Development of a multiplexed lateral flow assay for the serologic detection of HPV-associated head and neck cancer

Background: Among head and neck squamous cell carcinomas (HNSCCs), the incidence of oropharyngeal cancer (OPC) has been increasing in recent decades. Human papillomavirus (HPV) type 16 is associated with the majority of OPC. Circulating antibodies (Abs) to multiple HPV16 early antigens, including E2, E6, and E7, have been detected in patient sera, and are strongly associated with risk for OPC. However, HPV serology currently requires laboratory-based tests that are difficult to implement for large-scale cancer screening. Objective: The goal of this study was to develop and validate a point-of-care assay for rapid detection of circulating IgG to HPV16 early antigens. Methods: We measured Abs to HPV16 E2, E6, and E7 proteins using a lateral flow assay (LFA) in sera from 119 newly diagnosed OPC cases, 41 partners, and 81 healthy volunteers. The 119 patients with HPV-OPC were classified as HPV-positive based on in situ hybridization (ISH) or institutional p16 immunohistochemistry. The sensitivity and specificity of the LFA were determined by comparing to clinical diagnosis. Results: The specificity for each individual HPV16 E2, E6, and E7 antibodies was 95.1% (77/81), 96.3% (78/81), and 98.7% (80/81), respectively. The sensitivities of the individual HPV16 antibodies were as follows: E2, 47.9% (57/119), E6, 31.9% (38/119), and E7, 57.1% (68/119). The 3-biomarker panel (at least one positive for E2, E6, and E7 Abs) demonstrated a sensitivity of 76.5% (91/119) and a specificity of 92.6% (75/81). Conclusions: We developed a multiplexed lateral flow assay for the rapid detection of serologic responses to HPV16. Further research is required to determine the utility of these tests for HPV + HNSCC cancer screening, as higher specificity, and an assessment of the benefits of positive test results have yet to be evaluated in this context.