Yeast Assay for p53 Molecular Analysis in Bladder Cancer

描述（由申请人提供）：膀胱癌是美国第五大最常诊断的肿瘤，每年有超过57,400例新病例和12,500例死亡。其中85%的死亡来自25%的肌肉侵袭性疾病患者。发病率和死亡率的降低将来自于这组患者预后的改善。快速获取分子分析数据并了解其与临床结果的关系，对患者的管理和治疗具有不可估量的价值。该R21/R33申请的长期目标是：1)标准化基于档案的酵母功能测定（aYFA）的方案，这将导致广泛的临床应用；2)确定在检测和评估膀胱癌中p53功能时，分子分析可能提供的临床相关性。酵母功能试验是一种现有的检测人类癌症中p53突变的方法。在这个实验中，p53表达质粒和报告质粒都被共转染到酵母中。表达的野生型（wt） p53能够与p53应答元件结合，而突变型p53缺乏这种结合能力。这种结合和缺乏结合导致了交互活动的颜色差异。使用当前版本的酵母试验有一个严重的缺点：它是为研究来自细胞系和新鲜冷冻组织的mRNA而设计的。不幸的是，在大型临床试验中，鲜冻组织很少可用来比较遗传标记和患者结果。因此，基于档案的酵母菌功能分析可以证明非常适合于在更广泛的膀胱癌人群中进行快速、准确和治疗相关的分子分析。R21期的具体目标是优化技术和方法，以便在临床环境中更广泛地利用基于档案的酵母功能测定。该资助的R33期的具体目的是确定膀胱癌中不同p53突变（改变和功能）的临床相关性。基于档案的酵母菌功能分析技术具有快速和临床应用价值，有望对膀胱癌的分子分析产生影响。基于档案的酵母菌功能分析技术的创新之处在于，它将使临床医生，无论身处何地，都能够在实际治疗开始之前获得对患者结果有价值的分子分析信息。目前，由于需要新鲜冷冻组织来进行标准的酵母菌检测，因此临床医生几乎不可能及时获得个体患者的分子数据。这使得标准酵母菌测定法不太适合广泛使用，从而阻碍了未来的研究努力。该资助旨在比较膀胱癌患者的p53分子分析数据及其与患者临床预后的关系。已经报道的三组患者将构成R33试点研究的基础。基于档案的酵母功能测定技术将用于检测膀胱癌标本（膀胱切除术和TURBT），然后将其p53状态与患者临床结果进行比较。在此资助期间获得的结果将成为影响该疾病治疗策略的未来研究的基础。

DESCRIPTION (provided by applicant): Bladder cancer is the fifth most frequently diagnosed tumor in the United States, with more than 57,400 new cases and 12,500 deaths annually. Eighty-five percent of these deaths come from the 25% of patients who present with muscle-invasive disease. Reduction in morbidity and mortality will come from improving outcome in this group of patients. Having rapid access to molecular analysis data and knowing its relationship to clinical outcome could be invaluable to patient management and treatment. The long-term objectives of this R21/R33 application are to 1) standardize the protocol for an archival-based yeast functional assay (aYFA) that will lead to broad clinical utilization, and 2) determine what clinical relevance molecular analysis may provide when detecting and evaluating p53 functionality in bladder cancer. A yeast functional assay is an existing methodology that detects p53 mutations in human cancers. In this assay, both p53 expression plasmids and reporter plasmids are co-transfected into yeast. The expressed wild-type (wt) p53 is able to bind to a p53-response element, whereas mutant p53 lacks the ability to bind. This binding and lack of binding leads to color differences in transactivational activity. Use of this current version of the yeast assay has one serious drawback: it was designed for the study of mRNA from cell lines and fresh-frozen tissue. Unfortunately, in large clinical trials, fresh-frozen tissue is rarely available for comparing genetic markers to patient outcome. Therefore, an archival-based yeast functional assay could prove to be extremely suitable for rapid, accurate, and treatment-relevant molecular analysis in a wider bladder cancer population. The specific aim of the R21 phase of this grant is to optimize the technology and methodology for a broader utilization of the archival-based yeast functional assay in the clinical setting. The specific aim of the R33 phase of this grant is to determine the clinical relevance of different p53 mutations (alterations and functionality) in bladder cancer. It is expected that the archival-based yeast functional assay technology will impact the molecular analysis of bladder cancer by proving to be both rapid and clinically relevant. The archival-based yeast functional assay technology is innovative in that it would give clinicians, regardless of location, the ability to obtain molecular analysis information that could prove valuable to patient outcome prior to actual treatment initiation. Currently, with fresh-frozen tissue required to perform the standard yeast assay, timely accessibility of individual patient molecular data for the clinician is virtually impossible to obtain. This makes the standard yeast assay less than optimal for widespread use and subsequently hampers future research endeavors. This grant proposes to compare p53 molecular analysis data and its relationship to patient clinical outcomes in bladder cancer patients. Three groups of patients that have already been reported on will form the basis of the R33 pilot study. The archival-based yeast functional assay technology will be used in testing bladder cancer specimens (cystectomy and TURBT) and then comparing their p53 status to patient clinical outcomes. The results obtained during the period of this grant will form the basis for future studies that should affect treatment strategies for this disease.