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Follow-up of high-risk HPV positive women by combined cytology and bi-marker CADM1/MAL methylation analysis on cervical scrapes 8 Chapter 8 136 ABSTRACT Objectives Triage of HPV screen-positive women is needed to identify those with underlying cervical intraepithelial neoplasia grade 2/3 or worse (CIN2/3+). Presently, cytology on a physician-taken cervical scrape at baseline is mostly accepted as triage test, but needs follow-up testing in order not to miss severe disease. Here, we evaluated the performance of combined cytology and bi-marker CADM1/MAL methylation analysis as triage test on physician-taken cervical scrapes of HPV positive women. Methods In this post-hoc analysis, we used 364 left-over HPV positive cytology triage samples of participants of a randomized controlled trial (PROHTECT-3: n=46,001, age 33-63 years) performed in the population-based cervical screening program. Study endpoints were CIN2+ and CIN3+ detection. Cytology testing with and without methylation marker analysis was evaluated with regard to sensitivity, specificity, positive and negative predictive value, and referral rate. Results Bi-marker CADM1/MAL methylation positivity increased proportionally with severity of the underlying lesion. Overall, cytology and bi-marker CADM1/MAL methylation analysis yielded similar performances with regard to CIN3+ detection, yet in combination a significantly higher sensitivity for CIN3+ (88.7%) was obtained at a specificity of 53.6% and a colposcopy referral rate of 53.6%. The combined strategy detected all six cervical cancers, whereas triage by cytology alone failed to detect two of them. Conclusions Cytology and bi-marker CADM1/MAL methylation analysis perform complementary for CIN2+/ CIN3+ detection when used as triage tool on cervical scrapes of HPV positive women. This approach not only results in a higher CIN3+ sensitivity than cytology triage with an acceptable referral rate, but also seems to reduce the risk of missing cervical cancers and advanced high-grade lesions.