Abstract 3857: Comprehensive profiling of circulating tumor DNA in blood enables sensitive detection and genomic characterization of HPV cancer and precancer.

Abstract Background: Human papillomavirus (HPV) causes six cancer types, yet five lack population-level screening. We have developed HPV-DeepSeek, a blood-based, multi-feature HPV whole-genome sequencing assay targeting circulating tumor HPV DNA (ctHPVDNA). HPV-DeepSeek has detected HPV+ oropharynx cancer years prior to clinical diagnosis, establishing the feasibility of blood-based HPV+ cancer screening. Here we extend testing to HPV+ anal cancer (HPV+AC) with well-defined precancer stages, to comprehensively assess blood-based detection of HPV+ cancer and precancer. Methods: HPV-DeepSeek was applied to a pre-diagnostic cohort collected 2.8-8.6 years before HPV+AC diagnosis (N=6) and to a prospective cohort (SCAN-LITE, NCT06971276), comprising HPV+AC (N=10), anal intraepithelial neoplasia 3 (AIN3) (N=20), AIN2 (N=20), AIN1 (N=20), anal HPV-infection (N=20), anal HPV-negative with past HPV history (N=14), and healthy controls (N=60). For validation, 78 paired tissue samples were profiled with HPV-DeepSeek and 5 underwent PacBio long-read sequencing. Results: 4/6 pre-diagnostic samples tested positive, with detection lead time 2.8 to 7.2 years before cancer diagnosis. In the SCAN-LITE study, the sensitivity for HPV+AC was 100% (10/10). Profiling ctHPVDNA detected HPV-human integration in 6/10 cases and HPV-HPV rearrangements in 8/10, which were validated in paired tissue. PIK3CA mutations were found in 2/10 cases, and CNVs were detected in 3/10 cases at the chr3q hotspot. In precancers, ctHPVDNA was detectable with decreasing positivity by disease severity: 32.5% in AIN3/AIN2 and 12.5% in AIN1/infection. 1/14 (7.1%) HPV-negative control tested positive and was later diagnosed with HPV-infection at follow-up. All healthy controls were negative, yielding 100% specificity. No HPV integration, PIK3CA mutations, or CNVs were detected in precancer. While HPV+AC primarily showed HPV16 (10/10) with one case also harboring HPV35, precancer exhibited diverse genotypes. 20 genotypes were detected, including 6 multi-genotype infections found exclusively in high-grade precancers (AIN3/2). Fragmentomics analysis revealed distinct length profiles across stages, particularly di-nucleosome peaks enriched in HPV+AC while depleted in precancer, reflecting HPV epigenetic changes during carcinogenesis. Conclusions: HPV+AC is detectable in blood at and before clinical diagnosis. We also show, for the first time, that HPV precancer can be detected in blood, with increasing positivity as stages progress. Blood-based detection of HPV+ cancer hallmarks, including HPV integration, PIK3CA mutation, and CNV, differentiates cancer from precancer, while fragmentomics adds to stage-specific signatures. Together, these findings support the feasibility of blood-based HPV+ cancer and precancer screening with the potential of stage differentiation. Citation Format: Qin Wang, Samuli Eldorfs, Sangmi Sandra Lee, Yana Al-Inaya, Gjystina Lumaj, Eliana Epstein, Dipon Das, Emma Ricart, Harsharan Dhillon, Juniper Lake, Michael G. Drage, Shun Hirayama, Viktor Adalsteinsson, Benjamin T. Davis, Doga C. Gulhan, Daniel Faden. Comprehensive profiling of circulating tumor DNA in blood enables sensitive detection and genomic characterization of HPV cancer and precancer abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3857.