Whole genome RNA expression profiling of endoscopic biliary brushings provides data suitable for biomarker discovery in cholangiocarcinoma

Chapman, Michael H.; Tidswell, Robert; Dooley, James S.; Sandanayake, Neomal S.; Cerec, Virginie; Deheragoda, Maesha; Lee, Alvin J.X.; Swanton, Charles; Andreola, Fausto; Pereira, Stephen P.

doi:10.1016/j.jhep.2011.10.022

« Previous Next »Journal of Hepatology
Volume 56, Issue 4 , Pages 877-885, April 2012

Whole genome RNA expression profiling of endoscopic biliary brushings provides data suitable for biomarker discovery in cholangiocarcinoma

Michael H. Chapman
- UCL Institute of Hepatology, Royal Free Campus, University College London, United Kingdom
- Department of Gastroenterology, University College London Hospitals NHS Foundation Trust, United Kingdom
Robert Tidswell
- UCL Institute of Hepatology, Royal Free Campus, University College London, United Kingdom
James S. Dooley
- Centre for Hepatology, Royal Free Campus, University College London, United Kingdom
Neomal S. Sandanayake
- UCL Institute of Hepatology, Royal Free Campus, University College London, United Kingdom
- Department of Gastroenterology, University College London Hospitals NHS Foundation Trust, United Kingdom
Virginie Cerec
- UCL Institute of Hepatology, Royal Free Campus, University College London, United Kingdom
Maesha Deheragoda
- Department of Histopathology, University College London Hospitals NHS Foundation Trust, United Kingdom
Alvin J.X. Lee
- Translational Cancer Therapeutics Laboratory, Cancer Research UK London Research Institute, London, United Kingdom
Charles Swanton
- Translational Cancer Therapeutics Laboratory, Cancer Research UK London Research Institute, London, United Kingdom
Fausto Andreola
- UCL Institute of Hepatology, Royal Free Campus, University College London, United Kingdom
- Joint senior authors.
Stephen P. Pereira
- UCL Institute of Hepatology, Royal Free Campus, University College London, United Kingdom
- Department of Gastroenterology, University College London Hospitals NHS Foundation Trust, United Kingdom
- Corresponding author. Address: UCL Institute of Hepatology, UCL Medical School, Royal Free Campus, Rowland Hill Street, London NW3 2PF, United Kingdom. Tel.: +44 020 77940500x35622; fax: +44 020 7472 6225.
- Joint senior authors.

Received 8 July 2011; received in revised form 10 October 2011; accepted 25 October 2011. published online 14 December 2011.

Background & Aims

Molecular analyses of biliary brushings using microarray and qPCR have the potential to provide valuable information on the biology of biliary diseases. Microarray analysis of biliary strictures has rarely been applied to endoscopic biliary brushings.

Methods

Biliary brushings were obtained from patients with benign and malignant biliary disease at the time of ERCP. Microarray analysis of mRNA isolated using brushings from 10 patients was validated for a selection of genes by qPCR using the same source mRNA and a second fresh set of nine biliary brushings as well as surgical resection tissue. Cultured cholangiocytes were used to assess the impact of bile or X-ray contrast solution on RNA quality.

Results

RNA was of variable quantity (100–1500ng) and poor quality (Agilent RNA Integrity Number (RIN)<5, estimated to be fragments 100 to 600 base pairs long). Reliable qPCR results required primer pairs designed to produce amplicons <130bp. Differential gene expression by microarray analysis identified 1140 up-regulated genes and 1001 down-regulated genes between benign and malignant biliary strictures. The trends in a selection of 45 up-regulated genes, including various HOX genes, collagens, PVT1, MUC4, MUC5AC, and LEF1, were validated by qPCR using RNA from biliary strictures with a moderate to strong correlation coefficient between microarray and qPCR (r=0.41 to r=0.57). Immunohistochemistry of surgical resection tissues (n=23) showed elevated CD9, SERPINA3, and PNMA2 protein expression in cancer samples.