Donor-derived cell-free DNA and miRNA monitoring for the early prediction and diagnosis of liver allograft rejection and patient outcomes

Judit Julian¹Olga Millán^1,2Esther Titos³Pablo Ruiz⁴Yiliam Fundora Suárez⁵Alba Díaz⁶Jordi Colmenero^2,4Constantino Fondevila^2,7Joan Anton Puig-Butillé³Mercè Brunet^1,2*

• ¹Pharmacology and Toxicology, Biochemistry and Molecular Genetics, Biomedical Diagnostic Center (CDB), Hospital Clinic of Barcelona, Barcelona, Spain
• ²Biomedical Research Center in Hepatic and Digestive Diseases (CIBEREHD), Instituto de Salud Carlos III (ISCII), Madrid, Spain, Madrid, Spain
• ³Molecular Biology CORE Laboratory, Biomedical Diagnostic Centre (CDB), Hospital Clínic, Instituto de Investigaciones Biomédicas August Pi i Sunyer (IDIBAPS), University of Barcelona, Barcelona, Spain, Barcelona, Spain
• ⁴Liver Unit, Hospital Clinic of Barcelona, Instituto de Investigaciones Biomédicas August Pi I Sunyer (IDIBAPS), University of Barcelona, Barcelona, Spain,, Barcelona, Spain
• ⁵Department of General and Digestive Surgery, Hospital Clínic Barcelona, Instituto de InvestigacionesBiomédicas August Pi i Sunyer (IDIBAPS), University of Barcelona, Barcelona, Spain, Barcelona, Spain
• ⁶Pathological Department, Biomedical Diagnostic Centre (CDB), Hospital Clínic, Instituto de Investigaciones Biomédicas August Pi i Sunyer (IDIBAPS), University of Barcelona, Barcelona, Spain, Barcelona, Spain
• ⁷General & Digestive Surgery Service, Hospital Universitario La Paz, IdiPAZ, CIBERehd, Madrid, Spain, Madrid, Spain

The identification of noninvasive biomarkers for monitoring liver transplant (LT) recipients is crucial for the early detection of graft dysfunction and rejection. Donor-derived cell-free DNA (dd-cfDNA) and microRNAs (miRNAs) have been identified as promising biomarkers for assessing graft integrity. While the levels of dd-cfDNA have been validated for this use in kidney and heart transplantation, there are limited data regarding its potential in liver graft monitoring. Similarly, the expression levels of miRNAs, key regulators of immune responses and liver injury, have potential utility in distinguishing between rejection and other causes of graft dysfunction.In this prospective, observational study, we monitored the levels of dd-cfDNA and miRNAs by analysing 437 plasma samples from 64 LT recipients over a 12-month period, measuring the levels of dd-cfDNA and signature miRNAs at predefined time points and during episodes of graft dysfunction. The diagnostic performance of the levels of dd-cfDNA and signature miRNAs was assessed through receiver operating characteristic (ROC) curve analysis and logistic regression models.Results: dd-cfDNA levels were significantly elevated during acute rejection (AR) episodes, with a median 3.9-fold increase over those in stable patients. A diagnostic cut-off value of 9.88% yielded an area under the ROC curve (AUROC) of 0.812, a sensitivity of 100%, a specificity of 66.7%, a positive predictive value (PPV) of 17.5% and a negative predictive value (NPV) of 100%. Interestingly, patients with cholestasis also exhibited increased dd-cfDNA levels (3.0-fold vs. stable patients), suggesting that it could serve as a potential confounder in the diagnosis of transplant rejection. Plasma miRNA analysis demonstrated significant upregulation of the expression levels of miR-155-5p, miR-122-5p, and miR-181a-5p during rejection episodes, and the incorporation of these factors improved diagnostic accuracy when combined with the level of dd-cfDNA.Conclusions: dd-cfDNA and miRNA profiling represent promising noninvasive biomarkers for diagnosing liver graft rejection and dysfunction. The combined use of these biomarkers may result in increased diagnostic accuracy, reduce unnecessary biopsies, and allow personalized immunosuppressive management. Further studies in larger cohorts are needed to validate the clinical applicability of these compounds as diagnostic biomarkers.