HPV Genotype Landscape in Anal Squamous Cell Carcinoma Across Primary, Recurrent, and Metastatic Tumor Specimens.

BACKGROUND/OBJECTIVES: Anal squamous cell carcinoma (SCCA) is an HPV-driven malignancy with rising incidence, yet systematic HPV genotyping across recurrences and metastases is lacking. We characterized the complete HPV genotype distribution across the disease course. METHODS: We retrospectively analyzed 213 tumor specimens from 136 patients (82 female, 54 male) with histologically confirmed SCCA, spanning primary, recurrent, and metastatic disease (2009-2019 and from 2020 onwards). Patients contributed one or more specimens depending on the number of sampled disease events; specimens comprised 138 primary biopsies, 55 recurrence biopsies, 2 re-recurrence biopsies, and 18 distant metastases. HPV genotyping was conducted using a validated 28-type multiplex real-time PCR assay (Anyplex™ II HPV28, Seegene Inc.). RESULTS: HPV DNA was detected in 179/213 specimens (84.0%; 95% CI 78.9-88.7) and 125/136 patients (91.9%; 95% CI 86.8-96.3). Across 269 HPV type detections, 24 distinct types were identified, with HPV 16 predominating both per-specimen (90.5%) and per-detection (60.2%) values. Non-HPV-16/18 high-risk types accounted for one in four high-risk detections (54/219; 24.7%; 95% CI 19.4-30.8). HPV positivity differed significantly by specimen type: 90.6% in primary biopsies versus 70.9% in recurrences (p = 0.001) and 72.2% in metastases (p = 0.038). Co-infections occurred in 24.6% of HPV-positive specimens (95% CI 18.5-31.6), with up to eight types. In metastases, the spectrum narrowed to 4 of 24 types; two harbored HPV 33 as the sole high-risk type. CONCLUSIONS: SCCA harbors a substantially broader HPV type spectrum than previously recognized. One in four high-risk detections lies beyond HPV 16/18, and high-risk types other than HPV 16 were the sole high-risk genotype detected in individual metastatic specimens. HPV positivity rates are significantly lower across specimen types, while the metastatic type spectrum converges on a few high-risk genotypes. Broad-spectrum HPV genotyping beyond types 16 and 18 warrants adoption as standard practice in SCCA diagnostics and surveillance, and potential stratification for HPV-directed therapies.