Cui-zhu Wang

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.

U2AF1 Mutations in Chinese Patients with Acute Myeloid Leukemia and Myelodysplastic Syndrome

Somatic mutations of U2AF1 gene have recently been identified in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). In this study, we analyzed the frequency and clinical impact of U2AF1 mutations in a cohort of 452 Chinese patients with myeloid neoplasms. Mutations in U2AF1 were found in 2.5% (7/275) of AML and 6.3% (6/96) of MDS patients, but in none of 81 CML. All mutations were heterozygous missense mutations affecting codon S34 or Q157. There was no significant association of U2AF1 mutation with blood parameters, FAB subtypes, karyotypes and other gene mutations in AML. The overall survival (OS) of AML patients with U2AF1 mutation (median 3 months) was shorter than those without mutation (median 7 months) (P = 0.035). No difference in the OS was observed between MDS patients with and without U2AF1 mutations. Our data show that U2AF1 mutation is a recurrent event at a low frequency in AML and MDS.

Overexpression of miR-378 is frequent and may affect treatment outcomes in patients with acute myeloid leukemia

MicroRNA miR-378 plays important roles in tumorigenesis by enhancing cell survival, reducing apoptosis, promoting tumor growth, angiogenesis and promoting cell migration and invasion. Abnormal expression of miR-378 has been observed in various types of cancers. The aim of this study was to investigate the expression status of miR-378 and its clinical significance in patients with acute myeloid leukemia (AML) using real-time quantitative PCR. miR-378 overexpression was identified in 26 of 84 (31%) AML patients. The patients with miR-378 overexpression had lower hemoglobin level than those without miR-378 overexpression (66 versus 78 g/L, respectively, P=0.010). The frequency of miR-378 overexpression in FAB-M2 subtype was higher than other subtypes (44% versus 20%, P=0.032). Moreover, the frequency of miR-378 overexpression was higher in patients with t(8;21) than in others (64% versus 24%, P=0.012). The status of miR-378 expression was not correlated with the mutations of eight genes (FLT3-ITD, NPM1, C-KIT, IDH1/IDH2, DNMT3A, C/EBPA and U2AF1). The difference in relapse-free survival was observed between patients with and without miR-378 overexpression (P=0.049). These findings suggest that miR-378 up-regulation is a common event and might have an adverse impact on prognosis in AML.

Development of high-resolution melting analysis for the detection of the MYD88 L265P mutation

OBJECTIVE Recurrent L265P mutation of myeloid differentiation primary response gene 88 (MYD88) has been identified in a proportion of diffuse large B-cell lymphoma (DLBCL) and chronic lymphocytic leukemia. The present study aimed to establish a rapid, sensitive, and reliable method using high-resolution melting analysis (HRMA) to detect MYD88 L265P mutation in DLBCL. DESIGNS AND METHODS The sensitivity of HRMA in the detection of MYD88 L265P mutation was evaluated. MYD88 L265P mutation was further screened in 120 patients with DLBCL. The results of HRMA were validated by direct DNA sequencing. RESULTS For the detection of MYD88 L265P mutation, the reproducible maximal sensitivity of HRMA was 5% higher than that obtained by direct DNA sequencing (25%). Heterozygous MYD88 L265P mutations were identified in 11 (9.2%) DLBCL cases, all of which were diagnosed as non-germinal-center B cell (non-GCB) DLBCL. CONCLUSIONS The HRMA assay is a rapid, sensitive, reliable, and high-throughput method to screen MYD88 L265P mutation and could be used in clinical diagnostic laboratories.

Hypomethylation of PRAME promoter is associated with poor prognosis in myelodysplastic syndrome

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Aberrant hypomethylation of SALL4 gene in patients with myelodysplastic syndrome

The abnormalities of SALL4 gene, which encodes a zinc-finger transcription factor and is essential for developmental events, have been found to be involved in tumorigenesis. In this study, we investigated the methylation status of the CpG island of SALL4 promoter region in myelodysplastic syndrome (MDS) using methylation-specific PCR (MSP). Aberrant hypomethylation of SALL4 gene was found in 21.7% (18/83) of the cases analyzed. A significantly positive correlation was identified between the level of SALL4 transcript and the status of SALL4 hypomethylation (R=0.641, P<0.001). No correlation was found between SALL4 hypomethylation and clinical parameters. However, the frequency of SALL4 hypomethylation significantly increased in higher risk MDS (14% in Low/Int-1 versus 39% in Int-2/High, P=0.031). The association between SALL4 hypomethylation and the mutations in three methylation modifiers (IDH1, IDH2 and DNMT3A) was not observed. Although the estimated 50% survival time of the SALL4-hypomethylated group was shorter than that of SALL4-methylated group (11.0 months vs. 20.0 months), the difference was not statistically significant (P=0.430). These findings suggest that hypomethylation of SALL4 promoter is a common event in MDS.

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.

Development of a high-resolution melting analysis for the detection of the SF3B1 mutations.

SF3B1, located on chromosome 2q33.1, encodes a core component of RNA-splicing machinery, and its mutation has been described in myelodysplastic syndromes (MDS) characterized with ring sideroblasts (RS). To explore the reliability and sensitivity of the high-resolution melting analysis (HRMA) technique for the identification of the SF3B1 mutations, mutations in 92 patients with MDS were detected in this study. The sensitivity could reach 5%, obviously higher than the 25% of direct DNA sequencing. A low frequency (5.4%) of SF3B1 mutations were identified in patients with MDS, including three cases of K700E, one case of H662Q, and one case of K666M. Further, SF3B1 mutations were more frequently recurrent in the 33% of patients with MDS characterized with RS, whereas in other subtypes of MDS, only 2.3% of patients were detected with SF3B1 mutations (p=0.006). In conclusion, a rapid, reproducible, sensitive, and high-throughput HRMA assay has been established for the scanning of SF3B1 mutations.

Aberrant hypomethylation of DDX43 promoter in myelodysplastic syndrome

The gene DDX43 (DEAD (Asp-Glu-Ala-Asp) box polypeptide 43; also known as HAGE) was first identified as a cancer/testis antigen (CTA) gene in a human sarcoma cell line (Martelange et al, 2000) and has been found to be overexpressed in various solid cancers and haematological malignancies (Adams et al, 2002; Condomines et al, 2007; Roman-Gomez et al, 2007; Liggins et al, 2010; Mathieu et al, 2010). Recently, abnormal hypomethylation of DDX43 promoter was shown in chronic myeloid leukaemia (CML) and was associated with mRNA overexpression (Roman-Gomez et al, 2007). The present study aimed to analyse the methylation status of DDX43 promoter and its clinical and prognostic impacts in myelodysplastic syndrome (MDS) patients. Genomic DNA was isolated from bone marrow mononuclear cells (BMNCs) of 90 patients with primary MDS at diagnosis using the DNA Purification Kit (Gentra, Minneapolis, MN, USA) according to the manufacturer’s instructions. Total RNA from BMNCs was isolated using Trizol (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s recommendations. Samples were collected after obtaining informed consent. The diagnosis and classification of MDS were made according to the French-American-British (FAB) classification and the 2008 revision of the World Health Organization classification (Vardiman et al, 2009). Clinical parameters of all patients are provided in Table I. Follow-up data were obtained for 78 patients. Genomic DNA and total RNA from 24 cases with iron deficiency anaemia (IDA) were used as controls. Genomic DNA (1 lg) was modified according to the manufacturer’s protocol using the CpGenome DNA Modification kit (Chemicon, Ternecula, CA, USA). Amplification The DDX43 unmethylated sequence was amplified by methylation-specific real-time quantitative polymerase chain reaction (RQ-MSP) using the primers reported previously (Roman-Gomez et al, 2007). The relative quantification of unmethylated DDX43 sequence was calculated in relation to the reference Alu sequence (Qian et al, 2011). To further determine the comprehensive methylation status of DDX43 promoter and exon 1, bisulfite-modified DNA sequencing PCR was performed using the specific primers 5′-TTTTTTTTTGGAATAATGTTTTATTA-3′(forward) and 5′-TAACCCCACCTAT CCTACCCTAC-3′(reverse) (RomanGomez et al, 2007). PCR conditions were 95°C for 4 min, 40 cycles for 45 s at 94°C, 30 s at 57·2°C, 30 s at 72°C, followed by a melting program at 95°C for 15 s, 60°C for 60 s, 95°C for 15 s, and 60°C for 15 s. The 307-bp PCR products, which covered 21 CpG sites (+12 bp~+250 bp) within a CpG-rich region located in DDX43 promoter and exon 1, were cloned into pMD19-T Vector (TaKaRa, Dalian, China). Ten independent clones of each sample were sequenced (Sangon, Shanghai, China). Two micro-gram of total RNA was reverse-transcribed into cDNA using 10 lmol/l of random primers, 200 units of MMLV reverse transcriptase, 0·5 mmol/l of dNTPs, 10 mmol/l of dithiothreitol, and 25 units of RNase inhibitor. Real-time quantitative PCR (RQ-PCR) was performed to detect the expression of DDX43 transcript on a 7300 Thermo cycler (Applied Biosystems, Foster City, CA, USA), using 100 ng of cDNA in a 25 ll reaction volume with 0·2 mmol/l of dNTP, 4 mmol/l of MgCl2, 0·4 lmol/l of primers (Roman-Gomez et al, 2007), 1·2 ll of EvaGreen, and 1·0 U of Taq DNA Polymerase (MBI Fermentas, Amherst, NY, USA). The mRNA abundance of DDX43 gene was calculated relative to the expression of the housekeeping gene, ABL1. 23 IDA bone marrow specimens displayed slight hypomethylation of DDX43, which was confirmed by the presence of specific unmethylated products in the melting curves. Nunmethylation-DDX43 ratio of all controls was 0–100% (31·78 ± 27·00%). Thus, a Nunmethylation-DDX43 ratio equal to or above 112·78% (determined as the mean + 3 standard deviations) was chosen to define the DDX43 hypomethylation in MDS samples. Hypomethylation of DDX43 promoter was observed in 27 of 90 (30%) primary MDS patients (Table I). The RQ-MSP results were confirmed by evaluating the methylation density of DDX43 promoter in five samples from one control, two DDX43-hypermethylated and two DDX43-hypomethylated MDS according to RQ-MSP. The results revealed the high relevance between RQ-MSP and bisulfite DNA sequencing (Fig S1A and S1B). We also examined DDX43 expression in 27 MDS patients with available mRNA. A significantly positive correlation was observed between the level of DDX43 transcript and the level of DDX43 hypomethylation (R = 0·520, P = 0·005). Patients with DDX43 hypomethylation (n = 2) had significantly higher DDX43 expression than those with DDX43 methylation and than controls (P = 0·001 and 0·025, respectively). Haemoglobin level was the only clinical characteristic that showed a significant difference between patients with and without DDX43 methylation (Table I). Aberrant hypomethylation of DDX43 promoter was observed in each subtype of Correspondence