Abstract 750: Exploring PD1/PDL1 interactions and macrophage-tumor barriers in head and neck cancer: A spatial approach to immunotherapy response

Abstract With nearly a million new cases expected, the global death rate from mucosal head and neck cancer is projected to reach around 50% in 2024. In the U.S., patients with localized tumors have a five-year survival rate of 50-60%. While immune checkpoint inhibitors (ICIs) have shown promise in extending survival, significant challenges remain, particularly the limited effectiveness of PD-1/PD-L1 blockade therapies. Since many studies have shown that PD-L1 protein expression alone may not be a reliable predictor of response to ICI therapy, understanding the spatial context of PD-1/PD-L1 interactions might be crucial for identifying immune evasion mechanisms and predicting responses to ICIs in head and neck cancer. To address this, we aimed to take a more comprehensive approach by mapping PD-1/PD-L1 interactions across n=35 mucosal head and neck squamous cell carcinoma (HNSCC) tissue samples collected before ICI treatment, using a combination of high-plex spatial proteomics and the in situ Navinci Proximity Ligation Assay (isPLA). Formalin-fixed, paraffin-embedded (FFPE) tissue samples were stained using multiplex immunofluorescence and the Navinci Diagnostics isPLA assay to detect PD-1/PD-L1 interactions. After incubation with primary anti-PD1 and anti-PD-L1 antibodies, secondary probes tagged with oligonucleotides were applied, enabling PLA to highlight the interactions through a ligation step and an amplification process to enhance the visibility of the protein interactions. Finally, a detection solution was applied to visualize the amplification product at the sites of PD1/PD-L1 interaction.We identified distinct spatial patterns of PD-1/PD-L1 interactions at the tumor-stroma interface, particularly in progressive disease (PD) patients, who exhibited dense layers of isPLA+ macrophages, CD3e T cells, and tumor cells, suggesting an immunosuppressive barrier in this region. In contrast, complete response (CR) patients showed rare or absent barriers, with enriched immune cell aggregates, including B cells and T cells, near the tumor boundary. These findings were corroborated through neighborhood clustering analysis, revealing significant associations between isPLA+ regions and poor prognosis.The spatially resolved nature of our analysis underscores the critical role of PD-1/PD-L1 interactions in shaping the tumor microenvironment (TME) and highlights their potential as predictive biomarkers for ICI therapy efficacy. Unlike methods that detect mere colocalization, isPLA technology enables us to detect true PD-1/PD-L1 interactions, which are more likely to affect immune suppression directly. Our study also emphasizes the need for further exploration of macrophage and T cell subtypes, as well as PD-1/PD-L1 interactions across tumor-stroma boundaries in various cancers to improve immunotherapy strategies. Citation Format: Vahid Yaghoubi Naei, Rafael Tubelleza, James Monkman, Habib Sadeghirad, Meg L. Donovan, Tony Blick, Agata Wicher, Sara Bodbin, Subham Basu, Robert Stad, Catherine Barnett, Ken O’Byrne, Rahul Ladwa, Caroline Cooper, Majid Ebrahimi Warkiani, Brett GM Hughes, Arutha Kulasinghe. Exploring PD1/PDL1 interactions and macrophage-tumor barriers in head and neck cancer: A spatial approach to immunotherapy response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 750.