Validation of MyHPVscore: A High-Performance Human Papillomavirus Circulating Tumor DNA Laboratory-Developed Test.

As rates of human papillomavirus-positive (HPV+) oropharynx cancer increase, there is increasing need for accurate biomarkers for diagnosis, treatment, and surveillance. The analytical performance of MyHPVscore, a droplet digital PCR laboratory-developed test, was characterized to detect circulating tumor DNA from multiple high-risk HPV (hrHPV) types (16, 18, 31, 33, 35, and 39) in plasma from patients with HPV+cancer. Using Clinical and Laboratory Standards Institute guidelines, MyHPVscore was developed and validated in a Clinical Laboratory Improvement Amendments-certified laboratory. Analytical controls and plasma samples from patients with HPV+oropharyngeal squamous cell carcinoma and noncancer controls were used to evaluate sensitivity, specificity, linearity, and analyte stability. MyHPVscore demonstrated high analytical performance for detecting multiple hrHPV types. Limits of blank were 2.7 (HPV16) and 2.6 (other hrHPV types) positive droplets/reaction; limits of detection were 15.2 and 20.8 positive droplets/reaction, respectively. The linear range was 15 to 62,500 targets/mL plasma (HPV16) and 12 to 100,000 targets/mL (other hrHPV types) with strong correlation (R2 > 0.99). Analytes were stable for 96 hours at -20°C and yielded consistent qualitative results after >400 days of storage. No cross-reactivity was observed between hrHPV types or low-risk types (HPV6 and HPV11). These results support the use of MyHPVscore as a laboratory-developed test for detecting HPV+oropharynx cancer. This method establishes an approach and standard controls that can benchmark other emerging circulating tumor DNA assays.