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Human papillomavirus has been implicated in the carcinogenesis of squamous cell carcinoma of the anal canal. p16 expression and the presence of human papillomavirus DNA have been used to define human papillomavirus-positive patients, but neither approach has been validated against the standard of human papillomavirus E6/7 mRNA expression at this disease site. This study aimed to evaluate the acceptability of p16 immunohistochemistry as a surrogate to E6/7 mRNA expression in identifying human papillomavirus-mediated squamous cell carcinoma