胃癌的个体化治疗(鼓楼医院)

胃癌的个体化药物治疗

——鼓楼医院的经验

刘宝瑞

LiuBaoruiPh.DMD南京大学医学院附属鼓楼医院肿瘤中心DrumTowerHospitalCancerCenter,NanjingUniversityMedicalSchool南京大学临床肿瘤研究所ClinicalCancerInstituteofNanjingUniversity—南京大学医学院附属鼓楼医院肿瘤中心—住院病区日间化疗放射治疗生物治疗微创治疗温热治疗中药治疗南京大学临床肿瘤所细胞室药分室病理室标本室分子室动物室学科简介学科简介江苏省医学重点学科江苏省临床重点专科江苏省青年文明号江苏省医学分子技术重点实验室南京大学临床肿瘤研究所

目录个体化疗——简介肿瘤组织——为检查对象的个体化治疗外周血浆——为检查对象的个体化治疗

目录个体化疗——简介肿瘤组织——为检查对象的个体化治疗外周血浆——为检查对象的个体化治疗“标准化疗”的成就与无奈：当今肿瘤的化疗：

胃癌：FAMFAMtxEAPFPECFFOLFOXTFP

RR:28%-46%

均<50%

肺癌：RR30％-40%肿瘤“异质性”：有效率~毒副作用------个体差异……很大个体化疗：

细胞水平－药物敏感实验

基因水平－DNA水平（突变、甲基化）

－RNA水平（mRNA、miRNA）2004年ASCO预测：

未来5-10年将是由当今标准化疗向个体化疗的过渡期2005年ASCO预测：

药物基因指导下“个体化疗”是肿瘤化疗的一场革命2006年ASCO描述：

肿瘤的化疗已经迈入“个体化疗”的新时代2009年ASCO描述：

个体化医疗成为大会主题2010年以后态势：大样本的论文相继发表“药物相关基因指导下个体化疗”个体化疗药物遗传学（pharmacogenetics)药物基因组学（pharmacognomics）药物遗传学：研究与药物反应性/毒性相关的个体间DNA序列/基因多态性/甲基化的差异。药物基因组学：将全基因组技术（即基因表达数据）用于预测一个患病个体对一个/一组药物的敏感性或抵抗性。DPD:

二氢嘧啶脱氢酶

TS:

胸腺嘧啶核苷酸合成酶

OPRT:乳清酸磷酸核糖基转移酶

TP:胸(腺嘧啶脱氧核)苷磷酸化酶氟脲密啶

(5-FU)足叶乙甙（VP－16)-------------------------MDM2开普拓（CPT11)----------------------------WRNColorectalcancerstreatedwithirinotecan开普拓（CPT11)TopoI甲氨蝶呤（MTX)------------------------DHFR中国人群STAT3rs4796793位点多态性分布：

CC40.0%（IFNa敏感性较高）

CG46.7%GG13.3%干扰素（IFNa）------------------------STAT3吉西他滨（GEM）-------------------hENT1、hCNT3紫杉类

(paclitaxel，docetaxel)

1.Cytoskeletonandpaclitaxelsensitivityinbreastcancer:Theroleofβ–tubulinIntJCancer.2007;120,2078–20852.ClinicalsignificanceofclassIIIbeta-tubulinexpressionanditspredictivevalueforresistancetodocetaxel-basedchemotherapyingastriccancerIntJOncol.2006Feb;28(2):375-81

.Microtubule-AssociatedProtein-tauisaBifunctionalPredictorofEndocrineSensitivityandChemotherapyResistanceinEstrogenReceptorPositiveBreastCancerClinCancerRes2007;13(7):2061-7甲氨蝶呤（MTX)DHFRERCC1(excisionrepaircross-Complementing1)ERCC1mRNA水平与铂类的敏感性密切相关，可以当成铂类化疗效果的独立预测指标。BRCA1(breastcancer1)参与DNA修复，与铂类药物及作用于微管蛋白药物敏感性密切相关XRCC1

(X-rayrepaircross-complementinggroup1)XRCC1基因第399位密码子由CGGCAG的变异可以导致编码的氨基酸由ArgGln。变异型的DNA修复能力提高，对铂类敏感性下降。XPD(xerodermapigmentosumpomplementarygroupD)XPD基因第751位密码子由AAGCAG的变异导致氨基酸由LysGln。变异型的表型导致DNA修复能力的提高，铂类敏感性下降。GSTP1(Glutathione-S-transferaseP1)GSTP1基因密码子105位缬氨酸(Val)

转变为异亮氨基酸(Ile)

，这一氨基酸的替换导致酶活性升高,铂类敏感性下降。铂类药物疗效相关基因(总结）

目录个体化疗——简介肿瘤组织——为检查对象的个体化治疗外周血浆——为检查对象的个体化治疗

1、方法学建立——胃癌石蜡包埋组织检查微量的mRNA水平

2、开展胃癌化疗与疗效预后的随访

3、系统检查了如下基因状况

ERCC1mRNA&proteinexpressionTsmRNA&proteinexpressionOPRTmRNA&proteinexpression筛选提高化疗效果的

XRCC1Arg399GlnSNP基因标志

GSTP1Ile105ValSNPXPDLls751GlnSNP4、统计学分析：与疗效及生存时间的关系胃癌

——

生物标志筛选+临床研究

从1760例胃癌中筛选出完整随访信息的病人IF：4.0

他引次数：22胃癌

——

生物标志筛选+临床研究

5、发现：ERCC1mRNA与含铂方案疗效与预后有关

发现：XRCC1Arg399GlnSNP与含铂方案疗效与预后有关

发现：GSTP1Ile105ValSNP和XPDLls751GlnSNP无统计意义这一发现与西方人群有明显差异

IF：4.51

他引次数：196、进一步检查了如下基因mRNA水平

BRCA1mRNARAP80mRNASUMO调控途径核心基因PIAS1和PIAS4mRNA

发现：BRCA1水平高者含Doc化疗生存时间是低表达者的

2-3倍IF：14.6胃癌

——

生物标志筛选+临床研究

国科金7、癌性腹水开展了系列性基因标志的检查

ERCC1mRNA

BRCA1mRNAβ-tubulin

Ⅲ

mRNATs

mRNA、

OPRTmRNAcellfreemiR-152cellfreemiR-21cellfreemiR-146a…..胃癌

——

生物标志筛选+临床研究

23种miRNAs胃癌

——

生物标志筛选+临床研究

再次证实：BRCA1与紫衫疗效显著相关

ERCC1与铂类疗效显著相关

发现：miR-152

与紫衫疗效具有相关性

胃癌个体化治疗BREC-CHINA

——前瞻性临床研究BREC-AGC

胃癌“生物标志指导下个体化疗”的随机对照前瞻性研究伦理批文CRF报告表T2-T3RAP80(T1-T2BRCA1)T2-T3RAP80(T3BRCA1)Gem/CisDocetaxelDocetaxel/CisT1RAP80(T1-T3BRCA1)CONTROLDocetaxel/CisAdvancedNSCLC1:1EXPERIMENTAL肺癌治疗启动肺癌个体化化疗BREC-CHINA前瞻性多中心临床研究

目录个体化疗——简介肿瘤组织——为检查对象的个体化治疗外周血浆——为检查对象的个体化治疗新一代血清生物标志

------在肿瘤个体化药物治疗中的应用前景

刘宝瑞

Ph.DMD南京大学医学院附属鼓楼医院肿瘤中心南京大学临床肿瘤研究所目录

一、血清中含有大量肿瘤生物信息二、血清中分子靶向药物生物标志

三、血清中化疗药物疗效生物标志四、南京大学附属鼓楼医院的探索一、血清中含有大量肿瘤生物信息传统意义上的生物标志

目的：肿瘤诊断和随访回顾性预测治疗效果内容：血清蛋白标志CEAAFPCA125CA199CY211

CA242CA724PSAfPSA

一、血清中含有大量肿瘤生物信息2.新一类血清生物标志——药物疗效标志及预后标志

目的：前瞻性预测药物治疗效果及肿瘤患者的预后

predictivemarkerprognosticmarker

内容：外周血循环肿瘤细胞外周血循环特定的DNA

外周血循环特定的RNA

外周血循环特定的miRNA一、血清中含有大量肿瘤生物信息文献综述——循环肿瘤细胞及游离DNACirculatingnucleicacidsasapotentialsourceforcancerbiomarkers.Russia.CurrMolMed.2010Mar;10(2):142-65.SincetheassociationofcirculatingDNAlevelchangeswithtumorgrowthwasdiscoveredmanyattemptshavebeenmadetodevelopthesensitiveandrobustblood-basedtestsforearlytumordiagnostics.BothgenomicaswellasmitochondrialDNAquantificationinthecirculationhavebeenextensivelyevaluatedasadiagnosticandprognostictooltomonitorcancertherapy.Cell-freeDNA

bearingthesamegeneticandepigeneticchangesasthetumortissueswereshowntobedetectableinplasma/serumofcancerpatientsindicatingtheprincipalpossibilitytocreatetheminimallyinvasivediagnostictestsbasedontumor-specificDNAmarkers.ApartfromcirculatingDNA,tumor-derivedRNA

inplasma/serumwasfoundtobeapromisingapproachforthedevelopmentofcancermarkers.Resultsofthelasttwoyearsestablishthequantificationofthe

tumor-derivedmicroRNAs

inplasma/serumasanextremelypromisingapproachforcancerdiagnostics.一、血清中含有大量肿瘤生物信息文献综述——循环DNA~RNA~miRNA一、血清中含有大量肿瘤生物信息文献——血浆RNA与mRNA一、血清中含有大量肿瘤生物信息Chemosensitivityprofileassayofcirculatingcancercells:prognosticandpredictivevalueinepithelialtumors.Roma,Italy.IntJCancer2010;126(10):2437-47..Theprognosticvalueassociatedwiththedetectionofcirculatingtumorcells(CTCs)inmetastaticbreastcancerbytheCellSearchtechnologyraiseadditionalissuesregardingthebiologicalvalueofthisinformation.Wepostulatedthatadrug-resistanceprofileofCTCsmaypredictresponsetochemotherapyincancerpatientsandthereforecouldbeusedforpatientselection.Onehundred5patientswithdiagnosisofcarcinomawereenrolledinaprospectivetrial.CTCswereisolatedfromperipheralblood,andpositivesampleswereevaluatedforthe

expressionofapanelofgenesinvolvedinanticancerdrugsresistance.Sensitivityofthetest:abletopredicttreatmentresponsein98%ofpatients.Specificityofthetest:100%;nosamplefromhealthysubjectwaspositiveforthepresenceofCTCs.Weidentifiedadrug-resistanceprofileofCTCs,whichispredictiveofresponsetochemotherapy,independentoftumortypeandstageofdisease.Thisapproachmayrepresentafirststeptowardtheindividualizationofchemotherapyincancerpatients.文献——循环肿瘤细胞的个体化疗标志一、血清中含有大量肿瘤生物信息二、血清中分子靶向药物疗效标志

1.外周血EGFR突变

2.胸水EGFR突变

3.外周血Kras突变

4.外周血Her2mRNA水平

二、血清中分子靶向药物疗效标志

1.外周血EGFR突变（广州）

Detectionofepidermalgrowthfactorreceptormutationsinplasmabymutant-enrichedPCRassayforpredictionoftheresponsetogefitinibinpatientswithnon-small-celllungcancer.HeC,LiuM,ZhouC,GuangzhouChina.IntJCancer.2009；125(10):2393-9.SpecimensourceandmethodsforEGFRmutationanalysisarelimitedbytissueavailabilityandtechnicalfeasibilityinclinicalapplication.Therefore,thecurrentstudyisdesignedtoestablishablood-basedapproachfortheassessmentofEGFRmutationsinNSCLCpatients,inparticulartheadvancedstage,andtotestitsclinicalapplication.Plasmasampleswereobtainedfromtheenrolled134NSCLCpatients.ThedetectionrateoftheEGFRexon19deletionsandexon21L858Rwas49.3%(66/134)bytheblood-based,mutant-enrichedpolymerasechainreaction.Inthepairedtumorandplasmasamples,thedetectedmutanttypesofeachpairrespectivelybydirectsequencingandmutant-enrichedpolymerasechainreactionwereconcordantin17of18(94.4%).Inthepatientstreatedwithgefitinibasasecond-linetherapy,thosewithplasmaEGFRmutationhaveaprolongedmedianprogression-freesurvivalcomparedwiththosewithEGFRwildtype(7.609vs.2.877months,p=0.002).Oncomparingtheefficacyofgefitinibwiththatofdocetaxel,itwasfoundthatthemedianprogression-freesurvivalwassignificantlylongerforpatientstreatedwithgefitinibthanthosewithdocetaxelinthoseharboringplasmaEGFRmutation(7.609vs.3.192months,p=0.006).Theseresultssuggestthattheblood-basedEGFRmutationstesthastheabilitytoprovideareliableguidanceforclinicaldecisionmakingforthetreatmentoftheadvancedNSCLCpatients.二、血清中分子靶向药物疗效标志

1.外周血EGFR突变（北京）

EpidermalgrowthfactorreceptormutationsinplasmaDNAsamplespredicttumorresponseinChinesepatientswithstagesIIIBtoIVnon-small-celllungcancer.

BaiH,MaoL,WangHS,BeijingCancerHospitalJClinOncol.2009Jun1;27(16):2653-9PlasmaDNAsamplesandmatchedtumorsfrom230patientswithstagesIIIBtoIVNSCLCwereanalyzedforEGFRmutationsinexons19and21byusingdenaturinghigh-performanceliquidchromatography.WecomparedthemutationsintheplasmasamplesandthematchedtumorsanddeterminedanassociationbetweenEGFRmutationstatusandthepatients'clinicaloutcomesprospectively.In230patients,wedetected81EGFRmutationsin79(34.3%)ofthepatients'plasmasamples.Wedetectedthesamemutationsin63(79.7%)ofthematchedtumors.Sixteenplasma(7.0%)andfourteentumor(6.1%)samplesshoweduniquemutations.Inthe102patientswhofailedplatinum-basedtreatmentandwhoweretreatedwithgefitinib,22(59.5%)ofthe37withEGFRmutationsintheplasmasamples,whereasonly15(23.1%)ofthe65withoutEGFRmutations,achievedanobjectiveresponse(P=.002).PatientswithEGFRmutationshadasignificantlylongerprogression-freesurvivaltimethanthosewithoutmutations(P=.044)inplasma.EGFRmutationscanbereliablydetectedinplasmaDNAofpatientswithstagesIIIBtoIVNSCLCandcanbeusedasabiomarkertopredicttumorresponsetoTKIs.二、血清中分子靶向药物疗效标志

1.胸水EGFR突变（日本）

EGFRmutationstatusintumour-derivedDNAfrompleuraleffusionfluidisapracticalbasisforpredictingtheresponsetogefitinib.KimuraH,Japan.BrJCancer.2006；95(10):1390-5.Weobtained43samples,whichwasthecell-freesupernatantofpleuralfluid,fromJapaneseNSCLCpatients,andexaminedthemforEGFRmutations.Theepidermalgrowthfactorreceptormutationstatuswasdeterminedbyadirectsequencingmethod(exons18-21inEGFR).EGFRmutationsweredetectedin11cases(E746_A750delinsevencases,E746_T751delinsAinonecase,L747_T751delinonecase,andL858Rintwocases).AcomparisonbetweentheEGFRmutantstatusandtheresponsetogefitinibinthe27patientswhoreceivedgefitinibrevealedthatallsevenpatientswithpartialresponseandoneofthesevenpatientswithstablediseasehadanEGFRmutation.NoEGFRmutationsweredetectedinthepatientswithprogressivedisease.TheresultssuggestthatDNAinpleuraleffusionfluidcanbeusedtodetectEGFRmutationsandthattheEGFRmutationstatusmaybeusefulasapredictoroftheresponsetogefitinib.二、血清中分子靶向药物疗效标志

1.外周血及胸水EGFR突变（上海）

Predictionofepidermalgrowthfactorreceptormutationsintheplasma/pleuraleffusiontoefficacyofgefitinibtreatmentinadvancednon-smallcelllungcancer.JianG,SongwenZ,CaicunZ.JCancerResClinOncol.2010；136(9):1341-7.ThefreeDNAwasisolatedfromtheplasmaof56casesandpleuraleffusionofanother32casesofadvancedNSCLC.FivecommontypesofEGFRmutationswereanalyzedbyLightCyclePCRwithTaqman-MGBprobes.EGFRgenemutationswerefoundin22ofallthe88(25%)NSCLCpatients(23.2%of56plasmasamples,28.1%ofanother32pleuraleffusionsamples).TheEGFRmutationsintheserumandthepleuraleffusionfromadvancedNSCLCpatientscanbedetectedwithLightCyclePCRusingTaqman-MGBprobes.ThemutationshighlypredicttheefficacyofgefitinibinadvancedNSCLC.二、血清中分子靶向药物疗效标志2.外周血HER2mRNA（意大利）二、血清中分子靶向药物疗效标志DetectionofoccultHER2mRNA-positivetumorcellsintheperipheralbloodofpatientswithoperablebreastcancer:evaluationoftheirprognosticrelevance.ApostolakiS……Greece.BreastCancerResTreat.2009；117(3):525-34.ToevaluatewhetherHER2mRNAcouldbeusedasamarkerofcirculatingtumorcells(CTCs).AnestedRT-PCRassaywasdevelopedandusedforthedetectionofHER2mRNA-positiveCTCs.Bloodfrom216womenwithearlybreastcancerobtainedbeforeadjuvanttreatmentwastestedforHER2mRNA-positivecellstoassesstheirprognosticvalue.NestedRT-PCRforHER2mRNAshowedhighsensitivitywhereasnoHER2mRNA-positivecellscouldbeidentifiedinthebloodofhealthydonors.HER2mRNA-positiveCTCsweredetectedin53(24.5%)of216patientsandHER2mRNAdetectionwasassociatedwithreduceddisease-freesurvival(DFS;P<0.0001)andoverallsurvival(OS;P=0.004).Inmultivariateanalysis,detectionofHER2mRNA-positiveCTCsemergedasindependentprognosticfactorforDFS(P=0.0001)andOS(P=0.003).HER2mRNAcouldbeavaluableprognosticmarkerforthedetectionofCTCsinearly

breastcancerpatients.2.外周血HER2mRNA（希腊）二、血清中分子靶向药物疗效标志

2.外周血HER2mRNA(美国）CirculatingtumorcellsinHER-2positivemetastaticbreastcancerpatientstreatedwithtrastuzumabandchemotherapy.NunesRA,LiX,KangSP,Dana-FarberCancerInstitute,USA.IntJBiolMarkers.2009;24(1):1-10.ThegoalofthispaperistopresentasensitiveandspecificmethodologyofdetectingCTCsinwomenwithHER-2positivemetastaticbreastcancer,andtoexamineitsroleasamarkerthattracksdiseaseresponseduringtreatmentwithtrastuzumab-containingregimens.ThestudyincludedpatientswithHER-2-positivemetastaticbreastcancerenrolledontwodifferentclinicalprotocolsusingatrastuzumab-containingregimen.OurstudysupportstheprognosticandpredictiveroleofthedetectionofCTCsintreatmentofHER-2-positivemetastaticbreastcancerpatients.二、血清中分子靶向药物疗效标志OriginandprognosticvalueofcirculatingKRASmutationsinlungcancerpatients.GautschiO,HuegliB,ZieglerA,USA.CancerLett.2007;254(2):265-73.BecauseofthecurrentcontroversyontheoriginandclinicalvalueofcirculatingKRAScodon12mutationsinlungcancer,wescreened180patientsusingacombinedrestrictionfragment-lengthpolymorphismandpolymerasechainreaction(RFLP-PCR)assay.WedetectedKRASmutationsin9%plasmasamplesand0%matchedlymphocytes.PlasmaKRASmutationscorrelatedsignificantlywithpoorprognosis.WevalidatedthepositiveresultsinasecondlaboratorybyDNAsequencingandfoundmatchingcodon12sequencesinbloodandtumorin78%evaluablecases.TheseresultssupportthenotionthatcirculatingKRASmutationsoriginatefromtumorsandareprognosticallyrelevantinlungcancer.3.外周血Kras突变（美国）

Potentialclinicalsignificanceofaplasma-basedKRASmutationanalysisinpatientswithadvancednon-smallcelllungcancer.WangS,AnT,WangJ,BeijingCancerHospital&InstituteClinCancerRes.2010:1324-30.DNAextractedfromplasmaandmatchedtumortissueswereobtainedfrom273patientswithadvancedstageNSCLC.KRASmutationsincodon12and13weredetectedusingPCR-restrictionfragmentlengthpolymorphism.Mutationsinplasmaandmatchedtumorswerecompared.KRASmutationwasfoundin35(12.8%)plasmasamplesand30(11.0%)matchedtumortissues.TheconsistencyofKRASmutationsbetweenplasmaandtumorsis76.7%(23of30;kappa=0.668;P<0.001).Among120patientswhoreceivedEGFR-TKItreatment,theresponseratewasonly5.3%(1of19)forpatientswithplasmaKRASmutationcomparedwith29.7%forpatientswithnoKRASmutationinplasmaDNA(P=0.024).Themedianprogression-freesurvivaltimeofpatientswithplasmaKRASmutationwas2.5monthscomparedwith8.8monthsforpatientswithwild-typeKRAS(P<0.001).KRASmutationinplasmaDNAcorrelateswiththemutationstatusinthematchedtumortissuesofpatientswithNSCLC.PlasmaKRASmutationstatusisassociatedwithapoortumorresponsetoEGFR-TKIsinNSCLCandmaybeusedasapredictivemarkerinpatientsforsuchtreatment.二、血清中分子靶向药物疗效标志3.外周血Kras突变（北京）

K-rasmutationalstatuspredictspoorprognosisinunresectablepancreaticcancer.ChenH,TuH,MengZQFudanUniversity,Shanghai,China.EurJSurgOncol.2010；

36(7):657-62.Bloodsampleswerecollectedfrom91patientswithunresectablepancreaticcancerpriortotreatment.K-rasgenewasamplifiedfromthecirculatingplasmaDNA.Mutationsweredetectedbydirectsequencing.TherelationshipbetweenthetypesofK-rasgeneandprognosisofunresectablepancreaticcancerwasevaluated.K-Rascodon12mutationswerefoundin30of91(33%)plasmaDNAsamples,17mutationswerec.35G>A(p.G12D),11werec.35G>T(p.G12V)andonly2werec.34G>C(p.G12R)).K-rascodon12mutationscouldsignificantlyreflecttheclinicalparameters,includingTNMtumorstaging(P=0.033)andlivermetastasis(P=0.014).ThemediansurvivaltimeofpatientswithK-rasmutationswasshorterthanthatofpatientswithwild-typeK-rasgene(3.9monthsvs.10.2months,P<0.001).K-rascodon12mutationfromplasmaDNAwasanindependentnegativeprognosticfactorforsurvival(hazardratio,7.39;95%confidenceinterval,3.69-14.89).二、血清中分子靶向药物疗效标志3.外周血Kras突变（上海）

三、血清中化疗药物疗效标志1.常见化疗药物的疗效标志——举例2.血清中化疗药物疗效标志——推测

三、血清中化疗药物疗效标志1.常见化疗药物的疗效标志（肿瘤组织）铂类————BRCA1、ERCC1、XRCC1

紫杉类————BRCA1、TublinIII、TublinII

吉西他滨————hENT1、RRM1

开普拓————TopoI5-FU类————Ts、Tp、OPRT

足叶乙甙————MDM2

替莫唑胺————MGMT

培美曲赛————Ts三、血清中化疗药物疗效标志2.血清中化疗药物疗效标志举例

（潜在价值）TsmRNA水平与培美曲赛疗效相关PolymorphismsofXRCC1andsurvivalingastriccancerpatientsTsmRNA水平可以在外周血中检出，且已被证实为来源于肿瘤细胞提示：外周血TsmRNA水平是否可以反映培美曲赛的疗效？三、血清中化疗药物疗效标志2.血清中化疗药物疗效标志举例

（潜在价值）三、血清中化疗药物疗效标志

铂类————BRCA1、ERCC1、XRCC1

紫杉类————BRCA1、TublinIII、TublinII

吉西他滨————hENT1、RRM1

开普拓————TopoI5-FU类————Ts、Tp、OPRT

足叶乙甙————MDM2

替莫唑胺————MGMT

培美曲赛————Ts问题——血中ERCC1mRNA意义？血中BRCA1mRNA意义？血中hENT1mRNA意义？血中TopoImRNA意义？三、血清中化疗药物疗效标志miR-21&coloncancerSchetter,etal.JAMA,2008最近证实——（1）miR表达与化疗疗效有关（2）血中存在循环miR问题提出——

血中循环miR在个体化药物治疗中的意义？四、我们在血清/血浆个体化生物标志物领域的探索2008年起，探索建立了以PNA-PCR技术为基础的血清/血浆KRAS突变检测技术和EGFR突变检测技术。该技术具有严格的实验室质控，可进行半定量分析。PNA又称肽核酸，能有效抑制野生型DNAPCR扩增。同时联合测序或Taqman等技术，能够准确分型。灵敏度(Mt/Wt)：Exon19:1/500Exon20/21:1/5000KRAS:1/5000四、我们在血清/血浆个体化生物标志物领域的探索95例肠癌肿瘤组织与术前血浆KRAS突变结果比较肿瘤组织突变型野生型合计血浆突变型30535野生型174360合计474895以肿瘤原发灶KRAS突变作为评价标准：敏感性63.8%特异性85.7%准确性76.8%血浆Kras突变检测四、我们在血清/血浆个体化生物标志物领域的探索组织未测得突变，血浆测得KRAS突变突变部位病理类型分期脉管神经淋巴结Case1G12D升结肠溃疡型中分化腺癌IV(T3N1M1肺)-++Case2G12G直肠溃疡型中低分化腺癌IIIB(T3N1MO)+++Case3G12V横结肠浸润溃疡型中分化腺癌IIIB(T3N1MO)+-+Case4G12V乙状结肠溃疡型印戒细胞癌IV(T3N2M1盆腔)