URINARY BIOMARKERS OF RENAL CELL CARCINOMAS

描述（由申请人提供）：肾癌通常是无声的，经常是致命的。仅在2009年，美国就诊断出超过57,000例新的肾癌，几乎13,000例死亡可归因于这种疾病。早期诊断，触发手术切除，通常是治愈的。体征和症状出现较晚，当肾癌最终被诊断出来时，它通常已经转移到淋巴结或邻近器官，并且已经超过了治愈阶段，生存的机会很小。由于不相关的原因，在腹部成像（CT， MRI，超声）时可能会偶然发现。尽管如此，目前还没有筛查和诊断肾癌的方法，特别是在治疗阶段。未满足的需求是用于肾癌筛查的一种简单、高通量、具有成本效益的检测方法，其公共卫生意义在于每年防止13 000多人死亡，特别是在高危人群中。提出的研究将验证一个假设，即肾癌细胞表达和脱落或主动分泌蛋白质到尿液中，这些蛋白质是标志性的分子标志物（生物标志物），这些生物标志物是早期疾病指标，当肿瘤处于可治疗阶段时可以检测到。[在一篇新闻出版物中，我们已经表明，肾癌患者尿外泌体中的水通道蛋白1 （AQP1）和亲脂蛋白（ADFP）显著增加，从而提供了这种疾病的第一个敏感和特异性生物标志物。因此，我们进一步假设尿外泌体，即肾元各部分释放到尿液中的囊泡结构，将成为发现新的生物标志物蛋白的工具，并将作为肾细胞肿瘤发生和转移的非侵入性活检。总体目标是确定肾癌患者尿液外泌体中的蛋白质，这些蛋白质是这种疾病所特有的，并且可以发展为一种诊断测试，以筛查[全尿]，在早期可治愈阶段发现肾癌，并跟踪患者术后复发。具体目的是在发现阶段：1)在肾癌患者尿外泌体中发现与肾癌病原学相关的候选生物标志物蛋白；2)开发敏感和特异性的elisa来测量在Aim1中鉴定的新型生物标志物蛋白的水平；3)表征候选生物标志物蛋白以确定其诊断敏感性和特异性。独特的上调或下调蛋白将通过无偏自上而下和监督自下而上的质谱（LC/MS/MS）和尿液外泌体蛋白的Western blot蛋白质组学分析来鉴定。一旦通过质谱法在训练组患者中鉴定出蛋白质，将开发出精确但经济的基于elisa的免疫测定方法，可在当天给出结果，用于在验证组患者中进行[全尿和血清]蛋白质定量。总的来说，我们的研究将提高对肾癌的早期诊断，使其有很大的治愈机会，同时最大限度地提高未来的肾功能，[术后随访患者的复发情况，并确定未来选择性干预的目标]。

DESCRIPTION (provided by applicant): Renal cancer is typically silent and frequently fatal. In the United States in 2009 alone over 57,000 new kidney cancers were diagnosed and almost 13, 000 deaths were attributable to this disease. Early diagnosis, triggering surgical excision, is usually curative. Physical signs and symptoms occur late, and by the time renal cancer is consequently diagnosed, it has usually metastasized to lymph nodes or adjacent organs and advanced beyond a curative stage with minimal chance for survival. Incidental early discovery may occur fortuitously during abdominal imaging (CT, MRI, ultrasound) for unrelated causes. Nonetheless, there is presently no method for population screening and diagnosis of renal cancer, particularly at a curative stage. The unmet need is a simple, high through-put, cost-effective test for kidney cancer screening, with the public health significance of preventing over 13,000 deaths annually especially in at risk populations. The proposed research will test the hypothesis that renal cancer cells express and shed or actively secrete proteins into the urine that are signature molecular markers (biomarkers), and that these biomarkers are an early disease indicator, detectable when the tumor is at a treatable stage. [In an in press publication, we have shown that aquaporin 1 (AQP1) and adipophilin (ADFP) are significantly increased in urinary exosomes of patients with kidney cancer thus providing the first sensitive and specific biomarkers of this disease.] Therefore, it is further hypothesized that urine exosomes, vesicular structures released into the urine by all parts of the nephron, will be a [discovery tool for additional novel biomarker proteins and will serve as a non-invasive biopsy of kidney cell oncogenesis and metastasis]. The overall objective is to identify proteins in the urine exosomes of kidney cancer patients which are unique to this disease, and which can be developed into a diagnostic test to screen [whole urine] to detect kidney cancer at an early curable stage and to follow patients post-operatively for recurrence. The specific aims are a discovery stage to: 1) Identify candidate biomarker proteins in the urine exosomes of patients with kidney cancer which are pathogneumonic of the disease; a development stage to 2) Develop sensitive and specific ELISAs to measure levels of novel biomarker proteins identified in Aim1; and a validation stage to 3) Characterize the candidate biomarker proteins to determine their diagnostic sensitivity and specificity. Unique up- or down-regulated proteins will be identified by unbiased top-down and supervised bottom-up mass spectral (LC/MS/MS) and Western blot proteomic analysis of the urine exosomal proteins. Once proteins are identified in training sets of patients by mass spectrometry, precise but economical ELISA-based immunologic assays that give same-day results will be developed, for protein quantification [in whole urine and serum] in validation sets of patients. Overall, our study will improve the diagnosis of kidney cancer to a stage early enough to allow a significant chance for cure while maximizing future kidney function, [follow patients post-operatively for recurrence and identify future targets for selective intervention]. PUBLIC HEALTH RELEVANCE: Renal cancer is typically silent and frequently fatal. In the United States alone in 2009 there were over 57,000 new kidney cancers diagnosed and almost 13,000 deaths attributable to this disease. Physical signs and symptoms occur late, and by the time renal cancer diagnosed, it has usually metastasized to lymph nodes or adjacent organs and advanced beyond a curative stage with minimal chance for survival. The unmet need is a simple, high through-put, cost-effective test for kidney cancer screening and recurrence with the public health significance of preventing over 13,000 deaths annually. Our study will improve the diagnosis of kidney cancer at a stage early enough to allow a significant chance for cure, preserve remaining kidney function [and follow post-operative patients for recurrence and treatment while providing important insight into kidney cancer oncogenesis and metastasis].