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Vulvar squamous cell carcinoma (VSCC) pathogenesis is traditionally defined by the presence or absence of human papillomavirus (HPV), but the definition of these groups and their molecular characteristics remains ambiguous across studies. Here, we present a retrospective cohort analysis of 36 patients with invasive VSCC where HPV status was determined using RNA in situ hybridization (ISH) and polymerase chain reaction (PCR). Clinical annotation, p16 immunohistochemistry (IHC), programmed death ligand-1 (PD-L1) IHC, HPV16 circular E7 RNA