Rapid detection of donor cell free DNA in lung transplant recipients with rejections using donor-recipient HLA mismatch

Abstract

Fiberoptic bronchoscopy and transbronchial lung biopsy are currently the gold standard for detection of acute rejection following human lung transplantation (LTx). However, these surveillance procedures are expensive and invasive. Up to now, there are few new methods that have demonstrated clinical utility for detecting early stages of rejection following human lung transplantation. We optimized and technically validated a novel method to quantify donor-derived circulating cell free DNA (DcfDNA) that can be used as an early biomarker for lung allograft rejection. The method involves the initial development of a panel of probes in which each probe will specifically target a unique sequence on human leucocyte antigen (HLA) allele. After transplantation, donor/recipient specific probes are chosen based on the mismatched HLA loci, followed by droplet digital PCR (ddPCR) used as a quantitative assay to accurately track the trace amount of DcfDNA in an ample excess of recipient DNA background. The average false positive rate noted was about 1 per 800,000 molecules. Serially 2-fold diluted cfDNA, representing donor fractions of cfDNA, were spiked into a constant level of cfDNA representing the recipient cfDNA. The fraction of spiked cfDNA was measured and quantitative linearity was observed across seven serially diluted cfDNA samples. We were able to measure the minor portion of cfDNA as low as 0.2% of total cfDNA. We subsequently applied the method to a pilot set of 18 LTx recipients grouped into biopsy-proven acute rejection, bronchiolitis obliterans syndrome (BOS) or stable groups. Serial plasma samples were used to identify the percentage of DcfDNA over total cfDNA. The level of DcfDNA was significantly elevated in patients diagnosed with acute rejection (10.30 ± 2.80, n=18), compared to that from stable (1.71 ± 0.50, n=24) or from BOS patients (2.52 ± 0.62, n=20). In conclusion, we present results validating the application of digital PCR to quantify DcfDNA assay in primary clinical specimens, which demonstrate that DcfDNA can be used as an early non-invasive biomarker for acute lung allograft rejection.

The Future of Lung Transplantation

“…As this technology develops, the possibility of recognizing subclinical rejection months earlier by a single blood draw and allowing for initiation of early treatment may become a reality. 63,64 Further exploration of these pathways and molecules in the future may lead us to therapeutic targets or the development of on-site screening tools to better inform our decisions on whether to accept potential donor lungs before LTX occurs. Additionally, the platform in which these potential therapeutics or diagnostic testing could be used may very well be the EVLP circuit.…”

“…With early identification, adaptation of immunosuppressive drugs can be used, thereby curbing graft dysfunction and lending to a reduction in the incidence of CLAD. 63,64 With more precise definitions and classifications within the realm of CLAD, our hope is that future research will be able to better elicit the underlying mechanisms and therefore identify more targeted therapies with a goal of improving long-term LTX survival.…”

Damage-associated molecular patterns in trauma

“…Thus, cfDNA acts as potent immunothrombotic agents [265]. Increased levels of cfDNA in the form of nuclear DNA (nDNA), mtDNA, or NETs were found and correlated with detrimental outcomes in patients with sepsis [269,270], cancer [271], autoimmune disease [272], cardiopulmonary bypass surgery [273], and solid organ transplantation [274].…”

Quantitative PCR of INDEL s to measure donor‐derived cell‐free DNA —a potential method to detect acute rejection in kidney transplantation: a pilot study

“…Previous studies investigated Y‐chromosomal cfDNA in plasma and/or urine of female recipients of male organs . Another approach was to determine donor/recipient differential single‐nucleotide polymorphisms (SNP) or HLA mismatches by classical quantitative real‐time PCR (qPCR) , digital PCR or massively parallel sequencing (MPS) . CfDNA studies have been performed in heart , kidney , liver , lung and pancreas/kidney transplantation, and an association of increased levels of ddcfDNA in recipient plasma with episodes of AR has been found in most of those studies, however, with varying results for sensitivity and specificity.…”