Identification of Colon Cancer Protein Biomarkers in the Blood

描述(由申请人提供)： 通过检测外周血样本来检测结直肠癌(CRC)可以加强普通人群的筛查工作，并提高此类癌症患者的生存率。早期发现结直肠癌的患者存活率要高得多。此外，即使在被诊断为结直肠癌特定阶段的患者中，由于人们对其知之甚少的原因，其预后也可能存在巨大的差异。蛋白质组学分析方法的快速发展表明，有可能识别候选的CRC选择性生物标记蛋白，类似于前列腺癌的前列腺特异性抗原(PSA)或卵巢癌的CA125。确定合适的CRC生物标志物小组的合理方法是首先确定与正常结肠细胞相比，在结肠癌细胞中唯一存在或升高的蛋白质，或者释放到肿瘤细胞周围间质液体中的蛋白质。在确定了CRC选择蛋白之后，人们就可以研究哪些蛋白可能在外周血液中升高。这种对结直肠癌和正常肠上皮细胞进行全面而详细的比较分析的策略可以克服困扰许多血液筛查方法的关键障碍，这些方法经常被以下事实所困扰：仅有少数高丰度蛋白代表了血液蛋白质含量的90%以上。对于大多数分析方法，这些高丰度蛋白质提供了一层雾，掩盖了CRC选择性蛋白质或多肽。在这里，我们提出了一种策略和工作流程，其中结合了蛋白质组样品处理、纳米级液相色谱(Nano-LC)和质谱学(MS)方面的几个相互关联的分析进展，从而增强了纳米LC/MS方法在组织分析中的适用性。这些进展提供了(I)组织蛋白质的高度定量回收(Ii)高度重复性和灵敏的蛋白质表达谱，(Iii)比其他方法更多的蛋白质的定量，以及(Iv)对感兴趣的特定蛋白质的高度敏感的定量。采用这一分析策略，我们将(1)对来自结直肠癌和邻近正常结肠上皮的细胞进行比较蛋白质组学分析，以确定结直肠癌选择性蛋白质，(2)研究结直肠癌细胞可能分泌到细胞外环境中的蛋白质或多肽的补体，因为这些蛋白质或多肽可能更有可能进入外周血，以及(3)使用癌症手术前收集的患者配对血液样本，评估外周血中结直肠癌选择性蛋白质的量化策略。确定这一工作流程和策略的潜力对于拟议项目的合理后续工作至关重要，该项目将在一项全面的临床研究中寻找外周血中的CRC选择性蛋白。拟议研究的总体结果包括用于早期发现结直肠癌的基于血液的筛查工具，以及更全面地了解结直肠癌选择性标记物，这可以改善对患者预后的评估，或为了更有效的治疗而对疾病进行分层。

DESCRIPTION (provided by applicant): Detection of colorectal cancer (CRC) by examination of peripheral blood samples could enhance screening efforts of the general population and lead to better survival rates in those patients with this type of cancer. Patients have a much higher survival rate when CRC is detected at an early stage. Furthermore, even within the population of patients diagnosed with a specific stage of CRC, there can be dramatic differences in prognosis for reasons that are understood poorly. Rapid developments in proteomic analytical approaches suggest the potential to identify candidate CRC-selective biomarker proteins, akin to Prostate Specific Antigen (PSA) for prostate cancer or CA125 for ovarian cancer. A rational approach to identifying an appropriate CRC biomarker panel would be to first determine what proteins are uniquely present or elevated in colon cancer cells, compared to normal colon cells, or shed into the interstitial fluid around the tumor cells. Having identified CRC-selective proteins, one could then investigate which may be elevated in the peripheral blood. This strategy for comprehensive and detailed comparative analysis of CRC vs. normal intestinal epithelial cells could overcome key hindrances that beset many blood-screening approaches, which are often confounded by the fact that just a handful of high-abundance proteins represent more than 90% of the blood protein content. For most analytical approaches, these high abundance proteins provide a fog that obscures CRC-selective proteins or peptides. Here we propose a strategy and workflow that incorporate several interrelated analytical advances in proteomic sample processing, nano-scale liquid chromatography (nano-LC), and mass spectrometry (MS) that enhance the suitability of nano-LC/MS approaches for the analysis of tissues. These advances provide (i) highly quantitative recovery of tissue proteins (ii) highly reproducible and sensitive protein expression profiling, (iii) quantification of a significantly greater number of proteins than otherwise feasible, and (iv) highly sensitive quantification of specific proteins of interest. Employing this analytical strategy, we will (1) perform comparative proteomic analysis of cells derived from CRC vs. adjacent normal colon epithelium, in order to identify CRC-selective proteins, (2) investigate the complement of proteins or peptides that CRC cells may secrete into the extracellular environment, as these may have higher likelihood of making their way to peripheral blood, and (3) evaluating strategies to quantify CRC-selective proteins in the peripheral blood, using patient-matched blood samples collected prior to cancer surgery. Establishing the potential of this workflow and strategy would be essential for the logical follow-on to the proposed project, which would be to seek CRC-selective proteins in peripheral blood in a comprehensive clinical study. The overall outcomes of the proposed study include a blood based screening tool for the early detection of CRC, as well as a more comprehensive understanding of CRC-selective markers that could improve assessment of patient prognosis, or stratification of disease for more effective therapy.