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Transgenomic Eyes Dana-Farber's COLD-PCR for Cancer Screening Applications

NEW YORK (GenomeWeb News) – Transgenomic believes that a new PCR technology that it recently licensed from the Dana-Farber Cancer Institute could have promise down the road as a screening tool to detect early-stage cancer in blood samples.

The technology, called coamplification at lower denaturation temperature PCR, or COLD-PCR, was developed by Mike Makrigiorgos' lab at Dana-Farber. It preferentially amplifies segments of DNA that contain mutations, and is thereby able to increase the sensitivity of downstream mutation-detection methods by a factor of 10 to 100.

Transgenomic earlier this month signed an exclusive licensing agreement with Dana-Farber to use COLD-PCR combined with Sanger sequencing as well as in applications for mitochondrial DNA analysis. The company believes that it can increase the sensitivity of the approach even further in order to detect low-concentration mutations in the blood that could serve as early indicators of cancer.

Craig Tuttle, CEO of Transgenomic, stressed that the company's work in this area is very early stage, and that the level of circulating DNA mutations in cancer is still unknown. Nevertheless, he told GenomeWeb Daily News that preliminary studies with non-small cell lung cancer suggest that there is a "very high concordance" between mutations in tumor samples and those in blood samples.

One challenge, he noted, is that current technologies are not sensitive enough to identify very low levels of mutant DNA among large amounts of wildtype DNA.

"We're convinced that you can't get there for routine early screening based on the current technologies available," he said. The company's own Wave and Surveyor methods, for example, have a detection sensitivity of around 0.5 percent to 1 percent. Sanger sequencing, meantime, "can't see below 20 percent," while the jury is still out on next-generation sequencing methods. "There are some reports that they are at 5 percent, but we just don't know," Tuttle said. "We're looking at buying a second-gen analyzer so we can get to the bottom of that."

In the meantime, the company decided to cement an existing collaboration that it had with Makrogiorgos' lab at Dana-Farber regarding the COLD-PCR technology. In March, Transgenomic signed an option to license the technology in an agreement that was expected to run through January 2010.

Tuttle said that the company decided to lock down the license sooner due to interest from pharmaceutical clients "who are well aware of this concordance problem between circulating DNA in the blood and the tumors themselves. They were very interested in seeing if you can find these circulating DNA mutations in … some of the very key oncogenesis-related genes, and whether you can find those mutations earlier as a result of it."

So far, with KRAS testing, "we've seen an order of magnitude jump in sensitivity using COLD-PCR," Tuttle said. "Now, can we go one more? That would be great."

One outstanding question, he said, is how much more improvement will be necessary to make the approach viable for cancer screening. In the company's current studies, the concordance between tumor mutations and mutations in circulating DNA in the blood was "very high" — just under 80 percent — "but not enough to make this a screening technique."

Another consideration, he noted, is that these studies are all based on samples from patients with manifested tumors, so they don't provide a good indication of the level of circulating DNA mutations in early-stage cancer.

"We're working in an area now where I don't believe there's much of a literature portfolio suggesting how much DNA is really there," he said.

Other Applications, Future Plans

COLD-PCR takes advantage of the fact that during the denaturing step in PCR, DNA amplicons that contain mutations can be forced to form heteroduplexes with wildtype sequences, thus creating a mismatch at the mutation points.

"When a mismatch is formed, the double-stranded DNA duplex that contains the mismatch is thermodynamically more unstable than a sequence that has both strands that are wildtype," Dana-Farber's Makrigiorgos told GenomeWeb Daily News. With COLD-PCR, the temperature is adjusted so that it preferentially denatures these unstable heteroduplexes, which leaves most of the wild type homoduplexes in double-stranded form.

"As a result, during the process of PCR, the mutation-containing sequences will be preferentially duplicated," he said. "The final PCR product will contain a higher proportion of mutations, thereby one can easily find a mutation by one of many available technologies," including Sanger sequencing, high-resolution melting, next-generation sequencing, or denaturing high performance liquid chromatography — the technology behind Transgenomic's Wave platform.

"It's like magnifying a needle from a haystack, so that the needle can be detected," he said.

Makrigiorgos said that it will be difficult to pin down a timeline for the development of an early-stage screening test based on the COLD-PCR approach. "Although the technology is already worked out, for some applications one would have to do clinical validation studies, and for that it's difficult to predict at this point when and how rapidly that will come," he said.

He noted that there are several other promising application areas for the technology, including the detection of low-frequency resistance mutations in primary tumors, or as a way to avoid microdissection in cases where it is difficult to procure large amounts of sample.

Beyond the field of cancer, Makrigiorgos noted that the improved sensitivity of COLD-PCR also shows promise in prenatal diagnosis, where "you have the need to detect genetic alterations in a background of mainly wildtype alleles — maternal alleles. So that is a classical case where you may have 0.1 or 1 or 5 percent of fetal DNA, within the maternal DNA, that you want to detect."

He noted that other research groups are using COLD-PCR for additional applications. For example, a study published in Modern Pathology in March described how a team from the UK's Royal National Orthopaedic Hospital used COLD-PCR to increase the accuracy in diagnosing intramuscular myxoma — a type of rare soft tissue tumor that is difficult to distinguish from other types of lesions.

Makrigiorgos and colleagues Jin Li and Coren Milbury are currently developing a new version of the COLD-PCR "so that you effectively get a complete isolation of a mutation by the time you complete PCR."

Unlike the current version of COLD-PCR, which can be performed without any modification to existing PCR primers or thermal cyclers, the new version would require "some special oligonucleotides and primers that we are now in the process of validating," he said.

"In the original form of COLD-PCR we can enrich the mutations by a factor of 10 to 100. In future versions that are not yet published, we anticipate that it will be possible to get over 100-fold," he said. "Effectively it would be an isolation of the mutation."

Makrigiorgos said that his group is also looking at using the approach in combination with second-generation sequencing platforms.

"None of these technologies so far has reliably demonstrated that they can go below 1 to 5 percent of mutant abundance," he said. "We think that with COLD-PCR as a first step, followed by second-generation sequencing, one would have another leap in sensitivity while retaining the high throughput of the technology."

The researchers are currently exploring this approach with an Applied Biosystems/Life Technologies SOLiD and a Roche/454 FLX sequencer, but Makrigiorgos said it is too early to disclose any results.

Makrigiorgos said that he even sees promise for the approach with some of the single-molecule sequencing platforms from companies like Helicos and Pacific Biosciences that would not require PCR as the first step.

"Single-molecule sequencing is likely to still need some sort of fractionation of the genome in order to become more efficient," he noted. "Sequencing the entire genome each and every time you are applying third-generation sequencing is not efficient because 99 percent of the information is useless. People would still like to focus the resequencing on a specific portion of the genome."

In this case, he said, "we envision that COLD-PCR can be incorporated so that you achieve two things at once: reduction of the genome to the useful sequences, and selection of the mutants so that the depth of third-generation sequencing increases further."

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