Abstract 2937: Utilizing multiplex chromogenic IHC and digital image analysis to evaluate immune cell content and spatial distribution within NSCLC tumor tissue
Authors: Sherry Lorcan , Anderson Mark , Cowan Marianne , Dawson Lee , Bystry Richard , Green Sarah-Jane 
Published: July 2017

Abstract
Chromogenic-based immunohistochemistry (IHC) is an established technique utilized within oncology R&D, allowing cells to be visualized within the context of their tumor microenvironment. However, when quantifying chromogenic stained sections via digital image analysis, the number of targets is usually limited due to constraints in deconvolving more than 3 chromogens present on the same tissue section image. The purpose of this study was to apply a customized algorithm, including a 5-colour deconvolution process to triplex chromogen IHC plus hematoxylin stained Non-Small Cell Lung Carcinoma (NSCLC) tumor tissues in order to quantify immune cell content and spatial distribution within both tumor and stroma regions of interest (ROI).
A multiplex IHC assay was developed and applied to FFPE sections of NSCLC tumor tissue to stain epithelial tumor cells (Pan CK, yellow), CD8+ cells (purple) and FoxP3+ nuclei (brown), counterstained with hematoxylin (blue). Immunostained whole slide images were generated for image analysis. A customized algorithm, which included a 5-chromogen color deconvolution process, was utilized within the Indica Labs HALO platform to separate the four IHC chromogens (including counterstain) and a fifth color (black) representing carbon deposit artefacts. Nuclear objects were formed by applying weighted optical density values for the brown, purple and blue colors, which were positive for either CD8 or FoxP3 and counterstain. A classifier was developed to automatically segment tumor from stroma. Each positive cell type was identified, using defined size and shape parameters, and quantified within each ROI.
IHC multiplex staining highlighted CD8+ cells and FoxP3+ nuclei to be present in both tumor and stroma compartments. Analysis data demonstrated the number of tumor and stroma CD8+ cells was 120 and 554 cells per mm2, while the number of tumor and stroma FoxP3+ nuclei was 20 and 19 nuclei per mm2. This gave a CD8+:FoxP3+ ratio of 6 within the tumor and 29 within the stroma. The average distance of CD8+ cells in the tumor or stroma to their nearest FoxP3+ nuclei was 92µm and 174µm respectively. The average distance of CD8+ cells to their nearest tumor cell was 39µm while the average distance of FoxP3+ nuclei to their nearest tumor cell was 29µm.
This study demonstrated multiplex chromogenic IHC as a valuable approach for quantifying multiple cell types within the context of their tumor microenvironment. Furthermore, up to five chromogen colors can be separated utilizing a custom 5-plex analysis algorithm, leading to a more in-depth evaluation of immune cell types present and their spatial distribution. This propounds a greater potential for interpretation of therapeutic mechanistic responses within the tumor microenvironment.
Citation Format: Lorcan Sherry, Mark Anderson, Marianne Cowan, Lee Dawson, Richard Bystry, Sarah-Jane Green. Utilizing multiplex chromogenic IHC and digital image analysis to evaluate immune cell content and spatial distribution within NSCLC tumor tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2937. doi:10.1158/1538-7445.AM2017-2937