An Algorithm Measuring Donor Cell-Free DNA in Plasma of Cellular and Solid Organ Transplant Recipients That Does Not Require Donor or Recipient Genotyping.

Paul M K Gordon,Ryan E Lamont,Leo Dimnik,Umair Sajid,Jene Weatherhead,Debra Isaac,Shelly Wegener,Steven C Greenway,Richard T Pon,Steven R Martin,Varun Suresh,Aneal Khan,Nicholas Chang,Jillian S Parboosingh

doi:10.3389/fcvm.2016.00033

Abstract

Cell-free DNA (cfDNA) has significant potential in the diagnosis and monitoring of clinical conditions. However, accurately and easily distinguishing the relative proportion of DNA molecules in a mixture derived from two different sources (i.e., donor and recipient tissues after transplantation) is challenging. In human cellular transplantation, there is currently no useable method to detect in vivo engraftment, and blood-based non-invasive tests for allograft rejection in solid organ transplantation are either non-specific or absent. Elevated levels of donor cfDNA have been shown to correlate with solid organ rejection, but complex methodology limits implementation of this promising biomarker. We describe a cost-effective method to quantify donor cfDNA in recipient plasma using a panel of high-frequency single nucleotide polymorphisms, next-generation (semiconductor) sequencing, and a novel mixture model algorithm. In vitro, our method accurately and rapidly determined donor:recipient DNA admixture. For in vivo testing, donor cfDNA was serially quantified in an infant with a urea cycle disorder after receiving six daily infusions of donor liver cells. Donor cfDNA isolated from 1 to 2 ml of recipient plasma was detected as late as 24 weeks after infusion suggesting engraftment. The percentage of circulating donor cfDNA was also assessed in pediatric and adult heart transplant recipients undergoing routine endomyocardial biopsy with levels observed to be stable over time and generally measuring <1% in cases without moderate or severe cellular rejection. Unlike existing non-invasive methods used to define the proportion of donor cfDNA in solid organ transplant patients, our assay does not require sex mismatch, donor genotyping, or whole-genome sequencing and potentially has broad application to detect cellular engraftment or allograft injury after transplantation.

Highlights

The diagnosis of allograft rejection prompts an immediate change in management and is strongly associated with adverse outcomes
We were successful in replicating this part of the study from Snyder et al [9] and found a strong (R2 = 0.98) correlation between the expected and measured donor DNA concentrations but were dismayed by the cost, inefficiency, and complex bioinformatics required for this assay
Different centrifugation speeds for the separation of plasma from whole blood were tested with equivalence in Cell-free DNA (cfDNA) yield

Summary

Introduction

The diagnosis of allograft rejection prompts an immediate change in management and is strongly associated with adverse outcomes. The gold standard for the detection of solid organ transplant (SOT) rejection requires tissue biopsy and histologic evaluation. This approach has significant limitations including the logistics of organizing the biopsy at short notice and risks associated with an invasive procedure. Allogeneic hepatocyte transplantation is emerging as a successful strategy to bridge patients to liver transplant in metabolic diseases [2]. This approach is useful in small infants where liver transplantation is not immediately feasible due to size and increased risk of technical complications. There is currently no acceptable, noninvasive method for the in vivo detection of donor cells that is useable in humans

Methods

Results

Discussion

Conclusion