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Stanford Team Publishes Validation of Circulating DNA Sequencing for Heart Transplant Rejection Dx


NEW YORK (GenomeWeb) – A Stanford University method for sequencing circulating DNA to non-invasively detect heart transplant rejection has proved itself equal in accuracy to gold standard endomyocardial biopsy-based testing in a prospective study of 65 transplant patients.

The study, by a team led by Stanford's Stephen Quake and Kiran Khush, was published today in Science Translational Medicine.

Quake and Khush led the team that developed the sequencing approach, which is able to distinguish DNA from a donated organ from a recipient's own DNA and can detect spikes in the proportion of donor DNA indicative of transplant rejection, and published an early retrospective evaluation of it in 2011. At that time the group received a three-year grant from the NIH to conduct a prospective validation of the assay.

In their new study, the researchers describe the results of this validation, which found that the sequencing method could diagnose acute rejection accurately compared to results of current gold standard diagnostic biopsies, and was superior to gene expression-based rejection monitoring.

"We have shown the assay can be a very accurate marker of acute rejection," Khush told Clinical Sequencing News this week. However, she said, "any diagnostic test always deserves further validation, and really a clinical utility study … before it can be widely adopted."

She added that the group is looking to secure funding for such a project, but doesn't yet have concrete plans for a multi-center clinical validation.

The Stanford team's assay uses next-gen sequencing to distinguish between donor and recipient DNA by looking for differentiating SNPs. Both donor and recipient genotypes are collected to determine distinguishing SNPs. Then, after sequencing circulating cell-free DNA from the recipient's blood, the researchers count the number of reads at SNP positions that differ between donor and recipient in order to determine the proportion of donor DNA.

In their initial study on the method, the team established that elevated donor DNA levels could be used to diagnose rejection episodes.

According to Khush, the group's methodology has changed in many ways since their initial publication of the method. She said that in the initial experiments, the team performed DNA isolation and library preparation by hand but has since automated these steps.

Improvements in sequencing technology, from the Illumina GA to the Illumina HiSeq2000 and 2500, have also increased the accuracy of the assay, Khush said, as well as advances in algorithms used to analyze sequencing data.

"If you look back at our retrospective study, the threshold for calling a positive was much higher because the inherent error rate was much higher," Khush said.

In their prospective study, the team set out to test all heart transplant patients admitted at Stanford during the three-year period. Khush said that the group recruited a total of about 120 subjects, but for the purposes of the STM study only analyzed 63 of those — 21 pediatric and 44 adult cases.

The researchers collected a total of 565 serial blood samples from the study cohort on which they performed their cell-free DNA sequencing assay, comparing the results to diagnoses made by endomyocardial biopsy.

According to the study authors, after excluding samples with high technical errors and those collected within 14 days after transplant, the group calculated an area under the receiver operating characteristic curve for the sequencing assay of 0.83 at cell-free circulating DNA threshold of 0.25 percent.

The team reported that the sensitivity of the assay in comparison to biopsy is comparable to the rate of agreement between individual biopsy evaluations by different pathologists. "One could not expect a much better performance by any other test, even a 'perfect,' test," the group wrote.

The researchers also reported that the sequencing assay's performance appeared to improve with time after transplant, reaching an AUC of 0.91 at four months or longer after transplantation. In addition, the assay's concordance with biopsy was higher for younger patients and decreased with patient age.

Additionally, the group investigated the method's potential for early diagnosis of acute rejection, examining patterns of cell-free DNA in samples taken prior to moderate or severe rejection episodes. They found that the fraction of donor DNA was significantly elevated for samples collected up to five months before a rejection event.

According to Khush, the main next step toward bringing the assay to the clinic is to perform additional validation and clinical utility studies.

In its current form the test relies on shotgun sequencing, which Khush said is an advantage in that its breadth could allow for a multi-use assay to diagnose both acute rejection and other confounding conditions like viral or bacterial infections. "It's clinically relevant because sometimes patients present with non-specific symptoms and we don’t know if they have acute rejection or infection. They're certainly at risk for both since they are immunosupressed," Khush said.

In a recent study in Cell, she and her colleagues published data from an analysis of viral sequences in the circulating DNA of transplant patients using the same cell-free DNA method.

On the other hand, Khush said, in the context of clinical management of heart transplant patients, whole-genome sequencing may still be too cumbersome and costly to make sense, so adapting the assay to utilize other approaches like targeted sequencing may be another next step.

The Stanford team's method has been licensed by molecular diagnostics company CareDx, which markets a gene expression-based heart transplant rejection diagnostic called AlloMap. Earlier this year the firm acquired transplant diagnostic company ImmuMetrix, which was founded by Stanford's Quake, who now serves as a scientific adviser to CareDx.

Khush said that CareDx is likely planning to do more internal testing and development of the assay before it would be ready to launch it commercially.

CareDx did not respond by press time to a request for comment on its plans for commercializing the Stanford circulating DNA sequencing method.

Earlier this week the firm announced a supply agreement with Illumina for next-generation sequencing technologies. The company's President and CEO Peter Maag said at the time that Illumina's sequencing technology would support development and commercialization efforts as CareDx expands its product pipeline from the gene expression-based AlloMap to cell-free DNA-based tests.

According to Khush, the Stanford team is also exploring the utility of their assay for other types of solid organ transplants. As they recruited heart transplant patients for the prospective study, the researchers have also been recruiting lung cancer patients in parallel — about 80 so far — and are conducting a similar analysis of that data.

Lung transplants provide a trickier target, Khush said, because the grafts are exposed to the environment and thus cell-free donor DNA levels could rise in recipients' blood due not only to organ rejection but also to infection of the transplanted organ.

However, she said, considering the group's sequencing methodology, "it should be relevant for any solid organ transplant where you are looking for evidence of graft damage by assaying for donor cell death."