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PURPOSE: The incidence of human papillomavirus (HPV)-associated head and neck squamous cell carcinoma (HPV + HNSCC) is increasing in the United States. Currently, there are no early detection approaches for HPV + HNSCC. Two blood-based analytes for early detection and diagnosis of HPV + HNSCC, circulating tumor HPV DNA (ctHPVDNA) and HPV early protein antibodies (HPV Ab), show promise, yet current approaches lack adequate diagnostic accuracy for broad clinical utility. Further, performance metrics across various assays for detecting these analytes alone or in combination have not been compared head-to-head. To address these limitations and knowledge gaps, we developed a multifeature HPV whole-genome sequencing (WGS) liquid biopsy for improved low-level ctHPVDNA detection. We defined the performance characteristics of this WGS-based approach and compared it head-to-head with existing blood-based HPV detection approaches to determine the optimal single or combinatorial biomarker strategy for a future prospective study of HPV + HNSCC early detection.EXPERIMENTAL DESIGN: We tested blood samples from 304 participants: 152 patients with untreated incident HPV + HNSCC (77% stage I) and 152 general population control patients. We compared WGS-based ctHPVDNA detection, single-plex Droplet Digital PCR (ddPCR)-based ctHPVDNA detection, multiplex ddPCR-based ctHPVDNA detection, multiplex HPV Ab detection, and clinical standard-of-care tissue biopsy, benchmarked to gold-standard HPV + HNSCC tissue diagnosis. We then modeled the operational feasibility of these approaches as screening biomarkers for HPV + HNSCC.RESULTS: HPV WGS sensitivity and specificity were 98.7% and 98.7%, respectively. Single-plex ddPCR sensitivity and specificity were 94.2% and 98.6%, respectively. Multiplex ddPCR sensitivity and specificity were 90.6% and 96.3%, respectively. HPV Ab sensitivity and specificity were 86.4% and 96.3%, respectively. A combinatorial approach using both HPV WGS and HPV Ab yielded a sensitivity and specificity of 87.4% and 98.8%, respectively. In a head-to-head comparison, HPV WGS demonstrated significantly improved diagnostic accuracy compared with ddPCR (Youden index for HPV WGS, 0.99 vs. ddPCR, 0.90; P < 0.001), HPV Ab (HPV WGS, 0.99 vs. HPV Ab, 0.83; P < 0.001), and clinical workup (HPV WGS, 0.99 vs. clinical workup, 0.82; P < 0.001), which was maintained when evaluating only early-stage disease cases. For men ages 55 to 74, HPV WGS yielded the lowest number needed to screen (2,903 men) and the highest positive predictive value (2.6).CONCLUSIONS: HPV WGS-based ctHPVDNA detection demonstrated the highest sensitivity, specificity, and diagnostic accuracy and thus the lowest number needed to screen and highest positive predictive value compared with ddPCR-based ctHPVDNA detection, HPV Ab-based detection, and combinatorial approaches. These results highlight the promise of HPV WGS liquid biopsy for screening and early detection of HPV + HNSCC and the need for modeling and cost-effectiveness studies to evaluate and guide screening implementation.