A two-threshold algorithm using donor-derived cell-free DNA fraction and quantity to detect acute rejection after heart transplantation

Donor-derived cell-free DNA (dd-cfDNA) is a promising biomarker of acute rejection (AR) after heart transplantation (HTx). dd-cfDNA, measured as a fraction of total cfDNA, can be affected by changes in total cfDNA whereas dd-cfDNA quantity can mitigate this impact. This study investigated the performance of a 2-threshold algorithm (2TA) that combines dd-cfDNA fraction (dd-cfDNA%) and donor-quantity score (DQS). A total of 808 plasma samples were prospectively collected for dd-cfDNA testing from 187 adult HTx patients with contemporaneous endomyocardial biopsies. cfDNA was analyzed by a single nucleotide polymorphism-based next-generation sequencing workflow; dd-cfDNA% and DQS were measured using the sequencing reads and single nucleotide polymorphism genotypes. Both dd-cfDNA% and DQS were significantly higher in AR than in non-AR samples (P < 10^-14). Considering samples exceeding either dd-cfDNA% = 0.26% or DQS = 18 copies/mL as positive, the 2TA demonstrated 86.5% sensitivity and 83.6% specificity for AR detection and an area under the curve of 0.881. Compared to dd-cfdNA% alone, performance improved with a mean net reclassification index of 16.4% (standard deviation: 4.0%; P = .015) and a 37.3% reduction in the number of the false positive cases compared to the previously established cutoff of 0.15%. Combining dd-cfDNA fraction and quantity estimate in a 2TA may improve AR detection accuracy in HTx recipients compared with dd-cfDNA% alone.