By Ben Butkus
Life Technologies this week announced the expansion of its TaqMan Mutation Detection Assays based on competitive allele-specific TaqMan, or Cast, PCR technology.
The company now offers TaqMan Mutation Detection Assays for 241 of the most common mutations in 21 important cancer genes on top of the company's previous commercial assays, introduced about a year ago, for detecting mutations in the KRAS, BRAF, and EGFR genes.
In addition, a group of French researchers that was among the earliest users of Life Tech's CastPCR-based assays has recently published a study demonstrating the technology's high degree of sensitivity, specificity, and ease of use for detecting mutations in KRAS, BRAF, and EGFR genes in formalin-fixed paraffin-embedded tumor samples.
Life Tech unveiled the expanded assay portfolio at the American Association for Cancer Research meeting held this week in Chicago.
PCR Insider first reported on the development of the CastPCR method in September 2010, when a Life Tech scientist presented preliminary data on the assays' potential to specifically detect single rare mutant alleles in more than a million wild-type sequences in FFPE tumor samples (PCR Insider, 9/16/2010).
CastPCR combines allele-specific TaqMan primers and allele-specific minor groove binder as competitive blockers in order to suppress non-specific amplification from wild-type alleles. According to the company, this combination of technologies, along with optimized PCR conditions, results in an assay with 10-fold or higher sensitivity and specificity for detecting rare mutant alleles compared with other PCR-based assays, even existing TaqMan qPCR assays sold by Life Tech.
Last April, Life Tech launched an early-access program for CastPCR-based TaqMan Mutation Detection Assays for detecting somatic mutations in the KRAS, BRAF, and EGFR genes, ahead of a full commercial product launch in July 2011 (PCR Insider, 4/7/2011).
Since that time, Life Tech customers have been "extremely receptive" to the assays, and the company continued to develop additional assays for other important cancer-related genes, Sam Raha, head of next-gen qPCR and content at Life Tech, told PCR Insider last week.
"We've taken those and have expanded significantly our assay offerings for cancer mutations," Raha said. "Specifically, we are now launching assays tied to 241 of the most common mutations, now covering 21 of the most common cancer genes," including HRAS, NRAS, PIK3CA, KIT, PTEN, and TP53.
Prior to the CastPCR-based mutation-detection assays, Life Tech and predecessor companies have for years sold off-the-shelf, fully validated TaqMan assays for "millions" of DNA, RNA, and protein targets, Raha said. These assays are still extremely useful, he noted, adding that there was no risk of sales cannibalization from the new line of mutation-detection assays.
"The distinction is [that] in most of [the TaqMan assays] … you're looking at germline mutations or germline changes that have happened either at the DNA level or gene expression level," Raha said. In the case of the CastPCR-based assays, "they're really tied to somatic mutations," he said.
Another important distinction, Raha said, is that most of Life Tech's standard TaqMan qPCR assays have a sensitivity, depending on sample type, of around one mutation copy in a background of 100 or 1,000 wild-type or normal copies.
"With the CastPCR mutation-detection assays, you're really able to consistently look at a sensitivity of better than one mutation in a background of 1,000 and in most cases well over 10,000 [wild-type alleles]," Raha said. "And we have a lot of data showing we have the ability to look at one mutation in a background in more than 1 million wild-type copies."
For cancer research studies, "particularly when you're looking at circulating tumor cells or other heterogeneous samples, that level of sensitivity … makes these very unique," Raha said.
In a paper published last month in Experimental and Molecular Pathology, a group of researchers from France's INSERM, Université Paris Descartes, and Hôpital Européen Georges Pompidou have demonstrated that sensitivity by using the assays to successfully genotype KRAS, BRAF, and EGFR on sub-optimal FFPE tumor samples.
The research team reported that the CastPCR hydrolysis probes they used were "highly specific" and that the assays correctly typed 60 of 63 samples, missing only three deletions in EGFR that were not recognized by the assays used. The team also reported that the CastPCR technology "is less laborious and prevent[s] crossover of PCR products as compared to multistep methods;" and that the TaqMan Mutation Detection Assay "is an important technology to consider in the field of mutation detection for KRAS, BRAF, and EGFR point mutation screening."
In an e-mail to PCR Insider this week, Laurent-Puig further detailed his group's work with the mutation detection assays. Specifically, Laurent-Puig said that the group had already developed a set of TaqMan probes to detect KRAS, BRAF, and EGFR mutations in colon and lung cancer, and to date have provided "more than 3,000 diagnostic tests in France using this technology for colorectal and lung cancer patients. We [are] now developing [CastPCR] TaqMan probe tests in order to improve the sensitivity of our tests."
Another key point regarding the CastPCR mutation detection assays is how they will complement Life Tech's other molecular biology research tools. For instance, last year a Life Tech product manager told PCR Insider that the new mutation detection assays' high degree of sensitivity may actually complement the extreme sensitivity of another relatively new technology: digital PCR, which Life Tech offers through its OpenArray platform and recently launched QuantStudio 12K Flex comprehensive PCR platform.
Raha last week echoed that idea, and pointed out that the company in fact currently has "a number of collaborations with customers who are using CastPCR assays in a digital mode."
"The distinction is, with CastPCR, a customer orders it and runs it through their standard qPCR workflow that they would with TaqMan, so less than three hours from starting with their sample to getting a readout," Raha said.
Digital PCR, in comparison, is still a much more complex workflow, but using the two technologies together "would give you, in addition to the CastPCR sensitivity, the ability to very specifically count. With CastPCR or any alternative traditional qPCR assay, you get very good comparison, and an amount relative to something else. But it's not absolute as digital PCR is."
Laurent-Puig's group is also exploring the marriage of these technologies, although he and Université Paris Descartes collaborator Valérie Taly are using RainDance Technologies' new digital PCR platform, which competes with the Life Tech digital PCR products (PCR Insider, 3/29/2012).
Laurent-Puig told PCR Insider that combining TaqMan probes with droplet-based digital PCR "allows us to improve our sensitivity [even more] to one mutant allele among 200,000 wild-type alleles. The ultimate goal is to be able to detect the minority clone leading to the occurrence of resistance to targeted therapy." It is unclear whether Laurent-Puig and Taly are exploring digital PCR platforms from Life Tech or other competing vendors in addition to the RainDance platform.
The TaqMan Mutation Detection Assays are optimized for use on Life Tech qPCR platforms such as the ViiA 7 real-time PCR system; and the Applied Biosystems 7900 HT, 7500 Fast, and StepOnePlus systems. Raha said that assay users are not relegated to using a Life Tech qPCR system. "When you run it with our Life Tech or AB systems, we have application notes and we can provide support to the researcher. But they're pretty darn robust assays, which will really work on some of the competing platforms, as well," he said.
Life Tech also views its expanded mutation-detection assays as important tie-in products to other offerings, such as the company's recently launched AmpliSeq custom panels for targeted resequencing on its Ion Torrent PGM sequencer (PCR Insider, 3/29/2012).
"We are intentionally coupling this with … the AmpliSeq panel, and we just announced the expansion of that panel to more than 700 genes," Raha said. "And we've intentionally [highly correlated] those same targets. So someone can start with next-gen sequencing … and once they [identify] 10 or 20 or 50 or 100 real mutations that they want to go in and look at over and over again … we have the best one-two punch starting with the PGM and following on with these CastPCR assays."
Raha also noted that the recent expansion of its TaqMan mutation detection assays is the "first wave" in a series of similar expansions over the course of the coming year to achieve "a very high correlation" with the 700-some mutations covered in the AmpliSeq panels.
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