Background: The detection of donor-specific antibodies (DSA) has limited value as a diagnostic marker of late antibody-mediated rejection (ABMR), and the presence of circulating DSA does not necessarily indicate an ongoing rejection process. Here, we investigated whether and to which extent analysis of donor-derived cell-free DNA (dd-cfDNA) helps improve non-invasive monitoring of silent ABMR.
Methods: The study cohort consisted of 45 HLA class I and/or II DSA-positive kidney allograft recipients identified upon systematic cross-sectional alloantibody/ABMR screening (protocol biopsies in case of a positive DSA result). Twenty-five of these patients were diagnosed with ABMR (Banff 2017), and 20 recipients had no rejection. Cell-free DNA was extracted from 0.5 mL biobanked EDTA-plasma using Qiagen’s QIAamp Circulating Nucleic Acid Kit, followed by a double AMPure clean-up step to remove contaminating cell-derived DNA. Cell-free DNA was then analyzed by CareDx AlloSeq cfDNA assay sequencing.
Results: Overall, DSA-positive study subjects showed a significantly higher median percentage of dd-cfDNA than a control group of DSA-negative transplant recipients [0.78 (IQR: 0.52-1.95) versus 0.33 (0.27-1.12), P=0.002]. DSA-positive patients diagnosed with ABMR had by far higher levels than those without rejection [%dd-cfDNA: 1.90 (0.78-3.90) versus 0.52 (0.35-0.72); P<0.001] (Figure 1A). As shown in Figure 1B, receiver operating characteristic (ROC) curve analysis for ABMR prediction revealed an area under the curve (AUC; 0.89), which exceeded AUC levels computed for the mean fluorescence intensity of immunodominant DSA (0.77). In ROC analysis, dd-cfDNA showed several points of equal highest diagnostic accuracy (0.80) including one (threshold: 1.45%) with 100% specificity and 64% sensitivity.
Conclusions: Our results support the use of dd-cfDNA monitoring, as an adjunct to DSA analysis, as a reliable non-invasive tool to uncover silent ABMR.