Hepatic elastin content is predictive of adverse outcome in advanced fibrotic liver disease

Abstract

Aims: The aim of this study was to determine if elastin content in needle core native liver biopsies was predictive of clinical outcome in patients with chronic hepatitis C virus-related chronic liver disease.

Methods and results: Elastin contents in liver biopsies were determined by image analysis, technically validated in an independent centre, and correlated with outcome in patients with advanced (Ishak stage ≥5) chronic hepatitis C virus-related chronic liver disease. Elastin was robustly quantified in an operator-independent and laboratory-independent manner, with very strong correlation of elastin staining measured with two methods of image classification (r_s = 0.873, P < 0.00001). Elastin content (but not absolute scar content or Ishak stage) was predictive for future clinical outcomes. In a cohort of patients without sustained virological response, the median hepatic elastin content was 3.4%, and 17 patients (57%) progressed to a liver-related clinical outcome; 11 of the 15 patients (73%) with a hepatic elastin content of >3.4% progressed to a clinical outcome, as compared with only six of 15 (40%) with an elastin content of <3.4%. The difference in time to outcome was significant.

Conclusions: We describe a simple and reproducible method for elastin quantification in liver biopsies that provides potentially valuable prognostic information to inform clinical management.

Keywords: cirrhosis; elastin; hepatitis C virus; prognosis.

Figure 1

Quantification of elastin and fibrosis in biopsies of advanced (Ishak stage ≥5) hepatitis C virus infection. Biopsies from patients with chronic hepatitis C virus infections and advanced stage (Ishak stage ≥5) were stained with an antibody against elastin or with picrosirius red (PSR); representative whole‐slide images of a single case are shown (A and B, respectively; main scale bars 5 mm, inset 100 μm). Elastin and PSR contents were quantified by image analysis; there was no significant difference in elastin content between Ishak stages 5 and 6 (C, 2.51%, interquartile range (IQR) 2.07–4.36, and 3.61%, IQR 2.65–4.52, respectively; independent samples median test, P = 0.27] but there was a significant difference in PSR content between Ishak stages 5 and 6 (D, 18.80% ± 11.52% and 30.57% ± 11.24%, respectively, Welch two‐sample t‐test, P = 0.0012). There was a moderate positive correlation between elastin content and collagen content (E, r _s = 0.58, P = 0.000047, regression line from fitted linear model and 95% confidence intervals).

Figure 2

Independent validation of immunohistochemical elastin quantification and technical validation. Elastin contents in original whole‐slide images were independently quantified with an alternative machine learning‐based approach (A) to classify images into elastin‐immunopositive (lilac) and elastin‐immunonegative (green) tissue or blank (red) pixels. There was very strong rank correlation of elastin quantified with both methods (B, r _s = 0.87, P < 0.00001). Explant liver from a second liver centre from cases with a spectrum of primary aetiologies was independently stained with a different batch of the same primary anti‐elastin antibody by use of a non‐automated protocol to demonstrate centre‐agnostic and disease‐agnostic applicability (C, representative images); sections from the same blocks were also tinctorially stained to identify elastin (Elastic van Gieson) and demonstrate spatial equivalence in areas of fibrosis and internal elastic lamina of arteries as an internal positive control. Scale bars: 100 μm.

Figure 3

Elastin content functions as a tissue biomarker predictive of adverse liver‐related events. A, Patients whose liver biopsies had elastin content greater than the median (3.4%) according to primary classification more rapidly developed an adverse liver‐related event than those with biopsies whose elastin content was below this value; the difference in time to outcomes was significant (log rank: chi‐square 3.98; P = 0.046). B, weka quantification indicated that biopsies with elastin content greater than the optimum calculated cut‐off (6.2%) more rapidly developed an adverse liver‐related event than those with elastin content below this value; the difference in time to outcomes was significant (log rank: chi‐square 4.60; P = 0.0312).