Qualitative Analysis of Tissue Biomarkers and Pathways

描述（由申请人提供）：肿瘤生物标志物的定量测量在预测患者预后和治疗反应方面显示出很大的希望。最近，我们开发了一种用于评估组织切片上生物标志物表达的自动化系统（AQUA -自动定量分析）。AQUA提供免疫组织化学染色组织中生物标志物的定量分析和亚细胞定位。然而，AQUA受限于1)光学显微镜的分辨率和2)用于识别生物标志物的酶扩增的非线性效应。生物特异性治疗和相关药物诊断的进展将需要对每个患者肿瘤内的蛋白质表达进行线性定量测量。个体肿瘤分析也可以通过开发评估肿瘤相关通路功能的方法来推进，特别是通过研究特定的蛋白质-蛋白质相互作用。为了实现这一目标，我们提出以下具体目标：1：开发识别组织切片中特定蛋白质-蛋白质相互作用的方法。2：开发增加生物标志物表达动态范围的方法。为了评估远远低于光学显微镜分辨率的蛋白质-蛋白质相互作用，我们将开发结合异功能交联试剂、荧光能量共振转移（FRET）和催化酰胺扩增的新技术，使其适合用于组织切片。为了提高AQUA技术的动态范围，我们将采用最近描述的几种原位构建大荧光分子的方法（圣诞树，滚动圈和免疫-tail）。一旦这些技术被开发出来，它们将为组织中的生物标志物和途径提供新的线性、定量和高度特异性的评估，最终可用于推进个性化药物诊断。

DESCRIPTION (provided by applicant): Quantitative measurement of tumor biomarkers has shown great promise in predicting patient outcome and response to treatment. Recently, we have developed an automated system for assessing biomarker expression on tissue sections (AQUA - Automated Quantitative Analysis). AQUA provides quantitative analysis and sub-cellular localization of biomarkers on immunohistochemically stained tissues. AQUA, however, is limited by 1) the resolution of light microscopy and 2) the non-linear effects of enzymatic amplification used to identify biomarkers. The progression toward bio-specific therapies and associated pharmaco-diagnostics will require linear, quantitative measures of protein expression within each patient's tumor. Individual tumor profiling can also be advanced by developing methods for assessing the functioning of tumor-related pathways, in particular by studying specific protein-protein interactions. To acomplish this, we propose the following specific aims: 1: To develop methods for identifying specific protein-protein interactions in tissue sections. And 2: To develop methods for increasing the dynamic range of assessing biomarker expression. To assess protein-protein interactions - which are far below the resolution of light microscopy - we will develop new techniques that combine heterobifunctional crosslinking reagents, fluorescence energy resonance transfer (FRET), and catalyzed tyramide-amplification, making them suitable for use in tissue sections. To improve the dynamic range of the AQUA technology, we will adapt several recently described methods for building large fluorescent molecules in situ (Christmas trees, rolling-circle and ImmunoAT-tailing). Once these techniques are developed, they will provide new linear, quantitative, and highly specific assessments of biomarkers and pathways in tissues that can ultimately be used to advance personalized pharmaco-diagnostics.