Podium Presentation in Room 2 on Thursday at 11:20 (Chair: Richard Drake)
Authors: Jianhui Zhu (1), Junfeng Huang (2), Jie Zhang (1), Zhengwei Chen (2), Yu Lin (1), Gabriela Grigorean (1), Lingjun Li (2), Amit G. Singal (3), Neehar D. Parikh (1), David M. Lubman (1)
Introduction: Nonalcoholic steatohepatitis (NASH) is the most severe type of nonalcoholic fatty liver disease and has become a leading cause of hepatocellular carcinoma (HCC). Patients with NASH often present with obesity, making ultrasound screening more difficult. We lack adequate methods of surveillance for the early detection of HCC in patients with NASH. Site-specific glycopeptide analysis using mass spectrometry provides a great opportunity in discovery of cancer-related glyco-markers to aid in early detection of cancer. Serum haptoglobin (Hp), containing 4 N-glycosylation sites, has been demonstrated as a reporter molecule for aberrant glycosylation in the development of HCC. However, Hp N-glycosylation profiling and diagnostic performance in NASH-related HCC have not been well studied.
Objective: The objective of this study is to characterize site-specific N-glycan heterogeneity of serum Hp in NASH-related HCC versus cirrhosis patients for evaluation of the utility of Hp N-glycopeptide markers for early diagnosis of HCC in patients with NASH.
Methods: In total, 70 NASH patients (37 HCC and 33 cirrhosis cases) were analyzed, with Hp purified from 20 μL of serum in each patient, and 140 mass spectrometry (MS) data were collected using an LC-EThcD-MS/MS-based workflow. The workflow enables differentially quantitative determination of the microheterogeneity of site-specific N-glycopeptides in serum Hp between disease groups, which includes: 1) trypsin/GluC digestion and glycopeptide enrichment, 2) LC-EThcD-MS/MS analysis of glycopeptides on an Orbitrap Lumos mass spectrometer (Thermo), and 3) Data quantitation using Byologic and Byos softwares (Protein Metrics), where Byos is a novel data quantitation platform to integrate the quantitative results from a large data set, in this case 140 MS/MS data.
Results: Differential quantitative analysis revealed 5 significantly overexpressed N-glycopeptide at sites N184 and N241 in NASH HCC compared to cirrhosis (p<0.05). Evaluation of the diagnostic performance of the selected glycopeptide markers showed that the N-glycopeptides at sites of N184 and N241 bearing a mono-fucosylated tri-antennary glycan A3G3F1S3 had the best diagnostic performance (AUC=0.733 and 0.775, respectively) in distinguishing early NASH-related HCC from cirrhosis. When combined with AFP, the two panels improved the sensitivity of 59% in AFP alone to 73% for early detection of NASH-related HCC. Moreover, the N-glycopeptide at site N241 containing a mono-fucosylated tetra-antennary glycan A4G4F1S4 can significantly improve the sensitivity for AFP-negative HCC patients from 61% to 73%, suggesting that it could serve as a promising marker for monitoring AFP-negative HCC patients.
Conclusions: This noninvasive biomarker assay contributes a potentially useful tool for discovering new glyco-markers for early detection of NASH-related HCC.
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