The Role of Molecular Testing in Head and Neck Squamous Cell Carcinoma of Unknown Primary.

Squamous cell carcinomas of unknown primary (SCCUP), accounting for approximately 5% of head and neck malignancies, present significant clinical challenges in achieving disease control while minimizing therapy-related morbidities in the absence of a clear tumor origin. Biomarker assessment has traditionally focused on the detection of human papillomavirus and Epstein–Barr virus, which are strongly associated with oropharyngeal and nasopharyngeal carcinomas, respectively, and carry important diagnostic and therapeutic implications. Recent advances in molecular diagnostics have enabled integrative analysis of diverse targets, including tumor viral status and pathogenic genomic variants, by next-generation sequencing, improving specimen efficiency and analytic resolution of tumor-virus relationships, such as the identification of viral integration sites. In addition, emerging computational biomarkers, including mutational signature analysis and machine learning-assisted DNA methylation profiling, have shown promising performance in distinguishing squamous cell carcinomas arising from different sites, potentially providing valuable diagnostic support upon clinical validation. In parallel, ongoing clinical trials are investigating novel therapeutic regimens targeting major oncogenic alterations in head and neck cancer, including HRAS, PIK3CA, and CDKN2A mutations, with encouraging early results. This review summarizes recent progress in molecular testing for SCCUP, emphasizing the critical role of pathologists in ensuring analytical rigor and translating molecular findings into optimal patient care.