| Acute myeloid leukemia(AML)is a highly heterogeneous disease,which is characterized by the occurrence of abnormal blasts of different maturation in the bone marrow(BM),perturbing normal hematopoiesis.Current standard for the induction treament have remained largely unchanged for nearly 50 years,and AML still remains a disease of poor prognosis.5-year survival rate was only about 20%,most AML patients chemotherapy resistant or relapse.Many studies have found that CD123 is highly expressed on AML blasts and contrast to the low or not detectable expression on normal hematopoietic stem/progenitor cells(HSPCs).and CD123 overexpression is associated with poor prognosis of AML,and therefore CD123 has become a new ideal target for CART immunotherapy of AML.In the first part of this study,we retrospectively analyzed the relationship between CD123 expression and the prognosis in primary AML patients,to explore whether CD123 overexpression in AML patients are more likely to induce chemotherapy failure and have worse survival,so as to provide a theoretical basis for CD123 CART cells in the treatment of AML.the second part is the successful construction of the target CD123CAR lentiviral vector,developed the high transfection efficiency of CART123 cells,verified the CART123 cytotoxicity against CD123~+primary AML blasts,verified the effectiveness of CART123.So as to provide new treatment option for CD123~+refractory and recurrent AML patients.Part ? The study of expression and prognostic value of CD123 in acute myeloid leukemiaObjective Retrospective analysis the expression and distribution of CD123 and relationship between CD123 expression and the prognosis in 137 primary AML patients.Methods 137 cases of primary AML(excluding M3)were retrospectively analysis admitted to our hospital from January 2012 to December 2015,and collect basic clinical data including age,gender,initial peripheral blood WBC counts,BM blasts,cytogenetic and molecular abnormalities,transplantation,CR1 and CD123 expression.The expression of CD123 was detected by flow cytometry and was defined as positive of more than 20%of all AML blasts.IBM SPSS 21 statistical software was used for statistical analysis.Categorical date were expressed as percenttages(%).Continuous date were expressed as median.Clinical variables across groups were compared using chi square test or a two-sided Fisher exact test for categorical variables,and the nonparametric Mann-Whitney U test was applied for continuous variables.Survival data were analyzed by Kaplan-Meier method,and the differences between survival curves was compared by log-rank test.Logistic regression analysis was performed to evaluate complete remission rate(CR1).Univariate and multivariate analysis of OS and DFS by COX proportional hazards model.Regression analyses included OR or HR and 95%confidence intervals for risk assessment,and all tests were two-sided test.All statistical results P<0.05 was considered to be significant.Results 1.Among 137 patients,70(51.2%)cases were male and 67(48.9%)cases were female,and the median age was 46(13-72)years.A total of 61.3%(84/137)patients expressed CD123.According to FAB/WHO subtypes,the expression of CD123 in M1-M6 was 60.0%(3/5),48.5%(16/33),70.8%(34/48),68.4%(26/38),50.0%(4/8),unclassified 20.0%(1/5),respectively.Among the hundred percent of M1,M4 and M5were relatively high for CD123,whereas expression in M2 and M6 was somewhat lower.FAB subtype M4 expressed the highest percentage of positive cases,which was significantly different from M2(P=0.042).All cases were classified into risk groups based on cytogenetic and molecular abnormalities,and three groups of patients were 28,74 and 35,respectively,and no differences were found for CD123 expression between these three groups.2.The correlation between clinical features and CD123 expression was analyzed.The results showed that high WBC counts(P=0.001)and CR1(P=0.027)were significantly correlated with CD123 expression,whereas there was no significant correlation between the other clinical parameters and expression of CD123.3.Efficacy results show that CR1 rate was 62.0%of all cases,two groups were 54.8%(46/84)and 73.6%(39/53)respectively,and the difference was statistically significant(P=0.027).Logistic regression model analysis showed that the proportion of BM blasts(P=0.021)and CD123 expression(P=0.024)were independent factors of CR1in AML.The overall recurrence rate was 46.8%(52/85)to the end of follow-up,two groups were 50.8%(32/63)and 41.7%(20/48)respectively,no significant difference in recurrence rate between the two groups(P=0.340).4.Survival analysis showed that expression of CD123 in AML patients with significantly shorter OS(P=0.015),whereas no significant difference was observed in DFS between the two groups(P=0.086).CD123 was an independent prognostic factor for OS by multivariate analysis.137 patients was performed risk stratification,OS(P=0.035)and DFS(P=0.029)were found to be shorter in the CD123 expression group of the intermediate groups.Similarly,CD123 expression negatively affected OS(P=0.003)and DFS(P=0.006)in the group of age below 50 years.Conclusion 1.CD123 was widely expressed in AML blasts,and among M4expressed the highest CD123.CR1 and WBC counts were significantly correlation with percentage of CD123 expression.2.Proportion of BM blasts and CD123 were the risk factors of CR1 in newly diagnosed AML patients.Could be used to evaluate the effect of patient treatment.3.CD123 was an independent prognostic factor for OS,whereas has no impact on DFS.CD123 was a poor prognostic factor for DFS and OS in the intermediate groups and age?50 years.Part ? Study of the in vitro anti-leukemia effects by chimeric antigen receptor T cell targeting CD123Objective Construction of chimer antigen receptors(CARs)lentiviral vector targeting CD123,developed a CART123 cells with high transfection efficiency.CART123 was evaluated for the cytotoxicity to target CD123~+ primary AML cells in vitro,so as to confirm the validity of CART123,and to provide the experimental basis for clinical treatment of CD123~+ refractoryand recurrent AML patients.Methods Isolation of peripheral blood mononuclear cells(PBMCs)from healthy volunteers by Ficoll density gradient centrifugation,activated 2-3 days by CD3/CD28 combined with IL-2.T-lymphocytes was transduced with CD123 TBBz lentivirus vectors and then the engineered cells was expanded in vitro.CART123 cells transduction efficiency was evaluated by q PCR and phenotypes was detected by flow cytometry.The in vitro efficacy of CART123 was evaluated by flow cytometry Count Bright beads,and detected for upregulation of CD107?and production of cytokine by flow cytometry.Results 1.Both of the two lentivirus vectors were successfully constructed.CD123 TBBz can effectively transfect activated T lymphocyte cells,and CART123 cells has high transfection efficiency and appropriate CD4/CD8 cell subsets.2.We cocultured CART123 with CD123~+AML cells in vitro showed significant CD107? upregulation, cytokine secretion and robustly lysed CD123~+ primary AML samples.Conclusion The results showed that CART123 cells have high transfection efficiency,high amplification efficiency and the ability to lyse CD123~+ primary AML cells.These findings suggest that CART123 may be a promising immunotherapy for the treatment of high-risk AML. |
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