Lisanne Verhoef

Colposcopy referrals and CIN3 detection after triage by host cell DNA methylation and/or HPV genotyping in HPV positive women with low‐grade cytology from a population‐based Dutch primary HPV screening trial

AbstractHigh‐risk HPV (hrHPV)‐based screening has led to many unnecessary colposcopy referrals, mainly because of direct referral after low‐grade cytology (ASC‐US/LSIL). DNA methylation and genotyping tests on ASC‐US/LSIL samples have the potential to significantly improve the efficiency of screening. In this study, 12 triage strategies were constructed from FAM19A4/miR124‐2 or ASCL1/LHX8 methylation, HPV16/18 or HPV16/18/31/33/45 genotyping and 1‐year repeat cytology. The performance was evaluated on 215 hrHPV‐positive ASC‐US/LSIL samples from the IMPROVE trial (NTR5078). Performance was measured by colposcopy referral rate, positive predictive value (PPV) for detecting precancer (CIN3), and negative predictive value (NPV). To evaluate efficiency, strategies were ordered by the cumulative colposcopy referral rate after 1‐year cytology and compared by the marginal PPV to detect one additional CIN3 (mPPV). The most conservative strategy (referral when HPV16/18 and FAM19A4/miR124 methylation results are positive) had a direct referral rate of 5.2%, a cumulative referral rate after 1‐year cytology of 54.1%, and mPPV of 19.3%. Replacing HPV16/18 by HPV16/18/31/33/45 increased the cumulative 1‐year referral rate to 54.6%, and yielded an mPPV of 10.0%. Similar results were obtained for strategies with ASCL1/LHX8 methylation. Of all strategies, referral after an HPV16/18/31/33/45 positive, ASCL1/LHX8 methylation‐positive, and/or 1‐year cytology‐positive result yielded the highest direct and cumulative 1‐year colposcopy referral rates of 64.4% and 79.1%, respectively. The NPVs after 1‐year cytology varied between 98.1% and 99.4%, warranting a return to routine screening. Altogether, DNA methylation‐based triage strategies are recommended as they are discriminative for CIN3 and control the number of immediate colposcopy referrals.

Low methylation marker levels among human papillomavirus‐vaccinated women with cervical high‐grade squamous intraepithelial lesions

AbstractCervical 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.

Evaluation of Six Methylation Markers Derived from Genome-Wide Screens for Detection of Cervical Precancer and Cancer

Performance of DNA methylation analysis of ASCL1, LHX8, ST6GALNAC5, GHSR, ZIC1 and SST for the triage of HPV‐positive women: Results from a Dutch primary HPV‐based screening cohort

AbstractMethylation of host‐cell deoxyribonucleic acid (DNA) has been proposed as a promising biomarker for triage of high‐risk (hr) human papillomavirus (HPV) positive women at screening. Our study aims to validate recently identified host‐cell DNA methylation markers for triage in an hrHPV‐positive cohort derived from primary HPV‐based cervical screening in The Netherlands. Methylation markers ASCL1, LHX8, ST6GALNAC5, GHSR, ZIC1 and SST were evaluated relative to the ACTB reference gene by multiplex quantitative methylation‐specific PCR (qMSP) in clinician‐collected cervical samples (n = 715) from hrHPV‐positive women (age 29‐60 years), who were enrolled in the Dutch IMPROVE screening trial (NTR5078). Primary clinical end‐point was cervical intraepithelial neoplasia grade 3 (CIN3) or cancer (CIN3+). The single‐marker and bi‐marker methylation classifiers developed for CIN3 detection in a previous series of hrHPV‐positive clinician‐collected cervical samples were applied. The diagnostic accuracy was visualised using receiver operating characteristic (ROC) curves and assessed through area under the ROC curve (AUC). The performance of the methylation markers to detect CIN3+ was determined using the predefined threshold calibrated at 70% clinical specificity. Individual methylation makers showed good performance for CIN3+ detection, with highest AUC for ASCL1 (0.844) and LHX8 (0.830). Combined as a bi‐marker panel with predefined threshold, ASCL1/LHX8 yielded a CIN3+ sensitivity of 76.9% (70/91; 95% CI 68.3‐85.6%) at a specificity of 74.5% (465/624; 95% CI 71.1‐77.9%). In conclusion, our study shows that the individual host‐cell DNA methylation classifiers and the bi‐marker panel ASCL1/LHX8 have clinical utility for the detection of CIN3+ in hrHPV‐positive women invited for routine screening.

Assessing the risk of cervical neoplasia in the post‐HPV vaccination era

AbstractThis review is based on the recent EUROGIN scientific session: “Assessing risk of cervical cancer in the post‐vaccination era,” which addressed the demands of cervical intraepithelial neoplasia (CIN)/squamous intraepithelial lesion (SIL) triage now that the prevalence of vaccine‐targeted oncogenic high‐risk (hr) human papillomaviruses (HPVs) is decreasing. Change in the prevalence distribution of oncogenic HPV types that follows national HPV vaccination programs is setting the stage for loss of positive predictive value of conventional but possibly also new triage modalities. Understanding the contribution of the latter, most notably hypermethylation of cellular and viral genes in a new setting where most oncogenic HPV types are no longer present, requires studies on their performance in vaccinated women with CIN/SIL that are associated with nonvaccine HPV types. Lessons learned from this research may highlight the potential of cervical cells for risk prediction of all women's cancers.

DNA methylation testing for endometrial cancer detection in urine, cervicovaginal self‐samples and cervical scrapes

AbstractEndometrial cancer incidence is rising and current diagnostics often require invasive biopsy procedures. DNA methylation marker analysis of minimally‐ and non‐invasive sample types could provide an easy‐to‐apply and patient‐friendly alternative to determine cancer risk. Here, we compared the performance of DNA methylation markers to detect endometrial cancer in urine, cervicovaginal self‐samples and clinician‐taken cervical scrapes. Paired samples were collected from 103 patients diagnosed with stage I to IV endometrial cancer. Urine and self‐samples were collected at home. All samples were tested for nine DNA methylation markers using quantitative methylation‐specific PCR. Methylation levels measured in endometrial cancer patients were compared to unpaired samples of 317 healthy controls. Diagnostic performances were evaluated by univariable and multivariable logistic regression analysis, followed by leave‐one‐out cross‐validation. Each methylation marker showed significantly higher methylation levels in all sample types of endometrial cancer patients compared to healthy controls (P < .01). Optimal three‐marker combinations demonstrated excellent diagnostic performances with area under the receiver operating curve values of 0.95 (95% CI: 0.92‐0.98), 0.94 (0.90‐0.97) and 0.97 (0.96‐0.99), for endometrial cancer detection in urine, self‐samples and scrapes, respectively. Sensitivities ranged from 89% to 93% at specificities of 90% to 92%. Virtually equal performances were obtained after cross‐validation and excellent diagnostic performances were maintained for stage I endometrial cancer detection. Our study shows the value of methylation analysis in patient‐friendly sample types for endometrial cancer detection of all stages. This approach has great potential to screen patient populations at risk for endometrial cancer.

Evaluation of DNA Methylation Markers for Endometrial Cancer Risk-stratification Using Patient-collected Urine and Vaginal Samples and Clinician-collected Cervical Samples From Women With Postmenopausal Bleeding

The goal of this observational study is to investigate the clinical utility of DNA-methylation testing in urine and vaginal samples collected by patients and cervical samples collected by clinicians, to determine the risk of endometrial cancer in symptomatic women with postmenopausal bleeding. The study aims to answer the following research questions: * What is the diagnostic accuracy of DNA methylation testing in urine, vaginal and cervical samples compared to traditional TVUS for endometrial cancer detection? * What is the 2-year risk of EC among women testing negative on TVUS and/or DNA methylation tests or those testing positive on methylation only? Researchers will compare DNA methylation testing in patient-collected urine and vaginal samples as well as in clinician-collected cervical samples, with the traditional diagnostic pathway for women with PMB, which includes TVUS evaluation, and when indicated by abnormal TVUS findings, endometrial biopsy according to clinical guidelines. Participants will * take a urine and vaginal sample * have a cervical sample collected by a clinician * undergo TVUS evaluation according to clinical guidelines * If TVUS shows thickened endometrium (≥ 5 mm) and/or irregularity, an endometrial biopsy will be collected according to clinical guidelines * fill out a questionnaire regarding acceptability and preferences of sampling methods and complete a lifestyle questionnaire.

The Clinical Utility of DNA Methylation Testing in Patient-collected Urine and Vaginal Samples to Detect Endometrial Cancer: a Case-control Study

The goal of this observational case-control study is to investigate the use of DNA-methylation testing in patient-collected urine and vaginal samples to detect endometrial cancer. The study aims to answer the following questions: * Can DNA methylation testing in vaginal and full-void urine samples distinguish endometrial cancer cases from healthy controls? Researchers will compare patient-collected urine and vaginal samples from patients with diagnosed endometrial cancer (cases) to gynaecologically and oncologically healthy controls (controls). Participants will * take a urine and vaginal sample at the hospital. * answer a questionnaire regarding acceptability and preferences of self-sampling methods. * answer a lifestyle questionnaire.