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

使用质谱方法检测早期肝细胞癌的血清糖标记物

• 批准号: 8825456
• 负责人: David M. Lubman
• 金额: $ 45.25万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-01 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8825456
• 关键词: Affinity; Antibodies; Antibody Affinity; Biological Assay; Biological Markers; Blinded; Characteristics; Cirrhosis; Clinical; Complement; Data; Detection; Development; Diagnostic; Disease; Early Detection Research Network; Early Diagnosis; Enzyme-Linked Immunosorbent Assay; Excision; Frequencies; Glycoproteins; Hepatitis C virus; Incidence; Lectin; Level of Evidence; Link; Liver Cirrhosis; Liver diseases; Malignant Neoplasms; Mass Spectrum Analysis; Methodology; Methods; Monoclonal Antibodies; Patients; Pattern; Performance; Phase; Polysaccharides; Precipitation; Primary carcinoma of the liver cells; Protein Glycosylation; Proteins; Reproducibility; Resolution; Review Literature; Risk Factors; Running; Sample Size; Sampling; Screening for cancer; Serum; Serum Markers; Specificity; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Staging; Structure; Techniques; Testing; Ultrasonography; Validation; Work; alpha-Fetoproteins; base; carbohydrate structure; high risk; improved; mortality; novel; phase 2 study; screening; success; systematic review; tandem mass spectrometry; tumor; validation studies

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.

描述（由申请人提供）：肝细胞癌（HCC）是世界上第四大常见的癌症。 HCC的发病率和死亡率实际上是相同的，这表明早期发现HCC的新方法至关重要。肝硬化是HCC发展中最重要的因素。由于预计HCC的发生率将在未来十年中翻一番，因此可以将早期HCC与肝硬化区分开的高度特异性非侵入性测试可能会对患者的生存产生重大影响，而患者的生存率很高。我们已经开发了一种独特的凝集素阵列/质谱同位素标记方法，用于鉴定早期肝细胞癌（HCC）与血清中肝硬化的潜在糖蛋白标记。在我们以前的工作中已经确定了一些标记，其中一些标记已通过EDRN的第一阶段，基于ELISA测定法。在本文提出的工作中，我们计划开发一种基于MALDI QIT质量规格的基于早期HCC和Cirrhosis的聚糖测定法，并将其与基于ELISA的方法的结果进行比较，以改善检测HCC的整体性能。在这种方法中，使用免疫亲和力沉淀从血清中提取靶糖蛋白，去除聚糖，然后使用MALDI QIT MS对聚糖进行分析。该平台利用单克隆抗体来捕获和分离靶血清糖蛋白，可以使用基于质量规格的技术探测聚糖结构的改变。捕获的糖蛋白是脱脂型的，使用串联质谱法迅速分析了聚糖基团。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特定于疾病状态的较量的搜索。此外，它将聚糖变化与血清中的特定蛋白质相关联，为检测每种疾病状态的聚糖变化提供了高度选择性。结果表明，这种基于质量规格的方法是高度特异性的，可以检测早期HCC和肝硬化之间的聚糖结构的变化。该方法可以检测聚糖结构的变化，而当前标准ELISA仅检测糖蛋白表达的定量变化。质量规格方法还可以对多路复用样品使用MS/MS技术，以便可以歧视几种蛋白质的聚糖图案，并且可以同时运行几种测定。质量规格和ELISA方法将应用于EDRN提供的盲样样品集，以证明基于岩藻糖基化水平的变化和靶蛋白的糖蛋白水平的结合，可以鉴定早期HCC的标记，新型糖蛋白的糖蛋白水平在新的糖蛋白中会在1阶段生物标志物研究中经过较大的一阶段验证，在大型研究中经过较大的2期研究。