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

Abstract

Background. Lung transplant patients are vulnerable to various forms of allograft injury, whether from acute rejection (AR) (encompassing acute cellular rejection [ACR] and antibody-mediated rejection [AMR]), chronic lung allograft dysfunction (CLAD), or infection (INFXN). Previous research indicates that donor-derived cell-free DNA (dd-cfDNA) is a promising noninvasive biomarker for the detection of AR and allograft injury. Our aim was to validate a clinical plasma dd-cfDNA assay for detection of AR and other allograft injury and to confirm and expand on dd-cfDNA and allograft injury associations observed in previous studies. Methods. We measured dd-cfDNA fraction using a novel single-nucleotide polymorphism-based assay in prospectively collected plasma samples paired with clinical-pathologic diagnoses. dd-cfDNA fraction was compared across clinical-pathologic cohorts: stable, ACR, AMR, isolated lymphocytic bronchiolitis, CLAD/neutrophilic-responsive allograft dysfunction (NRAD), and INFXN. Performance characteristics were calculated for AR and combined allograft injury (AR + CLAD/NRAD + INFXN) versus the stable cohort. Results. The study included 195 samples from 103 patients. Median dd-cfDNA fraction was significantly higher for ACR (1.43%, interquartile range [IQR]: 0.67%–2.32%, P = 5 × 10 −6 ), AMR (2.50%, IQR: 2.06%–3.79%, P = 2 × 10 −5 ), INFXN (0.74%, IQR: 0.46%–1.38%, P = 0.02), and CLAD/NRAD (1.60%, IQR: 0.57%–2.60%, P = 1.4 × 10 −4 ) versus the stable cohort. Area under the receiver operator characteristic curve for AR versus stable was 0.91 (95% confidence interval [CI]: 0.83-0.98). Using a ≥1% dd-cfDNA fraction threshold, sensitivity for AR was 89.1% (95% CI: 76.2%-100.0%), specificity 82.9% (95% CI: 73.3%-92.4%), positive predictive value, 51.9% (95% CI: 37.5%-66.3%), and negative predictive value, 97.3% (95% CI: 94.3%-100%). For combined allograft injury area under the receiver operator characteristic curve was 0.76 (95% CI: 0.66-0.85), sensitivity 59.9% (95% CI: 46.0%-73.9%), specificity 83.9% (95% CI: 74.1%-93.7%), positive predictive value, 43.6% (95% CI: 27.6%-59.6%), and negative predictive value, 91.0% (95% CI: 87.9%-94.0%). Conclusions. These results indicate that our dd-cfDNA assay detects AR and other allograft injury. dd-cfDNA monitoring, accompanied by standard clinical assessments, represents a valuable precision tool to support lung transplant health and is appropriate for further assessment in a prospective randomized-controlled study.

Assessment of dd-cfDNA Levels in Clinically Stable Lung Allograft Recipients Beyond the Initial 2 y Posttransplant

“…However, dd-cfDNA% thresholds that define acute lung allograft injury have been comparable across different propriety platforms. 8,24 In conclusion, this study defines baseline levels of dd-cfDNA in clinically stable patients who are >2 y post-lung transplant. We show that median values of 0.45% with biologic variation of 73% up to 1.54 are the baseline in this population.…”

“…Levels in this time frame are similar to the baseline values of dd-cfDNA in patients within the first 2 y after lung transplantation (<0.5%). 7,10,24 We initially hypothesized that dd-cfDNA may increase over time because of allograft aging processes related to cellular senescence, telomere biology, and other mechanisms. Although there is a statistically significant increase in dd-cfDNA% in the subcohort of patients >5 y posttransplant, the clinical significance of this small change is unclear because these levels are still within the variability of the test in this population.…”

“…However, dd-cfDNA% thresholds that define acute lung allograft injury have been comparable across different propriety platforms. 8,24…”

Relative Change in Donor-Derived Cell-free DNA is Superior to Absolute Values for Diagnosis of Acute Lung Allograft Dysfunction

“…2 Dd-cfDNA% increases in the setting of acute cellular rejection, antibody-mediated rejection (AMR), and infection in several different cohorts of lung transplant recipients, all within the first 1 to 2 y posttransplant. [3][4][5][6] Recently, our group defined baseline dd-cfDNA% levels in a prospective, noninterventional cohort of 51 lung allograft recipients who did not have acute or chronic allograft dysfunction ≥2 y posttransplant. 7 The median dd-cfDNA% in this stable cohort was 0.45 (interquartile range, 0.26-0.69), intraindividual variation (CV I ) was 26%, and interindividual variation (CV G ) was 47%, resulting in a reference change value (RCV) of 73%.…”

“…Currently, absolute dd-cfDNA% thresholds of 0.85% to 1% have been used to delineate lung allograft injury. [3][4][5][6] However, use of absolute measurements may result in high rates of both false positive and false negative tests, depending on normal baseline dd-cfDNA% values. Population-based reference values based on absolute thresholds may have limited use in situations in which within-participant variability (CV I ) of a test is significantly less than the between-participant variability (CV G ).…”