Molecular Genetics Research On Gene Mutations And Monitoring Of Minimal Residual Disease In Acut Myeloid Leukemia

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.