Renske D. M. Steenbergen

Persistent high-risk human papillomavirus (hrHPV) infections are the principal cause of cervical cancer, and are increasingly associated with ecological instability of the cervicovaginal microbiome. Multiomics studies indicate that hrHPV persistence is linked to reduced Lactobacillus dominance, increased anaerobic taxa, and metabolic alterations that may compromise epithelial integrity and antiviral immunity. In this Perspective article, we introduce a sequential microbiome therapy (SMT) framework designed to restore cervicovaginal ecological balance and support viral clearance. SMT is a two-phase, strain-resolved strategy. The first phase targets dysbiotic communities using antimicrobials, biofilm-disrupting or microbiota-modulating approaches to create a window for ecological reset. The second phase introduces health-associated Lactobacillus species selected for their acidification capacity, competitive fitness, and ability to stabilize the mucosal environment. We outline the ecological principles underpinning SMT and discuss its development as a potentially scalable microbiome-directed strategy for restoring cervicovaginal health and managing hrHPV-associated disease within existing cervical cancer prevention frameworks.

Anal high‐grade squamous intraepithelial lesions (HSIL:AIN2/3) are precursors to anal cancer, and early detection and treatment through screening is crucial for effective cancer prevention. Current guidelines recommend anal swab‐based screening and highlight the need for objective biomarkers to improve risk stratification. While DNA methylation markers have shown strong potential in anal biopsies, their performance in anal swabs is reduced, likely due to sample‐type dependency. This study aimed to identify a DNA methylation classifier specifically derived from anal swabs of men who have sex with men living with HIV (MSMLWH) to enhance diagnostic accuracy. Genome‐wide methylation profiling was performed on anal swabs from 36 HSIL:AIN3 cases, 34 controls (p < 0.001). From these, a 3‐gene methylation classifier ( NOVA1 ; TBXT ; ZNF788 ) was identified, demonstrating excellent performance with an AUC of 0.80 (95% confidence interval: 0.72–0.88) in distinguishing HSIL:AIN3+ from ≤LSIL. This study identified a robust 3‐gene methylation classifier with strong potential for detecting HSIL:AIN3 and anal cancer when applied to anal swabs from MSMLWH.

Summary: Circular RNAs (circRNAs) are an emerging class of RNAs with biomarker potential, but their detection in liquid biopsies is challenging due to low abundance. We developed Ouro-seq, a novel long-read sequencing protocol optimized for full-length circRNA recovery. Applied to urine, cervico-vaginal self-samples from cervical cancer patients, and plasma from lung cancer patients and controls, Ouro-seq recovered 2-5 times more and substantially longer circRNA molecules than conventional methods. Plasma contained predominantly exonic circRNAs, while urine and cervico-vaginal samples were dominated by previously undercharacterized intergenic circRNAs. We also identified extensive alternative circularization and splicing events. Functional analysis revealed distinct specialization patterns: exonic circRNAs showed enhanced miRNA sponging potential, while circRNAs from unplaced genomic scaffolds demonstrated greater peptide-coding capacity. This study establishes Ouro-seq as a valuable tool for comprehensive circRNA characterization in low-yield clinical samples and advances circRNA biology understanding with potential biomarker discovery and disease monitoring applications. Motivation: While circular RNAs (circRNAs) constitute a minor fraction of total RNA, they may play critical roles in cancer development. CircRNA concentrations are typically too low for detection by Oxford Nanopore Long-Read Sequencing (LRS), particularly in samples with limited RNA content, such as liquid biopsies. Consequently, LRS-based circRNA analysis from liquid biopsies remains unexplored. To overcome these technical limitations, we developed an optimized circRNA enrichment method utilizing short-amplicon suppression, enabling circRNA profiling from urine, plasma, and cervico-vaginal samples.

Background: Histologically confirmed anal high-grade squamous intraepithelial lesions (hHSILs) are targets for diagnosis and treatment in anal cancer screening programs. However, the long-term natural history of untreated hHSILs remains poorly understood. Methods: In the APACHES study, 513 human immunodeficiency virus (HIV)–positive men who have sex with men and were aged ≥35 years underwent 3 annual visits for anal cytology, human papillomavirus (HPV) testing, and high-resolution anoscopy with biopsies to detect hHSILs. hHSILs were not treated but were monitored every 6 months for up to 5 years. Methylation markers, ASCL1 and ZNF582, were assessed in biopsy specimens at hHSIL diagnosis. The hHSIL regression probability was estimated using Cox proportional hazards models, adjusted hazard ratios, and Kaplan-Meier survival curves. Results: Of 127 untreated hHSILs, 21% regressed within 1 year, 41% within 2 years, and 75% within 5 years. None progressed to cancer during follow-up. Regression was significantly less likely for hHSILs with concurrent high-grade findings at anal cytology (adjusted hazard ratio, 0.41 [95% confidence interval, .19–.88]), 12-month persistent HPV-16 infection in anal swab samples (0.46 [.21–.97]), or positivity for both ASCL1 and ZNF582 (0.39 [.18–.82). The 5-year regression probabilities ranged from approximately 30% for ASCL1/ZNF582-positive hHSILs with high-grade cytology, to approximately 90% for ASCL1/ZNF582-negative hHSILs without high-grade cytology. hHSIL regression was not influenced by age, smoking, or HIV-related markers. Conclusions: Only one-quarter of untreated hHSILs persisted at 5 years, representing lesions with the highest anal cancer risk. The methylation markers ASCL1/ZNF582 were the strongest predictors of persistent hHSIL and, combined with HPV testing and cytology, may inform prioritization for diagnosis and treatment in anal cancer screening programs.

PURPOSE: The molecular analysis of urine cell-free DNA offers a noninvasive tool to advanced bladder cancer (BC) management. Assessment of somatic copy number aberration (SCNA) and DNA methylation analysis have emerged as promising approaches for BC detection. Here, we developed an integrated analysis to assess both SCNA and targeted methylation changes from the same template molecules, which we named the integrated sequencing-based copy number and methylation analysis in urine (iSECURE) method. MATERIALS AND METHODS: Urine samples of 30 patients with primary BC, 28 patients with recurrent BC, and 31 hematuria controls were collected at home. Copy number profiling and tumor fraction (TF) estimation were performed by shallow whole-genome enzymatic methyl sequencing. Methylation sequencing libraries were also used for the measurement of a previously validated three-gene methylation marker panel ( GALR1/HAND2/NRN1 ) by the quantitative methylation-specific polymerase chain reaction. Diagnostic accuracy was evaluated by logistic regression analysis using Youden's index for sensitivity and specificity and validated using leave-one-out cross-validation (LOOCV). RESULTS: TF and methylation levels were significantly higher in patients with BC compared with controls ( P < .0001). The individual assays yielded an AUC of 0.91 at 79% sensitivity and 97% specificity for the methylation marker panel and 0.81 at 74% sensitivity and 87% specificity for copy number profiling. The iSECURE workflow yielded a higher diagnostic accuracy with an AUC of 0.94 at 88% sensitivity (90% for primary tumors and 86% for recurrent tumors) and 94% specificity. LOOCV demonstrated an AUC of 0.91 at 84% sensitivity and 90% specificity. CONCLUSION: We developed a single workflow for copy number profiling and targeted methylation analysis, requiring less input DNA and hands-on time. The high accuracy of iSECURE in home-collected urine samples for detecting both primary and recurrent BC underscores its clinical potential.

DNA methylation analysis of the genes FAM19A4 and miR124‐2 has emerged as a promising triage strategy for high‐risk (hr) human papillomavirus (HPV)‐positive women in cervical screening. This study reports a prospective evaluation of the diagnostic accuracy of FAM19A4/miR124‐2 methylation in a large population‐based primary HPV‐based screening cohort with 18 months of follow‐up. In this prospective observational study, clinician‐collected cervical samples from 3848 consecutive hrHPV‐positive women who participated in the Dutch national primary HPV‐based screening program were tested with the QIAsure Methylation Test, a quantitative methylation‐specific PCR assessing FAM19A4 and miR124‐2 methylation. Through linkage to the Dutch Nationwide Pathology Databank (Palga), 11 cases of cervical carcinoma, 293 cervical intraepithelial neoplasia grade 3 (CIN3), and 29 adenocarcinoma in situ (AIS) were identified during 18 months of follow‐up. Clinical performance for CIN3, AIS, and cancer (CIN3+) detection was assessed based on (I) the threshold of the QIAsure Methylation Test, and (II) thresholds corresponding with clinical specificities of 70% and 80% in this hrHPV‐positive study population. The sensitivity and specificity of FAM19A4/miR124‐2 methylation for CIN3+ detection using the QIAsure Methylation Test threshold (I) were 57.4% (95% CI: 52.0–62.7) and 88.3% (95% CI: 87.2–89.5), respectively. At predefined specificities of 70% and 80%, FAM19A4/miR124‐2 methylation (II) demonstrated a CIN3+ sensitivity of 75.4% (95% CI: 70.7–80.0) and 66.1% (95% CI: 61.0–71.2), respectively. In conclusion, FAM19A4/miR124‐2 methylation analysis showed good triage performance in a primary HPV‐based screening setting. These findings support the use of methylation‐based testing as a triage strategy in primary HPV‐based screening programs, with an appropriate follow‐up policy for hrHPV‐positive women who test methylation‐negative.

Background DNA methylation analysis provides a promising triage strategy for cervical intraepithelial neoplasia (CIN) and cancer detection following Human Papillomavirus (HPV) testing on self-collected samples, including urine. Methods This study aimed to develop an entirely molecular cervical screening approach based on HPV and DNA methylation analysis in at-home collected first-void urine from healthy females (n = 69) and a referral population (n = 385; CIN-cancer). CIN3+ detection was analyzed by multivariate logistic regression. Results Here we show that urinary ASCL1/LHX8 methylation levels increase significantly in relation to disease severity, with AUC-values for CIN3+ of 0.81 (95% CI: 0.74–0.88) and 0.83 (95% CI: 0.74–0.92) in the training (n = 285) and validation cohort (n = 160), respectively. This corresponds to a validated CIN3+ sensitivity of 73.0% (95% CI: 57.0–84.6%) at 81.9% specificity (95% CI: 73.5–88.1%; ASCL1/LHX8 methylation and HPV16/18 genotyping have a similar CIN3+ sensitivity (75.0%; 95% CI: 62.8–84.2% vs 73.3%; 95% CI: 61.0–82.9%), with lower genotyping specificity. Combining ASCL1/LHX8 methylation with HPV16/18 genotyping yield a 85.0% sensitivity (95% CI: 73.9–91.9%) at 50.5% specificity (95% CI: 40.8–60.1%). Conclusions The ASCL1/LHX8 methylation test detects nearly all cancers and a majority of CIN3 in first-void urine, supporting the potential of full molecular screening in urine by primary HPV testing and methylation triage.

This chapter discusses the role of DNA methylation, an epigenetic mechanism that regulates gene expression, as a key event in cervical carcinogenesis, and highlights its potential as a biomarker for detecting cervical cancer and high-grade precancerous lesions. Methylation levels of specific genes (methylation markers) gradually increase with cervical disease severity, allowing several clinical applications. Methylation markers have been evaluated as alternative triage tools for high-risk (hr) human papillomavirus (HPV)-positive women, including those with borderline or mildly abnormal cytology results, and for use in specific populations such as women living with HIV. Methylation testing often outperforms traditional cytology-based triage methods and offers long-term reassurance against cervical cancer when the result is negative. Additionally, methylation markers have shown prognostic potential in predicting the natural course of high-grade precancerous lesions, as well as a promising performance for the detection of recurrent disease. While ongoing research focuses on validating methylation testing in self-collected samples, low-resource settings, and HPV-vaccinated populations, current evidence supports its potential to enhance early detection, risk stratification, and post-treatment monitoring in cervical cancer prevention and care.