November 2008: Volume 34, Number 11
Circulating Tumor Cells
A New Opportunity for Therapeutic Management of Cancer Patients
By Martin Fleisher, PhD, FACB
When cancer disseminates from the primary lesion to other vital organs, it becomes a devastating disease. In fact, it is the metastatic process that leads to 90% of cancer-related deaths. A complex multi-step event, this biological process requires tumor cells to break free of the primary solid tumor, penetrate into the blood or lymphatic circulation, and ultimately extravasate out of the circulation and into an organ or tissue distant from the primary lesion.
Implicit to the metastatic process is the generation of circulating tumor cells (CTC) that traverse the circulation and colonize distant organs. This ability of solid tumors to metastasize to a distant site has lead researchers to propose that solid tumors have a “leukemic” phase, in which unusually high amounts of lymphocytes are found in the peripheral blood (lymphoma).
More than 40 years ago, S.H. Seal, a research scientist at Memorial Sloan-Kettering Cancer Center, published a paper in which he described a method for filtering “free-floating cancer cells” from peripheral blood. He postulated that this technique would be helpful for studying the nature of the metastatic process. Only in the past decade, however, has measuring these cells in the peripheral blood of cancer patients become clinically relevant and technically feasible. Described here are the recent developments in CTC detection and future perspectives on the use of this technology for therapeutic target assessment and clinical management of patients.
What are CTC?
Epithelial tumors or carcinomas represent about 80% of all cancers. CTC originate from the epithelium and are not present in the circulating peripheral blood of individuals free of neoplastic disease. Derived from clones of the primary tumor, these cells can be detected before the primary tumor is identified and often persist even after the primary tumor has been removed.
The objective of CTC analysis is to capture, identify, and enumerate these cells in peripheral blood, and then characterize their genetic and pathobiological relevance to the primary tumor. Compared with the number of red blood cells and leukocytes in peripheral blood, the concentration of CTC is exceedingly low—about one CTC per 5–10 million red blood cells and leukocytes. Identifying and enriching the population of CTC in peripheral blood remains challenging, but researchers have developed novel techniques and instrumentation to capture them. Over the last 5 years, CTC analysis has gained clinical acceptance, and this new analytical procedure has been validated for use in CLIA-certified labs.
Overview of Methods and Techniques
Immunomagnetic enrichment. Cells of carcinoma origin elaborate a cell surface antigen called epithelial-cell adhesion molecule (EpCAM). This method uses plastic or ferromagnetic beads coated with antibodies that recognize EpCAM to capture and enrich CTC from peripheral blood. Other cancer cell surface markers have been used for CTC identification, such as EGFR, mammaglobin, MUC-1, HER-2, c-erbB2, and PMSA.
Since more than 90% of carcinomas are of epithelial origin, the presence of EpCAM-positive cells in the circulation strongly suggests the presence of cancer. This method identifies viable cells: their nuclear material stains positively with diamidino-2-phenylindole (DAPI) dye. Furthermore, the cells can be visualized and counted following treatment with an immunofluorescent phycoerythrin-conjugated antibody that recognizes cytokeratin in the cell’s cytoplasm. Epithelial cells of tumor origin can be differentiated from white blood cells since these cells express CD45 antigen on the cell membrane surface, which is detected by anti-CD45 antibody conjugated to allophycocyanin, a reddish fluorescent dye. Figure 1 depicts immunomagnetic selection of CTC from whole blood, and Figure 2 shows the actual microscopic presentation of CTC isolated and counted in the peripheral blood of patients.
Binding of the EpCAM Monoclonal Antibody (MAB) to EpCAM on the Cell Membrane of a CTC
The EpCAM monoclonal antibody (MAB) ferromagnetic conjugate binds to EpCAM on the cell membrane of a CTC. The interaction of cytokeratin antibody conjugate-PE, which binds to cytokeratin (CK-6, 8, 18) in the cytoplasm of the CTC, is also shown. Mononuclear cells, such as white blood cells, are identified by the CD45 antigen on the cell surface. In this analysis, EpCAM+ bound to the ferromagnetic conjugated MAB can be physically separated from mononuclear cells (CD45+) by a magnet, resulting in enrichment of CTC by more than 10,000- fold. Viable CTC (DAPI+) can also be identified and counted, since the cytoplasmic content of cytokeratin is immunofluorescently stained.
The Veridex CellSearch platform captures and assesses CTC and has been approved by the FDA as a independent predictor of overall survival and progression-free survival in patients with metastatic breast, colon, and prostate cancer when applied in discrete clinical context. The technology has been successfully standardized across multiple laboratories.
CTC microchips. Research labs are developing microchips and microfluidic devices to capture highly purified CTC from blood. One such microfluidic device captures CTC as whole blood flows past 78,000 EpCAM-coated microposts. It also counts and analyzes the cells for molecular markers, such as mutations in the EGFR gene in patients with non-small cell lung cancer.
Another device, manufactured by Biocept (San Diego), uses microfluidic channels and microposts to which EpCAM antibody is bound (See Figure at left). Once captured, the cells can be strained and counted or fixed for fluorescence in situ hybridization analysis of DNA targets. CTC can also be harvested from the device for PCR-based analysis.
Microscopic Presentation of CTC
This composite photo shows the actual microscopic presentation of CTC isolated and counted from the peripheral blood of patients. Specimens 104, 106, and 108-110 are designated as viable CTC because of morphology and staining by DAPI (red) and cytokeratin (green). CD45 staining is negative in each of the CTC cell images. Specimens 105 and 107 are not viable CTC because neither DAPI nor cytokeratin stains are contained within the cell membrane, and the cells do not have a normal morphology.
PCR-based assay. This assay measures free DNA in blood after extraction and PCR amplification with oligonucleotide primers to specific genes. Alternatively, RT-PCR and cDNA synthesis can also be used to measure mRNA. The assay identifies genes associated with a specific cancer by using primers designed to recognize the genes. While this method boasts a high analytical sensitivity, limitations of the technique include a high rate of false positives resulting from dying cancer cells, as well as problems with mRNA stability and copy number that appears to vary with the cell cycle.
Enrichment by filtration. Exciting new advancements have been made in filtration enrichment of CTC. Richard Cote and colleagues at the Biomedical Nanosciences Initiative, University of South California Keck School of Medicine, have developed filters with the precise pore size capable of retaining CTC while permitting the passage of red blood cells, leukocytes and platelets. Figure 3 shows clusters of CTC enriched from a patient with breast cancer that are resting on the filter matrix. To verify that cells isolated on the filter are CTC, the researchers treat them with an immunofluorescent dye to detect cytokeratin (green) within the cell cytoplasm and DAPI (blue) stain, indicating that the cells are nucleated CTC.
CTC Enrichment by Filtration
With a pore size of 7-8 µm (B), this filter permits smaller red blood cells, leukocytes, and platelets to pass, but catches CTC (A and C). When treated with immunofluorescent dyes, CTC stain blue/green (D).
Flow cytometry. To enhance the rate of detection and isolation of CTC from blood, some labs use flow cytometry to sort EpCAM-expressing cells from CD45-expressing white blood cells. Initially, nucleated cells are separated from blood by Ficoll-Hypaque density gradient centrifugation, and the collected cells are stained with EpCAM-PE antibody to identify CTC. The method also separates the CTC from CD45-positive white blood cells. Flow cytometry is considered a less traumatic method for CTC isolation since magnetic force is not used to pull CTC from whole blood, which may explain the higher yield of CTC by this method. In addition, with a fluorescence-activated cell sorter (FACS), CTC can be selectively isolated and the cells’ DNA and RNA can be analyzed.
Today, clinicians need early indications of therapeutic effectiveness to improve care of patients with cancer. A good prognostic test could help patients and their oncologists decide on the aggressiveness of therapy. In some cases, this would allow patients to avoid unnecessary treatment. For those resistant to certain therapies, this information would also allow clinicians to modify or change the treatment in a timely fashion.
In prospective studies designed to monitor therapeutic effectiveness in patients with breast, colon, or prostate cancer, researchers found CTC enumeration to be predictive of post-therapeutic response and prognostic prior to therapy. In one study of patients with breast cancer, researchers used 5 CTC/7.5 mL of blood as the lower cutoff limit. Patients with higher numbers of CTC demonstrated lower overall survival. In a cohort of patients with prostate cancer treated at Memorial Sloan Kettering, we demonstrated that CTC number was a strong independent predictor of survival as a continuous variable, with no threshold effect. In other studies evaluating patients with breast cancer, investigators used CTC to modify the staging system for advanced breast disease.
Another aspect of cancer treatment in which CTC analysis may be helpful is development of new cancer-directed drugs, where enumeration of cells may act as an intermediate endpoint to predict clinical outcomes. A biomarker that is predictive of treatment response would provide an early assessment of the effectiveness of a new drug. For example, in men with castrate-resistant prostate cancer, higher baseline CTC counts measured before initiation of therapy were observed in patients with bone metastases relative to those with soft tissue disease. The counts were modestly correlated to other measurements of tumor burden, such as PSA levels and bone involvement. In this study, baseline CTC counts were also strongly associated with survival, without a threshold effect, which increased further when others factors were accounted for, such as PSA levels.
In prospective studies of patients with metastatic prostate, breast, and colon cancer, baseline CTC number was a strong predictor of overall survival, and patients with persistent CTC shedding from the tumor after initiation of therapy had markedly inferior outcomes relative to those who did not. These data suggest that shedding of cells into the circulation represents an intrinsic property of the tumor biology and may provide unique information for prognosis and treatment response.
The emergence of CTC analysis could also have a significant impact on predicting which tumors will respond to a particular therapeutic intervention. This information would be particularly valuable when the clinician is choosing which therapy to use. In addition, because CTC retain specific genetic alterations, they could potentially provide a road map for characterizing a specific panel of markers predictive of tumor sensitivity. As a blood test, this information could be obtained with minimal patient discomfort.
It also may be possible to analyze molecular profile changes in CTC as a means to monitor treatment effectiveness. Such information could be valuable for streamlining development of new targeted therapies based on biologic effect, instead of maximum tolerated dose as is currently done. Not only could this type of analysis improve clinical trials of new drugs and speed their approval, most importantly it also could enable clinicians to administer the most efficacious, least toxic therapies to patients and improve outcomes.
CTC analysis, including enumeration, isolation, and genomic interrogation, has great potential for developing personalized therapies that will change the current way clinical trials are designed and how new targeted drugs are evaluated. In the future, I anticipate that CTC analysis will obviate the need for repeated invasive biopsies and bone marrow aspirations in patients with metastatic disease. Researchers have already demonstrated the potential of CTC analysis as an alternative to invasive biopsies in patients with metastatic prostate cancer.
As more advanced CTC technology becomes available, the importance of this assay in cancer patient management will grow as a tool to identify patients receiving therapy that is not working or to better manage patients with recurrent disease. I anticipate that CTC assays will resonate with oncologists accustomed to using serum-based circulating tumor markers in patient management, because it offers the potential to be used in real time to modify therapy and to help make sound clinical judgments, which is the goal that all involved in care of patients with cancer strive to achieve.
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Martin Fleisher, PhD, FACB, is chairman of the Department of Clinical Laboratories and chief and director of the Clinical Chemistry Service, Memorial Sloan-Kettering Cancer Center, New York. Dr. Fleisher’s research focuses on biomarker discovery and translational clinical investigations. He received AACC’s 2008 Morton K. Schwartz Award for Significant Contributions in Cancer Research Diagnostics.