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

Area of Science:

• Oncology
• Molecular Diagnostics
• Virology

Background:

• Rising incidence of human papillomavirus-positive (HPV+) oropharyngeal cancer necessitates improved diagnostic and monitoring tools.
• Current biomarkers lack the accuracy needed for timely diagnosis, treatment selection, and surveillance of HPV+ oropharyngeal squamous cell carcinoma (OPSCC).

Purpose of the Study:

• To characterize the analytical performance of MyHPVscore, a droplet digital PCR laboratory-developed test (LDT).
• To evaluate MyHPVscore's ability to detect circulating tumor DNA (ctDNA) from multiple high-risk HPV (hrHPV) types in plasma from patients with HPV+ oropharyngeal cancer.

Main Methods:

• MyHPVscore was developed and validated in a CLIA-certified laboratory following Clinical and Laboratory Standards Institute guidelines.
• Analytical performance was assessed using controls and plasma samples from HPV+ OPSCC patients and non-cancer controls, evaluating sensitivity, specificity, linearity, and analyte stability.
• Droplet digital PCR was employed to detect ctDNA from hrHPV types (16, 18, 31, 33, 35, 39).

Main Results:

• MyHPVscore demonstrated high analytical performance, with low limits of blank and detection for multiple hrHPV types.
• The assay exhibited a wide linear range (12-100,000 targets/mL) with strong correlation (R² > 0.99) and excellent analyte stability (>400 days storage).
• No cross-reactivity was observed between hrHPV types or with low-risk HPV types (HPV6, HPV11).

Conclusions:

• The analytical validation supports MyHPVscore as a reliable LDT for detecting HPV+ oropharyngeal cancer.
• This methodology provides a benchmark for developing and validating other circulating tumor DNA (ctDNA) assays for cancer diagnostics.
• MyHPVscore shows promise for improving the diagnosis, treatment, and surveillance of HPV+ oropharyngeal cancer.