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Stanford Team Tests Cell-Free DNA Assay to Monitor Lung Transplantation

NEW YORK (GenomeWeb) – A research team in Stephen Quake's Stanford University laboratory has developed a next-generation sequencing-based assay that analyzes circulating cell-free DNA in lung transplantation patients for signs of rejection and infection.

Reporting their work today in the Proceedings of the National Academy of the Sciences, the researchers compared their assay to transbronchial biopsy, the current gold standard, for monitoring transplant rejection in 51 patients. They found that their assay was "informative of acute and chronic rejection," with an area-under the curve of .9, a sensitivity of 100 percent and specificity of 73 percent at a threshold of 1 percent cell-free donor DNA.

Although lung transplantation is offered to patients with end-stage pulmonary disease, clinical outcome is poor, with a median survival of just 5.3 years. Complications following the surgery include injuries sustained during the procedure, infection, and two types of rejection: acute cellular rejection, which occurs in the first year post-transplant in about 35 percent of patients, and chronic rejection, which is the leading cause of death for transplant patients.

Currently, the only way to diagnose rejection is through transbronchial biopsy, which is invasive and has limited diagnostic abilities. Diagnosing infection typically requires ordering a suite of pathogen-specific tests.

As such, the Stanford team decided to develop an NGS-based test that could noninvasively monitor both rejection and infection. The test relies on shotgun sequencing of circulating cell-free DNA.

The method, which Stanford researcher Iwijn de Vlaminck described at last year's American Society for Human Genetics meeting, is based on a similar assay the group developed to detect heart transplant rejection last year.

Prior to performing transplantation, both the donor and recipient are genotyped via a SNP array. After the procedure, shotgun whole-genome sequencing is performed on circulating cell-free DNA from the patient. The researchers then use the SNPs to determine the proportion of cell-free DNA molecules originating from the donor.

To determine the level of donor DNA that constitutes a rejection, researchers first examined patients that had no evidence of rejection or infection. The researchers noted that immediately after transplantation, patients typically had high amounts of circulating donor DNA, but those levels dropped over time — similar to the results they found with heart transplantation patients. In addition, the levels of donor cfDNA differed depending on whether the patient was receiving two lungs or one.

Next, the researchers evaluated donor cfDNA in patients that had been diagnosed with rejection. From one patient that had been diagnosed with moderate rejection, the researchers found that donor cfDNA at the time of rejection diagnosis was 14.7 percent higher than baseline levels. In a patient diagnosed with severe rejection, donor cfDNA was 15.2 percent higher than baseline levels.

Looking at an aggregate of the sequencing data from the 398 time points of all 51 patients, the researchers found that it tracked with clinical observations of rejection. In addition, at some time points where there were not yet clinical signs of rejection, donor cfDNA was clearly elevated.

Looking at the concordance of the cfDNA test with transbronchial biopsy grades, the researchers found that the cfDNA test had an area under the curve of .9, 100 percent sensitivity, and 73 percent specificity at a donor cfDNA threshold level of 1 percent.

Finally, the researchers wanted to see whether they could find signs of pathogen infection. Cytomegalovirus is a common infection in organ transplant patients. The researchers found that they were able to identify sequences from the CMV genome assay. When compared to clinical testing for the virus, the cfDNA assay had an area under the curve of .91. The researchers also found evidence of infection by other viruses for which patients had not been screened clinically, including the community-acquired respiratory infection adenovirus; polyomavirus, which is a frequent cause of rejection in kidney transplantation but not routinely screened for in lung transplantation; and human herpes virus.

The assay "has the potential to become an important tool for lung transplant surveillance within the next few years, considering the high incidence of acute rejection and difficult-to-diagnose infectious complications; the numerous limitations of transbronchial biopsy in rejection surveillance; the lack of alternative, noninvasive tests of graft injury after lung transplantation; and the relatively low cost associated with the assay," the authors wrote.