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.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project--Cooperative Agreements (U01)
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Special Emphasis Panel (ZCA1-SRLB-4 (M1))
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Rinaudo, Jo Ann S
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Georgetown University
Internal Medicine/Medicine
Schools of Medicine
United States
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Sanda, Miloslav; Benicky, Julius; Wu, Jing et al. (2016) Increased sialylation of site specific O-glycoforms of hemopexin in liver disease. Clin Proteomics 13:24
Flowers, Sarah A; Zhou, Xin; Wu, Jing et al. (2016) Expression of the extracellular sulfatase SULF2 is associated with squamous cell carcinoma of the head and neck. Oncotarget 7:43177-43187
Singer, Mark S; Phillips, Joanna J; Lemjabbar-Alaoui, Hassan et al. (2015) SULF2, a heparan sulfate endosulfatase, is present in the blood of healthy individuals and increases in cirrhosis. Clin Chim Acta 440:72-8
Nayak, Aparajita; Pattabiraman, Nagarajan; Fadra, Numrah et al. (2015) Structure-function analysis of hepatitis C virus envelope glycoproteins E1 and E2. J Biomol Struct Dyn 33:1682-94
Yuan, Wei; Sanda, Miloslav; Wu, Jing et al. (2015) Quantitative analysis of immunoglobulin subclasses and subclass specific glycosylation by LC-MS-MRM in liver disease. J Proteomics 116:24-33
Goldman, Radoslav; Sanda, Miloslav (2015) Targeted methods for quantitative analysis of protein glycosylation. Proteomics Clin Appl 9:17-32
Chandler, Kevin Brown; Brnakova, Zuzana; Sanda, Miloslav et al. (2014) Site-specific glycan microheterogeneity of inter-alpha-trypsin inhibitor heavy chain H4. J Proteome Res 13:3314-29
Cole, Charles; Krampis, Konstantinos; Karagiannis, Konstantinos et al. (2014) Non-synonymous variations in cancer and their effects on the human proteome: workflow for NGS data biocuration and proteome-wide analysis of TCGA data. BMC Bioinformatics 15:28
Wu, Tsung-Jung; Shamsaddini, Amirhossein; Pan, Yang et al. (2014) A framework for organizing cancer-related variations from existing databases, publications and NGS data using a High-performance Integrated Virtual Environment (HIVE). Database (Oxford) 2014:bau022
Benicky, Julius; Sanda, Miloslav; Pompach, Petr et al. (2014) Quantification of fucosylated hemopexin and complement factor H in plasma of patients with liver disease. Anal Chem 86:10716-23

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