Abstract 3301: Immune monitoring for immuno-oncology applications
Authors: Lomas Woodrow E. , Gao Guo-Jian , Li Na , Saksena Suraj , Chattopadhyay Pratip K. 
Published: September 2020

Abstract
The immunotherapy revolution has spurred the development of many new drugs and drug regimens for patient treatment. A key challenge is to identify the factors that drive patient toxicities and responses to treatment, with a particularly acute need for predictive biomarkers that can discriminate patients destined to respond and fail treatment. Technologies to interrogate immune cells are now readily available, but important gaps remain in their application, which limit full realization of the promise of precision oncology.
One such technology, high parameter flow cytometry, represents a gold-standard for immune monitoring; however, antibody panel design and lack of standardization represent important application gaps that hinder broader use. Here, we present a new tool for panel design, Color Wheel, which builds antibody panels optimized for performance on the user's instrument. We used Color Wheel to develop six panels (cell lineage, immune checkpoint, checkpoint ligand, regulatory T-cell, B-cell, cytokine) that comprehensively interrogate the tumor microenvironment. We demonstrate the value of these panels in identifying differences between disease types, treatment responses, and patient groups in a variety of cancer settings. Furthermore, we have developed the immune checkpoint panel into a dried, ready to use (single test-per-tube) format to lower the barriers to access high parameter flow cytometry technology, and to drive workflow efficiency and assay standardization. We demonstrate excellent concordance between the dried and liquid (wet) versions of the high parameter multicolor panel.
A second technology, molecular cytometry, represents an exciting new approach to high dimensional immune analysis. The technology is capable of measuring at least 102 proteins simultaneously per cell, alongside 400 mRNA targets. An important application gap for this technology is lack of data indicating the sequencing depth needed to resolve cell types in an experiment. Here, we present results from a molecular cytometry experiment sequenced deeply, and then bioinformatically sub-sampled at different levels to identify the minimum level of sequencing needed for clear identification of cells, which can serve as a reference guide for users to save time and cost in their experiments. From the perspective of workflow efficiency, we also present preliminary performance data generated using a dried version of molecular cytometry panel(s).
Finally, our presentation includes an analysis of the rationale for choosing an immune monitoring technology, providing some guidelines that match study type and goals, cost, timelines, complexity, and labor with various single cell technologies.
Citation Format: Woodrow E. Lomas, Guo-Jian Gao, Na Li, Suraj Saksena, Pratip K. Chattopadhyay. Immune monitoring for immuno-oncology applications [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 3301.