The Molecular Diagnostic Study Of Acute Myeloid Leukemia

Purpose:To screen the genetic aberrations of de novo acute myeloid leukemia using cytogenetic and molecular cytogenetic techniques.Methods:Mononuclear cells acquired from the bone marrow of 205 patients with de novo acute myeloid leukemia were collected, the basic clinical data were recorded accordingly. Karyotype was analyzed using G-banding of chromosomes. Fusion genes and internal tandem duplication of FMS like tyrosine kinase 3 (FLT3-ITD) was detected by-poiymerase chain reaction. Mutations of nucleophosmin (NPM1), CCAAT enhancer binding factor a (CEBPa) and C-kit genes were detected by direct sequencing of PCR product. The clinical data were analyzed with the results of genetic screening.Results:Kayrotype was analyzed in 166 cases; results of fusion genes were available in 202 cases. FLT3-ITD mutation was found in 45 patients; NPM1 mutation was found in 34 patients; CEBPαmutation was found in 61 patients; C-kit mutation was found in 16 patients. The former three were all associated with normal karyotype, but with different FAB subtype respectively. The C-kit mutation was associated with t(8; 21) translocation.Conclusion:The combination of cytogenetic and molecular cytogenetic techniques can help to tell the background of patients with de novo acute myeloid leukemia. Purpose:To detect the IDH1 (Isocitrate dehydrogenase-1) and IDH2 (Isocitrate dehydrogenase-2) mutation rate and their clinical characteristics in 205 patients with acute myeloid leukemia (AML).Methods:DNA from Bone marrow mononuclear cells was obtained from 205 patients with acute myeloid leukemia. The 4th exon of IDH1 and IDH2 were amplified using polymerase chain reaction method and the DNA sequencing of purified products was applied. Finally, the clinical character of patients with IDH1 and IDH2 mutations was comprehended by specimens'library.Results:IDH1 mutations were detected in 10 (4.95%) of 205 subjects including Ml 2 cases, M2 6 cases, M3 1 case, M5 1 case. There were 6 R132H mutations,2 R132S mutations,2 R132G mutations. The relative age, chromosome analysis, fusion gene and FLT3, C-KIT, NPM1 mutations had no difference (P>0.05), however, the gender, FAB typing, CEBPa mutation and IDH1 mutation had significant correlation (P<0.05)。IDH1 mutations were detected in 15 (7.3%) of 205 subjects including M1 2 cases, M2 4 cases, M5 9 case. There were 10 R140Q mutations,1 R140W mutation,4 R172K mutations. The relative age, gender, chromosome analysis, fusion gene and FLT3, C-kit NPM1, CEBPa mutations had no difference (P>0.05).Conclusion:The IDH1 mutation was occurred principally in male patients or patients with AML-M2 and CEBPa mutation can not be observed in these patients. The relationship between IDH2 mutation and its clinical characteristic has not been discoved yet. It may be due to the limited amounts of samples in the study. To find the correlation between these mutations and the formation, development, treatment and prognosis of AML better, futher studies need to expand the sample size. Objective:To detect the DNMT3A mutation rate and its clinical characteristics in patients with acute myeloid leukemia (AML).Methods:DNA from bone marrow mononuclear cells was obtained from 205 patients with AML, then all exons of DNMT3A gene was amplified using polymerase chain reaction method and the DNA sequencing of purified products was applied. DNMT3A mutations were identified by SNPs detection, and were screened simultaneously in AML-NOD/SCID mouse model with leukemia. To explore the relative gene with DNMT3A mutation futher The genome expression profile in AML was analyzed by Cluster analysis using bioinformatics software,. Finally, the clinical character of patients with DNMT3A mutation was comprehended by specimens'library.Results:20(9.76%) among 205 AML patients including MO 1 case, M2 3 cases, M3 1 case, M4 1 case and M5 14 cases, showed DNMT3A mutation and the type of mutation contained R882H 14 cases, R882C 3 cases, F732V 1 cases, P904L 1case, R736C 1 case. 13(19.4%) among 67 AML patients with normal karyotype, and 65% mutation was detected in CN-AML. The same mutations were confirmed in SNP and NOD/SCID mouse model with AML. Among 109 cases patients with complete statistical data, the CR rate in DNMT3A mutation patients was 50%, in DNMT3A negative patients was 66.67%(P< 0.05), which had statistically significant difference. The gender, CEBPa, C-kit, IDH1 mutations, MLL expression and DNMT3A mutation had no difference (P>0.05), however, IDH2,FLT3 mutations, NPM1,IKAROS6 expression and DNMT3A mutation had significant correlation (P<0.01)Conclusion:DNMT3A was an independent risk factor for relapse and also a predictor for prognosis, the mutation of DNMT3A may change unique fingerprint of some leukemia subtypes. Objective:To evaluate the expression of IKAROS6 in Acute Myelogenous Leukemia and its clinical characteristicsMethods:The expression of IKAROS6 in 205 AML patients was amplified by PCR and certified by sequencing; the relationship between IKAROS6 and clinical characteristics or prognosis was investigated by analyzing the clinical and laboratory data; the differential gene between AML patients with IKAROS6 expression and AML patients without IKAROS6 expression was acquired by gene expression profiling.10 healthy volunteers were served as controls. The volunteers and patients were all informed the consent.Results:there was no IKAROS6 expression in control group, however, the expression of IKAROS6 was detected in 23 AML patients (accounts for 11.22% in total 205 AML patients), and the positive rate of IKAROS6 in M4/M5 was 19.77%(17/86), the positive rate of IKAROS6 in M1 was 5.56%(1/18), and was 4.23% in M2(3/71), positive rate of IKAROS6 in M3 was 7.14%(2/28). In 109 AML patients who had complete clinical and laboratory data, the complete remission(CR) rate of the patients with IKAROS6 expression (6/23,26.09%) was much lower than that of the patients without IKAROS6 expression(65/92,70.65%)(P< 0.01); the expression of IKAROS6 was related with sex and the mutation of NPM1,CEBPα,C-kit,IDH1,IDH2,FLT3(P<0.05),and the expression of IKAROS6 was also related with the mutation of DNMT3A and the expression of MLL(P<0.01).Conclusion:Abnormal expression of IKZF1 (mainly IKAROS6) is detected in AML, especially in M4 and M5, and the abnormal expression of IKZF1 is an independent prognosis index in AML patients. The results of cluster analysis suggested that:AML with IKAROS6 expression may be a new subtype of AML, and Targeting IKAROS6 may be of great potential in the specific AML diagnosis and treatment.