Donor-Derived, Cell-Free DNA: A New Biomarker for Detecting Renal Transplant Rejection

Released into the blood stream when cells lyse, cell-free DNA (cfDNA) is an analyte of much interest in molecular pathology. Currently used to screen for trisomies in pregnant women and avoid the risk of amniocentesis or chorionic villus sampling, laboratories have turned their attention to new disease states which can benefit from analysis of cfDNA. GRAIL Inc., Guardant and other companies are racing for a “liquid biopsy” test to detect cancer signatures in cfDNA. A closer analogue to the use of cfDNA in pregnancy, however, is in transplant patients.

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More than 190,000 people are living with a transplanted kidney in the U.S., and 20,000 new kidney transplants are performed each year. The probability of rejection ranges from 3 to 8 percent, and the current gold standard for diagnosis of rejection is a biopsy. However, biopsies when the patient is showing allograft dysfunction are indeterminate in up to 30 percent of cases. A real need exists for biomarkers that accurately, inexpensively, less invasively and quickly identify subjects at risk of rejection.

The promise of cfDNA

The use of cfDNA for rejection surveillance is promising. Levels of circulating, donor-derived cfDNA (dd-cfDNA) act as a marker of rejection and can help distinguish antibody-mediated rejection from nonantibody mediated rejection. DNA degrades into nucleosomal units of about 166 bases, and these cfDNAs are cleared from the blood by the liver and kidney (with a half-life of about 30 minutes). Studies have shown that dd-cfDNA levels are often very low in stable transplant recipients. In contrast, levels of dd-cfDNA rise as the transplanted kidney is injured in rejection, and decreases following successful treatment. Interestingly, this dd-cfDNA technique determines the fraction of donor-derived nucleotides at multiple single nucleotide polymorphism locations rather than directly genotyping the donor or the recipient.  Therefore, it is less reliable when previous allografts are still in place or if multiple organs have been transplanted.

CfDNA levels that indicate rejection

The potential clinical utility of dd-cfDNA in monitoring acute rejection in kidney transplant recipients has been demonstrated in the DART study  in 384 patients treated in 14 centers including Cleveland Clinic (with Emilio Poggio, MD, as site principal investigator). Kidney transplant recipients were prospectively enrolled within one to three months of their transplant and followed longitudinally for two years, or enrolled at the time of clinical suspicion of rejection.  In 107 biopsies of 102 patients, 27 had active rejection. Median dd-cfDNA was 5.3-fold higher in active versus no active rejection, and dd-cfDNA was found to be better than serum creatinine or renal biopsy in identifying acute antibody-mediated rejection.

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Based on a total of 1,272 blood samples collected, a dd-cfDNA level above 1.2 percent is out of range and potentially abnormal in a renal transplant patient. Several laboratories currently offer this test, and more tests (including tests for rejection in heart transplants) are expected on the market in 2019.