Understanding Immune Phenotypes And Their Spatial Relationships To Breast Adenocarcinoma In Ffpe Tissues

The relationship between elements of the immune system and breast tumors in situ requires an approach that leverages multiplexed immunohistochemistry (mIHC) with multispectral imaging to facilitate precise image analyses. To achieve this, we developed a novel 7-color mIHC assay, based on tyramide signal amplification, that allowed us to reliably interrogate CD3, CD4, CD8, FoxP3, CD68, and cytokeratin, in formalin-fixed, paraffin-embedded (FFPE) samples of human breast cancer. Imaging was performed using the multispectral Mantra system and inForm image analysis software. Using this specific mIHC panel and the cell segmenting and phenotyping tools in inForm, we were able to reliably identify cytotoxic T cells (CD3+ CD8+), helper T cells (CD3+ CD4+), regulatory T cells (CD3+ CD4+ FoxP3+), tumor associated macrophages (CD68+) and breast tumor cells (CK+). With cell phenotypes within the tumor microenvironment determined based on specific colocalized staining combinations, we then employed spatial point pattern analyses to examine spatial relationships between specific phenotypes. With this analysis, we were able to describe distances between cytotoxic T cells and regulatory T cells, cytotoxic T cells and tumor associated macrophages, as well as cytotoxic T cells and breast tumor cells. With this combined mIHC, multispectral imaging and advanced image analysis, we demonstrate a novel method that allows for unique tumor microenvironment assessments within in breast cancer. Through the preservation of tumor architecture available in archival FFPE tissues, these methods can advance our understanding of unique tumor microenvironment interactions, and could provide the ability to stratify responses to immunotherapies. Citation Format: Yi Zheng, Pallavi Thuse, Linying Liu, Edward C. Stack, Michael Campisano, Kent Johnson, Darryn Unfricht, Nara Narayanan, Clifford Hoyt, Milind Rajopadhye. Understanding immune phenotypes and their spatial relationships to breast adenocarcinoma in FFPE tissues. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2238.