SAN FRANCISCO (GenomeWeb) – A Cornell University research team aims to harness single-cell and cell-free DNA technologies to develop better, noninvasive methods to diagnose complications from kidney transplantation.
More than 15,000 kidney transplantations are performed each year in the US, and although such surgeries have become more successful — with a more than 90 percent success rate after one year — longevity is still limited. Current methods for identifying rejection require invasive biopsies and typically do not catch it in its early stages. Meanwhile, diagnosing infections is time consuming as physicians must test one pathogen at a time.
The Cornell researchers, led by Iwijn De Vlaminck, recently received a five-year, $2.3 million grant from the National Institutes of Health to come up with noninvasive and more sensitive genomic-based assays to identify kidney transplantation problems.
"The goal is to develop new technologies that dissect the heterogeneity of DNA molecules and cells that you can isolate from urine," De Vlaminck said. The researchers plan to use metagenomic and whole-genome sequencing to analyze cell-free DNA and single cells to identify markers of rejection and infection. In addition, De Vlaminck said a key component will be to use epigenetic marks identified in the cfDNA to trace the DNA fragment back to the tissue of origin. "We think that by measuring epigenetic marks, we can get a detailed view of the tissues that are being injured in transplantation due to either rejection or infection," he said.
De Vlaminck has been working on this problem for years. As a postdoctoral researcher in Stephen Quake's lab at Stanford University, he developed NGS-based techniques to identify rejection in both heart and lung transplant recipients.
In that work, De Vlaminck and colleagues also found that sequencing detected viral and bacterial DNA, and discovered that levels of annellovirus, which is abundant in humans but does not cause symptoms, can help distinguish when a patient is rejecting the graft organ.
Under the NIH grant, De Vlamnick plans to expand on this research. In collaboration with Manikkam Suthanthiran, a professor at Weill Cornell Medical College who focuses on transplantation immunology, the researchers will analyze more than 700 urine samples collected from a cohort of kidney transplant recipients.
In one study, the researchers plan to isolate single cells from the samples and perform RNA sequencing studies to look at gene expression profiles associated with rejection, infection, and graft inflammation. For this project, De Vlaminck said the team would be evaluating commercially available technologies for single-cell analysis, taking into consideration the number of cells that each platform can analyze as well as cost. "Those are the two biggest constraints," he said.
De Vlaminck's lab has also been developing methods to analyze cfDNA. While techniques to analyze cfDNA from plasma are commonly used for liquid biopsy applications in cancer and noninvasive prenatal testing, analyzing cfDNA from urine is less common and is slightly different, De Vlaminck said. The main difference is that urinary cfDNA is even more fragmented than cfDNA in blood, with fragments typically shorter than 100 base pairs, De Vlaminck said.
In a study published last year in Scientific Reports, De Vlaminck described a library preparation method for cfDNA that involves single-stranded ligation as opposed to more common double-stranded ligation methods. The single-stranded library prep is especially advantageous when analyzing urinary cfDNA, De Vlaminck said, because it does not require a size selection step. So, "it's sensitive to molecules with long overhangs, that are damaged, and that have nicks," he said, enabling a greater diversity of cfDNA fragments to be captured.
Following the Scientific Reports study describing the method, De Vlaminck's team has since applied the library prep approach to analyze cfDNA from 115 urine samples from 67 kidney transplant recipients. In a publication posted to the BioRxiv preprint server, the team demonstrated that the method worked reliably on just one milliliter of urine. Using a metagenomic sequencing approach, the researchers were able to detect pathogens responsible for some patients' urinary tract infections, viruses that are often difficult to detect, and even the presence of antibiotic resistance genes.
Detecting infection in kidney transplant recipients will be a key component of the upcoming research. Bacterial urinary tract infections affect more than 20 percent of kidney transplant recipients in the first year after transplantation and at least half of recipients within the three years following transplantation. In addition, some 5 percent to 8 percent of patients experience kidney damage caused by viral infections during the first three years. Currently, bacterial infections in transplant recipients are diagnosed by culturing the bacteria, which can frequently miss the culprit organism since many bacteria are not culturable. Culture-based methods also cannot detect viral infections.
By using a metagenomic sequencing approach, De Vlaminck said the researchers will be able to go beyond simply identifying the organisms that are present in a sample. Metagenomic sequencing will enable analysis of the "structure of genomes and how fast particular bacterial populations are growing and replicating," he said.
In addition, analyzing the epigenetic marks will help researchers pinpoint whether bacteria or viruses are more prevalent in a specific tissue. "It will enable more than just the detection of an infectious agent in the urinary tract," De Vlaminck said, including a better understanding of which tissues are infected and the host's response to that infection. Specifically, the researchers plan to focus on cytosine methylation and DNA binding factors to understand the cell and tissue types of the cfDNA.
De Vlaminck said that the researchers would first develop and hone all their different methods for analyzing single cells and cfDNA, after which the goal would be to apply the techniques on matched samples.
"In particular, metagenomic analysis with analysis of single cells could be very informative of infection biology and inflammation," he said.
He said that ultimately he hopes the research would shed light on the biology of transplantation and uncover biomarkers for rejection as well as ways to monitor infection that could lead to improvements in the way patients are treated. He said these techniques could be developed into commercial tests, citing previous work on heart transplantation that has since been commercialized by CareDx.
"Sequencing-based assays can lead to vast improvements over existing technologies," he said, particularly in the field of solid organ transplantation. "There's a big unmet need," he said. "Patients are often either over immunosuppressed or under suppressed," which can lead to either infection or rejection that "limits the lifespan of the procedure," he said. "There's a need for more precise tools to monitor these patients."