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Stanford Team Develops Non-invasive, DNA-based Transplant Rejection Test

By a GenomeWeb staff reporter

NEW YORK (GenomeWeb News) – Stanford University researchers have developed a non-invasive, sequencing-based strategy for gauging rejection in organ transplant recipients.

In a study appearing online last night in the Proceedings of the National Academy of Sciences, the team reported that levels of cell-free donor DNA jump in blood samples from transplant recipients experiencing rejection compared to those who aren't. From there, the researchers came up with a "genome transplant dynamics" strategy to test for transplant rejection by tracking donor DNA via genotyping and high-throughput sequencing — an approach they validated in a handful of heart transplant patients.

"Because transplanted organs have genomes that are distinct from the recipient's genome, we used high-throughput shotgun sequencing to develop a universal non-invasive approach to monitoring organ health," co-corresponding author Stephen Quake, a bioengineering researcher at Stanford, and co-authors wrote. "Our results demonstrate that cell-free DNA can be used to detect an organ-specific signature that correlates with rejection, and this measurement can be made on any combination of donor and recipient."

Individuals receiving heart transplants are typically monitored for rejection via endomyocardial biopsies that require nipping off small samples of transplanted heart tissue. Individuals deemed at risk of rejection are then treated with anti-rejection drugs. But, researchers explained, such biopsies are expensive, invasive, and sometimes have serious side effects. They argue that a non-invasive, blood-based alternative would not only spare patients the discomfort of biopsies but might also curb unnecessary treatment.

"Heart transplant recipients undergo at least 12 tissue biopsies during the first year after their transplant and two or three each year for about four additional years," co-corresponding author Hannah Valantine, a cardiovascular medicine researcher at Stanford, said in a statement. "The idea that we might now be able to diagnose rejection earlier and non-invasively is very, very exciting."

Valantine and others reported last year in the New England Journal of Medicine that they had identified a blood-based expression signature for predicting heart transplant rejection. That work spawned a US Food and Drug Administration approved test known as AlloMap, which tracks the expression of 20 genes to get clues about whether the transplant recipient's body is mounting an immune response against the new organ.

For the current study, the team took a slightly different tack, focusing on finding ways to look at the condition of the donated organ based on the amount of donor DNA seeping into the recipient's bloodstream.

Quake and his colleagues previously developed similar strategies for detecting chromosomal abnormalities in fetal DNA circulating in an expectant mother's blood samples.

To get a better handle on the typical levels of donor DNA present in transplant recipient blood samples — and determine how these change during transplant rejection, the team first used microfluidics-based digital PCR to test cell-free blood samples from nine heart transplant recipients.

Of these, three represented cases in which the donor and recipient belonged to the opposite sex — a situation that allowed the researchers to distinguish between donor and recipient DNA using a Y chromosome marker.

Based on these initial samples, and follow-up experiments involving 39 more sex-mismatched heart donor cases, the team found evidence supporting the notion that heart transplant rejection coincides with a rise in the donor DNA in a recipient's cell-free blood samples, up from a typical level of less than one percent of total detectable DNA.

In an effort to come up with a DNA-based test that's more broadly applicable to cases where the donor and recipient are of the same sex, the researchers first mixed DNA from two previously genotyped HapMap cell lines in combinations ranging from 1.5 to 7.5 percent "donor" DNA and sequenced the mixture by single-end sequencing with the Illumina GAII platform.

After demonstrating that SNP signatures can be used to distinguish "donor" from "recipient" DNA in these cell line mixtures, the team applied the strategy to samples from actual heart transplant cases.

Three of the samples came from women who had gotten heart transplants from male donors, while the remaining four recipients were men with hearts donated from other men. Two of the female recipients and all four of the male recipients had experienced transplant rejection.

Before sequencing cell-free DNA from transplant recipient blood samples, researchers genotyped donor and recipient DNA from banked cell samples using the Illumina Omni1-Quad BeadChip. They then used shotgun sequencing to sequence DNA from recipient blood samples, working out the ratio of donor and recipient DNA from SNP data.

Again, the researchers saw a spike in detectable donor DNA signatures in recipient blood samples that corresponded to transplant rejection. "In every case we could see an increase in donor DNA in the patient's blood before the biopsy itself showed any sign of rejection," Valantine said in a statement.

Though they emphasized that more research is needed to test additional patients and assess rejection cases that are intermediate or less severe, those involved in the study argue that the genome transplant dynamics, or GTD, approach could eventually curb the need for biopsies to test for transplant rejection.

In particular, they noted that it may be advantageous to use GTD in combination with AlloMap, which provides complementary information on recipient immune activity and has distinct sources of error compared to the GTD approach.

Moreover, the team argues that a similar method may be useful to test for rejection following other types of organ transplants as well. "As GTD is not particularly dependent on physiology specific to the heart, it also has the potential to be used in the setting of other solid organ transplants (such as kidney, lung, and liver), where DNA from the transplanted organ may also exist in the recipient's plasma," they concluded.

Quake, Valantine, and first author Thomas Snyder, a post-doctoral researcher in Quake's bioengineering lab at Stanford, have reportedly filed for a patent related to the new approach. The team has also secured a three-year, $2 million grant from the National Institutes of Health for prospective studies of heart and lung transplant rejection and plans to prospectively test GTD for predicting transplant rejection.

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