Abstract 2754: Exploring the interplay between TAM, PD-L1 and immunosuppressive molecules by a novel multiplex immunofluorescence assay in cancer immunotherapy trials
Authors: Adams Lisa , Asnani Deepika K. , Bender Jehovana , Chen Beiru , Tran Thai , Tangri Shabnam , Dakappagari Naveen , Bordeaux Jennifer 
Published: July 2021

Abstract
Purpose: Understanding the role of tumor-associated macrophages (TAM) and their complex interplay with immunosuppressive molecules within tumor microenvironment is critical for development of novel immunotherapies. PD-L1 expressing M2 macrophages predicted survival of PD-1 inhibitor treated NSCLC patients better than total tumor or stromal PD-L1 expression (Liu, 2019). Additionally, co-expression of immunosuppressive molecules within tumor microenvironment favorably correlated with survival of melanoma patients receiving PD-1 blockers (Bordeaux, 2018). Differential expression of macrophage subsets and immunosuppressive molecules can profoundly inhibit or promote tumor growth and its metastasis. Multiplex fluorescence immunohistochemistry (mFIHC) combined with hypothesis driven spatial profiling algorithms has been found to provide powerful predictors of immunotherapies in a systematic meta-analyses of over 8000 patients treated with PD1/L1 pathway inhibitors (Lu, 2019). Despite substantial evidence demonstrating mFIHC utility in tumor microenvironment exploration, there is a need for reliable tools to assess high volumes of patient samples in clinical trial testing.
Study Design: We designed a novel mFIHC assay to explore presence of different macrophage subsets and their spatial relationships with key immunosuppressive molecules and unique localization patterns. This assay incorporates antibodies to identify both tumor promoting (CD163+) and controlling (CD68+) macrophages and their co-localization with PD-L1, HLA-DR and IDO1. We successfully validated a clinical grade mFIHC assay using automated staining (Leica Bond RX), imaging (Vectra Polaris) and analyses (AQUA® Technology) workflow for seven tumor types selected for investigational immunotherapy trials.
Results: mFIHC assay met clinical trial assay validation criteria for sensitivity and specificity on known positive and negative controls. Excellent reproducibility was observed across instruments, operators and independent experiments. Results between multiplex assay and singleplex counterparts were highly concordant (r2 of ≥ 0.89). In the tumor ROI, the frequency of IDO1+ cells ranged from 2-25%. The expression of CD68+ and CD163+ cells ranged from 2-16% and 2-34% respectively. PD-L1+ cells range from 2-45% while HLA-DR+ cells range from 7-78%. Of the cell population expressing PD-L1, 5-24% were CD68+ macrophages and 7-30% were CD163+ macrophages for all tumor types. In either CD163 or CD68 positive cells, 50-70% were co-expressing HLA-DR.
Conclusion: This validated clinical grade mFIHC assay is currently being utilized in ongoing novel cancer immunotherapy trials known to modulate macrophages and/or reverse immunosuppression. We believe novel tools such as this will accelerate delivery of more effective therapies to patients.
Citation Format: Lisa Adams, Deepika K. Asnani, Jehovana Bender, Beiru Chen, Thai Tran, Shabnam Tangri, Naveen Dakappagari, Jennifer Bordeaux. Exploring the interplay between TAM, PD-L1 and immunosuppressive molecules by a novel multiplex immunofluorescence assay in cancer immunotherapy trials [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2754.