Yeast Assay for p53 Molecular Analysis in Bladder Cancer

描述(申请人提供)：膀胱癌是美国第五大最常见的诊断肿瘤，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。减少发病率和死亡率将来自于改善这类患者的预后。能够快速访问分子分析数据并了解其与临床结果的关系对于患者管理和治疗可能是非常宝贵的。这项R21/R33应用的长期目标是：1)标准化基于档案的酵母功能分析(AYFA)方案，这将导致广泛的临床应用；2)确定分子分析在检测和评估膀胱癌的P53功能时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。在本实验中，将P53表达载体和报告载体共转染酵母。表达的野生型(Wt)P53能够与P53反应元件结合，而突变型P53缺乏结合能力。这种结合和缺乏结合导致反式激活活性的颜色差异。使用当前版本的酵母菌试验有一个严重的缺陷：它是为研究细胞系和新鲜冰冻组织的mRNA而设计的。不幸的是，在大型临床试验中，鲜冻组织很少用于比较遗传标记和患者预后。因此，以档案为基础的酵母菌功能分析可能被证明非常适合在更广泛的膀胱癌人群中进行快速、准确和与治疗相关的分子分析。这项资助的R21阶段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使用基于档案的酵母菌功能分析。这项资助的R33阶段的具体目的是确定不同的p53突变(改变和功能)在膀胱癌中的临床相关性。基于档案的酵母菌功能分析技术被证明是快速和临床相关的，有望对膀胱癌的分子分析产生影响。基于档案的酵母功能分析技术是创新的，因为它将使临床医生无论在哪里都能够获得分子分析信息，这些信息可能在实际治疗开始之前被证明对患者的结果有价值。目前，由于需要新鲜冰冻组织进行标准的酵母菌检测，临床医生几乎不可能及时获得单个患者的分子数据。这使得标准的酵母化验不太适合广泛使用，从而阻碍了未来的研究努力。这项拨款建议比较膀胱癌患者的P53分子分析数据及其与患者临床结果的关系。已经报告的三组患者将构成R33试点研究的基础。基于档案的酵母功能分析技术将用于检测膀胱癌标本(膀胱切除术和TURBT)，然后将它们的P53状态与患者的临床结果进行比较。在这笔赠款期间获得的结果将构成未来研究的基础，这些研究应该影响这种疾病的治疗战略。

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.