Development of Glypican-3 as a biomarker for hepatocellular carcinoma

描述(由申请人提供)：迫切需要更好的血清学测试来早期发现、诊断和监测肝细胞癌(HCC)。目前使用的血清学检测(甲胎蛋白，AFP)的敏感性和特异性有限。多项研究表明，一种名为GPC3(GPC3)的蛋白质是肝癌的组织学和血清学标志。利用基因组学方法，我们发现GPC3在肝细胞癌中显著过度表达，而在非肿瘤肝脏中不表达。事实上，GPC3在肝细胞癌和非肿瘤肝脏中的差异表达程度仅次于AFP。因此，我们假设联合检测血清AFP和GPC3可以提高在肝细胞癌早期准确发现的能力，从而提高治疗干预的效果。事实上，我们的初步结果表明，AFP和GPC3的同时检测可以为肝癌的诊断和筛查有发生这种恶性肿瘤的风险的患者提供一种改进的检测方法。本提案的主要目的是改进和验证检测GPC3的酶联免疫吸附试验，并用该方法验证GPC3和AFP联合检测肝癌的临床应用价值。虽然检测GPC3的方法很常见，但创新之处在于发现了它对肝细胞癌的特异性，并有机会与另一种公认的生物标记物(AFP)进行联合分析，以提高该检测的临床实用性。我们将通过产生在ELISA中与来自1G12杂交瘤的当前抗体很好地配对的单抗来实现特定目标I和2的目标。此外，将对检测格式进行改进，以提高GPC3酶联免疫吸附试验的灵敏度。在初步研究中，我们已经实现了比以前版本的测试至少增加10倍的检测率。我们预计，我们将开发一种检测血清中GPC3的皮克级ELISA，通过增加该检测的敏感范围，可以在不牺牲特异性的情况下获得更高的检测肝癌的灵敏度。我们建议的另一个目标是使用这种改进的诊断测试作为监测治疗反应的工具。由于感染乙型或丙型肝炎病毒是肝细胞癌的主要危险因素，因此，改进早期发现这种癌症的诊断测试可能是处理这一重大公共卫生问题的有用组成部分。

DESCRIPTION (provided by applicant): There is an urgent need for better serological tests for the early detection, diagnosis and monitoring of hepatocellular carcinoma (HCC). The serological test that is currently used (alphafetoprotein, AFP) has limited sensitivity and specificity. Several studies have shown that a protein called Glypican-3 (GPC3) is a histological and serological marker of HCC. Using a genomics approach, we found GPC3 to be significantly over-expressed in HCC compared to non-tumor liver. In fact, GPC3 is second only to AFP in its magnitude of differential expression between HCC and non-tumor liver. Thus, we hypothesize that the combination of AFP and GPC3 measurements in serum could improve the ability to accurately detect HCC at its early stage, and therefore improve the efficacy of therapeutic interventions. Indeed, our preliminary results suggest that the simultaneous assessment of AFP and GPC3 could provide an improved test for HCC diagnosis and for the screening of patients at risk of developing this malignancy. The primary objective of this proposal is to improve and validate an ELISA for the detection of GPC3, and to use this assay to verify the clinical utility of combined analysis of GPC3 and AFP for the detection of HCC. While the methodology used to detect GPC3 is commonplace, the innovation lies in the discovery of it's specificity for HCC, and in the opportunity for combined analysis with another accepted biomarker (AFP) to improve the clinical utility of the test. We will accomplish the goals in Specific Aims I and 2 by generating monoclonal antibodies that pair well in ELISAs with the current antibody derived from the 1G12 hybridoma. In addition, refinements to the assay format will be made to improve the sensitivity of the GPC3 ELISA. In preliminary studies, we have already achieved at least a 10-fold increase in detection over the previous version of the test. We anticipate that we will develop an ELISA with picogram-level detection for GPC3 in serum, and that by increasing the sensitive range of the assay, greater sensitivity for detecting HCC can be achieved without sacrificing specificity. An additional goal of our proposal is to use this improved diagnostic test as a tool to monitor response to therapy. Since infection with Hepatitis B or Hepatitis C virus is a major risk factor for HCC, an improved diagnostic test for the early detection of this cancer could be a useful component of the management of this significant public health issue.