Abstract 2771: Comprehensive analysis of immuno oncology markers in the tumor microenvironment of solid tumor samples using GeoMxTM digital spatial profiler (DSP) and MultiOmyxTM hyperplexed immunofluorescence (IF)
Authors: Chandramohan Lakshmi , Au Qingyan , Nagy Mate , Parnell Erinn , Peters Tricia 
Published: July 2021

Abstract
In recent years, novel immunotherapy strategies have shown remarkable results in treating various advanced cancers. The efficacy of immunotherapeutics has been dictated by the dynamic and spatially heterogeneous tumor microenvironment (TME) which includes immune tumor infiltrates and immune checkpoint protein expression, in addition to tumor molecular profiles, leading to widely variable clinical outcomes and responses in patients. The local milieu as well as complex tumor-TME interactions vary widely between tumors of the same type and also within the same tumor and therefore highlights the need for enhanced analysis of tissue-immune content and their spatial context for predicting response to immunotherapies and biomarker discovery. In this study, we will apply a multi-faceted, highly multiplexed tissue analysis approach to quantitate and better characterize the spatial arrangement of key immuno oncology protein markers in a pan-cancer cohort of up to 35 FFPE samples from breast, head and neck, prostrate, non-small cell lung cancer (NSCLC), endometrial and colorectal indications using NanoString GeoMxTM Digital Spatial Profiler (DSP) and MultiOmyxTM Hyperplexed Immunofluorescence (IF) assay. For DSP analysis, each sample will be profiled for up to 52 immuno oncology (IO) markers included on NanoString Human IO protein panels. Selection of regions of interest will be guided by H&E staining, fluorescent markers (CD45, pan-CK and Syto 13). Protein profiling of tumor and TME regions will be achieved through segmenting by pan-CK+/CD 45+ followed by collection of indexed oligonucleotides and digital counting using the NanoString nCounter system. Data normalization and unsupervised clustering of IO protein expression across the ROIs will be performed to understand the spatial distribution of these IO biomarkers within the TME. An adjacent section of each tumor sample will be also stained and analyzed using MultiOmyx technology. MultiOmyx is a proprietary IF platform for the visualization and characterization of up to 60 protein biomarkers in a single FFPE section. Using the MultiOmyx IF assay in combination with proprietary algorithms, we will study the expression and spatial distribution of 26 IO markers to characterize different subtypes of immune cells and immune checkpoint inhibitors within the TME in the 35 FFPE tumor samples. Combination of MultiOmyx IF with Nanostring DSP provides a complementary and powerful solution to study the IO markers within the TME. Using the DSP nCounter output along with MultiOmyx cell classification and spatial analysis results, the researchers can simultaneously profile the high-plex protein expression, characterize the immunophenotypes and visualize the spatial distribution of tumor infiltrating immune cells at single-cell resolution within the TME.
Citation Format: Lakshmi Chandramohan, Qingyan Au, Mate Nagy, Erinn Parnell, Tricia Peters. Comprehensive analysis of immuno oncology markers in the tumor microenvironment of solid tumor samples using GeoMxTM digital spatial profiler (DSP) and MultiOmyxTM hyperplexed immunofluorescence (IF) [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 2771.