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

描述（由申请人提供）：尽管临床，经济和监管要求开发伴随诊断，但珍贵的新组织生物标志物已经转化为临床应用。临床验证研究必须在大量候选者中进行，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）生物标本的回顾性分析确定的。目前检测FFPE组织中蛋白质的金标准是免疫组织化学（IHC），但由于新的免疫组织化学测定法的开发和分析验证的高成本和较长的准备时间，该技术完全不足以支持在回顾性验证研究中对数百种候选生物标志物进行大规模测试。此外，即使采用多参数荧光检测，免疫组化的多重能力仍然有限，每次检测只能检测少量候选生物标志物。此外，基于免疫缺陷病毒的临床检测存在多种变异来源，导致实验室间一致性较差。此外，按照目前的部署，免疫组化分析结果最多是半定量的，导致难以解释中间结果，并阻碍了将多变量面板组装为诊断的能力。一种有潜力克服这一障碍的新兴技术是一种被称为多反应监测质谱法（MRM-MS）的靶向质谱法。虽然MRM能够在高多重水平上对多肽进行特异性、精确的定量，但许多分析物的灵敏度受到限制。为了解决这一限制，我们开发了一种新的平台，将肽免疫亲和富集与MRM偶联，从而产生高灵敏度的免疫MRM测定。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物的大规模测试的可行性，并且在此应用中，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，免疫mrm技术的分析验证将在使用存档的人类乳腺癌组织的模拟回顾性生物标志物验证研究中进行。

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.