Birgit I. Lissenberg‐Witte

Classification of high‐grade cervical intraepithelial neoplasia by p16ink4a, Ki‐67, HPV E4 and FAM19A4/miR124‐2 methylation status demonstrates considerable heterogeneity with potential consequences for management

AbstractHigh‐grade cervical intraepithelial neoplasia (CIN2 and CIN3) represents a heterogeneous disease with varying cancer progression risks. Biomarkers indicative for a productive human papillomavirus (HPV) infection (HPV E4) and a transforming HPV infection (p16ink4a, Ki‐67 and host‐cell DNA methylation) could provide guidance for clinical management in women with high‐grade CIN. This study evaluates the cumulative score of immunohistochemical expression of p16ink4a (Scores 0‐3) and Ki‐67 (Scores 0‐3), referred to as the “immunoscore” (IS), in 262 CIN2 and 235 CIN3 lesions derived from five European cohorts in relation to immunohistochemical HPV E4 expression and FAM19A4/miR124‐2 methylation in the corresponding cervical scrape. The immunoscore classification resulted in 30 lesions within IS group 0‐2 (6.0%), 151 lesions within IS group 3‐4 (30.4%) and 316 lesions within IS group 5‐6 (63.6%). E4 expression decreased significantly from CIN2 to CIN3 (P < .001) and with increasing immunoscore group (Ptrend < .001). Methylation positivity increased significantly from CIN2 to CIN3 (P < .001) and with increasing immunoscore group (Ptrend < .001). E4 expression was present in 9.8% of CIN3 (23/235) and in 12.0% of IS group 5‐6 (38/316). Notably, in a minority (43/497, 8.7%) of high‐grade lesions, characteristics of both transforming HPV infection (DNA hypermethylation) and productive HPV infection (E4 expression) were found simultaneously. Next, we stratified all high‐grade CIN lesions, based on the presumed cancer progression risk of the biomarkers used, into biomarker profiles. These biomarker profiles, including immunoscore and methylation status, could help the clinician in the decision for immediate treatment or a “wait and see” policy to reduce overtreatment of high‐grade CIN lesions.

Risk-stratification of HPV-positive women with low-grade cytology by FAM19A4/miR124-2 methylation and HPV genotyping

Abstract Background The introduction of primary HPV screening has doubled the number of colposcopy referrals because of the direct referral of HPV-positive women with a borderline or mild dyskaryosis (BMD) cytology (ASC-US/LSIL) triage test. Further risk-stratification is warranted to improve the efficiency of HPV-based screening. Methods This study evaluated the discriminative power of FAM19A4/miR124-2 methylation, HPV16/18 genotyping and HPV16/18/31/33/45 genotyping in HPV-positive women with BMD (n = 294) in two Dutch screening trials. Absolute CIN3+ risks and colposcopy referrals within one screening round were calculated. Results Methylation analysis discriminated well, yielding a CIN3+ risk of 33.1% after a positive result and a CIN3+ risk of 9.8% after a negative result. HPV16/18 and HPV16/18/31/33/45 genotyping resulted in a 27.6% and 24.6% CIN3+ risk after a positive result, and a 13.2% and 9.1% CIN3+ risk after a negative result. Colposcopy referral percentages were 41.2%, 43.2%, and 66.3% for FAM19A4/miR124-2 methylation, HPV16/18 and HPV16/18/31/33/45 genotyping, respectively. The CIN3+ risk after a negative result could be lowered to 2.8% by combining methylation and extended genotyping, at the expense of a higher referral percentage of 75.5%. Conclusion The use of FAM19A4/miR124-2 methylation and/or HPV genotyping in HPV-positive women with BMD can lead to a substantial reduction in the number of direct colposcopy referrals.

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.