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

Abstract

Surveillance after lung transplantation (LT) 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 LT recipients with diagnostic cohorts classified as: (1) Biopsy-confirmed or treated acute cellular rejection (ACR), (2) antibody-mediated rejection (AMR), (3) Obstructive chronic lung allograft dysfunction (CLAD), (4) allograft infection (INFXN), and (5) Stable healthy allografts (STABLE). Our principal findings: (1) dd-cfDNA level was elevated in ACR (median 0.91%; 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 did not reach statistical significance (p=0.07) due to limitations in sample size. (3) No difference in dd-cfDNA for allograft INFXN (0.39%; IQR: 0.18–0.67%) versus STABLE, that may relate to differences in “tissue injury” with spectrum of bronchial colonisation versus invasive infection. (4) No difference for dd-cfDNA in unilateral versus bilateral LT. (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.

Current Trends in Cell-Free DNA Applications. Scoping Review of Clinical Trials

“…(2021) showed that with a dd-cfDNA cut-off of 0.35%, sensitivity and specificity of dd-cfDNA for cardiac rejection were 0.76 and 0.83, respectively [36]. In lung transplantation, the optimal threshold for dd-cfDNA for aggregated rejection events representing allograft injury was determined as 0.85%, with sensitivity of 55.6% and specificity of 75.8% [37]. In tBPAR (biopsy-proven acute rejection requiring treatment after liver transplantation), the receiver operator characteristic curve analysis (ROC) showed that dd-cfDNA values reached 98.8% and were higher than in routine liver function tests [38].…”

Clinical Validation of a Plasma Donor-derived Cell-free DNA Assay to Detect Allograft Rejection and Injury in Lung Transplant

“…A previous study using a panel-based single-nucleotide polymorphism test did not observe this association. 12 However, in another study from the GRAfT consortium and National Heart, Lung, and Blood Institute, Jang et al 25 reported that median dd-cfDNA level during putative infection was elevated when associated with abnormal histopathologic findings (1.55%) or physiologic impairment (1.61%) but not when both were absent (0.53%). Our findings can also be considered consonant with a recent study by Bazemore et al 30 where in variability in plasma dd-cfDNA fraction related more to the identity of the specific isolated BAL pathogens and their associated risk for CLAD rather than their mere presence.…”

“…Other investigators have employed single-nucleotide polymorphism panel based dd-cfDNA tests to detect allograft injury, foregoing the need to genotype both donors and recipients. Khush et al 12 used a case-control design in which dd-cfDNA tests were paired with histopathologic assessment and reported associations between plasma dd-cfDNA and ACR and CLAD. Significant associations with AMR or infection were not observed, possibly because of limited sample size.…”

“…Significant associations with AMR or infection were not observed, possibly because of limited sample size. 12 In a pivotal study offering the first real-world experiences implementing plasma dd-cfDNA for surveillance after LT, Keller et al 13 reported high sensitivity and negative predictive value for the combined primary end point of acute lung allograft dysfunction (ie, either AR or infection events). However, the authors emphasized caution in interpretation of these results because of inherent limitations in study design.…”

Donor-Derived Cell-Free DNA for Acute Rejection Monitoring in Heart and Lung Transplantation

“…A threshold dd-cfDNA level of > 1% demonstrated 100% sensitivity and 73% specificity for the diagnosis of moderate or severe ACR with an area under the receiver-operating characteristic curve (AUC) of 0.9. Khush et al also performed a single-center, prospective cohort study analyzing 107 plasma dd-cfDNA samples in 38 patients with corresponding histopathology by TBBx and clinical data to adjudicate for ISHLT grade "probable" AMR [31]. The commercially available dd-cfDNA approach used targeted sequencing around a set of preestablished informative SNPs that were established from population genomic data.…”