LABORATORY CLINICAL INTERFACECirculating tumor cells (CTCs): Detection methods and their clinical relevance in breast cancer
Introduction
The outcome of breast cancer largely depends on the development of metastases in the course of the disease. Given this vital importance of metastases, means to detect and monitor their existence are continuously sought for. The detection of circulating tumor cells (CTCs) is one field of research focusing on a new method to detect metastatic disease earlier, less invasive and more reliably than currently available conventional methods, such as clinical presentation, radiographic evaluation and serum tumor markers do. CTCs are defined as tumor cells circulating in the peripheral blood of patients, shed from either the primary tumor or its metastases. Numerous efforts have been made to reliably detect and quantify CTCs in peripheral blood, but development of a suitable assay has proven to be difficult. Unfortunately, there is not one specific feature that universally distinguishes CTCs from blood cells. Ideally, a specific marker would be identified, which is expressed in every cell of every breast cancer type. In reality, different histological and molecular types of tumors express different arrays of markers, and marked heterogeneity of expression exists even within one histological distinct tumor type. Another challenge regarding sensitivity of assays is the fact that CTCs are rare events, with numbers as low as one CTC in 106–107 leukocytes.1 In spite of these challenging characteristics, the importance of detecting and enumerating CTCs in breast cancer has been established in several clinical studies, showing a correlation with decreased progression-free survival (PFS) and overall survival (OS).[2], [3] In addition to detection and enumeration, molecular characterization of CTCs provides a second much-anticipated application in oncology. Currently, we are dependent on the primary tumor for molecular characteristics in order to determine the type of therapy the patient will benefit from most. However, tumor genotype and/or phenotype may change in the course of treatment as indicated by therapy resistance. CTCs might function as a real-time biopsy of tumor load, and enable oncologists to make better-informed choices regarding therapy.
In addition to CTCs, disseminated tumor cells (DTCs), i.e., isolated tumor cells in bone marrow, are thought to reflect the metastatic potential of tumors. DTCs have also been correlated with prognosis[4], [5], but for their detection an invasive diagnostic procedure, bone marrow aspiration, is necessary. This requirement makes their implementation in the clinic more troublesome. By contrast, CTCs have the advantage of being readily available in peripheral blood and given this, together with mounting evidence supporting their clinical feasibility as reviewed here, the detection of CTCs is anticipated to gain clinical relevance shortly. For a comprehensive review on DTCs and their complementary role to CTCs, we refer to the recent review by Riethdorf et al.6
Here we will discuss the principals and technical aspects of the different techniques available for detecting CTCs, in addition to the most frequently used markers in these techniques. Furthermore, we will discuss clinical studies showing the utility of CTC detection in breast cancer patients with these techniques. Reviews focusing on the detection techniques of both DTCs and CTCs, as well as biological relevance, have been published recently.[7], [8]
Section snippets
Detection of circulating tumor cells
In general, methods for CTC detection can be divided into cytometric (i.e., whole-cell based) and nucleic-acid based techniques. Both techniques usually include an enrichment step and a detection step.
As CTCs are rare events occurring at rates as low as one cell per 106–107 leukocytes, enrichment is generally needed to increase sensitivity to an acceptable level. One type of enrichment relies upon the selection of target cells with tumor-specific markers (immunoseparation). Other methods for
Increasing assay sensitivity: enrichment techniques
As mentioned before, enrichment can be based on morphologic cell characteristics, such as size or density, or on immunoseparation, using magnetic beads, ferrofluids or rosettes (see Fig. 1, Fig. 2).
Cytometric methods
The presence of tumor cells in the bone marrow was first identified using conventional imaging techniques.19 Building on this, detecting tumor cells in the circulation was attempted using simple hematoxylin and eosin staining.20 This exhaustive method consisted of visually identifying large numbers of gradient-separated cells and comparing them with primary tumor cells morphologically. Nowadays, as previously mentioned, detection of CTCs occurs on a cytometric or a nucleic-acid basis.
Cytometric
Combined enrichment and detection techniques
CellSearch (Veridex™, Warren, PA) is a semi-automated technology by which whole blood is enriched for CTCs by adding ferrofluids loaded with antibodies directed towards EpCAM. Currently, CellSearch is the only FDA-approved assay for CTC detection. CTCs in the enriched population are stained with CK and DAPI using fluorescent antibodies, while hematopoietic cells are counterstained with CD45. The CK+/DAPI+/CD45- cells are then enumerated with an automated fluorescence microscope. The
Markers
The effectiveness of tumor cell enrichment and detection depends upon the choice of markers, tools to identify and characterize CTCs. Many different markers have been explored in the field of CTCs. To date, no one marker has proven to be ideal for the detection of breast cancer CTCs. This is not unexpected given the heterogeneity of the disease and the rarity of CTCs. In breast cancer, a wide array of markers has been studied, especially with nucleic-acid based techniques. In Table 3, we
Clinical applications of CTC detection
The presence of occult metastases cannot be deduced from the finding of CTCs alone, as CTCs must pass through several stages before forming a metastatic colony. Cells must extravasate from the circulation into target organs and subsequently proliferate whilst evading immunological response and overcoming metabolic difficulties. It has been estimated that only one in 10,000 CTCs is able to form a metastasis.50
Despite all this, the clinical usefulness of CTC detection has been demonstrated in
Neoadjuvant setting
For patients presenting with locally advanced breast cancer, i.e., tumors presenting with extensive regional lymph node involvement, skin involvement or a large size (>5 cm), resection of the primary tumor is frequently either not possible or only at the cost of an amputation of the breast. Systemic therapy given prior to management of the primary tumor, also known as neoadjuvant therapy, aims to reduce tumor size thereby rendering the residual tumor amendable for a breast-conserving resection.
Adjuvant setting
Adjuvant chemotherapy refers to systemic therapy after primary surgery for early stage breast cancer patients who are considered to have a high risk for metastatic disease developing from micro-metastases that are already present at initial presentation. The intent of adjuvant therapy is to cure patients by eradicating these micrometastases. Currently, it is not possible to adequately identify patients who do not harbor micrometastases and therefore should be spared from adjuvant therapy and
CTC detection in metastatic breast cancer
In metastatic disease, the intention of treatment is essentially palliative, striving to optimize quality rather than duration of life. Assessing prognosis in patients with metastatic breast cancer with CTCs can be helpful in the individualized management of these patients. In a multicenter, prospective study conducted by Cristofanilli et al.,78 CTC count was assessed using CellSearch in 177 progressive metastatic breast cancer patients who were to start a new line of systemic therapy. CTCs
Discussion
Circulating tumor cells are being recognized as a promising diagnostic tool in oncology, and thus many efforts have been made to detect them reliably. In breast cancer, several techniques (both cytometric and nucleic-acid based) have been explored in different settings yielding interesting results. However, in addition to independent confirmation of these results, several issues remain to be resolved.
Studies have shown remarkably varying CTC counts, ranging from <5 to thousands per ml in the
Conflicts of interest statement
None declared.
Acknowledgement
This study was supported in part by The Netherlands Genomics Initiative (NGI)/Netherlands Organization for Scientific Research (NWO).
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Cited by (0)
- c
Erasmus MC, Daniel den Hoed Cancer Center, Room G4-83, Groene Hilledijk 301, 3075 EA Rotterdam, The Netherlands. Tel.: +31 10 7041331; fax: +31 10 7041003.
- d
Erasmus MC, Josephine Nefkens Institute and Cancer Genomics Centre, Room BE-426, Dr. Molenwaterplein 50, 3015 GE Rotterdam, The Netherlands. Tel.: +31 10 7044369; fax: +31 10 7034627.
- e
Erasmus MC, Daniel den Hoed Cancer Center, Room D2-80a, Groene Hilledijk 301, 3075 EA Rotterdam, The Netherlands. Tel.: +31 10 7041418; fax: +31 10 7041005.