Cell-Free DNA Maps Tissue Injury and Correlates with Disease Severity in Lung Transplant Candidates

Abstract

Rationale: Plasma cell-free DNA levels correlate with disease severity in many conditions. Pretransplant cell-free DNA may risk stratify lung transplant candidates for post-transplant complications. Objectives: To evaluate if pretransplant cell-free DNA levels and tissue sources identify patients at high risk of primary graft dysfunction and other pre- and post-transplant outcomes. Methods: This multicenter, prospective cohort study recruited 186 lung transplant candidates. Pretransplant plasma samples were collected to measure cell-free DNA. Bisulfite sequencing was performed to identify the tissue sources of cell-free DNA. Multivariable regression models determined the association between cell-free DNA levels and the primary outcome of primary graft dysfunction and other transplant outcomes, including Lung Allocation Score, chronic lung allograft dysfunction, and death. Measurements and Main Results: Transplant candidates had twofold greater cell-free DNA levels than healthy control patients (median [interquartile range], 23.7 ng/ml [15.1-35.6] vs. 12.9 ng/ml [9.9-18.4]; P < 0.0001), primarily originating from inflammatory innate immune cells. Cell-free DNA levels and tissue sources differed by native lung disease category and correlated with the Lung Allocation Score (P < 0.001). High pretransplant cell-free DNA increased the risk of primary graft dysfunction (odds ratio, 1.60; 95% confidence interval [CI], 1.09-2.46; P = 0.0220), and death (hazard ratio, 1.43; 95% CI, 1.07-1.92; P = 0.0171) but not chronic lung allograft dysfunction (hazard ratio, 1.37; 95% CI, 0.97-1.94; P = 0.0767). Conclusions: Lung transplant candidates demonstrate a heightened degree of tissue injury with elevated cell-free DNA, primarily originating from innate immune cells. Pretransplant plasma cell-free DNA levels predict post-transplant complications.

Keywords: cell-free DNA; chronic lung allograft dysfunction; lung transplantation; primary graft dysfunction; transplantation.

Figure 1.

Patient flow diagram. Of the nine patients who withdrew consent, four transferred care to another facility. A total of 67 of the 186 patients had tissue-specific cell-free DNA analyzed. cfDNA = cell-free DNA; CLAD = chronic lung allograft dysfunction; GRAfT = Genomic Research Alliance for Transplantation; LAS = Lung Allocation Score; PGD = primary graft dysfunction; WGBS = whole-genome bisulfite sequencing.

Figure 2.

Concentrations of total cell-free DNA (cfDNA). (A) Comparison of median cfDNA values of patients with advanced lung disease compared with healthy control patients (HC). (B) Comparison of median cfDNA values of healthy control patients with patients who had different advanced lung disease diagnoses. Medians with interquartile ranges are displayed as horizontal lines. *P < 0.05; ****P < 0.0001. COPD/A1AT = chronic obstructive pulmonary disease/alpha-1 antitrypsin deficiency.

Figure 3.

Circos plot of DNA methylation CpG sites. Each red point represents a single CpG (methylation of a cytosine residue that is followed by a guanine residue). The mean relative methylation levels (from 0 to 1) of each signature CpG from all samples were plotted. The signature CpGs were ordered on the basis of their locations on the chromosomes.

Figure 4.

Cell-free DNA (cfDNA) levels by tissue of origin. (A) cfDNA levels by cellular/tissue origin in patients with advanced lung disease compared with healthy control patients (HC). (B) Median cfDNA levels by cellular/tissue of origin in the various advanced lung diseases compared with HC. Medians with interquartile ranges are displayed as horizontal lines. Gastrointestinal cells include hepatocytes, pancreatic cells, and upper and lower gastrointestinal tract cells; adaptive immune cells include B cells, CD4 T cells, and CD8 T cells. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. COPD/A1AT = chronic obstructive pulmonary disease/alpha-1 antitrypsin deficiency; GI = gastrointestinal; MEP = megakaryocyte/erythrocyte progenitor; NK = natural killer; ns = not significant.

Figure 5.

Cell-free DNA levels of Lung Allocation Score (LAS) and mean pulmonary artery pressure (mPAP) groups. (A) Median cfDNA levels by LAS: low = LAS <45; middle = LAS ⩾45 to <70; high = LAS ⩾70. (B) Median cfDNA levels by mPAP. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 6.

Comparison of cell-free DNA (cfDNA) levels and Lung Allocation Scores (LASs) of patients who developed post-transplant complications. (A) cfDNA levels in patients who developed primary graft dysfunction (PGD) compared with those who did not. (B) cfDNA levels in patients who developed chronic lung allograft dysfunction (CLAD) compared with those who did not. (C) cfDNA levels in patients who died compared with those who survived. (D) LASs in patients who developed PGD compared with those who did not. (E) LASs in patients who developed CLAD compared with those who did not. (F) LASs in patients who died compared with those who survived. Medians with interquartile ranges are displayed as horizontal lines. *P < 0.05; **P < 0.01; ***P < 0.001. ns = not significant.