Luciferase-Based Detection of Antibodies for the Diagnosis of HPV-Associated Head and Neck Squamous Cell Carcinoma

Burbelo, Peter D.; Chaturvedi, Adrija; Notkins, Abner L.; Gunti, Sreenivasulu

Using the LIPS technology, antibodies against wild type p53 were evaluated in serum samples from a cohort that included HV (n = 20), and CC (n = 20) and HNSCC (n = 20). Testing an extract containing only Renilla luciferase (Ruc) as a control protein revealed low levels of antibodies in the HV, CC and HNSCC subjects ( Figure 1A ). LIPS analysis of the Ruc-p53 protein target demonstrated relatively low antibody levels in HV, whereas a small number of the patients with CC and HNSCC had much higher antibody levels ( Figure 1B) . To determine the seropositivity of p53 antibodies in the cancer subjects, a cut-off value was assigned based on the antibody values corresponding to the mean plus three standard deviations of the 20 HV controls. As shown, 25% (5/20) of the CC and 20% (4/20) of HNSCC patients were p53 seropositive with a diagnostic specificity of 100% ( Figure 1B ). Three subjects with CC and two with HNSCC showed p53 seropositive autoantibodies that were just above the cut-off value. To determine whether the antibody signal detecting p53 antibodies might be enhanced, two cancer mutant variants of p53, p53-R175H, and p53-R273H, were tested ( Figure 1C,D) . However as shown, the antibody profile against p53-R175H and p53-R273H, for the most part, showed a similar profile as wild type p53 protein. Due to the known high prevalence of HPV-associated HNSCC, antibodies against E2, E7, and E6 HPV-16 proteins were analyzed. LIPS analysis of HPV-16 E2 antibodies revealed low levels in the HV and CC patients that were similar to the buffer blanks (Figure 2A and data not shown). However, eight (45%) of the HNSCC patients showed high E2 antibody levels that were approximately 20-100 times higher than the corresponding mean levels of the HV controls (Figure 2A ). LIPS analysis of antibodies against the second HPV-16 antigen, E7, also did not detect seropositives in the HV control or CC patients but detected seven (35%) seropositive HNSCC patients which overlapped the E2 seropositive subjects ( Figure 2B ). Testing of the E6 HPV antigen as a Renilla luciferase fusion protein revealed the same pattern with HV and CC showing seronegativity yet detected nine (45%) of the HNSCC as seropositive (data not shown). To further confirm this result, a different reporter, NanoLuc was employed to detect the HPV-16 E6 antibodies. As shown in Figure 2C , the NanoLuc-E6 test also revealed that nine (45%) HNSCC subjects that were seropositive. Testing of twenty SLE patients as another set of disease controls detected no E6 or E7 seropositivity further supporting the observation that the HPV-16 antibodies responses were associated with the HNSCC patients (data not shown). Inspection of the HPV antibody profile in the HNSCC subjects revealed that nine subjects were seropositive for E6, eight overlapping subjects were E2 seropositive and 7 subjects were seropositive for E7 protein. Lastly, the presence of nine E6 seropositive samples in the HNSCC group Due to the known high prevalence of HPV-associated HNSCC, antibodies against E2, E7, and E6 HPV-16 proteins were analyzed. LIPS analysis of HPV-16 E2 antibodies revealed low levels in the HV and CC patients that were similar to the buffer blanks (Figure 2A and data not shown) . However, eight (45%) of the HNSCC patients showed high E2 antibody levels that were approximately 20-100 times higher than the corresponding mean levels of the HV controls (Figure 2A ). LIPS analysis of antibodies against the second HPV-16 antig