Clinical And Biological Characteristics Of Acute Erythroid Leukemia

1. The clinical and biological studies of acute erythroid leukemiaObjectives To analyse systematically the clinical and laboratorial characteristics of 160 acute erythroid leukemia patients who received treatment in our hospital from 2003 to 2014 and to reveal the unique features of AEL patients in our study, improving the study on cytogenetics and molecular characteristics of AEL.Methods① Retrospective analysis was made on the clinical and biological data of 160 AEL patients, karyotypic analysis of 143 AEL, 40 MDS-E and 46 MDS patients were executed through RHG banding, and all these patients were classified into different risk groups according to its karyotypes on the basis of the IPSS and UKMRC proposals. 158 AEL cases were reclassified according to the WHO(2008) proposals, prognosis of patients with complete information were analysed. ② We chose 6 pairs of AEL patiens’ DNA samples,including de nove sample and the corresponding complete remission sample, and gave them whole-exome sequencing, in addition, about 41 cases of de nove AEL patients were investigated by Ion torrent, and gene expression profiling were carried out on 7 de nove AEL patients, all work was to analyze the characteristics of molecular biology.Results ①According to the 2001 WHO group criteria, 160 AEL patients were classified as follows, M6 a 148(92.5%), M6 b 12(7.5%). There were 92 male patients and 68 female patients among the AEL group, the ratio of male and female was 1.35:1, the median age was 50 years old(range, 13-83 years old), WBC 2.7×109/ml(range 0.52-182×109/ml),hemoglobin 74g/L(range 36-148g/L), and platelet 37×109/ml(range 4-715×109/ml),median bone marrow erythroid percentage 60%( 50%-97.5%), median bone marrow myeloblast percentage of all nucleated cells 16%(0.5%-43.5%), median bone marrow myeloblast percentage of nonerythroid cells 40%(20%-93.5%). The clinical features of M6 a was similar to M6 b. Except for erythroblasts percentage, their chracteristics of peripheral blood was alike too. Among 160 AEL patients, conventional cytogenetics analysis were performed on 143 cases. 86 patients have normal karyotype, accounting for about 60.1%, the abnormal karyotype cases’ number was 57, and the percentage was39.9%, including 28 complex karyotype patients. Comparing with western reports, the karyotype composition in our study showed unique features, the normal karyotype was the most frequent in our study but abnormal karyotype was the common in western countries,ethnic differences may contribute to this phenomenon.② 40 MDS-E, 46 MDS and 160 AEL patients were enrolled in our study, finally 158 AEL patients remained classifiable by adopting the WHO(2008) proposals. The new distribution included 108 N-AEL, 13 AML-MRC and 37 MDS-AEL. Comparing the clinical and laboratory characteristics between the groups, the male patients were slightly more than the female. Remarkable difference was observed on plt in PB between N-AEL and MDS-E, while other significant differences were not found among all groups in peripheral blood cells. In addition, the distribution of karyotype between thses N-AEL and MDS-AEL was also different, as well as MDS-E and MDS, normal chromosome was the main karyotype among AML-MRC and MDS-E.③ The whole-exon sequencing was progressed among 6 pairs AEL patients including de nove samples and complete remission samples, surprisingly, some somatic hypermutations were found, including 2 GATA2-muts and 4 CEBPA-muts. In order to have a more comprehensive view of molecular features of AEL, another 41 AEL de nove samples received Ion torrent detection on gene mutations which was common among other subtypes of AML. GATA2 and CEBPA were retested through PCR and sanger sequenceing method among 69 AEL patients, finally, we found 13 cases with GATA2-mut(18.8%) and 33 cases with CEBPA-mut(47.8%), in addition, RUNX1, NPM1, TET2,U2AF1, SETBP1 mutations were also detected.④ To probe the correlation between karyotype and mutations and prognosis, 71 AEL patients were classified into different groups by treatment modalities. Patients who underwent allogeneic bone marrow transplantation had a superior OS to those who received induction chemotherapy without HSCT(P<0.05). Patients with normal karyotype had a superior OS to those patients with none-complex karyotype, the complex karyotype patients’ OS was the shortest. According to IPSS, the higher risk stratification of IPSS are classified, the poorer prognostic will be. In this study, it seems that both GATA2-mut and CEBPA-mut had no effect on prognosis of AML.⑤ We compared the results of gene expression profiles of 7 AEL cases with that of 5normal cases and 10 none-AEL, the data of normal cases and none-AEL were downloaded from GEO data base. AEL patients’ gene expression profiles was different from that of normal human and none-AEL, they had a unique molecular characters.Conclusions① The clinical features of AEL in our study was similar to that of western patients, but had unique cyotogenetic charateristics. Cytogentic risk closely correlated with the prognosis. ② AEL patients in the study have unique molecular genetic features.GATA2-mut and CEBPA-mut were frequent in AEL. ③AEL patients have a unique gene expression profiles.2. Molecular cloning and preliminary functional study of GATA2-muts.Object① To clone the full-length coding sequences(CDS) of the GATA2-muts including P304 H,L321P, R330 X and wild type transcript which were afterwards inserted to eukaryotic expression vectors to perform the functional analyses. ② Preliminary functional study of GATA2-muts were progressed to shed light on deciphering the molecular mechanisms of leukemogenesis.Methods① In order to get plasmids PFLAG-GATA2-P304 H, PFLAG-GATA2-L321 P and PFLAG-GATA2-R330 X, plasmid PFLAG-GATA2 purchased from Addgene was used as template in the progress of site-directed mutagenesis, the CDS of GATA2-muts and wild type were cloned and then inserted to lentivirus LV5, all these plasimids or product of PCR were verified by sanger sequencing.② Lentivirus was packaged in 293 T cell line by using Calcium Phosphate Cell Transfection Kit, then, cell lines including 32 D and NIH3T3 were transfected by lentivirus carrying GATA2-muts and wild type. Western blot was progressed to verify the expression of target plasmids.③ We constructed some models with NIH3T3 cells which had a stable higher expression of GATA2-muts and wild type, respectively, and detected the subnuclear localization of the target proteins encoded by full-length CDS of GATA2-muts, and compared the difference of subnuclear between GATA2-mus and wild type.④ We constructed other models with 32 D cells which had a stable expression of GATA2-muts, as well as GATA2 wild type. Growth rate of the above cells was detected with the Cell Counting Kit 8(CCK-8) and growth curve was made. Growth ability of such cells in the methyl cellulose medium was detected too. All above experiments were carried out to explain the influences of GATA2 mutations.⑤ 32 D cells with stable expression of GATA2-muts and wild type were analyzed by Wright staining and Benzidine staining. Using fluorescent labeled monoclonal antibodies and flow cytometry, Ter119 was detected. Erythroid differentiation relevant β-globin,βh1-globin’s expression was conducted through RQ-PCR.⑥ Luciferase reporter gene assay with the GATA-luc reporter plasmid was used to detect the impact of the GATA2-muts on the normal transcriptional activities.⑦ To compare the difference of expression profile between 32D-GATA2-muts and wild type, RNA of 32 D cells with stable expression of GATA2-muts and wild type was extracted by Trizol method and received expression profile microarray.Results① The immunofluorescence results showed that wild-type GATA2 protein, as well as P304 H, L321 P, was found to localize in the nucleus. The GATA2-R330 X protein was found to localize not only in the nucleus, but also in the cytoplasm. ② There was no significant difference between GATA2 wild group and that of various mutants in cell proliferation, and all these GATA2 mutants and wild type could grow in colonies in the soft agar, but no significant difference was found. ③ 32 D cells with stable expression of GATA2 wild type or GATA2-muts received Wright staining and Benzidine staining, we did not observe obvious change among various mutants comparing with the wild type.Unlike the wright stain, it is interesting that the proportion of Benzidine staining positive cells of 32 D with GATA2-muts was much more than that in the vector group as well as the wild type group. ④ The cell surface sign Ter119 has a significant rise among 32 D with GATA2-muts detected by flow cytometer. ⑤ The elevation of expression level of β-globin and βh1-globin was detected through RQ-PCR method in group of 32 D with GATA2-muts.⑥ Proteins encoded by GATA2-R330 X could inhibit the transactivation ability of wild type GATA2 on the promoter of the GATA. The transactivation ability of all GATA2-muts included in this study declined comparing with wild type. ⑦ GATA2 mutation can lead to the alteration of gene expression assay.Conclution① Erythroid differentiation can be resulted from GATA2 mutation. ② GATA2-R330 X can lead to the abnormal localization of protein encoded by the gene.