Assessment of six markers for cervical (pre)cancer detection 45 2 INTRODUCTION Cervical cancer is caused by a persistent infection with human papillomavirus (HPV) 1. Cervical cancer develops through precursor lesions, called cervical intraepithelial neoplasia (CIN), which are classified into CIN1, CIN2, and CIN3 depending on the severity of the dysplasia. The latter is considered as the most advanced precursor of cervical cancer. Cervical screening programs aim to detect and treat precancerous stages in order to prevent cervical cancer. HPV testing is more sensitive for the detection of CIN3 and cervical cancer compared with cytology and has a high negative predictive value and reproducibility 2-4. Primary HPV testing has therefore been implemented or is scheduled for implementation in cervical screening programs in several countries. However, since most HPV infections are transient and do not cause clinically relevant disease 5, additional triage testing is required to identify HPV-positive women with progressive CIN lesions in need of treatment. Besides, triage testing is necessary to prevent over diagnosis and overtreatment of women without clinically relevant CIN lesions. Several triage strategies have been evaluated 6-13, with reflex cytology, repeat cytology, HPV16/18-genotyping, or a combination thereof being adopted in current HPV screening guidelines 14, 15. However, there is no consensus about the most appropriate triage strategy yet. An objective molecular test which could be automated and directly incorporated following a positive HPV result would be most beneficial. Several studies have demonstrated the potential of DNA methylation analysis as an alternative triage method for the detection of CIN3 or worse (CIN3+) in HPV-positive women 16-23. DNA methylation has been identified to play an important role in the development of cervical cancer 22-24. A strong association was found between the methylation levels of host-cell and/or viral genes and the severity of the lesion, reaching highest levels in cervical cancer 20, 25-27. Various DNA methylation marker panels have been proposed, including combinations of host-cell genes such as ASTN1, CADM1, DLX1, EPB41L3, FAM19A4, ITGA4, MAL, miR124-2, PAX1, POU4F3, RXFP3, SOX1, SOX17, ZNF671, and/or CpG sites in the late regions of various HPV genomes 17, 18, 27-31. In several studies these methylation markers have demonstrated to be a useful alternative to cytology or HPV16/18 genotyping for the detection of CIN3+ as a triage test 21. A recent longitudinal evaluation in HPV-positive women from a screening cohort demonstrated a similarly low long-term CIN3+ risk upon a negative methylation test result as was found for a negative cytology result, underscoring the good diagnostic potential of methylation markers 32, 33. Recently, we used genome-wide approaches to identify novel host-cell DNA methylation
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