Transcriptomics Identify Thrombospondin-2 as a Biomarker for NASH and Advanced Liver Fibrosis

Abstract

Background and aims: NAFLD is the most common liver disease worldwide. NASH, the progressive form of NAFLD, and advanced fibrosis are associated with poor outcomes. We searched for their noninvasive biomarkers.

Approach and results: Global RNA sequencing of liver tissue from 98 patients with biopsy-proven NAFLD was performed. Unsupervised hierarchical clustering well distinguished NASH from nonalcoholic fatty liver (NAFL), and patients with NASH exhibited molecular abnormalities reflecting their pathological features. Transcriptomic analysis identified proteins up-regulated in NASH and/or advanced fibrosis (stage F3-F4), including matricellular glycoprotein thrombospondin-2 (TSP-2), encoded by the thrombospondin 2 (THBS2) gene. The intrahepatic THBS2 expression level showed the highest areas under the receiver operating characteristic curves (AUROCs) of 0.915 and 0.957 for diagnosing NASH and advanced fibrosis, respectively. THBS2 positively correlated with inflammation and ballooning according to NAFLD activity score, serum aspartate aminotransferase and hyaluronic acid (HA) levels, and NAFLD Fibrosis Score (NFS). THBS2 was associated with extracellular matrix and collagen biosynthesis, platelet activation, caspase-mediated cleavage of cytoskeletal proteins, and immune cell infiltration. Serum TSP-2 expression was measured in 213 patients with biopsy-proven NAFLD, was significantly higher in NASH than in NAFL, and increased parallel to fibrosis stage. The AUROCs for predicting NASH and advanced fibrosis were 0.776 and 0.856, respectively, which were comparable to Fibrosis-4 index, serum HA level, and NFS in advanced fibrosis diagnosis. Serum TSP-2 level and platelet count were independent predictors of NASH and advanced fibrosis. Serum TSP-2 levels could stratify patients with NAFLD according to the risk of hepatic complications, including liver cancer and decompensated cirrhotic events.

Conclusions: TSP-2 may be a useful biomarker for NASH and advanced fibrosis diagnosis in patients with NAFLD.

FIG. 1

Hepatic transcriptome profiling of NAFLD patient samples revealed that THBS2 was significantly up‐regulated in NASH. (A) Unsupervised clustering based on the RNA‐sequencing data of liver tissues from 98 patients with biopsy‐proven NAFLD and the distribution of clinicopathological features. (B) Relative intrahepatic mRNA levels of 29 significantly up‐regulated genes in patients with NASH (n = 47) compared to patients with NAFL patients (n = 51). (C) mRNA fold change (NASH vs. NAFL) and P value of 29 significantly up‐regulated genes in patients with NASH compared to patients with NAFL. (D) Receiver operating characteristic curve indicating the performance of intrahepatic THBS2 mRNA levels in the diagnosis of NASH among patients with NAFLD. The AUROC is shown.

FIG. 2

Hepatic transcriptomic profiling of NAFLD patient samples identified THBS2 as strongly associated with liver fibrosis. (A‐H) Relative intrahepatic mRNA levels of eight genes based on fibrosis stage among 98 patients with biopsy‐proven NAFLD (*P < 0.05): THBS2 (A), LUM (B), LAMC3 (C), LAMA2 (D), AKR1B10 (E), COL4A4 (F), A2M (G), and C7 (H).

FIG. 3

Hepatic THBS2 levels were associated with NAFLD activity and fibrosis. (A‐C) Correlations between the intrahepatic THBS2 mRNA levels and serum HA levels (A), NFS (B), and serum AST levels (C) of 98 patients with biopsy‐proven NAFLD. (D) Relative intrahepatic THBS2 mRNA levels based on the NAS. (*P < 0.01). (E) Relative intrahepatic THBS2 mRNA levels based on the steatosis (left), inflammation (middle,) and ballooning (right) scores of the NAS (*P < 0.01). (F) Correlation of intrahepatic THBS2 mRNA levels with clinicopathological features, intrahepatic immune cell infiltration, and molecular pathways. (G) Correlation between intrahepatic THBS2 and COL1A1 (left) or COL1A2 (right) mRNA levels. Abbreviation: HS‐GAG, heparan sulfate glycosaminoglycan.

FIG. 4

Serum TSP‐2 levels discriminated NASH from NAFL among patients with NAFLD patients. (A) Relative serum TSP‐2 levels in 121 patients with NAFL and 92 patients with NASH (*P < 0.01). (B) Receiver operating characteristic curve of the performance of serum TSP‐2 levels in the diagnosis of NASH among 213 patients with biopsy‐proven NAFLD. The AUROC is shown. (C) Relative serum TSP‐2 levels based on the NAS (*P < 0.01). (D) Relative serum TSP‐2 levels based on the steatosis (left), inflammation (middle), and ballooning (right) scores of the NAS (*P < 0.01).

FIG. 5

Serum TSP‐2 levels identified advanced fibrosis patients among patients with NAFLD. (A) Relative serum TSP‐2 levels based on fibrosis staging in 213 patients with biopsy‐proven NAFLD (*P < 0.01). (B) Receiver operating characteristic curve of the performance of serum TSP‐2 levels, FIB‐4 index, HA, and NFS in the diagnosis of advanced liver fibrosis (F3‐F4) among patients with NAFLD. The AUROC is shown. (C‐E) Correlation of serum TSP‐2 levels with serum AST levels (C), serum HA levels (D), and NFS (E). (F,G) Kaplan‐Meier curves of the cumulative incidence rate of liver cancer (F) in 164 patients with NAFLD and decompensated cirrhotic events, including esophageal varices, ascites, and hepatic encephalopathy (G) in 163 patients with NAFLD stratified by serum TSP‐2 levels. Number of patients at risk is shown at each observation period.