微量残留病检测在急性白血病

1、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 insu

2、fficient cells to be detected by routine examination under the microscope.MRD检测的方法检测的方法MRD监测的临床意义监测的临床意义 MRD 检测在分层治疗中的意义检测在分层治疗中的意义FCM-MRD检测的结果检测的结果MRD检测方法检测方法a. PCR: 基因基因-定性、定性、 定量（定量（RQ-PCR）b.多参数多参数FCM：免疫标志免疫标志发病时寻找免疫标志和基因标志进行检测发病时寻找免疫标志和基因标志进行检测目的基因：目的基因：融合基因：融合基因：BCR/ABL,AML1/ETO,PML/RAR 基因重排：基因

3、重排： IgH，TCR(10-4-10-5) 基因突变：基因突变：FLT3-ITD,NPM1(10-5) 基因表达增加：基因表达增加：WT1,PRAME(10-3)优点：优点： 灵敏度高灵敏度高-10-5-10-6/5 copes /10-5 特异性强特异性强缺点缺点: 1. 融合基因：应用范围有限：融合基因：应用范围有限： ALL:10-30%，AML:30-50% 2. IgH/TCR : 90%, 操作复杂，费时操作复杂，费时,需要基因测序，特异引需要基因测序，特异引 物物/探针探针(每例患者特异）每例患者特异） 3.容易污染，出现假阳性。容易污染，出现假阳性。RQ-PCR检测检测MRD

4、特点特点优点：优点： 灵敏：灵敏：ALL-10-4 , AML-10-3-10-4 （获取细胞相关）（获取细胞相关） 定量单位：细胞定量单位：细胞% 快速：检测当天即可知结果快速：检测当天即可知结果 操作简便操作简便 应用范围广：应用范围广：90%缺点缺点： 表型的变化表型的变化:假阴性假阴性 受前体受前体B细胞细胞(Hematogones)的干扰：的干扰： 形态幼稚，表达形态幼稚，表达CD34,TDT 在小儿、化疗后、在小儿、化疗后、SCT后比例增加后比例增加5% 需要较高的分析水平和技能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应用不够广泛，需要建立标准化操作FCM MRD

5、检测的特点检测的特点定义：正常骨髓定义：正常骨髓/PB中不表达或表达比例较低的中不表达或表达比例较低的 免疫表型免疫表型 白血病相关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：白血病细胞的特异抗原：NG2(7.1)IM/MRD检测的抗原检测的抗原CD34,CD33-：正常分化抗原：表达于：正常分化抗原：表达于正常细胞的不同系列、分化阶段正常细胞的不同系列、分化阶段（非非白血病细胞所特异）白血病细胞所特异） 交叉系列抗原交叉系列抗原: B、T、髓、髓、NK细胞抗原细胞抗原 非同期抗原共表

6、达非同期抗原共表达: 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：异常的光散射信号：FSC和和SSC LAIP的分类的分类基础：熟悉正常细胞不同分化阶段抗原出现的基础：熟悉正常细胞不同分化阶段抗原出现的顺序顺序 和表达和表达量量的规律的规律Cytometry (communications in Clinical cytometry)38:139-152(1999)The effect of end-induction MRD on CIRCoustan-Smith E.BLOOD,2000;

7、96:2691Johns HopkinsMost 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第第8天形

8、态结果与天形态结果与29天天MRD关系关系 （N=1016）两组抗体两组抗体=90% Fraction of patients MRD + at day 29 Day 8 morphology Day 8 blood negative Day 8 blood positive M1 (25%) 2/51(3.9%) 70/260(27%)Totals 6/309(1.9%) 96/515(18.6%)Leukemia(2003)17,1566-1572 A Childrens Oncology Group Study（FCM-MRD) Blood.2008;111:5477 诱导缓解后诱导缓解

9、后MRD检测的意义（检测的意义（D29）, N=2134The effect of end-induction MRD on early and late relapseRelapse-free survivalYearsEFS of NCI SR patients with favorable genetic features.YearsYearsPrognostic significance of day-8 PB MRDCox multivariate analysisIdentification of a subgroup of patients with excellent outc

10、ome12% Best-prognosis patients:NCI-SR+DT or TEL-AML1+MRD- at both D8 and D29成人成人-ALL-FCMBlood.2003;10:4695 CD7/CD5/CD3CD4/CD8/CD3CD7/CD2/CD3CD7/CD34/CD38CD19/CD34/CD45CD10/CD13/CD19CD5/CD33/CD20CD34/CD38/CD19TDT/CD10/CD19CD34/CD22/CD19B-ALL N=73T-ALL N=29Spain0.05%N=440.5%,N=42Day +33Day +14MRD水平与水平

11、与RFS11.76%(n=12/102), Day 14 MRD-/0.03%, RFS OF 90% at 5 yearsUnivariateUnivariateMultivariateMultivariateWholeWholeCRCRVariableVariableP PP PP PAgeAge30/6030/600.0020.0020.050.05N NWBCWBC30000ul30000ul0.030.030.050.05N NPh Ch.Ph Ch.+/-+/-0.040.04N N0.040.04Time to mCRTime to mCRD14/D33D14/D330.0001

12、0.0001N NN ND35-MRDD35-MRD0.05%0.05%0.0010.0010.010.010.020.02Multivariate Analysis of the Prognostic Impact on RFSStatus of minimal residual disease after induction predictsoutcome in both standard and high-risk Ph-negative adult acutelymphoblastic leukaemia. The Polish Adult Leukemia GroupALL 4-20

13、02 MRD Study N=116British Journal of Haematology, 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-ALLKaplanM

14、eier estimates of relapse according to MRD evaluation after inductionImpact 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.Ri

15、sk and response-based classification of childhood B-ALLChildrens Oncology Group (COG)Blood: 2007; 109: 926935. Retrospective,CCG:1988-1995,POG:1986-1999,N=6238,Age:1-22yCOG risk classification schemeRisk factors: Age:10 y WBC:50000/ L Cytogenetics:TEL/AML1,Trisomies(4,10.17),BCR/ABL,MLL, Day-14 marr

16、ow response:M125% blast Day-29 MRD-FCM CNS/TD Childhood B-Precursor-ALL Age:10,WBC:50000/ L :High Risk :Standard RiskTEL/Tris,D8/15,29 BM,D29MRD, CNSorTD/MLL+, M1,M10.1%.-/nLow Risk-, M1,M10.1-1.0%.+/+SRHRD8/15 BM,D29BM/MRD,CNS orTD/MLL+,M1,M10.1-.0%.+/+HRHRRandomizedAugmentedAugmentedBCR/ABL.MLL+SE

17、R.DI1.0%+D43-M2,3/MRD1.0%,VHRRisk and response-based classification of childhood B-ALLBlood: 2007; 109: 926935. Pediatric Oncology Group (POG)Childrens Cancer Group (CCG)Risk and response-based classification of childhood B-ALLPrecursor-B-ALLPrecursor-B-ALLT-ALLT-ALLALLALLN=2854N=2854N=422N=422N=334

18、1N=3341No target54(2%)54(2%)29(7%)29(7%)88(3%)88(3%) 1 target2800(96%)2800(96%)393(93%)393(93%)3253(97%)3253(97%) 2 targets2662(93%)2662(93%)379(88%)379(88%)3089(92%)3089(92%) 3 targets2189(77%)2189(77%)281(67%)281(67%)2510(75%)2510(75%)No sensitive target173(6%)173(6%)38(9%)38(9%)217(6%)217(6%)1 se

19、nsitive target561(20%)561(20%)95(22%)95(22%)671(20%)671(20%) 2 sensitive targets2066(72%)2066(72%)260(62%)260(62%)2365(71%)2365(71%)IgH TCR基因的阳性率和敏感性基因的阳性率和敏感性T Flohr，Leukemia (2008) 22, 771782AIEOP-BFM ALL 2000PCR MRD-directed risk group stratificationAIEOP-BFM ALL 2000化疗流程化疗流程MRD-directed risk gro

20、up stratificationHR:PPR;NR,BCR/ABL,MLL/AF4QR-PCR 检测检测MRD流程流程Event-free survivalcumulative incidence of relapseBlood.2010;115:3206Molecular response to treatment redefines all prognostic factors in children and adolescents with B-cell precursor acute Lymphoblastic leukemia:results in 3184 patients of

21、 the AIEOP-BFM ALL 2000 studyTEL/AML1+favorable DNA index ( 1.16 and 1.6; B)Prognostic impact of PCR-MRDPh+ patientsPrognostic impact of PCR-MRDPrognostic impact of PCR-MRD in PH-pB-ALL patients within subgroups according to ALL-BFM 95 criteria(N=3184)SRIRHRResults of the univariate and multivariate

22、 analyses正常正常B细胞的分化规律细胞的分化规律北京大学人民医院血液病研究所北京大学人民医院血液病研究所 CD10 CD34 CD19CD10 CD34 CD19与与 CD45 CD45 关系关系CD22 CD20 CD19CD22 CD20 CD19与与 CD45CD45关系关系CD38 CD58 CD19CD38 CD58 CD19与与 CD45CD45关系关系I: CD34+CD10+ cIg- sIg- (Com-B-ALL)II:CD34-CD10+ cIg+ sIg- (pre-B-ALL)III: CD34-CD10- cIg+ sIg+(mature B-ALL)Not

23、e : no CD19+CD10-CD34+(Pro-B-ALL)成人与儿童成人与儿童B-ALLB-ALL各亚型中各亚型中LAIP LAIP 发生率发生率363/403(90.1)236/262(90.1)127/141(90.1)总总8/15(53.3)e8/12(69.2)c0/3(0)a成熟成熟 -/-59/75(78.7)d35/46(76.1)b24/29(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 +/-总总成人成人儿童儿童CD34/CD10 B-

24、ALL亚型亚型中国实验血液学杂志中国实验血液学杂志 2006；14：853Campana. Cytometry 38:139-152(1999)MRD+0.1%CD45-CD10st+CD34s+分析全部分析全部CD19+的细胞，的细胞，以以Hematogones作为内作为内对照对照观察每个双参数的图形，观察每个双参数的图形，无僵硬设门无僵硬设门优点：避免表型改变所优点：避免表型改变所致假阴性致假阴性不依赖于发病时的不依赖于发病时的IM缺点：不易掌握缺点：不易掌握4 4色色FCMFCM检测检测B-ALL MRDB-ALL MRD的临床意义的临床意义9898例：儿童例：儿童7878，成人，成人2

25、0 20 ，671671份标本，份标本，6.8 次/人，10.8月月 /人， 3.1月/次中国血液学杂志中国血液学杂志 2006；27：302有有无无总体总体初次初次IMIM7272例例2626例例9898BMBM复发复发12127 71919复发前复发前MRD+MRD+9 96 61515复发前复发前MRD-MRD-1 10 01 1复发前复发前MRD+%MRD+%90%90%100%100%93.70%93.70%复发前复发前4M NT4M NT2 21 13 3 两组四色抗体：通用：两组四色抗体：通用：CD34/CD10/CD45/CD19 个体化：个体化：CD38,CD123,CD13

26、,CD33,CD58,CD20/CD45/CD19 无无IM: CD20/CD22/CD45/CD19 or CD20/CD38/CD45/CD19连续监测，间隔连续监测，间隔3M。如。如MRD+？,需要证实时，在需要证实时，在2W。0102030405060010203040506070M RD+;50%,n=12M RD-:7.5%,n=40随 访时间 （月 ）累 积复 发 率发病发病1-31-3个月个月MRDMRD+ +和和MRDMRD-患者累计复发率患者累计复发率P=0.00112岁岁 女女 Pro-B-ALL 连续检测结果连续检测结果08.6.1108.6.1108.8.1908.8

27、.1909.3.2609.3.2609.6.3009.6.30LAIP+%LAIP+%38.0538.05（- -）（- -）0.030.03WT1WT14.24.20.020.020.090.090.110.11IgHIgH（+ +）（- -）（- -）（- -）09.7.909.7.909.8.2009.8.2009.9.2209.9.2209.11.309.11.3LAIP+%LAIP+%0.090.090.110.116.596.5934.1634.16WT1WT10.090.090.060.069.99.917.217.2IgHIgHN N（- -）N N（+ +）MRD+=0.45

28、%7色色-FCM-MRD-B-ALLCD10/CD34/CD123/CD38/CD58/CD45CD19CD19CD10CD10CD34CD38CD10CD58CD34CD45CD45CD123CD123CD10CD45CD45SSCSSCAML MRD检测检测NBM-CD34+细胞亚群分析细胞亚群分析CD7CD56CD3CD33CD38CD15CD1bCD13CD19HLA-DRCD117HLA-DRCD34CD34+/SSClowCD117+/SSClow相对比例（相对比例（%）实际比例实际比例（%）相对比例相对比例（%）实际比例（实际比例（%）CD11b+2.712.300.0120.0

29、113.361.840.0280.011CD19+6.663.970.0300.0211.230.720.0090.005CD56+8.184.910.0310.0215.894.670.0250.020CD7+12.244.400.0480.0266.832.180.0510.021CD15+20.535.120.0910.02829.594.720.2560.085CD9+33.4512.130.1120.06214.173.370.0760.028CD33+74.2326.730.2480.13043.0510.710.3190.130CD13+87.127.270.3400.15445

30、.4914.560.3970.199CD117+/CD34+89.145.920.3160.14251.959.850.3880.130HLA-DR+93.964.300.3330.14687.976.770.6600.293CD38+93.313.410.3650.13191.963.770.6940.304 正常骨髓正常骨髓CD34+/SSClow CD117+/SSClow细胞中的抗原表达（细胞中的抗原表达（xSD） 中华血液学杂志中华血液学杂志2008；29：121LAIP阳性患者中阳性患者中LAIP+细细胞比例（胞比例（%）NBM中中LAIP+细胞细胞比例（比例（%）对数差对数差*C

31、D34+CD7+44.09 (9.44-93.46)0.042 (0.016-0.109)3.02(2.35-3.35)CD34+CD56+41.65 (10.18-94.07)0.027 (0.005-0.087)3.19(2.58-3.54)CD34+CD11b+26.10 (4.17-83.68)0.007 (0.000-0.039)3.57(2.78-4.08)CD34+CD15+45.05 (12.92-83.43)0.090 (0.038-0.143)2.70(2.16-2.97)CD117+CD34+CD9+40.16 (16.00-56.83)0.093 (0.040-0.13

32、8)2.64(2.24-2.79)CD117+CD34-CD11b+41.65 (20.48-93.59)0.049 (0.004-0.145)2.93(2.62-3.28)CD117+CD34-CD15+35.92 (27.99-45.96)0.097 (0.038-0.263)2.57(2.46-2.68)CD117+CD34-CD9+36.65 (21.08-97.22)0.036 (0.019-0.351)3.01(2.77-3.43)CD117+CD15-11b+60.06 (21.96-85.86)0.025 (0.003-0.044)3.38(2.94-3.54)CD34+CD33-HLA-DR+63.53 (21.60-90.31)0.042 (0.004-0.182)3.18(2.71-3.33)610例例AML和正常骨髓中和