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.

Epigenomic Analysis Reveals the KCNK9 Potassium Channel as a Potential Therapeutic Target for Adenomyosis.

Adenomyosis is linked to dysmenorrhea and infertility. The pathogenesis of adenomyosis remains unclear, and little is known of the genetic and epigenetic changes in the eutopic endometrium in adenomyosis, which may predispose patients to the invasion and migration of endometrial tissues into the myometrium. Transcriptome studies have identified genes related to various cell behaviors but no targets for therapeutic intervention. The epigenetics of the eutopic endometrium in adenomyosis have rarely been investigated. Endometrial tissue was obtained from premenopausal women with (n = 32) or without adenomyosis (n = 17) who underwent hysterectomy aged 34-57 years at a tertiary hospital. The methylome and transcriptome were assessed by using a Methylation 450 K BeadChip array and Affymetrix expression microarray. Protein expression was examined by immunohistochemistry. Differential methylation analysis revealed 53 lowly methylated genes and 176 highly methylated genes with consistent gene expression in adenomyosis, including three genes encoding potassium ion channels. High expression of KCNK9 in the eutopic and ectopic endometria in patients with adenomyosis but not in normal controls was observed. Hormone-free, antibody-based KCNK9 targeting is a potential therapeutic strategy for adenomyosis-related dysmenorrhea, menorrhagia, and infertility.

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.

E2F6 functions as a competing endogenous RNA, and transcriptional repressor, to promote ovarian cancer stemness.

Ovarian cancer is the most lethal cancer of the female reproductive system. In that regard, several epidemiological studies suggest that long-term exposure to estrogen could increase ovarian cancer risk, although its precise role remains controversial. To decipher a mechanism for this, we previously generated a mathematical model of how estrogen-mediated upregulation of the transcription factor, E2F6, upregulates the ovarian cancer stem/initiating cell marker, c-Kit, by epigenetic silencing the tumor suppressor miR-193a, and a competing endogenous (ceRNA) mechanism. In this study, we tested that previous mathematical model, showing that estrogen treatment of immortalized ovarian surface epithelial cells upregulated both E2F6 and c-KIT, but downregulated miR-193a. Luciferase assays further confirmed that microRNA-193a targets both E2F6 and c-Kit. Interestingly, ChIP-PCR and bisulphite pyrosequencing showed that E2F6 also epigenetically suppresses miR-193a, through recruitment of EZH2, and by a complex ceRNA mechanism in ovarian cancer cell lines. Importantly, cell line and animal experiments both confirmed that E2F6 promotes ovarian cancer stemness, whereas E2F6 or EZH2 depletion derepressed miR-193a, which opposes cancer stemness, by alleviating DNA methylation and repressive chromatin. Finally, 118 ovarian cancer patients with miR-193a promoter hypermethylation had poorer survival than those without hypermethylation. These results suggest that an estrogen-mediated E2F6 ceRNA network epigenetically and competitively inhibits microRNA-193a activity, promoting ovarian cancer stemness and tumorigenesis.

[Research rules of famous veteran traditional Chinese medicine practitioners in treating infertility].

To explore the medication rules of famous veteran traditional Chinese medicine practitioners in treating infertility based on medical cases of infertility collected from book series of Hundred Traditional Chinese Medicine Clinicians of Hundred Years in China and Prescription Proven by Traditional Chinese Medicine Masters. Researchers extracted the information of prescriptions from these cases according to the inclusion and exclusion criteria. Then, Excel 2010, SPSS Clementine(ver.12.0) and SPSS(ver. 22.0) were adopted respectively for frequency analysis, association rules analysis, cluster analysis and factor analysis. Cluster analysis was carried out by Ochiai algorithm of binary variable data, which was a systematic clustering method. And principal component analysis was used for factor analysis. Besides, KMO test and Bartlett spherical test were used for factor adaptation test. Finally, 151 medical cases and 396 prescriptions were included in total. A total of 60 kinds of frequently used herbs were identified according to the results of frequency analysis for medication, they were mainly used for activating blood and resolving stasis, tonifying and clearing heat respectively. The association rules analysis found out 25 drug pair association rules and 14 3-drug combination association rules. A total of 15 medicine groups were extracted by cluster analysis. KMO test and Bartlett spherical test indicated that the method was suitable for factor analysis, and 21 common factors were respectively extracted by factor analysis. Association rules indicated the characteristics of the therapeutic methods, like tonifying Qi and replenishing blood. The famous veteran traditional Chinese medicine practitioners utilized modified Siwu Decoction for tonifying blood and preferred Atractylodis Macrocephalae Rhizoma(Baizhu) for tonifying Qi. The results of both cluster analysis and factor analysis demonstrated the characteristics of the therapies for tonifying kidney, activating blood, tonifying spleen and dispelling dampness. In addition, factor analysis could reflect the therapies for nourishing Yin, tonifying kidney, warming the meridian, dissipating cold, nourishing blood and dispelling blood stasis. These results of analysis comprehensively showed out the medication characteristics of famous veteran traditional Chinese medicine practitioners of strictly following the pathogenesis, making good use of classical formulas and providing proper compatibility. In conclusion, data mining techniques(including frequency analysis, association rules analysis, cluster analysis and factor analysis) were used to comprehensively analyze the medication rules of famous veteran traditional Chinese medicine practitioners in treating infertility, which is helpful for guiding the clinical practice of treating infertility with traditional Chinese medicine.

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.

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.

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.

Hypomethylation signature of tumor-initiating cells predicts poor prognosis of ovarian cancer patients.

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.

Paired boxed gene 1 expression: A single potential biomarker for differentiating endometrial lesions associated with favorable outcomes in patients with endometrial carcinoma.

AIM: Paired boxed gene 1 (PAX1) has previously been reported to be a methylation-silenced gene in cases of cervical and ovarian cancers. We evaluated the expression of PAX1 in normal endometrium, endometrial hyperplasia and endometrial carcinoma (EC), and investigated the prognostic value of PAX1 expression in patients with EC. METHODS: We conducted a hospital-based retrospective review of PAX1 distribution immunohistochemically in 201 samples of endometrium from biopsy or hysterectomy. PAX1 immunoreactivity was classified into low and high score groups based upon the extent and intensity of staining. RESULTS: There was intense intranuclear staining for PAX1 in premalignant endometrial lesions. A high PAX1 score was observed in a high percentage of samples of normal endometrium (93.3%), in endometrial hyperplasia without atypia (97.2%) and in endometrial atypical hyperplasia (87.5%), but this level was found in only one-third of the EC samples (30.1%). The PAX1 protein score was significantly higher in samples of premalignant endometrial lesions compared with those of EC (P < 0.001). Importantly, a higher PAX1 score in EC cases was correlated with good overall survival, with a hazard ratio of 0.22 for death (95% confidence interval, 0.05-0.96). CONCLUSIONS: PAX1 protein expression is a potential histopathology biomarker for the differential diagnosis of malignant and premalignant endometrial lesions. PAX1 is also a potential prognostic marker in cases of EC.

Concordance analysis of methylation biomarkers detection in self-collected and physician-collected samples in cervical neoplasm.

BACKGROUND: Non-attendance at gynecological clinics is a major limitation of cervical cancer screening and self-collection of samples may improve this situation. Although HPV testing of self-collected vaginal samples is acceptable, the specificity is inadequate. The current focus is increasing self-collection of vaginal samples to minimize clinic visits. In this study, we analyzed the concordance and clinical performance of DNA methylation biomarker (PAX1, SOX1, and ZNF582) detection in self-collected vaginal samples and physician-collected cervical samples for the identification of cervical neoplasm. METHODS: We enrolled 136 cases with paired methylation data identified from abnormal Pap smears (n = 126) and normal controls (n = 10) regardless of HPV status at gynecological clinics. The study group comprised 37 cervical intraepithelial neoplasm I (CIN1), 23 cervical intraepithelial neoplasm II (CIN2), 16 cervical intraepithelial neoplasm III (CIN3), 30 carcinoma in situ (CIS), 13 squamous cell carcinomas (SCCs) and seven adenocarcinomas (ACs)/adenosquamous carcinomas (ASCs). PAX1, SOX1 and ZNF582 methylation in study samples was assessed by real-time quantitative methylation-specific polymerase chain reaction analysis. We generated methylation index cutoff values for the detection of CIN3+ in physician-collected cervical samples for analysis of the self-collected group. Concordance between the physician-collected and self-collected groups was evaluated by Cohen's Kappa. Sensitivity, specificity and area under curve (AUC) were calculated for detection of CIN3+ lesions. Finally, we produced an optimal cutoff value with the best sensitivity from the self-collected groups. RESULTS: We generated a methylation index cutoff value from physician-collected samples for detection of CIN3+. There were no significant differences in sensitivity, specificity of PAX1, SOX1 and ZNF582 between the self-collected and physician-collected groups. The methylation status of all three genes in the normal control samples, and the CIN 1, CIN2, CIN3, CIS, ACs/ASCs and SCC samples showed reasonable to good concordance between the two groups (κ = 0.443, 0.427, and 0.609 for PAX1, SOX1, and ZNF582, respectively). In determining the optimal cutoff values from the self-collected group, ZNF582 showed the highest sensitivity (0.77; 95%CI, 0.65-0.87) using a cutoff value of 0.0204. CONCLUSIONS: Methylation biomarker analysis of the three genes for detection of CIN3+ lesions shows reasonable to good concordance between the self-collected and physician-collected samples. Therefore, self-collection of samples could be adopted to decrease non-attendance and improve cervical screening.

Downregulation of miR-29 contributes to cisplatin resistance of ovarian cancer cells.

Drug resistance is an obstacle to the treatment of ovarian cancer. Using a unique cell model, we have proven previously that a subpopulation of ovarian cancer cells is more resistant to cisplatin than are the original cells. MicroRNAs (miRNAs), small noncoding RNAs, are involved in many biological events in cancer cells. In our study, we explored whether miRNAs are involved in cisplatin resistance of ovarian cancer cells. Cisplatin-resistant cells expressed a lower level of miR-29a/b/c. Manipulation of microRNA-29 (miR-29) expression modulated cisplatin sensitivity of CP70, HeyC2, SKOV3 and A2780 ovarian cancer cells. Knockdown of miR-29a/b/c increased the ability of cells to escape cisplatin-induced cell death partly through upregulation of collagen type I alpha 1 (COL1A1) and increased the activation of extracellular signal-regulated kinase 1/2 and inactivation of glycogen synthase kinase 3 beta. When combined with cisplatin treatment, knockdown of miR-29 decreased the amount of the active form of caspase-9 and caspase-3. Ectopic expression of miR-29 alone or in combination with cisplatin treatment efficaciously reduced the tumorigenicity of CP70 cells in vivo. Our data show that downregulation of miR-29 increases cisplatin resistance in ovarian cancer cells. Taken together, these data suggest that overexpression of miR-29 is a potential sensitizer to cisplatin treatment that may have therapeutic implications.

Methylomics analysis identifies epigenetically silenced genes and implies an activation of ß-catenin signaling in 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 = 19) and a pool of DNA from cervical cancer tissues (n = 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 CIN3+ lesions. The sensitivity, specificity, and accuracy of POU4F3 for detecting CIN3+ 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 ß-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.

DNA methylation as a biomarker for the detection of hidden carcinoma in endometrial atypical hyperplasia.

OBJECTIVE: Women with atypical hyperplasia (AH) are often found to have endometrial carcinoma (EC) at hysterectomy. The purpose of this study was to evaluate whether the hypermethylation of specific genes found by methylomic approaches to the study of gynecologic cancers is a biomarker for EC in women with AH. METHODS: We evaluated the methylation of AJAP1, HS3ST2, SOX1, and PTGDR from 61 AH patients undergoing hysterectomy. Endometrial biopsy samples were analyzed by bisulfite conversion and quantitative methylation-specific polymerase chain reaction. A methylation index was used to predict the presence of cancer. To confirm the silencing effects of DNA methylation, immunohistochemical analysis of AJAP1, HS3ST2, and SOX1 was performed using tissue microarray. RESULTS: Fourteen (23%) patients had EC at hysterectomy. AJAP1, HS3ST2, and SOX1 were highly methylated in the EC patients' biopsy samples (p≤0.023). AJAP1, HS3ST2, and SOX1 protein expression was significantly higher in patients with AH only (p≤0.038). The predictive value of AJAP1, HS3ST2, and SOX1 methylation for EC was 0.81, 0.72, and 0.70, respectively. Combined testing of both AJAP1 and HS3ST2 methylation had a positive predictive value of 56%, methylation of any one of AJAP1, SOX1, or HS3ST2 had a 100% negative predictive value. CONCLUSIONS: Hypermethylation of AJAP1, HS3ST2, and SOX1 is predictive of EC in AH patients. Testing for methylation of these genes in endometrial biopsy samples may be a hysterectomy-sparing diagnostic tool. Validation of these new genes as biomarkers for AH screening in a larger population-based study is warranted.

High methylation rate of LMX1A, NKX6-1, PAX1, PTPRR, SOX1, and ZNF582 genes in cervical adenocarcinoma.

OBJECTIVE: This study aimed to investigate the status of DNA methylation of 6 genes, LMX1A, NKX6-1, PAX1, PTPRR, SOX1, and ZNF582, previously found from squamous cell carcinomas in adenocarcinomas (ACs) of the uterine cervix. METHODS: We assessed the methylation status of these genes in 40 ACs, cervical scrapings from 23 ACs, and 67 normal control cervices by real-time quantitative methylation-specific polymerase chain reaction. The results were validated by bisulfite pyrosequencing. RESULTS: The methylation levels of all the 6 genes in the ACs were significantly higher than those in normal cervical tissues, especially for PAX1, PTPRR, SOX1, and ZNF582. The odds ratios and 95% confidence intervals (CIs) of high methylation levels in PAX1, PTPRR, SOX1, and ZNF582 for the risk of developing an AC were 15.7 (95% CI, 7.0-40.6), 16.9 (95% CI, 7.6-43.0), 32.1 (95% CI, 12.1-124.3), and 25.4 (95% CI, 10.4-78.3), respectively (all P < 0.001). The methylation indices of PAX1, PTPRR, SOX1, and ZNF582 recovered from scrapings of ACs were significantly higher than in normal controls. The odds ratios of these indices for the risk of developing an AC in PAX1, PTPRR, SOX1, and ZNF582 were 6.2 (95% CI, 2.6-15.4), 12.1(95% CI, 3.8-46.4), 6.2 (95% CI, 2.6-15.8), and 20.6 (95% CI, 6.9-77.5), respectively (all P < 0.001). CONCLUSIONS: Cervical ACs carry aberrantly high methylation rates of PAX1, PTPRR, SOX1, and ZNF582--commonly methylated in squamous cell carcinomas--which might help for AC screening.

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.

Complete remission of heavily treated ovarian clear cell carcinoma with ARID1A mutations after pembrolizumab and bevacizumab combination therapy: a case report.

BACKGROUND: Patients with ovarian clear cell carcinoma (OCCC) have a poor prognosis because they show low sensitivity to platinum-based chemotherapy. New treatments for refractory OCCC are urgently needed. CASE PRESENTATION: We present a patient with refractory OCCC in whom conventional chemotherapy failed. Cachexia was induced by the disseminating recurrent tumors. Tumor tissue staining and genomic analysis revealed PD-L1 negativity, a low tumor burden, stable microsatellite instability, and two mutations in ARID1A. The patient was administered pembrolizumab combined with bevacizumab triweekly. Her serum CA-125 level decreased dramatically after the first cycle. A computerized tomography scan showed marked regression of the recurrent masses after 3 cycles, and the patient reached complete remission after 9 cycles. She showed good recovery from cachexia. We observed no marked side effects except for mild polyarthritis of the small joints. CONCLUSIONS: The therapeutic effect of checkpoint inhibitors combined with angiogenesis inhibitors is very promising in our patient with OCCC. Further clinical trials of tumors including ARID1A mutations are warranted.