Glycopeptides with Sialyl Lewis Antigen in Serum Haptoglobin as Candidate Biomarkers for Nonalcoholic Steatohepatitis Hepatocellular Carcinoma Using a Higher-Energy Collision-Induced Dissociation Parallel Reaction Monitoring-Mass Spectrometry Method

Abstract

Nonalcoholic steatohepatitis (NASH) is the fastest growing cause of hepatocellular carcinoma (HCC) in the United States. Changes in N -glycosylation on specific glycosites of serum proteins have been investigated as potential markers for the early detection of NASH-related HCC. Herein, we report a glycopeptide with a Sialyl Lewis structure derived from serum haptoglobin (Hp) as a potential marker for NASH related HCCs among 95 patients with NASH, including 46 cirrhosis, 32 early-stage HCC, and 17 late-stage HCC. Hp immuno-isolated from patient serum was analyzed using LC-HCD-PRM-MS/MS followed by data analysis via Skyline software. Two glycopeptides involving site N184 and four glycopeptides involving site N241 were significantly changed in patients with HCC vs NASH cirrhosis ( P < 0.05). The two-marker panel using N -glycopeptide N241_A4G4F2S4 showed the best performance for HCC detection when combined with α-fetoprotein (AFP), with an improved estimated area under the curve (AUC) = 0.898 (95% CI: 0.835, 0.951), compared to the AUC of 0.790(95% CI, 0.697 0.872) using AFP alone ( P = 0.048). At 90% specificity, the combination of N241_A4G4F2S4 + AFP had an improved sensitivity of 63.3%, compared to the sensitivity of 52.3% using AFP alone. When using three markers, the panel of AFP + N241_A2G2F1S2 + N241_A4G4F2S4 yielded an estimated AUC of 0.928 (95% CI: 0.877, 0.970). Our findings indicated that N241_A4G4F2S4 may play an important role in distinguishing HCC from NASH cirrhosis.

Systematic review: Glycomics as diagnostic markers for hepatocellular carcinoma

“…Additionally, the fucosylated Hp/Hp ratio (concentration of fucosylated Hp/concentration of total Hp) and the ELISA index (OD value of fucosylated Hp/OD value of total Hp) were significantly increased in HCC patients compared to (healthy) controls 36,37 . Whereas seven of these studies examined the aberrations in N‐glycosylation status of the Hp protein, 33,34,36,38–41 five studies focused on site‐specific N‐glycosylation patterns of the Hp‐β subunit 35,37,42–44 …”

“…As such, a 2‐marker panel (AFP + N241_A4G4F2S4) was developed to discriminate HCC from cirrhosis with an AUC of 0.90. In addition to AFP, this panel represented the bifucosylated tetra‐antennary glycopeptide at site N241 of Hp 42 . Notably, the bifucosylated tetra‐antennary glycan, with both core‐ and antennary‐ fucosylation, was increased in HCC compared to cirrhosis of all aetiologies (HBV‐, HCV‐, and alcohol‐related) 38,39 .…”

“…36,37 Whereas seven of these studies examined the aberrations in N-glycosylation status of the Hp protein, 33,34,36,[38][39][40][41] five studies focused on site-specific N-glycosylation patterns of the Hpβ subunit. 35,37,[42][43][44] According to a recent study, six fucosylated glycopeptides on serum Hp were able to differentiate between cirrhosis and HCC in patients with non-alcoholic steatohepatitis (NASH). As such, a 2-marker panel (AFP + N241_A4G4F2S4) was developed to discriminate HCC from cirrhosis with an AUC of 0.90.…”

Proteome Multimarker Panel for the Early Detection of Hepatocellular Carcinoma: Multicenter Derivation, Validation, and Comparison

“…Further, it is unknown whether the MRM-MS panel is useful for patients with other risk factors for HCC, such as alcoholic liver cirrhosis, or nonalcoholic fatty liver disease. Because the main etiological cause of HCC is shifting from CHB or CHC to NASH or ALD due to vaccination and antivirals, further validation in the ALD and NASH populations is required. , Also, the effectiveness of the MRM-MS panel in patients of various ethnicities is unverifiedall of our participants were Korean. Finally, no comparison with abdominal ultrasonography was performed in this study.…”

The role of N-glycosylation modification in the pathogenesis of liver cancer

“…Hp is known to have four N-glycosylation sites with various types of sugar chains and mostly sialylation modifications [ 142 ]. It has been reported that the N-glycan type of Hp is altered in patients with liver cancer compared with normal adults [ 143 ].…”