Analytical Validation of a Single-nucleotide Polymorphism-based Donor-derived Cell-free DNA Assay for Detecting Rejection in Kidney Transplant Patients

Abstract

Original Clinical Science-General Background. Early detection of rejection in kidney transplant recipients holds the promise to improve clinical outcomes. Development and implementation of more accurate, noninvasive methods to detect allograft rejection remain an ongoing challenge. The limitations of existing allograft surveillance methods present an opportunity for donor-derived cell-free DNA (dd-cfDNA), which can accurately and rapidly differentiate patients with allograft rejection from patients with stable organ function. Methods. This study evaluated the analytical performance of a massively multiplexed polymerase chain reaction assay that targets 13 962 single-nucleotide polymorphisms, characterized and validated using 66 unique samples with 1064 replicates, including cell line-derived reference samples, plasma-derived mixtures, and transplant patient samples. The dd-cfDNA fraction was quantified in both related and unrelated donor-recipient pairs. Results. The dd-cfDNA assay showed a limit of blank of 0.11%, a limit of detection and limit of quantitation of 0.15% for unrelated donors, and limit of blank of 0.23%, a limit of detection and limit of quantitation of 0.29% for related donors. All other metrics (linearity, accuracy, and precision) were observed to be equivalent between unrelated and related donors. The measurement precision of coefficient of variation was 1.8% (repeatability, 0.6% dd-cfDNA) and was <5% for all the different reproducibility measures. Conclusions. This study validates the performance of a single-nucleotide polymorphism-based massively multiplexed polymerase chain reaction assay to detect the dd-cfDNA fraction with improved precision over currently available tests, regardless of donorrecipient relationships.

A Practical Guide to the Clinical Implementation of Biomarkers for Subclinical Rejection Following Kidney Transplantation

“…Prospera is a ddcfDNA test commercialized by Natera, and uses a different technology platform than Allosure (see above). The analytical validity of this test was recently published 16. For the clinical evaluation, Sigdel et al reported on a retrospective cohort of 277 archived plasma samples from a single center, of which 217 were matched with paired biopsies (114 protocol biopsies, 103 indication biopsies; 38 with rejection and 72 with borderline changes) 17.…”

The Use of Donor-Derived Cell-Free DNA for Assessment of Allograft Rejection and Injury Status

“…Early rises of total dd-cfDNA levels during acute rejection have been observed in KT recipients [48,54]. These observations supported the premise for quantitative measurement and interpretation of dd-cfDNA as a tool to evaluate the relative health of a KT allograft and diagnose possible complications such as acute allograft rejection [45,46,53,55,56]. The measurement of dd-cfDNA in a transplant recipient involves blood being drawn into specialized tubes that preserve nucleic acids, followed by plasma isolation by centrifugation.…”

“…This is important because the incidence of rejection is known to be significantly greater in for-cause biopsies. Nevertheless, a study by Altug et al, that included six transplant patients confirmed the assay's analytical validity and performance with respect to detecting acute rejection in KT recipients, regardless of donor-recipient relationships [56].…”

Donor-Derived Cell Free DNA: Is It All the Same?

“…Two tests commercially available for patient management were evaluated, AlloSure (CareDx, Inc., Brisbane, CA) 4,5 and Prospera (Natera, Inc., San Carlos, CA). 6,7 Both have published analytical validation studies but have different degrees of clinical validation. AlloSure has large prospective, multicenter data, while Prospera was substantiated using a single-center, retrospective biobank.…”

“…8 Current publications around dd-cfDNA are discordant, with some suggesting superiority and use of a specific library preparation technology. 6 Yet both AlloSure and Prospera use SNP methodology and Illuminabased NGS platforms so it remains unclear whether these claims are true or not in the absence of a headto-head comparison. 5,6 The objective of this study is to provide an early real-life multi-center experience comparing results using the commercially available tests to assess these claims using paired values in renal allograft recipients.…”