Targeting checkpoint molecules expressed on immune cells has shown promising results in the treatment of non-small cell lung cancer (NSCLC). One interesting molecule that is frequently expressed on exhausted tumor-infiltrating lymphocytes (TIL) is lymphocyte activation gene-3 (LAG-3). We recently implemented chromogenic anti-LAG-3/CD3 dual immunohistochemistry (IHC) and digital image analysis to quantify LAG-3 positive T cells in NSCLC tissue. Here, we extended this work by establishing an anti-LAG-3/CD3/CD8/pan-Cytokeratin (pan-CK) fluorescent multiplex IHC (mIHC) assay followed by digital image analysis to examine the composition of T cells infiltrating NSCLC tissue in more detail. Tyramide signal amplification (TSA) based fluorescent 5-color mIHC (LAG-3/CD3/CD8/pan-CK + DAPI) was developed by Indivumed on the Leica BOND RX automated staining platform and applied to formalin-fixed paraffin-embedded (FFPE) NSCLC tissue samples. Image analysis was performed by OracleBio. Tumor and stroma regions of interest (ROI) were classified according to the pan-CK and DAPI signals. LAG-3, CD3 and CD8 single positive cells, as well as dual and triple positive cells, were then quantified in the tumor and stroma ROIs. Fluorescent mIHC allowed for a specific quantification of LAG-3, CD3 and CD8 single, LAG-3/CD3, LAG-3/CD8, CD3/CD8 dual and LAG-3/CD3/CD8 triple labeled cells in the tumor and stroma ROIs of the analyzed NSCLC samples. As part of the assay validation, similar ratios of the differently labeled cells, in particular LAG-3 positive T cells (LAG-3/CD3 dual positive), were observed with fluorescent mIHC and chromogenic IHC, demonstrating a good concordance between the two approaches. Among the analyzed NSCLC samples, different ratios of LAG-3 positive TILs in adeno- and non-adenocarcinoma samples were detected. Determining the ratios of dual or triple positive TIL subsets by mIHC in combination with digital image analysis allows for a clearer understanding of the lymphocyte composition within NSCLC tissue than a simple quantification of LAG-3, CD3 and CD8 single positive cells. Furthermore, such information will contribute to a deeper understanding of the role of LAG-3 positive immune cell sub-populations in the progression and treatment of NSCLC.

Citation Format: Malik Khenkhar, Daniel Biljes, Nickels Winkler, Philipp C. Uhlig, Hartmut Juhl, Alison L. Bigley, Lorcan Sherry. Utilization of fluorescent multiplex IHC and digital image analysis for studying LAG-3, CD3 and CD8 positive TIL subsets in NSCLC tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5686.