Cell-free DNA donor fraction analysis in pediatric and adult heart transplant patients by multiplexed allele-specific quantitative PCR: Validation of a rapid and highly sensitive clinical test for stratification of rejection probability.

Paula E North,Adam Vepraskas,Pippa Simpson,Mahua Dasgupta,Donna K Mahnke,Angela Thomm,Paul Daft,Mary Goetsch,Huan Ling Liang,Chris Rosenau,Karl D Stamm,Maria Angeles Baker,Emily Ziegler,Aoy Tomita-Mitchell,Michael E Mitchell,Vakhtang Tchantchaleishvili

doi:10.1371/journal.pone.0227385

Paula E North, Adam Vepraskas + Show 14 more

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https://doi.org/10.1371/journal.pone.0227385

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Abstract

Lifelong noninvasive rejection monitoring in heart transplant patients is a critical clinical need historically poorly met in adults and unavailable for children and infants. Cell-free DNA (cfDNA) donor-specific fraction (DF), a direct marker of selective donor organ injury, is a promising analytical target. Methodological differences in sample processing and DF determination profoundly affect quality and sensitivity of cfDNA analyses, requiring specialized optimization for low cfDNA levels typical of transplant patients. Using next-generation sequencing, we previously correlated elevated DF with acute cellular and antibody-mediated rejection (ACR and AMR) in pediatric and adult heart transplant patients. However, next-generation sequencing is limited by cost, TAT, and sensitivity, leading us to clinically validate a rapid, highly sensitive, quantitative genotyping test, myTAIHEART®, addressing these limitations. To assure pre-analytical quality and consider interrelated cfDNA measures, plasma preparation was optimized and total cfDNA (TCF) concentration, DNA fragmentation, and DF quantification were validated in parallel for integration into myTAIHEART reporting. Analytical validations employed individual and reconstructed mixtures of human blood-derived genomic DNA (gDNA), cfDNA, and gDNA sheared to apoptotic length. Precision, linearity, and limits of blank/detection/quantification were established for TCF concentration, DNA fragmentation ratio, and DF determinations. For DF, multiplexed high-fidelity amplification followed by quantitative genotyping of 94 SNP targets was applied to 1168 samples to evaluate donor options in staged simulations, demonstrating DF call equivalency with/without donor genotype. Clinical validation studies using 158 matched endomyocardial biopsy-plasma pairs from 76 pediatric and adult heart transplant recipients selected a DF cutoff (0.32%) producing 100% NPV for ≥2R ACR. This supports the assay’s conservative intended use of stratifying low versus increased probability of ≥2R ACR. myTAIHEART is clinically validated for heart transplant recipients ≥2 months old and ≥8 days post-transplant, expanding opportunity for noninvasive transplant rejection assessment to infants and children and to all recipients >1 week post-transplant.

Highlights

Noninvasive risk assessment for rejection in heart transplant recipients, both adult and pediatric, is an imperative and urgent clinical need
The myTAIHEART test uses multiplexed, high-fidelity amplification followed by allele-specific qPCR of a panel of 94 highly informative bi-allelic single nucleotide polymorphisms (SNPs) and two controls in a heart transplant recipient’s plasma, thereby distinguishing “donor specific” Cell-free DNA (cfDNA) originating from the engrafted heart from “self-specific” cfDNA originating from the recipient’s own native cells
As a one-time clinical requirement for initiation of myTAIHEART testing on any given transplant recipient, a sample of recipient whole blood, either pre- or post-transplant, is collected in an EDTA tube and without further processing sent to TAI Diagnostics for basic genotyping of the recipient’s genomic DNA at the 94 SNPs of interest

Summary

Introduction

Noninvasive risk assessment for rejection in heart transplant recipients, both adult and pediatric, is an imperative and urgent clinical need. EMB is the historical and still current gold standard for assessment of cardiac allograft acute cellular and antibody-mediated rejection (ACR and AMR) due to its direct histological visualization of myocardial and/or intravascular inflammatory infiltration and cellular injury. The incidence of “biopsy negative” rejection evidenced by hemodynamic compromise without demonstrable myocardial inflammation remains at approximately 20% [23,24,25,26] It is a problematic gold standard, and some centers seek to reduce incidence of its use, in infants and children, and in adults after the first year post-transplant [27]. To do this with improved confidence, noninvasive, relatively inexpensive testing alternatives with high negative predictive value (NPV) for significant rejection are needed to provide adequate, even increased, frequency of monitoring to detect rejection before it becomes clinically evident [28,29]

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