Development and Validation of an Assay to Quantify Plasma Circulating Tumor Human Papillomavirus DNA for 13 High-Risk Types that Cause 98% of HPV-Positive Cancers.

Purpose: Plasma circulating tumor HPV DNA (ctHPVDNA) persistence after curative-intent treatment may identify patients with HPV-positive cancers at risk for recurrence. Technical validation is required for use as an integral biomarker in a prospective clinical trial.

Methods: Development and analytical validation of a digital droplet PCR assay for detection and quantification of 13 high-risk HPV types (i.e., Cell-Free 13) was performed with oligonucleotides/plasmids encoding type-specific E6/E7 coding regions. Clinical performance, determinants of detection/quantification, and associations of pre-treatment ctHPVDNA with progression-free survival (PFS) were also evaluated in a prospective cohort of 272 head and neck cancer patients.

Results: Limit of detection, limit of quantification, and linear range of quantification were 5, 16 and 16-200,000 virus copies for all 13 high-risk HPV types. No cross-reactivity was detected across all 13 HPV types. At 10,000 copies, inter-assay coefficients of variation ranged from 0.3 to 4.6%. Multiplexing, DNA purification method, input plasma volume, total input cell-free (< 1800 ng) or genomic (< 700 ng) DNA did not affect HPV detection or quantification. The assay had a sensitivity of 91.7% (95%CI 87.3-94.9%) and specificity of 97.7% (95%CI 87.7-99.9%) for ctHPVDNA detection in the setting of newly diagnosed HPV-positive oropharyngeal cancer. Tumor and nodal stage categories, tumor viral load (ρ = 0.41, p < 0.05), and HPV integration status were associated with ctHPVDNA quantitative level. Pre-treatment ctHPVDNA greater than the median (231 copies/ml) was associated with worse PFS (HR = 2.14, 95%CI 1.16-3.97, p = 0.0156) in univariate analysis. However, this was no longer significant after adjustment for clinical covariates (HR_adj = 1.81, 95%CI 0.97-3.37, p = 0.0635).

Conclusion: Cell-Free 13 demonstrated excellent analytical performance and clinical sensitivity/specificity in HPV-positive oropharyngeal cancer. Pre-treatment ctHPVDNA may be associated with oncologic outcomes.