Department of Research & Development, Oncology, Prometheus Laboratories, 9410 Carroll Park Dr., San Diego, CA 92121, USA.
Proteome Sci. 2011 Dec 15;9(1):75. doi: 10.1186/1477-5956-9-75.
The clinical benefits associated with targeted oncology agents are generally limited to subsets of patients. Even with favorable biomarker profiles, many patients do not respond or acquire resistance. Existing technologies are ineffective for treatment monitoring as they provide only static and limited information and require substantial amounts of tissue. Therefore, there is an urgent need to develop methods that can profile potential therapeutic targets with limited clinical specimens during the course of treatment.
We have developed a novel proteomics-based assay, Collaborative Enzyme Enhanced Reactive-immunoassay (CEER) that can be used for analyzing clinical samples. CEER utilizes the formation of unique immuno-complex between capture-antibodies and two additional detector-Abs on a microarray surface. One of the detector-Abs is conjugated to glucose oxidase (GO), and the other is conjugated to Horse Radish Peroxidase (HRP). Target detection requires the presence of both detector-Abs because the enzyme channeling event between GO and HRP will not occur unless both Abs are in close proximity.
CEER was able to detect single-cell level expression and phosphorylation of human epidermal growth factor receptor 2 (HER2) and human epidermal growth factor receptor 1 (HER1) in breast cancer (BCa) systems. The shift in phosphorylation profiles of receptor tyrosine kinases (RTKs) and other signal transduction proteins upon differential ligand stimulation further demonstrated extreme assay specificity in a multiplexed array format. HER2 analysis by CEER in 227 BCa tissues showed superior accuracy when compared to the outcome from immunohistochemistry (IHC) (83% vs. 96%). A significant incidence of HER2 status alteration with recurrent disease was observed via circulating tumor cell (CTC) analysis, suggesting an evolving and dynamic disease progression. HER2-positive CTCs were found in 41% (7/17) while CTCs with significant HER2-activation without apparent over-expression were found in 18% (3/17) of relapsed BCa patients with HER2-negative primary tumors. The apparent 'HER2 status conversion' observed in recurrent BCa may have significant implications on understanding breast cancer metastasis and associated therapeutic development.
CEER can be multiplexed to analyze pathway proteins in a comprehensive manner with extreme specificity and sensitivity. This format is ideal for analyzing clinical samples with limited availability.
靶向肿瘤学药物的临床获益通常仅限于某些患者亚群。即使具有有利的生物标志物特征，许多患者也没有反应或产生耐药性。现有的技术在治疗监测方面效果不佳，因为它们仅提供静态和有限的信息，并且需要大量的组织。因此，迫切需要开发一种方法，以便在治疗过程中用有限的临床标本对潜在的治疗靶点进行分析。
我们开发了一种新型的基于蛋白质组学的测定方法，即协作酶增强反应免疫测定（CEER），可用于分析临床样本。CEER 利用在微阵列表面上的捕获抗体和另外两个检测抗体之间形成独特的免疫复合物。检测抗体之一与葡萄糖氧化酶（GO）结合，另一个与辣根过氧化物酶（HRP）结合。目标检测需要存在两个检测抗体，因为只有当两个抗体紧密接近时，GO 和 HRP 之间的酶通道事件才会发生。
CEER 能够在乳腺癌（BCa）系统中检测到单个细胞水平的人表皮生长因子受体 2（HER2）和人表皮生长因子受体 1（HER1）的表达和磷酸化。受体酪氨酸激酶（RTKs）和其他信号转导蛋白的磷酸化谱在差异配体刺激下的变化进一步证明了在多重阵列格式下极高的测定特异性。CEER 对 227 例 BCa 组织中的 HER2 分析显示，与免疫组织化学（IHC）结果相比，其准确性更高（83%对 96%）。通过循环肿瘤细胞（CTC）分析观察到，在复发性疾病中存在 HER2 状态改变的发生率很高，表明疾病进展是不断变化和动态的。在 HER2 阴性原发性肿瘤的复发性 BCa 患者中，HER2 阳性 CTC 的检出率为 41%（7/17），而 HER2 激活而无明显过表达的 CTC 的检出率为 18%（3/17）。在复发性 BCa 中观察到的明显“HER2 状态转换”可能对理解乳腺癌转移和相关治疗开发具有重要意义。
CEER 可以进行多重分析，以极其特异性和敏感性全面分析途径蛋白。这种格式非常适合分析可用样本有限的临床样本。
