Longitudinal biomarker progression and validation for predicting operational tolerance in a prospective multicenter liver transplantation immunosuppression withdrawal trial

Liver transplantation (LT) is a life-saving treatment for end-stage liver disease, but long-term immunosuppression (IS) is associated with significant side effects. Achieving operational tolerance (OT), where the graft is accepted without IS, remains a critical goal. Biomarkers play a pivotal role in understanding the complex mechanisms of OT, enabling personalized treatment strategies and improving patient outcomes. Additionally, machine learning techniques offer powerful tools for identifying predictive biomarkers and optimizing IS withdrawal protocols. This multicenter trial aimed to investigate the longitudinal evolution of genetic biomarkers during IS withdrawal and validate their predictive value for OT in LT recipients. A prospective, multicenter IS withdrawal trial was conducted with 91 LT patients. Tolerant (TOL) and non-tolerant (non-TOL) patients were compared, and longitudinal blood and liver samples were collected to analyze biomarkers. Generalized Additive Mixed Models (GAMMs) and logistic algorithms were employed to assess biomarker associations and predict OT. Of the 45 patients who completed the trial, 17 (37.8%) achieved OT. Molecular biomarker analysis revealed significant differences between TOL and non-TOL groups. Non-TOL patients exhibited higher baseline methylation of the FOXP3 regulatory T cell-specific demethylated region (TSDR) in whole blood. Longitudinal analysis showed distinct patterns in FOXP3, SENP6, miR31, and miR95 expression between groups. Notably, FOXP3 expression followed a U-shaped trajectory in TOL patients, decreasing during IS withdrawal and increasing post-withdrawal. Machine learning identified several key predictive biomarkers for OT. This study confirms the association between FOXP3 TSDR methylation and OT in LT patients and identifies FEM1C, miR31 and TFRC as promising predictive biomarkers. These findings highlight the potential for personalized IS withdrawal strategies, though further validation in larger cohorts is needed before clinical application.