VALIDATION AND QUANTIFICATION OF FFPE ANTIGEN RETRIEVAL BY PROTEOME ANALYSIS

通过蛋白质组分析验证和定量 FFPE 抗原回收

• 批准号: 7677468
• 负责人: Satya Prakash Saxena
• 金额: $ 75万
• 依托单位: CALIBRANT BIOSYSTEMS, INC.
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7677468
• 关键词: Actins; Antibodies; Antigens; Archives; Area; Back; Biliary; Bioinformatics; Biological; Biological Assay; Biological Markers; Biological Markers; Biological Products; Biopsy; Blood Cells; Blood capillaries; Breast; Breast Carcinoma; Buffers; California; Carcinoma; Cataloging; Catalogs; Cells; Clinical; Clinical Trials; Collagen; Control Groups; Coupling; Critical Pathways; DNA; Databases; Detection; Development; Diagnosis; Diagnostic; Digestion; Disease; Disease Outcome; Drug Approval Processes; Duct (organ) structure; Ductal Epithelium; E-Cadherin; Electrospray Ionization; Epithelial Cells; Epitopes; Evaluation; Fixatives; Formalin; Foundations; Functional disorder; Furuncles; Generations; Growth; Guidelines; Hepatocyte; Histopathology; Human; Human Resources; Immunohistochemistry; In Situ Hybridization; Individual; Industry; Inflammatory; Investigation; Ions; Isoelectric Focusing; Ki-67 Antigen; Knowledge; Kupffer Cells; Label; Lasers; Lead; Length; Libraries; Liquid Chromatography; Liver; Lymphoplasmacytic Infiltrate; Malignant Neoplasms; Mammary Gland Parenchyma; Maps; Mass Fragmentography; Measurement; Measures; Medical; Methodology; Microdissection; Modeling; Molecular Analysis; Molecular Profiling; Monitor; Morphology; Mucinous; Myosin Heavy Chains; Nature; Noninfiltrating Intraductal Carcinoma; Normal tissue morphology; Outcome; Paraffin; Paraffin Embedding; Patients; Peptide Library; Peptides; Phase; Phase I Clinical Trials; Ploidies; Population; Population Control; Preclinical Testing; Process; Progress Reports; Proteins; Proteome; Proteomics; RNA; Reaction; Recording of previous events; Reporting; Reproducibility; Research; Resources; Retrieval; Running; Sampling; Smooth Muscle; Solutions; Specimen; Staining method; Stains; Stromal Cells; Structure; System; TP53 gene; Techniques; Technology; Therapeutic; Tissue Banking; Tissue Banks; Tissue Fixation; Tissues; Trypsin; Two-Dimensional Polyacrylamide Gel Electrophoresis; Universities; Validation; Variant; anticancer research; base; calponin; capillary; cell type; clinical Diagnosis; comparative; cost; crosslink; expectation; human disease; improved; insight; laser capture microdissection; link protein; macromolecule; medical schools; mucoid; neoplastic cell; novel; outcome forecast; prevent; product development; prognostic; protein expression; public health relevance; response; technology development; therapy outcome; tissue fixing; tissue processing; tool; tumor; tumor eradication; validation studies

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.

描述(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有很长的历史，世界各地有大量的档案福尔马林固定和石蜡包埋(FFPE)组织库。这些FFPE组织收集，以及附带的临床和结果信息，为进行回溯性蛋白质生物标记物研究提供了宝贵的资源。除了目前蛋白质组技术(包括双向聚丙烯酰胺凝胶电泳法和多维液相色谱系统)对样品数量的限制外，缺乏从FFPE组织中提取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。在R41第一阶段研究期间，通过与南加州大学凯克医学院的施善荣博士和Clive R.Taylor博士合作，抗原检索(AR)与双子蛋白质组技术的结合不仅完成了对从FFPE组织中提取的蛋白质的质量和重复性的严格评估，以优化AR方法，而且还展示了利用存档的FFPE组织标本寻找生物标记物的重大机会。在R42第二阶段项目期间，CALIBRANT和USC团队之间拟议的协同工作旨在在模型和肿瘤FFPE组织中生成蛋白质型多肽库。这些蛋白型多肽文库代表了对可从FFPE组织中检索的抗原进行全球编目的第一步，并为后续的免疫组织化学(IHC)抗体开发提供了可用的表位数据库。除了为IHC从业者提供选择最佳AR条件/抗体组合的指南外，涉及使用蛋白质型多肽文库和相关抗体的比较蛋白质组学和验证研究将进一步提高定量IHC测量的重复性和灵敏度。由于IHC在临床诊断/预后和转化性癌症研究中的广泛应用，对定量IHC的需求继续上升。此外，使用高质量样本中的浓缩和精选细胞进行靶向蛋白质组学研究的最大期望在于在临床环境中以及在临床前测试和临床试验期间识别诊断、预后和预测性生物标记物，以及在生物制药行业发现和验证新的蛋白质靶标。FDA在2006年3月发布的关键路径机会报告不仅是关键路径倡议的第一个具体蓝图，关键路径倡议是通过应用新的战略和技术来简化药物审批过程的努力，而且还强调生物标记物的开发是“改善医疗产品开发的最重要领域之一”。 公共卫生相关声明：通过将Calbrant独特的组织蛋白质组能力与南加州大学的Clive R.Taylor博士在抗原检索-免疫组织化学方面的专业知识结合起来，拟议的研究不仅旨在通过创建蛋白质型多肽库来评估和优化定量免疫组织化学测量，而且还专注于进一步开发和展示一种新的生物标记物发现范式，以实现对存档的福尔马林固定和石蜡包埋组织标本的全面和比较蛋白质组学分析，以支持癌症研究、诊断和治疗。