Graft-derived cell-free DNA, a noninvasive early rejection and graft damage marker in liver transplantation: A prospective, observational, multicenter cohort study

Abstract

Background: Graft-derived cell-free DNA (GcfDNA), which is released into the blood stream by necrotic and apoptotic cells, is a promising noninvasive organ integrity biomarker. In liver transplantation (LTx), neither conventional liver function tests (LTFs) nor immunosuppressive drug monitoring are very effective for rejection monitoring. We therefore hypothesized that the quantitative measurement of donor-derived cell-free DNA (cfDNA) would have independent value for the assessment of graft integrity, including damage from acute rejection.

Methods and findings: Traditional LFTs were performed and plasma GcfDNA was monitored in 115 adults post-LTx at three German transplant centers as part of a prospective, observational, multicenter cohort trial. GcfDNA percentage (graft cfDNA/total cfDNA) was measured using droplet digital PCR (ddPCR), based on a limited number of predefined single nucleotide polymorphisms, enabling same-day turn-around. The same method was used to quantify blood microchimerism. GcfDNA was increased >50% on day 1 post-LTx, presumably from ischemia/reperfusion damage, but rapidly declined in patients without graft injury within 7 to 10 d to a median <10%, where it remained for the 1-y observation period. Of 115 patients, 107 provided samples that met preestablished criteria. In 31 samples taken from 17 patients during biopsy-proven acute rejection episodes, the percentage of GcfDNA was elevated substantially (median 29.6%, 95% CI 23.6%-41.0%) compared with that in 282 samples from 88 patients during stable periods (median 3.3%, 95% CI 2.9%-3.7%; p < 0.001). Only slightly higher values (median 5.9%, 95% CI 4.4%-10.3%) were found in 68 samples from 17 hepatitis C virus (HCV)-positive, rejection-free patients. LFTs had low overall correlations (r = 0.28-0.62) with GcfDNA and showed greater overlap between patient subgroups, especially between acute rejection and HCV+ patients. Multivariable logistic regression modeling demonstrated that GcfDNA provided additional LFT-independent information on graft integrity. Diagnostic sensitivity and specificity were 90.3% (95% CI 74.2%-98.0%) and 92.9% (95% CI 89.3%-95.6%), respectively, for GcfDNA at a threshold value of 10%. The area under the receiver operator characteristic curve was higher for GcfDNA (97.1%, 95% CI 93.4%-100%) than for same-day conventional LFTs (AST: 95.7%; ALT: 95.2%; γ-GT: 94.5%; bilirubin: 82.6%). An evaluation of microchimerism revealed that the maximum donor DNA in circulating white blood cells was only 0.068%. GcfDNA percentage can be influenced by major changes in host cfDNA (e.g., due to leukopenia or leukocytosis). One limitation of our study is that exact time-matched GcfDNA and LFT samples were not available for all patient visits.

Conclusions: In this study, determination of GcfDNA in plasma by ddPCR allowed for earlier and more sensitive discrimination of acute rejection in LTx patients as compared with conventional LFTs. Potential blood microchimerism was quantitatively low and had no significant influence on GcfDNA value. Further research, which should ideally include protocol biopsies, will be needed to establish the practical value of GcfDNA measurements in the management of LTx patients.

Fig 1. Flowchart displaying patient enrollment.

Charité-Berlin, Charité–Universitätsmedizin Berlin; SD, standard deviation; UKE-Eppendorf, University Medical Center Hamburg-Eppendorf; UMG-Goettingen, University Medical Center Göttingen.

Fig 2. Time course of plasma GcfDNA percentage during the first year after liver transplantation in patients without rejection, active infections, or interventions.

Boxes represent median with interquartile range, with whiskers showing the 5th–95th percentile. V4, 2–4 mo; V5, 4–8 mo; V6, 8–10 mo; and V7, 10–14 mo. The number of samples is given below each time point. The bold circle represents one patient with subclinical rejection without biopsy 1 wk earlier (see text). GcfDNA, graft-derived cell-free DNA; LTx, liver transplantation.

Fig 3. Plasma GcfDNA percentages during the first year after transplantation in stable patients and patients with either HCV or biopsy-proven acute rejection.

Boxes represent median with interquartile range, with whiskers showing the 5th–95th percentile and n’s showing the number of contributing values. The bold circle represents the patient with subclinical rejection without biopsy 1 wk earlier (see text). GcfDNA, graft-derived cell-free DNA; HCV, hepatitis C virus.

Fig 4. Receiver operator characteristic curve for GcfDNA.

All graft-derived cell-free DNA percentage values were considered (n = 282 samples from stable periods and n = 31 samples during biopsy-proven acute rejection). The upper and lower limits of the 95% CI are shown as dashed lines. AUC, area under the curve.