Background: After heart transplantation, Endomyocardial biopsy (EMBx) is used to monitor for acute rejection (AR). Unfortunately, EMBx is invasive and its conventional histologic interpretation has limitations. This is a validation study to assesses the performance of a sensitive blood biomarker— percent donor-derived cell-free DNA (%ddcfDNA) — for detection of AR in cardiac transplant recipients. Methods: This multicenter, prospective cohort study recruited heart transplant subjects and collected plasma samples contemporaneously with EMBx for %ddcfDNA measurement by shotgun sequencing. Histopathology data was collected to define AR, its two phenotypes (acute cellular rejection, ACR, and antibody-mediated rejection, AMR) and controls without rejection. The primary analysis was to compare %ddcfDNA levels (median and interquartile range - IQR) for AR, AMR and ACR to controls and to determine %ddcfDNA test characteristics using receiver-operator characteristics analysis. Results: The study included 171 subjects with median post-transplant follow-up of 17.7 months (IQR: 12.1-23.6), with 1,392 EMBx, and 1,834 ddcfDNA measures available for analysis. Median %ddcfDNA levels decayed after surgery to 0.13% (0.03-0.21) by 28 days. %ddcfDNA increased again with AR compared to controls values (0.38, IQR=0.31-0.83, vs. 0.03, IQR=0.01-0.14 p<0.001). The rise was detected 0.5 and 3.2 months before histopathological diagnosis of ACR and AMR. The area-under-the- receiver-operator characteristics curve (AUROC) for AR was 0.92. A 0.25 %ddcfDNA threshold had a negative predictive value (NPV) for AR of 99% and would have safely eliminated 81% of EMBx. %ddcfDNA showed distinctive characteristics comparing AMR to ACR, included 5-fold higher levels (pAMR ≥2 1.68, IQR=0.49-2.79 vs. ACR grade ≥2R 0.34, IQR=0.28-0.72), higher AUROC (0.95 vs. 0.85), higher guanosine-cytosine content, and higher percentage of short ddcfDNA fragments. Conclusions: %ddcfDNA detected AR with a high AUROC and NPV. Monitoring with ddcfDNA, demonstrated excellent performance characteristics for both ACR and AMR and led to earlier detection than the EMBx-based monitoring. This study supports the use of %ddcfDNA to monitor for AR in heart transplant patients and paves the way for a clinical utility study. Clinical Trial Registration: URL: http://clinicaltrials.gov Unique Identifier: NCT02423070

BackgroundAllograft failure is common in lung-transplant recipients and leads to poor outcomes including early death. No reliable clinical tools exist to identify patients at high risk for allograft failure. This study tested the use of donor-derived cell-free DNA (%ddcfDNA) as a sensitive marker of early graft injury to predict impending allograft failure.MethodsThis multicenter, prospective cohort study enrolled 106 subjects who underwent lung transplantation and monitored them after transplantation for the development of allograft failure (defined as severe chronic lung allograft dysfunction [CLAD], retransplantation, and/or death from respiratory failure). Plasma samples were collected serially in the first three months following transplantation and assayed for %ddcfDNA by shotgun sequencing. We computed the average levels of ddcfDNA over three months for each patient (avddDNA) and determined its relationship to allograft failure using Cox-regression analysis.FindingsavddDNA was highly variable among subjects: median values were 3·6%, 1·6% and 0·7% for the upper, middle, and low tertiles, respectively (range 0·1%–9·9%). Compared to subjects in the low and middle tertiles, those with avddDNA in the upper tertile had a 6·6-fold higher risk of developing allograft failure (95% confidence interval 1·6–19·9, p = 0·007), lower peak FEV1 values, and more frequent %ddcfDNA elevations that were not clinically detectable.InterpretationLung transplant patients with early unresolving allograft injury measured via %ddcfDNA are at risk of subsequent allograft injury, which is often clinically silent, and progresses to allograft failure.FundNational Institutes of Health.

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The %ddcfDNA assay is precise and reproducible across laboratories and in samples from 2 distinct types of heart transplant rejection. These findings pave the way for larger studies to assess the clinical utility of %ddcfDNA as a marker of acute rejection after heart transplantation.