The Expression And Function Of PD-1/TIM3 And Their Ligands In FLT3-ITD Positive Acute Myeloid Leukemia

Part I The clinical research of acute myeloid leukemia patients with FLT3-ITD mutationObjective:To analyze the genetic profile and MICM of acute myeloid leukemia(AML)patients with FLT3-ITD and/or FLT3-TKD;To explore correlations between FLT3and other mutations;To study clinical outcome of FLT3 mutations acute myeloid leukemia patients treated with allogeneic hematopoietic stem cell transplantation(allo-HSCT)and TKIs.Methods:A total of 373 AML patients in our center were retrospectively analyzed from September 2010 to September 2016;the clinical characteristics of patients carrying different mutation were compared;the clinical data of patients after treatment by allo-HSCT and TKIs collected and analyzed.Results:(1)Among 200 patients,19 patients with concurrence of both FLT3-ITD and FLT3-TKD mutations,149 patients with FLT3-ITD and 32 with FLT3-TKD.There was no significant difference in the gender,age,the hemogram parameter,primitive cell of bone marrow and FAB subtype(P>0.05)among the three groups.(2)200 cases of patients with FLT3 mutation in 101 patients(50.5%)with one or more other mutations,76 cases with NPM1(38%),34 cases with DNMT3A(17%),15 cases with CEBPA(7.5%),5 cases with C-KIT(2.5%).(3)(1)The overall survival time and complete remission time among FLT3-ITD,FLT3-ITD and FLT3-ITD~+/TKD~+AML patients were statistically significant(P=0.041,P=0.003).(2)The median survival time and complete remission time of patients with FLT3-ITD~+/TKD~-AML and FLT3-ITD~+/TKD~+AML were significantly longer than those treated with high-dose chemotherapy.(P=0.001,P=0.021;P=0.013,P=0.019).(3)The median survival time and the complete remission duration between FLT3 mutation positive AML patients who treated combined with Sorafinib or not was without difference(P=0.062;P=0.209).Conclusion:FLT3 gene mutation is a common gene mutation in AML patients,the patients with clinical manifestations and laboratory features of special,and often with other AML mutations coexist,should receive hematopoietic stem cell transplantation early,combined therapy with tyrosine kinase inhibitors.Patients with concurrence of both FLT3-ITD and FLT3-TKD mutations got poor prognosis,need to further explore the therapy..Part Ⅱ The expression and function of PD-1/TIM3 and their ligands in FLT3-ITD positive acute myeloid leukemiaObjective: To dynamic analysis the expression and the role of negative costimulatory molecules PD-1 and TIM3 T cells of FLT3-ITD~+ AML patients as well as their ligand Gal-9 and PD-L1 in the peripheral blood.To further explore whether PD1/PD-L1 and PD-1/PD-L1 and TIM 3/Gal-9 pathway involved in immune escape in AML.Methods: The clinical data of patients diagnosed with FLT3-ITD~+ AML by bone marrow MICM classification in our hospital were collected,anticoagulated peripheral blood before and after treatment was taken from each candidate.The anticoagulated peripheral blood from healthy volunteers was gathered as normal contral.We used the methods of immune fluorescence labeling and flow cytometry to dynamic analyze expression of PD-1/TIM3 on T cells.Secondly detected secretion of IFN-γand IL-2 on T cells.Lastly,we used the methods of ELISA to dynamic analyze the expression of PD-1/PD-L1 and TIM3/Gal-9 in plasma of AML patients.Results:(1)The proportion of CD4+T cells and CD8+T cells on lymphocytes in newly diagnosed FLT3-ITD~+ AML patients and wt FLT3 AML patients were remarkably decreased compared to control group(P=0.02,P=0.048;P=0.001,P=0.008);the levels of TIM3 on CD4+T cells and CD8+T cells in newly diagnosed FLT3-ITD~+AML patients and wt FLT3 AML patients were significantly increased compared with normal group(P=0.001,P=0.008;P=0.032,P=0.001);the levels of TIM3 on CD4+T cells in newly diagnosed FLT3-ITD~+ AML patients was significantly increased compared with wt FLT3 AML(P=0.002);the levels of PD-1 on CD4+T cells and CD8+T cells in newly diagnosed FLT3-ITD~+AML patients were significantly increased compared with normal group(P=0.04,P=0.004).(2)The secretion of IFN-γ and IL-2 in TIM3+T cells was significantly increased than TIM3-T cells(P<0.05).(3)The proportion of CD4+T cells and CD8+T cells on lymphocytes in FLT3-ITD~+ AML patients achieved complete remission(CR)after chemotherapy was significantly increased compared with the proportion before treatment and those without remission or relapse(P=0.007,P=0.007;P=0.003;P=0.043).The level of PD1 and TIM3 expression on T cells in patients achieved CR after chemotherapy was significantly increased compared with the proportion before treatment(P=0.007,P=0.018;P=0.044,P=0.001),compared with relapsed or refractory FLT3-ITD~+ AML patients were also obviously decreased(P=0.001,P=0.001;P=0.002,P=0.001).(4)The expression of PD-1 and TIM3 in plasma of newly diagnosed FLT3-ITD~+ AML patients was significantly increased than healthy volunteers(P=0.001,P=0.003),The level of TIM3 and PD1 in plasma of patients achieved CR after chemotherapy was significantly decreased compared with newly diagnosed and relapsed or refractory FLT3-ITD~+ AML patients(P=0.001,P=0.003;P=0.002,P=0.002).(5)The expression of Gal-9 and PD-L1 in plasma of newly diagnosed FLT3-ITD~+ AML patients was significantly increased than healthy volunteers(P=0.001,P=0.031),the level of PD-L1 and Gal-9 in plasma of patients achieved CR after chemotherapy was significantly decreased compared with newly diagnosed and relapsed or refractory FLT3-ITD~+ AML patients(P<0.001,P=0.046;P= 0.007,P=0.001).Conclusion: Dynamic analysis of changes in the proportions of T cells and the level of expression TIM3 and PD-1 in patients with FLT3-ITD~+ AML before and after chemotherapy prompt that PD-1 and TIM3 may play an important role in the pathogenesis of AML.PD-1/PD-L1 and TIM3/Gal-9 may be involved in the occurrence and development of AML by inhibiting T cell immune function by inhibiting the immune response of T cells and inducing immune escape.