Clinical validation of miRNA biomarkers to predict risk of primary non-function of fatty allografts following liver transplantation

Abstract

Abstract Background & Aims: Primary non-function (PNF) of the liver defines an irreversible graft failure without reasonable surgical or immunological causes. PNFs are unpredictable and constitute a life-threating condition that requires high-urgency re-transplantation. As part of a retrospective cohort study, we found PNF to be frequently associated with fatty allografts, and was associated with high mortality after re-transplantation. Based on findings from a fatty allograft PNF animal disease model, we assessed the clinical relevance of miRNA biomarker candidates for their usefulness to predict PNF in human orthotopic liver transplantation (OLT). Furthermore, we investigated their regulation following ischemia-reperfusion injury. Methods We recently performed genome wide scans to identify highly regulated miRNAs in a fatty allograft PNF animal disease model and selected 15 miRNAs for their marked tissue regulation (Kulik et al., 2024). We performed histopathology and assayed PNF-associated miRNAs by RT-qPCR in RNA extracts of FFPE tissue blocks of well documented clinical cases. Additionally, we investigated their regulation in pre- and intraoperative liver biopsies and blood samples from patients undergoing elective hepatobiliary surgery to define their function in reperfusion injury. Results We established clinical significance for 9 repressed and 2 induced miRNAs in PNF tissue extracts and show the grade of hepatic steatosis to significantly influence expression of miRNA-27b-3p, miRNA-122-3p, miRNA-125a-5p, miRNA-125b-5p and miRNA-192-5p. Following reperfusion injury, we discovered let-7b-5p, miRNA-122-5p, miRNA-125b-5p and miRNA-194-5p as significantly upregulated when pre- and intraoperative liver biopsies were compared. Strikingly, PNF-associated miRNAs are oppositely regulated in plasma samples of successful OLTs and liver resection cases post-surgery. Therefore, we demonstrate selectivity of the miRNA biomarker candidates. Moreover, and based on 21 independent studies, we confirmed the regulation of 8 bloodborne PNF-associated miRNAs in human acute liver failure cases. We therefore establish clinical relevance and predictive power across independent studies. Conclusions We identified miRNAs highly predictive of PNF and show their opposite regulation in liver biopsies and blood samples of successfully performed OLTs. Our findings will improve early identification of allografts at risk for PNF, its management and therefore warrant independent validation in clinical trials.