Serum Glyco-Markers of Early Hepatocellular Carcinoma Using a Mass Spec Approach

描述(申请人提供)：肝细胞癌是世界上第四常见的癌症。肝癌的发病率和死亡率几乎是相同的，这表明早期发现肝癌的新方法至关重要。肝硬变是肝细胞癌发生发展的最重要因素。由于肝细胞癌的发病率预计将在未来十年内翻一番，因此一种能够区分早期肝细胞癌和肝硬变的高度特异性的非侵入性测试可能会对高风险患者的生存产生重大影响。我们开发了一种独特的凝集素阵列/质谱仪同位素标记方法，用于在血清中识别早期肝细胞癌(HCC)和肝硬变的潜在糖蛋白标记物。在我们之前的工作中已经确定了几个标记，其中一些现在已经通过了基于ELISA法的EDRN盲法验证的第一阶段。在本文提出的工作中，我们计划开发一种基于MALDI QIT质谱学的早期肝细胞癌和肝硬变多糖的分析方法，并将其与基于ELISA的方法的结果进行比较，以提高检测肝细胞癌的整体性能。在该方法中，使用免疫亲和沉淀法从血清中提取目标糖蛋白，去除糖链，然后使用MALDI QIT MS分析糖链。该平台利用单抗捕获和分离目标血清糖蛋白，其上的糖链结构的变化可以通过基于质谱学的技术来探测。捕获的糖蛋白被脱糖基化，并用串联质谱仪快速分析糖基。抗体亲和力的使用选择了有限数量的候选，从而简化了使用质谱学搜索特定于疾病状态的多糖的过程。此外，它将糖链变化与血清中的特定蛋白质相关联，为检测每种疾病状态下的糖链变化提供了高度的选择性。这将表明，这种基于质谱学的方法具有高度的特异性，可以检测早期肝细胞癌和肝硬变之间的糖链结构的变化。该方法可以检测糖结构的变化，而目前的标准ELISA只检测糖蛋白表达的定量变化。质谱学方法还可以对多路样品使用MS/MS技术，这样就可以区分几种蛋白质的糖链模式，并同时进行几种分析。质谱仪和ELISA方法将被应用于EDRN提供的盲法样本集，以展示基于目标蛋白岩藻糖化水平和糖蛋白水平的变化来识别早期肝细胞癌标志物的潜力，其中新的糖蛋白将在第一阶段生物标志物研究中进行验证，然后在更大的第二阶段研究中进行验证。

DESCRIPTION (provided by applicant): Hepatocellular Carcinoma (HCC) is the fourth most common cancer in the world. The incidence and mortality rates for HCC are virtually identical, indicating that novel methods for the early detection of HCC are of utmost importance. Liver cirrhosis is the most important factor in the development of HCC. Since the incidence of HCC is expected to double over the next decade, a highly specific noninvasive test that can differentiate early HCC from cirrhosis could have major impact on survival of patients at high risk of developing this disease. We have developed a unique lectin array/mass spectrometry isotopic tag method for identifying potential glycoprotein markers of early stage hepatocellular cancer (HCC) versus cirrhosis in serum. Several markers have been identified in our previous work, some of which have now passed a first stage of EDRN blinded validation based on ELISA assays. In the work proposed herein, we plan to develop a MALDI QIT mass spec based assay of glycans from early HCC and cirrhosis and compare them to results from the ELISA based methods to improve the overall performance for detection of HCC. In this method, immunoaffinity precipitation is used to extract a target glycoprotein from serum, the glycans removed and then the glycans are profiled using MALDI QIT MS. This platform utilizes monoclonal antibodies to capture and isolate target serum glycoproteins on which alterations in glycan structure can be probed using a mass spec based technique. The captured glycoproteins are deglycosylated and the glycan groups rapidly analyzed using tandem mass spectrometry. The use of the antibody affinity selects a limited number of candidates thus simplifying the search for glycans that are specific to the disease state using mass spectrometry. In addition, it associates the glycan change to a specific protein in serum providing a high degree of selectivity for detection of the glycan change in each disease state. It will be shown that this mass spec based method is highly specific and can detect changes in glycan structure between early HCC and cirrhosis. The method can detect changes in glycan structure while the current standard ELISAs only detect quantitative changes in glycoprotein expression. The mass spec method can also use an MS/MS technique for multiplexed samples so that glycan patterns from several proteins can be discriminated and several assays can be run simultaneously. The mass spec and ELISA methods will be applied to blinded sets of samples provided by the EDRN to demonstrate the potential for identifying markers of early HCC based on a combination of changes in fucosylation levels and glycoprotein level of target proteins where novel glycoproteins will undergo validation in a phase 1 biomarker study followed by validation in a larger phase 2 study.