De-hong Wu

Overexpression of miR-216b: prognostic and predictive value in acute myeloid leukemia†

Accumulating studies have shown that miR‐216b acted as a tumor suppressor and was down‐regulated in solid tumors. However, little studies revealed the role or clinical implication of miR‐216b in blood cancers. Herein, we reported miR‐216b expression and its clinical significance in patients with acute myeloid leukemia (AML). In the current study, we analyzed bone marrow (BM) miR‐216b expression in 115 de novo AML patients examined by real‐time quantitative PCR. Notably, BM miR‐216b expression was significantly up‐regulated in AML patients, and could serve as a potential biomarker distinguishing AML from controls. No significant correlations of BM miR‐216 expression were found with sex, age, white blood cells, hemoglobin, platelets, BM blasts, French–American–British classifications, and karyotypes. Significantly, patients with high miR‐216b expression tended to have a lower frequency of FLT3‐ITD mutation and higher incidence of U2AF1 and IDH1/2 mutations. Moreover, complete remission (CR) rate and overall survival were negatively affected by BM miR‐216b overexpression among cytogenetically normal AML (CN‐AML). Cox regression analyses showed that high BM miR‐216b expression may act as an independent risk factor in CN‐AML patients. Among the follow‐up patients, BM miR‐216b level in CR phase was markedly lower than in diagnosis time, and was returned in relapse phase. Collectively, our findings indicated that miR‐216b overexpression was a frequent event in de novo AML, and independently conferred a poor prognosis in CN‐AML. Moreover, miR‐216b expression was a valuable biomarker correlated with disease recurrence in AML.

Methylation-associated DOK1 and DOK2 down-regulation: potential biomarkers for predicting adverse prognosis in acute myeloid leukemia†

DOK‐1 and DOK‐2 (DOK1/2) are closely related members of downstream of tyrosine kinase (DOK) family genes, which are found to be frequently rearranged in several hematopoietic cancers. However, the clinical implications of DOK1/2 in acute myeloid leukemia (AML) remain largely unknown. To investigate the clinical significance, real‐time quantitative PCR (RQ‐PCR) was carried out to detect DOK1/2 expressions in 125 de novo AML patients and 28 healthy controls. Real‐time quantitative methylation‐specific PCR (RQ‐MSP) and bisulfite sequencing PCR (BSP) were applied to detect DOK1/2 methylation level and density. DOK1/2 expressions were significantly down‐regulated in AML patients. The promoters of DOK1/2 were highly hypermethylated and negatively correlated with DOK1/2 expressions in AML patients. In addition, we also confirmed that DOK1/2 expressions could be restored by DOK1/2 demethylation using 5‐aza‐2′‐deoxycytidine in leukemia cell line THP‐1. Survival analyses showed that low‐expressed DOK1/2 were associated with markedly shorter overall survival and leukemia free survival in both whole‐cohort AML and non‐M3 AML patients. Multivariate analyses further revealed that DOK1/2 were act as independent prognostic factors in AML patients. These findings indicate that decreased DOK1/2 expressions associated with their promoter hypermethylations predict adverse prognosis in AML.

High bone marrow ID2 expression predicts poor chemotherapy response and prognosis in acute myeloid leukemia

Dysregulation of ID proteins is a frequent event in various human cancers and has a direct role in cancer initiation, maintenance, progression and drug resistance. Our previous study has revealed ID1 expression and its prognostic value in acute myeloid leukemia (AML). Herein, we further reported ID2 expression and its clinical significance in AML. Real-time quantitative PCR was performed to detect ID2 transcript level in bone marrow mononuclear cells of 145 de novo AML patients. ID2 expression was significantly up-regulated in AML patients compared with controls. ID2 overexpression occurred with the highest frequency in poor karyotype (10/17, 59%), lower in intermediate karyotype (35/83, 42%), and the lowest in favorable karyotype (7/40, 18%). Moreover, high ID2 expression correlated with lower complete remission (CR) rate, shorter overall survival, and acted as an independent prognostic biomarker in whole-cohort AML and non-M3-AML patients. Importantly, the prognostic value of ID2 expression in AML was validated by The Cancer Genome Atlas (TCGA) data. In the follow-up of patients, ID2 expression at CR phase was decreased than at the time of diagnosis, and was increased again at the time of relapse. These findings demonstrated that bone marrow ID2 overexpression was a frequent event in AML patients, and predicts poor chemotherapy response and prognosis.

Hypomethylation of let-7a-3 is associated with poor prognosis in myelodysplastic syndrome.

Abstract Abnormal methylation of let-7a-3 has been found in various cancers and may consequently affect their survival. In this study, real-time quantitative methylation specific PCR (RQ-MSP) was used to determine the unmethylation level of let-7a-3 in 95 patients with myelodysplastic syndrome (MDS). The hypomethylation of let-7a-3 promoter was detected in 22 of 95 (23.2%) patients with MDS compared to 4.2% (1/24) of controls (p= 0.0419). Moreover, the frequency of let-7a-3 hypomethylation was higher in older patients (≥70 years) than in younger patients (<70 years). No significant difference was observed in distribution of WHO, IPSS, and cytogenetic classification. However, hypomethylated patients had significantly shorter overall survival than those without hypomethylation (p= 0.007). Moreover both Kaplan–Meier and Multivariate Cox analyses confirmed that let-7a-3 hypomethylation was an independent prognostic risk factor in cohorts of MDS patients with lower-risk disease. Our study suggested that let-7a-3 hypomethylation may predict poor outcome in MDS.

SETBP1 mutations in Chinese patients with acute myeloid leukemia and myelodysplastic syndrome

BACKGROUND Somatic mutations in SETBP1 gene have recently been detected in hematologic malignancies. The present study aimed to explore the frequency and clinical correlations of SETBP1 mutations in patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). METHODS In this study, we used high-resolution melting analysis (HRMA) to detect the SETBP1 mutations in a cohort of 363 patients with AML or MDS. RESULTS A total of 1.2% (3/249) of AML and 1.8% (2/114) of MDS patients were found with heterozygous SETBP1 mutations. In AML, patients with SETBP1 mutations showed higher hemoglobin (P = 0.004) and were more frequently recurrent in AML-M4 subtype (P = 0.034). All five SETBP1 mutated patients had normal karyotypes. The patients with SETBP1 mutations had significantly higher incidences of concurrent SRSF2 mutations (P = 0.002). HRMA could detect SETBP1 mutations with 5% sensitivity, obviously higher than 25% of Sanger sequencing. CONCLUSIONS We established a rapid, inexpensive, high-throughput and sensitive method to screen SETBP1 mutations. SETBP1 mutations were a rare molecular event in AML and MDS patients.

Overexpression of CTNNB1 : Clinical implication in Chinese de novo acute myeloid leukemia

Activation of Wnt/β-catenin signaling played a crucial role in tumorigenesis, and β-catenin (CTNNB1) overexpression has been identified in numerous solid tumors. The present study was designed to determine CTNNB1 expression and its clinical significance in Chinese de novo acute myeloid leukemia (AML) patients. Real-time quantitative PCR was carried out to detect the pattern of CTNNB1 expression in 140 AML patients and 46 controls. The level of CTNNB1 transcript in AML patients was significantly up-regulated compared with controls (P < 0.001). CTNNB1high patients showed significantly older age than CTNNB1low patients (P < 0.05). The frequency of high CTNNB1 expression was significantly observed in patients with intermediate/poor karyotypes. CTNNB1high patients had a significantly lower complete remission (CR) rate than CTNNB1low patients (P = 0.004). Among cytogenetically normal AML (CN-AML), CTNNB1high patients presented significantly shorter overall survival (OS, P = 0.004) and leukemia-free survival (LFS, P = 0.038) than CTNNB1low patients. Multivariate analysis confirmed that CTNNB1 expression was an independent prognostic factor for OS among CN-AML. Moreover, CTNNB1 expression level significantly decreased after CR stage (P = 0.032) and increased in relapsed stage (P = 0.015). Our findings suggest that CTNNB1 is overexpressed and confers a poor prognosis in AML, and could be used as a biomarker in monitoring disease recurrence.

Decreased SCIN expression, associated with promoter methylation, is a valuable predictor for prognosis in acute myeloid leukemia

The present study was aimed to investigate SCIN expression as well as promoter methylation and further explore their clinical relevance in acute myeloid leukemia (AML) patients. Real‐time quantitative PCR was carried out to detect the expression level of SCIN in 119 AML patients and 37 healthy controls. Real‐time quantitative methylation‐specific PCR and bisulfite sequencing PCR were carried out to detect SCIN promoter methylation levels in 103 AML patients and 29 controls. As compared with controls, the level of SCIN transcript was significantly down‐regulated in AML patients (P = 0.001), and the level of methylated SCIN promoter was significantly higher in AML patients (P = 0.001). Moreover, the level of promoter methylation was weakly negatively correlated with SCIN expression in AML patients (R = −0.265, P = 0.027). Demethylation of SCIN promoter by 5‐aza‐2′‐deoxycytidine could restore its expression in leukemic cell line THP1. The age of SCINlow patients was significantly higher and C/EBPA mutation was significantly less than SCINhigh patients (P = 0.039 and 0.038, respectively). Moreover, the rate of complete remission (CR) of SCINlow patients was significantly lower than SCINhigh patients (P = 0.009). Kaplan‐Meier analysis showed that low SCIN expression was associated with shorter overall survival (P = 0.036). Cox regression analysis demonstrated low SCIN expression was an independent poor prognostic factor (P = 0.047). Furthermore, SCIN expression was restored in those patients who achieved CR after induction therapy (P = 0.003). These findings indicate that decreased SCIN expression associated with its promoter methylation is a valuable biomarker for predicting adverse prognosis in AML patients.

Dysregulation of miR - 200s clusters as potential prognostic biomarkers in acute myeloid leukemia

BackgroundIncreasing studies showed that miR-200 family (miR-200s) clusters are aberrantly expressed in multiple human cancers, and miR-200s clusters function as tumor suppressor genes by affecting cell proliferation, self-renewal, differentiation, division and apoptosis. Herein, we aimed to investigate the expression and clinical implication of miR-200s clusters in acute myeloid leukemia (AML).MethodsRT-qPCR was performed to detect expression of miR-200s clusters in 19 healthy donors, 98 newly diagnosed AML patients, and 35 AML patients achieved complete remission (CR).ResultsExpression of miR-200a/200b/429 cluster but not miR-200c/141 cluster was decreased in newly diagnosed AML patients as compared to healthy donors and AML patients achieved CR. Although no significant differences were observed between miR-200s clusters and most of the features, low expression of miR-200s clusters seems to be associated with higher white blood cells especially for miR-200a/200b. Of the five members of miR-200s clusters, low expression of miR-200b/429/200c was found to be associated with lower CR rate. Logistic regression analysis further revealed that low expression of miR-429 acted as an independent risk factor for CR in AML. Based on Kaplan–Meier analysis, low expression of miR-200b/429/200c was associated with shorter OS, whereas miR-200a/141 had a trend. Moreover, multivariate analysis of Cox regression models confirmed the independently prognostic value of miR-200b expression for OS in AML.ConclusionsExpression of miR-200a/200b/429 cluster was frequently down-regulated in AML, and low expression of miR-429 as an independent risk factor for CR, whereas low expression of miR-200b as an independent prognostic biomarker for OS.

Hypermethylation of ITGBL1 is associated with poor prognosis in acute myeloid leukemia: LIAN et al.

The current study was aimed to investigate integrin beta‐like 1 (ITGBL1) methylation pattern and its clinical relevance in patients with acute myeloid leukemia (AML). Real‐time methylation‐specific polymerase chain reaction (PCR; RQ‐MSP) and bisulfite sequencing PCR (BSP) were performed to detect the methylation of ITGBL1 promoter. Real‐time quantitative PCR (RT‐qPCR) was performed to analyze ITGBL1 transcript level. The results showed that ITGBL1 methylation level in 131 patients with AML was significantly higher than 29 controls (p < 0.001). The ITGBL1‐hypermethylated group tended to have a higher bone marrow (BM) blasts ( p = 0.076). Meanwhile, ITGBL1‐hypermethylated patients tended to have a lower complete remission (CR) rate ( p = 0.102). ITGBL1‐hypermethylated patients had significantly shorter overall survival (OS) and leukemia‐free survival (LFS) than ITGBL1 hypomethylated patients in whole AML cohort ( p = 0.009 and 0.043, respectively) and patients with nonacute promyelocytic leukemia (APL ; p = 0.023 and 0.039, respectively). Multivariate analysis confirmed that the ITGBL1 methylation served as an independent prognostic factor in both patients with whole‐cohort AML ( p = 0.030) and patients with non‐APL ( p = 0.020). Furthermore, the ITGBL1 methylation level was significantly decreased in follow‐up AML patients who achieved complete remission after induction therapy ( P = 0.001). ITGBL1 methylation negatively correlated with ITGBL1 expression in patients with AML ( R = −0.328, p = 0.008). Moreover, demethylation of ITGBL1 could increase the ITGBL1 expression in the K562 leukemic cell line ( p < 0.05). In conclusion, the ITGBL1 hypermethylation is a potential biomarker for predicting prognosis and monitoring disease status in patients with AML.

Methylation-independent ITGA2 overexpression is associated with poor prognosis in de novo acute myeloid leukemia: LIAN et al.

Previous studies have been indicated that integrin α2 (ITGA2) may be important in cell migration, invasion, survival, and angiogenesis. However, the correlation between ITGA2 expression and acute myeloid leukemia (AML) is still unclear. Real‐time quantitative polymerase chain reaction was carried out to analyze ITGA2 messenger RNA level. Methylation‐specific polymerase chain reaction (PCR) and bisulfite sequencing PCR were performed to detect the methylation of ITGA2 promoter. ITGA2 expression was significantly upregulated in 134 de novo AML patients compared with 33 controls (p = 0.007). ITGA2high group had markedly lower complete remission (CR) rate than ITGA2low group (p = 0.011). Furthermore, the overall survival in ITGA2high patients was significantly shorter than ITGA2low patients throughout AML cohort, non–acute promyelocytic leukemia (APL) and cytogenetic normal‐AML (p = 0.001, 0.002, and 0.044, respectively). Multivariate analysis confirmed that ITGA2 overexpression served as an independent prognostic factor in both whole‐cohort AML patients (p = 0.018) and non‐APL AML patients (p = 0.021). Besides, ITGA2 expression level was significantly decreased in AML patients after CR (p = 0.011), and was returned at the time of relapse phase (p = 0.021). Moreover, unmethylated ITGA2 promoter was identified in normal controls, leukemia cell lines, and primary leukemia cells with low or high ITGA2 expression. In conclusions, methylation‐independent ITGA2 overexpression is associated with poor prognosis in AML.