MRD检测在急性白血病分层治疗中的意义

北京大学人民医院北京大学血液病研究所微量残留病检测在急性白血病分层治疗中的意义MRD:TheleukemicpopulationundectablebymorpglogicmethodshasbeendefinedasMRDMinimalResidualDisease(MRD)MRDisatermusedwhenthereisevidence(immunophenotypic,molecular,orcytogenetic)thatleukemiccellsremainintheBMbutthereareinsufficientcellstobedetectedbyroutineexaminationunderthemicroscope.MRD检测的方法MRD监测的临床意义MRD检测在分层治疗中的意义FCM-MRD检测的结果MRD检测方法a.PCR:基因---定性、定量（RQ-PCR）b.多参数FCM：免疫标志发病时寻找免疫标志和基因标志进行检测目的基因：融合基因：BCR/ABL,AML1/ETO,PML/RAR基因重排：IgH，TCR(10-4-10-5)基因突变：FLT3-ITD,NPM1(10-5)基因表达增加：WT1,PRAME((10-3)优点：灵敏度高---10-5-10-6/5copes/10-5特异性强缺点:1.融合基因：应用范围有限：ALL:10-30%，AML:30-50%2.IgH/TCR:90%,操作复杂，费时,需要基因测序，特异引物/探针(每例患者特异）3.容易污染，出现假阳性。RQ-PCR检测MRD特点优点：灵敏：ALL-10-4,AML-10-3-10-4（获取细胞相关）定量单位：细胞%快速：检测当天即可知结果操作简便应用范围广：90%缺点：表型的变化:假阴性受前体B细胞(Hematogones)的干扰：形态幼稚，表达CD34,TDT在小儿、化疗后、SCT后比例增加5%需要较高的分析水平和技能应用不够广泛，需要建立标准化操作FCMMRD检测的特点定义：正常骨髓/PB中不表达或表达比例较低的免疫表型白血病相关的免疫表型(LAIP)（LeukemiaAssociatedImmunoPhenotype）FCM-MRD：白血病细胞的特异抗原：NG2(7.1)IM/MRD检测的抗原CD34,CD33---：正常分化抗原：表达于正常细胞的不同系列、分化阶段（非白血病细胞所特异）交叉系列抗原:B、T、髓、NK细胞抗原非同期抗原共表达:CD15/CD117CD34/CD64抗原表达量异常：表达强度过高、过低或不表达异常的光散射信号：FSC和SSCLAIP的分类基础：熟悉正常细胞不同分化阶段抗原出现的顺序和表达量的规律Cytometry(communicationsinClinicalcytometry)38:139-152(1999)诱导缓解后MRD水平与累计复发率Coustan-SmithE.BLOOD,2000;96:2691JohnsHopkinsMostinformationdisplaysNo.ofcases(%)ACD19/CD45/CD20/CD10(N=82)CD45vsCD1048(59)FSCvsCD1013(16)FSCvsCD2011(13)CD10vsCD204(5)76(93)BCD19/CD45/CD9/CD34(N=77)CD45vsCD3427(35)FSCvsCD3417(22)CD34vsCD916(21)72(94)A+B81(99)Leukemia(1999)13,558-567AChildren’sOncologyGroupStudy（FCM-MRD)Blood.2008;111:5477诱导缓解后MRD检测的意义（D29）,N=2134诱导后MRD水平对早期和晚期复发的影响Relapse-freesurvivalYearsNCISR伴好遗传学特性患者MRD与EFS关系YearsYears4,108天PBMRD的意义多变量分析鉴别预后非常好的一组患者NCI-SR+DTTEL-AML1+MRD-D8andD2912%成人-ALL-FCMBlood.2003;10:4695CD7/CD5/CD3CD4/CD8/CD3CD7/CD2/CD3CD7/CD34/CD38CD19/CD34/CD45CD10/CD13/CD19CD5/CD33/CD20CD34/CD38/CD19TDT/CD10/CD19CD34/CD22/CD19B-ALLN=73T-ALLN=29Spain0.05%N=430.05%N=440.5%N=210.5%,N=42Day+33Day+14MRD水平与RFS11.76%(n=12/102),Day14MRD-/0.03%,RFSOF90%at5yearsUnivariateMultivariateWholeCRVariablePPPAge30/600.0020.05NWBC30000ul0.030.05NPhCh.+/-0.04N0.04TimetomCRD14/D330.0001NND35-MRD0.05%0.0010.010.02对RFS的多变量分析Statusofminimalresidualdiseaseafterinductionpredictsoutcomeinbothstandardandhigh-riskPh-negativeadultacutelymphoblasticleukaemia.ThePolishAdultLeukemiaGroupALL4-2002MRDStudyN=116BritishJournalofHaematology,2008:142,227CD7/CD2/CD3CD7/CD5/CD3CD7/CD38/CD34CD7/CD4/CD8TDT/CD7/Ccd3CD7/CD1a/CD3cCD3/CD7/CD3CD10/CD20/CD19CD34/CD22/CD19CD34/CD38/CD19CD45/CD34/CD19TDT/CD10/CD19CD58/CD52/CD19CD33/CD13/CD19CD15/CD117/CD19CD65/CD56/CD19CD7/CD2/CD10B-ALLT-ALL诱导缓解后MRD与复发关系诱导后MRD状态与治疗的关系对复发率和leukaemiafree-survival的多变量分析Riskandresponse-basedclassificationofchildhoodB-ALLChildren’sOncologyGroup(COG)Blood:2007;109:926–935.Retrospective,CCG:1988-1995,POG:1986-1999,N=6238,Age:1-22yCOGriskclassificationschemeRiskfactors:Age:10yWBC:50000/LCytogenetics:TEL/AML1,Trisomies(4,10.17),BCR/ABL,MLL,Day-14marrowresponse:M15%.M2:5-25%,M3:25%blastDay-29MRD-FCMCNS/TDChildhoodB-Precursor-ALLAge:10,WBC:50000/L:HighRisk:StandardRiskTEL/Tris,D8/15,29BM,D29MRD,CNSorTD/MLL+,M1,M10.1%.-/nLowRisk-,M1,M10.1%-/n+-,D15M2/3,0.1-1.0%.+/+SRHRD8/15BM,D29BM/MRD,CNSorTD/MLL+,M1,M10.1%.-/nD15M2/3,0.1-.0%.+/+HRHRRandomizedAugmentedAugmentedBCR/ABL.MLL+SER.DI0.81/44Ch.NR,D29-M2/MRD1.0%+D43-M2,3/MRD1.0%,VHRRiskandresponse-basedclassificationofchildhoodB-ALLBlood:2007;109:926–935.PediatricOncologyGroup(POG)Children’sCancerGroup(CCG)Riskandresponse-basedclassificationofchildhoodB-ALLPrecursor-B-ALLT-ALLALLN=2854N=422N=3341Notarget54(2%)29(7%)88(3%)≥1target2800(96%)393(93%)3253(97%)≥2targets2662(93%)379(88%)3089(92%)≥3targets2189(77%)281(67%)2510(75%)Nosensitivetarget173(6%)38(9%)217(6%)1sensitivetarget561(20%)95(22%)671(20%)≥2sensitivetargets2066(72%)260(62%)2365(71%)IgHTCR基因的阳性率和敏感性TFlohr，Leukemia(2008)22,771–782AIEOP-BFMALL2000PCRMRD指导的危险度分层AIEOP-BFMALL2000化疗流程MRD指导的分层标准HR:PPR;NR,BCR/ABL,MLL/AF4QR-PCR检测MRD流程Event-freesurvivalcumulativeincidenceofrelapseBlood.2010;115:3206MolecularresponsetotreatmentredefinesallprognosticfactorsinchildrenandadolescentswithB-cellprecursoracuteLymphoblasticleukemia:resultsin3184patientsoftheAIEOP-BFMALL2000studyTEL/AML1+favorableDNAindex(1.16and1.6)PCR-MRD对预后好患者的影响PCR-MRD对Ph+患者的影响SRIRHRPCR-MRD对Ph-患者的影响多变量分析CML欧洲白血病网(ELN)最新推荐伊马替尼400mg/d初始治疗失败的定义:3个月未达到CHR,6个月未达到任何CyR,12个月未达到PCyR,18个月未达到CCyR,任何时间,丢失之前达到的CCyR或CHR疾病进展或出现耐药的Abl激酶突变推荐采用第二代TKI尼洛替尼治疗慢性期加速/急变期NCCN最新版治疗指南推荐对于伊马替尼400mg/d初始治疗患者，出现以下事件推荐患者接受尼洛替尼治疗:•3个月未达到血液学反应或者血液学复发,•6个月未达到任何CyR,•12个月未达到PCyR或者细胞遗传学复发,•18个月未达到CCyR或者细胞遗传学复发,CML患者CCR后BCR-ABLmRNA动态变化与imatinib应用QinYZ,LiuYR,ZhuHH,etal.IntJLabHematol2008;30:317“○”：CCR“●”：Ph+其中1例患者由CCR进展至急变期，BCR-ABL升高2.5log,但未检测出Ph染色体QinYZ,LiuYR.ZhuHH.etal.IntJLabHematol2008;30:317CML患者CCR后的BCR-ABLmRNA水平提示复发空心：CCR，实心：Ph+正常B细胞的分化规律北京大学人民医院血液病研究所CD10CD34CD19与CD45关系CD22CD20CD19与CD45关系CD38CD58CD19与CD45关系I:CD34+CD10+cIg-sIg-(Com-B-ALL)II:CD34-CD10+cIg+sIg-(pre-B-ALL)III:CD34-CD10-cIg+sIg+(matureB-ALL)Note:no