Abstract 659: Multiplex cocktails for immunotherapy targets: PD-L1 with tumor specific transcription factors

Abstract

Abstract Introduction Blocking the interaction between the programmed cell death (PD)-1 protein and one of its ligands, PD-L1, has been reported to generate impressive antitumor responses. Therapeutics targeting PD-L1 in several cancers are currently in clinical trials, and the U.S. FDA has granted accelerated approval for Keytruda (pembrolizumab) to treat patients with advanced (metastatic) non-small cell lung cancer (NSCLC). Various pathology scoring and interpretation methods have been put forth to assay PD-L1 expression, and variability of non-PD-L1 staining across tumors may have important prognostic aspects. PD-L1 expression on tumor cells can be upregulated via activation of CD8+ cytotoxic T lymphocytes. PD-L1 expression is also associated with certain subtypes of tumor-associated lymphocytes, macrophages and dendritic cells. In certain cancers, the cross reactivity of PD-L1 can make interpretation and scoring difficult. Thus, a strategy using IHC multiplex stains could help resolve challenging cases by cocktailing PD-L1 with target tissue transcription factor antibodies along with other potential markers. Method and Materials Formalin-fixed paraffin-embedded tissues and tissue microarrays for various tumor types including lung, bladder and melanoma were processed and cut at 4-5 microns. PD-L1 rabbit monoclonal antibody was cocktailed with the following antibodies: TTF-1 (lung cancer); CD163 (lung cancer); p40 + GATA3 (bladder cancer); and SOX10 (melanoma). Cocktails were detected with double stain detection systems using brown, red and blue chromogens for visualization. Sections were counterstained with a standard hematoxylin or Wiegert’s iron hematoxylin. Results PD-L1 and TTF-1 with or without CD163 (macrophage) and PD-L1 + CD163 were used in lung cancers. The nuclear staining of TTF-1 in lung adenocarcinoma (blue chromogen) helped define tumor positive PD-L1 (DAB) positive cells and CD163 (Fast Red) marked macrophages. PD-L1 combined with p40 and GATA3 was deployed for bladder cancers. PD-L1 membranous expression in tumor cells and co-expression of p40 and GATA3 gave robust nuclear staining in bladder tumor cells. Finally, SOX10 nuclear staining was observed in most melanoma cells and could be easily separated from PD-L1 membrane staining. Conclusion PD-L1 cocktailed with strategic nuclear or cytoplasmic antibodies can help discriminate tumor cells from non-tumor cells, and may facilitate quantitation or scoring methods for more accurate assessment of this key immunotherapy marker. Citation Format: David Altree-Tacha, Wei Yuan, George Yang. Multiplex cocktails for immunotherapy targets: PD-L1 with tumor specific transcription factors [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 659. doi:10.1158/1538-7445.AM2017-659