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Background: We screened out several hypermethylated solute carrier (SLC) family genes in acute myeloid leukemia by reduced representation bisulfite sequencing. SLC22A3 encodes an organic cation transport protein, which is critical for drug transportation and cellular detoxification. SLC22A3 is significantly downregulated and associated with tumor progression and worse prognosis in a variety of solid tumors. However, there are no data available regarding the role of SLC22 in AML. This study aimed to explore the regulatory mechanism of DNA methylation on SLC22A3 expression, as well as its clinical significance in AML prognosis. Results: SLC22A3 was identified as the sole prognosis-associated gene among SLCs based on TCGA and Beat AML databases. Bone marrow mononuclear cells (BMMNCs) from AML, MDS patients, and healthy donors were enrolled in this study. SLC22A3 methylation was significantly increased in AML compared with controls and MDS patients; meanwhile, the expression level of SLC22A3 was decreased. SLC22A3 hypermethylation presented an obvious association with some specific clinical characteristics and affected the survival time of AML patients as an independent risk indicator. SLC22A3 expression changed regularly as the disease complete remissions and relapses. Demethylation drug 5-aza-2′-deoxycytidine (DAC) activated transcription and increased mRNA expression of SLC22A3 in leukemia cell lines and AML fresh BMMNCs. Knockdown of SLC22A3 in leukemia cells enhanced cell proliferation and suppressed cell apoptosis. Data from public programs were used for auxiliary screening of probable molecular mechanisms of SLC22A3 in the antileukemia effect. Conclusions: Our results showed that increased methylation and decreased expression of SLC22A3 may be indicators of poor prognosis in AML. Methylation-silenced SLC22A3 expression may have potential guiding significance on antileukemia effect of DAC. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: CTNNA1 gene is a putative tumor suppressor for its roles in inhibiting proliferation and promoting apoptosis. Aberrant expression of CTNNA1 is regulated by epigenetic mechanisms including both promoter methylation and histone deacetylation in hematopoietic malignancies. However, the clinical relevance of CTNNA1 methylation remains rarely known in myelodysplastic syndrome (MDS). Methods: We investigated the methylation status of CTNNA1 promoter using methylation-specific PCR (MSP) and analyzed its clinical significance in Chinese MDS patients. Results: Aberrant hypermethylation of CTNNA1 gene was identified in 22% (18/83) of the patients. CTNNA1 expression was significantly correlated with promoter methylation status (p<0.05). No significant differences were observed in the age, sex, and blood parameters between patients with and without CTNNA1 hypermethylation (p>0.05). The frequency of CTNNA1 hypermethylation was significantly higher in patients with isolated del(5q) (3/4, 75%) than those with other abnormal karyotypes (4/23, 17%) and also than those with normal karyotypes (11/54, 20%) (p=0.042 and 0.040, respectively). The patients with higher IPSS risks (Int-2/High) had significantly increased incidence of CTNNA1 methylation than those with lower risks (Low/Int-1) (36% vs. 15%, p=0.049). Although the estimated 50% survival time of the CTNNA1-methylated group [median 13 months, 95% confidence interval (CI) 3-22 months] was shorter than that of CTNNA1-unmethylated group (median 24 months, 95% CI 7-41 months), the difference was not statistically significant (p=0.330). Conclusions: Our data confirm that aberrant CTNNA1 methylation is a common event and is associated with higher IPSS risk in MDS. [ABSTRACT FROM AUTHOR]

Background: Aberrant expression of SRY-box containing gene 17 ( SOX17) has been observed in several solid tumors. However, little is known about SOX17 expression in acute myeloid leukemia (AML). The purpose of this study was to investigate the alteration of SOX17 expression and to explore its clinical significance in AML. Methods: Real-time quantitative PCR (RQ-PCR) was performed to analyze the status of SO1X17 expression in 103 patients with de novo AML and 26 normal controls. The clinical relevance of SOX17 expression was analyzed in AML. Results: SOX17 level in AML was significantly down-regulated compared to controls (p<0.001). Receiver operating characteristic curve (ROC) curve analysis revealed that an area under the ROC curve (AUC) of 0.834 (95% CI 0.765-0.903; p<0.0001) or 0.789 (95% CI 0.690-0.888, p<0.001) in discriminating all patients or cytogenetically normal patients from controls, respectively. The cohort of AML patients was divided into two groups according to the cut-off value of 0.017 (60% sensitivity and 100% specificity, respectively). Cytogenetically normal patients with low SOX17 expression had significantly shorter OS than those with high SOX17 expression (median 4 vs. 25 months, respectively, p=0.035). Multivariate analysis confirmed low SOX17 expression as an independent risk factor. Conclusions: Our findings indicated that low SOX17 level may define an important risk factor in AML with normal cyotgenetics. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: The long non-coding RNA H19 plays a crucial role in solid tumor initiation and progression. However, the potential role of H19 and its clinical significance in acute myeloid leukemia (AML) remain largely elusive. Methods: H19 expression was detected by qPCR, and clinical significance in AML patients was further analyzed. The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) data for AML were used as validation cohorts. The roles of H19 in cell proliferation and apoptosis were determined by cell proliferation assay and flow cytometry analysis. Results: H19 expression was significantly increased in AML patients but not associated with embedded miR-675 expression. Moreover, H19 overexpression was not dependent on the methylation pattern in H19 differentially methylated region/imprinting control region. Strong association was observed between H19 overexpression and patients' characteristics including sex, higher white blood cells, older age, and intermediate karyotype, FLT3-ITD, and DNMT3A mutations. In addition, H19 overexpression correlated with lower complete remission (CR) rate and shorter overall survival, and further confirmed by multivariate analyses. Importantly, the prognostic effect of H19 expression was validated by TCGA and GEO data. In the follow-up of patients, H19 expression in CR phase was lower than diagnosis time and returned at relapse time. Loss-of-function experiments showed that H19 exhibited anti-proliferative and pro-apoptotic effects in leukemic cell HL60. Furthermore, H19 expression was positively correlated with potential downstream gene ID2 in AML. Conclusions: Our findings revealed that methylation-independent H19 was a prognostic and predictive biomarker in AML, and H19/ID2 played crucial roles in leukemogenesis with potential therapeutic target value. [ABSTRACT FROM AUTHOR]