| Objectives:1. To analyse systematically the clinical and laboratorial characteristics of 1208 newly diagnosed APL patients in the First Affiliated Hospital of Soochow University from January 2003 to January 2015, summarize the basic features of APL patients in our center.2. To analyse 470 APL patients with complete clinical data, explore additional chromosomal abnormalities effects on clinical characteristics and prognosis.3. We aim to fnid APL patients with positive STAT5B-RARα using FISH and RT-PCR technology, summarize its clinical characteristics and prognosis, clone STAT5B-RARα fusion gene, and study the biological function of STAT5B-RARα fusion gene.Methods:1. 1208 newly diagnosed APL patients in our center were studied, through collecting and analyzing basic laboratory data of MICM, clinical data, related gene mutation information by SPSS 21.0 software and Graphpad 5.0 software.2. 470 newly diagnosed APL cases with complete clinical data will be classified as simple t(15; 17) group, additional chromosomal abnormalities(ACAs) group, based on the karyotype analysis. We use the Kaplan-Meier method, chi-square test and Mann-Whitney U test to compare the differences of clinical and laboratorial characteristics among different groups. The ACAs group will be further grouped according to the structure and quantity of ACAs, and we analyze the prognostic differences between each additional chromosomal abnormalities and simple t(15; 17) group.3. We screened APL patients with STAT5B-RARα by FISH and RT-PCR, and summarize their clinical features and prognosis.The STAT5B-RARα fusion gene was cloned and ligated to the retroviral vector plasmid—LV5-GFP. Then the retrovirus was prepared by transfected target and package plasimids into 293 T cells by DNA-calucium phosphate method. The transfect efficiency on U937 cells was detected by measuring the expression of GFP through flow cytometry(FCM). The expression level of STAT5B-RARα in 293 T cells was measured by RT-PCR. CCK-8 test and cell colony formation assays(CFCs)were used to analyze the influence of STAT5B-RARα fusion gene on the proliferation of U937 cells.Results:1. The clinical and genetic features of 1208 newly diagnosed APL cases1208 APL patients(633 males/575 females,1.10/1),and the median age was 39 years(4-78 years old), mainly in 30-49 years. Among the 1028 APL patients, there were 1175 classic APL cases, 17 RARα rearrangement variant APL cases, and 16 APL cases with negative t(15;17) and negative PML-RARα fusion gene. Among three groups.,these 16 APL patients have more white blood cell counts and higher risk group ratio,and 17 RARα rearrangement variant APL cases more tend to be male amongKaryotype analysis was performed on 1142 APL patients with complete clinical data, there are 71.45%(816/1142) APL patients with t(15; 17) alone, 18.21%(208/1142) APL patients with ACAs, 6.83%(78/1142) APL cases with normal karyotype. Trisomy 8 is the most common additional chromosomal abnormality, accounting for 22.60%; In addition, there are 15 cases with 9q-, 12 cases with 7q-, 11 cases with +21, and the proportion was 7.21%, 5.77%, 5.29% respectively.The mutation analysis of APL patients showed that the mutation rate of FLT3-ITD was the highest among genes detected, reaching 32.67%. We analyzed 71 APL patients with complete mutation information,and found that 64.79%(46/71) cases carrying gene mutation, and of those 69.57%(32/46) APL cases carrying one mutation, 23.91%(11/46) patients carrying two mutations. Only 3 cases were detected three mutations. We analyze the effect of the mutations on clinical prognosis and found that, compared to APL patients with negative FLT3-ITD mutation, the APL cases with positive FLT3-ITD mutation have significantly more white blood cell count(P < 0.001), S type fusion gene(P < 0.05), and more patients in the high-risk group(P < 0.001), 5 years overall survival decreased significantly(P = 0.014); 5 year relapse free survival was no difference between the two groups(P = 0.055). In addition, FLT3-TKD, TET2, WT1, and NRAS mutations had no effect on the clinical outcome of APL patients.2. The effect of additional chromosomal abnormalities(ACAs)on the clinical characteristics and prognosisi of APL patientsCompared with t(15; 17) alone group, ACAs group tend to be male(50.4% vs. 66.7%, P = 0.004). Initial WBC, hemoglobin, platelet count, fusion gene types, and overall survival and relapse-free survival time et al show no significant differences between two groups. From analysis of structure of ACAs, +8 groups, 9q- group,- 7 / 7q- group, + 21 group, respectively were compared with t(15; 17) alone group, the overall survival time and relapse-free survival time had no significant difference; from analysis of number of ACAs, one ACA, two ACAs, three or more ACAs group, respectively with t(15; 17) alone group, the overall survival time, and relapse-free survival time show no obvious differences.3. Molecular cloning and function study of STAT5B-RARα fusion geneWe use FISH and RT-PCR technique to find 6 APL cases with positive STAT5B-RARα, and all 6 patients undergo ATRA / ATO therapy, 4 cases show no response to therapy, 1 case relapsed soon after achieving complete remission. Then we used c DNA of APL patients with positive STAT5B-RARα as template, using PCR amplified STAT5B-RARα fusion gene and constructed lentiviral vector plasmid LV5-GFP-STAT5B-RARα. The lentiviral vector plasmid was transfected into U937 cell lineby a calcium phosphate precipitation, and we successfully establish a stable cell strain. It showed that STAT5B-RARα could promote the proliferation of NU937 cells to a certain extent in vitro experiment.Conclusions:1. 1208 APL cases mainly focus on the young, and male and female incidence rate is basically the same. Among the 1028 APL patients, there were 1175 classic APL cases, 17 RARα rearrangement variant APL cases, and 16 APL cases with negative t(15;17) and negative PML-RARα fusion gene. Karyotype analysis showed that 71.45% of APL patients had simple t(15; 17); 18.21% of the patients had ACAs. Trisomy 8,accounting for 22.60%, is the most common ACAs, followed by 9q-(7.21%), 7q-(5.77%), +21(5.29%). The mutation analysis of APL patients showed that FLT3-ITD mutation rate was the highest, reaching 32.67%, which is a factor of poor prognosis in APL patients. WT1, FLT3-TKD mutation rate is higher, respectively, 11.11%, 14.46%. FLT3-TKD, TET2, WT1, and NRAS mutations had no significant effect on the clinical outcome of patients with APL. |
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