MSACL 2024 Abstract

INTRODUCTION:
N-linked glycosylation is one of the most common post-translational modifications. Changes in the N-glycan profile of a glycoprotein can occur in response to environmental and genetic factors and can correlate with disease stages. Alterations in N-glycosylation have demonstrated an immense potential to be used as diagnostic and therapeutic targets for many diseases. For example, the use of AFP-L3, a N-glycosylated protein has confirmed the importance of N-glycosylation in the clinic as a biomarker for HCC. Similarly, alterations in N-glycosylation have been established in other liver diseases like Metabolic-associated steatohepatitis (MASH). MASH is a progressive form of fatty liver disease and is characterized by inflammation, hepatocyte injury, and fibrosis. MASH is caused by an excess of fat in the liver which is linked to obesity and diet. In addition, MASH has been reported as a risk factor for cirrhosis and primary liver cancers: hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), and complications due to MASH are estimated to be the main reason for a liver transplant. Currently, there are no ideal biomarker candidates for the diagnosis of MASH or CCA.

OBJECTIVES:
Previous research has demonstrated that serum glycan profiles can be altered in MASH serum patients. Similarly, N-glycan alterations have been established in HCC. However, to date, there has been no examination of the N-glycan changes origin in tissue and the difference of these N-glycans between primary liver cancers (CCA and HCC). Here we characterize the N-glycome of MASH patients and primary liver cancer patients with an approach for biomarker identification using N-glycan modifications identified in serum and tissue.
METHODS: Our laboratory has developed methods that allow for in situ tissue and serum or plasma-based N-linked glycan analysis using matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS). We used this methodology to study the N-linked glycan modifications quantitatively and qualitatively in tissue and serum. All MALDI-IMS data obtained in MASH biopsies was correlated to a fibrosis score previously established by pathology. For CCA tissue and serum analysis, comparisons were made between samples from patients with CCA to patients at risk of developing CCA or other types of liver disease including HCC, cirrhosis, hepatitis, and fatty liver diseases. Finally, we generated a biomarker algorithm using the N-glycan observed in tissue and serum.

RESULTS:
N-glycan analysis in situ of MASH biopsies and CCA tissue was correlated to annotations made by pathology and revealed that N-glycan structures were specific areas of interest histologically. MASH biopsies had a prominent spatial correlation of fucosylated glycans in fibrotic areas but high mannose glycans in non-fibrotic areas. Similarly, in CCA tissue bisected fucosylated N-glycan structures were specific to the tumor region in CCA, while highly branched N-glycans were associated with HCC tumor regions. We expanded our tissue cohort using tissue microarrays that included tissues from over 150 patients with CCA, HCC, or non-transformed tissue. To extend this finding and to determine the translational potential of the N-glycan tissue changes, we analyzed a cohort of serum samples of 92 samples from patients with bile duct disease, hepatocellular carcinoma, or CCA. Importantly, many of the same glycan alterations that were observed in tissue were observed in serum, including alterations in bisected fucosylated N-glycans. The relative intensity of each N-glycan structure in tissue and serum was used to generate an algorithm that could be used as a biomarker of CCA. In our initial analysis, this biomarker algorithm quadrupled the sensitivity (at 90% specificity) of CCA detection as compared to the current “gold standard” biomarker of CCA (CA19-9). Overall, this study represents one of the only examples where alterations in N-linked glycan, or any macro-molecule for that matter, were identified in tissue, and serum and developed into a potential biomarker disease. Further, we explore this N-glycan modification in a transgenic model and confirm its value in liver disease progression.

CONCLUSION:
In conclusion, we identify specific N-glycan alterations that start early in liver disease and continue to be altered in primary liver cancer. Here, we propose the use of N-glycan alterations as strategies for the identification of biomarkers.