Development and validation of an assay to quantify plasma cell-free human papillomavirus DNA for 13 high-risk types that cause 98% of HPV-positive cancers.

person Michael Wotman MD Anderson Hematology/Oncology Fellowship, Houston, TX info_outline Michael Wotman, Weihong Xiao, Robyn Du, Bo Jiang, Suyu Liu, Maura L. Gillison

Authors person Michael Wotman MD Anderson Hematology/Oncology Fellowship, Houston, TX info_outline Michael Wotman, Weihong Xiao, Robyn Du, Bo Jiang, Suyu Liu, Maura L. Gillison Organizations MD Anderson Hematology/Oncology Fellowship, Houston, TX, The University of Texas MD Anderson Cancer Center, Houston, TX, Department of Thoracic/Head and Neck Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, Department of Biostatistics, The University of Texas MD Anderson Cancer Center, Houston, TX Abstract Disclosures Research Funding Cancer Prevention and Research Institute of Texas Background: Plasma cell-free HPV DNA (cfHPV) may be a valuable tool for identification of patients with local-regionally advanced HPV-positive cancers at risk for recurrence after chemoradiation. Technical validation is required for use as an integral biomarker in a prospective clinical trial. Methods: Cell-Free 13 (i.e., CF13) is a digital-droplet PCR assay for quantification of 13 high-risk HPV types and control ERV3 in plasma that considered both variant sequences into primer/probe design and a distance matrix in phylogenetic analysis in multiplex combinations. Oligonucleotides/plasmids encoding type-specific E6/E7 regions were used as positive controls. Limit of blank, detection, quantification (LoB, LoD, LoQ), linear range, inter- and intra-assay coefficients of variation (CoV), and type-specific cross-reactivity were determined as were sensitivity and specificity for an HPV-positive diagnosis in a cohort of 278 oropharyngeal/unknown primary/oral cavity cancers tested for HPV by E6/E7 mRNA qRT-PCR or p16 IHC and HPV in situ hybridization. Results: Under identical assay conditions, the LoB, LoD and linear range were <1, 5 and 5 to 200,000 virus copies without HPV type-specific cross-reactivity. Multiplexing had no effect on LoD or linearity. LoQ was 16 copies/ml plasma for all 13 HPV types. For 80 and 10,000 copies per ml, inter-assay CoV ranged from ~15-28% and ~3.2-4.6% and intra-assay CoV ranged from ~16-38% and 0.2-3.3%, respectively. cfDNA purification method (manual v automated; extraction efficiency 142% and 91% for 16 and 10,000 HPV16 copies/ml), input plasma volume (1, 2, or 4 ml), total background cfDNA (<1800ng) or genomic DNA (<700ng) did not affect quantification. For a diagnosis of HPV-positive cancer, sensitivity and specificity were 91.1% (214/235) and 97.7% (42/43), respectively. cfHPV was associated with gender, race, T stage, N stage, and primary treatment modality. When compared to below the median (≤230 copies/ml), cfHPV above the median was associated with worse progression-free survival (HR=2.26, 95% CI 1.23-4.17, p =0.009) in univariate analysis. However, this was no longer significant after adjustment for age, T stage, and M stage (HR adj =1.78, 95% CI 0.95-3.35, p =0.07). Conclusions: CF13 has high sensitivity, accuracy, precision, linearity, HPV type-specificity, and robustness, offering a rigorously validated assay applicable to ~98% of patients with HPV-positive cancer. CF13 had excellent clinical sensitivity and specificity for a diagnosis of HPV-positive OPC.