Cell-Free DNA to Detect Heart Allograft Acute Rejection

Abstract

Background: After heart transplantation, endomyocardial biopsy (EMBx) is used to monitor for acute rejection (AR). Unfortunately, EMBx is invasive, and its conventional histological interpretation has limitations. This is a validation study to assess 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 were collected to define AR, its 2 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 with controls and to determine %ddcfDNA test characteristics using receiver-operator characteristics analysis.

Results: The study included 171 subjects with median posttransplant follow-up of 17.7 months (IQR, 12.1-23.6), with 1392 EMBx, and 1834 %ddcfDNA measures available for analysis. Median %ddcfDNA levels decayed after surgery to 0.13% (IQR, 0.03%-0.21%) by 28 days. Also, %ddcfDNA increased again with AR compared with control values (0.38% [IQR, 0.31-0.83%], versus 0.03% [IQR, 0.01-0.14%]; P<0.001). The rise was detected 0.5 and 3.2 months before histopathologic diagnosis of ACR and AMR. The area under the receiver operator characteristic curve for AR was 0.92. A 0.25%ddcfDNA threshold had a negative predictive value for AR of 99% and would have safely eliminated 81% of EMBx. In addition, %ddcfDNA showed distinctive characteristics comparing AMR with ACR, including 5-fold higher levels (AMR ≥2, 1.68% [IQR, 0.49-2.79%] versus ACR grade ≥2R, 0.34% [IQR, 0.28-0.72%]), higher area under the receiver operator characteristic curve (0.95 versus 0.85), higher guanosine-cytosine content, and higher percentage of short ddcfDNA fragments.

Conclusions: We found that %ddcfDNA detected AR with a high area under the receiver operator characteristic curve and negative predictive value. 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 patients with heart transplant and paves the way for a clinical utility study. Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT02423070.

Keywords: allograft rejection; biomarkers; cardiac transplantation; cell-free DNA; graft injury.

Figure 1:. Study design

Patients were recruited from five regional transplant centers. Serial plasma samples were collected at the time of routine surveillance procedures after transplant (e.g. endomyocardial biopsy or echocardiogram) and when clinically indicated monitoring was performed (e.g. graft dysfunction or suspected rejection, Table I in the Supplement). A total of 165 patients were included in this analysis and 1,867 ddcfDNA measures.

Figure 2:. %ddcfDNA exponential decay early after transplant and longitudinal %ddcfDNA measures after transplant

A) Exponential decay of %ddcfDNA after transplant. Black dots represent individual %ddcfDNA measures. Decay parameters are in Table IIIb in the Supplement. B) Median %ddcfDNA overtime. Box-and-whisker plot is the median value (red dot), the lower quartile and the upper quartile of a given set of data. Median values are in Table IIIc in the Supplement.

Figure 3:. Correlation %ddcfDNA measures with biopsy graded rejection and allograft dysfunction by echocardiography

a) %ddcfDNA in relation to severity of acute cellular rejection (ACR) by histopathologic interpretation of endomyocardial biopsy. Grade 0 rejection includes both ACR grade 0 and pAMR 0. P-value obtained by generalized estimating equation approach comparing all categories. b) %ddcfDNA in relation to severity of antibody-mediated rejection (AMR) by histopathologic interpretation of the endomyocardial biopsy. Grade 0 rejection includes both ACR grades 0 or 1 and pAMR 0. P-value obtained by generalized estimating equation approach comparing all categories. c) %ddcfDNA in relation to severity of allograft dysfunction measured by echocardiography. Allograft dysfunction was defined as a reduction of left ventricular ejection fraction (LVEF) by ≥5%, and further stratified by severity based on the magnitude of LVEF decline as no (<5%), mild (5 – <10%), moderate (≥10 – <15%) or severe (≥15%) allograft dysfunction. P-value obtained by generalized estimating equation approach comparing all categories.

Figure 4:. Receiver operator characteristics curve for %ddcfDNA to detect endomyocardial biopsy-diagnosed acute rejection

a) The area under the receiver operator characteristics curve (AUROC) for %ddcfDNA overtime for acute rejection (AR), AMR and ACR. Maximal test performance characteristics were noted from day 28. ROC characteristics for each pre-specified time are presented in Table 3 and Table IV in the Supplement. b) AUROC of %ddcfDNA to detect acute rejection (AR), antibody mediated rejection (AMR, pAMR ≥1) or acute cellular rejection (ACR, grade ≥2R). Primary analysis excluded endomyocardial biopsies before day 28. 923 endomyocardial biopsies and concurrent ddcfDNA measures were included in this analysis. ROC characteristics are presented in Table 3.

Figure 5:. Distinct %ddcfDNA patterns in AMR and ACR

a – c %ddcfDNA against time plots for prototype subjects with acute cellular rejection, “0” = ACR grade 0 or grade 1 and pAMR0 d – f %ddcfDNA against time plots for prototype subjects with antibody-mediated rejection. “0” = ACR grade 0 or grade 1 and pAMR0 g. Trends of %ddcfDNA measures in all patients preceding a histopathologic diagnosis of AMR or ACR; mean and standard deviations are shown. For AMR, there is a longer lead-time and larger quantitative ddcfDNA release of ddcfDNA prior to diagnosis. In addition, 12/15 cases of AMR had elevations of ddcfDNA preceding the clinical diagnosis. In ACR the ddcfDNA elevations preceding clinical diagnosis are rarer (2/17), lower quantitatively and are detected for a shorter time preceding overt diagnosis. h. Different %ddcfDNA characteristics based on cfDNA length in ACR and AMR. Short DNA fragments were defined based on total read length < 100bp. In AMR, the % short cfDNA fragments is much higher than in ACR or controls without rejection.