HPV Serology and Circulating Viral DNA for Detection, Genotyping, and Risk-Stratification in p16+ Oropharyngeal Cancer

Purpose/Objective(s) The incidence of human papillomavirus-positive (HPV+) oropharyngeal cancer (OPC) has increased rapidly in North America. HPV serology has been proposed as a sensitive, scalable, and cost-effective early detection test. HPV seropositivity can precede clinical presentation of OPC by several years, so additional diagnostic and/or surveillance procedures may be necessary to optimize early cancer detection. The potential for HPV circulating tumor DNA (ctDNA) to confirm a diagnosis of OPC in seropositive individuals is poorly understood. Moreover, the concordance of baseline HPV serology and HPV ctDNA and their respective associations with cancer burden in OPC patients are not known. We hypothesized that plasma levels of HPV antibodies and HPV ctDNA are associated with disease burden in a cohort of p16+ OPC patients. Materials/Methods We analyzed pre-treatment peripheral blood plasma from 100 patients with non-metastatic p16+ OPC treated with definitive (chemo)radiotherapy. A multiplex ELISA was used for serologic evidence of HPV proteins (L1, E1, E2, E4, E6, E6) from 10 HPV genotypes, quantified by mean fluorescence intensity (MFI). Plasma HPV ctDNA was quantified by whole viral genome sequencing utilizing a custom HPV-targeted capture panel for 38 HPV genotypes (HPV-seq). Gross tumor volume (GTV) was obtained from the sum of all target contours on computed tomography simulation scans. Statistical tests Kruskal-Wallis and Pearson's r were used to compare the assays. Results Seropositivity and HPV ctDNA positivity were found in 100% of cases. Serological results indicated 10 genotypes, and HPV ctDNA results indicated 9 distinct genotypes; both assays identified HPV16 as the most prevalent genotype (98% for each). Among the 98 patients with detectable HPV16 within ctDNA, HPV16 E6 seropositivity demonstrated high sensitivity (95%), while E1 (50%), E4 (47%), E7 (66%), and L1 (63%) demonstrated considerably lower sensitivities. HPV ctDNA but not HPV16 E6 MFI was positively associated with disease burden as determined by T- and N-category (Table 1), and by GTV (ctDNA vs GTV, r=0.47 p=1.0e-06; E6 MFI vs GTV, r=-0.14 p=0.17). Conclusion This is the first report to compare HPV serology and HPV ctDNA sequencing in OPC patient plasma. Both tests were highly sensitive and could identify multiple oncogenic HPV genotypes. The level of HPV16 ctDNA but not HPV16 E6 antibodies in plasma was associated with disease burden. These findings have potential implications for early detection and prognostication.