HeterogeneityofEpidermalGrowthFactorReceptorStatus

andMutationsofKRAS/PIK3CAinCirculatingTumor

CellsofPatientswithColorectalCancer

ClinicalChemistryNovember7，2012Studyintroduction美國國家癌癥綜合治療聯(lián)盟（NCCN）《結(jié)直腸癌臨床實(shí)踐指南》(2011)明確指出：(1)所有轉(zhuǎn)移性結(jié)直腸癌患者都應(yīng)檢測(cè)KRAS基因狀態(tài)；(2)只有KRAS野生型患者才建議接受EGFR抑制劑（如愛必妥和帕尼單抗）治療。NCCN《非小細(xì)胞肺癌臨床實(shí)踐指南》(2011)明確指出：當(dāng)KRAS基因發(fā)生突變時(shí)，不建議使用EGFR-TKIs靶向治療藥物。衛(wèi)生部頒布的《結(jié)直腸癌診療規(guī)范（2010年版）》也明確指出：確定為復(fù)發(fā)或轉(zhuǎn)移性結(jié)直腸癌時(shí)，檢測(cè)腫瘤組織KRAS基因狀態(tài)，以確定合適的治療方案。1.

Amado

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G，

Wolf

M，

Peeters

M，Van

Cutsem

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al.

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2008.2.

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ASCO

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Bokemeyer

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ASCO

Annual

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2008:

abstract

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2008;26:374-9.5.

美國FDA相關(guān)網(wǎng)站：Patients4Celllines,cultureconditions,andfluorescenceinSituhybridizationanalysis(MCF,MDA-MB-468,BT-20,MDA-MB-468A)EnumerationofCTCsbyCS(FITC-thefourthchanneloftheCS)

SamplepreparationformolecularanalysisafterCellSearchdetectionIsolationofCTCsbymicromanipulationWhole-GenomeAmplificationofDNAfromsingletumorcellswiththeGenomePlexkitWGAofDNAfromsingletumorcellswiththeGenomiPhikitIdentificationofEGFRgeneamplificationsbyPCRonWGAproducts(LINE1reference,EGFRtarget)Sequencingofsingle-cellWGAproductsMaterialsandMethodsResults

ApplicabilityofWGAfromsingle-cellDNADetectionofEGFRexpressionandgeneamplificationsonsingleCTCsMutationalanalysisofsingleCTCsMutationsindownstreamgenesoftheEGFRsignalingpathwayDiscussion

GenomiPhi-amplifiedDNAwasmoresuitableThemeanandmediangeneamplificationrates(43.89-and40.29-fold)determinedonGenomiPhiWGAproductsaresimilartothosedeterminedbyqPCRonDNAextracts(approximately107cells)andbyFISHanalysis(30-to40-fold)andareconsistentwithpublisheddataforMDA-MB-468cellpopulations.Although30%–90%ofadvancedprimaryCRCCasesweredescribedtobepositiveforEGFRexpression,CTCswithincreasedEGFRexpressionlevelscouldbedetectedinonly7of33(21%)CTC-positivepatients.[1]ShankaranV,ObelJ,BensonAB3rd.PredictingresponsetoEGFRinhibitorsinmetastaticcolorectalcancer:currentpracticeandfuturedirections.Oncologist2010;15:157–67.InadditiontothelowfrequencyofEGFRpositivityinourpatientcohort,notallCTCsofindividualcasescouldbeclassifiedasEGFRoverexpressing,revealingasubstantialheterogeneityinEGFRlevelsamongCTCsfromthesamepatient.ThesevaryingexpressionlevelspresumablyreflectintratumoralheterogeneityofEGFRexpression.

Unliketheoverexpressionoftheimmunotherapytargethumanepidermalgrowthfactorreceptor2(HER2)inbreastcancerpatients,whichismostoftenconnectedwithanamplificationoftheHER2gene,thecorrelationofEGFRproteinlevelsandgeneamplificationandtheirmeaningforEGFRimmunotherapyresponseisstillcontroversial.AsalreadyshownforEGFRproteinexpression,wealsoobtainedaheterogeneousdistributionofEGFRgeneamplificationratesbetweenCTCsofthesamepatientaswellasofdifferentpatients.[1]NicholsonRI,GeeJM,HarperME.EGFRandcancerprognosis.EurJCancer2001;37(Suppl4):S9–15.

[2]CiardielloF,TortoraG.Epidermalgrowthfactorreceptor(EGFR)asatargetincancertherapy:

understandingtheroleofreceptorexpressionandothermoleculardeterminantsthatcouldinflu-

encetheresponsetoanti-EGFRdrugs.EurJCancer2003;39:1348–54.

[3]MoroniM,VeroneseS,BenvenutiS,MarrapeseG,Sartore-BianchiA,DiNicolantonioF,etal.Genecopynumberforepidermalgrowthfactorreceptor(EGFR)andclinicalresponsetoantiEGFRtreatmentincolorectalcancer:acohortstudy.LancetOncol2005;6:279–86.

ThemainnoveltyofthetechnologydescribedhereisthatitallowsmolecularanalysisofindividualCTCsaftertheyarecapturedandimmunostainedbyCS.Nevertheless,wecouldshowfeasibilityofseveraldownstreamapplicationstofurthercharacterizemolecularfeaturesofsingleCTCsdetectedwithCS,includingimmunocytochemistry,mutationalanalysis,andqPCR.GenomePlexkitGenomiPhikitManufactureSigma-AldrichGEHealthcareTechnique基于PCR技術(shù)-LMP不基于PCR技術(shù)-MDADNAamountsofWGAproductsMean8.9ugRange4.6–15.4ugMean1.54ugRange0.3–2.2ugAdequateDNAquality(atleast2of4PCRproductsaftermultiplexPCR)8of1111of11Reactions6of119of11ThemeanEGFRgeneamplificationstatus14.72Range0.56-40.35Median11.2243.89Range7.22–90.07Median40.29returnEGFRgeneexpressiondetectedbyCS(leftimages)correlateswithEGFRgeneamplificationratesdeterminedbyqPCRandFISH(righttables)withMCF-7(lowEGFRexpression=score0–1,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.continue10ngPurifiedproductcontinueToanalyzeCTCheterogeneitymolecularly,wefocusedonbloodsamples(n=5)withmorethan20

morphologicallyintactCTCsper7.5mL,whichexplainsinpartthelownumberofsamplesanalyzedbysingle-cellPCR.ThefailuretoanalyzeahighernumberofdetectedCTCsismainlyduetotheinabilitytotransferallCTCsundisturbedfromtheCellSearchcartridgeontoslidesandreidentifythemformicromanipulation.Thus,fromall33patientsanalyzedforEGFRexpressionofCTCs,onlyCTCsfrom3mCRCpatientscouldalsobeanalyzedforEGFRgeneamplificationbyqPCR.EGFRgeneamplificationratedeterminedbyqPCRin26analyzedCTCsfrompatients6,9,and26.Comp.;CK-PE,cytokeratinphycoerythrin;DAPI,4’,6-diamidino-2-phenylindole;APC,allophycocyanin;FITC,fluoresceinisothiocyanate.returnFortheestablishmentofatechniquetodetectmutationsonWGAproductsfromsinglecells,weusedMDA-MB-231cellscarryingap53mutation.ToinvestigatetheimpactofcontaminationofasingleCTCwithsurroundingleukocytesduringmicromanipulation,

weperformedamutationalanalysisonGenomiPhi

WGAproductsfromaMDA-MB-231singlecellsupplementedwith1–2leukocytes.Detectionofthep53R280KmutationinasingleMDA-MB-231cell(red).Additionofupto2leukocytes(green)orcell-freeliquidfromtheCScartridge(bluewaves)toasingleMDA-MB-231cellbymicromanipulationdidnotdisturbthedetectionofthep53mutation.returnPIK3CAmutationPIK3CAmutationE545APIK3CAmutationE542KPatient69of15CTCs60%6of93of9Patient91of5CTCs20%--------Patient181of3CTCs33%--------Patient263of11CTCs27%--------OthersCTCsnotmutated--------AKRAS(G12V)mutationfromCTCsAKRAS(G12V)mutationfromPrimarytumorPatient65of15(33%)CTCsMutationPatient9----Wild-typePatient18----Wild-typePatient26----Wild-type(B),CTCscarryingPIK3CA(n=9)andKRASmutation(n=5)

obtainedfrompatient6(totalanalyzedCTCs,n=15;wild-typeformofbothgenes,n=6)illustratethegenetic

heterogeneitypr