title: Validation of a Clinical-Grade Assay to Measure Donor-Derived Cell-Free DNA in Solid Organ Transplant Recipients.
abstract: The use of circulating cell-free DNA (cfDNA) as a biomarker in transplant recipients offers advantages over invasive tissue biopsy as a quantitative measure for detection of transplant rejection and immunosuppression optimization. However, the fraction of donor-derived cfDNA (dd-cfDNA) in transplant recipient plasma is low and challenging to quantify. Previously reported methods to measure dd-cfDNA require donor and recipient genotyping, which is impractical in clinical settings and adds cost. We developed a targeted next-generation sequencing assay that uses 266 single-nucleotide polymorphisms to accurately quantify dd-cfDNA in transplant recipients without separate genotyping. Analytical performance of the assay was characterized and validated using 1117 samples comprising the National Institute for Standards and Technology Genome in a Bottle human reference genome, independently validated reference materials, and clinical samples. The assay quantifies the fraction of dd-cfDNA in both unrelated and related donor-recipient pairs. The dd-cfDNA assay can reliably measure dd-cfDNA (limit of blank, 0.10%; limit of detection, 0.16%; limit of quantification, 0.20%) across the linear quantifiable range (0.2% to 16%) with across-run CVs of 6.8%. Precision was also evaluated for independently processed clinical sample replicates and is similar to across-run precision. Application of the assay to clinical samples from heart transplant recipients demonstrated increased levels of dd-cfDNA in patients with biopsy-confirmed rejection and decreased levels of dd-cfDNA after successful rejection treatment. This noninvasive clinical-grade sequencing assay can be completed within 3 days, providing the practical turnaround time preferred for transplanted organ surveillance.
authors.affiliation: CareDx, Inc., Brisbane, California. Electronic address: mgrskovic@caredx.com.
authors.forename: Marica
authors.initials: M
authors.lastname: Grskovic
authors.affiliation: CareDx, Inc., Brisbane, California.
authors.forename: David J
authors.initials: DJ
authors.lastname: Hiller
authors.affiliation: CareDx, Inc., Brisbane, California.
authors.forename: Lane A
authors.initials: LA
authors.lastname: Eubank
authors.affiliation: CareDx, Inc., Brisbane, California.
authors.forename: John J
authors.initials: JJ
authors.lastname: Sninsky
authors.affiliation: CareDx, Inc., Brisbane, California.
authors.forename: Cindy
authors.initials: C
authors.lastname: Christopherson
authors.affiliation: CareDx, Inc., Brisbane, California.
authors.forename: John P
authors.initials: JP
authors.lastname: Collins
authors.affiliation: CareDx, Inc., Brisbane, California.
authors.forename: Kathryn
authors.initials: K
authors.lastname: Thompson
authors.affiliation: CareDx, Inc., Brisbane, California.
authors.forename: Mindy
authors.initials: M
authors.lastname: Song
authors.affiliation: CareDx, Inc., Brisbane, California.
authors.forename: Yue S
authors.initials: YS
authors.lastname: Wang
authors.affiliation: CareDx, Inc., Brisbane, California.
authors.forename: David
authors.initials: D
authors.lastname: Ross
authors.affiliation: CareDx, Inc., Brisbane, California.
authors.forename: Mitchell J
authors.initials: MJ
authors.lastname: Nelles
authors.affiliation: CareDx, Inc., Brisbane, California.
authors.forename: James P
authors.initials: JP
authors.lastname: Yee
authors.affiliation: CareDx, Inc., Brisbane, California.
authors.forename: Judith C
authors.initials: JC
authors.lastname: Wilber
authors.affiliation: Advanced Heart Failure and Heart Transplantation Unit, Institute for Biomedical Research, University Hospital of Corunna, A Coruña, Spain.
authors.forename: Maria G
authors.initials: MG
authors.lastname: Crespo-Leiro
authors.affiliation: CareDx, Inc., Brisbane, California.
authors.forename: Susan L
authors.initials: SL
authors.lastname: Scott
authors.affiliation: CareDx, Inc., Brisbane, California.
authors.forename: Robert N
authors.initials: RN
authors.lastname: Woodward
mesh_terms: D000483:Alleles; D002460:Cell Line; D004247:DNA / Q000097:blood / Q000235:genetics*; D005260:Female; D005787:Gene Frequency; D005819:Genetic Markers; D005820:Genetic Testing / Q000379:methods* / Q000592:standards*; D005838:Genotype; D006084:Graft Rejection / Q000175:diagnosis / Q000235:genetics / Q000276:immunology; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008297:Male; D016377:Organ Transplantation; D020641:Polymorphism, Single Nucleotide; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014019:Tissue Donors; D066027:Transplant Recipients
chemical_list: D005819:Genetic Markers; D004247:DNA