Mutational Spectrum And Prognostic Factors Of Intermediate-risk Cytogenetics Acute Myeloid Leukemia With FLT3-ITD Mutation

Objective:This study aimed to investigate the mutational spectrum of intermediate-risk cytogenetics AML with FLT3-ITD mutation and clarify the influence of co-existence mutation and molecular structure of FLT3-ITD on patients’ prognosis.Finally to develop a new reliable prediction model.Methods:From December 2013 to February 2018,82 newly diagnosed de novo acute myeloid leukemia cases(no M3)with FLT3-ITD mutation comfirmed by first generation sequencing in the datebase of our center were collected.Next generation sequencing was applied for the bone marrow samples,which were obtained at the time of diagnosis,to screen mutational hotpots in 185 genes relevant to hematological malignancy.Cluster analysis was used for processing collaborative and exclusive pattern of co-existence mutations.The variant allele frequency[VAF=mutant/(mutant+wild)]of FLT3-ITD,number of clones,insertion site,complete remission(CR)rate,relapse free survival and overall survival were analyzed.Results:The function of co-existence mutations for patients of intermediate-risk cytogenetics AML with FLT3-ITD mutation mainly included histone methylation,DNA methylation,chromatin modification and signaling pathway.All these three groups of mutations might be the driver mutations,given their high frequency and burden in FLT3-ITD-mutated AML patients.The mutational spectrum of elder patients differed from younger patients and the frequency of co-existence mutations,especially NPM1,was higher in elder patients.Patients were classified into three categories based on FLT3 allelic ratio and presence of NPM1 mutant according to NCCN AML guideline.The 1-year OS were 30.0%,78.3%and 63.3%in favorable,intermediate and poor group(p=0.765;HR 0.969 95%CI 0.214-4.392,p=0.967 for intermediate group,HR 0.682,95%CI 0.139-3.341,p=0.637 for poor group),proving classification unavailable for Chinese patients.Median number of co-existence mutations was 8(range from 3 to 40).There were 14 patients harboring 3-5 mutations,50 patients for 6-10 mutations and 18 patients for more than 10 mutations.The OS and RFS were not dependent on the number of co-existence mutations(p>0.05 for OS,RFS).The pattern of co-existence mutations varied for different gene.NPM1 might act as driver mutation in FLT3-ITD-mutated AML,while NOTCH1 and SETD2 might be passage mutations.Median insertion length of FLT3-ITD was 53bp(range from 18-207bp).The locations of insertion included JM-S,JM-H,JM-Z,JM-B,p1-sheet and intron.The median VAF of FLT3-ITD was 0.4464(range from 0.0511 to 0.8310).VAF<0.33 was a protective factor for induction complete remission(HR:3.515,95%CI 1.055-11.716,p=0.041).However,0.33 was not a proper cutoff value of FLT3-ITD for our patients(p>0.05 for OS).Induction complete remission and HSCT were demonstrated to be protective factors of OS(Induction CR:HR:0.140,95%CI 0.070-0.279,p=0.000;HSCT:HR:0.283,95%CI 0.099-0.810,p=0.019),while combination of four gene mutations(USH2A,DNMT3A,TET2,ALK)to be risk factor against OS(p=0.049;HR:3.833,95%CI 1.394-10.543,p=0.009 for one mutation;HR:2.490,95%CI 0.949-6.533,p=0.064 for two mutations;HR:4.408,95%CI 1.364-14.244,p=0.013 for three mutations).The length of insertion>70bp and combination of four gene mutations were demonstrated to be risk factors against RFS(HR:4.116,95%CI 1.312-12.913,p=0.015 for the length of insertion;p=0.154;HR:7.111,95%CI 0.845-59.803,p=0.071 for one mutation;HR:7.838,95%CI 0.911-67.423,p=0.061 for two mutations;HR:26.915,95%CI 1.475-491.058,p=0.026 for three mutations).Conclusion:Our results suggest that gene mutations related to epigenetic and signaling pathway may act as driver factors in patients of intermediate-risk cytogenetics AML with FLT3-ITD mutation.The FLT3-ITD VAF<0.5 is an independent protective factor of induction remission.The combination of four gene mutations is an independent risk factor against OS,while induction complete remission and HSCT are independent protective factors of OS.The length of insertion>70bp and combination of four gene mutations are independent risk factors against RFS.