By Ben Butkus
DUBLIN – Life Technologies is developing a TaqMan-based qPCR method that can directly detect rare mutations such as those found in formalin-fixed paraffin-embedded tumor cells or rare circulating tumor cells in patients' blood, a Life Tech research scientist said this week.
Initial unpublished data from studies employing the method, called competitive allele-specific TaqMan, or Cast, PCR, suggest that it can detect single rare mutant alleles in up to a million wild-type sequences with a high degree of specificity, according to the scientist.
In addition, Life Tech scientists have successfully designed and validated CastPCR assays for more than 82 cancer-related SNPs for genes such as RAS, EGFR, KIT, pTEN, and p53, the company said.
In a presentation at Select Biosciences' qPCR Europe conference, held here this week, Mark Shannon, a senior staff scientist at Life Tech, discussed the development and applications of CastPCR.
Shannon outlined the need for better techniques for detecting rare tumor cells in blood, which have been reported as a surrogate marker for tumor response and linked to shorter survival in metastatic breast and lung cancer patients.
Current methods for identifying CTCs in the bloodstream generally involve cell-enrichment processes in order to identify cell surface markers using immunohistochemistry techniques.
"But we know that these cells also carry highly specific mutations," Shannon said. However, these mutations, and the cells that carry them, are found in extremely low numbers in the bloodstream – on the order of one in 100,000 or 1,000,000.
Other approaches for detecting these mutations have involved PCR-based microsatellite assays; digital PCR-based techniques such as the BEAMing technology developed at Johns Hopkins University and licensed to German firm Inostics; and protein-based assays.
All have promise, Shannon said, but scientists are currently still seeking a method that is highly specific; does not introduce detectable errors, such as can be caused by polymerases; and is easy to perform.
Enter CastPCR, which Shannon said Life Tech developed from another technique called allele-specific PCR, which employs selective PCR amplification of mutant alleles using an allele-specific primer. "This can be quite specific," Shannon said, "but not always."
One of the problems with allele-specific PCR, he said, is that it can still generate significant probe signal from mismatches, which occurs frequently enough to affect assay specificity.
CastPCR, meantime, is a homogenous assay that combines allele-specific TaqMan qPCR with allele-specific minor groove binder as blockers to effectively suppress non-specific amplification from wild-type alleles.
Basically, Shannon said, the method uses allele-specific primers; locus-specific primers; locus-specific TaqMan probes; and allele-specific competitors to achieve its high level of sensitivity and specificity.
Shannon said that Life Tech internal studies using plasmid constructs have revealed that the method has a 1,00,000-fold difference in specificity compared with allele-specific PCR.
"The question now is 'How does it perform with a large amount of wild-type DNA?'" Shannon said. "The potential is quite great for detecting a single copy in a large amount of DNA."
In order to find this out, Life Tech has been collaborating with researchers at the French National Institute of Health and Medical Research to test the specificity of the method in tumor cells. So far this study has revealed "good results," Shannon said, declining to disclose more details of the research since it has yet to be published.
"We are looking to now test blood samples, but this work is not yet completed," Shannon said.
In addition, the company has been working with scientists from the Institut Gustave-Roussy in France to detect colon cancer cells in FFPE samples for the purpose of correlating rare mutant alleles with disease. "This is where the tech really has a chance to shine," Shannon said.
Once again, Shannon said that early data from this collaboration look promising, but declined to provide additional detail except to say that their results have shown a high concordance with sequencing results.
Overall, Life Tech researchers have had a success rate of more than 90 percent for the more than 86 CastPCR assays they have developed so far, Shannon said.
He also pointed out the homogeneity of the assay as an important feature, saying that because the assay has no enrichment steps, there is no chance to lose rare DNA in the procedure.
In general, the method holds promise for early cancer detection and monitoring, prenatal analyses, and allelic gene expression analyses, Shannon said.
Shannon did not provide a timeline for commercial development of the method, and referred further questions about the technology to Caifu Chen, senior director of genomic assays R&D in Life Tech's Molecular Biology Systems division.
In an e-mail to PCR Insider, Chen also declined to discuss specifics of CastPCR, citing the early phase of the product's development. However, Chen did say that CastPCR "has generated a strong interest in the qPCR community, in particular [for] rare mutation detection."