Qualitative Analysis of Tissue Biomarkers and Pathways

描述(由申请人提供)：肿瘤生物标记物的定量测量在预测患者结局和治疗反应方面显示出巨大的前景。最近，我们开发了一种用于评估组织切片上生物标记物表达的自动化系统(Aqua-Automated Quantity Analysis)。AQUA为免疫组织化学染色的组织提供生物标记物的定量分析和亚细胞定位。然而，AQUA受到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的限制。向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、定量的测量。通过开发评估肿瘤相关通路的功能的方法，特别是通过研究特定的蛋白质-蛋白质相互作用，也可以促进个体肿瘤特征的形成。为了实现这一点，我们提出了以下具体目标：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。以及2：开发增加评估生物标记物表达的动态范围的方法。为了评估蛋白质之间的相互作用--这远远低于光学显微镜的分辨率--我们将开发新的技术，将异双功能交联剂、荧光能量共振转移(FRET)和催化酪胺放大结合在一起，使它们适合用于组织切片。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、滚圈和免疫AT尾巴)。一旦这些技术被开发出来，它们将为组织中的生物标记物和途径提供新的线性、定量和高度特异性的评估，最终可以用于推进个性化的药物诊断。

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.