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Heart Transplant Cell-Free DNA Test Enables Earlier Detection of Acute Rejection Than Biopsy


NEW YORK – The authors of a recent study on heart transplant rejection are hoping that their work could lead to improved organ rejection diagnostics, and could also help explain why African American patients tend to suffer poorer outcomes after heart transplant surgery compared to Caucasian patients.

In a paper published earlier this month in Circulation, researchers from the Genomic Research Alliance for Transplantation (GRAfT) described their findings from a multicenter, prospective cohort study, in which they assessed the performance of donor-derived cell-free DNA (ddcfDNA) as a biomarker in the blood of heart transplant patients to monitor for acute rejection (AR). They compared it to endomyocardial biopsy (EMBx), which is the current gold standard monitoring technique for heart transplant patients.

The researchers recruited 171 heart transplant patients who were monitored with EMBx post-transplant for a median of 17.7 months and measured ddcfDNA in their blood by shotgun sequencing. They also collected histopathology data to define AR and its two phenotypes — acute cellular rejection (ACR) and antibody-mediated rejection (AMR).

In endomyocardial biopsy, "the physician will get a piece of the heart and look under the microscope whether there is rejection or not. As you can imagine, this method is invasive and has other limitations," said Sean Agbor-Enoh, an instructor of medicine at the Johns Hopkins University School of Medicine and co-first author on the study. In his view, ddcfDNA testing certainly has the advantage of being less invasive, but also offers the advantage of being more sensitive than biopsy and finding additional injuries to the transplanted organ that a biopsy could miss.

Also, he added, "it picks up rejection — particularly rejection that is orchestrated by antibodies — two to four months before the biopsy shows any signs of rejection."

According to Agbor-Enoh, biopsy is conducted both as a routine test and as a clinically indicated test. For the former, patients go to the hospital at fixed times after transplantation and will undergo biopsy, whether they seem fine or not. "We call that surveillance biopsy," he said. "At Johns Hopkins, where I'm a physician, [patients] typically get somewhere between 12 to 20 of those surveillance biopsies within the first two years of transplantation. So it's a lot."

In addition to surveillance biopsy, patients also undergo clinically indicated biopsies, for example if they're showing symptoms of rejection or an echocardiograph indicates that something could be wrong, he said.

According to the study, the 1,392 EMBx and 1,834 ddcfDNA measurements showed that median ddcfDNA levels decayed to 0.13 percent by 28 days after surgery, but increased again if the patient underwent AR. The rise was detected between 0.5 and 3.2 months before a histopathological diagnosis of ACR and AMR. Further, they wrote, a 0.25 ddcfDNA percentage threshold indicated a negative predictive value for AR of 99 percent and would have safely eliminated 81 percent of EMBx for the patients in the study.

"In our mind, this test would be best used as surveillance," Agbor-Enoh said. "So, the patient gets this test, and if this test is negative, then you say there is no rejection— it is 99 percent good in telling you that there is no rejection. Now, if this test is positive, then you know there is something wrong."

A positive test would be a trigger for a biopsy, which would help a physician determine the exact type of rejection, and therefore determine the proper course of treatment, he added. So although this test wouldn't completely eliminate biopsy, it could help to avoid a significant number.

Importantly, the researcher added, ddcfDNA sequencing showed that AMR and ACR have distinctive characteristics. Specifically, AMR showed three distinct ddcfDNA patterns compared to ACR.

First, AMR showed higher percentages of ddcfDNA compared to ACR. For example, levels of ddcfDNA in pathological AMR1 were about 11 times higher than in ACR grade 1, and pathological AMR 2 and higher showed five times higher levels of ddcfDNA in the plasma of heart transplant patients than ACR grades 2 and higher.

The researchers also observed different trends in ddcfDNA before diagnosis of AMR versus ACR: a rise in ddcfDNA preceding diagnosis was not commonly observed with ACR, but was commonly detected before AMR diagnosis. They saw this rise in only two out of 17 patients with ACR but observed it in 12 out of 15 AMR episodes, with sufficient data to assess that levels of ddcfDNA increased at a median of 3.2 months before AMR diagnosis.

Finally, the fragment length of sequenced cfDNA was distinct in AMR and ACR. In AMR, the cfDNA fragments were shorter than in ACR or in controls with no rejection, the researchers said. Similarly, the percentage of guanosine-cytosine bases was highest in AMR compared to ACR or controls. And although ACR, AMR, and controls showed similar percentages of reads mapping to exons, both AMR and ACR showed a higher percentage of reads mapping to promoters and lower percentages of reads mapping to introns and intergenic regions compared to controls.

This study isn't the first evidence that ddcfDNA can be used to monitor heart transplant patients. Indeed, CareDx, Natera, and several other companies already market transplant diagnostics for various organs based on the detection of ddcfDNA. CareDx specifically markets a multimodal test called HeartCare that combines its AlloSure Heart ddcfDNA platform with its AlloMap Heart gene expression profiling test to monitor for graft injury. The difference between CareDx's technology and the new study, Agbor-Enoh pointed out, is that the GRAfT Consortium researchers sequenced about 3.2 million positions in the genome of the donor DNA, whereas commercially available tests sequence only a fraction of that number.

The researchers are hoping that this study provides additional clinical evidence for this type of testing so that doctors can feel more comfortable using the commercial tests to start monitoring their patients for rejection. Further, he added, though the researchers aren't planning to commercialize their own assay, they're hoping to collaborate with private companies "to ask the question whether their tests have a similar performance as this test that we used in this study. Then patients and doctors would have shovel-ready commercially available tests that can be used to take care of patients."

In an email, Sham Dholakia, CareDx's senior VP of medical affairs, noted that the investigators used a shotgun sequencing approach compared to CareDx's use of transplant-specific SNPs. He also said that the data "support the utility of ddcfDNA surveillance for heart transplant patients as we see clinical patient management continue to grow."

Natera declined to comment for this article. The company's ddcfDNA-based Prospera transplant test is currently used for monitoring kidney transplant rejection, but it has hinted at possibly using it for monitoring other organ transplants in the future.

Agbor-Enoh noted that sensitive tools such as cell-free DNA could be used to address the racial disparities in transplant outcomes. "Patients of African descent [and other minority patients] seem to have poorer outcomes after heart and after lung transplantation compared to patients who are white," he said. "The question becomes, if we now have sensitive tools that can pick up rejection and pick it up early, will these patients benefit from that?"

Right now, when rejection is found in these patients, it's sometimes found too late, he added. And even when treatment is administered, the patients don't recover well. The sensitivity of ddcfDNA could help pick up rejection in its earlier stages, he noted, which might prove to be useful in reducing the disparities in outcomes.

Agbor-Enoh — along with Inova Heart and Vascular Institute researcher Palak Shah and Stanford University researcher Hannah Valantine, who were co-first author and senior author on the Circulation study, respectively — is currently working on mechanistic studies using cell-free DNA to understand rejection in African-Americans compared to white patients. They're hoping to release preliminary findings in the next few months and lay the groundwork for a clinical study that could reduce the poorer outcomes in African-American patients after heart transplantation. 

Overall, Agbor-Enoh said he believes diagnostic medicine — and transplantation medicine in particular — is going to rely heavily on the detection of cell-free DNA in the future because of its sensitivity.

"[These tests] pick up disease before the patient shows any clinical sign or before it even shows up on biopsy," he said. "You can imagine, [for example], could we pick up dementia years before the patient shows any clinical signs of dementia? That, in my view, is the direction that I think medicine is going, already. And tools like this, like cell-free DNA, can really help us be able to identify patients who will develop significant diseases in the future."