Donor-derived, Cell-free DNA levels by Next Generation Targeted Sequencing are Elevated in Allograft Rejection after Lung Transplantation

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.