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Transgenomic Expects New Cold-PCR License to Extend Mutation-Detection Kit Sensitivity


By Ben Butkus

Molecular diagnostics firm Transgenomic said this week that it has expanded its license with the Dana-Farber Cancer Institute related to the coamplification at lower denaturation temperature, or Cold, PCR technique.

The expanded license gives Transgenomic access to an offshoot of Cold-PCR, called "improved and complete enrichment," or Ice Cold-PCR, that can detect certain mutations with greater sensitivity.

The new license also allows the company and its customers to exclusively analyze Cold-PCR products by pyrosequencing in addition to the Sanger sequencing and mitochondrial DNA analysis rights covered by the original license.

Transgenomic will use its expanded licensing rights to develop new kits that can detect mutations in common cancer-related genes such as KRAS and p53 directly from blood samples and then analyze the variants using multiple sequencing approaches, Transgenomic President and CEO Craig Tuttle told PCR Insider this week.

"The first market for this is with pharma companies looking at mutations in blood, in clinical trials," Tuttle said. "The one thing that interests me the most thus far [is that] we're seeing several KRAS mutations — not just one specific mutation — in blood, in lung cancers. I know we're seeing a fuller picture of the heterogeneity of the tumor."

Omaha, Neb.-based Transgenomic originally licensed exclusive rights to Cold-PCR from Dana-Farber in October 2009. Cold-PCR selectively amplifies mutant DNA by recognizing that mutant DNA strands denature at lower temperatures in a PCR reaction than normal DNA strands. As such, Cold-PCR amplifies mutated DNA with minimal amplification of normal DNA.

The enhanced analytic sensitivity of Cold-PCR 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; as well as studying mutated DNA in tumors directly, Transgenomic said.

The Ice Cold-PCR methodology, meantime, further enhances this sensitivity using a couple of techniques, according to Tuttle.

"One way is through the use of a locked nucleic acid base pair," Tuttle said. "We use an LNA … targeted to the predominant mutation site, and it basically locks out the wild type." In order to do this, Transgenomic purchases LNA primers from the technology's owner, Exiqon. Future diagnostic applications would require a license from Exiqon. Transgenomic has initiated discussions with Exiqon about this, but a license "will take some time," Tuttle said.

In addition, Ice Cold-PCR uses a reference strand engineered in such a way that when incorporated into PCR reactions in excess it binds rapidly to the amplicons. At a certain denaturation temperature, the reference strand-wild type duplexes remain and amplification of the wild type alleles is selectively inhibited. Meantime, reference strand-mutant duplexes are preferentially denatured and amplified.

One of the major benefits of this approach, Tuttle said, is that "we see all the mutations that would occur in that region of interest even though we're using a [locked nucleic acid], for example, at just one position."

Transgenomic is betting that the upshot of this will be mutation-detection kits and, eventually, companion diagnostic assays that can paint a broader picture of the mutations involved in certain cancer pathways.

Tuttle said that Transgenomic is currently working with "multiple" undisclosed pharma partners to explore these opportunities.

"In some cases, we're doing Ice Cold-PCR assays at their request and actually developing the assays for targets that aren't standard, in our opinion; and in other cases we're using the standard KRAS pathway genes [developing assays for] mutations in those," he said.

"Where they'd like to see us go is to develop Ice-Cold assays for p53," Tuttle added. "So we have that in development, as well."

The modified license does not necessarily mean that Transgenomic is abandoning conventional Cold-PCR, which works well for certain mutations and doesn't require the additional protocols and potential licenses of the Ice Cold-PCR technique.

"Our findings are that for temperature-lowering mutations, Cold-PCR works fine, and you still get that high level of sensitivity," Tuttle said. "But for temperature-neutral or temperature-increasing mutations, then you really have to use Ice Cold."

In either method, the resultant PCR-amplified fragments can be analyzed using a variety of methods, including Sanger sequencing, pyrosequencing, real-time PCR, or any next-generation sequencing technology.

The original license with Dana-Farber conferred to Transgenomic exclusive rights for Cold-PCR with Sanger sequencing; and the expanded license adds pyrosequencing to the mix. The company has also secured non-exclusive rights from other entities to analyze Cold-PCR products using real-time PCR or next-generation sequencing, Tuttle said.

"We are developing a KRAS Ice Cold-PCR kit that can be used on straight Sanger sequencing equipment," Tuttle said. "And then from that we'll just go through the mix of key assays, and continue developing those assays. Regarding pyrosequencing … we wanted to make sure we had access to the two key sequencing platforms used in the US and Europe [in order] to bring Ice Cold-PCR kits to the market."

Thus far, the company said that gene mutation assays using both techniques have obtained mutation detection levels that allow post-enrichment Sanger sequencing of somatic mutations that had initial concentrations of lower than 0.05 percent that of the wild-type allele.

In comparison, Sanger sequencing alone provides a typical detection level of 10 percent, according to Transgenomic.

New Revenue Stream

KRAS mutation detection kits based on Cold- and Ice Cold-PCR are expected to eventually replace Transgenomic's current Surveyor Scan KRAS mutation-detection kits, the sales of which have been "minimal," according to Tuttle, because several other comparable kits were on the market prior to Surveyor.

The Surveyor KRAS kits use Surveyor nuclease, a mismatch-specific endonuclease derived from celery that recognizes and cleaves all types of mismatches arising from the presence of SNPs or from small insertions or deletions.

Licensed from Fox Chase Cancer Center, this technology can identify all common mutations in KRAS coding exons 2 and 3 or just exon 2 with an analytic sensitivity of about 1 percent of mutant in a wild-type background — good, but not nearly as good as Cold- and Ice-Cold PCR.

Transgenomic sorely needs such an additional revenue source: Last week the company reported a 22 percent drop in fourth-quarter revenues and a 9 percent drop in full-year revenues amid a mounting net loss.

In late November, the company acquired Clinical Data's genetic and pharmacogenomic testing and biomarker development business, including a CLIA laboratory facility, for $15.5 million. The company noted in its Q4 financial statement that the acquisition is expected to generate $13 million in revenues annually and give it a larger presence with insurers and patients. It should also allow Transgenomic to offer its new mutation-detection assays, once they are developed, in its CLIA facility.

"Eventually, we will develop [Cold- and Ice Cold-PCR assays] for specific key cancer pathway genes that can be used as companion diagnostics," Tuttle said. "But we can develop them as assays; run them in our pharma lab; and as they become more widespread or move more toward a companion diagnostic, we can offer that test in our CLIA lab."

Have topics you'd like to see covered in PCR Insider? Contact the editor at bbutkus [at] genomeweb [.] com.