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记物的血清前列腺特异性抗原(PSA)水平的检测继续发展，以反映关于疾病特定亚型和与血清蛋白的自由结合复合体的新知识。虽然PSA检测有许多已知的优点，但随着美国人口老龄化，PSA是一种优秀的器官特异性标记物，但不是癌症特异性标记物的问题将继续恶化。近端液体被发现与给定的组织或器官相邻，代表着反映该组织生理状态的分泌蛋白质和脱落细胞的谱系。因此，近端体液正迅速成为癌症的蛋白质和遗传生物标记物的潜在来源。精浆和前列腺液(EPS)是前列腺的近端液体。在这项提案中，我们描述了前列腺癌近端积液的临床收集和表征，以满足对改进的前列腺癌生物标记物日益增长的需求。EPS代表前列腺在接受直肠指压按摩后分泌的液体，而这些液体又可以在检查后排出的尿液中收集。这种收集不会扰乱标准的泌尿科检查，也不会增加过度的就诊时间。目前，一种基于非编码RNA PCA3存在的用于前列腺癌检测的商业基因检测方法使用EPS尿液作为脱落上皮细胞的来源，该细胞在低速离心后收集作为沉淀物。从上皮细胞小球中鉴定出前列腺相关微RNA(MiRNA)物种的报道尚未见报道。剩余的尿液含有许多前列腺源性分泌蛋白，目前还没有得到很好的鉴定。在过去的一年里，弗吉尼亚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。来自健康临床对照、良性前列腺疾病和高危前列腺癌(低、中、高和转移)的250多个EPS尿液可用于这项研究。来自每种情况的EPS尿液的汇集样本被用于全面的蛋白质组学分析，导致鉴定出600多种前列腺浓缩的组成蛋白。我们还使用这些流体建立了一个从分泌的PSA和其他前列腺蛋白中提取的多糖的全面数据库，并正在评估沉淀的颗粒材料是否具有前列腺病特异性miRNA特征。我们假设，符合当前尿路检查实践的EPS收集的优化和标准化，以及这种液体的前列腺特异性成分的定义，将导致新的生物标记物的发现和前列腺癌新诊断方法的应用。这将在以下特定目标中实现：目的1.从表达的前列腺液中建立潜在蛋白质和miRNA生物标记物的前列腺特异性。目的2.确定什么是可接受的和不可接受的分泌性蛋白质含量和miRNA样本的标准目的3.建立前列腺切除术后和前列腺术前表达的前列腺液的标准操作程序。我们的目标是优化从EPS尿液中获得的临床信息，使用miRNA的细胞沉淀物和用于糖蛋白组学特征的前列腺液相。MiRNA和糖蛋白都是本质上稳定的分子。EPS尿液可以在前列腺癌和良性疾病的整个临床范围内收集。无论是医生还是患者，在DRE检查期间收集EPS尿液不会显著增加就诊时间或延误就诊时间。确定组成蛋白和细胞miRNAs，标准化采集程序，建立稳定性和储存参数，将有助于将基于EPS尿液的分析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诊断率和治疗率。不幸的是，PSA作为一种优秀的器官特异性标记物而不是癌症特异性标记物的问题越来越多，随着男性人口的老龄化，这个问题将继续恶化。我们建议将一种不同类型的液体命名为前列腺液，以改进目前的PSA检测。确定组成蛋白和细胞miRNAs，标准化采集程序，建立稳定性和储存参数，将有助于将基于EPS尿液的分析纳入主流临床应用。

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.