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Cynvenio to Offer Clinical PCR Testing of Isolated Circulating Tumor Cells for Select Targets

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Cynvenio Biosystems said this week that it expects to gain CLIA certification in the next few weeks for its circulating tumor cell technology. The company then plans to clinically validate its CTC sequencing service and a number of targeted PCR assays for isolated CTCs, which the company already offers in the research setting.

Cynvenio announced the research-use sequencing and PCR services, which couple isolation of CTCs from the blood with targeted sequencing using Life Technologies' Ion Torrent AmpliSeq Cancer Panel, or TaqMan PCR assays targeting 45 mutations in three genes — EGFR, KRAS, and BRAF — late last year. Paul Dempsey, the company's chief science officer, told PCR Insider 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 molecular analysis methods 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."

For PCR, Dempsey added, "each set of probes we are using also has to be validated in the lab. But the main threshold is to develop the standard workflow for PCR on our template. Then the validation for each primer set is an easier effort."

"We will definitely choose the targets [to offer clinically] first based on the models that we are focusing on within Cynvenio. So, since we are interested in breast cancer, PI3 kinase might be a more pressing target than EGFR," he added.

According to Dempsey, Cynvenio was involved in early testing of Life Technologies' castPCR reagents and benefitted from castPCR's ability to amplify a very weak signal in the cell line experiments it used to develop its CTC platform. Now that the current technology can isolate highly purified samples of CTCs, though, he said, the company is working with non allele-specific TaqMan reagents.

"There is no need to exclusively use reagents that are so specific now because a lot of the specificity is gained by recovering cells with high purity. We are validating our sequencing protocol down to 1 percent [sensitivity], and that is a relatively standard target for TaqMan reagents," he said.

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 without the need for additional pre-sequencing amplification.

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 believes it will be relatively easy to move forward with clinical PCR testing for groups interested in measuring a single marker or mutation.

"From our perspective, if we can see mutations with sequencing, PCR is a validation away, so it's easier to go in that direction than the other way around," Dempsey said.

"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," Dempsey said.

"We've been focused on sequencing initially because it was clear to us from the beginning that the molecular biology of CTCs is very poorly understood. We wanted to cast a wide net in order to develop an understanding of what this population is," he explained.

"That said, though, the way this becomes a test we run 5,000 times a day is to look for a particular druggable target's presence or absence to inform a clinical decision down the road," Dempsey said. "It's not necessary to do sequencing for that, so we've carried through PCR readouts because they are so much faster and cheaper and can be more accurate for different types of mutations, including point mutations."

Additionally, for groups potentially interested in doing their own molecular analyses, Cynvenio also offers just recovery and enumeration of CTCs. Or it will work with customers to create bespoke PCR panels or sequencing strategies.

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 molecular analysis.

"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 … To the best of my knowledge, we are the only group that can go from the patient to molecular output … in a seven-day turnaround."

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