DiaCarta, a four-year-old Bay Area startup eyeing point-of-care diagnostics, said this week that it has commercially launched a line of real-time PCR test kits for somatic mutations linked to cancer.
Called QClamp, the technology uses xenonucleic acid (XNA) to "clamp," or silence, amplification of non-mutant DNA in heterogeneous samples, thus exclusively amplifying mutant DNA. The tests, currently for research use only, can be run in under two hours on existing real-time PCR platforms without a DNA extraction step, on a variety of sample types, the company claims.
Heterogeneous biopsies have long been a thorny problem in molecular diagnostics; clinicians require high-resolution data on gene expression, but in standard real-time PCR and sequencing methods, unless a mutated gene makes up at least 10 percent of a sample, its signal is often swamped by super-abundant wild-type DNA.
"The problem with sequencing technology right now, if you take a tumor sample, it's like a big haystack with a few needles in it," Michael Powell, DiaCarta's chief science officer, told PCR Insider this week.
"What we do is we eliminate the haystack from the equation … we burn the haystack and amplify the needles," he said. A veteran of Boehringer Mannheim and Roche Diagnostics, Powell helped develop the XNA method, which binds wild-type DNA "like Velcro," he said. The method also improves mutation detection because "with our XNA clamp, if you get a single nucleotide polymorphism, the mismatch causes a 15 to 20 degree difference in [melt] temperature" — a 10-fold increase over other PCR methods, he said.
According to the company's website, its xenonucleic acids can be in the form of peptide nucleic acids, glycol nucleic acids, or another suitable modified backbone. DiaCarta now sells seven kits to detect common cancer mutations found in genes such as KRAS, NRAS, and EGFR.
"We have a BRAF assay [for] a melanoma mutation, a resistance mutation" similar to Roche's US Food and Drug Administration-approved Cobas 4800 BRAF V600 companion diagnostic for BRAF mutation-positive metastatic melanoma. However, product information on Roche's website claims its BRAF test detects mutations at less than a 5 percent mutation level, while DiaCarta claims its XNA method enables detection of less than 0.1 percent mutated DNA.
In addition to the XNA clamp, DiaCarta claims it has improved detection by using colorimetric dyes instead of hydrolysis-based probes to reveal mutant amplicons. "We're not using any sophisticated fluorescence chemistry on the probe … we're just measuring a fluorescent intercalating dye [SYBR Green]."
This real-time PCR version of the assay can be run on any existing commercial real-time PCR system, the company noted. However, DiaCarta is also currently developing a biotin-based detection method that could obviate the need for a real-time PCR system, instead relying on simpler detection methods.
Since "we only make mutant amplicons … we can also detect the mutant amplicon without using real-time PCR," said Powell. DiaCarta CEO and Founder Aiguo Zhang added that "if some lab doesn't have real-time PCR, it can use a regular thermocycler and get the amplicon [in] a biotin format, then use a capture plate" to perform standard ELISA. "Almost every lab has an ELISA reader," Zhang said.
What's more, DiaCarta is currently developing its QClamp technology into a strip-based test, somewhat similar to a pregnancy test, said Zhang. Powell added that the company is already able to perform V600E BRAF mutation detection on such a strip, although this version of the assay is not yet commercially available.
The company hopes to eventually "get to bedside detection of [cancer] mutations," said David Broecker, DiaCarta's chief commercial officer. "That's essentially the uniting vision of what we're trying to achieve."
DiaCarta has other commercial assays, a line of human papillomavirus tests that use branched DNA technology licensed from Siemens. One test can be used for cervical cancer screening, while another tests for cancer-linked HPV strains in saliva samples to determine head and neck cancer risk. These tests have CE Mark and Chinese FDA approvals, Zhang said, and are being used in China and other markets.
The QClamp technology is quite new, however, and, in terms of academic validation, Powell pointed out that "we have people working on it, they have results, but they haven't published yet."
At the moment DiaCarta is "really trying to focus on working with pharmaceutical and biotech companies and treatment centers to validate the technology so that we can turn around and start to publish papers and people can [see] the value of the technology and how it compares dramatically to what's already out there," Broecker said. "Then I think we have to decide how aggressive we want to be in terms of moving the platform forward, and that would trigger future investment, if that's a decision we want to make."
Meantime, Broecker said "because of some of our relationships and partnerships in some of the emerging markets like China with our HPV assay, we're going to be working with folks in emerging markets as well. We're a cash flow-positive company at the moment. At this stage our focus is on trying to generate more."