Correlation between Donor-Derived Cell-free DNA and Tissue Gene Expression in Heart Transplant Patients Undergoing for-cause Endomyocardial Biopsies.

INTRODUCTION The introduction of donor-derived cell-free DNA (dd-cfDNA) and the Molecular Microscope (MMDx) is changing how we diagnose rejection following heart transplantation (HT). This study aims to assess the accuracy of dd-cfDNA in detecting rejection as identified by MMDx and histology, with a focus on determining an optimal dd-cfDNA threshold to improve diagnostic performance. METHODS Single-center prospective study of HT recipients undergoing for-cause biopsies with paired MMDx results and dd-cfDNA levels. We employed a Receiver Operator Curve (ROC) to evaluate the performance of dd-cfDNA levels to detect rejection assessed by both histology and MMDx. We also assessed the correlation between dd-cfDNA levels and MMDx rejection scores. A mixed-effects model was applied to account for repeated dependent samples when appropriate. RESULTS 247 for-cause biopsies were identified with a median of 21 months from HT and a median of 11 days between dd-cfDNA and biopsy. 56.7% of the samples had dd-cfDNA levels ≥0.20%. MMDx identified rejection in 27.1% of biopsies, compared to 7.7% identified by histology. Elevated dd-cfDNA levels were associated with a fourfold increase in rejection rates by MMDx, mainly driven by a fivefold increase in ABMR detection when compared to histology. Dd-cfDNA demonstrated superior performance in predicting rejection by MMDx (AUC of 0.77; optimal cut-off dd-cfDNA value 0.30%). When incorporating a mixed-effects model, the predictive performance improved further, (AUC of 0.89; optimal cut-off value 0.26%). In contrast, prediction based on histology resulted in a lower AUC of 0.64. The correlation between dd-cfDNA levels and MMDx rejection scores was moderate (r=0.51; p<0.001). CONCLUSIONS In a for-cause biopsy population, elevated dd-cfDNA levels were more predictive of rejection on MMDx than on histology, suggesting that molecular techniques may detect rejection at earlier stages than traditional histological methods. A dd-cfDNA cutoff of 0.26% provided the highest predictive accuracy for rejection by MMDx when applied within a mixed-effects model for repeated measures.