CANCER GENETICS AND EPIGENETICS 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 Lisanne Verhoef 1 | Maaike C. G. Bleeker 1 | Nicole Polman 1 | Renske D. M. Steenbergen 1 | Chris J. L. M. Meijer 1 | Willem J. G. Melchers 2 | Ruud L. Bekkers 3,4 | Anco C. Molijn 5 | Wim G. Quint 5 | Folkert J. van Kemenade 6 | Johannes Berkhof 7 | Daniëlle A. M. Heideman 1 1 Amsterdam UMC, Vrije Universiteit Amsterdam, Pathology, Cancer Center Amsterdam, Amsterdam, The Netherlands 2 Radboud University Medical Center, Medical Microbiology, Nijmegen, The Netherlands 3 GROW School for Oncology and Developmental Biology, Maastricht University, Maastricht, The Netherlands 4 Department of Obstetrics and Gynaecology, Catharina Hospital, Eindhoven, The Netherlands 5 DDL Diagnostic Laboratory, NMDL-LCPL, Rijswijk, The Netherlands 6 Erasmus MC, University Medical Center, Pathology, Rotterdam, The Netherlands 7 Amsterdam UMC, Vrije Universiteit Amsterdam, Epidemiology and Data Science, Amsterdam, The Netherlands Correspondence Daniëlle A. M. Heideman, Amsterdam UMC, Vrije Universiteit Amsterdam, Pathology, Cancer Center Amsterdam, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands. Email: dam.heideman@amsterdamumc.nl Abstract Methylation of host-cell deoxyribonucleic acid (DNA) has been proposed as a promis- ing biomarker for triage of high-risk (hr) human papillomavirus (HPV) positive women at screening. Our study aims to validate recently identified host-cell DNA methyla- tion 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 previ- ous series of hrHPV-positive clinician-collected cervical samples were applied. The diagnostic accuracy was visualised using receiver operating characteristic (ROC) cur- ves and assessed through area under the ROC curve (AUC). The performance of the methylation markers to detect CIN3+ was determined using the predefined thresh- old 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 Abbreviations: ACTB, β-actin; AIS, adenocarcinoma in situ; ASCL1, achaete-scute family BHLH transcription factor 1; ASC-US, atypical squamous cells of undetermined significance; AUC, area under the curve; BMD, borderline or mild dyskaryosis; CI, confidence interval; CIN, cervical intraepithelial neoplasia; CIS, carcinoma in situ; CISOE-A, composition, inflammation, squamous, other and endometrium, and endocervical cylindrical epithelium, and adequacy; Cq, quantification cycle; DNA, deoxyribonucleic acid; GHSR, growth hormone secretagogue receptor; H&E, haematoxylin-eosin; HPV, human papillomavirus; hr, high risk; ICC, intraclass coefficient; LHX8, LIM homeobox 8; LSIL, low-grade squamous intraepithelial lesions; N, group size; n, number of; NILM, negative for intraepithelial lesion or malignancy; NPV, negative predictive value; PALGA, nationwide network and registry of histo- and cytopathology in the Netherlands; PPV, positive predictive value; qMSP, quantitative methylation-specific PCR; ROC, receiver operating characteristic; RR, relative risk; SST, somatostatin; ST6GALNAC5, ST6 N-acetylgalactosaminide alpha- 2,6-sialyltransferase 5; ZIC1, zinc finger of the cerebellum family member 1. Received: 18 May 2021 Revised: 17 August 2021 Accepted: 31 August 2021 DOI: 10.1002/ijc.33820 This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. © 2021 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC. 440 Int. J. Cancer. 2022;150:440–449. wileyonlinelibrary.com/journal/ijc