Cell-free DNA as a Biomarker After Lung Transplantation

February 13, 2020 updated by: Duke University

Cell-free DNA in the Blood and Lung Allograft Fluid: Understanding Association With Acute Rejection in Lung Transplant Recipients

The purpose of this investigation is to determine the association of the fraction of donor-derived cell-free DNA in plasma and lung fluid samples with acute rejection as proven by biopsy in lung transplant recipients.

Study Overview

Status

Completed

Intervention / Treatment

Detailed Description

Chronic lung disease is the third leading cause of death in the United States. Lung transplantation is an effective short-term intervention for select patients with advanced lung disease. Nearly 4,000 adult lung transplant operations were performed in 2014 according to International Society for Heart and Lung Transplantation (ISHLT) registry data(1). AR, however, is more common among recipients of lung, as compared with other solid organ, transplants with approximately 35% of lung recipients experiencing at least one AR episode within first year of transplantation(1). Importantly, AR is the principal risk factor for chronic allograft dysfunction; the leading cause of late death in lung transplant recipients the primary obstacle to improved long-term lung transplant outcomes(2-5).

AR is distinguished upon observation of perivascular or peribronchiolar lymphocytic infiltrates in transbronchial lung biopsy specimens(6). Prior studies have demonstrated even low grade AR events (AR events of minimal or mild severity) increase the risk of chronic graft dysfunction, yet most of these episodes are asymptomatic(4, 5). As such, the majority of lung transplant centers perform surveillance lung biopsies at three-month intervals throughout the first posttransplant year and some continue annual surveillance biopsies over the lifetime of the allograft. Accordingly, lung recipients incur an enormous burden of invasive procedures and the inherent risks associated with this practice. There is a critical unmet need, therefore, to establish a less invasive biomarker of AR to minimize the number of biopsies after lung transplantation.

It has been proposed that damage to the lung allograft in AR disrupts the endothelial barrier, causing release of donor-derived cell-free DNA (dd-cfDNA) into the recipient circulation(7). This hypothesis is based on studies establishing dd-cfDNA as a useful biomarker of AR in heart transplant recipients(8). Specifically, a prospective study of heart recipients demonstrated dd-cfDNA facilitated the diagnosis of AR with a sensitivity and specificity comparable to that of endomyocardial biopsy(8). Similarly, a proof-of-concept study in a small number of lung transplant recipients demonstrated dd-cfDNA significantly increased in association with moderate or severe lung AR and also suggested dd-cfDNA increases in association with signs of chronic graft dysfunction(9). Gaps in this prior work lie in defining the utility of dd-cfDNA as a biomarker for mild/minimal AR events, which are far more common than moderate/severe AR and confer a similar increase in risk for chronic graft failure. Additionally, no prior work has sought to understand whether dd-cfDNA may correlate with treatment response in AR or whether dd-cfDNA can be detected in the lung allograft fluid. As such, it remains unknown whether lung fluid, obtained through BAL may be a more sensitive matrix for detecting clinical events in lung recipients.

WORK ON cfDNA TO DATE IN MULTICENTER LUNG TRANSPLANT COHORT (CTOT-20) CTOT-20 is an NIH funded, multicenter, observational cohort study enrolling lung transplant recipients at the time of transplantation with longitudinal follow up over approximately 5 years. The study enrolled over 800 lung recipients from 2015 to 2018 at five North American lung transplant centers. As part of CTOT-20, protocol mandated peripheral blood samples are collected at 1, 3, 6, 9, and 12 months posttransplant. The five centers enrolling in CTOT-20 all follow similar clinical care practices that call for surveillance BAL and transbronchial lung biopsies at intervals generally corresponding to these blood collection time points. Importantly, all biopsies performed on CTOT-20 subjects are assessed for the presence and severity of AR according to international guidelines(6) by an experienced lung transplant pathologist at the collecting center and these results are entered into the CTOT-20 eCRF. As such, we have accrued a unique biorepository of well-phenotyped paired plasma and BAL samples from lung transplant recipients.

As part of an NIH-funded CTOT ancillary study, we isolated total cfDNA from 320 paired plasma and BAL samples on 126 CTOT-20 participants. The majority of these samples were collected within the first ~6 months of transplant. A total of 60 patients in this cohort have at least one AR event represented, all of which are minimal or mild in severity. cfDNA was isolated using an automated nucleic acid purification system (Maxwell RSC, Promega). Up to 1ml of plasma or BAL was used. cfDNA yields were quantified using the Quantifluor (Promega) fluorescent dye system and isolated cfDNA is being stored at -20°C. TapeStation has been performed on the cfDNA isolated from approximately one-third of the BAL samples. This limited analysis suggests the majority of the BAL samples contain largely very small fragment DNA, without significant contamination by genomic DNA.

Prior studies have demonstrated even low grade AR events (AR events of minimal or mild severity) increase the risk of chronic graft dysfunction, yet most of these episodes are asymptomatic(4, 5, 10). As such, the majority of lung transplant centers perform surveillance lung biopsies at three-month intervals throughout the first posttransplant year and some continue annual surveillance biopsies over the lifetime of the allograft. Accordingly, lung recipients incur an enormous burden of invasive procedures and the inherent risks associated with this practice. There is a critical unmet need, therefore, to establish a less invasive biomarker of AR to minimize the number of biopsies after lung transplantation.

For the current collaboration, isolated cfDNA from the subjects/samples detailed herein (126 subjects, 320 BAL samples paired with 320 plasma samples, 640 samples in total) will be transferred to CareDx for determination of the dd-cfDNA fraction using the Allosure platform(11). Additionally, as it is completely plausible that the donor will not always be the lower contributor to the BAL cfDNA, we request CareDx perform genomic alignment of the paired BAL and plasma samples to inform the selection of the unknown contributor that most likely represents the donor fraction.

Results on the fraction of dd-cfDNA from the transferred samples will be returned to Duke for integration with the clinical data and statistical analysis supported by our CTOT-20 statistical team with sharing of results in a collaborative manner with the CareDx team. A detailed statistical analysis plan will be developed in collaboration with CareDx prior to data analysis. In general, our analytic approach for the descriptive and inferential analyses to address the primary hypothesis/outcome will be as follows:

For the descriptive analyses, biopsies will binned into the following groups based on time of measurement: 20 to 60 days / 61 to 140 days / 141 to 220 days / >220 days (roughly windowing 1, 3, and 6 months posttransplant). To describe the distribution of fraction of dd-cfDNA, the mean (standard deviation), the 5-number summary, and the deciles of levels will be computed. The distribution of fraction of dd-cfDNA levels will be described using a split-time boxplot by AR status and by transplant type across the biopsy time points. Pearson's correlation coefficient will be used to describe the correlation between paired blood and BAL fraction of dd-cfDNA.

For the inferential analyses, to test whether fraction of dd-cfDNA is elevated in transplant patients at the time of an AR event, fraction of dd-cfDNA levels will be modeled as a function of AR status and time from transplant using a linear mixed effect model with a random intercept and a random slope on time. Linear mixed modeling will be used to account for dependencies in the observations. The model will also adjust for potential confounders including transplant type (entered as a binary indicator of bilateral vs. single), time from transplant to biopsy (entered as a continuous covariate, centered and scaled). Additionally, concurrent infection will be entered into the model as a binary indicator. The model will be fit twice, once for plasma and once for BAL.

Study Type

Observational

Enrollment (Actual)

126

Participation Criteria

Researchers look for people who fit a certain description, called eligibility criteria. Some examples of these criteria are a person's general health condition or prior treatments.

Eligibility Criteria

Ages Eligible for Study

18 years and older (ADULT, OLDER_ADULT)

Accepts Healthy Volunteers

No

Genders Eligible for Study

All

Sampling Method

Non-Probability Sample

Study Population

CTOT-20 Study Population (Patient population for the current analyses came from within this overall population). Adult lung transplant recipients undergoing lung transplant at each of the participating centers were eligible.

Description

Inclusion Criteria:

  • Subject must be able to understand and provide written informed consent and
  • Must be ≥18 years of age at the time of written informed consent.
  • Anticipated listing for lung transplantation OR within 45 days of having received a single or bilateral cadaveric donor lung transplant.
  • Enrollment must occur prior to the start of bronchoscopies eligible for research bronchoalveolar lavage (BAL) sampling.
  • Undergoing first lung transplant operation.
  • Transplant surgery to be performed or performed at enrolling center. Note: Concurrent participation in immune monitoring studies or interventional device trials are permitted.

Exclusion Criteria:

  • Multi-organ recipient.
  • Prior recipients of any solid organ transplant, including prior lung transplant.
  • Prior or concurrent recipient of bone marrow transplant.
  • HIV infection.
  • Any condition which the investigators feel would make it unlikely for the recipient to complete follow up procedures or complete the study.
  • Participation in an investigational drug trial at the time of enrollment visit.

Study Plan

This section provides details of the study plan, including how the study is designed and what the study is measuring.

How is the study designed?

Design Details

Cohorts and Interventions

Group / Cohort
Intervention / Treatment
Acute Rejection Cohort
The subset of samples corresponding to biopsy-proven acute rejection
Rejection-free Cohort
The subset of samples corresponding to a transbronchial biopsy free of acute cellular rejection.

What is the study measuring?

Primary Outcome Measures

Outcome Measure
Measure Description
Time Frame
Fraction dd-cfDNA, measured at the time of lung biopsy
Time Frame: Within the first 6 months after lung transplantation
The fraction of dd-cfDNA in plasma and BAL fluid measured at the time of concurrent clinical lung biopsy.
Within the first 6 months after lung transplantation

Secondary Outcome Measures

Outcome Measure
Measure Description
Time Frame
Fraction dd-cfDNA, measured at the time of PFT measurement.
Time Frame: Within the first 6 months after lung transplantation
The fraction of dd-cfDNA in plasma and BAL fluid measured at the time of concurrent clinical PFTs measurement.
Within the first 6 months after lung transplantation

Collaborators and Investigators

This is where you will find people and organizations involved with this study.

Sponsor

Collaborators

Investigators

  • Principal Investigator: Scott Palmer, MD, MHS, Duke University

Publications and helpful links

The person responsible for entering information about the study voluntarily provides these publications. These may be about anything related to the study.

General Publications

Study record dates

These dates track the progress of study record and summary results submissions to ClinicalTrials.gov. Study records and reported results are reviewed by the National Library of Medicine (NLM) to make sure they meet specific quality control standards before being posted on the public website.

Study Major Dates

Study Start (ACTUAL)

December 20, 2015

Primary Completion (ACTUAL)

November 30, 2019

Study Completion (ACTUAL)

November 30, 2019

Study Registration Dates

First Submitted

February 13, 2020

First Submitted That Met QC Criteria

February 13, 2020

First Posted (ACTUAL)

February 17, 2020

Study Record Updates

Last Update Posted (ACTUAL)

February 17, 2020

Last Update Submitted That Met QC Criteria

February 13, 2020

Last Verified

February 1, 2020

More Information

Terms related to this study

Other Study ID Numbers

  • Pro00078935

Plan for Individual participant data (IPD)

Plan to Share Individual Participant Data (IPD)?

NO

Drug and device information, study documents

Studies a U.S. FDA-regulated drug product

No

Studies a U.S. FDA-regulated device product

No

This information was retrieved directly from the website clinicaltrials.gov without any changes. If you have any requests to change, remove or update your study details, please contact register@clinicaltrials.gov. As soon as a change is implemented on clinicaltrials.gov, this will be updated automatically on our website as well.

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