The Promise of Circulating Tumor Cells

CTCs as a Window on Tumor Dynamics

“We know that circulating tumor cells travel in the blood to go from one place to another—that is the only way for them to go—so we know it is an important part of the metastatic process,” said Daniel Hayes, MD, professor in the Department of Internal Medicine at the University of Michigan, Ann Arbor. Hayes was one of the lead investigators in the CellSearch technology, approved by the FDA in 2004 for detecting CTCs from cancer patient blood samples.

Cynvenio BioSystem’s LiquidBiopsy Platform for recovery and characterization of CTCs. Courtesy of Cynvenio BioSystems, Inc.

CTC detection and analysis is seen as a window into the dynamics of solid tumors. Tumor tissue access is often not possible or involves highly invasive procedures. In contrast, the CTC assay is noninvasive and offers the potential for serial analysis over a short time period to help guide cancer prognosis and treatment decisions. Cells that detach from a tumor and find their way into the bloodstream are now detectable using a simple blood draw. So far, research has shown that detection of increased or decreased numbers of CTCs in a patient’s peripheral blood can correlate with tumor progression and response to therapy for certain tumor types such as breast, prostate, and colorectal cancer.^1-3

Although researchers hope to use CTC analyses in lieu of biopsies, more research is needed to understand the dynamics of CTCs, which can vary by tumor type and from patient to patient.

The tools to analyze CTCs continue to evolve as new platforms for capturing, counting, and analyzing these tumor-shed cells are developed. However, the potential utility of CTC detection is varied. Different types of CTCs exist and may signal the beginning of the metastatic process, when cells from the primary tumor travel to colonize other organs. Detection of CTCs may also be a way to monitor minimal residual disease. A change in CTC number might signal whether a patient is responding to therapy or has developed resistance.

The only circulating tumor cell assay currently approved by the FDA is the CellSearch Circulating Tumor Cell Test by New Jersey-based Veridex, LLC, a Johnson & Johnson company. The assay is licensed to detect CTCs in breast, prostate, and colorectal metastatic cancer patients to monitor the effectiveness of treatment, and is typically used in concert with other evaluation tests.⁴Studies have shown that the presence of CTCs in the blood of patients with these cancers can be associated with both decreased progression-free survival and overall survival.^1-3

The CellSearch system specifically detects CTCs that express the epithelial cell adhesion molecule EpCAM: magnetic particles attached to anti-EpCAM antibodies separate the EpCAM-positive CTCs from the rest of the blood sample. A limitation of the test is that because not all CTCs express the EpCAM marker, the assay can miss other types of CTCs. In addition, the assay cannot be used to detect tumor cells of nonepithelial origin, and the system is not sensitive enough to detect very low levels of CTCs.

CTC-Detection Technologies in Development

Other technologies to detect and analyze CTCs are in the works, including microchips to process small blood volumes, novel ways for selecting CTCs that express specific cell-surface markers, “unbiased” approaches to capture and analyze CTCs that are not biomarker-dependent, and automated tools for minimal CTC manipulation.Newer technologies are moving beyond epithelial biomarkers to detect other rare CTCs, and have greater sensitivity and specificity. For instance, new detection technologies can find tumor cells based on markers, and also remove white blood cells, leaving CTCs untouched, allowing for identification of CTCs that may not express known biomarkers.

Daniel Haber, MD, PhD

Researchers at Harvard University, Massachusetts General Hospital Cancer Center, and Shriner’s Hospital, Boston, recently demonstrated the ability of a new microfluid chip device to detect CTCs without the need for identification of specific tumor biomarkers on the cells. The CTC-iChip was first developed in 2007,⁵but this newer version is a more comprehensive way to detect and analyze CTCs. Study results were published in the April 3, 2013, issue of Science Translational Medicine.⁶

The iChip first filters out all cells from a blood sample that are not either putative CTCs or white blood cells. A microfluid chamber then lines up the remaining cells in a single file for sorting. To separate the CTCs from white blood cells, either positive or negative selection can be used: CTCs can be labeled and sorted using magnetic particles that attach to an epithelial marker on the surface of CTCs. Alternatively, white blood cells can be tagged and sorted away, leaving a population of unlabeled CTCs.

According to the study, this third-generation version of the iChip system can process large-volume blood samples rapidly, and both the positive selection method using a tumor-specific biomarker and the negative selection approach can recover more than 80% of tumor cells from different tumor types including prostate, breast, and lung. The CTC-iChip device was able to detect CTCs from melanoma and pancreatic cancer, two nonepithelial cancers that do not express EpCAM, as well as triple-negative breast cancer with no detectable EpCAM expression. The team also identified novel prostate CTC markers from samples from patients with prostate cancer. The iChip can also extract unlabeled tumor cells, to be used for both pathology at the cell level, as well as for genetic and molecular technologies such as genomic sequencing or RNA expression.

“This system is an ‘enabling tool’ that will allow a broad range of research and clinical applications,” said Daniel Haber, MD, PhD,director of the Massachusetts General Hospital Cancer Center and coauthor of the study. “Analysis of CTCs opens the window to studying cancer cells in transit through the blood, something that has tremendous implications for understanding and preventing the spread of cancer, but which had been limited by technological challenges in isolating and studying these rare cells.”

Other companies are also working on ways to characterize CTCs. Among them are California-based Cynvenio BioSystems, which has a microfluid device to capture CTCs, among other platforms for CTC detection and analysis. The company recently received its Clinical Laboratory Improvement Amendments (CLIA) certification. CLIA was established to verify laboratory quality standards and is regulated by the Centers for Medicare & Medicaid Services. Cynvenio already offers a service, available to researchers and clinical oncologists, to isolate and analyze CTCs, including whole-genome sequencing and PCR-based tests, from blood samples. “We have developed an automated platform to run our CTC device without any human handling and have been testing clinical patient samples for the last 3 years,” said André de Fusco, CEO and director of Cynvenio. Cynvenio is focusing on the genomics of CTCs and early detection, according to De Fusco.

Cynvenio’s microfluid chip for recovery of rare cells from a blood sample using immunomagnetic capture. Courtesy of Cynvenio BioSystems, Inc.

Many Challenges Remain

Another promising noninvasive assay in oncology is the detection of circulating tumor DNA in the peripheral blood to monitor patient progression and resistance to therapy.^7-9Unlike tumor biopsies, both CTC and circulating tumor DNA assays offer the ability to take consecutive samples over short time periods to gain “snapshots” of what is happening to tumors.In addition to the technical challenges that remain in detecting CTCs, there are biological challenges and questions: Can detection be consistently translated into meaningful information on the cancer? Thus far, successful detection of CTCs is not possible from all patient blood samples. This may be due to the sensitivity of the tools available or the biology of the patient. Approaches that do not require the use of biomarkers could overcome this issue. However, other questions remain, such as: Does detection of CTCs or changes in CTC counts always signify a negative prognosis? So far, this is not clear, and more research is needed to understand CTC biology.

Next Steps

How to best molecularly characterize CTCs is also not known. Researchers are developing ways to genotype CTCs for mutations; a potential goal is to detect mutations and track the emergence of new mutations, including resistance mutations that develop as a result of targeted therapy treatment. The full clinical relevance of CTCs in all cancer types remains to be seen.The CTC detection technology under development is not yet ready for routine clinical use. For instance, the iChip team is working on creating a version of the new system that can be produced on a large scale, and working to increase the purity and number of CTCs that can be analyzed.

Current applications of CTC technologies are limited to patients with advanced disease, but researchers would like to see the technology move toward earlier-stage cancer detection and monitoring. “Ultimately, there are applications for studying the process of blood-borne metastasis, and also potentially for early detection of invasive cancers,” said Haber. To detect early-stage cancer using a CTC assay, the field will have to continue to overcome the challenge of having a sensitive enough instrument to detect the presumably rare CTCs in cancer patients who have less tumor volume. Another hurdle is defining and characterizing a CTC and how these cells may be different during early-stage versus metastatic disease.

How far away is a sensitive assay to detect early-stage cancer? “We’re working on it,” said Haber.

References

1. Cristofanilli M, Budd GT, Ellis MJ, et al. Circulating tumor cells, disease progression, and survival in metastatic breast cancerN Engl J Med. 2004;351:781-791.
2. de Bono JS, Scher HI, Montgomery RB, et al. Circulating tumor cells predict survival benefit from treatment in metastatic castrationresistant prostate cancer.Clin Cancer Res. 2008;14(19):6302-6309.
3. Cohen SJ, Punt CJA, Iannotti N, et al. Relationship of circulating tumor cells to tumor response, progression-free survival, and overall survival in patients with metastatic colorectal cance.J Clin Oncol. 2008;26(19):3213-3221.
4. CellSearchâ„¢ Epithelial Cell Kit / CellSpotterâ„¢ Analyzer - K031588. http://www.fda.gov/MedicalDevices/ProductsandMedicalProcedures/ DeviceApprovalsandClearances/Recently-ApprovedDevices/ ucm081239.htm. Accessed May 15, 2013.
5. Nagrath S, Sequist LV, Maheswaran S. Isolation of rare circulating tumour cells in cancer patients by microchip technology.Nature. 2007;450(7173):1235-1239.
6. Ozkumur E, Shah AM, Ciciliano JC, et al. Inertial focusing for tumor antigen–dependent and –independent sorting of rare circulating tumor cells.Sci Transl Med. 2013;5(179):179ra47.
7. Dawson SJ, Tsui DWY, Murtaza M, et al. Analysis of circulating tumor DNA to monitor metastatic breast cancer.N Engl J Med. 2013;368(13):1199-1209.
8. Diaz LA Jr, Williams RT, Wu J, et al. The molecular evolution of acquired resistance to targeted EGFR blockade in colorectal cancers.Nature. 2012;486(7404):537-540.
9. Misale S, Yaeger R, Hobor S, et al. Emergence of KRAS mutations and acquired resistance to anti-EGFR therapy in colorectal cancer.Nature. 2012;486(7404):532-536.