Abstract 2676: Hyperplexed immunofluorescence analysis (Cell DIVETM) of immune-related tumor heterogeneity in stage III colorectal cancer
Authors: Stachtea Xanthi , Lindner Andreas , Salvucci Manuela , Cho Sanghee , Sood Anup , McDonough Elizabeth , Santamaria-Pang Alberto , Graf John , Dunne Philip , Lawler Mark , Prehn Jochen , Ginty Fiona , Longley Daniel 
Published: September 2020

Abstract
Colorectal cancer (CRC) has one of the highest Worldwide incidences and mortality rates. Genotoxic chemotherapy following surgery in stage III patients confers treatment benefit to less than 20% of the patients, with more than 50% of stage III patients going on to develop distant metastases. Currently, there are no predictive biomarkers that can identify which stage III patients will recur, which patients will benefit from chemotherapy and which should be redirected towards alternative therapeutic interventions. A major challenge in identifying such a universal biomarker is that CRC is a heterogeneous disease with multiple subtypes. In the current study, we assessed clinically-relevant immune cell populations in the tumour microenvironment (TME) of stage III tumours using a novel hyperplex in situ immunofluorescence imaging technology (Cell DIVETM, GE Healthcare, Issaquah, WA).
Tissue microarrays (TMAs) with up to three 1mm diameter cores per patient were prepared from 139 stage III CRC patients treated with adjuvant FOLFOX chemotherapy. Single sections (5 µm) were iteratively stained with Cy3- and Cy5-conjugated antibodies for immune cell markers as well as markers of cell death and metabolism. The images underwent illumination correction, DAPI-based registration and autofluorescence removal. After image quality control corrections, single cell segmentation was performed using a combination of DAPI [nuclear], pan-cytokeratin [epithelial], NaKATPase [membrane] and S6 [cytoplasmic] segmentation markers and an average of ~3,000 stromal cells and ~ 4,000 epithelial were segmented per tumour core.
A machine learning-based algorithm for immune cell classification and quantification was used to analyse the immune markers CD45, CD3, CD4, CD8, FOXP3 and PD1 to identify: cytotoxic T cells, T helper cells, regulatory T cells and potential relevance of immune checkpoint therapy. In the tumour tissues, the median proportion of CD3+ segmented cells was ~8%. Classified immune cells were counted within epithelial and stromal regions, with patients categorised as Low, Intermediate and High (based on &lt;25th, 25th - 75th and &gt;75th percentile, respectively) for each cell type. Preliminary survival analyses show that patients with ‘CD8 High' intratumoural cytotoxic T cells have better Disease-Free Survival compared to ‘CD8 Low' patients in this FOLFOX-treated cohort. By combining single-cell data with clinicopathological patient data, we aim to identify immune-, cell death- and metabolism-related signatures that can predict benefit from adjuvant FOLFOX chemotherapy for Stage III CRC patients.
Citation Format: Xanthi Stachtea, Andreas Lindner, Manuela Salvucci, Sanghee Cho, Anup Sood, Elizabeth McDonough, Alberto Santamaria-Pang, John Graf, Philip Dunne, Mark Lawler, Jochen Prehn, Fiona Ginty, Daniel Longley. Hyperplexed immunofluorescence analysis (Cell DIVETM) of immune-related tumor heterogeneity in stage III colorectal cancer [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 2676.