Cross-validation between anti-CD3/CD8/FOXP3/pan-Cytokerati n fluorescent multiplex IHC and chromogenic single IHC by digital image analysis for quantifying tumor -infiltrating lymphocytes in colorectal cancer patient samples

A rising need for the overall goal to decipher cancer development and progression is a better understanding of the dynamics of the tumor microenvironment (TME), which are grounded in the interactions and reciprocal manipulation of cancer, stromal, and immune cells. Tumor-infiltrating immune cells influence the TME, and analyses of immune cell types, densities and locations within the TME using fluorescent multiplex immunohistochemistry (mIHC) and digital image analysis appear promising for establishing prognostic indicators and in helping to identify more personalized anti-cancer therapies, but in order to be applied in the clinical setting the relevant assays require a precise validation. We developed a tyramide signal amplification (TSA)-based fluorescent mIHC assay for the detection of CD3, FOXP3, CD8, and pan-Cytokeratin (pan-CK) in formalin-fixed paraffin-embedded (FFPE) tissue on the Leica BOND RX automated staining platform. Algorithms for digital image analysis of chromogenic and fluorescent mIHC were developed using Visiopharm Oncotopix software, allowing for a distinct quantification of T cell populations within tumor and stroma regions of interest. To ensure the reliability and robustness of the mIHC assay an extensive assay validation was performed on FFPE colorectal cancer (CRC) tissue. Chromogenic IHC of the single markers was analyzed by a pathologist and compared to digital image analysis of chromogenic and mIHC. To ensure a suitable linearity of the assay titration series of the primary antibodies were performed. Furthermore, the repeatability and intermediate precision of the assay were determined. Finally, 50 individual FFPE CRC tissue samples were analyzed by fluorescent anti-CD3/FOXP3/CD8/pan-CK mIHC and digital image analysis. Good correlations were observed between chromogenic and mIHC, as well as between pathologist and digital image analysis, and an adequate specificity, accuracy, linearity, repeatability and intermediate precision of the assay could be demonstrated. Furthermore, the distributions and ratios of the differently labeled tumor-infiltrating T cell populations in 50 CRC tissue samples were in agreement with published literature and allowed for a classification of the samples regarding their immune phenotype. The reliable quantification of immune cell subsets in FFPE cancer tissue samples shown here provides an efficient way of analyzing the lymphocyte composition of the TME at a validation level that is comparable to chromogenic IHC and apparently suitable for an application in the clinical setting. The validated combination of mIHC and digital image analysis may therefore enable a classification of the immune status of CRC patient samples and could help to identify new targets for anti-cancer therapy. Citation Format: Daniel Biljes, Philipp Layer, Nickels Winkler, Nadine Fandrich-Dursun, Hartmut Juhl, Bernd Gromoll, Malik Khenkhar, Philipp C. Uhlig. Cross-validation between anti-CD3/CD8/FOXP3/pan-Cytokeratin fluorescent multiplex IHC and chromogenic single IHC by digital image analysis for quantifying tumor-infiltrating lymphocytes in colorectal cancer patient samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4572.