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Transgenomic Using STTR Grant to Explore Ice-Cold-PCR Detection of Pancreatic Cancer Mutations

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Transgenomic has been awarded a Phase I Small Business Technology Transfer grant from the National Institutes of Health to conduct initial studies on the feasibility of using Ice-Cold-PCR for the early detection of pancreatic cancer.

Under the one-year, $100,000 grant, administered by the NIH's National Center for Advancing Translational Sciences, Omaha, Neb.-based Transgenomic will use mouse models to test the ability of Ice-Cold-PCR to detect KRAS and Trp53 mutations in urine and blood samples, according to recently published grant information.

Researchers from the company will then induce pancreatitis in the mouse models, which will enable a "time-line" investigation of the link between early KRAS and Trp53 mutations and progression to stage I pancreatic cancer.

According to the grant, the researchers will then further validate their method in several tissues from well-characterized human pancreatic autopsy samples, including pancreas, blood, and urine. If Phase I yields promising results, the researchers hope to secure Phase II STTR funding to further study Ice-Cold-PCR detection of DNA mutations associated with early- and late-stage pancreatic cancer in humans.

"Improved and complete enrichment coamplification at lower denaturation temperature," or Ice-Cold-PCR, was invented in the laboratory of Mike Makrigiorgos at Dana Farber Cancer Institute, as an improvement upon the lab's original Cold-PCR assay technology.

Cold-PCR selectively amplifies mutant DNA by recognizing that mutant DNA strands denature at lower temperatures in a PCR reaction than normal DNA strands. The approach allows clinicians to use smaller amounts of sample for genetic analysis and less-intrusive sample collection methods such as a blood draw for serum or plasma preparation; urine; fine needle aspirates; or bronchial lavage. The approach also enables the study of mutated DNA directly from tumors.

The Ice Cold-PCR methodology, meantime, further enhances this sensitivity through the use of Exiqon's Locked Nucleic Acid technology and a reference strand that binds PCR amplicons to form duplexes that are preferentially denatured and amplified at a certain temperature.

The mutant amplicons can then be analyzed using a number of sequencing methods. Transgenomic has an exclusive license from Dana-Farber to analyze Ice-Cold-PCR products using pyrosequencing and Sanger sequencing.

Using Ice-Cold-PCR, Transgenomic scientists have been able to achieve 10,000-fold enrichment of mutant targets. Further, the assay can be engineered to enrich all relevant mutations in a particular gene for further downstream analysis.

"Anywhere there is a mutation in the range of the PCR, we will find it and enrich it to that level," Transgenomic CEO Craig Tuttle told PCR Insider this week. "It might be 5,000[-fold] in one case, and 15,000[-fold] in another, but we will get a good enrichment. By locking up the wild type, a lot of the product that you get is now mutant. So when you sequence, there is no issue."

The ultimate goal of the new NCATS-funded project is to develop a simple and highly sensitive diagnostic assay for the early detection of pancreatic ductal adenocarcinoma, Transgenomic said.

"We always wanted a blood-based technology to be able to monitor cancer, to look at treatment, predict treatment, and based on the mutation profile, to look for recurrence … and do all this in blood," Tuttle said. "That was really the initial goal … and then longer term to look at this for potentially screening. For example, if you do your annual blood workup and have circulating KRAS mutants, that's probably a bad thing. It isn't specific for where there is a cancer, but that's a nice overall potential gauge."

Transgenomic has signed a few other agreements over the past year to support its pursuit of this goal, and not just for pancreatic cancer. For instance, in November it inked a perpetual, worldwide, collaboration and distribution agreement with Paris-based ScreenCell for its isolation devices and dilution buffers for collecting circulating tumor cells in peripheral blood.

Specific terms of that agreement call for Transgenomic to exclusively distribute ScreenCell's technologies in combination with its own Blocker-Sequencing cancer mutation assays, Surveyor Scan cancer mutation kits, and Wave instrument systems; and eventually with Ice-Cold-PCR assays (PCR Insider, 11/10/2011).

And in March Transgenomic initiated a collaboration with MD Anderson Cancer Center to evaluate Ice-Cold-PCR for analyzing DNA isolated from circulating tumor cells in blood samples with advanced cancer (PCR Insider, 3/22/2012).

"We will look at all [patients] in their cancer clinic, take blood and tissue, and look at circulating free DNA and tumor cells from the blood … with the ScreenCell device, and try and compare what we see," Tuttle said. "We also want to validate that you can actually use a blood sample rather than biopsy. Our goal there is to find out if we can actually see tumor heterogeneity based on the blood sample."

Transgenomic has further work ongoing in the area of mutation detection for colon cancer and lung cancer. The company also has a non-exclusive license from Dana-Farber to analyze Ice-Cold-PCR products using next-generation sequencing technologies.

"We'll try all of these [sequencing] methods," Tuttle said. "We've got [Life Technologies] Ion Torrent [sequencers] up and running here, so we'll look at that to see if we can do a screen … and look in blood to monitor treatment."

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