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Biocept Laboratories Unveils New PCR Assay Technology to 'Select' Low-Level Mutations

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Biocept Laboratories, a San Diego-based oncology laboratory specializing in circulating tumor cells and biomarker analysis, has developed a pair of PCR-based assay technologies that it claims can identify and quantify single mutations in a background of between 10,000 and 100,000 wild-type sequences.

According to a company executive, the assays are more sensitive than any existing commercial mutation-detection assay, and offer the additional advantage of being allele-specific or able to identify unknown mutations in "hot spot" regions of a gene. The technology is also compatible with multiple sample types and readout modalities and can seamlessly integrate with downstream sequencing to increase its sensitivity greater than 1,000-fold.

Biocept has also demonstrated the ability to multiplex with the technology, and has validated it in house to detect low-level mutations in clinically relevant genes such as EGFR, KRAS, and BRAF. As such, the company is turning its attention to developing highly sensitive mutation-detection panels associated with non-small-cell lung cancer, which it hopes to offer through its CLIA lab later this year.

Biocept is also seeking collaborators for its assay technology. To that end, Lyle Arnold, chief scientific officer and senior vice president at the company, presented the technology during the Molecular Diagnostics Partnering Forum at Cambridge Healthtech Institute's Molecular Medicine Tri-Conference, held last week in San Francisco.

In an interview with PCR Insider following his presentation, Arnold said that Biocept developed its new assay technologies, called CEE-Selector and High-Tm Chimeric Selector, after the company evaluated "more than a dozen" existing commercial assays for one that would be able to detect rare genetic events with a sensitivity that it deemed appropriate for clinical deployment.

"For various reasons, the other platforms either weren't sensitive enough, or too cumbersome, or problematic in other ways," Arnold said.

The "CEE" in CEE-Selector stands for "Cell Enrichment and Extraction," a flagship technology that the company has been cultivating for the past few years for clinical applications. Biocept's CEE method captures circulating tumor cells from samples using a combination of reagents and microfluidic chambers. The cells can then be analyzed using direct-labeled DNA fluorescence in situ hybridization probes or by removing them for analysis with quantitative PCR or other methods.

The company currently offers one laboratory-developed test that uses the CEE technology — HER2 OncoCEE-BR — which uses FISH probes to determine the number of HER2 gene copies in a circulating tumor cell to help identify HER2-positive breast cancer and guide treatment.

While the CEE technology enables Biocept to detect and capture small numbers of circulating tumor cells in a vast background of normal blood cells, the Selector assay technology was designed to then detect rare genetic events in these cells — or potentially detect cell-free circulating mutant DNA.

"If you look at the sensitivity … of detecting a rare event, the question is, 'How low can you go?'" Arnold said. "With whole-genome sequencing, the sensitivity is about 1 percent," meaning it can detect a single mutation in a background of about 100 wild-type sequences. Meantime, hybridization arrays, mass spectrometry, and Sanger sequencing are all in the 10 percent range, Arnold noted.

In general, Arnold noted, PCR-based techniques have even better sensitivity, with techniques such as amplification refractory mutation system/Scorpion probes-based PCR hovering around 1 percent; and Life Technologies' competitive allele-specific TaqMan, or Cast-PCR, settling in between 1 percent and 0.1 percent — although Life Tech has claimed the ability to detect single mutants in a background of 1 million normal copies (PCR Insider, 4/7/2011).

Perhaps the most promising commercial low-level mutation-detection technique is digital PCR, which, depending on the technology, has been shown to be able to detect single mutant alleles in a background of 100,000 to 1 million or more wild-type copies.

"The problem with digital [PCR] is that you can't confirm it, because it's just an endpoint and [the DNA] goes to waste," Arnold said. "You can't recover those little microdroplets."

According to Arnold, Biocept has performed in-house experiments demonstrating that its Selector technology has a standard sensitivity of between one in 10,000 and one in 100,000 — but it also has other features that make it more versatile than the aforementioned assay technologies.

"In terms of the materials that we use, it's pretty much any organism, any target," Arnold said. "The real sweet spot for Selector is allele discrimination and mutations — essentially any genetic alteration where you're talking about resolution at a single nucleotide level, down to a single copy."

The assay can be run on any existing real-time PCR instrumentation; and, in regards to sample types, "we certainly focus on circulating tumor cells, and plasma, but it could be used with essentially anything that might be a source of nucleic acid," Arnold said.

Furthermore, Selector assays can be designed to be allele-specific or to interrogate a "footprint," or mutation hot spot — "for example, KRAS, within codons 12 and 13, you can have all of the KRAS 12 and 13 mutations within six nucleotides," Arnold said. "One Selector assay can detect any of the KRAS mutations, and if there were a new mutation that came up in that area, it could detect it."

"You can get readouts in qPCR, endpoint, and melt curve, but it is also an enrichment strategy so you can then move into mass spec, arrays, capillary [electrophoresis]," Arnold said. "It can be combined with digital PCR; and further integrated into sequencing — Sanger or next-gen. So it's a combination of target enrichment but it's also a real-time readout. All those things are built into the assay."

Arnold declined to provide a great amount of detail about how Selector assays work, as the company just recently filed patents covering the technology and has not yet published on it. However, he told PCR Insider that it essentially uses a blocking strategy, similar to that of locked nucleic acids or peptide nucleic acids; and also takes advantage of the variation in melting temperature that occurs between mutant and wild-type sequences.

"Imagine you have a huge amount of wild type … and a trace amount of mutant," Arnold said. "The idea is that you block the wild type and don't block the mutant, and allow the mutant to amplify up 100,000- to 1 million-fold and be the predominant species. Effectively, the presence of the blocker doesn't do anything at all to the mutant, so it amplifies completely. The wild type does not get amplified at all."

"The assay conditions are really kind of as challenging as it gets," Arnold added. "We're not doing any target selection or pre-amplification, so you enrich for the region you're interrogating. This is taking total nucleic acid and putting it directly into the assay. It's total genomic — we're not going in and fragmenting it, enriching it, pre-amplifying it — but if you do any of those things [the sensitivity] only gets better."

Yet another feature of the technology is the melting temperature difference that is created between the wild-type and mutant sequences. In the CEE-Selector version of the assay, a window of about 13 °C or 14 °C is created.

In Biocept's latest version, the High-Tm Chimeric Selector, the mutant Tm is around 52 °C while the wild-type is around 72 °C, for a difference of about 20 degrees.

"This is like something you could drive a 747 through, in terms of difference," Arnold said. "This higher Tm version will have more specificity and give fewer side products — primer-dimers, things associated with side reactions in PCR. Those things typically go away as you go to higher temperatures."

In addition, the High-Tm version results in cleaner amplification products for downstream sequencing; and it is attractive for multiplexing, "because now we have such a large window, we don't have to try and tune things perfectly. You can be a little sloppy, off by even 5 or 10 degrees, and it still works the same," Arnold said.

In other in-house experiments, Biocept has successfully "grafted" in sequencing primers for Ion Torrent sequencing to enable downstream NGS analysis. In addition, company researchers have shown selective enrichment of mutants of greater than 20,000-fold for downstream Sanger sequencing.

The Selector technology is co-owned by Biocept and Aegea Biotechnologies, a startup that Arnold founded. Arnold said that the companies are seeking collaborators for the technology, and that Aegea can provide partners with assay development services.

In the meantime, Biocept is moving forward with the technology as the basis for laboratory-developed tests in the area of oncology. First on the agenda is developing and validating a multiple-biomarker panel for NSCLC, which will eventually be administered in Biocept's CAP-accredited, CLIA-certified lab alongside its CEE and FISH offerings.

"We've demonstrated this works with the EGFR, KRAS, and BRAF mutation families … and determined that all the key hotspots within those gene families will multiplex," Arnold said. The NSCLC panel "will probably be five or six [hot-spot regions] right now, but we're hoping we can go to dozens."

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