Clinical And Mechanistic Study Of Low-dose Decitabine As Part Of Modified Bucy-conditioning In Improving Prognosis Of High-risk AML Patients

Part I.Clinical study of low-dose decitabine as part of modified Bu Cy-conditioning regimen in allogeneic stem cell transplantation for high-risk AML patientsObjective:To determine the efficacy and safety of low-dose decitabine as part of modified Bu Cy-conditioning regimen in allogeneic stem cell transplantation(allo-HSCT)for high-risk AML patients.Methods : Retrospectively analyzed 236 high-risk AML patients who received allo-HSCT from Aug 2012 to Oct 2017 in our hospital and paired the patients according to1:3.59 patients received low-dose decitabine(DAC)as part of modified Bu Cyconditioning(DAC group)while 177 patients received modified Bu Cy-conditioning(CON group).We compared the differences of two groups in the treatment-related toxicities,the incidence of GVHD,neutrophil and platelet recovery,cumulative relapse rate and survival.Results:Except for the status before transplantation(the percentage of non relapse before transplantation(NR)patients of DAC group is higher than CON group,P=0.026),there were no statistical differences in the clinical characteristics between two groups.The median follow-up of DAC group and CON group were 402(85-1504)days and 349(35-1404)days.Both of two groups median time of neutrophil and platelet recovery are 12 days and 13 days respectively.The treatment-related mortality(TRM)of DAC group is significantly lower than that of CON group(0 and 6.2%,P=0.05).There was no significant differences of the incidence of GVHD(whether a GVHD or c GVHD)between two groups.For NR patients,the median time from transplantation to relapse of two group were187 days and 86 days respectively,(P=0.313),while 2-years of relapse rate of two groups were10.29% and 49.24%,(P=0.005).2-years of overall survival(OS)of DAC group and CON group were 76.6% and 63.5%,respectively(P=0.018);2-years of leukemia free survival(LFS)of two group were 72.9% and 56%,respectively(P=0.007).For NRpatients,the 2-year of OS of DAC group was significantly higher than CON group(82.5%and 46.7%,P=0.011);2-year of LFS is 77% and 34.4%,respectivel(P=0.002).Multivariate analysis results showed that male,DAC group,without grade III-IV a GVHD,limited c GVHD were benefit for OS;while DAC group,without grade III-IV a GVHD,c GVHD and the status before transplantation were benefit for LFS.For AML-NR patients,DAC group and with c GVHD were benefit for OS and LFS.Conclusion: 1.This single-center respective pair-control study showed that DAC containing conditioning regimen could improve overall survival of high risk of NR patients with AML and decrease 2 years relapse rate.2.DAC containing conditioning regimen was safe because the transplant related mortality of DAC group was significantly lower than that of CON group.Part II.Preliminary mechanistic study of low-dose decitabine as part of modified Bu Cy-conditioning in allogeneic stem cell transplantation for high-risk AML patientsObjective : To define the preliminary mechanistic of low-dose DAC as part of modified Bu Cy-conditioning in allogeneic stem cell transplantation for high-risk AML patients.Methods : From May 2016 to December 2017,12 patients in our hospital with high-risk AML received low-dose DAC as part of modified Bu Cy-conditioning and 12 patients who received modified Bu Cy-conditioning were enrolled in the study by collecting the peripheral blood EDTA anticoagulant at preconditioning,the day of transplantation,post transplantation +2 week,+4 weeks,+6 weeks,+8 weeks,+12 weeks,+16weeks,+20 weeks,+24 weeks.1.The percentages of WT1+CTL ? NY-ESO-1+CTL ?SSX-2+CTL ? MAGE-A1+CTL,the immunophenotypes of lymphocytes,perforin and granzyme B were analyzed by flow cytometry(FCM);2.Comparing the differences between DAC and CON group,non relapse and relapse group in DAC group with the above-mentioned indicators;3.Comparing the tumor cell surface antigen of the leukemia cells in NR patients' bone marrow of two groups,which was detected by FCM before and after transplantation.Result:1.Dynamic detection of the lymphocyte subsets and their activations1.1 Comparison between DAC and CON group at the same timeThe percentage of CD3+ cell in DAC group is significantly higher than CON group at the day of transplantation and +12w while the percentage of CD3+CD8+cell in DAC group is significantly higher than CON group at +4w?+6w?+8w?+24w;The percentage of CD8+CD69+and CD3+TCR?? in DAC group is significantly higher than CON group at+12w?+16w and the percentage of CD8+DR+ at +12w?+16w?+20w is significantly higher than CON group.1.2 Comparison between Non relapse and Relapse group after transplantationThe percentage of CD3+cell in non relapse group is significantly higher than relapse group after transplantation at the day before transplantation?+20w?+24w;Meanwhile the percentage of CD3+CD8+ had statistical significances at +2w ? +6w ? +12w ? +16w(P<0.05).CD3+TCR?? cell at +12w ? +16w is significantly higher than relapse group.CD8+DR+ showed statistical significances at +12w ? +16w ? +20w ?+24w(P<0.05);Meanwhile the percentage of CD8+CD69+ is significantly higher than relapse group at +12w(P<0.05).2.Dynamic detection of the expression of perforin and granzyme B2.1 Comparison between DAC and CON group at the same timeDetection by flow cytometry technique(FCM): The percentage of CD8-G and CD8-P of DAC group is significantly higher than CON group at +20w and +6w ? +8w,respectively(P<0.05).2.2 Comparison between Non relapse and Relapse groupThe percentage of CD8-G is significantly higher than CON group at +2w?+6w?+20w?+24w(P<0.05);CD8-P had statistical significances at +12w?+20w(P<0.05).3.Dynamic detection of the Tumor-Associated Antigen-Specific T CellsA total of 17 patients(8 in DAC group while 9 in CON group)were enrolled.The percentage of WT1+CTL?SSX-2+CTL?MAGE-A1+CTL cell of DAC group increased more obvious than CON group at +8w-+24w.The percentage of NY-ESO-1+CTL had statistical significances at +16w?+20w?+24w(P<0.05).4.The tumor cell surface antigenA total of ten NR patients(6 in DAC group and 6 in CON group)detected by minimal residual disease(MRD),we found that the fluorescence intensity of costimulatory molecules(CD28)and adhesion protein(ICAM)expressed on the leukemia cells is significantly higher than CON group at +8w and +6w?+8w,respectively(P<0.05).Conclusions:1.DAC promoted the proliferation and differentiation of immune effector cells such as CTL,NK cell and ??T cell,increased the secretion levels of perforin and granzyme,then enhanced the effect of cell killing activity.2.DAC made tumor cell surface antigens easier to be identified and presented through upregulating the tumor cell surface antigen costimulatory molecules(CD28)and adhesion protein(ICAM).3.DAC upregulated the expression of tumor-associated antigen-specific T cell which enhanced the GVL effect.