Spatial immune signatures predict outcomes in HPV-positive oropharyngeal cancer: Results of a clinical trial

Background: The incidence of Human Papillomavirus (HPV)-associated oropharyngeal cancer (OPC) has been increasing in recent years. While patients with HPV-positive OPC generally demonstrate more favorable outcomes compared to those with HPV-negative disease, a subset still experiences locoregional recurrence and distant metastasis. These clinical challenges underscore the need for a better understanding of the tumor microenvironment (TME) and its role in modulating treatment response. We hypothesized that the composition and spatial dynamics of the TME differ between complete responders and partial/non-responders, and these differences may serve as predictive biomarkers. Methods: This retrospective study included patients with p16-positive OPC treated at two major cancer centers in Montreal between 2010 and 2023. Formalin-Fixed Paraffin-Embedded (FFPE) tissue blocks were retrieved, and tissue microarrays (TMAs) were constructed for high-dimensional immune profiling using imaging mass cytometry (IMC). Immune cell populations were analyzed across treatment response categories (complete vs. partial responders) and treatment stages (pre- vs. post-treatment). Deep learning-based cell segmentation was applied to quantify immune cell subsets, assess spatial architecture, and perform network and neighborhood analyses of the TME to identify potential predictive and prognostic immune signatures. Results: Tissue specimens were obtained from HPV-positive (p16+) OPC patients, of whom 79.6% were male, with a mean age of 62.5 years. IMC revealed distinct immune landscapes between response groups. In complete responders, treatment induced a robust recruitment of anti-tumor immune cells (e.g., CD8+ T cells and B cells), suggesting an activated immune phenotype post-treatment. In contrast, non-responders exhibited enrichment of immunosuppressive or tumor-promoting cell types following therapy. Spatial co-localization and cell-cell interaction analyses further indicated that B and T cells interactions may contribute to therapeutic success. Conclusion and Impact: This study provides novel insights into the spatial and cellular remodeling of the immune microenvironment in HPV-positive OPC before and after treatment. Our findings highlight the prognostic potential of IMC-based immune profiling and support the development of predictive biomarkers to guide the selection of patients who may benefit from neoadjuvant chemotherapy or immunomodulatory interventions.