Overexpression of MAPK/MAK/MRK overlapping kinase (MOK) has been found in various tumors. However, the mechanism underlying MOK upregulation remains unclear. A CpG island was identified in MOK promoter. In this study, we evaluated the expression and methylation status of MOK gene in acute myeloid leukemia (AML). Hypomethylation of MOK promoter was detected in 31.0% (45/145) of AML patients. The degree of MOK hypomethylation was significantly correlated with MOK expression in AML patients. MOK-hypomethylated patients had a trend towards lower WBCs. Receiver operating characteristic curve (ROC) analysis showed a good performance in distinguishing AML patients from controls with an area under the ROC curve (AUC) of 0.820 (P < 0.001). In summary, our results suggest MOK promoter hypomethylation is a common event and contributes to MOK overexpression in AML.

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.

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. (C) 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

髓系分化起始反应基因88(MYD88)位于染色体3p22,编码蛋白为Toll样受体(TLR)信号传导途径中的一个重要接头分子,结构上主要包括2个功能域:N端的死亡域(DD)和C端的Toll域(TIR),在天然免疫应答和炎症反应中发挥着重要的调控作用[1-2].最近的研究发现39%活化B细胞(ABC)型弥漫大B细胞淋巴瘤(DLBCL)患者中存在MYD88基因的获得性突变[3],其中位于TIR域的L265P突变发生率最高(29%).高分辨率熔解曲线分析(High resolution melting curve analysis,HRMA)是一种新型的突变分析技术,具有灵敏度高、特异性好、成本低廉、高通量检测的优点,已在临床上得到越来越多的应用[4].我们拟建立HRMA法对MYD88 L265P突变进行检测.

This study was aimed to investigate the expression pattern of gene PDLIM4 (PDZ and LIM domain 4) and analyze its clinical correlation with the patients suffered from acute myeloid leukemia (AML). The expression pattern of PDLIM4 in AML was detected by using EvaGreen real-time quantitative PCR (RQ-PCR). The results showed that the PDLIM4 transcript significantly decreased in 94 AML patients, compared with 21 controls (P < 0.01). The decrease of PDLIM4 transcript was found in 42 (45%) AML patients. PDLIM4 low-expression occurred among the subtypes of M1/M2/M3 more frequently than that in M4/M5/M6 (56% vs 20%, P < 0.01). AML patients with PDLIM4 low-expression had an overall survival (OS) higher than that in AML patients without PDLIM4 low-expression (P < 0.05). Analysis with receiver operating characteristic curve (ROC) displayed that PDLIM4 expression possesses the diagnostic value to differentiate AML from controls, with ROC curve area of 0.865 (95% CI: 0.801-0.930). It is concluded that reduced PDLIM4 expression is a common and favorable event for the good prognosis in AML, and can be used as a potential diagnostic biomarker of cancer.

Objective: To analyze the expression transcript level and promoter hypermethylation of PDZ and LIM domain 4(PDLIM4) gene and the clinical correlations in Chinese patients with chronic myeloid leukemia(CML).Methods: Real-time quantitative PCR(RQ-PCR) and real-time quantitative methylation-specific PCR(RQ-MSP) assays were used to detect the expression and methylation status of PDLIM4 gene in the bone marrow samples from CML patients.Results: Down-regulated PDLIM4 expression was found in 8/36(22%) CML cases,with significant statistical difference compared with all 21 controls(Fisher′s Exact Test,P=0.021).There was a significant correlation between PDLIM4 expression and bcr/abl fusion transcript(r=0.434,P=0.043) in CML patients.Hypermethylation of PDLIM4 promoter was detected in 13/59(22%) CML patients,but not in all 24 controls(Fisher′s Exact Test,P=0.016).PDLIM4 hypermethylation was not correlated with blood parameters(P>0.05).The frequencies of PDLIM4 promoter hypermethylation increased during the disease progression,as it was 16%(7/44),33%(2/6) and 44%(4/9) in chronic phase,accelerated phase and blast crisis,respectively.Conclusion: Hypermethylation of PDLIM4 promoter may be associated with the disease progression in CML.

The aim of this study was to investigate the expression level of the SALL4 gene and its clinical significance in patients with acute myeloid leukemia (AML) and chronic myeloid leukemia (CML). Real-time quantitative PCR (RQ-PCR) was performed to detect the expression level of SALL4 mRNA in bone marrow mononuclear cells (BMMNC) from 35 AML, 12 CML patients and 24 iron deficiency anemia patients as controls. The results indicated that the expression level of SALL4 in AML (0%-14%, median 1.43%) was obviously higher than that in controls (0% - 1%, median 0%) (P < 0.001). SALL4 expression was positive in 65.7% (23/35) AML patients. The frequency of SALL4 expression was in M2 (86.7%, 13/15) > M3 (75.0%, 6/8) > M1 (60.0%, 3/5) > M4 (14.3%, 1/7), and the difference among 4 groups was statistically significant (P = 0.008); there was no correlation of the frequency of SALL4 expression with the age, sex, white blood cell WBC count, hemoglobin concentration, platelet count and chromosomal abnormalities of AML patients (P > 0.05). All the 13 CML cases showed positive expression of SALL4 gene (1% - 128%, median 19.39%), which was higher than that in controls (P < 0.001). The analysis of receiver operating characteristic (ROC) curve showed the area under ROC curve (AUC) of AML and CML were 0.983 (95% confidence interval: 0.95 - 1.017) and 0.997 (95% confidence interval: 0.986 - 1.007) respectively. It is concluded that SALL4 expression is a common molecular event and can be considered as a molecular marker for assisting diagnosis of AML and CML.

The aim of this study was to investigate the expression status of the helicase antigen (HAGE) transcript and its clinical significance in patients with acute myeloid leukemia (AML) and chronic myeloid leukemia (CML). The expression of HAGE cDNA in bone marrow mononuclear cells from AML and CML patients was detected by using real-time quantitative PCR. The results indicated that overexpression of HAGE transcript (117.12% - 9842.70%, median 434.96%) was detected in 14.8% (11/74) AML patients. AML patients with HAGE cDNA expression were significantly older than those HAGE-negative patients (median 67 and 45 years, respectively, p = 0.001). HAGE cDNA expression was more frequently present among the patients with acute monoblastic leukemia (M(4) and M(5), 7 of 20, 35.0%), compared to the patients with acute non-monoblastic leukemia (M(1), M(2), M(3) and M(6), 4 of 54, 7.4%) (p = 0.007). 28.6% (8/28) cases with normal karyotypes showed HAGE cDNA overexpression, significantly higher than 7.5% (3 of 40) in those with chromosomal abnormalities (p = 0.041). Overexpression of HAGE transcript was found in 9 (34.6%) CML cases and more frequently observed at accelerated phase and blast crisis (4/4, 100%) than that at chronic phase (5/22, 22.7%) (p = 0.008). It is concluded that HAGE cDNA expression is relevant to specific subtypes of AML and to the progression of CML.

The purpose of this study was to detect the expression of RAGE-1 transcript in the bone marrow mononuclear cells (BMMNC) from patients with acute myeloid leukemia (AML) and to investigate the relationship of RAGE-1 expression level with clinical variables. The expression level of RAGE-1 gene in BMMNC from 94 newly diagnosed AML patients was measured using RQ-PCR. The relationship between RAGE-1 expression level and clinical parameters (age, sex, blood cell counts, diagnosis and prognosis) was investigated, and the levels of RAGE-1 expression were compared in patients before and after treatment. The results showed that overexpression of RAGE-1 transcript was found in 28% (26/94) AML patients (1.34 - 16.34, median 3.07). No significant difference was observed in sex, age, blood parameters and FAB subtypes between the groups with and without RAGE-1 overexpression. There was also no significant difference in the frequency of RAGE-1 overexpression among different cytogenetic risk groups and among the patients with different types of karyotypes. The level of RAGE-1 transcript significantly decreased in those patients obtained complete remission after treatment. The overall survival of AML patients with RAGE-1 overexpression was similar as that in those without RAGE-1 overexpression. It is concluded that RAGE-1 overexpression is a common event in AML, but has no impact on the prognosis of patients.

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.

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

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.

Objective: To study the methylation status of helicase antigen(HAGE) gene promoter and its clinical correlations in Chinese patients with chronic myeloid leukemia(CML).Methods: The methylation status of HAGE promoter in the bone marrow samples from 50 CML patients in different stages was detected using methylation-specific PCR(MSP) technique.Results: Hypomethylation of HAGE gene promoter was found in 13(26%) CML cases,but not in all 24 controls(P=0.007).There was no correlations between the hypomethylation of HAGE gene promoter with the age,sex,white blood cell count,hemoglobin concentration,platelet count,clinical stages,and chromosomal abnormalities of CML patients(P>0.05).The frequencies of HAGE promoter hypomethylation in patients in chronic phase or accelerated phase,and in blast crisis were 25%(9/36),25%(1/4) and 30%(3/10),respectively(P>0.05).Conclusion: Hypomethylation of HAGE gene promoter was a frequent molecular event in CML.

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.

Objective: The mutations of isocitrate dehydrogenase 1 (IDH1) gene have been identified in a proportion of hematologic malignancies including acute myeloid leukemia (AML). The aim of the present study was to explore the reliability of the high-resolution melting analysis (HRMA) for the identification of IDH1 R132 mutations in AMLDesigns and methods: We evaluated the sensitivity of HRMA in the detection of IDH1 R132 mutation and screened IDH1 mutations in 110 AML patients using HRMA. The results of HRMA were validated by direct DNA sequencing.Results: The reproducible sensitivity of HRMA was 5% for the detection of IDH1 R132 mutation, higher than 10% of direct DNA sequencing. Heterozygous IDH1 mutations were identified in 4 (3.6%) AML cases, which were R132H in 3 cases and R132S in 1 case confirmed by DNA sequencing.Conclusion: The HRMA is a rapid, accurate, reliable, high-throughput method to screen IDH1 gene mutations. (C) 2011 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Background: Several methods have been established to detect the JAK2 V617F mutation, a frequent event involved in the pathogenesis of myeloproliferative neoplasms (MPNs). High‐resolution melt (HRM) analysis is a newly established technique without the requirement of any gel‐based post‐PCR handling. Methods: An asymmetric PCR with unlabeled specific probe was developed and combined to HRM analysis o screen for JAK2 V617F mutation. Results: Heterozygous mutation was easily distinguished from homozygous JAK2 for the obvious shape change. Homozygous JAK2 mutant can be also well separated from wild‐type JAK2 in the presence of internal temperature calibrators. The easily recognizable and maximal sensitivity of HRM analysis was 5% for the detection of JAK2 V617F mutation, higher than 25% of direct sequencing. In the test of blind screening of 223 samples (111 Ph− MPNs, 60 Ph+ chronic myeloid leukemia, and 52 acute myeloid leukemia), JAK2 V617F mutations were found in 78 (70%) patients with MPNs, but in none with chronic and acute myeloid leukemia. HRM analysis of all cases was fully concordant with the results of PCR‐RFLP and direct sequencing. Conclusions: The HRM method with unlabeled probe could be used as convenient, sensitive and reliable diagnostic test for detection of JAK2 V617F mutation. J. Clin. Lab. Anal. 25:300–304, 2011. © 2011 Wiley‐Liss, Inc.

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.