52 Chapter 2 DISCUSSION DNA methylation of host-cell genes is a common event in cervical carcinogenesis 22, 23. In recent years, multiple promising host-cell DNA methylation markers for the detection of CIN3+ in HPV-positive women have been identified by targeted and genome-wide approaches 18, 19, 31, 34, 35, 41, 42. However, it is difficult to mutually compare the diagnostic performance of markers due to different techniques and the use of different study material. For the application in HPV-based cervical screening, this study evaluated six recently identified methylation markers for CIN3 detection by a direct head-to-head comparison on HPV-positive cervical samples. We demonstrated that all single markers, derived from two different genome-wide discovery screens, had a similarly good performance for the detection of CIN3 (AUC 0.774 - 0.886). Upon validation by LOOCV, nearly identical AUCs were obtained, indicating the stability of the prediction models. Three single markers (ASCL1, LHX8 and ZIC1) showed particularly good performance as quantified by cross-validated AUCs greater than 0.800 for CIN3 detection. LASSO logistic regression showed a bi-marker panel consisting of ASCL1 and LHX8 to be most discriminative for CIN3 detection with a cross-validated AUC of 0.882, resulting in a sensitivity of 83.4% and specificity of 82.4%. Of note, ASCL1 and LHX8 had a complementary performance resulting in the detection of all cervical cancers, including not only squamous cell carcinomas, but also adenocarcinomas and rare cancers. This further demonstrates the potential of methylation analysis, since non-squamous cell cancer histotypes are often missed by cytology. The concept of increased DNA hypermethylation during cervical carcinogenesis is reflected by the gradual increase of the methylation levels of all six markers with disease severity, reaching highest levels in cervical cancer. Data on a well-studied methylation marker panel with host-cell genes FAM19A4 (currently known as TAFA4) and miR124-2 recently showed that nearly all cervical cancers, including early-stage cancers, from a large worldwide series are detected by methylation analysis 43. Our findings support the high methylation positivity rate in cervical cancer. Though FIGO stage was only available for a subset of samples in our study, all cervical samples of women with known as FIGO stage I cancer (n = 26) scored methylation-positive for the bi-marker panel. These findings underscore the potential of the methylation test for use in population-based screening where most cervical cancers are early-stage. The six methylation markers are potentially not (fully) cervix-specific, as they have been described in other cancers as well 44, 45. Analysis of cervical samples may thereby have added value for the detection of
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