| Objective1. To analyze the clinical and laboratorial characteristics of 176 patients with de novo core-binding factor acute myeloid leukemia(CBF-AML) who received cytogenetics and molecular genetics analysis in the First Affiliated Hospital of Soochow University.2. To clone two kinds of rare FLT3-TKD mutants in core-binding factor acute myeloid leukemia and explore their roles in leukemogenesis via in vitro experiments.3. To investigate the frequency of C-KIT mutation and prognosis in t(8;21) acute myeloid leukemia with trisomy 4.Methods1. Cytogenetic studies were performed on 176 patients’ bone marrow samples at their diagnosis by standard R banding techniques. The karyotype obtained was described in accordance with the recommendations of the International System for Human Cytogenetic Nomenclature(ISCN 2005). We examined gene mutation detection of KIT, FLT3, JAK2, RAS and CBL by polymerase chain reaction(PCR) followed by direct bidirectional Sanger DNA sequencing in cells from 176 CBF-AML patients.2. PCR was performed to acquire two rare FLT3-TKD mutations which was afterwards inserted to a transfer plasmid-LV5, respectively. The mutants were transfected into Ba F3 cells by DNA-calcium phosphate, respectively. Growth curves of two mutant receptors as well as controls were made according to the assay of cell counting kit 8(CCK8). Growth ability of the cells in the methylcellulose and proliferation in culture without interleukin-3(IL-3) were also investigated to detect the malignant transformation ability of the Ba F3 cells expressing FLT3 mutant receptors.3. This retrospective study was conducted at the Department of Hematology, the First Affiliated Hospital of Soochow University from February 2005 to January 2013. 145 AML patients with t(8;21) were included in the present study. Detection of C-KIT mutation was performed on bone marrow samples of all patients at diagnosis. SPSS 19.0 was used for statistical analysis.Results1. The clinical and laboratorial characteristics of 176 newly diagnosed patients with CBF-AMLIn this study, we investigated the clinical and laboratorial characteristics of 176 CBF-AML patients including 139 patients with RUNX1-RUNX1T1 and 37 with CBFβ-MYH11 fusion transcripts. The patients consisted of 101 males and 75 females with a median age of 35(range, 11-77). Diagnosis and classification of AML were defined according to the French-American-British classification(FAB) and were revised for application of the World Health Organization(WHO 2008). The majority of t(8;21) AML patients were diagnosed as M2(84.2%) and most of the patients with inv(16) were M4Eo(64.9%). The analysis of baseline characteristics of the patients showed that patients with inv(16) had higher peripheral WBC counts, hemoglobin level and percentage of blast cell in bone marrow samples at diagnosis when compared with those of patients with t(8;21)(P < 0.05).174 cases out of 176 had cytogenetics analyzed successfully and 84 cases had additional chromosomal aberration. The most frequent additional chromosomal aberration was loss of X/Y identified in 55(31.3%) patients, followed by del(9q)(n = 10, 5.7%), trisomy 22(n = 10, 5.7%), trisomy 4(n = 7, 4.0%) and trisomy 8(n = 2, 1.1%). 14 patients were not detected t(8;21) or inv(16) by cytogenetics study, but RUNX1-RUNX1T1 or CBFβ-MYH11 fusion transcripts were detected by RT-PCR.Overall, 73 patients(41.5%) had no detected mutations, a single gene was mutated in 97(55.1%) patients, and more than one gene in six(3.4%) patients. The frequency of C-KIT mutation was 34.7%(n = 61) and most of the mutation occurred in exon 17(90.2%, 55/61). FLT3 mutation was presented in 12.5% of the patients(22/176),among which FLT3-ITD was 40.9%(9/22) and FLT3-TKD was 59.1%(13/22),respectively. Mutation of FLT3-TKD was predominantly occurred in exon 20(84.6%, 11/13), resulting in variable amino acid changes(D835Y, D835 E or D835H). More t(8;21) AML patients suffered C-KIT mutation than patients with inv(16)(39.6% vs. 16.2%)(P < 0.01). We also identified two rare FLT3-TKD mutants, namely FLT3 N676 K and FLT3 H671 Q, respectively.2. Molecular cloning and function analysis of the two rare FLT3-TKD in leukemogenesisThe mutated FLT3 was cloned and ligated to the LV5 vector plasmid. Ba F3 cell lines stably expressing various FLT3 constructs wereestablished. Through cell viability detection, we observed that the mutants could accelerate the proliferation of Ba F3 cell and make the cells grow cytokine-independently to some extent.3. Frequency of C-KIT mutation and prognosis in t(8;21) acute myeloid leukemia with trisomy 4In 145 t(8;21) AML patients with trisomy 4, 91.7%(11/12) of patients were identified with C-KIT mutation, which was significantly higher than those without trisomy 4(26.3% for the latter, P < 0.01). More importantly, the follow-up data showed that the patients with trisomy 4 correlated with a lower 3-year overall survival(OS)(15% vs. 56%, P < 0.01) and disease-free survival(DFS)(0% vs. 51%, P < 0.01) when compared with patients without trisomy 4. Furthermore, the subgroup of t(8;21)AML patients with both trisomy 4 and C-KIT mutation had a worse OS and DFS than the patients without trisomy 4 and C-KIT mutation(P < 0.05).Conclusion1. Loss of X/Y chromosome is the most frequent additional chromosomal aberration and C-KIT mutation is the most frequent gene mutation in CBF-AML, especially in t(8;21) AML. We also identified two rare FLT3-TKD mutants, namely FLT3 N676 K and FLT3 H671 Q.2. We successfully cloned the mutants and constructed the stable transfected cell lines. We observed that the mutants could accelerate the proliferation of Ba F3 cell and make the cells grow cytokine-independently to some extent.3. We firstly identified high frequency of C-KIT mutation in t(8;21) AML patients with trisomy 4. And patients with trisomy 4 demonstrate an adverse implication in t(8;21) AML patients, which indicates it may define a distinctive subtype of t(8;21) AML. |
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