Donor-derived, cell-free DNA levels by next-generation targeted sequencing are elevated in allograft rejection after lung transplantation

Abstract

Surveillance after lung transplantation is critical to the detection of acute cellular rejection (ACR) and prevention of chronic lung allograft dysfunction (CLAD). Therefore, we measured donor-derived cell-free DNA (dd-cfDNA) implementing a clinical-grade, next-generation targeted sequencing assay in 107 plasma samples from 38 unique lung transplantation recipients with diagnostic cohorts classified as: (1) biopsy-confirmed or treated ACR, (2) antibody-mediated rejection (AMR), (3) obstructive CLAD, (4) allograft infection (INFXN) and (5) Stable healthy allografts (STABLE). Our principal findings are as follows: (1) dd-cfDNA level was elevated in ACR (median 0.91%; interquartile range (IQR): 0.39-2.07%), CLAD (2.06%; IQR: 0.57-3.67%) and an aggregated cohort of rejection encompassing allograft injury (1.06%; IQR: 0.38-2.51%), compared with the STABLE cohort (0.38%; IQR: 0.23-0.87%) (p=0.02); (2) dd-cfDNA level with AMR was elevated (1.34%; IQR: 0.34-2.40%) compared to STABLE, although it did not reach statistical significance (p=0.07) due to limitations in sample size; (3) there was no difference in dd-cfDNA for allograft INFXN (0.39%; IQR: 0.18-0.67%) versus STABLE, which may relate to differences in "tissue injury" with the spectrum of bronchial colonisation versus invasive infection; (4) there was no difference for dd-cfDNA in unilateral versus bilateral lung transplantation; (5) "optimal threshold" for dd-cfDNA for aggregated rejection events representing allograft injury was determined as 0.85%, with sensitivity=55.6%, specificity=75.8%, positive predictive value (PPV)=43.3% and negative predictive value (NPV)=83.6%. Measurement of plasma dd-cfDNA may be a clinically useful tool for the assessment of lung allograft health and surveillance for "tissue injury" with a spectrum of rejection.

FIGURE 1

Biorepository plasma samples for donor-derived cell-free DNA (dd-cfDNA) analysis and associated diagnostic cohorts in lung transplant recipients. GTD: genome transplant dynamics; ACR: acute cellular rejection; CLAD: chronic lung allograft dysfunction; INFXN: allograft infection in absence of concurrent rejection.

FIGURE 2

Box plots representing median and 25–75th quartiles (interquartile range) for donor-derived cell-free DNA (dd-cfDNA) levels (log₁₀ y-axis) associated with (x-axis) stable healthy, acute cellular rejection (ACR), antibody-mediated rejection (AMR), chronic lung allograft dysfunction-obstructive phenotype (CLAD) and infection in absence of concurrent rejection (INFXN). Wilcoxon rank sum tests for cohorts with ACR (p=0.02), AMR (p=0.07), bronchiolitis obliterans syndrome (BOS) (p=0.02) and INFXN (p=0.56) compared with the stable cohort.

FIGURE 3

Individual donor-derived cell-free DNA (dd-cfDNA) levels (y-axis) for unique lung transplant patients associated with the cohorts (x-axis) of healthy stable, acute cellular rejection (ACR), antibody-mediated rejection (AMR), allograft infection in the absence of associated rejection (INFXN) and obstructive chronic lung allograft dysfunction (CLAD). Horizontal line depicts cohort median value.

FIGURE 4

The different donor-derived cell-free DNA (dd-cfDNA) levels (y-axis) across distinct clinical events (x-axis) for 19 unique patients with multiple associated plasma samples. Clinical events included: healthy stable, acute cellular rejection (ACR), antibody-mediated rejection (AMR), chronic lung allograft dysfunction (CLAD), allograft infection in absence of concurrent rejection (INFXN) and follow-up (F/U) testing 1–2 months after treatment. Individual patient dd-cfDNA levels demonstrated a trend for higher levels in association with AMR and CLAD clinical events.