Donor-Derived Cell-Free DNA Plus Tissue Gene Expression Profiling of Lung Allograft Injury

Abstract

<h3>Purpose</h3> Lung transplantation outcomes are limited by chronic lung allograft dysfunction, of which acute rejection is a significant risk factor. Acute rejection includes acute cellular rejection (ACR) and antibody-mediated rejection (AMR), and historically has been diagnosed through a combination of transbronchial biopsy histopathology, pulmonary function testing, and the presence of serum donor-specific antibodies. Donor-derived cell-free DNA (dd-cfDNA) has recently shown promise as a less invasive biomarker of allograft injury, but, in isolation, dd-cfDNA does not sufficiently delineate ACR from AMR from infection. Here we propose to combine dd-cfDNA analysis of allograft injury with in situ lung tissue gene expression to better characterize different forms of allograft injury following lung transplantation. <h3>Methods</h3> This is a single center retrospective cohort study of at least 20 lung transplant recipients with longitudinally matched plasma and transbronchial biopsies (n=100). All samples have previously been collected and stored. Plasma will be obtained from the institution's Total Transplant Care Protocol (TTCP) Biorepository, while formalin fixed paraffin embedded (FFPE) biopsy specimens will be obtained from the Department of Pathology through the institution's Tissue Archive Service (TAS). Samples will be assigned to specific clinical cohorts based on histopathology, radiology, microbiological studies and pulmonary function tests at the time of sample collection. Plasma dd-cfDNA fractions will be quantified using Allosure Lung (CareDx), and tissue gene expression profiles (GEP) will be captured with the nCounter Gene Expression Assay (Nanostring Technologies). Data will be analyzed with non-parametric tests and multivariate analyses to determine associations between dd-cfDNA levels and tissue gene expression patterns. <h3>Endpoints</h3> 1. Correlate plasma dd-cfDNA levels with acute allograft injury from ACR, AMR, or infection and determine the time course of dd-cfDNA expression during the first year after lung transplantation. 2. Validate the nCounter Gene Expression Assay platform and identify gene(s) associated with different types of acute rejection and/or infection.