Abstract 5135: Detection of IFNγ induced PDL1 expression by combined in situ RNA analysis and protein profiling from a single FFPE slide
Authors: Au Qingyan , Nguyen Kathy , Lazare Michael S. , Moler Edward J. , Hoe Nicholas 
Published: July 2016

PD1 ligands (PDL1) are often upregulated on the cell surface of many different tumors. The primary role of PDL1 in cancer is to inhibit T-cell mediated immune response. Two general mechanisms for PDL1 expression on tumor cells have been proposed. Innate immune resistance, in which PDL1 expression is induced by the constitutive oncogenic signaling, and adaptive immune resistance, in which PDL1 expression is induced by T-cells releasing interferon-γ (IFNγ) and activating the STAT signaling pathway. In order to differentiate between these two mechanisms, IFNγ mRNA expression is measured as an effective alternative to detecting IFNγ protein. Detection of cytokines by IHC is challenging as secreted proteins are widely diffused and the associated staining pattern appears to lack cellular specificity. RNAscope RNA in situ hybridization (ISH) assay is utilized to measure Interferon-γ (IFNγ) mRNA expression, and MultiOmyxTM multiplexed assay (demonstrated to stain up to 60 protein biomarkers) is utilized to measure CD3, CD4, CD8, CD56, CD68, PD1, and PDL1 protein expression. In this study, combined MO and RNAscope ISH assays, enabled identification of individual cells with characteristic mRNA and protein expression profile.

The MultiOmyx assay utilizes a pair of directly conjugated Cyanine dye-labeled (Cy3, Cy5) antibodies per round of staining. Each Cy-dye conjugated antibody recognizes different target proteins. Each round of staining is imaged and followed by novel dye inactivation chemistry, enabling repeated rounds of staining. RNAscope is a novel RNA ISH assay capable of single-molecule detection in individual cells, utilizing hybridization mediated signal amplification. The assay utilizes a pair of RNA target specific oligonucleotide probes, which sequentially hybridize to preamplifier, amplifier, and fluorophore label probes.

Utilizing MultiOmyx and RNAscope assays, this study proposed to profile both RNA and protein expression in lung, breast, melanoma, colorectal, esophageal, and prostate cancer samples.

Differentiating PDL1 expression induced in response to inflammatory signals produced by an activated T-cell, from PDL1 expression induced by constitutive oncogenic signaling, has potential implications in effectiveness of PD1 blockade therapy. According to the proposed mechanisms, PD1 blockade as a mono therapy may only benefit individuals with strong endogenous immune response. In individuals with weak endogenous immune response, combinational therapies consisting of both immune activation and PD1-pathway blockade may be more effective than either mono therapy alone.

Citation Format: Qingyan Au, Kathy Nguyen, Michael S. Lazare, Edward J. Moler, Nicholas Hoe. Detection of IFNγ induced PDL1 expression by combined in situ RNA analysis and protein profiling from a single FFPE slide. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5135.