Alliance of Glycobiologists for Detection of Cancer

描述(申请人提供)：这项建议旨在通过非侵入性方法检测处于可治疗阶段的肝细胞癌。为此，我们建议对肝脏分泌的N-糖蛋白(LNP)的部位特异性糖型进行定量，这些糖型与肝病进展到肝细胞癌有关。我们和其他人已经证明，蛋白质糖基化的变化伴随着肝细胞癌的发展。蛋白N-糖形式的主要再分布发生在癌前阶段，但我们最新的研究表明，结合珠蛋白糖肽的特定糖形式仅在肝细胞癌中增加。我们证明，结合珠蛋白的这些次要糖体可以在血清中检测到，并且与血清学金标准甲胎蛋白(AFP)相比，有可能提高对肝细胞癌的检测。这支持了我们的假设，即丰富的肝脏分泌的N-糖蛋白的次要部位特异性糖形式提供了在循环中可检测到的肝细胞癌特异性标记候选。我们建议完成对肝癌特异性N-糖链的鉴定；我们已经有证据表明，这些糖链具有多个岩藻糖基化和更多的分支。我们建议用最新优化的质谱学方法鉴定携带这些肝癌特异性N-糖链修饰的额外蛋白的糖肽。之所以这样做，是因为结合珠蛋白在一部分肝硬变患者中表达下调，可能需要其他蛋白来进行最佳检测。我们扩展了我们的信息学分析，对所有具有新的或取消的N-糖基化位点的多态和突变的人类N-糖蛋白进行分类。这些蛋白质变异体在人类基因组中过度表达，是与疾病相关的主要候选者，我们预计它们对癌症的发生有直接影响。我们相信，这些变异蛋白的公共可用资源将刺激糖蛋白组学预防癌症的研究。拟议的肝细胞癌特异性N-糖型检查是可行的，因为我们已经创建了肝细胞癌患者和肝硬变对照的样本库。这个资料库和其他优秀的QC资源对于分离肝细胞癌特有的肝脏分泌的N-糖蛋白的次要糖型是必不可少的；这些糖型在无疾病的受试者中是检测不到的。到研究结束时，我们将通过靶向LC-MS选择性反应监测(SRM)方法对选定的特定部位的蛋白质糖型进行定量。这些方法将以前所未有的准确性定量测定血清中与肝癌相关的部位特异性糖肽。我们这样做是因为我们相信，对特定糖蛋白多肽的特定糖形式进行量化可以提供最高的诊断准确率。定义临床适用的癌症标记物对疾病管理和患者健康具有潜在的深远影响。我们的研究有望对蛋白质糖基化对癌症发展的功能影响产生新的假说，并刺激一条全新的癌症预防和检测研究路线。 公共卫生相关性：美国的肝癌发病率正在稳步上升。癌症的早期发现可以提高患者的存活率。我们的研究旨在寻找新的生物标志物用于肝癌的早期检测，这将直接影响疾病的管理和预后。

DESCRIPTION (provided by applicant): This proposal aims to detect hepatocellular carcinoma (HCC) at a treatable stage by non-invasive methods. To this end, we propose to quantify site specific glycoforms of liver secreted N-glycoproteins (LNP) associated with the progression of liver disease to HCC. We and others have shown that changes in protein glycosylation accompany the development of HCC. Major re-distribution of protein N-glycoforms occurs at the premalignant stage of liver cirrhosis but our latest studies show that specific glycoforms of a glycopeptide of haptoglobin increase exclusively in HCC. We demonstrate that these minor glycoforms of haptoglobin are detectable in serum and have the potential to improve detection of HCC compared to the serologic gold standard, alpha fetoprotein (AFP). This supports our hypothesis that minor site specific glycoforms of abundant liver secreted N-glycoproteins provide HCC specific marker candidates detectable in the circulation. We propose to complete identification of the HCC specific N-glycans; we have already evidence that the glycans are characterized by multiple fucosylations and increased branching. We propose to identify glycopeptides of additional proteins carrying these HCC specific N-glycan modifications by newly optimized mass spectrometric methods. This is done because haptoglobin is downregulated in a fraction of cirrhotics and other proteins may be needed for optimal detection. We expand our informatic analysis to classify all polymorphic and mutant human N- glycoproteins with newly created or abolished N-glycosylation sites. These protein variants are over- represented in the human genome, represent prime candidates for association with diseases, and we expect that they have direct impact on carcinogenesis. We believe that a publically available resource of these variant proteins will stimulate glycoproteomic cancer prevention research. The proposed examination of HCC specific N-glycoforms is feasible because we have already created a repository of samples of HCC patients and cirrhotic controls. This repository, and additional outstanding QC resources, is essential for the isolation of the HCC specific minor glycoforms of liver secreted N-glycoproteins; these glycoforms are not detectable in disease free subjects. By the end of the study, we will have quantified the selected site specific protein glycoforms by targeted LC-MS selective reaction monitoring (SRM) methods. These methods will quantify the HCC associated site specific glycopeptides in serum with an unprecedented accuracy. We do this because we believe that quantification of specific glycoforms of specific glycoprotein peptides offers the highest diagnostic accuracy. Defining clinically applicable cancer markers has potentially far-reaching consequences for disease management and patient health. Our study is expected to generate new hypotheses on the functional impact of protein glycosylation on the development of cancer and to stimulate an entirely new line of cancer prevention and detection research. PUBLIC HEALTH RELEVANCE: The incidence of liver cancer in the United States is steadily increasing. Early detection of cancer improves patient survival. Our study seeks to identify new biomarkers for early detection of liver cancer, which would have a direct impact on the disease management and outcome.