VALIDATION AND QUANTIFICATION OF FFPE ANTIGEN RETRIEVAL BY PROTEOME ANALYSIS

描述（由申请人提供）：由于在组织病理学中使用福尔马林作为组织处理的标准固定剂的历史很长，世界各地有大量的福尔马林固定和石蜡包埋（FFPE）组织库。这些FFPE组织收集，附带临床和结果信息，为进行回顾性蛋白质生物标志物调查提供了宝贵的资源。除了当前蛋白质组学技术（包括二维聚丙烯酰胺凝胶电泳和多维液相色谱系统）对样品数量的限制外，缺乏从FFPE组织中检索蛋白质的优化方法进一步限制了对档案组织进行分子分析的能力。通过与博士合作。南加州大学（USC） Keck医学院的Shan-Rong Shi和Clive R. Taylor在R41 I期研究中，抗原检索（AR）与Gemini蛋白质组学技术的结合不仅完成了对FFPE组织中提取的蛋白质的质量和可重复性的严格评估，从而优化了AR方法；同时也展示了利用存档的FFPE组织收集来发现生物标志物的重要机会。在R42 II期项目中，Calibrant和USC团队提出的协同努力旨在在模型和肿瘤FFPE组织中生成蛋白型肽库。这些蛋白型肽文库代表了从FFPE组织中检索到的抗原全球编目的第一步，并为随后的免疫组织化学（IHC）抗体开发提供了可用的表位数据库。除了为免疫组化从业者选择最佳AR条件/抗体组合提供指导外，比较蛋白质组学和验证研究（包括使用蛋白质型肽库和相关抗体）将进一步提高免疫组化定量测量的可重复性和灵敏度。由于免疫组化在临床诊断/预后和转化性癌症研究中的广泛应用，对定量免疫组化的需求不断升级。此外，对靶向蛋白质组学研究的最大期望是利用从高质量标本中富集和选择的细胞，在临床环境、临床前测试和临床试验中识别诊断、预后和预测生物标志物，以及在生物制药工业中发现和验证新的蛋白质靶点。FDA于2006年3月发布的《关键路径机遇报告》不仅是关键路径倡议的第一份具体蓝图，该倡议旨在通过应用新策略和新技术来简化药物审批过程，而且还强调了生物标志物开发是“改善医疗产品开发的最重要领域之一”。

DESCRIPTION (provided by applicant): Because of the long history of the use of formalin as the standard fixative for tissue processing in histopathology, there are a large number of archival formalin-fixed and paraffin-embedded (FFPE) tissue banks worldwide. These FFPE tissue collections, with attached clinical and outcome information, present invaluable resources for conducting retrospective protein biomarker investigations. In addition to sample amount constraints imposed by current proteome techniques including two-dimensional polyacrylamide gel electrophoresis and multidimensional liquid chromatography system, the lack of optimized methodologies for retrieving proteins from FFPE tissues further restricts the ability to perform the molecular analysis of archival tissues. By collaborating with Drs. Shan-Rong Shi and Clive R. Taylor from the University of Southern California (USC) Keck School of Medicine during the R41 Phase I studies, the combination of antigen retrieval (AR) with Gemini proteomic technologies not only accomplished the rigorous evaluation of the quality and the reproducibility of proteins retrieved from FFPE tissues for the optimization of AR methodology, but also demonstrated significant opportunities in the pursuit of biomarker discovery using archived FFPE tissue collections. The proposed synergistic efforts between Calibrant and the USC team during the R42 Phase II project aim to generate proteotypic peptide libraries among model and tumor FFPE tissues. These proteotypic peptide libraries represent the first step toward globally cataloging antigens retrievable from FFPE tissues and presenting the available epitope database for subsequent immunohistochemistry (IHC) antibody development. Besides providing guidelines for practitioners of IHC to select the optimized AR condition/antibody combination, the comparative proteomic and validation studies involving the use of proteotypic peptide libraries and associated antibodies will provide further enhancements in the reproducibility and the sensitivity of quantitative IHC measurements. The demand for quantitative IHC continues to escalate due to the widespread utilization of IHC in clinical diagnosis/prognosis and translational cancer research. Furthermore, the greatest expectations for targeted proteomics research using enriched and selected cells from high quality specimens reside in the identification of diagnostic, prognostic, and predictive biological markers in the clinical setting and during preclinical testing and clinical trials, as well as the discovery and validation of new protein targets in the biopharmaceutical industry. The Critical Path Opportunity Report released by FDA in March 2006 not only serves as the first specific blueprint for the Critical Path Initiative, an effort to streamline the drug-approval process by applying new strategies and technologies, but also highlights biomarker development as one of the "most important areas for improving medical product development." Public Health Relevance Statement: By joining Calibrant's unique tissue proteome capabilities with the expertise of Dr. Clive R. Taylor at the University of Southern California in antigen retrieval-immunohistochemistry, the proposed research not only aims to evaluate and optimize quantitative immunohistochemistry measurements through the creation of proteotypic peptide libraries, but also focuses on further development and demonstration of a novel biomarker discovery paradigm for enabling comprehensive and comparative proteomic analysis of archived formalin-fixed and paraffin-embedded tissue collections in support of cancer research, diagnosis, and treatment.