Compartmentalized spatial profiling of the tumor microenvironment in head and neck squamous cell carcinoma identifies immune checkpoint molecules and tumor necrosis factor receptor superfamily members as biomarkers of response to immunotherapy.

Mucosal head and neck squamous cell carcinoma (HNSCC) are the seventh most common cancer, with approximately 50% of patients living beyond 5 years. Immune checkpoint inhibitors (ICIs) have shown promising results in patients with recurrent or metastatic (R/M) disease, however, only a subset of patients benefit from immunotherapy. Studies have implicated the tumor microenvironment (TME) of HNSCC as a major factor in therapy response, highlighting the need to better understand the TME, particularly by spatially resolved means to determine cellular and molecular components. Here, we employed targeted spatial profiling of proteins on a cohort of pre-treatment tissues from patients with R/M disease to identify novel biomarkers of response within the tumor and stromal margins. By grouping patient outcome categories into response or non-response, based on Response Evaluation Criteria in Solid Tumors (RECIST) we show that immune checkpoint molecules, including PD-L1, B7-H3, and VISTA, were differentially expressed. Patient responders possessed significantly higher tumor expression of PD-L1 and B7-H3, but lower expression of VISTA. Analysis of response subgroups indicated that tumor necrosis factor receptor (TNFR) superfamily members including OX40L, CD27, 4-1BB, CD40, and CD95/Fas, were associated with immunotherapy outcome. CD40 expression was higher in patient-responders than non responders, while CD95/Fas expression was lower in patients with partial response (PR) relative to those with stable disease (SD) and progressive disease (PD). Furthermore, we found that high 4-1BB expression in the tumor compartment, but not in the stroma, was associated with better overall survival (OS) (HR= 0.28, p-adjusted= 0.040). Moreover, high CD40 expression in tumor regions (HR= 0.27, p-adjusted= 0.035), and high CD27 expression in the stroma (HR= 0.2, p-adjusted=0.032) were associated with better survival outcomes. Taken together, this study supports the role of immune checkpoint molecu