Clinical Epigenetics

Discovery and technical validation of high-performance methylated DNA markers for the detection of cervical lesions at risk of malignant progression in low- and middle-income countries

Abstract Background Cervical cancer remains a leading cause of death, particularly in developing countries. WHO screening guidelines recommend human papilloma virus (HPV) detection as a means to identify women at risk of developing cervical cancer. While HPV testing identifies those at risk, it does not specifically distinguish individuals with neoplasia. We investigated whether a quantitative molecular test that measures methylated DNA markers could identify high-risk lesions in the cervix with accuracy. Results Marker discovery was performed in TCGA-CESC Infinium Methylation 450 K Array database and verified in three other public datasets. The panel was technically validated using Quantitative Multiplex-Methylation-Specific PCR in tissue sections (N = 252) and cervical smears (N = 244) from the USA, South Africa, and Vietnam. The gene panel consisted of FMN2, EDNRB, ZNF671, TBXT, and MOS. Cervical tissue samples from all three countries showed highly significant differential methylation in squamous cell carcinoma (SCC) with a sensitivity of 100% [95% CI 74.12–100.00], and specificity of 91% [95% CI 62.26–99.53] to 96% [95% CI 79.01–99.78], and receiver operating characteristic area under the curve (ROC AUC) = 1.000 [95% CI 1.00–1.00] compared to benign cervical tissue, and cervical intraepithelial neoplasia 2/3 with sensitivity of 55% [95% CI 37.77–70.84] to 89% [95% CI 67.20–98.03], specificity of 93% [95% CI 84.07–97.38] to 96% [95% CI 79.01–99.78], and a ROC AUC ranging from 0.793 [95% CI 0.68–0.89] to 0.99 [95% CI 0.97–1.00] compared to CIN1. In cervical smears, the marker panel detected SCC with a sensitivity of 87% [95% CI 77.45–92.69], specificity 95% [95% CI 88.64–98.18], and ROC AUC = 0.925 [95% CI 0.878–0.974] compared to normal, and high-grade squamous intraepithelial lesion (HSIL) at a sensitivity of 70% (95% CI 58.11–80.44), specificity of 94% (95% CI 88.30–97.40), and ROC AUC = 0.884 (95% CI 0.822–0.945) compared to low-grade intraepithelial lesion (LSIL)/normal in an analysis of pooled data from the three countries. Similar to HPV-positive, HPV-negative cervical carcinomas were frequently hypermethylated for these markers. Conclusions This 5-marker panel detected SCC and HSIL in cervical smears with a high level of sensitivity and specificity. Molecular tests with the ability to rapidly detect high-risk HSIL will lead to timely treatment for those in need and prevent unnecessary procedures in women with low-risk lesions throughout the world. Validation of these markers in prospectively collected cervical smear cells followed by the development of a hypermethylated marker-based cervical cancer detection test is warranted.

Evaluating PAX1/JAM3 methylation for triage in HPV 16/18-infected women

Referring all women who tested positive for human papillomavirus (HPV) 16/18 to colposcopy may lead to potential over-referral issues. Triage tests based on cytology results face challenges in achieving accurate diagnoses. Our study aims to assess the clinical effectiveness of PAX1/JAM3 methylation (CISCER) test as a triage method for HPV 16/18-positive women. From November 2021 to December 2022, a total of 334 women who tested positive for HPV 16/18 and were referred to colposcopy at The Second Affiliated Hospital of Zhejiang University School of Medicine were studied. The clinical utility of the CISCER test, cytology, and the combination of CISCER with cytology as potential triage tests was compared. We observed a significant increase in the methylation levels of PAX1 gene and JAM3 gene in women with cervical intraepithelial neoplasia (CIN) grade 2 or severe (CIN2+). The CISCER test demonstrated superior triage performance over cytology, even when used in combination with cytology, showing a high sensitivity of 89.0% (95% confidence interval [CI] 82.9-95.1%) and specificity of 95.3% (95% CI 92.6-98.0%). It achieved an area under the curve of 0.921 (95% CI 0.877-0.966) and an odds ratio of 164.02 (95% CI 68.64-391.95). The immediate CIN2+ risk based on positive CISCER results would be 89.0% (95% CI 80.8-94.1%), with an estimated average of 1.12 referrals needed to detect one CIN2+ case. Moreover, CISCER triaging successfully identified all cancer patients and did not miss any CIN3+ cases among women aged ≥ 30. The PAX1/JAM3 methylation detection exhibited excellent accuracy in identifying cervical precancerous lesions in HPV 16/18-positive women and could be considered as a triage tool to reduce excessive referrals for colposcopy and overtreatment.

MAL expression downregulation through suppressive H3K27me3 marks at the promoter in HPV16-related cervical cancers is prognostically relevant and manifested by the interplay of novel MAL antisense long noncoding RNA AC103563.8, E7 oncoprotein and EZH2

Abstract Background MAL (T-lymphocyte maturation-associated protein) is highly downregulated in most cancers, including cervical cancer (CaCx), attributable to promoter hypermethylation. Long noncoding RNA genes (lncGs) play pivotal roles in CaCx pathogenesis, by interacting with human papillomavirus (HPV)-encoded oncoproteins, and epigenetically regulating coding gene expression. Hence, we attempted to decipher the impact and underlying mechanisms of MAL downregulation in HPV16-related CaCx pathogenesis, by interrogating the interactive roles of MAL antisense lncRNA AC103563.8, E7 oncoprotein and PRC2 complex protein, EZH2. Results Employing strand-specific RNA-sequencing, we confirmed the downregulated expression of MAL in association with poor overall survival of CaCx patients bearing HPV16, along with its antisense long noncoding RNA (lncRNA) AC103563.8. The strength of positive correlation between MAL and AC103563.8 was significantly high among patients compared to normal individuals. While downregulated expression of MAL was significantly associated with poor overall survival of CaCx patients bearing HPV16, AC103563.8 did not reveal any such association. We confirmed the enrichment of chromatin suppressive mark, H3K27me3 at MAL promoter, using ChIP-qPCR in HPV16-positive SiHa cells. Subsequent E7 knockdown in such cells significantly increased MAL expression, concomitant with decreased EZH2 expression and H3K27me3 marks at MAL promoter. In silico analysis revealed that both E7 and EZH2 bear the potential of interacting with AC103563.8, at the same binding domain. RNA immunoprecipitation with anti-EZH2 and anti-E7 antibodies, respectively, and subsequent quantitative PCR analysis in E7-silenced and unperturbed SiHa cells confirmed the interaction of AC103563.8 with EZH2 and E7, respectively. Apparently, AC103563.8 seems to preclude EZH2 and bind with E7, failing to block EZH2 function in patients. Thereby, enhanced EZH2 expression in the presence of E7 could potentially inactivate the MAL promoter through H3K27me3 marks, corroborating our previous results of MAL expression downregulation in patients. Conclusion AC103563.8-E7-EZH2 axis, therefore, appears to crucially regulate the expression of MAL, through chromatin inactivation in HPV16-CaCx pathogenesis, warranting therapeutic strategy development.

Triage performance of PAX1m/JAM3m in opportunistic cervical cancer screening of non‒16/18 human papillomavirus-positive women: a multicenter prospective study in China

In this study, we aimed to validate the performance of the PAX1 and JAM3 methylation (PAX1 The triage performance of liquid-based cytology (LBC) and the PAX1 In total, 1851 participants had cervical histological outcomes and were included in the analysis. The sensitivity/specificity of the LBC test results with atypical squamous cells of undetermined significance or worse (LBC ≥ ASCUS) and the PAX1 In non-16/18 hrHPV(+) women, PAX1

PAX1 methylation as a robust predictor: developing and validating a nomogram for assessing endocervical curettage (ECC) necessity in human papillomavirus16/18-positive women undergoing colposcopy

Abstract Objective The major challenge in routine endocervical curettage (ECC) among Human Papillomavirus (HPV) 16/18-positive patients is that only a small fraction benefit. Nevertheless, current reported models often overestimate the validity and necessity of ECC, making it difficult to improve benefits for patients. This research hypothesized that assessing paired boxed gene 1 methylation levels (PAX1m) and clinical characteristics could enhance the predictive accuracy of detecting additional high-grade squamous intraepithelial lesions or worse (HSIL +) through ECC that were not identified by colposcopy-directed biopsy (CDB). Methods Data from 134 women with HPV16/18 positivity undergoing CDB and ECC between April 2018 and April 2022 were collected and analyzed. Quantitative methylation-specific polymerase chain reaction (qMSP) was utilized to measure PAX1m, expressed as ΔCp. Univariate and multivariate regression analyses were conducted to screen variables and select predictive factors. A nomogram was constructed using multivariate logistic regression to predict additional HSIL + detected by ECC. The discrimination, calibration, and clinical utility of the nomogram were evaluated using receiver operating characteristic curves (ROC) and the calibration plot. Results Age (odds ratio [OR], 5.654; 95% confidence interval [CI], 1.131–37.700), cytology (OR, 24.978; 95% CI, 3.085–540.236), and PAX1 methylation levels by grade (PAX1m grade) (OR, 7.801; 95% CI, 1.548–44.828) were independent predictive factors for additional detection of HSIL + by ECC. In HPV16/18-positive women, the likelihood of additional detection of HSIL + through ECC increased with the severity of cytological abnormalities, peaking at 43.8% for high-grade cytological lesions. Moreover, when cytological findings indicated low-grade lesions, PAX1 methylation levels were positively correlated with the additional detection of HSIL + by ECC (P value < 0.001). A nomogram prediction model was developed (area under curve (AUC) = 0.946; 95% CI, 0.901–0.991), demonstrating high sensitivity (90.9%) and specificity (90.5%) at the optimal cutoff point of 107. Calibration analysis confirmed the model’s strong agreement between predicted and observed probabilities. Conclusion The clinical nomogram presented promising predictive performance for the additional detection of HSIL + through ECC among women with HPV16/18 infection. PAX1 methylation level could serve as a valuable tool in guiding individualized clinical decisions regarding ECC for patients with HPV 16/18 infection, particularly in cases of low-grade cytological findings.

Molecular mechanisms by which C1orf112 promotes endometrial cancer progression and the development and validation of a clinical scoring model

To investigate the expression profile, clinical significance, and underlying molecular mechanisms of C1orf112 in endometrial cancer (EC). We performed bioinformatics analyses using data from The Cancer Genome Atlas to evaluate C1orf112 expression and its association with clinicopathological parameters in EC. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses, as well as protein-protein interaction network analysis, were conducted to explore the functional roles and regulatory pathways of C1orf112. A multi-omics integrative approach was used to elucidate the regulatory mechanisms underlying C1orf112 expression. Furthermore, we constructed and validated a prognostic scoring model incorporating C1orf112 expression. C1orf112 was found to be significantly highexpressed in EC tissues and its expression was closely associated with clinical stage, histological grade, and patient prognosis. Functional enrichment analyses indicated that C1orf112 and its co-expressed genes are primarily involved in cell cycle regulation, DNA replication, and the p53 signaling pathway. Notably, our bioinformatics predictions suggest that C1orf112 may be subject to bidirectional regulation by RNA-binding proteins, including LIN28B and SRRM4, potentially establishing a regulatory balance between oncogenic and tumor-suppressive pathways. Multi-omics analysis demonstrated that C1orf112 expression is co-regulated by genetic alterations and epigenetic modifications, with promoter DNA methylation levels showing a strong inverse correlation with transcriptional activity. However, further molecular and phenotypic validation is required to confirm these interactions. The prognostic scoring model incorporating C1orf112 expression exhibited robust predictive performance. Our findings highlight the potential clinical utility of C1orf112 as a diagnostic and prognostic biomarker in EC, and provide new insights into its regulatory molecular network. This study proposes a conceptual framework for understanding EC pathogenesis and guiding the development of targeted therapies. Nonetheless, further prospective clinical studies and mechanistic investigations are warranted to validate these findings.

The MLH1 polymorphism rs1800734 and risk of endometrial cancer with microsatellite instability

AbstractBoth colorectal (CRC, 15%) and endometrial cancers (EC, 30%) exhibit microsatellite instability (MSI) due to MLH1 hypermethylation and silencing. The MLH1 promoter polymorphism, rs1800734 is associated with MSI CRC risk, increased methylation and reduced MLH1 expression. In EC samples, we investigated rs1800734 risk using MSI and MSS cases and controls. We found no evidence that rs1800734 or other MLH1 SNPs were associated with the risk of MSI EC. We found the rs1800734 risk allele had no effect on MLH1 methylation or expression in ECs. We propose that MLH1 hypermethylation occurs by different mechanisms in CRC and EC.

Retrotransposon methylation profiles and survival in Black women with high-grade serous ovarian carcinoma

Retrotransposons (REs) constitute nearly half of the genome and include long terminal repeat (LTR) elements, Long INterspersed Elements (LINE), and Short INterspersed Elements (SINE). REs are typically silenced in somatic tissues via DNA methylation but can be reactivated through DNA hypomethylation, potentially impacting gene regulation. Here, we investigate genome-scale profiles of RE methylation in high-grade serous ovarian carcinoma (HGSOC) and associations with survival among Black women. Methylation levels of LTR, LINE-1, and Alu (type of SINE) in 200 HGSOC tumors were predicted using a random forest approach and clustered using multiple consensus algorithms. Associations between RE methylation clusters and survival were evaluated using Cox proportional hazard regression, adjusting for age, stage, and debulking status. We performed sensitivity analyses restricted to women with late-stage disease and with adjustment for BRCA1/BRCA2 mutations. Two RE methylation clusters were identified. Cluster 1 exhibited a more hypomethylated RE profile ("Active"), while Cluster 2 was more hypermethylated ("Repressed"). No statistically significant differences in patient or clinical characteristics were observed between clusters. Compared to the Active Cluster, the Repressed Cluster was associated with an increased risk of mortality (HR = 2.41; 95% CI 1.04-5.59) and had a lower proportion of T cells. This association was consistent in sensitivity analyses. A more hypermethylated RE profile was linked to worse survival among Black women with HGSOC, highlighting the potential of RE methylation as a prognostic biomarker. Further research is needed to understand the underlying biological mechanisms and their implications in ovarian cancer biology and treatment.

BRCA1 & BRCA2 methylation as a prognostic and predictive biomarker in cancer: Implementation in liquid biopsy in the era of precision medicine

BReast CAncer gene 1 (BRCA1) and BReast CAncer gene 2 (BRCA2) encode for tumor suppressor proteins which are critical regulators of the Homologous Recombination (HR) pathway, the most precise and important DNA damage response mechanism. Dysfunctional HR proteins cannot repair double-stranded DNA breaks in mammalian cells, a situation called HR deficiency. Since their identification, pathogenic variants and other alterations of BRCA1 and BRCA2 genes have been associated with an increased risk of developing mainly breast and ovarian cancer. Interestingly, HR deficiency is also detected in tumors not carrying BRCA1/2 mutations, a condition termed "BRCAness". One of the main mechanisms causing the BRCAness phenotype is the methylation of the BRCA1/2 promoters, and this epigenetic modification is associated with carcinogenesis and poor prognosis mainly among patients with breast and ovarian cancer. BRCA1 promoter methylation has been suggested as an emerging biomarker of great predictive significance, especially concerning Poly (ADP-ribose) Polymerase inhibitors (PARP inhibitor-PARPi) responsiveness, along with or beyond BRCA1/2 mutations. However, as its clinical exploitation is still insufficient, the impact of BRCA1/2 promoter methylation status needs to be further evaluated. The current review aims to gather the latest findings about the mechanisms that underline BRCA1/2 function as well as the molecular characteristics of tumors associated with BRCA1/2 defects, by focusing on DNA methylation. Furthermore, we critically analyze their translational meaning and the validity of BRCA methylation biomarkers in predicting treatment response. We believe that BRCA1/2 methylation alone or combined with other biomarkers in a clinical setting is expected to change the scenery in prognosis and predicting treatment response in multiple cancer types and is worthy of further attention. The quantitative BRCA1 promoter methylation assessment might predict treatment response in PARPi and analysis of BRCA1/2 methylation in liquid biopsy might define patient subgroups at different time points that may benefit from PARPi. Finally, we suggest a pipeline that could be implemented in liquid biopsy to aid precision pharmacotherapy in BRCA-associated tumors.

DNA methylation as a triage marker for colposcopy referral in HPV-based cervical cancer screening: a systematic review and meta-analysis

Abstract Background Screening plays a key role in secondary prevention of cervical cancer. High-risk human papillomavirus (hrHPV) testing, a highly sensitive test but with limited specificity, has become the gold standard frontline for screening programs. Thus, the importance of effective triage strategies, including DNA methylation markers, has been emphasized. Despite the potential reported in individual studies, methylation markers still require validation before being recommended for clinical practice. This systematic review and meta-analysis aimed to evaluate the performance of DNA methylation-based biomarkers for detecting high-grade intraepithelial lesions (HSIL) in hrHPV-positive women. Methods Hence, PubMed, Scopus, and Cochrane databases were searched for studies that assessed methylation in hrHPV-positive women in cervical scrapes. Histologically confirmed HSIL was used as endpoint and QUADAS-2 tool enabled assessment of study quality. A bivariate random-effect model was employed to pool the estimated sensitivity and specificity as well as positive (PPV) and negative (NPV) predictive values. Results Twenty-three studies were included in this meta-analysis, from which cohort and referral population-based studies corresponded to nearly 65%. Most of the women analyzed were Dutch, and CADM1, FAM19A4, MAL, and miR124-2 were the most studied genes. Pooled sensitivity and specificity were 0.68 (CI 95% 0.63–0.72) and 0.75 (CI 95% 0.71–0.80) for cervical intraepithelial neoplasia (CIN) 2+ detection, respectively. For CIN3+ detection, pooled sensitivity and specificity were 0.78 (CI 95% 0.74–0.82) and 0.74 (CI 95% 0.69–0.78), respectively. For pooled prevalence, PPV for CIN2+ and CIN3+ detection were 0.514 and 0.392, respectively. Furthermore, NPV for CIN2+ and CIN3+ detection were 0.857 and 0.938, respectively. Conclusions This meta-analysis confirmed the great potential of DNA methylation-based biomarkers as triage tool for hrHPV-positive women in cervical cancer screening. Standardization and improved validation are, however, required. Nevertheless, these markers might represent an excellent alternative to cytology and genotyping for colposcopy referral of hrHPV-positive women, allowing for more cost-effective screening programs.

Transcriptional epigenetic regulation of Fkbp1/Pax9 genes is associated with impaired sensitivity to platinum treatment in ovarian cancer

Abstract Background In an effort to contribute to overcoming the platinum resistance exhibited by most solid tumors, we performed an array of epigenetic approaches, integrating next-generation methodologies and public clinical data to identify new potential epi-biomarkers in ovarian cancer, which is considered the most devastating of gynecological malignancies. Methods We cross-analyzed data from methylome assessments and restoration of gene expression through microarray expression in a panel of four paired cisplatin-sensitive/cisplatin-resistant ovarian cancer cell lines, along with publicly available clinical data from selected individuals representing the state of chemoresistance. We validated the methylation state and expression levels of candidate genes in each cellular phenotype through Sanger sequencing and reverse transcription polymerase chain reaction, respectively. We tested the biological role of selected targets using an ectopic expression plasmid assay in the sensitive/resistant tumor cell lines, assessing the cell viability in the transfected groups. Epigenetic features were also assessed in 189 primary samples obtained from ovarian tumors and controls. Results We identified PAX9 and FKBP1B as potential candidate genes, which exhibited epigenetic patterns of expression regulation in the experimental approach. Re-establishment of FKBP1B expression in the resistant OVCAR3 phenotype in which this gene is hypermethylated and inhibited allowed it to achieve a degree of platinum sensitivity similar to the sensitive phenotype. The evaluation of these genes at a translational level revealed that PAX9 hypermethylation leads to a poorer prognosis in terms of overall survival. We also set a precedent for establishing a common epigenetic signature in which the validation of a single candidate, MEST, proved the accuracy of our computational pipelines. Conclusions Epigenetic regulation of PAX9 and FKBP1B genes shows that methylation in non-promoter areas has the potential to control gene expression and thus biological consequences, such as the loss of platinum sensitivity. At the translational level, PAX9 behaves as a predictor of chemotherapy response to platinum in patients with ovarian cancer. This study revealed the importance of the transcript-specific study of each gene under potential epigenetic regulation, which would favor the identification of new markers capable of predicting each patient’s progression and therapeutic response.

Risk stratification of HPV 16 DNA methylation combined with E6 oncoprotein in cervical cancer screening: a 10-year prospective cohort study

Abstract Background How to best triage human papillomavirus (HPV) positive women remains controversial in an era of HPV primary screening of cervical cancer. Here, we assessed the long-term risk stratification for triaging HPV 16 positive women by standalone HPV 16 methylation and combined with E6 oncoprotein. Methods  A total of 1742 women underwent screening with HPV DNA testing, cytology, and visual inspection with acetic acid (VIA) in 2005 and were followed for 10 years. Seventy-seven women with HPV 16 positivity determined by HPV genotyping test were examined via E6 oncoprotein detection and bisulfite pyrosequencing for quantitative methylation of L1 and LCR genes of HPV 16. Results The 10-year cumulative incidence rate (CIR) of cervical intraepithelial neoplasia grade 3 or severe (CIN3+) for HPV 16 positive women was 25.3% (95% CI 14.7–37.3%), which significantly increased in women with high methylation at six sites (CpG 5602, 6650, 7034, 7461, 31, and 37) and in women with positive E6 oncoprotein. A methylation panel based on the above six sites showed a competitive risk stratification compared to cytology (HR 11.5 vs. 8.1), with a higher 10-year CIR of CIN3+ in panel positives (57.2% vs 36.8%) and comparable low risk in panel negatives (5.7% vs 4.8%).The sensitivity and specificity for accumulative CIN3+ was 85.7% (95%CI 60.1–96.0%) and 78.4% (95%CI 62.8–88.6%) for a methylation panel and 57.1% (95%CI 32.6–78.6%) and 86.5% (95%CI 72.0–94.1%) for E6 oncoprotein. The AUC values of methylation standalone and the co-testing of methylation panel and E6 oncoprotein were around 0.80, comparable to 0.68 for cytology, 0.65 for viral load, and superior to 0.52 for VIA (p < 0.05). Conclusions Our findings indicated the promising use of HPV 16 methylation alone or combined with E6 oncoprotein for triaging HPV 16 positive women based on the long-term risk stratification ability.

Evaluation of a methylation classifier for predicting pre-cancer lesion among women with abnormal results between HPV16/18 and cytology

Abstract Background Although HPV testing and cytology detection are successful for cervical screening in China, additional procedures are urgently required to avoid misdiagnosis and overtreatment. In this multicenter study, we collected cervical samples during screening in clinics. A total of 588 women with HPV16/18+ and/or cytology result ≥HSIL+ (high-grade squamous intraepithelial lesion or worse) were referred to colposcopy for pathological diagnosis. Methylation of S5 was quantified by pyrosequencing. Results The S5 classifier separates women with ≥HSIL+ from <HSIL with a high area under the curve (AUC) of 0.86 (95% CI 0.840–0.910). The cutoff of 2.85 was conducted in our study. Remarkably, all cancer cases (n = 67) were detected by S5. The sensitivity of S5 for “≥HSIL+” was 89.1% (95% CI 86.2–92.4%), and the specificity was 76.6% (95% CI 72.2–78.9%). S5 could reduce unnecessary colposcopy referrals by 74% (95% CI 71.3–78.1%) with virtually no loss of sensitivity for HSIL+, and the follow-up data support the utility of the S5 classifier. Conclusion The S5 classifier with high sensitivity and specificity provided increasing diagnostic information for women with HPV16/18+ and/or cytology results and could reduce the numerous unnecessary colposcopy referrals and avoid overtreatment.

An epigenetic hypothesis for ovarian cancer prevention by oral contraceptive pill use

Abstract Background Ovarian cancer is the second most common gynecological cancer type after uterine cancers. In 2020, according to worldwide statistics, there were more than 313,000 new cases of ovarian cancer. Most concerning with ovarian cancer is the poor overall survival, with only 30% of patients surviving for longer than 5 years after diagnosis. The reason for this poor outcome includes late diagnosis due to non-specific symptoms and a lack of any highly effective biomarkers of the early stages of ovarian carcinogenesis. However, it is important to note that some modifiable lifestyle factors can be preventative [pregnancy, breastfeeding and combined oral contraceptives pill (COCP) use]. Results There is now increasing data reporting the role of epigenetic changes, which are detectable in ovarian cancer tumors, suggesting the possibility that epigenetics may also play a key role in the mechanism of long-term effective prevention of ovarian cancer. To our knowledge, there is a lack of high-quality data on the molecular mechanisms of ovarian cancer prevention, although several hypotheses have been proposed. Conclusions This review focusses on the evidence for a proposed novel hypothesis—that COCPs act as a chemoprevention through the impact on the epigenome of the cells of origin of ovarian cancer—fallopian tubes epithelium.

Early diagnosis of ovarian cancer based on methylation profiles in peripheral blood cell-free DNA: a systematic review

AbstractPatients diagnosed with epithelial ovarian cancer (OC) have a 5-year survival rate of 49%. For early-stage disease, the 5-year survival rate is above 90%. However, advanced-stage disease accounts for most cases as patients with early stages often are asymptomatic or present with unspecific symptoms, highlighting the need for diagnostic tools for early diagnosis. Liquid biopsy is a minimal invasive blood-based approach that utilizes circulating tumor DNA (ctDNA) shed from tumor cells for real-time detection of tumor genetics and epigenetics. Increased DNA methylation of promoter regions is an early event during tumorigenesis, and the methylation can be detected in ctDNA, accentuating the promise of methylated ctDNA as a biomarker for OC diagnosis. Many studies have investigated multiple methylation biomarkers in ctDNA from plasma or serum for discriminating OC patients from patients with benign diseases of the ovaries and/or healthy females. This systematic review summarizes and evaluates the performance of the currently investigated DNA methylation biomarkers in blood-derived ctDNA for early diagnosis of OC. PubMed’s MEDLINE and Elsevier’s Embase were systematically searched, and essential results such as methylation frequency of OC cases and controls, performance measures, as well as preanalytical factors were extracted. Overall, 29 studies met the inclusion criteria for this systematic review. The most common method used for methylation analysis was methylation-specific PCR, with half of the studies using plasma and the other half using serum. RASSF1A, BRCA1, and OPCML were the most investigated gene-specific methylation biomarkers, with OPCML having the best performance measures. Generally, methylation panels performed better than single gene-specific methylation biomarkers, with one methylation panel of 103,456 distinct regions and 1,116,720 CpGs having better performance in both training and validation cohorts. However, the evidence is still limited, and the promising methylation panels, as well as gene-specific methylation biomarkers highlighted in this review, need validation in large, prospective cohorts with early-stage asymptomatic OC patients to assess the true diagnostic value in a clinical setting.

MGMT epimutations and risk of incident cancer of the colon, glioblastoma multiforme, and diffuse large B cell lymphomas

Abstract Background Constitutional BRCA1 epimutations (promoter hypermethylation) are associated with an elevated risk of triple-negative breast cancer and high-grade serous ovarian cancer. While MGMT epimutations are frequent in colon cancer, glioblastoma, and B-cell lymphoma, it remains unknown whether constitutional MGMT epimutations are associated with risk of any of these malignancies. Methods We designed a nested case–control study, assessing potential associations between MGMT epimutations in blood from healthy individuals and subsequent risk of incident cancer. The study cohort was drawn from postmenopausal women, participating in the Women’s Health Initiative (WHI) study, who had not been diagnosed with either colon cancer, glioblastoma, or B-cell lymphoma prior to study entry. The protocol included n = 400 women developing incident left-sided and n = 400 women developing right-sided colon cancer, n = 400 women developing diffuse large B-cell lymphomas, all matched on a 1:2 basis with cancer-free controls, and n = 195 women developing incident glioblastoma multiforme, matched on a 1:4 basis. All cancers were confirmed in centralized medical record review. Blood samples, collected at entry, were analyzed for MGMT epimutations by massive parallel sequencing. Associations between MGMT methylation and incident cancers were analyzed by Cox proportional hazards regression. Results Analyzing epimutations affecting the key regulatory area of the MGMT promoter, the hazard ratio (HR) was 1.07 (95% CI 0.79–1.45) and 0.80 (0.59–1.08) for right- and left-sided colon cancer, respectively, 1.13 (0.78–1.64) for glioblastoma, and 1.11 (0.83–1.48) for diffuse large B-cell lymphomas. Sensitivity analyses limited to subregions of the MGMT promoter and to individuals with different genotypes of a functional SNP in the MGMT promoter (rs16906252), revealed no significant effect on HR for any of the cancer forms. Neither did we observe any effect of rs16906252 status on HR for any of the cancer forms among individuals methylated or non-methylated at the MGMT promoter. Conclusions Constitutional MGMT promoter methylation in normal tissue is not associated with an increased risk of developing colon cancer, glioblastoma, or B-cell lymphoma.

Targeting histone demethylases JMJD3 and UTX: selenium as a potential therapeutic agent for cervical cancer

Abstract Background The intriguing connection between selenium and cancer resembles a captivating puzzle that keeps researchers engaged and curious. While selenium has shown promise in reducing cancer risks through supplementation, its interaction with epigenetics in cervical cancer remains a fascinating yet largely unexplored realm. Unraveling the intricacies of selenium's role and its interaction with epigenetic factors could unlock valuable insights in the battle against this complex disease. Result Selenium has shown remarkable inhibitory effects on cervical cancer cells in various ways. In in vitro studies, it effectively inhibits the proliferation, migration, and invasion of cervical cancer cells, while promoting apoptosis. Selenium also demonstrates significant inhibitory effects on human cervical cancer-derived organoids. Furthermore, in an in vivo study, the administration of selenium dioxide solution effectively suppresses the growth of cervical cancer tumors in mice. One of the mechanisms behind selenium's inhibitory effects is its ability to inhibit histone demethylases, specifically JMJD3 and UTX. This inhibition is observed both in vitro and in vivo. Notably, when JMJD3 and UTX are inhibited with GSK-J4, similar biological effects are observed in both in vitro and in vivo models, effectively inhibiting organoid models derived from cervical cancer patients. Inhibiting JMJD3 and UTX also induces G2/M phase arrest, promotes cellular apoptosis, and reverses epithelial-mesenchymal transition (EMT). ChIP-qPCR analysis confirms that JMJD3 and UTX inhibition increases the recruitment of a specific histone modification, H3K27me3, to the transcription start sites (TSS) of target genes in cervical cancer cells (HeLa and SiHa cells). Furthermore, the expressions of JMJD3 and UTX are found to be significantly higher in cervical cancer tissues compared to adjacent normal cervical tissues, suggesting their potential as therapeutic targets. Conclusions Our study highlights the significant inhibitory effects of selenium on the growth, migration, and invasion of cervical cancer cells, promoting apoptosis and displaying promising potential as a therapeutic agent. We identified the histone demethylases JMJD3 and UTX as specific targets of selenium, and their inhibition replicates the observed effects on cancer cell behavior. These findings suggest that JMJD3 and UTX could be valuable targets for selenium-based treatments of cervical cancer.

Bromodomain inhibitor i-BET858 triggers a unique transcriptional response coupled to enhanced DNA damage, cell cycle arrest and apoptosis in high-grade ovarian carcinoma cells

Abstract Background Ovarian cancer has a specific unmet clinical need, with a persistently poor 5-year survival rate observed in women with advanced stage disease warranting continued efforts to develop new treatment options. The amplification of BRD4 in a significant subset of high-grade serous ovarian carcinomas (HGSC) has led to the development of BET inhibitors (BETi) as promising antitumour agents that have subsequently been evaluated in phase I/II clinical trials. Here, we describe the molecular effects and ex vivo preclinical activities of i-BET858, a bivalent pan-BET inhibitor with proven in vivo BRD inhibitory activity. Results i-BET858 demonstrates enhanced cytotoxic activity compared with earlier generation BETis both in cell lines and primary cells derived from clinical samples of HGSC. At molecular level, i-BET858 triggered a bipartite transcriptional response, comprised of a ‘core’ network of genes commonly associated with BET inhibition in solid tumours, together with a unique i-BET858 gene signature. Mechanistically, i-BET858 elicited enhanced DNA damage, cell cycle arrest and apoptotic cell death compared to its predecessor i-BET151. Conclusions Overall, our ex vivo and in vitro studies indicate that i-BET858 represents an optimal candidate to pursue further clinical validation for the treatment of HGSC.

Identification of a methylation panel as an alternative triage to detect CIN3+ in hrHPV-positive self-samples from the population-based cervical cancer screening programme

Abstract Background The Dutch population-based cervical cancer screening programme (PBS) consists of primary high-risk human papilloma virus (hrHPV) testing with cytology as triage test. In addition to cervical scraping by a general practitioner (GP), women are offered self-sampling to increase participation. Because cytological examination on self-sampled material is not feasible, collection of cervical samples from hrHPV-positive women by a GP is required. This study aims to design a methylation marker panel to detect CIN3 or worse (CIN3+) in hrHPV-positive self-samples from the Dutch PBS as an alternative triage test for cytology. Methods Fifteen individual host DNA methylation markers with high sensitivity and specificity for CIN3+ were selected from literature and analysed using quantitative methylation-specific PCR (QMSP) on DNA from hrHPV-positive self-samples from 208 women with CIN2 or less (< CIN2) and 96 women with CIN3+. Diagnostic performance was determined by area under the curve (AUC) of receiver operating characteristic (ROC) analysis. Self-samples were divided into a train and test set. Hierarchical clustering analysis to identify input methylation markers, followed by model-based recursive partitioning and robustness analysis to construct a predictive model, was applied to design the best marker panel. Results QMSP analysis of the 15 individual methylation markers showed discriminative DNA methylation levels between < CIN2 and CIN3+ for all markers (p < 0.05). The diagnostic performance analysis for CIN3+ showed an AUC of ≥ 0.7 (p < 0.001) for nine markers. Hierarchical clustering analysis resulted in seven clusters with methylation markers with similar methylation patterns (Spearman correlation> 0.5). Decision tree modeling revealed the best and most robust panel to contain ANKRD18CP, LHX8 and EPB41L3 with an AUC of 0.83 in the training set and 0.84 in the test set. Sensitivity to detect CIN3+ was 82% in the training set and 84% in the test set, with a specificity of 74% and 71%, respectively. Furthermore, all cancer cases (n = 5) were identified. Conclusion The combination of ANKRD18CP, LHX8 and EPB41L3 revealed good diagnostic performance in real-life self-sampled material. This panel shows clinical applicability to replace cytology in women using self-sampling in the Dutch PBS programme and avoids the extra GP visit after a hrHPV-positive self-sampling test.

PAX1 hypomethylation as a prognostic biomarker for radioresistance of cervical cancer

Abstract Background PAX1 gene methylation plays an important role in the development of cervical cancer. However, its prognostic value after radiotherapy for locally advanced cervical cancer is unknown, so this study aimed to investigate the value of PAX1 gene methylation for predicting the sensitivity of radiotherapy for cervical cancer. Methods We selected 125 patients with primary cervical cancer who underwent concurrent chemo-radiotherapy as the study population, quantitative methylation-specific polymerase chain reaction (QMSP) was used for detecting PAX1 methylation status of cervical exfoliated cells. Logistic regression model was used to analyze the risk factors associated with the short-term efficacy and to establish a prediction model of radiotherapy sensitivity based on PAX1 gene methylation. Cell viability after radiation of Hela and SiHa cells transfected with PAX1 or control vector was evaluated by CCK8. Furthermore, RNA-Seq analyses identified different expressed genes (DEGs) in PAX1 overexpressed SiHa cells. Gene Ontology (GO) and pathway enrichment analysis was carried out to determine the biological function of DEGs. Results PAX1 methylation level was associated with HPV16/18-positive rate. PAX1 hypomethylation was found to be a risk factor for tumor residual after chemo-radiotherapy. A nomogram containing the risk factors for PAX1 methylation status, lymph node metastasis, pathological type and tumor size was further constructed to predict the probability of tumor residual after chemo-radiotherapy (AUC = 0.823, 95% CI 0.736–0.910). High PAX1 protein level was more likely to cause radioresistance in both Hela and SiHa cells. Transcriptomic sequencing of PAX1 overexpressed and control cells identified 615 differentially expressed genes, and GO enrichment analysis suggested that PAX1 may be involved in the regulation of signaling receptor activity and response to viruses. Conclusion PAX1 hypomethylation status could be used as a promising biomarker to predict radioresistance in cervical cancer. This further provides a new idea for the individualized treatment strategy of simultaneous radiotherapy for cervical cancer.

Leveraging locus-specific epigenetic heterogeneity to improve the performance of blood-based DNA methylation biomarkers

Abstract Background Variation in intercellular methylation patterns can complicate the use of methylation biomarkers for clinical diagnostic applications such as blood-based cancer testing. Here, we describe development and validation of a methylation density binary classification method called EpiClass (available for download at https://github.com/Elnitskilab/EpiClass ) that can be used to predict and optimize the performance of methylation biomarkers, particularly in challenging, heterogeneous samples such as liquid biopsies. This approach is based upon leveraging statistical differences in single-molecule sample methylation density distributions to identify ideal thresholds for sample classification. Results We developed and tested the classifier using reduced representation bisulfite sequencing (RRBS) data derived from ovarian carcinoma tissue DNA and controls. We used these data to perform in silico simulations using methylation density profiles from individual epiallelic copies of ZNF154 , a genomic locus known to be recurrently methylated in numerous cancer types. From these profiles, we predicted the performance of the classifier in liquid biopsies for the detection of epithelial ovarian carcinomas (EOC). In silico analysis indicated that EpiClass could be leveraged to better identify cancer-positive liquid biopsy samples by implementing precise thresholds with respect to methylation density profiles derived from circulating cell-free DNA (cfDNA) analysis. These predictions were confirmed experimentally using DREAMing to perform digital methylation density analysis on a cohort of low volume (1-ml) plasma samples obtained from 26 EOC-positive and 41 cancer-free women. EpiClass performance was then validated in an independent cohort of 24 plasma specimens, derived from a longitudinal study of 8 EOC-positive women, and 12 plasma specimens derived from 12 healthy women, respectively, attaining a sensitivity/specificity of 91.7%/100.0%. Direct comparison of CA-125 measurements with EpiClass demonstrated that EpiClass was able to better identify EOC-positive women than standard CA-125 assessment. Finally, we used independent whole genome bisulfite sequencing (WGBS) datasets to demonstrate that EpiClass can also identify other cancer types as well or better than alternative methylation-based classifiers. Conclusions Our results indicate that assessment of intramolecular methylation density distributions calculated from cfDNA facilitates the use of methylation biomarkers for diagnostic applications. Furthermore, we demonstrated that EpiClass analysis of ZNF154 methylation was able to outperform CA-125 in the detection of etiologically diverse ovarian carcinomas, indicating broad utility of ZNF154 for use as a biomarker of ovarian cancer.

Constitutional epimutations in LTBP4, a component of the TGF-β signaling, and in BRCA1, as potential drivers of early-onset colorectal cancer

Abstract Background Constitutional primary monoallelic promoter methylation of hereditary cancer genes, although rare, may explain early-onset cancers without family history. Also, promoter methylation of a hereditary cancer gene secondary to a genetic alteration in a methylation regulatory region can cause a hereditary cancer syndrome. This study investigates constitutional promoter methylation as mechanism of inactivation of cancer predisposition genes in genetically unsolved familial and/or early-onset colorectal cancer (CRC) patients. Results Bisulfite-treated peripheral blood DNA from 46 early-onset/familial CRC patients was analyzed using the Illumina Infinium MethylationEPIC BeadChip. One early-onset CRC patient exhibited constitutional, likely monoallelic, methylation of CpG island 102 in LTBP4 , a gene involved in TGF-β signaling. Somatic methylation of this CpG island is common in CRC, and correlates with LTBP4 downregulation. LTBP4 double knockout mice develop colorectal adenomas and carcinomas, supporting the role of this gene in CRC predisposition. No additional cases with constitutional LTBP4 CpG island 102 methylation or enrichment of deleterious LTBP4 variants in CRC patients compared to controls were found. Another early-onset CRC patient exhibited mosaic BRCA1 promoter methylation, typically associated with increased breast and ovarian cancer risk. No somatic second hit in BRCA1 was detected in the patient’s tumor, and homologous recombination deficiency-associated features were inconclusive. Conclusions Our findings suggest that constitutional methylation of LTBP4 CpG island 102 may be associated with increased CRC risk. Identification of additional cases is needed to confirm the existence of a novel CRC predisposition syndrome driven by epigenetic inactivation of LTBP4 , potentially also linked to other clinical phenotypes associated with LTBP4 deficiency, such as pulmonary emphysema. Whether constitutional BRCA1 methylation contributes to CRC risk remains to be determined.

BRCA loss of function including BRCA1 DNA-methylation, but not BRCA-unrelated homologous recombination deficiency, is associated with platinum hypersensitivity in high-grade ovarian cancer

In high-grade ovarian cancer (HGOC), determination of homologous recombination deficiency (HRD) status is commonly used in routine practice to predict response to platinum-based therapy or poly (ADP-ribose) polymerase inhibitors (PARPi). Here we tested the hypothesis that BRCA loss of function (LOF) due to epigenetic or genetic aberrations is a better predictor for the clinical outcome than HRD. One hundred thirty-one HGOC tissues were tested for BRCA DNA-methylation, BRCA mutations, HRD and BRCA1 mRNA expression, followed by a comprehensive survival analysis. BRCA1-methylation was detected in 11% of the tumors, exclusively in BRCA1-wild-type (wt) HGOCs. BRCA1-methylated tumors (BRCA1-meth) had HRD-scores similar to those of BRCA-mutated (mut) tumors, and higher compared to unmethylated-BRCA-wt tumors (BRCA-wt-unmeth; P < 0.001). Platinum-refractory or -resistant HGOCs at first recurrence were all BRCA-unmeth cancers. Only one of the BRCA-mut cancers had a platinum-resistant recurrence. Thus, 99% of relapses in cancers with epigenetic or genetic BRCA-alterations were platinum-sensitive. Multivariate analysis confirmed BRCA-LOF as an independent predictor of progression-free survival (PFS) and overall survival (OS), whereas HRD-status had no predictive value for PFS and OS. Patients with BRCA-wt-unmeth cancers had the worst outcome compared to patients with cancers harboring epigenetic or genetic BRCA-alterations (PFS: P = 0.007; OS: P = 0.022). Most importantly, the BRCA-wt-unmeth subfraction of HRD-positive HGOCs exhibited the same poor survival as the entire HRD-negative cohort. In HGOC BRCA mutational status together with BRCA1-methylation exhibit the best predictive power for favorable clinical outcome and thus high sensitivity to platinum-based therapy, whereas BRCA-unrelated HRD positivity was not associated with improved platinum sensitivity.

BRCA1 promoter methylation predicts PARPi response in ovarian cancer: insights from the KOMET study

PARP inhibitors (PARPi) have become the new standard maintenance treatment for patients with advanced homologous recombination deficiency (HRD) ovarian cancer; they are also used upon platinum-sensitive relapse. HRD in ovarian cancer is primarily assessed through BRCA genes mutations and genomic scar scores, which are key biomarkers forecasting PARPi benefit. However, the role of BRCA1 promoter methylation in guiding clinical management is unclear. Evidence is needed to improve patient selection before initiating PARPi and to minimize PARPi-related toxicities. Our study aimed to determine the clinical relevance of BRCA1 promoter methylation for patients with ovarian carcinoma. The KOMET (Ovarian Cancer Methylation) study is a single-center retrospective study involving 88 ovarian cancer patients treated between January 2021 and July 2024. Methylation was assessed using Methylation specific high-resolution melting (MS-HRM). Outcomes were measured based on progression-free survival (PFS) from diagnosis and from the initiation of PARPi treatment, as well as overall survival (OS). A methylated BRCA1 promoter was detected in 17 out of 88 (19%) tumor tissues. Statistically, PFS from PARPi initiation was significantly different between the BRCA1 methylated promoter (BRCA1mp) group and the BRCA1 unmethylated promoter and HRD negative (BRCA1up HRD-) group (p value = 0.0003 log rank test; Hazard Ratio (HR), 95% CI 0.04-0.40). OS was also significantly different between these groups (p value = 0.047 log rank test; HR = 0.30, 95% CI 0.10-0.84), as was PFS from diagnosis (p value = 0.02 log rank test; HR = 0.43, 95% CI 0.21-0.89). BRCA1 promoter methylation in ovarian cancer is associated with a better response to PARPi and platinum salt chemotherapy than tumors without promoter methylation or classical homologous recombination deficiency. Patients with unmethylated BRCA1 promoters and HRD-negative tumors appeared to have a poorer prognosis in terms of PFS from diagnosis. BRCA1 methylation should be considered as a theragnostic biomarker for initiation of PARPi.

Genome-wide DNA methylome analysis identifies methylation signatures associated with survival and drug resistance of ovarian cancers

AbstractBackgroundIn contrast to stable genetic events, epigenetic changes are highly plastic and play crucial roles in tumor evolution and development. Epithelial ovarian cancer (EOC) is a highly heterogeneous disease that is generally associated with poor prognosis and treatment failure. Profiling epigenome-wide DNA methylation status is therefore essential to better characterize the impact of epigenetic alterations on the heterogeneity of EOC.MethodsAn epigenome-wide association study was conducted to evaluate global DNA methylation in a retrospective cohort of 80 mixed subtypes of primary ovarian cancers and 30 patients with high-grade serous ovarian carcinoma (HGSOC). Three demethylating agents, azacytidine, decitabine, and thioguanine, were tested their anti-cancer and anti-chemoresistant effects on HGSOC cells.ResultsGlobal DNA hypermethylation was significantly associated with high-grade tumors, platinum resistance, and poor prognosis. We determined that 9313 differentially methylated probes (DMPs) were enriched in their relative gene regions of 4938 genes involved in small GTPases and were significantly correlated with the PI3K-AKT, MAPK, RAS, and WNT oncogenic pathways. On the other hand, global DNA hypermethylation was preferentially associated with recurrent HGSOC. A total of 2969 DMPs corresponding to 1471 genes were involved in olfactory transduction, and calcium and cAMP signaling. Co-treatment with demethylating agents showed significant growth retardation in ovarian cancer cells through differential inductions, such as cell apoptosis by azacytidine or G2/M cell cycle arrest by decitabine and thioguanine. Notably, azacytidine and decitabine, though not thioguanine, synergistically enhanced cisplatin-mediated cytotoxicity in HGSOC cells.ConclusionsThis study demonstrates the significant association of global hypermethylation with poor prognosis and drug resistance in high-grade EOC and highlights the potential of demethylating agents in cancer treatment.Graphic abstract

Abnormal methylation characteristics predict chemoresistance and poor prognosis in advanced high-grade serous ovarian cancer

AbstractBackgroundPrimary or acquired chemoresistance is a key link in the high mortality rate of ovarian cancer. There is no reliable method to predict chemoresistance in ovarian cancer. We hypothesized that specific methylation characteristics could distinguish chemoresistant and chemosensitive ovarian cancer patients.MethodsIn this study, we used 450 K Infinium Methylation BeadChip to detect the different methylation CpGs between ovarian cancer patients. The differential methylation genes were analyzed by GO and KEGG Pathway bioinformatics analysis. The candidate CpGs were confirmed by pyrosequencing. The expression of abnormal methylation gene was identified by QRT-PCR and IHC. ROC analysis confirmed the ability to predict chemotherapy outcomes. Prognosis was evaluated using Kaplan–Meier.ResultsIn advanced high-grade serous ovarian cancer, 8 CpGs (ITGB6:cg21105318, cg07896068, cg18437633; NCALD: cg27637873, cg26782361, cg16265707; LAMA3: cg20937934, cg13270625) remained hypermethylated in chemoresistant patients. The sensitivity, specificity and AUC of 8 CpGs (ITGB6:cg21105318, cg07896068, cg18437633; NCALD: cg27637873, cg26782361, cg16265707; LAMA3: cg20937934, cg13270625) methylation to predict chemotherapy sensitivity were 63.60–97.00%, 46.40–89.30% and 0.774–0.846. PFS of 6 candidate genes (ITGB6:cg21105318, cg07896068; NCALD: cg27637873, cg26782361, cg16265707; LAMA3: cg20937934) hypermethylation patients was significantly shorter. The expression of NCALD and LAMA3 in chemoresistant patients was lower than that of chemosensitive patients. Spearman analysis showed that NCALD and LAMA3 methylations were negatively correlated with their expression.ConclusionsAs a new biomarker of chemotherapy sensitivity, hypermethylation of NCALD and LAMA3 is associated with poor PFS in advanced high-grade serous ovarian cancer. In the future, further research on NCALD and LAMA3 will be needed to provide guidance for clinical stratification of demethylation therapy.

DNA methylation-based profiling reveals distinct clusters with survival heterogeneity in high-grade serous ovarian cancer

AbstractHigh-grade serous ovarian cancer (HGSOC) is the most common type of epigenetically heterogeneous ovarian cancer. Methylation typing has previously been used in many tumour types but not in HGSOC. Methylation typing in HGSOC may promote the development of personalized care. The present study used DNA methylation data from The Cancer Genome Atlas database and identified four unique methylation subtypes of HGSOC. With the poorest prognosis and high frequency of residual tumours, cluster 4 featured hypermethylation of a panel of genes, which indicates that demethylation agents may be tested in this group and that neoadjuvant chemotherapy may be used to reduce the possibility of residual lesions. Cluster 1 and cluster 2 were significantly associated with metastasis genes and metabolic disorders, respectively. Two feature CpG sites, cg24673765 and cg25574024, were obtained through Cox proportional hazards model analysis of the CpG sites. Based on the methylation level of the two CpG sites, the samples were classified into high- and low-risk groups to identify the prognostic information. Similar results were obtained in the validation set. Taken together, these results explain the epigenetic heterogeneity of HGSOC and provide guidance to clinicians for the prognosis of HGSOC based on DNA methylation sites.

Chromatin accessibility changes at intergenic regions are associated with ovarian cancer drug resistance

Abstract Background Resistance to DNA damaging chemotherapies leads to cancer treatment failure and poor patient prognosis. We investigated how genomic distribution of accessible chromatin sites is altered during acquisition of cisplatin resistance using matched ovarian cell lines from high grade serous ovarian cancer (HGSOC) patients before and after becoming clinically resistant to platinum-based chemotherapy. Results Resistant lines show altered chromatin accessibility at intergenic regions, but less so at gene promoters. Clusters of cis-regulatory elements at these intergenic regions show chromatin changes that are associated with altered expression of linked genes, with enrichment for genes involved in the Fanconi anemia/BRCA DNA damage response pathway. Further, genome-wide distribution of platinum adducts associates with the chromatin changes observed and distinguish sensitive from resistant lines. In the resistant line, we observe fewer adducts around gene promoters and more adducts at intergenic regions. Conclusions Chromatin changes at intergenic regulators of gene expression are associated with in vivo derived drug resistance and Pt-adduct distribution in patient-derived HGSOC drug resistance models.

5-AZA-dC induces epigenetic changes associated with modified glycosylation of secreted glycoproteins and increased EMT and migration in chemo-sensitive cancer cells

Abstract Background Glycosylation, one of the most fundamental post-translational modifications, is altered in cancer and is subject in part, to epigenetic regulation. As there are many epigenetic-targeted therapies currently in clinical trials for the treatment of a variety of cancers, it is important to understand the impact epi-therapeutics have on glycosylation. Results Ovarian and triple negative breast cancer cells were treated with the DNA methyltransferase inhibitor, 5-AZA-2-deoxycytidine (5-AZA-dC). Branching and sialylation were increased on secreted N-glycans from chemo-sensitive/non-metastatic cell lines following treatment with 5-AZA-dC. These changes correlated with increased mRNA expression levels in MGAT5 and ST3GAL4 transcripts in ovarian cancer cell lines. Using siRNA transient knock down of GATA2 and GATA3 transcription factors, we show that these regulate the glycosyltransferases ST3GAL4 and MGAT5, respectively. Moreover, 5-AZA-dC-treated cells displayed an increase in migration, with a greater effect seen in chemo-sensitive cell lines. Western blots showed an increase in apoptotic and senescence (p21) markers in all 5-AZA-dC-treated cells. The alterations seen in N-glycans from secreted glycoproteins in 5-AZA-dC-treated breast and ovarian cancer cells were similar to the N-glycans previously known to potentiate tumour cell survival. Conclusions While the FDA has approved epi-therapeutics for some cancer treatments, their global effect is still not fully understood. This study gives insight into the effects that epigenetic alterations have on cancer cell glycosylation, and how this potentially impacts on the overall fate of those cells. Graphic abstract

CAMK2N1/RUNX3 methylation is an independent prognostic biomarker for progression-free and overall survival of platinum-sensitive epithelial ovarian cancer patients

Abstract Background To date, no predictive or prognostic molecular biomarkers except BRCA mutations are clinically established for epithelial ovarian cancer (EOC) despite being the deadliest gynecological malignancy. Aim of this biomarker study was the analysis of DNA methylation biomarkers for their prognostic value independent from clinical variables in a heterogeneous cohort of 203 EOC patients from two university medical centers. Results The marker combination CAMK2N1/RUNX3 exhibited a significant prognostic value for progression-free (PFS) and overall survival (OS) of sporadic platinum-sensitive EOC (n = 188) both in univariate Kaplan–Meier (LogRank p &lt; 0.05) and multivariate Cox regression analysis (p &lt; 0.05; hazard ratio HR = 1.587). KRT86 methylation showed a prognostic value only in univariate analysis because of an association with FIGO staging (Fisher’s exact test p &lt; 0.01). Thus, it may represent a marker for EOC staging. Dichotomous prognostic values were observed for KATNAL2 methylation depending on BRCA aberrations. KATNAL2 methylation exhibited a negative prognostic value for PFS in sporadic EOC patients without BRCA1 methylation (HR 1.591, p = 0.012) but positive prognostic value in sporadic EOC with BRCA1 methylation (HR 0.332, p = 0.04) or BRCA-mutated EOC (HR 0.620, n.s.). Conclusion The retrospective analysis of 188 sporadic platinum-sensitive EOC proved an independent prognostic value of the methylation marker combination CAMK2N1/RUNX3 for PFS and OS. If validated prospectively this combination may identify EOC patients with worse prognosis after standard therapy potentially benefiting from intensive follow-up, maintenance therapies or inclusion in therapeutic studies. The dichotomous prognostic value of KATNAL2 should be validated in larger sample sets of EOC.

DNA methylation for cervical cancer screening: a training set in China

Abstract Background Despite rapid improvements in DNA methylation tools for cervical cancer screening, few robust, exploratory studies have been performed using the combination of two host genes, EPB41L3 and JAM3, newly developed assays. Methods A review of abnormal liquid-based cytology and/or high-risk human papillomavirus (hrHPV) data from outpatient clinics in the study center from March 2018 to March 2019 was performed. Eligible patients with definitive histological pathology results were included, and their residual cytology samples were assessed for EPB41L3 and JAM3 methylation. The diagnostic accuracies of various screening strategies for definitive pathology and for cervical intraepithelial neoplasia (CIN) 2 or more severe lesions (CIN2+) were compared. Results In total, 306 patients were successfully tested; 301 cases with cervical histological pathology were included in the final analysis, including 118 (39.2%) and 183 (60.8%) cases of inflammation/CIN1 and CIN2+, respectively. Regarding CIN2+ detection, methylation status and hrHPV plus methylation had similar positive predictive values (0.930 and 0.954, respectively, p = 0.395). Additionally, hrHPV, methylation, and hrHPV plus methylation had similar negative predictive values (0.612, 0.679, and 0.655, p = 0.677) that were significantly higher than that of cytology alone (0.250, p values 0.012, 0.001, and 0.001, respectively). For 49 cases with negative hrHPV results, positive methylation alone was able to differentiate CIN2+ from inflammation/CIN1. Conclusions Methylation of both EPB41L3 and JAM3 is an accurate and feasible screening method for CIN2+.

Detection of ovarian cancer using plasma cell-free DNA methylomes

Abstract Background Ovarian cancer (OC) is a highly lethal gynecologic cancer, and it is hard to diagnose at an early stage. Clinically, there are no ovarian cancer-specific markers for early detection. Here, we demonstrate the use of cell-free DNA (cfDNA) methylomes to detect ovarian cancer, especially the early-stage OC. Experimental design Plasma from 74 epithelial ovarian cancer patients, 86 healthy volunteers, and 20 patients with benign pelvic masses was collected. The cfDNA methylomes of these samples were generated by cell-free methylated DNA immunoprecipitation and high-throughput sequencing (cfMeDIP-seq). The differentially methylated regions (DMRs) were identified by the contrasts between tumor and non-tumor groups, and the discrimination performance was evaluated with the iterative training and testing method. Results The DMRs identified for cfDNA methylomes can well discriminate tumor groups and non-tumor groups (ROC values from 0.86 to 0.98). The late-stage top 300 DMRs are more late-stage-specific and failed to detect early-stage OC. However, the early-stage markers have the potential to discriminate all-stage OCs from non-tumor samples. Conclusions This study demonstrates that cfDNA methylomes generated with cfMeDIP-seq could be used to identify OC-specific biomarkers for OC, especially early OC detection. To detect early-stage OC, the biomarkers should be directly identified from early OC plasma samples rather than mix-stage ones. Further exploration of DMRs from a k larger early-stage OC cohort is warranted.

Triage of hrHPV-positive women: comparison of two commercial methylation-specific PCR assays

Abstract Aim High-risk human papillomavirus (hrHPV)-based screening is becoming increasingly important, either by supplementing or replacing the traditional cytology-based cervical Pap smear. However, hrHPV screening lacks specificity, because it cannot differentiate between transient virus infection and clinically relevant hrHPV-induced disease. Therefore, reliable triage methods are needed for the identification of HPV-positive women with cervical intraepithelial neoplasia (CIN) in need of treatment. Promising tools discussed for the triage of these patients are molecular diagnostic tests based on epigenetic markers. Here, we compare the performance of two commercially available DNA methylation-based diagnostic assays—GynTect® and the QIAsure Methylation Test—in physician-taken cervical scrapes from 195 subjects. Findings Both GynTect® and the QIAsure Methylation Test detected all cervical carcinoma and carcinoma in situ (CIS). The differences observed in the detection rates between both assays for the different grades of cervical lesions (QIAsure Methylation Test: CIN1 26.7%, CIN2 27.8% and CIN3 74.3%; GynTect®: CIN1 13.3%, CIN2 33.3% and CIN3 60%) were not significant. Concerning the false-positive rates, significant differences were evident. For the healthy (NILM) hrHPV-positive group, the false-positive rates were 5.7% for GynTect® and 26.4% for QIAsure Methylation Test (p = 0.003) and for the NILM hrHPV-negative group 2.2% vs. 23.9% (p = 0.006), respectively. When considering hrHPV-positive samples only for comparison (n = 149), GynTect® delivered significantly higher specificity compared to the QIAsure Methylation Test for CIN2 + (87.6% vs. 67.4% (p &lt; 0.001)) and CIN3 + (84.1% vs. 68.2% (p = 0.002)). Overall our findings suggest that DNA methylation-based tests are suitable for the triage of hrHPV-positive women. With the goal to provide a triage test that complements the limited specificity of HPV testing in HPV-based screening, GynTect® may be preferable, due to its higher specificity for CIN2+ or CIN3+ .

Epigenetic age acceleration of cervical squamous cell carcinoma converged to human papillomavirus 16/18 expression, immunoactivation, and favourable prognosis

Abstract Background Ageing-associated molecular changes have been assumed to trigger malignant transformations and the epigenetic clock, and the DNA methylation age has been shown to be highly correlated with chronological age. However, the associations between the epigenetic clock and cervical squamous cell carcinoma (CSCC) prognosis, other molecular characteristics, and clinicopathological features have not been systematically investigated. To this end, we computed the DNA methylation (DNAm) age of 252 CSCC patients and 200 normal samples from TCGA and three external cohorts by using the Horvath clock model. We characterized the differences in human papillomavirus (HPV) 16/18 expression, pathway activity, genomic alteration, and chemosensitivity between two DNAm age subgroups. We then used Cox proportional hazards regression and restricted cubic spline (RCS) analysis to assess the prognostic value of epigenetic acceleration. Results DNAm age was significantly associated with chronological age, but it was differentiated between tumour and normal tissue (P &lt; 0.001). Two DNAm age groups, i.e. DNAmAge-ACC and DNAmAge-DEC, were identified; the former had high expression of the E6/E7 oncoproteins of HPV16/18 (P &lt; 0.05), an immunoactive phenotype (all FDRs &lt; 0.05 in enrichment analysis), CpG island hypermethylation (P &lt; 0.001), and lower mutation load (P = 0.011), including for TP53 (P = 0.002). When adjusted for chronological age and tumour stage, every 10-year increase in DNAm age was associated with a 12% decrease in fatality (HR 0.88, 95% CI 0.78–0.99, P = 0.03); DNAmAge-ACC had a 41% lower mortality risk and 47% lower progression rate than DNAmAge-DEC and was more likely to benefit from chemotherapy. RCS revealed a positive non-linear association between DNAm age and both mortality and progression risk (both, P &lt; 0.05). Conclusions DNAm age is an independent predictor of CSCC prognosis. Better prognosis, overexpression of HPV E6/E7 oncoproteins, and higher enrichment of immune signatures were observed in DNAmAge-ACC tumours.

DNA methylation-based detection and prediction of cervical intraepithelial neoplasia grade 3 and invasive cervical cancer with the WID™-qCIN test

AbstractBackgroundCervical screening using primary human papilloma virus (HPV) testing and cytology is being implemented in several countries. Cytology as triage for colposcopy referral suffers from several shortcomings. HPV testing overcomes some of these but lacks specificity in women under 30. Here, we aimed to develop and validate an automatable triage test that is highly sensitive and specific independently of age and sample heterogeneity, and predicts progression to CIN3+ in HPV+ patients.ResultsThe WID™-qCIN, assessing three regions in human genesDPP6,RALYL, andGSX1,was validated in both a diagnostic (case–control) and predictive setting (nested case–control), in a total of 761 samples. Using a predefined threshold, the sensitivity of the WID™-qCIN test was 100% and 78% to detect invasive cancer and CIN3, respectively. Sensitivity to detect CIN3+ was 65% and 83% for women &lt; and ≥ 30 years of age. The specificity was 90%. Importantly, the WID™-qCIN test identified 52% of ≥ 30-year-old women with a cytology negative (cyt−) index sample who were diagnosed with CIN3 1–4 years after sample donation.ConclusionWe identified suitable DNAme regions in an epigenome-wide discovery using HPV+ controls and CIN3+ cases and established the WID™-qCIN, a PCR-based DNAme test. The WID™-qCIN test has a high sensitivity and specificity that may outperform conventional cervical triage tests and can in an objective, cheap, and scalable fashion identify most women with and at risk of (pre-)invasive cervical cancer. However, evaluation was limited to case–control settings and future studies will assess performance and generalisability in a randomised controlled trial.

The CpG island methylator phenotype increases the risk of high-grade squamous intraepithelial lesions and cervical cancer

AbstractBackgroundHigh-risk human papillomavirus (HR-HPV) infection is the main cause of cervical cancer, but additional alterations are necessary for its development. Abnormal DNA methylation has an important role in the origin and dissemination of cervical cancer and other human tumors. In this work, we analyzed the methylation of eight genes (AJAP1, CDH1, CDH13, MAGI2, MGMT, MYOD1, RASSF1A and SOX17) that participate in several biological processes for the maintenance of cell normality. We analyzed DNA methylation by methylation-specific PCR (MSP) and HPV infection using the INNO‑LiPA genotyping kit in 59 samples diagnostic of normal cervical tissue (non-SIL), 107 low-grade squamous intraepithelial lesions (LSILs), 29 high-grade squamous intraepithelial lesions (HSILs) and 51 cervical cancers (CCs).ResultsWe found that all samples of LSIL, HSIL, and CC were HPV-positive, and the genotypes with higher frequencies were 16, 18, 51 and 56. In general, the genes analyzed displayed a significant tendency toward an increase in methylation levels according to increasing cervical lesion severity, except for the CDH13 gene. High CpG island methylator phenotype (CIMP) was associated with a 50.6-fold (95% CI 4.72–2267.3)-increased risk of HSIL and a 122-fold risk of CC (95% CI 10.04–5349.7).ConclusionsWe found that CIMP high was significantly associated with HSIL and CC risk. These results could indicate that CIMP together with HR-HPV infection and other factors participates in the development of HSIL and CC.

Inflammatory milieu and role of epigenetic modifications in high-grade serous ovarian cancer

Abstract High-grade serous ovarian cancer (HGSOC) is often diagnosed at advanced stages (III/IV), with approximately 80% of patients experiencing relapse due to therapeutic resistance. The disease progression is largely influenced by a dynamic tumor microenvironment (TME), which is marked by sustained inflammation, immune evasion, and epigenetic reprogramming. This review investigates the dual role of inflammatory pathways and epigenetic alterations in driving HGSOC progression and chemo-resistance. A comprehensive literature search of articles from 2000 to 2025 was conducted across PubMed, Google Scholar, and Research Rabbit using search terms including “HGSOC,” “epigenetics,” “inflammation,” and “chemoresistance.” Of 1,166 identified publications, 593 peer-reviewed studies comprising original research, clinical trials, meta-analyses, and reviews were critically analyzed. Findings reveal that chronic inflammation in the TME enhances tumor proliferation, immune suppression, epithelial-mesenchymal transition, and metastasis through cytokines, interferons, and chemokines. Epigenetic mechanisms such as DNA methylation, histone modifications, miRNA and lncRNA contribute to tumor plasticity and treatment failure. Emerging therapies, including histone deacetylase inhibitors, DNA methyltransferase inhibitors, and immune checkpoint inhibitors, anti-inflammatory drugs demonstrate potential in overcoming resistance when used in combination. Integrative treatment strategies that target both inflammatory signaling and epigenetic dysregulation offer a promising avenue for improving patient outcomes. Further clinical exploration of such combination therapies is warranted to address the urgent need for effective interventions in HGSOC.