Detection of antibodies to the HPV16 proteome in head and neck cancer patient sera.

Abstract

5545 Background: Human papillomavirus (HPV) type 16 is associated with a subset of oropharyngeal carcinomas (OPC). Of the 8 proteins encoded in the HPV genome, antibodies (Ab) to the early genes E6 and E7 have been detected in patients with HPV16-associated OPC, and are associated with improved prognosis. It is not known whether antibodies to other HPV-derived proteins are potential biomarkers of HPV-associated malignancies. Methods: Antibodies to the HPV16 and HPV18 antigens E1, E2, E4, E5, E6, E7, L1, L2 were quantified using a novel multiplexed bead array assay. Full-length genes were expressed as c-terminal GST fusion proteins in a cell-free transcription translation system and captured onto anti- GST Luminex beads. Blinded sera were obtained from head and neck cancer patients with known antibodies to HPV16 L1 (N=10) or HPV18 L1 (N=10) by Merck competitive Luminex immunoassay (cLIA). Healthy control samples were age- and sex-matched (n=20). The ratio of median fluorescent intensity (MFI) values for each antigen to control p21-GST fusion proteins was determined. P-values were calculated by Wilcoxon rank-sum test. Results: Antibody responses to multiple HPV-derived early genes were detected in patient sera. HPV16 E1, E2, E4, E6, E7, and L1 antibody levels (but not E5 or L2) were elevated in HPV16 L1 Ab+ patients (defined by Merck assay) compared to healthy control samples. Median MFI ratios were: E1 39 vs. 2.0, p<0.0001; E2 2.7 vs. 1.4, p<0.0001; E4 9.1 vs. 1.4, p<0.001; E6 10 vs. 2.1, p<0.0001; E7 3.9 vs. 2.0, p<0.01; and L1 10.8 vs. 2.4, p<0.0001. For patients with HPV18 L1 Abs defined by Merck assay, only Abs to HPV18 E1 (11.9 vs. 2.3, p<0.001); E2 (20.7 vs. 1.9, p=0.0016), and L1 (9.8 vs. 2.7, p<0.0001) were specifically detected. There was no cross-reactivity detected between HPV16 and HPV18-specific antibodies. Conclusions: We developed a custom multiplexed bead array for the rapid detection of HPV16 and HPV18-specific antibodies in patient sera. Patients with head and neck cancers have detectable antibodies to multiple early genes derived from HPV. These antibodies are potential biomarkers for HPV-associated malignancies. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Merck Merck