Elevated core-fucosylated IgG is a new marker for hepatitis B virus-related hepatocellular carcinoma

Abstract

Immunoglubulin G (IgG) and its abnormal glycosylations are associated with carcinogenesis. The present study investigates the relationship between cancer-derived IgG and clinicopathological characteristics in hepatocellular carcinoma (HCC) and assesses the value of serum N-glycosylated IgG in diagnosing and monitoring hepatitis B virus (HBV)-related HCC. Tissue microarray analysis of 90 HCC tissues showed that HCC patients with IgG immunopositivity had higher levels of core-fucosylated α fetoprotein (AFP-L3), larger tumors, and a higher incidence of portal vein tumor thrombus. HCC-derived IgG stimulated the growth of liver cancer cells in vitro. HCC patients presented a significantly increased fraction of Lens culinaris agglutinin binding IgG (core-fucosylated IgG, IgG-L3) among total serum IgG. The clinical diagnostic performance of serum IgG-L3% was evaluated in 3 case-control studies (1 training set and 2 validation cohorts), including 293 patients with HCC, 131 with liver cirrhosis, 132 HBV carriers, and 151 healthy controls. IgG-L3% had better general diagnostic performance than AFP in the training set and validation cohort 1 (accuracy: 81.33-85.11% versus 63.33-78.61%). In validation cohort 2, where we aimed to assess the efficiency of IgG-L3% in patients with AFP-negative HCC, the diagnostic accuracy of IgG-L3% was 72.54-73.60%. Finally, a longitudinal evaluation based on 31 HCC patients demonstrated that IgG-L3% decreased in 24 patients after curative surgery. The remaining 7 patients showed elevated IgG-L3% and post-operative recurrence. HCC patients with higher IgG-L3% had poor survival during a 3-year follow up. We conclude that HCC-derived IgG is correlated with progressive behavior of HCC. Therefore, elevated core-fucosylated IgG is a new diagnostic and prognostic marker in HBV-related HCC.

Keywords: IgG; Lens culinaris agglutinin (LCA); diagnosis; hepatocellular carcinoma (HCC); non-invasive biomarker; tissue array.

Figures

Figure 1.

IgG expression is associated with serum AFP levels, tumor size, and incidence of portal vein tumor thrombus in patients with HCC. (A) Immunohistochemical staining for IgG was performed in tissue array of HBV-related HCC including 90 paired samples of HCC carcinoma and adjacent noncancerous liver tissue; the right panel shows representative tissues with positive IgG (a) and negative IgG (b) staining. (B, C) Serum AFP levels (B) and serum AFP-L3% (C) were measured in HCC patients included in the tissue array with different levels of IgG. (D) The association between IgG immunopositivity and tumor size/tumor capsule/PVTT was analyzed. *P < 0.05

Figure 2.

HCC cells secrete IgG. IgG in supernatants of 3 hepatoma cell lines, MHCC97-H, CSQT-2 and SMMC-7721, was measured as described in Patients and Methods.

Figure 3.

IgG from HCC sera stimulates proliferation of HCC cells in vitro. Eight HCC cell lines were treated with IgG purified from sera of HCC patients (HCC-IgG) or healthy individuals (Healthy-IgG) by protein G/A spin columns. Cell proliferation was evaluated over 5 days by CCK8 assay. *P < 0.05

Figure 4.

IgG-L3% is increased in HCC. HCC tissues were co-stained for IgG and LCA. (A) A representative liver tissue sample. (B, C) Serum IgG-L3 was measured with IgG LCA-blotting (B) and scattering immunoassay after LCA chromatography (C) as described in Patients and Methods. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5.

Recruitment and exclusion process for the clinical study population.

Figure 6.

IgG-L3% has better HCC diagnostic efficacies than AFP. The area under the curve (AUC) shows diagnostic efficacies of IgG-L3% and AFP (A): HCC versus non-HCC; (B): HCC versus LC) in the training set (left panel) and validation 1 cohort (right panel). (C) Sensitivity, specificity, and accuracy of the 2 parameters. AFP, α-fetoprotein; HCC, hepatocellular carcinoma; LC, liver cirrhosis.

Figure 7.

IgG-L3% shows good diagnostic efficacy in identifying HCC with serum AFP ≤ 20 ng/ml. (A) IgG-L3% was determined in different groups of patients in validation 2 cohort: healthy volunteers, HBV carriers, LC, and HCC with AFP ≤ 20 ng/mL. (B-C) AUC shows the diagnostic efficacy of IgG-L3% in discriminating HCC from non-HCC (B) and LC (C). Detailed sensitivity, specificity and accuracy of the 2 parameters are presented in (D). ***P < 0.001. AFP, α-fetoprotein; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; LC, liver cirrhosis

Figure 8.

HCC patients with a high serum level of IgG-L3% show poor prognosis. (A) IgG-L3% was measured in 31 HCC patients before and 6 months after radical surgery. (B) Kaplan-Meier analysis of overall survival time in 55 HCC patients with 3-year follow-up.