The role of aldehyde dehydrogenase 1 (ALDH1) as an ovarian cancer stem cell marker and its clinical significance have rarely been explored. We used an Aldefluor assay to isolate ALDH1-bright (ALDH1(br)) cells from epithelial ovarian cancer cell lines and characterized the properties of the stem cells. ALDH1(br) cells were enriched in ES-2 (1.3%), TOV-21G (1.0%), and CP70 (1.2%) cells. Both ALDH1(br) and ALDH1(low) cells repopulated stem cell heterogeneity, formed spheroids, and grew into tumors in immunocompromised mice, although these processes were more efficient in ALDH1(br) cells. In the ES-2 and CP70 cells, ALDH1(br) cells conferred more chemoresistance, and were more enriched in CD44 (by 1.74-fold and 5.18-fold, respectively) than in CD133 (by 1.39-fold and 1.17-fold, respectively), compared with ALDH1(low) cells. Immunohistochemical staining for ALDH1 on a tissue microarray containing 84 epithelial ovarian cancer samples revealed that patients with higher ALDH1 expression (>50%) had poor overall survival, compared with those with lower ALDH1 (P = 0.004) and yielded an odds ratio of death of 2.43 (95% CI = 1.12 to 5.28) by multivariate analysis. The results did not support ALDH1 alone as an ovarian cancer stem cell marker, but demonstrated that ALDH1 is associated with CD44 expression, chemoresistance, and poor clinical outcome. The use of a combination of ALDH1 with other stem cell markers may help define ovarian cancer stem cells more stringently.

BACKGROUND: DNA methylation may be used a potential biomarker for detecting cervical cancer. The authors of this report used quantitative methylation analysis of 4 genes in a full spectrum of cervical lesions to test its potential clinical application. METHODS: This hospital-based, retrospective, case-control study was conducted in 185 patients and included patients who had a normal uterine cervix (n ¼ 53), cervical intraepithelial neoplasm type 1 (CIN1) (n ¼ 37), CIN2 (n ¼ 22), CIN3 (n ¼ 24), carcinoma in situ (CIS) (n ¼ 22), squamous cell carcinoma (SCC, n ¼ 20), and adenocarcinoma (AC) (n ¼ 7). Methylation levels of the genes sex-determining region Y, box 1 (SOX1); paired box gene 1 (PAX1); LIM homeobox transcription factor 1a (LMX1A), and NK6 transcription factor-related locus 1 (NKX6-1) were determined by using real-time methylation-specific polymerase chain reaction (PCR) amplification. Cutoff values of the percentage of methylation reference (PMR) for different diagnoses were determined to test the sensitivity and specificity and to generate receiver operating characteristic (ROC) curves. Two-sided Mann-Whitney U tests were used to test differences in PMR between groups. RESULTS: The PMRs of the 4 genes were significantly higher in CIN3 and worse (CIN3þ) lesions than the PMRs in specimens of normal cervix and CIN1 or CIN2 (P < .001). ROC curve analysis demonstrated that the sensitivity, specificity, and accuracy for detecting CIN3þ lesions were 0.88, 0.82, and 0.95, respectively, for SOX1; 0.78, 0.91, and 0.89, respectively, for PAX1; 0.77, 0.88, and 0.90, respectively, for LMX1A; and 0.93, 0.97, and 0.97, respectively, for NKX6-1. CONCLUSIONS: The current results indicated that quantitative PCR-based testing for DNA methylation of 4 genes holds great promise for cervical cancer screening and warrants further population-based studies using standardized DNA methylation testing. Cancer 2010;116:4266-74.

This study demonstrates the potential use of methylated BHLHE22/CDO1/CELF4 panel for endometrial cancer screening of cervical scrapings. Clin Cancer Res; 23(1); 263-72. ©2016 AACR.

BackgroundDespite of the trend that the application of DNA methylation as a biomarker for cancer detection is promising, clinically applicable genes are few. Therefore, we looked for novel hypermethylated genes for cervical cancer screening.Methods and FindingsAt the discovery phase, we analyzed the methylation profiles of human cervical carcinomas and normal cervixes by methylated DNA immunoprecipitation coupled to promoter tiling arrays (MeDIP-on-chip). Methylation-specific PCR (MSP), quantitative MSP and bisulfite sequencing were used to verify the methylation status in cancer tissues and cervical scrapings from patients with different severities. Immunohistochemical staining of a cervical tissue microarray was used to confirm protein expression. We narrowed to three candidate genes: DBC1, PDE8B, and ZNF582; their methylation frequencies in tumors were 93%, 29%, and 100%, respectively. At the pre-validation phase, the methylation frequency of DBC1 and ZNF582 in cervical scraping correlated significantly with disease severity in an independent cohort (n = 330, both P<0.001). For the detection of cervical intraepithelial neoplasia 3 (CIN3) and worse, the area under the receiver operating characteristic curve (AUC) of ZNF582 was 0.82 (95% confidence interval  = 0.76–0.87).ConclusionsOur study shows ZNF582 is frequently methylated in CIN3 and worse lesions, and it is demonstrated as a potential biomarker for the molecular screening of cervical cancer.

Using DNA methylation biomarkers in cancer detection is a potential direction in clinical testing. Some methylated genes have been proposed for cervical cancer detection; however, more reliable methylation markers are needed. To identify new hypermethylated genes in the discovery phase, we compared the methylome between a pool of DNA from normal cervical epithelium (n 5 19) and a pool of DNA from cervical cancer tissues (n 5 38) using a methylation bead array. We integrated the differentially methylated genes with public gene expression databases, which resulted in 91 candidate genes. Based on gene expression after demethylation treatment in cell lines, we confirmed 61 genes for further validation. In the validation phase, quantitative MSP and bisulfite pyrosequencing were used to examine their methylation level in an independent set of clinical samples. Fourteen genes, including ADRA1D, AJAP1, COL6A2, EDN3, EPO, HS3ST2, MAGI2, POU4F3, PTGDR, SOX8, SOX17, ST6GAL2, SYT9, and ZNF614, were significantly hypermethylated in CIN31 lesions. The sensitivity, specificity, and accuracy of POU4F3 for detecting CIN31 lesions were 0.88, 0.82, and 0.85, respectively. A bioinformatics function analysis revealed that AJAP1, EDN3, EPO, MAGI2, and SOX17 were potentially implicated in b-catenin signaling, suggesting the epigenetic dysregulation of this signaling pathway during cervical cancer development. The concurrent methylation of multiple genes in cancers and in subsets of precancerous lesions suggests the presence of a driver of methylation phenotype in cervical carcinogenesis. Further validation of these new genes as biomarkers for cervical cancer screening in a larger population-based study is warranted.

Women with advanced stage ovarian cancer (OC) have a five-year survival rate of less than 25%. OC progression is associated with accumulation of epigenetic alterations and aberrant DNA methylation in gene promoters acts as an inactivating ?hit? during OC initiation and progression. Abnormal DNA methylation in OC has been used to predict disease outcome and therapy response. To globally examine DNA methylation in OC, we used next-generation sequencing technology, MethylCap-sequencing, to screen 75 malignant and 26 normal or benign ovarian tissues. Differential DNA methylation regions (DMRs) were identified, and the Kaplan?Meier method and Cox proportional hazard model were used to correlate methylation with clinical endpoints. Functional role of specific genes identified by MethylCap-sequencing was examined in in vitro assays. We identified 577 DMRs that distinguished (p < 0.001) malignant from non-malignant ovarian tissues; of these, 63 DMRs correlated (p < 0.001) with poor progression free survival (PFS). Concordant hypermethylation and corresponding gene silencing of sonic hedgehog pathway members ZIC1 and ZIC4 in OC tumors was confirmed in a panel of OC cell lines, and ZIC1 and ZIC4 repression correlated with increased proliferation, migration and invasion. ZIC1 promoter hypermethylation correlated (p < 0.01) with poor PFS. In summary, we identified functional DNA methylation biomarkers significantly associated with clinical outcome in OC and suggest our comprehensive methylome analysis has significant translational potential for guiding the design of future clinical investigations targeting the OC epigenome. Methylation of ZIC1, a putative tumor suppressor, may be a novel determinant of OC outcome.

ObjectiveWe hypothesized that DNA methylation of development-related genes may occur in endometrial cancer (EC)/ovarian cancer (OC) and may be detected in cervical scrapings.MethodsWe tested methylation status by quantitative methylation-specific polymerase chain reaction for 14 genes in DNA pools of endometrial and OC tissues. Tissues of EC/normal endometrium, OC/normal ovary, were verified in training set using cervical scrapings of 10 EC/10 OC patients and 10 controls, and further validated in the testing set using independent cervical scrapings in 30 EC/30 OC patients and 30 controls. We generated cutoff values of methylation index (M-index) from cervical scrapings to distinguish between cancer patients and control. Sensitivity/specificity of DNA methylation biomarkers in detecting EC and OC was calculated.ResultsOf 14 genes, 4 (PTGDR, HS3ST2, POU4F3, MAGI2) showed hypermethylation in EC and OC tissues, and were verified in training set. POU4F3 and MAGI2 exhibited hypermethylation in training set were validated in independent cases. The mean M-index of POU4F3 is 78.28 in EC and 20.36 in OC, which are higher than that in controls (6.59; p<0.001 and p=0.100, respectively), and that of MAGI2 is 246.0 in EC and 12.2 in OC, which is significantly higher that than in controls (2.85; p<0.001 and p=0.480, respectively). Sensitivity and specificity of POU4F3/MAGI2 were 83%–90% and 69%–75% for detection of EC, and 61% and 62%–69% for the detection of OC.ConclusionThe findings demonstrate the potential of EC/OC detection through testing for DNA methylation in cervical scrapings.

DNA methylation contributes to tumor formation, development and metastasis. Epigenetic dysregulation of stem cells is thought to predispose to malignant development. The clinical significance of DNA methylation in ovarian tumor-initiating cells (OTICs) remains unexplored. We analyzed the methylomic profiles of OTICs (CP70sps) and their derived progeny using a human methylation array. qRT-PCR, quantitative methylation-specific PCR (qMSP) and pyrosequencing were used to verify gene expression and DNA methylation in cancer cell lines. The methylation status of genes was validated quantitatively in cancer tissues and correlated with clinicopathological factors. ATG4A and HIST1H2BN were hypomethylated in OTICs. Methylation analysis of ATG4A and HIST1H2BN by qMSP in 168 tissue samples from patients with ovarian cancer showed that HIST1H2BN methylation was a significant and independent predictor of progression-free survival (PFS) and overall survival (OS). Multivariate Cox regression analysis showed that patients with a low level of HIST1H2BN methylation had poor PFS (hazard ratio (HR), 4.5; 95% confidence interval (CI), 1.4-14.8) and OS (HR, 4.3; 95% CI, 1.3-14.0). Hypomethylation of both ATG4A and HIST1H2BN predicted a poor PFS (HR, 1.8; 95% CI, 1.0-3.6; median, 21 months) and OS (HR, 1.7; 95% CI, 1.0-3.0; median, 40 months). In an independent cohort of ovarian tumors, hypomethylation predicted early disease recurrence (HR, 1.7; 95% CI, 1.1-2.5) and death (HR, 1.4; 95% CI, 1.0-1.9). The demonstration that expression of ATG4A in cells increased their stem properties provided an indication of its biological function. Hypomethylation of ATG4A and HIST1H2BN in OTICs predicts a poor prognosis for ovarian cancer patients.