Detection of early graft rejection (EGR) is a great deal in the field of transplantation (Tx). A number of biomarkers have been used to determine the state of graft rejection. However, unique biomarkers of graft rejection have yet to be identified. Recently, donor derived cell free DNA (dd-cf-DNA) has been shown to detect allograft rejection in heart and lung Tx. In this study we tested the feasibility of xenograft-derived cell‐free‐DNA (xd-cf‐DNA) as a biomarker for EGR in cardiac xenotransplantation (XTx).

Methods: Heterotopic cardiac XTx was performed using genetically engineered pigs to baboons. Cell‐free DNA was extracted from pre-and post‐Tx plasma samples (n=16) of 4 baboons for library preparation and shortgun sequencing. The sequence reads were aligned to pig and baboon genome, masking overlapping regions between the two genomes. Poor and low quality reads such as PCR duplicates, misalignments, and reads mapping to both baboon and pig genomes. The remaining baboon (b) and pig (p) reads were utilized to compute the % of xd‐cf‐DNA.

Results: The mean sequencing depth was 27M±26K, of which [sim]42% remained after removing poor quality reads. Pig and baboon cf-DNA showed similar physical characteristics to human cf-DNA (peak length [sim]160 bp, a 10 bp periodicity,[sim]60% AT content). As expected, before XTx, the xd-cf-DNA was low (0.07%±0.03%), and was high at >92% in pigs's plasma. It was observed that xd-cf‐DNA was elevated (10-15%) immediately post XTx followed by an exponential decay. The % of xd‐cf‐DNA was elevated (14% and 31%) in 2 of baboon after XTx who had shown decrease heart contractility and wall motion in intra-abdominal ultrasound and later confirmed with histopathology‐proven rejection episodes.

Conclusion: The xd‐cf‐DNA in XTx demonstrated similar kinetics to human dd‐cf‐DNA in Tx. Our results demonstrate that xenograft rejection was appropriately accompanied by an increase in xd‐cf‐DNA. A larger sample size will be useful to better define the utility of xd-cf‐DNA in XTx.

CITATION INFORMATION: Singh A, Agbor-Enoh S, Chan J, Groham S, Corcoran P, Lewis B, Thomas M, Ayares D, Horvath K, Valantine H, Mohiuddin M. Early Detection of Xenograft Rejection or Injury by Donor Derived Cell Free DNA in Pig to Baboon Cardiac Xenotransplantation. Am J Transplant. 2017;17 (suppl 3).

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