Zhen Qian

Recurrent DNMT3A R882 Mutations in Chinese Patients with Acute Myeloid Leukemia and Myelodysplastic Syndrome

Somatic mutations of DNMT3A gene have recently been reported in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). We examined the entire coding sequences of DNMT3A gene by high-resolution melting analysis and sequencing in Chinese patients with myeloid malignancies. R882 mutations were found in 12/182 AML and in 4/51 MDS, but not in either 79 chronic myeloid leukemia (CML), or 57 myeloproliferative neoplasms (MPNs), or 4 chronic monomyelocytic leukemia. No other DNMT3A mutations were detected in all patients. R882 mutations were associated with old age and more frequently present in monoblastic leukemia (M4 and M5, 7/52) compared to other subtypes (5/130). Furthermore, 14/16 (86.6%) R882 mutations were observed in patients with normal karyotypes. The overall survival of mutated MDS patients was shorter than those without mutation (median 9 and 25 months, respectively). We conclude that DNMT3A R882 mutations are recurrent molecular aberrations in AML and MDS, and may be an adverse prognostic event in MDS.

IDH1 and IDH2 mutation analysis in Chinese patients with acute myeloid leukemia and myelodysplastic syndrome

The somatic mutations of isocitrate dehydrogenase genes (IDH1 and IDH2) have been identified in a proportion of hematologic malignancies. We examined IDH1 R132 and IDH2 R140/R172 mutations by high resolution melting analysis and direct sequencing in Chinese patients with different myeloid malignancies including 198 acute myeloid leukemia (AML), 82 myelodysplastic syndrome (MDS), 85 chronic myeloid leukemia, and 57 myeloproliferative neoplasms. IDH1 and IDH2 mutations were found in four (2.0%) and ten (5.0%) AML and in two (2.4%) and three (3.6%) MDS cases, but not in other patients. IDH1 and IDH2 mutations were heterozygous and mutually exclusive. IDH1/2 mutations were significantly more frequently observed in cytogenetically normal AML or MDS compared to those without mutations. There was no difference in overall survival of both AML and MDS patients with or without IDH1/2 mutations (P = 0.177 and 0.407, respectively). In conclusion, IDH1/2 mutations are recurrent but rare molecular aberrations in Chinese AML and MDS.

Rapid detection of JAK2 V617F mutation using high-resolution melting analysis with LightScanner platform

BACKGROUND Detection of the JAK2 mutation has recently been included under the essential diagnostic criteria for myeloproliferative neoplasm (MPN). High-resolution melt (HRM) curve analysis, a nongel-based, automated system, is introduced as a means of mutation scanning without the requirement of any post-PCR handling. METHODS We studied the sensitivity and reproducibility of LightScanner™ platform in the detection of JAK2 V617F mutation and the availability for diagnostic use in MPN. RESULTS The reproducible sensitivity of HRM analysis with LightScanner™ platform was 5% for the detection of JAK2 V617F mutation. In the test of blind screening of 105 samples (48 Ph- MPN and 57 Ph+ chronic myeloid leukemia), the identical judgement was interpreted by two blinded investigators. HRM analysis of all cases was fully concordant with the results of PCR-RFLP and direct sequencing. CONCLUSIONS The HRM method developed here is an extremely sensitive, accurate and reliable technique and allows high-throughput, fast pre-screening to select for sequencing only those specimens that most likely contain mutant JAK2 V617F allele(s).

Dysregulation of miR-124-1 predicts favorable prognosis in acute myeloid leukemia

OBJECTIVE MicroRNA miR-124 has been suggested as a tumor suppressor for its role in inhibiting cell growth, inducing differentiation and promoting apoptosis. The present study was aimed to investigate the expression status of miR-124-1 and its clinical relevance in patients with acute myeloid leukemia (AML). DESIGNS AND METHODS Real-time quantitative PCR was performed to detect the expression level of miR-124-1 in AML patients. The clinical significance of miR-124-1 expression in AML was investigated. RESULTS miR-124-1 underexpression was identified in 30 (36%) of 83 AML patients. No significant difference could be observed in sex, age and blood parameters between the patients with and without miR-124-1 underexpression. The frequency of miR-124-1 underexpression was higher in the patients with t(15;17) than in others (62% versus 30%, P = 0.040). The status of miR-124-1 expression was not correlated with the mutations of nine genes (FLT3-ITD, NPM1, C-KIT, IDH1/IDH2, DNMT3A, N/K-RAS and C/EBPA). The patients with miR-124-1 underexpression had borderline longer overall survival and relapse-free survival than those without miR-124-1 underexpression (P = 0.052 and 0.045, respectively). CONCLUSIONS These findings suggest that miR-124-1 underexpression is a common event and might have a favorable impact on prognosis in AML.

Methylation status of DDIT3 gene in Chronic Myeloid Leukemia

BackgroundDNA-damage-inducible transcript 3 (DDIT3), a candidate tumor suppressor gene (TSG), has been found involved in the regulation of cellular growth and differentiation. The epigenetic changes of TSGs are recently recognized as an abnormal mechanism contributing to the development of chronic myeloid leukemia (CML). The aim of this study was to investigate the methylation status of DDIT3 gene in CML patients.MethodsThe methylation status of DDIT3 promoter was detected in the bone marrow mononuclear cells from 53 patients with CML using methylation-specific PCR (MSP). The expression levels of DDIT3 and bcr/abl transcript were determined by real-time quantitative PCR (RQ-PCR). Clinical data of these patients were collected and analyzed.ResultsThe aberrant methylation of DDIT3 gene promoter was found in 35 of 53 (66%) CML cases. Correlation was not found between DDIT3 promoter hypermethylation and the age, sex, hemoglobin concentration, platelet counts, chromosomal abnormalities, bcr/abl transcript, and staging of CML patients (P > 0.05), but found between DDIT3 promoter hypermethylation and WBC counts of CML cases (R = 0.781, P < 0.001). The level of DDIT3 transcript in CML patients was significantly lower than that in controls (median 3.28 vs 19.69, P < 0.001), however, there was no difference in the level of DDIT3 transcript between methylation-positive CML cases (0.05-65.32, median 2.13) and methylation- negative CML cases (0.12-126.04, median 3.92) (P > 0.05).ConclusionOur results demonstrate that aberrant methylation of DDIT3 occurs in CML frequently.

Abnormal methylation of GRAF promoter Chinese patients with acute myeloid leukemia

The epigenetic disturbances are recognized as an alternative mechanism contributing to the pathogenesis of acute myeloid leukemia (AML). GTPase regulator associated with focal adhesion kinase (GRAF), a putative tumor suppressor gene, was revealed with mutations and promoter methylation in AML and myelodysplastic syndrome. In this study, we investigated the methylation status of GRAF promoter in Chinese AML patients. Aberrant methylation of GRAF promoter was detected in 66.7% (88/132) of the cases analyzed. The methylation of GRAF gene could be detected in all FAB subtypes and in all cytogenetic risk groups. There were no significant differences in clinical features, FAB subtypes and cytogenetic risk groups between patients with and without GRAF methylation. GRAF transcript was significantly lower in AML group compared to controls (3.30 vs 56.06, P<0.001). Both patients with methylated GRAF gene and those without methylated GRAF gene had significantly lower GRAF transcript than controls (P<0.001). Furthermore, GRAF transcript was significantly lower in patients with methylated GRAF than those without methylated GRAF (1.64 vs 6.42, P=0.005). These findings suggest that the hypermethylation of GRAF promoter might be one of early events in the development of AML.

Detection of SRSF2-P95 mutation by high-resolution melting curve analysis and its effect on prognosis in myelodysplastic syndrome.

Hotspot mutations of serine/arginine-rich splicing factor 2 (SRSF2) gene have been identified in a proportion of hematologic malignancies including myelodysplastic syndrome (MDS). The aim of the present study was to develop a new approach to screen SRSF2 mutation and analyze the clinical relevance of SRSF2 mutations in Chinese MDS. A protocol based on high-resolution melting analysis (HRMA) was established to screen SRSF2-P95 mutation in 108 MDS patients and was compared with Sanger sequencing. The clinical relevance of SRSF2 mutations was further evaluated. HRMA identified five (4.6%) cases with SRSF2 mutation, completely validated by Sanger sequencing without false positive or negative results. The sensitivities of HRMA and Sanger sequencing were 10% and 25% for the detection of SRSF2-P95H mutation, respectively, against the background of wild-type DNA. Patients with SRSF2 mutation had shorter overall survival time than those with wild-type SRSF2 in both the whole cohort of cases and those with normal karyotype (P = 0.069 and 0.023, respectively). Multivariate analysis confirmed SRSF2 mutation as an independent risk factor in both patient populations. We established a fast, high-throughput, and inexpensive HRMA-based method to screen SRSF2 mutation, which could be used in clinical diagnostic laboratories. SRSF2 mutations were significantly associated with mortality rate in the MDS affected Chinese.

Aberrant hypomethylation of SALL4 gene is associated with intermediate and poor karyotypes in acute myeloid leukemia

OBJECTIVE SALL4 gene has been identified to stimulate the expansion of hematopoietic stem cell (HSCs) and enhance the self-renewal of HSCs. Overexpression of SALL4 has been found in several cancers. The present study was aimed to investigate the methylation status of SALL4 promoter region in acute myeloid leukemia (AML). DESIGNS AND METHODS The methylation status of SALL4 promoter was analyzed in 84 patients with AML using methylation-specific PCR (MSP) and its clinical significance was evaluated. RESULTS Aberrant hypomethylation of SALL4 gene, which was correlated with SALL4 expression, was found in 17.8% (15/84) cases. The patients with SALL4 hypomethylation had significantly older age and higher WBCs than those without SALL4 hypomethylation. The incidence of SALL4 hypomethylation was higher in M1 subtype than in M2 and other subtypes (50%, 26% and 6%, respectively, P=0.001). SALL4 hypomethylation was associated with cytogenetically intermediate and poor groups. Although survival time of the SALL4-hypomethylated AML was shorter than that of SALL4-methylated group (4 months vs 9 months), the difference was not statistically significant (P=0.356). CONCLUSIONS Hypomethylation of SALL4 promoter is a common event and is associated with the intermediate and poor karyotypes in AML.

Low miR-34c expression is associated with poor outcome in de novo acute myeloid leukemia.

MicroRNA‐34c (miR‐34c) has been found to play important roles in tumorigenesis. However, little is known about miR‐34c expression and the impact on prognosis in acute myeloid leukemia (AML).

Aberrant methylation of CCAAT/enhancer binding protein zeta promoter in acute myeloid leukemia

The aberrant changes of tumor suppressor genes (TSGs) are now recognized as an important mechanism contributing to the development of acute myeloid leukemia (AML). CCAAT/enhancer binding protein zeta (C/EBPζ), a candidate TSG, has been found to be involved in cancers including AML. We detected the methylation status of C/EBPζ promoter in 133 patients with AML using the methylation-specific polymerase chain reaction (MS-PCR) and examined C/EBPζ transcript in 32 patients using real-time quantitative PCR. The abnormal methylation of C/EBPζ gene promoter was found in 62 (46.6%) AML cases. No correlation was found between C/EBPζ promoter hypermethylation and the age, sex, WBC counts, platelet counts and FAB subtypes of AML patients (P>0.05). The trend that the frequency of C/EBPζ methylation increased as karyotype became more adverse was observed (R=0.167, P=0.075). There was a significant correlation between C/EBPζ expression and C/EBPζ methylation in AML patients (R=0.606, P=0.002). Our data suggest that the aberrant methylation of C/EBPζ promoter may be involved in AML.