张波液体活检在肠癌精准医疗中的应用

张波液体活检在肠癌精准医疗中的应用第1页/共57页结直肠癌的发生率和死亡率http://globocan.iarc.fr/Pages/fact_sheets_population.aspx第2页/共57页19992000200120022003200420052006200720082009201020112012卡培他滨单药美国依立替康

(+5-FU+叶酸)依立替康

(+5-FU+亚叶酸)奥沙利铂(+5-FU+亚叶酸)卡培他滨联合治疗BEV+氟嘧啶为基础的CTBEV+氟嘧啶为基础的CTBEV+CT西妥昔单抗+化疗(EGFR/

KRAS野生型)帕尼单抗+FOLFOX(EGFR/

KRAS野生型)欧盟奥沙利铂(+5-FU+亚叶酸)瑞戈非尼

三/四线阿柏西普

(二线)西妥昔单抗+FOLFIRI(KRAS野生型)西妥昔单抗+化疗(EGFR/

RAS野生型)2014无分子标记物KRAS标记物RAS标记物mCRC治疗方案的更新-从KRAS到RAS分子标记物的发展史第3页/共57页Metastaticcolorectalcancerpatienttriagetoanti-EGFRtreatmentinclinicalpractice第4页/共57页结直肠癌分子靶向治疗相关检测瑞格菲尼西妥昔/帕尼单抗Aspirin第5页/共57页结直肠癌分子检测概略IHC:MMRLynchsyndrome预防：LS；治疗：5-FU；预后：MSI；靶向治疗；预后第6页/共57页K-RAS突变第7页/共57页KRASmutationsinvariouscancertypes第8页/共57页KRASrecurrentmutationsincancers第9页/共57页KRAS突变在原发及转移结肠癌灶

第10页/共57页K-RAS基因突变检测Cetuximab(西妥昔)andpanitumumab(帕尼)aremonoclonalantibodiesthatbindtotheextracellulardomainofEGFR,therebyinhibitingdownstreamsignalingandresultingindecreasedcellproliferationandmigration.K-RASforpredictingresponsetoanti-EGFRantibodies,cetuximaborpanitumumab.codons12oftheK-RASgenealmostneverbenefitedfromtreatmentwiththeseantibodies.However,15–20%ofpatientswithwild-typeK-RASshowanobjectiveresponsewithantibodyaloneand35–40%,whentreatedwithcetuximabandirinotecan.G13Dmaybeanexception.administrationofcetuximabtopatientswiththismutationwasassociatedwithasignificantlybetteroutcomethanthatseeninpatientswithothertypesofK-RASmutations.第11页/共57页Anti-epidermalgrowthfactorreceptor(EGFR)treatmentofMetastaticcolorectalcancer(mCRC)patients瑞格菲尼西妥昔/帕尼单抗第12页/共57页KRAS基因突变检测的意义KRAS基因突变与anti-EGFR靶向治疗：G13D例外：需进一步证实；However,codon12and13mutationsmaydifferintermsoftheirclinicalimpact.Indeed,evidencederivedretrospectivelyinasmallcohort(n=32)ofchemotherapy-refractorymCRCpatientssuggeststhatpatientswithtumorsharboringG13Dmutations(thethirdmostfrequentKRASmutationinCRC)maybenefitfromanti-EGFRantibodytherapy.KRAS基因突变的预后意义；第13页/共57页KRAS检测的有关问题质控：与敏感性定量：averylowfrequencyofKRASmutationscouldnotimpairanti-EGFRtherapiesactivity.肿瘤异质性：KRAS突变克隆；第14页/共57页Clinicalimpactofminormutantsubpopulations―KRAS突变定量化averylowfrequencyofKRASmutationscouldnotimpairanti-EGFRtherapiesactivity.第15页/共57页结直肠癌治疗与ExtendedRAS检测KRAS

(exon2codons12/13)beanegativepredictivebiomarkerforEGFR-directedmonoclonalantibodiesamongpatientswithcolorectalcancer.ExtendedRASanalysisincludingadditionalRASmutations(KRASexons3/4andNRASexon1/2/3/4).ExtendedRASanalysisshouldbeconsideredbeforeinitiatinganti-EGFRtherapytopatientsofmetastaticCRC.KRASexons2/3/4andNRASexon1/2/3/4第16页/共57页InadditiontotestingformutationsinKRASexon2(codons12and13)asrecommendedpreviously,beforetreatmentwithanti-EGFRantibodytherapy,patientswithmCRCshouldhavetheirtumortestedformutationsin:●KRASexons3(codons59and61)and4(codons117and146)●NRASexons2(codons12and13),3(codons59and61),and4(codons117and146).第17页/共57页ExtendedRASanalysis

ExtendedRAS:KRAS(beyondexon2)andNRASKRAS:Exon2codon(12and13),exon3(codon61)andexon4(codons117and146);NRAS:exon2(codons12and13),exon3(codon61),andexon4(codons117and146);第18页/共57页Rasfamily第19页/共57页Rasmutationandactivation第20页/共57页PRIMEstudy,primaryendpointsof(PFSandOS)efficacyresultsaccordingtoRASmutationstatus第21页/共57页PRIMEstudy第22页/共57页FIRE3study第23页/共57页FIRE3study第24页/共57页KRAS检测的拓展KRAS基因拷贝数(KRASgenecopynumber,GCN)：AnincreaseinKRASgenecopynumber(GCN)hasbeenassociatedwithamoreactive‘mutation-like’phenotype.KRASGCNisasmallsubset(2%)ofwild-typetumors(0.67%)andmutuallyexclusivewithKRASmutations.highCGNofwild-typeKRASmaynotrespondtocetuximabadministration,andmayalsobeacquiredduringtreatmentwithEGFRinhibitors.KRAS基因12,13密码外突变：61,146;KRAS同源基因突变：Neuroblastoma-RAS(NRAS)status:NRASmutationrateinCRCis3–5%andmostmutationsoccurincodon61,ratherthancodon12or13.mutationsinNRASandKRASaremutuallyexclusive.NRASmutationshaveasignificantlylowerresponseratethanwildtype.第25页/共57页Dynamicmonitoringanddrugsusing第26页/共57页Circulatingtumormarkers第27页/共57页第28页/共57页RAS检测:一线治疗决策的重要因素1.VanCutsemE,etal.AnnOncol2014;25(suppl3):iii1–iii9

2.NCCNclinicalpracticeguidelines;ColonCancer,Version2.2015.Availableat/professionals/physician_gls/pdf/colon.pdf.(accessedApril2015)

3.ErbituxSmPCJune/2014;4.VectibixSmPCFebruary/2015“Theavailabilityofanexpanded

RASstatusisaprerequisiteforanyuseofananti-EGFRantibody”NCCN20152ESMO20141“Thepanelstronglyrecommendsgenotypingoftumortissue(eitherprimarytumorormetastasis)inallpatientswithmetastaticcolorectalcancerforRAS(KRASexon2andnon-exon2;NRAS)andBRAFatdiagnosisofstageIVdisease”2014/2015指南推荐

CetuximabandpanitumumabareapprovedinpatientswithRASwtmCRC.3,4

CetuximabandpanitumumabarenotindicatedforthetreatmentofpatientswithmCRCwhosetumorshaveRASmutationsorforwhomRAStumorstatusisunknown3,4第29页/共57页肠癌原发灶与转移灶：基因突变不一致原发灶转移灶N=107对53%51%21%21%13%6%6%12%56%50%21%19%10%5%5%9%KopetzS,etal.ASCO2014(Abstractno.3509);adaptedfromupdatedinformationpresentedatmeeting:

http:///content/94598?media=sl(accessedJune302015)第30页/共57页既往治疗过的CRC原发灶与转移灶不一致原发灶和转移灶切除之间的任何化疗都导致:2.7倍高的不一致率

(95%CI1.3–6.0,p=0.005)9080706050403020100012+14%31%30%化疗线数突变配对-所有检测基因DiscordantConcordant突变数KopetzS,etal.ASCO2014(Abstractno.3509);adaptedfromupdatedinformationpresentedatmeeting:

http:///content/94598?media=sl(accessedJune302015)第31页/共57页CRC肿瘤基因异质性SottorivaA,etal.NatGenet2015;47:209–21615例肠癌样本的349个腺体高度肿瘤内基因异质性（ITH）第32页/共57页KRAS检测的方法Asageneralrule,amutationfrequencyof40%andaclusterofthreemutationtypes(p.G12D,p.G12Vandp.G13D)inprimarytumorsandmetastasescanbeconsideredbenchmarksforroutineKRASanalyses.KRASmutationalstatusbydirectsequencing;High-resolutionmeltinganalysis;TheraScreenkit;KRASmutationalstatusbycobas;StripAssay;Pyrosequencing;Next-generationsequencing;第33页/共57页CTCAdaptedfromFleischhackerM,SchmidtB.NatMed2008;14:914–915肿瘤特异性变化（如突变）肿瘤肿瘤细胞释放DNA循环肿瘤DNA正常DNACTC血管生物标志物检测创新技术:液体活检*TheliquidbiopsyRASIVDisawaitingaCEmarkand,therefore,itisnotcurrentlybeingmarketed检测方法：BEAMing，qtPCR，coldPCR，ARMS……第34页/共57页Digital

PCR（数字PCR）PCR扩增前对样品进行微滴化处理突变型和野生型特异性的探针绝对定量灵敏度<0.1%第35页/共57页二代测序仪(部分)Humangenome=3Gigabases(Gb)

第36页/共57页生物信息学分析

数据库比对1、所有类型突变及比例2、拷贝数变化3、外来基因组4、全基因组变化第37页/共57页ASCO2014默克雪兰诺与SysmexInostics签订合作协议默克-Sysmex：液体活检技术*WCGC2015首次公布一致性数据:ScottR,etal.WCGC2015(AbstractNo.P-273)ECC2015增加一致性数据:JonesFS,etal.ECC2015(AbstractNo.2012)中国：液体活检*科研用试剂盒已上市，BEAMing平台待搭建技术开发与验证*上市2015年2月第一个RAS液体活检中心启动*LaunchofaCE-markedIVDforRASusingliquidbiopsiesbySysmexInostics,incollaborationwithMerckKGaA(Darmstadt,Germany),isexpectedin2015(RUOkitalreadyavailable)第38页/共57页SchematicofBEAMing第39页/共57页Photographofatypicalmicroemulsion第40页/共57页Densityplotsofflow-cytometricdataobtainedfromBEAMing第41页/共57页DensityplotsofBEAMingwithgenomicDNAorRT-PCRproductsastemplate第42页/共57页DetectionandvalidationofvariantspresentinaminorfractionoftheDNApopulation第43页/共57页配对的血和组织†

标本来自31例III/IV期CRC患者*cfDNA血检测使用OncoBEAM™33-mutationRASpanel(SysmexInostics)

†使用诊断时获得的原发灶或转移灶FFPE肿瘤标本

‡标准检测患者的RAS突变，同时用液体活检方法测血标本也是突变

§标准检测患者的RAS野生，同时用液体活检方法测血标本也是野生液体活检*对比标准组织检测：RAS检测高一致率1.ScottR,etal.WCGC2015(AbstractNo.P-273)‡§*LaunchofaCE-markedIVDforRASusingliquidbiopsiesbySysmexInostics,incollaborationwithMerckKGaA(Darmstadt,Germany),isexpectedin2015(RUOkitalreadyavailable)n=13n=16n=8n=13n=21n=29第44页/共57页ECC2015荟萃分析:液体活检*和组织检测mCRC的RAS突变具有高一致率JonesFS,etal.ECC2015(AbstractNo.2012)纳入的两项研究使用OncoBEAM™ExpandedRASpanel来检测血浆RAS突变，Sanger或焦磷酸测序检测原发灶或转移灶FFPE标本中的RAS突变

组织标本与血标本配对比较mCRC病例:46例新诊断患者和22例进展/复发患者一致率,

n/N(%)欧洲和亚太数据荟萃IV期(mCRC)阳性一致率（敏感性）36/39(92.3)阴性一致率（特异性）29/29(100)总体一致率65/68(95.6)RAS突变:血(55%)vs组织标本(57%)*LaunchofaCE-markedIVDforRASusingliquidbiopsiesbySysmexInostics,incollaborationwithMerckKGaA(Darmstadt,Germany),isexpectedin2015(RUOkitalreadyavailable)第45页/共57页其他KRAS血检测研究：

特异性高，敏感性差别较大敏感性高的方法多采用BEAMing或定量PCR第46页/共57页最初西妥昔单抗治疗有效，后进展诊断获得性耐药

用BEAMing法定量分析血里KRAS(Q61H)突变DNA液体活检未来的潜在用途*MisaleS,etal.Nature2012;486:532‒536DetectionofcirculatingKRASmutantDNAinasinglepatientwithacquiredresistancetocetuximabtherapy;thresholdpercentageofmutationunknown;BEAM:beads,emulsification,amplification,andmagnetics*TheliquidbiopsyRASIVDisawaitingaCEmarkand,therefore,itisnotcurrentlybeingmarketedPD,progressivedisease第47页/共57页液体活检发现mCRC患者抗EGFR治疗获得性耐药机制1.BettegowdaC,etal.SciTranslMed2014;6:224ra24;

2.SiravegnaG,etal.NatMed2015;21:795–801;mCRC患者循环肿瘤DNA中发现抗EGFR治疗获得性耐药突变1mCRC患者血检测发现抗EGFR耐药相关基因改变2第48页/共57页液体活检测出的mCRC耐药突变*研究方法疾病进展时测出的(K)RAS突变n/N%Diazetal,2012**1PCRligation/BEAMing9/2437.5Misaleetal,2012**2NGS/BEAMing2/366.7Morellietal,2015**3BEAMing27/6243.5Misaleetal,

20144BEAMing2/450.0Siravegnaetal,20155ddPCR11/1668.8**KRASonly*LaunchofaCE-markedIVDforRASusingliquidbiopsiesbySysmexInostics,incollaborationwithMerckKGaA(Darmstadt,Germany),isexpectedin2015(RUOkitalreadyavailable)ddPCR,dropletdigitalPCR;NGS,next-generationsequencing;PCR,polymerasechainreaction1.DiazL,etal.Nature2012;486:537–540;

2.MisaleS,etal.Nature2012;486:532‒536;

3.MorelliM,etal.AnnOncol2015;26:731–736;

4.MisaleS,etal.SciTranslMed2014;6:224ra26;

5.SiravegnaGetal.NatMed2015;21:795–801第49页/共57页液体活检*:快速和最小创伤的检测1.DiehlF,etal.ProcNatlAcadSciUSA2005;102:16368–16373;

2.DiehlF,etal.NatMed2008;14:985–990液体活检*最小创伤快速获得结果精确敏感1,2避免肿瘤异质性带来的选择偏倚1,2获得最新的突变状态临床获益诊断时能为患者带来最佳的个体化治疗决策提供患者最新的突变状态未来可能性动态监测生物标志物和疗效变化促进精准医疗发展*LaunchofaCE-markedIVDforRASusingliquidbiopsiesbySysmexInostics,incollaborationwithMerckKGaA(Darmstadt,Germany),isexpectedin2015(RUOkitalreadyavailable)第50页/共57页KRAS突变等位基因与抗EGFR治疗的关系肿瘤负荷CEAKRASp.Q61L突变等位基因(%)肿瘤负荷(基线%)或CEA(ng/ml×10–1)基线1stCT扫描1线PDFolfoxiri+PanitPanitFolfiri节律化疗2010,12,242011,02,21CT扫描:PR2011,06,23最后抗EGFR治疗2011,07,012011,10,062011,11,07肿瘤负荷CEAKRASp.Q61L突变等位基因(%)肿瘤负荷(基线%)或CEA(ng/ml×10–1)基线1stCT扫描1线PDFolfoxiri+CetuxCetuxFolfiri+BevBev2011,12,152012,04,12CT扫描:CR无可预测疾病2013,01,24最后抗EGFR治疗2013,08,222014,06,19CT扫描:PDFolfox+Bev肿瘤负荷CEAKRASp.Q61L突变等位基因(%)肿瘤负荷(基线%)或CEA(ng/ml×10–1)基线1stCT扫描1线PD手术手术Folfoxiri+CetuxFolfoxiri+CetuxFolFOX+BevBev2012,04,192012,06,08CT扫描:PR2012,08,16~2012,10,11手术：原发病灶和肝转移灶2013,06,182013,08,212014,07,03FolFOX+Bev2013,06,13最后抗EGFR治疗2013,10,22手术：肝转移灶肿瘤负荷CEAKRASp.Q61L突变等位基因(%)肿瘤负荷(基线%)或CEA(ng/ml×10–1)基线1stCT扫描1线PD手术Folfoxiri+PanitFolFOX+BevBev2011,04,042011,06,15CT扫描:PR手术：结肠转移灶2012,06,282012,10,052014,04,232012,03,07最后抗EGFR治疗2013,09,172012,12射频治疗疾病进展期患者接受抗EGFR治疗时，循环DNA出现KRAS突变等位基因，停止治疗后KRAS突变等位基因衰减*CEA:癌胚抗原;Panit:帕尼单抗;Cetux:西妥昔单抗;Bev:贝伐单抗;CR:完全缓解;PR:部分缓解;PD:疾病进展AOUP-CRC04AOUP-CRC01AOUP-CRC03AOUP-CRC06SiravegnaG,etal.NatMed.2015Jul;21(7):795-801.第51页/共57页是否能持续监测，避免突变，找到最可能从西妥昔单抗再给药中获益的患者?*1–3不同的治疗西妥昔单抗治疗时间RASmutantctDNAlevels初始RAS野生型mCRC患者...…可能在西妥昔单抗治疗过程中发生RAS突变……换方案治疗后RAS突变可能检测不到……患者对西妥昔单抗再治疗敏感？第52页/共57页FIRE-4研究:

II期随机对照西妥昔单抗再治疗(德国)1Availableat1.http://www.aio-portal.de/index.php/studien.html;预计完成时间:2022年1月主要终点:西妥昔单抗三线治疗OS前瞻性研究敏感和耐药标记物第53页/共57页液体活检