Clinical Research Of Allogeneic Hematopoietic Stem Cell Transplantation For Patients With Myeloid Neoplasms And RUNX1 Mutations

Objective To investigate the survival and prognostic factors of allogeneic hematopoietic stem-cell transplantation(allo-HSCT)for patients with myeloid neoplasms and RUNX1 mutations.To study the preliminary function of representative RUNX1 mutations in vivo and vitro,and explore the role of RUNX1 mutations in myeloid differentiation.Methods From July 2014 to April 2018,the clinical data of forty-two AML/MDS patients with RUNX1 mutations in the First Affiliated Hospital of Soochow University were retrospectively analyzed.The clinical characteristic features and distributions of the mutations frequently observed with RUNX1 mutations were summarized,and the prognosis of allo-HSCT for these patients was also analyzed.In addition,we constructed the lentiviral vector which expressed the RUNX1 mutations including R201X,R204X,N136K,S141X and wild type.Lentivirus was packaged,293T cell line and human cord blood CD34+cells were transfected by lentivirus which carried RUNX1 mutations and wild type.The subnuclear localization of the proteins encoded by RUNX1 mutations and wild type by applying immunofluorescence technology.We further sorted human cord blood CD34+cells which stably expressed the RUNX1-muts and wild type in order to perform colony-forming unit(CFU)assay and the liquid culture assay with myeloid differentiation.Results Among 42 AML/MDS patients with RUNX1 mutations,27 were male and 15 were female.The median age was 43.5(16-68)years old.The numbers of acute myeloid leukemia(AML)and myelodysplastic syndrome(MDS)patients were 30 and 12,respectively.There were 31 patients in allo-HSCT group and 11 patients in chemotherapy group.RUNX1 mutations co-occurred with many other gene mutations,and the most frequent mutated gene was FLT3(26.2%,11/42).Interestingly,FLT3 mutations only occurred in AML patients compared with MDS patients(P=0.014),while ASXL1(25%,3/12)mutations were observed as the most frequent co-mutations in MDS patients.One-year overall survival(OS)of allo-HSCT and chemotherapy patients were(70.6±9.0)%and(61.0±9.4)%respectively.One year disease-free survival(DFS)of allo-HSCT and chemotherapy patients were(34.4±16.7)%and(22.4±15.3)%respectively.OS and DFS between allo-HSCT and chemotherapy patients were compared.There were significant differences in 1-OS(?2=4.843,P=0.036)and(?2=4.320,P=0.047)between the HSCT and chemotherapy patients.In univariable analyses,age>45 years at transplantation was a poor prognostic factor on OS[HR=4.819(95%CI 1.145-20.283),P=0.032]and DFS[HR=5.945(95%CI 1.715-20.604),P=0.005].Also,complex chromosome karyotype abnormality was a poor prognostic factor on OS[HR=5.572(95%CI 1.104-28.113),P=0.038].The immunofluorescence results indicated that the wild-type RUNX1 protein and the RUNX1-N136K protein localized in the nucleus,while the RUNX1-R201X,RUNX1-S141X and RUNX1-R204X protein were found to localize in the cytoplasm.The CFU assay showed that the number of total cells in S141X group was significantly lower than RUNX1 wild group(P<0.05).The percentage of myeloid CFUs showed a significant decrease in R201X and R204X groups compared to RUNX1 wild group(P<0.05).Moreover,the percentage of CFU-GEMM was significantly higher than RUNX1 wild group(P<0.05).Compared to RUNX1 wild group,the differentiation into myeloid progenitor cells and granulocytes in R201X and R204X groups was significantly decreased with the liquid culture assay(P<0.05).Conclusion The prognosis of the myeloid neoplasm patients with RUNX1 mutations could be improved by allogeneic hematopoietic stem-cell transplantation.Age(>45 years)at transplantation and complex chromosome karyotype abnormality were poor prognostic factors in allo-HSCT for myeloid neoplasms patients with RUNX1 mutations.RUNX1-R201X,RUNX1-R204X and RUNX1-S141X proteins had abnormal localizations,and RUNX1-R201X and RUNX1-R204X could lead to myeloid differentiation inhibition and self-renewal enhancement in hematopoietic stem cells(HSCs),which susequently resulting in the decrease of myeloid progenitor cells and granulocytes differentiation.