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

Date Published: April 25, 2017

Publisher: Public Library of Science

Author(s): Ekkehard Schütz, Anna Fischer, Julia Beck, Markus Harden, Martina Koch, Tilo Wuensch, Martin Stockmann, Björn Nashan, Otto Kollmar, Johannes Matthaei, Philipp Kanzow, Philip D. Walson, Jürgen Brockmöller, Michael Oellerich, Amit Singal

Abstract: BackgroundGraft-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 findingsTraditional 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.ConclusionsIn 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.

Partial Text: Noninvasive monitoring of graft integrity after liver transplantation (LTx) is needed because of the imprecision and other limitations of traditional methods used to assess liver damage and detect rejection [1]. LTx patients must be continuously monitored for rejection episodes that—if detected—require immunosuppressant drug (ISD) adjustments. A major limitation of standard of care management is that, currently, suspected rejection episodes can be confirmed only by invasive biopsy. However, using serial biopsies to repeatedly assess graft integrity—to adjust ISD treatment and thereby individualize treatment—is often clinically impossible as well as impractical, cost-prohibitive, and a major burden for patients. Biopsies also have limited sensitivity and specificity as well as turn-around times that limit their usefulness for making rapid ISD dosing decisions. A number of conventional liver function tests (LFTs) are routinely used to assess graft function, but they are not diagnostic for assessing acute cellular or antibody-mediated rejection after LTx [2,3].

The results from this prospective, observational, multicenter study confirm previous preliminary reports from our group, that plasma GcfDNA measured in LTx patients is a very useful marker of graft integrity, identifying patients with acute rejection better than conventional LFTs and with a clinically acceptable turn-around time. Changes observed in AST, ALT, γ-GT, and bilirubin showed more overlap between patients with bpar, those with HCV infection, and stable patients. The results of ROC analysis indicate a higher diagnostic sensitivity at 95% specificity for GcfDNA percentage compared to conventional LFTs.



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