Affinity Based Strategies to Fast Track Development of Colon Cancer Biomarkers

简介(申请人提供)：结直肠癌(CRC)是美国最常见的癌症之一，在癌症相关死亡原因中排名第二。该项目建议进行广泛的蛋白质组和血糖筛查以及有针对性的分析，以发现早期发现和诊断结直肠癌的生物标志物。我们的目标是通过添加彼此或现有标记一起产生良好特异性的高度敏感的标记来改进结肠筛查。我们将在定制的高密度抗体微阵列(多达6000种不同的分析物)上询问独特的诊断前血浆样本、原发结直肠癌肿瘤组织和结直肠癌病例的血浆，以发现新的蛋白质组和血糖生物标志物。平行阵列将与血浆或组织裂解液孵育，以进行蛋白质组学比较或糖基化(通过与不同凝集素的孵育)。这种微阵列方法采用了具有无与伦比的灵敏度的技术，使询问能够低至循环蛋白质组的低皮摩尔浓度。在“发现”阵列上通过统计阈值的生物标志物候选将被保留，并在较小的阵列上使用新样本进行重新检查(以降低错误发现率)--我们将这一步骤称为“预验证”，以表明潜在的生物标志物将在多个样本集中显示为具有统计意义，但尚未受到正式表征或验证。这一独特的分类过程允许对潜在的生物标记物进行快速分类，使我们能够只关注最有希望的生物标记物。然后，将使用更传统的ELISA法和基于凝集素的方法对最佳候选者进行评估。我们的方法将专门针对在结直肠癌过程中调节细胞凋亡、增殖、血管生成、炎症/前列腺素、胰岛素抵抗、Toll样受体(TLR)、转化生长因子(TGF)β和STAT信号通路失控的蛋白质组和糖基化改变，以发现早期发现和诊断的生物标志物。这些包括针对21个MAPK、12个转化生长因子-β和许多STAT通路靶标的300多个磷酸特异性抗体及其匹配的非磷酸特异性抗体。

DESCRIPTION (provided by applicant): Colorectal cancer (CRC) is one of the most common cancers in the United States and the second ranked cause of cancer related death. This project proposes to perform both broad proteomic and glycomic screens and targeted analyses to discover early detection and diagnostic biomarkers of CRC. The goal is to improve colon screening by the addition of highly sensitive markers that together with each other or existing markers yield good specificity. We will interrogate unique prediagnostic plasma samples, tissue from primary CRC tumors, and plasma from CRC cases on custom high-density antibody microarrays (up to 6000 different analytes) to discover novel proteomic and glycomic biomarkers. Parallel arrays will be incubated with plasma or tissue lysate for proteomic comparison or glycosylation (via incubation with different lectins). This microarray approach employs technology with unparalleled sensitivity, enabling interrogation down to the low picomolar concentration of the circulating proteome. Biomarker candidates that pass the statistical threshold on a "discovery" array will be retained and be re-examined using new samples on smaller arrays (to reduce the false discovery rate) - a step we will refer to as "pre-validation" to indicate that a potential biomarker would have shown up as statistically significant in multiple sample sets but was not yet subjected to formal characterization or validation. This unique triage process allows for rapid sorting of potential biomarkers, allowing us to focus on only the most promising. The best candidates will then be evaluated with more conventional ELISA and lectin based methods. Our approaches will specifically target proteomic and glycomic changes in proteins critical for the regulation of apoptosis, proliferation, angiogenesis, inflammation/prostaglandins, insulin resistance, toll-like receptor (TLR), transforming growth factor (TGF)-¿ and STAT signaling pathway deregulation during CRC to discover biomarkers of early detection and diagnosis. These include over 300 phospho-specific antibodies and their matched non-phospho-specific counterparts to 21 MAPK, 12 TGF-¿, and many STAT pathway targets.