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Liquid Biopsy Dx Looks for Donor-Derived DNA as Measure of Chronic Lung Transplant Rejection

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NEW YORK (GenomeWeb) – In the world of organ transplantation, there are three types of rejection that doctors and organ recipients have to worry about: hyperacute rejection, which takes place minutes after transplantation; acute rejection, which generally occurs in the first six to 12 months after transplant; and chronic rejection, which takes place over the long term as the organ slowly loses its function.

Lung transplant patients, in particular, have a very high incidence of chronic rejection, which leads to the shortest survival rate of any other solid organ transplantation. And the therapies that have been devised have largely been ineffective, likely because the rejection was discovered too late, according to a new study in EBioMedicine, a journal published by The Lancet.

"The clinical course of [chronic lung allograft dysfunction, or CLAD] is therefore progressive with irreversible allograft injury that ultimately leads to allograft failure," the authors — led by senior author and National Heart, Lung, and Blood Institute researcher Hannah Valantine — wrote. "Perhaps interventions at earlier stages before allograft injury becomes irreversible may delay or even prevent the development of CLAD and improve lung transplant outcomes. To date, no reliable clinical predictive biomarker exists."

Another problem, according to the researchers, is that the biomarkers that have been proposed remain limited because sample collection requires invasive procedures such as bronchoscopy, or because the biomarkers themselves have poor specificity or sensitivity, and/or the detection of CLAD has a significant time-lag to the irreversible clinical manifestations.

Chronic rejection is the Achilles heel of transplants, Valantine noted in an interview, adding, "We are pretty good at preventing acute rejection and diagnosing acute rejection, but we are really bad about diagnosing or predicting chronic rejection and that's what the patients are dying of."

To overcome those problems, the team has proposed a new approach based on a newly popular idea: liquid biopsy. The hypothesis is that allograft injury early after transplantation is predictive of CLAD and other poor outcomes, and observations in the clinic have shown that early post-transplant complications like primary graft dysfunction have a strong relationship with CLAD. This suggests that allograft injury early after transplantation is a precursor of CLAD, according to the researchers.

Further, even though lung transplant patients undergo monitoring with bronchoscopies, transbronchial biopsy, spirometry, and other testing, CLAD still occurs at a very high rate. But with a blood sample from the transplant recipient, a clinician could test for donor-derived cell-free DNA (ddcfDNA).

"What this test does is that we genotype the donor and the recipient and we look for SNPs that are different between donor and recipient. And then we take a sample of blood from the recipient after the transplant and we look for fragments of DNA that are coming from the donor," Valantine said. "And so that leads us to be able to calculate the percentage of the circulating DNA that's coming from the donor as a percentage of the total circulating DNA, and that gives us a readout of the amount of damage. If the heart or the lung is being damaged even slightly, little fragments of DNA get into the circulation and it's those fragments of DNA that are distinct from those of the recipient's DNA that we're able to pick up."

Right after a transplant, there is a high level of donor-derived DNA, but that goes down to virtually zero within 24 hours and ideally remains stable, she added. Subsequent blood tests can then detect if that stable situation is changing, and if there is a rise in the donor-derived DNA that's a signal that there is some damage going on to the transplant, indicating the early stages of rejection. 

For the study, Valantine and her colleagues monitored 106 lung transplant patients for the development of CLAD or death. They serially collected plasma samples from the patients in the early post-transplant period, and quantified the percentage of ddcfDNA. They found that the average percent of ddcfDNA in the first three months after transplant, which they called avddDNA, was variable between patients, correlating with clinical risk factors like older age and primary graft dysfunction.

"Patients with high avddDNA, signifying high early post-transplant allograft injury, showed 6.6-fold higher hazard of developing CLAD or death than those with lower avddDNA levels. More than half of these patients showed no clinical complication over the early post-transplant period," the authors wrote. "Patients with high avddDNA also showed lower lung function than those with low avddDNA levels. At a molecular level, they continued to show high ddcfDNA [percentage] levels beyond the early post-transplant period. Surprisingly, only one-third of the high ddcfDNA [percentage] levels were detected clinically or by monitoring tools. The rest were clinically unrecognized."

The bottom line, Valantine said, is that it's virtually impossible to identify early which patients are going to develop chronic rejection versus those that are not. And if they're followed using the usual pulmonary function tests, "those things become abnormal very late, and it's too late and you cannot reverse it," she added. "So, a unique aspect of this test is that level of donor-derived cell-free DNA measured in the first three months after transplant predicts those patients who are going to develop this chronic rejection distinct from those who are not. That's the beauty of this test. It's not so much about acute rejection. It's about this long-term outcome."

Indeed, the researchers concluded that avddDNA is a potential predictive biomarker for CLAD and death, and if it's validated, the blood test could be used to stratify transplant patients' risk for CLAD as early as three months after transplant in a non-invasive manner. If the test determines that a patient is at risk for chronic rejection, they then have the option for another transplant, to increase their dosage of immunosuppressive drugs, or to change their drug regimen altogether.

The next step, according to Valantine, is to refine the test and make it faster. Right now, it takes about 2.5 to three days to get a result, which isn't fast enough, she said. Ideally, results would arrive on a physician's desk 12 to 24 hours after a blood draw.

Valantine also noted that the team plans to do another study looking at what happens if the dosage of immunosuppressive drugs is increased earlier in order to stave off rejection. "If you have this elevated level of donor-derived DNA, what happens if you just simply use the current drugs that we have, but increase them early? Will that, in fact, be able to reverse the course?" she said. "We know that when we introduce those drugs later, it does nothing and the patients still declines. So, this test gives us an opportunity to treat earlier, which might make a difference for the patient."

Further, because the test is noninvasive, patients can be monitored as often as required by a clinician. Chronic rejection monitoring is especially intensive in the first three months after transplant, Valantine said. A blood test makes it possible to identify the patients most at risk for rejection and stratify them for continued monitoring. Eventually, she even envisions developing the technology into an at-home finger-prick test, much like a blood-sugar measurement test for diabetics.

If measuring ddcfDNA to check for organ rejection sounds familiar, that's because it's the basis for molecular diagnostics firm CareDx's kidney transplant monitoring test AlloSure, which was launched in October 2017. The Brisbane, California-based company uses ddcfDNA in blood to test for active rejection as a measure of kidney injury. CareDx has licensed the patents that cover ddcfDNA use for transplantation diagnostics from Stanford University, where it was originally developed by Valantine and her colleagues.

In a July 2017 study published in the Journal of the American Society of Nephrology, a team led by researchers at Baylor University and Washington University School of Medicine and including investigators from CareDx found that the elevation of ddcfDNA was significantly associated with active antibody-mediated rejection in kidney transplants.

Indeed, Valantine noted, the test her group is developing for chronic rejection could eventually apply to organs other than the lung. But the researchers chose to begin with lung transplants because those patients tend to develop chronic rejection within the first two years, whereas chronic rejection doesn't crop up in heart transplant patients, for example, until about four or five years after transplant.

No one is sure why the lungs are more prone to chronic rejection, but one reason might be that the it's the organ most in contact with the outside environment, Valantine speculated. "It is prone to infection, it is prone to environmental insults, and that results in an increased likelihood of damage to the airways in the lung. And somehow or other infection often triggers rejection," she said.

It's still too early to say how or when such a test could be commercialized, but CareDx is keeping an eye out. The NHLBI is leading the way in terms of ddcfDNA studies in lung transplantation, "and we have been following the work that they've been doing," said Robert Woodward, the company's senior vice president of R&D.

Further, he added, when AlloSure was developed, the clinical validation studies evaluated the use of the test for ddcfDNA measurement for acute T cell-mediated rejection, acute antibody-mediated rejection, and chronic active antibody-mediated rejection. "So, chronic rejection is something that we would expect AlloSure to pick up, especially in the early post-transplant period that was defined in the study," and for as many years post-transplant as necessary, Woodward said.

As such, the lung diagnostic as described in the new study would likely fit well with AlloSure and with CareDx's other products. But no plans have yet been made for licensing or any other similar arrangements, as it's still too early.

Woodward also noted that this study demonstrates the value of standardized diagnostic testing protocols — the collection of data from a series of time points meant to derive a prognostic factor. "I think [that] because there is a series of time points at which the test was run that enabled [the results] … the study points out the value of standardized surveillance tests in these patients," he said.