Assessing the feasibility of a multimodal liquid biopsy for the diagnosis of HPV-associated oropharyngeal squamous cell carcinoma

James S. Lewis, Saskia Naegele, Vasileios Efthymiou, Mitra Mehrad, Kim A. Ely, Tim Waterboer, Krystle A.Lang Kuhs, Seth J. Davis, Kelsey Richard, Dipon Das, Daniel L. Faden

Research output: Contribution to journalArticlepeer-review


Objectives: In this feasibility study, we explored the combined use of circulating tumor human papillomavirus (HPV) DNA (ctHPVDNA) and HPV serology as diagnostic tests for HPV-associated oropharyngeal squamous cell carcinoma (OPSCC). Methods: Among patients with research-banked serum or plasma at diagnosis, IgG antibodies to oncoproteins from HPV types 16, 18, 31, 33, 35, 45, 52, and 58 were detected with multiplex serology. Positivity for HPV 16 was defined based on detection of combinations of anti-E6, E1, E2, and E7 and for other high-risk types on detection of anti-E6 and anti-E7. Circulating tumor HPV DNA was detected by custom digital droplet polymerase chain reaction (ddPCR) assays for HPV types 16, 18, 33, 35, and 45. p16 immunohistochemistry and high-risk HPV RNA in situ hybridization (ISH) using a cocktail of 18 high-risk HPV types were performed on tissue. Results: Of 75 patients, 67 (89.3%) were HPV-associated (p16 and HPV RNA ISH positive) and 8 (10.7%) were HPV-independent. All 8 HPV-independent patients were seronegative and negative for ctHPVDNA (100% specificity). Serology was positive in 53 (79.1%) of 67 HPV-associated patients, while ddPCR was positive for ctHPVDNA in 59 (88.6%) of 67 HPV-associated patients. Requiring both tests to be positive resulted in a sensitivity of 50 (74.6%) of 67 while combining assays (either positive) improved sensitivity to 62 (92.6%) of 67. Conclusions: Compared to HPV RNA ISH, HPV serology and ctHPVDNA are sensitive and highly specific biomarkers for HPV-associated OPSCC at the time of presentation.

Original languageEnglish
Pages (from-to)570-578
Number of pages9
JournalAmerican Journal of Clinical Pathology
Issue number6
StatePublished - Jun 1 2024

Bibliographical note

Publisher Copyright:
© 2024 The Author(s).


  • circulating tumor DNA
  • human papillomavirus
  • oropharyngeal squamous cell carcinoma
  • p16
  • serology

ASJC Scopus subject areas

  • Pathology and Forensic Medicine


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