Evaluating the density of immune cell subtypes and their relative spatial positioning in cancers has become an important tool in understanding response to immune checkpoint inhibitors. The purpose of this study was to investigate protein expression of PD-L1, PD-1, CD3, CD4, CD8, CD68, FoxP3, and Ki-67 with four multiplex immunohistochemical assays (Ki-67+CD8, CD3+CD4+Ki-67, FoxP3+PD-1+CD8, and CD68+PD-L1+CD3) using DAB, red, and green chromogens within formalin-fixed, paraffin embedded non-small cell lung (NSCLC) tissues. The Ki-67+CD8 multiplex was developed to evaluate the density of cytotoxic T cells and the percentage that are proliferating. The CD3+CD4+Ki-67 multiplex characterizes the frequency of CD4+ T cells and evaluate proliferation in this subset. The FoxP3+PD-1+CD8 multiplex evaluates the regulatory T cell subset and evaluates PD-1 expression. The CD68+PD-L1+CD3 multiplex characterizes PD-L1 expression on macrophages and T cells. Cell densities were evaluated in the center of tumor and invasive margin regions, and image analysis was performed to quantitate each stain as a single agent and co-expression within each multiplex assay.

Citation Format: Lisa M. Dauffenbach, Gela C. Sia, Jianping Zheng, Natalia Jun, Eric P. Olsen, Christopher A. Kerfoot. Multiplex immunohistochemical staining of PD-L1, PD-1, CD3, CD4, CD8, CD68, FoxP3, and Ki-67 and image analysis of tumor and invasive margin in human FFPE NSCLC 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 1669. doi:10.1158/1538-7445.AM2017-1669