Evolution of testing for the diagnosis of human papillomavirus (HPV) status in head and neck squamous cell carcinoma: Where from and where to?

• The various test methods used for the detection of human papillomavirus (HPV) in head and neck squamous cell carcinomas are reviewed. • Methods include p16 immunohistochemistry, HPV DNA PCR, HPV E6/E7 mRNA RT-PCR, DNA in situ hybridization and RNA in situ hybridization. • A combined testing approach is recommended - screening with p16 immunohistochemistry and confirming with HPV DNA PCR. • Liquid biopsies to detect HPV biomarkers demonstrate promising clinical utility to aid diagnosis. • Plasma HPV circulating tumour DNA is a leading biomarker, while HPV16 E6 antibodies and oral HPV DNA could also serve as adjunct tests for diagnosis. Human papillomavirus (HPV) is causally associated with head and neck squamous cell carcinomas with the strongest association in the oropharynx. HPV-associated oropharyngeal carcinomas have a different pathogenesis with distinct clinical features and better prognosis than HPV-negative oropharyngeal carcinomas which impacts staging and prognosis. It is, therefore, of clinical significance to accurately determine the HPV status, particularly in oropharyngeal carcinomas. In this review, the different test methods that are used for characterizing HPV status in head and neck squamous cell carcinomas, both conventional methods (p16 immunohistochemistry, HPV DNA in-situ hybridization, HPV DNA PCR, HPV E6/E7 mRNA RT-PCR, HPV RNA in-situ hybridization) as well as emerging novel approaches (HPV circulating tumour DNA, HPV16 E6 antibodies, oral HPV DNA/mRNA PCR), are discussed. Currently, a combined testing approach is favoured, using a sequential strategy of screening with p16 immunohistochemistry and confirming with HPV DNA PCR. HPV RNA in-situ hybridization could potentially serve as a single test owing to its good sensitivity and specificity. The use of liquid biopsies is gaining momentum with HPV circulating tumour DNA as the frontrunner in demonstrating promising clinical utility for early detection in HPV-associated oropharyngeal carcinomas. HPV16 E6 antibodies and oral HPV DNA PCR has potential utility as adjunct tests to aid diagnosis. In this rapidly evolving HPV testing landscape, we as clinicians and laboratorians must evolve and advocate for access to cost-effective accurate HPV testing globally.