Advanced development of immuno-MRM technology to analyze archived cancer tissues

免疫 MRM 技术的先进发展可用于分析存档的癌症组织

• 批准号: 8547605
• 负责人: AMANDA G PAULOVICH
• 金额: $ 43.1万
• 依托单位: FRED HUTCHINSON CANCER RESEARCH CENTER
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8547605
• 关键词: Address; Advanced Development; Affinity; Antigens; Archives; Biological Assay; Biological Markers; Biopsy; Cancer Patient; Cells; Characteristics; Clinic; Clinical; Clinical Pathology; Clinical Trials; Companions; Couples; Development; Diagnostic; Diagnostic tests; Digestion; Economics; Emerging Technologies; Fine needle aspiration biopsy; Fluorescence; Formalin; Goals; Gold; Human; Immunohistochemistry; Individual; Laboratories; Lead; Malignant Neoplasms; Mass Spectrum Analysis; Methods; Outcome; Paraffin Embedding; Patients; Peptides; Performance; Pharmaceutical Preparations; Plasma; Procedures; Process; Proteins; Reporting; Retrieval; Running; Sampling; Signal Transduction; Source; Technology; Testing; Time; Tissues; Translating; Trypsin; Validation; Variant; base; biobank; cancer diagnosis; cancer therapy; cost; cost effective; malignant breast neoplasm; multiple reaction monitoring; multiplex detection; new technology; novel; novel diagnostics; polypeptide; public health relevance; validation studies

DESCRIPTION (provided by applicant): Despite a clinical, economic, and regulatory imperative to develop companion diagnostics, precious few new tissue biomarkers have been translated into clinical use. Clinical validation studies must be performed on large numbers of candidates for a single novel biomarker of clinical utility to be identified. The handful of biomarkers that have successfully reached the clinic were identified mostly through retrospective analysis of archival formalin-fixed paraffin embedded (FFPE) biospecimens. The current gold standard for detecting proteins in FFPE tissues is immunohistochemistry (IHC), but this technology is wholly inadequate to support large-scale testing of hundreds of candidate biomarkers in retrospective validation studies, due to the high costs and long lead time for the development and analytical validation of new IHC assays. Furthermore, even with multi-parameter fluorescence detection, the multiplex capabilities of IHC remain limited and would only allow testing of small numbers of candidate biomarkers in each assay. Additionally, multiple sources of variation in IHC-based clinical assays have resulted in poor inter-laboratory concordance. Furthermore, as currently deployed, IHC assay results are semi-quantitative at best, leading to difficulties interpreting intermediate results, and hampering the ability to assemble multivariate panels as diagnostics. An emerging technology that has the potential to overcome this barrier is a targeted form of mass spectrometry called multiple reaction monitoring mass spectrometry (MRM-MS). While MRM enables specific, precise quantification of polypeptides at high multiplex levels, sensitivity is limiting for many analytes. To address thi limitation, we have developed a novel platform that couples peptide immuno-affinity enrichment to MRM, resulting in highly sensitive immuno-MRM assays. We recently established the feasibility of using this emerging immuno-MRM technology for large-scale testing of cancer biomarker candidates in plasma, and in this application we will perform advanced development of the immuno-MRM technology platform for application to small numbers of human cells derived from FFPE tissues. In Aim 1, a standard operating procedure will be developed that supports analytically robust multiplex MRM and immuno-MRM quantification in FFPE cancer tissues. In Aim 2, analytical validation of the immuno-MRM technology will be performed in an emulated retrospective biomarker validation study using archived human breast cancer tissues.

描述（由申请人提供）：尽管临床、经济和监管方面都迫切需要开发伴随诊断，但很少有新的组织生物标志物已转化为临床使用。必须对大量候选者进行临床验证研究，以确定具有临床实用性的单一新型生物标志物。少数已成功进入临床的生物标志物主要是通过对档案福尔马林固定石蜡包埋 (FFPE) 生物样本的回顾性分析来鉴定的。目前检测 FFPE 组织中蛋白质的黄金标准是免疫组织化学 (IHC)，但由于新 IHC 检测的开发和分析验证成本高昂且周期长，该技术完全不足以支持回顾性验证研究中数百种候选生物标志物的大规模测试。此外，即使采用多参数荧光检测，IHC 的多重功能仍然有限，并且仅允许在每次测定中测试少量候选生物标志物。此外，基于 IHC 的临床检测中存在多种变异来源，导致实验室间一致性较差。此外，按照目前的部署，IHC 测定结果充其量是半定量的，导致解释中间结果的困难，并阻碍了组装多变量面板作为诊断的能力。 一种有潜力克服这一障碍的新兴技术是一种有针对性的质谱分析形式，称为多反应监测质谱分析 (MRM-MS)。虽然 MRM 能够以高多重水平对多肽进行特异性、精确定量，但许多分析物的灵敏度受到限制。为了解决这一限制，我们开发了一种新颖的平台，将肽免疫亲和富集与 MRM 结合起来，从而实现高度灵敏的免疫 MRM 测定。我们最近确定了使用这种新兴的免疫 MRM 技术大规模测试血浆中的癌症生物标志物候选物的可行性，在该应用中，我们将进行免疫 MRM 技术平台的高级开发，以应用于源自 FFPE 组织的少量人类细胞。 在目标 1 中，将开发一个标准操作程序，支持 FFPE 癌症组织中分析稳健的多重 MRM 和免疫 MRM 定量。 在目标 2 中，将使用存档的人类乳腺癌组织在模拟回顾性生物标志物验证研究中进行免疫 MRM 技术的分析验证。