Abstract Background High-resolution anoscopy (HRA) to prevent anal cancer is complex and screening capacity is limited. Previously, DNA methylation analysis of anal high-grade squamous intraepithelial lesion (HSIL) biopsies was shown to distinguish between HSIL with an increased and a low cancer risk, supporting personalized treatment. Here, methylation analysis on anal swabs was assessed to detect underlying HSIL with an increased cancer risk. Methods A cross-sectional series of paired anal swabs and biopsies of 215 persons with human immunodeficiency virus and swabs of 19 cancer patients were tested for 6 methylation markers. Data were analyzed by logistic regression analysis. The primary endpoint was methylation-positive biopsy HSIL (M+ HSIL), indicating increased cancer risk. Test performance of methylation markers, human papillomavirus (HPV), and/or cytology, and cancer detection and HRA referral, were calculated. Results Anal cancer swabs showed highest methylation. ZNF582 and panels ASCL1/ZNF582 and LHX8/ZNF582 yielded an area under the curve of 0.68–0.70 to detect underlying M+ HSIL. Methylation at 80% sensitivity corresponded to 43% fewer patients requiring HRA, without missing any cancers and detecting 79% of HPV-16–positive HSIL-AIN3. Methylation/HPV and cytology/HPV co-testing performed similarly. Conclusions Methylation levels in anal swabs reflect underlying anal disease. Methylation analysis could reduce HRA referrals substantially, while maintaining a high sensitivity for M+ HSIL and detecting all cancers. These results encourage screening on anal swabs to preselect patients needing HRA.
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