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（由申请人提供）：由于将福尔马林用作组织病理学组织加工的标准固定剂的历史悠久，因此在全球范围内有大量的福尔马林固定档案和石蜡包裹（FFPE）组织库。这些FFPE组织收集及其附着的临床和结果信息，呈现出可回顾性蛋白质生物标志物研究的宝贵资源。除了由当前蛋白质组技术（包括二维聚丙烯酰胺凝胶电泳）和多维液态色谱系统（包括二维聚丙烯酰胺凝胶电泳）所施加的样品量约束外，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Drs合作。在R41阶段研究期间，南加州大学凯克医学院的Shan-Rong Shi和Clive R. Taylor，将抗原检索（AR）与Gemini蛋白质组学技术的结合不仅实现了对FFPE组织的质量和蛋白质的质量的严格评估，而且还取决于FFPE组织的质量和可重复性，而且还实现使用存档的FFPE组织收集发现。在R42 II期项目中，校准剂与USC团队之间提出的协同工作旨在在模型和肿瘤FFPE组织之间产生蛋白质型肽库。这些蛋白质型肽文库代表了可从FFPE组织检索的全球分类抗原的第一步，并介绍了可用的表位数据库，以进行随后的免疫组织化学（IHC）抗体的发展。除了为IHC的实践者提供优化的AR条件/抗体组合的指南外，还涉及使用蛋白质型肽库和相关抗体的比较蛋白质组学和验证研究，还将提供进一步的增强能力以及定量IHC测量的敏感性。由于IHC在临床诊断/预后和转化癌症研究中的广泛利用，对定量IHC的需求继续升级。此外，对使用高质量标本的富集和选定细胞进行靶向蛋白质组学研究的最大期望是在临床环境中，临床前测试和临床试验中的诊断，预后和预测性生物学标志物的鉴定，以及在生物素养工业中的发现和新蛋白质靶标的。 FDA在2006年3月发布的关键路径机会报告不仅是关键路径计划的第一个特定蓝图，这是通过应用新策略和技术来简化药物批准过程的努力，还突出了生物标志物作为“改善医疗产品开发的最重要领域之一”。 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生物标志物发现范式，用于对福尔马林固定和石蜡包含的组织收集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。