Despite the advances in early detection and treatment of cancer, patients continue to die of the disease even when they seek care at an early stage. For patients with breast cancer, it is now possible to detect circulating tumor cells (CTCs) in the bloodstream and disseminated tumor cells (DTCs) in the bone marrow by using immunocytochemical and molecular methods. CTCs and DTCs have been found to share similar genotypic and phenotypic characteristics with so-called breast cancer stem cells, a finding that could potentially explain the eventual relapse of disease in a patient previously considered to have been cured by primary therapy. In some studies, the presence of CTCs or DTCs at the time of diagnosis of breast cancer is an independent adverse prognostic variable. However, before CTC/DTC testing can achieve standard-of-care status, there must be improvement in the sensitivity, precision, and reproducibility of the detection methods.
Circulating tumor cells
Disseminated tumor cells
In the United States, cancer is the second leading cause of death, with lung, colon, prostate, and breast cancers accounting for the majority of fatal cases.1 Despite advances in early detection and treatment, many patients continue to die of the disease even if it was originally diagnosed at an early stage. Current opinion favors that minimal residual disease, after the completion of primary therapy, ultimately leads to disease relapse and distant metastases. This concept dates back to 1869, when Ashworth2 first reported on the existence of circulating tumor cells (CTCs). In 1889, Paget3 described the theory of “seed and soil” based on his observations of meta-static breast cancer.
In the early 1990s, molecular methods were first used to detect circulating prostate cancer and melanoma tumor cells using real-time polymerase chain reaction techniques (RT-PCR).4,5 At the same time, the expanded use of immunohistochemical analysis in bone marrow and lymph nodes further enhanced the detection of micrometastasis. This was followed by novel techniques to detect CTCs in peripheral blood and disseminated tumor cells (DTCs) in bone marrow. CTC and DTC assays use unique cell-separation strategies and ultrasensitive tumor cell detection methods and have now been applied to the study of breast, lung, colorectal, gastric, esophageal, pancreatic, prostatic, gallbladder, head and neck, bladder, ovarian, and cervical cancers.6
CTC and DTC Detection Methods
A variety of methods have been developed to detect CTCs in blood and DTCs in bone marrow samples of patients with breast cancer Table 1.7–11 Although the peripheral blood and bone marrow normally do not contain significant numbers of benign epithelial cells, the extremely low concentration of malignant epithelial cells in positive blood or bone marrow samples, at about 1 in 106 to 107 total nucleated cells, still makes them extremely difficult to detect. Thus, to enhance the identification of rare CTCs and DTCs, methods designed to increase their concentration, including differential centrifugation, Ficoll enrichment, and cell separation by immunomagnetic techniques, have been the cornerstone of commercially developed and novel early-stage technologies.7–11 A major issue confronting all approaches is the problem of the loss of malignant cells owing to the inherent fragility of CTCs and DTCs.12 The positive detection of CTCs and DTCs has used a variety of methods, including immunohistochemical analysis, immunofluorescence, fluorescence in situ hybridization (FISH), flow cytometry, Southern blot, Northern blot, PCR, RT-PCR, cell culture, and proteomic techniques.7–11 For breast cancer, a variety of individual and multiplex biomarkers for CTC and DTC identification have been used, including cytokeratins, MUC1, GA 733-2, mammaglobin, maspin, and carcinoembryonic antigen.7–11 In addition, several studies and approaches have also used biomarkers such as CD45 to exclude circulating leukocytes and actin to exclude platelets.7–11
The CellSearch System (Veridex, Raritan, NJ) is a semiautomated device for the detection of CTCs expressing the epithelial cell adhesion molecule (EpCAM) with antibody-coated magnetic beads as an enrichment method Figure 1.10 The captured cells are then labeled with fluorescent monoclonal antibodies specific for leukocytes (CD45) and epithelial cells (cytokeratins 8, 18, and 19). Slides are then analyzed for fluorescent marker–labeled CTCs using an automated microscope. CTCs are defined as CK+/CD45– nucleated cells and are counted using the CellSpotter Analyzer (Veridex). This method is now US Food and Drug Administration (FDA)-approved for detection of CTCs in patients with metastatic breast, colon, and prostate cancers.10
The initial CellSearch-based study on a large cohort of patients showed that, for breast cancer, a high CTC count at the time of diagnosis was a significant adverse independent prognostic factor.10 This study also showed that if the initial chemotherapy given to a patient with a positive CTC assay in the metastatic breast cancer setting failed to reduce the number of CTCs to less than 5 cells per 7.5 mL of blood, the disease would undoubtedly progress while undergoing that treatment regimen and the patient’s prognosis was grave.10
Although a number of additional institutions have confirmed this original study, to date, no randomized prospective clinical trial has been conducted that has validated this finding by showing that changing therapy in the face of a failure to reduce CTC levels to less than 5 cells per 7.5 mL of blood could result in an improved patient outcome. Currently, 2 trials are ongoing that have been designed to test whether sequential CTC measurements using CellSearch can improve disease outcome when therapy is changed owing to a rising CTC count.
The CellSearch technique features high intraobserver and interobserver concordance and high interinstrument concordance.12,13 In the CellSearch clinical trials, several apparently healthy people who had standard CTC determinations as control subjects were found to have CTCs. It is not known how many of these people subsequently were diagnosed with malignant disease. Using the CellSearch method, CTCs have been detected in peripheral blood samples of patients with a wide variety of metastatic epithelial malignancies.13 However, the sensitivity of the CellSearch technique has been a subject of concern for some investigators. In a recent study, the detection of 5 or more CTCs in 7.5 mL blood in 43 (52%) of 83 patients with stage IV metastatic breast cancer before the initiation of first-line chemotherapy confirmed the original observations that the CellSearch technique was highly predictive for progression-free and overall survival14 and provided more helpful information than conventional imaging procedures.15 However, another recent study has caused some concern about the CellSearch technique in that it reported that the so-called normal genotype of invasive breast cancer, which accounts for approximately 10% of all cases, is typically negative for EpCAM expression and may thus be a cause of false-negative CellSearch-based CTC determinations.16
The AdnaTest BreastCancerSelect (AdnaGen, Langenhagen, Germany) is another CTC test that features a CTC-enrichment procedure using a proprietary mixture of immunomagnetic beads coated with 1 of 3 antibodies to epithelial surface antigens Figure 2.17,18 After messenger RNA (mRNA) extraction, RNA quantity and integrity is confirmed (Agilent Bioanalyzer, Agilent Technologies, Santa Clara, CA). The “number” of CTCs is indirectly determined by a semiquantitative RT-PCR method using probes for 3 epithelial cell–associated mRNAs: MUC1, HER2, and the surface glycoprotein GA 733-2. To avoid excessive dilution of extracted CTC mRNA, the amount of mRNA extracted from platelets is estimated by determining the relative actin mRNA level, which reflects the extent of platelet contamination. This mRNA-based test claims to have a higher sensitivity than the whole-cell morphologic detection–based CellSearch assay. Although the test has not, to date, passed regulatory approval, the company began a dialogue with the FDA in May 2008. This test is commercialized in Europe for breast and colorectal cancers, and marketing in the United States is expected to commence in 2009 (OncoVista, San Antonio, TX).
Membrane Microfilter Assay
This method uses filters to concentrate the larger CTCs for microscopic identification by pathologists and to separate them from circulating leukocytes and platelets. Polycarbonate filters, the first filters used for this method, are a relatively easy, efficient, and inexpensive technique for CTC enrichment.19 More recently, a microfabrication technique using parylene C has produced a specialized microfilter for separation of circulating CTCs.20 After separation, RT-PCR is performed on the capturing membrane using electrolysis. Although there are presently no published clinical trials using this method, it has significant promise given the apparent high efficiency of the technique. It has not, to date, been commercialized.
The CellSearch System. The figure outlines the CellSearch procedure for determining circulating tumor cells. Adapted with permission from Veridex.
Microfluidic CTC Chip
The CTC chip is a microchip technology that uses a microfluidic device designed to improve CTC capture efficiency Figure 3.21 The CTC chip consists of an array of microposts coated with anti-EpCAM antibodies. A laminar flow system passes whole blood from the patient through the coated posts. The captured CTCs are subsequently identified by fluorescence microscopy after CTC staining with a nuclear marker for DNA content and an anticytokeratin epithelial cytoplasmic marker. To prevent the inadvertent counting of leukocytes, negative selection with an anti-CD45 antibody is used.
The AdnaTest BreastCancerSelect System. The figure outlines the AdnaTest procedure for determining circulating tumor cells. RT-PCR, real-time polymerase chain reaction. Adapted with permission from AdnaGen NA.
In its initial study, the CTC chip achieved a high cell yield (>99%), featuring a large number of isolated CTCs with high purity (>47%) that were believed to be secondary to the use of a single-step technology from whole blood without the additional preparatory steps of centrifugation, washing, and incubation that result in loss and/or destruction of a significant proportion of CTCs in other methods.21 The CTC chip results were positive in cases of metastatic lung, prostate, pancreatic, breast, and colon cancer in 115 (99.1%) of 116 samples, with a range of 5 to 1,281 CTCs per milliliter and approximately 50% purity.21 CTCs were also identified in 7 (100%) of 7 patients with early-stage prostate cancer. In a preliminary study of a small cohort of patients with metastatic cancer, the CTC chip results appeared to correlate with the clinical course of disease as measured by standard radiographic methods.21 This test has not, as yet, been commercialized.
Fiberoptic Array Scanning Technology
A fiberoptic array scanning technology (also called FAST) using fluorescence cytometry combined with an automated digital microscopy imaging system featuring laser-printing optics that scan 300,000 cells per second has been used to detect immunofluorescently labeled CTCs on a glass slide.22,23 This method is in an early stage of development, and large-scale clinical trials have not been reported.
The microfluidic circulating tumor cell (CTC) chip. A, The workstation setup for CTC separation. The sample is continually mixed on a rocker and pumped through the chip using a pneumatic pressure–regulated pump. B, The CTC chip with microposts etched in silicon. C, Whole blood flowing through the microfluidic device. D, Scanning electron microscope image of a captured NCI-H1650 lung cancer cell spiked into blood (pseudo colored red). The inset shows a high-magnification view of the cell. Adapted with permission from Nature Publishing Group and Nagrath et al.21
Epithelial Immunospot (EPISPOT)
The EPISPOT approach detects viable tumor cells by taking advantage of their ability to produce and secrete proteins after a 48-hour cell culture Figure 4.24,25 CD45 is used to negatively select leukocytes. Dying or dead CTCs that do not produce or secrete epithelial-associated proteins are not identified as CTCs in this method. The EPISPOT assay has successfully identified metastatic breast cancer cells cultured from blood and bone marrow using MUC1 and CK19 as the identifying proteins.24,25 The test has not, to date, been evaluated in large-scale clinical trials or undergone formal commercial development.
Laser Scanning Cytometry
The laser scanning cytometry technique identifies CTCs in peripheral blood by the use of an automated laser scanning cytometer.26 Positive CTC identification is achieved by using fluorescent-labeled anti-EpCAM, and negative selection is performed with an anti-CD45 antibody. In a recent clinical study of patients who received neoadjuvant treatment for breast cancer, nearly all subjects were positive for CTCs at the start of therapy and the CTC count correlated with tumor size.26 In addition, an initial decrease in cell numbers highly correlated with the final tumor size reduction. To date, the laser scanning cytometry approach has not been formally commercialized for CTC detection.
Other Homebrew Techniques
Flow cytometry has been implemented for detecting CTCs and DTCs in patients with metastatic cancer, although it has a low sensitivity.7 Routine (nonfluorescence) microscopy using immunohistochemical analysis for epithelial markers after enrichment techniques featuring differential centrifugation approaches has been used with variable results.7,8,27–29
Morphologic vs Molecular Detection of CTCs
The relative advantages and disadvantages of morphologic cell count vs nonmorphologic molecular approaches (RT-PCR) have been debated in numerous reviews. Slide-based cell counting features a high specificity, but a number of investigators believe that this method lacks sensitivity when compared with quantitative mRNA techniques. This likely reflects the fragility of CTCs in general.
The EPISPOT assay procedure. Day 1, the membranes of the EPISPOT plates are coated with a specific antibody. Days 2 to 3, the cells are seeded in each well and cultured for 48 hours. During this incubation period, the released specific proteins are directly immunocaptured by the immobilized antibody on the bottom of the well. Plates are then washed, and cells are removed. The presence of the released protein is revealed by the addition of a fluorochrome-conjugated antibody. Day 4, fluorescent immunospots are counted with an automated reader. One immunospot corresponds to the fingerprint left only by 1 viable cell releasing the marker protein. Adapted with permission from the American Association of Cancer Research and Alix-Panabières et al.25
Increasing the detection rate of CTCs would be desirable because it would allow the test to be more useful in that more patients would be positive at the start of their treatment. At this point, in addition to obtaining prognostic information, the patients could subsequently be monitored for therapy response.
Molecular methods that use RT-PCR to amplify the target mRNAs of tumor cells can also distinguish them from circulating leukocytes and platelets.30,31 A wide variety of mRNA epithelial cell and more specific breast cancer cell biomarkers such as CK19, CK20, MUC1, mammaglobin, and others have been used, with multimarker assays offering greater sensitivity than single-probe assays. Among the main advantages of molecular methods is their enhanced sensitivity and large assortment of available primers. However, the extraction, storage, and preparation of the relatively unstable mRNA can lead to loss of sensitivity and misidentification of tumor-specific markers.25
Other issues with PCR-based tests include the false-positive values caused by low-level expression of the marker by noncancerous cells, heterogeneity in the expression levels of particular target transcripts that cannot be predicted, and false-negative values due to down-regulation of the expression of a single target gene (often overcome when multimarker approaches are used). Another potential issue that may challenge the accuracy of PCR-based CTC detection is the fact that amplifiable cell free nucleic acids can be present in blood and in the debris that drains from primary tumors to their regional lymph nodes.32 The PCR method may also detect mRNA associated with benign and malignant cells, such as the identification of tyrosinase mRNA in lymph nodes containing only benign nevus cell nests that are actually not positive for meta-static melanoma. Finally, PCR can detect viable and nonviable CTCs, which may be important when the CTC test is being used as a monitor of the response to therapy.5–9,24–31
Breast Cancer–Relevant Biomarker Measurements on CTCs and DTCs
Recently, a number of investigators have reported preliminary findings concerning the measurement of biomarkers on CTCs and DTCs and differences between these results and those for the patient’s primary tumors. The measurement of CTC HER2 status has been controversial.33,34 Mostly using the CellSearch method and FISH-based determination of HER2 gene amplification status, some studies have found that CTCs maintain the same HER2 status as the primary tumor, whereas other reports have claimed that CTCs may be HER2+ in cases in which the primary tumor was originally HER2–.33,34 The methodological differences in assessing HER2 status in the primary tumor vs in the CTCs may at least partially account for these discrepant results.
The different CTC techniques have influenced the capability of performing HER2 testing, with the CellSearch method requiring a slide-based HER2 test such as FISH or immunocytochemistry. The RT-PCR–based techniques, with or without immunomagnetic-based cellular enrichment, claimed enhanced sensitivity for the detection of HER2+ CTCs based on their relative HER2 mRNA measurements.17,18 Recent studies have shown that HER2 overexpression on CTCs and DTCs was predictive of a poor clinical outcome in patients with stage I through III breast cancer.18,35 The overexpression of HER2 is predictive of the presence of CTCs in patients with early-stage breast cancer.36 HER2+ DTCs might identify additional patients who can benefit from anti-HER2 targeted therapy. Finally, there has been very limited consideration of estrogen and progesterone receptor determination on CTCs and DTCs,37 which might prove useful in predicting the transition from hormone receptor–positive to hormone receptor–negative disease.
CTCs vs DTCs
It is now widely held that the metastatic propensity of a newly diagnosed primary breast cancer is determined early in tumor progression.38 The presence of DTCs in bone marrow at early stages of breast cancer has been extensively analyzed.7 Although the clinical significance of the detection of DTCs in bone marrow in breast cancer is well studied, the impact on the wide variety of other solid tumors that can also feature bone marrow DTCs early or late in the clinical course of these diseases is not as well understood.7
In an analysis of pooled data from several prospective studies, the detection of bone marrow DTCs in breast cancer was associated with a significantly higher risk of recurrence and disease-specific death.39 In these studies, a variety of detection techniques were used, mostly depending on immunohistochemical analysis to identify the rare malignant epithelial cells among the hematopoietic and stromal elements. Nevertheless, in the 2007 recommendations update from the American Society of Clinical Oncology (ASCO) on the use of tumor markers in breast cancer, it was concluded that data were insufficient to recommend assessment of bone marrow micrometastases for management of patients with breast cancer.40 The ASCO group concluded that, in contrast with the insignificant impact of bone marrow DTC positivity in patients treated with systemic adjuvant chemotherapy, the presence of bone marrow DTCs in patients who did not receive adjuvant systemic therapy did predict a statistically significant higher risk of relapse. However, the difference in distant disease–free survival between patients who had DTCs vs patients who did not was very small.40 The group further concluded that bone marrow DTCs in patients with small, low-grade, node-negative breast cancers did not forecast a sufficiently worse prognosis that could be used to adjust recommendations for adjuvant therapy.40
Prognostic Significance of CTCs
By the end of 2008, more than 550 published reports describing a wide variety of techniques had considered the potential prognostic significance of the presence of CTCs on the outcome of epithelial malignancies. The CellSearch assay is FDA approved in the United States as a prognostic test for breast, colon, and prostate cancers. However, although the negative prognostic impact associated with the detection of CTCs during the course of breast cancer is generally accepted, this relatively complex and expensive test competes with a variety of homebrew prognostic assays and a growing list of approved and unapproved commercialized multigene predictors of clinical outcome.41 Thus, the future commercial success of CTC tests as stand-alone prognostic tests would appear to be limited.
Predictive Significance of CTCs
It is generally accepted that the future expanded use of CTC testing for breast cancer lies in the use of the test to predict therapy efficacy and resistance and serve as a monitor of treatment response. However, despite the prognostic significance of validated data, the ASCO tumor markers group concluded that, as of 2007, the measurement of CTCs was not to be used to influence treatment decisions in patients with breast cancer.40 The group further held that the CellSearch could not be recommended for use until additional validation confirms its clinical value.40
Given this resistance to large-scale clinical adoption based on the original data approved by the FDA, investigators have recommended that prospective, randomized breast cancer clinical trials be developed to verify the cost/benefit ratio of the test. The Southwest Oncology Group and the Breast Cancer Intergroup of North America are now conducting a prospective trial in which patients with metastatic breast cancer who have a positive CTC count using the CellSearch system after 1 cycle of first-line chemotherapy will be randomly assigned to remaining on that therapy until clinical and/or radiographic evidence signals progression or switching therapy at that time to a different chemotherapeutic agent (Southwest Oncology Group protocol S0500).41 As trials that have included CTC measurements in their biomarker protocols proceed in various clinical settings, the critical data needed to confirm the clinical usefulness of CTCs for the ongoing management of breast cancer will be obtained and the future of CTC testing will be decided.
CTC/DTC vs Positron Emission Scanning
The introduction of positron emission tomography–based scanning (PET-CT) for the detection of recurrent and metastatic breast cancer created a competitive environment in which functional imaging was compared with in vitro molecular diagnostics. Published studies designed to coevaluate PET-CT scanning with CTC testing are limited. In 1 study, a significant correlation among PET-CT scan, serum CA 27.29 levels, and CTC counts was found.42 In this study, CA 27.29 and CTC counts had low sensitivity and a negative predictive value for the detection of metastatic disease seen on PET-CT scan.42 However, clinical outcome was not evaluated in this study, and the relative ability for each test to predict therapy response was not assessed.
Summary and Future Clinical Implications
Since the introduction of CTC testing, substantial progress has been made in the field, including the FDA approval of the CellSearch assay. Nevertheless, numerous significant technical and commercial challenges need to be overcome before CTC testing achieves widespread acceptance and becomes incorporated into routine breast cancer management.
Among the important technical issues is the desire to expand CTC usefulness by increasing the number of positive patients eligible for clinical monitoring. The goal to capture just a single circulating malignant cell in a 7.5-mL volume of whole blood, which may feature more than 10 billion benign blood cells, is a major challenge. The fact that the sample volume required to be processed in a typical CTC test is in the milliliter range, whereas typical microdevice measuring systems are normally used to process much smaller sample volumes, is another concern, especially when sample dilution is also required. Some investigators have approached this issue by attempting improvements in the cell capture technology by increasing efficiency of CTC recovery and more effectively separating away from contaminating blood cells and bone marrow elements. This approach will clearly increase the CTC detection sensitivity.
The competition between tests that count captured cells such as the CellSearch technique and tests that feature target gene amplification such as by RT-PCR must also be played out to verify which approach is the most accurate and clinically useful. Of recent controversy is the emerging data surrounding the testing of captured breast cancer cells for important management-associated biomarkers such as HER2 and whether these results have scientific and clinical management merit.
The commercial development of CTC testing has also been controversial. The initial commercial launch of the CellSearch assay featured a decentralized approach in which hospital-based and physician office laboratories were to acquire the testing equipment and necessary reagents and perform the CTC test. More recently, commercial laboratory–based centralized testing has been introduced. The selection of centralized testing similar to the Oncotype DX assay (Genomic Health, Redwood City, CA),43 which introduces sample degradation issues, or decentralized testing in which instruments and reagent kits are sold to customers is of paramount importance.
CTC and DTC testing seems to have significant future potential value in the clinical management of breast cancer, including the identification of patients at high risk for relapse, the stratification of patients to specific adjuvant therapies, and the monitoring of response to treatment in the metastatic and neoadjuvant settings. Further optimization and standardization of CTC and DTC detection techniques; improvements in test sensitivity, specificity, and reproducibility; and, most important, the demonstration of a significant impact on patient outcome in prospective randomized trials could lead to the inclusion of CTC and DTC detection in daily clinical practice.
Molecular profiling and predictive value of circulating tumor cells in patients with metastatic breast cancer: an option for monitoring response to breast cancer related therapies [published online ahead of print August 5, 2008]. Breast Cancer Res Treat. doi:10.1007/s10549-008-0143x.
Isolation by size of epithelial tumor cells in peripheral blood of patients with breast cancer: correlation with real-time reverse transcriptase–polymerase chain reaction results and feasibility of molecular analysis by laser microdissection. Hum Pathol. 2006;37:711–718.
Prognostic value of the molecular detection of circulating tumor cells using a multimarker reverse transcription–PCR assay for cytokeratin 19, mammaglobin A, and HER2 in early breast cancer. Clin Cancer Res. 2008;14:2593–2600.
View protocol abstract: S05005 [A randomized phase III trial to test the strategy of changing therapy versus maintaining therapy for metastatic breast cancer patients who have elevated circulating tumor cell levels at first follow-up assessment]. Southwest Oncology Group Web site. http://www.swog.org/visitors/ViewProtocolDetails.asp?ProtocolID=2046. Accessed March 13, 2009.
Correlation among [18F] fluorodeoxyglucose positron emission tomography/computed tomography, cancer antigen 27.29, and circulating tumor cell testing in metastatic breast cancer [published correction appears in Clin Breast Cancer. 2008;8:457]. Clin Breast Cancer. 2008;8:357–361.