Swab-Derived Viral Load as a Biomarker of Tumor Biology in HPV-Associated Oropharyngeal Squamous Cell Carcinoma.

Abstract

Site-specific identification of human papillomavirus (HPV)-driven oropharyngeal squamous cell carcinoma (HPV+ OPSCC) remains a major diagnostic challenge. We developed a site-directed mucosal swab assay designed to detect and quantify tumor-derived HPV DNA shed into the oropharyngeal surface overlying HPV-associated tumors. Patients with biopsy-proven HPV+ OPSCC or oral cavity SCC as controls were enrolled after providing informed consent. Swabs of the tumor site as well as the contralateral normal mucosa were collected. Swab-derived DNA was extracted and analyzed by quantitative PCR targeting HPV16 and HPV genotyping with Sanger sequencing. Multivariable linear regression models were used to evaluate associations between swab-based viral load and clinicopathologic features. In a prospective cohort of 30 patients with HPV+ OPSCC, tumor-side swabs detected HPV DNA in 30 of 30 cases. Swabs obtained from the contralateral mucosa showed substantially lower HPV DNA levels, demonstrating strong spatial specificity for the tumor-bearing region. The assay limit of detection was determined to be ~10 HPV16 copies per reaction (1 viral genome per 500 diploid cells). All negative control patients with oral cavity SCC had negative results. Regression models revealed node-positive status to be independently associated with low quantitative HPV16 viral load (fold change = 0.013, 95% CI: 0.001-0.178). By enabling minimally invasive, anatomically targeted detection of HPV-driven cancer at the primary site, this swab-based assay has the potential to shorten the diagnostic pathway for HPV+ OPSCC while providing quantitative viral load measurements that may serve as a surrogate biomarker for intratumoral biology in HPV+ OPSCC.