Detect NPM1 And FLT3-ITD Mutation In Acute Myeloid Leukemia And Assess Its Clinical Significance

Objective :To establish high-resolution melting curve (HRM) and denaturing high performance liquid chromatography (DHPLC) technology to detecte NPM1 and FLT3-ITD mutation in newly diagnosed acute myeloid leukemia (AML) patients .Preliminary study the relationship between NPM1 ,FLT3-ITD mutations and the clinical characteristics of AML,then to supply reference index for the clinical stratified treatment and molecular targeted therapy for AML, also for assess prognosis . Method: 1.Using PCR with HRM, PCR combined with CE, sequencing technology to detect of NPM1 gene mutation in 103 cases of AML patients , compared with each other to verify the sensitivity and specificity of PCR combined with HRM technology in detection of NPM1 mutations.2.Using PCR with DHPLC, PCR combined with CE, sequencing technology to quantitative detect of FLT3-ITD allele gene mutation in 103 cases of AML patients,and compared the results witht the results of CE for validation of the mehod of DHPLC,and finally sequence the presence of mutations.3.Analysis the 86 patients with available data in newly diagnosed 103 AML patients,combined the NPM1, FLT3-ITD mutation test results with clinical information ,discuss the general clinical characteristics,leukemia immuophenotyping,karyotype analysis,clinical characteristics and prognosis of patients with NPM1, FLT3-ITD mutation gene.Results:1.We have establish the PCR combind with HRM techology to detect the NPM1 gene mutation in AML patients successfully,and the speciality and sensitiveness is 100%.And the Positive findings consistent with the sequencing results.2.We have also establish the PCR combind with DHPLC techology to relatively qutify the FLT3-ITD gene mutation in AML patients successfully,and there is no statistic mean between the quantitative results used CE method and ours. And the Positive findings consistent with the sequencing results. 3.31 patients in 103 were detected NPM1 mutation ,and the detection rate was 30.1%., the NPM1 mutation detection rate is 47.6% in patients with normal karyotype in 86 AML patients who we have the aviable information. We found three kinds of mutation in NPM1 gene mutations:A mutant, B mutant, D mutation type .4.20 patients with FLT3-ITD mutations were detected in 103 cases of AML patients, the detection rate was 19.4%. There are 6 cases of patients with low percentage of mutations,8 cases of medium-scale mutations,6 cases of high proportion of mutations in those 20 cases with FLT3-ITD mutaion. Sequenced them and found all the FLT3-ITD mutations is a single insert fragment ,ranging from12bp to 107bp.In AML patients with normal karyotype,FLT3-ITD mutation detection rate was 26.2% in 86 AML patients who we have the aviable information.5.The NPM1 mutated patients have higher white blood cell in peripheral blood than the patients with wild-type patiens(median 21.4×109/L vs 8.4×109/L,P=0.034),and also associated with low expression of CD34(P=0.022).And the FLT3-ITD mutated patients also have higher white blood cell in peripheral blood in peripheral blood than the patients with wild-type patiens(median 23.1×109/L vs 9.5×109/L, P=0.037), also high in bone marrow blast cells (median 0.76 vs 0.4, P=0.023), the immunophenotype is also associated with high expression of CD7(P=0.024).6.There is no statistics in the complete remission rate between the NPM1 mutated and wild type patients(p=0.883),there is difference between the NPM1 mutated and wild type patients in complete remission after first chemotherapy in statistics(P=0.042).But there is also no difference between the FLT3-ITD mutated and wild-typed patients in the CR and CR1 in statistics(p=0.43,p=0.072 ) .7.The following four groups:NPM1-/FLT3-ITD-,NPM1+/FLT3-ITD-,NPM1-/FLT3-ITD+,NPM1+/FLT3-ITD+ accounted for 57.0%,23.3%,12.8% and 6.9%,corresponding to the CR rate was 81.6%,85%,54.5% and 66.6%, the RR within one year was 42.5%, 17.6%,50%,25%,and the CR of the group of NPM1 +/FLT3-ITD- is the highest and the RR within one year is the lowest, and the CR of the group of NPM1-/FLT3-ITD+ is the lowest and the RR within one year is the highest. Conclusion: We can rapid detect of NPM1 gent mutation in AML patients with PCR combined with HRM,also can percisely quantitative detect of FLT3-ITD gene mutation in AML patients with PCR combined with.DHPLC.Comprehensive analyzed, the prognosis of NPM1+/FLT3-ITD- patients is best.And the patients with NPM1 and FLT3-ITD mutation have special clinical characteristic .To detect of NPM1 and FLT3-ITD mutations in AML can provide one of independent guildline for layered treatment.