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Objective: To assess the clinical value of DNA methylation measurement in exfoliated cervical cells for distinguishing high-grade squamous intraepithelial lesions (HSIL) from other cervical abnormalities., Methods: A total of 276 patients were enrolled, and general clinical information was collected. Exfoliated cervical cells were obtained to assess human papillomavirus (HPV) infection, conduct ThinPrep cytology tests (TCT), and measure methylation levels of JAM3 (△CtJ) and PAX1 (△CtP). Logistic regression was performed to identify factors significantly associated with HSIL diagnosis. A conditional inference tree model and the area under the curve (AUC) were employed to evaluate the efficacy of JAM3 and PAX1 methylation in detecting HSIL., Results: Independent risk factors for HSIL diagnosis included △CtJ, △CtP, atypical squamous cells of undetermined significance (ASCUS), and HPV16 infection. The conditional inference tree indicated that 96.4% of patients were non-HSIL when △CtJ > 11.66, and 99.1% were non-HSIL when △CtP > 10.97. The diagnostic performance of △CtJ/△CtP surpassed that of TCT/HPV alone. Among six methods, the combination of △CtP, TCT, and high-risk HPV (hr-HPV) testing achieved the highest sensitivity (91.2%), positive predictive value (50.0%), negative predictive value (98.6%), and AUC (0.932)., Conclusion: In women with hr-HPV infection, DNA methylation analysis of cervical cytology outperformed traditional TCT or HPV testing. The combination of △CtP with TCT and HPV may offer the most accurate screening approach for HSIL., Competing Interests: Declarations. Ethics approval and consent to participate: This study was conducted in accordance with the Declaration of Helsinki and approved by the ethics committee of the Third Xiangya Hospital of Central South University (No. 23137). All participants provided written informed consent. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Global methylation analysis of gene promoters is promising for detection of high-grade squamous intraepithelial lesions or worse (HSIL+) in high-risk human papillomavirus (hrHPV)-positive women. However, diagnostic performance of methylation data at individual CpG-sites is limited. We explored methylation for predicting HSIL+ in self- and clinician-collected samples from Papua New Guinea. Methylation of EPB41L3 (1-6 CpG-sites), hTERT (1-10 CpG-sites) and FAM19A4 (1-5 CpG-sites) was assessed through pyrosequencing from 44 HPV+ samples (4 cancers, 19 HSIL, 4 low-grade squamous intraepithelial lesions (LSIL), 17 normal). New primers were designed for FAM19A4 directed to the first exon region not explored previously. In clinician-collected samples, methylation at CpG-sites 4 and 5 of EPB41L3 were the best HSIL predictors (AUC >0.83) and CpG-site 4 for cancer (0.925). Combination of EPB41L3 sites 2/4 plus FAM19A4 site 1 were the best HSIL+ markers [100% sensitivity, 63.2% specificity]. Methylation at CpG-site 5 of FAM19A4 was the best HSIL predictor (0.67) in self-collected samples, and CpG-sites 1 and 3 of FAM19A4 for cancer (0.77). Combined, FAM19A4 site 1 plus HPV 16/18 detection yielded sensitivity of 82.6% and specificity of 61.9%. In conclusion, methylation at individual CpG-sites of EPB41L3 and FAM19A4 outperformed global analysis and improved HSIL+ detection, warranting further investigation., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests. AJV, JG, JB, GMM, PJT, SGB, JMK have received subsidized test kits for research from Cepheid. MS, JMLB, GT, DH have received donated test kits for research from Roche, Abbott, Seegene, Cepheid, Aus Diagnostics and Becton Dickinson. AJV and MS jointly lead the Elimination of Cervical Cancer in theWesternPacific (ECCWP) program with philanthropic funding support from the Minderoo Foundation and the Frazer Family Foundation; and equipment, tests and consumables donated by Cepheid for HPV-based cervical screening in Papua New Guinea and Vanuatu. SMG is a member of the Global Advisory Board HPV Merck, and has led investigator-initiated grants from Merck on HPV in young women. All other authors report no potential conflicts., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Cervical cancer screening programs, including triage tests, need redesigning as human papillomavirus (HPV)-vaccinated women are entering the programs. Methylation markers offer a potential solution to reduce false-positive rates by identifying clinically relevant cervical lesions with progressive potential. In a nested case-control study, 9242 women who received the three-dose HPV16/18-vaccine at ages 12-15 or 18 in a community-randomized trial were included. Subsequently, they were re-randomized for either frequent or infrequent cervical cancer screening trials. Over a 15-year post-vaccination follow-up until 2022, 17 high-grade squamous intraepithelial lesion (HSIL) and 15 low-grade (LSIL) cases were identified at the 25-year screening round, alongside 371 age and community-matched HPV16/18-vaccinated controls. Methylation analyses were performed on cervical samples collected at age 25, preceding histologically confirmed LSIL or HSIL diagnoses. DNA methylation of viral (HPV16/18/31/33) and host-cell genes (EPB41L3, FAM19A4, and miR124-2) was measured, along with HPV-genotyping. No HPV16/18 HSIL cases were observed. The predominant HPV-genotypes were HPV52 (29.4%), HPV59/HPV51/HPV58 (each 23.5%), and HPV33 (17.7%). Methylation levels were generally low, with no significant differences in mean methylation levels of viral or host-cell genes between the LSIL/HSIL and controls. However, a significant difference in methylation levels was found between HSIL cases and controls when considering a combination of viral genes and EPB41L3 (p value = .0001). HPV-vaccinated women with HSIL had HPV infections with uncommon HPV types that very rarely cause cancer and displayed low methylation levels. Further investigation is warranted to understand the likely regressive nature of HSIL among HPV-vaccinated women and its implications for management., (© 2024 The Author(s). International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

Cervical cancer (CC) was considered to be the most common gynaecological cancer, with an estimated 342,000 deaths worldwide each year, as the majority of patients were diagnosed at an advanced stage of the disease. The purpose of this study was to evaluate the predictive value of multi-locus methylation assay for the early detection of CC. The cervical exfoliated cell samples from 492 HPV-positive females with cervical lesions were collected and subjected to methylation detection of gene FAM19A4, EPB41L3 and PAX1 after bisulfite conversion. The levels of gene methylation in patients with different severity of cervical lesions were evaluated and compared. The receiver-operating characteristic (ROC) curve was established and efficacy indexes such as sensitivity, specificity and area under the curve (AUC) were calculated to assess the diagnostic value of DNA methylation detection at multiple gene loci for CC. The methylation levels of FAM19A4, EPB41L3 and PAX1 were significantly increased with the grade of cervical squamous intraepithelial lesions. The sensitivities of FAM19A4, EPB41L3 and PAX1 alone for high-grade squamous intraepithelial lesion (HSIL) and CC diagnosis were 84.6%, 86.3% and 88.0%, respectively; when three markers were combined by a logistic regression model, the sensitivity was 88.0%, with a high specificity of 97.7% and AUC of 0.957 (95% CI 0.937-0.977). Methylation status of FAM19A4, EPB41L3 and PAX1 were highly specific and effective for monitoring the progression of cervical lesions and the tri-gene methylation assay could be used as a triage tool for CC early screening., (© 2024. The Author(s).)

People living with HIV (PLWH) are at highest risk of anal cancer and will benefit from optimized screening for early disease detection. We compared host DNA methylation markers in high-grade squamous intraepithelial lesions (HSIL) versus samples negative for intraepithelial lesions (NILM) or low-grade intraepithelial lesions (LSIL) in PLWH. We recruited PLWH identifying as male aged ≥18 years undergoing high-resolution anoscopy (HRA) in Seattle, Washington, 2015-2016. Anal brush samples were collected for HPV detection, genotyping, and pyrosequencing methylation (host genes ASCL1, PAX1, FMN2, and ATP10A); clinical data were abstracted from medical records. We assessed associations between methylation and presence and extent of HSIL using generalized estimating equation logistic regression, adjusting for age, CD4 count and HIV viral load. Marker panels using HPV DNA and methylation were also evaluated to predict prevalent HSIL. We analyzed 125 samples from 85 participants (mean age 50.1; standard deviation 11.0 years). ASCL1 (adjusted odds ratio [aOR] per 1 unit increase mean percent methylation: 1.07, 95% CI: 1.01-1.13) and FMN2 (aOR per 1 unit increase mean percent methylation: 1.14, 95% CI: 1.08-1.20) methylation were significantly associated with HSIL versus NILM/LSIL. ASCL1 (aOR: 1.06, 95% CI: 1.01-1.11) and FMN2 (aOR: 1.13, 95% CI: 1.08-1.17) methylation were positively associated with increasing HSIL extent. A panel combining methylation (ASCL1 and FMN2) and HPV DNA (HPV16, HPV18, and HPV31) demonstrated best balance of sensitivity (78.2%) and specificity (73.9%) for HSIL detection compared with methylation or HPV alone. Increasing levels of DNA methylation of ASCL1 and FMN2 were positively associated with HSIL detection in PLWH. Host gene methylation testing shows promise for HSIL screening and triage., (© 2024 UICC.)

Background: In this research, an meta-analysis was performed for assessment of the associations between O6-methyguanine-DNA methyltransferase (MGMT) promoter hypermethylation possessing low-grade intraepithelial lesion (LSIL), high-grade intraepithelial lesion (HSIL), cervical cancer (CC), and clinicopathological characters of CC., Methods: Literature selection were conducted through searching PubMed, Web of science, EMBASE, China National Knowledge Infrastructure and Wanfang databases (up to November 2018). An assessment of associations between MGMT methylation and LSIL, HSIL, CC risk and clinicopathological characteristics was performed through pooled odds ratios (ORs) with relevant 95% confidence intervals (CIs). Subgroup analyses, meta-regressions and Galbraith plots were conducted to conduct an exploration on the possible sources of heterogeneity. The genome-wide DNA methylation array studies were extracted from Gene Expression Omnibus (GEO) databases for validation of these outcomes., Results: In this meta-analysis of 25 published articles, MGMT hypermethylation gradually elevated the rates among control group (12.16%), LSIL (20.92%), HSIL (36.33%) and CC (41.50%) specimens. MGMT promoter methylation was significant associated with the increased risk of LSIL by 1.74-fold (P<0.001), HSIL by 3.71-fold (P<0.001) and CC by 7.08-fold (P<0.001) compared with control. A significant association between MGMT promoter methylation with FIGO stage was also found (OR = 2.81, 95% CI: 1.79-4.41, p<0.001). The results of GEO datasets showed that 5 CpG sites in MGMT with a great diagnostic value for the screening of cervical cancer., Conclusion: The meta-analysis indicated the association between MGMT promoter hypermethylation and squamous intraepithelial lesion and cervical cancer. MGMT methylation detection might have a potential value to be an epigenetic marker for the clinical diagnosis of cervical cancer., Competing Interests: The authors have declared that no competing interests exist.

Background: DNA methylation has been suggested as one of the epigenetic changes promoting carcinogenesis. The aim of this study was to prospectively evaluate the methylation status of CADM 1, MAL and hsa-miR-124 genes in high-grade squamous intraepithelial lesion (HSIL) liquid-based cytology (LBC) samples with a histological correlation., Methods: Seventy histologically confirmed cases of HSIL paired with prior screening LBC diagnosis of HSIL within a 3-month interval were selected. Histologically, the lesions were reviewed and assessed including: (a) number of blocks harbouring dysplastic squamous epithelium; (b) number of blocks containing glandular extension of dysplastic epithelium; and (c) the depth of glandular extension (which was assessed semi-quantitatively as graded 1-3). Human papillomavirus (HPV) subtyping was performed from residual LBC materials using the LINEAR ARRAY HPV Genotyping Test and in-house polymerase chain reaction targeting the HPV E1 gene. The detection of methylation silencing of tumour suppressor genes CADM1, MAL and hsa-miR-124 was performed by multiplex methylation-specific real-time polymerase chain reaction., Results: A positive methylation status was detected in 41 cases (58.6%). The number of blocks with HSIL varied from one to 13. Glandular extension was seen in 44 cases with the number of blocks involved ranging from one to 10. The depth of HSIL glandular extension varied., Conclusion: The DNA methylation test allows HSIL lesions to be divided into two distinct groups of methylated HSIL in significantly older patients and unmethylated HSIL in younger patients. This study was not able to prove that methylation status in cervical HSIL correlates with the size of the lesion (measured by the number of blocks involved) or with HSIL propensity for endocervical glandular extension, nor with HPV type or multi-infection., (© 2019 John Wiley & Sons Ltd.)