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、经济和法规要求开发配套诊断，但很少有新的组织生物标记物被转化为临床使用。必须对大量候选者进行临床验证研究，才能确定具有临床实用价值的单一新生物标志物。少数已成功进入临床的生物标记物主要是通过对档案福尔马林固定石蜡包埋(FFPE)生物标本的回顾分析来鉴定的。目前检测FFPE组织中蛋白质的金标准是免疫组织化学(IHC)，但由于新的IHC分析方法的开发和分析验证的高成本和长时间，这项技术完全不足以支持在回溯性验证研究中对数百个候选生物标志物的大规模测试。此外，即使使用多参数荧光检测，IHC的多重能力仍然有限，只能在每次检测中检测少量的候选生物标记物。此外，在基于IHC的临床检测中，多种来源的变异导致实验室间的一致性很差。此外，正如目前部署的那样，IHC检测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。一种有可能克服这一障碍的新兴技术是一种被称为多反应监测质谱学(MRM-MS)的靶向形式的质谱学。虽然MRM能够在高多重水平下对多肽进行特定、准确的定量，但对许多分析物来说，灵敏度是有限的。为了解决这一局限性，我们开发了一种新的平台，将多肽免疫亲和浓缩与MRM相结合，从而产生高灵敏度的免疫-MRM分析。我们最近确定了将这种新兴的免疫-MRM技术用于血浆中癌症生物标志物候选的大规模测试的可行性，在这一应用中，我们将对免疫-MRM技术平台进行高级开发，以应用于来自FFPE组织的少量人类细胞。在目标1中，将制定一个标准操作程序，以支持在FFPE癌组织中进行分析稳健的多重MRM和免疫MRM量化。在目标2中，免疫-MRM技术的分析验证将在一项模拟的回溯性生物标志物验证研究中进行，该研究使用存档的人类乳腺癌组织。