Abstract 6615: Novel digital pathology tools for exploring role of myeloid cells in modulating next generation immunotherapies
Authors: Van Krieken Richard , Adams Lisa , Chen Beiru , Pacia Emmanuel , Dakappagari Naveen , Bourke-Martin Brian , Bordeaux Jennifer 
Published: June 2023

Abstract
Purpose: Deeper understanding of the immune landscape in tumor microenvironments is critical for creation of next generation immunotherapies (I-O). While significant investments were made to understand the role of T-cells, the contribution of myeloid cells in limiting the effectiveness of I-O therapies is not well understood primarily due to limitations posed by conventional immunohistochemistry (IHC) analysis that typically provides information on a single target. We developed a clinical grade multiplex fluorescence IHC (mFIHC) assay to simultaneously evaluate different subsets of myeloid cells (e.g., M1, M2 and MDSC) as well as PD-L1 receptors associated with these subsets as the latter is known to have profound positive impact on patients treated with PD-1 blockers (Liu et al., CCR, 2019). In addition, we developed an algorithm to identify the microenvironment using imaging datasets from AQUA® (Automated Quantitative Analysis) and HALO to understand the spatial relationship between tumor cells and myeloid cell subsets as well as immune checkpoint (IC) expression. mFIHC combined with hypothesis driven spatial profiling algorithms (e.g., AQUA Technology) were found to provide superior predictors of I-O therapies in a systematic meta-analysis of over 8000 patients treated with PD1/L1 pathway blockers (Lu et al., JAMA Oncol 2019). Furthermore, spatial profiling by AQUA has proven to robustly identify powerful biomarker predictors (PD1/PDL1) in melanoma (Johnson et al., CCR, 2018). Implementation of mFIHC coupled with robust image analysis may provide great insight into immune surveillance, mechanisms of resistance and patient stratification.
Study Design: We designed a novel mFIHC assay (CD68, CD163, HLA-DR, IDO-1, PD-L1, PanMel-CK) and evaluated various myeloid and tumor cell types on tissue sections (e.g., non-small cell lung cancer, gastric carcinoma, breast carcinoma, and renal cell carcinoma). Following the initial evaluation, the mFIHC assay was validated for clinical trial application using automated staining (Leica Bond RX) and imaging (PhenoImager HT). We conducted image analysis using AQUA and HALO technologies.
Results: Analysis of samples by AQUA and HALO revealed unique myeloid microenvironments in the examined tissue types. Microenvironment data from AQUA and HALO were assessed by analyzing enrichment of macrophage phenotypes in areas dense with checkpoint markers. Our analysis revealed unique myeloid cell clusters in the assessed tumor microenvironments.
Conclusion: We anticipate this hypothesis driven clinical trial grade mFIHC assay design combined with novel analyses algorithms will aid in the better understanding of the immunomodulatory effects of myeloid cells in several cancer types and the development of next generation I-O therapies.
Citation Format: Richard Van Krieken, Lisa Adams, Beiru Chen, Emmanuel Pacia, Naveen Dakappagari, Brian Bourke-Martin, Jennifer Bordeaux. Novel digital pathology tools for exploring role of myeloid cells in modulating next generation immunotherapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6615.