Host-cell DNA methylation biomarkers in cervical cancer screening: towards molecular screening on self-collected samples

The implementation of primary HPV-based screening has significantly improved the efficacy of cervical cancer screening. However, the majority of HPV infections is transient and will be cleared within months following infection and does not lead to the development of cancer. The use of DNA methylation biomarkers involved in cervical carcinogenesis may result in a more accurate cervical cancer screening. In this thesis we investigated the potential use of DNA methylation markers as a molecular tool in HPV-based cervical cancer screening. In Chapter 2, we described the triage performance of six host-cell DNA methylation markers (i.e., ASCL1, LHX8, ST6GALNAC5, GHSR, SST and ZIC1) for detection of CIN3 and cervical cancer in a cross-sectional series of 577 hrHPV-positive clinician-collected cervical samples. This study demonstrated that all six methylation markers exhibited comparable high performance for triaging hrHPV-positive women using clinician-collected cervical samples, with complementarity among the markers. In Chapter 3, we validated 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. The cohort included 715 hrHPV-positive clinician-collected cervical samples of women aged 29-60 years with the primary endpoint of CIN3 or cancer (CIN3+). 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. In Chapter 4, we analysed the performance of methylation markers ASCL1/LHX8 as potential molecular triage markers for hrHPV-positive self-collected samples. This study suggest that the ASCL1/LHX8 methylation marker panel is a feasible direct triage method for detecting CIN3+ in hrHPV-positive women who participate in routine screening through self-sampling. In Chapter 5, we investigated a direct cell conversion protocol as an alternative to the commonly used bisulphite protocol. The results of this study show that the direct cell conversion protocol had a high success rate and good analytical performance on clinician-collected cervical samples. This practical workflow may provide the basis for automation to aid effective high-throughput DNA methylation analysis and support a fully molecular solution to cervical cancer screening. In Chapter 6, we evaluated various molecular secondary triage methods for hrHPV-positive women with ASC-US/LSIL cytology to identify CIN3+. The study utilised 194 HPV-positive, ASC-US/LSIL clinician-collected cervical samples from the Dutch IMPROVE screening trial (NTR5078) that had been tested for FAM19A4/miR124-2 and ASCL1/LHX8 methylation, as well as had been genotyped for 14 hrHPV types. This study underscores the potential of incorporating methylation analysis, possibly combined with HPV genotyping, as a secondary triage test for HPV-positive women with ASC-US/ LSIL cytology. The proposed strategies enhance the efficiency of cervical screening by significantly reducing the number of colposcopy referrals at baseline. In Chapter 7, we assessed the potential of host-cell methylation markers in identifying vaccinated women with clinically relevant disease, performing a pilot study with FAM19A4 and miR124-2 host-cell methylation markers on 403 cervical samples of vaccinated women aged 25 years. This study showed that in vaccinated women host-cell methylation levels were low, with in nearly all vaccinated HSIL cases levels inalterably like controls. It is plausible that among the HPV-vaccinated women most cervical lesions would regress without the need of treatment. However, these results on the potential of host-cell methylation markers to identify vaccinated women with spontaneously regressive lesions from those who will progress, require validation in larger HPV-vaccinated cohorts with an extended age range and follow-up period. Finally, Chapter 8 provides a general discussion of the findings presented in this thesis, highlighting the expectations of host-cell DNA methylation markers in the prevention of cervical cancer.