198 Chapter 9 using clinician-collected cervical samples, with complementarity among the markers. Chapter 3 validates the triage performance of the methylation markers described in Chapter 2 in an independent hrHPV-positive cohort derived from primary HPV-based cervical cancer screening in the Netherlands. Until that time, most if not all studies evaluating host-cell DNA methylation markers had been performed in referral populations or case-control settings. This study utilised bio-banked samples from the Dutch IMPROVE screening trial (NTR5078), nested within the population-based screening setting, that was carried out in 2015-2016. It aimed to reflect the new primary HPV-based screening program introduced in 2017 in the Netherlands, signifying that the results could be representative for routine screening. The cohort included 715 hrHPV-positive cliniciancollected cervical samples of women aged 29-60 years with the primary endpoint of CIN3 or cancer (CIN3+). The study validates that methylation levels of the markers ASCL1, LHX8, ST6GALNAC5, GHSR, SST and ZIC1 increase with the severity of the underlying cervical disease. The performance for CIN3+ detection was comparable to the study described in Chapter 2, with highest AUC for ASCL1 (0.844) and LHX8 (0.830). When combined as a bi-marker panel, with the predefined threshold that was set in Chapter 2, ASCL1/LHX8 detected CIN3+ with a 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%). Utilising revision diagnoses with biomarkers p16INK4A and Ki-67 provided similar results, supporting the application of methylation analysis as a triage tool. This study confirms the clinical usefulness of the individual host-cell DNA methylation markers and the bi-marker panel ASCL1/LHX8 for the detection of CIN3+ in hrHPV-positive women invited for routine screening. The implementation of primary HPV-based screening provides the opportunity of using self-sampling as an alternative sampling method for all women invited for screening, which can potentially overcome barriers to cervical cancer screening and increase coverage. However, the current triage method for hrHPV-positive women, cytology, is less reliable on self-collected samples and necessitates recalling hrHPV-positive women who participated with self-sampling for clinician-based sampling, resulting in a 18% loss to follow-up. Hence, there is a need for molecular triage markers that can be directly applied to self-samples. Chapter 4 analyses the performance of methylation markers ASCL1/LHX8 as potential molecular triage markers for hrHPV-positive self-collected samples. Previous research indicated the potential use of host-cell DNA methylation analysis on self-collected samples to triage hrHPV-positive women. However, these data were mainly restricted to under-/never-screened women and referral populations. The
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