General introduction and thesis outline 29 1 women without clinically relevant infections. Several strategies have been proposed, yet a consensus on the optimal triage test remains elusive. Each proposed strategy carries its own set of strengths and limitations, both in terms of clinical performance and screeningrelated burden. Although current triage methods, such as cytology and HPV16/18 genotyping, demonstrate acceptable clinical performance, there exists a demand for alternative methods applicable to both clinician-collected and self-collected cervical screening samples. This need is underscored by contemporary challenges, including reducing over-referral and overtreatment, the implementation of self-sampling as a primary screening tool, the call for automation, and the inclusion of vaccinated women in the screening program. DNA methylation markers emerge as promising objective biomarkers capable of identifying women with clinically relevant cervical disease, offering potential solutions to the aforementioned challenges. Chapter 2 evaluates six novel host-cell DNA methylation markers derived from two genome-wide discovery screens. The assessment focuses on the detection of cervical cancer and CIN3 using hrHPV-positive clinician-collected cervical samples. Building on the findings of Chapter 2, Chapter 3 validates the host-cell DNA methylation markers, for detection of CIN3+ on clinician-collected cervical samples. The validation is conducted on a large and independent cohort of hrHPV-positive women who participated in primary HPV-based screening. Chapter 4 delves into an analysis of the best-performing markers in Chapter 3, namely ASCL1 and LHX8, for the detection of CIN3+ in self-collected samples from hrHPV-positive women who underwent primary HPV self-sampling for cervical cancer screening. Chapter 5 centres on the evaluation of a bisulphite conversion protocol directly applicable to clinician-collected cervical samples. This protocol is designed to eliminate the need for prior DNA isolation, aiming to establish an automated laboratory workflow for increased efficiency. Chapter 6 explores the utilisation of FAM19A4/miR124-2 methylation, ASCL1/LHX8 methylation and HPV genotyping analysis as a secondary triage tool in hrHPV-positive women with ASC-US/LSIL cytology, aiming to enhance the efficiency of the triage process. Chapter 7 explores FAM19A4/miR124-2 methylation in a pilot series of cervical samples from young women who have been vaccinated against HPV. Chapter 8 provides a general discussion, summarising the results of this thesis in light of existing evidence. It discusses the clinical implications of the findings and explores potential avenues for further research. Finally, Chapter 9 offers a concise summary of the thesis, providing a quick overview of the key contributions and findings presented in each chapter.
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