Serum proteomic profiling in patients with drug-induced liver injury

Abstract

Background: Idiosyncratic drug-induced liver injury (DILI) is a complex disorder that is difficult to predict, diagnose and treat.

Aim: To describe the global serum proteome of patients with DILI and controls.

Methods: A label-free, mass spectrometry-based quantitative proteomic approach was used to explore protein expression in serum samples from 74 DILI patients (collected within 14 days of DILI onset) and 40 controls. A longitudinal analysis was conducted in a subset of 21 DILI patients with available 6-month follow-up serum samples.

Results: Comparison of DILI patients based on pattern, severity and causality assessment of liver injury revealed many differentially expressed priority 1 proteins among groups. Expression of fumarylacetoacetase was correlated with alanine aminotransferase (ALT; r = 0.237; P = 0.047), aspartate aminotransferase (AST; r = 0.389; P = 0.001) and alkaline phosphatase (r = -0.240; P = 0.043), and this was the only protein with significant differential expression when comparing patients with hepatocellular vs. cholestatic or mixed injury. In the longitudinal analysis, expression of 53 priority 1 proteins changed significantly from onset of DILI to 6-month follow-up, and nearly all proteins returned to expression levels comparable to control subjects. Ninety-two serum priority 1 proteins with significant differential expression were identified when comparing the DILI and control groups. Pattern analysis revealed proteins that are components of inflammation, immune system activation and several hepatotoxicity-specific pathways. Apolipoprotein E expression had the greatest power to differentiate DILI patients from controls (89% correct classification; AUROC = 0.97).

Conclusion: This proteomic analysis identified differentially expressed proteins that are components of pathways previously implicated in the pathogenesis of idiosyncratic drug-induced liver injury.

Figure 1

Pathway analysis of priority 1 proteins with significant differential expression when comparing DILI patients with hepatocellular and cholestatic patterns of liver injury. Classification of the 25 priority 1 proteins with significant (q < 0.05) differential expression when comparing patients with hepatocellular and cholestatic DILI demonstrated involvement in inflammatory and immune system response pathways, activation of the coagulation cascade and included proteins previously implicated in liver proliferation and development of cirrhosis.

Figure 2

Pathway analysis of priority 1 proteins with significant differential expression when comparing baseline and 6-month follow-up samples from patients with DILI included in the longitudinal analysis. Classification of the 53 priority 1 proteins with significant (q < 0.05) differential expression when comparing patients with DILI at baseline (within 14 days of DILI onset) and after a 6-month follow-up period revealed proteins with functions related to inflammation and oxidative stress, including those involved in liver steatosis/steatohepatitis/hepatitis and hepatocyte necrosis/cell death. Expression of nearly all differentially expressed proteins returned to ‘normal’ expression levels throughout the 6-month follow-up period (using protein expression in the control group as a reference standard).

Figure 3

Pathway analysis of priority 1 proteins differentially expressed between DILI patients and controls and identification of proteins with the greatest ability to differentiate patients with DILI vs. controls. Ingenuity Pathway Analysis software was used to classify the 92 priority 1 proteins with significant (q < 0.05) differential expression when comparing DILI patients and controls (Panel A), and pattern analysis showed involvement in many pathways previously implicated in the pathogenesis of DILI, including several directly related to hepatotoxicity (liver inflammation, steatosis, cholestasis, necrosis/cell death, and hepatitis). Diagnostic power of priority 1 proteins and clinical characteristics was explored by linear discriminant analysis and assessment of AUROC (Panel B; grey bars). Apolipoprotein E was identified as having the greatest power to differentiate DILI patients and controls (89% of patients classified correctly; AUROC = 0.97), and consideration of expression of several additional proteins and age increased the percentage of patients classified correctly to 96% and the AUROC to 0.99. The diagnostic utility of ALT alone (black bar) in differentiating patients from controls is also shown (73% correct classification; AUROC = 0.99).