Characterization of Antibody Repertoires in Patients with HPV-Related HNSCC Undergoing Definitive Radiation with Immunotherapy

Abstract

Integrating immune checkpoint inhibitor (ICI) immunotherapy agents into the definitive treatment of head and neck squamous cell carcinoma (HNSCC) has been challenging. It is apparent that understanding how these therapies affect specific components of the immune response is critical to improve outcomes. The role of B cells has been increasingly recognized, especially in HPV+ HNSCC. As antibodies are one of the chief downstream products of B cells, we sought to evaluate the antibody repertoires of patients with HPV+ HNSCC undergoing definitive radiation therapy (RT) and ICI (RT-ICI). Serum samples from patients with p16+ HNSCC undergoing RT-ICI were collected for the KEYCHAIN clinical trial (NCT03383094). We analyzed 8 samples from 4 patients collected pre-treatment and 3 months following treatment using the HuProt Human Proteome Microarray, which samples >20,000 human proteins. Genes encoding surface proteins (GESPs) were obtained from The Cancer Surfaceome Atlas. Mutational data were obtained from AACR Project GENIE. For murine studies, C3H mice were injected with 5x105 cells of AT-84-E7-OVA syngeneic SCC into the right flank. Anti-PD-L1 ICI (aPD-L1) was given via intraperitoneal injection every 3 days for 3 doses. RT was 12 Gy in 1 fraction. Cell surface markers in lymph nodes (LNs) were analyzed by flow cytometry. In total, we detected antibodies against 10959 unique antigens in the pre-treatment serum, of which 14% and 11% were shared by 3 and 4 patients, respectively. Following completion of RT-ICI, we detected antibodies against 11019 unique antigens, of which 20% and 18% were shared by 3 and 4 patients, respectively. Of these, 5824 (53%) antigens were not detected in the pre-treatment serum, and therefore represent antibodies against "newly detected" antigens. We found 777 antigens that were newly detected and shared by at least 2 patients. Analysis of these antigens revealed enrichment in pathways such as, "response to oxygen levels." We next found that 114 (14.7%) of these represented GESPs. We integrated mutational analysis of the most frequently mutated genes in >1,800 HNSCC samples and found 2/114 GESPs were shared. Using a murine model of HPV+ HNSCC, we found that treatment with RT and aPD-L1 led to the greatest frequency of germinal center (GC) B cells in tumor-draining and non-tumor-draining LNs. In patients with p16-positive HNSCC, proteomic analysis of antibody repertoires revealed many antigens against which antibodies were formed during RT-ICI that are shared between patients. Intriguingly, GC formation, which is the nidus for B cell responses, in locoregional LNs was greatest with RT-ICI. These findings support the further investigation of B-cell mediated responses in HPV+ HNSCC.