombusevolution furthermore net burden was shown tobe positively correlated with the infarct size in patientsundergoing primary percutaneous coronary interventionspostmi this is supported by increased levels of mpodna and ne in the lesion site therefore nets couldpotentially be considered as a novel biomarker in atherosclerosis diabetes mellitusinduced vasculopathy it has beenshown that neutrophils form peripheral blood of diabetesmellitus dm patients showed increased spontaneousnetosis interestingly metformin reduces the deleterious effects of netosis in a mechanism independentlyfrom glucose control one recent study showed that month treatment with metformin in predm patients reduced levels of components of nets whereas glycaemiccontrol with other medication such as insulin saw nodifference when compared with placebo controls thiscahilog zhao wu alam eguchi weng and mahas been attributed to a direct effect of metformin oninhibiting the activation of nadph oxidase musculoskeletal systemrheumatoid arthritis neutrophils from the peripheralblood and synovial fluid of patients with rheumatoid arthritisra exhibit increased netosis compared with healthy controls and patients with osteoarthritis the externalisation ofcitrullinated proteins during the process of netosis wasfound to initiate and perpetuate the aberrant immune responsein ra moreover the autoantibodies and inflammatory cytokines commonly seen in ra promote netosis resulting in avicious cycle of tissue destruction gout gout is an inflammatory disease that involvesthe deposition of monosodiumurate msu crystals injoints during acute gout there is increased movement ofneutrophils into the synovial fluid msu is a known neutrophil stimulus and it has been shown that acute gout isassociated with an increase in il1β levels a keyplayer in net formationgutulcerative colitis there is prominent neutrophil infiltration in the colon mucosa in ulcerative colitis uc and this correlates with disease severity in uc the inflammatory environment promotes neutrophil activation andil1β expression in contrast nets do not seem toplay a key role in crohn™s disease this may explain whymesalazine a known inhibitor of il1β production and thefirstline treatment for uc flareups is not therapeutic incrohn™s patients per se reproductive systempreeclampsia placentas from women diagnosed withpreeclampsia showed increased neutrophil infiltration andnetosis when compared with nonhypertensive pregnantcontrols [ ] and are probably involved in causingwidespread damage to the maternal endothelium placental and endothelial injury during pregnancy aberrantneutrophil activity during pregnancy is also associated withother severe complications including recurrent foetal loss one recent study indicated neutrophils in pregnant womenseem to have an increased propensity to undergo netosissecondary to an increase in granulocyte colonystimulating 0crole of neutrophil netosis in an injuryfactor during pregnancy progesterone has been shown toattenuate neutrophilmediated ros production whereas 17βestradiol induces intracellular ros generation in a dosedependent manner associated an injury associated with netosis fig examples of recent publications on potential therapeutic compounds targeting netosis are summarised in table netosis as a therapeutic targettargeting critical steps in net formation and degradation is critical for developing treatment strategies for netosistlr inhibitorsdexamethasone dex has been shown in vitro toreduce netosis in neutrophils that are stimulated withstaphylococcus aureus but not in those stimulated withpma the tlrs involved in s aureusinduced net formation seem to be tlr2 and tlr4 as agonists of thesereceptors rescued dex inhibition interestingly althoughfig therapeutic strategies targeting net formation stimulation of neutrophil receptors eg fc γ receptor tolllike receptor by microanisms orsterile signals leads to release of calcium ca2 from the endoplasmic reticulum er cellular ca2 overload results in activation of protein kinase c pkcassembly of the nicotinamide adenine dinucleotide phosphate nadph oxidase complex andor mitochondrial activation thus stimulating reactive oxygenspecies ros production oxidative stress promotes myeloperoxidase mpodependent migration of granular neutrophil elastase ne into the nucleus tocleave histones subsequent activation of peptidylarginine deiminase pad induces histone citrullination to cause chromatin decondensation the last stepinvolves nuclear membrane degradation and extrusion of a mixture of chromatin and granular proteins into extracellular space whereby extracellular dnaseeventually digests and removes neutrophil extracellular traps nets in this regard modulation of critical steps in net formation and degradation shown byblocking arrows might be beneficial for the treatment of inflammatory disorders figure modified and reproduced with permission fcγr fc γ receptortlr tolllike receptor 0cdex reduced net formation it did not significantly affectros production calcineurin inhibitorscalcineurin is a calciumdependent serinethreonineprotein phosphatase that is important for neutrophil activity many stages of netosis induction depend upon calcium mobilisation hence modulators of the calcineurinpathway are potential pharmacological inhibitors of netformation cyclosporine a csa an antagonist of thecalcineurin pathway has been shown to reduce the effectof key physiological activators of neutrophils this effecton netosis may in part explain csa™s efficacy in ra and steroidresistant uc patients pmainducednetosis seems to be calciumindependent as this wasnot inhibited by csa cahilog zhao wu alam eguchi weng and mapad inhibitorsusing a murine model of atherosclerosis knight andcolleagues have shown that pharmacological inhibition ofpad4 using weeks of daily clamidine injections reduced netinduced vascular damage with delayed plaqueprogression in the carotid arteries the same groupalso showed that pad inhibitors reduce disease activity inmurine models of sle by reducing endothelial dysfunction it is worth mentioning that the possibility of padinhibition as a therapeutic avenue to be pursued in netinduced an damage in glomerulonephritis has beenrecently challenged by the work of gordon and colleagueson murine models on sle with pad4 deletion theyshowed that this did not reduce endan damage asmeasured by proteinuria suggesting that mechanismsother than net formation are implicated in this complexautoimmune conditionros scavengersdnase therapythe mitochondria are a powerful source of ros ros scavengers such as nacetyl cysteine nac reducenet formation and severity of sle in patients troloxand tempol are two antioxidants which have also beenshown to prevent netosis of pmastimulated humanneutrophils in a dosedependent manner and have beenrecommended for treatment of autoimmune and inflammatory pathologies dnase therapy has been proposed to improve outcomes in cf patients through reducing mucous viscosityhowever it appears that recombinant dnase does notreduce the load of dnaprotein complexes seen innetosis one solution to this is to combine elastase withdnase in order to degrade histones and provide dnaseaccess to chromatin this combination has been shown toenhance solubilisation of sputum drug classstudymain findingstable potential therapeutic approaches targeting netosistlr inhibitorswan t et al calcineurin inhibitorsgupta ak et al dexamethasone reduced netosis in neutrophils stimulated with s aureusagonists of tlr2 and tlr4 rescued dexamethasone inhibitionros production was unaffected by dexamethasonecyclosporine a reduced the effect of key physiological stimuli that activate neutrophilssuch as il8 and suppressed netosisros scavengerspatel et al vorobjeva nv and pinegin bvnacetyl cysteine reduced net formation and severity of sle in patientsantioxidants trolox and tempol prevent netosis of in stimulated human neutrophils in apad inhibitorsknight js et al weeks of daily clamidine injections reduced netinduced vascular damage and area ofdosedependent mannerlesions in a murine model of atherosclerosispad inhibition dampens disease activity by reducing endothelial dysfunction in a murinemodel of slednase therapypapayannopoulos v staab delastase combined with dnase therapy enhances solubilisation of sputum in cystic fibrosistetrahydroisoquinolines martinez ne et al tetrahydroisoquinolines selectively target net overproduction at micromolarzychlinsky a patientsconcentrations possibly at multiple stages of net formation without compromisingnormal neutrophil function 0crole of neutrophil netosis in an injurytetrahydroisoquinolinesin contrary to the aforementioned mechanisms ofnetosis inhibitors tetrahydroisoquinolines thiqs area new class of net formation inhibitors that do not targetros formation or granular protein activity as functionalneutrophils are paramount to maintaining immune reactivity this difference offers an advantage to selectively targetnet overproduction without impairing normal functionthe exact molecular mechanisms of thiqs are yet to bedetermined however it is known that thiq inhibition ofnetosis take place at micromolar concentrations and possibly at different stages of net formation conclusionwhen regulated as part of normal physiology netsare antimicrobial and fundamental to the innate immunesystem dysregulated net formation contributes to thepathogenesis of a plethora of diseases this review hassummarised the role of netosis in pathologies of multiplebody systems as well as highlighted the stages of netosisthat has so far been investigated as emerging pharmacological targets these putative strategies seem to hold therapeutic potential and warrant further investigationauthors™ contributionsdm designed and reviewed the manuscript zc andhz wrote the first draft of the paper all authors readrevised and approved the final manuscriptcompliance with ethical standardscompeting interests the authors declare that they haveno competing interestsethics approval and consent to participate notapplicableconsent for publication not applicableopen access this is licensed under a creativecommons attribution international license whichpermits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons licence and indicateif changes were made the images or other third partymaterial in this are included in the 's creativecommons licence unless indicated otherwise in a creditline to the material if material is not included in the's creative commons licence and your intended useis not permitted by statutory regulation or exceeds thepermitted use you will need to obtain permission directlyfrom the copyright holder to view a copy of this licencevisit httpcreativecommonslicensesby40references vandenabeele p l galluzzi t vanden berghe and g kroemer molecular mechanisms of necroptosis an ordered cellularexplosion nature reviews molecular cell biology “ httpsdoi101038nrm2970 lewis hd j liddle je coote sj atkinson md barker bdbax kl bicker rp bingham m campbell yh chen cwchung pd craggs rp davis d eberhard g joberty kelind k locke c maller k martinod c patten o polyakovace rise m rüdiger rj sheppard dj slade p thomas jthorpe g yao g drewes dd wagner pr thompson rkprinjha and dm wilson inhibition of pad4 activity issufficient to disrupt mouse and human net formation naturechemical biology “ httpsdoi101038nchembio1735 galluzzi lorenzo ilio vitale stuart a aaronson john m abramsdieter adam patrizia agostinis emad s alnemri et al molecular mechanisms of cell death recommendations of the nomenclature committee on cell death cell death and differentiation “ httpsdoi101038s414180170012 gupta s and mj kaplan the role of neutrophils andnetosis in autoimmune and renal diseases nature reviews nephrology “ httpsdoi101038nrneph201671 papayannopoulos v neutrophil extracellular traps in immunity and disease nature reviews immunology “httpsdoi101038nri2017105 kobayashi sd and fr deleo role of neutrophils ininnate immunity a systems biologylevel approach wiley interdisciplinary reviews systems biology and medicine “httpsdoi101002wsbm32 metzler kd c goosmann a lubojemska a zychlinsky andv papayannopoulos a myeloperoxidasecontaining complex regulates neutrophil elastase release and actin dynamics duringnetosis cell reports “ httpsdoi101016jcelrep201406044 tessarz p and t kouzarides histone core modificationsregulating nucleosome structure and dynamics nature reviewsmolecular cell biology “ httpsdoi101038nrm3890 yousefi s c mihalache e kozlowski i schmid and husimon viable neutrophils release mitochondrial dna toform neutrophil extracellular traps cell death and differentiation “ httpsdoi101038cdd200996 wang haiting ting li sheng chen gu yueying and shuang ye neutrophil extracellular trap mitochondrial dna and its 0cautoantibody in systemic lupus erythematosus and a proofofconcept trial of metformin arthritis rheumatology “ kenny ef a herzig r kruger a muth s mondal prthompson v brinkmann hv bernuth and a zychlinsky diverse stimuli engage different neutrophil extracellular trappathways elife httpsdoi107554elife24437 hakkim a bg furnrohr k amann b laube ua abed vbrinkmann m herrmann re voll and a zychlinsky impairment of neutrophil extracellular trap degradation is associatedwith lupus nephritis proceedings of the national academy of sciences of the united states of america “ httpsdoi101073pnas0909927107 farrera c and b fadeel macrophage clearance of neutrophil extracellular traps is a silent process journal of immunology “ httpsdoi104049jimmunol1300436 maueroder c a mahajan s paulus s gosswein j hahn dkienhofer mh biermann et al menageatrois the ratio ofbicarbonate to co2 and the ph regulate the capacity of neutrophilsto form nets frontiers in immunology httpsdoi103389fimmu201600583 behnen m s moller a brozek m klinger and t laskay extracellular acidification inhibits the rosdependent formation ofneutrophil extracellular traps frontiers in immunology httpsdoi103389fimmu201700184 amulic b sl knackstedt u abu abed n deigendesch cjharbort be caffrey v brinkmann fl heppner pw hindsand

cancer290

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: netosis is a type of regulated cell death dependent on the formation of neutrophil extracellular traps net where netlike structures of decondensed chromatin andproteases are produced by polymorphonuclear pmn granulocytes these structuresimmobilise pathogens and restrict them with antimicrobial molecules thus preventing theirspread whilst nets possess a fundamental antimicrobial function within the innate immunesystem under physiological circumstances increasing evidence also indicates that netosisoccurs in the pathogenic process of other disease type including but not limited to atherosclerosis airway inflammation alzheimer™s and stroke here we reviewed the role ofnetosis in the development of an injury including injury to the brain lung heart kidneymusculoskeletal system gut and reproductive system whilst therapeutic agents in blockinginjuries induced by netosis in its primitive stages were also discussed this review providesnovel insights into the involvement of netosis in different an injuries and whilstpotential therapeutic measures targeting netosis remain a largely unexplored area thesewarrant further investigationkey words netosis neutrophil an injury cell death inflammation cell death is commonly segregated into necrosisand apoptosis apoptosis being programmed cell death anaesthetics pain medicine and intensive care department of surgeryand cancer faculty of medicine imperial college london chelsea andwestminster hospital fulham road london sw10 9nh uk department of anesthesiology shanghai fengxian district central hospital shanghai jiao tong university affiliated sixth people™s hospitalsouth campus fengxian district shanghai china to whom correspondence should be addressed at anaesthetics painmedicine and intensive care department of surgery and cancer faculty of medicine imperial college london chelsea and westminsterhospital fulham road london sw10 9nh uk emaildmaimperialacukfor instance during development and physiological cellular turnover whilst necrosis predominantly takesplace in an unregulated manner netosis like necrosis is a mode of cell death that involves the loss ofmembrane integrity during netosis decondensationof chromatin is thought to be initiated by peptidyl arginine deiminase pad4 its subsequent releasetogether with granule contents is vital in the innateimmune response to infection and inflammation recentstudies suggest that net formation is of central topathogenesis of an injury this review will summarise the current understanding of the molecular mechanisms of netosis and the therapeutic approaches underdevelopment targeting netinduced an injury the authors this is an open access publication 0cmolecular mechanism of netformationalthough netosis is closely associated with netformation not all net formation requires the process ofcell death to take place beforehand according to nomenclature committee on cell death the term ˜netosis™should only be used in the context of cell death and notjust based on the presence of net formation two main pathways of net formation have beendescribed and categorised according to their dependenceon the activity of nicotinamide adenine dinucleotide phosphate nadph oxidase pathway fig nadph oxidasedependent net formationthe nadph oxidasedependent molecular pathwayof net formation begins with activation of neutrophilsurface receptors by stimuli derived from pathogenic ornonpathogenic sources such as cholesterol or urate andends with cellular lysis these stimuli trigger calciumrelease from the endoplasmic reticulum er resulting inthe activation of protein kinase c pkc and the assemblyof the nadph oxidase complex generating reactive oxygen species ros following this neutrophil elastasene a protease stored in the cytoplasmic granules migrates to the nucleus in a myeloperoxidase mpodependent manner and cleaves histones to initiate chromatindecondensation this is promoted by the citrullinationof histone arginine residues by peptidylarginine deiminaseiv pad4 finally mixing of the chromatin andgranule proteins takes place as cellular membranes arebroken down interestingly there have been reports ofmitochondrial dna mtdna instead of nuclear dnabeing the source of the dna fibres in nets with observations of mtdna being released from granulocytes inresponse to disease states such as trauma and systemiclupus erythematosus sle [ ] moreover it seemsthat histone citrullination is not always required for netformation as observed by kenny and colleagues in theirstudy of neutrophils activated by the pkc agonist phorbol12myristate 13acetate pma this highlights the diversity of pathways for net formation following their induction degradation of nets can take place through severalpathways for example by dnases or endocytosed bymacrophages factors that influence net formation include phco2 and hco3ˆ’ levels which modulate neutrophil activation this explains why nets form more readily in thecahilog zhao wu alam eguchi weng and maperiphery of an inflammatory site where the ph is morealkaline this may influence treatment efficacy asnets can seal off the affected area an acidic environmenthas been hypothesised to reduce nadph oxidasedependent net formation by reducing neutrophil glycolysis nadph oxidasedependent net formation also requires neutrophils to be in the cell cycle necessitating theactivation of cyclindependent kinases cdk phosphorylating the retinoblastoma proteinnadph oxidaseindependent net formationthis mechanism of net formation is more relevant inthe context of infection as inducers of netosis here arecalcium ionophore a23187 and the potassium ionophorenigericin which are products of streptomyceschartreusensis and streptomyces hygroscopicus respectively how this pathway leads to net release ispoorly understoodnetosis and inflammationnets under physiological conditions are central topathogen clearance when there is excessive formation orsuboptimal nets are able to initiate further destructivesignalling through interaction with other tissue constituentsand the immune system moreover the antimicrobial histones and peptides within the net structure impose adirect cytotoxic effect on tissues to date there havebeen numerous accounts of netosis being present indiseases of major ans understanding of netosis inpathophysiology may offer unique opportunities for clinical managementnetosis in an injurythere is an expanding body of research describingnetosis in infectious and noninfectious an injurysummarised in fig although it is valid that in thesescenarios nuclear dna released during necrotic cell deathcan contribute to tissue injury and exacerbate the extent ofan damage here we focus on the contribution by aberrant net formation and the implication of understandingits underlying pathogenesis for therapeutic interventions 0crole of neutrophil netosis in an injuryfig type of cell death for neutrophil in an injury during solid an injury neutrophils could be prompted to undergo caspasedependent apoptosiswhich results in controlled dissolution of cell into apoptotic bodies containing cellular debris to prevent immune and inflammatory responses neutrophilextracellular traps nets form via two pathways the first is through lytic netosis a cell death pathway characterised by nuclear delobulation anddisintegration of the nuclear envelope which precedes loss of cellular polarisation chromatin decondensation and plasma membrane rupture the secondmechanism involves the nonlytic form of netosis which is not associated with cellular death but prompts expulsion of nuclear chromatin together withrelease of granule proteins through degranulation these components can assemble in the extracellular space into nets leaving behind enucleated cytoplaststhat continue to ingest microanisms in addition neutrophils could undergo unregulated necrosis that does not involve specific molecular pathwayswith uncontrolled release of cellular debris acting as dangerassociated molecular patterns damps to trigger proinflammatory responsebrainalzheimer™s disease alzheimer™s disease ad is acommon disorder of neurodegeneration characterised bygradual loss of cognitive functions in ad patients neutrophils have been observed to invade the brain parenchyma and release nets causing destruction of neural cellsand the bloodbrain barrier stroke it is well known that the adaptive immunesystem is altered after a stroke predisposing patients toinfections [“] interestingly netosis has also beendescribed as significantly impaired up until on day inthose with an ischaemic stroke though netosis inhaemorrhagic stroke patients has yet to be documented ithas been noted that the generation of ros a keyrequirement for chromatin decondensation is suppressedin these patients for up to days lungcystic fibrosis it has been well established that chronic infections in cystic fibrosis cf patients are due to thehighly viscous mucus production harbouring microbialgrowth although impaired clearance of mucus has beenprincipally named responsible there is increasing evidencethat the high viscosity is also due vast amounts of freedna found in sputum samples which was in concordance with the high concentration of neutrophil and 0ccahilog zhao wu alam eguchi weng and mafig involvement of netosis in an injury accumulating evidences now point to an important role of netosis in infectious and noninfectious solidan injury neutrophil invasion into brain parenchyma and release of neutrophil extracellular traps nets have been established in the pathophysiology ofalzheimer™s disease to cause destruction to neural cells and bloodbrain barrier abnormal netosis activity and reactive oxygen species ros response akey element to netosis initiation were observed in stroke patients the degree of neutrophil infiltration net formationcomponent eg cellfreenucleosomes and netosis have been found to correlate with the severity of a range of lung diseases including cystic fibrosis acute lung injury aliacute respiratory distress syndrome ards and lung infection netosis was also shown to be involved in allergic asthma chronic obstructive pulmonarydisease and pulmonary hypertension wherein degree of net formation correlates with disease severity during liver ischaemiareperfusion tolllikereceptordependent net release has been suggested to mediate liver inflammation and injury conversely deficiency in net release was reported indecompensated cirrhotic liver disease and could explain susceptibility to bacterial peritonitis infection in those patients net formation and netosis havefurther been implicated in atherosclerosis and myocardial infarction wherein net was found in thrombi and infarct lesion and correlate with disease severityin rheumatoid arthritis enhanced net release and netosis are observed in synovial tissue rheumatoid nodules and skin whilst proinflammatory cytokinesand autoantibodies further aggravate neutrophil infiltration and netosis neutrophils could also be potently activated by monosodiumurate msu crystals ingout joints and point to a potential role of netnetosis in gout pathogenesis moreover neutrophil activation and net deposition were also observed incolon mucosa of ulcerative colitis excessive neutrophil activation net formation and netosis could also be responsible different pregnancyrelateddisorders including preeclampsia wherein net deposition and netosis in the intervillous space may damage maternal endothelium and impair foetaloxygen exchangenets found in cf lungs the source was believed to befrom necrotic neutrophils but is now considered to besecondary to netosis additionally net productionwas found to be promoted by bacterial infection in cfairways and was defective in clearance of the bacteriapseudomonas aeruginosa nets are also named as a facilitative factor for biofilm formation there is evidencethat surfactant protein d spd responsible foropsonising pathogens and dying cells for clearance byalveolar macrophages is essential for net clearancethrough binding directly to the chromatin within the netsspd levels are decreased in cf patients and the level ofdecrease is proportional to the degree of inflammationthrough accumulation of nets 0crole of neutrophil netosis in an injurylung infection neutrophils migrated into the affectedsite and initiate the cascade of antimicrobial mechanismsincluding net generation to combat microanismsthis happens more readily in the lungs compared with inother tissues with neutrophils found to exist in higherconcentrations in pulmonary vasculature compared withsystemic blood vessels a prominent pathway leadingto net formation in infection is through the interaction oflipopolysaccharide lps with tolllike receptor tlr4found on neutrophils in patients with communityacquired pneumoniacap increased levels of cellfree nucleosomes in serumsamples used as surrogate markers of netosis werefound this was associated with prolonged hospitalisationand a greater 30day allcause mortality this findingsuggests nets could function as a novel marker of prognosis in capacute lung injury and acute respiratory distresssyndrome the degree of neutrophil influx into the lungsand net release during ali and ards positively correlates with disease progression and severity with neutrophil depletion conferring protection in a transfusionrelated ali animal model netosis seems to be akey component of ventilatorinduced lung injury vili as evidenced by detection of citrullinated histone3suggesting that this was a mode of cell death independentfrom apoptosis and necrosis the authors suggestedthat this may be due to increased levels of il1β andhmgb1 which have been both shown to be able to inducenetosisallergic asthma patients with neutrophilic asthmahave a greater severity of disease and reduced response tocorticosteroid treatment compared with the eosinophilictype the increased expression of neutrophilchemoattractant il8 in airway smooth muscle cells couldbe contributing to disease severity through inducingnetosischronic obstructive pulmonary disease netosis hasbeen documented as an integral part of chronic obstructivepulmonary disease copd pathophysiology unlike asthma where neutrophils are important in certain subtypesnetosis has been directly linked to disease severity incopd [ ] tlr4 expression one of the main potentiators for net formation is increased during copd exacerbations pulmonary hypertension nets are also able to potentiate dysregulated angiogenesis as seen in patients withchronic thromboembolic pulmonary hypertension and idiopathic pulmonary hypertension as plasma levels of dnane and mpo levels are significantly elevated moreovernets also seem to destabilise intercellular junctions andincrease endothelial cell motility through direct contactwith endothelial cells nets were found to induce theactivity of the proinflammatory transcription factor nfκbby approximately 3fold moreover nets increase thesurface expression of von willebrand factor and plateletadhesion thereby producing a prothrombotic state kidneyglomerulonephritides nets have been visualised upon immunostaining renal biopsies from patients with sleand antineutrophil cytoplasmic antibodiesassociated vasculitis aav and may be at least partially responsiblefor activating complement pathways resulting in diseaseexacerbations these autoimmune conditions alsoseem to affect the patient™s ability to degrade nets amplifying their deleterious inflammatory effects increscentic glomerulonephritis neutrophilmediated glomerular damage is worsened by addition of extracellularmpo which could have been released during netosis netosis could also contribute to the loss of immunetolerance through further externalisation of crucialautoantigens during cell death haemolytic uraemic syndrome hus plasma from affected patients exhibited a greater capacity to undergonetosis compared with healthy patients the ensuingdamage has been linked to the proinflammatory cytokinesil6 and il8 released from glomerular epithelial cellsupon stimulation by nets this potentiates microvasculature inflammation and thrombosis precipitating renal failure liverdecompensated cirrhotic liver disease a deficiency innet release has been demonstrated to play a role in theonset of endstage liver disease as neutrophils incirrhotic patients are found to have defective ros production which commonly triggers net release thismay also partially explain why these have a predispositionto recurrent bacterial infections and increased rates ofdecompensatory complications such as spontaneous bacterial peritonitis sbp this is corroborated by defectivenet release from ascitic fluid neutrophils in cirrhoticpatients compared with controls in vitro cirrhoticpatients with sbp were also found to have an increase in 0cpro and antiinflammatory cytokines in peripheral bloodand ascitic fluid ischaemiareperfusion injury ischaemiareperfusioninjury iri is an inherent consequence of liver transplantation hypovolaemia or trauma and results in the release ofdamageassociated molecular patterns damps which inturn cause net formation in a tlrdependent mannerexacerbating inflammation treatment with apeptidylargininedeiminase pad4 inhibitor ordnase has been shown to be significantly hepatoprotectivefollowing liver iri cardiovascular systematherosclerosis nets are a wellknown constituent ofatherosclerotic lesions mpo has been strongly associated with diminishing the cardioprotective effects ofhighdensity lipoprotein cholesterol hdlc through oxidation reactions and an increased enzymatic activity islinked to increased plaque rupture other proteinsfound in nets such as cathelinrelated antimicrobial peptide cramp have also been shown to contribute todisease progression moreover in vitro studies showthat hypercholesterolemia triggers net formation alargescale study in patients with suspected coronary arterydisease revealed that the markers of netosis such asextracellular dna are independently associated with disease severity coronary specimens from patients afteran acute myocardial infarction mi showed the presenceof nets in both fresh and lytic thrombi therefore suggesting netosis happens in the early stages of thrombusevolution furthermore net burden was shown tobe positively correlated with the infarct size in patientsundergoing primary percutaneous coronary interventionspostmi this is supported by increased levels of mpodna and ne in the lesion site therefore nets couldpotentially be considered as a novel biomarker in atherosclerosis diabetes mellitusinduced vasculopathy it has beenshown that neutrophils form peripheral blood of diabetesmellitus dm patients showed increased spontaneousnetosis interestingly metformin reduces the deleterious effects of netosis in a mechanism independentlyfrom glucose control one recent study showed that month treatment with metformin in predm patients reduced levels of components of nets whereas glycaemiccontrol with other medication such as insulin saw nodifference when compared with placebo controls thiscahilog zhao wu alam eguchi weng and mahas been attributed to a direct effect of metformin oninhibiting the activation of nadph oxidase musculoskeletal systemrheumatoid arthritis neutrophils from the peripheralblood and synovial fluid of patients with rheumatoid arthritisra exhibit increased netosis compared with healthy controls and patients with osteoarthritis the externalisation ofcitrullinated proteins during the process of netosis wasfound to initiate and perpetuate the aberrant immune responsein ra moreover the autoantibodies and inflammatory cytokines commonly seen in ra promote netosis resulting in avicious cycle of tissue destruction gout gout is an inflammatory disease that involvesthe deposition of monosodiumurate msu crystals injoints during acute gout there is increased movement ofneutrophils into the synovial fluid msu is a known neutrophil stimulus and it has been shown that acute gout isassociated with an increase in il1β levels a keyplayer in net formationgutulcerative colitis there is prominent neutrophil infiltration in the colon mucosa in ulcerative colitis uc and this correlates with disease severity in uc the inflammatory environment promotes neutrophil activation andil1β expression in contrast nets do not seem toplay a key role in crohn™s disease this may explain whymesalazine a known inhibitor of il1β production and thefirstline treatment for uc flareups is not therapeutic incrohn™s patients per se reproductive systempreeclampsia placentas from women diagnosed withpreeclampsia showed increased neutrophil infiltration andnetosis when compared with nonhypertensive pregnantcontrols [ ] and are probably involved in causingwidespread damage to the maternal endothelium placental and endothelial injury during pregnancy aberrantneutrophil activity during pregnancy is also associated withother severe complications including recurrent foetal loss one recent study indicated neutrophils in pregnant womenseem to have an increased propensity to undergo netosissecondary to an increase in granulocyte colonystimulating 0crole of neutrophil netosis in an injuryfactor during pregnancy progesterone has been shown toattenuate neutrophilmediated ros production whereas 17βestradiol induces intracellular ros generation in a dosedependent manner associated an injury associated with netosis fig examples of recent publications on potential therapeutic compounds targeting netosis are summarised in table netosis as a therapeutic targettargeting critical steps in net formation and degradation is critical for developing treatment strategies for netosistlr inhibitorsdexamethasone dex has been shown in vitro toreduce netosis in neutrophils that are stimulated withstaphylococcus aureus but not in those stimulated withpma the tlrs involved in s aureusinduced net formation seem to be tlr2 and tlr4 as agonists of thesereceptors rescued dex inhibition interestingly althoughfig therapeutic strategies targeting net formation stimulation of neutrophil receptors eg fc γ receptor tolllike receptor by microanisms orsterile signals leads to release of calcium ca2 from the endoplasmic reticulum er cellular ca2 overload results in activation of protein kinase c pkcassembly of the nicotinamide adenine dinucleotide phosphate nadph oxidase complex andor mitochondrial activation thus stimulating reactive oxygenspecies ros production oxidative stress promotes myeloperoxidase mpodependent migration of granular neutrophil elastase ne into the nucleus tocleave histones subsequent activation of peptidylarginine deiminase pad induces histone citrullination to cause chromatin decondensation the last stepinvolves nuclear membrane degradation and extrusion of a mixture of chromatin and granular proteins into extracellular space whereby extracellular dnaseeventually digests and removes neutrophil extracellular traps nets in this regard modulation of critical steps in net formation and degradation shown byblocking arrows might be beneficial for the treatment of inflammatory disorders figure modified and reproduced with permission fcγr fc γ receptortlr tolllike receptor 0cdex reduced net formation it did not significantly affectros production calcineurin inhibitorscalcineurin is a calciumdependent serinethreonineprotein phosphatase that is important for neutrophil activity many stages of netosis induction depend upon calcium mobilisation hence modulators of the calcineurinpathway are potential pharmacological inhibitors of netformation cyclosporine a csa an antagonist of thecalcineurin pathway has been shown to reduce the effectof key physiological activators of neutrophils this effecton netosis may in part explain csa™s efficacy in ra and steroidresistant uc patients pmainducednetosis seems to be calciumindependent as this wasnot inhibited by csa cahilog zhao wu alam eguchi weng and mapad inhibitorsusing a murine model of atherosclerosis knight andcolleagues have shown that pharmacological inhibition ofpad4 using weeks of daily clamidine injections reduced netinduced vascular damage with delayed plaqueprogression in the carotid arteries the same groupalso showed that pad inhibitors reduce disease activity inmurine models of sle by reducing endothelial dysfunction it is worth mentioning that the possibility of padinhibition as a therapeutic avenue to be pursued in netinduced an damage in glomerulonephritis has beenrecently challenged by the work of gordon and colleagueson murine models on sle with pad4 deletion theyshowed that this did not reduce endan damage asmeasured by proteinuria suggesting that mechanismsother than net formation are implicated in this complexautoimmune conditionros scavengersdnase therapythe mitochondria are a powerful source of ros ros scavengers such as nacetyl cysteine nac reducenet formation and severity of sle in patients troloxand tempol are two antioxidants which have also beenshown to prevent netosis of pmastimulated humanneutrophils in a dosedependent manner and have beenrecommended for treatment of autoimmune and inflammatory pathologies dnase therapy has been proposed to improve outcomes in cf patients through reducing mucous viscosityhowever it appears that recombinant dnase does notreduce the load of dnaprotein complexes seen innetosis one solution to this is to combine elastase withdnase in order to degrade histones and provide dnaseaccess to chromatin this combination has been shown toenhance solubilisation of sputum drug classstudymain findingstable potential therapeutic approaches targeting netosistlr inhibitorswan t et al calcineurin inhibitorsgupta ak et al dexamethasone reduced netosis in neutrophils stimulated with s aureusagonists of tlr2 and tlr4 rescued dexamethasone inhibitionros production was unaffected by dexamethasonecyclosporine a reduced the effect of key physiological stimuli that activate neutrophilssuch as il8 and suppressed netosisros scavengerspatel et al vorobjeva nv and pinegin bvnacetyl cysteine reduced net formation and severity of sle in patientsantioxidants trolox and tempol prevent netosis of in stimulated human neutrophils in apad inhibitorsknight js et al weeks of daily clamidine injections reduced netinduced vascular damage and area ofdosedependent mannerlesions in a murine model of atherosclerosispad inhibition dampens disease activity by reducing endothelial dysfunction in a murinemodel of slednase therapypapayannopoulos v staab delastase combined with dnase therapy enhances solubilisation of sputum in cystic fibrosistetrahydroisoquinolines martinez ne et al tetrahydroisoquinolines selectively target net overproduction at micromolarzychlinsky a patientsconcentrations possibly at multiple stages of net formation without compromisingnormal neutrophil function 0crole of neutrophil netosis in an injurytetrahydroisoquinolinesin contrary to the aforementioned mechanisms ofnetosis inhibitors tetrahydroisoquinolines thiqs area new class of net formation inhibitors that do not targetros formation or granular protein activity as functionalneutrophils are paramount to maintaining immune reactivity this difference offers an advantage to selectively targetnet overproduction without impairing normal functionthe exact molecular mechanisms of thiqs are yet to bedetermined however it is known that thiq inhibition ofnetosis take place at micromolar concentrations and possibly at different stages of net formation conclusionwhen regulated as part of normal physiology netsare antimicrobial and fundamental to the innate immunesystem dysregulated net formation contributes to thepathogenesis of a plethora of diseases this review hassummarised the role of netosis in pathologies of multiplebody systems as well as highlighted the stages of netosisthat has so far been investigated as emerging pharmacological targets these putative strategies seem to hold therapeutic potential and warrant further investigationauthors™ contributionsdm designed and reviewed the manuscript zc andhz wrote the first draft of the paper all authors readrevised and approved the final manuscriptcompliance with ethical standardscompeting interests the authors declare that they haveno competing interestsethics approval and consent to participate notapplicableconsent for publication not applicableopen access this is licensed under a creativecommons attribution international license whichpermits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons licence and indicateif changes were made the images or other third partymaterial in this are included in the 's creativecommons licence unless indicated otherwise in a creditline to the material if material is not included in the's creative commons licence and your intended useis not permitted by statutory regulation or exceeds thepermitted use you will need to obtain permission directlyfrom the copyright holder to view a copy of this licencevisit httpcreativecommonslicensesby40references vandenabeele p l galluzzi t vanden berghe and g kroemer molecular mechanisms of necroptosis an ordered cellularexplosion nature reviews molecular cell biology “ httpsdoi101038nrm2970 lewis hd j liddle je coote sj atkinson md barker bdbax kl bicker rp bingham m campbell yh chen cwchung pd craggs rp davis d eberhard g joberty kelind k locke c maller k martinod c patten o polyakovace rise m rüdiger rj sheppard dj slade p thomas jthorpe g yao g drewes dd wagner pr thompson rkprinjha and dm wilson inhibition of pad4 activity issufficient to disrupt mouse and human net formation naturechemical biology “ httpsdoi101038nchembio1735 galluzzi lorenzo ilio vitale stuart a aaronson john m abramsdieter adam patrizia agostinis emad s alnemri et al molecular mechanisms of cell death recommendations of the nomenclature committee on cell death cell death and differentiation “ httpsdoi101038s414180170012 gupta s and mj kaplan the role of neutrophils andnetosis in autoimmune and renal diseases nature reviews nephrology “ httpsdoi101038nrneph201671 papayannopoulos v neutrophil extracellular traps in immunity and disease nature reviews immunology “httpsdoi101038nri2017105 kobayashi sd and fr deleo role of neutrophils ininnate immunity a systems biologylevel approach wiley interdisciplinary reviews systems biology and medicine “httpsdoi101002wsbm32 metzler kd c goosmann a lubojemska a zychlinsky andv papayannopoulos a myeloperoxidasecontaining complex regulates neutrophil elastase release and actin dynamics duringnetosis cell reports “ httpsdoi101016jcelrep201406044 tessarz p and t kouzarides histone core modificationsregulating nucleosome structure and dynamics nature reviewsmolecular cell biology “ httpsdoi101038nrm3890 yousefi s c mihalache e kozlowski i schmid and husimon viable neutrophils release mitochondrial dna toform neutrophil extracellular traps cell death and differentiation “ httpsdoi101038cdd200996 wang haiting ting li sheng chen gu yueying and shuang ye neutrophil extracellular trap mitochondrial dna and its 0cautoantibody in systemic lupus erythematosus and a proofofconcept trial of metformin arthritis rheumatology “ kenny ef a herzig r kruger a muth s mondal prthompson v brinkmann hv bernuth and a zychlinsky diverse stimuli engage different neutrophil extracellular trappathways elife httpsdoi107554elife24437 hakkim a bg furnrohr k amann b laube ua abed vbrinkmann m herrmann re voll and a zychlinsky impairment of neutrophil extracellular trap degradation is associatedwith lupus nephritis proceedings of the national academy of sciences of the united states of america “ httpsdoi101073pnas0909927107 farrera c and b fadeel macrophage clearance of neutrophil extracellular traps is a silent process journal of immunology “ httpsdoi104049jimmunol1300436 maueroder c a mahajan s paulus s gosswein j hahn dkienhofer mh biermann et al menageatrois the ratio ofbicarbonate to co2 and the ph regulate the capacity of neutrophilsto form nets frontiers in immunology httpsdoi103389fimmu201600583 behnen m s moller a brozek m klinger and t laskay extracellular acidification inhibits the rosdependent formation ofneutrophil extracellular traps frontiers in immunology httpsdoi103389fimmu201700184 amulic b sl knackstedt u abu abed n deigendesch cjharbort be caffrey v brinkmann fl heppner pw hindsand Answer:

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"annual meeting of the european associationfor the study of diabetes“ september sindex of oral presentationsop diabetes complications new insights from cutting edge epidemiologyop news on the insulin secretion frontop insulin sensitivity and biomarkersop central actions in diabetesop glucoselowering therapies and the liverop uncomplicating the pathogenesis of diabetes complications inhumansop smoke on the water is bat still hotop charting human beta cell failure in type diabetesop novel agents in type diabetesop developing better insulinsop from diagnostics to the endstage of diabetic kidney diseaseop nafld is it all about the liverop diabetic retinopathy see what's newop taking the long view of diabetesop pregnancy in diabetes prediction and outcomesop signals and networks in beta cell failureop broken heart in diabetesop unlocking the potential of digital healthop decoding the heritable basis of type diabetesop feeding the pipeline from drugs to surgeryop sglt2 inhibitors at the heart of the matterop new treatments for nafld hope or hypeop addressing potential new treatments of diabetic kidney diseaseop glucagon and hormones beyondop incretin based therapiesop unusual forms of diabetesop macrovascular complications and beyondop linking inflammation to metabolismop what's new in automated insulin deliveryop understanding the mechanisms of diabetic kidney diseaseop novel aspects of diabetic neuropathyop reducing the burden of hypoglycaemiaop what exercise doesop back to the future risk markers in diabetesop diet not only quantity mattersop on the road to human islet failure in type diabetesop a deep dive into the mechanisms of diabetesop triggers and drivers of beta cell failure in type diabetesop gastroentero pancreatic factors anoids mice and menop new aspects of novel therapiesop fatty mattersop diabetes care is expensiveop developing beta cellsop modelling metabolism lessons from animalsop diabetic foot new developments in wound healingop challenges in delivering diabetes care new solutionsop thinking about diabetes complications in the brainop insulin secretion in various subgroupsindex of poster sessionsps diabetes and early deathps living with chronic diabetes complicationsps micro and macrovascular complications of diabetesps global view on diabetes complicationsps type diabetes treatment irlps unusual forms of diabetesps molecular insights into glucose abnormalitiesps pathophysiology of glucose homeostasisps the inner workings of the pancreasps islets and antibodies in type diabetesps markers and phenotypes of glucose traitsps global aspects on the epidemiology of type diabetesps risk factors for type diabetesps prevalence of type diabetes around the worldps risk factors in type diabetesps islet transplants revisitedps islets in type diabetes new playersps beta cells under stressps to live and let die a beta cell perspectiveps job description insulin secretionps further down the road to human islet failure in type diabetesps sitting and exercising does it allps the ins and outs of carbohydrate metabolismps pregnancy in vitro and in vivo studiesps pregnancy epidemiologyps pregnancy who is at riskps incremental studies on gut hormonesps the fundamentals of insulin resistanceps studies on insulin resistanceps treatment of hyperglycaemia in pregnancyps pancreatic hormonesps insulin secretion in mice and menps something more about obesityps more about metabolismps inflammation in type diabetesps models of prediabetes and diabetesps models of obesity and insulin resistanceps lipid metabolismps adipokine signallingps drugs and environment in obesityps weight loss interventionsps brain mattersps sglt2 inhibitors clinical aspectsps different aspects of sglt2 inhibitorsps basic aspects of incretinbased therapiesps clinical outcome of incretinbased therapies 0cdiabetologiaps glycaemic control and incretinbased therapiesps various clinical aspects of incretinbased therapiesps various aspects of nutrition and dietps oral therapies metformin sensitizers and other nonsecretagoguesps novel agents to treat diabetes and its consequencesps novel glucoselowering agents in type diabetesps key issues in improving outcomes in people with diabeteseducation and costsps how to improve diabetes careps the impact of new basal insulinsps insulin therapy real world studiesps insulin therapy fast acting insulin analoguesps the challenges of insulin therapy in type diabetesps different aspects of insulin therapyps the continued advance of continuous glucose monitoringps insulin pump therapyps automated insulin deliveryps the varied use of technologies in type diabetesps novel applications of technology in diabetesps novel therapies to reduce hypoglycaemiaps mechanisms and clinical consequences of hypoglycaemiain diabetesps emerging topics in hypoglycaemiaps investigating diabetes distress and depressionps aspects of quality of life and well beingps digital health in type diabetesps is telehealth the answer to improving care in diabetesps predicting prognosis of diabetic kidney diseaseps clinical aspects of diabetic kidney diseaseps the rock and role of experimental kidney diseaseps new tools to view diabetic retinopathyps diabetic retinopathy screening and interventionps focus on diabetic foot ulcersps hypertension and vascular diseaseps cure the pain of diabetic neuropathyps understanding clinical neuropathyps from artificial intelligence to treatment of diabetic footps from biomarkers to genetics of diabetic kidney diseaseps treatment of nafld and diabetes from food to pharmacologyps mechanisms and prevalence of nafldps lipids everywhere lipid metabolism in the liver and the heartps all about coronary arteries and diabetesps lipids and glucose not so good for the heartps cardiac complications of mice rats and cellsps atherosclerotic complications stemming from cells to the kidneyps stiff arteries and how to avoid themps cardiac function and dysfunctionps cardiovascular complications in humans through and throughps diabetes and neoplasiaps contemplating cognitive dysfunction in diabetesps endothelial cell circulation and the heartps tradition no nontraditional complications of diabetes 0cdiabetologiaop diabetes complications new insightsfrom cutting edge epidemiologycirculating metabolites significantly improve the prediction of renaldysfunction in type diabetesm scarale1 s de cosmo1 c prehn2 f schena3 j adamski2 vtrischitta4 c menzaghi11fondazione irccs œcasa sollievo della sofferenza san giovannirotondo italy 2helmholtz zentrum m¼nchen germany 3universityof bari bari italy 4sapienza university roma italy and aims chronic kidney disease ckd mainly indicated by a reduced glomerular filtration rate gfr remains one of theleading causes of reduced lifespan in patients with type diabetest2d discovering novel biomarkers able to predict low gfr will helpidentify highrisk patients to be targeted to more aggressive and burdensome preventive and treatment strategiesmaterials and methods we measured serum metabolites byabsoluteidqtm p180 kit biocrates life sciences ag innsbruckaustria and investigated their association with egfr calculated with theckdepi formula in a discovery sample of patients with t2d cases and controls with egfr60 and ‰¥70mlmin173m2 respectively a threshold p value of 28x104 ie followingbonferroni's rule was used as statistical significance in a model comprising age sex smoking bmi hba1c diabetes duration albumintocreatinine ratio acr and ongoing treatments metabolites associatedin the discovery sample were validated threshold p value of 005numberof surviving validation metabolites in a second cohort comprising diabetic patients cases and controls for egfr60 or ‰¥70mlmin173m2 respectively standardized values of each validated metabolitesweighted for the effect size ie observed in the discovery samplewere then summed up in a metabolic score metscore to be used as agfr prediction tool to this purpose metscore was used on top of anestablished clinical model comprising sex age bmi hba1c and acrand then discrimination [δ area under the receiver operating characteristic roc curve auc and the relative integrated discriminationimprovement ridi] and reclassification [the categoryfree net reclassification index cnri] measures were evaluatedresults thirteen metabolites six acylcarnitines six biogenic amines andone amino acid were independently associated to low egfr [ors range for 1sd increase p range 13x107 20x104] in the discoverysample all of them but one a biogenic amine were validated in thereplication sample [ors range for 1sd increase p range32x1018 43x106 below the threshold of 0051242x103] theauc of the abovementioned clinical model was and in the discovery the replication and the pooled sample respectively the addition of metscore on top of the clinical model improvedboth discrimination and reclassification measures in the discovery δauc4 p14x103 ridi29 p20x1011 ½cnri54p15x1014 the replication δ auc39 p16x103 ridi28p38x108 ½cnri30 p22x1010 and the pooled samples δauc39 p40x106 ridi29 p22x1017 ½cnri35p19x108conclusion we have discovered and validated metabolites that arestrongly associated with low egfr in patients with t2d a metscorecomprising these metabolites improves an established clinical prediction model of low egfr in terms of both discrimination and reclassification encouraged by these findings we are now investigating the ability ofmetscore to improve prediction of gfr decline in prospective cohorts oft2d with the aim of improving risk stratification and therefore refiningprevention efforts of kidney dysfunction in diabetic patientssupported by italian ministry of health rf201302356459disclosure m scarale noneassociation between insulinlike growth factor binding protein2 andinsulin sensitivity metformin and mortality in patients with newlydiagnosed type diabetesmr kristiansen12 js nielsen12 i brandslund3 da olsen3 jvstidsen2 sk nicolaisen4 r hjortebjerg25 j frystyk561danish centre for strategic research in type diabetes dd2odense 2steno diabetes center odense odense 3irs lillebaelthospital biochemistry and immunology vejle 4department ofclinical epidemiology aarhus 5department of clinical researchuniversity of southern denmark odense 6department ofendocrinology odense university hospital odense denmark and aims insulinlike growth factor binding protein2igfbp2 is engaged in metabolism circulating concentrations ofigfbp2 are positively correlated to insulin sensitivity overexpressionof igfbp2 protects against obesity and diabetes in mice and metforminincreases igfbp2 gene expression indicating that igfbp2 is a target ofmetformin action interestingly igfbp2 appears to predict mortalityindependently of insulin sensitivity this study aimed to investigate theassociation between indices of insulin sensitivity metformin treatmentand mortality in patients with newly diagnosed type diabetes t2dmaterials and methods in this crosssectional study we included newlydiagnosed patients with t2d enrolled in the danish centre for strategicresearch in type diabetes dd2 cohort patients were continuouslyenrolled from to throughout denmark and followed usingdanish healthcare registries unbound fractions of igfbp2 were determinedin serum from fasting drug na¯ve n864 and metformin treated ‰¥ twoprescriptions months prior enrollment patients n558 using an inhouseassay developed on the simoa platform values are given as medians iqrassociation was analyzed using a pearson™s regressioncox regression amultivariable model was used to adjust for age bmi and homasresults a total of patients with median age of medianbmi of and median diabetes duration of yearswere included igfbp2 level was positively correlated with homasr2026 and p0005 and inversely correlated with cpeptide r2018and p0005 both associations persisted following adjustments for ageand bmi the igfbp2 level in metformin treated patients was slightlylower ngml than in drug na¯ve patients ngml p0026 a total of patients suffered from one or morecomorbidities from charlson comorbidity index their igfbp2 levelswere higher than patients with no comorbidity vs ngml p0001 during a median of years offollowup a total of patients died igfbp2 level was significantly higher at baseline in patients that died vs not died vs ngml p0001 igfbp2 was associated withmortality with a hazard ratiohr ci per doubling in proteinconcentration of p0001 this association was notobserved when analyzing patients without comorbidities but was significant in patients with other comorbidities hr p0001conclusion this is the first larger study to confirm that igfbp2 isassociated with indices of insulin sensitivity but is not largely affectedby metformin treatment interestingly increased igfbp2 level is associated with high mortality rates but the association was mainly driven bythe presence of comorbidities at baselinesupported by university of southern denmark and region of southerndenmarkdisclosure mr kristiansen nonebuilding clinical risk score systems for predicting allcause andcardiovascularspecific mortality among type diabetes patientscs liu1 tc li2 cc lin1 ci li11china medical university hospital taichung 2china medicaluniversity taichung taiwan 0c and aims no prior prediction model for mortality considered the effect of glycemic variability and blood pressure variabilitywhich have been broadly reported as the important clinical predictors ofmortality especially in diabetes patients the aim of this study was todevelop and validate risk score systems with considering the effects ofglycemic and blood pressure variability on allcause and cardiovascularspecific mortality in persons with type dmmaterials and methods this is a retrospective cohort study consistingof type diabetic patients aged years during allparticipants were randomly allocated into two groups derivation andvalidation sets in ratio and were followed up until death or august cox proportional hazards regression were used to develop allcauseand cardiovascularspecific mortality prediction model prediction modelperformance was assessed by the area under the receiver operating characteristics curve aurocresults overall deaths were identified after a mean of years offollowup the prediction accuracy measured by auroc of and 15year allcause mortality based on a model containing the identifiedtraditional risk factor biomarkers and variability in fasting plasmaglucose and hba1c and diastolic blood pressure variability were and respectively in derivation set and the corresponding values forcardiovascularspecific mortality were and respectively the predictionaccuracy in the validation set for allcause mortality were and respectively and for cardiovascularspecific mortality were and respectivelyconclusion our prediction model considering glycemic and blood pressure variability had good accuracy of prediction of cardiovascularspecific and allcause mortality in patients with type diabetessupported by ministry of science and technology of taiwandisclosure c liu noneincident cardiovascular disease by clustering of favourable riskfactors in type diabetes the eurodiab prospectivecomplications studys soulimane1 yd vogtschmidt12 m toeller3 b balkau4 nchaturvedi5 jh fuller6 ss soedamahmuthu121department of medical and clinical psychology center of research onpsychological and somatic disorders corps tilburg universitynetherlands 2institute for food nutrition and health university ofreading reading uk 3heinrichheineuniversity d¼sseldorfd¼sseldorf germany 4clinical epidemiology universit© parissaclayuvsq inserm cesp villejuif france 5institute of cardiovascularscience university college of london london uk 6department ofepidemiology and public health eurodiab london uk and aims the incidence of cardiovascular diseases cvdis up to eight times higher in people with type diabetes t1d greaterclustering of adverse risk factors is thought to contribute to excess cvdrisks in type diabetes though not explored in t1d the aim of this studywas to examine a cvd risk reduction for those in the most favourablethird of individual risk factors compared to the least favourable two thirdsand b cvd risk reduction by clustering of favourable cvd risk factorsmaterials and methods we analysed data of participants from theeurodiab prospective complications study a european t1d cohortrecruited in countries between were men with a meanage of ± years we studied seven cvd risk factors namely hba1csmoking bmi combined systolic and diastolic bp ldl cholesterolphysical activity pa and diet table cox proportional hazards analyses were used to calculate hazard ratios hr [95ci] of incident cvdfor each cvd risk factor adjusted for age sex retinopathy comparingthose in the most favourable tertiles with the least favourable two tertilesdiabetologiawe then scored each individual by the number of risk factors for whichthey occupied the most favourable tertilesresults there were incident cvd cases after a mean followup of± years multivariable cox models showed that participants withthe most favourable hba1c57 [39mmolmol] had a significantlylower cvd risk hr [95ci] [] than the least favourabletwo tertiles nonsignificant inverse associations were found withfavourable bmi [] pa [] diet score[] and bp [] no associations were foundwith smoking or ldlcholesterol greater clustering of favourablecvd risk factors was associated with a lower risk of cvd in univariatemodels with a significant linear trend in multivariate models the resultswere partly attenuated with the lowest hr of [ ] in peoplewith clustering of favourable cvd risk factors tableconclusion greater clustering of favourable cvd risk factors was associated with a lower risk of incident cvd in people with t1d with a doseresponse relationship hba1c remained the most protective factor againstcvd in t1d targeting combined risk factors could be more effective inpreventing cvd risk than targeting single risk factorssupported by welcome trust the european community and diabetesukdisclosure s soulimane nonebidirectional association between type diabetes and obstructivesleep apnoea a metaepidemiological studyt karagiannis1 e athanasiadou1 a tsapas12 e bekiari11clinical research and evidencebased medicine unit aristotleuniversity of thessaloniki thessaloniki greece 2harris manchestercollege university of oxford oxford uk and aims individual epidemiological studies suggest acomplex relationship between type diabetes and obstructive sleepapnea we aimed to assess whether there is a bidirectional associationbetween the two conditions by conducting a metaanalysis of longitudinalcohort studiesmaterials and methods we included cohort studies that evaluated theassociation between type diabetes and obstructive sleep apnea in eitherdirection published until january we pooled cohortspecific estimates by means of random and fixed effects metaanalyses and calculatedodds ratios ors with confidence intervals cis to measure theassociation of prevalent obstructive sleep apnea with incident type diabetes and of prevalent type diabetes with incident obstructive sleepapnearesults out of records identified through the search cohortstudies were included in the metaepidemiological analysis ten studiesevaluated the association between prevalent obstructive sleep apnea andincident type diabetes one study assessed the association betweenprevalent type diabetes and incident obstructive sleep apnea while fourstudies evaluated a bidirectional association duration of study followupranged between and years median years the random effectsmetaanalysis for prevalent obstructive sleep apnea and incident type diabetes patients yielded an or of ci to 0cdiabetologiaresults were consistent in the fixed effects metaanalysis figureprevalent type diabetes increased the odds of incident obstructive sleepapnea patients with an or of ci to and ci to for the randomeffects and fixedeffects metaanalysis respectively metaanalyses of effect estimates adjusted forconfounding factors were similar to those of the main analysisconclusion pooled evidence from large cohort studies suggests thatpresence of obstructive sleep apnea at baseline is associated withincreased risk for developing type diabetes while presence of type diabetes is associated with increased risk for developing obstructive sleepapnea thus effective management of either condition could preventdevelopment of the otherfigure odds ratio for developing type diabetes in patients with obstructive sleep apnea versus those without obstructive sleep apneaalzheimer hr [ ic ] vascular dementia hr [ ic ] and nonvascular dementia hr [ ic ] when a 3years landmark analysis was conducted the associations remained similar for vascular and nonvascular dementia but disappeared for alzheimer™s diseasesconclusion the association of t2d with neurodegenerative diseasesdiffer by type of dementia the strongest detrimental association wasobserved for vascular dementia moreover t2d patients with polycaemic control have an increased risk of developing vascular andnonvascular dementiadisclosure c celismorales nonesupported by greece and the european social fund esfdisclosure t karagiannis noneglycated haemoglobin type diabetes and the links to dementia andits major sub types findings from the swedish national diabetesregisterc celismorales1 s franz©n2 am svensson3 n sattar1 sgudbjornsdottir21institute of cardiovascular and medical sciences university ofglasgow glasgow uk 2department of molecular and clinicalmedicine university of gothenburg gothenburg sweden 3swedishnational diabetes register gothenburg sweden and aims type diabetes t2d has been associated withhigh dementia risk however the links to different dementia subtypes isunclear we examined to what extent t2d associated with alzheimervascular and non vascular dementia incidence and whether such associations differed by glycaemic controlmaterials and methods in this swedish national diabetes registerstudy we included patients with t2d and matchedcontrols the outcomes were incidence of alzheimer vascular and nonvascular dementia the association of t2d with dementia was stratifiedby baseline glycated haemoglobin hba1c concentrations cox regression was used to study the excess risk of outcomesresults the followup median years t2d patientsand controls developed dementia the strongest association was observed for vascular dementia here patients with t2d had ahr of [ ci ] compared to controls the association oft2d with nonvascular dementia was more modest hr [ ci ] however risk of alzheimer was lower in t2d patientscompared to controls hr [ ci ] when the analyseswere stratified by circulating concentrations of hba1c a doseresponseassociation was observed compare to patients with t2d with hba1c mmolmol those with hba1c mmolmol had a higher risk of 0cop news on the insulin secretion frontwhat makes beta cells 1st responders and are they temporallyconsistentv kravets we schleicher jm dwulet am davis rkpbenningerbioengineering university of colorado aurora usa and aims calcium ca2 uptake drives glucosestimulated insulin secretion from the pancreatic cells functionalsubpopulations of cells disproportionally control the oscillatory phaseof ca2 uptake which is disrupted with ageing and in diabetes less isknown about cells which impact the 1st phase of ca2 uptake disruptedin early diabetes here we determine whether œ1stresponder cells thatlead the 1st phase of ca2 uptake are the same as œhub cells that coordinate oscillatory ca2 2nd phase we study what makes cell a1st responder and whether 1st responders are a transient state or a distincttemporally stable subpopulationmaterials and methods we used mipcreer gcamp6s mouse modelwhich expresses ca2sensitive gfp specifically in cells weperformed simultaneous recording of ca2 dynamics and gap junctionpermeability in individual islets we stimulated islets with glucosekatp channel blocker glibenclamide and kcl based on ca2 dynamicswe defined the of cells responding to the glucose stimulation soonerthan the rest of the islet as œ1st responders and the of cellsresponding slower as œlast responders we tested their temporal consistency over and hours we used laser ablation to remove specificcells from the islet we performed computational modelling of the isletelectrophysiologyresults we found that ca2 wave coordination of the 1st responders wasnot greater than the isletaverage and hence they are not overlapping withhighlycoordinated œhub cells in fact according to our gap junctionpermeability data 1st responders had lower than average electricalcoupling p00157 furthermore our computational model showedlower electrical coupling conductance in both 1st and last respondersp00447 p00279 this may be explained by our finding that1st responders are located at the islet™s periphery at ± of the islet™sradius we found 1st responders to be consistent under glibenclamidestimulation cells which respond first to the glucose remained in the15th percentile of the time response distribution when stimulated withglibenclamide sem this is consistent with our computationalresults 1st responders had lower katp conductance hence highermembrane depolarization probability p00086 glucose elevationswith 1h period showed that 1st responders remained consistent withreaction time within the of the reaction time distribution for all cellswith an elevation period of hours their reaction time shifted to thesecond quartile of the distribution and with hours to the medianunlike 1st responders last responder cells were not consistent at any timeinterval ablation of the 1st responders discoordinated but did notdisrupt the ca2 response of the islet a different cell took over the roleof the 1st responder postablation this new 1st responder was a cell whichoriginally preablation was within a leading 7th percentile of the timeresponse distribution sem conclusion in conclusion 1st responders are distinct from œhub cellsubpopulation have higher membrane depolarization probability and areless strongly coupled to other cells after the laser ablation of a1st responder new 1st responder taking on it™s role comes from a poolof original leading cells while initially consistent over a short 1h periodof time 1st responders may be losing temporal consistency over longertime periodssupported by nr01 dk102950 dk106412 jdrf 3pdf2019741andisclosure v kravets nonediabetologiabetaarrestin is absolutely required for the potentiation of insulinsecretion by gipma ravier1 j obeid1 m leduc1 s costes1 p gilon2 s dalle1 gbertrand11igf univ montpellier cnrs inserm montpellierfrance 2universit© catholique de louvain brussels belgium and aims the scaffold protein betaarrestin2 arrb2 isknown to uncouple g protein coupled receptors gpcrs from the gprotein and to recruit new signaling pathways such as the erk12pi3k fak‹¯ in non beta cells arrb2 interacts with a wide rangeof gpcrs but its interaction with the gip receptor gipr is still unclearour aim is to determine if arrb2 is involved in the signaling of thegipr in pancreatic beta cellsmaterials and methods the experiments were carried out in beta cellsfrom fivemonthold arrb2 and arrb2 male mice camp productioncampsepac endogenous pka akar3 and erk12 ekaractivations [ca2] in the cytosol [ca2]c fura2lr and in the endoplasmic reticulum [ca2]er d4er were assessed by live cell imagingin mouse pancreatic beta cells epac2 epac2gfp recruitmentbeneath the plasma membrane was monitored by total internal reflectionfluorescence microscopy factin depolymerisation was evaluated byphalloidin staining alexa fluor 488conjugated phalloidin and thep h o s p h o r y l a t i o n o f f o c a l a d h e s i o n k i n a s e f a k b yimmunofluorescenceresults insulin secretion from arrb2 islets was reduced by compared to arrb2 islets in response to gip 100pm10nm p001when arrb2 arrb2gfp was reexpressed in arrb2 beta cells insulin secretion in response to gip was restored to a similar level thanin arrb2 islets surprisingly upon gip stimulation 10pm10nm thecamp production pka activation and epac2 recruitment were similarin arrb2and arrb2 beta cells both [ca2]c and [ca2]er remainedcomparable finally the activation of erk12 was also similarin arrb2 and arrb2 beta cells by contrast the factin depolymerisationinduced by 10nm gip was significantly reduced p001 in arrb2 beta cells pi3kγ and fak have been reported to be involved in factindepolymerisation in response to gip and glucose respectively and to berequired for optimal insulin secretion as expected the pi3kγ inhibitoras604850 1μmoll reduced factin depolymerisation p001by gip stimulation in arrb2 beta cells but no additional effect wasobserved in arrb2 beta cells moreover gipinduced fak activationwas also reduced by in arrb2 beta cellsconclusion our study revealed that arrb2 is required for the potentiation of insulin secretion by gip through factin depolymerisation probably via fak activation and pi3kγ recruitment but independently fromthe canonical camp signalling pka and epac2 and the erk12 pathway therefore any variation in the expression of arrb2 as observed indiabetic states should functionally affect the incretin effect produced bygipsupported by soci©t© francophone du diabete sfddisclosure ma ravier nonepancreatic beta cellselective deletion of the mitofusins and mfn1and mfn2 impairs glucosestimulated insulin secretion in vitro andin vivoga rutter1 e geiadou1 t rodriguez2 c muralidharan3 mmartinez3 p chabosseau1 a tomas1 g carrat1 a di gregorio2 ileclerc1 ak linnemann31cell biology functional genomics faculty of medicine imperialcollege london london uk 2national heart and lung instituteimperial college london london uk 3center for diabetes andmetabolic diseases indiana university school of medicineindianapolis usa 0cdiabetologia and aims mitochondrial metabolism of glucose is essential for the initiation of insulin release from pancreatic beta cellsalthough altered in subjects with type diabetes whether mitochondrialultrastructure and the proteins controlling the fission and fusion of theseanelles are important for glucose recognition is unclear here wegenerated mice with beta cellselective adultrestricted deletionof mfn1 and mfn2 essential for mitochondrial fusion and studied theimpact on insulin secretion and glucose homeostasis in vivo and in vitromaterials and methods c57bl6 mice bearing mfn1 and mfn2 alleleswith floxp sites were crossed to transgenic animals carrying aninducible cre recombinase under pdx1 promoter control pdxcreertrecombination was achieved by daily tamoxifen injections for one weekislets were isolated and used for live beta cell fluorescence imaging ofcytosolic cal520 or mitochondrial rgeco free ca2 concentrationand membrane potential tetramethyl rhodamine methyl ester tmrmusing spinning disc confocal microscopy nikon ti2 mitochondrialnetwork characteristics were quantified using super resolution fluorescence zeiss lsm and transmission electron microscopy intravitalimaging was performed in mice injected with an adenoassociated virusto express the cytosolic ca2 sensor gcamp6s selectively in beta cellsunder the control of the rat insulin promoter using multiphoton microscopy leica tcs sp8 dive blood flow through the islet was visualisedsimultaneously after injection of fluorescent albumin647results mitochondrial length was sharply to ± of controlsp00001 reduced in the mfn12 ko mice and these animals displayedhigher fasting glycaemia than control littermates at weeks vs mmoll p005 in vivo an increase in circulating glucose levelswas also observed p005 at min and p001 at min and wasassociated with a substantial fivefold decrease in plasma insulin min p00001 postintraperitoneal glucose injection mitochondrialca2 accumulation and membrane potential were significantly reducedp001 in response to high glucose in the ko animals examined byintravital imaging of the exteriorised pancreas antiparallel changes incytosolic ca2 and mitochondrial membrane potential observed incontrol animals were largely suppressed after mfn12 deletionconclusion mitochondrial fusion and fission cycles are essential in thebeta cell to maintain normal mitochondrial bioenergetics and glucosesensing both in vitro and in the living mouse such cycles may bedisrupted in some forms of diabetes to impair mitochondrial functionand consequently insulin secretio

cancer291

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: "annual meeting of the european associationfor the study of diabetes“ september sindex of oral presentationsop diabetes complications new insights from cutting edge epidemiologyop news on the insulin secretion frontop insulin sensitivity and biomarkersop central actions in diabetesop glucoselowering therapies and the liverop uncomplicating the pathogenesis of diabetes complications inhumansop smoke on the water is bat still hotop charting human beta cell failure in type diabetesop novel agents in type diabetesop developing better insulinsop from diagnostics to the endstage of diabetic kidney diseaseop nafld is it all about the liverop diabetic retinopathy see what's newop taking the long view of diabetesop pregnancy in diabetes prediction and outcomesop signals and networks in beta cell failureop broken heart in diabetesop unlocking the potential of digital healthop decoding the heritable basis of type diabetesop feeding the pipeline from drugs to surgeryop sglt2 inhibitors at the heart of the matterop new treatments for nafld hope or hypeop addressing potential new treatments of diabetic kidney diseaseop glucagon and hormones beyondop incretin based therapiesop unusual forms of diabetesop macrovascular complications and beyondop linking inflammation to metabolismop what's new in automated insulin deliveryop understanding the mechanisms of diabetic kidney diseaseop novel aspects of diabetic neuropathyop reducing the burden of hypoglycaemiaop what exercise doesop back to the future risk markers in diabetesop diet not only quantity mattersop on the road to human islet failure in type diabetesop a deep dive into the mechanisms of diabetesop triggers and drivers of beta cell failure in type diabetesop gastroentero pancreatic factors anoids mice and menop new aspects of novel therapiesop fatty mattersop diabetes care is expensiveop developing beta cellsop modelling metabolism lessons from animalsop diabetic foot new developments in wound healingop challenges in delivering diabetes care new solutionsop thinking about diabetes complications in the brainop insulin secretion in various subgroupsindex of poster sessionsps diabetes and early deathps living with chronic diabetes complicationsps micro and macrovascular complications of diabetesps global view on diabetes complicationsps type diabetes treatment irlps unusual forms of diabetesps molecular insights into glucose abnormalitiesps pathophysiology of glucose homeostasisps the inner workings of the pancreasps islets and antibodies in type diabetesps markers and phenotypes of glucose traitsps global aspects on the epidemiology of type diabetesps risk factors for type diabetesps prevalence of type diabetes around the worldps risk factors in type diabetesps islet transplants revisitedps islets in type diabetes new playersps beta cells under stressps to live and let die a beta cell perspectiveps job description insulin secretionps further down the road to human islet failure in type diabetesps sitting and exercising does it allps the ins and outs of carbohydrate metabolismps pregnancy in vitro and in vivo studiesps pregnancy epidemiologyps pregnancy who is at riskps incremental studies on gut hormonesps the fundamentals of insulin resistanceps studies on insulin resistanceps treatment of hyperglycaemia in pregnancyps pancreatic hormonesps insulin secretion in mice and menps something more about obesityps more about metabolismps inflammation in type diabetesps models of prediabetes and diabetesps models of obesity and insulin resistanceps lipid metabolismps adipokine signallingps drugs and environment in obesityps weight loss interventionsps brain mattersps sglt2 inhibitors clinical aspectsps different aspects of sglt2 inhibitorsps basic aspects of incretinbased therapiesps clinical outcome of incretinbased therapies 0cdiabetologiaps glycaemic control and incretinbased therapiesps various clinical aspects of incretinbased therapiesps various aspects of nutrition and dietps oral therapies metformin sensitizers and other nonsecretagoguesps novel agents to treat diabetes and its consequencesps novel glucoselowering agents in type diabetesps key issues in improving outcomes in people with diabeteseducation and costsps how to improve diabetes careps the impact of new basal insulinsps insulin therapy real world studiesps insulin therapy fast acting insulin analoguesps the challenges of insulin therapy in type diabetesps different aspects of insulin therapyps the continued advance of continuous glucose monitoringps insulin pump therapyps automated insulin deliveryps the varied use of technologies in type diabetesps novel applications of technology in diabetesps novel therapies to reduce hypoglycaemiaps mechanisms and clinical consequences of hypoglycaemiain diabetesps emerging topics in hypoglycaemiaps investigating diabetes distress and depressionps aspects of quality of life and well beingps digital health in type diabetesps is telehealth the answer to improving care in diabetesps predicting prognosis of diabetic kidney diseaseps clinical aspects of diabetic kidney diseaseps the rock and role of experimental kidney diseaseps new tools to view diabetic retinopathyps diabetic retinopathy screening and interventionps focus on diabetic foot ulcersps hypertension and vascular diseaseps cure the pain of diabetic neuropathyps understanding clinical neuropathyps from artificial intelligence to treatment of diabetic footps from biomarkers to genetics of diabetic kidney diseaseps treatment of nafld and diabetes from food to pharmacologyps mechanisms and prevalence of nafldps lipids everywhere lipid metabolism in the liver and the heartps all about coronary arteries and diabetesps lipids and glucose not so good for the heartps cardiac complications of mice rats and cellsps atherosclerotic complications stemming from cells to the kidneyps stiff arteries and how to avoid themps cardiac function and dysfunctionps cardiovascular complications in humans through and throughps diabetes and neoplasiaps contemplating cognitive dysfunction in diabetesps endothelial cell circulation and the heartps tradition no nontraditional complications of diabetes 0cdiabetologiaop diabetes complications new insightsfrom cutting edge epidemiologycirculating metabolites significantly improve the prediction of renaldysfunction in type diabetesm scarale1 s de cosmo1 c prehn2 f schena3 j adamski2 vtrischitta4 c menzaghi11fondazione irccs œcasa sollievo della sofferenza san giovannirotondo italy 2helmholtz zentrum m¼nchen germany 3universityof bari bari italy 4sapienza university roma italy and aims chronic kidney disease ckd mainly indicated by a reduced glomerular filtration rate gfr remains one of theleading causes of reduced lifespan in patients with type diabetest2d discovering novel biomarkers able to predict low gfr will helpidentify highrisk patients to be targeted to more aggressive and burdensome preventive and treatment strategiesmaterials and methods we measured serum metabolites byabsoluteidqtm p180 kit biocrates life sciences ag innsbruckaustria and investigated their association with egfr calculated with theckdepi formula in a discovery sample of patients with t2d cases and controls with egfr60 and ‰¥70mlmin173m2 respectively a threshold p value of 28x104 ie followingbonferroni's rule was used as statistical significance in a model comprising age sex smoking bmi hba1c diabetes duration albumintocreatinine ratio acr and ongoing treatments metabolites associatedin the discovery sample were validated threshold p value of 005numberof surviving validation metabolites in a second cohort comprising diabetic patients cases and controls for egfr60 or ‰¥70mlmin173m2 respectively standardized values of each validated metabolitesweighted for the effect size ie observed in the discovery samplewere then summed up in a metabolic score metscore to be used as agfr prediction tool to this purpose metscore was used on top of anestablished clinical model comprising sex age bmi hba1c and acrand then discrimination [δ area under the receiver operating characteristic roc curve auc and the relative integrated discriminationimprovement ridi] and reclassification [the categoryfree net reclassification index cnri] measures were evaluatedresults thirteen metabolites six acylcarnitines six biogenic amines andone amino acid were independently associated to low egfr [ors range for 1sd increase p range 13x107 20x104] in the discoverysample all of them but one a biogenic amine were validated in thereplication sample [ors range for 1sd increase p range32x1018 43x106 below the threshold of 0051242x103] theauc of the abovementioned clinical model was and in the discovery the replication and the pooled sample respectively the addition of metscore on top of the clinical model improvedboth discrimination and reclassification measures in the discovery δauc4 p14x103 ridi29 p20x1011 ½cnri54p15x1014 the replication δ auc39 p16x103 ridi28p38x108 ½cnri30 p22x1010 and the pooled samples δauc39 p40x106 ridi29 p22x1017 ½cnri35p19x108conclusion we have discovered and validated metabolites that arestrongly associated with low egfr in patients with t2d a metscorecomprising these metabolites improves an established clinical prediction model of low egfr in terms of both discrimination and reclassification encouraged by these findings we are now investigating the ability ofmetscore to improve prediction of gfr decline in prospective cohorts oft2d with the aim of improving risk stratification and therefore refiningprevention efforts of kidney dysfunction in diabetic patientssupported by italian ministry of health rf201302356459disclosure m scarale noneassociation between insulinlike growth factor binding protein2 andinsulin sensitivity metformin and mortality in patients with newlydiagnosed type diabetesmr kristiansen12 js nielsen12 i brandslund3 da olsen3 jvstidsen2 sk nicolaisen4 r hjortebjerg25 j frystyk561danish centre for strategic research in type diabetes dd2odense 2steno diabetes center odense odense 3irs lillebaelthospital biochemistry and immunology vejle 4department ofclinical epidemiology aarhus 5department of clinical researchuniversity of southern denmark odense 6department ofendocrinology odense university hospital odense denmark and aims insulinlike growth factor binding protein2igfbp2 is engaged in metabolism circulating concentrations ofigfbp2 are positively correlated to insulin sensitivity overexpressionof igfbp2 protects against obesity and diabetes in mice and metforminincreases igfbp2 gene expression indicating that igfbp2 is a target ofmetformin action interestingly igfbp2 appears to predict mortalityindependently of insulin sensitivity this study aimed to investigate theassociation between indices of insulin sensitivity metformin treatmentand mortality in patients with newly diagnosed type diabetes t2dmaterials and methods in this crosssectional study we included newlydiagnosed patients with t2d enrolled in the danish centre for strategicresearch in type diabetes dd2 cohort patients were continuouslyenrolled from to throughout denmark and followed usingdanish healthcare registries unbound fractions of igfbp2 were determinedin serum from fasting drug na¯ve n864 and metformin treated ‰¥ twoprescriptions months prior enrollment patients n558 using an inhouseassay developed on the simoa platform values are given as medians iqrassociation was analyzed using a pearson™s regressioncox regression amultivariable model was used to adjust for age bmi and homasresults a total of patients with median age of medianbmi of and median diabetes duration of yearswere included igfbp2 level was positively correlated with homasr2026 and p0005 and inversely correlated with cpeptide r2018and p0005 both associations persisted following adjustments for ageand bmi the igfbp2 level in metformin treated patients was slightlylower ngml than in drug na¯ve patients ngml p0026 a total of patients suffered from one or morecomorbidities from charlson comorbidity index their igfbp2 levelswere higher than patients with no comorbidity vs ngml p0001 during a median of years offollowup a total of patients died igfbp2 level was significantly higher at baseline in patients that died vs not died vs ngml p0001 igfbp2 was associated withmortality with a hazard ratiohr ci per doubling in proteinconcentration of p0001 this association was notobserved when analyzing patients without comorbidities but was significant in patients with other comorbidities hr p0001conclusion this is the first larger study to confirm that igfbp2 isassociated with indices of insulin sensitivity but is not largely affectedby metformin treatment interestingly increased igfbp2 level is associated with high mortality rates but the association was mainly driven bythe presence of comorbidities at baselinesupported by university of southern denmark and region of southerndenmarkdisclosure mr kristiansen nonebuilding clinical risk score systems for predicting allcause andcardiovascularspecific mortality among type diabetes patientscs liu1 tc li2 cc lin1 ci li11china medical university hospital taichung 2china medicaluniversity taichung taiwan 0c and aims no prior prediction model for mortality considered the effect of glycemic variability and blood pressure variabilitywhich have been broadly reported as the important clinical predictors ofmortality especially in diabetes patients the aim of this study was todevelop and validate risk score systems with considering the effects ofglycemic and blood pressure variability on allcause and cardiovascularspecific mortality in persons with type dmmaterials and methods this is a retrospective cohort study consistingof type diabetic patients aged years during allparticipants were randomly allocated into two groups derivation andvalidation sets in ratio and were followed up until death or august cox proportional hazards regression were used to develop allcauseand cardiovascularspecific mortality prediction model prediction modelperformance was assessed by the area under the receiver operating characteristics curve aurocresults overall deaths were identified after a mean of years offollowup the prediction accuracy measured by auroc of and 15year allcause mortality based on a model containing the identifiedtraditional risk factor biomarkers and variability in fasting plasmaglucose and hba1c and diastolic blood pressure variability were and respectively in derivation set and the corresponding values forcardiovascularspecific mortality were and respectively the predictionaccuracy in the validation set for allcause mortality were and respectively and for cardiovascularspecific mortality were and respectivelyconclusion our prediction model considering glycemic and blood pressure variability had good accuracy of prediction of cardiovascularspecific and allcause mortality in patients with type diabetessupported by ministry of science and technology of taiwandisclosure c liu noneincident cardiovascular disease by clustering of favourable riskfactors in type diabetes the eurodiab prospectivecomplications studys soulimane1 yd vogtschmidt12 m toeller3 b balkau4 nchaturvedi5 jh fuller6 ss soedamahmuthu121department of medical and clinical psychology center of research onpsychological and somatic disorders corps tilburg universitynetherlands 2institute for food nutrition and health university ofreading reading uk 3heinrichheineuniversity d¼sseldorfd¼sseldorf germany 4clinical epidemiology universit© parissaclayuvsq inserm cesp villejuif france 5institute of cardiovascularscience university college of london london uk 6department ofepidemiology and public health eurodiab london uk and aims the incidence of cardiovascular diseases cvdis up to eight times higher in people with type diabetes t1d greaterclustering of adverse risk factors is thought to contribute to excess cvdrisks in type diabetes though not explored in t1d the aim of this studywas to examine a cvd risk reduction for those in the most favourablethird of individual risk factors compared to the least favourable two thirdsand b cvd risk reduction by clustering of favourable cvd risk factorsmaterials and methods we analysed data of participants from theeurodiab prospective complications study a european t1d cohortrecruited in countries between were men with a meanage of ± years we studied seven cvd risk factors namely hba1csmoking bmi combined systolic and diastolic bp ldl cholesterolphysical activity pa and diet table cox proportional hazards analyses were used to calculate hazard ratios hr [95ci] of incident cvdfor each cvd risk factor adjusted for age sex retinopathy comparingthose in the most favourable tertiles with the least favourable two tertilesdiabetologiawe then scored each individual by the number of risk factors for whichthey occupied the most favourable tertilesresults there were incident cvd cases after a mean followup of± years multivariable cox models showed that participants withthe most favourable hba1c57 [39mmolmol] had a significantlylower cvd risk hr [95ci] [] than the least favourabletwo tertiles nonsignificant inverse associations were found withfavourable bmi [] pa [] diet score[] and bp [] no associations were foundwith smoking or ldlcholesterol greater clustering of favourablecvd risk factors was associated with a lower risk of cvd in univariatemodels with a significant linear trend in multivariate models the resultswere partly attenuated with the lowest hr of [ ] in peoplewith clustering of favourable cvd risk factors tableconclusion greater clustering of favourable cvd risk factors was associated with a lower risk of incident cvd in people with t1d with a doseresponse relationship hba1c remained the most protective factor againstcvd in t1d targeting combined risk factors could be more effective inpreventing cvd risk than targeting single risk factorssupported by welcome trust the european community and diabetesukdisclosure s soulimane nonebidirectional association between type diabetes and obstructivesleep apnoea a metaepidemiological studyt karagiannis1 e athanasiadou1 a tsapas12 e bekiari11clinical research and evidencebased medicine unit aristotleuniversity of thessaloniki thessaloniki greece 2harris manchestercollege university of oxford oxford uk and aims individual epidemiological studies suggest acomplex relationship between type diabetes and obstructive sleepapnea we aimed to assess whether there is a bidirectional associationbetween the two conditions by conducting a metaanalysis of longitudinalcohort studiesmaterials and methods we included cohort studies that evaluated theassociation between type diabetes and obstructive sleep apnea in eitherdirection published until january we pooled cohortspecific estimates by means of random and fixed effects metaanalyses and calculatedodds ratios ors with confidence intervals cis to measure theassociation of prevalent obstructive sleep apnea with incident type diabetes and of prevalent type diabetes with incident obstructive sleepapnearesults out of records identified through the search cohortstudies were included in the metaepidemiological analysis ten studiesevaluated the association between prevalent obstructive sleep apnea andincident type diabetes one study assessed the association betweenprevalent type diabetes and incident obstructive sleep apnea while fourstudies evaluated a bidirectional association duration of study followupranged between and years median years the random effectsmetaanalysis for prevalent obstructive sleep apnea and incident type diabetes patients yielded an or of ci to 0cdiabetologiaresults were consistent in the fixed effects metaanalysis figureprevalent type diabetes increased the odds of incident obstructive sleepapnea patients with an or of ci to and ci to for the randomeffects and fixedeffects metaanalysis respectively metaanalyses of effect estimates adjusted forconfounding factors were similar to those of the main analysisconclusion pooled evidence from large cohort studies suggests thatpresence of obstructive sleep apnea at baseline is associated withincreased risk for developing type diabetes while presence of type diabetes is associated with increased risk for developing obstructive sleepapnea thus effective management of either condition could preventdevelopment of the otherfigure odds ratio for developing type diabetes in patients with obstructive sleep apnea versus those without obstructive sleep apneaalzheimer hr [ ic ] vascular dementia hr [ ic ] and nonvascular dementia hr [ ic ] when a 3years landmark analysis was conducted the associations remained similar for vascular and nonvascular dementia but disappeared for alzheimer™s diseasesconclusion the association of t2d with neurodegenerative diseasesdiffer by type of dementia the strongest detrimental association wasobserved for vascular dementia moreover t2d patients with polycaemic control have an increased risk of developing vascular andnonvascular dementiadisclosure c celismorales nonesupported by greece and the european social fund esfdisclosure t karagiannis noneglycated haemoglobin type diabetes and the links to dementia andits major sub types findings from the swedish national diabetesregisterc celismorales1 s franz©n2 am svensson3 n sattar1 sgudbjornsdottir21institute of cardiovascular and medical sciences university ofglasgow glasgow uk 2department of molecular and clinicalmedicine university of gothenburg gothenburg sweden 3swedishnational diabetes register gothenburg sweden and aims type diabetes t2d has been associated withhigh dementia risk however the links to different dementia subtypes isunclear we examined to what extent t2d associated with alzheimervascular and non vascular dementia incidence and whether such associations differed by glycaemic controlmaterials and methods in this swedish national diabetes registerstudy we included patients with t2d and matchedcontrols the outcomes were incidence of alzheimer vascular and nonvascular dementia the association of t2d with dementia was stratifiedby baseline glycated haemoglobin hba1c concentrations cox regression was used to study the excess risk of outcomesresults the followup median years t2d patientsand controls developed dementia the strongest association was observed for vascular dementia here patients with t2d had ahr of [ ci ] compared to controls the association oft2d with nonvascular dementia was more modest hr [ ci ] however risk of alzheimer was lower in t2d patientscompared to controls hr [ ci ] when the analyseswere stratified by circulating concentrations of hba1c a doseresponseassociation was observed compare to patients with t2d with hba1c mmolmol those with hba1c mmolmol had a higher risk of 0cop news on the insulin secretion frontwhat makes beta cells 1st responders and are they temporallyconsistentv kravets we schleicher jm dwulet am davis rkpbenningerbioengineering university of colorado aurora usa and aims calcium ca2 uptake drives glucosestimulated insulin secretion from the pancreatic cells functionalsubpopulations of cells disproportionally control the oscillatory phaseof ca2 uptake which is disrupted with ageing and in diabetes less isknown about cells which impact the 1st phase of ca2 uptake disruptedin early diabetes here we determine whether œ1stresponder cells thatlead the 1st phase of ca2 uptake are the same as œhub cells that coordinate oscillatory ca2 2nd phase we study what makes cell a1st responder and whether 1st responders are a transient state or a distincttemporally stable subpopulationmaterials and methods we used mipcreer gcamp6s mouse modelwhich expresses ca2sensitive gfp specifically in cells weperformed simultaneous recording of ca2 dynamics and gap junctionpermeability in individual islets we stimulated islets with glucosekatp channel blocker glibenclamide and kcl based on ca2 dynamicswe defined the of cells responding to the glucose stimulation soonerthan the rest of the islet as œ1st responders and the of cellsresponding slower as œlast responders we tested their temporal consistency over and hours we used laser ablation to remove specificcells from the islet we performed computational modelling of the isletelectrophysiologyresults we found that ca2 wave coordination of the 1st responders wasnot greater than the isletaverage and hence they are not overlapping withhighlycoordinated œhub cells in fact according to our gap junctionpermeability data 1st responders had lower than average electricalcoupling p00157 furthermore our computational model showedlower electrical coupling conductance in both 1st and last respondersp00447 p00279 this may be explained by our finding that1st responders are located at the islet™s periphery at ± of the islet™sradius we found 1st responders to be consistent under glibenclamidestimulation cells which respond first to the glucose remained in the15th percentile of the time response distribution when stimulated withglibenclamide sem this is consistent with our computationalresults 1st responders had lower katp conductance hence highermembrane depolarization probability p00086 glucose elevationswith 1h period showed that 1st responders remained consistent withreaction time within the of the reaction time distribution for all cellswith an elevation period of hours their reaction time shifted to thesecond quartile of the distribution and with hours to the medianunlike 1st responders last responder cells were not consistent at any timeinterval ablation of the 1st responders discoordinated but did notdisrupt the ca2 response of the islet a different cell took over the roleof the 1st responder postablation this new 1st responder was a cell whichoriginally preablation was within a leading 7th percentile of the timeresponse distribution sem conclusion in conclusion 1st responders are distinct from œhub cellsubpopulation have higher membrane depolarization probability and areless strongly coupled to other cells after the laser ablation of a1st responder new 1st responder taking on it™s role comes from a poolof original leading cells while initially consistent over a short 1h periodof time 1st responders may be losing temporal consistency over longertime periodssupported by nr01 dk102950 dk106412 jdrf 3pdf2019741andisclosure v kravets nonediabetologiabetaarrestin is absolutely required for the potentiation of insulinsecretion by gipma ravier1 j obeid1 m leduc1 s costes1 p gilon2 s dalle1 gbertrand11igf univ montpellier cnrs inserm montpellierfrance 2universit© catholique de louvain brussels belgium and aims the scaffold protein betaarrestin2 arrb2 isknown to uncouple g protein coupled receptors gpcrs from the gprotein and to recruit new signaling pathways such as the erk12pi3k fak‹¯ in non beta cells arrb2 interacts with a wide rangeof gpcrs but its interaction with the gip receptor gipr is still unclearour aim is to determine if arrb2 is involved in the signaling of thegipr in pancreatic beta cellsmaterials and methods the experiments were carried out in beta cellsfrom fivemonthold arrb2 and arrb2 male mice camp productioncampsepac endogenous pka akar3 and erk12 ekaractivations [ca2] in the cytosol [ca2]c fura2lr and in the endoplasmic reticulum [ca2]er d4er were assessed by live cell imagingin mouse pancreatic beta cells epac2 epac2gfp recruitmentbeneath the plasma membrane was monitored by total internal reflectionfluorescence microscopy factin depolymerisation was evaluated byphalloidin staining alexa fluor 488conjugated phalloidin and thep h o s p h o r y l a t i o n o f f o c a l a d h e s i o n k i n a s e f a k b yimmunofluorescenceresults insulin secretion from arrb2 islets was reduced by compared to arrb2 islets in response to gip 100pm10nm p001when arrb2 arrb2gfp was reexpressed in arrb2 beta cells insulin secretion in response to gip was restored to a similar level thanin arrb2 islets surprisingly upon gip stimulation 10pm10nm thecamp production pka activation and epac2 recruitment were similarin arrb2and arrb2 beta cells both [ca2]c and [ca2]er remainedcomparable finally the activation of erk12 was also similarin arrb2 and arrb2 beta cells by contrast the factin depolymerisationinduced by 10nm gip was significantly reduced p001 in arrb2 beta cells pi3kγ and fak have been reported to be involved in factindepolymerisation in response to gip and glucose respectively and to berequired for optimal insulin secretion as expected the pi3kγ inhibitoras604850 1μmoll reduced factin depolymerisation p001by gip stimulation in arrb2 beta cells but no additional effect wasobserved in arrb2 beta cells moreover gipinduced fak activationwas also reduced by in arrb2 beta cellsconclusion our study revealed that arrb2 is required for the potentiation of insulin secretion by gip through factin depolymerisation probably via fak activation and pi3kγ recruitment but independently fromthe canonical camp signalling pka and epac2 and the erk12 pathway therefore any variation in the expression of arrb2 as observed indiabetic states should functionally affect the incretin effect produced bygipsupported by soci©t© francophone du diabete sfddisclosure ma ravier nonepancreatic beta cellselective deletion of the mitofusins and mfn1and mfn2 impairs glucosestimulated insulin secretion in vitro andin vivoga rutter1 e geiadou1 t rodriguez2 c muralidharan3 mmartinez3 p chabosseau1 a tomas1 g carrat1 a di gregorio2 ileclerc1 ak linnemann31cell biology functional genomics faculty of medicine imperialcollege london london uk 2national heart and lung instituteimperial college london london uk 3center for diabetes andmetabolic diseases indiana university school of medicineindianapolis usa 0cdiabetologia and aims mitochondrial metabolism of glucose is essential for the initiation of insulin release from pancreatic beta cellsalthough altered in subjects with type diabetes whether mitochondrialultrastructure and the proteins controlling the fission and fusion of theseanelles are important for glucose recognition is unclear here wegenerated mice with beta cellselective adultrestricted deletionof mfn1 and mfn2 essential for mitochondrial fusion and studied theimpact on insulin secretion and glucose homeostasis in vivo and in vitromaterials and methods c57bl6 mice bearing mfn1 and mfn2 alleleswith floxp sites were crossed to transgenic animals carrying aninducible cre recombinase under pdx1 promoter control pdxcreertrecombination was achieved by daily tamoxifen injections for one weekislets were isolated and used for live beta cell fluorescence imaging ofcytosolic cal520 or mitochondrial rgeco free ca2 concentrationand membrane potential tetramethyl rhodamine methyl ester tmrmusing spinning disc confocal microscopy nikon ti2 mitochondrialnetwork characteristics were quantified using super resolution fluorescence zeiss lsm and transmission electron microscopy intravitalimaging was performed in mice injected with an adenoassociated virusto express the cytosolic ca2 sensor gcamp6s selectively in beta cellsunder the control of the rat insulin promoter using multiphoton microscopy leica tcs sp8 dive blood flow through the islet was visualisedsimultaneously after injection of fluorescent albumin647results mitochondrial length was sharply to ± of controlsp00001 reduced in the mfn12 ko mice and these animals displayedhigher fasting glycaemia than control littermates at weeks vs mmoll p005 in vivo an increase in circulating glucose levelswas also observed p005 at min and p001 at min and wasassociated with a substantial fivefold decrease in plasma insulin min p00001 postintraperitoneal glucose injection mitochondrialca2 accumulation and membrane potential were significantly reducedp001 in response to high glucose in the ko animals examined byintravital imaging of the exteriorised pancreas antiparallel changes incytosolic ca2 and mitochondrial membrane potential observed incontrol animals were largely suppressed after mfn12 deletionconclusion mitochondrial fusion and fission cycles are essential in thebeta cell to maintain normal mitochondrial bioenergetics and glucosesensing both in vitro and in the living mouse such cycles may bedisrupted in some forms of diabetes to impair mitochondrial functionand consequently insulin secretio Answer:

Colon_Cancer

high throughput methods in biological and biomedical fields acquire alarge number of molecular parameters or omics data by a single experimentcombining these omics data can significantly increase the capability for recoveringfinetuned structures or reducing the effects of experimental and biological noise indataresultsfhclust for identifying patient subgroups from different omics information eg geneexpression mirna expression methylation in particular hierarchical structures of patientdata are obtained in each omic or view and finally their topologies are merged byconsensus matrix one of the main aspects of this methodology is the use of ameasure of dissimilarity between sets of observations by using an appropriate metricfor each view a dendrogram is obtained by using a hierarchical clustering based on afuzzy equivalence relation with łukasiewicz valued fuzzy similarity finally a consensusmatrix that is a representative information of all dendrograms is formed by combiningmultiple hierarchical agglomerations by an approach based on transitive consensusmatrix construction several experiments and comparisons are made on real data egglioblastoma prostate cancer to assess the proposed approachs fuzzy logic allows us to introduce more flexible data agglomerationtechniques from the analysis of scientific literature it appears to be the first time that amodel based on fuzzy logic is used for the agglomeration of multiomic data theresults suggest that fhclust provides better prognostic value and clinical significancecompared to the analysis of singleomic data alone and it is very competitive withrespect to other techniques from literaturekeywords multiomics data data integration hierarchical clustering fuzzy similarityfuzzy aggregation the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriatecredit to the original authors and the source provide a link to the creative commons licence and indicate if changes weremade the images or other third party material in this are included in the ™s creative commons licence unlessindicated otherwise in a credit line to the material if material is not included in the ™s creative commons licence and yourintended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directlyfrom the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40 the creativecommons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to the data madeavailable in this unless otherwise stated in a credit line to the data 0cciaramella bmc bioinformatics 21suppl page of nowadays high throughput methods in biological and biomedical fields acquire a largenumber of molecular parameters by a single experiment in particular such measuredparameters are collected in œomics datasets eg genomics transcriptomics methylomics among multiple measured parameters dna genome sequence rna expressionand dna methylation are representative instances for individually analysing suchdata several methodologies have been introduced in literature even though recentlya number of studies pointed out the best performance coming from the integration ofmultiomics data for instance analysing each omic or view in the machine learningjargon set separately fundamental patterns can be detected from data however somefinetuned structures such as cancer subtypes can be highlighted by both gene expression and dna methylation information so that multiomics analysis can reduce theeffects of experimental and biological noise in data from literature three kinds ofintegration methodologies emerge¢ early integration builds a single featurebased matrix by concatenating each omic¢ intermediate integration builds a joint representation of data given the views¢ late integration each omic is analysed separately and the solutions are integratedin general late integration methods and in particular clustering are preferred whenthe analysis combines continuous and discrete data together for a review of integrationapproaches and their comparisons the reader may refer to in this work a multiviewclustering methodology named fhclust is introduced see fig for its general schemafor identifying patient subgroups from different omics information or datasets eg geneexpression mirna expression methylation specifically for each omic dataset a fuzzybased hierarchical clustering approach is adopted and finally the results are mergedby consensus matrix the idea behind the proposed approach comes from observingthat a hierarchical clustering dendrogram can be associated with a łukasiewicz fuzzydataset ie view and applies a singleomic analysisfig proposed approach a data preparation b data normalization and feature selection c multiomicshierarchical agglomerations d data integration e clustering and visualization 0cciaramella bmc bioinformatics 21suppl page of similaritybased equivalence relation so that a consensus matrix that is the representative information of all dendrograms is derived by combining multiple hierarchicalagglomerations following an approach based on transitive consensus matrix constructionmethodscluster analysis or clustering is an unsupervised technique that aims at agglomerating aset of patterns in homogeneous groups or clusters [ ] hierarchical clustering hc isone of several different available techniques for clustering which seeks to build a hierarchyof clusters and it can be of two types namely agglomerative where each sample starts inits own cluster and pairs of clusters are merged as one moves up the hierarchy or divisivewhere all samples start in one cluster and splits are performed recursively as one movesdown the hierarchy thus hc aims at grouping similar objects into a cluster and werethe endpoint is a set of clusters where each cluster is distinct from each other and theobjects within each cluster are broadly similar to each other hc can be performed eitheron a distance matrix or raw data agglomerative hc starts by treating each observationas a separate cluster and it repeatedly executes the following two steps identifies thetwo clusters that are closest together and merges the two most similar clusters thisprocess continues until all the clusters are merged togetherthe main output of hc is a dendrogram which shows the hierarchical relationshipbetween the clusters distances many distance metrics have been developed and thechoice should be made based on theoretical concerns from the domain of studylater on it is necessary to determine how the distance is computed eg singlelinkagecompletelinkage averagelinkage as with distance metrics the choice of linkage criteria should be based on theoretical considerations from the application domainin nonfuzzy clustering or hard clustering data is divided into distinct clusters andeach data point can only belong to exactly one cluster in fuzzy clustering data pointscan potentially belong to multiple clusters for example in hard clustering given someparameters a œsymptom can be in a mutually exclusive way present or absent red orblue whereas in fuzzy clustering that œsymptom could simultaneously be of somegrade red and some other grade blue in fig a comparison between hard and fuzzycategorisation is shown the reader can refer to for a recent comparison betweenhard and fuzzy clustering in this work we introduce a data integration methodologybased on fuzzy concepts in particular we associate a dendrogram to a fuzzy equivalencerelation ie łukasiewicz valued fuzzy similarity so that a consensus matrix in a multiview clustering that is the representative information of all dendrograms can be obtainedfrom multiple hierarchical agglomerations [ ] the main steps of fuzzy agglomerationcan be summarised as follows characterisation of membership functions computation of a fuzzy similarity matrix or dendrogram for all models at a giventime construction of a consensus matrix for all hierarchical agglomerationsmembership functionswhen dealing with clustering tasks fuzzy logic fl permits to obtain a soft clusteringinstead of an hard clustering of data specifically data points can belong to more 0cciaramella bmc bioinformatics 21suppl page of fig hard vs fuzzy in symptom risk example a hard categorization b fuzzy categorizationthan one cluster simultaneously the fundamental concept in fl upon which all thesubsequent theory is constructed is the notion of fuzzy set a generalisation of a crisp setfrom classical set theorya fuzzy set generalises a crisp set by allowing its characteristic function ie itsmembership function assuming values in the interval [ ] rather than in the set in this way a given item belongs to the fuzzy set with a degree of truthranging from do not belong at all ie its membership function assumes value to completely belong ie the membership function assumes value in fl applications fuzzy sets make it possible to represent qualitative nonnumeric values ielinguistic variables such as high medium low for approximate reasoninginference or fuzzy control systems linguistic variables can be represented by fuzzy setsthrough a transformation step called fuzzification and it is achieved by using different types of membership functions representing the degree of truth to whicha given input sample belongs to a fuzzy set see œmembership functions sectionin supplementary material 0cciaramella bmc bioinformatics 21suppl page of fuzzy similarity matrixa measure of similarity or dissimilarity defines the resemblance between two samples orobjects similarity measure is a significant means for measuring uncertain informationfuzzy similarity measure is a measure that depicts the closeness among fuzzy sets and hasbeen used for dealing issues of pattern recognition and clustering analysisa binary fuzzy relation that is reflexive symmetric and transitive is known as a similarity relation fuzzy similarity relations are the generalisation of equivalence relationsin binary crisp relations to binary fuzzy relations in details a fuzzy similarity relationcan be considered to effectively group elements into crisp sets whose members are similar to each other to some specified grade and it is a generalization of classical equivalencerelation as described in œfuzzy similarity section in supplementary material in orderto introduce the fuzzy similarity in the following we focus on the properties of thełukasiewicz tnorm tl and the biresiduum in this way we obtain a fuzzy equivalencerelation that can be used for building dendrogram for more details in the derivation ofthese results see œfuzzy similarity section in supplementary materialdendrogram and consensus matrixif a similarity relation is mintransitive ie t min then it implies the existence ofthe dendrogram see œdendrogram and consensus matrix section in supplementarymaterial for details the mintransitive closure of a relation matrix r can be easilycomputed and the overall process is described in algorithm the last ingredient to accomplish an agglomerative clustering is a dissimilarity relationhere we considered the following result lemma letting r be a similarity relation with the elements rcid2x ycid3 ˆˆ[ ] and lettingd be a dissimilarity relation which is obtained from r bydx y ˆ’ rcid2x ycid3then d is ultrametric iif r is mintransitivein other words we have a onetoone correspondence between mintransitive similaritymatrices and dendrogram and between ultrametric dissimilarity matrices and dendrograms finally after the dendrograms have been obtained each time a consensus matrixie the representative information of all dendrograms is obtained by combining thetransitive closures ie maxmin operation the overall approach is described inalgorithm the overall workflow of the proposed approach is summarised in fig in particular for each omic data set xi a fuzzification step is adopted for obtaining thenew data set yi see supplementary material successively adopting a fuzzy similaritymeasure the similarity matrix si is computed and to guarantee the transitive closure ofthe matrix a new matrix ci is computed see algorithm finally all the ci matricesare collected for obtaining the consensus matrix a and the overall final dendrogram seealgorithm in fig we show an example that summarize a realistic agglomeration result we plotin figs 4abc three input hierarchies obtained on datasets that should be combinedin this case four sequences of patients are considered namely s1 s2 s3 and s4 respectively in fig 4d we show the final result by agglomerating dendrograms we observe that 0cciaramella bmc bioinformatics 21suppl page of fig workflow of the fuzzy based hierarchical clusteringthe output hierarchy contains clusters s1 s2 s3 and s1 s2 s3 s4 at different levels andeach of these clusters eg s1 s2 s3 are repeated at least in two out of the three inputdendrograms moreover it is worth stressing that the proposed approach based on theagglomeration of dendrograms can also be applied with commonly used metrics egeuclidean distance in fig we show a comparison between the dendrograms obtainedby using an euclidean metric and a similarity based approach ie łukasiewicz tnormrespectively in this realistic example we simulate three omic data sets with rows ienumber of patients and columns ie features we split the single datasets in twopartitions or clusters such that the first rows are random samples from a standard normal distribution with variance and the other rows have the same distribution withfig combination algorithm abc input dendrograms d combined hierarchy 0cciaramella bmc bioinformatics 21suppl page of fig crisp hierarchical clustering vs fuzzy based hierarchical clustering a dendrogram of euclidean basedhierchical clustering b dendrogram of similarity based hierachical clusteringalgorithm mintransitive closure input relation si output transitive relation ci sti elaborate compute sˆ— if sˆ—ielse ci sti si ˆª si —¦ sicid8 si replace si with sˆ—i sˆ—i and go to step i and the algorithm terminatesvariance obtaining a sort of an overlap we observe that both methods find two separated clusters but the similarity based approach in fig 5b permits to obtain a perfectseparation of the source partitionsresults and discussionin the following we describe the behaviour of the proposed methodology on multiomicsbenchmark datasetsalgorithm combination of dendrograms input ci ‰¤ i ‰¤ l l input similarity matrices dendrograms output similarity matrix dendrogram a aggregate the similarity matrices to a final similarity matrixa aggregate c1 c2 cla let aˆ— be the identity matrixb for each ci calculate e aˆ— aˆ— ˆª aˆ— —¦ cic if aˆ— is not changed a aˆ— and goto step else goto step 1b create the final dendrogram from aomics datasetswe consider multiomics cancer datasets available from the cancer genome atlastcga tcga is a large multiomic repository of data on thousands of cancerpatients all datasets contain three omics gene expression mirna expression and 0cciaramella bmc bioinformatics 21suppl page of table datasets description three omics are provided for each dataset respectively dna geneexpression mirna and methylationcases dnaoridatasetamlbiccoadgbmkirclihcluscskcmovsarclnrfmirnaorilnmethyorilnmultiomicsorilnrfrfrfthe number of features at each variable selection method is shown ori original variable dimension ln logarithm andnormalisation and rf random forest based on mean decrease gini indexdna methylation1 in table are summarised the main properties of the datasetsnamely acute myeloid leukemia aml breast invasive carcinoma bic colon adenocarcinoma coad glioblastoma multiforme gbm kidney renal clear cell carcinoma kirc liver hepatocellular carcinoma lihc lung squamous cell carcinomalusc skim cutaneous melanoma skcm ovarian serous cystadenocarcinoma ovsarcoma sarc the number of patients ranges from for aml to for bicmultiview clustering algorithmsfor validating the effectiveness of our model we compared it against several categories ofmultiview clustering algorithms2¢ kmeans and spectral clustering techniques ¢ lracluster it is a lowrank approximation based integrative probabilistic model¢ pins perturbation clustering for data integration and disease subtyping pinsis able to address subtype discovery as well as integration of multiple data types thealgorithm is built upon the resilience of patient connectivity and cluster ensembles toensure robustness against noise and biasto fast find the shared principal subspace across multiple data types¢ snf similarity network fusion snf allows for discovery of disease subtypesthrough integration of several types of highthroughput data on a genomic scale snfcreates a fused network of patients using a metric fusion technique and thenpartitions the data using spectral clustering snf appears to be the state of the art inthis area and has proven to be very powerful however the unstable nature ofkernelbased clustering makes the algorithm sensitive to small changes in molecularmeasurements or in its parameter settings¢ mcca multi canonical correlation analysis mcca which extends theapplication of canonical correlation analysis cca to more than two views is oneof the most widely used dimension reduction method for finding linear relationsbetween two or more multidimensional random variables1row data are available at httpacgtcstauacilmulti_omic_benchmarkdownloadhtml2httpsgithubcomshamirlabmultiomicscancerbenchmark 0cciaramella bmc bioinformatics 21suppl page of evaluation metricsin order to assess the performance of each method we adopt three evaluation metricsthat are the logrank test the enrichment of clinical labels in the clusters and the methods runtime the logrank test assumes that if clusters of patients have significantlydifferent survival they are different in a biologically meaningful way for the enrichmentof clinical labels in clusters six clinical labels are considered gender age at diagnosispathologic tumor pathologic metastases pathologic lymph nodes and pathologic stagethe four latter parameters are discrete pathological parameters measuring the progression of the tumor metastases and cancer in lymph nodes and the total progressionpathologic stage enrichment for discrete parameters was calculated using the χ2 testfor independence and for numeric parameters using kruskalwallis test not all clinicalparameters were available for all cancer types so a total of clinical parameters wereavailable for testing to derive a pvalue for the logrank test the χ2 test for independence the kruskalwallis test and the statistic for these three tests is assumed to have χ2distribution preprocessingtcga datasets were preprocessed as follows patients and features with more than missing values were removed and missing values were imputed using knearest neighborimputation the sequence features were logtransformed the features with highestvariance from geneexpression and methylation omics were selected in the mirna omicfeatures with zero variance were filtered all features were then normalized to have zeromean and standard deviation for methylation we selected the features with maximal variance in each dataset and also adopted the standard pipeline proposed in whose procedure filters out the probes from the x and y chromosomes or probes that areknown to have common snps at the cpg sitea further unsupervised variable selection step has been performed by using the meandecrease gini based on random forest the mean decrease in gini is the average of a variable total decrease in node impurity weighted by the proportion of samplesfig mean performance of the algorithms on ten multiomics cancer datasets the xaxis measures thedifferential survival between clusters mean log10 of logrank™s test pvalue and the yaxis is the meannumber of clinical parameters enriched in the clusters 0cciaramella bmc bioinformatics 21suppl page of fig performance of the algorithms on ten multiomics cancer datasets for each plot the xaxis measuresthe differential survival between clusterslog10 of logrank™s test pvalue and the yaxis is the number ofclinical parameters enriched in the clusters red vertical lines indicate the threshold for significantly differentsurvival pvalue cid2 reaching that node in each individual decision tree in the forest this is effectively a measure of how important a variable is for estimating the value of the target variable acrossall of the trees that make up the forest a higher mean decrease in gini indicates highervariable importance therefore the most important variables to the model is the highestin the plot with the largest mean decrease in gini values conversely the least importantvariable is the lowest in the plot with the smallest mean decrease in gini values by following this strategy we cutoff all those variables whose importance is zero the numberof variable cutoff at each step is summarised in table experimental resultsin the experiments for all methods the number of searched clusters is selected in therange [ ˆ’ ] to determine the number of clusters for a method we used the œelbowmethod to automatically pick out the optimal elbow rather than choose it manually weused as approximation the second derivative of a vector vv [i ] v [i ˆ’ ] ˆ’ 2v[ i] in particular we consider the index i that brings this expression to a maximum or minimum depending on whether v increases or decreases for all methods we adhered tothe guidelines for usage and parameter selection given by the developers in some casestable performance on ten multiomics number of clinical parameters enriched in the clustersfhclustcrisp hclustkmeansspectrallraclusterpinssnfmccaamlbiccoadgbmkirclihcluscskcmovsarcmeans 0cciaramella bmc bioinformatics 21suppl page of table performance on ten multiomics differential survival between clusters log10 of logrank™stest pvaluefhclustcrisp hclustkmeansspectrallraclusterpinssnfmccaamlbiccoadgbmkirclihcluscskcmovsarcmeanswhere no information was provided by the authors we devised parameter selection methods we performed the same process pipeline used in for evaluating the performanceof our method all methods were run on a multicore intelr xeonr cpu e52620v3 240ghz with gb ram in the following the obtained experimental results aredescribedfigure shows the average performance for multiomics data and for each singleomicseparately across all cancer types and fig shows the performance on the different cancer datasets all algorithms show quite similar performance in either differential survivalor enriched clinical parameters with respect to survival our fhclust method achievedthe overall best prognostic value sum of ˆ’log10 pvalues while pins and mcca ranked second and third respectivelyin table the differential survival between clusters mean ˆ’log10 of logrank™s testpvalue are reported spectral achieved the highest total number of significant clinical parameters with parameters fhclust along with lracluster and kmeansplaced themselves second with parameters snf achieved the third position with parameterswith respect to survival table fhclust outperformed its competitors achieving parameters mcca pins and snf have achieved good results with and enriched parameters respectivelywe also counted the number of datasets for which a method solution obtains significantly different survival these results are reported in table all methods that weredeveloped for multiomics data had at least four cancer types with significantly differentsurvival in this case fhclust and pins had different cancer subtypes for which itsclustering had significantly different prognosis fhclust spectral clustering and mccahad enrichment in cancer typeson average fhclust pins and mcca had better prognostic value but found lessenriched clinical labels as compared to spectral clustering methodtable for each benchmarked algorithm the number of cancer subtypes for which its clusteringhad significantly different prognosis first row and had at least one enriched clinical label secondrow are shownsignificant different survivalsignificant clinical enrichmentfhclustkmeansspectrallraclusterpinssnfmcca 0cciaramella bmc bioinformatics 21suppl page of table number of clusters chosen by the benchmarked algorithms on ten multiomics cancerdatasetsfhclustcrisp hclustkmeansspectrallraclusterpinssnfmccaamlbiccoadgbmkirclihcluscskcmovsarcmeansthe number of clusters found for each dataset are presented in table ranging from to because of the good methods performance in the previous analysis partitioning thedata into a relatively high number of clusters could indicate that clustering cancer patientsinto more clusters improves prognostic value and clinical significanceconcerning with methods computational burden their run times are reported intable fhclust takes on average seconds per dataset while spectral and snf gotlower timing the worst method takes roughly minutes per dataset see fig finally fig shows the benchmarked methods performance for singleomic datamoreover for each dataset and method the single omic that gave the best results forsurvival and clinical enrichment are also shown these results suggest that fhclust provides better prognostic value and clinical significance on multiomics data compared tothe analysis of singleomic data used separately nevertheless the interested reader mayrefer to the supplementary material for details on additional results concerning singleomics we also stress that the proposed method differently from other methods suchas snf does not need any hyperparameter tuning moreover clustering is embeddedin the data integration and vice versa and the use of fuzzy concepts ie tnormsfrom one hand permits to obtain a generalisation of the clustering approaches whereason the other hand gives the possibility to apply an inference system eg mamdanifor a quantitative and qualitative measure eg œhigh œmedium œlow in cancer riskassessmentsin this work we proposed a multiview clustering methodology for identifying patientsubgroups from different omics data in biological and biomedical fields combining theseomics data can significantly increase data mining capabilities one of the main aspects oftable runtime in seconds of the algorithms on ten multiomics cancer datasetsovfhclustcrisp hclustkmeansspectrallraclusterpinssnfmccacoadskcmbicamlgbmkirclihcluscsarcmeans 0cciaramella bmc bioinformatics 21suppl page of fig computational time comparisonsthis methodology is the use of a measure of dissimilarity between sets of observations byusing an appropriate metric and a consensus matrix that is a representative agglomerateinformation of all the dendrograms as emerged from the analysis of the scientific literature to the best of our knowledge our work concerns for the first time a model based onfuzzy logic used for the agglomeration of multiomic data the use of fuzzy logic allowsus to introduce more flexible data mining features also related to approximate reasoningseveral experiments and comparisons have been made on real data eg glioblastomaprostate cancer to assess the proposed methodology the results suggest that fhclustprovides better prognostic value and clinical significance compared to analysis of singleomic data alone fuzzy logic concepts and in particular membership functions permitsfig summarized performance of the algorithms across ten cancer datasets for each plot the xaxismeasures the total differential prognosis between clusters sum across all datasets of “log10 of logrank™s testpvalue and the yaxis is the total number of clinical parameters enriched in the clusters across all cancertypes a“c results for singleomic datasets d results when each method uses the single omic that achievesthe highest significance in survival e same with respect to enrichment of clinical labels 0cciaramella bmc bioinformatics 21suppl page of to develop a fuzzy inference model ie mamdani fuzzy cognitive maps for easilyobtaining a model for a quantitative and qualitative risk assessment of the cancer themodel based on approximate reasoning can be particularly useful for embedded devicesin future work it could be possible to improve results for multiomics analysis ina number of ways for instance more accurate feature selection algorithms couldbe adopted for improving the overall performance on one hand the integration oflabelled data could improve the feature selection step on the other hand some specific feature extraction strategies could be adopted indeed approaches based on thesignal analysis of gene expression data eg nonlinear principal component analysis compressive sensing could possibly further improve the performance [ ]in future it is possible to foresee a different weight for each omic data in order toobtain a more robust similarity and parametric similarity measures can be adoptedeg uninorm for generalizing the concept of and and or connections betweenclusterssupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12859020035676additional file supplementary materialabbreviationsfhclust fuzzyhierarchical clustering dna deoxyribonucleic acid rna ribonucleic acid hierarchical clustering hccrisp hc crisp hierarchical clustering fl fuzzy logic tcga the cancer genome atlas aml acute myeloid leukemia bicbreast invasive carcinoma coad colon adenocarcinoma gbm glioblastoma multiforme kirc kidney renal clear cellcarcinoma lihc liver hepatocellular carcinoma lusc lung squamous cell carcinoma skcm skim cutaneousmelanoma ov ovarian serous cystadenocarcinoma sarc sarcoma pins perturbation clustering for data integrationand disease subtyping lracluster low rank approximation based multiomics data clustering snf similarity networkfusion mcca multi canonical correlation analysisabout this supplementthis has been published as part of bmc bioinformatics volume supplement proceedings from the 13thbioinformatics and computational biology international conference bbcc2018 the full contents of the supplement areavailable online at httpsbmcbioinformaticsbiomedcentralcomssupplementsvolume21supplement10authors™ contributionsac originally designed the methodology ac and dn worked on the developing of the method and the design of theexperiments ac dn and as contributed for interpreting and for analysing the results all authors contributed forwriting the manuscript read and approved the final manuscriptfundingpublication costs are funded by a grant from the dipartimento di scienze e tecnologie università degli studi di napoliœparthenope tecniche di machine learning e soft computing per l™elaborazione di dati multivariati softmulan piciaramellaavailability of data and materialscode and data of the proposed approach are available on multiomicscancerbenchmark github repositoryethics approval and consent to participateno ethics approval was required for the studyconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1dipartimento di scienze e tecnologie università degli studi di napoli œparthenope centro direzionale c4 island naples italy 2hitachi rail sts via argine naples italypublished august 0cciaramella bmc bioinformatics 21suppl page of referencescamastra f di taranto md staiano a statistical and computational methods for genetic diseases an overviewcomput math meth med 20152015 id “serra a fratello m fortino v raiconi g tagliaferri r greco d mvda a multiview genomic data integrationmethodology bmc bioinformatics “rappoport n shamir r multiomic and multiview clustering algorithms review and cancer benchmark nucleicacids res “reddy ck aggarwal cc data clustering boca raton chapman and hallcrc camastra f ciaramella a son lh riccio a staiano a fuzzy similaritybased hierarchical clustering for atmosphericpollutants prediction lncs “ciaramella a staiano a on the role of clustering and visualization techniques in gene microarray data algorithmsbora dj gupta ak int j emerg trends technol comput sci “napolitano f pinelli m raiconi g tagliaferri r ciaramella a staiano a miele g clustering and visualizationapproaches for human cell cycle gene expression data analysis int j approx reason “ciaramella a cocozza sand clustering of genomic data neural netw “iorio f miele g napolitano f pinelli m raiconi g tagliafer

cancer292

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: high throughput methods in biological and biomedical fields acquire alarge number of molecular parameters or omics data by a single experimentcombining these omics data can significantly increase the capability for recoveringfinetuned structures or reducing the effects of experimental and biological noise indataresultsfhclust for identifying patient subgroups from different omics information eg geneexpression mirna expression methylation in particular hierarchical structures of patientdata are obtained in each omic or view and finally their topologies are merged byconsensus matrix one of the main aspects of this methodology is the use of ameasure of dissimilarity between sets of observations by using an appropriate metricfor each view a dendrogram is obtained by using a hierarchical clustering based on afuzzy equivalence relation with łukasiewicz valued fuzzy similarity finally a consensusmatrix that is a representative information of all dendrograms is formed by combiningmultiple hierarchical agglomerations by an approach based on transitive consensusmatrix construction several experiments and comparisons are made on real data egglioblastoma prostate cancer to assess the proposed approachs fuzzy logic allows us to introduce more flexible data agglomerationtechniques from the analysis of scientific literature it appears to be the first time that amodel based on fuzzy logic is used for the agglomeration of multiomic data theresults suggest that fhclust provides better prognostic value and clinical significancecompared to the analysis of singleomic data alone and it is very competitive withrespect to other techniques from literaturekeywords multiomics data data integration hierarchical clustering fuzzy similarityfuzzy aggregation the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriatecredit to the original authors and the source provide a link to the creative commons licence and indicate if changes weremade the images or other third party material in this are included in the ™s creative commons licence unlessindicated otherwise in a credit line to the material if material is not included in the ™s creative commons licence and yourintended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directlyfrom the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40 the creativecommons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to the data madeavailable in this unless otherwise stated in a credit line to the data 0cciaramella bmc bioinformatics 21suppl page of nowadays high throughput methods in biological and biomedical fields acquire a largenumber of molecular parameters by a single experiment in particular such measuredparameters are collected in œomics datasets eg genomics transcriptomics methylomics among multiple measured parameters dna genome sequence rna expressionand dna methylation are representative instances for individually analysing suchdata several methodologies have been introduced in literature even though recentlya number of studies pointed out the best performance coming from the integration ofmultiomics data for instance analysing each omic or view in the machine learningjargon set separately fundamental patterns can be detected from data however somefinetuned structures such as cancer subtypes can be highlighted by both gene expression and dna methylation information so that multiomics analysis can reduce theeffects of experimental and biological noise in data from literature three kinds ofintegration methodologies emerge¢ early integration builds a single featurebased matrix by concatenating each omic¢ intermediate integration builds a joint representation of data given the views¢ late integration each omic is analysed separately and the solutions are integratedin general late integration methods and in particular clustering are preferred whenthe analysis combines continuous and discrete data together for a review of integrationapproaches and their comparisons the reader may refer to in this work a multiviewclustering methodology named fhclust is introduced see fig for its general schemafor identifying patient subgroups from different omics information or datasets eg geneexpression mirna expression methylation specifically for each omic dataset a fuzzybased hierarchical clustering approach is adopted and finally the results are mergedby consensus matrix the idea behind the proposed approach comes from observingthat a hierarchical clustering dendrogram can be associated with a łukasiewicz fuzzydataset ie view and applies a singleomic analysisfig proposed approach a data preparation b data normalization and feature selection c multiomicshierarchical agglomerations d data integration e clustering and visualization 0cciaramella bmc bioinformatics 21suppl page of similaritybased equivalence relation so that a consensus matrix that is the representative information of all dendrograms is derived by combining multiple hierarchicalagglomerations following an approach based on transitive consensus matrix constructionmethodscluster analysis or clustering is an unsupervised technique that aims at agglomerating aset of patterns in homogeneous groups or clusters [ ] hierarchical clustering hc isone of several different available techniques for clustering which seeks to build a hierarchyof clusters and it can be of two types namely agglomerative where each sample starts inits own cluster and pairs of clusters are merged as one moves up the hierarchy or divisivewhere all samples start in one cluster and splits are performed recursively as one movesdown the hierarchy thus hc aims at grouping similar objects into a cluster and werethe endpoint is a set of clusters where each cluster is distinct from each other and theobjects within each cluster are broadly similar to each other hc can be performed eitheron a distance matrix or raw data agglomerative hc starts by treating each observationas a separate cluster and it repeatedly executes the following two steps identifies thetwo clusters that are closest together and merges the two most similar clusters thisprocess continues until all the clusters are merged togetherthe main output of hc is a dendrogram which shows the hierarchical relationshipbetween the clusters distances many distance metrics have been developed and thechoice should be made based on theoretical concerns from the domain of studylater on it is necessary to determine how the distance is computed eg singlelinkagecompletelinkage averagelinkage as with distance metrics the choice of linkage criteria should be based on theoretical considerations from the application domainin nonfuzzy clustering or hard clustering data is divided into distinct clusters andeach data point can only belong to exactly one cluster in fuzzy clustering data pointscan potentially belong to multiple clusters for example in hard clustering given someparameters a œsymptom can be in a mutually exclusive way present or absent red orblue whereas in fuzzy clustering that œsymptom could simultaneously be of somegrade red and some other grade blue in fig a comparison between hard and fuzzycategorisation is shown the reader can refer to for a recent comparison betweenhard and fuzzy clustering in this work we introduce a data integration methodologybased on fuzzy concepts in particular we associate a dendrogram to a fuzzy equivalencerelation ie łukasiewicz valued fuzzy similarity so that a consensus matrix in a multiview clustering that is the representative information of all dendrograms can be obtainedfrom multiple hierarchical agglomerations [ ] the main steps of fuzzy agglomerationcan be summarised as follows characterisation of membership functions computation of a fuzzy similarity matrix or dendrogram for all models at a giventime construction of a consensus matrix for all hierarchical agglomerationsmembership functionswhen dealing with clustering tasks fuzzy logic fl permits to obtain a soft clusteringinstead of an hard clustering of data specifically data points can belong to more 0cciaramella bmc bioinformatics 21suppl page of fig hard vs fuzzy in symptom risk example a hard categorization b fuzzy categorizationthan one cluster simultaneously the fundamental concept in fl upon which all thesubsequent theory is constructed is the notion of fuzzy set a generalisation of a crisp setfrom classical set theorya fuzzy set generalises a crisp set by allowing its characteristic function ie itsmembership function assuming values in the interval [ ] rather than in the set in this way a given item belongs to the fuzzy set with a degree of truthranging from do not belong at all ie its membership function assumes value to completely belong ie the membership function assumes value in fl applications fuzzy sets make it possible to represent qualitative nonnumeric values ielinguistic variables such as high medium low for approximate reasoninginference or fuzzy control systems linguistic variables can be represented by fuzzy setsthrough a transformation step called fuzzification and it is achieved by using different types of membership functions representing the degree of truth to whicha given input sample belongs to a fuzzy set see œmembership functions sectionin supplementary material 0cciaramella bmc bioinformatics 21suppl page of fuzzy similarity matrixa measure of similarity or dissimilarity defines the resemblance between two samples orobjects similarity measure is a significant means for measuring uncertain informationfuzzy similarity measure is a measure that depicts the closeness among fuzzy sets and hasbeen used for dealing issues of pattern recognition and clustering analysisa binary fuzzy relation that is reflexive symmetric and transitive is known as a similarity relation fuzzy similarity relations are the generalisation of equivalence relationsin binary crisp relations to binary fuzzy relations in details a fuzzy similarity relationcan be considered to effectively group elements into crisp sets whose members are similar to each other to some specified grade and it is a generalization of classical equivalencerelation as described in œfuzzy similarity section in supplementary material in orderto introduce the fuzzy similarity in the following we focus on the properties of thełukasiewicz tnorm tl and the biresiduum in this way we obtain a fuzzy equivalencerelation that can be used for building dendrogram for more details in the derivation ofthese results see œfuzzy similarity section in supplementary materialdendrogram and consensus matrixif a similarity relation is mintransitive ie t min then it implies the existence ofthe dendrogram see œdendrogram and consensus matrix section in supplementarymaterial for details the mintransitive closure of a relation matrix r can be easilycomputed and the overall process is described in algorithm the last ingredient to accomplish an agglomerative clustering is a dissimilarity relationhere we considered the following result lemma letting r be a similarity relation with the elements rcid2x ycid3 ˆˆ[ ] and lettingd be a dissimilarity relation which is obtained from r bydx y ˆ’ rcid2x ycid3then d is ultrametric iif r is mintransitivein other words we have a onetoone correspondence between mintransitive similaritymatrices and dendrogram and between ultrametric dissimilarity matrices and dendrograms finally after the dendrograms have been obtained each time a consensus matrixie the representative information of all dendrograms is obtained by combining thetransitive closures ie maxmin operation the overall approach is described inalgorithm the overall workflow of the proposed approach is summarised in fig in particular for each omic data set xi a fuzzification step is adopted for obtaining thenew data set yi see supplementary material successively adopting a fuzzy similaritymeasure the similarity matrix si is computed and to guarantee the transitive closure ofthe matrix a new matrix ci is computed see algorithm finally all the ci matricesare collected for obtaining the consensus matrix a and the overall final dendrogram seealgorithm in fig we show an example that summarize a realistic agglomeration result we plotin figs 4abc three input hierarchies obtained on datasets that should be combinedin this case four sequences of patients are considered namely s1 s2 s3 and s4 respectively in fig 4d we show the final result by agglomerating dendrograms we observe that 0cciaramella bmc bioinformatics 21suppl page of fig workflow of the fuzzy based hierarchical clusteringthe output hierarchy contains clusters s1 s2 s3 and s1 s2 s3 s4 at different levels andeach of these clusters eg s1 s2 s3 are repeated at least in two out of the three inputdendrograms moreover it is worth stressing that the proposed approach based on theagglomeration of dendrograms can also be applied with commonly used metrics egeuclidean distance in fig we show a comparison between the dendrograms obtainedby using an euclidean metric and a similarity based approach ie łukasiewicz tnormrespectively in this realistic example we simulate three omic data sets with rows ienumber of patients and columns ie features we split the single datasets in twopartitions or clusters such that the first rows are random samples from a standard normal distribution with variance and the other rows have the same distribution withfig combination algorithm abc input dendrograms d combined hierarchy 0cciaramella bmc bioinformatics 21suppl page of fig crisp hierarchical clustering vs fuzzy based hierarchical clustering a dendrogram of euclidean basedhierchical clustering b dendrogram of similarity based hierachical clusteringalgorithm mintransitive closure input relation si output transitive relation ci sti elaborate compute sˆ— if sˆ—ielse ci sti si ˆª si —¦ sicid8 si replace si with sˆ—i sˆ—i and go to step i and the algorithm terminatesvariance obtaining a sort of an overlap we observe that both methods find two separated clusters but the similarity based approach in fig 5b permits to obtain a perfectseparation of the source partitionsresults and discussionin the following we describe the behaviour of the proposed methodology on multiomicsbenchmark datasetsalgorithm combination of dendrograms input ci ‰¤ i ‰¤ l l input similarity matrices dendrograms output similarity matrix dendrogram a aggregate the similarity matrices to a final similarity matrixa aggregate c1 c2 cla let aˆ— be the identity matrixb for each ci calculate e aˆ— aˆ— ˆª aˆ— —¦ cic if aˆ— is not changed a aˆ— and goto step else goto step 1b create the final dendrogram from aomics datasetswe consider multiomics cancer datasets available from the cancer genome atlastcga tcga is a large multiomic repository of data on thousands of cancerpatients all datasets contain three omics gene expression mirna expression and 0cciaramella bmc bioinformatics 21suppl page of table datasets description three omics are provided for each dataset respectively dna geneexpression mirna and methylationcases dnaoridatasetamlbiccoadgbmkirclihcluscskcmovsarclnrfmirnaorilnmethyorilnmultiomicsorilnrfrfrfthe number of features at each variable selection method is shown ori original variable dimension ln logarithm andnormalisation and rf random forest based on mean decrease gini indexdna methylation1 in table are summarised the main properties of the datasetsnamely acute myeloid leukemia aml breast invasive carcinoma bic colon adenocarcinoma coad glioblastoma multiforme gbm kidney renal clear cell carcinoma kirc liver hepatocellular carcinoma lihc lung squamous cell carcinomalusc skim cutaneous melanoma skcm ovarian serous cystadenocarcinoma ovsarcoma sarc the number of patients ranges from for aml to for bicmultiview clustering algorithmsfor validating the effectiveness of our model we compared it against several categories ofmultiview clustering algorithms2¢ kmeans and spectral clustering techniques ¢ lracluster it is a lowrank approximation based integrative probabilistic model¢ pins perturbation clustering for data integration and disease subtyping pinsis able to address subtype discovery as well as integration of multiple data types thealgorithm is built upon the resilience of patient connectivity and cluster ensembles toensure robustness against noise and biasto fast find the shared principal subspace across multiple data types¢ snf similarity network fusion snf allows for discovery of disease subtypesthrough integration of several types of highthroughput data on a genomic scale snfcreates a fused network of patients using a metric fusion technique and thenpartitions the data using spectral clustering snf appears to be the state of the art inthis area and has proven to be very powerful however the unstable nature ofkernelbased clustering makes the algorithm sensitive to small changes in molecularmeasurements or in its parameter settings¢ mcca multi canonical correlation analysis mcca which extends theapplication of canonical correlation analysis cca to more than two views is oneof the most widely used dimension reduction method for finding linear relationsbetween two or more multidimensional random variables1row data are available at httpacgtcstauacilmulti_omic_benchmarkdownloadhtml2httpsgithubcomshamirlabmultiomicscancerbenchmark 0cciaramella bmc bioinformatics 21suppl page of evaluation metricsin order to assess the performance of each method we adopt three evaluation metricsthat are the logrank test the enrichment of clinical labels in the clusters and the methods runtime the logrank test assumes that if clusters of patients have significantlydifferent survival they are different in a biologically meaningful way for the enrichmentof clinical labels in clusters six clinical labels are considered gender age at diagnosispathologic tumor pathologic metastases pathologic lymph nodes and pathologic stagethe four latter parameters are discrete pathological parameters measuring the progression of the tumor metastases and cancer in lymph nodes and the total progressionpathologic stage enrichment for discrete parameters was calculated using the χ2 testfor independence and for numeric parameters using kruskalwallis test not all clinicalparameters were available for all cancer types so a total of clinical parameters wereavailable for testing to derive a pvalue for the logrank test the χ2 test for independence the kruskalwallis test and the statistic for these three tests is assumed to have χ2distribution preprocessingtcga datasets were preprocessed as follows patients and features with more than missing values were removed and missing values were imputed using knearest neighborimputation the sequence features were logtransformed the features with highestvariance from geneexpression and methylation omics were selected in the mirna omicfeatures with zero variance were filtered all features were then normalized to have zeromean and standard deviation for methylation we selected the features with maximal variance in each dataset and also adopted the standard pipeline proposed in whose procedure filters out the probes from the x and y chromosomes or probes that areknown to have common snps at the cpg sitea further unsupervised variable selection step has been performed by using the meandecrease gini based on random forest the mean decrease in gini is the average of a variable total decrease in node impurity weighted by the proportion of samplesfig mean performance of the algorithms on ten multiomics cancer datasets the xaxis measures thedifferential survival between clusters mean log10 of logrank™s test pvalue and the yaxis is the meannumber of clinical parameters enriched in the clusters 0cciaramella bmc bioinformatics 21suppl page of fig performance of the algorithms on ten multiomics cancer datasets for each plot the xaxis measuresthe differential survival between clusterslog10 of logrank™s test pvalue and the yaxis is the number ofclinical parameters enriched in the clusters red vertical lines indicate the threshold for significantly differentsurvival pvalue cid2 reaching that node in each individual decision tree in the forest this is effectively a measure of how important a variable is for estimating the value of the target variable acrossall of the trees that make up the forest a higher mean decrease in gini indicates highervariable importance therefore the most important variables to the model is the highestin the plot with the largest mean decrease in gini values conversely the least importantvariable is the lowest in the plot with the smallest mean decrease in gini values by following this strategy we cutoff all those variables whose importance is zero the numberof variable cutoff at each step is summarised in table experimental resultsin the experiments for all methods the number of searched clusters is selected in therange [ ˆ’ ] to determine the number of clusters for a method we used the œelbowmethod to automatically pick out the optimal elbow rather than choose it manually weused as approximation the second derivative of a vector vv [i ] v [i ˆ’ ] ˆ’ 2v[ i] in particular we consider the index i that brings this expression to a maximum or minimum depending on whether v increases or decreases for all methods we adhered tothe guidelines for usage and parameter selection given by the developers in some casestable performance on ten multiomics number of clinical parameters enriched in the clustersfhclustcrisp hclustkmeansspectrallraclusterpinssnfmccaamlbiccoadgbmkirclihcluscskcmovsarcmeans 0cciaramella bmc bioinformatics 21suppl page of table performance on ten multiomics differential survival between clusters log10 of logrank™stest pvaluefhclustcrisp hclustkmeansspectrallraclusterpinssnfmccaamlbiccoadgbmkirclihcluscskcmovsarcmeanswhere no information was provided by the authors we devised parameter selection methods we performed the same process pipeline used in for evaluating the performanceof our method all methods were run on a multicore intelr xeonr cpu e52620v3 240ghz with gb ram in the following the obtained experimental results aredescribedfigure shows the average performance for multiomics data and for each singleomicseparately across all cancer types and fig shows the performance on the different cancer datasets all algorithms show quite similar performance in either differential survivalor enriched clinical parameters with respect to survival our fhclust method achievedthe overall best prognostic value sum of ˆ’log10 pvalues while pins and mcca ranked second and third respectivelyin table the differential survival between clusters mean ˆ’log10 of logrank™s testpvalue are reported spectral achieved the highest total number of significant clinical parameters with parameters fhclust along with lracluster and kmeansplaced themselves second with parameters snf achieved the third position with parameterswith respect to survival table fhclust outperformed its competitors achieving parameters mcca pins and snf have achieved good results with and enriched parameters respectivelywe also counted the number of datasets for which a method solution obtains significantly different survival these results are reported in table all methods that weredeveloped for multiomics data had at least four cancer types with significantly differentsurvival in this case fhclust and pins had different cancer subtypes for which itsclustering had significantly different prognosis fhclust spectral clustering and mccahad enrichment in cancer typeson average fhclust pins and mcca had better prognostic value but found lessenriched clinical labels as compared to spectral clustering methodtable for each benchmarked algorithm the number of cancer subtypes for which its clusteringhad significantly different prognosis first row and had at least one enriched clinical label secondrow are shownsignificant different survivalsignificant clinical enrichmentfhclustkmeansspectrallraclusterpinssnfmcca 0cciaramella bmc bioinformatics 21suppl page of table number of clusters chosen by the benchmarked algorithms on ten multiomics cancerdatasetsfhclustcrisp hclustkmeansspectrallraclusterpinssnfmccaamlbiccoadgbmkirclihcluscskcmovsarcmeansthe number of clusters found for each dataset are presented in table ranging from to because of the good methods performance in the previous analysis partitioning thedata into a relatively high number of clusters could indicate that clustering cancer patientsinto more clusters improves prognostic value and clinical significanceconcerning with methods computational burden their run times are reported intable fhclust takes on average seconds per dataset while spectral and snf gotlower timing the worst method takes roughly minutes per dataset see fig finally fig shows the benchmarked methods performance for singleomic datamoreover for each dataset and method the single omic that gave the best results forsurvival and clinical enrichment are also shown these results suggest that fhclust provides better prognostic value and clinical significance on multiomics data compared tothe analysis of singleomic data used separately nevertheless the interested reader mayrefer to the supplementary material for details on additional results concerning singleomics we also stress that the proposed method differently from other methods suchas snf does not need any hyperparameter tuning moreover clustering is embeddedin the data integration and vice versa and the use of fuzzy concepts ie tnormsfrom one hand permits to obtain a generalisation of the clustering approaches whereason the other hand gives the possibility to apply an inference system eg mamdanifor a quantitative and qualitative measure eg œhigh œmedium œlow in cancer riskassessmentsin this work we proposed a multiview clustering methodology for identifying patientsubgroups from different omics data in biological and biomedical fields combining theseomics data can significantly increase data mining capabilities one of the main aspects oftable runtime in seconds of the algorithms on ten multiomics cancer datasetsovfhclustcrisp hclustkmeansspectrallraclusterpinssnfmccacoadskcmbicamlgbmkirclihcluscsarcmeans 0cciaramella bmc bioinformatics 21suppl page of fig computational time comparisonsthis methodology is the use of a measure of dissimilarity between sets of observations byusing an appropriate metric and a consensus matrix that is a representative agglomerateinformation of all the dendrograms as emerged from the analysis of the scientific literature to the best of our knowledge our work concerns for the first time a model based onfuzzy logic used for the agglomeration of multiomic data the use of fuzzy logic allowsus to introduce more flexible data mining features also related to approximate reasoningseveral experiments and comparisons have been made on real data eg glioblastomaprostate cancer to assess the proposed methodology the results suggest that fhclustprovides better prognostic value and clinical significance compared to analysis of singleomic data alone fuzzy logic concepts and in particular membership functions permitsfig summarized performance of the algorithms across ten cancer datasets for each plot the xaxismeasures the total differential prognosis between clusters sum across all datasets of “log10 of logrank™s testpvalue and the yaxis is the total number of clinical parameters enriched in the clusters across all cancertypes a“c results for singleomic datasets d results when each method uses the single omic that achievesthe highest significance in survival e same with respect to enrichment of clinical labels 0cciaramella bmc bioinformatics 21suppl page of to develop a fuzzy inference model ie mamdani fuzzy cognitive maps for easilyobtaining a model for a quantitative and qualitative risk assessment of the cancer themodel based on approximate reasoning can be particularly useful for embedded devicesin future work it could be possible to improve results for multiomics analysis ina number of ways for instance more accurate feature selection algorithms couldbe adopted for improving the overall performance on one hand the integration oflabelled data could improve the feature selection step on the other hand some specific feature extraction strategies could be adopted indeed approaches based on thesignal analysis of gene expression data eg nonlinear principal component analysis compressive sensing could possibly further improve the performance [ ]in future it is possible to foresee a different weight for each omic data in order toobtain a more robust similarity and parametric similarity measures can be adoptedeg uninorm for generalizing the concept of and and or connections betweenclusterssupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12859020035676additional file supplementary materialabbreviationsfhclust fuzzyhierarchical clustering dna deoxyribonucleic acid rna ribonucleic acid hierarchical clustering hccrisp hc crisp hierarchical clustering fl fuzzy logic tcga the cancer genome atlas aml acute myeloid leukemia bicbreast invasive carcinoma coad colon adenocarcinoma gbm glioblastoma multiforme kirc kidney renal clear cellcarcinoma lihc liver hepatocellular carcinoma lusc lung squamous cell carcinoma skcm skim cutaneousmelanoma ov ovarian serous cystadenocarcinoma sarc sarcoma pins perturbation clustering for data integrationand disease subtyping lracluster low rank approximation based multiomics data clustering snf similarity networkfusion mcca multi canonical correlation analysisabout this supplementthis has been published as part of bmc bioinformatics volume supplement proceedings from the 13thbioinformatics and computational biology international conference bbcc2018 the full contents of the supplement areavailable online at httpsbmcbioinformaticsbiomedcentralcomssupplementsvolume21supplement10authors™ contributionsac originally designed the methodology ac and dn worked on the developing of the method and the design of theexperiments ac dn and as contributed for interpreting and for analysing the results all authors contributed forwriting the manuscript read and approved the final manuscriptfundingpublication costs are funded by a grant from the dipartimento di scienze e tecnologie università degli studi di napoliœparthenope tecniche di machine learning e soft computing per l™elaborazione di dati multivariati softmulan piciaramellaavailability of data and materialscode and data of the proposed approach are available on multiomicscancerbenchmark github repositoryethics approval and consent to participateno ethics approval was required for the studyconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1dipartimento di scienze e tecnologie università degli studi di napoli œparthenope centro direzionale c4 island naples italy 2hitachi rail sts via argine naples italypublished august 0cciaramella bmc bioinformatics 21suppl page of referencescamastra f di taranto md staiano a statistical and computational methods for genetic diseases an overviewcomput math meth med 20152015 id “serra a fratello m fortino v raiconi g tagliaferri r greco d mvda a multiview genomic data integrationmethodology bmc bioinformatics “rappoport n shamir r multiomic and multiview clustering algorithms review and cancer benchmark nucleicacids res “reddy ck aggarwal cc data clustering boca raton chapman and hallcrc camastra f ciaramella a son lh riccio a staiano a fuzzy similaritybased hierarchical clustering for atmosphericpollutants prediction lncs “ciaramella a staiano a on the role of clustering and visualization techniques in gene microarray data algorithmsbora dj gupta ak int j emerg trends technol comput sci “napolitano f pinelli m raiconi g tagliaferri r ciaramella a staiano a miele g clustering and visualizationapproaches for human cell cycle gene expression data analysis int j approx reason “ciaramella a cocozza sand clustering of genomic data neural netw “iorio f miele g napolitano f pinelli m raiconi g tagliafer Answer:

Colon_Cancer

" among eukaryotic anisms alternative splicing is an important process that can generate multipletranscripts from one same precursor messenger rna which greatly increase transcriptome and proteome diversitythis process is carried out by a superprotein complex defined as the spliceosome specifically splicing factor branchpoint binding protein sf1bbp is a single protein that can bind to the intronic branchpoint sequence bpsconnecting the ² and ² splice site binding complexes during early spliceosome assembly the molecular functionof this protein has been extensively investigated in yeast metazoa and mammals however its counterpart inplants has been seldomly reportedresults to this end we conducted a systematic characterization of the sf1 gene family across plant lineages inthis work a total of sequences from plant species were identified phylogenetic relationships of thesesequences were constructed and subsequent bioinformatic analysis suggested that this family likely originatedfrom an ancient gene transposition duplication event most plant species were shown to maintain a single copy ofthis gene furthermore an additional rna binding motif rrm existed in most members of this gene family incomparison to their animal and yeast counterparts indicating that their potential role was preserved in the plantlineage our analysis presents general features of the gene and protein structure of this splicing factor familyand will provide fundamental information for further functional studies in plantskeywords alternative splicing expression profile phylogenetics plants promoter splicing factor correspondence fyzhunjfueducn kailu zhang zhen feng jingfang yang and feng yang contributedequally to this work1coinnovation center for sustainable forestry in southern china college ofbiology and the environment nanjing forestry university nanjing jiangsu province chinafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0czhang bmc plant biology page of in eukaryotes canonical splicing removes noncoding intronic sequences and assembles the coding elements intomature mrnas while alternative splicing as generatesdifferent multiple transcripts that encode proteins withdistinct structures and functions by differential usage ofexons or splice site [ ] the resulting transcripts ofas greatly contribute to posttranscriptional regulationbiological complexity and proteome diversity in eukaryotes [ ] given that on average there are approximately exons in each transcript in the humantranscriptome and the degenerative nature of corresponding splice sites premrna splicing is sophistically catalysed by the spliceosome spliceosome is amultimegadalton protein complex which consists offive u1 u2 u4 u5 and u6 small nuclear ribonucleoprotein ps snrnps and over spliceosomalproteins furthermore the early assembly of spliceosome complex e or the commitment complex is anatpindependent process and contains u1 snrnps sf1and u2 snrnp auxiliary factors u2af large and u2afsmallthe prespliceosome complex a is formed by replacing sf1 withsf3b155sap155 of u2 snrnps [ ] stepwiseassembly of the following spliceosome during the splicing reaction has been reported as well [ ] however splice site recognition is a critical step during earlyassembly of the spliceosome the current model describes the binding of u1 snrnp and u1 snrna to ashort stretch of nucleotides at the ² splice site of splicing factor sf1mammalian branch point bindingprotein mbbp at the branch point and of u2 snrnpauxiliary factors at the ² splice site these threeciselements are necessary but usually insufficient to define a specific exon“intron boundary thus additionalsplicing enhancers or silencers located at exons and introns may allow the recognition of genuine splice sitesduring early spliceosome assembly [ ] subsequentlysubunitsimportantly sf1 preferentially binds to the intronbranch point sequence bps which is adjacent to thebinding site polypyrimidine tract py of u2af largesubunits mammal u2af65 and fission yeast u2af59bridging u1 and u2af to form an intermediate lariatstructure [ ] in particular sf1 is characterized bythe presence of two types of rna binding motifs at thenterminus a k homologyquaking khqua2 domain which originated from the human heterogeneousribonucleoprotein hnrnp k protein [ ] and oneor two zinc knuckle motifs cx2cx4hx4c x represents any amino acid sf1 also contains a prolinerichregion at cterminus [ ] intriguingly the yeast khdomain specifically binds to the bps of premrnas witha glyproarggly motif and the variable loop of thekh domain and is necessary for spliceosomeassembly the first but not the second zinc knuckledomain in yeast has been demonstrated to bind rnawith high affinity moreover the stability of thesf1“u2af65“rna complex is further affected by thephosphorylation status of several sf1 serine residuesser20 ser80 and ser82 in vitro the prolinerichregion of sf1 interacts with u1 snrnp prp40fbp11 inyeast and human [ ] in regards to its interactionpartner the u2af large subunit the nterminal of sf1interacts with its noncanonical rna recognition motifsrrm or u2af homology motif[ ]whereas the other two rrms of u2af large subunitbind to the py region uhmsaccharomycescerevisiae common fruita previous study in fission yeast schizosaccharomycespombe suggests that the initial corecognition of thebranch site and ² splice site is pivotal for correct splicing of target premrnas because of the importance of splice site recognition for gene expression andprotein diversity sf1 has been demonstrated to play essential roles in a number of eukaryotic species includinghuman homo sapiens mice mus musculus buddingyeastflydrosophila melanogaster and roundworm caenorhabditis elegans [ ] for example in humansmissense mutation of splicing factors which are responsible for splice site recognition such as sf1 has beenlinked to tumourigenesis similarly heterozygoussf1 ˆ’ knockdown mice are susceptible to colontumourigenesis induced by an anotrophic carcinogenazoxymethane and sf1 has been found to associatewith betacatenintcf4 complex suggesting its role incarcinogenesis in contrast knockdown of sf1 suppresses the development of germ cell tumours in mice indicating its tissue dependency in cancer researchfurthermore the molecular function of sf1 has been extensively studied in yeast for instance a sf1 mutantstrain causes frequent exon skipping in fission yeast additionally sf1 has been proposed to recognize suboptimal sequences in specific introns and lead to nuclearaccumulation of premrna with aberrant splicing however increasing evidence indicates that this proteinis a regulator of splice site recognition and does not reduce general splicing specifically during alternative splicing by targeting a subset of genes [ ] thishypothesis is supported by the fact that knockdown ofsf1 in both yeast and human extracts only slightly affects the splicing outcome rnai targeting of thisgene has been demonstrated to not affect the splicingpattern of several splicing marker genes tested in comparison to studies in human and yeast few reports have been published related to plant sf1 genessimilar functions of the arabidopsis sf1 gene were proposed in an early study in this plant sf1homologue is reportedly responsible for the splicing of a 0czhang bmc plant biology page of to maintain itsgroup of transcripts the lossoffunction mutant atsf1“ of this gene leads to abnormal development earlyflowering and dwarfism and aba or heat stress sensitivity in arabidopsis [ ] subsequently the domainstructure and its functional relationships have been substantially investigated and the rrm domain is considered crucialfunction in plantsmoreover sf1 may have a different mechanism of ²splice site recognition in plant because the plant sf1 homologs contain a different rrm domain compared withfungal and metazoan counterparts [ ] on the otherhand a study found that atsf1 may be likely to play afunctional role in the cytoplasm because it was found toshuttle between the nucleus and cytoplasm however no related investigations have been conducted onthe phylogenetic analysis of plant sf1 genes and theirregulatory mechanisms although it is a highly conserved family and has conserved functions in eukaryotesplant sf1 genes may have overlapping and distinct rolescompared to the mammalian genes hence studying thephylogenetic relationship and regulatory mechanism ofplant sf1 genes may make us understand the evolutionary history characteristics an expression profile of thisgene family and predict specific functions in plants thiscan lay the foundation for further functional studies inviridiplantae to this end we systematically identified sf1 sequences from plant species ranging fromalgae to higher plants meanwhile the gene and proteinstructure potential regulation at promoter regions andexpression pattern of these genes were further investigated in this study we hypothesize that plant sf1 isstructurally different from its counterparts in animalsand yeast but it is conserved among lower and higherplants indicating its specific role in alternative splicingin branch point recognitionthalianasf1methodssequence acquisition and identification of plant sf1genesthe arabidopsisprotein sequenceat5g51300 was used to search similar sequences inall available plant species from the phytozome v121databasehttpsphytozomejgidoegovpzportalhtml by running the blastp program with an evaluecutoff 1e10 the other parameters were the default settings then the retrieved protein sequences wereexamined and filtered using the hmmer score defaultsettings which contained pf16275 splicing factorkhomology domain kh_1 and pf00076 rna recognition motif rrm_1 finally putative sf1 sequencesfrom plant species were identified detailed information including groups plant species common namesand number of sf1 homologs reported for each planthelixhairpinsf1hhdomainpf00013species for subsequent analysis are listed in table s1subcellular location prediction of identified sf1 proteinswas carried out using wolf psort httpswolfpsorthgcjp construction of molecular phylogenetic tree of plant sf1genesprotein sequences of the aforesaid plant sf1 genes wereextracted from phytozome v121 database for phylogenetic relationship analysis the sequences with the longestcoding sequences were chosen for genes with multipledifferent splicing isoforms then multiple sf1 proteinsequences were aligned with the muscle v38 softwarewith default settings the molecular phylogenetictree of plant sf1 genes was then constructed using themaximum likelihood method ml jtt g i modelvia phyml v30 program with the following parametersinitial tree bionj discrete gamma model yes numberof categories gamma shape parameter proportion of invariant subtree patterns aliasing no figtree v143 was used to visualize and edit the phylogenetic treegene structure protein domain and multiple em for motifelicitation meme analysisrequired genomic cdna and peptide sequences and allsf1 gene structures were downloaded from the phytozome v121 database corresponding intron phases weregenerated using the online program gene structure display server gsds20httpgsdscbipkueducn correlation analysis of sf1 exons were performedby using the piece2 webserver httpwwwbioinfogenomenetpiecesearchphp tdsourcetags_pctim_aiomsg sf1 protein sequences were used to search formatching pfam families using the hmmer websitehttpswwwebiacuktoolshmmer then protein domain patterns were drawn by using tbtools software according to the full pfam resultanttableconserved motifs of plant sf1 cdna sequences andprotein sequences were analysed on the meme onlineprogram httpmemesuitetoolsmeme considering a maximum of the most preserved motifs predicted for each sequence and leaving other settings onthe default parametersmotif prediction in promoter regions of plant sf1 genesthe 15kb ²flanking sequences of plant sf1 geneswere extracted from genomic data available in phytozome database prediction of plant putative ciselementswas performed with the online server plantcarehttpbioinformaticspsbugentbewebtoolsplantcarehtml motifs related to tissuespecific expressioninternal hormones and external environmental stress response were selected for further analysis and discussion 0czhang bmc plant biology page of expression analysis base on microarray datasets and geneexpression experimentsexpression data of arabidopsis s tuberosum g max slycopersicum p trichocarpa and b distachyon includingtissue specificity and stress responses were extracted fromthe efp browser series of the bioanalytic resource forplant biology httpbarutorontoca expressionvalues of selected plant sf1 genes were log transformedlg to generate visualize expression difference heatmapsby using bar heatmapper tool program httpbarutorontocantoolscgibinntools_heatmappercgigene expression experimentstotal rna of samples from different plant tissues wereextracted by rneasy mini kit qiagen usa and subsequently reversed transcribed into cdna by fastkinggdna dispelling rt supermix fastking tiangenchina according to the manufacturer™s instruction rtpcr amplification were programmed asfollowings °c min °c s °c s °c s cycles °c min sybr premix ex taqtm accuratebiotechnology co ltd hunan china was used forquantitative realtime rtpcr analysis which was conducted on the stepone plus realtime pcr system following optimized program °c s °c s °c s cycles the data were normalized to the expression of internal reference genes table s6 and the transcript abundance was determined by the comparativect value method analysis of proteinprotein interaction network andstructural conservationa proteinprotein interaction network was generated bythe string website httpsstringdb withrepresentative protein sequences from arabidopsis thefollowing basic settings were employed meaning of network edges evidence line colour indicates the type ofinteraction evidence and active interaction sourcesexperimentsthere are three domains in the arabidopsis sf1 protein the phosphorylation and u2af65 binding of thenterminal domain of splicing factor during ² splicesite recognition of homo sapiens pdbid 2m0g identity evalue 7e17 was similar to that of the khomology domain the structure for recognition of theintron branch site rna by splicing factor of homo sapiens pdbid 1k1g identity evalue 9e27 canbe used as the template for the splicing factor helixhairpin domain therefore homology modelling wasperformed with modeller based on two crystalstructures the amino acid conservation scores were calculated using the consurf web server based on the mlmethod input attributes were the 3d model andmultiple sequence alignment figure s4 related figureswere created based on pymol with default settings analysis gene structure evolution with orthologue groupof sf1 genesreconstruction of the evolutionary history of the structure of the plant sf1 family of orthologous genes wascarried out by searching at5g513001 in the piece severhttpwwwbioinfogenomenetpieceindexphpthis provided an exonintron display for orthologousgenes from gene structure data sets linked to the phylogenetic treeresultssequence identification and phylogenetic analysis of theplant sf1 gene familyto identify sf1 gene family members in plants we carried out a blastp search using the arabidopsis atsf1at5g51300 amino acid sequence against the phytozome database v121 after filtering the sequence without sf1 signature or truncated sequences a total of sequences from plant species were retrieved whichwere roughly classified as algae bryophyta basicangiosperm monocots and eudicots table s1specifically the only species with four copies of plantsf1s was eutrema salsugineum salt cress table s1 inparticular three copies of sf1 genes were observed infive species including panicum virgatum switchgrasstriticum aestivum common wheat daucus carotacarrot kalanchoe laxiflora milky widow™s thrill andsalix purpurea purple osier willow additionally plant species contained two copies and species including the model plant arabidopsis possessed only onecopy of plant sf1s respectively the relatively largernumber of sf1 genes and higher number of plant speciesin this work demonstrated the universality and complexity of the sf1 gene family the retrieved sequences of plant species provided us with more complete information to analyse the phylogenetic relationship of the sf1gene family subsequently a rooted phylogenetic treewas constructed based on the abovementioned protein sequences by using the maximum likelihoodmethod the tree™s bootstrap threshold “ was represented by a colour gradient fig in general all sf1protein sequences were clustered into four major cladesincluding alga in yellow other land plants in greenmonocots in pink and eudicots in blue and one species amborella trichopoda belonged to basic angiosperm shown in colourless the phylogenetic tree ofsf1s figs and left panel with clear topology andoverall high bootstrap values was similar to evolutionarytrend from lower plants to higher plants reported inother studies for example the genes of algae in the yellow branch were representative members of the lineage 0czhang bmc plant biology page of fig circular phylogenetic tree of the sf1 gene family available in plants the phylogenetic tree of sf1 genes in plants was constructed basedon maximumlikelihood with jtt g model by using phyml v3037 a total of protein sequences from plant species were chosen tocalculate the phylogenetic relationship for tree construction bootstrap values are labelled at each major branch the corresponding informationof each transcript such as species name common name number of identified transcripts and their transcript id nomenclature are shown intable s1 taxonomies based on apgiv system 0czhang bmc plant biology page of fig see legend on next page 0czhang bmc plant biology page of see figure on previous pagefig gene structure comparisons and conserved motif identification among plant sf1 genes from left panel to right panel verticalphylogenetic tree genomic anization and identified cdna conserved motifs by meme analysis intron phase and are shown on thegene structure the conserved sequence of identified motifs represented by different coloured boxes are listed below some long genes werereduced to onehalf of their original length to fit this picturethat diverged before the evolution of land plants whichwas the basal part of the phylogeny in the blue branchfive sequences from kalanchoe with higher bs valuesformed a subclade showing their closer evolutionary relationships additionally cagra3782 s00261p fromcapsella grandiflora and carubv10025900m from c rubella formed a subclade with the arabidopsis sequencesbecause they all belong to brassicaceae which is consistent with the apg iv system fig and table s1 usually some homologous sf1 sequences from the samespecies were clustered in the same small branch next toeach otherthese species included cashew soybeanapple woodland strawberry quinoa carrot coloradoblue columbine maize common wheat cereal grassmoss and bog moss fig and table s1 in contrastsome other homologous sf1 members from the samespecies were clustered into the different subclades suchas purple osier willow poplar eastern cottonwood saltcress potato diploid kalanchoe milky widow™s thrillhall™s panicgrass switchgrass green algae and volvoxfig and table s1gene structure and conserved motif analysisit is necessary to compare the exonintron anizationand conserved motifs of the plant sf1 gene family toclarify their evolutionary process and potential functionthe gene structure models of sf1 genes were attachedto the phylogenetic tree fig and the correspondingintron phase of each was also displayed fig tables2 figure middle panel shows that the gene lengthand structure of each member of the sf1 family exhibitssignificant differences for example the gene structureof members of sf1 family genes did not containintron sequences this subset accounts for of thetotal number of members fortyeight sequences of sf1genes had exon1 intron anizations accounting for of all genes in particular some genes from algaehad multiple exons including vocar0008 s02941p volvox carteri which contained the most exons exonsmoreover different gene structures were also observedat the same subbranch for instance two sequencesfrom zea mays maize zm00008a037777_p01 exonsand zm00008a007621_p01 exons were observed tohave distinctive gene structures although the dissimilation of gene structure of each member of sf1s was substantial we found that the length of cdss did notsignificantly change fig thus whether it influencesthe differentiation of their gene function needs to befurther investigated further investigation on conservedmotifs by using multiple em for motif elicitationmeme search tool demonstrated that most sf1 genes sequences exhibited similar sequence signatures andthe same order and all contained the analysed motifsexcept one sequence of micromonas pusilla hada different position fig right panel although no obvious differences in identified conserved motifs werefound among basal angiosperm monocots and eudicotssequences from the same species were found to have different motifs fig for example aqcoe5g4069001pand aqcoe7g0393001p from the eudicot aquilegiacoerulea had motifs and motifs respectively thesame situation was found in d carota dcar_006843dcar_008506 and dcar_004968 had motifs motifs and motifs respectively intriguingly the cdslength of dcar_008506 was the longest notably somesequences from algae and moss had fewer conservedmotifs for examplein bryophyta the sequences ofphyscomitrella patens pp3c7_10890v31p and pp3c11_24710v31p sphagnum fallax sphfalx0015s00771pand sphfalx0010s01971p and marchantia polymorphamapoly0009s01891p had nine motifs in algal plantsthe sequences of and from micromonashad only motifs and motifs respectively moreoveralthough the sequences of volvox carteri vocar0007s03451p and vocar0008 s02941p and chlamydomonasandcre09g386731t11 contained multiple exons they had motifs indicating their sequence variation had little influence on function classes further correlation analysisof the sf1 exon regions were carried out to elucidate thegainloss of introns correlations between transcripts ofplant sf1s are shown in fig providing additional information for phylogenetic analysis for example thereis more similarity between pgsc0003dmt400081859and migutd025312 because of multiple exact matchesbetween the exons of the two transcriptscre12g553750t11reinhardtiianalysis of protein domain and conserved motifs inpeptidesthe protein domains were analysed by using the aboveselected peptide sequences from plant species thepeptides™ annotations were splicing factorrelated andconserved protein motifs were predicted according tothe retrieved peptide sequences by meme analysisfig consequently all sf1s were found having sf1_hh nterminal domain on the nterminal ofthe 0czhang bmc plant biology page of fig analysis of gene structure evolution with orthologue group of sf1 genes exonintron structure and intron phase right panel are linked tothe plant species tree left panel genes with red colour represent the members of the plant sf1 genes different coloured lines mean differentexon comparison results between species 0czhang bmc plant biology page of fig comparisons of protein domains and conserved motif identification among plant sf1 genes protein domain middle panel and identifiedprotein conserved motifs right panel identified by meme analysis are shown against the vertical phylogenetic tree left panel the conservedsequence of identified motifs represented by different coloured boxes are listed below 0czhang bmc plant biology page of peptides followed by a kh domain and a cterminal domain namely an rna recognition motif rrm fig middle panel interestingly in algae peptides from mpusilla v carteri vocar0008 s02941p andc reinhardtii cre09g386731t11 had two rrm domains the amino acid lengths of sf1 proteins rangedfrom aa to aa and most of them possessed to amino acids table s3 consistently most ofthem are approximately to amino acids inlength subcellular location prediction showed that themajority of sf1 proteins were had nuclear localization table s3 moreover proteins of 147m014250 ricinus communis and migutf011911pmimulus guttatus were located in the vacuoles proteins of traes_2dl_6f03f05fa4 t aestivum and m pusilla were predicted to be cytoplasmic proteins of gsmua_achr5p25100_001 musa acuminataand cre09g386731t11 c reinhardtii were located inthe chloroplast and endoplasmic reticulum respectivelymeme analysis for sf1 peptide sequences was used topredict a total of conserved motifs which are presented as coloured boxes and cover most of the proteinfig right panel further analysis showed that peptides had all motifs accounting for approximately of all sf1 protein sequences analysed in the studyinterestingly all sequences from moss have conservedmotifs in the analysis suggesting the conservation ofsf1 proteins in bryophyta furthermore almost all eudicots had conserved motifs”except anacardium occidentalegrandifloracagra3782 s00261p which lacked motif and malusdomesticavescamrna211921v10hybrid and brassica rapa brarac014811p which lacked motif ”while most monocots had eight conserved motifs in contrast algal plantsonly possess approximately half of the predicted motifs due to their peptides with integrant protein domainsimplying the least degree of conservation and divergenceof plant sf1 proteins in algae t motifs that all algaeshared were motif motif motif and motif anaoc0018 s04251pmdp0000558834fragariaand canalysis of promoter and tissuespecific expression of sf1genesto further analyse the regulation of plant sf1 genes atthe transcriptional level the 15kb upstream sequencesof plant sf1 genes were obtained from the phytozomedatabase then the ciselements of each promoter wereidentified by using the plantcare program table s4 consequently a total of motifs were predictedgenerally eight ciselements related to tissuespecificexpression among them were selected fig and tables4 including hdzip1 for differentiation of the palisademesophyll cells the ryelement which regulates seedspecific expression the aaca_motif and gcn4_motif waspresentfurther hdzipinvolved in endosperm expression and the catboxccgtccbox doct and oct for meristem expression further analysis showed that there were only promoters of sf1 genes which had tissuespecific regulatory ciselements particularly the catbox and ccgtccbox turned up at the highest frequency and greatestabundance in the promoters of sf1 genes both of themregulate meristemspecific expression and play key rolesduring development and growth of plants consistentlypurple false brome brachypodium distachyon of monocots not only had a catbox and ccgtccbox butwas also highly expressed in young leaves internode adventitious roots and roots fig and figure s2however no motifs were found to link the highexpression of two sf1s of glycine max soybean insam and roottip figure s1 additionally the aaca_motif was only detected in solanum tuberosumpgsc0003dmp400032853 of potato suggesting itsspecific role in regulating endospermspecific negativeexpressioninpodel03g1132001p of populus deltoideseasterncottonwood and spipo17g0046100 of spirodela polyrhiza greater duckweed the ryelement was detectedin the promoter of the dicot model plant arabidopsisand low expression was also reported in dry seed inarabidopsis fig suggesting that the ryelement isinvolved in seedspecific negative expression of arabidopsis moreover expression levels in the same tissuetype showed significant differences during differentgrowth stages for example the expression level in stamen of flower stage of arabidopsis was obviouslyhigher than that of the other flower development stageshowever the expression levels of different growth stagesof solanum lycopersicum were not only similar butlower and no motifs were found in the promoter in tomato figs and s1 furthermore different expressionpatterns were detected in several sf1 genes with multiple copies figs s1 and s6 for instance similar tissue expression profiles were detected in two sf1trichocarpahomologuespotri001g1264001 and potri003g1072001 and themonocotandzm00008a037777_p01 figure s1 and s5 in contrasttwo sf1 genes of s tuberosum showed differential expression patterns similar to in g max figs and s1zm00008a007621_p01dicot populuszea maysfrom theanalysis of promoter and internal and external hormonesexpression of sf1 genesin longterm evolution and development plants havegradually formed mechanisms of adaptation and resistance to adversity to maintain their life and sustaingrowth to understand the regulatory mechanisms of internal and external stimuli on plant sf1s cisacting elements involved in hormone and stress were studied with 0czhang bmc plant biology page of fig see legend on next page 0czhang bmc plant biology page of see figure on previous pagefig analysis of motifs related to tissue specificity in the plant sf1 promoter regions eight cisacting motifs are represented in different colortriangles positions of these identified motifs are labeelled along the kb ²flanking regions of each sf1 gene the line solid and dottedrepresents regions with basic pairs and regions of no sequences or annexed base n respectively symbols on above the line represent the motifsat the plus strand whereas symbols on below the line represent the motifs at the minus strand function of motifs aacamotif involved inendospermspecific negative expression catbox cisacting regulatory element related to meristem expression ccgtccbox cisactingregulatory element related to meristem specific activation doct cisacting regulatory element related to meristem specific activationgcn4_motif cisregulatory element involved in endosperm expression hdzip1 element involved in differentiation of the palisade mesophyllcells ryelement cisacting regulatory element involved in seedspecific regulation the black vertical lines represent break at that particularbranch oct cisacting regulatory element related to meristem specific activationlowtemperatureltr droughtthe plantcare database fig table s4 finally hormone and stressrelated motifs were selected from promoter sequences of plant sf1s there are hormonerelated motifs including abscisic acid abreauxin auxre auxrecore tgabox tgaelementethylene ere gibberellin garemotif pbox tatcbox meja cgtcamotif tgacgmotif and salicylic acid tcaelement and five stressrelated motifsincludingmbswound wunmotif and anoxic are gcmotif motifs almost each sf1 sequence had a great diversity ofciselements in its promoter regions except some sequences such as araha13031 s00021 and traes_2al_3d67296921 which did not contain a single motif dueto the sequences contain ˜n™ or no promoter suggestingthat multiple hormonesmediated signalling pathwaysare closely related to sf1 plants resistance analysisshowed that more than half of sf1 promoters containedabre cgtcamotif tgacgmotif and are respectively moreover external hormone signals also affect theabundance of sf1 transcripts figure s3 for examplein arabidopsis at5g513001 mj methyl jasmonateinhibited its expression fig and treatment withother hormones like acc a precursor of ethylene iaaauxin

cancer293

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: " among eukaryotic anisms alternative splicing is an important process that can generate multipletranscripts from one same precursor messenger rna which greatly increase transcriptome and proteome diversitythis process is carried out by a superprotein complex defined as the spliceosome specifically splicing factor branchpoint binding protein sf1bbp is a single protein that can bind to the intronic branchpoint sequence bpsconnecting the ² and ² splice site binding complexes during early spliceosome assembly the molecular functionof this protein has been extensively investigated in yeast metazoa and mammals however its counterpart inplants has been seldomly reportedresults to this end we conducted a systematic characterization of the sf1 gene family across plant lineages inthis work a total of sequences from plant species were identified phylogenetic relationships of thesesequences were constructed and subsequent bioinformatic analysis suggested that this family likely originatedfrom an ancient gene transposition duplication event most plant species were shown to maintain a single copy ofthis gene furthermore an additional rna binding motif rrm existed in most members of this gene family incomparison to their animal and yeast counterparts indicating that their potential role was preserved in the plantlineage our analysis presents general features of the gene and protein structure of this splicing factor familyand will provide fundamental information for further functional studies in plantskeywords alternative splicing expression profile phylogenetics plants promoter splicing factor correspondence fyzhunjfueducn kailu zhang zhen feng jingfang yang and feng yang contributedequally to this work1coinnovation center for sustainable forestry in southern china college ofbiology and the environment nanjing forestry university nanjing jiangsu province chinafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0czhang bmc plant biology page of in eukaryotes canonical splicing removes noncoding intronic sequences and assembles the coding elements intomature mrnas while alternative splicing as generatesdifferent multiple transcripts that encode proteins withdistinct structures and functions by differential usage ofexons or splice site [ ] the resulting transcripts ofas greatly contribute to posttranscriptional regulationbiological complexity and proteome diversity in eukaryotes [ ] given that on average there are approximately exons in each transcript in the humantranscriptome and the degenerative nature of corresponding splice sites premrna splicing is sophistically catalysed by the spliceosome spliceosome is amultimegadalton protein complex which consists offive u1 u2 u4 u5 and u6 small nuclear ribonucleoprotein ps snrnps and over spliceosomalproteins furthermore the early assembly of spliceosome complex e or the commitment complex is anatpindependent process and contains u1 snrnps sf1and u2 snrnp auxiliary factors u2af large and u2afsmallthe prespliceosome complex a is formed by replacing sf1 withsf3b155sap155 of u2 snrnps [ ] stepwiseassembly of the following spliceosome during the splicing reaction has been reported as well [ ] however splice site recognition is a critical step during earlyassembly of the spliceosome the current model describes the binding of u1 snrnp and u1 snrna to ashort stretch of nucleotides at the ² splice site of splicing factor sf1mammalian branch point bindingprotein mbbp at the branch point and of u2 snrnpauxiliary factors at the ² splice site these threeciselements are necessary but usually insufficient to define a specific exon“intron boundary thus additionalsplicing enhancers or silencers located at exons and introns may allow the recognition of genuine splice sitesduring early spliceosome assembly [ ] subsequentlysubunitsimportantly sf1 preferentially binds to the intronbranch point sequence bps which is adjacent to thebinding site polypyrimidine tract py of u2af largesubunits mammal u2af65 and fission yeast u2af59bridging u1 and u2af to form an intermediate lariatstructure [ ] in particular sf1 is characterized bythe presence of two types of rna binding motifs at thenterminus a k homologyquaking khqua2 domain which originated from the human heterogeneousribonucleoprotein hnrnp k protein [ ] and oneor two zinc knuckle motifs cx2cx4hx4c x represents any amino acid sf1 also contains a prolinerichregion at cterminus [ ] intriguingly the yeast khdomain specifically binds to the bps of premrnas witha glyproarggly motif and the variable loop of thekh domain and is necessary for spliceosomeassembly the first but not the second zinc knuckledomain in yeast has been demonstrated to bind rnawith high affinity moreover the stability of thesf1“u2af65“rna complex is further affected by thephosphorylation status of several sf1 serine residuesser20 ser80 and ser82 in vitro the prolinerichregion of sf1 interacts with u1 snrnp prp40fbp11 inyeast and human [ ] in regards to its interactionpartner the u2af large subunit the nterminal of sf1interacts with its noncanonical rna recognition motifsrrm or u2af homology motif[ ]whereas the other two rrms of u2af large subunitbind to the py region uhmsaccharomycescerevisiae common fruita previous study in fission yeast schizosaccharomycespombe suggests that the initial corecognition of thebranch site and ² splice site is pivotal for correct splicing of target premrnas because of the importance of splice site recognition for gene expression andprotein diversity sf1 has been demonstrated to play essential roles in a number of eukaryotic species includinghuman homo sapiens mice mus musculus buddingyeastflydrosophila melanogaster and roundworm caenorhabditis elegans [ ] for example in humansmissense mutation of splicing factors which are responsible for splice site recognition such as sf1 has beenlinked to tumourigenesis similarly heterozygoussf1 ˆ’ knockdown mice are susceptible to colontumourigenesis induced by an anotrophic carcinogenazoxymethane and sf1 has been found to associatewith betacatenintcf4 complex suggesting its role incarcinogenesis in contrast knockdown of sf1 suppresses the development of germ cell tumours in mice indicating its tissue dependency in cancer researchfurthermore the molecular function of sf1 has been extensively studied in yeast for instance a sf1 mutantstrain causes frequent exon skipping in fission yeast additionally sf1 has been proposed to recognize suboptimal sequences in specific introns and lead to nuclearaccumulation of premrna with aberrant splicing however increasing evidence indicates that this proteinis a regulator of splice site recognition and does not reduce general splicing specifically during alternative splicing by targeting a subset of genes [ ] thishypothesis is supported by the fact that knockdown ofsf1 in both yeast and human extracts only slightly affects the splicing outcome rnai targeting of thisgene has been demonstrated to not affect the splicingpattern of several splicing marker genes tested in comparison to studies in human and yeast few reports have been published related to plant sf1 genessimilar functions of the arabidopsis sf1 gene were proposed in an early study in this plant sf1homologue is reportedly responsible for the splicing of a 0czhang bmc plant biology page of to maintain itsgroup of transcripts the lossoffunction mutant atsf1“ of this gene leads to abnormal development earlyflowering and dwarfism and aba or heat stress sensitivity in arabidopsis [ ] subsequently the domainstructure and its functional relationships have been substantially investigated and the rrm domain is considered crucialfunction in plantsmoreover sf1 may have a different mechanism of ²splice site recognition in plant because the plant sf1 homologs contain a different rrm domain compared withfungal and metazoan counterparts [ ] on the otherhand a study found that atsf1 may be likely to play afunctional role in the cytoplasm because it was found toshuttle between the nucleus and cytoplasm however no related investigations have been conducted onthe phylogenetic analysis of plant sf1 genes and theirregulatory mechanisms although it is a highly conserved family and has conserved functions in eukaryotesplant sf1 genes may have overlapping and distinct rolescompared to the mammalian genes hence studying thephylogenetic relationship and regulatory mechanism ofplant sf1 genes may make us understand the evolutionary history characteristics an expression profile of thisgene family and predict specific functions in plants thiscan lay the foundation for further functional studies inviridiplantae to this end we systematically identified sf1 sequences from plant species ranging fromalgae to higher plants meanwhile the gene and proteinstructure potential regulation at promoter regions andexpression pattern of these genes were further investigated in this study we hypothesize that plant sf1 isstructurally different from its counterparts in animalsand yeast but it is conserved among lower and higherplants indicating its specific role in alternative splicingin branch point recognitionthalianasf1methodssequence acquisition and identification of plant sf1genesthe arabidopsisprotein sequenceat5g51300 was used to search similar sequences inall available plant species from the phytozome v121databasehttpsphytozomejgidoegovpzportalhtml by running the blastp program with an evaluecutoff 1e10 the other parameters were the default settings then the retrieved protein sequences wereexamined and filtered using the hmmer score defaultsettings which contained pf16275 splicing factorkhomology domain kh_1 and pf00076 rna recognition motif rrm_1 finally putative sf1 sequencesfrom plant species were identified detailed information including groups plant species common namesand number of sf1 homologs reported for each planthelixhairpinsf1hhdomainpf00013species for subsequent analysis are listed in table s1subcellular location prediction of identified sf1 proteinswas carried out using wolf psort httpswolfpsorthgcjp construction of molecular phylogenetic tree of plant sf1genesprotein sequences of the aforesaid plant sf1 genes wereextracted from phytozome v121 database for phylogenetic relationship analysis the sequences with the longestcoding sequences were chosen for genes with multipledifferent splicing isoforms then multiple sf1 proteinsequences were aligned with the muscle v38 softwarewith default settings the molecular phylogenetictree of plant sf1 genes was then constructed using themaximum likelihood method ml jtt g i modelvia phyml v30 program with the following parametersinitial tree bionj discrete gamma model yes numberof categories gamma shape parameter proportion of invariant subtree patterns aliasing no figtree v143 was used to visualize and edit the phylogenetic treegene structure protein domain and multiple em for motifelicitation meme analysisrequired genomic cdna and peptide sequences and allsf1 gene structures were downloaded from the phytozome v121 database corresponding intron phases weregenerated using the online program gene structure display server gsds20httpgsdscbipkueducn correlation analysis of sf1 exons were performedby using the piece2 webserver httpwwwbioinfogenomenetpiecesearchphp tdsourcetags_pctim_aiomsg sf1 protein sequences were used to search formatching pfam families using the hmmer websitehttpswwwebiacuktoolshmmer then protein domain patterns were drawn by using tbtools software according to the full pfam resultanttableconserved motifs of plant sf1 cdna sequences andprotein sequences were analysed on the meme onlineprogram httpmemesuitetoolsmeme considering a maximum of the most preserved motifs predicted for each sequence and leaving other settings onthe default parametersmotif prediction in promoter regions of plant sf1 genesthe 15kb ²flanking sequences of plant sf1 geneswere extracted from genomic data available in phytozome database prediction of plant putative ciselementswas performed with the online server plantcarehttpbioinformaticspsbugentbewebtoolsplantcarehtml motifs related to tissuespecific expressioninternal hormones and external environmental stress response were selected for further analysis and discussion 0czhang bmc plant biology page of expression analysis base on microarray datasets and geneexpression experimentsexpression data of arabidopsis s tuberosum g max slycopersicum p trichocarpa and b distachyon includingtissue specificity and stress responses were extracted fromthe efp browser series of the bioanalytic resource forplant biology httpbarutorontoca expressionvalues of selected plant sf1 genes were log transformedlg to generate visualize expression difference heatmapsby using bar heatmapper tool program httpbarutorontocantoolscgibinntools_heatmappercgigene expression experimentstotal rna of samples from different plant tissues wereextracted by rneasy mini kit qiagen usa and subsequently reversed transcribed into cdna by fastkinggdna dispelling rt supermix fastking tiangenchina according to the manufacturer™s instruction rtpcr amplification were programmed asfollowings °c min °c s °c s °c s cycles °c min sybr premix ex taqtm accuratebiotechnology co ltd hunan china was used forquantitative realtime rtpcr analysis which was conducted on the stepone plus realtime pcr system following optimized program °c s °c s °c s cycles the data were normalized to the expression of internal reference genes table s6 and the transcript abundance was determined by the comparativect value method analysis of proteinprotein interaction network andstructural conservationa proteinprotein interaction network was generated bythe string website httpsstringdb withrepresentative protein sequences from arabidopsis thefollowing basic settings were employed meaning of network edges evidence line colour indicates the type ofinteraction evidence and active interaction sourcesexperimentsthere are three domains in the arabidopsis sf1 protein the phosphorylation and u2af65 binding of thenterminal domain of splicing factor during ² splicesite recognition of homo sapiens pdbid 2m0g identity evalue 7e17 was similar to that of the khomology domain the structure for recognition of theintron branch site rna by splicing factor of homo sapiens pdbid 1k1g identity evalue 9e27 canbe used as the template for the splicing factor helixhairpin domain therefore homology modelling wasperformed with modeller based on two crystalstructures the amino acid conservation scores were calculated using the consurf web server based on the mlmethod input attributes were the 3d model andmultiple sequence alignment figure s4 related figureswere created based on pymol with default settings analysis gene structure evolution with orthologue groupof sf1 genesreconstruction of the evolutionary history of the structure of the plant sf1 family of orthologous genes wascarried out by searching at5g513001 in the piece severhttpwwwbioinfogenomenetpieceindexphpthis provided an exonintron display for orthologousgenes from gene structure data sets linked to the phylogenetic treeresultssequence identification and phylogenetic analysis of theplant sf1 gene familyto identify sf1 gene family members in plants we carried out a blastp search using the arabidopsis atsf1at5g51300 amino acid sequence against the phytozome database v121 after filtering the sequence without sf1 signature or truncated sequences a total of sequences from plant species were retrieved whichwere roughly classified as algae bryophyta basicangiosperm monocots and eudicots table s1specifically the only species with four copies of plantsf1s was eutrema salsugineum salt cress table s1 inparticular three copies of sf1 genes were observed infive species including panicum virgatum switchgrasstriticum aestivum common wheat daucus carotacarrot kalanchoe laxiflora milky widow™s thrill andsalix purpurea purple osier willow additionally plant species contained two copies and species including the model plant arabidopsis possessed only onecopy of plant sf1s respectively the relatively largernumber of sf1 genes and higher number of plant speciesin this work demonstrated the universality and complexity of the sf1 gene family the retrieved sequences of plant species provided us with more complete information to analyse the phylogenetic relationship of the sf1gene family subsequently a rooted phylogenetic treewas constructed based on the abovementioned protein sequences by using the maximum likelihoodmethod the tree™s bootstrap threshold “ was represented by a colour gradient fig in general all sf1protein sequences were clustered into four major cladesincluding alga in yellow other land plants in greenmonocots in pink and eudicots in blue and one species amborella trichopoda belonged to basic angiosperm shown in colourless the phylogenetic tree ofsf1s figs and left panel with clear topology andoverall high bootstrap values was similar to evolutionarytrend from lower plants to higher plants reported inother studies for example the genes of algae in the yellow branch were representative members of the lineage 0czhang bmc plant biology page of fig circular phylogenetic tree of the sf1 gene family available in plants the phylogenetic tree of sf1 genes in plants was constructed basedon maximumlikelihood with jtt g model by using phyml v3037 a total of protein sequences from plant species were chosen tocalculate the phylogenetic relationship for tree construction bootstrap values are labelled at each major branch the corresponding informationof each transcript such as species name common name number of identified transcripts and their transcript id nomenclature are shown intable s1 taxonomies based on apgiv system 0czhang bmc plant biology page of fig see legend on next page 0czhang bmc plant biology page of see figure on previous pagefig gene structure comparisons and conserved motif identification among plant sf1 genes from left panel to right panel verticalphylogenetic tree genomic anization and identified cdna conserved motifs by meme analysis intron phase and are shown on thegene structure the conserved sequence of identified motifs represented by different coloured boxes are listed below some long genes werereduced to onehalf of their original length to fit this picturethat diverged before the evolution of land plants whichwas the basal part of the phylogeny in the blue branchfive sequences from kalanchoe with higher bs valuesformed a subclade showing their closer evolutionary relationships additionally cagra3782 s00261p fromcapsella grandiflora and carubv10025900m from c rubella formed a subclade with the arabidopsis sequencesbecause they all belong to brassicaceae which is consistent with the apg iv system fig and table s1 usually some homologous sf1 sequences from the samespecies were clustered in the same small branch next toeach otherthese species included cashew soybeanapple woodland strawberry quinoa carrot coloradoblue columbine maize common wheat cereal grassmoss and bog moss fig and table s1 in contrastsome other homologous sf1 members from the samespecies were clustered into the different subclades suchas purple osier willow poplar eastern cottonwood saltcress potato diploid kalanchoe milky widow™s thrillhall™s panicgrass switchgrass green algae and volvoxfig and table s1gene structure and conserved motif analysisit is necessary to compare the exonintron anizationand conserved motifs of the plant sf1 gene family toclarify their evolutionary process and potential functionthe gene structure models of sf1 genes were attachedto the phylogenetic tree fig and the correspondingintron phase of each was also displayed fig tables2 figure middle panel shows that the gene lengthand structure of each member of the sf1 family exhibitssignificant differences for example the gene structureof members of sf1 family genes did not containintron sequences this subset accounts for of thetotal number of members fortyeight sequences of sf1genes had exon1 intron anizations accounting for of all genes in particular some genes from algaehad multiple exons including vocar0008 s02941p volvox carteri which contained the most exons exonsmoreover different gene structures were also observedat the same subbranch for instance two sequencesfrom zea mays maize zm00008a037777_p01 exonsand zm00008a007621_p01 exons were observed tohave distinctive gene structures although the dissimilation of gene structure of each member of sf1s was substantial we found that the length of cdss did notsignificantly change fig thus whether it influencesthe differentiation of their gene function needs to befurther investigated further investigation on conservedmotifs by using multiple em for motif elicitationmeme search tool demonstrated that most sf1 genes sequences exhibited similar sequence signatures andthe same order and all contained the analysed motifsexcept one sequence of micromonas pusilla hada different position fig right panel although no obvious differences in identified conserved motifs werefound among basal angiosperm monocots and eudicotssequences from the same species were found to have different motifs fig for example aqcoe5g4069001pand aqcoe7g0393001p from the eudicot aquilegiacoerulea had motifs and motifs respectively thesame situation was found in d carota dcar_006843dcar_008506 and dcar_004968 had motifs motifs and motifs respectively intriguingly the cdslength of dcar_008506 was the longest notably somesequences from algae and moss had fewer conservedmotifs for examplein bryophyta the sequences ofphyscomitrella patens pp3c7_10890v31p and pp3c11_24710v31p sphagnum fallax sphfalx0015s00771pand sphfalx0010s01971p and marchantia polymorphamapoly0009s01891p had nine motifs in algal plantsthe sequences of and from micromonashad only motifs and motifs respectively moreoveralthough the sequences of volvox carteri vocar0007s03451p and vocar0008 s02941p and chlamydomonasandcre09g386731t11 contained multiple exons they had motifs indicating their sequence variation had little influence on function classes further correlation analysisof the sf1 exon regions were carried out to elucidate thegainloss of introns correlations between transcripts ofplant sf1s are shown in fig providing additional information for phylogenetic analysis for example thereis more similarity between pgsc0003dmt400081859and migutd025312 because of multiple exact matchesbetween the exons of the two transcriptscre12g553750t11reinhardtiianalysis of protein domain and conserved motifs inpeptidesthe protein domains were analysed by using the aboveselected peptide sequences from plant species thepeptides™ annotations were splicing factorrelated andconserved protein motifs were predicted according tothe retrieved peptide sequences by meme analysisfig consequently all sf1s were found having sf1_hh nterminal domain on the nterminal ofthe 0czhang bmc plant biology page of fig analysis of gene structure evolution with orthologue group of sf1 genes exonintron structure and intron phase right panel are linked tothe plant species tree left panel genes with red colour represent the members of the plant sf1 genes different coloured lines mean differentexon comparison results between species 0czhang bmc plant biology page of fig comparisons of protein domains and conserved motif identification among plant sf1 genes protein domain middle panel and identifiedprotein conserved motifs right panel identified by meme analysis are shown against the vertical phylogenetic tree left panel the conservedsequence of identified motifs represented by different coloured boxes are listed below 0czhang bmc plant biology page of peptides followed by a kh domain and a cterminal domain namely an rna recognition motif rrm fig middle panel interestingly in algae peptides from mpusilla v carteri vocar0008 s02941p andc reinhardtii cre09g386731t11 had two rrm domains the amino acid lengths of sf1 proteins rangedfrom aa to aa and most of them possessed to amino acids table s3 consistently most ofthem are approximately to amino acids inlength subcellular location prediction showed that themajority of sf1 proteins were had nuclear localization table s3 moreover proteins of 147m014250 ricinus communis and migutf011911pmimulus guttatus were located in the vacuoles proteins of traes_2dl_6f03f05fa4 t aestivum and m pusilla were predicted to be cytoplasmic proteins of gsmua_achr5p25100_001 musa acuminataand cre09g386731t11 c reinhardtii were located inthe chloroplast and endoplasmic reticulum respectivelymeme analysis for sf1 peptide sequences was used topredict a total of conserved motifs which are presented as coloured boxes and cover most of the proteinfig right panel further analysis showed that peptides had all motifs accounting for approximately of all sf1 protein sequences analysed in the studyinterestingly all sequences from moss have conservedmotifs in the analysis suggesting the conservation ofsf1 proteins in bryophyta furthermore almost all eudicots had conserved motifs”except anacardium occidentalegrandifloracagra3782 s00261p which lacked motif and malusdomesticavescamrna211921v10hybrid and brassica rapa brarac014811p which lacked motif ”while most monocots had eight conserved motifs in contrast algal plantsonly possess approximately half of the predicted motifs due to their peptides with integrant protein domainsimplying the least degree of conservation and divergenceof plant sf1 proteins in algae t motifs that all algaeshared were motif motif motif and motif anaoc0018 s04251pmdp0000558834fragariaand canalysis of promoter and tissuespecific expression of sf1genesto further analyse the regulation of plant sf1 genes atthe transcriptional level the 15kb upstream sequencesof plant sf1 genes were obtained from the phytozomedatabase then the ciselements of each promoter wereidentified by using the plantcare program table s4 consequently a total of motifs were predictedgenerally eight ciselements related to tissuespecificexpression among them were selected fig and tables4 including hdzip1 for differentiation of the palisademesophyll cells the ryelement which regulates seedspecific expression the aaca_motif and gcn4_motif waspresentfurther hdzipinvolved in endosperm expression and the catboxccgtccbox doct and oct for meristem expression further analysis showed that there were only promoters of sf1 genes which had tissuespecific regulatory ciselements particularly the catbox and ccgtccbox turned up at the highest frequency and greatestabundance in the promoters of sf1 genes both of themregulate meristemspecific expression and play key rolesduring development and growth of plants consistentlypurple false brome brachypodium distachyon of monocots not only had a catbox and ccgtccbox butwas also highly expressed in young leaves internode adventitious roots and roots fig and figure s2however no motifs were found to link the highexpression of two sf1s of glycine max soybean insam and roottip figure s1 additionally the aaca_motif was only detected in solanum tuberosumpgsc0003dmp400032853 of potato suggesting itsspecific role in regulating endospermspecific negativeexpressioninpodel03g1132001p of populus deltoideseasterncottonwood and spipo17g0046100 of spirodela polyrhiza greater duckweed the ryelement was detectedin the promoter of the dicot model plant arabidopsisand low expression was also reported in dry seed inarabidopsis fig suggesting that the ryelement isinvolved in seedspecific negative expression of arabidopsis moreover expression levels in the same tissuetype showed significant differences during differentgrowth stages for example the expression level in stamen of flower stage of arabidopsis was obviouslyhigher than that of the other flower development stageshowever the expression levels of different growth stagesof solanum lycopersicum were not only similar butlower and no motifs were found in the promoter in tomato figs and s1 furthermore different expressionpatterns were detected in several sf1 genes with multiple copies figs s1 and s6 for instance similar tissue expression profiles were detected in two sf1trichocarpahomologuespotri001g1264001 and potri003g1072001 and themonocotandzm00008a037777_p01 figure s1 and s5 in contrasttwo sf1 genes of s tuberosum showed differential expression patterns similar to in g max figs and s1zm00008a007621_p01dicot populuszea maysfrom theanalysis of promoter and internal and external hormonesexpression of sf1 genesin longterm evolution and development plants havegradually formed mechanisms of adaptation and resistance to adversity to maintain their life and sustaingrowth to understand the regulatory mechanisms of internal and external stimuli on plant sf1s cisacting elements involved in hormone and stress were studied with 0czhang bmc plant biology page of fig see legend on next page 0czhang bmc plant biology page of see figure on previous pagefig analysis of motifs related to tissue specificity in the plant sf1 promoter regions eight cisacting motifs are represented in different colortriangles positions of these identified motifs are labeelled along the kb ²flanking regions of each sf1 gene the line solid and dottedrepresents regions with basic pairs and regions of no sequences or annexed base n respectively symbols on above the line represent the motifsat the plus strand whereas symbols on below the line represent the motifs at the minus strand function of motifs aacamotif involved inendospermspecific negative expression catbox cisacting regulatory element related to meristem expression ccgtccbox cisactingregulatory element related to meristem specific activation doct cisacting regulatory element related to meristem specific activationgcn4_motif cisregulatory element involved in endosperm expression hdzip1 element involved in differentiation of the palisade mesophyllcells ryelement cisacting regulatory element involved in seedspecific regulation the black vertical lines represent break at that particularbranch oct cisacting regulatory element related to meristem specific activationlowtemperatureltr droughtthe plantcare database fig table s4 finally hormone and stressrelated motifs were selected from promoter sequences of plant sf1s there are hormonerelated motifs including abscisic acid abreauxin auxre auxrecore tgabox tgaelementethylene ere gibberellin garemotif pbox tatcbox meja cgtcamotif tgacgmotif and salicylic acid tcaelement and five stressrelated motifsincludingmbswound wunmotif and anoxic are gcmotif motifs almost each sf1 sequence had a great diversity ofciselements in its promoter regions except some sequences such as araha13031 s00021 and traes_2al_3d67296921 which did not contain a single motif dueto the sequences contain ˜n™ or no promoter suggestingthat multiple hormonesmediated signalling pathwaysare closely related to sf1 plants resistance analysisshowed that more than half of sf1 promoters containedabre cgtcamotif tgacgmotif and are respectively moreover external hormone signals also affect theabundance of sf1 transcripts figure s3 for examplein arabidopsis at5g513001 mj methyl jasmonateinhibited its expression fig and treatment withother hormones like acc a precursor of ethylene iaaauxin Answer:

Colon_Cancer

" phytolaccaceae species in china are not only ornamental plants but also perennial herbs that areclosely related to human health however both largescale fulllength cdna sequencing and reference genevalidation of phytolaccaceae members are still lacking therefore singlemolecule realtime sequencing technologywas employed to generate fulllength transcriptome in invasive phytolacca americana and noninvasive exotic picosandra based on the transcriptome data rtqpcr was employed to evaluate the gene expression stability in thetwo plant species and another indigenous congener p acinosaresults total of gb and gb clean reads of p americana and p icosandra were generated including and full length nonchimeric flnc reads respectively transcript clustering analysis of flnc readsidentified and consensus isoforms including and highquality ones after removingredundant reads and transcripts were obtained based on structure analysis total and alternative splicing variants and simple sequence repeats ssr as well as and completecoding sequences were detected separately furthermore and lncrna were predicted and and transcripts were annotated respectively subsequently seven reference genes in the two plant species and anative species p acinosa were selected and evaluated by rtqpcr for gene expression analysis when tested indifferent tissues leaves stems roots and flowers 18s rrna showed the highest stability in p americana whetherinfested by spodoptera litura or not ef2 had the most stable expression in p icosandra while ef1α was the mostappropriate one when attacked by s litura ef1α showed the highest stability in pacinosa whereas gapdh wasrecommended when infested by s litura moreover ef1α was the most stable one among the three plant specieswhenever germinating seeds or flowers only were consideredcontinued on next page correspondence yiwangynueducn1yunnan key laboratory of plant reproductive adaption and evolutionaryecology yunnan university kunming china2laboratory of ecology and evolutionary biology state key laboratory forconservation and utilization of bioresources in yunnan yunnan universitykunming chinafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cliu bmc plant biology page of continued from previous page fulllength transcriptome of p americana and p icosandra were produced individually based on thetranscriptome data the expression stability of seven candidate reference genes under different experimentalconditions was evaluated these results would facilitate further exploration of functional and comparative genomicstudies in phytolaccaceae and provide insights into invasion success of p americanakeywords phytolaccaceae smrt sequencing fulllength transcriptome analysis reference gene evaluation rtqpcr phytolacca americana is a member of the phytolaccaceae family and is native to northeast america becauseof its ornamental and medicinal applications it was introduced into china in unfortunately it hasevolved into an invasive species and spread to mostareas of the country especially in central and southernchina compared to noninvasive exotic congener p icosandra and native congener p acinosa p americana isof interest because it exhibits multiple biological activities such as plant pesticides antimicrobial propertyheavy metal accumulation capacity [“]in order to investigate the mechanisms of various bioactivities of p americana further transcriptomewidestudy is necessary to facilitate reports have showed thatjasmonic acidinduced and cadmiumtreated transcriptome data of p americana have been obtained by illumina hiseq and illumina hiseq platformrespectively [ ] howeverthese data were bothachieved by second generation sequencing sgs whichcould not produce fulllength transcripts genomic dataof p americana was available at the sra under projectprjna544344 but it™s raw reads without coding sequences prediction and functional annotation third generation sequencing tgs is known for itskilobasesized long reads and is an outstanding strategyfor better understanding rna processing for exampleit can be used to analyze different transcript isoformsregulated by alternative splicing which greatly increasesthe repertoire of proteins lead to genetic and functionaldiversity and is prevalent in most eukaryotic anisms the long reads could also provide sequence information on genecoding regions for functional analysis atthe transcriptional level and thus can be applied to refine an assembled genome for better annotation however tgs could not quantify gene expression forthe moment and have a relatively high error rate thansgs the combination of tgs and sgs are able to solvethis problem and are highly recommended by most researchers with the transcriptome and genome data availablefunctional genomics research is being performed whichrelied heavily on gene expression analysis reversetranscription quantitative real time pcr rtqpcr hasbeen reported to be a very sensitive and accurate technique to analyze gene expression level but it requiresappropriate reference gene as an internal control tonormalize mrna levels between different samples foran exact comparison of gene expression [ ] anideal reference gene should be expressed at a constantlevel across various experimental conditions howeverstudies have shown that no single reference gene is universal for all experimental conditions [“] therefore it™s necessary to estimate the stability of referencegenes under particular experimental condition beforeusing them for gene expression analysisin the present study to provide highquality and morecomplete assemblies in genome and transcriptome studies of phytolaccaceae a hybrid sequencing approachcombining the sgs and tgs technologies was carriedout first fulllength transcriptome of the invasive plantspecies of pameracana and an noninvasive exotic conicosandra was generated by singlemoleculegener prealtimesmrtsplicingevents simple sequence repeats ssr coding sequencesprotein annotations and long noncoding sequenceswere analyzed respectively at transcription level furtherthe stability of reference genes was evaluated in two phytolaccaceae species mentioned above and one nativecongener p acinosa by rtqpcr in order to facilitatefuture research on functional gene expressionsequencing alternativeresultsto classify the plant species these three phytolaccaceaemembers p americana p icosandra and pacinosa wereidentified by pcr and followed by sequences alignmentbased on sequences of second internal transcribed spacer its2 and the intergenic spacer of photosystem iiprotein d1 gene and trnahis gene of chloroplast genome psbatrnh table s1 the sequences of its2 andpsbatrnh in p americana that we employed had theidentity of with the sequences reported by chen in p icosandra the sequences of its2 had identity and the sequences of psbatrnh had identity with the results of chen™s in pacinosa 0cliu bmc plant biology page of similarity of its2 and identify of psbatrnh werefound isoforms afterremoving redundantsmrt sequencing data outputusing the pacific biosciences™ smrt sequencing protocol gb clean reads of invasive species p americana were obtained after preprocessing on the basis offull passes and sequence quality circular consensus sequences ccs with fulllength rate were obtained including fulllengthnonchimeric flnc sequences and highqualityconsensussequences from the high quality consensus isoforms transcripts alternative splicing events ssr complete coding sequences lnc rnasand annotated transcripts in p americana wereachieved similarly gb clean reads in p icosandrawere identified and ccs with fulllength rate flnc sequences as well as highquality consensus isoforms were filtered subsequently transcripts and alternative splicingevents were obtained what™s more ssrs complete coding sequences lnc rnas and annotated transcripts were identified in picosandratable transcriptome analysisbased on the structure of achieved transcripts and alternative splicing events were identified in pamericana and p icosandra respectively transcripts of bp in total in p americana wereemployed for ssr analysis based on the sequence lengththat was more than bp including ssrs and ssrcontaining sequences similarly transcripts bp in total in picosandra wereemployed for ssr analysis and ssrs togetherwith ssrcontaining sequences were identifiedtable summary of fulllength transcriptome sequencingclean reads gbccsflncflnc flncccsconsensus isoformhigh quality consensus isoformtranscriptsalternative splicingssrcomplete coding sequenceslncrnaannotated transcriptsp americanap icosandrathe detail information about the number of sequencescontaining more than one ssr the number of ssrspresent in compound formation and the number of different types of ssrs were shown in table in additiontotal of complete coding sequences cds in pamericana and cds in p icosandra were identified by using transdecoder the length distribution ofpredicted proteins was shown in fig s1in pdatabasespecifically nrwith the eight protein databases sequence alignmentswere performed to annotate predicted proteins in total transcripts in p americana and tranicosandra were annotated separatelyscriptstable the number of annotated protein sequencesin p americana was similar with p icosandra under aparticularncbi nonredundant protein analysis revealed that approximately transcripts in p americana and transcripts in picosandra showed the highest sequencesimilarity with beta vulgaris fig go gene ontology assignment also suggested that similar amount ofsequences in the two plant species belonged to the sameterm and many were classified into cell part and cellterm of cellular component catalytic activity and binding of molecular function and metabolic process andcellular process of biological process fig cogclusters of orthologous groups of proteins annotationshowed that a large number of predicted proteins in thetwo plant species were linked to functional class r general function prediction only j translation ribosomalstructure and biogenesis t signal transduction mechanisms g carbohydrate transport and metabolism ando posttranslational modification protein turnoverchaperones fig s2 the result of eggnog evolutionary genealogy of genes nonsupervised orthologousgroup annotation indicated that most of the annotatedproteins in the two plant species were belonging to thefunctional class s function unknown fig s3 kogeukaryotic ortholog groups functional classificationsuggested that r general function prediction only ando posttranslational modification protein turnover andchaperones were the most abundant functional categories in the two plant species fig s4 these results indicated that most of the sequences obtained were trulyfunctional proteins and had a similar functional classification in p americana and its congener picosandraeven though more work is needed to identify sequencesthat regulated or involved in the invasion success of pamericana the annotation of predicted proteins provided necessary information for further studiesbesides the transcripts encoding proteins long noncoding rnas lncrnas were achieved lncrnas arereported to be key regulators in plant biological prolncrna in pameracana andcesses the number ofpicosandra was predicted by cpc coding potential 0cliu bmc plant biology page of table ssrs obtained from transcripts with more than bpsearching itemtotal number of sequences examinedtotal size of examined sequences bptotal number of identified ssrsnumber of ssr containing sequencesnumber of sequences containing more than ssrnumber of ssrs present in compound formationnumber of mono nucleotide ssrnumber of di nucleotide ssrnumber of tri nucleotide ssrnumber of tetra nucleotide ssrnumber of penta nucleotide ssrnumber of hexa nucleotide ssrcalculator cnci codingnoncoding index pfamand cpat coding potential assessment tool respectively in total lncrna in pamericana and lncrna in picosandra were predicted by all these fourmethods fig subsequentlytranscription factorstfs that are key components involved in the transcriptional regulatory system were predicted in p americana tfs of types were filtered and in picosandra tfs of types were predicted thesetwo plant species shared the first types of tfs butthe number of each type tf was not similar especiallyrlkpelle_dlsv c3h snf2 and camk_camklchk1 indicating the particular functions on transcriptregulation fig amplification performance of rtqpcrprimers designed for rtqpcr were evaluated by pcrfirst the primers which produced single ampliconwithout primer dimer were chosen for melting curveanalysis only primers which produced a single fragmentefficiencywereqpcr amplificationchosenforp americanap icosandraassessment the qpcr efficiency of each primer pair wasgenerated from a 10fold serial dilution of pooled cdnaand was shown in table the threshold cycle ct values of each reference genewere employed to evaluate expression level under different experimental conditions fig average ct valuesfor all the seven candidate reference genes ranged from to in which ef1α showed the highest expression level and 28s rrna had the lowest expression levelit was also suggested that ct values of βactin and tubulin fluctuated significantly across all the experimentalsamplesstability of candidate reference genesforto determine the appropriate reference genesnormalization in different experimental conditions theexpression data was analyzed by genorm normfinderand bestkeeper respectively table s2when expression stability of reference genes wereanalyzed in different tissues leaves stems roots andflowers of p americana 18s rrna and ef2 oftable number of proteins annotated via differential protein databasedatabasesp americanaannotated number ‰¤ length length ‰¥ p icosandraannotated number ‰¤ length length ‰¥ coggokeggkogpfamswissproteggnognrall 0cliu bmc plant biology page of fig homologous species distribution of p americana and p icosandra annotated based on the nr database a p americana b p icosandrapamericana were identified as the most suitable reference genes by genorm and normfinder and 18s rrnawas also suggested by bestkeeper pairwise variation valueof v23 was below the cutoff value of which meansthe combination of two reference genes were most suitable for gene expression normalization fig whentested in picosandra ef1α was recommended for normalizing gene expression analysis not only by genorm butalso by normfinder ef2 was also suggested by genormand bestkeeper in pacinosa ef1α was the best reference gene suggested by genorm and bestkeeper but 18srrna was recommended by normfinder the use of tworeference genes was suitable because pairwise variationvalue of v23 was below when pooled the data of different tissues from pamericana and picosandra togetheref2 was shown to be the most stable gene by all the threemethods when investigated the expression stability ofreference genes in different tissues of pamericana andpacinosa 18s rrna showed the best expression stabilityby genorm and normfinder while ef2 was referred asthe most stable one by bestkeeper however the combination of five reference genes was recommended bygenorm for v56 which was less than when thedata of different tissues from picosandra and pacinosawas put together ef1α was identified as the best oneby genorm and normfinder whereas ef2 was suggested to be the best stability reference gene by bestkeeper when set the data of these three plant speciesas a pool ef1α was suggested to be the most stableone by genorm and normfinder while ef2 was alsorecommended by genorm and bestkeeper accordingto these results it is very important to select the appropriate reference gene when analyze the gene expressionlevel among plant species 0cliu bmc plant biology page of fig classification of the transcripts annotated by the gene ontology gowhen analyzed the data among germinating seeds28s rrna and ef1α were identified as the best reference genes by genorm while 18s rrna was recommended by normfinder and gapdh was suggested bybestkeeper three reference genes were sufficient tonormalize gene expression for v34 was below inflowers only of these three plant species ef1α was confirmed by all the three methods the genorm analysisshowed that the value of v45 was below so fourreference genes in combination were suggested thesefig venn diagram of the number of lncrnas predicted by cpc cnci cpat and pfam a p americana b p icosandra 0cliu bmc plant biology page of fig classification of predicted transcription factorsresults indicated that when focusing on particular tissuesof different plant species the selection of reference genewas also very essentialwhen plants were infested by slitura 18s rrnashowed the most expression stability suggested by genorm and normfinder in different tissues of pamericanawhile ef1α wasby bestkeeper therevealedcombination of two reference genes was suggested bygenorm due to the value of v23 was less than 18srrna was also recommended by genorm in s liturainfested picosandra and ef1α was shown to be themost stable one by normfinder and bestkeeper fourreference genes in combination were recommended bygenorm 18s rrna was also identified as the besttable primers for rtqpcr analysisgene nametubulingene descriptiontubulin ef1αelongation factor 1alphaprimer sequence ²²f gtaaggaagccgagaattgr tcaacaacagtgtcagagaf tgaagaaggtcggatacaatr gtagacatcctggagtgggaphdglyceraldehyde3phosphate dehydrogenasef tggtgctaagaaggttattatcef2elongation factor 18s rrna18s rrnaβactinactin728s rrna28s rrnar2 linear regression coefficientr gagtgaacggtggtcataf gtatcaccatcaagtcaactgr acaatcaaccacaacaaggf acttcctcttctcgtatcattr tgttcagcatagactgtgaf atgctatccttcgtctggr tactcttggctgtctctgf tacgattggttacggacatr ttctcatcaacaacagcatatlength bppcr efficiency r2 0cliu bmc plant biology page of together 18s rrna showed the best stability in genormand normfinder while the expression stability of ef1αwas suggested by bestkeeper in pamericana and picosandra 18s rrna was identified as the best referencegene by all the three algorithms in pamericana andpacinosa 18s rrna and βactin were suggested bygenorm in picosandra and pacinosa while gapdhand ef2 were recommended by normfinder and bestkeeper respectively when take all the data of s liturainfested plant species into account 18s rrna exhibitedthe most stable expression suggested by genorm andnormfinder while ef2 was the gene with the most constant expression identified by bestkeeperdiscussionfulllength transcripts are fundamental resources forstructuralfunctional and comparative genomics research [ ] smrt sequencing has been acknowledged by enabling the generation of multikilobasesequences to improve genome and transcriptome assembly the fulllength cdna sequences generated areable to characterize the posttranscriptional processsuch as alternative splicing lncrna prediction and coding sequences for further gene functional studies basedon the fulllength transcriptome data generated about gb of clean data were obtained for pamericana andpicosandra respectively table accordinglythenumber of ccs flnc consensus isoforms highqualityfig rna transcription levels of seven candidate reference genesin p americana picosandra and p acinosa the expression level ofcandidate reference genes in total samples n was presentedas cycle threshold number ctvalue and explained by box andwhisker plots the asterisks represented the minimum and maximumct value the squares indicated the 25th and 75th percentiles andthe median was represented by a bar across the squarereference gene by genorm in plant species pacinosawhile tubulin was suggested by normfinder and 28srrna was recommended by bestkeeper the combination of three reference genes was appropriate by genorm when analyzed the data oftwo plant speciesfig pairwise variation analyzed by genorm to determine the optimal number of reference genes for accurate normalization a threshold valueof was suggested for valid normalization if the value of vnn pairwise variation is less than then n reference genes in combinationare recommended for gene normalization if the value of vnn is more than then vn 1n should be taken into account pam pamericana pic p icosandra pac p acinosa lsrf different tissues of leaves stems roots and flowers gs germinating seeds of these three plantspecies f flowers of these three plant species lsr different tissues of leaves stems and roots i infested by s litura of third instar 0cliu bmc plant biology page of isoforms transcripts alternative splicing events ssrscomplete coding sequeces lncrnas and annotated transcripts were analyzed providing basic transcriptomic information for further studiesreports have showed that fulllength transcriptome ofzea mays have greatly helped in refining gene annotation and revealed the complexity of gene expression inmaize similar analysis has also been conducted inshum bicolor what™s more the world expansioncapability of cydia pomonella has been informed according to its genome information molecularmechanism of rapid growth and invasive adaptation ofan invasive species mikania micrantha has also been investigated according to itsreference genome therefore the fulllength transcriptome data of pamericana and picosandra will contribute to the genomic research and provide insights into invasive mechanism ofpamericana through comparative genomics study inphytolaccaceae speciesgenereliesonanalysisexpressionaccurate relative quantification of rtqpcr for furtherrobustnormalization by stably expressed reference genes tominimize error in the experimental process therefore suitable reference genes for the normalization ofrelative gene expression data in three phytolaccaceaespecies pamericana picosandra and pacinosa weresought under a diverse set of conditions these resultsdemonstrated the importance of validating referencegenes under the relevant experimental conditions forexamplein different tissues leaves stems roots andflowers of pamericana 18s rrna and ef2 were recommended to be the bestsuited reference genes and similar results were found in s liturainfested pamericanahowever even though the appropriate reference genesin picosandra were ranked according to the analyzed results of the three methods all the pairwise variationvalues were above the cutoff value of while thecombination of 18s rrna βactin ef1α and ef2 weremost suitable in s liturainfested picosandra ef2 andef1α have been considered as the ideal reference genesin pacinosa whereas the combination of 18s rrna βactin and gapdh were recommended after s litura infestation researches have also showed that no singlereference gene is stably expressed among different tissues of an anism such as the reference gene selectionin amygdalus persica solanum lycopersicum and glycine max [ ] what™s more our results alsosuggested that reference genes identified based on transcriptome data should be confirmed by experimentalevidence in jainduced transcriptome of p americana28s rrna showed stable expression between exogenousjatreated and control plants ja signal pathway ofplants can be induced by lepidopteran herbivores infestation however 18s rrna and ef2 were identifiedas the most stable expression reference genes in pamericana after s litura infestationin order to conductthe gene expression analysisamong different plant species of phytolaccaceae the dataof the three plant species were also compared togetherwhen compared the data in germinating seeds of threeplant species various genes were recommended by thethree methods the combination of plant species underother experimental conditions showed that the pairwisevalues of almost all the combination were higher thanthe cutoff value of exceptthe combination ofpamericana and pacinosa where five reference geneswere recommended for data normalization as well as thecombination of sliturainfested pamericana and sliturainfested picosandra where three reference geneswere suggested these results indicated that no particular gene was expressed constantly across different plantspecies even though these plants are congeners therefore reference genes should be employed appropriatelyunder the relevant experimental conditionsthe research has provided transcriptomewide fulllength isoforms of pamericana and picosandraproviding insights into invasive success of pamericanaguidelines for selecting appropriate reference genesunder different tissues in one plant species or amongvaried plant species were recommended further no particular gene was expressed constantly under differentexperimental conditions indicating the necessity of reference gene identification these results would facilitatethe exploration of functional and comparative genomicsstudies in phytolaccaceae to better understand plantbiologymethodsplant and insect materialsplants of p americana °²n °²e p icosandra°²n °²e and p acinosa °²n °²eused in this study which was named m k and q firstwere collected in yunnan china sampling was permitted when conducted complying with locallegislationthe formal identification of the samples were conductedby chao chen botany major of laboratory of ecologyand evolutionary biology state key laboratory for conservation and utilization of bioresources in yunnanyunnan university according to flora of china vol5“ flora of north america vol43“ chinese virtual herbarium httpwwwcvhaccn and global plants on jstor httpplantsjstor dna identification was also employed according tothe its2 region of nuclear ribosomal dna one of themost widely used dna fragments in plant molecularsystematics at the generic and species levels and the 0cliu bmc plant biology page of chloroplast psbatrnh intergenic region all voucher specimens were maintained at an experimental fieldof laboratory of ecology and evolutionary biology statekey laboratory for conservation and utilization of bioresources in yunnan yunnan universitytissues of leaves stems roots and flowers from oneindividual plant of p americana or p icosandra werecollected individually from the wild in yunnan provinceand no permission is needed for collecting theses samples each sample was flash frozen in liquid nitrogen andstored at ˆ’ °c for further experimentsshop101732681taobaocomthird instar larvae of spodoptera litura were purchased from henan jiyuan baiyun industry co ltdchinaand then werereared on artificial diet in a climate chamber h at °c with light and h at °c without light for further usefor reference gene evaluation seeds of p americanap icosandra and p acinosa were collected first from thewild in yunnan province and no permission is neededthe seeds were sown separately in agar plates andcultivated in the climate chamber after d five germinating seeds of one plant species were collected togetheras one sample for subsequent experiments each plantspecies have three replications two weeks later othergerminating seeds of each species were transplanted intoplastic pots cm diameter and cm height withsoil jiangsu peilei matrix technology development coltd china and cultivated with adequate water in artificial chambers with same conditions as described abovefour months later leaves stems roots and flowers ofeach plant species were collected individually simultaneously six larvae s litura of third instar were employedto infest on p americana p icosandra or p acinosawith one insect per leaf control treatments were herbivore free after h infestation leaves stems and rootsof these three plant species were harvested individuallyall samples collected were flash frozen in liquid nitrogenand stored in ˆ’ °c for subsequent assays and threereplicates were conducted for each treatmentnucleic acid extraction and assaysgenomic dna was isolated from the leaves of differentplant species following protocols provided by dnaquickplant system tiangen biotech co ltd beijing chinathen it was employed as the pcr template for plantspecies identificationpurekitplanttotal rnas from different tissues was prepared usingrnapreppolysaccharides polyphenolicsrich tiangen biotech co ltd beijingchina according to the manufacturer™s instructionsthe rna quality and purity were measured by using ananophotometer n60 implen germany and the agilent bioanalyzer system agilent technologies causa samples only with a ratio of to a ratio between and and a rin value morethan were chosen for the sequencing library construction an equal amount of total rnas from four different tissues of the same plant species were mixed asone sample for fulllength transcriptome sequencingtotal rnas from the samples collected for referencegene evaluation was also extracted individually as described above for each sample cdna was prepared byusing μg of total rna following the recommendedinstructions of fastquant rt kit with gdnase tiangenbiotech co ltd beijing chinapacbio cdna library preparation and smrt sequencingfulllength cdna was synthesized by using the smarter„¢ pcr cdna synthesis kit clontech ca usathe generated cdna was then reamplified using pcrafter end repairing smrt adaptor with a hairpin loopstructure was ligated to the cdna via exonucleasedigesting the cdna library was constructed after quality measurement of the cdna library smrt sequencingwas performed using the pacific bioscience sequel platform following the provided protocolillumina cdna library construction and secondgenarationsequencingthe extracted mrna was purified using oligo dtattached magnetic beads fragmentation was conducted inthe nebnext first strand synthesis reaction bufferfirststrand cdna was acquired based on the randomhexamers and then the secondstrand cdna was synthesized with dntps rnase h and primestar gxldna polymerase the synthesized cdna was purifiedwith ampure xp beads after end repairing adding polya and adaptor ligation ampure xp beads were used forsize selection the generated cdna was then amplifiedfor building cdna libraries the qualified libraries werepair end sequenced on illumina nova platformquality filtering and error correction of long readsraw smrt sequencing reads were filtered by removingpolymerase reads less than bp and sequence accuracyless than after removing adaptor subreads were obtained clean data was produced with subreads morethan bp ccss were produced from clean data withparameters of full passes and accuracy over after examining the coexistence of ² and ² adaptorsand poly a tail fulllength transcripts were selectedduring the processes of library preparation the chimericsequences formed by the direct linkage of two cdnatemplate strands due to the low concentrations ofadaptor or smrtbell are called artificial chimeric sequences the nonchimeric sequences in the fulllength 0cliu bmc plant biology page of transcripts are the fulllength nonchimeric flncsequencesas smrt sequencing generates a high error rate it isnecessary to perform error correction iterative clustering was used first to obtain consensus isoforms and thefulllength consensus sequences from iterative clusteringfor error correction were refined using quiver [ ]moreover the raw illumina sgs reads were filtered toremove adaptor sequences and low quality reads anderror correction of lowquality isoforms was conductedusing the sgs reads with the software proovread inbriefly the short reads of illumina rnaseq data weremapped to the low quality isoforms and then the basein the low quality isoform was replaced by the particularbase that had the maximum number

cancer294

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: " phytolaccaceae species in china are not only ornamental plants but also perennial herbs that areclosely related to human health however both largescale fulllength cdna sequencing and reference genevalidation of phytolaccaceae members are still lacking therefore singlemolecule realtime sequencing technologywas employed to generate fulllength transcriptome in invasive phytolacca americana and noninvasive exotic picosandra based on the transcriptome data rtqpcr was employed to evaluate the gene expression stability in thetwo plant species and another indigenous congener p acinosaresults total of gb and gb clean reads of p americana and p icosandra were generated including and full length nonchimeric flnc reads respectively transcript clustering analysis of flnc readsidentified and consensus isoforms including and highquality ones after removingredundant reads and transcripts were obtained based on structure analysis total and alternative splicing variants and simple sequence repeats ssr as well as and completecoding sequences were detected separately furthermore and lncrna were predicted and and transcripts were annotated respectively subsequently seven reference genes in the two plant species and anative species p acinosa were selected and evaluated by rtqpcr for gene expression analysis when tested indifferent tissues leaves stems roots and flowers 18s rrna showed the highest stability in p americana whetherinfested by spodoptera litura or not ef2 had the most stable expression in p icosandra while ef1α was the mostappropriate one when attacked by s litura ef1α showed the highest stability in pacinosa whereas gapdh wasrecommended when infested by s litura moreover ef1α was the most stable one among the three plant specieswhenever germinating seeds or flowers only were consideredcontinued on next page correspondence yiwangynueducn1yunnan key laboratory of plant reproductive adaption and evolutionaryecology yunnan university kunming china2laboratory of ecology and evolutionary biology state key laboratory forconservation and utilization of bioresources in yunnan yunnan universitykunming chinafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cliu bmc plant biology page of continued from previous page fulllength transcriptome of p americana and p icosandra were produced individually based on thetranscriptome data the expression stability of seven candidate reference genes under different experimentalconditions was evaluated these results would facilitate further exploration of functional and comparative genomicstudies in phytolaccaceae and provide insights into invasion success of p americanakeywords phytolaccaceae smrt sequencing fulllength transcriptome analysis reference gene evaluation rtqpcr phytolacca americana is a member of the phytolaccaceae family and is native to northeast america becauseof its ornamental and medicinal applications it was introduced into china in unfortunately it hasevolved into an invasive species and spread to mostareas of the country especially in central and southernchina compared to noninvasive exotic congener p icosandra and native congener p acinosa p americana isof interest because it exhibits multiple biological activities such as plant pesticides antimicrobial propertyheavy metal accumulation capacity [“]in order to investigate the mechanisms of various bioactivities of p americana further transcriptomewidestudy is necessary to facilitate reports have showed thatjasmonic acidinduced and cadmiumtreated transcriptome data of p americana have been obtained by illumina hiseq and illumina hiseq platformrespectively [ ] howeverthese data were bothachieved by second generation sequencing sgs whichcould not produce fulllength transcripts genomic dataof p americana was available at the sra under projectprjna544344 but it™s raw reads without coding sequences prediction and functional annotation third generation sequencing tgs is known for itskilobasesized long reads and is an outstanding strategyfor better understanding rna processing for exampleit can be used to analyze different transcript isoformsregulated by alternative splicing which greatly increasesthe repertoire of proteins lead to genetic and functionaldiversity and is prevalent in most eukaryotic anisms the long reads could also provide sequence information on genecoding regions for functional analysis atthe transcriptional level and thus can be applied to refine an assembled genome for better annotation however tgs could not quantify gene expression forthe moment and have a relatively high error rate thansgs the combination of tgs and sgs are able to solvethis problem and are highly recommended by most researchers with the transcriptome and genome data availablefunctional genomics research is being performed whichrelied heavily on gene expression analysis reversetranscription quantitative real time pcr rtqpcr hasbeen reported to be a very sensitive and accurate technique to analyze gene expression level but it requiresappropriate reference gene as an internal control tonormalize mrna levels between different samples foran exact comparison of gene expression [ ] anideal reference gene should be expressed at a constantlevel across various experimental conditions howeverstudies have shown that no single reference gene is universal for all experimental conditions [“] therefore it™s necessary to estimate the stability of referencegenes under particular experimental condition beforeusing them for gene expression analysisin the present study to provide highquality and morecomplete assemblies in genome and transcriptome studies of phytolaccaceae a hybrid sequencing approachcombining the sgs and tgs technologies was carriedout first fulllength transcriptome of the invasive plantspecies of pameracana and an noninvasive exotic conicosandra was generated by singlemoleculegener prealtimesmrtsplicingevents simple sequence repeats ssr coding sequencesprotein annotations and long noncoding sequenceswere analyzed respectively at transcription level furtherthe stability of reference genes was evaluated in two phytolaccaceae species mentioned above and one nativecongener p acinosa by rtqpcr in order to facilitatefuture research on functional gene expressionsequencing alternativeresultsto classify the plant species these three phytolaccaceaemembers p americana p icosandra and pacinosa wereidentified by pcr and followed by sequences alignmentbased on sequences of second internal transcribed spacer its2 and the intergenic spacer of photosystem iiprotein d1 gene and trnahis gene of chloroplast genome psbatrnh table s1 the sequences of its2 andpsbatrnh in p americana that we employed had theidentity of with the sequences reported by chen in p icosandra the sequences of its2 had identity and the sequences of psbatrnh had identity with the results of chen™s in pacinosa 0cliu bmc plant biology page of similarity of its2 and identify of psbatrnh werefound isoforms afterremoving redundantsmrt sequencing data outputusing the pacific biosciences™ smrt sequencing protocol gb clean reads of invasive species p americana were obtained after preprocessing on the basis offull passes and sequence quality circular consensus sequences ccs with fulllength rate were obtained including fulllengthnonchimeric flnc sequences and highqualityconsensussequences from the high quality consensus isoforms transcripts alternative splicing events ssr complete coding sequences lnc rnasand annotated transcripts in p americana wereachieved similarly gb clean reads in p icosandrawere identified and ccs with fulllength rate flnc sequences as well as highquality consensus isoforms were filtered subsequently transcripts and alternative splicingevents were obtained what™s more ssrs complete coding sequences lnc rnas and annotated transcripts were identified in picosandratable transcriptome analysisbased on the structure of achieved transcripts and alternative splicing events were identified in pamericana and p icosandra respectively transcripts of bp in total in p americana wereemployed for ssr analysis based on the sequence lengththat was more than bp including ssrs and ssrcontaining sequences similarly transcripts bp in total in picosandra wereemployed for ssr analysis and ssrs togetherwith ssrcontaining sequences were identifiedtable summary of fulllength transcriptome sequencingclean reads gbccsflncflnc flncccsconsensus isoformhigh quality consensus isoformtranscriptsalternative splicingssrcomplete coding sequenceslncrnaannotated transcriptsp americanap icosandrathe detail information about the number of sequencescontaining more than one ssr the number of ssrspresent in compound formation and the number of different types of ssrs were shown in table in additiontotal of complete coding sequences cds in pamericana and cds in p icosandra were identified by using transdecoder the length distribution ofpredicted proteins was shown in fig s1in pdatabasespecifically nrwith the eight protein databases sequence alignmentswere performed to annotate predicted proteins in total transcripts in p americana and tranicosandra were annotated separatelyscriptstable the number of annotated protein sequencesin p americana was similar with p icosandra under aparticularncbi nonredundant protein analysis revealed that approximately transcripts in p americana and transcripts in picosandra showed the highest sequencesimilarity with beta vulgaris fig go gene ontology assignment also suggested that similar amount ofsequences in the two plant species belonged to the sameterm and many were classified into cell part and cellterm of cellular component catalytic activity and binding of molecular function and metabolic process andcellular process of biological process fig cogclusters of orthologous groups of proteins annotationshowed that a large number of predicted proteins in thetwo plant species were linked to functional class r general function prediction only j translation ribosomalstructure and biogenesis t signal transduction mechanisms g carbohydrate transport and metabolism ando posttranslational modification protein turnoverchaperones fig s2 the result of eggnog evolutionary genealogy of genes nonsupervised orthologousgroup annotation indicated that most of the annotatedproteins in the two plant species were belonging to thefunctional class s function unknown fig s3 kogeukaryotic ortholog groups functional classificationsuggested that r general function prediction only ando posttranslational modification protein turnover andchaperones were the most abundant functional categories in the two plant species fig s4 these results indicated that most of the sequences obtained were trulyfunctional proteins and had a similar functional classification in p americana and its congener picosandraeven though more work is needed to identify sequencesthat regulated or involved in the invasion success of pamericana the annotation of predicted proteins provided necessary information for further studiesbesides the transcripts encoding proteins long noncoding rnas lncrnas were achieved lncrnas arereported to be key regulators in plant biological prolncrna in pameracana andcesses the number ofpicosandra was predicted by cpc coding potential 0cliu bmc plant biology page of table ssrs obtained from transcripts with more than bpsearching itemtotal number of sequences examinedtotal size of examined sequences bptotal number of identified ssrsnumber of ssr containing sequencesnumber of sequences containing more than ssrnumber of ssrs present in compound formationnumber of mono nucleotide ssrnumber of di nucleotide ssrnumber of tri nucleotide ssrnumber of tetra nucleotide ssrnumber of penta nucleotide ssrnumber of hexa nucleotide ssrcalculator cnci codingnoncoding index pfamand cpat coding potential assessment tool respectively in total lncrna in pamericana and lncrna in picosandra were predicted by all these fourmethods fig subsequentlytranscription factorstfs that are key components involved in the transcriptional regulatory system were predicted in p americana tfs of types were filtered and in picosandra tfs of types were predicted thesetwo plant species shared the first types of tfs butthe number of each type tf was not similar especiallyrlkpelle_dlsv c3h snf2 and camk_camklchk1 indicating the particular functions on transcriptregulation fig amplification performance of rtqpcrprimers designed for rtqpcr were evaluated by pcrfirst the primers which produced single ampliconwithout primer dimer were chosen for melting curveanalysis only primers which produced a single fragmentefficiencywereqpcr amplificationchosenforp americanap icosandraassessment the qpcr efficiency of each primer pair wasgenerated from a 10fold serial dilution of pooled cdnaand was shown in table the threshold cycle ct values of each reference genewere employed to evaluate expression level under different experimental conditions fig average ct valuesfor all the seven candidate reference genes ranged from to in which ef1α showed the highest expression level and 28s rrna had the lowest expression levelit was also suggested that ct values of βactin and tubulin fluctuated significantly across all the experimentalsamplesstability of candidate reference genesforto determine the appropriate reference genesnormalization in different experimental conditions theexpression data was analyzed by genorm normfinderand bestkeeper respectively table s2when expression stability of reference genes wereanalyzed in different tissues leaves stems roots andflowers of p americana 18s rrna and ef2 oftable number of proteins annotated via differential protein databasedatabasesp americanaannotated number ‰¤ length length ‰¥ p icosandraannotated number ‰¤ length length ‰¥ coggokeggkogpfamswissproteggnognrall 0cliu bmc plant biology page of fig homologous species distribution of p americana and p icosandra annotated based on the nr database a p americana b p icosandrapamericana were identified as the most suitable reference genes by genorm and normfinder and 18s rrnawas also suggested by bestkeeper pairwise variation valueof v23 was below the cutoff value of which meansthe combination of two reference genes were most suitable for gene expression normalization fig whentested in picosandra ef1α was recommended for normalizing gene expression analysis not only by genorm butalso by normfinder ef2 was also suggested by genormand bestkeeper in pacinosa ef1α was the best reference gene suggested by genorm and bestkeeper but 18srrna was recommended by normfinder the use of tworeference genes was suitable because pairwise variationvalue of v23 was below when pooled the data of different tissues from pamericana and picosandra togetheref2 was shown to be the most stable gene by all the threemethods when investigated the expression stability ofreference genes in different tissues of pamericana andpacinosa 18s rrna showed the best expression stabilityby genorm and normfinder while ef2 was referred asthe most stable one by bestkeeper however the combination of five reference genes was recommended bygenorm for v56 which was less than when thedata of different tissues from picosandra and pacinosawas put together ef1α was identified as the best oneby genorm and normfinder whereas ef2 was suggested to be the best stability reference gene by bestkeeper when set the data of these three plant speciesas a pool ef1α was suggested to be the most stableone by genorm and normfinder while ef2 was alsorecommended by genorm and bestkeeper accordingto these results it is very important to select the appropriate reference gene when analyze the gene expressionlevel among plant species 0cliu bmc plant biology page of fig classification of the transcripts annotated by the gene ontology gowhen analyzed the data among germinating seeds28s rrna and ef1α were identified as the best reference genes by genorm while 18s rrna was recommended by normfinder and gapdh was suggested bybestkeeper three reference genes were sufficient tonormalize gene expression for v34 was below inflowers only of these three plant species ef1α was confirmed by all the three methods the genorm analysisshowed that the value of v45 was below so fourreference genes in combination were suggested thesefig venn diagram of the number of lncrnas predicted by cpc cnci cpat and pfam a p americana b p icosandra 0cliu bmc plant biology page of fig classification of predicted transcription factorsresults indicated that when focusing on particular tissuesof different plant species the selection of reference genewas also very essentialwhen plants were infested by slitura 18s rrnashowed the most expression stability suggested by genorm and normfinder in different tissues of pamericanawhile ef1α wasby bestkeeper therevealedcombination of two reference genes was suggested bygenorm due to the value of v23 was less than 18srrna was also recommended by genorm in s liturainfested picosandra and ef1α was shown to be themost stable one by normfinder and bestkeeper fourreference genes in combination were recommended bygenorm 18s rrna was also identified as the besttable primers for rtqpcr analysisgene nametubulingene descriptiontubulin ef1αelongation factor 1alphaprimer sequence ²²f gtaaggaagccgagaattgr tcaacaacagtgtcagagaf tgaagaaggtcggatacaatr gtagacatcctggagtgggaphdglyceraldehyde3phosphate dehydrogenasef tggtgctaagaaggttattatcef2elongation factor 18s rrna18s rrnaβactinactin728s rrna28s rrnar2 linear regression coefficientr gagtgaacggtggtcataf gtatcaccatcaagtcaactgr acaatcaaccacaacaaggf acttcctcttctcgtatcattr tgttcagcatagactgtgaf atgctatccttcgtctggr tactcttggctgtctctgf tacgattggttacggacatr ttctcatcaacaacagcatatlength bppcr efficiency r2 0cliu bmc plant biology page of together 18s rrna showed the best stability in genormand normfinder while the expression stability of ef1αwas suggested by bestkeeper in pamericana and picosandra 18s rrna was identified as the best referencegene by all the three algorithms in pamericana andpacinosa 18s rrna and βactin were suggested bygenorm in picosandra and pacinosa while gapdhand ef2 were recommended by normfinder and bestkeeper respectively when take all the data of s liturainfested plant species into account 18s rrna exhibitedthe most stable expression suggested by genorm andnormfinder while ef2 was the gene with the most constant expression identified by bestkeeperdiscussionfulllength transcripts are fundamental resources forstructuralfunctional and comparative genomics research [ ] smrt sequencing has been acknowledged by enabling the generation of multikilobasesequences to improve genome and transcriptome assembly the fulllength cdna sequences generated areable to characterize the posttranscriptional processsuch as alternative splicing lncrna prediction and coding sequences for further gene functional studies basedon the fulllength transcriptome data generated about gb of clean data were obtained for pamericana andpicosandra respectively table accordinglythenumber of ccs flnc consensus isoforms highqualityfig rna transcription levels of seven candidate reference genesin p americana picosandra and p acinosa the expression level ofcandidate reference genes in total samples n was presentedas cycle threshold number ctvalue and explained by box andwhisker plots the asterisks represented the minimum and maximumct value the squares indicated the 25th and 75th percentiles andthe median was represented by a bar across the squarereference gene by genorm in plant species pacinosawhile tubulin was suggested by normfinder and 28srrna was recommended by bestkeeper the combination of three reference genes was appropriate by genorm when analyzed the data oftwo plant speciesfig pairwise variation analyzed by genorm to determine the optimal number of reference genes for accurate normalization a threshold valueof was suggested for valid normalization if the value of vnn pairwise variation is less than then n reference genes in combinationare recommended for gene normalization if the value of vnn is more than then vn 1n should be taken into account pam pamericana pic p icosandra pac p acinosa lsrf different tissues of leaves stems roots and flowers gs germinating seeds of these three plantspecies f flowers of these three plant species lsr different tissues of leaves stems and roots i infested by s litura of third instar 0cliu bmc plant biology page of isoforms transcripts alternative splicing events ssrscomplete coding sequeces lncrnas and annotated transcripts were analyzed providing basic transcriptomic information for further studiesreports have showed that fulllength transcriptome ofzea mays have greatly helped in refining gene annotation and revealed the complexity of gene expression inmaize similar analysis has also been conducted inshum bicolor what™s more the world expansioncapability of cydia pomonella has been informed according to its genome information molecularmechanism of rapid growth and invasive adaptation ofan invasive species mikania micrantha has also been investigated according to itsreference genome therefore the fulllength transcriptome data of pamericana and picosandra will contribute to the genomic research and provide insights into invasive mechanism ofpamericana through comparative genomics study inphytolaccaceae speciesgenereliesonanalysisexpressionaccurate relative quantification of rtqpcr for furtherrobustnormalization by stably expressed reference genes tominimize error in the experimental process therefore suitable reference genes for the normalization ofrelative gene expression data in three phytolaccaceaespecies pamericana picosandra and pacinosa weresought under a diverse set of conditions these resultsdemonstrated the importance of validating referencegenes under the relevant experimental conditions forexamplein different tissues leaves stems roots andflowers of pamericana 18s rrna and ef2 were recommended to be the bestsuited reference genes and similar results were found in s liturainfested pamericanahowever even though the appropriate reference genesin picosandra were ranked according to the analyzed results of the three methods all the pairwise variationvalues were above the cutoff value of while thecombination of 18s rrna βactin ef1α and ef2 weremost suitable in s liturainfested picosandra ef2 andef1α have been considered as the ideal reference genesin pacinosa whereas the combination of 18s rrna βactin and gapdh were recommended after s litura infestation researches have also showed that no singlereference gene is stably expressed among different tissues of an anism such as the reference gene selectionin amygdalus persica solanum lycopersicum and glycine max [ ] what™s more our results alsosuggested that reference genes identified based on transcriptome data should be confirmed by experimentalevidence in jainduced transcriptome of p americana28s rrna showed stable expression between exogenousjatreated and control plants ja signal pathway ofplants can be induced by lepidopteran herbivores infestation however 18s rrna and ef2 were identifiedas the most stable expression reference genes in pamericana after s litura infestationin order to conductthe gene expression analysisamong different plant species of phytolaccaceae the dataof the three plant species were also compared togetherwhen compared the data in germinating seeds of threeplant species various genes were recommended by thethree methods the combination of plant species underother experimental conditions showed that the pairwisevalues of almost all the combination were higher thanthe cutoff value of exceptthe combination ofpamericana and pacinosa where five reference geneswere recommended for data normalization as well as thecombination of sliturainfested pamericana and sliturainfested picosandra where three reference geneswere suggested these results indicated that no particular gene was expressed constantly across different plantspecies even though these plants are congeners therefore reference genes should be employed appropriatelyunder the relevant experimental conditionsthe research has provided transcriptomewide fulllength isoforms of pamericana and picosandraproviding insights into invasive success of pamericanaguidelines for selecting appropriate reference genesunder different tissues in one plant species or amongvaried plant species were recommended further no particular gene was expressed constantly under differentexperimental conditions indicating the necessity of reference gene identification these results would facilitatethe exploration of functional and comparative genomicsstudies in phytolaccaceae to better understand plantbiologymethodsplant and insect materialsplants of p americana °²n °²e p icosandra°²n °²e and p acinosa °²n °²eused in this study which was named m k and q firstwere collected in yunnan china sampling was permitted when conducted complying with locallegislationthe formal identification of the samples were conductedby chao chen botany major of laboratory of ecologyand evolutionary biology state key laboratory for conservation and utilization of bioresources in yunnanyunnan university according to flora of china vol5“ flora of north america vol43“ chinese virtual herbarium httpwwwcvhaccn and global plants on jstor httpplantsjstor dna identification was also employed according tothe its2 region of nuclear ribosomal dna one of themost widely used dna fragments in plant molecularsystematics at the generic and species levels and the 0cliu bmc plant biology page of chloroplast psbatrnh intergenic region all voucher specimens were maintained at an experimental fieldof laboratory of ecology and evolutionary biology statekey laboratory for conservation and utilization of bioresources in yunnan yunnan universitytissues of leaves stems roots and flowers from oneindividual plant of p americana or p icosandra werecollected individually from the wild in yunnan provinceand no permission is needed for collecting theses samples each sample was flash frozen in liquid nitrogen andstored at ˆ’ °c for further experimentsshop101732681taobaocomthird instar larvae of spodoptera litura were purchased from henan jiyuan baiyun industry co ltdchinaand then werereared on artificial diet in a climate chamber h at °c with light and h at °c without light for further usefor reference gene evaluation seeds of p americanap icosandra and p acinosa were collected first from thewild in yunnan province and no permission is neededthe seeds were sown separately in agar plates andcultivated in the climate chamber after d five germinating seeds of one plant species were collected togetheras one sample for subsequent experiments each plantspecies have three replications two weeks later othergerminating seeds of each species were transplanted intoplastic pots cm diameter and cm height withsoil jiangsu peilei matrix technology development coltd china and cultivated with adequate water in artificial chambers with same conditions as described abovefour months later leaves stems roots and flowers ofeach plant species were collected individually simultaneously six larvae s litura of third instar were employedto infest on p americana p icosandra or p acinosawith one insect per leaf control treatments were herbivore free after h infestation leaves stems and rootsof these three plant species were harvested individuallyall samples collected were flash frozen in liquid nitrogenand stored in ˆ’ °c for subsequent assays and threereplicates were conducted for each treatmentnucleic acid extraction and assaysgenomic dna was isolated from the leaves of differentplant species following protocols provided by dnaquickplant system tiangen biotech co ltd beijing chinathen it was employed as the pcr template for plantspecies identificationpurekitplanttotal rnas from different tissues was prepared usingrnapreppolysaccharides polyphenolicsrich tiangen biotech co ltd beijingchina according to the manufacturer™s instructionsthe rna quality and purity were measured by using ananophotometer n60 implen germany and the agilent bioanalyzer system agilent technologies causa samples only with a ratio of to a ratio between and and a rin value morethan were chosen for the sequencing library construction an equal amount of total rnas from four different tissues of the same plant species were mixed asone sample for fulllength transcriptome sequencingtotal rnas from the samples collected for referencegene evaluation was also extracted individually as described above for each sample cdna was prepared byusing μg of total rna following the recommendedinstructions of fastquant rt kit with gdnase tiangenbiotech co ltd beijing chinapacbio cdna library preparation and smrt sequencingfulllength cdna was synthesized by using the smarter„¢ pcr cdna synthesis kit clontech ca usathe generated cdna was then reamplified using pcrafter end repairing smrt adaptor with a hairpin loopstructure was ligated to the cdna via exonucleasedigesting the cdna library was constructed after quality measurement of the cdna library smrt sequencingwas performed using the pacific bioscience sequel platform following the provided protocolillumina cdna library construction and secondgenarationsequencingthe extracted mrna was purified using oligo dtattached magnetic beads fragmentation was conducted inthe nebnext first strand synthesis reaction bufferfirststrand cdna was acquired based on the randomhexamers and then the secondstrand cdna was synthesized with dntps rnase h and primestar gxldna polymerase the synthesized cdna was purifiedwith ampure xp beads after end repairing adding polya and adaptor ligation ampure xp beads were used forsize selection the generated cdna was then amplifiedfor building cdna libraries the qualified libraries werepair end sequenced on illumina nova platformquality filtering and error correction of long readsraw smrt sequencing reads were filtered by removingpolymerase reads less than bp and sequence accuracyless than after removing adaptor subreads were obtained clean data was produced with subreads morethan bp ccss were produced from clean data withparameters of full passes and accuracy over after examining the coexistence of ² and ² adaptorsand poly a tail fulllength transcripts were selectedduring the processes of library preparation the chimericsequences formed by the direct linkage of two cdnatemplate strands due to the low concentrations ofadaptor or smrtbell are called artificial chimeric sequences the nonchimeric sequences in the fulllength 0cliu bmc plant biology page of transcripts are the fulllength nonchimeric flncsequencesas smrt sequencing generates a high error rate it isnecessary to perform error correction iterative clustering was used first to obtain consensus isoforms and thefulllength consensus sequences from iterative clusteringfor error correction were refined using quiver [ ]moreover the raw illumina sgs reads were filtered toremove adaptor sequences and low quality reads anderror correction of lowquality isoforms was conductedusing the sgs reads with the software proovread inbriefly the short reads of illumina rnaseq data weremapped to the low quality isoforms and then the basein the low quality isoform was replaced by the particularbase that had the maximum number Answer:

Colon_Cancer

" rectus sheath block rsb is known to attenuate postoperative pain and reduce perioperative opioidconsumption thus a retrospective study was performed to examine the effects of bilateral rectus sheath blockbrsb in cytoreductive surgery crs combined with hyperthermic intraperitoneal chemotherapy hipecmethods a total of patients undergoing crshipec at our hospital were included patient information andanaesthesiarelated indicators were collected from the electronic medical record emr system all subjects weredivided into the following two groups the g group general anaesthesia and the gr group rsb combined withgeneral anaesthesia patients in the gr group received ropivacaine for brsb before surgery the primaryoutcomes included the total amount of remifentanil and rocuronium the total consumption of dezocine aftersurgery the visual analogue scale vas score and the patientcontrolled intravenous analgesia pcia input dose at h t6 h t7 h t8 h t9 and h t10 after surgery other outcomes were also recorded such aspatient demographic data the intraoperative heart rate hr and mean arterial pressure map and postoperativecomplicationsresults compared with the g group the gr group showed a shorter time to tracheal extubation p adecreased total amount of remifentanil and rocuronium p and a reduced vas score pcia input dose andnumber of pcia boluses at h h and h after surgery p however at h and h after surgery therewere no differences in the vas score of pain at rest or during motion between the two groups p moreoverthe incidence of hypertension emergence agitation delayed recovery hypercapnia and nausea and vomiting waslower in the gr group than in the g group p there were no differences in the changes in map and hrduring the surgery between the two groups p no complications associated with nerve block occurred brsb could provide shortterm postoperative analgesia reduce perioperative opioid consumption andreduce the incidence of postoperative complications it is an effective and safe procedure in crshipeckeywords cytoreductive surgery hyperthermic intraperitoneal chemotherapy rectus sheath block generalanaesthesia analgesia correspondence trmzltz126comdepartment of anesthesiology beijing shijitan hospital capital medicaluniversity beijing china the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cwang bmc anesthesiology page of radical cytoreductive surgery crs combined withhyperthermic intraperitoneal chemotherapy hipec isconsidered a standard for the treatment of peritonealcancer such as rectal cancer ovarian cancer peritonealpseudomyxoma and peritoneal mesothelioma thistechnique could prolong the longterm survival of patients with a decreased recurrence rate although thepositive results of this treatment have been proven inprevious studies [“] because of the large peritonealsurface area involved in this kind of surgery crshipecis time consuming and complex which presents agreat challenge for the anaesthesiologist in terms of perioperative managementdue to the stable respiratory and circulatory supportgeneral anaesthesia is the preferred choice in this surgery however long periods of general anaesthesia leadto drug accumulation in the body followed by increasedanaesthesiarelated complications including delayed recovery respiratory inhibition and cognitive dysfunction consequently exploring better anaesthesia methodsfor this surgery is still a major concerna new approach called ultrasoundguided bilateral rectus sheath block brsb has been proven to amelioratepostoperative pain and reduce the consumption of morphine [“] nonetheless there have been no reportson the application of general anaesthesia combined withbrsb in patients undergoing crshipec based on theinformation presented above this retrospective observational study was conducted to examine the efficacy andsafety of brsb in patients treated with crs and hipecmethodssubjectsall patients who underwent crs and hipec at beijingshijitan hospital between august and december were retrieved from the institutional database inthis study the exclusion criteria were as follows laparoscopic surgery with crshipec intraoperativeblood loss volume greater than ml mechanicventilation required after surgery and use of analgesictechniques apart from brsb and general anaesthesiaaccording to this standard a total of patients wereincluded and divided into the following groups generalanaesthesia g group n and general anaesthesiacombined with posterior rsb gr group n anaesthesia methodgeneral anaesthesia was consistently induced in all patients with intravenous propofol mgkg sufentanil μgkg and rocuronium mgkg invasive arterialpressure and central venous pressure were monitored byradial artery puncture flotracvigileo® edwards lifesciences irvine ca usa and internaljugular veinsitetargeteffectpuncture respectively after anaesthesia induction anaesthesia was maintained with sevoflurane and remifenconcentration “ ngmltanilkeeping the bispectral index bis between and rocuronium mgkg wasintermittently used tomaintain muscle relaxation in the gr group before anaesthesia induction patients received brsb under ultrasound guidance the puncture site was placed at theouter edge of the bilateral rectus abdominis at the levelof the umbilicus fig a a total of ropivacaine ml was injected into each side the spindleshapedspread of ropivacaine was observed between the posterior sheath of the rectus abdominis and the rectus abdominis itself implying success of the procedure fig b c patientcontrolled intravenous analgesia pciawas applied in both groups after the surgery sufentanil μgkg palonosetron hydrochloride mg was diluted to ml the dose was mlh and asingle dose was mlh with a 15min lockout intervalafter the surgery all patients were sent to the surgicalintensive care unit sicu if the visual analogue scalevas score at rest after surgery was ‰¥ dezocine mgwas used as a rescue analgesicdata collectionall the indicators we needed were obtained from theemr system the records included patient demographicdata patient medical history american society of anesthesiologists asa grade and new york heart association nyha grade the hr and map were recordedat the time before brsb t1 the time of anaesthesiat2 the time of skin incision t3 the time of peritoneal thermochemotherapy t4 and the end of surgeryt5 in addition the duration of the surgery time totracheal extubation the time after skin closure totalamount of remifentanil and muscle relaxants total fluidvolume urine volume and the total volume of allogeneicerythrocytes and plasma infused during the surgery wereall recorded moreover after surgery the occurrence ofhypertension the systolic blood pressure dropped bymore than of baseline blood pressure beforeanesthesia or the sbp mmhg during surgery nausea and vomiting hypoxemia spo2 or pao2 mmhg hypercapnia paco2 mmhg and emergence agitation during the recovery period were recorded the recovery period was considered as the timefrom switching off inhalation anaesthetics remifentaniland muscle relaxant to recovery of the patients™ abilitiesto command movement orientation as well as conscious state when the recovery period of patients is beyond min it was considered as delayed recovery thevas score for pain at rest and during motion the pciainput dose and the number of boluses at h t6 ht7 h t8 h t9 and h t10 after surgery 0cwang bmc anesthesiology page of the incomplete datachemotherapy crshipec during the year from to in our hospital one hundred and six patientsreceived brsb seventeen patients were excluded because ofincluding patientsundergoing intraoperative haemorrhage blood ml and other patients received mechanic ventilationbecause of acute respiratory distress syndrome ardsallergic shock and cardiac insufficiency thus patients with brsb were eventually obtained finally patients without brsb were randomly selected to analysis in this study fig statistical analysisspss software was used for statistical analysisnormal distribution data were recorded as the mean ±standard deviation sd and analysed by independentsamples t test for comparison between the two groupsnonnormally distributed data are presented as themedian range and were analysed by kruskalwallistest chisquared test or fisher™s exact test was used forcategorical data a p value of was considered statistically significantresultscharacteristics of study populationin total patients were included in the study thebaseline demographic and surgical variables of patientsare presented in table there were no significant differences in age sex body mass index bmi basic diseases asa grade nyha grade total surgery time totalfluid volume urine volume total volume of allogeneicerythrocyte infusion or total volume of plasma p however the time to tracheal extubation was shorter inthe gr group than in the g group p the totalamount of both remifentanil and rocuronium used wasless in the gr group than in the g group p thus posterior rsb could reduce the use of remifentaniland rocuronium during surgerychanges in haemodynamic parametersthe changes in hr and map are presented in table there were no significant differences in hr or map atany point in time t1 to t5 between the two groupsp the results suggest that brsb did not affectthe haemodynamics ofthe patient undergoing crshipecpainrelated indicatorstable shows the postoperative vas score the pca input dose and the number of pca boluses at h t6 ht7 h t8 h t9 and h t10 after surgeryas well as the dose of dezocine used as a rescue analgesic from t6 to t8 compared with the g group thegr group showed significantly decreased vas scores offig ultrasoundguided brsbas well as the dose of dezocine used as a rescue analgesic were also recordedin addition brsbrelatedcomplications such as peritoneal punctureinternalan injury and systemic toxicity were all recordeda total of patients underwent cytoreductive surintraperitonealcombined withgeryhyperthermic 0cwang bmc anesthesiology page of fig flow chart showing patient consecutive enrolment and analysis abbreviations crshipec cytoreductive surgery and hyperthermicintraperitoneal chemotherapy ga general anesthesia brsb bilateral rectus sheath block ards acute respiratory distress syndrome vas visualanalogue scalepain at rest and during motion p however at h and h after surgery there were no significant differences in the vas scores of pain at rest and duringmotion between the two groups p from t6 tot10 the pcia input dose and the number of pca boluses were also obviously reduced in the gr group compared with the g group p in addition as arescue analgesic the dose of dezocine after surgery inthe gr group was significantly lower than that in the ggroup p postoperative adverse eventsadverse events that occurred in the sicu are presentedin table after surgery there were cases withhypertension cases of emergence agitation cases ofdelayed recovery cases of hypercapnia and cases ofnausea and vomiting in the g group fewer cases of allof these events occurred in the gr group p there were no differences in the incidence of hypoxemiabetween the two groups p there were no complications associated with nerve block in either group 0cwang bmc anesthesiology page of table demographic and surgical variables mean ± sdage ysex malefemalebmi kgm2medical historydiabetes mellitus n yesnohypertension n yesnocoronary heart disease n yesnoasa grade iiiiiinyha grade iiitotal surgery time mintime to tracheal extubation minremifentanil mgrocuronium mgtotal fluid volume mlurine volume mltotal volume of allogeneic erythrocyte infusion mltotal volume of plasma mlg group n ± ± ± ± ± ± ± ± ± ± gr group n ± ± ± ± ± ± ± ± ± ± p value asa american society of anesthesiologists bmi body mass index calculated as weight in kilograms divided by height in metres squared nyha new york heartassociation g general anaesthesia gr bilateral rectus sheath block combined with general anaesthesia before bilateral rectus sheath block t1 the time ofanaesthesia t2 the time of skin incision t3 the time of peritoneal thermochemotherapy t4 and the end of surgery t5discussionin this retrospective study we examined the efficacy andsafety of brsb combined with general anaesthesia in patients undergoing crshipec regarding efficacy theresults show that ultrasoundguided brsb significantlyreduced the total dose of remifentanil used during thesurgery and shortened the time to tracheal catheter extraction which is consistent with the findings of previous studies of other surgeries [ ] in addition rsbreduced the total dose of rocuronium in this studytable haemodynamic parameters in both groups mean ± sdindextimepointgn ± grn ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± pvaluemmhghrbpmt1t2t3t4t5t1t2t3t4t5map mean arterial pressure hr heart rate g general anaesthesia gr bilateralrectus sheath block combined with general anaesthesiawhich may be associated with the high concentration ofropivacaine used in the studyrsb also effectively relieved postoperative pain in thisstudy we found that the vas scores of pain at rest andduring motion were all lower in the gr group than inthe g group at h after surgery however at h and h after surgery there were no differences in the vasscores of pain at rest and during motion between thetwo groups suggesting that the analgesic effects of asingle brsb remained within h after surgery thisresult may be different from the findings of others cho reported that at h after surgerythere were no differences in the vas scores of painat rest and during motion between the rsb and nonrsb groups the discrepant results may be related todifferences in the concentration of ropivacaine andthe physical constitution of patients a high concentration can prolong the duration of action of a localanaesthetic in this study we selected not ropivacaine additionallythese patientsundergoing crshipec may have been adaptive topain furthermore compared with the control groupthe rsb group showed a reduced totalinfused doseof sufentanil as pcia number of pca boluses within h after surgery and total dose of dezocine used asa rescue analgesic after surgery these results furtherprove the role of rsb in providing shortterm postoperative analgesia 0cwang bmc anesthesiology page of table painrelated indicators in both groups median [range]vas score of pain at rest [median range]t6t7t8t9t10vas score of pain during motion [median range]t6t7t8t9t10total infused dose of pcia [ml median range]t6t7t8t9t10cumulative number of pcia boluses [median range]t6t7t8t9t10total dose of dezocine as a rescue analgesic mggn [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ± grn [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ± p value vas visual analogue scale pcia patientcontrolled intravenous analgesia g general anaesthesia gr bilateral rectus sheath block combined with generalanaesthesia the time at h after surgery t6 h after surgery t7 h after surgery t8 h after surgery t9 and h after surgery t10table postoperative adverse events in both groupsadverse eventsn gn grn hypertensionemergence agitation delayed recoveryhypoxemiahypercapnia nausea and vomiting peritoneal punctureinternal an injurysystemic toxicity g general anaesthesia gr bilateral rectus sheath block combined withgeneral anaesthesiawe also examined the safety of rsb during the surgery ultrasoundguided brsb had no significant effectson the haemodynamics of patients during surgery compared with general anaesthesia alone in terms of postoperative adverse events the results show that comparedwith the control group the rsb group showed a reducedincidence of hypertension emergence agitation delayedrecovery hypercapnia and nausea and vomiting whichmight be correlated with the decreased analgesic andmuscle relaxant doses no rsbrelated complications occurred in any patient these data indicate that rsbcould reduce the risk of complications associated withgeneral anaesthesia and is safe for patientsrsb an established technique has regained popularityin clinical applications [“] previous studies havedemonstrated that this technique could achieve relaxation of the anterior abdominal wall [ ] bashandyreported that anterior branches of the t7t12 thoracicnerve and the l1 lumbar nerve travelled through thepvalue“““ 0cwang bmc anesthesiology page of plane of the transverse abdominis muscle entered the rectus abdominis sheath and distributed on the surface of theskin the main process of rsb is to inject local anaesthetics between the rectus abdominis and the posteriorsheath of the rectus abdominis therefore rsb exerteda good effect in terms of perioperative analgesia for medianabdominal incisions a midline incision is required inthis kind of surgery thus based on these results rsbcould meet the need for analgesia in these patientsin addition for a long time epidural analgesia eawas thought to be an effective method for abdominalsurgery [“] studies have proved that epidural analgesia could maintain a good analgesic effect and reduceperioperative opioid consumption including in this typeof surgery [ ] however the safety of ea in crshipec remains controversial especially regarding effectson coagulation and circulatory function coagulationdysfunction and profound fluid loss are the main characteristics of patients with peritoneal cancer [ ]which might limit the administration of eaalthough epidural catheter is standard of care insolanki™s guideline in our hospital epidural catheter in not the standard of care we performed generalanesthesia combined with epidural anesthesia in somepatients to reduce the consumption of intravenous drugsand provide perfect analgesia but coagulation dysfunction and profound fluid loss are the main characteristicsof patients with peritoneal cancer in our previous studywe found that the mean arterial pressure of patientsundergoing epidural anesthesia was difficult to be maintained in the surgery besides there were many patientswith coagulation dysfunction before surgery who werenot suitable for the epidural anesthesia these results wefound in clinical were similar with others™ researcheskajdi and colleagues reported a case of epidural haematoma in their study godden found that the incidence of hypotension in the ea group was obviouslyhigher than that in the rsb group consequentlyrsb could be a better choice than ea in crshipecadditionally there are some limitations to this studyfirst all the data of this study were collected from theemr system as this was a retrospective study the findings are not as persuasive as those of a randomized controlled study we plan to conduct prospective studies toexplore the comprehensive influence of rsb in this surgery second we only examined the application of brsbestablished with a single injection which provides only ashortterm analgesic effect the efficacy of continuousanalgesia with brs catheters in crshipec remains unclear and needs further exploration third in our the results are initially presented according to the different aspects the primary outcome of this study is thetotal consumption of remifentanil during the surgeryother indicators were belonged to second outcomessin brsb could provide good postoperativeanalgesia reduce perioperative opioid consumption andreduce the incidence of postoperative complicationsthis is an easily applicable and safe procedure in crshipecabbreviationsrsb rectus sheath block brsb bilateral rectus sheath blockcrs cytoreductive surgery hipec hyperthermic intraperitonealchemotherapy emr electronic medical record vas visual analogue scalepcia patientcontrolled intravenous analgesia hr heart rate map meanarterial pressure bis bispectral index sicu surgical intensive care unitasa american society of anesthesiologists nyha new york heartassociation spo2 pulse oximetry paco2 partial pressure of carbon dioxidepao2 oxygen partial pressure bmi body mass indexacknowledgementsi would like to express my heartfelt thanks to the staff of the informationdata center of beijing shijitan hospital affiliated to capital medical universityauthors™ contributionswsh study design data collection writing paper lpf gt data collectionand data analysis gl coordinated the study and manuscript revision ltzstudy design and manuscript revision all authors read and approved thefinal manuscript all authors ensure the accuracy of the manuscript andagree to take personal responsibility for their contributionsfundingno fundingavailability of data and materialsthe datasets used andor analyzed during the current study are availablefrom the corresponding author on reasonable requestethics approval and consent to participatethis study was approved by the ethics committee of beijing shijitan hospitalaffiliated to capital medical university approval code research ethicsno69 this study is retrospective only anonymous data sources were usedand informed consent was not requiredconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsreceived january accepted july referencesklaver ce musters gd bemelman wa punt cj verwaal vj dijkgraaf mgaalbers ag van der bilt jd boerma d bremers aj adjuvanthyperthermic intraperitoneal chemotherapy hipec in patients with coloncancer at high risk of peritoneal carcinomatosis the colopec randomizedmulticentre trial bmc cancer 201515undefined428van oudheusden tr braam hj nienhuijs sw wiezer mj van ramshorst bluyer md lemmens ve de hingh ih cytoreduction and hyperthermicintraperitoneal chemotherapy a feasible and effective option for colorectalcancer patients after emergency surgery in the presence of peritonealcarcinomatosis ann surg oncol “passot g vaudoyer d villeneuve l kepenekian v beaujard ac bakrin ncotte e gilly fn glehen o what made hyperthermic intraperitonealchemotherapy an effective curative treatment for peritoneal surfacemalignancy a 25year experience with procedures j surg oncol“arjonasánchez a barrios p boldoroda e camps b carrascocampos jmartín vc garcíafadrique a gutiérrezcalvo a morales r ortegapérez g hipect4 multicentre randomized clinical trial to evaluate safety andefficacy of hyperthermic intraperitoneal chemotherapy hipec with 0cwang bmc anesthesiology page of huepenbecker sp cusworth se kuroki lm lu p samen cd woolfolk cdeterding r wan l helsten dl bottros m continuous epiduralinfusion in gynecologic oncology patients undergoing exploratorylaparotomy the new standard for decreased postoperative pain and opioiduse gynecol oncol “teoh da hutton mj else s walker a lee a mack la epidural analgesia aprospective analysis of perioperative coagulation in cytoreductive surgeryand hyperthermic intraperitoneal chemotherapy am j surg “schmidt c creutzenberg m piso p hobbhahn j bucher m perioperativeanaesthetic management of cytoreductive surgery with hyperthermicintraperitoneal chemotherapy anaesthesia “kajdi me beckschimmer b held u kofmehl r lehmann k ganter mtanaesthesia in patients undergoing cytoreductive surgery withhyperthermic intraperitoneal chemotherapy retrospective analysis of asingle centre threeyear experience world j surg oncol solanki sl mukherjee s agarwal v thota rs balakrishnan k shah sb desain garg r ambulkar rp bhorkar nm society of oncoanaesthesia andperioperative care consensus guidelines for perioperative management ofpatients for cytoreductive surgery and hyperthermic intraperitonealchemotherapy crshipec indian j anaesth “ godden ar marshall mj grice as daniels ir ultrasonography guidedrectus sheath catheters versus epidural analgesia for open colorectal cancersurgery in a single centre ann r coll surg engl “publisher™s notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsmitomycin c used during surgery for treatment of locally advancedcolorectal carcinoma bmc cancer baratti d kusamura s iusco d gimondi s pietrantonio f milione mguaglio m bonomi s grassi a virzì s hyperthermic intraperitonealchemotherapy hipec at the time of primary curative surgery in patientswith colorectal cancer at high risk for metachronous peritoneal metastasesann surg oncol “chua tc robertson g liauw w farrell r yan td morris dl intraoperativehyperthermic intraperitoneal chemotherapy after cytoreductive surgery inovarian cancer peritoneal carcinomatosis systematic review of currentresults j cancer res clin oncol “li y zhou yf liang h wang hq hao jh zhu zg wan ds qin lx cui szji jf chinese expert consensus on cytoreductive surgery andhyperthermic intraperitoneal chemotherapy for peritoneal malignanciesworld j gastroenterol “ willschke h bosenberg a marhofer p johnston s kettner sc wanzel o kaprals ultrasonographyguided rectus sheath block in paediatric anaesthesiaanew approach to an old technique br j anaesth “azemati s khosravi mb an assessment of the value of rectus sheath blockfor postlaparoscopic pain in gynecologic surgery j minim invasive gynecol“ dingeman rs barus lm chung hk clendenin dj lee cs tracy s johnsonvm dennett kv zurakowski d chen c ultrasonographyguided bilateralrectus sheath block vs local anesthetic infiltration after pediatric umbilicalhernia repair a prospective randomized clinical trial jama surg “ relland lm tobias jd martin d veneziano g beltran rj mckee c bhalla tultrasoundguided rectus sheath block caudal analgesia or surgical siteinfiltration for pediatric umbilical herniorrhaphy a prospective doubleblinded randomized comparison of three regional anesthetic techniques jpain res 201710undefined2629“ xu l hu z shen j pm mq efficacy of usguided transversus abdominisplane block and rectus sheath block with ropivacaine anddexmedetomidine in elderly highrisk patients minerva anestesiol “ cho s kim yj jeong k moon hs ultrasoundguided bilateral rectus sheathblock reduces early postoperative pain after laparoscopic gynecologicsurgery a randomized study j anesth “li t ye q wu d li j yu j doseresponse studies of ropivacaine in bloodflow of upper extremity after supraclavicular block a doubleblindrandomized controlled study bmc anesthesiol bell jc rylah bg chambers rw peet h mohamed f moran bjperioperative management of patients undergoing cytoreductive surgerycombined with heated intraperitoneal chemotherapy for peritoneal surfacemalignancy a multiinstitutional experience ann surg oncol “landmann a visoiu m malek mm development of a novel technique forbilateral rectus sheath nerve blocks under laparoscopicguidance j pediatrsurg “kumar a wilson ga engelhardt te ultrasound guided rectus sheathblockade compared to perioperative local anesthetic infiltration in infantsundergoing supraumbilical pyloromyotomy saudi journal of anaesthesia“ bashandy gm elkholy ah reducing postoperative opioid consumption byadding an ultrasoundguided rectus sheath block to multimodal analgesiafor abdominal cancer surgery with midline incision anesthesiol pain med201443e18263 dowidar aerm ezz haa shama aae eloraby ma postoperative analgesiaof ultrasound guided rectus sheath catheters versus continuous woundcatheters for colorectal surgery a randomized clinical trial ˜† egypt journalof anaesth “karaarslan e topal a avci o tuncer uzun s research on the efficacy of therectus sheath block method agri “ piccioni f casiraghi c fumagalli l kusamura s baratti d deraco m arientif langer m epidural analgesia for cytoreductive surgery withperitonectomy and heated intraperitoneal chemotherapy int j surg 16pt a99“ vesterandersen m lundstrøm lh møller mh the association betweenepidural analgesia and mortality in emergency abdominal surgery apopulationbased cohort study acta anaesthesiol scand httpsdoi101111aas13461 0c"

cancer295

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: " rectus sheath block rsb is known to attenuate postoperative pain and reduce perioperative opioidconsumption thus a retrospective study was performed to examine the effects of bilateral rectus sheath blockbrsb in cytoreductive surgery crs combined with hyperthermic intraperitoneal chemotherapy hipecmethods a total of patients undergoing crshipec at our hospital were included patient information andanaesthesiarelated indicators were collected from the electronic medical record emr system all subjects weredivided into the following two groups the g group general anaesthesia and the gr group rsb combined withgeneral anaesthesia patients in the gr group received ropivacaine for brsb before surgery the primaryoutcomes included the total amount of remifentanil and rocuronium the total consumption of dezocine aftersurgery the visual analogue scale vas score and the patientcontrolled intravenous analgesia pcia input dose at h t6 h t7 h t8 h t9 and h t10 after surgery other outcomes were also recorded such aspatient demographic data the intraoperative heart rate hr and mean arterial pressure map and postoperativecomplicationsresults compared with the g group the gr group showed a shorter time to tracheal extubation p adecreased total amount of remifentanil and rocuronium p and a reduced vas score pcia input dose andnumber of pcia boluses at h h and h after surgery p however at h and h after surgery therewere no differences in the vas score of pain at rest or during motion between the two groups p moreoverthe incidence of hypertension emergence agitation delayed recovery hypercapnia and nausea and vomiting waslower in the gr group than in the g group p there were no differences in the changes in map and hrduring the surgery between the two groups p no complications associated with nerve block occurred brsb could provide shortterm postoperative analgesia reduce perioperative opioid consumption andreduce the incidence of postoperative complications it is an effective and safe procedure in crshipeckeywords cytoreductive surgery hyperthermic intraperitoneal chemotherapy rectus sheath block generalanaesthesia analgesia correspondence trmzltz126comdepartment of anesthesiology beijing shijitan hospital capital medicaluniversity beijing china the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cwang bmc anesthesiology page of radical cytoreductive surgery crs combined withhyperthermic intraperitoneal chemotherapy hipec isconsidered a standard for the treatment of peritonealcancer such as rectal cancer ovarian cancer peritonealpseudomyxoma and peritoneal mesothelioma thistechnique could prolong the longterm survival of patients with a decreased recurrence rate although thepositive results of this treatment have been proven inprevious studies [“] because of the large peritonealsurface area involved in this kind of surgery crshipecis time consuming and complex which presents agreat challenge for the anaesthesiologist in terms of perioperative managementdue to the stable respiratory and circulatory supportgeneral anaesthesia is the preferred choice in this surgery however long periods of general anaesthesia leadto drug accumulation in the body followed by increasedanaesthesiarelated complications including delayed recovery respiratory inhibition and cognitive dysfunction consequently exploring better anaesthesia methodsfor this surgery is still a major concerna new approach called ultrasoundguided bilateral rectus sheath block brsb has been proven to amelioratepostoperative pain and reduce the consumption of morphine [“] nonetheless there have been no reportson the application of general anaesthesia combined withbrsb in patients undergoing crshipec based on theinformation presented above this retrospective observational study was conducted to examine the efficacy andsafety of brsb in patients treated with crs and hipecmethodssubjectsall patients who underwent crs and hipec at beijingshijitan hospital between august and december were retrieved from the institutional database inthis study the exclusion criteria were as follows laparoscopic surgery with crshipec intraoperativeblood loss volume greater than ml mechanicventilation required after surgery and use of analgesictechniques apart from brsb and general anaesthesiaaccording to this standard a total of patients wereincluded and divided into the following groups generalanaesthesia g group n and general anaesthesiacombined with posterior rsb gr group n anaesthesia methodgeneral anaesthesia was consistently induced in all patients with intravenous propofol mgkg sufentanil μgkg and rocuronium mgkg invasive arterialpressure and central venous pressure were monitored byradial artery puncture flotracvigileo® edwards lifesciences irvine ca usa and internaljugular veinsitetargeteffectpuncture respectively after anaesthesia induction anaesthesia was maintained with sevoflurane and remifenconcentration “ ngmltanilkeeping the bispectral index bis between and rocuronium mgkg wasintermittently used tomaintain muscle relaxation in the gr group before anaesthesia induction patients received brsb under ultrasound guidance the puncture site was placed at theouter edge of the bilateral rectus abdominis at the levelof the umbilicus fig a a total of ropivacaine ml was injected into each side the spindleshapedspread of ropivacaine was observed between the posterior sheath of the rectus abdominis and the rectus abdominis itself implying success of the procedure fig b c patientcontrolled intravenous analgesia pciawas applied in both groups after the surgery sufentanil μgkg palonosetron hydrochloride mg was diluted to ml the dose was mlh and asingle dose was mlh with a 15min lockout intervalafter the surgery all patients were sent to the surgicalintensive care unit sicu if the visual analogue scalevas score at rest after surgery was ‰¥ dezocine mgwas used as a rescue analgesicdata collectionall the indicators we needed were obtained from theemr system the records included patient demographicdata patient medical history american society of anesthesiologists asa grade and new york heart association nyha grade the hr and map were recordedat the time before brsb t1 the time of anaesthesiat2 the time of skin incision t3 the time of peritoneal thermochemotherapy t4 and the end of surgeryt5 in addition the duration of the surgery time totracheal extubation the time after skin closure totalamount of remifentanil and muscle relaxants total fluidvolume urine volume and the total volume of allogeneicerythrocytes and plasma infused during the surgery wereall recorded moreover after surgery the occurrence ofhypertension the systolic blood pressure dropped bymore than of baseline blood pressure beforeanesthesia or the sbp mmhg during surgery nausea and vomiting hypoxemia spo2 or pao2 mmhg hypercapnia paco2 mmhg and emergence agitation during the recovery period were recorded the recovery period was considered as the timefrom switching off inhalation anaesthetics remifentaniland muscle relaxant to recovery of the patients™ abilitiesto command movement orientation as well as conscious state when the recovery period of patients is beyond min it was considered as delayed recovery thevas score for pain at rest and during motion the pciainput dose and the number of boluses at h t6 ht7 h t8 h t9 and h t10 after surgery 0cwang bmc anesthesiology page of the incomplete datachemotherapy crshipec during the year from to in our hospital one hundred and six patientsreceived brsb seventeen patients were excluded because ofincluding patientsundergoing intraoperative haemorrhage blood ml and other patients received mechanic ventilationbecause of acute respiratory distress syndrome ardsallergic shock and cardiac insufficiency thus patients with brsb were eventually obtained finally patients without brsb were randomly selected to analysis in this study fig statistical analysisspss software was used for statistical analysisnormal distribution data were recorded as the mean ±standard deviation sd and analysed by independentsamples t test for comparison between the two groupsnonnormally distributed data are presented as themedian range and were analysed by kruskalwallistest chisquared test or fisher™s exact test was used forcategorical data a p value of was considered statistically significantresultscharacteristics of study populationin total patients were included in the study thebaseline demographic and surgical variables of patientsare presented in table there were no significant differences in age sex body mass index bmi basic diseases asa grade nyha grade total surgery time totalfluid volume urine volume total volume of allogeneicerythrocyte infusion or total volume of plasma p however the time to tracheal extubation was shorter inthe gr group than in the g group p the totalamount of both remifentanil and rocuronium used wasless in the gr group than in the g group p thus posterior rsb could reduce the use of remifentaniland rocuronium during surgerychanges in haemodynamic parametersthe changes in hr and map are presented in table there were no significant differences in hr or map atany point in time t1 to t5 between the two groupsp the results suggest that brsb did not affectthe haemodynamics ofthe patient undergoing crshipecpainrelated indicatorstable shows the postoperative vas score the pca input dose and the number of pca boluses at h t6 ht7 h t8 h t9 and h t10 after surgeryas well as the dose of dezocine used as a rescue analgesic from t6 to t8 compared with the g group thegr group showed significantly decreased vas scores offig ultrasoundguided brsbas well as the dose of dezocine used as a rescue analgesic were also recordedin addition brsbrelatedcomplications such as peritoneal punctureinternalan injury and systemic toxicity were all recordeda total of patients underwent cytoreductive surintraperitonealcombined withgeryhyperthermic 0cwang bmc anesthesiology page of fig flow chart showing patient consecutive enrolment and analysis abbreviations crshipec cytoreductive surgery and hyperthermicintraperitoneal chemotherapy ga general anesthesia brsb bilateral rectus sheath block ards acute respiratory distress syndrome vas visualanalogue scalepain at rest and during motion p however at h and h after surgery there were no significant differences in the vas scores of pain at rest and duringmotion between the two groups p from t6 tot10 the pcia input dose and the number of pca boluses were also obviously reduced in the gr group compared with the g group p in addition as arescue analgesic the dose of dezocine after surgery inthe gr group was significantly lower than that in the ggroup p postoperative adverse eventsadverse events that occurred in the sicu are presentedin table after surgery there were cases withhypertension cases of emergence agitation cases ofdelayed recovery cases of hypercapnia and cases ofnausea and vomiting in the g group fewer cases of allof these events occurred in the gr group p there were no differences in the incidence of hypoxemiabetween the two groups p there were no complications associated with nerve block in either group 0cwang bmc anesthesiology page of table demographic and surgical variables mean ± sdage ysex malefemalebmi kgm2medical historydiabetes mellitus n yesnohypertension n yesnocoronary heart disease n yesnoasa grade iiiiiinyha grade iiitotal surgery time mintime to tracheal extubation minremifentanil mgrocuronium mgtotal fluid volume mlurine volume mltotal volume of allogeneic erythrocyte infusion mltotal volume of plasma mlg group n ± ± ± ± ± ± ± ± ± ± gr group n ± ± ± ± ± ± ± ± ± ± p value asa american society of anesthesiologists bmi body mass index calculated as weight in kilograms divided by height in metres squared nyha new york heartassociation g general anaesthesia gr bilateral rectus sheath block combined with general anaesthesia before bilateral rectus sheath block t1 the time ofanaesthesia t2 the time of skin incision t3 the time of peritoneal thermochemotherapy t4 and the end of surgery t5discussionin this retrospective study we examined the efficacy andsafety of brsb combined with general anaesthesia in patients undergoing crshipec regarding efficacy theresults show that ultrasoundguided brsb significantlyreduced the total dose of remifentanil used during thesurgery and shortened the time to tracheal catheter extraction which is consistent with the findings of previous studies of other surgeries [ ] in addition rsbreduced the total dose of rocuronium in this studytable haemodynamic parameters in both groups mean ± sdindextimepointgn ± grn ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± pvaluemmhghrbpmt1t2t3t4t5t1t2t3t4t5map mean arterial pressure hr heart rate g general anaesthesia gr bilateralrectus sheath block combined with general anaesthesiawhich may be associated with the high concentration ofropivacaine used in the studyrsb also effectively relieved postoperative pain in thisstudy we found that the vas scores of pain at rest andduring motion were all lower in the gr group than inthe g group at h after surgery however at h and h after surgery there were no differences in the vasscores of pain at rest and during motion between thetwo groups suggesting that the analgesic effects of asingle brsb remained within h after surgery thisresult may be different from the findings of others cho reported that at h after surgerythere were no differences in the vas scores of painat rest and during motion between the rsb and nonrsb groups the discrepant results may be related todifferences in the concentration of ropivacaine andthe physical constitution of patients a high concentration can prolong the duration of action of a localanaesthetic in this study we selected not ropivacaine additionallythese patientsundergoing crshipec may have been adaptive topain furthermore compared with the control groupthe rsb group showed a reduced totalinfused doseof sufentanil as pcia number of pca boluses within h after surgery and total dose of dezocine used asa rescue analgesic after surgery these results furtherprove the role of rsb in providing shortterm postoperative analgesia 0cwang bmc anesthesiology page of table painrelated indicators in both groups median [range]vas score of pain at rest [median range]t6t7t8t9t10vas score of pain during motion [median range]t6t7t8t9t10total infused dose of pcia [ml median range]t6t7t8t9t10cumulative number of pcia boluses [median range]t6t7t8t9t10total dose of dezocine as a rescue analgesic mggn [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ± grn [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ± p value vas visual analogue scale pcia patientcontrolled intravenous analgesia g general anaesthesia gr bilateral rectus sheath block combined with generalanaesthesia the time at h after surgery t6 h after surgery t7 h after surgery t8 h after surgery t9 and h after surgery t10table postoperative adverse events in both groupsadverse eventsn gn grn hypertensionemergence agitation delayed recoveryhypoxemiahypercapnia nausea and vomiting peritoneal punctureinternal an injurysystemic toxicity g general anaesthesia gr bilateral rectus sheath block combined withgeneral anaesthesiawe also examined the safety of rsb during the surgery ultrasoundguided brsb had no significant effectson the haemodynamics of patients during surgery compared with general anaesthesia alone in terms of postoperative adverse events the results show that comparedwith the control group the rsb group showed a reducedincidence of hypertension emergence agitation delayedrecovery hypercapnia and nausea and vomiting whichmight be correlated with the decreased analgesic andmuscle relaxant doses no rsbrelated complications occurred in any patient these data indicate that rsbcould reduce the risk of complications associated withgeneral anaesthesia and is safe for patientsrsb an established technique has regained popularityin clinical applications [“] previous studies havedemonstrated that this technique could achieve relaxation of the anterior abdominal wall [ ] bashandyreported that anterior branches of the t7t12 thoracicnerve and the l1 lumbar nerve travelled through thepvalue“““ 0cwang bmc anesthesiology page of plane of the transverse abdominis muscle entered the rectus abdominis sheath and distributed on the surface of theskin the main process of rsb is to inject local anaesthetics between the rectus abdominis and the posteriorsheath of the rectus abdominis therefore rsb exerteda good effect in terms of perioperative analgesia for medianabdominal incisions a midline incision is required inthis kind of surgery thus based on these results rsbcould meet the need for analgesia in these patientsin addition for a long time epidural analgesia eawas thought to be an effective method for abdominalsurgery [“] studies have proved that epidural analgesia could maintain a good analgesic effect and reduceperioperative opioid consumption including in this typeof surgery [ ] however the safety of ea in crshipec remains controversial especially regarding effectson coagulation and circulatory function coagulationdysfunction and profound fluid loss are the main characteristics of patients with peritoneal cancer [ ]which might limit the administration of eaalthough epidural catheter is standard of care insolanki™s guideline in our hospital epidural catheter in not the standard of care we performed generalanesthesia combined with epidural anesthesia in somepatients to reduce the consumption of intravenous drugsand provide perfect analgesia but coagulation dysfunction and profound fluid loss are the main characteristicsof patients with peritoneal cancer in our previous studywe found that the mean arterial pressure of patientsundergoing epidural anesthesia was difficult to be maintained in the surgery besides there were many patientswith coagulation dysfunction before surgery who werenot suitable for the epidural anesthesia these results wefound in clinical were similar with others™ researcheskajdi and colleagues reported a case of epidural haematoma in their study godden found that the incidence of hypotension in the ea group was obviouslyhigher than that in the rsb group consequentlyrsb could be a better choice than ea in crshipecadditionally there are some limitations to this studyfirst all the data of this study were collected from theemr system as this was a retrospective study the findings are not as persuasive as those of a randomized controlled study we plan to conduct prospective studies toexplore the comprehensive influence of rsb in this surgery second we only examined the application of brsbestablished with a single injection which provides only ashortterm analgesic effect the efficacy of continuousanalgesia with brs catheters in crshipec remains unclear and needs further exploration third in our the results are initially presented according to the different aspects the primary outcome of this study is thetotal consumption of remifentanil during the surgeryother indicators were belonged to second outcomessin brsb could provide good postoperativeanalgesia reduce perioperative opioid consumption andreduce the incidence of postoperative complicationsthis is an easily applicable and safe procedure in crshipecabbreviationsrsb rectus sheath block brsb bilateral rectus sheath blockcrs cytoreductive surgery hipec hyperthermic intraperitonealchemotherapy emr electronic medical record vas visual analogue scalepcia patientcontrolled intravenous analgesia hr heart rate map meanarterial pressure bis bispectral index sicu surgical intensive care unitasa american society of anesthesiologists nyha new york heartassociation spo2 pulse oximetry paco2 partial pressure of carbon dioxidepao2 oxygen partial pressure bmi body mass indexacknowledgementsi would like to express my heartfelt thanks to the staff of the informationdata center of beijing shijitan hospital affiliated to capital medical universityauthors™ contributionswsh study design data collection writing paper lpf gt data collectionand data analysis gl coordinated the study and manuscript revision ltzstudy design and manuscript revision all authors read and approved thefinal manuscript all authors ensure the accuracy of the manuscript andagree to take personal responsibility for their contributionsfundingno fundingavailability of data and materialsthe datasets used andor analyzed during the current study are availablefrom the corresponding author on reasonable requestethics approval and consent to participatethis study was approved by the ethics committee of beijing shijitan hospitalaffiliated to capital medical university approval code research ethicsno69 this study is retrospective only anonymous data sources were usedand informed consent was not requiredconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsreceived january accepted july referencesklaver ce musters gd bemelman wa punt cj verwaal vj dijkgraaf mgaalbers ag van der bilt jd boerma d bremers aj adjuvanthyperthermic intraperitoneal chemotherapy hipec in patients with coloncancer at high risk of peritoneal carcinomatosis the colopec randomizedmulticentre trial bmc cancer 201515undefined428van oudheusden tr braam hj nienhuijs sw wiezer mj van ramshorst bluyer md lemmens ve de hingh ih cytoreduction and hyperthermicintraperitoneal chemotherapy a feasible and effective option for colorectalcancer patients after emergency surgery in the presence of peritonealcarcinomatosis ann surg oncol “passot g vaudoyer d villeneuve l kepenekian v beaujard ac bakrin ncotte e gilly fn glehen o what made hyperthermic intraperitonealchemotherapy an effective curative treatment for peritoneal surfacemalignancy a 25year experience with procedures j surg oncol“arjonasánchez a barrios p boldoroda e camps b carrascocampos jmartín vc garcíafadrique a gutiérrezcalvo a morales r ortegapérez g hipect4 multicentre randomized clinical trial to evaluate safety andefficacy of hyperthermic intraperitoneal chemotherapy hipec with 0cwang bmc anesthesiology page of huepenbecker sp cusworth se kuroki lm lu p samen cd woolfolk cdeterding r wan l helsten dl bottros m continuous epiduralinfusion in gynecologic oncology patients undergoing exploratorylaparotomy the new standard for decreased postoperative pain and opioiduse gynecol oncol “teoh da hutton mj else s walker a lee a mack la epidural analgesia aprospective analysis of perioperative coagulation in cytoreductive surgeryand hyperthermic intraperitoneal chemotherapy am j surg “schmidt c creutzenberg m piso p hobbhahn j bucher m perioperativeanaesthetic management of cytoreductive surgery with hyperthermicintraperitoneal chemotherapy anaesthesia “kajdi me beckschimmer b held u kofmehl r lehmann k ganter mtanaesthesia in patients undergoing cytoreductive surgery withhyperthermic intraperitoneal chemotherapy retrospective analysis of asingle centre threeyear experience world j surg oncol solanki sl mukherjee s agarwal v thota rs balakrishnan k shah sb desain garg r ambulkar rp bhorkar nm society of oncoanaesthesia andperioperative care consensus guidelines for perioperative management ofpatients for cytoreductive surgery and hyperthermic intraperitonealchemotherapy crshipec indian j anaesth “ godden ar marshall mj grice as daniels ir ultrasonography guidedrectus sheath catheters versus epidural analgesia for open colorectal cancersurgery in a single centre ann r coll surg engl “publisher™s notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsmitomycin c used during surgery for treatment of locally advancedcolorectal carcinoma bmc cancer baratti d kusamura s iusco d gimondi s pietrantonio f milione mguaglio m bonomi s grassi a virzì s hyperthermic intraperitonealchemotherapy hipec at the time of primary curative surgery in patientswith colorectal cancer at high risk for metachronous peritoneal metastasesann surg oncol “chua tc robertson g liauw w farrell r yan td morris dl intraoperativehyperthermic intraperitoneal chemotherapy after cytoreductive surgery inovarian cancer peritoneal carcinomatosis systematic review of currentresults j cancer res clin oncol “li y zhou yf liang h wang hq hao jh zhu zg wan ds qin lx cui szji jf chinese expert consensus on cytoreductive surgery andhyperthermic intraperitoneal chemotherapy for peritoneal malignanciesworld j gastroenterol “ willschke h bosenberg a marhofer p johnston s kettner sc wanzel o kaprals ultrasonographyguided rectus sheath block in paediatric anaesthesiaanew approach to an old technique br j anaesth “azemati s khosravi mb an assessment of the value of rectus sheath blockfor postlaparoscopic pain in gynecologic surgery j minim invasive gynecol“ dingeman rs barus lm chung hk clendenin dj lee cs tracy s johnsonvm dennett kv zurakowski d chen c ultrasonographyguided bilateralrectus sheath block vs local anesthetic infiltration after pediatric umbilicalhernia repair a prospective randomized clinical trial jama surg “ relland lm tobias jd martin d veneziano g beltran rj mckee c bhalla tultrasoundguided rectus sheath block caudal analgesia or surgical siteinfiltration for pediatric umbilical herniorrhaphy a prospective doubleblinded randomized comparison of three regional anesthetic techniques jpain res 201710undefined2629“ xu l hu z shen j pm mq efficacy of usguided transversus abdominisplane block and rectus sheath block with ropivacaine anddexmedetomidine in elderly highrisk patients minerva anestesiol “ cho s kim yj jeong k moon hs ultrasoundguided bilateral rectus sheathblock reduces early postoperative pain after laparoscopic gynecologicsurgery a randomized study j anesth “li t ye q wu d li j yu j doseresponse studies of ropivacaine in bloodflow of upper extremity after supraclavicular block a doubleblindrandomized controlled study bmc anesthesiol bell jc rylah bg chambers rw peet h mohamed f moran bjperioperative management of patients undergoing cytoreductive surgerycombined with heated intraperitoneal chemotherapy for peritoneal surfacemalignancy a multiinstitutional experience ann surg oncol “landmann a visoiu m malek mm development of a novel technique forbilateral rectus sheath nerve blocks under laparoscopicguidance j pediatrsurg “kumar a wilson ga engelhardt te ultrasound guided rectus sheathblockade compared to perioperative local anesthetic infiltration in infantsundergoing supraumbilical pyloromyotomy saudi journal of anaesthesia“ bashandy gm elkholy ah reducing postoperative opioid consumption byadding an ultrasoundguided rectus sheath block to multimodal analgesiafor abdominal cancer surgery with midline incision anesthesiol pain med201443e18263 dowidar aerm ezz haa shama aae eloraby ma postoperative analgesiaof ultrasound guided rectus sheath catheters versus continuous woundcatheters for colorectal surgery a randomized clinical trial ˜† egypt journalof anaesth “karaarslan e topal a avci o tuncer uzun s research on the efficacy of therectus sheath block method agri “ piccioni f casiraghi c fumagalli l kusamura s baratti d deraco m arientif langer m epidural analgesia for cytoreductive surgery withperitonectomy and heated intraperitoneal chemotherapy int j surg 16pt a99“ vesterandersen m lundstrøm lh møller mh the association betweenepidural analgesia and mortality in emergency abdominal surgery apopulationbased cohort study acta anaesthesiol scand httpsdoi101111aas13461 0c" Answer:

Colon_Cancer

" excessive perioperative fluid administration may result in iatrogenic endothelial dysfunction andtissue edema transducing inflammatory markers into the bloodstream colloids remain longer in the circulationrequiring less volume to reach similar hemodynamic endpoints compared to crystalloids thus we tested thehypothesis that a goaldirected colloid regimen attenuates the inflammatory response compared to a goaldirectedcrystalloid regimemethods patients undergoing moderate to highrisk open abdominal surgery were randomly assigned to goaldirected lactated ringer™s solution n or a hydroxyethyl starch n fluid regimen our primaryoutcome was perioperative levels of pro and antiinflammatory cytokines secondary outcome was perioperativelevels of white blood cell count wbc creactive protein crp procalcitonin pct and lipopolysaccharidebindingprotein lbp measurements were performed preoperatively immediate postoperatively on postoperative day onetwo and fourresults the areas under the curve of interleukin il p il p il p and tumor necrosisfactor α p levels did not differ significantly between the groups wbc crp and pct values were alsocomparable lbp although significantly higher in the crystalloid group remained in the normal range patientsassigned to crystalloids received a median iqr amount of ml “ of crystalloid patients assigned tocolloids received ml “ of crystalloid and ml “ of colloid cytokine and inflammatory marker levels did not differ between goaldirected crystalloid and colloidadministration after moderate to highrisk abdominal surgerytrial registration clinicaltrialsgov nct00517127 registered 16th august correspondence barbarakabonmeduniwienacat1department of anaesthesia general intensive care medicine and painmedicine medical university of vienna spitalgasse vienna austriafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cobradovic bmc anesthesiology page of is crucialintroductionvolume replacementin the perioperativeperiod and has great impact on postoperative outcome fluid restriction may cause hypotension and hypoperfusion leading to an dysfunction on the otherhand excessive fluid administration leads to destructionof the endothelial surface layer and consequently to tissue edema with harmful side effects [ ]fluidgdtbasedtherapygoaldirectedonoptimization of flowrelated hemodynamic parametersimproves clinical outcome in low to highrisk surgicalpatients compared to fixed fluid protocols [ ] specifically gdt enhances cardiac performance and gutmicrocirculation while avoiding iatrogenic hyperhydration [ ] in addition to hypervolemia the inflammatoryaggravatesdegradation of the endothelial barrier the socalled glycocalyx inflammation leads to cytokine release andmay thus worsen outcome for example high postoperative interleukin il levels are independently associatedwith postoperative complications response dueto surgicaltraumaso far in most previously performed gdt studieshemodynamic algorithms were based on colloid bolusadministration to improve hemodynamic variables colloids better maintain the intravascular oncotic pressure and provide a higher volume effect when used incase of hypovolemia goaldirected colloid administration reduces intraoperative fluid requirement and improves cardiac performance compared to crystalloids[ ] whether this translates into better outcomespecifically in a decreased postoperative inflammatoryresponse is still a matter of research the comparisonbetween colloid versus crystalloid based fluid regimenswas still lacking therefore we tested the primary hypothesis that perioperative levels of pro and antiinflammatory cytokines il il il and tumor necrosis factor alpha tnf α are reduced by goaldirectedcolloid versus crystalloid administration during the firstfour postoperative days in patients undergoing moderateto highrisk open abdominal surgery in addition wemeasured white blood cell wbc count creactive protein crp procalcitonin pct and lipopolysaccharidebinding protein lbp levelsmaterials and methodsthis prospective randomized controlled trial was conducted at the department of anesthesia intensive caremedicine and pain medicine medical university ofvienna vienna austria the institutional review boardof the medical university of vienna approved it as partof a large multicenter outcome trial evaluating the effectof goaldirected crystalloid and colloid on postoperativecombined morbidity and complications the ethicalcommittee of medical university of vienna viennaaustria provided ethical approval for this trial the trialwas conducted in accordance with the declaration ofhelsinki and good clinical practice and registered atclinicaltrialsgov nct00517127 and eudract “ a written informed consent was obtainedfrom all patients the authors have followed the applicable consort guidelinesfor this single center substudy consecutive eligiblepatients were included patients aged to yearsundergoing elective moderate to highrisk open abdominal surgery with american society of anesthesiologistsasa physical status iiii were included we excludedpatients with severe obesity body mass index bmi kgmˆ’ cardiac insufficiency ejection fraction ef coronary artery disease with angina severe chronicobstructive pulmonary disease autoimmune diseases coagulopathies renal insufficiency creatinine clearance mlminˆ’ or renal replacement therapy symptoms of infection or sepsis and preoperative crp higher than mgdlˆ’ allpatientsreceivedprotocolpreoperativelyantimicrobialprophylaxis using a single dose of a 2nd generationcephalosporine according to our clinicalstandardsanesthetic management wasstandardized standardmonitoring included electrocardiography ecg invasiveblood pressure surveillance pulse oximetry and esophageal core temperature monitoring a central venouscatheter was inserted when deemed clinically necessarywe used balanced anesthesia with sevoflurane none ofour patients received locoregional anesthesia accordingto patients™ requirements additional fentanyl and nondepolarizing neuromuscular blocking were administeredventilatory rate was adjusted to maintain endtidal carbon dioxide partial pressure etco2 of “ mmhgnormothermia was maintained with forced air warmingpatients were randomized to crystalloid lactatedringer™s solution or colloid hydroxyethyl starch voluven fresenius kabi germany grouprandomization was based on computergenerated codesto conceal allocation sealed opaque envelopes wereopened only shortly before induction of anesthesiaall patients were given “ mlkgˆ’ oflactatedringer™s solution during induction of anesthesia followedby “ mlkgˆ’ per hour for maintenance normalized toideal body weight ibw throughout surgery we calculated ibw according to the robinson formula thereafter the randomized fluid crystalloid or colloidwas esophageal dopplerguided cardiac q deltex medical group plc chichester uk according to a standardalgorithm this method is based on corrected aorticflow time ftc as well as stroke volume sv and allowsdistinguishing whether a patient is a fluid responder or 0cobradovic bmc anesthesiology page of not if mean arterial pressure map was below mmhg and no signs of hypovolemia were detected vasopressors were administratedpatients were transferred to postanesthetic care unitpacu or intensive care unit icu at the discretion ofthe attending anesthesiologist fluid management wasstandardized for the first postoperative hours in whichpatients received mlkgˆ’ ibw crystalloid per hourmeasurementsdemographic and morphometric data were recorded aswell as asa score medical history type of surgery andpreoperative laboratory values duration of anesthesiaand surgery were recorded we also recorded intraoperative fluid requirements estimated blood loss transfusion requirements and urinary output for evaluation ofanesthetic management the total amount of fentanylendtidal sevoflurane concentration core temperatureandnotedhemodynamic parameters such as map heart ratehr ftc sv and cardiac output co were recorded at10min intervals the application of phenylephrine usewas notedicu admission werepostoperativethe primary outcomes were the areas under the curveaucs of postoperative levels of pro and antiinflammatory cytokines il il il and tnf α andtheir differences between the crystalloid and the colloidgroup secondary outcomes were aucs of wbc crppct and lpb and their differences between the groupsall blood samples for parameteranalyses were obtainedbefore surgery as baseline values t0 immediately postoperatively t1 as well as on postoperative days onetwo and four t2 t3 and t4 respectively for analysisof il il il and tnf α blood samples were centrifuged within h at g for min and plasma wasimmediately stored at ˆ’ °c for later enzymelinkedimmunosorbent assay elisa analyses the serum concentrations of il il il and tnf α were determined according to the manufacturer™sinstructionshuman sil6 instant elisa human il8nap1 instant elisa and human sil10 instant elisaebioscience vienna austria wwwebiosciencecom human tnf α duoset rd systems minneapolis minnesota wwwrndsystemscom for that purpose opticaldensity was measured with a victor microplate readerat a wavelength of nm multiple testing of sampleson different plates revealed an intraassay variability of for il for il for il for tnf α andan interassay variability of for il for il for il and for tnf αfor investigation of wbc crp pct and lpb separateblood samples were obtained their analysis took placeimmediately after blood sampling as routine laboratoryanalysessample size calculation and statistical analysissample size calculations for our trial were based on thestudy of steppan and colleagues they observed amean standard deviation sd of pgmlˆ’ in il h after surgery in abdominal surgery patients assuming a similar coefficient of variation sdmean for each of the four cytokines primarily plannedfor evaluation in our study we calculated a total of patients in order to obtain a power to detect a reduction in any of the cytokines at an overall significance level with powergroups were primarily compared for balance in patients™ demographic data intraoperative characteristicsand postoperative variables absolute standardized differences asd were calculated for patients™ baseline covariates subsequent measurements ofintraoperativeparameters were first averaged within each patient andthen averaged among the patients in each treatmentgroup for descriptive analysis normal distribution wasassessed with qq plots and kolmogorowsmirnow testsnormally distributed variables were with unpaired twotailed ttests otherwise the in case of normally distributed values wilcoxon ranksum test was used for notnormally distributed continuous data paired comparisons between baseline data and postoperative data wereperformed with paired sample ttest or wilcoxonsignedrank test as applicable nominal data were analyzed with chisquare or fisher™s exact test for low expected cell counts data were presented as means ± sdmedians iqr or as numbers percentage as applicableadjustment for multiple testing was performed with thebonferroni method a p value was considered statistically significantanalysis was conducted with spss software version armonk ny ibm corp r for macintosh version321 r core team r a language and environmentfor statistical computing r foundation forstatistical computing vienna austria was used to calculate asdresultsa total of patients were included between november and october in the colloid group and in the crystalloid group fig at t0 all values weremeasured overall in the crystalloid group and inthe colloid group of the preplanned blood sampleswere collected and analyzedpatient™s baseline characteristics did not differ exceptfor height bmi with a slightly higher bmi in the colloidgroup type of surgery and crp also higher in the colloid group table duration of anesthesia and surgerywere comparable between both groups patients assignedto crystalloid administration received a median of ml “ crystalloids whereas patients assigned 0cobradovic bmc anesthesiology page of fig consort patient flow chartto the colloid group received ml “ ofcrystalloid solution and ml “ of colloidsblood loss transfusion requirements and urinary outputdid not differ between the groups anesthetic management map and hr did not differ between the groupsftc sv and co were significantly higher in the colloidgroup compared to the crystalloid group ftc ms“ versus ms “ p sv ml“ versus ml to p and co ± lminˆ’ versus ± lminˆ’ p thenumber of patients requiring vasopressor support wascomparable between groups the incidence of postoperative icu admissions did not differ in the crystalloid versus in the colloid group p table baseline values of il il il and tnf α in thecrystalloid group were comparable to values in the colloid group il pgmlˆ’ “ versus pgmlˆ’ “ p il pgmlˆ’ “ versus pgmlˆ’ “ p il pgmlˆ’ “ versus pgmlˆ’ “ p tnf α pgmlˆ’ “ versus pgmlˆ’ “ p immediatepostoperative values of il and were significantlyhigher compared to baseline values in both groupsp for all measurements while tnf α did notshow any significant increase in the crystalloid p and the colloid group p fig aucs of il il il and tnf α did not differ significantly between the groups table wbc values at baseline were glˆ’ “ in thecrystalloid versus glˆ’ “in the colloidgroup p crp pct and lbp baseline valueswere also comparable in both groupscrp mgdlˆ’ “ versus mgdlˆ’ “p pct ngmlˆ’ “ versus ngmlˆ’ “ p lbp mcglˆ’ “ versus mcglˆ’ “ p immediate postoperative values of wbc and pctwere significantly higher compared to the baseline valuesin both groups p for all measurements fig 0cobradovic bmc anesthesiology page of table baseline characteristicsage yrsweight kgheight cmbmi kgmˆ’ gender no menwomenasa score no iiiiiimedical history no pulmonary diseasecardiovascular diseasediabetes type idiabetes type iitype of surgery no colorectalliverpancreaticcrystalloidsn ± ± ± ± colloidsn ± ± ± ± asd preoperative laboratory valuescrp mgdlˆ’ ± ± patient characteristics data are presented as means ± sd or as counts for thecategorical outcomesabbreviations asd absolute standardized differences absolute difference inmeans or proportions divided by the pooled sd asd values of and represent small median and large differencesbmi body mass index m male f female asa american society ofanesthesiologists crp creactive protein sd standard deviationthe aucs for wbc crp pct and lbp for the timeperiods from t1 to t4 did not differ significantly between the groups fig table however lbpshowed significantly higher levels in the crystalloidgroup in the immediate postoperative period comparedto the colloid group mcglˆ’ “ versus mcglˆ’ “ p at all other postoperativetime points there were no significant differences betweenthe groups fig discussionthis trial is a substudy of a large multicenter randomized trial evaluating the effect of goaldirected crystalloidversus goaldirected colloid fluid administration on acomposite of serious complications after moderate tohighrisk open abdominal surgery the overall trial concluded that colloids did not decrease the composite ofmajorincomplicationsare ourresultsconcordance as they did not show any differences inperioperative pro and antiinflammatory markers between a crystalloid and a colloid fluid regimendespite multimodal care and enhanced recovery programs it still remains challenging to blunt the inflammatory response to surgery systemic inflammationafter abdominal surgery impairs outcome and thereforemany attempts have been made to alter the inflammatory response several factors influence the perioperative inflammatoryresponse such as the underlying disease type and invasiveness of surgery as well as type of anesthesia [“] themost important factor is the magnitude of surgical traumaand tissue damage which induce proliferation and activation of immune competent cells in turn triggering cytokine and inflammatory marker release so far veryfew trials have specifically investigated the influence offluid therapy and differences in terms of the type of fluidon the extent of inflammatory marker releaseto investigate the potential influence of goaldirected hydroxyethyl starch versus a lactated ringer™s solution fluid regimen on inflammatory response pro il il and tnf α and antiinflammatory cytokine il serum levels were measured during the perioperativeperiod additionally we measured wbc crp pct andlbpgenerally the most commonly measured biomarkersare crp and wbc if levels of crp are above mgdlˆ’ after postoperative day four a postoperative infection can be suspected crp levels in our studygroups increased on the first postoperative day droppingon the fourth postoperative day to nearly mgdlˆ’ inboth study groupswbcs are an imprecise marker to detect postoperativecomplications after major abdominal surgery amore sensitive parameter in predicting postoperativecomplications after major abdominal surgery is il surgical trauma and hypoperfusion of the colon aremain sources of il release in colorectal surgery noblett demonstrated that gdt during elective colorectal surgery significantly reduced il levels in comparison to a control group yates showed no differencesof il and il levels between goaldirected colloidand crystalloid fluid therapy during the first h in asubgroup of patients undergoing colorectal surgery although patients in the crystalloid group received significant more volume amount as compared to the colloid groupthere was no significant difference inhemodynamic variables our patients showed similar courses of il and il levels in the immediatepostoperative period in contrast to the trial of yateswho measured cytokine levels up to the first h aftersurgery we extended our measurement period to fourpostoperative days we showed comparable circulating 0cobradovic bmc anesthesiology page of table intraoperative dataduration of anesthesia minduration of surgery minfluid managementtotal fluid intake mlacrystalloid mlcolloid mlestimated blood loss mltransfusion yesno urinary output mlanesthesia managementfentanyl mcgtwa et sevoflurane core temperature °cicu admission yesno hemodynamictwa map mmhgtwa hr beatsminˆ’twa ftc mstwa sv mltwa co lminˆ’ phenylephrine yesno crystalloidsn ± ± “ “ “ “ “ [ ] “ ± ± ± “ “ ± colloidsn ± ± “ “ “ “ [ ] “ “ ± ± ± “ “ ± p value ° ° °° °°intraoperative data are presented as means ± sd medians iqr or as counts for the categorical outcomes means were compared with an unpaired twosided ttests ormannwhitneyu tests as appropriate medians with wilcoxon ranksum tests and counts with chisquare or fisher™s exact tests ° represents statisticalsignificance p abbreviations et end tidal icu intensive care unit twa time weighted average map mean arterial pressure hr heart rate ftc corrected flow time sv strokevolume co cardiac output sd standard deviationa total fluid intake includes baseline fluid boluses antibiotics analgesics and additional fluid administered at the discretion of attending anesthesiologistfig ad pro and antiinflammatory cytokines il il il and tnf α over time a il b il c il and d tnf α data arepresented as medians iqr abbreviations il “ interleukin tnf α “ tumor necrosis factor alpha pod “ postoperative day 0cobradovic bmc anesthesiology page of table areas under the curve of inflammatory markerspcrystalloidsn “ “ “colloidsn “ “ “ “ “ “ “ “auc il pgmlˆ’ 1dauc il pgmlˆ’ 1dauc il pgmlˆ’ 1dauc tnf α pgmlˆ’ 1d “auc wbc glˆ’1dauc crp mgdlˆ’ 1dauc pct ngmlˆ’ 1dauc lbp mcglˆ’ 1dtable areas under the curve of il il il and tnf α as well as wbccrp pct and lbp are presented as medians iqr medians were comparedwith wilcoxon ranksum testsabbreviations il interleukin tnf α tumor necrosis factor alpha wbc whiteblood cells pct procalcitonin lbp lipipopolysaccharidebinding protein “ “ “ “il and il levels between a gdt crystalloid and colloid administration these surrogates of inflammatoryresponse imply that gut perfusion during surgery waswell preserved with both types of fluid and suggest thatthe type of fluid might be of minor importance as longas the fluid is administered in a goaldirected fashionthe fact that tnf α levels in both groups remainedstable over the entire measured period further supportsour theory the course of tnf α levels during the perioperative period was in accordance with the study ofin which fluid therapy was guided withszakmanypicco versusin majorvenous pressurecentralabdominal surgery in patients at risk for postoperativecomplications as tnf α per se triggers glycocalyxdegradation we anticipate that tnf α did not influence glycocalyx shedding and thus possible fluid shifts inour study populationpct is an early predictive marker for systemic inflammation after abdominal surgery values above ngmlˆ’ are associated with postoperative complicationssuch as pneumonia or anastomotic leakage in ourstudy median pct levels did not exceed ngmlˆ’ atany measured time point these results are in concordance with our main study where infectious complications rate were held low and did not differ between thegroups moreover as pct production can also beinduced by tissue hypoperfusion we might assume thatgoaldirected fluid administration contributed to lowpct values by optimizing cardiac performance furthermore we measured lbp a prognostic markerfor bacterial infections patients in the crystalloidgroup showed significantly higher levels immediatelyafter surgery however the measured values remainedwithin the normal range therefore this difference ismost likely not to of clinical importancethe vascular endothelium is one of the earliest sitesinvolved in the inflammatory response syndrome an adequate perioperative fluid management has a major impact on the integrity of the glycocalyx with goaldirected fluid management individualized and time appropriate fluid resuscitation can be achieved enablingfig ad inflammatory markers wbc crp pct and lbp over time a wbc b crp c pct and d lbp data are presented as medians iqrabbreviations wbc “ white blood cells crp “ creactive protein pct “ procalcitonin lbp “ lipopolysaccharidebinding protein pod “postoperative day ˜… represents significant difference in lbp in the immediate postoperative period between the crystalloid and the colloidgroup p 0cobradovic bmc anesthesiology page of preservation of endothelial surface layer and sufficientan perfusion thus improving postoperative outcomesafter major surgery patients in the colloid group received significantly lessfluid ml confirming the previously publishedfluid sparing effect of colloids however clinical significance of this difference may be questionable during aperioperative period of nearly five hours further ourhemodynamic data showed significantly higher values ofsv and co with colloid administration though the absolute difference of ml in sv most likely has onlylimited clinical relevance it might very well be that thesignificant differences in sv and co are the result ofour number of patients included in this substudyassurrogatesfirst limitation of our study is that we measured inflammatory markers that reflect the inflammatory responseand not direct markers ofglycocalyx degradation like syndecan1 therefore wecannot draw any s about the preservation ofthe endothelial surface layer in our patients secondlywe did not control postoperative fluid management during the postoperative followup period a further limitation isthe time between patient enrolment andsubmission of our current results due to the fact thatthe main trial has been published recently a delay of oursubmission occurred nevertheless our results canstill be extrapolated to current clinical practicein summary goaldirected hydroxyethyl starch administration did not attenuate the inflammatory responseexpressed by cytokine levels of il il il and tnfα in patients undergoing moderate to highrisk open abdominal surgery wbc crp and ptc values did not differ between the different fluid regimes as wellabbreviationsasa american society of anesthesiologists asd absolute standardizeddifference auc area under the curve bmi body mass index co cardiacoutput crp creactive protein ecg electrocardiography ef ejectionfraction elisa enzymelinked immunosorbent assay etco2 endtidalcarbon dioxide ftc corrected flow time gdt goaldirected therapyhr heart rate ibw ideal body weight icu intensive care unitil interleukin iqr interquartile range lbp lipopolysaccharidebindingprotein map mean arterial pressure pacu postanesthetic care unitpct procalcitonin picco pulse contour cardiac output spss statisticalpackage for the social sciences sv stroke volume tnf α tumor necrosisfactor alpha twa time weighted average wbc white blood cell countacknowledgementsassistance with this we thank bianca tudor md department ofanesthesia intensive care medicine and pain medicine medical university ofvienna spitalgasse vienna austria for her support in laboratoryskills performing elisasauthors™ contributionsall authors have read and approved the manuscript mo patientrecruitment data acquistion and prepratation of the manuscript ak studyprotocol writing and preparation of the manuscript bk study protocolwriting and preparation of the manuscript statistical analyisis gr dataanalysis revision of the manuscript ok data analysis preparation of themanuscript oz patient recruitment data acquisition data managementab patient recruitment data acquisition revsion the manuscript cr dataacquistion revision of the manuscript as patient recruitment revision ofthe manuscript ef study protocol writing and preparation of the manuscriptfundingmedical university of viennapartially funded by fresenius kabi deltex medical provided oesophagealdoppler monitors and disposablesthe sponsors were not involved in protocol development data acquisitionor data analysisavailability of data and materialsthe datasets used andor analysed during the current study are availablefrom the corresponding author on reasonable requestbarbarakabonmeduniwienacatethics approval and consent to participatethe main trial was approved by the local ethics committee of the medicaluniversity of vienna in ek and was registered atclinicaltrialsgov nct00517127 and eudract “ a writteninformed consent was obtained from all patients prior to participationconsent for publicationnot applicablecompeting intereststhe authors declare no competing interestsauthor details1department of anaesthesia general intensive care medicine and painmedicine medical university of vienna spitalgasse vienna austria2department of outcomes research and general anesthesiologyanesthesiology institute euclid avenue cleveland clinic cleveland ohusa 3department of anesthesiology and general intensive care franziskushospital nikolsdorfergasse vienna austria 4department ofgynecology klinik ottakring montleartstrasse vienna austria5department of surgery medical university of vienna spitalgasse vienna austriareceived july accepted august referencesbrandstrup b tonnesen h beierholgersen r effects of intravenousfluid restriction on postoperative complications comparison of twoperioperative fluid regimens a randomized assessorblinded multicentertrial ann surg “ myles ps bellomo r corcoran t restrictive versus liberal fluidtherapy for major abdominal surgery new engl j med “chappell d jacob m hofmannkiefer k conzen p rehm m a rationalapproach to perioperative fluid management anesthesiology “lowell ja schifferdecker c driscoll df benotti pn bistrian brpostoperative fluid overload not a benign problem crit care med “sun y chai f pan c romeiser jl gan tj effect of perioperative goaldirected hemodynamic therapy on postoperative recovery following majorabdominal surgerya systematic review and metaanalysis of randomizedcontrolled trials critical care calvovecino jm ripollesmelchor j mythen mg effect of goaldirected haemodynamic therapy on postoperative complications in lowmoderate risk surgical patients a multicentre randomised controlled trialfedora trial br j anaesth “noblett se snowden cp shenton bk han af randomized clinical trialassessing the effect of doppleroptimized fluid management on outcomeafter elective colorectal resection br j surg “ 0cobradovic bmc anesthesiology page of alphonsus cs rodseth rn the endothelial glycocalyx a review of thevascular barrier anaesthesia “ pearse rm harrison da macdonald n effect of a perioperativecardiac outputguided hemodynamic therapy algorithm on outcomesfollowing major gastrointestinal surgery a randomized clinical trial andsystematic review jama “publisher™s notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationskimberger o arnberger m brandt s goaldirected colloidadministration improves the microcirculation of healthy andperianastomotic colon anesthesiology “kolarova h ambruzova b svihalkova sindlerova l klinke a kubala lmodulation of endothelial glycocalyx structure under inflammatoryconditions mediat inflamm rettig tc verwijmeren l dijkstra im boerma d van de garde emnoordzij pg postoperative interleukin6 level and early detection ofcomplications after elective major abdominal surgery ann surg “ gan tj soppitt a maroof m goaldirected intraoperative fluidadministration reduces length of hospital stay after major surgeryanesthesiology “jacob m chappell d rehm m clinical update perioperative fluidmanagement lancet “feldheiser a pavlova v bonomo t balanced crystalloid comparedwith balanced colloid solution using a goaldirected haemodynamicalgorithm br j anaesth “ orbegozo cortes d gamarano barros t njimi h vincent jl crystalloidsversus colloids exploring differences in fluid requirements by systematicreview and metaregression anesth analg “kabon b sessler di kurz a effect of intraoperative goaldirectedbalanced crystalloid versus colloid administration on major postoperativemorbidity a randomized trial anesthesiology “ robinson jd lupkiewicz sm palenik l lopez lm ariet m determination ofideal body weight for drug dosage calculations am j hosp pharm “steppan j hofer s funke b sepsis and major abdominal surgery leadto flaking of the endothelial glycocalix j surg res “ wilmore dw from cuthbertson to fasttrack surgery years of progress inreducing stress in surgical patients ann surg “ desborough jp the stress response to trauma and surgery br j anaesth“ veenhof aa sietses c von blomberg bm the surgical stress responseand postoperative immune function after laparoscopic or conventional totalmesorectal excision in rectal cancer a randomized trial int j color dis “ gilliland he armstrong ma carabine u mcmurray tj the choice ofanesthetic maintenance technique influences the antiinflammatory cytokineresponse to abdominal surgery anesth analg “ wichmann mw huttl tp winter h immunological effects oflaparoscopic vs open colorectal surgery a prospective clinical study archsurg “ watt dg han pg mcmillan dc routine clinical markers of themagnitude of the systemic inflammatory respon

cancer296

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: " excessive perioperative fluid administration may result in iatrogenic endothelial dysfunction andtissue edema transducing inflammatory markers into the bloodstream colloids remain longer in the circulationrequiring less volume to reach similar hemodynamic endpoints compared to crystalloids thus we tested thehypothesis that a goaldirected colloid regimen attenuates the inflammatory response compared to a goaldirectedcrystalloid regimemethods patients undergoing moderate to highrisk open abdominal surgery were randomly assigned to goaldirected lactated ringer™s solution n or a hydroxyethyl starch n fluid regimen our primaryoutcome was perioperative levels of pro and antiinflammatory cytokines secondary outcome was perioperativelevels of white blood cell count wbc creactive protein crp procalcitonin pct and lipopolysaccharidebindingprotein lbp measurements were performed preoperatively immediate postoperatively on postoperative day onetwo and fourresults the areas under the curve of interleukin il p il p il p and tumor necrosisfactor α p levels did not differ significantly between the groups wbc crp and pct values were alsocomparable lbp although significantly higher in the crystalloid group remained in the normal range patientsassigned to crystalloids received a median iqr amount of ml “ of crystalloid patients assigned tocolloids received ml “ of crystalloid and ml “ of colloid cytokine and inflammatory marker levels did not differ between goaldirected crystalloid and colloidadministration after moderate to highrisk abdominal surgerytrial registration clinicaltrialsgov nct00517127 registered 16th august correspondence barbarakabonmeduniwienacat1department of anaesthesia general intensive care medicine and painmedicine medical university of vienna spitalgasse vienna austriafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cobradovic bmc anesthesiology page of is crucialintroductionvolume replacementin the perioperativeperiod and has great impact on postoperative outcome fluid restriction may cause hypotension and hypoperfusion leading to an dysfunction on the otherhand excessive fluid administration leads to destructionof the endothelial surface layer and consequently to tissue edema with harmful side effects [ ]fluidgdtbasedtherapygoaldirectedonoptimization of flowrelated hemodynamic parametersimproves clinical outcome in low to highrisk surgicalpatients compared to fixed fluid protocols [ ] specifically gdt enhances cardiac performance and gutmicrocirculation while avoiding iatrogenic hyperhydration [ ] in addition to hypervolemia the inflammatoryaggravatesdegradation of the endothelial barrier the socalled glycocalyx inflammation leads to cytokine release andmay thus worsen outcome for example high postoperative interleukin il levels are independently associatedwith postoperative complications response dueto surgicaltraumaso far in most previously performed gdt studieshemodynamic algorithms were based on colloid bolusadministration to improve hemodynamic variables colloids better maintain the intravascular oncotic pressure and provide a higher volume effect when used incase of hypovolemia goaldirected colloid administration reduces intraoperative fluid requirement and improves cardiac performance compared to crystalloids[ ] whether this translates into better outcomespecifically in a decreased postoperative inflammatoryresponse is still a matter of research the comparisonbetween colloid versus crystalloid based fluid regimenswas still lacking therefore we tested the primary hypothesis that perioperative levels of pro and antiinflammatory cytokines il il il and tumor necrosis factor alpha tnf α are reduced by goaldirectedcolloid versus crystalloid administration during the firstfour postoperative days in patients undergoing moderateto highrisk open abdominal surgery in addition wemeasured white blood cell wbc count creactive protein crp procalcitonin pct and lipopolysaccharidebinding protein lbp levelsmaterials and methodsthis prospective randomized controlled trial was conducted at the department of anesthesia intensive caremedicine and pain medicine medical university ofvienna vienna austria the institutional review boardof the medical university of vienna approved it as partof a large multicenter outcome trial evaluating the effectof goaldirected crystalloid and colloid on postoperativecombined morbidity and complications the ethicalcommittee of medical university of vienna viennaaustria provided ethical approval for this trial the trialwas conducted in accordance with the declaration ofhelsinki and good clinical practice and registered atclinicaltrialsgov nct00517127 and eudract “ a written informed consent was obtainedfrom all patients the authors have followed the applicable consort guidelinesfor this single center substudy consecutive eligiblepatients were included patients aged to yearsundergoing elective moderate to highrisk open abdominal surgery with american society of anesthesiologistsasa physical status iiii were included we excludedpatients with severe obesity body mass index bmi kgmˆ’ cardiac insufficiency ejection fraction ef coronary artery disease with angina severe chronicobstructive pulmonary disease autoimmune diseases coagulopathies renal insufficiency creatinine clearance mlminˆ’ or renal replacement therapy symptoms of infection or sepsis and preoperative crp higher than mgdlˆ’ allpatientsreceivedprotocolpreoperativelyantimicrobialprophylaxis using a single dose of a 2nd generationcephalosporine according to our clinicalstandardsanesthetic management wasstandardized standardmonitoring included electrocardiography ecg invasiveblood pressure surveillance pulse oximetry and esophageal core temperature monitoring a central venouscatheter was inserted when deemed clinically necessarywe used balanced anesthesia with sevoflurane none ofour patients received locoregional anesthesia accordingto patients™ requirements additional fentanyl and nondepolarizing neuromuscular blocking were administeredventilatory rate was adjusted to maintain endtidal carbon dioxide partial pressure etco2 of “ mmhgnormothermia was maintained with forced air warmingpatients were randomized to crystalloid lactatedringer™s solution or colloid hydroxyethyl starch voluven fresenius kabi germany grouprandomization was based on computergenerated codesto conceal allocation sealed opaque envelopes wereopened only shortly before induction of anesthesiaall patients were given “ mlkgˆ’ oflactatedringer™s solution during induction of anesthesia followedby “ mlkgˆ’ per hour for maintenance normalized toideal body weight ibw throughout surgery we calculated ibw according to the robinson formula thereafter the randomized fluid crystalloid or colloidwas esophageal dopplerguided cardiac q deltex medical group plc chichester uk according to a standardalgorithm this method is based on corrected aorticflow time ftc as well as stroke volume sv and allowsdistinguishing whether a patient is a fluid responder or 0cobradovic bmc anesthesiology page of not if mean arterial pressure map was below mmhg and no signs of hypovolemia were detected vasopressors were administratedpatients were transferred to postanesthetic care unitpacu or intensive care unit icu at the discretion ofthe attending anesthesiologist fluid management wasstandardized for the first postoperative hours in whichpatients received mlkgˆ’ ibw crystalloid per hourmeasurementsdemographic and morphometric data were recorded aswell as asa score medical history type of surgery andpreoperative laboratory values duration of anesthesiaand surgery were recorded we also recorded intraoperative fluid requirements estimated blood loss transfusion requirements and urinary output for evaluation ofanesthetic management the total amount of fentanylendtidal sevoflurane concentration core temperatureandnotedhemodynamic parameters such as map heart ratehr ftc sv and cardiac output co were recorded at10min intervals the application of phenylephrine usewas notedicu admission werepostoperativethe primary outcomes were the areas under the curveaucs of postoperative levels of pro and antiinflammatory cytokines il il il and tnf α andtheir differences between the crystalloid and the colloidgroup secondary outcomes were aucs of wbc crppct and lpb and their differences between the groupsall blood samples for parameteranalyses were obtainedbefore surgery as baseline values t0 immediately postoperatively t1 as well as on postoperative days onetwo and four t2 t3 and t4 respectively for analysisof il il il and tnf α blood samples were centrifuged within h at g for min and plasma wasimmediately stored at ˆ’ °c for later enzymelinkedimmunosorbent assay elisa analyses the serum concentrations of il il il and tnf α were determined according to the manufacturer™sinstructionshuman sil6 instant elisa human il8nap1 instant elisa and human sil10 instant elisaebioscience vienna austria wwwebiosciencecom human tnf α duoset rd systems minneapolis minnesota wwwrndsystemscom for that purpose opticaldensity was measured with a victor microplate readerat a wavelength of nm multiple testing of sampleson different plates revealed an intraassay variability of for il for il for il for tnf α andan interassay variability of for il for il for il and for tnf αfor investigation of wbc crp pct and lpb separateblood samples were obtained their analysis took placeimmediately after blood sampling as routine laboratoryanalysessample size calculation and statistical analysissample size calculations for our trial were based on thestudy of steppan and colleagues they observed amean standard deviation sd of pgmlˆ’ in il h after surgery in abdominal surgery patients assuming a similar coefficient of variation sdmean for each of the four cytokines primarily plannedfor evaluation in our study we calculated a total of patients in order to obtain a power to detect a reduction in any of the cytokines at an overall significance level with powergroups were primarily compared for balance in patients™ demographic data intraoperative characteristicsand postoperative variables absolute standardized differences asd were calculated for patients™ baseline covariates subsequent measurements ofintraoperativeparameters were first averaged within each patient andthen averaged among the patients in each treatmentgroup for descriptive analysis normal distribution wasassessed with qq plots and kolmogorowsmirnow testsnormally distributed variables were with unpaired twotailed ttests otherwise the in case of normally distributed values wilcoxon ranksum test was used for notnormally distributed continuous data paired comparisons between baseline data and postoperative data wereperformed with paired sample ttest or wilcoxonsignedrank test as applicable nominal data were analyzed with chisquare or fisher™s exact test for low expected cell counts data were presented as means ± sdmedians iqr or as numbers percentage as applicableadjustment for multiple testing was performed with thebonferroni method a p value was considered statistically significantanalysis was conducted with spss software version armonk ny ibm corp r for macintosh version321 r core team r a language and environmentfor statistical computing r foundation forstatistical computing vienna austria was used to calculate asdresultsa total of patients were included between november and october in the colloid group and in the crystalloid group fig at t0 all values weremeasured overall in the crystalloid group and inthe colloid group of the preplanned blood sampleswere collected and analyzedpatient™s baseline characteristics did not differ exceptfor height bmi with a slightly higher bmi in the colloidgroup type of surgery and crp also higher in the colloid group table duration of anesthesia and surgerywere comparable between both groups patients assignedto crystalloid administration received a median of ml “ crystalloids whereas patients assigned 0cobradovic bmc anesthesiology page of fig consort patient flow chartto the colloid group received ml “ ofcrystalloid solution and ml “ of colloidsblood loss transfusion requirements and urinary outputdid not differ between the groups anesthetic management map and hr did not differ between the groupsftc sv and co were significantly higher in the colloidgroup compared to the crystalloid group ftc ms“ versus ms “ p sv ml“ versus ml to p and co ± lminˆ’ versus ± lminˆ’ p thenumber of patients requiring vasopressor support wascomparable between groups the incidence of postoperative icu admissions did not differ in the crystalloid versus in the colloid group p table baseline values of il il il and tnf α in thecrystalloid group were comparable to values in the colloid group il pgmlˆ’ “ versus pgmlˆ’ “ p il pgmlˆ’ “ versus pgmlˆ’ “ p il pgmlˆ’ “ versus pgmlˆ’ “ p tnf α pgmlˆ’ “ versus pgmlˆ’ “ p immediatepostoperative values of il and were significantlyhigher compared to baseline values in both groupsp for all measurements while tnf α did notshow any significant increase in the crystalloid p and the colloid group p fig aucs of il il il and tnf α did not differ significantly between the groups table wbc values at baseline were glˆ’ “ in thecrystalloid versus glˆ’ “in the colloidgroup p crp pct and lbp baseline valueswere also comparable in both groupscrp mgdlˆ’ “ versus mgdlˆ’ “p pct ngmlˆ’ “ versus ngmlˆ’ “ p lbp mcglˆ’ “ versus mcglˆ’ “ p immediate postoperative values of wbc and pctwere significantly higher compared to the baseline valuesin both groups p for all measurements fig 0cobradovic bmc anesthesiology page of table baseline characteristicsage yrsweight kgheight cmbmi kgmˆ’ gender no menwomenasa score no iiiiiimedical history no pulmonary diseasecardiovascular diseasediabetes type idiabetes type iitype of surgery no colorectalliverpancreaticcrystalloidsn ± ± ± ± colloidsn ± ± ± ± asd preoperative laboratory valuescrp mgdlˆ’ ± ± patient characteristics data are presented as means ± sd or as counts for thecategorical outcomesabbreviations asd absolute standardized differences absolute difference inmeans or proportions divided by the pooled sd asd values of and represent small median and large differencesbmi body mass index m male f female asa american society ofanesthesiologists crp creactive protein sd standard deviationthe aucs for wbc crp pct and lbp for the timeperiods from t1 to t4 did not differ significantly between the groups fig table however lbpshowed significantly higher levels in the crystalloidgroup in the immediate postoperative period comparedto the colloid group mcglˆ’ “ versus mcglˆ’ “ p at all other postoperativetime points there were no significant differences betweenthe groups fig discussionthis trial is a substudy of a large multicenter randomized trial evaluating the effect of goaldirected crystalloidversus goaldirected colloid fluid administration on acomposite of serious complications after moderate tohighrisk open abdominal surgery the overall trial concluded that colloids did not decrease the composite ofmajorincomplicationsare ourresultsconcordance as they did not show any differences inperioperative pro and antiinflammatory markers between a crystalloid and a colloid fluid regimendespite multimodal care and enhanced recovery programs it still remains challenging to blunt the inflammatory response to surgery systemic inflammationafter abdominal surgery impairs outcome and thereforemany attempts have been made to alter the inflammatory response several factors influence the perioperative inflammatoryresponse such as the underlying disease type and invasiveness of surgery as well as type of anesthesia [“] themost important factor is the magnitude of surgical traumaand tissue damage which induce proliferation and activation of immune competent cells in turn triggering cytokine and inflammatory marker release so far veryfew trials have specifically investigated the influence offluid therapy and differences in terms of the type of fluidon the extent of inflammatory marker releaseto investigate the potential influence of goaldirected hydroxyethyl starch versus a lactated ringer™s solution fluid regimen on inflammatory response pro il il and tnf α and antiinflammatory cytokine il serum levels were measured during the perioperativeperiod additionally we measured wbc crp pct andlbpgenerally the most commonly measured biomarkersare crp and wbc if levels of crp are above mgdlˆ’ after postoperative day four a postoperative infection can be suspected crp levels in our studygroups increased on the first postoperative day droppingon the fourth postoperative day to nearly mgdlˆ’ inboth study groupswbcs are an imprecise marker to detect postoperativecomplications after major abdominal surgery amore sensitive parameter in predicting postoperativecomplications after major abdominal surgery is il surgical trauma and hypoperfusion of the colon aremain sources of il release in colorectal surgery noblett demonstrated that gdt during elective colorectal surgery significantly reduced il levels in comparison to a control group yates showed no differencesof il and il levels between goaldirected colloidand crystalloid fluid therapy during the first h in asubgroup of patients undergoing colorectal surgery although patients in the crystalloid group received significant more volume amount as compared to the colloid groupthere was no significant difference inhemodynamic variables our patients showed similar courses of il and il levels in the immediatepostoperative period in contrast to the trial of yateswho measured cytokine levels up to the first h aftersurgery we extended our measurement period to fourpostoperative days we showed comparable circulating 0cobradovic bmc anesthesiology page of table intraoperative dataduration of anesthesia minduration of surgery minfluid managementtotal fluid intake mlacrystalloid mlcolloid mlestimated blood loss mltransfusion yesno urinary output mlanesthesia managementfentanyl mcgtwa et sevoflurane core temperature °cicu admission yesno hemodynamictwa map mmhgtwa hr beatsminˆ’twa ftc mstwa sv mltwa co lminˆ’ phenylephrine yesno crystalloidsn ± ± “ “ “ “ “ [ ] “ ± ± ± “ “ ± colloidsn ± ± “ “ “ “ [ ] “ “ ± ± ± “ “ ± p value ° ° °° °°intraoperative data are presented as means ± sd medians iqr or as counts for the categorical outcomes means were compared with an unpaired twosided ttests ormannwhitneyu tests as appropriate medians with wilcoxon ranksum tests and counts with chisquare or fisher™s exact tests ° represents statisticalsignificance p abbreviations et end tidal icu intensive care unit twa time weighted average map mean arterial pressure hr heart rate ftc corrected flow time sv strokevolume co cardiac output sd standard deviationa total fluid intake includes baseline fluid boluses antibiotics analgesics and additional fluid administered at the discretion of attending anesthesiologistfig ad pro and antiinflammatory cytokines il il il and tnf α over time a il b il c il and d tnf α data arepresented as medians iqr abbreviations il “ interleukin tnf α “ tumor necrosis factor alpha pod “ postoperative day 0cobradovic bmc anesthesiology page of table areas under the curve of inflammatory markerspcrystalloidsn “ “ “colloidsn “ “ “ “ “ “ “ “auc il pgmlˆ’ 1dauc il pgmlˆ’ 1dauc il pgmlˆ’ 1dauc tnf α pgmlˆ’ 1d “auc wbc glˆ’1dauc crp mgdlˆ’ 1dauc pct ngmlˆ’ 1dauc lbp mcglˆ’ 1dtable areas under the curve of il il il and tnf α as well as wbccrp pct and lbp are presented as medians iqr medians were comparedwith wilcoxon ranksum testsabbreviations il interleukin tnf α tumor necrosis factor alpha wbc whiteblood cells pct procalcitonin lbp lipipopolysaccharidebinding protein “ “ “ “il and il levels between a gdt crystalloid and colloid administration these surrogates of inflammatoryresponse imply that gut perfusion during surgery waswell preserved with both types of fluid and suggest thatthe type of fluid might be of minor importance as longas the fluid is administered in a goaldirected fashionthe fact that tnf α levels in both groups remainedstable over the entire measured period further supportsour theory the course of tnf α levels during the perioperative period was in accordance with the study ofin which fluid therapy was guided withszakmanypicco versusin majorvenous pressurecentralabdominal surgery in patients at risk for postoperativecomplications as tnf α per se triggers glycocalyxdegradation we anticipate that tnf α did not influence glycocalyx shedding and thus possible fluid shifts inour study populationpct is an early predictive marker for systemic inflammation after abdominal surgery values above ngmlˆ’ are associated with postoperative complicationssuch as pneumonia or anastomotic leakage in ourstudy median pct levels did not exceed ngmlˆ’ atany measured time point these results are in concordance with our main study where infectious complications rate were held low and did not differ between thegroups moreover as pct production can also beinduced by tissue hypoperfusion we might assume thatgoaldirected fluid administration contributed to lowpct values by optimizing cardiac performance furthermore we measured lbp a prognostic markerfor bacterial infections patients in the crystalloidgroup showed significantly higher levels immediatelyafter surgery however the measured values remainedwithin the normal range therefore this difference ismost likely not to of clinical importancethe vascular endothelium is one of the earliest sitesinvolved in the inflammatory response syndrome an adequate perioperative fluid management has a major impact on the integrity of the glycocalyx with goaldirected fluid management individualized and time appropriate fluid resuscitation can be achieved enablingfig ad inflammatory markers wbc crp pct and lbp over time a wbc b crp c pct and d lbp data are presented as medians iqrabbreviations wbc “ white blood cells crp “ creactive protein pct “ procalcitonin lbp “ lipopolysaccharidebinding protein pod “postoperative day ˜… represents significant difference in lbp in the immediate postoperative period between the crystalloid and the colloidgroup p 0cobradovic bmc anesthesiology page of preservation of endothelial surface layer and sufficientan perfusion thus improving postoperative outcomesafter major surgery patients in the colloid group received significantly lessfluid ml confirming the previously publishedfluid sparing effect of colloids however clinical significance of this difference may be questionable during aperioperative period of nearly five hours further ourhemodynamic data showed significantly higher values ofsv and co with colloid administration though the absolute difference of ml in sv most likely has onlylimited clinical relevance it might very well be that thesignificant differences in sv and co are the result ofour number of patients included in this substudyassurrogatesfirst limitation of our study is that we measured inflammatory markers that reflect the inflammatory responseand not direct markers ofglycocalyx degradation like syndecan1 therefore wecannot draw any s about the preservation ofthe endothelial surface layer in our patients secondlywe did not control postoperative fluid management during the postoperative followup period a further limitation isthe time between patient enrolment andsubmission of our current results due to the fact thatthe main trial has been published recently a delay of oursubmission occurred nevertheless our results canstill be extrapolated to current clinical practicein summary goaldirected hydroxyethyl starch administration did not attenuate the inflammatory responseexpressed by cytokine levels of il il il and tnfα in patients undergoing moderate to highrisk open abdominal surgery wbc crp and ptc values did not differ between the different fluid regimes as wellabbreviationsasa american society of anesthesiologists asd absolute standardizeddifference auc area under the curve bmi body mass index co cardiacoutput crp creactive protein ecg electrocardiography ef ejectionfraction elisa enzymelinked immunosorbent assay etco2 endtidalcarbon dioxide ftc corrected flow time gdt goaldirected therapyhr heart rate ibw ideal body weight icu intensive care unitil interleukin iqr interquartile range lbp lipopolysaccharidebindingprotein map mean arterial pressure pacu postanesthetic care unitpct procalcitonin picco pulse contour cardiac output spss statisticalpackage for the social sciences sv stroke volume tnf α tumor necrosisfactor alpha twa time weighted average wbc white blood cell countacknowledgementsassistance with this we thank bianca tudor md department ofanesthesia intensive care medicine and pain medicine medical university ofvienna spitalgasse vienna austria for her support in laboratoryskills performing elisasauthors™ contributionsall authors have read and approved the manuscript mo patientrecruitment data acquistion and prepratation of the manuscript ak studyprotocol writing and preparation of the manuscript bk study protocolwriting and preparation of the manuscript statistical analyisis gr dataanalysis revision of the manuscript ok data analysis preparation of themanuscript oz patient recruitment data acquisition data managementab patient recruitment data acquisition revsion the manuscript cr dataacquistion revision of the manuscript as patient recruitment revision ofthe manuscript ef study protocol writing and preparation of the manuscriptfundingmedical university of viennapartially funded by fresenius kabi deltex medical provided oesophagealdoppler monitors and disposablesthe sponsors were not involved in protocol development data acquisitionor data analysisavailability of data and materialsthe datasets used andor analysed during the current study are availablefrom the corresponding author on reasonable requestbarbarakabonmeduniwienacatethics approval and consent to participatethe main trial was approved by the local ethics committee of the medicaluniversity of vienna in ek and was registered atclinicaltrialsgov nct00517127 and eudract “ a writteninformed consent was obtained from all patients prior to participationconsent for publicationnot applicablecompeting intereststhe authors declare no competing interestsauthor details1department of anaesthesia general intensive care medicine and painmedicine medical university of vienna spitalgasse vienna austria2department of outcomes research and general anesthesiologyanesthesiology institute euclid avenue cleveland clinic cleveland ohusa 3department of anesthesiology and general intensive care franziskushospital nikolsdorfergasse vienna austria 4department ofgynecology klinik ottakring montleartstrasse vienna austria5department of surgery medical university of vienna spitalgasse vienna austriareceived july accepted august referencesbrandstrup b tonnesen h beierholgersen r effects of intravenousfluid restriction on postoperative complications comparison of twoperioperative fluid regimens a randomized assessorblinded multicentertrial ann surg “ myles ps bellomo r corcoran t restrictive versus liberal fluidtherapy for major abdominal surgery new engl j med “chappell d jacob m hofmannkiefer k conzen p rehm m a rationalapproach to perioperative fluid management anesthesiology “lowell ja schifferdecker c driscoll df benotti pn bistrian brpostoperative fluid overload not a benign problem crit care med “sun y chai f pan c romeiser jl gan tj effect of perioperative goaldirected hemodynamic therapy on postoperative recovery following majorabdominal surgerya systematic review and metaanalysis of randomizedcontrolled trials critical care calvovecino jm ripollesmelchor j mythen mg effect of goaldirected haemodynamic therapy on postoperative complications in lowmoderate risk surgical patients a multicentre randomised controlled trialfedora trial br j anaesth “noblett se snowden cp shenton bk han af randomized clinical trialassessing the effect of doppleroptimized fluid management on outcomeafter elective colorectal resection br j surg “ 0cobradovic bmc anesthesiology page of alphonsus cs rodseth rn the endothelial glycocalyx a review of thevascular barrier anaesthesia “ pearse rm harrison da macdonald n effect of a perioperativecardiac outputguided hemodynamic therapy algorithm on outcomesfollowing major gastrointestinal surgery a randomized clinical trial andsystematic review jama “publisher™s notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationskimberger o arnberger m brandt s goaldirected colloidadministration improves the microcirculation of healthy andperianastomotic colon anesthesiology “kolarova h ambruzova b svihalkova sindlerova l klinke a kubala lmodulation of endothelial glycocalyx structure under inflammatoryconditions mediat inflamm rettig tc verwijmeren l dijkstra im boerma d van de garde emnoordzij pg postoperative interleukin6 level and early detection ofcomplications after elective major abdominal surgery ann surg “ gan tj soppitt a maroof m goaldirected intraoperative fluidadministration reduces length of hospital stay after major surgeryanesthesiology “jacob m chappell d rehm m clinical update perioperative fluidmanagement lancet “feldheiser a pavlova v bonomo t balanced crystalloid comparedwith balanced colloid solution using a goaldirected haemodynamicalgorithm br j anaesth “ orbegozo cortes d gamarano barros t njimi h vincent jl crystalloidsversus colloids exploring differences in fluid requirements by systematicreview and metaregression anesth analg “kabon b sessler di kurz a effect of intraoperative goaldirectedbalanced crystalloid versus colloid administration on major postoperativemorbidity a randomized trial anesthesiology “ robinson jd lupkiewicz sm palenik l lopez lm ariet m determination ofideal body weight for drug dosage calculations am j hosp pharm “steppan j hofer s funke b sepsis and major abdominal surgery leadto flaking of the endothelial glycocalix j surg res “ wilmore dw from cuthbertson to fasttrack surgery years of progress inreducing stress in surgical patients ann surg “ desborough jp the stress response to trauma and surgery br j anaesth“ veenhof aa sietses c von blomberg bm the surgical stress responseand postoperative immune function after laparoscopic or conventional totalmesorectal excision in rectal cancer a randomized trial int j color dis “ gilliland he armstrong ma carabine u mcmurray tj the choice ofanesthetic maintenance technique influences the antiinflammatory cytokineresponse to abdominal surgery anesth analg “ wichmann mw huttl tp winter h immunological effects oflaparoscopic vs open colorectal surgery a prospective clinical study archsurg “ watt dg han pg mcmillan dc routine clinical markers of themagnitude of the systemic inflammatory respon Answer:

Colon_Cancer

" inflammatory pseudotumour has been used to describe an inflammatory or fibrosing tumoral processof an undetermined cause that may involve a variety of an systems including the lungs spleen liver lymphnodes pancreas and extrahepatic bile duct with potential for recurrence and persistent local growth in this we report a patient with a big mass of uncertain nature and behaviorcase presentation a 60yearold woman presented with a 1week history of abdominal pain fever and jaundicesix months before she had had right upper quadrant pain that was interpreted as biliary colic a contrastenhancedct scan showed a big mass of soft tissue with diffuse infiltration of the gallbladder displacement of the transversecolon hepatic flexure and duodenum for diagnostic distinction between a chronic inflammatory disease or aneoplasm exploratory laparotomy was required intraoperative exploration disclosed a big mass of hard textureinvolving the gallbladder with multiple concrements hepatoduodenal ligament right and transverse mesocolonstomach and duodenumcholecystectomy was performed preserving adjacent ans with macroscopic desmoplastic reactionhistopathologic examination of the gallbladder showed a spindle cell proliferation with diffuse chronicinflammatory infiltrate of lymphocytes plasma cells and hyalinized fibrous stroma no vascular invasion or cellularatypia were evident inflammatory pseudotumour is a rare condition and diagnostic distinction from a chronicinflammatory disease or other neoplasm is only possible by histopathologic examination there is a limited numberof case reports in the literature indicating tumor location in the gallbladderkeywords inflammatory pseudotumor gallbladder inflammatory pseudotumour is a rare lesion that hasbeen described in various ans and tissues intraabdominal variants of the disease are reported to occurmost frequently in the liver spleen mesentery and extrahepatic bile duct the location of the gallbladder iseven more uncommon correspondence acd3202yahooesdepartment of surgery and pathology puerto real university hospital c¡dizspainmalignant transformations and recurrences of inflammatory pseudotumour have been reported years aftersurgery therefore longterm followup is necessary evenfor patients successfully treated by surgical resectionelevated igg4 serum levels have been reported in association with this illness as well as abundant igg4 positivity in tumor infiltrating plasma cells signs suggestiveof an igg4related disease a high serum igg4 concentrations thus provides a useful means of distinguishingthis disorder from other differential diagnoses the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0ccalvo bmc gastroenterology page of pharmacologic treatments have also been reported forigg4associated inflammatory pseudotumor and thereare even cases of complete resolution of the disease withsteroids treatments however in the presented case thiscondition did not occur so treatment was exclusivelysurgicalcase presentationa 60yearold woman presented to the emergencyroom with abdominal pain fever pruritus and jaundicesince week the patient had a history of smoking anda family history of pancreatic canceron physical examination a hard and painful mass wasidentified on the right hypochondrium blood laboratoryexamination showed extrahepatic cholestasis enzymestotal bilirubin mgdl direct bilirubin mgdlast ul alt ul ggt ul ldh ul alkaline phosphatase ul in anamnesis thepatient referred to abdominal pain occurring during thelast months located in the right upper quadrant whichhad been interpreted as biliary colic by her generalpractitionertumour markers and blood count showed no alterations viral serology autoimmunity antibodies metanephrines and urine normetanephrine were within thenormal rangea large mass associated with the gallbladder was identified by abdominal ultrasound contrastenhanced ctscan disclosed a large soft tissue mass originating fromthe gallblader with homogenous contrast enhancementand without clear infiltration of the hepatic parenchymathe mass displaced the transverse colon hepatic flexureand duodenum no lymphadenopathies were identifiedin the hepatoduodenal ligament pancreas retroduodenum or celiac axis fig 1a b the gallbladder was distended contained stones and had a regular lumenwhile there was slight dilatation of the intrahepatic bileduct on magnetic resonance imaging fig 1csmallerwith these findings a diagnostic distinction between a chronic inflammatory disease or a neoplasticprocess was necessary the biopsy of the mass wasperformed under ultrasonographic control histopathologic examination showed spindle cells and some inflammatory cells ofsize and absence ofxanthic cells the tumor showed a mesenchymal aspect that was confirmed by absence of epithelial cellson pancytokeratin staining while some histiocyteswere recognized in summary the pathology diagnosiswas a mesenchymal process that could be reactive ormalignant the microbiological study of the bile obtained from gallbladder punctured showed a nonpurulent gram™s stain and negative cultures for bothaerobic and anaerobic germs the oral endoscopy andbiopsies of the second part of the duodenum didn™tshow any pathological conditionexploratory laparotomy was decided and cholecystectomy could be performed preserving the adjacent ans with macroscopic desmoplastic reaction the masswas peeled off the transverse colon first and the secondpart of the duodenum and common bile duct fig the histopathological examination offibrousspecimen disclosed sclerosingthe resectiontissue withfig axial and coronal computed tomography images showing a large mass of diffuse soft tissues originating from the gallbladder anddisplacing the duodenum transverse colon and hepatic flexure a b in magnetic resonance imaging the gallbladder was distended andcontained stones associated with a slight dilatation of the intrahepatic bile duct c 0ccalvo bmc gastroenterology page of stains were performedto achieve a definitive classification complementaryimmunohistochemicalandshowed positive staining for smooth muscle actin in themuscular layer ofthe gallbladder and vessel wallscd34 in the vascular lumen cd68 in histiocytes andremained negative for anaplastic lymphoma kinasealk and pancytokeratin panck masson™s trichrome stain showed intense positivity on collagen fibers less than of the tumor cells sample were ki67positive and plasma cells were igg4 positive per highpower field taken together these findings confirmed thediagnosis of inflammatory pseudotumor of the gallbladder with sclerosing cholangitis associated with a normallevel of serum of immunoglobulin g4 of mgdl “ mgdlno local recurrence was detected at the threeyearsfollowup on ct scandiscussion and sthe term inflammatory pseudotumour has been used todescribe an inflammatory or fibrosing tumoural processof undetermined cause that may involve a variety ofan systems including the lungs spleen liver lymphnodes pancreas and extrahepatic bile duct with potentialfor recurrence and persistent local growth [ ] thereis a limited number of case reports in the literature indicating gallbladder location [ ]fig on laparotomy a large and hard mass was identified in thegallbladder with stones displacing but not infiltrating right andtransverse mesocolon stomach duodenum andhepatoduodenal ligamenthistiocytes chronic lymphocytic inflammatory infiltrateand plasma cells with isolated eosinophils and no epithelial malignancy the mass presented as an expansivegrowth from the outer portion of the muscular layer ofthe gallbladder to the surrounding fatty tissuethe definitive pathological diagnosis was inflammatorypseudotumour of the gallbladder with chronic sclerosingcholangitis fig a bfig histopathologic examination disclosed a thickened gallbladder wall a with spindle cells and proliferation of connective fibrous tissuewithout signs of celular atypia b and inflammatory cells including lymphocites plasma cells and hyalinized fibrous tissue without vascularinvasion c few plasma cells were igg4 positive in relation to the whole inflammatory cell infiltrate d 0ccalvo bmc gastroenterology page of consent for publicationwe confirm in this statement that a written consent to publish thisinformation was obtained from study participant and the proof of consentto publish from study participants can be provided at any timethe authors have in their possession the informed consent of the patientbiomed central consent formcompeting intereststhe authors declare that they have no competing interestsreceived january accepted august referencesbehranwala ka straker p wan a fisher c thompson jn inflammatorymyofibroblastic tumour of the gallbladder world j surg oncol sinha l hasan a sngh ak bhadani pp jha an singh pk kumar minflammatory myofibroblastic tumor involving liver gallbladder pylorus andduodenum a rare case presentation int j surg case rep “badea r veres aa andreica v inflammatory myofibroblastic tumor ofthe gallbladder imaging aspects j med ultrason “abrantes cf silva mr oliveira rc eloy c cipriano ma castro lpinflammatory myofibroblastic tumour arising incidentally as a polypoidlesion in the gallbladder j bras patol med lab v51 n p “koea jb broadhurst gw rodgers ms inflammatory pseudotumor ofthe liver demographics diagnosis and the case for nonoperativemanegement j am coll surg “sato y kojima m takata k immunoglobulin g4relatedlymphadenopathy with inflammatory pseudotumorlike features med molmorphol “hamano h kawa s horiuchi a high serum igg4 concentrations inpatients with sclerosing pancreatitis n engl j med “aldhahab h mcnabbbaltar j albusafi s barkun an immunoglobulin g4related pancreatic and biliary disease can j gastroenterol “lee ys lee sh lee mg immunoglobulin g4related diseasemimicking unresectable gallbladder c¡ncer gut liver “ muduly d deo sv shukla nk inflammatory myofibroblastic tumor ofgall bladder trop gastroenterol “publisher™s notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsinflammatory pseudotumour appear to be more common in noneuropean populations they usually occur ininfancy and young adults but can occur in the elderly []the igg4 serum level should be determined due to acommon association of elevated serum igg4 with this illness and some authors describe abundant igg4 positivityin plasma cells as suggestive of igg4related disease []high serum igg4 concentrations might provide afromuseful means of distinguishing this disorderother lesions [“]the histopathological examinations showed sclerosingfibrous tissue with histiocytes chronic lymphocytic inflammatory infiltrate and plasma cells with isolated eosinophils compatible with igg4related disease howeveras igg4 serum levels were within normal range and theigg4 tissue expression was very weak there is no strongevidence of a clear association with igg4related diseaseinflammatory pseudotumour can generally be considered to be a relatively rare disease of undefined originwith a great variety of symptoms causing diagnosticchallenges in the distinction of chronic inflammatorydisease and neoplasminflammatory pseudotumoris defined as nonneoplastic but is currently considered as a tumour withlowgrade malignant transformations it has been reported in the liver urinary bladder kidney breast stomach pancreas spleen and retroperitoneum there is alsoa limited number of case reports in the literature indicating the gallbladder location these patients must beobserved with close and regular longterm followup asrecurrences have been reported to occur four to yearsafter surgery []acknowledgementsangela hens head of the pathology department of puerto real universityhospital for providing her pathological knowledgemario bruno for providing the new corrections in the translation of thismanuscriptpd dr med ulrich f wellner consultant surgeon pancreatic surgery andresearch clinic of surgery uksh campus l¼beck germany for providingthe latest english language correctionsauthors™ contributionsac the first author directed the operation and wrote the paper js hasparticiped in the design of the report and copy edited the manuscript adhas participated in the operation mc has participated in the operation gmhas made the pathological diagnosis and part of the literature review allauthors read and approved the final version of the manuscript all authors ofthis manuscript are in agreement with its content and are not beingpublished or under consideration in another scientific journalfundingnot applicableavailability of data and materialsdata sharing is not applicable to this as no data sets were generatedor analysed during the current studyethics approval and consent to participatenot applicable 0c"

cancer297

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: " inflammatory pseudotumour has been used to describe an inflammatory or fibrosing tumoral processof an undetermined cause that may involve a variety of an systems including the lungs spleen liver lymphnodes pancreas and extrahepatic bile duct with potential for recurrence and persistent local growth in this we report a patient with a big mass of uncertain nature and behaviorcase presentation a 60yearold woman presented with a 1week history of abdominal pain fever and jaundicesix months before she had had right upper quadrant pain that was interpreted as biliary colic a contrastenhancedct scan showed a big mass of soft tissue with diffuse infiltration of the gallbladder displacement of the transversecolon hepatic flexure and duodenum for diagnostic distinction between a chronic inflammatory disease or aneoplasm exploratory laparotomy was required intraoperative exploration disclosed a big mass of hard textureinvolving the gallbladder with multiple concrements hepatoduodenal ligament right and transverse mesocolonstomach and duodenumcholecystectomy was performed preserving adjacent ans with macroscopic desmoplastic reactionhistopathologic examination of the gallbladder showed a spindle cell proliferation with diffuse chronicinflammatory infiltrate of lymphocytes plasma cells and hyalinized fibrous stroma no vascular invasion or cellularatypia were evident inflammatory pseudotumour is a rare condition and diagnostic distinction from a chronicinflammatory disease or other neoplasm is only possible by histopathologic examination there is a limited numberof case reports in the literature indicating tumor location in the gallbladderkeywords inflammatory pseudotumor gallbladder inflammatory pseudotumour is a rare lesion that hasbeen described in various ans and tissues intraabdominal variants of the disease are reported to occurmost frequently in the liver spleen mesentery and extrahepatic bile duct the location of the gallbladder iseven more uncommon correspondence acd3202yahooesdepartment of surgery and pathology puerto real university hospital c¡dizspainmalignant transformations and recurrences of inflammatory pseudotumour have been reported years aftersurgery therefore longterm followup is necessary evenfor patients successfully treated by surgical resectionelevated igg4 serum levels have been reported in association with this illness as well as abundant igg4 positivity in tumor infiltrating plasma cells signs suggestiveof an igg4related disease a high serum igg4 concentrations thus provides a useful means of distinguishingthis disorder from other differential diagnoses the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0ccalvo bmc gastroenterology page of pharmacologic treatments have also been reported forigg4associated inflammatory pseudotumor and thereare even cases of complete resolution of the disease withsteroids treatments however in the presented case thiscondition did not occur so treatment was exclusivelysurgicalcase presentationa 60yearold woman presented to the emergencyroom with abdominal pain fever pruritus and jaundicesince week the patient had a history of smoking anda family history of pancreatic canceron physical examination a hard and painful mass wasidentified on the right hypochondrium blood laboratoryexamination showed extrahepatic cholestasis enzymestotal bilirubin mgdl direct bilirubin mgdlast ul alt ul ggt ul ldh ul alkaline phosphatase ul in anamnesis thepatient referred to abdominal pain occurring during thelast months located in the right upper quadrant whichhad been interpreted as biliary colic by her generalpractitionertumour markers and blood count showed no alterations viral serology autoimmunity antibodies metanephrines and urine normetanephrine were within thenormal rangea large mass associated with the gallbladder was identified by abdominal ultrasound contrastenhanced ctscan disclosed a large soft tissue mass originating fromthe gallblader with homogenous contrast enhancementand without clear infiltration of the hepatic parenchymathe mass displaced the transverse colon hepatic flexureand duodenum no lymphadenopathies were identifiedin the hepatoduodenal ligament pancreas retroduodenum or celiac axis fig 1a b the gallbladder was distended contained stones and had a regular lumenwhile there was slight dilatation of the intrahepatic bileduct on magnetic resonance imaging fig 1csmallerwith these findings a diagnostic distinction between a chronic inflammatory disease or a neoplasticprocess was necessary the biopsy of the mass wasperformed under ultrasonographic control histopathologic examination showed spindle cells and some inflammatory cells ofsize and absence ofxanthic cells the tumor showed a mesenchymal aspect that was confirmed by absence of epithelial cellson pancytokeratin staining while some histiocyteswere recognized in summary the pathology diagnosiswas a mesenchymal process that could be reactive ormalignant the microbiological study of the bile obtained from gallbladder punctured showed a nonpurulent gram™s stain and negative cultures for bothaerobic and anaerobic germs the oral endoscopy andbiopsies of the second part of the duodenum didn™tshow any pathological conditionexploratory laparotomy was decided and cholecystectomy could be performed preserving the adjacent ans with macroscopic desmoplastic reaction the masswas peeled off the transverse colon first and the secondpart of the duodenum and common bile duct fig the histopathological examination offibrousspecimen disclosed sclerosingthe resectiontissue withfig axial and coronal computed tomography images showing a large mass of diffuse soft tissues originating from the gallbladder anddisplacing the duodenum transverse colon and hepatic flexure a b in magnetic resonance imaging the gallbladder was distended andcontained stones associated with a slight dilatation of the intrahepatic bile duct c 0ccalvo bmc gastroenterology page of stains were performedto achieve a definitive classification complementaryimmunohistochemicalandshowed positive staining for smooth muscle actin in themuscular layer ofthe gallbladder and vessel wallscd34 in the vascular lumen cd68 in histiocytes andremained negative for anaplastic lymphoma kinasealk and pancytokeratin panck masson™s trichrome stain showed intense positivity on collagen fibers less than of the tumor cells sample were ki67positive and plasma cells were igg4 positive per highpower field taken together these findings confirmed thediagnosis of inflammatory pseudotumor of the gallbladder with sclerosing cholangitis associated with a normallevel of serum of immunoglobulin g4 of mgdl “ mgdlno local recurrence was detected at the threeyearsfollowup on ct scandiscussion and sthe term inflammatory pseudotumour has been used todescribe an inflammatory or fibrosing tumoural processof undetermined cause that may involve a variety ofan systems including the lungs spleen liver lymphnodes pancreas and extrahepatic bile duct with potentialfor recurrence and persistent local growth [ ] thereis a limited number of case reports in the literature indicating gallbladder location [ ]fig on laparotomy a large and hard mass was identified in thegallbladder with stones displacing but not infiltrating right andtransverse mesocolon stomach duodenum andhepatoduodenal ligamenthistiocytes chronic lymphocytic inflammatory infiltrateand plasma cells with isolated eosinophils and no epithelial malignancy the mass presented as an expansivegrowth from the outer portion of the muscular layer ofthe gallbladder to the surrounding fatty tissuethe definitive pathological diagnosis was inflammatorypseudotumour of the gallbladder with chronic sclerosingcholangitis fig a bfig histopathologic examination disclosed a thickened gallbladder wall a with spindle cells and proliferation of connective fibrous tissuewithout signs of celular atypia b and inflammatory cells including lymphocites plasma cells and hyalinized fibrous tissue without vascularinvasion c few plasma cells were igg4 positive in relation to the whole inflammatory cell infiltrate d 0ccalvo bmc gastroenterology page of consent for publicationwe confirm in this statement that a written consent to publish thisinformation was obtained from study participant and the proof of consentto publish from study participants can be provided at any timethe authors have in their possession the informed consent of the patientbiomed central consent formcompeting intereststhe authors declare that they have no competing interestsreceived january accepted august referencesbehranwala ka straker p wan a fisher c thompson jn inflammatorymyofibroblastic tumour of the gallbladder world j surg oncol sinha l hasan a sngh ak bhadani pp jha an singh pk kumar minflammatory myofibroblastic tumor involving liver gallbladder pylorus andduodenum a rare case presentation int j surg case rep “badea r veres aa andreica v inflammatory myofibroblastic tumor ofthe gallbladder imaging aspects j med ultrason “abrantes cf silva mr oliveira rc eloy c cipriano ma castro lpinflammatory myofibroblastic tumour arising incidentally as a polypoidlesion in the gallbladder j bras patol med lab v51 n p “koea jb broadhurst gw rodgers ms inflammatory pseudotumor ofthe liver demographics diagnosis and the case for nonoperativemanegement j am coll surg “sato y kojima m takata k immunoglobulin g4relatedlymphadenopathy with inflammatory pseudotumorlike features med molmorphol “hamano h kawa s horiuchi a high serum igg4 concentrations inpatients with sclerosing pancreatitis n engl j med “aldhahab h mcnabbbaltar j albusafi s barkun an immunoglobulin g4related pancreatic and biliary disease can j gastroenterol “lee ys lee sh lee mg immunoglobulin g4related diseasemimicking unresectable gallbladder c¡ncer gut liver “ muduly d deo sv shukla nk inflammatory myofibroblastic tumor ofgall bladder trop gastroenterol “publisher™s notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsinflammatory pseudotumour appear to be more common in noneuropean populations they usually occur ininfancy and young adults but can occur in the elderly []the igg4 serum level should be determined due to acommon association of elevated serum igg4 with this illness and some authors describe abundant igg4 positivityin plasma cells as suggestive of igg4related disease []high serum igg4 concentrations might provide afromuseful means of distinguishing this disorderother lesions [“]the histopathological examinations showed sclerosingfibrous tissue with histiocytes chronic lymphocytic inflammatory infiltrate and plasma cells with isolated eosinophils compatible with igg4related disease howeveras igg4 serum levels were within normal range and theigg4 tissue expression was very weak there is no strongevidence of a clear association with igg4related diseaseinflammatory pseudotumour can generally be considered to be a relatively rare disease of undefined originwith a great variety of symptoms causing diagnosticchallenges in the distinction of chronic inflammatorydisease and neoplasminflammatory pseudotumoris defined as nonneoplastic but is currently considered as a tumour withlowgrade malignant transformations it has been reported in the liver urinary bladder kidney breast stomach pancreas spleen and retroperitoneum there is alsoa limited number of case reports in the literature indicating the gallbladder location these patients must beobserved with close and regular longterm followup asrecurrences have been reported to occur four to yearsafter surgery []acknowledgementsangela hens head of the pathology department of puerto real universityhospital for providing her pathological knowledgemario bruno for providing the new corrections in the translation of thismanuscriptpd dr med ulrich f wellner consultant surgeon pancreatic surgery andresearch clinic of surgery uksh campus l¼beck germany for providingthe latest english language correctionsauthors™ contributionsac the first author directed the operation and wrote the paper js hasparticiped in the design of the report and copy edited the manuscript adhas participated in the operation mc has participated in the operation gmhas made the pathological diagnosis and part of the literature review allauthors read and approved the final version of the manuscript all authors ofthis manuscript are in agreement with its content and are not beingpublished or under consideration in another scientific journalfundingnot applicableavailability of data and materialsdata sharing is not applicable to this as no data sets were generatedor analysed during the current studyethics approval and consent to participatenot applicable 0c" Answer:

Colon_Cancer

" african americans aa are at high risk for colorectal cancer crc studies report a “ increasein crc risk with physical inactivity obesity and metabolic syndrome activation of the wntcatenin ctnnb1signaling pathway plays a critical role in colorectal carcinogenesis accumulating evidence also indicates a role ofwntctnnb1 signaling in obesity and metabolic diseasesaim to examine the association between obesity catenin expression and colonic lesions in african americansmethods we reviewed the pathology records of colorectal specimens from to crcs advanced adenomas and normal colon tissues tissue microarrays tma were constructed from these samplesimmunohistochemistry ihc for ctnnb1 catenin clone catenin1 was performed on the constructed tmasthe ihc results were evaluated by pathologists and the nuclear intensity staining was scored from to bmisex age location of the lesion and other demographic data were obtainedresults positive nuclear staining in normal advanced adenoma and crc was and respectively p crc was asso ciated with positive status for nuclear ctnnb1 intensity adjusted or 95ci “p for positive nuclear staining compared to noncrc samples normal or advanced adenoma nuclearstaining percentage has a fair diagnostic ability for crc with an auc of 95ci “overweightobese patients bmi did not show a significant difference in p nuclear ctnnb1 staining positive in normal weight vs positive in overweightobese the association between nuclear intensityand crc was not different between normal and overweight patients p for interaction the positive nuclearctnnb1status in crc stage iii and iv of all crc was not different from stage i and ii vs respectively p continued on next page correspondence b_shokranihowardedu hashktorabhowardedu1department of medicine department of pathology and cancer centerhoward university college of medicine geia avenue nwwashington dc usafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cshokrani bmc gastroenterology page of continued from previous page in our study advanced adenoma and crc were associated with activation of catenin in physically fitoverweight and obese patients thus obesity and wntcatenin pathway seem to be independent in africanamerican patients wntcatenin signaling pathway has a potential to be used as an effector in coloncarcinogenic transformation whether or not bmi is a modifier of this pathway needs to be investigated furtherkeywords catenin colorectal cancer advanced adenoma african americans colorectal cancer crc is one of the most commoncancers in the industrialized world lifestyle and epidemiological factors associated with an increased risk ofcrc include physical inactivity obesity and metabolicsyndrome in the united states approximately twothirds of the adult population are overweight or obesewhich represents a putative risk factor for multiple target an malignancies including crc there is evidence to suggest that excess adiposity is associated with up to greater risk of crc comparedwith normal weight individuals and that physical activity may decrease colorectal cancer risk althoughexcessive accumulation of white adipose tissue wat isthe key feature of adiposity obesity is clinically definedby a bmi over kgm2 which does not take fat contentinto account it is also known that most crcs arise froma genetic and morphological adenoma to carcinomatransition also it is widely accepted that both crcsand colorectal adenomas cras share similar etiologicalcauses which explains why cras which are amongstthe mostfindings in all crcscreening participants are present in more than ofgeneral asymptomatic populations consequentlyrisk algorithms have been applied to use bmi as a predictor variable to stratify individuals according to theirrisk of colorectal neoplasia however the underlyingmechanisms that might explain the association and themagnitude ofthe connection between excess bodyweight and crc remain unclearfrequent pathologicalin the obesitycancer relationship multiple biologicalprocesses including insulininsulinlike growth factorigf1 insulin resistance sexual hormones estrogensand proinflammatory cytokines tnfα il6 and crpactively participate all these elements create a favorable environment for carcinogenesis and a decrease inapoptosisas a separate molecular pathway activation of thewnt signaling pathway plays a critical role in colorectalcarcinogenesis wnt ligands are a family of proteinsfor normal cell development that are importantcatenin ctnnb1 is a major mediator of the wntpathway that is traditionally classified into canonical catenindependent and noncanonical cateninindependent wnt canonical pathway utilizes a group ofcell surface receptors called frizzled frz to activateseveral pathways the most important one involving catenin and apc in the absence of wnt signalingapc causes degradation of catenin preventing its accumulation in the cytoplasm by forming a complex withcatenin which leads to the phosphorylation and eventually destruction of catenin by the proteasome signaling by wnt blocks this process allowing cateninto migrate from the cytoplasm to the nucleus once inthe nucleus catenin upregulates cmyc cyclin d1and other genes which increase cellular proliferation therefore continuous wnt signaling can be seenin cells with loss of apc metabolic syndromeassociated conditionssuch asobesity and type ii diabetes are influenced by geneticand functional variations in the wnt signaling pathway wnt signaling when activated represses the terminal differentiation during adipogenesis whereby preadipocytes take on the characteristics of mature adipocytes a cascade of transcriptional events like the induction of catenin ensues which in turn inducesenhancer binding proteinα cebpa and peroxisomeproliferatoractivated receptorÎ pparg the excessive accumulation of wat features adiposity butobesity does not take fat content into account recently genetic factors linked to fat mass and adipositywere reported to be associated with increased obesityrisk in young obese individuals wholeexome sequencing revealed rare gainoffunction mutations inctnnb1catenin the cateninregulated transcription of an adipocytederived chemokine calledserum amyloid a3 saa3 leads to the formation of a catenin“tcf complex in mature adipocytes that promotes the proliferation of preadipocytes in wat andthereby increases obesity and the risk for metabolic syndrome other data also suggest that obesity and lack ofphysical activity are associated with a higher risk forcolorectal cancer [ ] these findings have importantimplications especially in the obese and physically inactive african american population that may haveunderlying predisposing mutations to colorectal cancer the aim of this study is to assess the catenin expression profile in colorectal premalignant and malignant lesions in correlation with obesity as determined by 0cshokrani bmc gastroenterology page of body mass index bmi or waist circumference wc inafrican american populationmethodspatients and clinical datacolorectaltissue samples submitted to the surgicalpathology laboratory at howard university hospitalfrom january to december were retrieved from the pathology archive system powerpath„¢a total of samples were included in the study consisting of tissue samples from crc n advancedadenoma n and normal colon n patients™data included age sex height weight and waist circumference body mass index bmi was calculated in thestudy table the protocol of this study was approvedby the howard university institutional review boardirbtissue microarray tma constructionhematoxylineosin stained slides he slides weremade from paraffinembedded blocks the he slideswere reviewed by two pathologists to confirm the pathological diagnosis and to mark areas of interest multipleareas from more than one block were marked to ensurea good representability of the sample on the tma fivetma paraffin blocks each containing cores of mmin diameter each and mm distance from each otherwere constructed tissuespecific and an system controls were included in each tmaimmunohistochemical ihc analysis of ctnnb1 cateninthe constructed tmas were stained for catenin theimmunostaining was carried out as follows dako monoclonal mouse antihuman betacatenin cateninc1intended for laboratory application to identify qualitatively by light microscopy catenin positive cells in normal and neoplastic tissues was used at a dilution of using the envision dab code k4006 detectionsystem the deparaffinized tmas were treated prior tothe ihc staining procedure target retrievalinvolvedimmersion of tissue sections in a preheated buffer solution and maintaining heat in a water bath “ °cfor greater adherence of tissue sections to glass slidessilanized slides dako code s3003 were used targetretrieval solution code s1700 or 10x concentratecode s1699 using a 20min heating protocol was usedthe cellular staining pattern of anticatenin is mainlymembranous especially at the cellcell boundaries positive nuclear staining and diffuse cytoplasmic staining arealso reported in cancer cells fig evaluation and assessment of the catenin expressiontwo pathologists interpreted the ihc slides cateninexpression status was assessed based on the pattern ofstaining nuclear cytoplasmic and membranous intensity to and percentage of staining to the staining would be considered negative if there wasweak or no nuclear expression or positive if there wasmoderate or strong nuclear expressionstatistical analysisdistribution of continuous and categorical variables weretested by kruskalwallis and chisquare test betweendifferent groups respectively we used logistic regression analysis to test association between the staining andrisk of crc after adjusting for age and gender areaunder the curve auc was calculated for variables withsignificant association with crc using receiver operative characteristics curve all statistical analyses wereperformed by stata statacorp college station txresultsepidemiological characteristics and bmi in normaladvanced adenoma and crcthe bmi was calculated for individual patients and normal subjects as represented in table crc patientswere older p while our healthy normal population was mostly overweight higher bmi was moreclosely associated with advanced adenoma and crchowever the differences were not significant table advanced adenomas and crcs were associated withpositive nuclear ctnnb1we assessed whether alterations in wntctnnb1 catenin signaling plays any role colon carcinogenesispositive catenin nuclear stains were seen in normaladvanced adenomas and crcs were and respectively p table based on the designationtable epidemiological characteristics and bmi in normal advanced adenoma and crc patientsage median iqrmale n bmi kgm2 median iqroverweight n normaln “ “ advanced adenoman “ “ crcn “ “ p value 0cshokrani bmc gastroenterology page of fig immunostain for catenin in three individuals normal a × advanced adenoma b × and cancer c d × and × respectively showing membranous staining in the normal cytoplasmic and membranous staining in adenoma with no evidence of nuclearexpression arrow showing lack of nuclear staining and nuclear and cytoplasmic staining in cancer arrow showing nuclear stainingof œn intensity  which is associated with higher risk ofcancer crcs were associated with positive status fornuclear ctnnb1 intensity age gender adjusted or 95ci “ p for positive nuclearstaining compared to noncrc samples normal or advanced adenoma fig nuclear staining percentagehas also a fair diagnostic ability for crc with an auc of 95ci “ table fig overweight and obese patients show a trend withpositive nuclear ctnnb1 expressionpositive nuclear ctnnb1 staining was in normalweight and in overweightobese bmi patientsthis difference pointed to trend that was not statisticallysignificantassociation between nuclear intensity and crc in normaland overweight patientsthe association between nuclear intensity and crcwas not statistically significant different between normal weight and overweight patientsinteraction tables and the positive nuclearctnnb1 status in crc stage iii and iv of allcrc was not different from stage i and ii vs respectively p p fortable catenin nuclear and cytoplasmic expression tabulated as intensity and percentage in normal advanced adenoma andcrcnormaln “ “cnc intensity n intensity advanced adenoma n “ “ crcn “ “ c cytoplasmic n nuclearc intensity and n intensity mean intensity and aboveoverall p valuep value for advanced adenoma vs normalp value for crc vs other 0cshokrani bmc gastroenterology page of fig catenin nuclear and cytoplasmic expression in normal advanced adenoma and crcdiscussionone of the important risk factors in colorectal cancer isobesity [ ] catenin is an ecadherin binding proteinthat mediates cellcell adhesion and plays a role inthe canonical wnt signaling pathway that controls thecoordinated expansion and differentiation of the intestinal crypt stem cells degradation of catenin byphosphorylation followed by alteration of destructioncomplex apc gsk3 and axin results in inactivation if wnt pathway in our study we found thatwas associated with an increased adjusted or of 95ci “ p for positive nuclear staining compared to noncrc samples normal or advanced adenomathe gatekeeper gene apc is a negative regulator of catenin and is mutated in approximately of sporadic and hereditary colon cancers there are severalmutations that can cause an accumulation of cateninin tumor cells such as mutations of the apc gene pointmutations in gsk3 or mutations in catenin gene itself [“]our positive nuclear staining in crc and its association with the positive status for nuclear ctnnb1intensity compared to noncrc samples are in contrastto a study by brabletz which showed that catenin is localized in the cytoplasm and membrane ofthe tumor cells whereas in our study it was mainly concentrated in the cytoplasm and the nucleus they alsomentioned that there was positive nuclear staining at theinvasive front as catenin is involved in tumor progression such is not the case in our studyindeed evenwhen considering nuclear staining in our specimensthere was no statistically significant differences betweenstage iiiiv cases™ staining versus stages iii crc caseslevels of staining the fact that catenin is expressedearly in the african american specimens analyzed heremight partially explain the aggressive nature of crc inthis population in addition we showed that there is uniform membranous staining in normal and increasingcytoplasmic and nuclear staining in advanced adenomasand crcs this confirms that the decrease in membranous staining begins with dysplastic changes leading to atable association of bmi with catenin nuclear intensity in advanced adenoma casesadvanced adenoma with catenin expression n in intensity and no nuclear intensity n bmi median interquartileoverweight n nuclear intensity negativen “ nuclear intensity n “ p value 0cshokrani bmc gastroenterology page of table association of bmi with catenin nuclear intensity in crc casescrc with catenin expression with high nuclear intensity and without negativebmi median interquartileoverweight n nuclear intensity negativen “ nuclear intensity n “ p valueprogressive disappearance atcrcsthe membrane levelinas we mentioned above a major risk factor for crc isobesity which continues to expand as a pandemicworldwide the american cancer society cancerprevention study ii states that there is an increased incidence of crc esophageal adenocarcinoma and othercancers with obesity in our study we showed that of advanced adenoma patients and of crc patients were overweight with bmi in comparison toadvanced adenoma the percentage was lower in cancerperhaps due to the late stage of cancer and weight lossin the interim table there are several mechanismsby which obesity is believed to promote crc this includes increase in leptin levels that cause an increase ingrowth and proliferation of colon cancer cells altered adipokine levels altered gut microbiome apartfrom increased steroid hormones and growth factors insulin is however the established biochemical linkand the main pathway involved is pik3aktmtorpathway elevated igf1 and insulin act through the insulin receptors and phosphotidylinositide3 kinase in addition to the above findings we also found thatoverweight and obese patients bmi did not showa significantly increased expression p of nuclearctnnb1 positive in normal weight vs positive in overweightobese morikawa found that inobesepositivity wasnuclear ctnnb1patientsassociated with significantly better cancerspecific survival suggesting that wnt signaling acts as a switch andwhen it is on adipogenesis is repressed kennell demonstrated that activated frz1 frizzled promotes catenin stabilityinhibits apoptosis and adipogenesisross also showed that wnt signaling acts as a molecular switch that controls adipogenesis upregulationof wnt signaling maintains preadipocytes in an undifferentiated state and when wnt signaling is prevented theydifferentiate into adipocytes [ ]although in our study there was no association between nuclear intensity and crc between normal andoverweight patients p for interaction there is accumulating evidence to show that the state of chronicinflammation incited by obesity might play a role in promoting colorectal carcinogenesis [ ] of the manymarkers tnfα is important [ ] as it activateswnt signaling through the induction of gsk3 phosphorylation resulting in increased nuclear localization ofcatenin in addition to tnfα other humoralagents associated with obesity might also be contributingto the activation of wnt signaling like il1 and adiponectin which is decreased in the obese state and is notan inflammatory cytokine that can modulate gsk3catenin signaling pathway although multiplemechanisms may be operating in parallel and contributing to the protumorigenic milieu wnt is a pivotaltumorigenic pathway aberrations of which isfig the putative relationship between obesity and colorectal cancer evolution pathways by cellular ctnnb1 status based on the data by thecurrent study our study suggests that there is no association between obesity and ctnnb1 expression 0cshokrani bmc gastroenterology page of important in the evolution of most sporadic crc insummary there is positive nuclear staining in crcs which was associated with the positive status fornuclear ctnnb1 intensity adjusted or 95ci “ p for positive nuclear staining compared to noncrc samples normal or advanced adenoma this shows that advanced adenomas and crcswere associated with activation of catenin in physically fit overweight and obese patients fig advanced adenoma and crc were associated with activation of catenin in physically fit overweight andobese patients thus participation of obesity and wntpathway seem to be independent crc factors in africanamerican patientsinflammationdriven activation ofwnt signaling as a potential pathway linking obesity tothe development of crc needs to be investigated furtherin the african american population this might provideinsights into the identification of new therapeutic targetsto reduce the burden of obesityassociated crcabbreviationscrc colorectal cancer wat white adipose tissue aa african americansacknowledgementsnot applicableauthors™ contributionsconceived and designed experiments bs ha performed experiments bsel ha hb analyzed data ha hb mn th aa and zs contributed reagentsmaterialsanalysis tools zs ha hb bs and el wrote and edited manuscriptbs and ha provided statistical analysis mn all authors have read andapproved the manuscriptfundingthis project was supported in part by grant from the national institute onminority health and health disparities of the national institutes of healthunder award numbersg12md007597 the funder had no role in designing or execution of thisstudyavailability of data and materialsall data generated or analyzed during this study are included in thispublished ethics approval and consent to participatethis retrospective and chart review study was conducted according to theworld medical association declaration of helsinki and was approved by theinternal review board of howard university since the chart review was donethrough unidentifiable approach no consent form needed for this studyconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interests related to thismanuscriptauthor details1department of medicine department of pathology and cancer centerhoward university college of medicine geia avenue nwwashington dc usa 2division of pulmonary allergy and criticalcare medicine university of pittsburg pittsburg pa usareceived april accepted august referencesresearch iafco estimated cancer incidence mortality and prevalenceworlwide in lyon globocan haggar fa boushey rp colorectal cancer epidemiology incidencemortality survival and risk factors clin colon rectal surg “oxentenko as body size and incident colorectal cancer a prospectivestudy of older women cancer prev res phila “ ma y obesity and risk of colorectal cancer a systematic review ofprospective studies plos one 201381e53916slattery ml physical activity and colorectal cancer sports med “ wong mc lam ty tsoi kk hirai hw chan vc ching jy chan fk sung jja validated tool to predict colorectal neoplasia and inform screening choicefor asymptomatic subjects gut “sung jjy wong mcs lam tyt tsoi kkf chan vcw cheung w ching jyla modified colorectal screening score for prediction of advanced neoplasiaa prospective study of subjects jgastroenterolhepatol “liu z dietinduced obesity elevates colonic tnfalpha in mice and isaccompanied by an activation of wnt signaling a mechanism for obesityassociated colorectal cancer j nutr biochem “najdi r holcombe rf waterman ml wnt signaling and coloncarcinogenesis beyond apc j carcinog macdonald bt tamai k he x wntbetacatenin signaling componentsmechanisms and diseases dev cell “ denysenko t wntbetacatenin signaling pathway and downstreammodulators in low and highgrade glioma cancer genomics proteomics“ willert k nusse r betacatenin a key mediator of wnt signaling curr opingenet dev “ vella a camilleri m pharmacogenetics potential role in the treatment ofdiabetes and obesity expert opin pharmacother “ christodoulides c adipogenesis and wnt signalling trendsendocrinol metab “ world health anization [httpwwwwhointmediacentrefactsheetsfs311en] chen m lu p ma q cao y chen n li w zhao s chen b shi j sun yshen h sun l shen j liao q zhang y hong j gu w liu r ning g wangw wang j ctnnb1betacatenin dysfunction contributes to adiposity byregulating the crosstalk of mature adipocytes and preadipocytes sci adv20206eaax9605 morikawa t kuchiba a yamauchi m meyerhardt ja shima k nosho kchan at giovannucci e fuchs cs ogino s association of ctnnb1 betacatenin alterations body mass index and physical activity with survival inpatients with colorectal cancer jama “ morikawa t prospective analysis of body mass index physical activityand colorectal cancer risk associated with betacatenin ctnnb1 statuscancer res “ ashktorab h kupfer ss brim h carethers jm racial disparity ingastrointestinal cancer risk gastroenterology “kaler p augenlicht l klampfer l activating mutations in betacatenin incolon cancer cells alter their interaction with macrophages the role of snailplos one 201279e45462enzo mv the wntbetacatenin pathway in human fibroticlikediseases and its eligibility as a therapeutic target mol cell ther stamos jl weis wi the betacatenin destruction complex cold springharb perspect biol 201351a007898 chung gg tissue microarray analysis of betacatenin in colorectalcancer shows nuclear phosphobetacatenin is associated with a betterprognosis clin cancer res “ miyoshi y activation of the betacatenin gene in primaryhepatocellular carcinomas by somatic alterations involving exon cancerres “fukuchi t betacatenin mutation in carcinoma of the uterineendometrium cancer res “ brabletz t variable betacatenin expression in colorectal cancersindicates tumor progression driven by the tumor environment proc natlacad sci u s a “ 0cshokrani bmc gastroenterology page of berger na obesityassociated gastrointestinal tract cancer from beginningto end cancer “ calle ee the american cancer society cancer prevention study iinutrition cohort rationale study design and baseline characteristicscancer “frezza ee wachtel ms chirivainternati m influence of obesity on the riskof developing colon cancer gut “ berger na obesity and cancer pathogenesis ann n y acad sci “ guo s insulin signaling resistance and the metabolic syndrome insightsfrom mouse models into disease mechanisms j endocrinol 20142202t1“t23 ross se inhibition of adipogenesis by wnt signaling science “kennell ja macdougald oa wnt signaling inhibits adipogenesis throughbetacatenindependent and independent mechanisms j biol chem “liu z yingka y inflammation driven activation of wnt pathway a potentialmechanism responsible for obesity associated colorectal cancer obes resopen j “tilg h moschen ar adipocytokines mediators linking adipose tissueinflammation and immunity nat rev immunol “fischerposovszky p wabitsch m hochberg z endocrinology of adiposetissue an update horm metab res “ oguma k activated macrophages promote wnt signalling throughtumour necrosis factoralpha in gastric tumour cells embo j “ wang y adiponectin modulates the glycogen synthase kinase3betabetacatenin signaling pathway and attenuates mammary tumorigenesis ofmdamb231 cells in nude mice cancer res “ renehan ag roberts dl dive c obesity and cancer pathophysiologicaland biological mechanisms arch physiol biochem “publisher™s notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"

cancer298

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: " african americans aa are at high risk for colorectal cancer crc studies report a “ increasein crc risk with physical inactivity obesity and metabolic syndrome activation of the wntcatenin ctnnb1signaling pathway plays a critical role in colorectal carcinogenesis accumulating evidence also indicates a role ofwntctnnb1 signaling in obesity and metabolic diseasesaim to examine the association between obesity catenin expression and colonic lesions in african americansmethods we reviewed the pathology records of colorectal specimens from to crcs advanced adenomas and normal colon tissues tissue microarrays tma were constructed from these samplesimmunohistochemistry ihc for ctnnb1 catenin clone catenin1 was performed on the constructed tmasthe ihc results were evaluated by pathologists and the nuclear intensity staining was scored from to bmisex age location of the lesion and other demographic data were obtainedresults positive nuclear staining in normal advanced adenoma and crc was and respectively p crc was asso ciated with positive status for nuclear ctnnb1 intensity adjusted or 95ci “p for positive nuclear staining compared to noncrc samples normal or advanced adenoma nuclearstaining percentage has a fair diagnostic ability for crc with an auc of 95ci “overweightobese patients bmi did not show a significant difference in p nuclear ctnnb1 staining positive in normal weight vs positive in overweightobese the association between nuclear intensityand crc was not different between normal and overweight patients p for interaction the positive nuclearctnnb1status in crc stage iii and iv of all crc was not different from stage i and ii vs respectively p continued on next page correspondence b_shokranihowardedu hashktorabhowardedu1department of medicine department of pathology and cancer centerhoward university college of medicine geia avenue nwwashington dc usafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cshokrani bmc gastroenterology page of continued from previous page in our study advanced adenoma and crc were associated with activation of catenin in physically fitoverweight and obese patients thus obesity and wntcatenin pathway seem to be independent in africanamerican patients wntcatenin signaling pathway has a potential to be used as an effector in coloncarcinogenic transformation whether or not bmi is a modifier of this pathway needs to be investigated furtherkeywords catenin colorectal cancer advanced adenoma african americans colorectal cancer crc is one of the most commoncancers in the industrialized world lifestyle and epidemiological factors associated with an increased risk ofcrc include physical inactivity obesity and metabolicsyndrome in the united states approximately twothirds of the adult population are overweight or obesewhich represents a putative risk factor for multiple target an malignancies including crc there is evidence to suggest that excess adiposity is associated with up to greater risk of crc comparedwith normal weight individuals and that physical activity may decrease colorectal cancer risk althoughexcessive accumulation of white adipose tissue wat isthe key feature of adiposity obesity is clinically definedby a bmi over kgm2 which does not take fat contentinto account it is also known that most crcs arise froma genetic and morphological adenoma to carcinomatransition also it is widely accepted that both crcsand colorectal adenomas cras share similar etiologicalcauses which explains why cras which are amongstthe mostfindings in all crcscreening participants are present in more than ofgeneral asymptomatic populations consequentlyrisk algorithms have been applied to use bmi as a predictor variable to stratify individuals according to theirrisk of colorectal neoplasia however the underlyingmechanisms that might explain the association and themagnitude ofthe connection between excess bodyweight and crc remain unclearfrequent pathologicalin the obesitycancer relationship multiple biologicalprocesses including insulininsulinlike growth factorigf1 insulin resistance sexual hormones estrogensand proinflammatory cytokines tnfα il6 and crpactively participate all these elements create a favorable environment for carcinogenesis and a decrease inapoptosisas a separate molecular pathway activation of thewnt signaling pathway plays a critical role in colorectalcarcinogenesis wnt ligands are a family of proteinsfor normal cell development that are importantcatenin ctnnb1 is a major mediator of the wntpathway that is traditionally classified into canonical catenindependent and noncanonical cateninindependent wnt canonical pathway utilizes a group ofcell surface receptors called frizzled frz to activateseveral pathways the most important one involving catenin and apc in the absence of wnt signalingapc causes degradation of catenin preventing its accumulation in the cytoplasm by forming a complex withcatenin which leads to the phosphorylation and eventually destruction of catenin by the proteasome signaling by wnt blocks this process allowing cateninto migrate from the cytoplasm to the nucleus once inthe nucleus catenin upregulates cmyc cyclin d1and other genes which increase cellular proliferation therefore continuous wnt signaling can be seenin cells with loss of apc metabolic syndromeassociated conditionssuch asobesity and type ii diabetes are influenced by geneticand functional variations in the wnt signaling pathway wnt signaling when activated represses the terminal differentiation during adipogenesis whereby preadipocytes take on the characteristics of mature adipocytes a cascade of transcriptional events like the induction of catenin ensues which in turn inducesenhancer binding proteinα cebpa and peroxisomeproliferatoractivated receptorÎ pparg the excessive accumulation of wat features adiposity butobesity does not take fat content into account recently genetic factors linked to fat mass and adipositywere reported to be associated with increased obesityrisk in young obese individuals wholeexome sequencing revealed rare gainoffunction mutations inctnnb1catenin the cateninregulated transcription of an adipocytederived chemokine calledserum amyloid a3 saa3 leads to the formation of a catenin“tcf complex in mature adipocytes that promotes the proliferation of preadipocytes in wat andthereby increases obesity and the risk for metabolic syndrome other data also suggest that obesity and lack ofphysical activity are associated with a higher risk forcolorectal cancer [ ] these findings have importantimplications especially in the obese and physically inactive african american population that may haveunderlying predisposing mutations to colorectal cancer the aim of this study is to assess the catenin expression profile in colorectal premalignant and malignant lesions in correlation with obesity as determined by 0cshokrani bmc gastroenterology page of body mass index bmi or waist circumference wc inafrican american populationmethodspatients and clinical datacolorectaltissue samples submitted to the surgicalpathology laboratory at howard university hospitalfrom january to december were retrieved from the pathology archive system powerpath„¢a total of samples were included in the study consisting of tissue samples from crc n advancedadenoma n and normal colon n patients™data included age sex height weight and waist circumference body mass index bmi was calculated in thestudy table the protocol of this study was approvedby the howard university institutional review boardirbtissue microarray tma constructionhematoxylineosin stained slides he slides weremade from paraffinembedded blocks the he slideswere reviewed by two pathologists to confirm the pathological diagnosis and to mark areas of interest multipleareas from more than one block were marked to ensurea good representability of the sample on the tma fivetma paraffin blocks each containing cores of mmin diameter each and mm distance from each otherwere constructed tissuespecific and an system controls were included in each tmaimmunohistochemical ihc analysis of ctnnb1 cateninthe constructed tmas were stained for catenin theimmunostaining was carried out as follows dako monoclonal mouse antihuman betacatenin cateninc1intended for laboratory application to identify qualitatively by light microscopy catenin positive cells in normal and neoplastic tissues was used at a dilution of using the envision dab code k4006 detectionsystem the deparaffinized tmas were treated prior tothe ihc staining procedure target retrievalinvolvedimmersion of tissue sections in a preheated buffer solution and maintaining heat in a water bath “ °cfor greater adherence of tissue sections to glass slidessilanized slides dako code s3003 were used targetretrieval solution code s1700 or 10x concentratecode s1699 using a 20min heating protocol was usedthe cellular staining pattern of anticatenin is mainlymembranous especially at the cellcell boundaries positive nuclear staining and diffuse cytoplasmic staining arealso reported in cancer cells fig evaluation and assessment of the catenin expressiontwo pathologists interpreted the ihc slides cateninexpression status was assessed based on the pattern ofstaining nuclear cytoplasmic and membranous intensity to and percentage of staining to the staining would be considered negative if there wasweak or no nuclear expression or positive if there wasmoderate or strong nuclear expressionstatistical analysisdistribution of continuous and categorical variables weretested by kruskalwallis and chisquare test betweendifferent groups respectively we used logistic regression analysis to test association between the staining andrisk of crc after adjusting for age and gender areaunder the curve auc was calculated for variables withsignificant association with crc using receiver operative characteristics curve all statistical analyses wereperformed by stata statacorp college station txresultsepidemiological characteristics and bmi in normaladvanced adenoma and crcthe bmi was calculated for individual patients and normal subjects as represented in table crc patientswere older p while our healthy normal population was mostly overweight higher bmi was moreclosely associated with advanced adenoma and crchowever the differences were not significant table advanced adenomas and crcs were associated withpositive nuclear ctnnb1we assessed whether alterations in wntctnnb1 catenin signaling plays any role colon carcinogenesispositive catenin nuclear stains were seen in normaladvanced adenomas and crcs were and respectively p table based on the designationtable epidemiological characteristics and bmi in normal advanced adenoma and crc patientsage median iqrmale n bmi kgm2 median iqroverweight n normaln “ “ advanced adenoman “ “ crcn “ “ p value 0cshokrani bmc gastroenterology page of fig immunostain for catenin in three individuals normal a × advanced adenoma b × and cancer c d × and × respectively showing membranous staining in the normal cytoplasmic and membranous staining in adenoma with no evidence of nuclearexpression arrow showing lack of nuclear staining and nuclear and cytoplasmic staining in cancer arrow showing nuclear stainingof œn intensity  which is associated with higher risk ofcancer crcs were associated with positive status fornuclear ctnnb1 intensity age gender adjusted or 95ci “ p for positive nuclearstaining compared to noncrc samples normal or advanced adenoma fig nuclear staining percentagehas also a fair diagnostic ability for crc with an auc of 95ci “ table fig overweight and obese patients show a trend withpositive nuclear ctnnb1 expressionpositive nuclear ctnnb1 staining was in normalweight and in overweightobese bmi patientsthis difference pointed to trend that was not statisticallysignificantassociation between nuclear intensity and crc in normaland overweight patientsthe association between nuclear intensity and crcwas not statistically significant different between normal weight and overweight patientsinteraction tables and the positive nuclearctnnb1 status in crc stage iii and iv of allcrc was not different from stage i and ii vs respectively p p fortable catenin nuclear and cytoplasmic expression tabulated as intensity and percentage in normal advanced adenoma andcrcnormaln “ “cnc intensity n intensity advanced adenoma n “ “ crcn “ “ c cytoplasmic n nuclearc intensity and n intensity mean intensity and aboveoverall p valuep value for advanced adenoma vs normalp value for crc vs other 0cshokrani bmc gastroenterology page of fig catenin nuclear and cytoplasmic expression in normal advanced adenoma and crcdiscussionone of the important risk factors in colorectal cancer isobesity [ ] catenin is an ecadherin binding proteinthat mediates cellcell adhesion and plays a role inthe canonical wnt signaling pathway that controls thecoordinated expansion and differentiation of the intestinal crypt stem cells degradation of catenin byphosphorylation followed by alteration of destructioncomplex apc gsk3 and axin results in inactivation if wnt pathway in our study we found thatwas associated with an increased adjusted or of 95ci “ p for positive nuclear staining compared to noncrc samples normal or advanced adenomathe gatekeeper gene apc is a negative regulator of catenin and is mutated in approximately of sporadic and hereditary colon cancers there are severalmutations that can cause an accumulation of cateninin tumor cells such as mutations of the apc gene pointmutations in gsk3 or mutations in catenin gene itself [“]our positive nuclear staining in crc and its association with the positive status for nuclear ctnnb1intensity compared to noncrc samples are in contrastto a study by brabletz which showed that catenin is localized in the cytoplasm and membrane ofthe tumor cells whereas in our study it was mainly concentrated in the cytoplasm and the nucleus they alsomentioned that there was positive nuclear staining at theinvasive front as catenin is involved in tumor progression such is not the case in our studyindeed evenwhen considering nuclear staining in our specimensthere was no statistically significant differences betweenstage iiiiv cases™ staining versus stages iii crc caseslevels of staining the fact that catenin is expressedearly in the african american specimens analyzed heremight partially explain the aggressive nature of crc inthis population in addition we showed that there is uniform membranous staining in normal and increasingcytoplasmic and nuclear staining in advanced adenomasand crcs this confirms that the decrease in membranous staining begins with dysplastic changes leading to atable association of bmi with catenin nuclear intensity in advanced adenoma casesadvanced adenoma with catenin expression n in intensity and no nuclear intensity n bmi median interquartileoverweight n nuclear intensity negativen “ nuclear intensity n “ p value 0cshokrani bmc gastroenterology page of table association of bmi with catenin nuclear intensity in crc casescrc with catenin expression with high nuclear intensity and without negativebmi median interquartileoverweight n nuclear intensity negativen “ nuclear intensity n “ p valueprogressive disappearance atcrcsthe membrane levelinas we mentioned above a major risk factor for crc isobesity which continues to expand as a pandemicworldwide the american cancer society cancerprevention study ii states that there is an increased incidence of crc esophageal adenocarcinoma and othercancers with obesity in our study we showed that of advanced adenoma patients and of crc patients were overweight with bmi in comparison toadvanced adenoma the percentage was lower in cancerperhaps due to the late stage of cancer and weight lossin the interim table there are several mechanismsby which obesity is believed to promote crc this includes increase in leptin levels that cause an increase ingrowth and proliferation of colon cancer cells altered adipokine levels altered gut microbiome apartfrom increased steroid hormones and growth factors insulin is however the established biochemical linkand the main pathway involved is pik3aktmtorpathway elevated igf1 and insulin act through the insulin receptors and phosphotidylinositide3 kinase in addition to the above findings we also found thatoverweight and obese patients bmi did not showa significantly increased expression p of nuclearctnnb1 positive in normal weight vs positive in overweightobese morikawa found that inobesepositivity wasnuclear ctnnb1patientsassociated with significantly better cancerspecific survival suggesting that wnt signaling acts as a switch andwhen it is on adipogenesis is repressed kennell demonstrated that activated frz1 frizzled promotes catenin stabilityinhibits apoptosis and adipogenesisross also showed that wnt signaling acts as a molecular switch that controls adipogenesis upregulationof wnt signaling maintains preadipocytes in an undifferentiated state and when wnt signaling is prevented theydifferentiate into adipocytes [ ]although in our study there was no association between nuclear intensity and crc between normal andoverweight patients p for interaction there is accumulating evidence to show that the state of chronicinflammation incited by obesity might play a role in promoting colorectal carcinogenesis [ ] of the manymarkers tnfα is important [ ] as it activateswnt signaling through the induction of gsk3 phosphorylation resulting in increased nuclear localization ofcatenin in addition to tnfα other humoralagents associated with obesity might also be contributingto the activation of wnt signaling like il1 and adiponectin which is decreased in the obese state and is notan inflammatory cytokine that can modulate gsk3catenin signaling pathway although multiplemechanisms may be operating in parallel and contributing to the protumorigenic milieu wnt is a pivotaltumorigenic pathway aberrations of which isfig the putative relationship between obesity and colorectal cancer evolution pathways by cellular ctnnb1 status based on the data by thecurrent study our study suggests that there is no association between obesity and ctnnb1 expression 0cshokrani bmc gastroenterology page of important in the evolution of most sporadic crc insummary there is positive nuclear staining in crcs which was associated with the positive status fornuclear ctnnb1 intensity adjusted or 95ci “ p for positive nuclear staining compared to noncrc samples normal or advanced adenoma this shows that advanced adenomas and crcswere associated with activation of catenin in physically fit overweight and obese patients fig advanced adenoma and crc were associated with activation of catenin in physically fit overweight andobese patients thus participation of obesity and wntpathway seem to be independent crc factors in africanamerican patientsinflammationdriven activation ofwnt signaling as a potential pathway linking obesity tothe development of crc needs to be investigated furtherin the african american population this might provideinsights into the identification of new therapeutic targetsto reduce the burden of obesityassociated crcabbreviationscrc colorectal cancer wat white adipose tissue aa african americansacknowledgementsnot applicableauthors™ contributionsconceived and designed experiments bs ha performed experiments bsel ha hb analyzed data ha hb mn th aa and zs contributed reagentsmaterialsanalysis tools zs ha hb bs and el wrote and edited manuscriptbs and ha provided statistical analysis mn all authors have read andapproved the manuscriptfundingthis project was supported in part by grant from the national institute onminority health and health disparities of the national institutes of healthunder award numbersg12md007597 the funder had no role in designing or execution of thisstudyavailability of data and materialsall data generated or analyzed during this study are included in thispublished ethics approval and consent to participatethis retrospective and chart review study was conducted according to theworld medical association declaration of helsinki and was approved by theinternal review board of howard university since the chart review was donethrough unidentifiable approach no consent form needed for this studyconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interests related to thismanuscriptauthor details1department of medicine department of pathology and cancer centerhoward university college of medicine geia avenue nwwashington dc usa 2division of pulmonary allergy and criticalcare medicine university of pittsburg pittsburg pa usareceived april accepted august referencesresearch iafco estimated cancer incidence mortality and prevalenceworlwide in lyon globocan haggar fa boushey rp colorectal cancer epidemiology incidencemortality survival and risk factors clin colon rectal surg “oxentenko as body size and incident colorectal cancer a prospectivestudy of older women cancer prev res phila “ ma y obesity and risk of colorectal cancer a systematic review ofprospective studies plos one 201381e53916slattery ml physical activity and colorectal cancer sports med “ wong mc lam ty tsoi kk hirai hw chan vc ching jy chan fk sung jja validated tool to predict colorectal neoplasia and inform screening choicefor asymptomatic subjects gut “sung jjy wong mcs lam tyt tsoi kkf chan vcw cheung w ching jyla modified colorectal screening score for prediction of advanced neoplasiaa prospective study of subjects jgastroenterolhepatol “liu z dietinduced obesity elevates colonic tnfalpha in mice and isaccompanied by an activation of wnt signaling a mechanism for obesityassociated colorectal cancer j nutr biochem “najdi r holcombe rf waterman ml wnt signaling and coloncarcinogenesis beyond apc j carcinog macdonald bt tamai k he x wntbetacatenin signaling componentsmechanisms and diseases dev cell “ denysenko t wntbetacatenin signaling pathway and downstreammodulators in low and highgrade glioma cancer genomics proteomics“ willert k nusse r betacatenin a key mediator of wnt signaling curr opingenet dev “ vella a camilleri m pharmacogenetics potential role in the treatment ofdiabetes and obesity expert opin pharmacother “ christodoulides c adipogenesis and wnt signalling trendsendocrinol metab “ world health anization [httpwwwwhointmediacentrefactsheetsfs311en] chen m lu p ma q cao y chen n li w zhao s chen b shi j sun yshen h sun l shen j liao q zhang y hong j gu w liu r ning g wangw wang j ctnnb1betacatenin dysfunction contributes to adiposity byregulating the crosstalk of mature adipocytes and preadipocytes sci adv20206eaax9605 morikawa t kuchiba a yamauchi m meyerhardt ja shima k nosho kchan at giovannucci e fuchs cs ogino s association of ctnnb1 betacatenin alterations body mass index and physical activity with survival inpatients with colorectal cancer jama “ morikawa t prospective analysis of body mass index physical activityand colorectal cancer risk associated with betacatenin ctnnb1 statuscancer res “ ashktorab h kupfer ss brim h carethers jm racial disparity ingastrointestinal cancer risk gastroenterology “kaler p augenlicht l klampfer l activating mutations in betacatenin incolon cancer cells alter their interaction with macrophages the role of snailplos one 201279e45462enzo mv the wntbetacatenin pathway in human fibroticlikediseases and its eligibility as a therapeutic target mol cell ther stamos jl weis wi the betacatenin destruction complex cold springharb perspect biol 201351a007898 chung gg tissue microarray analysis of betacatenin in colorectalcancer shows nuclear phosphobetacatenin is associated with a betterprognosis clin cancer res “ miyoshi y activation of the betacatenin gene in primaryhepatocellular carcinomas by somatic alterations involving exon cancerres “fukuchi t betacatenin mutation in carcinoma of the uterineendometrium cancer res “ brabletz t variable betacatenin expression in colorectal cancersindicates tumor progression driven by the tumor environment proc natlacad sci u s a “ 0cshokrani bmc gastroenterology page of berger na obesityassociated gastrointestinal tract cancer from beginningto end cancer “ calle ee the american cancer society cancer prevention study iinutrition cohort rationale study design and baseline characteristicscancer “frezza ee wachtel ms chirivainternati m influence of obesity on the riskof developing colon cancer gut “ berger na obesity and cancer pathogenesis ann n y acad sci “ guo s insulin signaling resistance and the metabolic syndrome insightsfrom mouse models into disease mechanisms j endocrinol 20142202t1“t23 ross se inhibition of adipogenesis by wnt signaling science “kennell ja macdougald oa wnt signaling inhibits adipogenesis throughbetacatenindependent and independent mechanisms j biol chem “liu z yingka y inflammation driven activation of wnt pathway a potentialmechanism responsible for obesity associated colorectal cancer obes resopen j “tilg h moschen ar adipocytokines mediators linking adipose tissueinflammation and immunity nat rev immunol “fischerposovszky p wabitsch m hochberg z endocrinology of adiposetissue an update horm metab res “ oguma k activated macrophages promote wnt signalling throughtumour necrosis factoralpha in gastric tumour cells embo j “ wang y adiponectin modulates the glycogen synthase kinase3betabetacatenin signaling pathway and attenuates mammary tumorigenesis ofmdamb231 cells in nude mice cancer res “ renehan ag roberts dl dive c obesity and cancer pathophysiologicaland biological mechanisms arch physiol biochem “publisher™s notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c" Answer:

Colon_Cancer

" vibrio cholerae are oxidasepositive bacteria that are classified into various serotypes based on the osurface antigen v cholerae serotypes are divided into two main groups the o1 and o139 group and the nono1nono139 group o1 and o139 v cholerae are related to cholera infection whereas nono1nono139 v choleraenovc can cause choleralike diarrhea a pubmed search revealed that only cases of necrotizing fasciitis causedby novc have been recorded in the scientific literature to date we report the case of a japanese woman whodeveloped necrotizing fasciitis caused by novc after traveling to taiwan and returning to japancase presentation a 63yearold woman visited our hospital because she had experienced left knee pain for thepast days she had a history of colon cancer stage iv t3n3 m1a and had received chemotherapy she hadvisited taiwan days previously where she had received a massage she was diagnosed with septic shock owingto necrotizing fasciitis she underwent fasciotomy and received intensive care she recovered from the septic shockhowever after weeks she required an aboveknee amputation for necrosis and infection her condition improvedand she was discharged after weeks in the hospitals with the increase in tourism it is important for clinicians to check patients™ travel history cliniciansshould be alert to the possibility of necrotizing fasciitis in patients with risk factors necrotizing fasciitis caused bynovc is severe and requires early fasciotomy and debridement followed by intensive postoperative carekeywords necrotizing fasciitis vibrio cholerae taiwan massage septic shock polymyxin b correspondence kei610805gmailcom1emergency department minaminara general hospital ooazafukugamiooyodocho yoshinogun nara japanfull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0ctsuruta bmc infectious diseases page of vibrio cholerae are curved gramnegative rod gnrbacteria that are oxidase positive they are classified intovarious serotypes based on the o surface antigen vcholerae serotypes are divided into two main groups theo1 and o139 group and the nono1nono139 group o1 and o139 v cholerae are related to cholera infection whereas nono1nono139 v cholerae novccan cause choleralike diarrhea novc are found as autochthonous microbes in coastal and marine environments outbreaks of choleralike illness caused bynovc have been reported in the united states o141and o75 former czechoslovakia o37 sudan o37peru o10 o12 and mexico o14 [“] moreovernovc can cause a range of extraintestinal infectionsincluding bacteremia meningitis pneumonia peritonitischolangitis salpingitis and softtissue infection seafood including oysters fishes shrimps clams musselsand apple snail is the most common source of infection a pubmed search revealed that only cases of necrotizing fasciitis caused by novc have beenreported in the scientific literature to date we reportthe case of a patient who developed necrotizing fasciitisand septic shock caused by novc which necessitatedan aboveknee amputation of her left legcase presentationa 63yearold woman visited minaminara general hospital in nara japan because she had experienced leftknee pain for days prior to her visit she had been diagnosed with colon cancer stage iv t3n3 m1a years and months previously and had undergone surgery and received chemotherapy her most recent doseof chemotherapy was administered days before herinitial consultation she had visited taiwan days previously where she had received a massage after themassage she developed gradually worsening pain in herlower left leg on presentation she was able to walkunaided and she reported her history of colon cancerand recent travel as we suspected that the pain in herleg could be due to necrotizing fasciitis we requestedmagnetic resonance imaging mri of her left lowerleg the images showed a swollen soleus muscle andposterior tibial muscle and the t2weighted imageshowed hyperintensity of the muscle tissue fig after the mri our patient™s condition deteriorated andthe following vital signs were observed blood pressurebp mmhg heart rate beatsmin respiratory rate breathsmin and temperature °cthe results of arterial blood gas analysis were as folˆ’ mmhglows ph paco2 mmhg hco3base excess be ˆ’ meql and lactate mmoll the patient™s laboratory test results were as followscreactive protein crp mgdl blood ureanitrogen bun mgdl creatinine mgdlprocalcitonin ngml nterminal probrain natriuretic peptide ntprobnp pgml and fibrinfibrinogen degradation products fdp μgmlinitiatedadministeredlowvenovenoushemodiafiltrationsurgery her blood pressure wasintravenous infusion of meropenem and noradrenaline wasand the patient underwentemergency surgery before the surgery the compartment pressure of her left leg was measured by simpleneedle manometry the pressures were as follows mmhg mmhg mmhg and mmhg in theanterior lateral superficial posterior and deep posterior compartments respectively some muscle tissuesin the anterior and deep posterior compartments werenecrotic for double incision fasciotomy a relaxationincision was made on her left knee and theaffected area was irrigated and debrided fig aftertheandtherefore wepolymyxin b directhemoperfusion pmxdhp to trap endotoxins andcontinuoususinghemofeel ch13 w toray medical co ltdurayasu japan as a slightly curved gnr that wasoxidase positive was detected in her blood we diagnosed her with necrotizing fasciitis and septic shockcaused by vibrio species we changed the antibioticsfrom meropenem to ceftriaxonelevofloxacin andminocycline we used the pmxdhp once again andtapered the dose of noradrenalin gradually wediscontinued noradrenalin on day postoperativelyon day postoperatively the anism was identifiedas novc theantibiotics wasconfirmed postoperatively on day and we discontinued levofloxacin table although the patient™sgeneral condition improved there was a discharge ofpus from the postoperative wound on day postoperatively a second debridement was performedseveral muscles in the patient™s left leg including theanterior tibial muscle had become necrotic and thenecrosis had spread to her knee on day postoperatively an aboveknee amputation was performedher vital signs and laboratory data obtained since admission are shown in fig her condition improvedand she was discharged weeks after admissionsusceptibility ofdiscussion and sixteen cases of necrotizing fasciitis caused by novchave been previously reported table [“] themajority of patients were exposed to seawater or hadan injury in rare cases vigorous massage is one ofthe risk factors of necrotizing fasciitis howeverthe patient in the present case had a risk of novcinfection because of colon cancer and immunosuppression due to chemotherapy as she received chemotherapy within a month thusthein this case 0ctsuruta bmc infectious diseases page of fig t2weighted magnetic resonance images of the patient™s lower legs a coronal image b axial image these images show that the soleusand posterior tibial muscles on the left lower leg indicated by red arrows are swollen and inflamedfig photographs of lesions in the patient™s leg the patient™s leg before surgery shows multiple large blisters 0ctsuruta bmc infectious diseases page of table susceptibility of antibioticsantibioticsampicillinminimal inhibitory concentrations piperacillinceftazidimeimipenemcilastatinamoxicillinclavulanategentamicinminocyclinechloramphenicolsulfamethoxazoletrimethoprimlevofloxacinfosfomycins s s ‰¦s s s ‰¦s ‰¦s ‰¦s ‰¦r the novc remains unknown assource ofthepatient did not report any exposure to sea water oreating seafood the only potential cause of injury toher left leg was the massage she received thereforewe speculate that the massage might have been thesource ofthe novc based on the circumstantialevidence we administered blood purification therapyusing pmxdhp and venovenous hemodiafiltrationfor septic shock although no previous studies havereported the use of pmxdhp for novc a studyreported the use of pmx for v vulnificus thirdgenerationtetracyclineandfluoroquinolone were used for severe vibrio infections tetracycline combined with the fluoroquinoloneorcephalosporinfollowed by oral fluoroquinolones or doxycycline wasrecommended for invasive novc infections [ ]an in vitro study revealed that cefotaxime and minocycline have a synergistic effect in the treatment forcephalosporinsaparenteralthirdgenerationfig change of vital signs and laboratory data during the hospital admission a changes in the patient™s vital signs during days “ ofhospitalization b changes in patient™s blood biochemistry during days “ of hospitalization atiii antithrombin iii crp creactive proteinfdp fibrinfibrinogen degradation products map mean arterial pressure nad noradrenaline 0ctsuruta bmc infectious diseases page of age sex underlying diseasestable clinical characteristics of patients with nono vibrio cholerae necrotizing fasciitisyear ofreportsourcesurgery amputation multiple debridementand antibiotics ticarcillinclavulanate imipenemgentamicin clindamycinrisk factors mdiabetes mellitussurvived usatreatmentoutcome country oantigen epidemiologicexposureexposure of achronic plantar ulcerto sand in abathhouse mcirrhosissurgery cefotaxime minocycline cefotaximesurvivedtaiwan f mcirrhosis congestiveheart failurecirrhosis diabetesmellitus mhepatitis csurgery ceftriaxonediedtaiwansurgical debridement ceftazidime doxycyclinediedtaiwan o56surgery antibiotics thirdgeneration cephalosporindoxycyclinediedtaiwanhandling seafood mhepatitis steroidssurgery antibiotics thirdgeneration cephalosporindoxycyclinesurvivedtaiwan mcirrhosissurgery clindamycin ceftazidime tetracyclinesurvivedtaiwansurgery antibioticssurgery antibioticsdiedtaiwandiedtaiwan m m mcirrhosis hepatitis cdiabetes mellituscirrhosis hepatitis bhepatitis c diabetesmellituscirrhosis diabetesmellitusexposure to seawaterprobable woundinfectionconsumption of rawseafoodseawater exposureinsect bite woundinfectionminor abrasionexposed to seawaterseawater exposuresurgery antibioticsdiedtaiwanseawater exposure mcirrhosissurgery antibioticssurvivedtaiwanseawater exposure mcopdsurgery antibioticssurvivedtaiwan mhiv hepatitis ccirrhosis mdiabetes mellitus michthyosis cellulitisnone mcopd chronic constructive pulmonary diseasesurgical debridement daptomycin levofloxacinsurviveditalyo137surgical debridement piperacillintazobactamfosfomycinsurgical debridement piperacillintazobactamtigecycline metronidazolesurgical debridementpenicillin gentamicin metronidazolesurvived austriadiedaustriaseawater exposuresurvived croatia o8seawater exposurev cholerae infections as patients with novcbacteremia require antibiotic treatment for at least month we administered ceftriaxone and minocycline for month necrotizing softtissue infectionscaused by novc are more lethal than those causedby v vulnificus to conclude we treated a woman with necrotizingfasciitis and septic shock caused by novc this caseillustrates that early fasciotomy and debridement arenecessary forsevere necrotizing fasciitis caused bynovc and prolonged intensive care may be requiredafter surgeryo139 vibrio cholerae ntprobnp nterminal probrain natriuretic peptidepmxdhp polymyxin b direct hemoperfusionacknowledgementsnoneauthors™ contributionskt tu tw kn and ku treated the patient kt tu hf reviewed the literatureand mainly wrote this report kn tw ku reviewed the literature andmodified this paper based on specialty orthopedics emergency departmentinfectious disease all authors have read and approved the manuscriptfundingnoneabbreviationsbun blood urea nitrogen fdp fibrinogen degradation productsgnr gramnegative rod mri magnetic resonance imaging novc nono1availability of data and materialsall data are included in this published 0ctsuruta bmc infectious diseases page of ethics approval and consent to participatethis case report was approved by the ethics review committee atminaminara general hospital and was conducted in accordance with thedeclaration of helsinki consent for participation is not applicableconsent for publicationwritten informed consent was obtained from the patient for publication ofthis case report and any accompanying images a copy of the writtenconsent is available for review by the editor of this journalcompeting intereststhe authors declare that they have no competing interestsauthor details1emergency department minaminara general hospital ooazafukugamiooyodocho yoshinogun nara japan 2orthopedic departmentminaminara general hospital nara japan 3infectious diseases departmentminaminara general hospital nara japan 4department of emergency andcritical care medicine nara medical university nara japanreceived march accepted august referencesgardner ad venkatraman kv the antigens of the cholera group of vibriosj hyg lond “ hirk s huhulescu s allerberger f lepuschitz s rehak s weil s necrotizing fasciitis due to vibrio cholerae nono1nono139 after exposureto austrian bathing sites wien klin wochenschr “ dobrović k rudman f ottaviani d crnek sÅ¡ leoni f Å¡krlin j a rare case ofnecrotizing fasciitis caused by vibrio cholerae o8 in an immunocompetentpatient wien klin wochenschr “jain akc varma ak mangalanandan kh kumar h bal a surgical outcome ofnecrotizing fasciitis in diabetic lower limbs j diab foot comp “ikeda t kanehara s ohtani t furukawa f endotoxin shock due to vibriovulnificus infection eur j dermatol “su ba tang hj wang yy liu yc ko wc liu cy in vitro antimicrobialeffect of cefazolin and cefotaxime combined with minocycline against vibriocholerae nono1 nono139 j microbiol immunol infect “tsai yh huang tj hsu rww weng yj hsu wh huang kc necrotizing softtissue infections and primary sepsis caused by vibriovulnificus and vibrio cholerae nono1 j trauma “publisher™s notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations morris jg nono group vibrio cholerae a look at the epidemiology of anoccasional pathogen epidemiol rev “li m shimada t morris jg jr sulakvelidze a sozhamannan s evidence forthe emergence of nono1 and nono139 vibrio cholerae strains withpathogenic potential by exchange of oantigen biosynthesis regions infectimmun “dalsgaard a albert mj taylor dn shimada t meza r serichantalergs o characterization of vibrio cholerae nono1 serogroups obtained froman outbreak of diarrhea in lima peru j clin microbiol “isaacmárquez ap lezamadávila cm eslavacampos c navarroocaña acraviotoquintana a serotypes of vibrio cholerae nono1 isolated fromwater supplies for human consumption in campeche mexico and theirantibiotic susceptibility pattern mem inst oswaldo cruz “hughes jm hollis dg gangarosa ej weaver re noncholera vibrioinfections in the united states clinical epidemiologic and laboratoryfeatures ann intern med “deshayes s daurel c cattoir v parienti jj quilici ml de la blanchardière anono1 nono139 vibrio cholerae bacteraemia case report and literaturereview springerplus mubarak sj owen ca doubleincision fasciotomy of the leg fordecompression in compartment syndromes j bone joint surg am “ wagner pd evans sd dunlap j ballonlanda g necrotizing fasciitis andseptic shock caused by vibrio cholerae acquired in san diego californiawest j med “ko w chuang y huang g hsu sy infections due to nono1 vibrio choleraein southern taiwan predominance in cirrhotic patients clin infect dis “ cheng nc tsai jl kuo ys hsueh pr bacteremic necrotizing fasciitis causedby vibrio cholerae serogroup o56 in a patient with liver cirrhosis j formosmed assoc “tsai yh hsu rww huang kc chen ch cheng cc peng kt systemicvibrio infection presenting as necrotizing fasciitis and sepsis a series ofthirteen cases j bone joint surg am “ changchien ch bacteraemic necrotizing fasciitis with compartmentsyndrome caused by nono1 vibrio cholerae j plast reconstr aesthetic surg“ maraki s christidou a anastasaki m scoulica e nono1 nono139 vibriocholerae bacteremic skin and soft tissue infections infect dis lond “ ottaviani d leoni f rocchegiani e canonico c masini l pianetti a unusual case of necrotizing fasciitis caused by vibrio cholerae o137 j clinmicrobiol “ 0c"

cancer299

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: " vibrio cholerae are oxidasepositive bacteria that are classified into various serotypes based on the osurface antigen v cholerae serotypes are divided into two main groups the o1 and o139 group and the nono1nono139 group o1 and o139 v cholerae are related to cholera infection whereas nono1nono139 v choleraenovc can cause choleralike diarrhea a pubmed search revealed that only cases of necrotizing fasciitis causedby novc have been recorded in the scientific literature to date we report the case of a japanese woman whodeveloped necrotizing fasciitis caused by novc after traveling to taiwan and returning to japancase presentation a 63yearold woman visited our hospital because she had experienced left knee pain for thepast days she had a history of colon cancer stage iv t3n3 m1a and had received chemotherapy she hadvisited taiwan days previously where she had received a massage she was diagnosed with septic shock owingto necrotizing fasciitis she underwent fasciotomy and received intensive care she recovered from the septic shockhowever after weeks she required an aboveknee amputation for necrosis and infection her condition improvedand she was discharged after weeks in the hospitals with the increase in tourism it is important for clinicians to check patients™ travel history cliniciansshould be alert to the possibility of necrotizing fasciitis in patients with risk factors necrotizing fasciitis caused bynovc is severe and requires early fasciotomy and debridement followed by intensive postoperative carekeywords necrotizing fasciitis vibrio cholerae taiwan massage septic shock polymyxin b correspondence kei610805gmailcom1emergency department minaminara general hospital ooazafukugamiooyodocho yoshinogun nara japanfull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0ctsuruta bmc infectious diseases page of vibrio cholerae are curved gramnegative rod gnrbacteria that are oxidase positive they are classified intovarious serotypes based on the o surface antigen vcholerae serotypes are divided into two main groups theo1 and o139 group and the nono1nono139 group o1 and o139 v cholerae are related to cholera infection whereas nono1nono139 v cholerae novccan cause choleralike diarrhea novc are found as autochthonous microbes in coastal and marine environments outbreaks of choleralike illness caused bynovc have been reported in the united states o141and o75 former czechoslovakia o37 sudan o37peru o10 o12 and mexico o14 [“] moreovernovc can cause a range of extraintestinal infectionsincluding bacteremia meningitis pneumonia peritonitischolangitis salpingitis and softtissue infection seafood including oysters fishes shrimps clams musselsand apple snail is the most common source of infection a pubmed search revealed that only cases of necrotizing fasciitis caused by novc have beenreported in the scientific literature to date we reportthe case of a patient who developed necrotizing fasciitisand septic shock caused by novc which necessitatedan aboveknee amputation of her left legcase presentationa 63yearold woman visited minaminara general hospital in nara japan because she had experienced leftknee pain for days prior to her visit she had been diagnosed with colon cancer stage iv t3n3 m1a years and months previously and had undergone surgery and received chemotherapy her most recent doseof chemotherapy was administered days before herinitial consultation she had visited taiwan days previously where she had received a massage after themassage she developed gradually worsening pain in herlower left leg on presentation she was able to walkunaided and she reported her history of colon cancerand recent travel as we suspected that the pain in herleg could be due to necrotizing fasciitis we requestedmagnetic resonance imaging mri of her left lowerleg the images showed a swollen soleus muscle andposterior tibial muscle and the t2weighted imageshowed hyperintensity of the muscle tissue fig after the mri our patient™s condition deteriorated andthe following vital signs were observed blood pressurebp mmhg heart rate beatsmin respiratory rate breathsmin and temperature °cthe results of arterial blood gas analysis were as folˆ’ mmhglows ph paco2 mmhg hco3base excess be ˆ’ meql and lactate mmoll the patient™s laboratory test results were as followscreactive protein crp mgdl blood ureanitrogen bun mgdl creatinine mgdlprocalcitonin ngml nterminal probrain natriuretic peptide ntprobnp pgml and fibrinfibrinogen degradation products fdp μgmlinitiatedadministeredlowvenovenoushemodiafiltrationsurgery her blood pressure wasintravenous infusion of meropenem and noradrenaline wasand the patient underwentemergency surgery before the surgery the compartment pressure of her left leg was measured by simpleneedle manometry the pressures were as follows mmhg mmhg mmhg and mmhg in theanterior lateral superficial posterior and deep posterior compartments respectively some muscle tissuesin the anterior and deep posterior compartments werenecrotic for double incision fasciotomy a relaxationincision was made on her left knee and theaffected area was irrigated and debrided fig aftertheandtherefore wepolymyxin b directhemoperfusion pmxdhp to trap endotoxins andcontinuoususinghemofeel ch13 w toray medical co ltdurayasu japan as a slightly curved gnr that wasoxidase positive was detected in her blood we diagnosed her with necrotizing fasciitis and septic shockcaused by vibrio species we changed the antibioticsfrom meropenem to ceftriaxonelevofloxacin andminocycline we used the pmxdhp once again andtapered the dose of noradrenalin gradually wediscontinued noradrenalin on day postoperativelyon day postoperatively the anism was identifiedas novc theantibiotics wasconfirmed postoperatively on day and we discontinued levofloxacin table although the patient™sgeneral condition improved there was a discharge ofpus from the postoperative wound on day postoperatively a second debridement was performedseveral muscles in the patient™s left leg including theanterior tibial muscle had become necrotic and thenecrosis had spread to her knee on day postoperatively an aboveknee amputation was performedher vital signs and laboratory data obtained since admission are shown in fig her condition improvedand she was discharged weeks after admissionsusceptibility ofdiscussion and sixteen cases of necrotizing fasciitis caused by novchave been previously reported table [“] themajority of patients were exposed to seawater or hadan injury in rare cases vigorous massage is one ofthe risk factors of necrotizing fasciitis howeverthe patient in the present case had a risk of novcinfection because of colon cancer and immunosuppression due to chemotherapy as she received chemotherapy within a month thusthein this case 0ctsuruta bmc infectious diseases page of fig t2weighted magnetic resonance images of the patient™s lower legs a coronal image b axial image these images show that the soleusand posterior tibial muscles on the left lower leg indicated by red arrows are swollen and inflamedfig photographs of lesions in the patient™s leg the patient™s leg before surgery shows multiple large blisters 0ctsuruta bmc infectious diseases page of table susceptibility of antibioticsantibioticsampicillinminimal inhibitory concentrations piperacillinceftazidimeimipenemcilastatinamoxicillinclavulanategentamicinminocyclinechloramphenicolsulfamethoxazoletrimethoprimlevofloxacinfosfomycins s s ‰¦s s s ‰¦s ‰¦s ‰¦s ‰¦r the novc remains unknown assource ofthepatient did not report any exposure to sea water oreating seafood the only potential cause of injury toher left leg was the massage she received thereforewe speculate that the massage might have been thesource ofthe novc based on the circumstantialevidence we administered blood purification therapyusing pmxdhp and venovenous hemodiafiltrationfor septic shock although no previous studies havereported the use of pmxdhp for novc a studyreported the use of pmx for v vulnificus thirdgenerationtetracyclineandfluoroquinolone were used for severe vibrio infections tetracycline combined with the fluoroquinoloneorcephalosporinfollowed by oral fluoroquinolones or doxycycline wasrecommended for invasive novc infections [ ]an in vitro study revealed that cefotaxime and minocycline have a synergistic effect in the treatment forcephalosporinsaparenteralthirdgenerationfig change of vital signs and laboratory data during the hospital admission a changes in the patient™s vital signs during days “ ofhospitalization b changes in patient™s blood biochemistry during days “ of hospitalization atiii antithrombin iii crp creactive proteinfdp fibrinfibrinogen degradation products map mean arterial pressure nad noradrenaline 0ctsuruta bmc infectious diseases page of age sex underlying diseasestable clinical characteristics of patients with nono vibrio cholerae necrotizing fasciitisyear ofreportsourcesurgery amputation multiple debridementand antibiotics ticarcillinclavulanate imipenemgentamicin clindamycinrisk factors mdiabetes mellitussurvived usatreatmentoutcome country oantigen epidemiologicexposureexposure of achronic plantar ulcerto sand in abathhouse mcirrhosissurgery cefotaxime minocycline cefotaximesurvivedtaiwan f mcirrhosis congestiveheart failurecirrhosis diabetesmellitus mhepatitis csurgery ceftriaxonediedtaiwansurgical debridement ceftazidime doxycyclinediedtaiwan o56surgery antibiotics thirdgeneration cephalosporindoxycyclinediedtaiwanhandling seafood mhepatitis steroidssurgery antibiotics thirdgeneration cephalosporindoxycyclinesurvivedtaiwan mcirrhosissurgery clindamycin ceftazidime tetracyclinesurvivedtaiwansurgery antibioticssurgery antibioticsdiedtaiwandiedtaiwan m m mcirrhosis hepatitis cdiabetes mellituscirrhosis hepatitis bhepatitis c diabetesmellituscirrhosis diabetesmellitusexposure to seawaterprobable woundinfectionconsumption of rawseafoodseawater exposureinsect bite woundinfectionminor abrasionexposed to seawaterseawater exposuresurgery antibioticsdiedtaiwanseawater exposure mcirrhosissurgery antibioticssurvivedtaiwanseawater exposure mcopdsurgery antibioticssurvivedtaiwan mhiv hepatitis ccirrhosis mdiabetes mellitus michthyosis cellulitisnone mcopd chronic constructive pulmonary diseasesurgical debridement daptomycin levofloxacinsurviveditalyo137surgical debridement piperacillintazobactamfosfomycinsurgical debridement piperacillintazobactamtigecycline metronidazolesurgical debridementpenicillin gentamicin metronidazolesurvived austriadiedaustriaseawater exposuresurvived croatia o8seawater exposurev cholerae infections as patients with novcbacteremia require antibiotic treatment for at least month we administered ceftriaxone and minocycline for month necrotizing softtissue infectionscaused by novc are more lethal than those causedby v vulnificus to conclude we treated a woman with necrotizingfasciitis and septic shock caused by novc this caseillustrates that early fasciotomy and debridement arenecessary forsevere necrotizing fasciitis caused bynovc and prolonged intensive care may be requiredafter surgeryo139 vibrio cholerae ntprobnp nterminal probrain natriuretic peptidepmxdhp polymyxin b direct hemoperfusionacknowledgementsnoneauthors™ contributionskt tu tw kn and ku treated the patient kt tu hf reviewed the literatureand mainly wrote this report kn tw ku reviewed the literature andmodified this paper based on specialty orthopedics emergency departmentinfectious disease all authors have read and approved the manuscriptfundingnoneabbreviationsbun blood urea nitrogen fdp fibrinogen degradation productsgnr gramnegative rod mri magnetic resonance imaging novc nono1availability of data and materialsall data are included in this published 0ctsuruta bmc infectious diseases page of ethics approval and consent to participatethis case report was approved by the ethics review committee atminaminara general hospital and was conducted in accordance with thedeclaration of helsinki consent for participation is not applicableconsent for publicationwritten informed consent was obtained from the patient for publication ofthis case report and any accompanying images a copy of the writtenconsent is available for review by the editor of this journalcompeting intereststhe authors declare that they have no competing interestsauthor details1emergency department minaminara general hospital ooazafukugamiooyodocho yoshinogun nara japan 2orthopedic departmentminaminara general hospital nara japan 3infectious diseases departmentminaminara general hospital nara japan 4department of emergency andcritical care medicine nara medical university nara japanreceived march accepted august referencesgardner ad venkatraman kv the antigens of the cholera group of vibriosj hyg lond “ hirk s huhulescu s allerberger f lepuschitz s rehak s weil s necrotizing fasciitis due to vibrio cholerae nono1nono139 after exposureto austrian bathing sites wien klin wochenschr “ dobrović k rudman f ottaviani d crnek sÅ¡ leoni f Å¡krlin j a rare case ofnecrotizing fasciitis caused by vibrio cholerae o8 in an immunocompetentpatient wien klin wochenschr “jain akc varma ak mangalanandan kh kumar h bal a surgical outcome ofnecrotizing fasciitis in diabetic lower limbs j diab foot comp “ikeda t kanehara s ohtani t furukawa f endotoxin shock due to vibriovulnificus infection eur j dermatol “su ba tang hj wang yy liu yc ko wc liu cy in vitro antimicrobialeffect of cefazolin and cefotaxime combined with minocycline against vibriocholerae nono1 nono139 j microbiol immunol infect “tsai yh huang tj hsu rww weng yj hsu wh huang kc necrotizing softtissue infections and primary sepsis caused by vibriovulnificus and vibrio cholerae nono1 j trauma “publisher™s notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations morris jg nono group vibrio cholerae a look at the epidemiology of anoccasional pathogen epidemiol rev “li m shimada t morris jg jr sulakvelidze a sozhamannan s evidence forthe emergence of nono1 and nono139 vibrio cholerae strains withpathogenic potential by exchange of oantigen biosynthesis regions infectimmun “dalsgaard a albert mj taylor dn shimada t meza r serichantalergs o characterization of vibrio cholerae nono1 serogroups obtained froman outbreak of diarrhea in lima peru j clin microbiol “isaacmárquez ap lezamadávila cm eslavacampos c navarroocaña acraviotoquintana a serotypes of vibrio cholerae nono1 isolated fromwater supplies for human consumption in campeche mexico and theirantibiotic susceptibility pattern mem inst oswaldo cruz “hughes jm hollis dg gangarosa ej weaver re noncholera vibrioinfections in the united states clinical epidemiologic and laboratoryfeatures ann intern med “deshayes s daurel c cattoir v parienti jj quilici ml de la blanchardière anono1 nono139 vibrio cholerae bacteraemia case report and literaturereview springerplus mubarak sj owen ca doubleincision fasciotomy of the leg fordecompression in compartment syndromes j bone joint surg am “ wagner pd evans sd dunlap j ballonlanda g necrotizing fasciitis andseptic shock caused by vibrio cholerae acquired in san diego californiawest j med “ko w chuang y huang g hsu sy infections due to nono1 vibrio choleraein southern taiwan predominance in cirrhotic patients clin infect dis “ cheng nc tsai jl kuo ys hsueh pr bacteremic necrotizing fasciitis causedby vibrio cholerae serogroup o56 in a patient with liver cirrhosis j formosmed assoc “tsai yh hsu rww huang kc chen ch cheng cc peng kt systemicvibrio infection presenting as necrotizing fasciitis and sepsis a series ofthirteen cases j bone joint surg am “ changchien ch bacteraemic necrotizing fasciitis with compartmentsyndrome caused by nono1 vibrio cholerae j plast reconstr aesthetic surg“ maraki s christidou a anastasaki m scoulica e nono1 nono139 vibriocholerae bacteremic skin and soft tissue infections infect dis lond “ ottaviani d leoni f rocchegiani e canonico c masini l pianetti a unusual case of necrotizing fasciitis caused by vibrio cholerae o137 j clinmicrobiol “ 0c" Answer: