Noninvasive detection of graft injury after heart transplant using donor-derived cell-free DNA: A prospective multicenter study

Abstract

Standardized donor-derived cell-free DNA (dd-cfDNA) testing has been introduced into clinical use to monitor kidney transplant recipients for rejection. This report describes the performance of this dd-cfDNA assay to detect allograft rejection in samples from heart transplant (HT) recipients undergoing surveillance monitoring across the United States. Venous blood was longitudinally sampled from 740 HT recipients from 26 centers and in a single-center cohort of 33 patients at high risk for antibody-mediated rejection (AMR). Plasma dd-cfDNA was quantified by using targeted amplification and sequencing of a single nucleotide polymorphism panel. The dd-cfDNA levels were correlated to paired events of biopsy-based diagnosis of rejection. The median dd-cfDNA was 0.07% in reference HT recipients (2164 samples) and 0.17% in samples classified as acute rejection (35 samples; P = .005). At a 0.2% threshold, dd-cfDNA had a 44% sensitivity to detect rejection and a 97% negative predictive value. In the cohort at risk for AMR (11 samples), dd-cfDNA levels were elevated 3-fold in AMR compared with patients without AMR (99 samples, P = .004). The standardized dd-cfDNA test identified acute rejection in samples from a broad population of HT recipients. The reported test performance characteristics will guide the next stage of clinical utility studies of the dd-cfDNA assay.

Keywords: biomarker; clinical research/practice; heart (allograft) function/dysfunction; heart transplantation/cardiology.

Figure 1

CONSORT diagrams for Donor‐Derived Cell‐Free DNA‐Outcomes AlloMap Registry (D‐OAR). A, Reference population (Box A) includes all 2164 D‐OAR samples from patients with no clinical signs or symptoms of rejection and no biopsy‐based evidence of rejection (no rejection [NR] = no acute cellular rejection [ACR] grade ≥ 2R or antibody‐mediated rejection [AMR] grade ≥ pAMR1). Eight hundred forty‐one samples (Box B) had biopsy paired with donor‐derived cell‐free DNA (dd‐cfDNA) results, of which 18 had AMR, including 2 mixed rejections (Box C), 17 had ACR (Box D), and 806 had NR (ACR grade 0R or 1R and AMR grade pAMR0, Box E). B, The 2405 D‐OAR samples that did not have a rejection diagnosis (Box F) include 31 graft dysfunction samples (left ventricular ejection fraction [LVEF] < 40 or drop in LVEF of ≥ 25% from previous visits, Box G) and 2374 no graft dysfunction samples (Box H). C, The 110 samples from Cedars‐Sinai patients (Box I) include 11 associated with biopsy evidence of AMR (Box J) and 99 with NR (Box K)

Figure 2

The donor‐derived cell‐free DNA (dd‐cfDNA) levels in heart transplant reference population. A, Analysis includes 2164 Donor‐Derived Cell‐Free DNA‐Outcomes AlloMap Registry samples. The median dd‐cfDNA level was 0.07% (IQR, 0.03%‐0.14%). The 97.5th percentile was 1.29%. B, The dd‐cfDNA levels by time posttransplant. Levels of dd‐cfDNA in the reference population are stable from 55 days to 2 years posttransplant (P = .182)

Figure 3

The donor‐derived cell‐free DNA (dd‐cfDNA) level correlates with acute rejection (AR). A, Sample sizes and median dd‐cfDNA levels for samples with (AR) and without (NR) AR. B, Sample sizes and median dd‐cfDNA levels by ACR grade, for patients not diagnosed with AMR. C, Sample sizes and median dd‐cfDNA levels by AMR grade, including patients with mixed rejection. D, Sample sizes and median dd‐cfDNA levels for samples associated with surveillance biopsy with NR, ACR grade ≥ 2R, and AMR grade ≥ pAMR1 (including mixed rejection). E, Sample sizes and median dd‐cfDNA levels for samples associated with for‐cause biopsy with NR, ACR grade ≥ 2R, and AMR grade ≥ pAMR1 (including mixed rejection)

Figure 4

Association of donor‐derived cell‐free DNA (dd‐cfDNA) with graft dysfunction without biopsy evidence of rejection. Thirty‐one patient samples with either no biopsy or no acute rejection (grade 0 or grade 1R acute cellular rejection and pAMR0) within 3 days of the dd‐cfDNA sample. Graft dysfunction was defined as left ventricular ejection fraction (LVEF) < 40% (green cross, n = 12, median 0.07%), a drop in LVEF ≥ 25% from the prior visit (blue triangle, n = 11, median 0.08%), or both (red circle, n = 8, median 0.53%). Left, LVEF. Right, drop in LVEF. One patient (without a time‐matched biopsy) had dd‐cfDNA of 1.4% measured 4 days before a biopsy revealed grade 3R rejection

Figure 5

Donor‐derived cell‐free DNA (dd‐cfDNA) correlates with antibody‐mediated rejection (AMR) in the Cedars‐Sinai study. Sample sizes and median dd‐cfDNA levels for samples from patients with AMR grade pAMR0 and AMR grade ≥ pAMR1. Samples with pAMR1 or higher have elevated levels of dd‐cfDNA