Methylation 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.
As cervical screening transitions from cytology to primary human papillomavirus (HPV) testing worldwide, effective triage tests are increasingly needed. Here, the authors report on the performance of host‐cell DNA methylation biomarkers ASCL1, LHX8, ST6GALNAC5, GHSR, ZIC1, and SST in an HPV‐positive cohort derived from primary HPV‐based screening in The Netherlands. All markers exhibited significant differences in methylation levels between cervical intraepithelial neoplasia grade 3 or worse (CIN3/CIN3+) and CIN1, CIN2, and women with normal histology. The robust triage performance for CIN3+ as compared to cytology and HPV16/18 genotyping highlights the potential of methylation biomarker‐based triage for HPV‐positive women.
Keywords: cervical intraepithelial neoplasia; clinical performance; cytology; diagnostic accuracy; epigenetic; genotyping; primary HPV screening.
© 2021 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.