Use of donor-derived-cell-free DNA as a marker of early allograft injury in primary graft dysfunction (PGD) to predict the risk of chronic lung allograft dysfunction (CLAD)

doi:10.1016/j.healun.2021.02.008

The Journal of Heart and Lung Transplantation

Volume 40, Issue 6, June 2021, Pages 488-493

https://doi.org/10.1016/j.healun.2021.02.008 Get rights and content

BACKGROUND

Primary graft dysfunction (PGD) is a risk factor for chronic lung allograft dysfunction (CLAD). However, the association between PGD and degree of allograft injury remains poorly defined. In this study, we leverage a novel biomarker for allograft injury, percentage donor-derived cell-free DNA (%ddcfDNA), to study the association between PGD, degree of allograft injury, and the development of CLAD.

METHODS

This prospective cohort study recruited 99 lung transplant recipients and collected plasma samples on days 1, 3, and 7 for %ddcfDNA measurements. Clinical data on day 3 was used to adjudicate for PGD. %ddcfDNA levels were compared between PGD grades. In PGD patients, %ddcfDNA was compared between those who developed CLAD and those who did not.

RESULTS

On posttransplant day 3, %ddcfDNA was higher in PGD than in non-PGD patients (median [IQR]: 12.2% [8.2, 22.0] vs 8.5% [5.6, 13.2] p = 0.01). %ddcfDNA correlated with the severity grade of PGD (r = 0.24, p = 0.02). Within the PGD group, higher levels of %ddcfDNA correlated with increased risk of developing CLAD (log OR(SE) 1.38 (0.53), p = 0.009). PGD patients who developed CLAD showed ∼2-times higher %ddcfDNA levels than patients who did not develop CLAD (median [IQR]: 22.4% [11.8, 27.6] vs 9.9% [6.7, 14.9], p = 0.007).

CONCLUSION

PGD patients demonstrated increased early posttransplant allograft injury, as measured by %ddcfDNA, in comparison to non-PGD patients, and these high %ddcfDNA levels were associated with subsequent development of CLAD. This study suggests that %ddcfDNA identifies PGD patients at greater risk of CLAD than PGD alone.

Section snippets

Study design

We conducted an observational analysis that included subjects from two ongoing prospective cohort studies. The first study, Genome transplant dynamics (GTD) (NCT01985412), is a single center study that began in 2010 at Stanford University Hospital to evaluate the utility of plasma %ddcfDNA to monitor for acute rejection. The second study, Genomic Research Alliance for Transplantation (GRAfT) (NCT0243070), began recruitment in 2015 at three centers (the Johns Hopkins Hospital and University of

Cohort description

Ninety-nine subjects met criteria for inclusion in our analysis; however, 13 of these patients did not have available day 3 CXR or ABG data necessary to adjudicate for PGD. 86 patients were therefore included in our final analysis (Supplementary Figure S1). The mean (SD) age at transplantation was 54.3 (14.4) years and the mean (SD) Lung Allocation Score (LAS) was 44.9 (12.1). Interstitial lung disease was the most common indication for transplant (46.4%) and 70.2% of patients underwent

Discussion

This study reports two important findings supporting the use of %ddcfDNA in patients with PGD. First, from Day 1 of transplantation, %ddcfDNA correlated with the presence and severity of PGD. Second, in patients with PGD, %ddcfDNA correlated with the subsequent risk of developing CLAD. Tanaka et al. previously reported an inverse correlation between %ddcfDNA and oxygenation in a cohort of 15 patients undergoing living donor-lobar lung transplantation as well as higher levels of %ddcfDNA in

Disclosure statement

Dr. Errol Bush has received consulting fees from VeraMedica Institute for occupational/environmental exposure litigation. Dr. Kiran Khush reports receiving grants and personal fees as a scientific advisor and participant on a speaker's bureau from CareDx Inc. outside of the scope of the submitted work. Dr. Sean Agbor-Enoh receives funding from the Cystic Fibrosis Foundation, the National Institute of Health Distinguished Scholar Program and the National Heart, Lung and Blood Institute

References (25)

GI Snell et al.
Report of the ISHLT working group on primary lung graft dysfunction, part I: definition and grading-A 2016 consensus group statement of the international society for heart and lung transplantation
J Heart Lung Transplant
(2017)
D Kreisel et al.
Short- and long-term outcomes of 1000 adult lung transplant recipients at a single center
J Thorac Cardiovasc Surg
(2011)
EM Gielis et al.
Cell-free DNA: An upcoming biomarker in transplantation
Am J Transplant
(2015)
S Agbor-Enoh et al.
Donor-derived cell-free DNA predicts allograft failure and mortality after lung transplantation
EBioMedicine
(2019)
GM Verleden et al.
Chronic lung allograft dysfunction: definition, diagnostic criteria, and approaches to treatment-A consensus report from the pulmonary council of the ISHLT
J Heart Lung Transplant
(2019)
S Agbor-Enoh et al.
Applying rigor and reproducibility standards to assay donor-derived cell-free DNA as a non-invasive method for detection of acute rejection and graft injury after heart transplantation
J Heart Lung Transplant
(2017)
S Agbor-Enoh et al.
Late manifestation of alloantibody-associated injury and clinical pulmonary antibody-mediated rejection: Evidence from cell-free DNA analysis
J Heart Lung Transplant
(2018)
JG Penfield et al.
Transplant surgery injury recruits recipient MHC class II-positive leukocytes into the kidney
Kidney Int
(1999)
TK Waddell et al.
Major histocompatibility complex expression and lung ischemia-reperfusion in rats
Ann Thorac Surg
(1996)
M Gunasekaran et al.
Donor-derived exosomes with lung self-antigens in human lung allograft rejection
Am J Transplant
(2017)

T Mohanakumar et al.

A novel mechanism for immune regulation after human lung transplantation

J Thorac Cardiovasc Surg

(2019)

L Zaffiri et al.

Collagen type-V is a danger signal associated with primary graft dysfunction in lung transplantation

Transpl Immunol

(2019)

Cited by (23)

Short airway telomeres are associated with primary graft dysfunction and chronic lung allograft dysfunction
2023, Journal of Heart and Lung Transplantation
Primary graft dysfunction (PGD) is a major risk factor for chronic lung allograft dysfunction (CLAD) following lung transplantation, but the mechanisms linking these pathologies are poorly understood. We hypothesized that the replicative stress induced by PGD would lead to erosion of telomeres, and that this telomere dysfunction could potentiate CLAD.
In a longitudinal cohort of 72 lung transplant recipients with >6 years median follow-up time, we assessed tissue telomere length, PGD grade, and freedom from CLAD. Epithelial telomere length and fibrosis-associated gene expression were assessed on endobronchial biopsies taken at 2 to 4 weeks post-transplant by TeloFISH assay and nanoString digital RNA counting. Negative-binomial mixed-effects and Cox-proportional hazards models accounted for TeloFISH staining batch effects and subject characteristics including donor age.
Increasing grade of PGD severity was associated with shorter airway epithelial telomere lengths (p = 0.01). Transcriptomic analysis of fibrosis-associated genes showed alteration in fibrotic pathways in airway tissue recovering from PGD, while telomere dysfunction was associated with inflammation and impaired remodeling. Shorter tissue telomere length was in turn associated with increased CLAD risk, with a hazard ratio of 1.89 (95% CI 1.16-3.06) per standard deviation decrease in airway telomere length, after adjusting for subject characteristics.
PGD may accelerate telomere dysfunction, potentiating immune responses and dysregulated repair. Epithelial cell telomere dysfunction may represent one of several mechanisms linking PGD to CLAD.
Differentiation between lung allograft rejection and infection using donor-derived cell-free DNA and pathogen detection by metagenomic next-generation sequencing
2023, Heliyon
In lung transplant recipients (LTRs), the primary causes of mortality are rejection and infection, which often present similar symptoms, making differentiation challenging. This study aimed to explore the diagnostic efficacy of plasma donor-derived cell-free DNA (dd-cfDNA) in conjunction with metagenomic next-generation sequencing (mNGS) for pathogen detection in differentiation between lung allograft rejection and infection in LTRs experiencing new-onset pulmonary complications.
We conducted a retrospective study on 188 LTRs who underwent lung or heart-lung transplantation at our institution from 2015 to 2021. The LTRs were categorized into three groups: stable, rejection, and infection. We measured plasma dd-cfDNA levels and utilized both mNGS and culture methods to identify pathogens in the bronchoalveolar lavage fluid (BALF).
The rejection group exhibited the highest levels of plasma dd-cfDNA (median 1.34 %, interquartile range [IQR] 1.06–2.19 %) compared to the infection group (median 0.72 %, IQR 0.62–1.07 %) and the stable group (median 0.69 %, IQR 0.58–0.78 %) (both p < 0.001). Within the infection group, a significantly higher level of dd-cfDNA was observed in the cytomegalovirus infection subgroup (p < 0.001), but not in the fungal (p > 0.05) or bacterial infection subgroups (p > 0.05), when compared to the stable group. Elevated dd-cfDNA levels, in combination with negative mNGS results, strongly indicated lung allograft rejection, with a positive predictive value and negative predictive value of 88.7 % and 99.2 %, respectively.
Plasma dd-cfDNA in combination with BALF pathogen detection by mNGS shows satisfactory accuracy in differentiating lung allograft rejection from infectious complications.
Chronic lung allograft dysfunction in 2022, past and updates
2023, Revue des Maladies Respiratoires
Les résultats à court terme de la transplantation pulmonaire se sont améliorés mais la survie à long terme reste en deçà de celle observée dans les autres transplantations d’organes solides.
La principale cause de mortalité tardive est la dysfonction chronique du greffon pulmonaire (DCG) qui affecte près de la moitié des receveurs à 5 ans de la greffe. Des facteurs de risques immunologiques et non immuns ont été identifiés. Ces facteurs vont activer le système immunitaire inné et adaptatif entraînant des processus lésionnels et de réparations altérées aboutissant à une fibrose touchant les petites voies aériennes ou le tissu interstitiel. Plusieurs phénotypes de DCG sont maintenant identifiés en fonction du profil fonctionnel respiratoire et de l’imagerie. En dehors de la retransplantation, uniquement possible pour une minorité de patients, il n’existe pas à ce jour de traitement permettant d’inverser le processus de la DCG.
Des espoirs thérapeutiques existent, comme les traitements anti-fibrosants ou la photophérèse. Les travaux de recherche ont par ailleurs permis d’identifier de nombreux biomarqueurs qui pourraient s’avérer être pertinents comme cibles thérapeutiques.
Les mécanismes physiopathologiques de la DCG sont mieux compris aujourd’hui mais le défi thérapeutique reste entier.
While short-term results of lung transplantation have improved considerably, long-term survival remains below that achieved for other solid organ transplants.
The main cause of late mortality is chronic lung allograft dysfunction (CLAD), which affects nearly half of the recipients 5 years after transplantation. Immunological and non-immune risk factors have been identified. These factors activate the innate and adaptive immune system, leading to lesional and altered wound-healing processes, which result in fibrosis affecting the small airways or interstitial tissue. Several phenotypes of CLAD have been identified based on respiratory function and imaging pattern. Aside from retransplantation, which is possible for only small number of patients, no treatment can reverse the CLAD process.
Current therapeutic research is focused on anti-fibrotic treatments and photopheresis. Basic research has identified numerous biomarkers that could prove to be relevant as therapeutic targets.
While the pathophysiological mechanisms of CLAD are better understood than before, a major therapeutic challenge remains.
Elevated cell-free DNA in respiratory viral infection and associated lung allograft dysfunction
2022, American Journal of Transplantation
Respiratory viral infection (RVI) in lung transplant recipients (LTRs) is a risk for chronic lung allograft dysfunction (CLAD). We hypothesize that donor-derived cell-free DNA (%ddcfDNA), at the time of RVI predicts CLAD progression. We followed 39 LTRs with RVI enrolled in the Genomic Research Alliance for Transplantation for 1 year. Plasma %ddcfDNA was measured by shotgun sequencing, with high %ddcfDNA as ≥1% within 7 days of RVI. We examined %ddcfDNA, spirometry, and a composite (progression/failure) of CLAD stage progression, re-transplant, and death from respiratory failure. Fifty-nine RVI episodes, 38 low and 21 high %ddcfDNA were analyzed. High %ddcfDNA subjects had a greater median %FEV₁ decline at RVI (−13.83 vs. −1.83, p = .007), day 90 (−7.97 vs. 0.91, p = .04), and 365 (−20.05 vs. 1.09, p = .047), compared to those with low %ddcfDNA and experienced greater progression/failure within 365 days (52.4% vs. 21.6%, p = .01). Elevated %ddcfDNA at RVI was associated with an increased risk of progression/failure adjusting for symptoms and days post-transplant (HR = 1.11, p = .04). No difference in %FEV₁ decline was seen at any time point when RVIs were grouped by histopathology result at RVI. %ddcfDNA delineates LTRs with RVI who will recover lung function and who will experience sustained decline, a utility not seen with histopathology.
Comparison of donor-derived cell-free DNA between single versus double lung transplant recipients
2022, American Journal of Transplantation
Citation Excerpt :
The primary clinical endpoint of this study was acute rejection, a composite outcome of acute cellular rejection (ACR) grade A2 or higher, ACR grade 1 with allograft dysfunction (>10% decline in forced expiratory volume in 1 second (FEV1)), and clinical antibody mediated rejection (AMR). All endpoints were adjudicated by centralized multidisciplinary adjudication committees blinded to %dd-cfDNA data as previously described.2,6,8 Endpoints were adjudicated using center level data to remain consistent with usual care practices.
Plasma donor-derived cell-free DNA (dd-cfDNA) is a sensitive biomarker for the diagnosis of acute rejection in lung transplant recipients; however, differences in dd-cfDNA levels between single and double lung transplant remains unknown. We performed an observational analysis that included 221 patients from two prospective cohort studies who had serial measurements of plasma dd-cfDNA at the time of bronchoscopy and pulmonary function testing, and compared dd-cfDNA between single and double lung transplant recipients across a range of disease states. Levels of dd-cfDNA were lower for single vs. double lung transplant in stable controls (median [IQR]: 0.15% [0.07, 0.44] vs. 0.46% [0.23, 0.74], p < .01) and acute rejection (1.06% [0.75, 2.32] vs. 1.78% [1.18, 5.73], p = .05). Doubling dd-cfDNA for single lung transplant to account for differences in lung mass eliminated this difference. The area under the receiver operating curve (AUC) for the detection of acute rejection was 0.89 and 0.86 for single and double lung transplant, respectively. The optimal dd-cfDNA threshold for the detection of acute rejection was 0.54% in single lung and 1.1% in double lung transplant. In conclusion, accounting for differences in dd-cfDNA in single versus double lung transplant is key for the interpretation of dd-cfDNA testing in research and clinical settings.
Variability in plasma donor-derived cell-free DNA levels with CLAD more than 5-years after Lung Transplantation: Pilot data
2022, Transplantation Reports
Citation Excerpt :
Diagnosis of CLAD is determined by clinical and physiologic criteria since transbronchial biopsies usually lack adequate sensitivity to provide histopathologic confirmation [3]. Donor-derived cell-free DNA (dd-cfDNA) has recently emerged as a valuable plasma biomarker that has been clinically validated for detection of acute cellular and antibody mediated rejection (AMR) in LT [4–6]. To date, studies have principally focused on estimation of the donor fraction (%dd-cfDNA) expressed as percentage of background (host) cell-free DNA (cfDNA), with thresholds validated for %dd-cfDNA during the earlier post-LT epochs.
Chronic lung allograft dysfunction (CLAD) is a highly prevalent and devastating complication in lung transplant (LT), culminating in increased allograft failure, morbidity, and mortality. Determination of the fraction of plasma donor-derived cell-free DNA (dd-cfDNA) has emerged as a valuable noninvasive monitoring tool after LT; however, the increased variance of host cfDNA caused by infection and other inflammation can complicate the approach.
In a retrospective pilot study, we analyzed both the fraction of dd-cfDNA (%dd-cfDNA) and absolute quantity of dd-cfDNA (cp/mL) in recipients with CLAD ≥ 5-year post-LT with co-morbid conditions (gastro-esophageal reflux, antibody-mediated rejection, or chronic infection) designated as complicated (C-CLAD) and uncomplicated (U-CLAD) cohorts.
Median time post-LT was 2,149 days (1,899-2,920). The median %dd-cfDNA for the C-CLAD (N=5) cohort was 1.79% (IQR: 1.04-2.29) and significantly elevated compared to the U-CLAD cohort (N=7, 0.49%; 0.28-0.88) (p=0.018). Absolute dd-cfDNA was also significantly higher in C-CLAD (43.2 cp/mL; 27.9-89.3) than the in U-CLAD cohort (19.6 cp/mL; 8.1-27.9) (p=0.048).
We report a heretofore undescribed dichotomy of dd-cfDNA levels with CLAD ≥ 5-years, related specifically to elevation in allograft quantity as opposed to alteration in host plasma cfDNA. Further, dd-cfDNA analysis in association with co-morbid conditions in C-CLAD may offer insights for potential treatment and alleviation of molecular injury. Measurement of longitudinal absolute quantity dd-cfDNA may provide additional value for future clinical study design of pathobiology and CLAD treatment algorithms.

View all citing articles on Scopus

View full text

Original Clinical ScienceUse of donor-derived-cell-free DNA as a marker of early allograft injury in primary graft dysfunction (PGD) to predict the risk of chronic lung allograft dysfunction (CLAD)

BACKGROUND

METHODS

RESULTS

CONCLUSION

Section snippets

Study design

Cohort description

Discussion

Disclosure statement

J Heart Lung Transplant

J Thorac Cardiovasc Surg

Am J Transplant

EBioMedicine

J Heart Lung Transplant

J Heart Lung Transplant

J Heart Lung Transplant

Kidney Int

Ann Thorac Surg

Am J Transplant

J Thorac Cardiovasc Surg

Transpl Immunol

Original Clinical Science
Use of donor-derived-cell-free DNA as a marker of early allograft injury in primary graft dysfunction (PGD) to predict the risk of chronic lung allograft dysfunction (CLAD)