Anal cancer causes significant morbidity as the second most common solid malignancy in People with HIV (PWH) with >2,000 new cases per year. Our data show that anal cancer incidence is rapidly growing in the US. This trend is even more pronounced in HIV infected men who have sex with men where the lifetime risk is 10% and is projected to increase despite advances in early detection and prevention. Anal Cancer is more prevalent in PWH above 50 years of age compared to those under 50. Prevention of anal cancer centers around identification and ablation of high-grade squamous intraepithelial lesions (HSIL), the immediate precursors of carcinoma. HSIL are highly aggressive in PWH, with 1% per year progressing to invasive carcinoma. Treatment efficacy is limited for PWH, however, as 50% of patients with treated HSIL will have high-grade lesions detected in the same anatomic quadrant. There are indications that treatment failure rates increase with age, but the data is limited and biomarkers for predicting treatment failure are needed and may be age dependent. Objective: The goal of this project is to understand whether age disparities are associated with HSIL aggressiveness and to identify biomarkers of aggressiveness. Specific Aims: (1) To determine the failure rate of treatment with increasing age; (2) and to determine the extent of clonality in anal high-grade squamous intraepithelial lesions (HSIL) across age groups and test the association of clonality with lesion aggressiveness in people with HIV. Study Design: We propose to study and sequence HSIL from a tissue bank of >2,000 specimens from an ethnically diverse cohort of PWH harboring HPV-associated HSIL. We will first determine the frequency of lesions found in the same anatomic quadrant after ablation in people above 50 years of age and those below 50. This will establish if treatment is failing with age and if it is correlated to the number of recurrent lesions. We will randomly select 200 HSIL unselected to outcome by age to use for the prediction of HSIL that will recur after ablation to improve risk stratification.