A Cost Effective Liquid Biopsy Technology Using Donor Derived Cell Free Dnato Diagnose Rejection in Thoracic Organ Transplant Recipients

Abstract

<h3>Purpose</h3> Solid organ transplant recipients are periodically reviewed to assess the health of transplanted organ. Surgical biopsy is considered as gold standard to monitor rejections as histological changes associated with rejection can be identified by direct microscopy or after immunohistochemical staining with rejection specific biomarker, C4d. However, a number of inconsistencies exist while interpreting the sections as criteria for quantitative scoring of endothelial enlargement and C4d-positivity in patients without clinical evidence of rejection and C4d-negativity in patients with clinical evidence of rejection is not established. Besides the above repeated biopsies can expose the organs to risk of scarring, which in turn can affect its optimal function. <h3>Methods</h3> To mitigate these inconsistencies and risks we have developed a cost effective liquid biopsy technology to diagnose allograft rejection. We first screen an array of single-nucleotide polymorphisms (SNPs) in donor and recipient genomic DNA to discriminate and identify donor specific SNPs. As these SNPs are representative of donor derived cell free DNA (dd-cfDNA), quantification of them by real time PCR in total cell free DNA (total cfDNA) fraction of recipients can be considered as indicator for allograft health. By using this technology we performed a pilot study on 12 heart or lung allograft recipients. 9ml of whole blood collected in PAXGENE tubes from the patients before biopsy were processed to extract total cfDNA and quantify the abundance of dd-cfDNA in the total cfDNA fraction by real time qPCR. <h3>Results</h3> Data analysis indicated presence of dd-cfDNA in 2/12 samples, which correlated with histopathological investigations that identified minimal acute rejection in one patient and cell mediated rejection in the other. One more patient was identified to be undergoing minimal cellular rejection one month post transplant. However, we could not detect dd-cfDNA signal in the plasma. Based on these data, our liquid biopsy technology is able to diagnose rejection in 66.6% (2/3) of patients with a negative prediction at 100% (9/9). <h3>Conclusion</h3> A cost effective Liquid Biopsy Technology using dd-cfDNA quantification by real time qPCR which has excellent correlation with conventional biopsies. Large randomised control studies need to be conducted to further validate this.