Prostate cancer (PCa) is one of the leading causes of death among men in the developed countries. Nevertheless, current methods of diagnosis for PCa have a high rate of false positive results due to a low specificity and, therefore, to many unnecessary prostatic biopsies (PBs). Moreover, in many PBs a non-cancerous pathology of the prostate is found. A particularly clinically relevant benign condition is High Grade Intraepithelial Neoplasia (HGPIN), recognized as the most probable precursor to PCa. When found in PB, it is associated with an increased probability of the existence of an undetected PCa. For this reason, its encounter in a first PB guarantees an intensive surveillance over the years, including multiple repeat PBs. However, only in a small part of these patients an aggressive form of PCa will be eventually found. In summary, the great majority of the PBs practiced nowadays are unnecessary, causing pointless discomfort to the patient and extra expenses to the health care system. In the last years, a lot of effort has been put into the identification of new biomarkers for PCa that would improve the current situation. Because of the location of the prostate in the body, in direct contact with the urethra, desquamated cells and secreted products, including exosomes, can be detected in urine. The main aim of this thesis is the identification of new biomarkers for PCa in urine, in order to develop a non-invasive method for the early and accurate diagnosis of PCa, both in a first PB setting and in patients already diagnosed with HGPIN. Exosomes have been proposed as a potential new source for biomarkers, since they contain molecules representing their tissues of origin. In order to identify new PCa biomarkers in urinary exosomes, we have first established a reliable vesicles isolation method, and then the obtained vesicles were characterized by electronic microscopy, Western blot, and nanoparticle tracking analysis. Next, we carried out a discovery phase by label-free LC-MS/MS (n=24). Analysis of the data was carried out measuring the ion peak intensities and applying several different statistic approaches, in order to increase the reliability of the results. Once we had a list of biomarker candidates, these were validated following a targeted proteomics approach (Selected Reaction Monitoring, SRM). Finally, we have identified a profile of 15 differentially expressed protein biomarkers for PCa. Two of these validated proteins, corresponding to one alpha and one beta integrin subunits, were selected for further characterization using in vitro models, based on biological interest. This characterization proved the intracellular location of these protein biomarkers in late endosomes. In the context of repeat PBs due to a previous diagnosis of HGPIN, we have assayed a set of known RNA-based PCa biomarkers from urine sediment. As a result, we have described a set of three multiplex models with different combinations of the genes CDH1, PSMA, PSGR and KLK3. All these models outperform the currently existent methods for the detection of PCa in men with HGPIN, and their use in clinical practice could potentially save a high number of unnecessary repeat PBs. In the future, the results derived from this study could improve current PCa detection, distinguishing aggressive from clinically insignificant PCa and other benign conditions and, therefore, avoiding PCa related over-diagnosis and over-treatment.
|Date of Award||27 Nov 2014|
|Supervisor||Andreas Doll (Director), Juan Morote Robles (Director) & Marina Rigau Resina (Director)|