Circulating cell-free DNA as a biomarker of tissue injury: Assessment in a cardiac xenotransplantation model

Abstract

Background: Observational studies suggest that cell-free DNA (cfDNA) is a biomarker of tissue injury in a range of conditions including organ transplantation. However, the lack of model systems to study cfDNA and its relevance to tissue injury has limited the advancements in this field. We hypothesized that the predictable course of acute humoral xenograft rejection (AHXR) in organ transplants from genetically engineered donors provides an ideal system for assessing circulating cfDNA as a marker of tissue injury.

Methods: Genetically modified pig donor hearts were heterotopically transplanted into baboons (n = 7). Cell-free DNA was extracted from pre-transplant and post-transplant baboon plasma samples for shotgun sequencing. After alignment of sequence reads to pig and baboon reference sequences, we computed the percentage of xenograft-derived cfDNA (xdcfDNA) relative to recipient by counting uniquely aligned pig and baboon sequence reads.

Results: The xdcfDNA percentage was high early post-transplantation and decayed exponentially to low stable levels (baseline); the decay half-life was 3.0 days. Post-transplantation baseline xdcfDNA levels were higher for transplant recipients that subsequently developed graft loss than in the 1 animal that did not reject the graft (3.2% vs 0.5%). Elevations in xdcfDNA percentage coincided with increased troponin and clinical evidence of rejection. Importantly, elevations in xdcfDNA percentage preceded clinical signs of rejection or increases in troponin levels.

Conclusion: Cross-species xdcfDNA kinetics in relation to acute rejection are similar to the patterns in human allografts. These observations in a xenotransplantation model support the body of evidence suggesting that circulating cfDNA is a marker of tissue injury.

Trial registration: ClinicalTrials.gov NCT02423070.

Keywords: acute humoral xenograft rejection; biomarker; cell-free DNA; cell-free DNA models; genetically engineered donors; tissue injury; xenotransplantation.

Figure 1:. Study design

Genetically engineered pig’s heart were transplanted to weight-matched baboons. Recipient baboons were monitored prospectively for xenograft rejection (Supplementary Materials). Plasma samples were collected prospectively prior to clinical examination to assay for xenograft-derived cfDNA. Briefly, cfDNA ((containing pig (pink) and baboon (gray) cfDNA)) was isolated from plasma, used to make cfDNA libraries (12 PCR cycles plus addition of indexes = red bars at the end) prior to NGS. Reads were aligned to baboon or pig reference genomes; baboon and pig specific sequences were counted and used to compute %xdcfDNA. The serial xdcfDNA levels was assessed in relation to xenograft rejection.

Figure 2:. Kinetics of plasma cell-free DNA

(A) Cell-free DNA was extracted from prospectively collected plasma samples and quantified using a florescent DNA quantification Assay. The x-axis intervals were selected based on observed kinetics of cfDNA amount in relation to transplant surgery within individual NHPs. Error bards represent standard deviation. (B) The percentage of xdcfDNA quantified by shotgun sequencing and plotted over days after transplant surgery; only non-rejection time-points were included. A first-order decay model (red solid line) is shown ((R² =0.62, Y₀=26.9, half-life=3.0 days, plateau (baseline)=1.5%))

Figure 3:. Individual NHP xdcfDNA and troponin trends

The xdcfDNA (solid black line, left y-axis) or troponin (dashed black line, right y-axis) against days post-transplantation; negative x-axis values represent pre-transplantation; positive x-axis represents post-transplantation. (A)= NHP 1 with no rejection event, (B-F) NHP 2–6 has rejection events, rejection time-points denoted “R”. * in (D) denotes cardiac arrest, (G) NHP 7 with one-post-transplant xdcfDNA assessment due to short follow-up during the study period.

Figure 4:. The xdcfDNA in relation to xenograft rejection

(A) Comparing xdcfDNA at rejection time-points (n=11) to non-rejection time-points (n=27), p-value determined by Mann Whitney U test. (B) Linear regressing troponin on xdcfDNA. Regression equation is provided. P-value determined assuming the null hypothesis with a slope of 0.

Figure 5:. Structure of xenograft-derived cell-free DNA

(A) Length distribution: end coordinates of properly aligned paired reads were obtained from the baboon (blue), pig (red) or human (gray) reference sequences to deduce the length of the cell-free DNA fragments. The frequency of each length fragment was plotted. Peak length fragment noted. (b) Relative nucleotide composition (y-axis in percentage) at +/−25 bases relative to end (“0”) of properly aligned baboon (blue), pig (red), or human (gray) cell-free reads were computed for each of the 4 nucleotides (A, T, C or G) and plotted over the nucleotide position relative to the cell-free DNA cleavage site designated as 0 (x-axis); negative and positive coordinates denotes 5’ side or 3’ side in relation to the cleavage site