Donor-derived cell-free DNA accurately detects acute rejection in lung transplant patients, a multicenter cohort study

Abstract

Background: Acute rejection, which includes antibody-mediated rejection and acute cellular rejection, is a risk factor for lung allograft loss. Lung transplant patients often undergo surveillance transbronchial biopsies to detect and treat acute rejection before irreversible chronic rejection develops. Limitations of this approach include its invasiveness and high interobserver variability. We tested the performance of percent donor-derived cell-free DNA (%ddcfDNA), a non-invasive blood test, to detect acute rejection.

Methods: This multicenter cohort study monitored 148 lung transplant subjects over a median of 19.6 months. We collected serial plasma samples contemporaneously with TBBx to measure %ddcfDNA. Clinical data was collected to adjudicate for acute rejection. The primary analysis consisted of computing the area-under-the-receiver-operating-characteristic-curve of %ddcfDNA to detect acute rejection. Secondary analysis determined %ddcfDNA rule-out thresholds for acute rejection.

Results: ddcfDNA levels were high after transplant surgery and decayed logarithmically. With acute rejection, ddcfDNA levels rose six-fold higher than controls. ddcfDNA levels also correlated with severity of lung function decline and histological grading of rejection. %ddcfDNA area-under-the-receiver-operating-characteristic-curve for acute rejection, AMR, and ACR were 0.89, 0.93, and 0.83, respectively. ddcfDNA levels of <0.5% and <1.0% showed a negative predictive value of 96% and 90% for acute rejection, respectively. Histopathology detected one-third of episodes with ddcfDNA levels ≥1.0%, even though >90% of these events were coincident to clinical complications missed by histopathology.

Conclusions: This study demonstrates that %ddcfDNA reliably detects acute rejection and other clinical complications potentially missed by histopathology, lending support to its use as a non-invasive marker of allograft injury.

Keywords: cell-free DNA; early diagnosis; rejection.

Figure 1. Flowchart of study design.

Prospective transplant patients were approached (n = 182). Subjects were excluded if they declined to participate (n = 7), did not undergo a transplantation (n = 23), or died within one month of transplantation (n = 4). Transplant patients who were analyzed (n = 148) underwent a %ddcfDNA assessment (via serial plasma samples) as well as had clinical data, including histopathology, collected to bin them into either the “acute rejection” or “control” endpoint. %ddcfDNA from “controls” and “acute rejection” subjects were compared to determine performance of %ddcfDNA, levels of donor-derived cell-free DNA.

Figure 2. Median %ddcfDNA vs. time curve post-transplantation.

Median %ddcfDNA levels are shown over 24 months post-transplantation. %ddcfDNA, levels of donor-derived cell-free DNA.

Figure 3. Correlation of %ddcfDNA with ACR and allograft dysfunction.

%ddcfDNA is measured by first excluding data before day 45. %ddcfDNA levels are shown for (a) grade 0, 1 and 2 ACR (per ISHLT criteria) and b) allograft dysfunction (as measured by FEV1 decline) categorized as “no”, “mild”, or “moderate/severe”. Number of subjects with each complication is shown in Table 2. %ddcfDNA, levels of donor-derived cell-free DNA.

Figure 4. %ddcfDNA trends between acute rejection phenotypes.

(a) %ddcfDNA levels are shown for controls, acute rejection, ACR and AMR. (b) Measuring the performance sensitivity and specificity of using %ddcfDNA to detect acute rejection, ACR and AMR. ACR, acute cellular rejection; AMR, antibody-mediated rejection; %ddcfDNA, levels of donor-derived cell-free DNA.

Figure 5. Comparing the time course of elevated %ddcfDNA to time of diagnosis.

Shown are the number of episodes that showed a ≥ 1% rise in %ddcfDNA. ACR, acute cellular rejection; AMR, antibody-mediated rejection; %ddcfDNA, levels of donor-derived cell-free DNA. Donor-specific antibodies (DSA) was positive for AMR patients is shown.