Patricia F. Fitzpatrick Dimond Ph.D. Technical Editor of Clinical OMICs President of BioInsight Communications
With one test on the market, researchers are developing tools that leverage CTCs as biomarkers.
Cancer cell gene signatures have received a lot of attention lately as biomarkers for certain forms of cancer. Circulating tumor cells (CTCs) isolated from patients’ blood is also gaining ground as validation study results accumulate and physician acceptance increases.
The accessibility of CTCs in peripheral blood eliminates the need for tumor biopsies, a major driving force behind innovation in this area. Furthermore, there is the hope that peripheral blood detection of early metastatic cancer might eventually be possible.
Veridex, marketer of the only FDA-approved CTC test, launched its CTC CellSearch platform in 2004. It is sanctioned for the assessment of metastatic breast, colon, and prostate cancers. CTC assessment is gaining acceptance as a prognostic marker among physicians, according to Veridex.
“We launched the test in December 2004, so we’ve been out there for six years,” points out Bob McCormack, head of technology innovation. “All of the original findings for the three metastatic disorders have been validated multiple times. The test is well received and growing, and we are getting positive insurance coverage decisions based on the number of physicians ordering the test.”
Veridex’ CellSearch captures, stains, and counts tumor cells from a 7.5 mL patient blood sample. It is enabled by magnets, Dr. McCormack told GEN. The kit isolates tumor cells from peripheral blood by capturing them onto nanoparticles, followed by fluorescent staining.
“We have conjugated the EpCAM, an epithelial-cell-specific antibody, to ferrofluid nanoparticles targeted to the tumor cell. In adults, about 80% of cancers are epithelial in origin. The anti-EpCAM antibody, or epithelial cell adhesion molecule, works particularly well in the blood because normal individuals don’t have circulating epithelial cells.”
In the next step, Dr. McCormack said, to determine whether the cells have been captured, “we visualize the cells with a second antibody, an anticytokeratin antibody conjugated to a fluorochrome. We also treat the cells with an anti-CD45 antibody to distinguish white blood cells from CTCs.
“Then, following concentration of the blood sample down to a 200 microliter volume, it is placed into a ‘magnest,’ a magnet that causes everything captured by the magnetic particles to rise to the same plane,” Dr. McCormack added. “These are placed on cell tracts, which can read the entire chamber through four colors to give us all the images we need. The images can be enumerated for true tumor cells.”
Veridex says several prospective multicenter clinical studies have validated CTCs as strong, independent predictors of both progression-free survival and overall survival in metastatic prostate cancer patients. CellSearch provides more accurate information about a patient’s prognosis 2–5 weeks after the initiation of therapy than prostate-specific antigen (PSA) testing, the firm reported.
Results of a 2009 study published in The Lancet Oncology and conducted in 164 men with progressive castration-resistant metastatic disease showed that changes in CTC number 4, 8, and 12 weeks after chemotherapy were strongly associated with mortality risk, whereas changes in PSA were weakly or not associated.
Tests in Development
In January Veridex entered into an agreement with Massachusetts General Hospital (MGH) to establish a center of excellence for research on CTC technologies. The overall goal of the agreement is to develop and commercialize novel, quicker technologies for capturing and characterizing CTCs with improved specificity as well as sensitivity.
MGH scientists along with researchers at Harvard Medical School, Shriners Childrens’ Hospital in Boston, and the National Human Genomics Research Institute had developed a microfluidic in which patient blood samples were processed over a silicon chip covered with antibody-coated microscopic posts. This original chip proved challenging to manufacture reliably and cost effectively. Additionally, the smooth flow of blood around the microposts limited the number of CTCs that came into contact with the antibody-covered surfaces.
The researchers found that passing samples through a chamber lined with a herringbone (HB) pattern of grooves worked better because it allowed for rapid mixing of indpendent fluid streams.
Similarly to Veridex’ approved device, the HB chip employs positive selection of CTCs using the EpCAM antibody to capture the cells. But instead of the magnetic nanoparticles Veridex uses, cells are captured onto antibody-coated microposts arrayed in this herringbone pattern under precisely controlled laminar flow conditions. In its previous incarnation, the chip successfully identified CTCs in the peripheral blood of patients with metastatic lung, prostate, pancreatic, and breast cancers at an approximately 50% purity.
The HB chip that replaces the collaborators’ original CTC chip is expected to provide more comprehensive and easily accessible data from captured tumor cells. “The earlier versions of the CTC chip required hand-counting of thousands of microscopic images, which was sufficient for the initial proof-of-principle studies but far too time-intensive for handling high volumes of patient samples,” explained Shannon Stott, Ph.D., of the MGH Center for Engineering in Medicine. “We also were limited in our ability to analyze cellular factors that could be markers for important properties of the tumors.”
Dr. Stott continued, “We are also working to create a plug-and-play version of the machine that will be easy to use clinically, exploring options for large-scale production of the CTC chip, and continuing to optimize the device to increase its speed and efficiency.”
In another approach, researchers described direct measurement of CTCs in the presence of white blood cells on nanoparticles using surface enhanced Raman spectroscopy (SERS). To differentiate between white blood cells and epithelial cells, the Veridex test and CTC chip test treat patient blood samples with anti-CD45 antibodies, which selectively bind to white blood cells allowing them to be visualized and discounted as CTCs in the final enumeration analysis. In the SERS method, nanoparticles with epidermal growth factor peptide as a targeting ligand were used.
The SERS nanoparticles successfully identified CTCs in the peripheral blood of 19 patients with squamous cell carcinoma of the head and neck, with a range of 1–720 CTCs per milliliter of whole blood. The research was conducted by scientists at Emory University and Quest Diagnostics and published in the journal Cancer Research in a paper appearing January 2011.
To date the Veridex test remains the only FDA-approved diagnostic for CTCs. Dr. McCormack said that in terms of the competitive environment, “what’s important is that analytical validation instrumentation and reagents undergo multiple evaluations. There are 20-plus large biotech companies, small biotechs, and academic labs that are developing technologies to enter into the CTC space. The trick is to go beyond a novel observation or different way of doing it.
“It’s a long, long road to get to an FDA approved test,” Dr. McCormack remarked. While technologies for CTC isolation may take a long time to develop and validate, their increasing application in predicting cancer treatment responses, monitoring tumor progression, and identifying novel drug cancer targets may make it worth developers’ time and effort.
Patricia F. Dimond, Ph.D. (firstname.lastname@example.org), is a principal at BioInsight Consulting.