Decentralized donor-derived cell-free DNA testing for cardiac allograft rejection: clinical validation and additive value beyond standard-of-care monitoring
G Coutance, C Delacroix, M F Scuppa, A Certain, A Giarraputo, J Bastian, A C Martin, P Rouvier, P Bruneval, P Leprince, E Marijon, X Jouven, P Achouh, S Varnous, A LoupyAbstract
Background
Donor-derived cell-free DNA (dd-cfDNA) has emerged as a validated non-invasive biomarker for cardiac allograft rejection. While centralized assays have demonstrated robust clinical utility, evidence for decentralized platforms remains limited. Decentralized testing could improve access to molecular rejection monitoring across transplant centers of varying sizes and resources.
Purpose
This study evaluated the clinical performance of a decentralized donor derived cell-free DNA (dd-cfDNA) assay and its additive value on top of standard-of-care (SOC) monitoring for cardiac allograft rejection.
Methods
This case-enriched prospective cohort included 320 heart transplant recipients from two high-volume transplant centers, enrolled from October 2021 to October 2023. All biopsy-proven acute rejection episodes (ACR ≥2R and/or AMR ≥pAMR1) were included alongside randomly-selected controls. From 1542 plasma samples collected at endomyocardial biopsy (EMB), dd-cfDNA was quantified using a decentralized next-generation sequencing assay targeting 202 single nucleotide polymorphisms in 416 samples from 187 patients. EMBs were evaluated per ISHLT criteria by pathologists blinded to dd-cfDNA results. SOC monitoring was assessed using a previously-validated clinical score which included five predictive variables: time post-transplant, pre-transplant sensitizing event, circulating anti-HLA DSAs with mean fluorescence intensity ≥3000, acute graft dysfunction, and history of rejection. Mixed-effects logistic regression models were used and validated using bootstrap resampling.
Results
Acute rejection occurred in 55 patients (12.8%; 5 ACR≥2R, 50 AMR≥pAMR1). Rejecting patients had markedly higher dd-cfDNA levels (median 1.75% vs 0.17%, p<0.001). dd-cfDNA levels correlated with microvascular inflammation severity (r=0.52, p<0.001) and increased progressively with MVI extension (Jonckheere-Terpstra z=7.75, p<0.001). NT-proBNP and troponin T showed no significant associations with rejection in univariable (NT-proBNP: OR=1.00, 95%CI 1.00-1.00, p=0.99; Troponin T: OR=1.00, 95%CI 0.997-1.001, p=0.33) or multivariable models. dd-cfDNA remained independently linked to rejection after adjusting for SOC predictors (OR 2.96, 95%CI 2.28-3.82, p<0.001). Adding dd-cfDNA to SOC monitoring improved discrimination (AUC 0.79→0.89, p=0.002), calibration (slope 1.31→1.50), and reclassification (continuous NRI 0.81, 95%CI 0.13-1.49, p=0.019; continuous IDI 0.18, 95%CI 0.09-0.27, p<0.001).
Conclusions
Decentralized dd-cfDNA testing significantly enhances risk stratification for cardiac allograft rejection beyond standard-of-care monitoring. Implementation of this assay enables risk-stratified surveillance protocols that reduce unnecessary invasive procedures while maintaining diagnostic safety. Successful decentralized implementation extends molecular rejection monitoring access across transplant centers of varying sizes and resources.