Abstract 2462: Novel digital pathology algorithms to explore the role of tumor microenvironment in next generation immuno-oncology clinical trials utilizing 17 plex fluorescent immunohistochemistry

Abstract Purpose: A deeper understanding of the intricacies between tumor microenvironments (TMEs) and immune cell infiltrates is critical for creation of next generation immune modulating therapies. We previously validated a clinical grade, 17 plex tumor immune landscape assay which characterizes T cells, B cells, myeloid cells, plasma cells, and tumor cells across five major cancer types. This assay was developed on the Orion platform, which images up to 18 markers concurrently and allows for same slide hematoxylin and eosin (H and E). Using the Orion platform, we characterized the TME through two approaches: cellular neighborhoods and clustering based on H and E and segmented fluorescent immunohistochemistry data. Our findings elucidate innovative strategies for leveraging high dimensional data from Orion to uncover clinically useful insights, opening new avenues for understanding mechanism of response or resistance in next generation immune oncology (IO) clinical trials Study Design: We designed a mFIHC assay (Granzyme B, Ki67, CD3e, CD20, CD4, CD163, CD8a, CD56, PDL1, PD1, LAG3, FOXP3, CD11b, CD138, SOX10, Cytokeratin and Hoechst) to evaluate expression of immune markers on non small cell lung cancer, breast carcinoma, colorectal cancer, prostate cancer and melanoma. After the initial evaluation, we validated the mFIHC assay for clinical trial use with automated staining (IntelliPath FLX) and imaging (Orion). Image analysis was conducted using HALO technologies. Results: HALO analysis revealed distinct tumor immune microenvironments in the examined tissues. Microenvironment data were assessed by analyzing immune cell infiltration alongside immune activation markers. Our analysis revealed unique cell clusters in these microenvironments. Conclusion: Our clinically validated 17 plex mFIHC assay, combined with advanced spatial algorithms, offers improved characterization of the TME. We expect these novel algorithms to contribute to gaining novel insights into tumor biology and development of next generation IO therapies. Citation Format: Richard Van Krieken, Jaspreet Kaur, James Santos, Naveen Dakappagari, Jennifer Bordeaux. Novel digital pathology algorithms to explore the role of tumor microenvironment in next generation immuno-oncology clinical trials utilizing 17 plex fluorescent immunohistochemistry [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 2462.