DNA Methylation of Window of Implantation Genes in Cervical Secretions Predicts Ongoing Pregnancy in Infertility Treatment.

Window of implantation (WOI) genes have been comprehensively identified at the single cell level. DNA methylation changes in cervical secretions are associated with in vitro fertilization embryo transfer (IVF-ET) outcomes. Using a machine learning (ML) approach, we aimed to determine which methylation changes in WOI genes from cervical secretions best predict ongoing pregnancy during embryo transfer. A total of 2708 promoter probes were extracted from mid-secretory phase cervical secretion methylomic profiles for 158 WOI genes, and 152 differentially methylated probes (DMPs) were selected. Fifteen DMPs in 14 genes (BMP2, CTSA, DEFB1, GRN, MTF1, SERPINE1, SERPINE2, SFRP1, STAT3, TAGLN2, TCF4, THBS1, ZBTB20, ZNF292) were identified as the most relevant to ongoing pregnancy status. These 15 DMPs yielded accuracy rates of 83.53%, 85.26%, 85.78%, and 76.44%, and areas under the receiver operating characteristic curves (AUCs) of 0.90, 0.91, 0.89, and 0.86 for prediction by random forest (RF), naïve Bayes (NB), support vector machine (SVM), and k-nearest neighbors (KNN), respectively. SERPINE1, SERPINE2, and TAGLN2 maintained their methylation difference trends in an independent set of cervical secretion samples, resulting in accuracy rates of 71.46%, 80.06%, 80.72%, and 80.68%, and AUCs of 0.79, 0.84, 0.83, and 0.82 for prediction by RF, NB, SVM, and KNN, respectively. Our findings demonstrate that methylation changes in WOI genes detected noninvasively from cervical secretions are potential markers for predicting IVF-ET outcomes. Further studies of cervical secretion of DNA methylation markers may provide a novel approach for precision embryo transfer.

Cervical Secretion Methylation Is Associated with the Pregnancy Outcome of Frozen-Thawed Embryo Transfer.

The causes of implantation failure remain a black box in reproductive medicine. The exact mechanism behind the regulation of endometrial receptivity is still unknown. Epigenetic modifications influence gene expression patterns and may alter the receptivity of human endometrium. Cervical secretions contain endometrial genetic material, which can be used as an indicator of the endometrial condition. This study evaluates the association between the cervical secretion gene methylation profile and pregnancy outcome in a frozen-thawed embryonic transfer (FET) cycle. Cervical secretions were collected from women who entered the FET cycle with a blastocyst transfer (36 pregnant and 36 non-pregnant women). The DNA methylation profiles of six candidate genes selected from the literature review were measured by quantitative methylation-specific PCR (qMSP). Bioinformatic analysis of six selected candidate genes showed significant differences in DNA methylation between receptive and pre-receptive endometrium. All candidate genes showed different degrees of correlation with the pregnancy outcomes in the logistic regression model. A machine learning approach showed that the combination of candidate genes' DNA methylation profiles could differentiate pregnant from non-pregnant samples with an accuracy as high as 86.67% and an AUC of 0.81. This study demonstrated the association between cervical secretion methylation profiles and pregnancy outcomes in an FET cycle and provides a basis for potential clinical application as a non-invasive method for implantation prediction.

BHLHE22 Expression Is Associated with a Proinflammatory Immune Microenvironment and Confers a Favorable Prognosis in Endometrial Cancer.

Endometrial cancer (EC) rates are rising annually. Additional prediction markers need to be evaluated because only 10-20% of EC cases show an objective response to immune-checkpoint inhibitors (ICIs). Our previous methylomic study found that BHLHE22 is hypermethylated in EC tissues and can be detected using a Pap-smear sample. BHLHE22, a basic helix loop helix transcription factor family member, is known as a transcriptional repressor and is involved in cell differentiation. However, the role of BHLHE22 in EC remains poorly understood. Herein, we analyzed BHLHE22 expression in 54 paired cancer and normal endometrial tissue samples, and confirmed with databases (TCGA, GTEx, and human protein atlas). We found that BHLHE22 protein expression was significantly downregulated in EC compared with normal endometrium. High BHLHE22 expression was associated with microsatellite-instable subtype, endometrioid type, grade, and age. It showed a significant favorable survival. BHLHE22 overexpression inhibited the proliferation and migration of EC cells. Functional enrichment analysis showed that BHLHE22 was significantly associated with immune-related pathways. Furthermore, BHLHE22 was positively correlated with proinflammatory leukocyte infiltration and expression of chemokine genes in EC. In conclusion, BHLHE22 regulates immune-related pathways and modulates the immune microenvironment of EC.

Epigenomic Profiling of Epithelial Ovarian Cancer Stem-Cell Differentiation Reveals GPD1 Associated Immune Suppressive Microenvironment and Poor Prognosis.

Intraperitoneal metastasis is a challenging clinical scenario in epithelial ovarian cancer (EOC). As they are distinct from hematogenous metastasizing tumors, epithelial ovarian cancer cells primarily disseminate within the peritoneal cavity to form superficially invasive carcinomas. Unfavorable pharmacokinetics for peritoneal tumors and gut toxicity collectively lead to a narrow therapeutic window and therefore limit the opportunities for a favorable clinical outcome. New insights into tumor metastasis in the peritoneal microenvironment are keenly awaited to develop new therapeutic strategies. Epithelial ovarian cancer stem cell (OCSC) seeding is considered to be a critical component of the peritoneal spread. Using a unique and stepwise process of the OCSC differentiation model may provide insight into the intraperitoneal metastasis. The transcriptome and epigenome of OCSC differentiation were characterized by expression array and MethylCap-Seq. The TCGA, AOCS, and KM-Plotter databases were used to evaluate the association between survival outcomes and the methylation/expression levels of candidate genes in the EOC datasets. The STRING database was used to investigate the protein-protein interaction (PPI) for candidates and their associated genes. The infiltration level of immune cells in EOC patients and the association between clinical outcome and OCSCs differentiation genes were estimated using the TIDE and TIME2.0 algorithms. We established an EOC differentiation model using OCSCs. After an integrated transcriptomics and methylomics analysis of OCSCs differentiation, we revealed that the genes associated with earlier OCSC differentiation were better able to reflect the patient's outcome. The OCSC differentiation genes were involved in regulating metabolism shift and the suppressive immune microenvironment. High GPD1 expression with high pro-tumorigenic immune cells (M2 macrophage, and cancer associated fibroblast) had worst survival. Moreover, we developed a methylation signature, constituted by GNPDA1, GPD1, GRASP, HOXC11, and MSLN, that may be useful for prognostic prediction in EOC. Our results revealed a novel role of epigenetic plasticity OCSC differentiation and suggested metabolic and immune intervention as a new therapeutic strategy.

NKX6-1 mediates cancer stem-like properties and regulates sonic hedgehog signaling in leiomyosarcoma.

BACKGROUND: Leiomyosarcoma (LMS), the most common soft tissue sarcoma, exhibits heterogeneous and complex genetic karyotypes with severe chromosomal instability and rearrangement and poor prognosis. METHODS: Clinical variables associated with NKX6-1 were obtained from The Cancer Genome Atlas (TCGA). NKX6-1 mRNA expression was examined in 49 human uterine tissues. The in vitro effects of NXK6-1 in LMS cells were determined by reverse transcriptase PCR, western blotting, colony formation, spheroid formation, and cell viability assays. In vivo tumor growth was evaluated in nude mice. RESULTS: Using The Cancer Genome Atlas (TCGA) and human uterine tissue datasets, we observed that NKX6-1 expression was associated with poor prognosis and malignant potential in LMS. NKX6-1 enhanced in vitro tumor cell aggressiveness via upregulation of cell proliferation and anchorage-independent growth and promoted in vivo tumor growth. Moreover, overexpression and knockdown of NKX6-1 were associated with upregulation and downregulation, respectively, of stem cell transcription factors, including KLF8, MYC, and CD49F, and affected sphere formation, chemoresistance, NOTCH signaling and Sonic hedgehog (SHH) pathways in human sarcoma cells. Importantly, treatment with an SHH inhibitor (RU-SKI 43) but not a NOTCH inhibitor (DAPT) reduced cell survival in NKX6-1-expressing cancer cells, indicating that an SHH inhibitor could be useful in treating LMS. Finally, using the TCGA dataset, we demonstrated that LMS patients with high expression of NKX6-1 and HHAT, an SHH pathway acyltransferase, had poorer survival outcomes compared to those without. CONCLUSIONS: Our findings indicate that NKX6-1 and HHAT play critical roles in the pathogenesis of LMS and could be promising diagnostic and therapeutic targets for LMS patients.

TET1 reprograms the epithelial ovarian cancer epigenome and reveals casein kinase 2α as a therapeutic target.

Ten-eleven translocation methylcytosine dioxygenase-1, TET1, takes part in active DNA demethylation. However, our understanding of DNA demethylation in cancer biology and its clinical significance remain limited. This study showed that TET1 expression correlated with poor survival in advanced-stage epithelial ovarian carcinoma (EOC), and with cell migration, anchorage-independent growth, cancer stemness, and tumorigenicity. In particular, TET1 was highly expressed in serous tubal intraepithelial carcinoma (STIC), a currently accepted type II EOC precursor, and inversely correlated with TP53 mutations. Moreover, TET1 could demethylate the epigenome and activate multiple oncogenic pathways, including an immunomodulation network having casein kinase II subunit alpha (CK2α) as a hub. Patients with TET1high CK2αhigh EOCs had the worst outcomes, and TET1-expressing EOCs were more sensitive to a CK2 inhibitor, both in vitro and in vivo. Our findings uncover the oncogenic and poor prognostic roles of TET1 in EOC and suggest an unexplored role of epigenetic reprogramming in early ovarian carcinogenesis. Moreover, the immunomodulator CK2α represents a promising new therapeutic target, warranting clinical trials of the tolerable CK2 inhibitor, CX4945, for precision medicine against EOC. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Distinct methylation profile of mucinous ovarian carcinoma reveals susceptibility to proteasome inhibitors.

Mucinous type of epithelial ovarian cancer (MuOC) is a unique subtype with a poor survival outcome in recurrent and advanced stages. The role of type-specific epigenomics and its clinical significance remains uncertain. We analyzed the methylomic profiles of 6 benign mucinous adenomas, 24 MuOCs, 103 serous type of epithelial ovarian cancers (SeOCs) and 337 nonepithelial ovarian cancers. MuOC and SeOC exhibited distinct DNA methylation profiles comprising 101 genes, 81 of which exhibited low methylation in MuOC and were associated with the response to glucocorticoid, ATP hydrolysis-coupled proton transport, proteolysis involved in the cellular protein catabolic process and ion transmembrane transport. Hierarchical clustering analysis showed that the profiles of MuOC were similar to colorectal adenocarcinoma and stomach adenocarcinoma. Genetic interaction network analysis of differentially methylated genes in MuOC showed a dominant network module is the proteasome subunit beta (PSMB) family. Combined functional module and methylation analysis identified PSMB8 as a candidate marker for MuOC. Immunohistochemical staining of PSMB8 used to validate in 94 samples of ovarian tumors (mucinous adenoma, MuOC or SeOC) and 62 samples of gastrointestinal cancer. PSMB8 was commonly expressed in MuOC and gastrointestinal cancer samples, predominantly as strong cytoplasmic and occasionally weak nuclei staining, but was not expressed in SeOC samples. Carfilzomib, a second-generation proteasome inhibitor, suppressed MuOC cell growth in vitro. This study unveiled a mucinous-type-specific methylation profile and suggests the potential use of a proteasome inhibitor to treat MuOC.

GATA3 as a master regulator and therapeutic target in ovarian high-grade serous carcinoma stem cells.

Ovarian high-grade serous carcinoma (HGSC) is the most lethal gynecological malignancy. Prevailing evidences suggest that drug resistance and recurrence of ovarian HGSC are caused by the presence of cancer stem cells. Therefore, targeting cancer stems is appealing, however, all attempts to date, have failed. To circumvent this limit, we analyzed differential transcriptomes at early differentiation of ovarian HGSC stem cells and identified the developmental transcription factor GATA3 as highly expressed in stem, compared to progenitor cells. GATA3 expression associates with poor prognosis of ovarian HGSC patients, and was found to recruit the histone H3, lysine 27 (H3K27) demethylase, UTX, activate stemness markers, and promote stem-like phenotypes in ovarian HGSC cell lines. Targeting UTX by its inhibitor, GSKJ4, impeded GATA3-driven stemness phenotypes, and enhanced apoptosis of GATA3-expressing cancer cells. Combinations of gemcitabine or paclitaxel with GSKJ4, resulted in a synergistic cytotoxic effect. Our findings provide evidence for a new role for GATA3 in ovarian HGSC stemness, and demonstrate that GATA3 may serve as a biomarker for precision epigenetic therapy in the future.

Epigenetic loss of heparan sulfate 3-O-sulfation sensitizes ovarian carcinoma to oncogenic signals and predicts prognosis.

Precision medicine requires markers for therapeutic guidance. The purpose of this study was to determine whether epithelial ovarian cancer (EOC) epigenetics can lead to the identification of biomarkers for precision medicine. Through integrative methylomics, we discovered and validated the epigenetic signature of NEFH and HS3ST2 as an independent prognostic factor for type II EOC in our dataset (n = 84), and two independent methylomics datasets (total n = 467). Integrated transcriptomics dataset (n = 1147) and tissue microarrays (n = 54) of HS3ST2 also related to high-methylation statuses and the EOC prognosis. Mechanistic explorations of HS3ST2 have assessed responses to oncogenic stimulations such as IL-6, EGF, and FGF2 in cancer cells. The combination of HS3ST2 and various oncogenic ligands also confers the worse outcome. 3-O-sulfation of heparan sulfate by HS3ST2 makes ovarian cancer cells intrinsically sensitive to oncogenic signals, which sheds new light on the application of HS3ST2 as a companion diagnostic for targeted therapy using kinase inhibitors or therapeutic antibodies.

Active DNA Demethylase, TET1, Increases Oxidative Phosphorylation and Sensitizes Ovarian Cancer Stem Cells to Mitochondrial Complex I Inhibitor.

Ten-eleven translocation 1 (TET1) is a methylcytosine dioxygenase involved in active DNA demethylation. In our previous study, we demonstrated that TET1 reprogrammed the ovarian cancer epigenome, increased stem properties, and activated various regulatory networks, including metabolic networks. However, the role of TET1 in cancer metabolism remains poorly understood. Herein, we uncovered a demethylated metabolic gene network, especially oxidative phosphorylation (OXPHOS). Contrary to the concept of the Warburg effect in cancer cells, TET1 increased energy production mainly using OXPHOS rather than using glycolysis. Notably, TET1 increased the mitochondrial mass and DNA copy number. TET1 also activated mitochondrial biogenesis genes and adenosine triphosphate production. However, the reactive oxygen species levels were surprisingly decreased. In addition, TET1 increased the basal and maximal respiratory capacities. In an analysis of tricarboxylic acid cycle metabolites, TET1 increased the levels of α-ketoglutarate, which is a coenzyme of TET1 dioxygenase and may provide a positive feedback loop to modify the epigenomic landscape. TET1 also increased the mitochondrial complex I activity. Moreover, the mitochondrial complex I inhibitor, which had synergistic effects with the casein kinase 2 inhibitor, affected ovarian cancer growth. Altogether, TET1-reprogrammed ovarian cancer stem cells shifted the energy source to OXPHOS, which suggested that metabolic intervention might be a novel strategy for ovarian cancer treatment.

In vitro drug testing using patient-derived ovarian cancer organoids.

BACKGROUND: Ovarian cancer is the most lethal gynecological cancer. As the primary treatment, chemotherapy has a response rate of only 60-70% in advanced stages, and even lower as a second-line treatment. Despite guideline recommendations, which drugs will be most effective remains unclear. Thus, a strategy to prioritize chemotherapy options is urgently needed. Cancer organoids have recently emerged as a method for in vitro drug testing. However, limited clinical correlations have been assessed with test results from cancer organoids, particularly in gynecological cancers. We therefore aimed to generate patient-derived organoids (PDOs) of ovarian cancer, to assess their drug sensitivities and correlations with patient clinical outcomes. METHODS: PDOs were generated from fresh tumors obtained during surgical resection, which was then cultured under matrix gel and appropriate growth factors. Morphological and molecular characterization of PDOs were assessed by phase contrast microscopy and paraffin-embedded histopathology. Expressions of PAX8, TP53, WT1, CK7, and CK20 were tested by immunohistochemical staining and compared with parental tumor tissues and the human protein atlas database. PDOs were subjected to in vitro drug testing to determine drug sensitivity using Titer-Glo® 3D Cell Viability Assay. PDO viability was measured, and area under the curve calculated, to compare responses to various compounds. Correlations were calculated between selected patients' clinical outcomes and in vitro drug testing results. RESULTS: We established 31 PDOs. Among them, 28 PDOs can be expanded, including 15, 11, and 2 from ovarian, endometrial, and cervical cancers, respectively. The PDOs preserved the histopathological profiles of their originating tumors. In vitro drug testing of 10 ovarian cancer PDOs revealed individual differential responses to recommended drugs, and interpersonal heterogeneity in drug sensitivity, even with the same histology type. Among four patients who were platinum sensitive, resistant, or refractory, PDO drug responses correlated well with their clinical courses. CONCLUSION: In vitro drug testing using ovarian cancer organoids is feasible and correlates well with patient clinical responses. These results may facilitate development of precision chemotherapy and personalized screening for repurposed or new drugs.

Genome-wide analysis of cervical secretions obtained during embryo transfer reveals the association between deoxyribonucleic acid methylation and pregnancy outcomes.

OBJECTIVE: To study whether the methylation status of cervical secretions can reflect the ability of the endometrium to allow embryo implantation. DESIGN: Case-control study. SETTING: In vitro fertilization centers. PATIENT(S): Women undergoing embryo transfer cycles, in which at least 1 good-quality embryo was transferred. INTERVENTION(S): Collection of cervical secretions during the procedure of embryo transfer. MAIN OUTCOME MEASURE(S): Methylation profiles of cervical secretions in relation to pregnancy outcomes. RESULT(S): Genome-wide methylation profiles differ between cervical secretions from pregnancy and nonpregnancy cycles. Clustering analysis on the basis of the top 2,000 differentially methylated probes of cervical secretions from 28 pregnancy and 29 nonpregnancy cycles correctly categorized 86.0% of the samples in terms of conceptional status, which was verified in selected genes by quantitative methylation-specific polymerase chain reaction and validated in another independent sample set. The combination of selected genes was estimated to predict pregnancy outcomes with a maximal area under the receiver operating characteristic curve of 0.83. CONCLUSION(S): The methylation profiles of cervical secretions were associated with pregnancy outcomes in embryo transfer cycles. Although not clinically useful at present, deoxyribonucleic acid methylation in cervical secretions may shed new light on the less invasive assessment of endometrial receptivity.

Endometrial Cancer Detection Using a Cervical DNA Methylation Assay (MPap) in Women with Abnormal Uterine Bleeding: A Multicenter Hospital-Based Validation Study.

BACKGROUND: We describe a DNA methylation assay, named MPap test, using cervical scraping as an alternative technique for endometrial cancer detection. METHODS: A multicenter hospital-based, two-stage validation study was conducted to validate the cancer detection performance of the MPap test. The MPap value was determined from the DNA methylation status of two genes (BHLHE22, CDO1) and combined with two other clinical variables (age, BMI). The cutoff threshold of the MPap value was established in stage 1 and validated in stage 2. A total of 592 women with abnormal uterine bleeding were enrolled from five medical centers throughout Taiwan. RESULTS: In stage 1, the sensitivity, specificity, and positive and negative predictive values of the MPap test for detecting endometrial cancer were 92.9%, 71.5%, 39.8%, and 98.0%, respectively. These values were validated in stage 2, being 92.5%, 73.8%, 40.2%, and 98.1%. Moreover, MPap outperformed transvaginal ultrasound in sensitivity and negative predictive values for detecting endometrial cancer. When we applied the algorithm for triage of endometrial cancer detection by MPap in the Taiwan National Health Insurance dataset, we found that it may reduce invasive procedures by 69~73%. CONCLUSIONS: MPap may provide a feasible alternative for endometrial cancer detection and can be considered as a triage test to reduce unnecessary invasive procedures.

Combined genetic mutations and DNA-methylated genes as biomarkers for endometrial cancer detection from cervical scrapings.

BACKGROUND: Endometrial cancer is a common gynecologic cancer. Noninvasive molecular biomarkers for triage of high-risk patients for invasive procedures are needed. Based on the success of cytological Pap smear screening, cervical scrapings are a good source of DNA for molecular testing. In addition to genetic lesions, DNA methylation is a promising biomarker. We assessed the usefulness of combining genetic and epigenetic biomarkers from cervical scrapings to detect endometrial carcinomas. METHODS: We performed a retrospective case-control study of 96 consecutive cervical scrapings from patients with abnormal uterine bleeding who underwent surgery for diagnostic evaluation. Thirty and 16 cases were diagnosed with type I and type II endometrial cancers, respectively. The remaining non-cancer cases included normal endometrium (n = 12), benign uterine lesions (n = 20), and endometrial hyperplasia (n = 18). Quantitative methylation-specific PCR and mass spectrometry were used for DNA methylation and genetic mutation analysis. Logistic regression was used to evaluate the clinical performance of these candidate biomarkers. RESULTS: We tested the effectiveness of the methylation status of four genes (BHLHE22, CDO1, TBX5, and HAND2) in endometrial cancer detection. The area under the receiver operating characteristic curves ranged from 0.703 to 0.878, and panels of hypermethylated BHLHE22/CDO1/HAND2 (87.0% sensitivity and 86.0% specificity) and BHLHE22/CDO1/TBX5 (89.1% sensitivity and 80.0% specificity) showed significant differences and could distinguish benign from malignant endometrial lesions. The sensitivity and specificity in endometrial cancer detection for BHLHE22/CDO1 were 84.8% and 88.0%, respectively. Both type I and II endometrial carcinomas could be detected using a BHLHE22/CDO1-based methylation profile, suggesting that they may have common epigenomes. Moreover, PTEN and TP53 mutations were found in 63.3% of type I and 93.6% of type II endometrial cancers. Unexpectedly, PTEN and TP53 mutations were commonly found in cervical scrapings of the normal endometrium (25% and 33.3%, respectively) and in cases with benign uterine lesions (10% and 50%, respectively). Finally, combinations of any one mutation of PTEN and TP53 mutations had a sensitivity of 91.3%, but a specificity of only 42.0%. CONCLUSIONS: Adding PTEN/TP53 mutation testing to BHLHE22/CDO1-based methylation testing did not improve the detection of endometrial cancer.

Sulforaphane inhibition of monocyte adhesion via the suppression of ICAM-1 and NF-kappaB is dependent upon glutathione depletion in endothelial cells.

Sulforaphane (SFN) is an isothiocyanate found in cruciferous vegetables. We here report that SFN is a potent inhibitor of LPS-induced monocyte adhesion, and also blocks the gene expression of the adhesion molecule, ICAM-1, at non-toxic concentrations. Downstream of ICAM-1, NF- kappaB activity was also found to be abolished in a dose-and time-dependent by SFN in LPS-treated endothelial cells (ECs). SFN exerts its suppressive effects on NF- kappaB activity in these cells by preventing the degradation of IkappaB-alpha. Interestingly, the inhibition of P65 translocation and IkappaB-alpha degradation was reversed slightly after 12 hours pretreatment. The intracellular GSH levels in SFN-treated ECs were observed to be reduced, the time course coincident with the suppression of P65 translocation and IkappaB-alpha degradation. NAC and GSH reverse the inhibitory effects of SFN upon p65 translocation and IkappaB-alpha degradation when preincubated with this agent. Furthermore, the use of BSO to decrease intracellular GSH levels further enhanced the effects of SFN. These data thus suggest that the anti-inflammatory mechanisms of SFN are dependent upon intracellular glutathione level.

The feasibility of detecting endometrial and ovarian cancer using DNA methylation biomarkers in cervical scrapings.

OBJECTIVE: We hypothesized that DNA methylation of development-related genes may occur in endometrial cancer (EC)/ovarian cancer (OC) and may be detected in cervical scrapings. METHODS: We 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. RESULTS: Of 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. CONCLUSION: The findings demonstrate the potential of EC/OC detection through testing for DNA methylation in cervical scrapings.

Methylomics of nitroxidative stress on precancerous cells reveals DNA methylation alteration at the transition from in situ to invasive cervical cancer.

Epigenetic dysregulation is important in cervical cancer development, but the underlying mechanism is largely unknown. Increasing evidence indicates that DNA methylation is sensitive to changes in microenvironmental factors, such as nitric oxide (NO) in the chronic inflammatory cervix. However, the epigenomic effects of NO in cancer have not been investigated. In this study, we explored the methylomic effects of nitroxidative stress in HPV-immortalized precancerous cells. Chronic NO exposure promoted the acquisition of malignant phenotypes such as cell growth, migration, invasion, and anchorage-independent growth. Epigenetic analysis confirmed hypermethylation of PTPRR. Whole-genome methylation analysis showed BOLA2B, FGF8, HSPA6, LYPD2, and SHE were hypermethylated in cells. The hypermethylation BOLA2B, FGF8, HSPA6, and SHE was confirmed in cervical scrapings from invasive cancer, but not in CIN3/CIS, CIN2 and CIN1 (p=0.019, 0.023, 0.023 and 0.027 respectively), suggesting the role in the transition from in situ to invasive process. Our results reveal that nitroxidative stress causes epigenetic changes in HPV-infected cells. Investigation of these methylation changes in persistent HPV infection may help identify new biomarkers of DNA methylation for cervical cancer screening, especially for precancerous lesions.

Integrated Epigenomics Analysis Reveals a DNA Methylation Panel for Endometrial Cancer Detection Using Cervical Scrapings.

PURPOSE: Endometrial cancer is a common gynecologic cancer whose incidence is increasing annually worldwide. Current methods to detect endometrial cancer are unreliable and biomarkers are unsatisfactory for screening. Cervical scrapings were reported as a potential source of material for molecular testing. DNA methylation is a promising cancer biomarker, but limited use for detecting endometrial cancer. EXPERIMENTAL DESIGN: We analyzed two methylomics databases of endometrioid-type endometrial cancer. Using nonnegative matrix factorization algorithm clustered the methylation pattern and reduced the candidate genes. We verified in pools DNA from endometrial cancer tissues and cervical scrapings, and validated in 146 cervical scrapings from patients with endometrioid-type endometrial cancer (n = 50), uterine myoma (n = 40), and healthy controls (n = 56) using quantitative methylation-specific PCR (QMSP). The logistic regression was used to evaluate the performance of methylation signal and gene combination. RESULTS: We filtered out 180 methylated genes, which constituted four consensus clusters. Serial testing of tissues and cervical scrapings detected 14 genes that are hypermethylated in endometrial cancer. Three genes, BHLHE22, CDO1, and CELF4, had the best performance. Individual genes were sensitivity of 83.7%-96.0% and specificity of 78.7%-96.0%. A panel comprising any two of the three hypermethylated genes reached a sensitivity of 91.8%, specificity of 95.5%, and odds ratio of 236.3 (95% confidence interval, 56.4-989.6). These markers were also applied to cervical scrapings of type II endometrial cancer patients, and detected in 13 of 14 patients. CONCLUSIONS: 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.