Absolute Quantification of Donor Derived Cell Free DNA in Heart Transplant Patients

Abstract

PurposeCirculating donor-derived cell-free DNA (dd-cfDNA) is a validated biomarker of allograft rejection. Current assays express dd-cfDNA as a percentage of total cell-free DNA (donor fraction). However, a number of factors can influence recipient cfDNA levels (e.g. BMI, infection) unrelated to the allograft. Thus, we assessed the performance of absolute quantification to detect acute rejection (AR) in heart transplant recipients.MethodsWe developed a method to quantify absolute levels of dd-cfDNA expressed as copies/mL. We measured both dd-cfDNA fraction (%) and absolute quantity (copies/mL) using a clinically available SNP-based massively multiplexed PCR dd-cfDNA assay (ProsperaTM, Natera, San Carlos, CA). These values were compared with diagnosis of AR by histology (defined as ACR >1R and pAMR >0) and test performance was assessed for sensitivity, specificity, negative predictive value and positive predictive value.ResultsOut of 447 samples collected from 150 heart-transplant only patients, 29 had AR and 418 did not. Both dd-cfDNA fraction and absolute quantity were significantly higher in samples with AR compared to samples with no AR. Table 1 characterizes the performance of donor fraction and quantification to distinguish AR. Median dd-cfDNA fraction for AR was 0.62% (IQR: 0.09-1.43) and significantly higher than no AR at 0.04% (0.01-0.11, p < 0.01). Median absolute dd-cfDNA for AR was 78.8 copies/mL (5.3-125.6) and significantly higher than no AR at 2.7 copies/mL (1.37-6.90, p=1.67e-09).ConclusionAbsolute quantification of dd-cfDNA may increase the accuracy to discriminate rejection when compared with dd-cfDNA expressed as a percentage of total cfDNA. Circulating donor-derived cell-free DNA (dd-cfDNA) is a validated biomarker of allograft rejection. Current assays express dd-cfDNA as a percentage of total cell-free DNA (donor fraction). However, a number of factors can influence recipient cfDNA levels (e.g. BMI, infection) unrelated to the allograft. Thus, we assessed the performance of absolute quantification to detect acute rejection (AR) in heart transplant recipients. We developed a method to quantify absolute levels of dd-cfDNA expressed as copies/mL. We measured both dd-cfDNA fraction (%) and absolute quantity (copies/mL) using a clinically available SNP-based massively multiplexed PCR dd-cfDNA assay (ProsperaTM, Natera, San Carlos, CA). These values were compared with diagnosis of AR by histology (defined as ACR >1R and pAMR >0) and test performance was assessed for sensitivity, specificity, negative predictive value and positive predictive value. Out of 447 samples collected from 150 heart-transplant only patients, 29 had AR and 418 did not. Both dd-cfDNA fraction and absolute quantity were significantly higher in samples with AR compared to samples with no AR. Table 1 characterizes the performance of donor fraction and quantification to distinguish AR. Median dd-cfDNA fraction for AR was 0.62% (IQR: 0.09-1.43) and significantly higher than no AR at 0.04% (0.01-0.11, p < 0.01). Median absolute dd-cfDNA for AR was 78.8 copies/mL (5.3-125.6) and significantly higher than no AR at 2.7 copies/mL (1.37-6.90, p=1.67e-09). Absolute quantification of dd-cfDNA may increase the accuracy to discriminate rejection when compared with dd-cfDNA expressed as a percentage of total cfDNA.