Distinct Anaerobic Microbial Signature Differentiates Anal Squamous Cell Carcinoma From High-Grade Squamous Intraepithelial Lesions: A Prospective Metagenomic Study

Background: Anal squamous cell carcinoma (ASCC) develops from high-grade squamous intraepithelial lesions (HSIL), but only a subset of patients with HSIL progress to invasive cancer, suggesting that factors beyond persistent human papillomavirus (HPV) infection contribute to disease progression. The anal microbiome may be a contributing factor; however, direct comparisons between HSIL and treatment-naive ASCC remain limited. Methods: Anal canal swabs were collected from adults with histologically confirmed HSIL or nonmetastatic ASCC prospectively enrolled at Montefiore Medical Center from September 2022 through October 2024. Shotgun metagenomic sequencing was performed after human-read removal. Microbial diversity and community composition were compared between disease groups. Differentially abundant species were identified using ANCOM-BC2 and validated using adjusted centered log-ratio regression. Longitudinal changes and MetaCyc pathways associated with persistent ASCC-enriched species were also evaluated. Multivariable analyses adjusted for age, sex, tobacco use, HIV status, and HPV status. Results: Among 121 participants, 102 had HSIL and 19 had ASCC. Observed richness was lower in ASCC (adjusted beta = -21.28; p = 0.035), while Shannon and inverse Simpson diversity did not differ. Microbial community composition differed by disease group using Bray-Curtis (R2 = 0.100; p = 0.001), Jaccard (R2 = 0.133; p = 0.002), and Aitchison distances (R2 = 0.172; p = 0.001). Of 60 species identified by ANCOM-BC2, 58 were validated, including six enriched in ASCC: Fusobacterium nucleatum, Parvimonas micra, Peptococcus niger, Porphyromonas asaccharolytica, Porphyromonas levii, and Porphyromonas sp. CAG:1061. All six remained associated with ASCC longitudinally, and five demonstrated greater baseline-to-month-6 decreases in ASCC than HSIL. Twenty-six MetaCyc pathways were linked to the persistent ASCC-enriched species and were also enriched in ASCC, highlighting shared functions in amino acid metabolism, anaerobic degradation, and carbohydrate and energy metabolism. Conclusion: ASCC was characterized by lower observed richness and enrichment of a distinct anaerobic microbial signature that persisted across longitudinal sampling and declined following treatment initiation. These taxa were linked to a shared functional profile dominated by amino acid metabolism and anaerobic metabolic pathways. These findings identify microbial features associated with invasive anal cancer and provide a foundation for evaluating their relationship with disease progression, tumor burden, and treatment response.