A novel diagnostic test for lung transplant rejection – circulating cell-free methylated DNA

Overview

Lung transplantation is a complex medical procedure for those with advanced lung disease. Average survival following lung transplantation is shorter than for any other solid organ. The median survival time of a lung transplant recipient is only 8 years due to a high incidence (over 75% at 10 years) of chronic rejection (also called chronic lung allograft dysfunction – CLAD). The clinical course of CLAD is progressive with irreversible lung injury that ultimately leads to lung failure. The median survival time following diagnosis of CLAD is only 3 years [1-3].  Interventions before lung injury becomes irreversible may delay or even prevent the development of CLAD and improve lung transplant outcomes. Ideally, a non-invasive blood test that could quantitate cumulative allograft injury would provide an opportunity to detect the early onset of CLAD, or, predict future development, unlike current semi-quantitative testing such as histopathology, spirometry and BAL monitoring. Methylation of DNA encoding genes and transcriptional enhancers is essential to silencing gene expression. Conversely, demethylation, which can occur throughout life, enables transcription factors to bind DNA and induce gene expression. Thus, methylation patterns are ideal for identifying cell types in mixed populations as they reflect the transcriptome, function and ontogeny of cells [4]. 

Aims and Hypotheses

Identify methylation markers specific to transplant rejection.

Surprisingly, no published studies have assessed genome wide methylation patterns on cfDNA from lung transplant patients. The team is in a unique position with access to an extensive biobank of plasma and bronchoalveolar lavage fluid (BALF) from 445 transplant recipients from 2011 to 2019 (70 developing CLAD). Leveraging this impressive resource, the team will carry out for the first time a genome wide methylation analysis on cfDNA using Illumina EPIC Human Methylation 850K arrays. The source of the cfDNA will be from the lung; with BALF having high amounts of cfDNA, especially in transplant patients with rejection [5]. 

Research plan and methods

The team will analyse methylation patterns of cfDNA in BALF from 25 transplant recipients with CLAD and 25 transplant recipients without CLAD.  cfDNA will be isolated from BALF and genome wide epigenetics assessed in the cfDNA using the Illumina EPIC Human Methylation 850K array with coverage of nearly 1 million methylation sites. Epigenetic patterns will be analysed using Minifi (https://bioconductor.org/packages/release/bioc/html/minfi.html) to QC data, normalise and identify differential methylation regions (DMRs) associated with CLAD and clinical parameters associated with lung transplant rejection. The sample size (n=50) has 98.8 % power to detect less than 6.5 % methylation differences between groups [6].  DMRs will be analysed with respect to clinical parameters associated with rejection severity, including CLAD, lung function (FEV1% predicted, which is defined as FEV1% of the patient divided by the average FEV1% in the population for any person of the same age, height, gender, and race, as shown in Figure 1) and time to death. 

Expected outcomes

The team will identify DMRs associated with transplant rejection. DMR’s will provide a unique dataset to identify pathways associated with rejection and enable the development of diagnostic assays to monitor lung transplant rejection. DMRs will enable individual lung transplant patients to be assessed on cell types within the lung (for example epithelial cells vs immune cells), ratio of donor and recipient cells and chronological age of cells, all important determinants of susceptibility and outcome in patients with lung fibrosis. DMRs of highest significance not represented in tissue wide methylation datasets (ie lung transplant rejection specific DMRs) will be used to develop a PCR based assay to quantitate the DMR.

Clinical significance/commercial impact

Early detection of lung rejection would allow for the early institution of measures to prevent and treat this process potentially leading to improved survival rates in patient’s currently undergoing lung transplantation at the TPCH and worldwide.  The aim is to develop a rapid, non-invasive test useful in a clinical setting to inform clinicians on rejection status enabling fine-tuning of immunosuppression. In addition, epigenetic changes are reversible and this study will inform the discovery of new drug targets.  Currently lung transplantation has the lowest survival rate of all other organ allografts, owing largely to the development of CLAD. This is a significant issue and requires immediate attention to increase survival rates of patients as well as their quality of life.

Keywords

• lung
• methylation
• transplantation

Contact

Contact the supervisor for more information: Dr Joanne Voisey.