Plasma donor-derived cell-free DNA kinetics after kidney transplantation using a single tube multiplex PCR assay

Abstract

Background: After transplantation, cell-free DNA derived from the donor organ (ddcfDNA) can be detected in the recipient's circulation. We aimed to quantify ddcfDNA levels in plasma of kidney transplant recipients thereby investigating the kinetics of this biomarker after transplantation and determining biological variables that influence ddcfDNA kinetics in stable and non-stable patients.

Materials and methods: From 107 kidney transplant recipients, plasma samples were collected longitudinally after transplantation (day 1-3 months) within a multicenter set-up. Cell-free DNA from the donor was quantified in plasma as a fraction of the total cell-free DNA by next generation sequencing using a targeted, multiplex PCR-based method for the analysis of single nucleotide polymorphisms. A subgroup of stable renal transplant recipients was identified to determine a ddcfDNA threshold value.

Results: In stable transplant recipients, plasma ddcfDNA% decreased to a mean (SD) ddcfDNA% of 0.46% (± 0.21%) which was reached 9.85 (± 5.6) days after transplantation. A ddcfDNA threshold value of 0.88% (mean + 2SD) was determined in kidney transplant recipients. Recipients that did not reach this threshold ddcfDNA value within 10 days after transplantation showed a higher ddcfDNA% on the first day after transplantation and demonstrated a higher individual baseline ddcfDNA%.

Conclusion: In conclusion, plasma ddcfDNA fractions decreased exponentially within 10 days after transplantation to a ddcfDNA threshold value of 0.88% or less. To investigate the role of ddcfDNA for rejection monitoring of the graft, future research is needed to determine causes of ddcfDNA% increases above this threshold value.

Fig 1. Sequido workflow.

Step 1: Multiplex PCR amplification of 1027 SNP containing amplicons followed by a universal PCR step adding platform-specific sequencing adapters and sample molecular identifiers (MID) after which the library is ready for sequencing. Step 2: Next generation sequencing on an Illumina MiSeq or Illumina NextSeq 500/550 instrument. Step 3: Processing of generated sequencing data resulting in a ddcfDNA% for each sample. PCR: polymerase chain reaction, NGS: next generation sequencing, ddcfDNA: donor-derived cell-free DNA.

Fig 2. Principle of the Sequido assay.

Differences in the expected and observed variant allele frequencies (f_v) at a SNP position for different donor cell-free DNA percentages in a sample. For this certain SNP, the donor has a homozygous constitution for the variant allele (TT) and the recipient is homozygous for the wild type allele (AA). The expected variant allele frequencies (f_v) are depicted for a ddcfDNA% of 100% (grey circle), 60% (blue circle), 40% (green circle) and 20% (orange circle). The expected variant allele frequency for every ddcfDNA% mixture is visualized in the frameworks with identical colors, according to the recipient (AA) and donor (TT) allele constitution in this example. Δ: difference in the expected and observed variant allele frequencies for a certain ddcfDNA%; f_v: variant allele frequency; A: wild type allele; T: variant allele; ddcfDNA: donor-derived cell-free DNA.

Fig 3. Fit ddcfDNA% curves of kidney transplant recipients.

Fit ddcfDNA% curves from 42 stable kidney transplant recipients are shown in red. The median ddcfDNA% fit curve is shown in blue. ddcfDNA: donor-derived cell-free DNA.

Fig 4. Fit ddcfDNA% curves from all kidney transplant recipients.

Fit ddcfDNA% curves from all kidney transplant recipients (exponential kinetic: n = 90; abnormal kinetic: n = 16) during the unstable post-transplantation phase before day 10. One recipient was excluded because of a lack of sufficient samples to define the ddcfDNA% kinetic. The reference median fit curve of the subgroup of stable renal transplant recipients is shown in red in both graphs. ddcfDNA: donor-derived cell-free DNA.

Fig 5. ddcfDNA% at day 1 and individual baseline ddcfDNA%.

Differences in ddcfDNA% at day 1 (A) and median baseline ddcfDNA% (B) between recipients who reached a ddcfDNA% below the threshold value of 0.88% before day 10 and recipients who did not reach the threshold value by that time. Individual median baseline ddcfDNA% were determined for each recipient thereby taking the median of all stabilized measurements (below the ddcfDNA threshold value established in the group of stable transplant recipients). One recipient never reached a ddcfDNA% below 0.88%. Plasma samples from the first day after transplantation were not available from 6 recipients (n = 2 not stable before day 10; n = 4 stable before day 10). p < 0.05 with a Student’s t-test. ddcfDNA: donor-derived cell-free DNA; n = number of recipients.