Risk Factors And Clonal Evolution Mechanism Of Early Relapse After Allogeneic Hematopoietic Stem Cell In Acute Myeloid

ObjectiveAt present,allogeneic hematopoietic stem cell transplantation(allo-HSCT)represents a curative option for intermediate-and high-risk adult acute leukemia.Despite substantial improvements in non-relapse mortality(NRM)over the years,relapse of acute leukemia remains the leading cause of failure after allo-HSCT.The risk factors related to relapse have been widely studied,but the risk factors related to the time of relapse are rarely reported.The purpose of this study was to investigate the clinical characteristics,related risk factors of relapse and early relapse after allo-HSCT in acute myeloid leukemia(AML),and to study the clonal evolution at relapse.Methods1.Patients:a total of 846 patients with acute myeloid leukemia(except M3)who underwent allo-HSCT in the First Affiliated Hospital of Soochow University from January 2016 to February 2020 were analyzed.The overall relapse after allo-HSCT,the risk factors that may lead to relapse,and its impact on prognosis were analyzed.In addition,the patients who relapsed within 6 months after transplantation were defined as the early relapse group,and the patients relapsed after 6 months were defined as the late relapse group.The clinical characteristics and molecular genetics between the two groups were compared.And the evolution of molecular genetics in patients with AML was dynamically tracked during initial diagnosis,pre-transplantation and post-transplantation relapse.2.Statistical analysis of clinical data:SPSS24.0 software was used for statistical analysis.The clinical features and continuous variables of laboratory examination were compared between groups by independent sample t-test,and the differences between classified data were analyzed by Fisher test or chi-square test.GraphPadPrism8.0 software was used for survival analysis,and Log-Rank test was used to compare the overall survival of each group.The difference was statistically significant(P<0.05).The cumulativerelapse rate was analyzed by R4.0.4 software,death was regarded as competitive risk,and competitive risk model(Fine And Gray)was used to analyze.The risk factors of relapse were studied by Cox regression model with time-dependent variables.The forest map is created in GraphPadPrism8.0 software.The "fish map"(fishplot)of R4.0.4 was used for visualization,and the clonal evolution of the tumor was tracked in detail.Results1.Overall relapse of patients:124 of 846 patients were found to have relapsed.The cumulative relapse rates of 1 year,3 years and 5 years after transplantation were 12.8%,20.4%and 22.2%,respectively.60 patients(48.4%)relapsed within 6 months after transplantation,and 64 patients(51.6%)relapsed 6 months after transplantation.2.Risk factors related to relapse after transplantation:(1)Analysis of clinical related variables:using univariate regression analysis,the newly diagnosed patients developed primary drug resistance after the first induction chemotherapy(HR=1.979,95%CI:1.251-3.131,P=0.004),and were in a state of non-remission at the time of transplantation(HR=3.963,95%CI:2.377-6.608,P<0.001)may be an important risk factor related to relapse.Multivariate regression analysis showed that extramedullary infiltration before transplantation(HR=2.890,95%CI:0.995-2.740,P=0.004)and non-remission of the disease at the time of transplantation(HR=2.800,95%CI:1.587-4.941,P<0.0001)were risk factors for relapse after allo-HSCT.(2)Molecular genetics related variable analysis:univariate analysis showed that TP53(HR=4.8,95%CI:2.3-9.9,P<0.001),BCORL1(HR=6.8,95%CI:2.1-22,P=0.0011),ETV6(HR=2.3,95%CI:1-5.2,P=0.049),JAK2(HR=4.2,95%CI:1.7-10).P=0.0019)mutations are risk factors for relapse after transplantation.In multivariate analysis,an increased risk of relapse was observed in patients with BCORL1(HR=6.51,95%CI:1.23-34.41,P=0.003),JAK2(HR=4.39,95%CI:1.54-12.51,P=0.01),SF3B1(HR=5.47,95%CI:1.54-19.43,P=0.01)and TP53(HR=4.43,95%CI:1.91-10.25,P<0.001)mutations.3.Survival after relapse:1-year,3-year and 5-year overall survival rates of 846 patients were 87.3%,80.6%and 73.1%,respectively.According to the different time period from transplant to relapse,there was no significant difference in overall survival between patients with relapse within 3 months after transplantation and patients with relapse within 3 to 6 months(P<0.05).The one-year overall survival rate of patients with relapse within 3 months was 19.08%,within 6 months was 28.5%,while differing from patients with relapse within 6 months and 6 months after transplantation.Therefore,we define early relapse as the time from allogeneic transplantation to relapse less than 6 months.4.Prognostic risk factors:(1)Clinical related variable analysis:multivariate regression analysis,results:the disease was not in remission at the time of transplantation(HR=1.830,95%CI:1.012-3.308,P=0.045)and primary chemotherapy resistance(HR=1.629,95%CI:1.006-2.638,P=0.047)significantly affected the overall survival of patients after transplantation.(2)Molecular genetics related variable analysis:univariate regression analysis showed that mutations in TP53may also increase the mortality of recurrent patients after transplantation(HR=3.4;95%CI:1.6-7.3).The mutations in TET2(HR=0.4;95%CI:0.16-0.99 and Pseudomonas 0.047)may reduce the risk of death in patientsrelapsing after transplantation.Multivariate analysis showed thatmutations in TP53(HR=4.44;95%CI:1.89-10.44,P<0.001)was a risk factor for death after transplantation,whilemutations in TET2(HR=0.36;95%CI:0.13-0.94)were associated with a reduced risk of disease relapseafter transplantation.5.Predictors of early relapse after transplantation:(1)Analysis of clinical related variables:124 patients with relapse were divided into early relapse group(≤6 months)and late relapse group(>6 months).The analysis showed that the time from initial diagnosis to transplantation(>120d)(P<0.001),the disease state in non-remission before transplantation(P<0.001),primary chemotherapy resistance(P<0.021)was the risk factors for early relapse after transplantation.Multivariate regression analysis showed that primary chemotherapy resistance(OR=3.178;95%CI:1.060-9.533)may be a risk factor for early relapse after transplantation.(2)Molecular genetics related variable analysis:with univariate regression analysis,we compared 52 gene mutations,including TP53,DNMT3A,IDH1,FLT3-ITD,etc,where no significant difference were found.When the mutant genes with P<0.5 were included in multivariate regression analysis,no significant differences were found.6.Analysis of clonal evolution:(1)Molecular genetic characteristics of patients with relapse after transplantation:47 patients with paired diagnosis and second-generation sequencing at the time of relapse were collected,and 36 patients recorded gene mutations different from those at first diagnosis.Only gene mutation was obtained in 16 patients with relapse,only loss of gene mutation was found in 17 patients,and both gene mutation and loss were found in 3 patients.WT1,ETNK1,JAK2,JAK3,SETD2,GATA2,KIT,TET2,DNMT3A,TP53,IDH1,FLT3-ITD,etc.are the new mutations that appear at the time of relapse,including those obtained separately or screened during treatment.(2)Comparison of the genetic characteristics of relapse after transplantation with those before transplantation:in 47 patients with hematopoietic stem cell transplantation,there were positive gene mutations before hematopoietic stem cell transplantation.By analyzing and comparing the results of gene mutation in the three stages from pre-transplantation to relapse,it was found that there were three kinds of change patterns.New mutations were found during relapse after transplantation(including DNMT3A,CEBPA single mutation,FLT3 point mutation,GATA2,TP53,WT1,TET2,etc.).Conclusion1.Extramedullary infiltration before transplantation and non-remissionat the time of transplantation may increase the risk of relapse after transplantation.AML patients with BCORL1,JAK2,SF3B1 and TP53 mutations had a significantly higher risk of relapse after transplantation than those with negative mutations.2.The disease of non-remission at the time of transplantation and primary chemotherapy resistance may lead to poor prognosis of AML patients after transplantation.TP53 is a risk factor for death after transplantation,while TET2 may reduce the risk of death after transplantation.3.There is a significant difference in the prognosis of AML with relapse within 6 months and 6 months later,so we define early relapse as relapse from allogeneic hematopoietic stem cell transplantation less than 6 months.4.Primary chemotherapy resistance may be a risk factor for early relapse after transplantation,and there is no significant difference in mutations between early and late relapse group.