2117 - Anal Microbiome Profiles Differ Between HSIL and Treatment-Naïve Anal Squamous Cell Carcinoma

Purpose/Objective(s): Progression from high-risk HPV infection or High-grade squamous intraepithelial lesions (HSIL) to anal squamous cell carcinoma (ASCC) occurs in only a subset of patients, the factors driving this remain poorly understood. The anal microbiome may contribute to both anal carcinogenesis and response to chemoradiation. The purpose of this study was to analyze the microbiome dynamics in patients with HSIL and ASCC as a prespecified component of an ongoing prospective clinical trial.

Materials/Methods: Anal swabs were collected at enrollment for patients with HSIL and throughout treatment for patients with ASCC between September 2022–October 2024. Shotgun metagenomic sequencing was performed and reads underwent taxonomic profiling to the species' level. Core taxa were defined using relative abundance and prevalence thresholds. Alpha diversity was compared using Wilcoxon tests and multivariable linear regression adjusting for age, sex, tobacco use, HIV status, and HPV status. Beta diversity was assessed using Bray–Curtis, Jaccard, and Aitchison distances with PERMANOVA. Differential abundance was evaluated using false discovery rate–corrected testing and ANCOM-BC2 with covariate adjustment.

Results: A total of 121 participants were included, comprising 102 with HSIL and 19 with ASCC. Patients with ASCC were older (mean 62.4 vs. 52.8 years, p = 0.023). HIV positivity (90.2% vs. 52.6%, p < 0.001) was higher in HSIL patients, while tobacco use was similar. Among ASCC patients, 89.4% had stage II–III disease. Alpha diversity measures (Shannon, inverse Simpson) were similar between groups before and after adjustment, but observed richness was higher in HSIL compared to ASCC (p = 0.009; adjusted p = 0.038). Beta diversity differed significantly by disease group across multiple metrics, including Bray–Curtis (p = 0.001), Aitchison (p = 0.001), and Jaccard distances (p = 0.002), with no differences in dispersion for Bray–Curtis or Aitchison distances. Core microbiome composition was largely shared (377 species), with 74 taxa unique to HSIL and 79 unique to ASCC. An exploratory random forest classifier demonstrated good discrimination between HSIL and ASCC (AUC 0.849; 95% CI 0.809–0.888), with Porphyromonas somerae, P. asaccharolytica, Campylobacter ureolyticus, Parvimonas micra, and Fusobacterium nucleatum among the most important taxa.

Conclusion: ASCC is associated with decreased richness and enrichment of pro-inflammatory and oncogenesis-associated taxa including Fusobacterium, Porphyromonas, and Campylobacter species which are distinct from the taxa found in HSIL patients. These findings support a potential role for the anorectal microbiome in anal carcinogenesis. Future work will characterize longitudinal microbiome dynamics during treatment for ASCC, assess their impact on treatment response and toxicity, and explore the anal microbiome as a potentially modifiable patient characteristic.