Abstract 1725: Tissue-based biomarker analysis of DLBCL using multiplex immunofluorescence AQUA (Automated Quantitative Analysis) algorithms
Authors: Santos James Paul , Levy Jacob , Ruma Kristen , Yang David , Woolfenden Steve , Lim Jimmie , Li Xun , Valencia Ariana , Deeds James , Thiruvamoor Ramu , Li Xin , Pacia Emmanuel , McLaughlin Margaret , Tran Thai , Choi Sarah , Bordeaux Jennifer 
Published: June 2022

Abstract
Purpose: Deeper understanding of immune landscape of DLBCL tumor microenvironment is critical for exploration and development of next generation immunotherapies. Although conventional immunohistochemical (IHC) analysis can provide information on the immune landscape or target expression, it is generally limited to single marker analysis. Advantages of multiplex fluorescence IHC (mFIHC) include the ability to assess different cell types, their spatial relationship as well as variable antigen expression on tumor cells. As such, we developed quantitative mFIHC panels using AQUA® (Automated Quantitative Analysis) technology to evaluate T and B cell populations and their functional status to generate detailed spatial information of immune checkpoint (IC) markers. mFIHC combined with hypothesis driven spatial profiling algorithms (e.g., AQUA Technology) were 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). Implementation of mFIHC coupled with robust image analysis may provide great insight into DLBCL immune surveillance, mechanism of resistance and patient stratification.
Study Design: We designed two novel mFIHC assays to (1) characterize various B cell populations (CD19, CD20, CD22, PAX5, CD3, TIM3), and (2) evaluate T cell functional status (CD8, Granzyme B, PD1, PDL1, TIM3, Tumor). We successfully validated clinical grade mFIHC assays using automated staining (Leica Bond RX), imaging (Vectra Polaris) and analyses (AQUA Technology) workflow.
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. Between these two panels, over 200 unique parameters were evaluated. The prevalence of CD19, CD20, CD22, and PAX5 ranged from 30% to &gt;90% positive for the DLBCL samples tested and were overall highly concordant with each other.
Conclusion: The validated mFIHC assays examine B cell antigen expression in DLBCL and their interaction with CD3+ T cells and further characterize CD8+ T cells and immune checkpoint expression. These panels may be used to further understand the complex immune cell and tumor cell spatial biology in the context of clinical trials.
Citation Format: James Paul Santos, Jacob Levy, Kristen Ruma, David Yang, Steve Woolfenden, Jimmie Lim, Xun Li, Ariana Valencia, James Deeds, Ramu Thiruvamoor, Xin Li, Emmanuel Pacia, Margaret McLaughlin, Thai Tran, Sarah Choi, Jennifer Bordeaux. Tissue-based biomarker analysis of DLBCL using multiplex immunofluorescence AQUA (Automated Quantitative Analysis) algorithms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1725.