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Stanford Team Tests NGS of Circulating cfDNA to Diagnose Organ Transplantation Rejection and Infection

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NEW YORK (GenomeWeb) – Researchers at Stanford University are exploring next-generation sequencing of circulating cell-free DNA as a means to noninvasively diagnose rejection and infection in organ transplant recipients.

The group has tested the whole-genome technique on heart, lung, and kidney transplant recipients and thinks that such a test could eventually compete with current gold standards for monitoring transplant rejection, which are invasive, expensive, and subjective.

In addition, the team has found that the same sequencing technique can identify the presence of pathogens to detect infection, and they are also studying immune repertoire sequencing to monitor immune system fitness.

Iwijn de Vlaminck, a postdoctoral student in Stephen Quake's laboratory at Stanford University, discussed the work in a presentation at the American Society of Human Genetics meeting in San Diego last month and also in a subsequent interview with GenomeWeb.

The Stanford team first demonstrated in a publication in Science Translational Medicine in June that sequencing circulating cell-free DNA could identify rejection in heart transplant recipients.

Eventual commercialization of a sequencing-based transplant rejection diagnostic could potentially come from CareDx, de Vlaminck said. The company bought startup ImmuMetrix in May, which was co-founded by Quake, and has licensed the Stanford team's method.

Since the Science Translational Medicine study, the group has tested the technique in lung and kidney transplant patients, de Vlaminck told GenomeWeb.

Being able to diagnose lung rejection noninvasively is especially critical, de Vlaminck said, because there are currently no noninvasive markers and rejection diagnosis must be done by lung biopsy or a pulmonary test measuring lung capacity.  In addition, median survival rate for single-lung transplants is about 4.6 years and for double-lung recipients it's 6.6 years.

"There is a great need for new noninvasive markers for graft surveillance," de Vlaminck said.

The group tested the technique on 51 patients, seven of whom had received a single transplant and 44 of whom had received bilateral transplants. They sequenced the whole genomes of recipients' plasma DNA to about 1x coverage.

Before sequencing, both the donor and recipient had been genotyped. Immediately after transplantation the levels of donor DNA were high, but then dropped. Over the course of time, the researchers observed that in patients that experienced rejection events, the amount of donor DNA rose again, peaking in conjunction with rejection events that were clinically diagnosed by biopsy.

The group found that the sequencing method has an area under curve score, a common metric used to gauge test performance, of 0.9, which de Vlaminck said was slightly better than the AUC for monitoring heart transplant rejection.

Another interesting finding, de Vlaminck noted during his ASHG presentation, was that sequencing detected not only human DNA, but viral and bacterial DNA as well. In a publication in Cell last year, the group sequenced cell-free DNA from 656 plasma samples taken from 96 transplant recipients and found that antivirals and immunosuppressant drugs alter the virome of patients.

Specifically, de Vlaminck said, it appears that levels of anellovirus, which is abundant in humans but does not cause symptoms, fluctuate in transplant patients before and after transplantation and are dependent on their dosage of immunosuppressants and whether they are rejecting the organ or not.

When a patient is on high levels of immunosuppressants, anellovirus load increases, de Vlaminck said. But during rejection events, even if the patient is still taking immunosuppressants, anellovirus load increases.

During organ rejection events, the levels of anellovirus actually decrease, de Vlaminck said, likely because the immune system has kicked back in. The next goal is to establish a threshold for diagnosing rejection, and then one sequencing-based test could potentially be used to look for two different biomarkers to monitor rejection — specifically, levels of donor DNA and anellovirus load.

Another potential measure of rejection is also infection, de Vlaminck said. Some infections can directly or indirectly cause injury to the graft tissue, so being able to detect various pathogens could help monitor and diagnose infection.

"Infection can trigger rejection and also cause graft damage," he said. "Based on our observations, it will be important to extend the test to do not only surveillance [of donor DNA level] but also to monitor infection."

He said that while the 1x shotgun sequencing approach is able to detect the presence of bacterial and viral species, "the abundance of those sequences are low," and the group is looking for ways to enrich those sequences. One way would be to design probes that can capture those sequences and to do that at the same time as the whole-genome shotgun sequencing.

Detecting infection is especially important for lung transplants, de Vlaminck noted. In work performed since the Cell publication, he said the group has "been able to pick up signals from various types of infections before being clinically diagnosed at a great frequency in that patient population."

Another avenue of research the group is pursuing is immune repertoire sequencing. "Initially, we were interested to see if we could use it to gauge the level of immune suppression," de Vlaminck said. Currently, adult transplant recipients receive the same dosage of immunosuppressants, but not everyone responds to the drug in the same way. The goal is to develop a method to measure a person's "level of immune fitness."

For instance, de Vlaminck said, there is an inverse correlation between anellovirus load and a patient's dosage of immune suppressants. But, while this is an indirect measure of immune fitness, another option is to sequence the immune repertoire, which would enable the detection of "immune system activation during graft rejection."

While this work is still early in research, de Vlaminck said he thought that immune repertoire sequencing of plasma could serve as a complementary test to shotgun sequencing.

"Information from both should allow physicians to make better treatment decisions, which is the ultimate goal," he said.

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