Study On The Prognosis Evaluation And Treatment Strategy Of Acute Leukemia

Acute leukemia(AL)is a highly heterogeneous hematological malignant with a high recurrence rate.It is divided into acute myelocytic leukemia(AML)and acute lymphoblastic leukemia(ALL)according to the cell lineage.Although the progress of traditional chemotherapy and targeted therapy has increased the complete remission rate of AL,the treatment of relapse or refractory AL is still an urgent problem to be solved.Therefore,it is extremely urgent to improve the assessment accuracy of AL prognosis and explore new treatment strategies for AL.This thesis focuses on clinical study on the prognosis evaluation and treatment strategies of AL,including the prognosis value of CD56 in AML,the study of PADI protein inhibitor inducing apoptosis of AML cells and its mechanism,and the clinical trial study of humanized CD19-CAR-T cell therapy for relapsed or refractory B-ALL.Part Ⅰ Prognostic value of CD56 in patients with de novo acute myelocytic leukemiaBackground: There are many factors affecting the prognosis of AML.Genetic abnormality is the most commonly stratification factor for AML.However,the prognosis of patients at the same genetic risk level varies,suggesting that multiple factors affect the development of the disease.Leukocyte differentiation antigen(cluster of differentiation,CD)is a cell surface marker that appears or disappears during different differentiation stages or activation condition of normal blood cells.In leukemia cells,CD markers may express abnormally.For example,the NK cell marker CD56,sometimes over expressed on AML cells.Therefore,analyzing the prognosis value of CD56 expression on AML may provide a more accurate stratification basis,and may be a therapeutic target.Methods: Bone marrow samples of 89 AML and 46 control cases were collected.Multiparameter flow cytometry(FCM)was used to detect the CD56 expression in bone marrow cells and the prognosis value of CD56 in AML was analyzed.1.We compared the CD56 expression on bone marrow cells between AML and controls,the difference between AML before and after treatment;2.Follow up study was conducted in AML patients to analyze the correlation between CD56 expression and treatment response or survival prognosis;3.To verify whether CD56 is an independent prognostic factor of AML through univariate and multivariate analysis.Results:1.The expression level of CD56 in AML patients was significantly higher than that in the control group(P<0.05),while it is dramatically decreased in AML after treatment(P <0.001);2.A cutoff point of CD56 was calculated by X-Tile program at 24.62,which divided AML patients into two group.29.21%(25)patients classified into the CD56-High group,and others classified into CD56-Low group.The overall survival(OS)of patients in CD56-High group is significantly poorer(P = 0.015);3.The results of the Cox proportional hazard model show that CD56 was an independent risk factor affecting the OS of AML patients(P = 0.010).Conclusion: CD56 antigen tends to be abnormally expressed in AML cells,and highly expression of CD56 is associated with poor clinical outcome,suggesting that CD56 can be used as a stratification maker for AML patients,and it may be a treatment target.Part Ⅱ PADI inhibitor BB-Cl-Amidine effectively kill AML cells by inducing ER stressBackground: Epigenetic changes play an important role in the occurrence and development of AML,such as DNA methylation and histone modification.Some demethylation or histone acetylation modulator drugs have been widely used in clinical practice.Although there are certain effects generated by the current drugs at some extent,not all patients can benefit from it,also the problem of side effect.Peptidylarginine deminase(PADI)protein is a member of the histone demethylase family.Among the subtypes of PADI proteins,the expression of PADI2 and PADI4 increased in AML patients,suggesting the potential role of PADI protein in AML.Inhibition of PADI2 or PADI4 is expected to become a new target for the treatment of AML.Methods: With DMSO as the control,the pan-PADI inhibitor BB-Cl-Amidine(BB-Cl-A)and the PADI4 specific inhibitor GSK484 were used for AML cell lines in vitro:1.GEPIA2 database,CCLE gene expression data and RT-q PCR method analyzed the PADIs’ expression in AML cell lines;2.Proliferation activity of AML cells were detected by CCK8 experiment;3.Cell death of AML cells were detected by Annexin V/PI Flow cytometry;4.The phosphorylation of PERK and e IF2α was detected by western blot(WB).The gene expression level of ATF4,CHAC1,DDIT3 and DDIT4,which were related to apoptosis,was analyzed by RT-q PCR.Results:1.Gene expression data and RT-q PCR results showed that PADI2 and PADI4 are the main subtypes of PADI proteins expressed in AML patients and AML cell lines;2.CCK8 and Annexin V/PI test results showed that BB-Cl-A can does dependently inhibit the proliferation of 6 AML cell lines,and this inhibition effect was dose-dependent.Compared with the control group,BB-Cl-A significantly induced AML cells apoptosis and which can be rescued by Z-VAD-FMK,an apoptosis inhibitor.But GSK484 has no obvious effect on the growth and apoptosis of AML cell lines.3.BB-Cl-A promoted the phosphorylation of PERK-e IF2α pathway and up regulated the m RNA expression of ATF4,CHAC1,DDIT3 and DDIT4.Conclusion: The above data indicate that PAID2 plays a major role in AML.BB-Cl-A may activate ER stress by inhibiting PADI2,thereby inducing apoptosis of AML cells.Therefore,PADI2 plays an important role in maintaining the homeostasis of the endoplasmic reticulum and inhibiting cell apoptosis.Targeting PADI2 by BB-Cl-A may be a new strategy to treat AML.Part Ⅲ A clinical study of humanized CD19-CAR-T cell in the treatment of relapsed or refractory acute B lymphocytic leukemiaBackground: B-ALL is a type of AL with an incidence of about 25%.However,because of the variable resistance mechanisms of tumor cells,traditional treatment strategies are helpless in relapsed or refractory B-ALL.Chimeric antigen receptor T-cell(CAR-T)is a novel immunotherapy.As a tumor specific antigen sequence is transferred into T lymphocytes which separated from patient,the CAR-T cells could specifically recognize tumor cells.Due to the strong positivity and high stability of CD19 expression in ALL malignant cells,CAR-T cell targeting CD19 have great potential for the treatment of B-ALL.Thus,we conducted a clinical trial study to treat relapsed or refractory B-ALL with CD19-CAR-T cell and analyzed its efficacy and safety.(Clinical trial platform,NCT02349698).Methods: CD19-CAR-T cells were prepared and used to treat 15 patients with relapsed or refractory B-ALL,then observe the treatment effect and the occurrence of side effects,evaluating the effectiveness and safety of CAR-T cell therapy:1.T cells of patient were isolated and CD19-CAR-T cells were prepared by genetic engineering technology;2.CAR-T cells were injected into patient.Bone marrow morphology,flow cytometry,and gene detection were used to evaluate the treatment response.Meanwhile,cytokine release syndrome(CRS)and neurotoxicity should be monitored;4.Follow-up study to observe the patient’s condition and survival.Results:1.The median time of CD19-CAR-T cell expansion peak was 11 days(8-68 days)after infusion.The CR rate of CAR-T cell therapy was 93%(14 cases).The disease-free survival(DFS)rate at 3rd month after CAR-T cell infusion was 100%,and the 12 th month was 40%;2.93%(14 cases)of patients experienced CRS,of which 71%(10 cases)were diagnosed as mild CRS(grade 1 and 2),and 29%(4 cases)were severe CRS(grade 3 and 4).20%(3 cases)of patients developed neurotoxicity;3.Interleukin-6(IL-6)rapidly increased in patients with severe CRS.The main side effects of CRS and neurotoxicity were well controlled by using Tocilizumab,hormones,and plasma exchange.Conclusion: The humanized CD19-CAR-T cell therapy can effectively increase the CR rate of relapsed or refractory B-ALL with controllable side effects.But the technology needs further observation and research to maintain long-term survival.