SPECIFIC DETECTION OF GRAFT DERIVED DNA IN THE PLASMA DURING ANTIBODY MEDIATED REJECTION: DEVELOPMENT OF A NEW DIAGNOSTIC TOOL

JB Whitlam^1,2, L Ling², FL Ierino¹, J Kanellis³, D Bruno², H Slater², DA Power¹

¹Department of Nephrology, Austin Health, Heidelberg, Victoria; ²Murdoch Childrens Research Institute, Royal Children’s Hospital, Parkville, Victoria; ³Department of Nephrology, Monash Medical Centre, Clayton, Victoria.

Aim: To study the diagnostic potential of a new method for measuring DNA released from kidney allografts in response to immunologic injury.

Background: Cell-free DNA (cfDNA) is a novel biomarker of graft injury. Release of DNA from damaged cells in a kidney transplant (graft derived cell-free DNA; gdcfDNA) can be distinguished from recipient cfDNA and quantified. We have developed a novel method that exploits copy number variation to measure the absolute concentration of gdcfDNA using digital droplet PCR without prior donor or recipient genotyping. Unlike other approaches that measure gdcfDNA as a proportion of total cfDNA concentration (tcfDNA), called graft fraction (GF), this approach provides an absolute measurement of gdcfDNA and is simpler, quicker and more cost-effective.

Methods: A panel of 31 copy number variation assays was run on cfDNA extracted from plasma of 38 adult kidney transplant recipients undergoing biopsy for acute graft dysfunction. GdcfDNA and tcfDNA concentrations were measured and GF calculated. The results were correlated with diagnostic biopsy histopathology.

Results: Biopsy results included 18 normal, 8 acute antibody mediated rejection (aAMR), 7 other rejection (OR) and 5 other non-rejection pathologies (“Other”). GdcfDNA was significantly elevated in aAMR compared with all other groups (p<0.01). GF was higher in aAMR (p<=0.01) compared to all groups except “Other” (p=0.1). For aAMR diagnosis, gdcfDNA appears to be a better measure than GF.

Conclusions: In contrast to other methods for measurement of cfDNA, this novel approach permits absolute quantification of both gdcfDNA and tcfDNA. Plasma gdcfDNA was raised in aAMR but no other pathologies, suggesting a role for this biomarker in diagnosis of aAMR. This technology warrants large-scale clinical trials of its efficacy.