Abstract LB-281: Development of a sensitive and quantitative PD-L1 immunoassay superior to IHC with application in human FFPE tissue samples (2015)

Abstract Introduction: Cancer immunotherapy approaches and targets are rapidly expanding for the treatment of many different types of cancer. The programmed cell death-1 receptor (PD-1) and its ligand PD-L1 have garnered a great deal of interest lately, partially due to current therapeutic agents demonstrating a long and durable clinical response with low toxicities in several cancer types. Binding of the PD-1 receptor on activated T-cells to PD-L1 expressing tumor cells reduces T-cell activation, thereby evading an immune response against tumor progression. PD-L1 expression by immunohistochemistry (IHC) has been shown to be a prognostic and potential predictive biomarker for response to both anti-PD-L1 and anti-PD-1 therapy, however, currently the IHC assay is not standardized and the definitions used for positivity are variable and subjective due to a visual scoring system. A lack of sensitivity of the IHC assay may partially explain the observed response to therapy in patients whose tumors were identified as IHC = 0/PD-L1 negative. In an attempt to offer a more sensitive and quantitative assay, we developed a PD-L1 protein expression assay using the VeraTag technology. The PD-L1 VeraTag assay utilizes the release of a unique fluorescent reporter (VeraTag), which is measured with high sensitivity via capillary electrophoresis to accurately and objectively quantify the amount of PD-L1 protein expression in FFPE samples. Methods: The anti-PD-L1 rabbit monoclonal antibody E1L3N (Cell Signaling Technologies) was utilized in the VeraTag assay together with a goat anti-rabbit secondary antibody conjugated to the VeraTag reporter. FFPE cancer cell line lines were used to optimize antigen retrieval, primary antibody concentration and signal/background ratio, with emphasis on the lower end of the dynamic range. Results: VeraTag measurements of PD-L1 protein expression correlated to both IHC and PD-L1 gene expression (CCLE, R squared = 0.7212) in FFPE cancer cell lines. The PD-L1 protein expression by VeraTag and IHC was compared across a group of FFPE squamous cell carcinoma of the head and neck (SCCHN) and HER2- and HER2+ breast samples. VeraTag assays for the measurement of the HER-family of receptors (HER1, HER2 and HER3 total, HER1-HER1 homodimer, HER2-HER3 heterodimer, phospho-HER3 and HER3-PI3 kinase complex) were evaluated for correlation to PD-L1 protein expression in these two cancer types. There was good agreement between the VeraTag and IHC measurements of PD-L1 protein expression, with the added advantage of the VeraTag assay providing an ∼5-fold range of PD-L1 expression within the IHC = 0 category. Conclusions: We have developed a sensitive and quantitative measurement of PD-L1 protein expression in FFPE human SCCHN and breast cancer samples utilizing the VeraTag technology. Measurement of PD-L1 expression from clinical samples with the VeraTag assay is warranted. Citation Format: Gerald Wallweber, Ahmed Chenna, Roy Ravanera, David Stathas, Weidong Huang, Christos Petropoulos. Development of a sensitive and quantitative PD-L1 immunoassay superior to IHC with application in human FFPE tissue samples. [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 LB-281. doi:10.1158/1538-7445.AM2015-LB-281