Abstract 3396: Optimization of an assay for the detection of PD-L1 by immunohistochemistry in formalin-fixed, paraffin-embedded human tissue and cell lines
Authors: Dauffenbach Lisa M. , Sia Gela C. , Cash Patricia A. , Girees Sherif K. , Zheng Jianping , Lim Ryan S. , Olsen Eric P. , Kerfoot Christopher A. 
Published: August 2015

Abstract
Programmed Death-1 (PD-1) is a member of the CD28 family of receptors and plays a role in cellular immune response. It is expressed on activated T-cells and B-cells and binds to two ligands, Programmed Death Ligand 1 (PD-L1) and Programmed Death Ligand 2 (PD-L2). PD-1 negatively regulates T-cell functions through interactions with its primary ligand PD-L1, which is variably expressed by a variety of tumors, including melanoma, glioblastoma, and carcinomas of the lung, breast, colon, ovary, and renal cells. Many tumor cells express PD-L1 and as a result, the interaction between PD-1 and PD-L1 has been demonstrated to be an important target for therapeutic intervention. Anti-PD-1 therapeutics such as pembrolizumab and nivolumab have demonstrated clinical activity in melanoma, non-small cell lung cancer and other types of cancer. The purpose of this study was to investigate 7 commercially available anti-PD-L1 antibodies in human tissue and cell lines. Antibodies included 1 mouse monoclonal (clone 29E.2a3), 2 rabbit monoclonals (EPR1161(2) and E1L3N), 3 rabbit polyclonals, and 1 goat polyclonal from 6 manufacturers. An initial screen in human tonsil was performed to ensure appropriate sensitivity and specificity in staining of the crypt epithelium. Only 2 antibodies yielded staining that matched the expected specificity and were selected for further evaluation: rabbit clone EPR1161(2) from Abcam and rabbit clone E1L3N from Cell Signaling. Antibody specificity was evaluated in a positive cell line (MDA-MB-231) and negative cell line (MCF-7). Both antibodies demonstrated appropriate specificity in these cell lines. Antibody sensitivity was evaluated in a variety of human tumor tissues provided by Mosaic Laboratories’ tissue bank. PD-L1 localization was expected to be membranous and cytoplasmic in tonsil crypt epithelium, inflammatory cells and tumor cells. Immunohistochemistry with both antibodies demonstrated staining in tumor cells, lymphocytes and macrophages. Immunohistochemistry with clone E1L3N often demonstrated stronger staining in a greater percentage of cancer cells than EPR1161(2). Of interest, clone E1L3N demonstrated specific staining of some peripheral nerve fibers that was not observed with clone EPR1161(2). In conclusion, 2 commercially available antibodies, rabbit clone EPR1161(2) from Abcam and rabbit clone E1L3N from Cell Signaling are appropriate for immunohistochemistry, of which rabbit clone E1L3N demonstrates greater sensitivity in tumor tissue.
Citation Format: Lisa M. Dauffenbach, Gela C. Sia, Patricia A. Cash, Sherif K. Girees, Jianping Zheng, Ryan S. Lim, Eric P. Olsen, Christopher A. Kerfoot. Optimization of an assay for the detection of PD-L1 by immunohistochemistry in formalin-fixed, paraffin-embedded human tissue and cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3396. doi:10.1158/1538-7445.AM2015-3396