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

描述（由申请人提供）：肝细胞癌（HCC）是世界上第四大常见癌症。HCC的发病率和死亡率几乎相同，这表明早期发现HCC的新方法至关重要。肝硬化是HCC发生的最重要因素。由于HCC的发病率预计在未来十年翻一番，一种高度特异性的非侵入性检测可以区分早期HCC和肝硬化，这可能对发展为这种疾病的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/质谱同位素标记方法，用于鉴定血清中早期肝细胞癌（HCC）与肝硬化的潜在糖蛋白标志物。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分析的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质谱的早期HCC和肝硬化聚糖检测方法，并将其与基于ELISA方法的结果进行比较，以提高HCC检测的整体性能。该方法采用免疫亲和沉淀法从血清中提取目标糖蛋白，去除多糖，然后使用MALDI QIT ms对多糖进行分析。该平台利用单克隆抗体捕获和分离目标血清糖蛋白，并使用基于质谱的技术检测多糖结构的变化。将捕获的糖蛋白去糖基化，并使用串联质谱快速分析多糖基团。抗体亲和力的使用选择了有限数量的候选者，从而简化了使用质谱法寻找特定于疾病状态的聚糖。此外，它将多糖变化与血清中的特定蛋白质联系起来，为检测每种疾病状态下的多糖变化提供了高度的选择性。结果表明，这种基于质谱的方法具有高度特异性，可以检测早期HCC和肝硬化之间聚糖结构的变化。该方法可以检测到多糖结构的变化，而目前的标准elisa只能检测糖蛋白表达的定量变化。质谱法还可以对多路样品使用质谱/质谱技术，这样可以区分来自几种蛋白质的聚糖模式，并且可以同时进行几种分析。质谱仪和ELISA方法将应用于EDRN提供的盲法样品集，以证明基于靶蛋白聚焦水平和糖蛋白水平变化的组合识别早期HCC标志物的潜力，其中新的糖蛋白将在1期生物标志物研究中进行验证，然后在更大的2期研究中进行验证。

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.