Background Human papillomavirus-independent, TP53 wild-type verruciform acanthotic vulvar intraepithelial neoplasia [HPVi(p53wt) vaVIN] is a precursor lesion for HPVi(p53wt) vulvar squamous cell carcinoma (VSCC) characterized by acanthotic or verruciform architecture, altered squamous maturation, minimal cytologic atypia, negative p16, and wild-type/non-aberrant p53 expression. Although cytokeratin 17 (CK17) positivity can sometimes aid in the distinction between reactive processes and vaVIN, it does not perfectly distinguish these categories and diagnostic challenges remain. Design A search of the pathology archive at a large academic institution identified 4 patients with verrucous lesions meeting morphologic criteria for vaVIN arising in lichen sclerosus. By immunohistochemistry, these lesions were negative for p16, positive for CK17, and exhibited non-aberrant p53 expression. Each lesion showed at least focal positivity for low-risk HPV by RNA in situ hybridization (ISH; see Figure), raising the differential diagnosis of condyloma acuminatum or viral verruca. To further investigate this uncharacteristic feature, lesional tissue samples from each patient were assessed for the presence of HPV DNA using hybrid capture-based next-generation sequencing (NGS) with the Twist Biosciences Comprehensive Viral Research Panel (CVRP), which targets 143 HPV subtypes (including all those covered by the HPV RNA ISH assays). In addition, whole-exome sequencing (WES) using the Twist Biosciences Exome 2.0 Panel was utilized to compare mutation profiles of lesional and normal tissues. Results Hybrid capture NGS with CVRP generated at least 7,416,001 unique reads per sample, none of which showed significant alignment to any of the targeted HPV genomes (HPV-negative). WES showed that all 4 lesions harbored mutations associated with HPVi vulvar neoplasia, including 2 cases with HRAS hotspot mutations, 2 cases with PIK3CA hotspot mutations, and 2 cases with deep deletion (two-copy loss) of CDKN2A. See Table for further details. Conclusions All 4 lesions meeting criteria for vaVIN were HPV-negative by hybrid capture NGS despite the presence of nuclear positivity for low-risk HPV types by RNA ISH. The vaVIN diagnoses were substantiated by detection of mutations previously reported in HPVi(p53wt) vaVIN/VSCC. This series highlights the importance of thorough pathologic examination of vulvar lesions and underscores pitfalls in interpretation of RNA ISH studies for low-risk HPV.