Methylation markers for cervical cancer prevention

The implementation of primary HPV-based screening has greatly improved the efficacy of cervical cancer screening. The majority of HPV infections is however self-limiting and does not lead to the development of cancer. The use of biomarkers involved in cervical carcinogenesis may result in a more accurate screening for and management of CIN lesions. In this thesis we aimed to assess the value of host cell DNA methylation markers in the detection and classification of cervical premalignant disease, and the management of women at risk for cervical cancer. Part I: Methylation markers in cervical cancer screening In Chapter 2, we described the FAM19A4/miR124-2 methylation positivity rate (98.3%) in a large worldwide series of 519 invasive cervical carcinomas. These results indicate that FAM19A4/miR124-2 methylation has a very high sensitivity for cervical cancer and that a negative test is likely to rule out the presence of cervical cancer. In Chapter 3, we evaluated cross-sectional and long-term triage performance of FAM19A4/miR124-2 methylation analysis in a Dutch population-based HPV-positive screening cohort. This study underlines that methylation analysis has a good triage performance on baseline Dutch screening samples and may be used to build robust triage algorithms with more objective stratification of women referred for colposcopy versus re-testing compared with cytology. In Chapter 4, we studied the discriminative power of FAM19A4/miR124-2 methylation and HPV genotyping in 294 HPV-positive women with BMD cytology. This study shows that the use of FAM19A4/miR124-2 methylation and/or HPV genotyping in HPV-positive women with BMD can lead to a substantial reduction in the number of direct colposcopy referrals. In Chapter 5, we aimed to validate methylation markers ASCL1 and LHX8 for primary methylation analysis in an South African cohort of 411 women living with HIV (WLHIV). Our results confirmed that primary methylation analysis of ASCL1 and LHX8 genes is an full- molecular alternative to cytology- or HPV-based screening in LMIC, without the need for additional triage testing. Part II: Methylation and immunohistochemical markers for improved classi- fication and clinical management of cervical precursor lesions In Chapter 6, we described FAM19A4/miR124-2 methylation and HPV16/18 genotyping patterns in 1061 HPV-positive women <30 years. We found a high specificity of the FAM19A4/miR124-2 methylation test for non-productive, transforming CIN2/3 lesions in women <30 years, providing clinicians supportive information about the need for treatment of CIN2/3 in young HPV-positive women. In Chapter 7, we demonstrated considerable heterogeneity of p16ink4a, Ki-67, HPV E4 staining and FAM19A4/miR124-2 methylation analysis within a series of 497 high- grade CIN. We presented biomarker profiles with presumed cancer progression risks based on the p16ink4a/Ki-67 immunoscores and methylation status. These biomarker profiles could help the clinician in the decision for immediate treatment or a ‘wait-and- see’ policy to reduce overtreatment of high-grade CIN. In Chapter 8, we hypothesized that the concomitant presence of high methylation levels in cervical scrapes with both productive (E4) and transforming characteristics (increased p16ink4a/Ki-67 expression) in WLHIV might indicate the rapid aggressive course of HPV infections towards cancer in these women. In Chapter 9, we evaluated the value of ASCL1/LHX8 methylation analysis in post- treatment monitoring of women treated for CIN2/3. We showed that a negative ASCL1/LHX8 methylation test in follow-up resulted in a low rCIN2/3 risk and could serve as an objective test of cure and safe alternative for HPV and/or cytology screening in the post-treatment monitoring of WLHIV. Finally, Chapter 10 provides a general discussion and overview of the clinical indications of methylation analysis presented in this thesis, in the context of the available literature, and also points out future perspectives.