Using Both the Fraction and Quantity of Donor-Derived Cell-Free DNA to Detect Kidney Allograft Rejection.

Early detection of allograft rejection is critical to the successful management of transplant recipients. Tissue biopsy has been the “gold standard” for diagnosis of active rejection (AR), but is invasive and has poor reproducibility. Conventional noninvasive biomarkers, such as changes in serum creatinine, are available for detectingAR, but are limited due to low sensitivity and specificity. Thus, there is a need for new noninvasive markers that have high accuracy for detecting AR. Donor-derived cell-free DNA fraction (dd-cfDNA[%]) is a promising noninvasive biomarker for detecting allograft rejection. However, dd-cfDNA(%) can be artificially depressed by high levels of circulating cfDNA, which can occur in patients who are obese, have had recent surgery, have medical complications, or received certain medications. This can potentially lead to false negative results. Recently, two studies provided preliminary evidence indicating that the absolute quantity of dd-cfDNA may show better performance for detecting AR than dd-cfDNA(%). In this study we present our results from an assay that utilizes a new two-threshold algorithm that combines both dd-cfDNA(%) and absolute quantity of dd-cfDNA (copies/ml), with the goal of increasing test sensitivity, particularly through improved detection in patients where cfDNA levels are high. The study included 41 patients undergoing allograft management at the University of California at Los Angeles Medical Center, who received dd-cfDNA testing as part of routine clinical care between November 6, 2019 and September 18, 2020. Patients who were under 18, pregnant, had an organ transplant other than kidney, or a blood transfusionwithin 2weeks of enrollment were excluded. Ethics approval was provided by the University of California at Los Angeles Institutional Review Board (IRB12–000991). The study was performed in full adherence to the Declaration of Helsinki, and clinical and research activities reported are consistentwith the Principles of theDeclaration of Istanbul as outlined in the Declaration of Istanbul on Organ Trafficking and Transplant Tourism. Laboratory testing was performed at Natera Inc.’s Clinical Laboratory Improvement Amendments–certified andCollege ofAmericanPathologists–accredited laboratory (San Carlos, CA), as previously described, amplifying cfDNA using massively multiplexed-PCR, targeting 13,926 single nucleotide polymorphisms. dd-cfDNA(%) was measured according to a previously publishedmethodology; the absolute concentration of dd-cfDNA was calibrated to give the quantity of dd-cfDNA (cp/ml). The new two-threshold algorithm combined the previously validated dd-cfDNA(%) cutoff ($1%) and a previously established ddcfDNA quantity cutoff of $78 cp/ml. Samples exceeding either threshold were considered at high risk for AR. Matched biopsy results (for cause biopsies occurring within 4 weeks of dd-cfDNA testing) were available for 16 patients; 14 out of 16 occurred within 2 weeks of the dd-cfDNA test. Biopsy samples were analyzed and graded by pathologists according to standard practice using Banff 2017 classification. Samples without biopsy were classified as not having AR, on the basis of clinical assessment (stable according to serum creatinine and other clinical indicators). AR was found in nine of 16 (56%) biopsies performed, with five classified as T cell–mediated rejection, one as antibody-mediated rejection, and three as mixed type (antibody-mediated rejection/T cell–mediated rejection). We calculated sensitivity and specificity for each algorithm for the 41 patients in the sample. The original method, on the basis of the $1% dd-cfDNA cutoff, had a sensitivity of seven out of nine (77.8%; 95% confidence interval [95%