Analytical and Clinical Validation of a Serum microRNA RT-qPCR Assay for Detection of Acute Cellular Rejection in...

Background: Acute cellular rejection (ACR) remains a significant cause of graft dysfunction after liver transplantation and requires timely detection. Liver biopsy, the diagnostic gold standard for ACR, is invasive and unsuitable for frequent monitoring, while liver enzyme tests lack specificity for detecting ACR. Circulating microRNAs (miRNAs), including miR-122 and miR-885, have been previously identified as predictive biomarkers of ACR in liver transplant recipients. HepatoTrack™ is a serum-based miRNA RT-qPCR assay designed for noninvasive assessment of ACR using these biomarkers. This study evaluated the analytical performance and clinical validity of HepatoTrack™ for diagnosing ACR in liver transplant recipients. Methods: HepatoTrack™ uses 100 μL of serum and a one-step RT-qPCR workflow. It quantifies miR-122 and miR-885 normalized to miR-23a, with synthetic cel-miR-39 included as an exogenous control. Analytical validation assessed performance characteristics. Clinical performance was evaluated in a cohort of liver transplant recipients undergoing for-cause liver biopsy and used for model development and validation. Results: Analytical validation demonstrated robust assay performance. The HepatoTrack™ Prediction Score (HPS) algorithm was trained using 47 subjects from the training cohort based on a linear regression model incorporating longitudinal miRNA changes. In the independent testing cohort (n = 37), HPS achieved a sensitivity of 92.9%, specificity of 73.9%, positive predictive value (PPV) of 68.4%, and negative predictive value (NPV) of 94.4% for biopsy-confirmed ACR. HPS achieved an area under the curve (AUC) of 0.831 compared with 0.731 for ALT and 0.660 for AST. Conclusions: HepatoTrack™ support the analytical and clinical validity for detection of biopsy-confirmed acute cellular rejection in liver transplant recipients. The assay provides a noninvasive molecular test to aid in the diagnosis of acute cellular rejection and may complement existing post-transplant diagnostic evaluation.