Applying rigor and reproducibility standards to assay donor-derived cell-free DNA as a non-invasive method for detection of acute rejection and graft injury after heart transplantation

Abstract

Background: Use of new genomic techniques in clinical settings requires that such methods are rigorous and reproducible. Previous studies have shown that quantitation of donor-derived cell-free DNA (%ddcfDNA) by unbiased shotgun sequencing is a sensitive, non-invasive marker of acute rejection after heart transplantation. The primary goal of this study was to assess the reproducibility of %ddcfDNA measurements across technical replicates, manual vs automated platforms, and rejection phenotypes in distinct patient cohorts.

Methods: After developing and validating the %ddcfDNA assay, we subjected the method to a rigorous test of its reproducibility. We measured %ddcfDNA in technical replicates performed by 2 independent laboratories and verified the reproducibility of %ddcfDNA patterns of 2 rejection phenotypes: acute cellular rejection and antibody-mediated rejection in distinct patient cohorts.

Results: We observed strong concordance of technical-replicate %ddcfDNA measurements across 2 independent laboratories (slope = 1.02, R2 > 0.99, p < 10-6), as well as across manual and automated platforms (slope = 0.80, R2 = 0.92, p < 0.001). The %ddcfDNA measurements in distinct heart transplant cohorts had similar baselines and error rates. The %ddcfDNA temporal patterns associated with rejection phenotypes were similar in both patient cohorts; however, the quantity of ddcfDNA was significantly higher in samples with severe vs mild histologic rejection grade (2.73% vs 0.14%, respectively; p < 0.001).

Conclusions: The %ddcfDNA assay is precise and reproducible across laboratories and in samples from 2 distinct types of heart transplant rejection. These findings pave the way for larger studies to assess the clinical utility of %ddcfDNA as a marker of acute rejection after heart transplantation.

Keywords: allograft rejection; automated; cell-free DNA; heart transplantation; reproducibility.

Conflict of interest statement

Disclosures

The authors have no conflicts to disclose.

Copyright © 2017. Published by Elsevier Inc.

Figures

Figure 1:. Development and reproducibility of a local sequencing and computational workflow to determine %ddcfDNA

a) Experimental design is adapted from NIH panel discussions on reproducing genomic data (www.videocast.nih.gov) suggesting three steps: Step 1 is to develop and validate a local computation workflow. Step 2 is to perform internal validation using sequence files and biosamples previously analyzed. Step 3 (external validation) is to apply the analysis to an external cohort with similar characteristics to the internal validation cohort. (b) Cell-free DNA from two genotyped healthy subjects (one assigned as donor, the other as recipient) were mixed at predefined ratios and sequenced to measure %ddcfDNA. The measured %ddcfDNA was regressed on the expected %ddcfDNA (slope=1.20, r2 =0.97, p-value = 0.032). (c) Serial %ddcfDNA measurements of a GTD subject plotted against months after transplant. Quiescent (0R, 1R) or acute rejection (2R) states are indicated. Solid and dashed lines represent NIH and Stanford results respectively. (d) The linear correlation between %ddcfDNA determined at the NIH (y-axis) and Stanford (x-axis) using replicate plasma samples (n=39). The linear regression line (slope=1.02, r2 = 0.998 and p-value << 10 −6) testing the hypothesis that the slope equals zero is shown.

Figure 2:. Reproducibility of %ddcfDNA patterns of rejection phenotypes in distinct patient cohorts

Serial %ddcfDNA against time plots of two representative HTRs from the NIH cohort: (a) No rejection; (b) ≥ grade 2R ACR. Corresponding grading of EMB as: No-to-mild rejection (0R or 1R) or moderate to severe rejection (≥2R) are labeled for each %ddcfDNA measurement analyzed. Figures (c) and (d) are representative time plots of two subjects from the NIH cohort with donor-specific antibodies (DSA) against HLA Class II DQ antigens displaced as the mean florescence intensity (MFI-right y-axis) represented in dotted line. Graft function as measured by the left ventricular ejection fraction (upper left y-axis) is shown in the dashed line. The %ddcfDNA (lower left-y-axis) is shown in solid black line. pAMR label in (d) represents time of histopathologic and clinical diagnosis of AMR and initiation of treatment.

Figure 3:. Reproducibility of %ddcfDNA measurement on a high throughput automated workflow

(a) An automated workflow was assembled with platforms for DNA isolation (QiaSymphony, Qiagen), library preparation (Sciclone G3 NGS workstation, Perkin Elmer), and high throughput sequencing (HiSeq 3000, Illumina). The workflow was coupled to an automated computational workflow with minimal manual manipulation. (b) Cell-free DNA extracted from 8 plasma samples using the semi-automated platform and the traditional manual platform (QIAAmp Circulating Nucleic Acid Kit, Qiagen) were quantified by quantitative PCR and compared using the Student t-test. The horizontal line represents the mean and the error bars represent the standard deviation. (c) The %ddcfDNA for samples analyzed on the automated and manual platforms. Linear regression of the two sets of measurements showed a slope =0.80, r2=0.92 and p-value

Figure 4:. Correlation between %ddcfDNA and severity of graft injury

a) The %ddcfDNA values were grouped as

Figure 5:. Determining the optimal sequencing depth for precise %ddcfDNA measurement.

(a) A pair of donor and recipient genotypes and the hg19 reference was utilized to simulate two reference genomes. Mock master files of 200 million reads (≈160 bp) per file were generated, each file containing a different fraction of donor reads (0%, 0.05%, 0.25%, 0.5%, 1.25%, 2.5%, 5%). A subset (1, 5, 1o, 15… million) of each master mock file was randomly sampled to determine %ddcfDNA. The analysis was repeated 20 times for each read depth, with replacement after each random sampling. (b) A plot of measured %ddcfDNA against read depth for expected %ddcfDNA of 0.05%. The solid dots represent the mean of the twenty %ddcfDNA values for each read depth. The dashed line shows the expected (true) %ddcfDNA of 0.05%. Error bars represent the standard deviation. (c) A plot of the coefficient of variance (standard deviation/mean) against read depth for expected %ddcfDNA of 0.05%. The arrow points to a read depth of 10 million, beyond which the CV showed less variation.