46 Chapter 2 markers associated with CIN3 and cervical cancer. Methylation markers GHSR, SST and ZIC1 were identified using a methyl-binding domain-enriched DNA-based discovery screen on high-risk HPV-transformed cell lines and cervical tissue specimen 34 and methylation markers ASCL1, LHX8 and ST6GALNAC5 were discovered using an array-based methylation discovery screen on HPV-positive self-collected cervico-vaginal material 34, 35. These six methylation markers showed to be most promising for the triage of HPV-positive women in subsequent verification and validation series using a multiplex quantitative methylation-specific PCR (qMSP). DNA methylation markers GHSR, SST and ZIC1 resulted in AUCs of 0.87, 0.86 and 0.89, respectively, for CIN3+ detection in HPV-positive cervical samples 34. DNA methylation markers ASCL1, LHX8 and ST6GALNAC5 resulted in a combined AUC of 0.88 and 0.90 for CIN3+ detection in HPV-positive lavage- and brush self-samples, respectively 35. These markers were initially evaluated in different cervical sample series, comprising cervical samples and self-collected cervico-vaginal material. This study was designed as a next step to assess and compare the clinical performance of these methylation markers on the same cohort. The six recently identified host-cell DNA methylation markers were evaluated on HPV-positive cervical samples as a molecular triage method for the detection of CIN3 and cervical cancer for the application in HPVbased cervical screening. MATERIAL AND METHODS CLINICAL SPECIMENS A series of HPV-positive cervical samples (n = 527) obtained from screening or gynaecologic outpatient populations was used, comprising 175 samples from women who were histologically diagnosed with CIN3 (median age 36.5 years) and 352 samples from control women (median age 38.0 years). The controls consisted of women with no evidence of CIN2+ classified as CIN1 or less (≤CIN1), including 225 women with no histology, 64 with normal histology (CIN0) and 63 with histologically proven CIN1. The histological diagnosis of CIN2 comprises a heterogeneous disease category and was therefore not evaluated in this study 36. In addition, 50 HPV-positive cervical samples from women diagnosed with cervical cancer were used (median age 47.0 years; squamous cell carcinoma n = 40; adenocarcinoma n = 4; adenosquamous carcinoma n = 2; clear cell carcinoma n = 1; neuroendocrine carcinoma n = 2; gastric-type mucinous adenocarcinoma n = 1). HPV testing was performed using clinically validated high-risk HPV DNA assays 37. This study followed the ethical guidelines of the Institutional Review Board of VU University Medical Centre and University Medical Centre Groningen.
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