By Aaron Krol
March 18, 2016 | It’s a telling moment for liquid biopsies. The idea that molecular tests on blood samples―usually for the diagnosis and treatment of cancer―can yield the same information as much more invasive tissue biopsies is now well-established. Both circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) can be extracted from blood draws, albeit at miniscule levels, and can give a reliable read on the mutations a tumor is carrying. But there is no consensus yet on how the tiny volumes of these biomarkers can best be parsed from the huge background noise of healthy cells and DNA, and that means there’s a lot of room for new biotech companies to sprout up with their own patented approaches.
One of these companies, which has developed its own tools for analyzing both CTCs and ctDNA, is Biocept of San Diego, Calif. “We’re one of the only companies out there to have a dual platform that’s looking at both tumor components,” says Raaj Trivedi, Biocept’s Vice President for Commercial Operations. “I think there’s now good evidence that they’re both extremely complementary.”
Trivedi, along with Chief Scientific Officer Lyle Arnold, met with Diagnostics World last week at the Molecular Medicine Tri-Conference in San Francisco, where they had come to demonstrate the Biocept workflow for an audience of diagnostics experts from industry, academia, and healthcare providers. Their company now has tests covering all the major solid tumors, including lung, breast, colon, prostate, and gastric cancers and melanoma―at the moment mainly for choosing targeted therapies, but increasingly also to monitor the course of disease after treatment has started. The products are not first-line diagnostics, but second-line tests to define a tumor’s molecular subtypes.
Twin Technologies
The challenge for any liquid biopsy is to separate cancerous material from the mass of white blood cells and other normal matter found in any serum sample. Biocept takes two entirely different approaches to this problem for CTCs and for ctDNA, although it’s able to collect both from a single tube of blood.
For CTCs, the company has designed a microfluidic chip the size of a standard microscope slide, studded with around 9,000 tiny “posts” coated with streptavidin. Streptavidin, which forms immensely tight bonds with the molecule biotin, is commonly used to capture proteins that have been engineered with biotin outgrowths; in Biocept’s workflow, those proteins are antibodies that attach to well-known tumor antigens. When the Biocept lab extracts cells from a blood sample, it mixes those cells with a cocktail of these biotinylated antibodies. “We come in with roughly ten antibodies that have been screened across dozens of cell lines in all kinds of tumor types,” says Arnold, adding in one or two extra antibodies for certain cancer types with distinctive profiles. (Melanoma especially gets a very unique mixture, because melanoma cells differ so strongly from other CTCs.)
When the cell-and-antibody mixture is run through the chip, CTCs disproportionately latch onto the posts and stay in place after washing. “Cells are enriched for CTCs within this microchannel, about 20 to 50 thousand-fold,” Arnold says. “So if you start out with about 50 million cells, at that level of enrichment we can see individual cancer cells.” While there are still large numbers of healthy cells on the chip at the end of the process, the signal-to-noise ratio is dramatically improved. The entire slide can be put under a microscope for imaging and molecular tests like FISH (fluorescent in situ hybridization), which not only start to characterize the proteins expressed in each CTC, but also further narrow down Biocept’s focus to cancerous cells only, for more detailed rounds of imaging.
Meanwhile, each blood sample Biocept receives also yields a layer of free-floating DNA―including a very small percentage of ctDNA. Biocept also has a solution for boosting the signal from this valuable cancer marker.
Some companies get at ctDNA more or less through brute force: targeting a few key gene regions, then sequencing the entire sample at such extreme depth of coverage that even the tiniest fraction of cancerous material can be seen in the data. Biocept, however, has invented an extra sample preparation step that ensures almost all the DNA it sequences comes from the mutant cells shed by tumors. Before performing PCR (polymerase chain reaction), the chemical reaction that creates thousands of copies of the sample DNA fragments for sequencing, their workflow blocks the healthy wildtype DNA from duplicating.
This is accomplished with molecules Biocept calls “target selectors.” A target selector contains several pieces―among them a fluorescent label that lets lab workers track the process in real time on a standard PCR machine―but the crucial piece is a series of three DNA elements, which fit against the sample DNA almost like a strip of Velcro with a snap fastener at the end.
First, there’s a long DNA sequence that guides the target selector to a region of the genome where common and therapeutically important cancer mutations can be found. That’s the “Velcro,” binding tightly to wildtype and cancer DNA alike. Next to this long sequence, there’s a short bridge that doesn’t attach to the sample DNA at all, allowing the final element to swing open and shut. Finally, there’s the “snap fastener,” a much shorter DNA sequence―as little as eight DNA letters―that overlaps with the exact space where the key cancer mutations might occur.
This final sequence is an exact match to the wildtype, so when it encounters healthy DNA, it snaps shut. But the sequence is short enough that even a one-letter mutation in the sample DNA will prevent it from binding, like an irregularity in a snap fastener’s groove. When a target selector encounters mutated ctDNA, this entire end piece swings free, leaving only the “Velcro” in place. As a result, during the PCR process, healthy DNA is blocked off from the enzymes that duplicate the sample, but ctDNA is still accessible.
“We’ve shown that we selectively amplify for the mutant over a million-fold,” says Arnold. “So now it’s a piece of cake to do analysis on the back end.”
A Tool for Oncologists
Biocept goes after two different sets of molecular targets with its combination approach. By treating and imaging CTCs, its tests can identify surface proteins like estrogen and progesterone receptors, which mark out certain cancers for treatment with drugs like tamoxifen, as well as immune-related proteins like PDL-1 that can help guide the use of investigative immunotherapies. Meanwhile, by sequencing ctDNA, Biocept looks for mutations to genes like EGFR, ALK, and BRAF, all of which can indicate that a tumor is vulnerable to certain targeted drugs. In designing its test menus, Biocept relies on the Tier 1 guidelines of the National Comprehensive Cancer Network.
As a provider of laboratory developed tests, which do not go through the FDA approval process, Biocept does not make treatment recommendations; it just tells oncologists about its molecular findings, and lets them decide what next steps to take. But, says Trivedi, “at some point we definitely want to consider more regulatory approval.” If Biocept can validate its tests as co-diagnostics with cancer drugs, he says, they would be easy to distribute as software-integrated kits for clinical labs to run on their own―either in the U.S. or, more likely, in Europe. “They plug-and-play really well with a standard laboratory workflow, and that gives us the option to distribute.”
Another natural evolution Trivedi sees for Biocept’s products is increasing adoption for patients who have already started treatment, to track whether new sub-populations of cancer cells are developing that might require different drug regimens. Right now this makes up a small minority of Biocept’s business, Trivedi says, “[but] I think as more new therapies come out, the monitoring concept is becoming much more important.”
One area Trivedi does not expect Biocept to pursue any time soon is diagnosing new cases of cancer, or screening healthy patients for the disease. These applications for liquid biopsy have attracted huge enterprises, which see enormous upside in catching cancer earlier than any current diagnostics allow, but the field is also a legal and ethical minefield.
Biocept has formed research partnerships with several major oncology hospitals, including MD Anderson Cancer Center and Baylor College of Medicine, which have helped introduce the technology to doctors. The company is also pursuing agreements with insurers, to help overcome the reimbursement issues that have been a persistent obstacle for liquid biopsies; it has relationships with the national PPOs FedMed, Fortified Provider Network, and Three Rivers Provider Network, among others.