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

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.

Cross-sectional study.

Academic tertiary referral center.

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.

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.

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.