Whole genome RNA expression profiling for the identification of novel biomarkers in the diagnosis and prognosis of biliary tract cancer.
Doctoral thesis, UCL (University College London).
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.
|Title:||Whole genome RNA expression profiling for the identification of novel biomarkers in the diagnosis and prognosis of biliary tract cancer|
|Open access status:||An open access version is available from UCL Discovery|
|UCL classification:||UCL > School of Life and Medical Sciences > Faculty of Medical Sciences > Institute of Hepatology|
Archive Staff Only