Proximal Prostate Fluids for Protein and miRNA Biomarkers

描述（由申请人提供）：作为前列腺癌生物标志物的血清中前列腺特异性抗原（PSA）水平的检测不断发展，以反映关于疾病特异性亚型和游离与血清蛋白结合复合物的新知识。虽然有许多已知的优点，PSA测试，有据可查的问题与PSA是一个很好的器官特异性标志物，但不是一个癌症特异性标志物，将继续与美国的老龄化人口复合。近端流体被发现邻近给定的组织或器官，并代表一个库的分泌蛋白和脱落细胞反映的生理状态的组织。因此，近端液体正迅速成为癌症蛋白质和遗传生物标志物的潜在来源。精浆和前列腺分泌物（EPS）是前列腺的近端液体。在该提案中，我们描述了前列腺近端液体的临床收集和表征，以满足对改进的前列腺癌生物标志物日益增长的需求。EPS代表前列腺在直肠前列腺按摩后分泌的液体，反过来可以在检查后的排尿中收集。这种收集不会破坏标准的泌尿系统检查，也不会增加过多的就诊时间。目前，基于非编码RNA PCA 3的存在，用于前列腺癌检测的商业遗传测定使用EPS尿作为在低速离心后作为沉淀物收集的脱落上皮细胞的来源。尚未报道从上皮细胞团块中鉴定前列腺相关microRNA（miRNA）种类。剩余的尿液含有许多前列腺源性分泌蛋白，尚未得到很好的表征。在过去的一年里，弗吉尼亚州前列腺中心的临床医生和研究人员一直在收集和扩大现有的EPS尿样生物储存库，以及在前列腺切除术前获得的更纯的前列腺液。来自健康诊所对照、良性前列腺疾病和风险分层的前列腺癌（低、中、高和转移性）的超过250个EPS尿液可用于本研究。将来自每种条件的EPS尿液的合并样品用于全面的蛋白质组学分析，从而鉴定出超过600种前列腺富集的组成蛋白。我们还使用这些液体来建立来自分泌的PSA和其他前列腺蛋白的聚糖的综合数据库，并且正在评估沉淀的颗粒材料的前列腺疾病特异性miRNA特征。我们假设EPS收集的优化和标准化与当前泌尿科检查实践一致，并定义该液体的前列腺特异性组分，将导致发现新的生物标志物和应用新的前列腺癌诊断检测。这将通过以下具体目标实现：目标1。建立前列腺特异性的潜在蛋白质和miRNA生物标志物从前列腺分泌物。目标2.确定构成分泌蛋白含量和miRNA Aim 3的可接受和不可接受样品的标准。建立DRE后和前列腺切除术前前列腺分泌物采集的标准操作规程。我们的目标是优化从EPS尿液中获得的临床信息，使用细胞沉淀物的miRNA和前列腺蛋白液相的糖蛋白质组学表征。miRNA和糖蛋白都是固有的稳定分子。可以在前列腺癌和良性疾病的整个临床范围内收集EPS尿液。在门诊检查期间DRE时收集EPS尿液不会显著增加医生或患者的访视时间或延迟。定义组成蛋白和细胞miRNA、标准化收集程序以及建立稳定性和储存参数将有助于将基于EPS尿液的测定纳入广泛的临床应用。 公共卫生相关性：对50岁以上男性血液中前列腺特异性抗原水平的检测提高了前列腺癌的检测和治疗率。不幸的是，PSA作为一种优秀的器官特异性标志物，但不是一种特异性癌症标志物，存在越来越多的问题，这一问题将随着男性人口的老龄化而继续恶化。我们建议描述一种不同类型的液体，称为前列腺分泌物，以改善目前的PSA检测。定义组成蛋白和细胞miRNA、标准化收集程序以及建立稳定性和储存参数将有助于将基于EPS尿液的测定纳入主流临床应用。

DESCRIPTION (provided by applicant): 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.