Abstract 3390: Validating the RNAscope for molecular profiling of key biomarkers associated with gemcitabine resistance
Authors: Ghazaly Essam A. , Gnanaranjan Chathunissa , Greenhalf Bill , Blagden Sarah P. , Mullen Peter , Harrison David , Gribben John G. 
Published: August 2015

Abstract
Introduction: Acelarin® is the first anti-cancer ProTide to enter the clinic and has achieved a very high rate of disease control in a Phase I study (ProGem1) of patients with advanced progressive solid tumours. It is a first-in-class nucleotide analogue specifically designed to bypass key cancer resistance pathways associated with gemcitabine, including altered expression of human equilibrative nucleoside transporter 1 (hENT1), deoxycytidine kinase (dCK) and cytidine deaminase (CDA). We have used a cytological RNA-based assay, RNAscope® and quantitative Reverse Transcriptase Polymerase Chain Reaction (qPCR) analysis to quantify these three biomarkers in seven cancer cell lines, and reviewed and tested a panel of commercially available antibodies against each protein. The aim was to select and validate a method suitable for stratifying tumours according to their molecular signature and allow the identification of patients unlikely to derive clinical benefit from gemcitabine.
Methods: Cells from seven cancer lines (BxPC-3, MiaPaCa-2, PANC-1, SK-MES-1, NCI-H1703, OVCAR-3 and OVCAR-8) were prepared in formalin-fixed paraffin-embedded blocks. In addition, transiently transfected MiaPaCa-2 cells overexpressing hENT1, dCK and CDA were prepared. Expression of mRNA was detected using RNAscope and quantified by SpotStudio as well as by qPCR. Antibodies were examined by Western-blot analysis, reverse phase protein arrays (RPPA) and immunohistochemistry. Protein expression by immunofluorescence was quantified using Aqua HistoRx technology.
Results: RNAscope detected mRNA for CDA at low levels in all cell lines. dCK and hENT1 were detected at low (in BxPC-3) to high (in NCI-H1703) expression levels across the cell lines. The qPCR method quantified precisely the amount of RNA, which demonstrated a positive and significant correlation with RNAscope data (Spearman correlation rho = 0.76, p = 0.037 for CDA, rho = 0.67, p = 0.08 for dCK, rho = 0.67, p = 0.08). Most antibodies were rejected because of low specificity as observed by Westerns, RPPA and immunohistochemistry. In addition, both techniques identified the gene over-expression in the transfected cell lines where the expression was up to 100 fold higher in transfected cells compared to their wild-type pairs.
Conclusions:
Selection of antibodies against hENT1, CDA and DCK for clinical use remains challenging. The RNAscope technology was validated, allowing reliable detection of biomarkers thought to be associated with cancer cell resistance to gemcitabine.
Citation Format: Essam A. Ghazaly, Chathunissa Gnanaranjan, Bill Greenhalf, Sarah P. Blagden, Peter Mullen, David Harrison, John G. Gribben. Validating the RNAscope for molecular profiling of key biomarkers associated with gemcitabine resistance. [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 3390. doi:10.1158/1538-7445.AM2015-3390