Abstract 6193: Exploring interplay between tumor intrinsic features and immune cell phenotypes in multi-tumor tissue microarrays by a single step 15-plex fluorescence immunohistochemistry and imaging on the Orion platform
Authors: Van Krieken Richard , Bender Jehovana O. , Kekchidou Sophia , Burgin Michelle , Ruma Kristen , Dakappagari Naveen , Bordeaux Jennifer 
Published: March 2024

Abstract
Purpose: Deeper understanding of the intricacies between tumor microenvironments and immune cell infiltrates is critical for creation of next generation therapies. However, current immunohistochemistry protocols in clinical practice are restricted to one to six markers (cell types) and rely on multiple rounds of antigen retrieval and sequential staining over two days. This current workflow has high risk for tissue and signal loss. Hence, we explored utility of a single step staining workflow using 15 distinct antibodies conjugated novel flours (Argo Dye System) of formalin fixed paraffin embedded (FFPE) tumor microarrays and imaged on the Orion microscope (Rarecyte). Fidelity of the staining was assessed by comparing data from the Orion platform to internally developed clinical grade assays. The implementation of higher plex imaging will enable greater insight into immune surveillance, mechanisms of resistance and patient stratification while minimizing patient tissue required for analysis.
Study Design: We utilized a custom panel developed by RareCyte (Hoescht, CD3e, CD4, CD8a, CD20, CD68, CD163, FOXP3, Ki67, Pan-CK, PD1, PDL1, CD45, Granzyme B, LAG3) to evaluate expression of immune markers on a pan-tumor and melanoma TMA including Hodgkin’s lymphoma, ovarian cancer, esophageal carcinoma, hepatic cell carcinoma, head and neck cancer. Serial sections were stained with 6-plex clinical grade assays, and correlation scores were determined between the 15-plex Orion and 6-plex assays utilizing TSA-based amplification.
Results: In this study, the performance of Orion platform was assessed by quantitative and qualitative measures. Linear regression between the 6-plex clinical grade assays and 15-plex Orion workflow were calculated for a panel of biomarkers including CD3, CD4, CD8, CD68, CD163, CK, FOXP3, GZMB, Ki67, and PD1. Our results demonstrated that the Orion performed similarly to clinical grade assays both in terms of proportion of cells and fluorescence intensity (R2 ~ 0.7-0.98, Slope ~ 0.7-1.1).
Conclusion: Our results suggest that the Orion platform offers a reliable and efficient solution for comprehensive biomarker analysis. Our results demonstrate that this platform can successfully capture the expression of 15 biomarkers on a single FFPE slide which offers workflow advantages over traditional TSA-based fIHC multiplexing that is limited to detection of 6 biomarkers on a single slide. The ability to analyze a wider range of biomarkers through a single platform has the potential to enhance the efficiency of biomarker analysis, leading to improved diagnostic and therapeutic outcomes in clinical practice.
Citation Format: Richard Van Krieken, Jehovana O. Bender, Sophia Kekchidou, Michelle Burgin, Kristen Ruma, Naveen Dakappagari, Jennifer Bordeaux. Exploring interplay between tumor intrinsic features and immune cell phenotypes in multi-tumor tissue microarrays by a single step 15-plex fluorescence immunohistochemistry and imaging on the Orion platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6193.