The field of circulating tumor cells (CTCs) is exploding with competing technologies, all offering different approaches to isolating and characterizing CTCs from blood. CTCs are incredibly rare. Out of ten billion blood cells, only a handful may be CTCs. They are believed to be the mechanism behind cancer metastasis, and there is a great deal of interest in using them to diagnose, monitor, and predict the course of the disease. While CTC technologies are just now reaching maturity and becoming available for clinical use, their history dates back to 1869, with the discovery of CTCs by Thomas Ashworth. The first methods for isolating CTCs in the 1950s were based on filtration. Many CTC technologies now make use of technologies like immunomagnetic separation and microfluidics. Refinements on these techniques and innovative new approaches have improved clinical utility and marketability for CTC-based liquid biopsies.
An automated assay for multiple myeloma
Menarini’s acquisition of the CellSearch platform from Janssen Diagnostics in 2017 set the stage for development of the company’s advanced liquid biopsy platform. The CellSearch Circulating Tumor Cell Test was the first analytically validated blood test cleared by the FDA for metastatic breast, prostate, and colorectal cancers. It separates circulating tumor cells from a blood sample using magnetic nanoparticles with antibodies targeting epithelial cell adhesion molecules. These are then separated from the blood, tagged with antibodies that can identify CTCs, and analyzed by fluorescence. Menarini’s DEPArray™ digital cell-sorting and isolation system adds single-cell isolation functionality to the existing CellSearch platform.
Menarini is now expanding its applications into multiple myeloma, a blood cancer ideally suited for a liquid biopsy approach. In a 2018 publication in the British Journal of Hematology, researchers from Menarini and Janssen reported the results of an automated assay characterizing circulating multiple myeloma cells (CMMCs) from peripheral blood in patients with plasma cell disorders. The assay uses CD138 and CD38 for capture and detection, respectively. They counted CMMCs from 1000 patient samples including newly diagnosed patients and those with smoldering multiple myeloma. The CMMC counts correlated with markers of disease burden such as percentage of bone marrow plasma cells and serum M protein, and those markers decreased when the disease entered remission.
Steven Gross is head of CellSearch assay development at Menarini and one of the authors of the paper. He says that the platform is an important advance for multiple myeloma patients because it offers a potential way to monitor progression of disease and response to therapy without invasive bone marrow biopsies. “Right now, the only way to look at the actual plasma cells is through bone marrow aspirate for clinical diagnosing and staging,” says Gross. Another application is minimal residual disease. “So if a patient is being treated, and if the number of CMMCs drops down dramatically, this would be a good way to monitor them to see whether or not they continue to do well under treatment, and if CMMCs begin to rise, it would be a very quick way to know whether they are no longer responding to treatment.”
Combining microfluidics and immunomagnetic separation
Fluxion CEO Jeff Jensen says that when he and his co-founders started the company, they looked at CellSearch and other CTC analysis technologies with an eye to improving on what was already available. “Every approach focuses on some attribute of cancer cells that is unique, that isn’t shared with the rest of the blood cells,” says Jensen. Those would include physical attributes like size, density, and deformability, as well as functional attributes like expression of surface proteins.
“We looked at all of these and felt the best opportunity to optimize both sensitivity of isolation and the specificity and purity of the resulting population of recovered cells was to focus on immunomagnetic separation—which is effectively what CellSearch was doing—but we wanted to combine it with microfluidics,” says Jensen.
Jensen adds that the challenge of CTCs is that their relative abundance in the sample is so low that bulk separation techniques typically aren’t sensitive enough. Their system uses magnetic beads to capture the target cells, and separates them in microfluidic channels.
An example of an application of the Fluxion system is lung cancer. Typically a patient is initially screened for lung cancer with a CT scan. CT scans have a high rate of false positives, so many patients enter a waiting period of 3 to 6 months before they get another scan. “Obviously, with lung cancer, the longer you wait before you start treatment, the worse the outcomes get,” says Jensen. “What we’re working on is taking someone who falls into that grey area and doing a blood test that would indicate whether they should stay on a ‘wait and see’ approach, or be moved to biopsy and more aggressive treatment.”
A scalable solution
Many CTC assays have been highly successful for individual patients or in small, scale studies. However, scaling the assay up for market requires that it can be mass produced. “We now have a system where we can screen and monitor patients, and we can make it so that we can mass produce it and can bring it to the people. We can really analyze tens of thousands of patients,” says Rolf Muller, CEO of Biofluidica.
Biofluidica’s liquid scan platform is based on a microfluidic chip. The chip is built on a capture bed comprised of 50 to 500 channels coated with antibodies. The antibodies are targeted to specific CTCs in the blood and are chemically immobilized in the channels. Biofluidica’s robotic platform can process eight patient samples at once, and each instrument can be run three times a day, according to Muller.
Biofluidica has gathered clinical data in a number of cancers including pancreatic cancer, colorectal cancer, ovarian cancer, and breast cancer. According to Muller, the company is now testing its platform in clinical trials in acute myeloid leukemia (AML) and acute lymphocytic leukema (ALL). And it is expanding beyond cancer to look at circulating fetal cells in maternal blood as a potential replacement for amniocentesis. Some researchers have studied cell-free fetal DNA in maternal blood. However, Muller says, “Cell-free DNA can find only about 5% of defects in prenatal testing. It is not a full replacement of amniocentesis. We can find, with our CTC system, fetal cells in the mother’s blood in sufficient amounts to do FISH or RNA-seq analysis.”
Muller adds that Biofluidica’s technology is distinguished from competitors in that it involves no pre-processing of blood where CTCs could be lost, and that the company’s device is more selective and sensitive than bead-based or similar systems. “Recovery is very high and background is very low,” Muller says.
Functional enrichment for separation
Vitatex uses a cell adhesion matrix (CAM)-based platform to functionally enrich CTCs in order to separate them from blood. This is in contrast to more common separation methods based on physical properties of CTCs like size or a surface receptor. In the CAM workflow, patient samples are added to CAM-coated plates or tubes. The CTCs adhere and are fluorescently labeled and analyzed by flow or image cytometry. They can also be studied for gene expression, gene mutations, or cultured in plates.
Vitatex has demonstrated clinical proof of concept for early detection of metastasis in breast, colon, lung, pancreatic, and ovarian cancer. The technology can also be applied to cancer prognosis, by monitoring a drop or increase in CTCs. This information can then be used to select a therapy using CTCs in culture that are treated with a panel of potential therapies.
“A lot of attention has been shifted into genetic testing of plasma cell-free DNA,” says Wen-Tien Chen, PhD, president and CSO of Vitatex. “I think most of us still believe circulating tumor cells fundamentally are responsible for the worst outcome for a cancer patient, which is metastasis.”
In addition to providing a tool for treatment of individual patients in the clinic, this approach can be harnessed for use in pharmaceutical development to select effective pipeline drugs at the preclinical stage. Chen says that Vitatex is currently looking for pharmaceutical companies to partner with for CTC drug response testing.
Separation by image analysis
While most approaches to analyzing CTCs are based on some method of separating the cells from the blood or pre-enriching the cells before the analytical step, that’s not the approach at Epic Sciences. “Very few groups have taken the stance that all of those systems have a pre-bias,” says Pascal Bamford, PhD, former CSO of Epic Sciences. “They’re all pre-sorting by size, shape, or a certain antigen expression.”
Epic puts all of the cells into a medium and uses digital imaging as a “post-enrichment” process, according to Bamford. “We’ve basically built a system that we knew could be globally deployable. While proprietary, there are no complicated widgets or difficult manual steps in the process of plating cells onto glass slides.” The next step is staining for specific antigens or biomarkers of interest. Then every cell is scanned. Tens of millions of cells are indexed for each patient. The “separation” is carried out by the algorithms.
“We’re literally able to find one single cell among 50 million objects,” says Bamford.
Epic recently unveiled a liquid biopsy test that predicts prostate cancer sensitivity to PARP inhibitors. The test is paired with an investigational PARP inhibitor in development by BeiGene, in a Phase II clinical trial. The assay measures chromosomal instability in CTCs by identifying distinct morphology feature sets.
Epic’s image analysis algorithms are created through machine learning combined with more traditional code written based on measured cell properties. This approach allows a detailed understanding of the algorithms and avoids the “black box” effect associated with other neural network methods.“I think we’re fortunate that we’re in a wave of AI and machine learning. We have a vast array of tools and computational power at our disposal. It’s really feeding those tools with comprehensive and representative data where I think a lot of companies and a lot of efforts fall down, and that’s another key area of our focus. In terms of machine learning and artificial intelligence algorithms, the state of the art is very competent and we’ve been applying it extensively to the tests we develop,” says Bamford.
Novel Technology for Liquid Biopsy
A Paragon Genomics poster (“Determining genetic predispositions using CleanPlex® Hereditary Cancer Panel for a rapid and streamlined amplicon-based NGS workflow”) shows how CleanPlex UMI technology used in liquid biopsy applications demonstrates high sensitivity with low false positive rates for the detection of low-frequency alleles, even at low DNA inputs, according to a company official, who adds that it has a simple workflow. Liquid biopsy presents challenges as low fractions of mutant DNA can be masked by artifacts or background noise, leading to false negatives. The CleanPlex UMI Lung Cancer Panel using an input of just 50ng of cell-free DNA detected nearly all mutations at 0.1% minor allele frequency (mAF) and at 0.25% mAF with 100% positive predictive value (PPV), says the company spokesperson.