PRO-C3, a Serological Marker of Fibrosis, During Childhood and Correlations With Fibrosis in Pediatric NAFLD

Abstract

Nonalcoholic fatty liver disease (NAFLD) is a common chronic liver disease in children and may lead to cirrhosis requiring liver transplant. Thus, prompt diagnosis of advanced fibrosis is essential. Our objectives were to examine PRO-C3 (a neo-epitope pro-peptide of type III collagen formation) levels across childhood/adolescence and associations with advanced fibrosis in pediatric NAFLD. This cross-sectional study included 88 children and adolescents with biopsy-proven NAFLD (mean age: 13.9 ± 2.9 years, 71% male) and 65 healthy participants (11.8 ± 4.5 years, 38% male). PRO-C3, and the bone remodeling biomarkers C-terminal telopeptide of type I collagen (CTX-I; bone resorption) and osteocalcin (N-MID; bone formation), were measured in serum by enzyme-linked immunosorbent assay. Fibrosis was assessed by liver biopsy in participants with NAFLD, who were categorized as having advanced (Ishak score ≥ 3) or none/mild fibrosis (Ishak score ≤ 2). Overall, PRO-C3 was similar in participants with NAFLD (median [interquartile range]: 20.6 [15.8, 25.9] ng/mL) versus healthy participants (19.0 [13.8, 26.0] ng/mL), but was significantly lower in older adolescents ≥ 15 years old (16.4 [13.0, 21.2] ng/mL) compared with children ≤ 10 years old (22.9 [18.1, 28.4] ng/mL; P < 0.001) or 11-14 years old (22.4 [18.3, 31.2] ng/mL; P < 0.001). PRO-C3 was also directly correlated with levels of CTX-I and N-MID (r = 0.64 and r = 0.62, respectively; both P < 0.001). Among participants with NAFLD, PRO-C3 was higher in those with advanced fibrosis (median [IQR]: 28.5 [21.6, 37.6]) compared with none/mild fibrosis (20.3 [18.2, 22.8]; P = 0.020) in models adjusted for age, sex, and body mass index z-score. However, associations were attenuated after additionally adjusting for bone-remodeling CTX-I (P = 0.09) or N-MID (P = 0.08). Conclusion: Collectively, these findings show that PRO-C3 levels are higher in children with advanced fibrosis in NAFLD, but are also influenced by age and pubertal growth spurt, assessed by bone remodeling biomarkers, and therefore may not be a reliable biomarker for liver fibrosis in pediatric NAFLD until late adolescence.

FIG. 1

Trends in PRO‐C3 levels according to participant age among participants with NAFLD (n = 87) and healthy participants (n = 65). Within each plot, trends are stratified by sex, as indicated by circles (boys) and triangles (girls) and blue (boys) versus yellow (girls) trend lines. All trend lines were estimated by locally weighted smoothing regression (method = “loess”) in ggplot2. One participant with an outlier value for PRO‐C3 (61.6 ng/mL) was excluded from the plot.

FIG. 2

Scatter plots showing correlations between PRO‐C3 and the bone biomarkers CTX‐I (A) and N‐MID (B) among participants with NAFLD (n = 88) and healthy participants (n = 65). Correlation coefficients and P values were calculated by Spearman correlation. All trend lines were estimated by linear regression (method = “lm”) in ggplot2.

FIG. 3

PRO‐C3 levels according to fibrosis stage based on Ishak scores in children and adolescents with NAFLD and healthy controls. Participants were categorized with advanced fibrosis based on fibrosis scores ≥ 3 (n = 8) or none/mild fibrosis based on scores ≤ 2 (n = 80). P values were calculated for pairwise comparisons using the nonparametric Dunn’s test.

FIG. 4

PRO‐C3 levels in children and adolescents with NAFLD and healthy controls according to other histological variables: steatosis (A), lobular inflammation (B), portal inflammation (C), and ballooning (D). P values were calculated by Kruskal‐Wallis one‐way analysis of covariance tests. Medians and IQRs for PRO‐C3 according to grade of each histological variable are provided in Supporting Table S3.