By Ben Butkus
Life Technologies this week launched an early-access program for a newly developed line of TaqMan assays for detecting somatic mutations in genes associated with colorectal, lung, pancreatic, and breast cancer, among others.
The TaqMan Mutation Detection Assays, based on a technology dubbed competitive allele-specific TaqMan, or Cast, PCR, are sensitive enough to detect single mutant molecules in a background of 1 million normal copies – at least a 10-fold greater sensitivity than currently available mutation detection assays, the company said.
Life Tech is now seeking cancer researchers as early-access customers in advance of a full commercial launch in July, at which time the company will also release new data analysis software to support the assays, the company said.
As reported by PCR Insider in September, the company developed the CastPCR method to improve upon another method, called allele-specific PCR, and enable the detection of rare mutations such as those found in formalin-fixed paraffin-embedded tissue tumor cells.
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 Life Tech, 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 as compared with other PCR-based assays.
"We do have a lot of TaqMan assays for gene expression and genotyping, but we feel that the customers are interested in somatic mutations," Sejal Desai, a Life Tech product manager, told PCR Insider this week. "And we don't have [assays for] somatic mutations in our collection so far. So we wanted to start building the pipeline in this area."
According to Desai, detecting somatic mutations requires assays with extremely high degrees of sensitivity and specificity. "If you just use a standard TaqMan or homebrewed assay, which a lot of customers are doing, sensitivity can often be a challenge," Desai said.
She added that other commercially available products – though she didn't specify which – typically claim a mutant allele detection sensitivity of around 1 percent; whereas the new CastPCR-based TaqMan assays have demonstrated sensitivity of 0.1 percent, or 10-fold greater.
The first TaqMan Mutation Detection Assays are for detecting mutations in the KRAS, BRAF, and EGFR genes, most strongly associated with colorectal, lung, pancreatic, and breast cancers; but also implicated in leukemia, non-Hodgkin's lymphoma, melanoma, and papillary thyroid carcinoma.
At the September conference, Life Tech scientist Mark Shannon revealed that the company had already developed and internally validated CastPCR assays for more than 80 cancer-related genes.
However, Desai said this week that Life Tech "had to start developing assays somewhere … and the first three genes are based on research publications and what our customers are interested in. But certainly there are many other targets that are interesting in the area of cancer."
Life Tech has already conducted some external validation of the assays with one early-access laboratory affiliated with France's Institut National de la Sante et de la Recherche Medicale.
In a statement, Pierre Laurent-Puig, director of UMR-S775 INSERM laboratory, said that using the new assays "has enabled us to detect cancer-relevant DNA mutations more robustly even within abundant backgrounds of normal material."
Laurent-Puig, who is also a professor at Descartes University Medical School and head of clinical oncogenetics at the European Georges Pompidou Hospital, added that the level of sensitivity of the new TaqMan assays is "superior to classical methods we have tested and lends itself well to clinical research applications, including the initial identification of mutations."
Desai said that Laurent-Puig's lab has tested all three of the currently available assays, and that Life Tech is currently developing "additional collaborations" with the lab.
Life Tech is now seeking additional early-access cancer researchers to continue validating the new assays, and the company will "roll out the product, the full launch, in July," Desai said.
The new assays are optimized for use on Life Tech PCR platforms such as the ViiA 7 real-time PCR system; and the Applied Biosystems 7900 HT, 7500 Fast, and StepOnePlus systems.
"We always test our assays on our own instruments … and it is difficult for me to comment on how they will work on other real-time systems," Desai said. "But the cycling conditions and the workflow optimization is based on our instruments."
The new assays may even complement another recently launched Life Tech product designed to help researchers detect rare alleles: the OpenArray real-time qPCR system for digital PCR applications.
In November, Life Tech launched its first commercial research kit on the OpenArray system, but the new TaqMan assays may represent additional content for that system.
"Digital PCR is a great technology for these kinds of applications, and we are working on both of these technologies," Desai said. But the content that goes on a digital PCR platform still has to be a good assay. So if you have an assay with lower sensitivity and specificity, it's going to affect the performance of digital PCR. I think it really goes hand in hand."
The CastPCR assays are currently available for research use only. However, the assays do have diagnostic potential. In September the company presented unpublished data demonstrating the potential use of the assays in detecting rare circulating tumor cells from a patient's blood.
This week, Life Tech reiterated in a statement that the assays "will also support the development of emerging technologies and future products for detecting circulating tumor cells," citing recent work by UK scientists demonstrating that such cells can help determine aggressiveness levels and potential treatments for cancer.
"The potential is there, but right now it is only approved for research use," Desai said. "But because of the high sensitivity, it might be interesting to see if there is application in something like circulating tumor cells."
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