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Cynvenio Offering NGS Analysis of Isolated Circulating Tumor Cells Using AmpliSeq Panel

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Cynvenio Biosystems expects to gain CLIA certification this year for its circulating tumor cell recovery technology and sequencing service using the Ion Torrent PGM, which the company is already performing for several academic and pharmaceutical industry customers in the research setting.

Cynvenio announced the service, which couples isolation of CTCs from the blood with targeted sequencing using the AmpliSeq Panel, late last year. Paul Dempsey, the company's chief science officer, told Clinical Sequencing News that the team is now hoping to receive CLIA certification for its isolation technology within the next few weeks. Following that, the company will validate its sequencing method for clinical use as well.

"We expect certification in the next four or so weeks," Dempsey said. "That will initially cover just the recovery stages and enumeration [CTCs] … and we are now conducting validation for the sequencing step as well."

"The CLIA approval is necessary to report test outcomes to patients through their doctors, but for research we already have a number of customers in academic and pharma using the test," he said.

According to Dempsey, while Cynvenio has worked with Foundation Medicine on CTC sequencing research, the company realized "early on" that it would need to develop an in-house sequencing strategy to be able to glean meaningful molecular information from the small numbers of tumor cells its platform is able to isolate.

Cynvenio has said that in internal research its system has delivered CTC recovery rates of more than 90 percent directly from whole blood with non-target capture of less than 80 cells/mL of blood. This yields samples with enough purity to be sequenced directly using the AmpliSeq standard methodology without the need for additional pre-sequencing amplification and with a seven-day turnaround.

Nonetheless, Dempsey said, "We found early on that getting good molecular information from blood has a number of challenges. We weren't able to find anyone able to do this, so all these steps down to doing the sequencing we developed in house."

Dempsey didn't detail the company's protocols, but said Cynvenio is using the AmpliSeq panel in an essentially unmodified state. "The version 2 has got a lot of the targets that are of value to the customers we are working with now," he said. "Down the road, for specific pathways or drug categories, there will likely be bespoke panels that will be much more targeted, but right now AmpliSeq is a very well-curated, well-characterized panel."

"Our effort has been to figure out how to get sequencing to work with as few as 200 cells without genome amplification and accommodating some of the noise that is inevitable with all NGS platforms," he added. "Once we have a handle on that we can design any panel we want."

According to Cynvenio, the company's CTC isolation technology relies on an automated platform that processes multiple 7.5mL blood samples in under three hours, yielding isolated CTC cell pellets. Dempsey said the isolation is based mainly on EPCAM antibodies, but he stressed that the company augments this with other targeting strategies to enhance the selection of target cells. This can change based on a particular customer's interest, he said.

"It is fundamentally EPCAM-based, but it can be elaborated on," he said. "We do have other [antibodies] that we are comfortable with and have qualified in our lab that we can deploy if people are interested. And we also have a service to quantify markers that are not in our library, so if someone came along and had a specific antibody to another population of cells, we could qualify that internally with relatively short turnaround."

While the company has been working primarily on developing its sequencing service thus far, Dempsey said Cynvenio also plans to offer PCR testing for isolated CTCs for groups interested in measuring a single marker or mutation.

"The broader profile you get with sequencing has discovery capabilities and monitoring capabilities. But if you are looking at drugs and specific treatments, there will always be a smaller class of mutations that need to be defined to make those decisions. We think both tools are essentially valuable," Dempsey said.

Additionally, for groups potentially interested in doing their own molecular analyses, Cynvenio also offers just recovery and enumeration of CTCs.

Massimo Cristofanilli of Thomas Jefferson University is one of the company's research customers and collaborators. He told CSN this week that his team conducted a study with Cynvenio to measure how closely sequencing data from isolated CTCs using the company's service matched to data from tumor tissue samples.

"Our interests are from the therapeutic point of view, to identify mutations we can target with new drugs," Cristofanilli said. "The first step was to prove that CTCs actually reflect the tumor; that we could use this liquid biopsy … so we did a small study, which showed that it was a match between CTCs and tissue."

"Now we want to expand this because many patients have tissue that is not easy to access for genomic analysis. And also importantly, you don't always have an opportunity to sample tissue repeatedly over time."

"So the first results [with Cynvenio] have been extremely encouraging," he said.

Cynvenio is not the only company to have developed technology for isolating CTCs from blood, but Dempsey said the company believes it is the first to offer the full package of isolation and sequencing.

"All [the platforms] have strengths and weaknesses," he said. "But it was clear to us from the beginning that the [cell isolation] platform on its own is not sufficient. On one side, we had to work on stabilizing blood samples, so now we have a protocol to stabilize blood for 72 hours, while many other platforms have to process within two hours. That is hugely important [for clinical use]."

"Then once you get the cells, you still have a mixed population, and it's very small in number. We couldn't give 200 cells to Foundation Medicine, for example, and expect them to be able to sequence that without whole genome amplification, so then we started our own effort to bring sequencing in-house. To the best of my knowledge, we are the only group that can go from the patient to molecular output — to sequenced output — in a seven-day turnaround."

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