title: 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.
authors.affiliation: Department of Laboratory Medicine, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA. Electronic address: jzou@mdanderson.org.
authors.forename: Jun
authors.initials: J
authors.lastname: Zou
authors.affiliation: HLA Laboratory, Barnes-Jewish Hospital, St. Louis, MO 63110, USA.
authors.forename: Brian
authors.initials: B
authors.lastname: Duffy
authors.affiliation: Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Slade
authors.affiliation: Department of Pediatrics, Division of Hematology and Oncology, Washington University School of Medicine, Saint Louis, MO, USA; Center for Genome Sciences and Systems Biology, Washington University School of Medicine, Saint Louis, MO, USA.
authors.forename: Andrew Lee
authors.initials: AL
authors.lastname: Young
authors.affiliation: Department of Surgery, Washington University School of Medicine, St. Louis, MO 63110, USA.
authors.forename: Nancy
authors.initials: N
authors.lastname: Steward
authors.affiliation: Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA.
authors.forename: Ramsey
authors.initials: R
authors.lastname: Hachem
authors.affiliation: Norton Thoracic Institute Research Laboratory, St. Joseph's Hospital & Medical Center, Phoenix, AZ 85013, USA.
authors.forename: T
authors.initials: T
authors.lastname: Mohanakumar
mesh_terms: D000208:Acute Disease; D000328:Adult; D000368:Aged; D015415:Biomarkers / Q000032:analysis*; D001774:Blood Chemical Analysis / Q000379:methods*; D001989:Bronchiolitis Obliterans / Q000175:diagnosis*; D004247:DNA / Q000032:analysis*; D003937:Diagnosis, Differential; D005260:Female; D006084:Graft Rejection / Q000175:diagnosis* / Q000235:genetics; D006680:HLA Antigens / Q000235:genetics*; D006648:Histocompatibility; D006650:Histocompatibility Testing; D006801:Humans; D016040:Lung Transplantation; D008297:Male; D008875:Middle Aged; D014019:Tissue Donors; D066027:Transplant Recipients; D055815:Young Adult
chemical_list: D015415:Biomarkers; D006680:HLA Antigens; D004247:DNA
keywords: Acute rejection; Donor cell free DNA; Droplet digital PCR; HLA; Lung transplantation