Detection of prostate specific antigen (PSA) levels in serum as a biomarker of prostate cancer continues to evolve to reflect new knowledge about disease-specific isoforms and free versus bound complexes with serum proteins. While there are many known advantages to PSA testing, the well documented problems with PSA being an excellent organ-specific marker, but not a cancer-specific marker, will continue to be compounded with the aging population of the U.S. Proximal fluids are found adjacent to a given tissue or organ and represent a repertoire of secreted proteins and shed cells reflective of the physiological state of that tissue. Hence, proximal fluids are rapidly emerging as a potential source of protein and genetic biomarkers for cancers. Seminal plasma and expressed-prostatic secretion (EPS) fluids are proximal fluids of the prostate. In this proposal, we describe the clinical collection and characterization of prostate proximal fluids to meet the increasing demand for improved prostate cancer biomarkers. EPS represents the fluid being secreted by the prostate following a digital rectal prostate massage, which in turn can be collected in voided urine post-exam. This collection is not disruptive to a standard urological exam, nor does it add excessive time to the visit. Currently, a commercial genetic assay for prostate cancer detection, based on the presence of a non-coding RNA, PCA3, uses EPS urines as a source of shed epithelial cells collected as a sediment after low speed centrifugation. Identification of prostate-associated microRNA (miRNA) species from the epithelial cell pellet has not been reported. The remaining urine, which contains many prostate-derived secreted proteins, has not been well characterized. Over the past year, clinicians and researchers at the Virginia Prostate Center have been collecting and expanding an existing biorepository of EPS urine samples, as well as more pure prostatic fluids obtained prior to prostatectomy. Over 250 EPS urines from healthy clinic controls, benign prostatic disease, and risk stratified prostate cancers (low, intermediate, high and metastatic) are available for this study. A pooled sample of EPS urines from each condition was used for comprehensive proteomic analysis, leading to identification of over 600 prostate enriched constituent proteins. We are also using these fluids to establish a comprehensive database of glycans from secreted PSA and other prostatic proteins, and the sedimented pellet material is being evaluated for prostate disease specific miRNA signatures. We hypothesize that optimization and standardization of EPS collection consistent with current urological exam practices, and definition of the prostate specific components of this fluid, will lead to the discovery of new biomarkers and application of new diagnostic assays for prostate cancer. This will be accomplished in the following Specific Aims: Aim 1. Establish the prostate specificity of potential protein and miRNA biomarkers from expressed prostatic secretions. Aim 2. Determine standards for what constitutes an acceptable and unacceptable sample for secreted protein content and miRNA Aim 3. Establish a standard operating procedure for collection of post-DRE and pre-prostatectomy expressed prostatic secretions. Our goal is to optimize the clinical information obtained from EPS urines, using both the cellular sediment for miRNA and the prostatic protein fluid phase for glycoproteomic characterizations. Both miRNA and glycoproteins are inherently stable molecules. The EPS urines can be collected across the entire clinical spectrum of prostatic cancers and benign disease. Collection of EPS urine at the time of DRE during office examinations does not add significant time or delays to these visits, either for the physician or patient. Defining the constituent proteins and cellular miRNAs, standardization of collection procedures and establishing stability and storage parameters will facilitate incorporation of EPS urine based assays into widespread clinical use. PUBLIC HEALTH RELEVANCE: Testing of prostate specific antigen levels in the blood of men over 50 has improved prostate cancer detection and treatment rates. Unfortunately, there are an increasing number of problems with PSA being an excellent organ specific marker, but not a specific cancer marker, a problem that will continue to worsen with the aging male population. We propose to characterize a different type of fluid termed expressed prostatic secretions for improving current PSA testing. Defining the constituent proteins and cellular miRNAs, standardization of collection procedures and establishing stability and storage parameters will facilitate incorporation of EPS urine based assays into mainstream clinical use.