急性髓细胞白血病分子遗传学研究及微小残留病的检测
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摘要
本论文包括两部分：（一）正常核型急性髓细胞白血病分子遗传学研究；（二）多参数流式细胞仪检测急性髓细胞白血病微小残留病的临床意义
第一部分正常核型急性髓细胞白血病分子遗传学研究
目的：
分析正常核型急性髓细胞白血病的分子遗传学特征、初步探索TET2基因突变及不同基因突变模式与预后的关系、评估两类突变在AML的发生率及其与在疾病发生中的作用。
方法：
病例来源于2005~2010年期间在苏州大学附属第一医院住院诊断和治疗的正常核型急性髓细胞白血病患者373例，采用患者的骨髓标本提取基因组DNA，再以PCR方法扩增目标DNA，直接进行DNA测序，分析TET2、DNMT3A、IDH1、IDH2、EZH2、CBL、ASXL1、MLL-PTD、NPM1、WT1、RUNX1、c-KIT、FLT3-ITD、FLT3-TKD、N-RAS和JAK2V617F基因突变在AML中的作用。
结果：
(1).在373例正常核型AML患者中，TET2突变60例（16.1%）、FLT3-ITD突变118例（31.6%）、FLT3-TKD突变23例（6.2%）、c-KIT突变9例（2.4%）、NPM1突变141例（37.8%）、WT1突变42（11.3%）、RUNX1突变22例（5.9%）、ASXL1突变43例（11.5%）、MLL-PTD突变14例（3.8%）、NRAS突变29例（7.8%）、IDH1突变29例（7.8%）、IDH2突变46例（12.3%）、EZH2突变6例（1.6%）、DNMT3A突变55例（14.7%）、CBL和JAK2V617F未发现基因突变。373例正常核型AML患者发生基因突变的患者总计为287例，突变率为77%。
(2). TET2突变与高龄、高血红蛋白相关，而与性别、初诊白细胞水平、血小板计数、骨髓原始细胞比例、FAB亚型等方面未见明显相关性。我们发现TET2基因突变与DNMT3A （P=0.041）、RUNX1（P<0.001）基因突变具有相关性，而与IDH2(P=0.021)和IDH1/2(P=0.006)基因突变具有互斥性。 NPM1突变与DNMT3A（P<0.0001）、IDH1(P<0.0001)、IDH2(P=0.001)基因突变高度相关，而与RUNX1（P=0.003）突变具有互斥性。IDH2突变与WT1突变具有互斥性（P=0.01）；而DNMT3A基因突变与NRAS突变具有相关性（P=0.01）。
(3).单独考虑一个基因如TET2和NPM1突变，其突变型和野生型的中位生存时间无明显差异，但在NPM1阳性组中，TET2突变型和野生型的中位生存时间为9.9个月和27.0个月（P=0.023）；在NPM1m+/FLT3-ITDm-的患者中，TET2突变组与未突变组的中位生存时间分别为9.5个月和32.2个月（P=0.013）。DNMT3A突变组与未突变组的中位生存时间分别为14.0个月和24.5个月（P=0.036）；在NPM1阴性组中，DNMT3A突变组的中位生存时间为8.2个月，DNMT3A阴性组的中位生存时间为24.4个月（P=0.003）；NPM1m-/DNMT3Am+与NPM1m+/DNMT3Am-的患者相比较，发现其中位生存时间分别为8.0个月和23.3个月（P=0.01）。FLT3-ITD突变型与野生型的中位生存时间分别为16.0个月和26.0个月（P=0.01）。
(4).我们对NPM1以外的13种急性髓细胞白血病基因突变进行积分，建立基因突变预后积分系统（mutation prognostic scoring system，MPSS），它们分别为TET2、FLT3-ITD、FLT3-TKD、c-KIT、WT1、RUNX1、ASXL1、MLL-PTD、NRAS、IDH1、IDH2、EZH2、DNMT3A。每一个突变积1分，依据积分来分组，MPSS≤1为低危组，MPSS>1为高危组；低危组和高危组患者的中位无复发生存时间分别为32.8个月和11.7个月（P<0.0001）、中位总生存时间分别为28.1个月和16.7个月（P=0.007）。
结论：
正常核型AML患者中TET2、DNMT3A突变均提示预后不良； NPM1m+和NPM1m+/FLT3-ITDm-均不能修正TET2基因突变所致的不良预后；TET2、FLT3-ITD、FLT3-TKD、c-KIT、WT1、RUNX1、ASXL1、MLL-PTD、NRAS、IDH1、IDH2、EZH2和DNMT3A基因突变，其基因突变达到或超过2个者预后不良。
第二部分多参数流式细胞仪检测急性髓细胞白血病微小残留病的临床意义
目的:
研究多参数流式细胞术监测白细胞病细胞测微小残留（MRD）在急性髓细胞白血病（AML）中的临床价值，探索最合适的MRD阈值，讨论其在判断疾病复发、预后及指导个体化治疗等方面的作用。
方法:
回顾性研究于2003~2011年期间，在苏州大学附属第一医院诊断和治疗的252例急性髓细胞白血病（M3除外）患者，在患者诱导和巩固治疗阶段，采用多参数流式细胞术连续监测患者体内白血病细胞微小残留病。
结果:
(1)第1次标准方案诱导化疗后，ROC分析确定MRD阈值为1.5×10-2，Cox比例风险模型显示相对于MRD阴性组，MRD阳性组的RR值为2.41。在缓解后采用联合方案化疗的患者中，MRD阳性组和MRD阴性组的中位RFS分别为(19.45±3.74)个月和（56.46±4.28）个月，差异有统计学意义（P<0.01）；中位OS也显著不同（分别为（29.37±4.47）个月和（77.97±4.30）个月，P<0.01)。第1次达完全缓解时，ROC分析所确定的MRD阈值为3.0×10-3，Cox比例风险模型显示相对于MRD阴性组，MRD阳性组的RR值为1.75。缓解后采取联合化疗，阳性组和阴性组患者的中位RFS分别为(28.36±3.40)个月和（55.70±4.32）个月，差异有统计学意义（P<0.01)；中位OS分别为（39.30±3.73）个月和（70.19±4.34）个月，差异有统计学意义（P<0.01）。
(2)巩固治疗阶段，采取复发前的最大MRD值对复发状态进行ROC曲线分析，复发风险明显增加的MRD阈值为4.3×10-3，其敏感性为78.6%，特异性为75%。以此阈值把MRD分组阳性组和阴性组，其复发率分别为55.5%和25.8%(P=.001)。Cox比例风险模型显示MRD阳性组的复发相对危险度（relative risk，RR）是MRD阴性组的4.57倍（P<0.01）。持续缓解时间：联合化疗组，MRD阴性组平均持续缓解时间为65.19±5.58个月；MRD阳性组平均持续缓解时间为24.05±3.02个月（P<0.01)。总生存时间：联合化疗组，MRD阴性组平均总生存时间为84.88±6.01个月；MRD阳性组平均总生存时间为46.65±5.40个月（P<0.01)。在移植组中，MRD阴性组平均总生存时间是70.83±4.72个月；而在MRD阳性组总生存时间为45.38±4.31个月（P<0.01）。
结论:
在急性髓细胞白血病诱导及巩固治疗阶段，MRD偏高或持续升高，提示疾病复发风险增加，无复发生存时间和总生存时间明显缩短。因此，MRD的检测可以预测AML的复发、判断预后和指导个体化治疗。
This thesis will comprise two parts：Part I: Molecular genetics research on genemutations in acute myeloid leukemia with normal karyotype; Part II: Clinical significanceof monitoring minimal residual disease by multi-parameter flow cytometry in acutemyeloid leukemia.
PART I Molecular genetics research on gene mutations in acut myeloidleukemia with normal karyotype.
Objective:
To analyze the molecular genetics characteristics of acute myeloid leukemia withnormal karyotype, to explore the relationship between TET2gene mutations or differentgenetic mutation patterns and prognosis, and to assess the incidence of two types ofmutations in AML, and learn more about leukemogenesis.
Methods:
A total of373acute myeloid leukemia (AML) with normal karyotype diagnosed andtreatment in the First Affiliated Hospital of Soochow University during2005to2010,which were recruited in this research to assess the genetic mutation patterns. The genomicDNA is extracted from bone marrow cell and amplified by PCR. The analysis of TET2,DNMT3A, IDH1, IDH2, EZH2, CBL, ASXL1, MLL-PTD, NPM1, WT1, RUNX1, c-KIT,FLT3-ITD, FLT3-TKD, N-RAS and JAK2V617F gene mutations by massively DNAsequencing.
Results:
(1). A total of16.1%of patients had TET2mutations,31.6%had FLT3internal tandem duplications (ITDs),6.2%had FLT3tyrosine kinase domain mutations,2.4%hadc-KIT mutations,37.8%had NPM1mutations,11.3%had WT1mutations,5.9%hadRUNX1mutations,11.5%had ASXL1mutations,3.8%had MLL partial tandemduplications (PTDs),7.8%had IDH1mutations,7.8%had NRAS mutations,12.3%hadIDH2mutations,1.6%had EZH2mutations,14.7%had DNMT3A mutations and nomutations were fand of CBL and JAK2V617F. In conclusion, there are77%(287/373) genemutations hide in normal karyotype AML patients.
(2). TET2mutation in primary AML patients was closely associated with older age,higher Hemoglobin, but not with gender, initial white blood cell, platelet count, bonemarrow blast, FAB subtype and normal karyotype. We found that the TET2gene mutationswere associated with DNMT3A (P=0.041) and RUNX1(P <0.001) mutations, but mutuallyexclusive with IDH2(P=0.021), or IDH1/2(P=0.006) gene mutations. NPM1mutationgene mutations were highly correlated with DNMT3A mutations (P <0.0001), IDH1mutations (P <0.0001) and IDH2mutations (P=0.001), but mutually exclusive withRUNX1mutations (P=0.003). IDH2mutations and WT1mutations were mutually exclusive(P=0.01); DNMT3A mutations were associated with NRAS mutations (P=0.01).
(3). In the NPM1m+patients, TET2mutations were associated with shorter medianOS in contrast to TET2wild type (9.9vs.27.0months, P=0.023). However, the TET2andNPM1mutations were not associated with shorter OS when one of them is consideredindividually. Interestingly, TET2mutations is an unfavorable prognostic factor, it is closelyassociated with shorter median OS in contrast to TET2wild type (9.5vs.32.2months,P=0.013) in NPM1m+/FLT3-ITDm-group. DNMT3A mutations were associated withshorter median OS in contrast to DNMT3A wild type (14.0months and24.5months,respectively (P=0.036). Further studies showed that DNMT3A mutations and wild typehad different median OS (8.2vs.24.4months, P=0.003) in NPM1m-group. Similarly, inthe NPM1m-/DNMT3Am+and NPM1m+/DNMT3Am-group, the median OS was8.0months and23.3months, respectively (P=0.01). FLT3-ITD mutations were associatedwith a shorter median OS compare to FLT3-ITD wild-type (16.0vs.26.0months, P= 0.01).
(4). Thirtee kinds of AML common gene mutations which including TET2, FLT3-ITD,FLT3-TKD, c-KIT, WT1, RUNX1, ASXL1, MLL-PTD, NRAS, IDH1, IDH2, EZH2andDNMT3A mutations were considered to establish an mutation prognostic scoring system.Each mutation denotes one score to the mutation prognostic scoring system. Based on thisintegration system, patients with MPSS≤1belongs to the low-risk group and the MPSS>1belongs to high-risk group. Excitedly, low-risk and high-risk groups had an obviouslymedian EFS (32.8vs.11.7months, P<0.0001) and OS (28.1vs.16.7months, P=0.007).
Conclusion: TET2and DNMT3A mutations were unfavorable prognostic factor innormal karyotype AML, and its negative impact was cannot amend by NPM1m+orNPM1m+/FLT3-ITDm-. Mutation prognostic scoring system based on TET2, FLT3-ITD,FLT3-TKD, c-KIT, WT1, RUNX1, ASXL1, MLL-PTD, NRAS, IDH1, IDH2, EZH2andDNMT3A mutations indicate that high-risk group (MPSS>1) was an obviously negativeprognostic factor.
PART II Clinical significance of monitoring minimal residual disease bymulti-parameter flow cytometry in acute myeloid leukemia.
Objective:
Our aim is to find the optimal threshold of MRD, to explore the clinical value ofMRD in adult acute myeloid leukemia which monitored by multi-parameter flowcytometry and to discuss whether the optimal threshold could predict relapse, prognosisand guide individual treatment.
Methods:
In a retrospective study, a total of252adult acute myeloid leukemia patients (withoutM3), from the First Affiliated Hospital of Soochow University during2003to2011, wererecruited. The MRD of252adult acute myeloid leukemia patients which were detected bymulti-parameter flow cytometry monitored consecutively during the introduction and consolidation therapy.
Results:
(1).The cut-off value (1.5×10-2) was calculated from ROC curve after the firststandard induction chemotherapy, with which the AML patients were separated into twogroups. Cox proportional hazards model analysis showed that the relative risk (RR) of thepositive group was2.41times compared with the negative group (1.45~4.03,95%C.I.).The clinical outcome of positive and negative groups with continuing chemotherapies wassignificantly different in terms of median RFS (56.46±4.28months vs.19.45±3.74months；P<0.01) and OS (77.97±4.30months vs.29.37±4.47months; P<0.01). Similarly,the cut-off value was3.0×10-3after the first CR. Cox proportional hazards model analysisshowed that the RR of positive group was1.75times compared with MRD-negative group(1.11~2.77,95%C.I.). The clinical outcome of positive and negative groups withcontinuing chemotherapies was significantly different in terms of RFS (28.36±3.40monthsvs.55.70±4.32months, P<0.01), and OS (39.30±3.73months vs.70.19±4.34months, P<0.01) as well.
(2). The maximum MRD of individual was analyzed by receiver operatingcharacteristic curves to determine the cut-off point as4.3×10-3that the sensitivity andspecificity were78.6%and75%(P<0.01), respectively. With this cut-off value, AMLpatients also separated into two groups (MRD positive and negative groups) with relapserates of55.5%versus25.8%(P<0.01), respectively. Cox proportional hazards modelanalysis showed that MRD-positive group, relative risk is4.75times than MRD-negativegroup (P<0.01). The distinct outcome of MRD-negative and MRD-positive group wasclearly distinguished in terms of median RFS (65.19±5.58months versus24.05±3.02months； P<0.01), OS (84.88±6.01months versus46.65±5.40months; P<0.01) inchemotherapy group. Identically, MRD-negative and MRD-positive group have aconspicuous OS (70.83±4.72months versus45.38±4.31months; P<0.01) intransplantation group.
Conclusion:
In conclusion, AML patients who had a positive or consecutively positive of MRDstrongly suggest that the risk of relapse is increasing significantly with a shorterrelapse-free survival and overall survival time. So, we can conclude that MRD monitoringby multi-parameter flow cytometry in acute myeloid leukemia was a clinical useful indexmonitored during the course of treatment, which could predict relapse, prognosis and guideindividual treatment.
第一部分正常核型急性髓细胞白血病分子遗传学研究
目的：
分析正常核型急性髓细胞白血病的分子遗传学特征、初步探索TET2基因突变及不同基因突变模式与预后的关系、评估两类突变在AML的发生率及其与在疾病发生中的作用。
方法：
病例来源于2005~2010年期间在苏州大学附属第一医院住院诊断和治疗的正常核型急性髓细胞白血病患者373例，采用患者的骨髓标本提取基因组DNA，再以PCR方法扩增目标DNA，直接进行DNA测序，分析TET2、DNMT3A、IDH1、IDH2、EZH2、CBL、ASXL1、MLL-PTD、NPM1、WT1、RUNX1、c-KIT、FLT3-ITD、FLT3-TKD、N-RAS和JAK2V617F基因突变在AML中的作用。
结果：
(1).在373例正常核型AML患者中，TET2突变60例（16.1%）、FLT3-ITD突变118例（31.6%）、FLT3-TKD突变23例（6.2%）、c-KIT突变9例（2.4%）、NPM1突变141例（37.8%）、WT1突变42（11.3%）、RUNX1突变22例（5.9%）、ASXL1突变43例（11.5%）、MLL-PTD突变14例（3.8%）、NRAS突变29例（7.8%）、IDH1突变29例（7.8%）、IDH2突变46例（12.3%）、EZH2突变6例（1.6%）、DNMT3A突变55例（14.7%）、CBL和JAK2V617F未发现基因突变。373例正常核型AML患者发生基因突变的患者总计为287例，突变率为77%。
(2). TET2突变与高龄、高血红蛋白相关，而与性别、初诊白细胞水平、血小板计数、骨髓原始细胞比例、FAB亚型等方面未见明显相关性。我们发现TET2基因突变与DNMT3A （P=0.041）、RUNX1（P<0.001）基因突变具有相关性，而与IDH2(P=0.021)和IDH1/2(P=0.006)基因突变具有互斥性。 NPM1突变与DNMT3A（P<0.0001）、IDH1(P<0.0001)、IDH2(P=0.001)基因突变高度相关，而与RUNX1（P=0.003）突变具有互斥性。IDH2突变与WT1突变具有互斥性（P=0.01）；而DNMT3A基因突变与NRAS突变具有相关性（P=0.01）。
(3).单独考虑一个基因如TET2和NPM1突变，其突变型和野生型的中位生存时间无明显差异，但在NPM1阳性组中，TET2突变型和野生型的中位生存时间为9.9个月和27.0个月（P=0.023）；在NPM1m+/FLT3-ITDm-的患者中，TET2突变组与未突变组的中位生存时间分别为9.5个月和32.2个月（P=0.013）。DNMT3A突变组与未突变组的中位生存时间分别为14.0个月和24.5个月（P=0.036）；在NPM1阴性组中，DNMT3A突变组的中位生存时间为8.2个月，DNMT3A阴性组的中位生存时间为24.4个月（P=0.003）；NPM1m-/DNMT3Am+与NPM1m+/DNMT3Am-的患者相比较，发现其中位生存时间分别为8.0个月和23.3个月（P=0.01）。FLT3-ITD突变型与野生型的中位生存时间分别为16.0个月和26.0个月（P=0.01）。
(4).我们对NPM1以外的13种急性髓细胞白血病基因突变进行积分，建立基因突变预后积分系统（mutation prognostic scoring system，MPSS），它们分别为TET2、FLT3-ITD、FLT3-TKD、c-KIT、WT1、RUNX1、ASXL1、MLL-PTD、NRAS、IDH1、IDH2、EZH2、DNMT3A。每一个突变积1分，依据积分来分组，MPSS≤1为低危组，MPSS>1为高危组；低危组和高危组患者的中位无复发生存时间分别为32.8个月和11.7个月（P<0.0001）、中位总生存时间分别为28.1个月和16.7个月（P=0.007）。
结论：
正常核型AML患者中TET2、DNMT3A突变均提示预后不良； NPM1m+和NPM1m+/FLT3-ITDm-均不能修正TET2基因突变所致的不良预后；TET2、FLT3-ITD、FLT3-TKD、c-KIT、WT1、RUNX1、ASXL1、MLL-PTD、NRAS、IDH1、IDH2、EZH2和DNMT3A基因突变，其基因突变达到或超过2个者预后不良。
第二部分多参数流式细胞仪检测急性髓细胞白血病微小残留病的临床意义
目的:
研究多参数流式细胞术监测白细胞病细胞测微小残留（MRD）在急性髓细胞白血病（AML）中的临床价值，探索最合适的MRD阈值，讨论其在判断疾病复发、预后及指导个体化治疗等方面的作用。
方法:
回顾性研究于2003~2011年期间，在苏州大学附属第一医院诊断和治疗的252例急性髓细胞白血病（M3除外）患者，在患者诱导和巩固治疗阶段，采用多参数流式细胞术连续监测患者体内白血病细胞微小残留病。
结果:
(1)第1次标准方案诱导化疗后，ROC分析确定MRD阈值为1.5×10-2，Cox比例风险模型显示相对于MRD阴性组，MRD阳性组的RR值为2.41。在缓解后采用联合方案化疗的患者中，MRD阳性组和MRD阴性组的中位RFS分别为(19.45±3.74)个月和（56.46±4.28）个月，差异有统计学意义（P<0.01）；中位OS也显著不同（分别为（29.37±4.47）个月和（77.97±4.30）个月，P<0.01)。第1次达完全缓解时，ROC分析所确定的MRD阈值为3.0×10-3，Cox比例风险模型显示相对于MRD阴性组，MRD阳性组的RR值为1.75。缓解后采取联合化疗，阳性组和阴性组患者的中位RFS分别为(28.36±3.40)个月和（55.70±4.32）个月，差异有统计学意义（P<0.01)；中位OS分别为（39.30±3.73）个月和（70.19±4.34）个月，差异有统计学意义（P<0.01）。
(2)巩固治疗阶段，采取复发前的最大MRD值对复发状态进行ROC曲线分析，复发风险明显增加的MRD阈值为4.3×10-3，其敏感性为78.6%，特异性为75%。以此阈值把MRD分组阳性组和阴性组，其复发率分别为55.5%和25.8%(P=.001)。Cox比例风险模型显示MRD阳性组的复发相对危险度（relative risk，RR）是MRD阴性组的4.57倍（P<0.01）。持续缓解时间：联合化疗组，MRD阴性组平均持续缓解时间为65.19±5.58个月；MRD阳性组平均持续缓解时间为24.05±3.02个月（P<0.01)。总生存时间：联合化疗组，MRD阴性组平均总生存时间为84.88±6.01个月；MRD阳性组平均总生存时间为46.65±5.40个月（P<0.01)。在移植组中，MRD阴性组平均总生存时间是70.83±4.72个月；而在MRD阳性组总生存时间为45.38±4.31个月（P<0.01）。
结论:
在急性髓细胞白血病诱导及巩固治疗阶段，MRD偏高或持续升高，提示疾病复发风险增加，无复发生存时间和总生存时间明显缩短。因此，MRD的检测可以预测AML的复发、判断预后和指导个体化治疗。
This thesis will comprise two parts：Part I: Molecular genetics research on genemutations in acute myeloid leukemia with normal karyotype; Part II: Clinical significanceof monitoring minimal residual disease by multi-parameter flow cytometry in acutemyeloid leukemia.
PART I Molecular genetics research on gene mutations in acut myeloidleukemia with normal karyotype.
Objective:
To analyze the molecular genetics characteristics of acute myeloid leukemia withnormal karyotype, to explore the relationship between TET2gene mutations or differentgenetic mutation patterns and prognosis, and to assess the incidence of two types ofmutations in AML, and learn more about leukemogenesis.
Methods:
A total of373acute myeloid leukemia (AML) with normal karyotype diagnosed andtreatment in the First Affiliated Hospital of Soochow University during2005to2010,which were recruited in this research to assess the genetic mutation patterns. The genomicDNA is extracted from bone marrow cell and amplified by PCR. The analysis of TET2,DNMT3A, IDH1, IDH2, EZH2, CBL, ASXL1, MLL-PTD, NPM1, WT1, RUNX1, c-KIT,FLT3-ITD, FLT3-TKD, N-RAS and JAK2V617F gene mutations by massively DNAsequencing.
Results:
(1). A total of16.1%of patients had TET2mutations,31.6%had FLT3internal tandem duplications (ITDs),6.2%had FLT3tyrosine kinase domain mutations,2.4%hadc-KIT mutations,37.8%had NPM1mutations,11.3%had WT1mutations,5.9%hadRUNX1mutations,11.5%had ASXL1mutations,3.8%had MLL partial tandemduplications (PTDs),7.8%had IDH1mutations,7.8%had NRAS mutations,12.3%hadIDH2mutations,1.6%had EZH2mutations,14.7%had DNMT3A mutations and nomutations were fand of CBL and JAK2V617F. In conclusion, there are77%(287/373) genemutations hide in normal karyotype AML patients.
(2). TET2mutation in primary AML patients was closely associated with older age,higher Hemoglobin, but not with gender, initial white blood cell, platelet count, bonemarrow blast, FAB subtype and normal karyotype. We found that the TET2gene mutationswere associated with DNMT3A (P=0.041) and RUNX1(P <0.001) mutations, but mutuallyexclusive with IDH2(P=0.021), or IDH1/2(P=0.006) gene mutations. NPM1mutationgene mutations were highly correlated with DNMT3A mutations (P <0.0001), IDH1mutations (P <0.0001) and IDH2mutations (P=0.001), but mutually exclusive withRUNX1mutations (P=0.003). IDH2mutations and WT1mutations were mutually exclusive(P=0.01); DNMT3A mutations were associated with NRAS mutations (P=0.01).
(3). In the NPM1m+patients, TET2mutations were associated with shorter medianOS in contrast to TET2wild type (9.9vs.27.0months, P=0.023). However, the TET2andNPM1mutations were not associated with shorter OS when one of them is consideredindividually. Interestingly, TET2mutations is an unfavorable prognostic factor, it is closelyassociated with shorter median OS in contrast to TET2wild type (9.5vs.32.2months,P=0.013) in NPM1m+/FLT3-ITDm-group. DNMT3A mutations were associated withshorter median OS in contrast to DNMT3A wild type (14.0months and24.5months,respectively (P=0.036). Further studies showed that DNMT3A mutations and wild typehad different median OS (8.2vs.24.4months, P=0.003) in NPM1m-group. Similarly, inthe NPM1m-/DNMT3Am+and NPM1m+/DNMT3Am-group, the median OS was8.0months and23.3months, respectively (P=0.01). FLT3-ITD mutations were associatedwith a shorter median OS compare to FLT3-ITD wild-type (16.0vs.26.0months, P= 0.01).
(4). Thirtee kinds of AML common gene mutations which including TET2, FLT3-ITD,FLT3-TKD, c-KIT, WT1, RUNX1, ASXL1, MLL-PTD, NRAS, IDH1, IDH2, EZH2andDNMT3A mutations were considered to establish an mutation prognostic scoring system.Each mutation denotes one score to the mutation prognostic scoring system. Based on thisintegration system, patients with MPSS≤1belongs to the low-risk group and the MPSS>1belongs to high-risk group. Excitedly, low-risk and high-risk groups had an obviouslymedian EFS (32.8vs.11.7months, P<0.0001) and OS (28.1vs.16.7months, P=0.007).
Conclusion: TET2and DNMT3A mutations were unfavorable prognostic factor innormal karyotype AML, and its negative impact was cannot amend by NPM1m+orNPM1m+/FLT3-ITDm-. Mutation prognostic scoring system based on TET2, FLT3-ITD,FLT3-TKD, c-KIT, WT1, RUNX1, ASXL1, MLL-PTD, NRAS, IDH1, IDH2, EZH2andDNMT3A mutations indicate that high-risk group (MPSS>1) was an obviously negativeprognostic factor.
PART II Clinical significance of monitoring minimal residual disease bymulti-parameter flow cytometry in acute myeloid leukemia.
Objective:
Our aim is to find the optimal threshold of MRD, to explore the clinical value ofMRD in adult acute myeloid leukemia which monitored by multi-parameter flowcytometry and to discuss whether the optimal threshold could predict relapse, prognosisand guide individual treatment.
Methods:
In a retrospective study, a total of252adult acute myeloid leukemia patients (withoutM3), from the First Affiliated Hospital of Soochow University during2003to2011, wererecruited. The MRD of252adult acute myeloid leukemia patients which were detected bymulti-parameter flow cytometry monitored consecutively during the introduction and consolidation therapy.
Results:
(1).The cut-off value (1.5×10-2) was calculated from ROC curve after the firststandard induction chemotherapy, with which the AML patients were separated into twogroups. Cox proportional hazards model analysis showed that the relative risk (RR) of thepositive group was2.41times compared with the negative group (1.45~4.03,95%C.I.).The clinical outcome of positive and negative groups with continuing chemotherapies wassignificantly different in terms of median RFS (56.46±4.28months vs.19.45±3.74months；P<0.01) and OS (77.97±4.30months vs.29.37±4.47months; P<0.01). Similarly,the cut-off value was3.0×10-3after the first CR. Cox proportional hazards model analysisshowed that the RR of positive group was1.75times compared with MRD-negative group(1.11~2.77,95%C.I.). The clinical outcome of positive and negative groups withcontinuing chemotherapies was significantly different in terms of RFS (28.36±3.40monthsvs.55.70±4.32months, P<0.01), and OS (39.30±3.73months vs.70.19±4.34months, P<0.01) as well.
(2). The maximum MRD of individual was analyzed by receiver operatingcharacteristic curves to determine the cut-off point as4.3×10-3that the sensitivity andspecificity were78.6%and75%(P<0.01), respectively. With this cut-off value, AMLpatients also separated into two groups (MRD positive and negative groups) with relapserates of55.5%versus25.8%(P<0.01), respectively. Cox proportional hazards modelanalysis showed that MRD-positive group, relative risk is4.75times than MRD-negativegroup (P<0.01). The distinct outcome of MRD-negative and MRD-positive group wasclearly distinguished in terms of median RFS (65.19±5.58months versus24.05±3.02months； P<0.01), OS (84.88±6.01months versus46.65±5.40months; P<0.01) inchemotherapy group. Identically, MRD-negative and MRD-positive group have aconspicuous OS (70.83±4.72months versus45.38±4.31months; P<0.01) intransplantation group.
Conclusion:
In conclusion, AML patients who had a positive or consecutively positive of MRDstrongly suggest that the risk of relapse is increasing significantly with a shorterrelapse-free survival and overall survival time. So, we can conclude that MRD monitoringby multi-parameter flow cytometry in acute myeloid leukemia was a clinical useful indexmonitored during the course of treatment, which could predict relapse, prognosis and guideindividual treatment.
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1. Swerdlow SH, Campo E, Harris NL, et al, eds. WHO Classification of Tumours ofHaematopoietic and Lymphoid Tissues. Lyon, France: IARC Press;2008.
2. Byrd JC, Mrózek K, Dodge RK, et al. Pretreatment cytogenetic abnormalities arepredictive of induction success, cumulative incidence of relapse, and overall survival inadult patients with de novo acute myeloid leukemia: results from Cancer and LeukemiaGroup B (CALGB8461). Blood2002;100:4325-36.
3. Mrózek K, Marcucci G, Paschka P, Whitman SP, Bloomfield CD. Clinical relevance ofmutations and gene expression changes in adult acute myeloid leukemia with normalcytogenetics: are we ready for a prognostically prioritized molecular classification?Blood2007;109:431-48.
4. D hner H. Implication of the molecular characterization of acute myeloid leukemia.Hematology Am Soc HematolEduc Program.2007;2007:412-9.
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