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Chronix Biomedical Develops Organ Transplant Rejection Assay on Bio-Rad ddPCR Platform

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Scientists from molecular diagnostics firm Chronix Biomedical and University Medical Center Göttingen in Germany have developed an assay to measure circulating cell-free graft DNA as an early indicator of organ transplant rejection.

The assay uses Bio-Rad's QX100 Droplet Digital PCR system to absolutely quantify cell-free DNA released into the bloodstream by dying cells from the transplanted organ as a percentage of total circulating organ-derived DNA in a patient's plasma.

In a pilot study, the assay was able to quantify less than 2 percent of graft cell-free DNA in the plasma of solid organ recipients with a one-day turnaround time, and was also able to track increasing percentages of graft cfDNA in organ rejection patients much earlier than conventional testing methods.

Chronix and the Göttingen researchers are now beginning larger validation studies with the goal of developing a so-called "liquid biopsy" for organ transplant monitoring that might be adopted by hospitals and transplant centers, Chronix CEO Howard Urnovitz recently told PCR Insider.

Further, Urnovitz said, Chronix hopes to employ droplet digital PCR in a similar fashion as part of an assay for minimal residual disease for breast and other cancers, and offer that test through its CLIA lab, expected to open by 2015.

Currently, the most common method for detecting organ rejection in transplant patients is to monitor levels of creatinine, aspartate aminotransferase, and bilirubin in the bloodstream. However, in many cases more than 50 percent of transplanted organ function may be lost by the time physicians are able to assess the levels of these molecules.

"The problem in transplantation is that … at the present time [there are] no good biomarkers that give you reliable information on … rejection and early organ damage," Michael Oellerich, a professor of clinical chemistry at University Medical Center Göttingen and head of the recent pilot study, told PCR Insider.

"We of course [monitor patients] for many years, and can only primarily detect toxicity, but it doesn't tell you very much about efficacy … the effect on the immune cells … and the susceptibility to immunosuppressive drugs might vary from patient to patient," Oellerich added. "That is why we thought coupling this interesting new technology, droplet digital PCR, with circulating cell-free DNA [was] an excellent opportunity to monitor transplant patients."

Chronix, headquartered in San Jose, Calif., with laboratories in Göttingen, Germany and Brookings, SD, is focused in general on developing diagnostic tests based on cell-free DNA. The company has used a variety of nucleic acid analysis technologies – mostly next-generation sequencing – to identify and validate cell-free DNA markers of disease.

For instance, as reported in June by PCR Insider sister publication Clinical Sequencing News, Chronix presented data at the American Society for Clinical Oncology meeting in Chicago from a study in which it used target enrichment and sequencing to identify patient-specific tumor markers in circulating cell-free DNA in post-surgical breast cancer patients as a strategy to monitor minimal residual disease.

"We've used almost every sequencer there is – NGS from [Roche] 454, [Life Tech] ABI Solid, Illumina HiSeq 2000, and we'll be getting the [Life Tech] Ion Proton shortly," Urnovitz said.

However, even though NGS has also proved useful for indentifying biomarkers in cell-free DNA from transplant patients, "for a transplantation test, we needed to have something that can be done in one day," Urnovitz said. "Even with Ion Proton, it would take a couple of days. That's why the team decided to give droplet digital PCR a go."

Furthermore, the cost of sequencing – though rapidly dropping – is still not low enough to make it ideal for use in a common diagnostic or disease monitoring assay, according to Ekkehard Schütz, CTO of Chronix and senior author on the study. Digital PCR – and particularly droplet-based digital PCR – fits the bill. The company chose to work with Bio-Rad because its droplet digital PCR was the most mature product on the market at the time the study began.

"There are a couple of advantages of the Bio-Rad system," Schütz said. "You don't need to use a $150 chip, even if you only have one or two samples … you can do one sample if you wish, and the costs are still not increasing like crazy. The other thing is … our experience is that it really gives us a robust readout. And that's what we really need most – it's something you can do with a simple quantitative PCR, but you want to have the precision and reproducibility. We have to distinguish between [a few low] percentages of graft DNA, and for that there is no doubt you need a digital PCR system."

Schutz said that a blood sample from a healthy patient will contain something on the order of 1,500 to 2,000 copies of circulating DNA per milliliter. In organ transplant patients, he added, "we have found, if everything is normal, around 1 percent of the circulating DNA comes from the graft. In liver it will be a little more because the organ is bigger, then perhaps we have something like 3 percent. But for kidney and heart it's around or below 1 percent, if the organ is healthy."

The group presented the results of its pilot study in a poster at the American Association of Clinical Chemistry annual meeting held earlier this month in Houston, where it received a National Academy of Biochemistry Distinguished Abstract Award.

The researchers used plasma samples from four patients soon after liver transplantation and from 11 stable liver transplant recipients. They selected a number of SNPs from public databases, especially considering those with high minor allelic frequencies, and established a total of 37 TaqMan assays.

Next, they extracted cell-free DNA from plasma samples and subjected it to library preparation and PCR on a Roche LightCycler 480 to determine useful heterologous SNPs. Finally, they used those SNPs for graft DNA quantification using the Bio-Rad QX100 Droplet Digital platform.

Among their top-line findings, the researchers saw that about 5 percent of the total cell-free DNA on average was of donor origin, but saw values of greater than 15 percent in patients in the first five days of a transplant procedure.

In addition, one patient with a biopsy-proven rejection at day 43 post-transplantation showed a steep increase in graft cell-free DNA to more than 50 percent on day 32 – several days before conventional aspartate aminotransferase and bilirubin tests detected a significant increase.

Led by Oellerich's lab at University Medical Center Göttingen, Chronix and collaborators are now moving forward with a larger validation study with "as many transplant patients as possible," Urnovitz said. And, for the time being, the company plans to move forward with droplet digital PCR as the detection technology.

"From a business perspective we are looking to get this into … [the] roughly 240 transplant centers [in the US]," Urnovitz said. "We want to try and get this so it's actually in the laboratories of the places that are actually doing transplants, because this information is time critical."

Urnovitz added, however, that Chronix "doesn't have the sales force and reach to be able to get there," meaning the company will likely need to partner with a larger company. Ideally, he said, "we'll provide them with the reagents, and they'll have the ddPCR in their laboratories. We think this is the fastest way to start using this game-changing technology for the betterment of outcomes. So we're probably looking at a licensing deal."

In addition, Chronix plans to use droplet digital PCR for its minimal residual disease test, "which we should be offering once we open our CLIA lab next," Urnovitz said. Chronix told Clinical Sequencing News earlier this year that the company was targeting the summer of 2014 to establish this CLIA lab, and he told PCR Insider that the lab will be open "no later than the first quarter of 2015."

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