%0 Thesis
%9 Doctoral
%A Chapman, M.H.
%B UCL Institute of Hepatology
%D 2011
%F discovery:1310148
%I UCL (University College London)
%P 387
%T Whole genome RNA expression profiling for the identification of novel biomarkers in the diagnosis and prognosis of biliary tract cancer
%U https://discovery.ucl.ac.uk/id/eprint/1310148/
%X Biliary tract cancer (BTC) is difficult to diagnose, in part related to the lack of  reliable tumour markers. The aim of this project was to use whole genome  RNA expression profiling in order to identify novel biomarkers for diagnosis  and prognosis in biliary tract cancer.  Chapter 1 summarises clinical aspects of BTC as well as current diagnostic  and prognostic tests.  Chapter 2 addresses the identification of circulating tumour cells for the  diagnosis of BTC. It includes details of a study investigating measurement of  circulating cytokeratin 19 fragments (CYFRA 21-1), demonstrating that  CYFRA 21-1 is a more specific, but less sensitive diagnostic marker than  CA19-9, and predicts a poor prognosis in BTC.  Chapter 3 investigates the potential for using RNA isolated from archived  formalin fixed, paraffin embedded (FFPE) surgical and explanted liver tissues  from patients with primary sclerosing cholangitis (PSC) with and without  cholangiocarcinoma, for use in whole genome RNA expression analysis. We  demonstrate that, although technically possible, the rarity of samples and  RNA degradation that occurs as a result of the tissue processing, are such  that further evaluation using these materials is not feasible at this time.  Chapter 4 addresses and validates methodology for isolating RNA from  samples of biliary brushings taken at the time of endoscopic retrograde cholangiopancreatography (ERCP). We demonstrate that RNA isolated from  biliary brushings is of low quantity and degraded, and that this degradation  occurs in vivo. However, we demonstrate that such RNA is still useful for  downstream applications such as quantitative real time PCR and is therefore  suitable for whole genome RNA expression analysis using microarray  technology.  Chapter 5 describes the methods and results obtained from using whole  genome RNA expression analysis using microarray of RNA isolated from  ERCP biliary brushings. The results are presented as a shortlist of candidate  genes requiring further validation.  Chapter 6 provides results of qPCR studies performed in order to validate the  gene expression profile identified by microarray. A selection of candidate  genes are investigated using TaqMan Array and SYBR Green qPCR and  demonstrate a high correlation with the pattern of expression shown by  microarray.  Chapter 7 investigates whether a selection of the genes identified in  malignant biliary brushings are similarly upregulated in fresh frozen surgical  resection material from patients with benign and malignant biliary diseases. In  addition, we provide evidence for gene translation and upregulation at the  protein level by immunohistochemistry for a selection of the protein products.  Chapter 8 discusses the main conclusions drawn from the work as well as  potential future studies.