Quantitative Donor-Derived Cell-Free DNA Levels Reflect the Variability in Lung Allograft Cellular Injury

Abstract

<h3>Purpose</h3> Recently we clinically validated a donor-derived cell-free DNA fraction (%dd-cfDNA) test for detecting acute rejection (AR) in lung transplant recipients (LTR). The sensitivity and specificity to detect AR was 89.1% and 82.9% respectively. We now assess the clinical validity of using quantitative absolute dd-cfDNA levels for detecting AR and other graft injury. <h3>Methods</h3> We measured %dd-cfDNA and absolute dd-cfDNA (cp/mL) in 195 prospectively collected plasma samples paired with clinical-pathologic diagnoses using a massively-multiplexed PCR SNP-based assay (Prospera™, Natera, Inc.). Absolute dd-cfDNA levels were compared for the clinical-pathologic cohorts: acute cellular rejection (ACR), antibody-mediated rejection (AMR), chronic lung allograft dysfunction or neutrophilic-responsive allograft dysfunction (CLAD/NRAD), infection (INFXN), and STABLE (no graft injury). Performance characteristics were calculated for AR (ACR+AMR) vs STABLE. Linear regression analysis compared absolute dd-cfDNA to %dd-cfDNA. <h3>Results</h3> Absolute dd-cfDNA levels were significantly higher for injury cohorts, AR, and combined allograft injury (AR+CLAD/NRAD+INFXN) vs STABLE (TABLE1). Using absolute dd-cfDNA to distinguish AR from STABLE, the area under the receiver operating characteristic curve was 0.87 (95% CI:0.78-0.95). The associated sensitivity and specificity was 79.5% (95% CI:71.3-96.6) and 80.3% (95% CI:70.2-90.4), using a 90 cp/mL threshold. There was a strong association between absolute dd-cfDNA and %dd-cfDNA for STABLE (R²=0.74, p=9.6 × 10⁻⁵⁹) and combined allograft injury (R²=0.61, p=6.5 × 10⁻³⁷). <h3>Conclusion</h3> Absolute dd-cfDNA levels have clinical validity for detecting AR and other forms of allograft injury and are strongly associated with %dd-cfDNA. Further research will investigate whether absolute dd-cfDNA measurement offers additional precision for allograft cellular injury assessment and longitudinal responses to therapy beyond %dd-cfDNA.