Detection of NPM1 Mutations in Acute Myeloid Leukemia by using Drop-Off Droplet Digital PCR and its Clinical Application.

BACKGROUND: Nucleophosmin 1 (NPM1) mutations, which occur in 25 - 30% of acute myeloid leukemia (AML) and 50 - 60% of AML with normal karyotype, have been identified as an important marker for stratification of prog-nosis in AML. This study aimed to establish a new quantitative polymerase chain reaction (PCR) technique, the drop-off droplet digital PCR (ddPCR), for rapid and sensitive detection of NPM1 mutations in AML. METHODS: We established the drop-off ddPCR system and verified its performance. NPM1 mutations were screened in 130 AML patients by drop-off ddPCR and were validated by Sanger sequencing and next-generation sequencing (NGS). Then, the NPM1 mutation burden was dynamically monitored in five patients. RESULTS: The limit of blank (LOB) of drop-off ddPCR established for NPM1 mutation was 3.36 copies/µL, and the limit of detection (LOD) was 5.00 - 5.37 copies/µL in 50 ng DNA, and the sensitivity was about 0.05%, which had good linearity. Drop-off ddPCR identified 33/130 (25.4%) NPM1 mutated cases, consistent with Sanger sequencing. In 18 NPM1 positive cases selected randomly, NGS identified fourteen with type A mutation, two with type D mutation, and two with rare type mutations. The mutation burden of NPM1 mutation analyzed by NGS was consistent with the drop-off ddPCR. The sequential samples were detected for measurable residual disease (MRD) monitoring in 5 patients showed that the NPM1 mutation burden was consistent with clinical remission and recurrence. Compared with traditional ddPCR, drop-off ddPCR was also suitable for MRD monitoring. CONCLUSIONS: In this study, we established a drop-off ddPCR method for detecting three common mutations in AML with good sensitivity and repeatability, which can be used to screen mutations in newly diagnosed AML patients and for MRD monitoring after remission to guide treatment.

DNA methylation-mediated differential expression of DLX4 isoforms has opposing roles in leukemogenesis.

BACKGROUND: Previously, we reported the expression of DLX4 isoforms (BP1 and DLX7) in myeloid leukemia, but the functional role of DLX4 isoforms remains poorly understood. In the work described herein, we further determined the underlying role of DLX4 isoforms in chronic myeloid leukemia (CML) leukemogenesis. METHODS: The expression and methylation of DLX4 isoforms were detected by real-time quantitative PCR (RT-qPCR) and real-time quantitative methylation-specific PCR (RT-qMSP) in patients with CML. The functional role of DLX4 isoforms was determined in vitro and in vivo. The molecular mechanism of DLX4 isoforms in leukemogenesis was identified based on chromatin immunoprecipitation with high-throughput sequencing (ChIP-Seq)/assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-Seq) and RNA sequencing (RNA-Seq). RESULTS: BP1 expression was increased in patients with CML with unmethylated promoter, but DLX7 expression was decreased with hypermethylated promoter. Functionally, overexpression of BP1 increased the proliferation rate of K562 cells with S/G2 promotion, whereas DLX7 overexpression reduced the proliferation rate of K562 cells with G1 arrest. Moreover, K562 cells with BP1 overexpression increased the tumorigenicity in NCG mice, whereas K562 cells with DLX7 overexpression decreased the tumorigenicity. Mechanistically, a total of 91 genes including 79 messenger RNAs (mRNAs) and 12 long noncoding RNAs (lncRNAs) were discovered by ChIP-Seq and RNA-Seq as direct downstream targets of BP1. Among the downstream genes, knockdown of RREB1 and SGMS1-AS1 partially revived the proliferation caused by BP1 overexpression in K562 cells. Similarly, using ATAC-Seq and RNA-Seq, a total of 282 genes including 151 mRNA and 131 lncRNAs were identified as direct downstream targets of DLX7. Knockdown of downstream genes PTPRB and NEAT1 partially revived the proliferation caused by DLX7 overexpression in K562 cells. Finally, we also identified and validated a SGMS1-AS1/miR-181d-5p/SRPK2 competing endogenous RNA (ceRNA) network caused by BP1 overexpression in K562 cells. CONCLUSIONS: The current findings reveal that DNA methylation-mediated differential expression of DLX4 isoforms BP1 and DLX7 plays opposite functions in leukemogenesis. BP1 plays an oncogenic role in leukemia development, whereas DLX7 acts as a tumor suppressor gene. These results suggest DLX4 as a therapeutic target for antileukemia therapy.

EZH2 dysregulation: Potential biomarkers predicting prognosis and guiding treatment choice in acute myeloid leukaemia.

Accumulating studies have proved EZH2 dysregulation mediated by mutation and expression in diverse human cancers including AML. However, the expression pattern of EZH2 remains controversial in acute myeloid leukaemia (AML). EZH1/2 expression and mutation were analysed in 200 patients with AML. EZH2 expression was significantly decreased in AML patients compared with normal controls but not for EZH1 expression. EZH2 mutation was identified three of the 200 AML patients (1.5%, 3/200), whereas none of the patients harboured EZH1 mutation (0%, 0/200). EZH2 expression and mutation were significantly associated with -7/del(7) karyotypes. Moreover, lower EZH2 expression was associated with older age, higher white blood cells, NPM1 mutation, CEBPA wild-type and WT1 wild-type. Patients with EZH2 mutation showed shorter overall survival (OS) and leukaemia-free survival (LFS) than patients without EHZ2 mutation after receiving autologous or allogeneic haematopoietic stem cell transplantation (HSCT). However, EZH2 expression has no effect on OS and LFS of AML patients. Notably, in EZH2 low group, patients undergone HSCT had significantly better OS and LFS compared with patients only received chemotherapy, whereas no significant difference was found in OS and LFS between chemotherapy and HSCT patients in EZH2 high group. Collectively, EZH2 dysregulation caused by mutation and under-expression identifies specific subtypes of AML EZH2 dysregulation may be acted as potential biomarkers predicting prognosis and guiding the treatment choice between transplantation and chemotherapy.

Establishment and molecular characterization of decitabine-resistant K562 cells.

The clinical activity of decitabine (5-aza-2-deoxycytidine, DAC), a hypomethylating agent, has been demonstrated in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) patients. However, secondary resistance to this agent often occurs during treatment and leads to treatment failure. It is important to clarify the mechanisms underlying the resistance for improving the efficacy. In this study, by gradually increasing concentration after a continuous induction of DAC, we established the DAC-resistant K562 cell line (K562/DAC) from its parental cell line K562. The proliferation and survival rate of K562/DAC was significantly increased, whereas the apoptosis rate was remarkably decreased than that of K562 after DAC treatment. In K562/DAC, a total of 108 genes were upregulated and 118 genes were downregulated by RNA-Seq. In addition, we also observed aberrant expression of DDX43/H19/miR-186 axis (increased DDX43/H19 and decreased miR-186) in K562/DAC cells. Ectopic expression of DDX43 in parental K562 cells rendered cells resistant to the DAC. Taken together, we successfully established DAC-resistant K562 cell line which can serve as a good model for investigating DAC resistance mechanisms, and DDX43/H19/miR-186 may be involved in DAC resistance in K562.

Reduced protocadherin17 expression in leukemia stem cells: the clinical and biological effect in acute myeloid leukemia.

BACKGROUND: Leukemia stem cell (LSC)-enriched genes have been shown to be highly prognostic in acute myeloid leukemia (AML). However, the prognostic value of tumor suppressor genes (TSGs) that are repressed early in LSC remains largely unknown. METHODS: We compared the public available expression/methylation profiling data of LSCs with that of hematopoietic stem cells (HSCs), in order to identify potential tumor suppressor genes in LSC. The prognostic relevance of PCDH17 was analyzed on a cohort of 173 AML patients from The Cancer Genome Atlas (TCGA), and further validated in three independent cohorts (n = 339). RESULTS: We identified protocadherin17 (PCDH17) and demonstrated that it was significantly down-regulated and hypermethylated in LSCs compared with HSCs. Our analyses of primary AML patient samples also confirmed these deregulations. Clinically, low PCDH17 expression was associated with female sex (P = 0.01), higher WBC (P < 0.0001), higher percentages of blasts in bone marrow (BM) and peripheral blood (PB) (P = 0.04 and < 0.001, respectively), presence of FLT3-internal tandem duplications (P = 0.002), mutated NPM1 (P = 0.02), and wild-type TP53 (P = 0.005). Moreover, low PCDH17 expression predicted worse overall survival (OS) in four independent cohorts as well as in the molecularly defined subgroups of AML patients. In multivariable analyses, low PCDH17 expression retained independent prognostic value for OS. Biologically, PCDH17 expression-associated gene signatures were characterized by deregulations of EMT- and Wnt pathway-related genes. CONCLUSIONS: PCDH17 gene was silenced by DNA methylation in AML. Low PCDH17 expression is associated with distinct clinical and biological features and improves risk stratification in patients with AML.

Down-regulation of miR-29c is a prognostic biomarker in acute myeloid leukemia and can reduce the sensitivity of leukemic cells to decitabine.

BACKGROUND: MicroRNA-29c (miR-29c) is abnormally expressed in several cancers and serves as an important predictor of tumor prognosis. Herein, we investigate the effects of abnormal miR-29c expression and analyze its clinical significance in acute myeloid leukemia (AML) patients. In addition, decitabine (DAC) has made great progress in the treatment of AML in recent years, but DAC resistance is still common phenomenon and the mechanism of resistance is still unclear. We further analyze the influences of miR-29c to leukemic cells treated with DAC. METHODS: Real-time quantitative PCR (RQ-PCR) was carried out to detect miR-29c transcript level in 102 de novo AML patients and 25 normal controls. miR-29c/shRNA-29c were respectively transfected into K562 cells and HEL cells. Cell viability after transfection was detected by cell counting Kit-8 assays. Flow cytometry was used to detect apoptosis. RESULTS: MiR-29c was significantly down-regulated in AML (P < 0.001). Low miR-29c expression was frequently observed in patients with poor karyotype and high risk (P = 0.006 and 0.013, respectively). Patients with low miR-29c expression had a markedly shorter overall survival (OS) than those with high miR-29c expression (P < 0.001). Multivariate analysis confirmed the independent prognostic value of low miR-29c expression in both the whole cohort as well as the cytogenetically normal AML (CN-AML) subset. Over-expression of miR-29c in K562 treated with DAC inhibited growth, while silencing of miR-29c in HEL promoted growth and inhibited apoptosis. MiR-29c overexpression decreased the half maximal inhibitory concentration (IC50) of DAC in K562, while miR-29c silencing increased the IC50 of DAC in HEL. The demethylation of the miR-29c promoter was associated with its up-regulated expression. Although miR-29c demethylation was also observed in DAC-resistant K562 (K562/DAC), miR-29c expression was down-regulated. MiR-29c transfection also promoted apoptosis and decreased the IC50 of DAC in K562/DAC cells. CONCLUSIONS: Our results suggest that miR-29c down-regulation may act as an independent prognostic biomarker in AML patients, and miR-29c over-expression can increase the sensitivity of both non-resistant and resistant of leukemic cells to DAC.

Hypomethylation-mediated H19 overexpression increases the risk of disease evolution through the association with BCR-ABL transcript in chronic myeloid leukemia.

Previous study has revealed that H19 expression is required for efficient tumor growth induced by BCR-ABL in chronic myeloid leukemia (CML). Herein, we further determined H19 expression and its clinical implication in patients with CML. H19 expression and methylation were detected by real-time quantitative PCR and real-time quantitative methylation-specific PCR, and then clinical implication of H19 expression was further analyzed. H19 expression was significantly up-regulated in CML patients (p < 0.001). H19 expression with an area under receiver operating characteristic curve value of 0.824 might serve as a promising biomarker in distinguishing CML patients from controls. The patients with high H19 expression had a tendency of higher white blood cells and BCR-ABL transcript than those with low H19 expression. H19 overexpression occurred with the higher frequency in blast crisis stage (11/11, 100%), lower in accelerated phase (3/5, 60%), and chronic phase (42/62, 66%) stages. Moreover, paired patients during disease progression with increased BCR-ABL transcript also showed a significant upregulation of H19 expression. Meanwhile, H19 expression was decreased in follow-up patients who achieved complete molecular remission after tyrosine kinase inhibitors-based therapy. Epigenetic studies showed that H19 differentially methylated region/imprinting control region (DMR/ICR) was hypomethylated and associated with H19 expression in CML patients. Moreover, demethylation of H19 DMR/ICR reactivated H19 expression in K562 cells. Collectively, H19 overexpression, a frequent event in CML, was associated with higher BCR-ABL transcript involving in disease progression. Moreover, H19 DMR/ICR hypomethylation in CML may be one of the mechanisms mediating H19 overexpression.

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

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.

TET2 expression is a potential prognostic and predictive biomarker in cytogenetically normal acute myeloid leukemia.

TET2 (Ten-Eleven Translocation 2) gene is a member of TET family that can modify DNA through catalyzing the conversion of 5-methylcytosine (5-mC) into 5-hydroxymethylcytosine (5-hmC). Although TET2 mutation has been disclosed in a variety of hematopoietic malignancies, the prognostic implication of TET2 expression in acute myeloid leukemia (AML) remains largely elusive. In this study, real-time quantitative PCR was carried out to detect the level of TET2 transcript in 134 de novo AML patients and 35 healthy donors. TET2 mRNA level was significantly down-regulated in AML patients compared with controls (p = 0.010). Among the French-American-British (FAB) subtypes, the incidence of TET2 under-expression in M0/M1 subtypes was significantly higher than in the other subtypes M2/M3/M4/M5/M6 (p = 0.017), and also markedly higher than in the other granulocyte subtypes M2/M3 (p = 0.005). TET2 low-expressed patients showed a significantly higher frequency of NPM1 mutations than TET2 high-expressed patients. Although there was no significant difference in complete remission rate between two groups (low and high TET2 expression), patients with low TET2 expression had markedly shorter overall survival (OS) in both non-M3 and cytogenetically normal AML (CN-AML) (p = 0.016 and 0.044, respectively). Furthermore, multivariate analysis confirmed the prognostic value of TET2 expression on OS among CN-AML patients (p = 0.049). Importantly, TET2 expression in complete remission (CR) time was significantly higher than newly diagnosis time (p = 0.001), and was returned to lower level when in relapse time (p < 0.001). These findings indicated that down-regulation of TET2 expression was a common event and acted as a prognostic and predictive biomarker in CN-AML patients.

Methylation-independent CHFR expression is a potential biomarker affecting prognosis in acute myeloid leukemia.

CHFR acts as a tumor suppressor gene, which is frequently inactivated caused by its promoter hypermethylation in various solid tumors. Although a recent study showed that CHFR hypermethylation was a frequent event in acute myeloid leukemia (AML) and correlated with adverse clinical outcome, herein, we found that CHFR methylation was a rare event in patients with myeloid malignancies (including AML, chronic myeloid leukemia, and myelodysplastic syndromes), but its expression may serve as an independent prognostic biomarker in AML. CHFR expression was assessed by real-time quantitative PCR, whereas CHFR methylation was detected by methylation-specific PCR and bisulfite sequencing PCR. In AML patients, lower CHFR expression was associated with lower complete remission (CR) rate, and CHFR expression was significantly increased in CR after chemotherapy. Moreover, patients with lower CHFR expression showed shorter overall survival and leukemia-free survival, and multivariate analysis confirmed that lower CHFR expression was an independent risk factor in AML. Importantly, the prognostic value of CHFR expression was validated using the published Gene Expression Omnibus datasets. Notably, CHFR promoter was nearly unmethylated in patients with myeloid malignancies. Our findings revealed that lower CHFR expression was independently associated with unfavorable prognosis in AML. Moreover, aberrant CHFR promoter methylation was a rare event in myeloid malignances.

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.

BACKGROUND: Increasing 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). METHODS: RT-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). RESULTS: Expression 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. CONCLUSIONS: Expression 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.

Epigenetic dysregulation of ID4 predicts disease progression and treatment outcome in myeloid malignancies.

Promoter hypermethylation-mediated inactivation of ID4 plays a crucial role in the development of solid tumours. This study aimed to investigate ID4 methylation and its clinical relevance in myeloid malignancies. ID4 hypermethylation was associated with higher IPSS scores, but was not an independent prognostic biomarker affecting overall survival (OS) in myelodysplastic syndrome (MDS). However, ID4 hypermethylation correlated with shorter OS and leukaemia-free survival (LFS) time and acted as an independent risk factor affecting OS in acute myeloid leukaemia (AML). Moreover, ID4 methylation was significantly decreased in the follow-up paired AML patients who achieved complete remission (CR) after induction therapy. Importantly, ID4 methylation was increased during MDS progression to AML and chronic phase (CP) progression to blast crisis (BC) in chronic myeloid leukaemia (CML). Epigenetic studies showed that ID4 methylation might be one of the mechanisms silencing ID4 expression in myeloid leukaemia. Functional studies in vitro showed that restoration of ID4 expression could inhibit cell proliferation and promote apoptosis in both K562 and HL60 cells. These findings indicate that ID4 acts as a tumour suppressor in myeloid malignancies, and ID4 methylation is a potential biomarker in predicting disease progression and treatment outcome.

The prognostic implication of SRSF2 mutations in Chinese patients with acute myeloid leukemia.

Recently, somatic mutations in SRSF2 gene have been discovered in a proportion of hematologic malignancies including acute myeloid leukemia (AML). This study was aimed to investigate SRSF2 mutations in Chinese AML patients. High-resolution melting analysis (HRMA) was developed to screen SRSF2 mutations in 249 cases with AML, and then direct DNA sequencing was used to verify the results of HRMA. In this study, 3.6 % (9/249) of Chinese AML patients were found with heterozygous SRSF2 mutations. Patients with SRSF2 mutations were older than those with wild-type SRSF2 (P = 0.014). No differences in the sex, blood parameters, French-American-British classification (FAB) subtypes, and karyotypes were observed between AML patients with and without SRSF2 mutations. Although the overall survival (OS) of SRSF2-mutated patients was inferior to those without mutations in both whole AML patients (median 4 vs. 11 months, respectively; P = 0.006) and cytogenetically normal patients (median 2 vs. 12 months, respectively; P = 0.008), multiple analysis disclosed that SRSF2 mutation was not an independent prognostic factor in AML patients. These results suggest that SRSF2 mutation occurs at a low frequency in aged AML patients and might not be associated with adverse prognosis in Chinese AML patients.

DLX4 hypermethylation is a prognostically adverse indicator in de novo acute myeloid leukemia.

Hypermethylation of distal-less homeobox 4 (DLX4) has been increasingly identified in several cancers. Our study was aimed to determine the role of DLX4 methylation in regulating DLX4 expression and further analyze its clinical significance in de novo acute myeloid leukemia (AML) patients. DLX4 methylation level was detected by real-time quantitative methylation-specific PCR and bisulfite sequencing PCR. Treatment with 5-aza-2'-deoxycytidine (5-aza-dC) was used for demethylation studies. Clinical significance of DLX4 methylation was obtained by the comparison between the patients with and without DLX4 methylation. DLX4 was significantly methylated in AML patients compared with controls (P < 0.001). DLX4 methylation was negatively associated with DLX7 (the shorter DLX4 isoform) (R = -0.202, P = 0.021) but not BP1 (the longer DLX4 isoform) (R = -0.049, P = 0.582) expression in AML patients. DLX7 and BP1 messenger RNA (mRNA) were significantly increased after 5-aza-dC treatment in leukemic cell lines THP1 and Kasumi-1. DLX4 methylated patients showed significantly higher frequency of U2AF1 mutation compared with DLX4 unmethylated patients (P = 0.043). Both all AML and non-M3 patients with DLX4 methylation presented significantly lower complete remission rate than those with DLX4 unmethylation (P = 0.001 and <0.001, respectively). DLX4 methylated cases had significantly shorter overall survival than DLX4 unmethylated cases among both all AML (P = 0.003), non-M3 AML (P = 0.001), and cytogenetically normal AML (P = 0.032). Multivariate analysis confirmed that DLX4 methylation was independent risk factor in both all AML and non-M3 patients. Our study indicates that DLX4 hypermethylation is negatively associated with DLX7 expression and predicts poor clinical outcome in de novo AML patients.

Let-7a-3 hypomethylation is associated with favorable/intermediate karyotypes but not with survival in acute myeloid leukemia.

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.

Hypermethylation of DLX4 predicts poor clinical outcome in patients with myelodysplastic syndrome.

BACKGROUND: Hypermethylation of DLX4 (distal-less homeobox 4) has been disclosed in a variety of cancers. Our work was aimed to examine the pattern of DLX4 methylation and further investigate its clinical relevance in patients with myelodysplastic syndrome (MDS). METHODS: Real-time quantitative methylation-specific PCR and bisulfite sequencing PCR were carried out to detect the level of DLX4 methylation. Clinical significance of DLX4 methylation was analyzed between the DLX4 hypermethylated and non-hypermethylated patients. RESULTS: DLX4 was significantly hypermethylated in MDS patients than controls (p<0.001). No significant differences were observed between the hypermethylated and non-hypermethylated MDS patients in white blood cells, platelets, age, WHO classifications, FAB classifications, IPSS risks, and common gene mutations (p>0.05). However, DLX4 hypermethylated patients tended to have higher hemoglobin (HB) than DLX4 non-hypermethylated patients (p=0.079). Moreover, there was a trend that male patients, poor karyotype patients, and IPSS Int-2/High patients had a higher frequency of DLX4 hypermethylation (p=0.067, 0.065, and 0.068). DLX4 hypermethylated patients had significantly shorter overall survival than DLX4 non-hypermethylated patients (p=0.004). Multivariate analysis confirmed the prognostic value of DLX4 methylation in MDS patients (p<0.001). CONCLUSIONS: Our study indicated that DLX4 hypermethylation was a frequent event and acted as an independent prognostic biomarker in de novo MDS patients.

Reduced miR-215 expression predicts poor prognosis in patients with acute myeloid leukemia.

OBJECTIVE: Abnormal expression of microRNA-215 has been identified in a variety of solid cancers. However, little is known about the expression pattern of microRNA-215 in acute myeloid leukemia. This study was to investigate the status of microRNA-215 expression and further analyze its clinical significance in acute myeloid leukemia. METHODS: Real-time quantitative polymerase chain reaction assay was performed to evaluate the expression level of microRNA-215 in 113 patients with acute myeloid leukemia. Besides, the relationship between microRNA-215 levels and clinical and pathological factors was explored. RESULTS: Compared with the healthy individuals, microRNA-215 expression in acute myeloid leukemia patients was significantly down-regulated (P= 0.001). MicroRNA-215 low-expressed patients had higher white blood cells than microRNA-215 high-expressed patients (P= 0.014). The incidence of FLT3/ITD mutation in the patients with low microRNA-215 expression was significantly higher than those with high microRNA-215 expression (P= 0.025). MicroRNA-215 low-expressed patients had significantly shorter overall survival than microRNA-215 high-expressed patients in both non-M3 acute myeloid leukemia patients and cytogenetically normal patients (P= 0.017 and P= 0.044, respectively). Meanwhile, multivariate analysis confirmed the adverse prognostic value of microRNA-215 expression in acute myeloid leukemia patients with non-M3 subtypes. CONCLUSIONS: Our study demonstrates that reduced microRNA-215 expression is a common event and is associated with poor clinical outcome in acute myeloid leukemia.

Down-Regulation of miR-186 Correlates with Poor Survival in de novo Acute Myeloid Leukemia.

BACKGROUND: MicroRNA-186 (miR-186) plays an important role in the pathogenesis of several cancers. Our study was intended to investigate the expression status and the prognostic implication of miR-186 in acute myeloid leukemia (AML). METHODS: Real-time quantitative PCR was carried out in 112 de novo AML patients and 28 controls. RESULTS: The level of miR-186 expression in AML was significantly down-regulated compared to normal controls (p < 0.001). Patients with low miR-186 expression presented significantly older age than those with high miR-186 expression (p = 0.004). MiR-186high patients had a significantly higher frequency of CEBPA mutation than miR-186low patients (20% and 4%, respectively, p = 0.022). In addition, miR-186low patients had a significantly lower complete remission (CR) rate (30% vs. 53%, respectively, p = 0.028) than miR-186high patients. Moreover, miR-186low patients showed significantly shorter overall survival (OS) time than miR-186high patients in both whole AML and non-M3 patients (p = 0.023 and 0.026, respectively). Additionally, the adverse prognostic impact of miR-186 down-regulation was also shown in both whole AML and non-M3 patients without CEBPA mutation (p = 0.017 and 0.023, respectively). CONCLUSIONS: Our study suggests that miR-186 down-regulation is a frequent event and predicts poor prognosis in de novo AML patients.

Epigenetic inactivation of DLX4 is associated with disease progression in chronic myeloid leukemia.

Aberrant DNA methylation of various genes has been identified to be associated with disease progression in chronic myeloid leukemia (CML). Our study was intended to investigate DLX4 methylation pattern in different clinical stages of CML and further determine its role in regulating DLX4 expression. Real-time quantitative methylation-specific PCR and bisulfite sequencing PCR were applied to detect DLX4 methylation. 5-aza-2'-deoxycytidine (5-aza-dC) was used for demethylation studies. DLX4 was significantly hypermethylated in CML patients (P = 0.002) especially in blastic phase (BC) stage (P < 0.001) as compared with controls. Moreover, DLX4 methylation level in BC stage was significantly higher than in chronic phase (CP) stage (P < 0.001). DLX4 methylation density was significantly increased during the progression of CML among the tested two patients (P < 0.001). DLX4 hypermethylation occurred with the highest incidence in BC stage (83%), lower incidence in acute phase (AP) stage (43%), and the lowest incidence in CP stage (26%) (P = 0.001). Moreover, t(9; 22) with additional alteration cases had significantly higher frequency of DLX4 hypermethylation compared with the other cytogenetics (P = 0.010). Significantly negative correlation was observed between DLX4 methylation and DLX4-TV2 (the shorter DLX4 isoform) expression (R = -0.382, P = 0.001, n = 78) but not between DLX4 methylation and BP1 (the longer DLX4 isoform) expression (R = 0.134, P = 0.244, n = 78) in CML patients. Both DLX4-TV2 and BP1 mRNA were significantly increased after 5-aza-dC treatment in K562 cell line (P < 0.001). Our study indicated that hypermethylation of DLX4 correlated with disease progression of CML. Moreover, DLX4 expression was regulated by its methylation in CML.