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 of a human leukocyte 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.

Keywords: Acute rejection; Donor cell free DNA; Droplet digital PCR; HLA; Lung transplantation.

Figure 1

Primers and probes of digital PCR targeting to HLA-DRB1 region. Primers are shown by the blue arrow and the amplicons are highlighted in the black box. The specific probes are specifically designed to target a unique sequence shown in red.

Figure 2

(A). The ddPCR reactions were carried out with one isolated genomic DNA containing DRB1*03 at a fixed concentration shown in green, spiked with another isolated genomic DNA sample containing DRB1*04 shown in blue that are serially diluted. The copy number of targeted alleles was measured. Scatterplots of three representing assays (1, 3, 5) with corresponding probes (HEX 03 and FAM 04) showing the segregating droplets. (B). The sensitivity of ddPCR was tested using genomic DNA (HLA-DRB1*11/11). The copy number of targeted allele (DR11) was measured at each sample with indicated concentration.

Figure 3

(A). 1-D plot of individual probe binding to the panel of different HLA-DR alleles. The ddPCR reactions were carried out with the individual probe against a panel of highly concentrated genomic DNA containing all eight alleles (HLA-DR 01, 03, 04, 07, 08, 11, 13 and 15). (B). CV (%) of each assay with individual probe. The CV is obtained using 8 repetitions within the same run. (C). The absolute copy number of each allele measured from homozygous (HLA-DR04/04) and heterozygous (HLA-DR04/08) genomic DNA with similar amount (2ng).

Figure 4

Quantification with cfDNA. Serially diluted cfDNA representing donor DNA, was spiked into a constant level of cfDNA representing the recipient DNA. The fraction of the spiked DNA was tested and spotted at high (A) and low levels (B) of background cfDNA.

Figure 5

(A). The levels of DcfDNA in patient who developed acute rejection compared to that from stable or BOS positive patients. B. The time course of the level of donor cfDNA in stable patient who developed BAL culture positive infection (B).