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

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.