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Originally published as JHC exPRESS on April 14, 2008.
doi:10.1369/jhc.2008.951111
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Volume 56 (7): 667-675, 2008
Copyright ©The Histochemical Society, Inc.
Phenotypic and Genetic Characterization of Circulating Tumor Cells by Combining Immunomagnetic Selection and FICTION Techniques
Immunology Division, Department of Health Sciences, Faculty of Experimental Sciences, University of Jaén, Jaén, Spain (MC,FW,JR-M,JJG); Division of Oncology, Center for Applied Medical Research (CIMA), Pamplona, Spain (CP,IZ,RC,AC); and Department of Genetics (IZ) and Department of Histology and Pathology (RC,AC), University of Navarra, Pamplona, Spain
Correspondence to: José J. Gaforio, PhD, MD, Immunology Division, Department of Health Sciences, Faculty of Experimental Sciences, Campus las Lagunillas, University of Jaén, 23071 Jaén, Spain. E-mail: jgaforio{at}ujaen.es
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The presence of circulating tumor cells (CTCs) in breast cancer patients has been proven to have clinical relevance. Cytogenetic characterization of these cells could have crucial relevance for targeted cancer therapies. We developed a method that combines an immunomagnetic selection of CTCs from peripheral blood with the fluorescence immunophenotyping and interphase cytogenetics as a tool for investigation of neoplasm (FICTION) technique. Briefly, peripheral blood (10 ml) from healthy donors was spiked with a predetermined number of human breast cancer cells. Nucleated cells were separated by double density gradient centrifugation of blood samples. Tumor cells (TCs) were immunomagnetically isolated with an anti-cytokeratin antibody and placed onto slides for FICTION analysis. For immunophenotyping and genetic characterization of TCs, a mixture of primary monoclonal anti-pancytokeratin antibodies was used, followed by fluorescent secondary antibodies, and finally hybridized with a TOP2A/HER-2/CEP17 multicolor probe. Our results show that TCs can be efficiently isolated from peripheral blood and characterized by FICTION. Because genetic amplification of TOP2A and ErbB2 (HER-2) in breast cancer correlates with response to anthracyclines and herceptin therapies, respectively, this novel methodology could be useful for a better classification of patients according to the genetic alterations of CTCs and for the application of targeted therapies. (J Histochem Cytochem 56:667–675, 2008)
Key Words: breast cancer • circulating tumor cells • cytokeratin expression • FICTION • ERBB2 (HER-2/neu) gene • immunomagnetic selection • TOP2A gene
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Introduction |
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BREAST CANCER is the most frequent type of cancer in women (Parkin et al. 2005
). Because of its high incidence and good prognosis, breast cancer is the most prevalent cancer in the world today. Approximately 4.4 million women who were diagnosed with breast cancer within the last 5 years are still alive (Parkin et al. 2005). Despite the improvement in detection and treatment, 
30% of newly diagnosed women with breast cancer will die. In most cases, death results from the dissemination of cancer cells through lymphatic or blood vessels and the development of distant metastases.
The ErbB2 oncogene (also known as c-erbB2/HER-2/neu) is the most frequently amplified oncogene in breast cancer (20–35% of invasive breast tumors) (Pauletti et al. 1996; Press et al. 1997; Ross and Fletcher 1999), which correlates with poor clinical outcome (Järvinen and Liu 2003). ErbB2 is localized at 17q12-q21 and encodes a 185-kDa tyrosine kinase protein that belongs to the epidermal growth factor (EGF) receptor family. Although there is no known ligand for ErbB2, heterodimerization with other members of the EGF receptor family causes a strong mitotic response (Olayioye et al. 2000; Yarden and Sliwkowski 2001). Patients with ErbB2 gene amplification or protein overexpression are eligible for trastuzumab (Herceptin) therapy.
TOP2A, a gene that codes for the protein topoisomerase-II
, is another key gene for breast cancer. TOP2A is located at the ErbB2 region and has been found altered in almost 90% of primary breast cancers with ErbB2 amplification (Järvinen et al. 1999,2000). However, although 40% of those cases have a coamplification of both genes, in another 40% of the tumors, TOP2A has been found deleted (Järvinen and Liu 2003). Breast cancer cells with high topoisomerase-II expression show a better response to anthracyclines (such as doxorubicin, epirubicin, or idarubicin) (Järvinen and Liu 2003,2006; Hannemann et al. 2006). Therefore, the determination of genomic amplification of either ErbB2 or TOP2A in breast cancer patients is critical for administering an effective targeted therapy.
Breast cancer has been shown to shed tumor cells into the circulation, even at the earliest stages of primary tumor development (Gaforio et al. 2003). Pretlow et al. (2000) have shown that circulating tumor cells (CTCs) isolated from peripheral blood (PB) of patients with cancer are able to develop metastasis when xenotransplanted into nude mice. Thus, the early detection of CTCs may have important therapeutic and prognostic implications. The number of CTCs in PB from breast cancer patients has clinical relevance (Gaforio et al. 2003; Cristofanilli et al. 2005; Cristofanilli and Mendelsohn 2006; Müller et al. 2006; Camara et al. 2007), not just as an indicator of overall survival, but also as a disease progression marker and/or metastatic marker. However, little attention has been paid to the cytogenetic features of such cells.
In 1992, Weber-Matthiesen et al. (1992) developed a new technique for simultaneous immunophenotyping and interphase cytogenetics analyses using cell lines. The method was referred to as fluorescence immunophenotyping and interphase cytogenetics as a tool for investigation of neoplasm (FICTION). This technique was shown to be very useful in the study of hematological neoplasms (Nylund et al. 1993; Haferlach et al. 1997; Temple et al. 2004; Young et al. 2006). In this study, we show the development of a novel method that combines immunomagnetic selection of tumor cells (TCs) with cytogenetic characterization by FICTION analysis. With this method, we were able to isolate TCs from a 10-ml blood sample, immunophenotype them (with anti-cytokeratin antibodies), and analyze ErbB2, TOP2A, and CEP17 gene copy number.
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Materials and Methods |
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Materials
Cell culture media MEM with Earle's salt and RPMI-1640 and fetal calf serum (FCS) were obtained from PAA Laboratories (Pasching, Austria). Penicillin-streptomycin, PBS, formaldehyde 37% solution (formalin), Igepal, Histopaque 1077, Histopaque 1119, poly-L-lysine–coated glass slides, and Fast Red TR/Naphthol AS-MX substrate were purchased from Sigma (St. Louis, MO). The Carcinoma Cell Enrichment and Detection Kit, the MACS MS Columns, anti-cytokeratin 7/8 antibody (isotype: mouse IgG2a) conjugated to FITC, and anti-FITC antibody (Isotype: mouse IgG1) conjugated to alkaline phosphatase were obtained from Miltenyi Biotec (Bergisch Gladbach, Germany). Mouse anti-human AE1-AE3 antibody was purchased from BioGenex (San Ramon, CA). This is a biclonal mouse antibody cocktail that recognizes cytokeratins 1/2/3/4/5/6/7/8 (clone: AE1; immunoglobulin class: IgG1) and cytokeratins 10/14/15/16/19 (clone: AE3; immunoglobulin class: IgG1). Secondary antibodies Alexa Fluor 350 rabbit anti-mouse IgG (H+L) and Alexa Fluor 350 goat anti-rabbit IgG (H+L) and the Slow Fade Light Antifade Kit were obtained from Invitrogen (Eugene, OR). The LSI TOP2A/HER-2/CEP17 multicolor probe was purchased from Abbott Molecular (Vysis; Des Plaines, IL). Sodium chloride-sodium citrate buffer (SSC) was obtained from MP Biomedicals Europe (Illkirch, France). Methanol, acetone, and ethanol absolute were purchased from Panreac Quimica (Barcelona, Spain). Acetic acid (glacial), Mayer's hematoxylin, and Kaiser's glycerol gelatin were obtained from Merck (Darmstadt, Germany).
Cell Lines
The human breast cancer cell lines MCF-7 and MDA-MB-231, known to have no ErbB2 amplification, were obtained from the American Type Cultured Collection (ATCC; Rockville, MD). SK-Br-3, a human breast cancer cell line with ErbB2 amplification, was obtained from Eucellbank (Barcelona, Spain). MCF-7 and MDA-MB-231 cells were grown in MEM with Earle's salt, supplemented with 10% (v/v) heat-inactivated FCS and 1% of a stock solution of penicillin-streptomycin. SK-Br-3 was grown in RPMI 1640, supplemented with 10% (v/v) heat-inactivated FCS and 1% of a stock solution of penicillin-streptomycin. Cells in the exponential growth phase were used for all experiments.
Blood Sample Processing and Density Gradient Separation
PB samples (10 ml) from nine healthy volunteers were obtained in heparinized tubes (BD Vacutainer; Becton Dickinson, Heidelberg, Germany) and spiked with 1000 MCF-7, MDA-MB-231, or SK-Br-3 breast cancer cells. As negative controls, we processed three different samples without breast cancer cells. All samples were obtained with previous informed consent of healthy donors. A double-density ficoll gradient was prepared by placing 5 ml Histopaque 1077 in a tube containing an equal volume of Histopaque 1119. Blood samples were carefully placed onto the upper layer of Histopaque 1077, and tubes were centrifuged at 700 x g for 30 min at 20C. The mononuclear cell fraction (plasma/1077 interphase) and granulocyte fraction (1077/1119 interphase) were isolated, and both fractions were mixed and washed in 50 ml PBS by centrifugation at 200 x g for 15 min, according to previous publications (Gaforio et al. 2003).
Cell Enrichment by Immunomagnetic Separation
Tumor cells were immunoseparated using the Carcinoma Cell Enrichment and Detection Kit, with some modifications. Briefly, after cell permeabilization, the cell pellet was resuspended in 40 ml dilution buffer plus 5 ml MACS CellPerm Solution and incubated for 5 min at room temperature. Cells were fixed by adding 5 ml CellFix Solution for 30 min at room temperature. Cells were washed twice, resuspended in 600 µl MACS CellStain Solution, incubated with 200 µl Fc-Blocking Reagent, and immunomagnetically labeled with 200 µl MACS anti-cytokeratin microbeads (microbeads conjugated to a monoclonal anticytokeratin 7/8 antibody; clone: CAM5.2).
After a 45-min incubation at room temperature, cells were washed, resuspended in 1 ml CellStain Solution, and placed onto a MACS MS column. Unlabeled cells were washed off the column with 3 x 500 µl dilution buffer. The column was removed from the magnetic field, and the retained cells (magnetic-positive cell population) were eluted with 1 ml dilution buffer. The magnetically enriched cell fractions were spun down onto poly-L-lysine–coated glass slides in a cytocentrifuge (Hettich; Tuttlingen, Germany) at 1500 rpm for 10 min. Slides were air-dried overnight at room temperature and stored at –20C without fixation.
LSI TOP2A/HER-2/CEP17 Probes
The LSI TOP2A/HER-2/CEP17 multicolor probe includes a TOP2A probe labeled with SpectrumOrange, an ERBB2 (HER-2/neu) probe labeled with SpectrumGreen, and a chromosome enumeration probes CEP17, labeled with SpectrumAqua.
FICTION
Previously published protocols for FICTION were used (Weber-Matthiesen et al. 1992), with some modifications. After thawing, slides were fixed in an ice-cold mixture of methanol and acetone (1:1) for 5 min and air dried. Slides were hydrated in PBS for 5 min and incubated for 30 min with a blocking solution (10% rabbit serum in PBS) before primary antibody incubation. The slides were washed in PBS for 5 min and incubated with the biclonal mouse anti-AE1-AE3 antibody overnight at 4C, diluted 1:200 in PBS containing 10% FCS. The revelation was conducted by applying two sequential layers of secondary antibodies (1:50 dilution in PBS for both of them, 30 min at room temperature): Alexa Fluor 350 rabbit anti-mouse IgG and goat anti-rabbit IgG. After fluorescent immunophenotyping, slides were coverslipped under PBS for evaluation under a Zeiss Axioplan 2 epifluorescence microscope (Carl Zeiss; Jena, Germany), with appropriate filter sets. ISIS software (MetaSystems; Altslussheim, Germany) was used for evaluation throughout.
After assessment of positive (tumor) cells on the slides, samples were fixed with Carnoy's solution (3:1 methanol: acetic acid fixative), rinsed in distilled water for 1 min, fixed in 1% formaldehyde, and rinsed again in distilled water for 1 min. After dehydration in an ethanol series (70%, 80%, and 100%) and air dried, slides were codenatured with the LSI TOP2A/HER-2/CEP17 multicolor probe for 5 min at 85C and hybridized overnight in a humidified chamber at 37C. Posthybridization wash was carried out at 72C in 2x SSC/0.3% Igepal, pH 7, for 2 min, followed by another wash at room temperature. Finally, slides were mounted with the Slow Fade Light Antifade Kit. Microscopic evaluation was carried out with the microscope and software imaging system described above.
A minimum number of 50 morphologically intact and non-overlapping nuclei were scored in every sample to determine the number of hybridization signals for each ErbB2, TOP2A, and CEP17 probe. Both absolute copy numbers and the relative copy number ratio (ratio between the mean number of ErbB2 or TOP2A signals and the mean number of chromosome 17 centromere signals) were determined. A [ErbB2 or TOP2A]/CEP17 ratio 
1.5 was considered gene amplification. Similarly, ratios 
0.7 were considered gene deletions.
Analysis of Cell Recovery
To determine the number of cells recovered, we applied the double-density ficoll gradient and immunomagnetic separation describe previously, followed by immunocytochemistry (using anti-cytokeratin antibodies) for quantification of positive cells. Briefly, we used 10 ml of blood from healthy volunteers that was spiked with 1000 cells (MCF-7, MDA-MB-231, or SK-Br-3 cell lines). After double-density ficoll gradient, immunomagnetic labeling, and separation, cells were subjected to cytospin and stored at –20C without fixation. After thawing, slides were stained with 100 µl of anti-cytokeratin 7/8 conjugated to FITC (isotype: mouse IgG2a) for 10 min at room temperature and further labeled with 10 µl anti-FITC antibody conjugated to alkaline phosphatase (isotype: mouse IgG1) for 10 min at room temperature. Slides were washed in PBS for 5 min, and cytokeratin-expressing cells were detected by incubation with Fast Red TR/Naphthol AS-MX substrate solution for 15 min in a moist chamber. Slides were washed in PBS, counterstained with Mayer's hematoxylin, and mounted with Kaiser's glycerol gelatin. Cytokeratin-expressing cells (CK+) were counted separately by two expert researchers. All experiments were done in triplicate.
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Efficiency of the TCs Immunoselection Method
Cell recovery was in the range of 60–80% for the three cell lines studied (Table 1 ), which is similar to what was described in previous studies (Gaforio et al. 2003
). All tumor cells (CK+) showed a strong cytoplasmic staining pattern, and the cell morphology was consistent with a malignant phenotype (Figures 1A
–1C). According to previous publications, although tumor cells were specifically immunoselected, some white blood cells could be seen in the positive tumor fraction (Figures 1A–1C). Immunophenotyping was also conducted with mouse anti-cytokeratins antisera followed by two layers of fluorescent antibodies (Alexa Fluor 350 rabbit anti-mouse and Alexa Fluor 350 goat anti-rabbit). This procedure rendered an excellent labeling of tumor cells for the three lines analyzed (Figures 1D–1F).
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FICTION Analysis of the Selected TCs
Breast cancer cells were unequivocally distinguished among the white blood cells by their blue color provided by the immunofluorescent labeling (Figures 2A –2C). The three tumor cell lines displayed a well-preserved morphology (Figures 2A–2C).
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Hybridization signals for ErbB2, TOP2A, and CEP17 were also clearly observed in both tumor cells and leukocytes (Figures 2A–2F). All leukocytes showed two signals for ErbB2, TOP2A, and CEP17, thus serving as internal controls. Quantitative results of the number of signals found in tumor cells are given in Table 2 . The most common genetic pattern for the cell line MCF-7 was the presence of three copies of chromosome 17 (CEP17) but two copies of ErbB2 (mean, 2.02 ± 0.24 copies/cell) and TOP2A (mean, 2.01 ± 0.26 copies/cell; Figure 2D; Table 2). In the case of MDA-MB-231 cells, two patterns were basically found. The predominant one (accounting for 91.33% of the cells) showed trisomy of CEP17. The other one was tetrasomic for CEP17 (which was observed in 8.67% of the cells). In general, neither of these populations presented ErbB2 or TOP2A amplifications or deletions (average ratio with respect to CEP17 being 1.01 for both genes; Table 1). The cell line SK-Br-3 showed many copies of ErbB2, TOP2A, and CEP17 (Figure 2F). The average copy number for ErbB2 was 25.87 ± 7.39, whereas for TOP2A, it was 9.48 ± 2.55. Gene amplification for ErbB2 (3.81-fold relative to CEP17) was found in this cell line. In negative control samples, no hybridization signals or blue-positive cells were detected.
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Discussion |
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Studies from our group and from others have shown that the number of CTCs in breast cancer patients correlates with clinical outcome (Gaforio et al. 2003
; Cristofanilli et al. 2005; Müller et al. 2006). The presence of more than five CTCs in a 7.5-ml blood volume determines a bad prognosis and decreased overall survival, irrespective of the treatment received by the patients (Cristofanilli et al. 2004). These remarkable data could be even more clinically relevant if the precise genetic alterations of such cells were accurately determined. We showed here the feasibility of analyzing TCs by immunomagnetic separation and FICTION techniques to examine gene copy number of ErbB2, TOP2A, and CEP17 in CK+ (i.e., tumor) cells.
Few studies have applied molecular techniques to identify genetic signatures of CTCs. The analysis by RT-PCR for mammaglobin and B305D-C in CTCs showed a sensitivity of 70% and a specificity of 81% for the diagnosis of invasive breast carcinoma (Reinholz et al. 2005). Several studies analyzing genetic defects in disseminated tumor cells isolated from bone marrow showed several chromosomal alterations (Klein et al. 1999; Schmidt-Kittler et al. 2003) and amplification of the ErbB2 gene by fluorescence in situ hybridization (FISH) (Müller et al. 1996; Schardt et al. 2005). However, the molecular characterization of CTCs in PB would be preferable to more invasive procedures, such as bone marrow aspirations.
The FICTION technique was developed to assign tumor cells to a cytogenetically defined clone and, at the same time, to determine their specific cell lineage (Weber-Matthiesen et al.1992,1993). FICTION has been proven to be clinically meaningful in hematological neoplasms (Nylund et al 1993; Haferlach et al. 1997; Temple et al. 2004; Young et al. 2006). In solid tumors, three studies have applied FICTION to correlate the presence of genetic alterations with the expression of a particular protein (Terada et al. 2000; Zhang et al. 2000; Dettori et al. 2003). Zhang et al. (2000) used this technique in six breast carcinoma cell lines to study the relationship between estrogen receptor (ER) expression and deletion of the estrogen receptor gene (ESR). Terada et al. (2000) studied, using FICTION, 105 patients with gastric tumors, showing that the frequency of positive cells for proliferating cell nuclear antigen (PCNA) and chromosome 17 numerical aberrations were indicators of metastatic potential. Dettori et al. (2003) evaluated, using FICTION, the immunoexpression of a mitochondrial membrane antigen with a simultaneous visualization of numerical chromosomal aberrations in 18 patients with thyroid tumors.
In this study, we showed for the first time that breast cancer cells isolated from PB can be analyzed by FICTION to simultaneously examine ErbB2/TOP2A/CEP17 copy number and cytokeratins 1-8/10/14-16/19 in these cells. To ensure a high specificity of the assay in the analysis of tumor cells, we incorporated the following steps: (a) the first milliliters of blood collected from the samples were discarded to avoid contamination with squamous cells; (b) Fc receptors from cells were blocked to avoid nonspecific binding, previously to the incubation with the anti-cytokeratin mAb; and (c) application of double CK immunolabeling with an anti-CAM5.2 (cytokeratin 7/8) monoclonal antibody (for immunoselection) and with a biclonal anti-AE1/AE3 (anti-pancytokeratin) antibody (for further immunophenotyping).
The genetic amplification of both ErbB2 and TOP2A in SK-Br-3 cells was previously published using FISH (Szöllösi et al. 1995; Järvinen et al. 1999,2000; Forozan et al. 2000), comparative genomic hybridization (Forozan et al. 2000; Lottner et al. 2005), and spectral karyotyping (Kytölä et al. 2000), and is coincident with our results. The deletion of ErbB2 in MCF-7 cells (Szöllösi et al. 1995; Shadeo and Lam 2006) and the normal ErbB2 copy number in MDA-MB-231 cells (Satya-Prakash et al. 1981; Grushko et al. 2002; Lottner et al. 2005) were also previously documented and are in keeping with our data. However, no data showing the TOP2A status in MCF-7 and MDA-MB-231 cells were previously reported.
Ideally, the technique we describe here could be useful for early detection of ErbB2/TOP2A-amplified disseminated breast cancer and to monitor, in a relatively easy way, the response to herceptin/anthracycline-based therapies. However, many questions arise from our study that should be addressed in future studies. First of all, breast cancer patients should be tested to determine the clinical value of this technique. In addition, it is currently unknown whether ErbB2/TOP2A amplifications or deletions in CTCs will represent the general pattern of aberrations of the primary tumor. ErbB2 is amplified in 20–35% of invasive breast carcinomas, and TOP2A has been found altered in almost 90% of ErbB2-amplified tumors. However, several tumors with extra copies of ErbB2 contain tumor cells with amplification and deletion of the TOP2A gene (Järvinen et al. 1999). It has been suggested that this finding may have therapeutical implications, because TOP2A deletions confer resistance to topoisomerase II inhibitors (Järvinen and Liu 2003). This could be an explanation for the gradual loss of efficacy of topoisomerase II inhibitors, which is common in breast cancer treatment. Meng et al. (2004) found that 9 of 24 patients whose primary tumors were ErbB2 negative showed ErbB2 amplification in their CTCs. However, other studies have shown that ErbB2 status was similar between breast cancer metastases and the primary tumor (Tanner et al. 2001; Dirix et al. 2005). In this scenario, one can think that disseminated circulating cells may come from subclones of tumor cells and may not be a representative sample of the whole tumor.
Despite these caveats, which warrant further studies, the presence of ErbB2/TOP2A-amplified CTCs could serve as a surrogate marker to monitor targeted therapy. A rapid drop in the number of CTCs and cytokeratin-19 mRNA levels has been described in patients after herceptin treatment (Bozionellou et al. 2004). It is likely that the monitoring of the number of cells carrying the specific target against which drugs were designed (whether is herceptin or anthracyclines) would be suitable for testing the efficacy of such therapy. Although we describe here a FICTION method for the analysis of ErbB2/TOP2A/CEP17 status in TCs from PB, many other breast cancer targets could be analyzed using a similar technology. For instance, a recent paper showed that node-negative breast cancer patients with 11q deletion might benefit from anthracycline-based chemotherapy (Climent et al. 2007).
In summary, we showed the feasibility of a new methodology that combines immunomagnetic selection and FICTION for ErbB2/TOP2A/CEP17, which could allow genetic characterization of CTCs from PB.
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Acknowledgments |
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This study was supported by the "Fundación de Investigación Médica Mutua Madrileña" (to JJG) and partially funded by an ISCIII-RETIC RD06/0020 grant (to AC).
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Footnotes |
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Received for publication February 22, 2008; accepted March 27, 2008
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