Abstract Introduction: Prostate cancer is diagnosed utilizing anywhere from 10-12 transrectal prostate needle biopsy specimens (PNB). The decision to biopsy is driven in part by the PSA test, which is known to have marginal accuracy in appropriate patient selection. As a result, between 40 to 60% of patients will have a benign biopsy with approximately 20% having false negative results. Exosomal RNA is stable in urine and can be easily extracted from a random urine sample. A novel molecular assay with an improved ability to predict the likelihood of having prostate cancer on a needle biopsy is being developed through isolation of RNA from urine exosomes and subsequent identification of an accurate prognostic gene signature. Methods: A multicenter study was designed to collect first catch random urine, without a prior digital rectal exam (DRE) from men presenting for a PNB. Urine samples were processed at Exosome Diagnostic's clinical laboratory under quality control using the EXO70 Urine Microvesicle RNA Isolation Kit. The exosomal RNA was reverse transcribed and qPCR assays for specific genes performed. The results of those assays along with collected clinical data and PNB histopathology were subjected to statistical analysis to derive ROC curves. Results: Urine samples from 258 men scheduled for a PNB were tested. Triaged gene lists previously shown to be important in prostate cancer were compiled and univariately assessed with respect to discrimination of PNB results. A series of single and multiple gene expression profiles representing anywhere from 1 to 3 gene signatures relative to a reference gene were identified. Multivariate models including several gene signatures with a ROC AUC performance > 0.7 were found. Interestingly, a 3 gene signature composed of PCA3, ERG and KLK3 provided a very good performance with an AUC of 0.76 (N = 122). Conclusions: Prostate cancer cells release exosomes / microvesicles that can be isolated from a random urine sample. The exosomal RNA is stable and can be efficiently isolated and used for tumor profiling even without a prior DRE. This urine exosome platform presents a robust model for a quick and simple test that can assist in the initial biopsy decision process. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B34. Citation Format: Johan Skog, Mikkel Noerholm, Stefan Bentink, Charlotte Romain, Jillian Fishbeck, Ian Sinclair, Anna Scott, Romy Mueller, Tina Koestler, Susan Belzer. Development of a urine microvesicle/exosome RNA biomarker panel to identify prostate cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B34.