Abstract
Bronchoscopy and transbronchial lung biopsy are currently the gold standard for detection of acute rejection and chronic dysfunction (bronchiolitis obliterans syndrome [BOS]) following human lung transplantation (LTx). However, these procedures are expensive and invasive. Up to now, few new methods have demonstrated clinical utility, therefore development of noninvasive biomarkers of rejection are needed.
We developed, optimized and validated a novel method to rapidly quantify donor-derived circulating cell free DNA (DcfDNA) as a potential non-invasive biomarker for lung allograft rejection. The method involves the development of a panel of probes that specifically target a unique sequence on human leucocyte antigen (HLA) alleles. After Tx, donor/recipient probes are chosen based on the mismatched HLA loci, followed by droplet digital PCR used as a quantitative assay to accurately track the trace amount of DcfDNA in a 100–1000 fold excess of recipient background DNA.
With as low as 30pg genomic DNA, we are able to provide an absolute measurement of the copy numbers of target DNA molecule. Each probe was specific as the average false positive rate was about 1 per 100,000 reads. With constant level of background cfDNA mimicking recipient cfDNA, another serially diluted cfDNA was used as donor cfDNA were spiked in. The fraction of spiked cfDNA was measured and calculated. A quantitative linearity was observed spanning all dilutions. The fraction of spiked cfDNA was accurately measured down to 0.2% of total cfDNA. We applied this method to a pilot set of sixty sera from nineteen LTx recipients grouped into biopsy-proven acute rejection, BOS or stable. The level of DcfDNA was significantly elevated in patients with acute rejection (7.9±2.2%, n=16), compared to stable (2.76±1.4%, n=22) or BOS (1.70±0.35, n=22). We also demonstrate significant elevations of DcfDNA in the stable or BOS patients undergoing severe lung infection (4.6±1.82, n=16), suggesting a higher level of turnover rate in the grafted organ during infection.
We developed and validated a clinically feasible application of digital PCR to quantify DcfDNA, which can be used as a non-invasive biomarker for acute lung allograft rejection and infection.