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BackgroundCurrent markers for prostate cancer, such as PSA lack specificity. Therefore, novel biomarkers are needed. Unfortunately, the complexity of body fluids often hampers biomarker discovery. An attractive alternative approach is the isolation of small vesicles, i.e. exosomes, ∼100 nm, which contain proteins that are specific to the tissue from which they are derived and therefore can be considered as treasure chests for disease-specific biomarker discovery.Materials and MethodsExosomes were isolated from 2 immortalized primary prostate epithelial cells (PNT2C2 and RWPE-1) and 2 PCa cell lines (PC346C and VCaP) by ultracentrifugation. After tryptic digestion, proteomic analyses utilized a nanoLC coupled with an LTQ-Orbitrap operated in tandem MS (MS/MS) mode. Accurate Mass and Time (AMT) tag approach was employed for peptide identification and quantitation. Candidate biomarkers were validated by Western blotting and Immunohistochemistry.ResultsProteomic characterization resulted in the identification of 248, 233, 169, and 216 proteins by at least 2 peptides in exosomes from PNT2C2, RWPE-1, PC346C, and VCaP, respectively. Statistical analyses revealed 52 proteins differently abundant between PCa and control cells, 9 of which were more abundant in PCa. Validation by Western blotting confirmed a higher abundance of FASN, XPO1 and PDCD6IP (ALIX) in PCa exosomes.ConclusionsIdentification of exosomal proteins using high performance LC-FTMS resulted in the discovery of PDCD6IP, FASN, XPO1 and ENO1 as new candidate biomarkers for prostate cancer.

Extracellular vesicles (including the subclass exosomes) secreted by cells contain specific proteins and RNA that could be of interest in determining new markers. Isolation/characterization of PCa-derived exosomes from bodily fluids enables us to discover new markers for this disease. Unfortunately, isolation with current techniques (ultracentrifugation) is labor intensive and other techniques are still under development. The goal of our study was to develop a highly sensitive time-resolved fluorescence immunoassay (TR-FIA) for capture/detection of PCa-derived exosomes. In our assay, biotinylated capture antibodies against human CD9 or CD63 were incubated on streptavidin-coated wells. After application of exosomes, Europium-labeled detection antibodies (CD9 or CD63) were added. Cell medium from 37 cell lines was taken to validate this TR-FIA. Urine was collected (after digital rectal exam) from patients with PCa (n 5 67), men without PCa (n 5 76). As a control, urine was collected from men after radical prostatectomy (n 5 13), women (n 5 16) and patients with prostate cancer without digital rectal exam (n 5 16). Signal intensities were corrected for urinary PSA and creatinine. This TR-FIA can measure purified exosomes with high sensitivity and minimal background signals. Exosomes can be measured in medium from 37 cell lines and in urine. DRE resulted in a pronounced increase in CD63 signals. After DRE and correction for urinary PSA, CD9 and CD63 were significantly higher in men with PCa. This TR-FIA enabled us to measure exosomes with high sensitivity directly from urine and cell medium. This TR-FIA forms the basis for testing different antibodies directed against exosome membrane markers to generate disease-specific detection assays.Prostate-specific antigen (PSA, KLK3) is a protein that is commonly used in daily practice to aid urologists in diagnosing prostate cancer (PCa). Although PSA has a high sensitivity, it lacks specificity and therefore causes unnecessary biopsies. Furthermore, PSA is a poor prognostic marker.1 To increase specificity and distinguish between the clinically insignificant cancers and the ones that are clinically relevant, novel markers have to be identified. Recent studies have shown that extracellular vesicles and particularly the vesicles from endosomal origin, referred to as exosomes, could help us in identifying novel tissue-specific markers. Moreover, also their presence and number might be indicative of disease. 2-4Quantifying the number of exosomes and characterizing them on single particle level remains challenging. To determine the number of exosomes in body fluids or measure an exosomal marker of interest, purification and concentration steps are often needed. Isolation of exosomes is most commonly performed by ultracentrifugation, filtration, precipitation or antibody-based capture technologies. Most of these protocols are still under development, labor intensive and limited with respect to efficient isolation or purity of the final exosomal preparation. 5 Measuring the numbe...

Study Type – Therapy (meta‐analysis)
Level of Evidence 1a OBJECTIVE To review the evidence for the use of α‐blockers after extracorporeal shock wave lithotripsy (ESWL) in enhancing the effectiveness of renal and ureteric stone clearance. METHODS We searched MEDLINE, Embase and the Cochrane Library up to January 2009. All randomized controlled trials in which α‐blockers were evaluated after ESWL were eligible for the analysis. Outcome measures assessed were clearance rate (primary) and expulsion time (secondary). Two authors independently assessed study quality and extracted data. All data were analysed using RevMan 5. RESULTS Of the 29 identified papers, seven trials with a total of 484 patients met the predefined criteria. These studies evaluated the effectiveness of the α‐blocker tamsulosin, and studied clearance rate as the primary outcome. There was large heterogeneity between trials, but their methodological quality was adequate. The pooled absolute risk difference of clearance rate was 16% (95% confidence interval 5–27%) in favour of the tamsulosin group, i.e. an average of six patients have to be treated with tamsulosin after ESWL to achieve clearance in one. Subgroup analysis for the six studies that used a dose of 0.4 mg tamsulosin showed a pooled risk difference of 19 (10–29)%. The expulsion time was analysed in three studies and the pooled mean difference was 8  (−3–20) days in favour of the tamsulosin group. Pain and analgesic usage was reported to be lower with tamsulosin. Adverse effects of tamsulosin, mainly dizziness, were reported in eight patients (3%). CONCLUSIONS Treatment with tamsulosin after ESWL appears to be effective in assisting stone clearance in patients with renal and ureteric calculi. To make a definite clinical recommendation to use tamsulosin after ESWL for renal and ureteric calculi, a high quality confirmatory trial is warranted.

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