Abstract

Gallbladder cancer (GBC) is a public health problem in Chile, with the highest reported gallbladder cancer incidence in the world. GBC is the second cause of cancer deaths in Chilean women and the third in men, and the mortality rates have been increasing since last 30 years for both sexes. The prognosis for gallbladder cancer patients is dismal; survival after a GBC diagnosis is very low, with an overall 5-year relative survival of 10.3% and median survival of 3.4 months. If gallbladder cancer is detected at an early stage, the 5-year survival is approximately 90%. Therefore, early diagnosis is crucial for reducing the mortality of GBC. Unfortunately, current clinical and radiologic examination is not sufficient to detect GBC, being only found at early stages when the gallbladder is removed for other reasons, such as gallstones disease. For this reason, biomarkers are urgently needed for the early diagnosis of GBC. Proteins, as functional mediators of the biological processes, may reveal precisely the physiology signs associated to oncogenesis, and proteomic studies have been conducted in many other types of cancer for the discovery of novel, disease-related biomarkers. The progress in proteomic technology, including innovative techniques for profiling and absolute protein quantification, offers a valuable opportunity for biomarker discovery and proteomic analyses of readily accessible biofluids such as sera and urine. Our research proposal is aimed to obtain a comprehensive massspectrometry- based proteomic profile of gallbladder cancer and validate potential biomarkers in tissues and body fluids. Hypotheses: We hypothesize that the tumour cells in gallbladder cancer overexpress a repertoire of proteins as compared to gallstone related gallbladder tissue. We further hypothesize that some of these proteins are either secreted or shed into the extracellular space and can be detected in serum and/or urine and serve at potential biomarkers for early detection of gallbladder cancer. General Objective: To identify and validate potential protein biomarkers of gallbladder cancer in tumour, serum and urine samples. Specific aim 1: “To carry out a quantitative proteome and secretome analysis of gallbladder tumour tissue, non-tumour tissue and GBC cell lines (Phase 1 biomarker development)”. Methods: Potential biomarkers from GBC secretome will be identify in gallbladder cancer cell lines using a quantitative proteomic analysis based on SILAC strategy. In addition, iTRAQ-based quantitative proteomics will be carrying out to identify differentially expressed proteins in GBC tissues (compared to non-neoplastic gallbladder), which may have high potential to serve as potential biomarkers. Specific aim 2: “To validate over-expressed proteins in a different cohort of gallbladder cancer by immunohistochemistry (IHC) in TMAs”. Methods: Five potential biomarkers selected from the quantitative proteome and secretome profile will be validate on formalin-fixed paraffin-embedded (FFPE) gallbladder tissues (Tissue microarray format) by IHC. Specific aim 3: “To validate Phase II biomarkers in a different cohort of serum and urine samples from patients with GBC and symptomatic gallstones patients (not associated GBC)”. Methods: Multiple Reaction Monitoring (MRM)-based assays will be used in this step to monitor and quantify protein markers in body fluids such as serum and urine. Expected outcomes: We expect to identify a panel of secreted and GBC specific potential biomarkers. Our results will allow us to understand better the GBC protein profile in GBC patients. The evaluation of selected potential biomarkers by IHC in TMAs will be useful for further validation of them in urine and serum (Phase 2 biomarker development). Then, we expect to identify sufficient and high quality potential biomarkers to be tested in subsequent full scale Phase 3 and 4 biomarkers validation studies. As a final clinical application of the present proposal, we expect to generate diagnostic tools for surgeons in order to prioritize cholecistectomies in a subset of patients with higher risk of GBC.