Assessment of intra-tumoural colorectal cancer prognostic biomarkers using RNA in situ hybridisation

// Arthur Morley-Bunker 1 , John Pearson 2 , Margaret J. Currie 1 , Helen Morrin 1, 3 , Martin R. Whitehead 4 , Tim Eglinton 5 and Logan C. Walker 1 1 Mackenzie Cancer Research Group, Department of Pathology and Biomedical Science, University of Otago, Christchurch, New Zealand 2 Biostatistics and Computational Biology Unit, University of Otago, Christchurch, New Zealand 3 Cancer Society Tissue Bank, Department of Pathology and Biomedical Science, University of Otago, Christchurch, New Zealand 4 Canterbury Health Laboratories, Christchurch Hospital, Christchurch, New Zealand 5 Department of Surgery, University of Otago, Christchurch, New Zealand Correspondence to: Logan C. Walker, email: Logan.walker@otago.ac.nz Keywords: colorectal cancer, gene expression, RNA in situ hybridisation, prognostic markers Abbreviations: TCGA: The Cancer Genome Atlas Network; TMA: tissue microarrays; FFPE: formalin fixed paraffin embedded; CNA: copy number alterations; AJCC: American Joint Committee on Cancer Received: June 27, 2018      Accepted: February 01, 2019      Published: February 15, 2019 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. Results from these techniques are limited by tumour sample heterogeneity and the lack of correlation between mRNA transcript abundance and corresponding protein levels. The aim of this research was to investigate the clinical utility of the RNA in situ hybridisation technique, RNAscope ® , for measuring intra-tumoural gene expression of potential prognostic markers in a colorectal cancer cohort. Two candidate gene markers ( GFI1 and TNFRSF11A ) assessed in this study were identified from a previous study led by the The Cancer Genome Atlas (TCGA) Network, and analysis was performed on 112 consecutively collected, archival FFPE colorectal cancer tumour samples. Consistent with the TCGA Network study, we found reduced GFI1 expression was associated with high-grade and left-sided tumours, and reduced TNFRSF11A expression was associated with metastasis and high nodal involvement. RNAscope ® combined with image analysis also enabled quantification of GFI1 and TNFRSF11A mRNA expression levels at the single cell level, allowing cell-type determination. These data showed that reduced mRNA transcript abundance measured in patients with poorer prognosis occurred in carcinoma cells, and not lymphocytes, stromal cells or normal epithelial cells. To our knowledge, this is the first study to assess the intra-tumoural expression patterns of GFI1 and TNFRSF11A and to validate their microarray expression profiles using RNAscope. We also demonstrate the utility of RNAscope ® technology to show that expression differences are derived from carcinoma cells rather than from cells located in the tumour microenvironment.