Digital Droplet PCR for Rapid Quantification of Donor DNA in the Circulation of Transplant Recipients as a Potential Universal Biomarker of Graft Injury

J. Beck, S. Bierau, S. Balzer, R. Andag, P. Kanzow, J. Schmitz, J. Gaedcke, O. Moerer, J.E. Slotta, P. Walson, O. Kollmar, M. Oellerich, and E. Schütz December 2013

www.clinchem.org/content/59/12/1732.full

© Copyright 2013 by the American Association for Clinical Chemistry

© Copyright 2009 by the American Association for Clinical Chemistry

IntroductionIntroduction

Graft-derived cell-free DNA (GcfDNA) Released by apoptotic graft cells. Low percentages of total cfDNA population. Increased percentages detected during rejection (1). Sensitive quantification methods needed. Next-generation sequencing possible (1). Downsides:

- Long turn-around time and high costs.- Described methods require donor/graft genomic DNA.

1.Snyder TM, Khush KK, Valantine HA, Quake SR. Universal noninvasive detection of solid organ transplant rejection. Proc Natl Acad Sci USA 2011;108:6229–34.

© Copyright 2009 by the American Association for Clinical Chemistry

IntroductionIntroduction

Droplet digital PCR Accurate absolute quantification of template

molecules by separation of target molecules and counting statistics.

GcfDNA quantification by droplet digital PCR Use SNPs with heterologous alleles in donor and

recipient. Pre-selected panel with minor allele frequencies of

>0.4 highest probability for heterologous allele ⇒state.

© Copyright 2009 by the American Association for Clinical Chemistry

Question 1Question 1

What is the probability of a SNP (population allele frequency = 0.5) being AA/BB or AA/AB in unrelated recipient/donor combinations?

© Copyright 2009 by the American Association for Clinical Chemistry

Materials and MethodsMaterials and Methods

Patients 8 patients followed after liver transplantation (LTx). Stable outpatients: n=10 LTx, n=8 heart transplantation.

(HTx), n=9 kidney transplantation (KTx).

cfDNA Extraction & Preamplification cfDNA was extracted from 1 mL of plasma (1000-1500

genome equivalents). Preamplification using the NEBNext Ultra DNA Library Prep

Kit (New England Biolabs).

Digital Droplet PCR Using the QX100 ddPCR system (Bio-Rad) according to

vendor’s instructions. Input DNA: 30 – 100 ng.

© Copyright 2009 by the American Association for Clinical Chemistry

Question 2Question 2

Why is a pre-amplification of the cfDNA necessary?

© Copyright 2009 by the American Association for Clinical Chemistry

Main ResultsMain Results

Performance characteristics of ddPCR assays

Intra-assay imprecision: CV of <15% (range 4–14%) at 2% minor allele content, determined for 13 assays in a series of 9 repetitions in 1 run.

Recovery: 1.87% (94% of spiked value) over 13 SNP assays, with an SD of 0.24% (13%).

© Copyright 2009 by the American Association for Clinical Chemistry© Copyright 2009 by the American Association for Clinical Chemistry

Figure 3. Results from preamplified cfDNA libraries and direct detection, displayed as ratio of the result in preamplified samples to either genomic DNA or direct cfDNA measurement from the same blood draw. It can be seen that the preamplification did not introduce a bias into the SNP detection method. Ratios of the copy number ratios with SDs are given.

Main Results: Main Results: Assessment of Assessment of pre-amplification pre-amplification biasbias

© Copyright 2009 by the American Association for Clinical Chemistry© Copyright 2009 by the American Association for Clinical Chemistry

Figure 3. The first screening step uses real-time PCR and recipient’s genomic DNA. All SNPs with heterozygous genotypes in the recipient are eliminated. The next selection step uses ddPCR and preamplified cfDNA as template; here the final informative assay set for the patient is defined. An informative assay detects an SNP homozygous in the recipient with the graft having at least one heterologous allele. The percentages and numbers are calculated for a minor allele frequency of 0.5 and can vary between individual patients.

Main Main Results: Results: Assay Assay selection selection workflowworkflow

© Copyright 2009 by the American Association for Clinical Chemistry

Main ResultsMain ResultsGraft cfDNA in clinical samples

Percentage of GcfDNA in stable patients < 10%.- LTx: mean 3.5% (1.0% - 8.5%).- KTx: mean 1.2% (0.2% - 3.5%).- HTx: mean 0.9% (0.1% - 3.4%).- Higher LTx levels reflect larger graft volume and

higher regeneration rate of hepatocytes.

Very high amounts ( 90%) in samples taken ∼immediately after reperfusion.

GcfDNA levels in complication-free patients in stable range (<15%) after 10 days.

© Copyright 2009 by the American Association for Clinical Chemistry© Copyright 2009 by the American Association for Clinical Chemistry

Figure 4. GcfDNA measured in the circulation of 9 stable KTx, 8 stable HTx, and 10 stable LTx patients (mean and SD are given). The number of ddPCR SNP assays used for each patient is given below the abscissa.

Stable Stable patientspatientsGcfDNAGcfDNA

© Copyright 2009 by the American Association for Clinical Chemistry© Copyright 2009 by the American Association for Clinical Chemistry

Figure 5. The variance in the pattern of decrease of GcfDNA seen in 3 LTx patients during the first days posttransplantation. In 1 case there was a rapid decrease, in another there was an initial persistence at higher values, followed by a slightly delayed sharp decrease, whereas the third patient (LTx8) showed a steady, but slow, decrease of GcfDNA.

Decrease of GcfDNA after TransplantationDecrease of GcfDNA after Transplantation

© Copyright 2009 by the American Association for Clinical Chemistry

Main ResultsMain Results Graft cfDNA during rejection and cholestasis

episodes Patient with biopsy proven rejection at day 43.

- GcfDNA increased gradually with marked peaks (day 38 and 58).

Patient with late rejection proven by biopsy at day 144.- GcfDNA levels were measured on day 145 and

were found to be increased (19%).

Patient with cholestasis episode.- Unspecific increase of biomarkers AST, GGT,

and bilirubin.- GcfDNA remains in stable range.

© Copyright 2009 by the American Association for Clinical Chemistry© Copyright 2009 by the American Association for Clinical Chemistry

Figure 6. GcfDNA content showed a marked and steady increase from day 31 onward, with an initial peak of approximately 60% at day 38. During this period, the conventional biomarker AST was more variable. (Note that the ordinate scale for the GcfDNA is 10-fold of Fig. 4.) (B), Late time course of patient LTx3: a late acute rejection episode was diagnosed at day 144 postsurgery. The percentage of GcfDNA was 19% at day 145 and showed a steep increase to 55% at day 151. (C), The late time course of patient LTx1 is shown, including an episode of cholestasis. It is noteworthy that GcfDNA did not rise despite increases in conventional liver function tests.

Time-course of GcfDNA during clinical eventsTime-course of GcfDNA during clinical events

© Copyright 2009 by the American Association for Clinical Chemistry

Question 3Question 3

What are the advantages of measuring graft-derived DNA compared to conventional test for graft dysfunction in liver transplant recipients?

© Copyright 2009 by the American Association for Clinical Chemistry

ConclusionConclusion

A novel cost-effective graft injury test based on automated ddPCR with adequate same-day turnaround has been developed.

GcfDNA directly interrogates organ integrity. GcfDNA is obtained from simple blood draw

enabling a more frequent “liquid biopsy.” Test may be useful to monitor optimal

immunosuppression therapy. Can result in reducing risk of chronic rejection by

guiding to more timely intervention.

© Copyright 2009 by the American Association for Clinical Chemistry

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