Abstract 2645: Intratumoral assessment of candidate colorectal cancer prognostic markers using RNA in situ hybridization

Abstract

Abstract Genome-wide expression studies using microarrays and RNAseq have increased our understanding of colorectal cancer development. Translating potential gene biomarkers from these studies for clinical utility has typically relied on PCR-based technology and immunohistochemistry. However, these techniques often suffer from limitations because of tumor sample heterogeneity, or a lack of correlation between protein and corresponding mRNA transcript levels. RNAscope® (Advanced Cell Diagnostics) is an RNA in situ hybridization method that overcomes these limitations by enabling single-molecule detection, while preserving cellular and tissue morphology. The aim of this research was to investigate the clinical utility of RNAscope to measure gene expression of two potential prognostic markers (GFI1 and TNFRSF11A) that were identified from a previously reported study from The Cancer Genome Atlas (TCGA) Network. We analyzed 112 consecutively collected archival formalin-fixed, paraffin-embedded tumors from a collection of well-characterized colorectal cancer cases. Consistent with TCGA Network data, we found significant associations for reduced GFI1 and TNFRSF11A mRNA expression and clinicopathologic features in our cohort. Reduced GFI1 expression measured by RNAscope was associated with high-grade tumor (P = 0.02) and left-sided tumor (P = 0.01). Reduced TNFRSF11A expression was associated with metastasis (P = 0.04) and high nodal involvement (P = 0.02). RNAscope was combined with semiautomated image analysis to provide quantification of mRNA expression at the single-cell level, allowing for cell-type determination of mRNA expression levels within a tumor. These data showed that GFI1 and TNFRSF11A were expressed at a significantly higher level in carcinoma cells compared to noncarcinoma cells (lymphocytes, stromal cells and normal cells). Thus, we were able to demonstrate that reduced expression measured in patients with poorer prognosis was not due to contamination of tumor samples with noncarcinoma cells. Limited information is available regarding TNFRSF11A's involvement in colorectal cancer. Gene knockdown of TNFRSF11A in colorectal cell line models showed changes in cell growth properties. To our knowledge, this is the first study to assess the intercellular expression patterns of GFI1 and TNFRSF11A as candidate prognostic markers in colorectal tumor. Furthermore, we show that intratumoral expression analysis of candidate genes by RNAscope is an informative technology for validating results from genome-wide expression studies. Citation Format: Arthur Morley-Bunker, John Pearson, Margaret Currie, Helen Morrin, Martin Whitehead, Tim Eglinton, Logan Walker. Intratumoral assessment of candidate colorectal cancer prognostic markers using RNA in situ hybridization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2645.