Heterogeneity of Epidermal Growth Factor Receptor Statusand Mutations of KRAS / PIK3CA in Circulating TumorCells of Patients with Colorectal Cancer ClinicalChemistryNovember7，2012在结直肠癌循环肿瘤细胞中和的突变以及异质性的研究在结直肠癌循环肿瘤细胞中和的突变以及异质性的研究Studyintroduction美国国家癌症综合治疗联盟（NCCN）结直肠癌临床实践指南(2011)明确指出：(1)所有转移性结直肠癌患者都应检测KRAS基因状态；(2)只有KRAS野生型患者才建议接受EGFR抑制剂（如爱必妥和帕尼单抗）治疗。NCCN非小细胞肺癌临床实践指南(2011)明确指出：当KRAS基因发生突变时，不建议使用EGFR-TKIs靶向治疗药物。 卫生部颁布的结直肠癌诊疗规范（2010年版）也明确指出：确定为复发或转移性结直肠癌时，检测肿瘤组织KRAS基因状态，以确定合适的治疗方案。1.AmadoRG，WolfM，PeetersM，VanCutsemEetal.JournalofClinicalOncology.April2008.2.VanCutsemetal.ASCOAnnualMeeting2008:abstract2.3.Bokemeyeretal.ASCOAnnualMeeting2008:abstract4000.4.Livreetal.JClinOncol2008;26:374-9.5.美国FDA相关网站：在结直肠癌循环肿瘤细胞中和的突变以及异质性的研究EGFR是一种跨膜酪氨酸激酶受体，与配体结合后可引起下游信号传导通路的活化， 如 ：KRASBRAF(4%12%) RAS-RAF-MEK-Erk/MAPK PIK3CA(14%20%) P13K-AKT-PKC-IKK 在许多实体瘤中 EGFR均有不同程度的表达，表达率最高为头颈部肿瘤 ， 达 95%100%。 CRC次之 ， 为 72%89%， 胃 癌表达率为 41%64%，并且 EGFR表达率高的肿瘤恶性度高 ，侵袭力强 ， 预后差 。而且在同一患者中不同的CTCs中，EGFR的表达也存在着异质性。1GoldsteinNS,ArminM.EpidermalgrowthfactorreceptorimmunohistochemicalreactivityinpatientswithAmericanJointCommitteeonCancerStageIVcolonadenocarcinoma:ImplicationsforastandardizedscoringsystemJ.Cancer,2001,92(5):1331-1346.2BergstromJD,WestermarkB,HeldinNE.Epidermalgrowthfactorre-ceptorsignalingactivatesmetinhumananaplasticthyroidcarcinomacellsJ.ExperimentalCellResearch,2000,259(1):292-299.3DenningMF,DlugoszAA,ChengC,etal.Cross-talkbetweenepidermalgrowthfactorreceptorandproteinkinaseCduringcalciumnduceddifferentiationofkeratinoytesJ.ExperimentalDermatology,2000,9(3):192-199.在结直肠癌循环肿瘤细胞中和的突变以及异质性的研究Patients 4 Cell lines , culture conditions , and fluorescence in Situ hybridization analysis (MCF,MDA-MB-468,BT-20,MDA-MB-468A)Enumeration of CTCs by CS(FITC- the fourth channel of the CS) Sample preparation for molecular analysis after Cell Search detection Isolation of CTCs by micromanipulation Whole-Genome Amplification of DNA from single tumor cells with the GenomePlex kitWGA of DNA from single tumor cells with the GenomiPhi kit Identification of EGFR gene amplifications by PCR on WGA products (LINE1 reference,EGFR target)Sequencing of single-cell WGA products MaterialsandMethods在结直肠癌循环肿瘤细胞中和的突变以及异质性的研究ResultsApplicabilityofWGAfromsingle-cellDNADetectionofEGFRexpressionandgeneamplificationsonsingleCTCsMutationalanalysisofsingleCTCsMutationsindownstreamgenesoftheEGFRsignalingpathway在结直肠癌循环肿瘤细胞中和的突变以及异质性的研究DiscussionGenomiPhi-amplifiedDNAwasmoresuitableThemeanandmediangeneamplificationrates(43.89-and40.29-fold)determinedonGenomiPhiWGAproductsaresimilartothosedeterminedbyqPCRonDNAextracts(approximately107cells)andbyFISHanalysis(30-to40-fold)andareconsistentwithpublisheddataforMDA-MB-468cellpopulations.在结直肠癌循环肿瘤细胞中和的突变以及异质性的研究Wedetected2CTCsin49%ofpatientswithmCRC(24of49)and22%ofpatientswithnmCRC(7of32).Theseresultsaresimilartopreviousfindingsrevealingdetectionratesof2CTCsin33%61%ofpatientswithmCRC(17,30)and26%ofpatientswithnmCRC.1CohenSJ,PuntCJ,IannottiN,SaidmanBH,SabbathKD,GabrailNY,etal.Relationshipofcirculatingtumorcellstotumorresponse,progression-freesurvival,andoverallsurvivalinpatientswithmetastaticcolorectalcancer.JClinOncol2008;26:321321.2SastreJ,MaestroML,PuenteJ,VeganzonesS,AlfonsoR,RafaelS,etal.Circulatingtumorcellsincolorectalcancer:correlationwithclinicalandpathologicalvariables.AnnOncol2008;19:9358.58:15.在结直肠癌循环肿瘤细胞中和的突变以及异质性的研究Although30%90%ofadvancedprimaryCRCCasesweredescribedtobepositiveforEGFRexpression,CTCswithincreasedEGFRexpressionlevelscouldbedetectedinonly7of33(21%)CTC-positivepatients.1ShankaranV,ObelJ,BensonAB3rd.PredictingresponsetoEGFRinhibitorsinmetastaticcolorectalcancer:currentpracticeandfuturedirections.Oncologist2010;15:15767.在结直肠癌循环肿瘤细胞中和的突变以及异质性的研究InadditiontothelowfrequencyofEGFRpositivityinourpatientcohort,notallCTCsofindividualcasescouldbeclassifiedasEGFRoverexpressing,revealingasubstantialheterogeneityinEGFRlevelsamongCTCsfromthesamepatient.ThesevaryingexpressionlevelspresumablyreflectintratumoralheterogeneityofEGFRexpression.在结直肠癌循环肿瘤细胞中和的突变以及异质性的研究Unliketheoverexpressionoftheimmunotherapytargethumanepidermalgrowthfactorreceptor2(HER2)inbreastcancerpatients,whichismostoftenconnectedwithanamplificationoftheHER2gene,thecorrelationofEGFRproteinlevelsandgeneamplificationandtheirmeaningforEGFRimmunotherapyresponseisstillcontroversial.AsalreadyshownforEGFRproteinexpression,wealsoobtainedaheterogeneousdistributionofEGFRgeneamplificationratesbetweenCTCsofthesamepatientaswellasofdifferentpatients.1NicholsonRI,GeeJM,HarperME.EGFRandcancerprognosis.EurJCancer2001;37(Suppl4):S915.2CiardielloF,TortoraG.Epidermalgrowthfactorreceptor(EGFR)asatargetincancertherapy:understandingtheroleofreceptorexpressionandothermoleculardeterminantsthatcouldinflu-encetheresponsetoanti-EGFRdrugs.EurJCancer2003;39:134854.3MoroniM,VeroneseS,BenvenutiS,MarrapeseG,Sartore-BianchiA,DiNicolantonioF,etal.Genecopynumberforepidermalgrowthfactorreceptor(EGFR)andclinicalresponsetoantiEGFRtreatmentincolorectalcancer:acohortstudy.LancetOncol2005;6:27986.在结直肠癌循环肿瘤细胞中和的突变以及异质性的研究Foraresponsetoanti-EGFRtherapies,anormalfunctionofdownstreamelementsofthesignalingpathway(e.g.,KRAS,BRAF,andPIK3CA)isessential.Toovercometheselimitations,wesuccessfullyestablishedaprotocolforthemutationalanalysisofDNAfromsingleCTCsdetectedbyCS.在结直肠癌循环肿瘤细胞中和的突变以及异质性的研究ThemainnoveltyofthetechnologydescribedhereisthatitallowsmolecularanalysisofindividualCTCsaftertheyarecapturedandimmunostainedbyCS.Nevertheless,wecouldshowfeasibilityofseveraldownstreamapplicationstofurthercharacterizemolecularfeaturesofsingleCTCsdetectedwithCS,includingimmunocytochemistry,mutationalanalysis,andqPCR.在结直肠癌循环肿瘤细胞中和的突变以及异质性的研究GenomePlex kit GenomiPhi kit ManufactureSigma-AldrichGEHealthcareTechnique基于PCR技术-LMP不基于PCR技术-MDADNAamountsofWGAproductsMean8.9ugRange4.615.4ugMean1.54ugRange0.32.2ugAdequateDNAquality(atleast2of4PCRproductsaftermultiplexPCR)8of1111of11Reactions6of119of11ThemeanEGFRgeneamplificationstatus14.72Range0.56-40.35Median11.2243.89Range7.2290.07Median40.29return在结直肠癌循环肿瘤细胞中和的突变以及异质性的研究EGFRgeneexpressiondetectedbyCS(leftimages)correlateswithEGFRgeneamplificationratesdeterminedbyqPCRandFISH(righttables)withMCF-7(lowEGFRexpression=score01,EGFRamplificationrate0.55/0.7),MDA-MB-468A(moderateEGFRexpression=score2,EGFRamplificationrate1.83/notanalyzed),BT-20(strongEGFRexpression=score3,EGFRamplificationrate6.43/8.2),andMDA-MB-468(strongEGFRexpression=score3,EGFRamplificationrate38.65/30)cells.TheEGFRqPCRwasperformedonDNAextractsfromapproximately107CellsaswellasonWGAproductsfrom10singlecellsafterCS.continue10ng Purifiedproduct在结直肠癌循环肿瘤细胞中和的突变以及异质性的研究Atleast2CTCsweredetectedin24of49(49%)patientswithmCRCand7of32(22%)patientswithnmCRC.Wefurtherassessed741CTCsfrom33patientswithCRC(27mCRC,6nmCRC)forEGFRproteinexpression.Altogether,1EGFR-positiveCTCcouldbeobservedin7of33(21%)patients,withonly2of33patients(6%)possessingstronglyEGFR-positiveCTCs.WhereasallCTCsdetectedinnmCRCpatientswereEGFR-negative,increasedEGFRlevelswereobservedin7of27(26%)patientswithmCRC.Furthermore,EGFRwasdifferentlyexpressedbetweenCTCsfromthesamepatients,rangingforexamplefromEGFR-negativetostronglyEGFR-positive.HeterogeneityinEGFRexpressionintheCTCpopulationofpatient9.continue在结直肠癌循环肿瘤细胞中和的突变以及异质性的研究continue在结直肠癌循环肿瘤细胞中和的突变以及异质性的研究ToanalyzeCTCheterogeneitymolecularly,wefocusedonbloodsamples(n=5)withmorethan20morphologicallyintactCTCsper7.5mL,whichexplainsinpartthelownumberofsamplesanalyzedbysingle-cellPCR.ThefailuretoanalyzeahighernumberofdetectedCTCsismainlyduetotheinabilitytotransferallCTCsundisturbedfromtheCellSearchcartridgeontoslidesandreidentifythemformicromanipulation.Thus,fromall33patientsanalyzedforEGFRexpressionofCTCs,onlyCTCsfrom3mCRCpatientscouldalsobeanalyzedforEGFRgeneamplificationbyqPCR.EGFRgeneamplificationratedeterminedbyqPCRin26analyzedCTCsfrompatients6,9,and26.Comp.;CK-PE,cytokeratinphycoerythrin;DAPI,4,6-diamidino-2-phenylindole;APC,allophycocyanin;FITC,fluoresceinisothiocyanate.return在结直肠癌循环肿瘤细胞中和的突变以及异质性的研究FortheestablishmentofatechniquetodetectmutationsonWGAproductsfromsinglecells,weusedMDA-MB-231cellscarryingap53mutation.ToinvestigatetheimpactofcontaminationofasingleCTCwithsurroundingleukocytesduringmicromanipulation,weperformedamutationalanalysisonGenomiPhiWGAproductsfromaMDA-MB-231singlecellsupplementedwith12leukocytes.Detectionofthep53R280KmutationinasingleMDA-MB-231cell(red).Additionofupto2leukocytes(green)orcell-freeliquidfromtheCScartridge(bluewaves)toasingleMDA-MB-231cellbymicromanipulationdidnotdisturbthedetectionofthep53mutation.return在结直肠癌循环肿瘤细胞中和的突变以及异质性的研究GeneCodons CTCs analyzed KRAS12,1359CandetectedBRAF60044NotbedetectedPIK3CA542,554,104739CandetectedPatient IDMutated gene Patient6=1mutatedgenePatient9=1mutatedgenePatient18=1mutatedgenePatient22-deletePatient26=1mutatedgenecontinue第一步第二步在结直肠癌循环肿瘤细胞中和的突变以及异质性的研究PIK3CA mutationPIK3CA mutation E545A PIK3CA mutation E542KPatient69of15CTCs60%6of93of9Patient91of5CTCs20%-Patient181of3CTCs33%-Patient263of11CTCs27%-OthersCTCsnotmutated-A KRAS (G12V) mutation from CTCsA KRAS (G12V) mutation fromPrimary tumor Patient65of15(33%)CTCs MutationPatient9-Wild-typePatient18-Wild-typePatient26-Wild-type(B),CTCscarryingPIK3CA(n=9)andKRASmutation(n=5)obtainedfrompatient6(totalanalyzedCTCs,n=15;wild-typeformofbothgenes,n=6)illustratethegeneticheterogeneitypresentinaCTCpopulation.(C),PIK3CAgenestatusinanalyzedCTCsfrompatients9,18,22,and26.MUT,mutation;WT,wildtype.return在结直肠癌循环肿瘤细胞中和的突变以及异质性的研究Patients Thelocalethicscommitteeapprovedthisstudy,andwritteninformedconsentwasobtainedfromallpar-ticipants.Bloodsamples(7.5mL)32patientsnmCRC49patientsmCRCTheUniversityMedicalCenterHamburg-EppendorfTheMedicalUniversityofGrazSTUDYSTUDYSAMPLESSAMPLESTREATTREATcontinue在结直肠癌循环肿瘤细胞中和的突变以及异质性的研究continue在结直肠癌循环肿瘤细胞中和的突变以及异质性的研究continue+在结直肠癌循环肿瘤细胞中和的突变以及异质性的研究continue在结直肠癌循环肿瘤细胞中和的突变以及异质性的研究WeperformedfurthermolecularanalysisonCTCsfrom5patientswithmCRChavingadvancedstage(DukesD)diseaseA期癌肿浸润深度限于直肠壁内，未穿出深肌层，且无淋巴结转移。B期癌肿侵犯浆膜层，亦可侵入浆膜外或肠外周围组织，但尚能整块切除， 无淋巴结转移。C期癌肿侵犯肠壁全层或未侵犯全层，但伴有淋巴结转移：C1期癌肿伴有癌灶附近肠旁及系膜淋巴结转移；C2期癌肿伴有系膜根部淋巴结转移，尚能根治切除。D期癌肿伴有远处器官转移、局部广泛浸润或淋巴结广泛转移不能根治性 切除。return在结直肠癌循环肿瘤细胞中和的突变以及异质性的研究WGA方法return在结直肠癌循环肿瘤细胞中和的突变以及异质性的研究