MRD检测在急性白血病分层治疗中的意义解读

1、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

2、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-10-5-10-6/5 copes /10-

3、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

4、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

5、序 和表达量的规律Cytometry (communications in Clinical cytometry)38:139-152(1999)诱导缓解后MRD水平与累计复发率Coustan-Smith E.BLOOD,2000;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

6、/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 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诱导后 MRD水平对早期和晚期复发的影响Relapse-free survivalYears NCI SR 伴好遗传学特性患者MRD与EFS关系YearsYears4,108 天PB MRD的意义多变

7、量分析鉴别预后非常好的一组患者NCI-SR+DTTEL-AML1+MRD- D8and D2912%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RFS11.76%(n=12/102), Day 14 MR

8、D-/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/D330.00010.0001N NN ND35-MRDD35-MRD0.05%

9、0.05%0.0010.0010.010.010.020.02对RFS的多变量分析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=116British Journal of Haematology, 2008:142, 227CD7/CD2/CD3

10、CD7/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leukaemia free-survival的多变量分析Risk and respons

11、e-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 marrow response:M12

12、5% 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+SER.DI1.0%+D43-M2,

13、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=3341N=3341No target

14、54(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 sensitive target56

15、1(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T Flohr，Leukemia (2008) 22, 771782AIEOP-BFM ALL 2000PCR MRD指导的危险度分层AIEOP-BFM ALL 2000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-free survivalcumulative

16、 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 the AIEOP-BFM ALL 2000 studyTEL/AML1+favorable DNA index ( 1.16 and 1.6)PCR-MRD对预后好患者的影响

17、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

18、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-ABL mRNA动态变化与imatinib应用Qin YZ,Liu YR,Zhu HH,et al. Int J Lab Hematol 2008;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Qin YZ,Liu YR.Zhu HH.et al. Int J Lab Hematol 2008;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

19、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)e8/12(69.2)c0

20、/3(0)a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 +/-总成人儿童CD34/CD10 B-ALL亚型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

21、依赖于发病时的IM缺点：不易掌握4色FCM检测B-ALL MRD的临床意义98例：儿童78，成人20 ，671份标本，6.8 次/人，10.8月 /人， 3.1月/次中国血液学杂志 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：CD34/CD10/CD45/CD19 个体

22、化：CD38,CD123,CD13,CD33,CD58,CD20/CD45/CD19 无IM: CD20/CD22/CD45/CD19 or CD20/CD38/CD45/CD19连续监测，间隔3M。如MRD+？,需要证实时，在2W。01 02 03 04 05 06 001 02 03 04 05 06 07 0M R D + ;5 0 %,n = 1 2M R D - :7 .5 %,n = 4 0随访 时 间 （月）累积 复 发 率发病1-3个月MRD+和MRD-患者累计复发率P=0.00112岁 女 Pro-B-ALL 连续检测结果08.6.1108.6.1108.8.1908.8.1

23、909.3.2609.3.2609.6.3009.6.30LAIP+%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%7

24、色-FCM-MRD-B-ALLCD10/CD34/CD123/CD38/CD58/CD45CD19CD19CD10CD10CD34CD38CD10CD58CD34CD45CD45CD123CD123CD10CD45CD45SSCSSCAML MRD检测NBM-CD34+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+2.712.300.0120.0113.361.8

25、40.0280.011CD19+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+87.127.270.3400.15445.4914.560

26、.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CD34+/SSClow CD117+/SSClow细胞中的抗原表达（xSD） 中华血液学杂志2008；29：121LAIP阳性患者中阳性患者中LAIP+细胞比例细胞比例（%）NBM中中LAIP+细胞比例细胞比例（%）对数差对数差*CD34+CD7+44.09 (9.44-93.46)0.042

27、 (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 (0.040-0.138)2.64(2.24-2.79)CD117+CD34-CD1

28、1b+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)0.0

29、42 (0.004-0.182)3.18(2.71-3.33)610例AML和正常骨髓中LAIP细胞的表达差异中华血液学杂志2007；28：73119种抗体，四色组合，LAIP+=86%诊断时AML M2患者特点：SSC小CD117+CD33+CD13-HLA-DR-LAIP: CD117+CD34-CD33+HLA-DR-和CD117+CD34-CD33+CD13- 诊断后23个月（CR） MRD（+）=0.25%诊断后27个月（CR）MRD（+）=0.80%051 01 52 02 502 55 07 51 0 0M R D ( - ) , N = 3 6M R D ( + ) , N = 1 2p = 0 . 0 1 1 8 m o n t h sP e r c e n t r e la p s e - f r e e s u r v iv a l01 02 03 002 55 07 51 0 0M R D ( - ) ,N = 4 7M R D ( + ) ,N = 1 1p