Ling Aye

Surgery is a standard treatment for early-stage human papillomavirus–associated head and neck cancer (HPV+HNC), yet adjuvant therapy decisions rely on clinicopathologic features with limited prognostic accuracy. Circulating tumor HPV DNA is a promising biomarker, but current assays lack the sensitivity needed for detecting minimal residual disease after surgery. This study evaluated the prognostic value of an HPV whole-genome sequencing assay, HPV-DeepSeek, and compared this with existing methods and clinical standards. One hundred three patients with stage I to IV HPV+HNC treated with surgery were prospectively enrolled (NCT06730412). Blood samples were collected before surgery, postoperatively, and during surveillance and were analyzed using HPV-DeepSeek and droplet digital polymerase chain reaction (PCR) assays. After a median follow-up of 27 months, patients with circulating tumor HPV DNA detected after surgery had worse 2-year disease-free and overall survival than those without detection. Detection after completion of all treatment was also associated with poorer outcomes. Minimal residual disease status was a stronger predictor of recurrence than standard clinicopathologic criteria. Molecular recurrence was identified up to 17.5 months earlier than clinical recurrence and nearly twice as early as with droplet digital PCR. These findings demonstrate that sensitive HPV whole-genome sequencing enables accurate detection of minimal residual disease, early identification of recurrence, and improved postoperative risk stratification, supporting its potential as a tool for guiding personalized adjuvant therapy in HPV+HNC.

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.See related commentary by Haring et al., p. 3359See related article by Sim et al., p. 3494

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.