Abstract 1525: Integration of high-plex tumor-Immune phenotyping and checkpoint interactions for deeper spatial characterization of human cancer tissues

Abstract Background: Immune evasion is a key cancer hallmark, and one of the primary mechanisms is the PD-1/PD-L1 checkpoint axis. While immunotherapies that target these interactions have reshaped cancer treatment, the variable efficacy in patients is not fully understood. Due to the weak correlation between PD-1/PD-L1 expression measured by single-plex assays and observed clinical outcomes, there is a need to understand protein interactions at the cellular and subcellular level. Proximity Ligation Assays, detecting spatial interactions between ligand-receptor targets, provide insights into the activation of signaling pathways. Coupled with high-plex spatial phenotyping with tumor and immune profiling biomarkers, the integration of cellular and functional information will be beneficial for a deeper characterization of the tumor microenvironment (TME), understanding immune responses, and identifying spatial signatures for patient stratification and targeted treatment strategies. Methods: In this study, we phenotyped biopsies from head and neck cancer patients enrolled in immune checkpoint inhibitor therapies. We integrated Naveni® PD-1/PD-L1 proximity ligation assays and a customizable high-plex PhenoCode™ Signature Panel (CD3ε/CD8/CD20/CD68/PanCK) for high-throughput profiling of the TME utilizing the PhenoImager® HT 2. 0 platform. Comprehensive bioinformatic analyses were conducted for whole-slide segmentation, identifying cellular phenotypes, spatial neighborhoods, functional interactions, and distinct spatial signatures via the open-source image analysis software QuPath. Results: Our analyses revealed key differences in the localization of the PD-1/PD-L1 interactions within the TME of head and neck cancers. Areas of PD-1/PD-L1 interactions are associated with immune cell types on the periphery of the tumor and some within the tumor-infiltrating lymphocytes. The PhenoCode Signature panels helped resolve the localization of PD-1/PD-L1 interactions to immune cell types within the TME and provided a dual workflow for spatial co-localization of interacting receptor/ligand interactions and cell phenotypes within the TME. Conclusions: By combining spatial immune profiling with Akoya’s PhenoCode Signature panels and protein-protein interaction data from Navinci’s Naveni® PD-1/PD-L1 assay, deep correlative insights of the TME can be applied to improve predictions of clinical outcomes. This innovative approach enriches our comprehension of underlying mechanisms and stands as a promising tool for refining patient selection and optimizing treatment outcomes in immunotherapy. Citation Format: Sara Bodbin, Ning Ma, Aditya Pratapa, Nadya Nikulina, James Monkman, Niyati Jhaveri, Hampus Elofsson, Subham Basu, Agata Zieba-Wicher, Arutha Kulasinghe. Integration of high-plex tumor-Immune phenotyping and checkpoint interactions for deeper spatial characterization of human cancer tissues abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 1525.