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 investigates the performance of a two-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 (SNP)-based next-generation sequencing workflow; dd-cfDNA% and DQS were measured using the sequencing reads and SNP genotypes. Both dd-cfDNA% and DQS were significantly higher in AR compared to non-AR samples (p<10 -14 ). Considering samples exceeding either dd-cfDNA%=0.26% or DQS=18 cp/mL as positive, the 2TA demonstrated 86.5% sensitivity and 83.6% specificity for AR detection and an AUC of 0.881. Compared to dd-cfdNA% alone, performance improved with a mean net reclassification index (NRI) of 16.4% (SD: 4.0%; p=0.015) and a 37.3% reduction of the false positive rate 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 to dd-cfDNA% alone.