| OBJECTIVE:(1) Establish the molecular diagnosis platform of common gene mutations in myeloid malignancies using high-resolution melting analysis (HRMA).(2) Explore the clinical relevance of these gene muations.METHODES:(1) Collection of clinical cases, cell separation and DNA extraction: Bone marrows were collected from 221 cases with acute myeloid leukemia (AML),88 myelodysplastic syndrome (MDS) and 142 myeloproliferative neoplasm (MPN) which were diagnosed accoriding to FAB and WHO cirtieria. Bone marrow mononuclear cells were separated using Ficoll-Hypaque and genomic DNA was extracted using DNA Purification Kit.(2) PCR amplification:Different PCR systems were established to amplify different mutated genes (C-KIT, IDH1, IDH2, DNMT3A, U2AF1 and JAK2) according to the specific mutated sites of different genes in 7300 Thermo cycler.(3) HRMA detection:PCR products were transferred to the LightscannerTM platform for scanning and were analyzed using software package. The sensitivity of HRMA was evaluated. Gene mutations in the detected samples were further analyzed.(4) DNA sequencing:The sensitivity of direct DNA sequencing was evaluated for different gene mutations. The samples which were identified positive in clinical cases were further sequenced.(5) Analysis of clinical relevance of gene mutations:The correlation between gene mutations and sex, age, hematologic parameters, chromosomal groups, FAB subtypes and WHO subtypes was analyzed with Spearman's rank correlation. Chi-square analysis or Fisher exact test was carried out to compare the difference of categorical variables between patient groups. Mann-Whitney's U-test was carried out to compare the difference of continuous variables between patient groups. Survival was analyzed according to the Kaplan-Meier method (univariant analysis) and the Cox regression method (multivariate analysis).RESULTS:(1) C-KIT mutation:â‘ HRMA could distinguish C-KIT mutation with the maximal sensitivity of 5% in a background of wild-type DNA, higher than that of direct DNA sequencing (10%).â‘¡In 160 AML patients, 9 (6%) cases were identified with C-KIT mutations, which were further confirmed by DNA sequencing, including 4 D816V mutations,4 N822K mutations, and 1 dual muations of D816H and N822K.8 mutations occurred in the patients with AML-M2 subtype, among which 7 cases had the translocation t(8;21); 2 had normal karyotype. Among 25 cases with t(8;21), the frequency of C-KIT mutation was 28%. There was no difference in sex, age and blood parameters between mutated and wild-type groups.(2) IDH1/IDH2 mutation:â‘ HRMA could distinguish IDH1 R132H, IDH2 R140Q and IDH2 R172K mutations with the maximal sensitivity of 5%,2% and 2%, respectively, higher than that obtained by direct DNA sequencing (10%).â‘¡IDH1 mutations were found in 4 of 198 AML patients (2.0%) and IDH2 mutations were found in 10 cases (5.0%). All mutations were heterozygous. IDH1 and IDH2 mutations were mutually exclusive. IDH1 mutations included 3 R132H and 1 R132S. while IDH2 mutations were R140Q (n=7) and R172K (n=3). AML patients with IDH1/IDH2 mutations had significantly older age than those with wild-type IDHs (P=0.002). There were no difference in sex, blood parameters and FAB subtypes between cases with and without mutations (P>0.05). IDH mutations were significantly more frequently observed in cytogenetically normal AML (12/85,14.1%) than in cytogenetically abnormal AML (2/99, 2.0%) (P=0.004).â‘¢2 of 82 (2.4%) MDS cases were identified with IDH1 mutations (one R132H and one R132S) and 3 (3.6%) with IDH2 mutations (all R140Q). IDH1 and IDH2 mutations were also heterozygous and mutually exclusive. No significant defference in sex, age, hemoglobin and platelets was observed between MDS patients with and without IDH1/IDH2 mutations (P>0.05). MDS cases with IDH1/IDH2 mutations had higher white blood cell (WBC) counts than those without mutations (P=0.010). All IDH1/IDH2 mutations were observed in cases with favorable risks according to karyotype classification, however, statistical difference was not observed (P=0.407). The frequency of IDH1/IDH2 mutations were higher in MDS patients with normal karyotype (5/52,9.6%) than those with abnormal karyotype (0/30,0%) (P=0.153). In addition, all IDH mutations were identified in patients at Low and Int-1 risks according to IPSS classification. Multivariate analysis showed that age and IPSS grouping were independent prognostic factors. The overall survival (OS) of MDS patients with and without IDH1/IDH2 mutations did not differ.â‘£No IDH1 or IDH2 mutations were detected in 85 chronic myeloid leukemia (CML) and 57 Ph-MPNs.(3) DNMT3A mutation:â‘ HRMA could easily distinguish R882H mutation with the maximal sensitivity of 2%, higher than that obtained by DNA sequencing (10%).â‘¡R882 mutations were identified in 12 (6.6%) of 182 AML patients. All mutations were heterozygous, including R882H (n=8), R882C (n=3) and R882P (n=1). No correlation was observed between R882 mutations and gender or WBC counts. AML patients with DNMT3A R882 mutations were more prevalent at older age and present with significantly higher median platelet counts at diagnosis compared to those without mutations. DNMT3A R882 mutations were found more frequently among monoblastic leukemia (M4 and M5,7 of 52,13.4%) compared to non-monoblastic leukemia (M1, M2, M3 and M6,5 of 130, 3.8%) (P=0.041). Among the patients with normal karyotypes,13.2%(10 of 76) cases showed R882 mutation, significantly higher than 2.2%(2 of 92) in those with chromosomal abnormalities (P=0.007).â‘¢4 (7.8%) heterozygous DNMT3A R882 mutations were also identified in MDS, including 3 R882H and 1 R882C mutations. The difference in age, hematologic parameters and WHO classifications was not seen between patients with and without mutations. All 4 patients were identified with normal karyotype and were classified in intermediate-risk group according to IPSS classification. The OS of MDS patients with DNMT3A mutation (median 9 months) was shorter than those without mutation (median 25 months) (P=0.047).â‘£No DNMT3A R882 mutations were detected in 79 CML and 57 Ph-MPNs.(4) U2AF1 mutation:â‘ HRMA could easily distinguish U2AF1 S34 and Q157 mutations with the maximal sensitivity of 5%, higher than that obtained by DNA sequencing (10%).â‘¡U2AF1 mutations were identified in 4 (1.8%) of 221 AML patients. All mutations were heterozygous, including 2 S34Y and 2 Q157 (1 Q157P and 1 Q157R) mutations. There were no difference in sex, age, blood parameters, FAB subtypes, and karyotype classification between cases with and without mutations (P>0.05). The OS of AML patients with U2AF1 mutation (median 1 months) was shorter than those without mutation (median 7 months) (P=0.034).â‘¢4 (4.5%) cases were identified with heterozygous S34 mutations (3 S34F and 1 S34Y). No significance in sex, blood parameters, WHO subtypes, and IPSS classification was observed between MDS patients with and without U2AF1 mutations (P>0.05). The cases with U2AF1 mutations had younger age than those without mutations (.P=0.059). No difference in OS was observed between patients with and without U2AF1 mutations (P=0.954).â‘£No U2AF1 mutations were detected in 79 CML and 57 Ph-MPNs.(5) JAK2 mutation:â‘ HRMA assay was able to distinguish 5% of JAK2 V617F mutant, more sensitive than that obtained by DNA sequencing (10%).â‘¡Out of 48 cases with Ph-negative MPN, all 3 (100%) with polycythemia vera (PV) were positive for the presence of JAK V617F heterozygous mutation,34 (70%) with essential thrombocythemia (ET) were positive for JAK V617F mutation (6 homozygous and 28 heterozygous), and 3 (60%) with primary myelofibrosis (PMF) were positive (1 homozygous and 2 heterozygous). JAK2 V617F mutation was not found in all patients with CML. HRMA detection of all cases was fully concordant with the results of direct sequencing. Both the diagnostic specificity and sensitivity were 100%.CONCLUSIONS:(1) HRMA is a high-throughput technique for the detection of gene mutation and can be utilized to screen gene mutation in a large scale samples for good specificity, high sensitivity and low cost.(2) Heterozygous IDH1 and IDH2 mutations are present in Chinese AML and MDS patients and mainly observed in cytogenetically normal cases. IDH1/IDH2 mutations may have no prognostic impact on patients with MDS.(3) DNMT3A R882 mutations are recurrent molecular aberrations in AML and MDS. R882 mutations are all heterozygous and mainly observed in cytogenetically normal patients. DNMT3A R882 muation may be an adverse prognostic event in MDS.(4) U2AF1 mutations, a molecular event of low frequency in Chinese AML and MDS, occur at S34 and Q157 and are all heterozygous. The overall survival of AML patients with U2AF1 mutation was shorter than those without mutation. |
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