微量残留病检测在急性白血病

微量残留病检测在急性白血病分层治疗中的意义,MRD:The leukemic population undectable by morpglogic methodshas been defined as MRD,Minimal Residual Disease (MRD),MRD is a term used when there is evidence( immunophenotypic, molecular, or cytogenetic) that leukemic cells remain in the BM but there are insufficient cells to be detected by routine examination under the microscope.,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,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-10-5-10-6/5 copes /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,FCM MRD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)（Leukemia Associated ImmunoPhenotype）,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/CD117 CD34/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SSC,LAIP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 (communications in Clinical cytometry)38:139-152(1999),The effect of end-induction MRD on CIR,Coustan-Smith E.BLOOD,2000;96:2691,Johns Hopkins,Most information displays No. of cases(%) A CD19/CD45/CD20/CD10 (N=82)CD45 vs CD10 48(59)FSC vs CD10 13(16)FSC vs CD20 11(13)CD10 vs CD20 4(5) 76(93)B CD19/CD45/CD9/CD34 (N=77) CD45 vs CD34 27(35) FSC vs CD34 17(22)CD34 vs CD9 16(21) 72(94)A+B 81(99),Leukemia(1999)13,558-567,第8天形态结果与29天MRD关系 （N=1016）,两组抗体=90%,Leukemia(2003)17,1566-1572,A Childrens Oncology Group Study（FCM-MRD),Blood.2008;111:5477,诱导缓解后MRD检测的意义（D29）, N=2134,The effect of end-induction MRD on early and late relapse,Relapse-free survival,Years,EFS of NCI SR patients with favorable genetic features.,Years,Years,Prognostic significance of day-8 PB MRD,Cox multivariate analysis,Identification of a subgroup of patients with excellent outcome,12% Best-prognosis patients:NCI-SR+DT or TEL-AML1+MRD- at both D8 and D29,成人-ALL-FCM,Blood.2003;10:4695,CD7/CD5/CD3CD4/CD8/CD3CD7/CD2/CD3CD7/CD34/CD38,Spain,MRD水平与RFS,11.76%(n=12/102), Day 14 MRD-/0.03%, RFS OF 90% at 5 years,Multivariate Analysis of the Prognostic Impact on RFS,Status of minimal residual disease after induction predictsoutcome in both standard and high-risk Ph-negative adult acutelymphoblastic leukaemia. The Polish Adult Leukemia GroupALL 4-2002 MRD Study N=116,British Journal of Haematology, 2008:142, 227,CD7/CD2/CD3CD7/CD5/CD3CD7/CD38/CD34CD7/CD4/CD8TDT/CD7/Ccd3CD7/CD1a/CD3cCD3/CD7/CD3,CD10/CD20/CD19CD34/CD22/CD19CD34/CD38/CD19CD45/CD34/CD19TDT/CD10/CD19CD58/CD52/CD19CD33/CD13/CD19CD15/CD117/CD19CD65/CD56/CD19CD7/CD2/CD10,B-ALL,T-ALL,KaplanMeier estimates of relapse according to MRD evaluation after induction,Impact of MRD status after induction on outcome accordingto the treatment actually received.,Univariate and multivariate analysis of prognostic factorsassociated with increased risk of relapse and decreasedleukaemia free-survival.,Risk and response-based classification of childhood B-ALL,Childrens Oncology Group (COG),Blood: 2007; 109: 926935.,Retrospective,CCG:1988-1995,POG:1986-1999,N=6238,Age:1-22y,COG risk classification scheme,Risk factors: Age:10 y WBC:50000/L Cytogenetics:TEL/AML1,Trisomies(4,10.17),BCR/ABL,MLL, Day-14 marrow response:M125% blast Day-29 MRD-FCM CNS/TD,Childhood B-Precursor-ALL,Age:10,WBC:50000/L,:High Risk,:Standard Risk,TEL/Tris,D8/15,29 BM,D29MRD, CNSorTD/MLL,+, M1,M10.1%.-/n,Low Risk,-, M1,M10.1-1.0%.+/+,SR,HR,D8/15 BM,D29BM/MRD,CNS orTD/MLL,+,M1,M10.1-.0%.+/+,HR,HR,Randomized,Augmented,Augmented,BCR/ABL.MLL+SER.DI1.0%+D43-M2,3/MRD1.0%,VHR,Risk and response-based classification of childhood B-ALL,Blood: 2007; 109: 926935.,Pediatric Oncology Group (POG),Childrens Cancer Group (CCG),Risk and response-based classification of childhood B-ALL,IgH TCR基因的阳性率和敏感性,T Flohr，Leukemia (2008) 22, 771782,AIEOP-BFM ALL 2000,PCR MRD-directed risk group stratification,AIEOP-BFM ALL 2000化疗流程,MRD-directed risk group stratification,HR:PPR;NR,BCR/ABL,MLL/AF4,QR-PCR 检测MRD流程,Event-free survival,cumulative incidence of relapse,Blood.2010;115:3206,Molecular response to treatment redefines all prognostic factors in children and adolescents with B-cell precursor acute Lymphoblastic leukemia:results in 3184 patients of the AIEOP-BFM ALL 2000 study,TEL/AML1+,favorable DNA index ( 1.16 and 1.6; B),Prognostic impact of PCR-MRD,Ph+ patients,Prognostic impact of PCR-MRD,Prognostic impact of PCR-MRD in PH-pB-ALL patients within subgroups according to ALL-BFM 95 criteria(N=3184),SR,IR,HR,Results of the univariate and multivariate analyses,正常B细胞的分化规律,北京大学人民医院血液病研究所,CD10 CD34 CD19与 CD45 关系,CD22 CD20 CD19与 CD45关系,CD38 CD58 CD19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+(mature B-ALL),Note : no CD19+CD10-CD34+(Pro-B-ALL),成人与儿童B-ALL各亚型中LAIP 发生率,363/403(90.1),236/262(90.1),127/141(90.1),总,8/15(53.3)e,8/12(69.2)c,0/3(0)a,成熟 -/-,59/75(78.7)d,35/46(76.1)b,24/29(82.8),Pre -/+,238/255(93.3),148/159(93.1),90/96(93.8),Com +/+,58/58(100),45/45(100),13/13(100),Pro +/-,总,成人,儿童,CD34/CD10,B-ALL,亚型,中国实验血液学杂志 2006；14：853,Campana. Cytometry 38:139-152(1999),MRD+0.1%,CD45-CD10st+CD34s+,分析全部CD19+的细胞，以Hematogones作为内对照观察每个双参数的图形，无僵硬设门优点：避免表型改变所致假阴性不依赖于发病时的IM缺点：