Nanopore Sequencing for HPV in Oropharyngeal Squamous Cell Carcinoma and Benign Tonsil Specimens

OBJECTIVES: Human papillomavirus (HPV) accounts for the majority of oropharyngeal squamous cell carcinoma (OPSCC) cases in the United States but understanding the prevalence of high-risk HPV in oropharyngeal tissue remains poor. We evaluated nanopore sequencing, a novel rapid, and long-read sequencing platform, on OPSCC tissue and tested it in benign tonsils from pediatric and young adult patients. STUDY DESIGN: Cross-sectional study. SETTING: Academic tertiary referral center. METHODS: Formalin-fixed, paraffin-embedded (FFPE) specimens of HPV-positive and negative OPSCC diagnosed from 2013 to 2023 were sequenced on the nanopore MinION platform using an amplicon-based approach. Nanopore sequencing was subsequently performed on FFPE tissue from benign tonsillectomy in patients ages 30 and younger performed from 2016 to 2023. RESULTS: In 54 OPSCC cases, nanopore sequencing detected HPV DNA in all 27 HPV-positive specimens with 96% of cases exhibiting high abundance (HPV reads >1000). HPV DNA was also detectable in all 27 HPV-negative specimens, but 89% had a low abundance. By interpreting high HPV abundance as HPV-positive, nanopore sequencing demonstrated a 96% sensitivity, 89% specificity, 90% positive predictive value, and 96% negative predictive value rate. In 150 benign tonsillectomy cases, 27% had HPV DNA detected by nanopore sequencing, all in low abundance, including 37% of patients younger than 10 years old. HPV DNA sequences detected belonged to subtype 16. CONCLUSIONS: Nanopore sequencing demonstrated high sensitivity and specificity for HPV DNA in FFPE OPSCC tissue. High-risk HPV prevalence in pediatric and young adults may be higher than previously studied.