M. Nguyen1,2, D. James1,2, N. Mavila1,2, S. Zhou1, L. Wang1, K. Wang1,2 1Children’s Hospital Los Angeles,Pediatric Surgery,Los Angeles, CA, USA 2Saban Research Institute,Developmental Biology And Regenerative Medicine,Los Angeles, CA, USA
Introduction:
Biliary atresia (BA) is a severe neonatal liver disease caused by progressive fibro-obliteration of the biliary tree. BA is the leading indication for liver transplantation in children. Pathologically, BA is marked with intrahepatic ductular reactions and variable degrees of biliary fibrosis. We recently demonstrated the expansion of a population of myofibroblastic cells which co-express the stem cell marker PROMININ-1 (PROM1) and COLLAGEN-1α1 within regions of developing biliary fibrosis in experimental Rhesus rotavirus-induced BA (Mavila et al, Hepatology, 2014). In this study, we hypothesized that serum PROM1 levels in BA patients is a biomarker for severity of disease or liver fibrosis.
Methods:
Liver and blood specimens were obtained with parental informed consent from patients undergoing liver biopsy at CHLA under a study protocol approved by the Institutional Review Board. Ishak fibrosis scores were determined by two pathologists who were blinded to patient diagnosis and PROM1 expression levels. Serum levels were measured using a PROM1 ELISA assay kit (MyBioSource, San Diego). Statistical analyses of PROM1 serum levels were performed using Student's t-test. Quantitative Real-Time PCR (qPCR) was performed using cDNA with Light-Cycler Taqman Master (Roche) and probes from the Universal Probe Library (Roche Applied Science) using intron spanning and gene specific primers. Relative expression levels were calculated by Δ–Δ Ct method.
Results:
Serum PROM1 levels correlated with liver tissue mRNA levels of PROM1 (R2 = 0.73). Serum PROM1 levels were significantly higher in patients with late-stage BA (n=4, p=0.02). BA patients who required liver transplantation expressed a greater than 2-fold increase in PROM1 serum levels compared to patients undergoing initial Kasai procedure (2.02±1.14 vs. 0.70±0.07 μg/ml, n=11, p=0.05). A similar trend in PROM1 levels was seen in liver samples, with a 14-fold increase in RNA expression in liver explants compared to native livers (n=3, p=0.05). Serum PROM1 levels also correlated with total bilirubin levels (linear regression analysis: R2 = 0.63). Ishak scores of ≥4 were associated with higher serum PROM1 levels than <4 (1.84 vs 0.69 μg/mL).
Conclusion:
Serum PROM1 levels correlate with the diagnosis of BA as well as the degree of biliary obstruction and fibrosis. Thus, serum PROM1 levels could be used potentially as a biomarker for BA-associated fibrosis.