Profiling Autoantibodies in Non-small Cell Lung Cancer with Fusion Protein-Peptid

描述（由申请人提供）：在美国，每年约有135,000例非小细胞肺癌（NSCLC）新病例被诊断出来，但这些患者中只有20%-25%能够在足够早期的阶段被诊断出来，从而通过手术或其他方法治愈。当与放射成像结合使用时，对非小细胞肺癌进行敏感和特异性的血液检查可能是一种临床上有价值的、具有成本效益的早期诊断工具。早在转移发生之前，免疫系统就通过分泌自身抗体对肿瘤相关蛋白作出反应。外周血中这组重要的循环肿瘤标志物的自身抗体谱分析使我们能够识别合适的靶肽。利用来自非小细胞肺癌细胞系cDNA的T7噬菌体展示文库，我们开发了一个由8个肽组成的蛋白质微阵列，用于简单的捕获免疫测定（Zhong et al. 2006， J. Thoracic oncology . 1(6), 513）。当用血液样本检测时，该阵列可以识别非小细胞肺癌中存在的肿瘤相关抗体，但在疾病的早期和晚期，其灵敏度和特异性均超过90%。当用于测量来自癌症患者队列和风险匹配对照的血清中肿瘤相关抗体时，该测试提供的预测准确性超过了目前可用的循环NSCLC相关蛋白标记物。本应用程序的总体目的是实现三个目标：1)将编码我们最初发现的八种肽的DNA序列亚克隆到两个融合表达载体中；2)制备目标1中描述的融合肽的微阵列，并证明它们能够测量自身抗体谱，从而以前所未有的灵敏度和特异性区分癌症和正常血液样本。3)最后，我们将获得30例非小细胞肺癌和30例风险对照组的队列，并使用蛋白质微阵列验证上述肽作为真正的肺癌生物标志物。纯化肽的微阵列将用于多靶点免疫测定的所有技术和临床性能指标的严格优化和标准化。我们最初的临床应用将是早期发现肺癌，尽管在肺癌管理中的多种应用是合理的。我们的数据显示了可行性和概念证明，支持进一步开发和测试这种方法的基本原理。随后的II期申请将导致使用一个适当的平台进行明确的标记物验证试验，用于NSCLC的临床筛查。因此，本应用程序的主要目标是开发一种可以快速转化为临床实践的新型非小细胞肺癌血液检测方法。公共卫生相关性：用于早期发现肺癌的血液检查可以提供一种具有成本效益的筛查方法，并与CT扫描相结合，将成为降低这种疾病严重死亡率的可行策略。

DESCRIPTION (provided by applicant): Approximately 135,000 new cases of non-small cell lung cancer (NSCLC) are diagnosed each year in the USA, yet only 20%-25% of those patients will be diagnosed at a sufficiently early stage to be cured by surgical or other procedures. When used in concert with radiographic imaging, a sensitive and specific blood test for NSCLC could be a clinically valuable and cost-effective tool for early diagnosis. The immune system responds to tumor-associated proteins by secreting autoantibodies long before metastasis occurs. Autoantibody profiling for this important group of circulating tumor markers in peripheral blood has lead us to the identification of appropriate target peptides. Using a T7 phage display library derived from the cDNA of a NSCLC line, we have developed a panel of eight peptides which were formatted as a protein microarray for use in a simple capture immunoassay (Zhong et al. 2006, J. Thoracic Oncol. 1(6), 513). When probed with a blood sample, the array can identify the presence of tumor-associated antibodies in NSCLC but not in normals with both sensitivity and a specificity exceeding 90% at early and later stages of the disease. As used to measure tumor-associated antibodies in serum from a cohort of cancer patients and risk- matched controls, this test affords predictive accuracy that exceeds that of currently available circulating NSCLC- associated protein markers. The overall purpose of this application is to achieve three aims: 1) subclone the DNA sequences encoding the eight peptides identified in our initial discovery effort into two fusion-expression vectors, 2) prepare microarrays of the fusion peptides described in aim 1 and demonstrate that they enable measurement of autoantibody profiles that can distinguish cancer from normal blood samples with unprecedented sensitivity and specificity. 3) Finally, we will acquire a cohort of 30 NSCLC and 30 at risk controls and validate the above peptides as bona fide lung cancer biomarkers using protein microarrays. Microarrays of the purified peptides will be used for rigorous optimization and standardization of all technical and clinical performance measures for the multi-target immunoassay. Our initial clinical application will be early detection of lung cancer, although multiple applications in lung cancer management are rational. Our data shows feasibility and proof of concept that supports the rationale for further development and testing of this approach. A subsequent Phase II application will lead to use of an appropriate platform for definitive marker validation trials for application to clinical screening of NSCLC. Thus the primary goal of this application is to develop a novel blood test for NSCLC that can be rapidly translated into clinical practice. PUBLIC HEALTH RELEVANCE: A blood test for early detection of lung cancer could provide a cost-effective screening approach, and in concert with CT scanning would enable a viable strategy for reducing the severe mortality rate of this disease.