Zhao-qun Deng

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) (Pu200a=u200a0.035). 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.

Overexpressed let-7a-3 is associated with poor outcome in acute myeloid leukemia.

Dysregulation of microRNA let-7a-3 has been identified in several solid tumors and is associated with prognosis of patients. However, the pattern of let-7a-3 expression and the impact on prognosis has not yet been studied in acute myeloid leukemia (AML). The purpose of this study is to investigate the expression status of let-7a-3 and its clinical significance in AML patients using real-time quantitative PCR. Overexpression of let-7a-3 was identified in 25 of 102 (25%) de novo AML. There was no significant difference in age, blood parameters, FAB/WHO subtypes, karyotype risks and nine gene mutations (FLT3-ITD, NPM1, C-KIT, IDH1/IDH2, DNMT3A, C/EBPA and N/K-RAS) between patients with and without let-7a-3 overexpression (P>0.05). The patients with let-7a-3 overexpression had similar rates of complete remission (CR) as those without let-7a-3 overexpression (50% vs. 56%, P=0.693). Although the overall survival (OS) of AML patients with let-7a-3 overexpression (median 12 months,) was shorter than those without overexpression (median 25 months), the difference was not statistically significant (P=0.228). However, among those 51 obtained CR, patients with let-7a-3 overexpression had significantly shorter OS than those without let-7a-3 overexpression (P=0.029). The difference in relapse-free survival (RFS) was also significant between two groups (P=0.005). These findings suggest that let-7a-3 overexpression is a common event and is associated with poor clinical outcome in AML.

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.

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

OBJECTIVEnMicroRNA 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).nnnDESIGNS AND METHODSnReal-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.nnnRESULTSnmiR-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).nnnCONCLUSIONSnThese findings suggest that miR-124-1 underexpression is a common event and might have a favorable impact on prognosis in AML.

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

OBJECTIVEnRecurrent 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.nnnDESIGNS AND METHODSnThe 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.nnnRESULTSnFor 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.nnnCONCLUSIONSnThe HRMA assay is a rapid, sensitive, reliable, and high-throughput method to screen MYD88 L265P mutation and could be used in clinical diagnostic laboratories.

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 (Pu200a=u200a0.069 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 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.

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 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

Methylation of CTNNA1 promoter: Frequent but not an adverse prognostic factor in acute myeloid leukemia

The reduced expression of CTNNA1 gene, a putative tumor suppressor gene, has been found in several cancers including acute myeloid leukemia (AML). CTNNA1 expression is regulated by methylation and histone deacetylation. However, the clinical significance of CTNNA1 methylation in AML is rarely known. The present study was aimed to investigate the methylation status of CTNNA1 promoter region using methylation-specific PCR (MSP) and its clinical relevance in Chinese AML patients. Patients with CTNNA1 hypermethylation had significantly lower level of CTNNA1 transcript than those without CTNNA1 hypermethylation (P=0.031). The relationship of CTNNA1 methylation with clinical parameters was evaluated. Aberrant hypermethylation of CTNNA1 gene was found in 23.9% (37/155) AML cases. The status of CTNNA1 methylation was not correlated with the mutations of seven genes (FLT3-ITD, NPM1, C-KIT, IDH1/IDH2, DNMT3A, N/K-RAS and C/EBPA). There was no significant difference in the rates of complete remission (CR) between patients with and without CTNNA1 methylation. Although the overall survival (OS) time of the CTNNA1-methylated AML was shorter than that of CTNNA1-unmethylated group (6 months vs 9 months), the difference was not statistically significant (P=0.681). Our data suggest that CTNNA1 methylation is a recurrent event but has no influence on prognosis in AML.