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Aberrant methylation of let-7a-3 promoter has been observed in various malignancies. However, the clinical relevance of let-7a-3 methylation remains poorly known in acute myeloid leukemia (AML). This study was to investigate the let-7a-3 methylation status and to explore its clinical significance in AML. let-7a-3 promoter was significantly hypomethylated in AML patients compared to controls (median 4.51 vs 0.49) (P = 0.0003). Receiver operating characteristic curve (ROC) analysis discriminated all patients or cytogenetically normal patients from controls with an areas under the ROC curve (AUC) of 0.737 or 0.783, respectively (P < 0.001). Patients with favorable/intermediate karyotypes had significantly higher let-7a-3 unmethylation than controls. Patients with DNMT3A mutations had a trend of high level of let-7a-3 unmethylation than did those with wild-type DNMT3A (median 6.76 vs 3.66, P = 0.096). There was no significant difference in overall survival between patients with and without hypomethylated let-7a-3 (median 12 vs 5 months, P = 0.103). No correlation was observed between the level of let-7a-3 expression and let-7a-3 unmethylation in AML samples (R = 0.197, P = 0.150). However, the level of let-7a-3 expression was increased in a dose-dependent manner in THP-1 line treated with 5-aza-dC, while the methylation density of let-7a-3 promoter decreased with 5-aza-dC dose. Our findings suggest that let-7a-3 hypomethylation is associated with favorable and intermediate karyotypes but not a prognostic predictor for AML patients. Let-7a-3 expression may be partially regulated by promoter methylation.

In recent years, many researches have shown that OCT4 is overexpressed in both germ cell tumors and somatic cancers. Meanwhile, OCT4 has relationship with poor prognosis in a lot of solid tumors, such as hepatocellular carcinoma, gastric cancer, and esophageal cancer. In our study, we investigated the expression status of OCT4 and its clinical significance in patients with acute myeloid leukemia (AML) using real-time quantitative PCR. The receiver operating characteristic (ROC) curve reveals that the level of OCT4 expression could be available for a potential diagnostic biomarker for differentiating AML from controls with an area under the ROC curve (AUC) of 0.915 (95 % confidence interval (CI) 0.837-0.992; P < 0.001). At the cutoff value of 0.56, the sensitivity and the specificity are 75.9 and 81.2 %, respectively. The amount of white blood cell (WBC) of patients with high OCT4 expression is higher than that of patients with low OCT4 expression (18.2 × 10(9) versus 2.7 × 10(9) L(-1), P = 0.001). Among those patients who are less than 70 years old, patients with OCT4 high expression have significantly shorter overall survival (OS) than those without OCT4 high expression (P = 0.048). These findings suggest that OCT4 high expression is a common event and may have an adverse impact on prognosis in AML.

DNA methyltransferase 3A (DNMT3A), a member of de novo methyltransferases, has been found with overexpression in several cancers including acute myeloid leukemia (AML). The present study was aimed to investigate the methylation status of DNMT3A intragenic differentially methylated region 2 (DMR2) using real-time quantitative methylation-specific PCR (RQ-MSP) and analyze its clinical significance in AML. Aberrant hypomethylation of DNMT3A gene was found in 55.3% (84/152) of AML cases, but the status of DNMT3A hypomethylation was not correlated with the expression of four DNMT3A isoforms as well as DNMT3A mutation. There was no significant difference in the rates of complete remission (CR) between patients with and without DNMT3A hypomethylation. However, the patients with DNMT3A hypomethylation had shorter overall survival (OS) time than those without DNMT3A hypomethylation (7 months vs. 11 months, P=0.034). Moreover, the patients with DNMT3A hypomethylation also showed significantly shorter OS than those without DNMT3A hypomethylation in cytogenetically normal AML (CN-AML) (7 months vs. 25 months, P=0.011). Multivariate analysis confirmed the independent adverse impact of DNMT3A hypomethylation in CN-AML. Our data suggest that DNMT3A DMR2 hypomethylation is a negative prognostic hallmark in CN-AML., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Deregulation of secreted frizzled-related protein 1 (SFRP1) has been found in many types of cancer. However, the pattern of SFRP1 expression in acute myeloid leukemia (AML) is still unclear. This study determined SFRP1 expression in patients with AML. SFRP1 expression was decreased markedly in patients with AML compared to controls (p < 0.001). White blood cell (WBC) counts increased as SFRP1 expression decreased in AML (p = 0.016). Patients with low SFRP1 expression showed a different distribution of French-American-British (FAB) subtypes M1/M2/M3 from those with high SFRP1 expression (p = 0.031). NPM1 mutation was mainly observed in patients with low SFRP1 expression (p = 0.011). There was a weak trend that patients with AML with low SFRP1 expression had shorter overall survival (OS) than those with high SFRP1 expression (p = 0.103). Our results indicate that reduced SFRP1 expression is found more frequently in the less well-differentiated subgroups of AML and is associated with NPM1 mutation in AML.

To investigate the clinical significance of ID1 expression in Chinese de novo AML patients. Real-time quantitative PCR was carried out to detect the status of ID1 expression in 102 de novo AML patients and 28 controls. ID1 transcript level was significantly increased in AML compared to normal controls (p=0.029). The age in the patients with high ID1 expression is significantly older than in those with low ID1 expression (p=0.044). ID1 overexpression occurred with the highest frequency in the patients with poor karyotype (7/7, 100%), lower frequency in the patients with intermediate karyotype (28/60, 47%), and the lowest frequency in the patients with favorable karyotype (12/31, 39%). Both whole AML and non-M3 patients with high ID1 expression had significantly lower rate of complete remission than those with low ID1 expression (p=0.007 and 0.038). ID1 high-expressed patients showed significantly shorter overall survival (OS) than ID1 low-expressed patients in both whole AML and non-M3 according to Kaplan-Meier analysis (p=0.007 and 0.040). However, multivariate analysis indicated that ID1 overexpression was not an independent risk factor in both whole AML and non-M3 patients. However, the adverse impact of ID1 overexpression on outcome was revealed by both Kaplan-Meier analysis and multivariate analysis in the non-M3 patients less than 60 years old. Our study reveals that ID1 overexpression may be associated with higher risk karyotype classification and act as an independent risk factor in young non-M3 patients.

Background: Aberrant expression of miR-378 has been observed in various malignancies including acute myeloid leukemia (AML). However, the mechanism regulating of miR-378 expression remains unknown. This study was aimed to investigate miR-378 methylation and to explore its clinical significance in AML., Methods: Methylation status of miR-378 5'-flanking region was investigated by real-time quantitative methylation-specific PCR (RQ-MSP) and bisulfite-sequencing PCR (BSP). The expression of miR-378 was evaluated by real-time quantitative PCR (RQ-PCR). The correlation between expression of miR-378 and 5'-flanking region methylation was analyzed using 5-aza-2'-deoxycytidine (5-aza-dC) treatment., Results: miR-378 5'-flanking region was significantly hypomethylated in AML patients compared to controls (median 0.109 vs. 0.058) (P=0.048). miR-378 expression was correlated with miR-378 5'-flanking region in leukemic cell line treated with 5-aza-dC, but not in AML patients. The level of miR-378 hypomethylation significantly increased in M2 subtype compared to other subtypes. Moreover, patients with t(8;21) harbored the highest level of miR-378 hypomethylation. However, there was no significant difference in overall survival between patients with high and low miR-378 hypomethylation. The association of miR-378 expression with methylation was not observed in AML patients, but miR-378 expression in THP-1 line was increased while methylation status of miR-378 5-flanking region was decreased after 5-aza-dC treatment., Conclusions: Our findings suggest that miR-378 is reactivated by demethylation after 5-aza-dC treatment. 5'-flanking region of miR-378 is hypomethylated in AML especially in those with t(8;21).

This study was designed to learn the expression status of miR-24 and its clinical relevance in patients with acute myeloid leukemia (AML). We detected the miR-24 expression levels using real-time quantitative PCR in 84 AML patients and investigated the clinical significance of miR-24 expression in AML. There was no difference in clinical parameters between cases with miR-24 high expression and with miR-24 low expression. The frequency of miR-24 high expression was higher in patients with t(8;21) than in others (82% (9/11) versus 44% (32/72), P=0.026). The levels of miR-24 expression had no correlation with the mutations of nine genes (FLT3-ITD, NPM1, C-KIT, IDH1/IDH2, DNMT3A, N/K-RAS and C/EBPA). Meanwhile, among the group who obtained CR, the cases with miR-24 high expression had no difference in overall survival (OS) and relapse-free survival (RFS) than those with miR-24 low expression (P=0.612 and 0.665, respectively). These findings implicated that miR-24 high regulation is a common event in AML with t(8;21), and it might serve as a novel and selective therapeutic target for the treatment of AML with t(8;21).