Abstract 310: Immunophenotyping tumors by novel multiplex immunofluorescence and AQUA (Automated Quantitative Analyses) algorithms to guide development of immunotherapies
Authors: Kim Ju Young , Hoerter John , Roscoe Nathan , Fathman John , Santos James , Iaconis Lori , Santos Justin , Qiu Minhua , Levy Jacob , Tolley Valerie , Pacia Emmanuel , Carolan Tom , Li Xun , Barzaghi-Rinaudo Patrizia , Schwartz Brian , Newlin Robbin , Colman Karyn , Schumacher Andy , Knee Deborah , Bender Steven , McLaughlin Margaret , Savchenko Alexander , Tran Thai , Tangri Shabnam , Dakappagari Naveen , Bordeaux Jennifer 
Published: September 2020

Abstract
Purpose: Deeper understanding of immune landscape of the tumor microenvironment is critical for exploring and development of next generation immunotherapies. Multiplex fluorescence immunohistochemistry (mFIHC) combined with hypothesis driven spatial profiling algorithms (e.g., AQUA Technology) was found to provide the most powerful predictors of immunotherapies in a systematic meta-analyses of over 8000 patients treated with PD1/L1 pathway blockers (Lu et al., JAMA Oncol 2019).
Study Design: To guide clinical development of next generation and/or combination immunotherapies, we built four novel mFIHC assays to simultaneously explore presence of major immune cell lineages, their spatial relationships and functional attributes. The first assay incorporates antibodies to identify major immune cells lineages (e.g., T-, B-, NK-/T- and subtypes of myeloid cells), the second assay is designed to understand T-cell proliferation (via co-expression of Ki67) or suppression (via co-expression of FoxP3), the third method is focused on quantifying the expression of immune checkpoint inhibitors (e.g., LAG3, PD1 and PD-L1), while, the final assay is designed to explore the IFNγ mediated adaptive resistance and immunosuppression (via co-expression of HLA-DR and IDO1). We will describe the successful development of these clinical grade mFIHC assays that utilize automated staining (Leica Bond RX), imaging (Vectra Polaris) and analyses (AQUA® Technology) workflows on tissue microarrays (TMAs) representing multiple regions of interest including tumor, tumor margin, tumor-associated fibrosis, various adjacent normal tissues, lymphoid structures and/or associated inflammation, and metastatic lesions collected from 100 colorectal, and pancreatic cancer patients.
Results: Sensitivity, accuracy and specificity were confirmed for all mFIHC assays on known positive and negative controls. Excellent reproducibility (less than 35% CV) and precision were observed across instruments, operators and independent experiments for all markers. TMA work is underway and the latest results will be presented.
Conclusion: The validated mFIHC assays helped classify unique immune phenotypes and their interactions. These assays, combined with comprehensive selection of tissues from the tumor microenvironment across multiple cancer subtypes, are expected to enable clinical development of next generation cancer therapies.
Citation Format: Ju Young Kim, John Hoerter, Nathan Roscoe, John Fathman, James Santos, Lori Iaconis, Justin Santos, Minhua Qiu, Jacob Levy, Valerie Tolley, Emmanuel Pacia, Tom Carolan, Xun Li, Patrizia Barzaghi-Rinaudo, Brian Schwartz, Robbin Newlin, Karyn Colman, Andy Schumacher, Deborah Knee, Steven Bender, Margaret McLaughlin, Alexander Savchenko, Thai Tran, Shabnam Tangri, Naveen Dakappagari, Jennifer Bordeaux. Immunophenotyping tumors by novel multiplex immunofluorescence and AQUA (Automated Quantitative Analyses) algorithms to guide development of immunotherapies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 310.