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Molecular Oncology, Markers, Clinical Correlates |
1 Medical Oncology Department, 2 Unité Mixte de Recherche 144 Centre National de la Recherché Scientifique, 3 Department of Tumor Biology, and 4 Surgery Department, Institut Curie, Paris, France
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Experimental Design: Cytospun Ficoll fractions of peripheral blood and bone marrow were obtained simultaneously in 114 breast cancer patients at different stages of the disease (I to IV) before treatment with chemotherapy. The pancytokeratin (CK) monoclonal antibody A45-B/B3 (anti-CKs 8, 18, and 19) was used for epithelial cell detection. Immunostained cells were detected by an automated cellular imaging system (ChromaVision Medical System).
Results: CK+ cells were detected in 28 (24.5%) patients in blood and in 67 (59%) patients in bone marrow. Twenty-six (93%) patients with CK-positive cells in blood also had positive bone marrow (P < 0.001). Positive cells were detected in peripheral blood in 3/39 (7.5%) operable breast cancers (stage I/II), 9 of 36 (25%) locally advanced breast cancers (stage III), and 16 of 39 (41%) patients with metastatic disease (stage IV; P = 0.017). In the subgroup of nonmetastatic patients (n = 75), prognostic factors for poor disease-free survival were: absence of estrogen receptor; presence of CK+ cells in bone marrow (P = 0.012); clinical nodal involvement; large tumor size (T4); and presence of tumor emboli. Presence of circulating CK+ cells in the peripheral blood was not statistically correlated with disease-free survival. On multivariate analysis, independent indicators for disease-free survival were: absence of estrogen receptor (P = 0.043) and presence of CK+ cells in bone marrow (P = 0.076).
Conclusions: The clinical relevance of circulating epithelial cells as a prognostic factor is not supported by the present data, especially in comparison with tumor cells in the bone marrow. However, this method of detection may be useful to monitor the efficacy of treatment in advanced or metastatic breast cancer.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The aim of the present study was to evaluate the feasibility of identifying CK-expressing cells in peripheral blood with an automated-assisted immunohistochemical detection system and to compare the results with the same detection method in BM samples. We conducted this study on blood and BM samples from breast cancer patients with stage I to IV disease before chemotherapy.
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PATIENTS AND METHODS |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The median age was 51 years (range: 31–78 years). Forty-five of the 75 nonmetastatic patients received neoadjuvant chemotherapy and 30 received adjuvant chemotherapy based on 4–6 cycles of docetaxel-doxorubicin or epirubicin, cyclophosphamide +/- 5-fluorouracil. Twelve patients who received neoadjuvant chemotherapy were treated for inflammatory breast cancer (T4d). Twenty-four patients received repeated high-dose chemotherapy with peripheral blood stem cell reinfusion: 10 for inflammatory breast cancer and 14 for high axillary nodal involvement (>8N+). All patients with positive hormone receptors received adjuvant tamoxifen. Thirty-nine metastatic patients received chemotherapy: as first-line treatment in 85% (33 patients) or second- or third-line treatment in 15% (6 patients). Twenty-five stage IV patients (64%) had clinically documented bone metastasis.
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1 million cells) was cytocentrifuged onto polylysine- coated slides at 580 x g twice for 3 min (Hettich Universal 16A cytocentrifuge; Ref. 22
). The supernatant was carefully removed from each slide after the first cytocentrifugation, and the slides were allowed to dry in air overnight. Slides were stored at room temperature before staining or were stored at -20°C and then at -80°C until staining. A median number of 10 x 106 MNCs (range: 5–35) and 40 x 106 MNCs (range: 5–530) were recovered after Ficoll in blood and BM samples, respectively.
ICC Staining.
The pancytokeratin (CK) monoclonal antibody A45-B/B3 (Micromet, Munich, Germany and ChromaVision, San Juan Capistrano, CA), which recognizes several CK epitopes CK 8, CK 18, and CK 19, was applied for cell detection (23)
. The immunostaining procedure was standardized by using a Cadenza automat (Shandon). Before staining, cytospots were fixed with 4% paraformaldehyde for 5 min, then dried for 15 min at room temperature. Endogenous alkaline phosphatase was then blocked with Tris-buffered saline solution (Sigma) with 2% AB serum (15 min; Sanofi Diagnostics Pasteur). This solution was used to dilute primary and secondary antibodies. After blocking, the slides were incubated with the primary antibody A45 B/B3 for 40 min (2 µg/ml). Control slides were incubated under the same conditions with a mouse monoclonal anti-FITC IgG1 (1/1250; Sigma Immuno Chemicals). Slides were incubated for 20 min with secondary polyclonal rabbit antimouse antibody (Dako). After each step, the slides were rinsed for 5 min in Tris-buffered saline 1x solution. Immune complexes were revealed by the alkaline phosphatase-antialkaline phosphatase technique (1/50;Dako) for 25 min (24)
. The chromogen reaction was performed for 20 min with a colorimetric substrate of fuchsin solution (2.5% in 2 N HCl; New Fuchsin, Sigma) with 4% NaNO2, 8% ß-naphthol (Sigma), and 2% levamisole (Dako). Cells were counterstained with Mayer hematoxylin (1 min; Sigma) and diluted to one-third in distilled water. The specimen was then rinsed under running water for 5 min and then in Tris-buffered saline. Slides were coverslipped using Faramount mounting medium (Dako). A total of 3 x 106 mononuclear cells (three slides) was evaluated for each patient and for each sample (BM and blood sample). The number of MNCs evaluated was based on a statistical model established on the hypothetical assumption that a highly sensitive antibody recognizes all epithelial cells in a pure BM sample examination of 3 x 106 BM-derived cells to provide a probability of detecting one epithelial cell/106 MNC with 95% confidence interval (6
, 25)
. Negative controls, stained with anti-FITC monoclonal mouse antibody, were performed on an equivalent number of cells (i.e., three slides, 3 x 106 mononuclear cells) for each patient.
Positive controls were obtained with BM from normal donors undergoing orthopedic surgery (Cochin Hospital), spiked with SKBR3 or MCF7 cell lines, 10 to 102 for 106 mononuclear cells/cytospot. One positive control slide and one negative control slide were added to each series of 20 stained slides in the automated device.
CK+ Cell Detection by Digital Microscopy.
The automated cellular imaging system (ChromaVision Medical Systems, Inc., San Juan Capistrano, CA) is a computerized microscope, which includes an image processing system that has been optimized for the detection of rare carcinoma cells in specimens (26)
. The application software supplied with the instrument starts by scanning a microscope slide at low magnification (x10). The instrument then returns to objects originally identified by their stain for a second analysis at higher magnification (x40 or x60). In this case, more sophisticated image analysis of color and morphometric characteristics is performed to exclude cellular debris, large clumps, and cells with morphological features typical of normal hematological mononuclear cells as opposed to CK+ carcinoma cells. Cellular objects that meet color- and morphometry-based criteria for probable tumor cells are collected and presented as montage images for review and classification by a pathologist or another investigator (J-Y. P.). Independently, slides of BM specimens were manually blind reviewed by a pathologist. Pictures of CK+ cells detected manually by the pathologist were digitized on his own microscope and stored. After comparison of the results of automated cellular imaging system and the pathologist’s examination (A. V-S.), in the case of discrepancy (15% of the cases), a consensus was established between the observers based on joint evaluation. Criteria for evaluation of immunostained cells in BM were adapted from Borgen et al. (27)
based on the results of the European ISHAGE Working Group for standardization of tumor cell detection. These criteria are summarized in Table 2
. Blood specimens were analyzed with the automated cellular imaging system and were reviewed by a single investigator (J-Y. P.), blinded to the patient characteristics.
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2 tests for categorical variables and t tests for continuous variables (or Mann-Whitney test for nonparametric variables). Survival time and disease-free survival time were measured from the date of BM aspiration until the date of death or last follow-up. Survival curves were determined using a Kaplan-Meier product-limit method (28)
. Statistical significance between groups was assessed using the log-rank test. Multivariate analysis was carried out to assess the relative influence of prognostic factors on disease-free survival, using the Cox proportional hazards model in a forward stepwise procedure (29)
. Statistical analyses were performed by Statview software (SAS Institute, Inc.). |
RESULTS |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Only 2 patients had positive blood with negative BM (Table 3)
. CK+ cells were detected in peripheral blood in 3 of 39 (7.5%) patients with operable breast cancer (stage I/II), 9 of 36 (25%) patients with locally advanced breast cancer (stage III), and 16 of 39 (41%) patients with metastatic disease (stage IV; Table 4
). In four blood samples, 1 or 2 positive immunostained cells were detected on control slides and were therefore classified as negative cases. In BM, the percentage of positive cases ranged from 33% in stage I and II to 77% in stage IV (Table 4)
. Thirty seven (49%) nonmetastatic patients were considered to be positive for BM micrometastatic cell detection, according to our classification adapted from Borgen et al. (27)
: 11 were classified as A; 26 as B; 16 as C; and 22 as D. The BM classification for the 114 patients was 30 as A, 37 as B, 18 as C, and 29 as D.
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. The mean and median number of CK+ cells in BM-positive patients with positive blood were 129 and 23, respectively, versus 49 and 5 in BM-positive patients with negative blood (P = 0.05, Mann-Whitney).
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). The presence of CK+ cells in the BM in nonmetastatic patients was correlated with premenopausal status (P = 0.024), high tumor size (T4; P = 0.025), ER-negative tumors (P = 0.026), and tumor emboli (P = 0.043). Age was not significantly different in the two groups: 50 years for CK+ in BM versus 53 in CK- (P = 0.18, t test). For the whole population (n = 114), the presence of CK+ in blood and/or BM was strongly correlated with clinical metastatic disease (Table 4)
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Survival.
Median follow-up was 28 months, and 31 deaths have occurred in these 114 patients. Four deaths have occurred on nonmetastatic patients (n = 75). The overall survival prognosis cannot be assessed due to this small number of events and short follow-up in nonmetastatic patients. Thirteen patients developed distant metastases and 6 developed local relapses. Prognostic factors were evaluated for disease-free survival. The same prognostic factors were observed for metastasis-free survival (data not shown).
In the 114 patients, on univariate analysis, poor prognostic factors for overall survival were clinical metastatic stage (stage IV; P < 0.0001), presence of CK+ cells in BM (P = 0.0004; Fig. 3
), presence of CK+ cells in peripheral blood (P = 0.0004; Fig. 4
), and premenopausal status (P = 0.047; Table 5
). On multivariate analysis, metastatic clinical stage (P < 0.0001) and presence of CK+ cells in BM (P = 0.051) were independent factors of poor prognosis (Table 6)
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), clinical nodal involvement (0.016), large tumor size (T4; P = 0.029), and presence of tumor emboli (P = 0.048; Table 7
). Presence of circulating CK+ cells in peripheral blood was not statistically significant (Fig. 6)
. On multivariate analysis, the independent indicator for disease-free survival was absence of ER (P = 0.043). The second factor in the model was presence of CK+ cells in BM (P = 0.076; Table 8
).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the present study, most of the cases positive for blood were observed in metastatic patients or in patients with locally advanced breast cancer. The incidence of CK+ cells in patients at an operable stage was low (7.5%). In a large study on 155 patients before high-dose chemotherapy with autologous stem cell support, tumor cells were detected by ICC staining of peripheral blood cytospins in 17% of cases, mostly in stage IV patients (31) . The incidence of CK-19 mRNA-positive cells was 30% in peripheral blood in early breast cancer and 52% in metastatic breast cancer in Stathopoulou’s study (15) . The overall sensitivity of detection is, however, lower in blood than in BM. CK+ cells may indeed not circulate continuously, and the small volume of blood may result in sampling errors.
In our study with ICC and automated detection, we obtained a level of detection comparable with that observed with PCR techniques. Slade et al. (32) compared a semiquantitative RT-PCR assay to ICC for the detection of CK 19-positive cells in the peripheral blood of breast cancer patients. In this study, PCR was not significantly superior to ICC, 49.6% of these samples were positive by RT-PCR, and 42% were positive by ICC. Automated-assisted screening appears to increase the sensitivity of epithelial cell detection by allowing the routine examination of a larger number of BM or PB cells (26 , 33) . This sensitivity can be potentially increased by other techniques such as immunomagnetic bead enrichment of larger sample volumes. The tumor-rich magnetic fraction can be processed by RT-PCR, ICC, or in situ hybridization. This should lead to more accurate quantification and molecular characterization of these tumor cells (34) .
The use of density centrifugation in the present study is likely to result in the loss of many circulating tumor cell (CTC; particularly CTC clumps). Immunomagnetic separation, followed by specific RT-PCR (35) , allows semiquantitative detection of circulating mammary cells without density centrifugation. A significant correlation was found in the latter study between the frequency of CTC and the clinical stage of breast tumors: 24% in the group with localized disease (versus 16% in the present study) and 45% in the group with metastatic disease (versus 41% in the present study). In Witzig’s series (33) , using immunomagnetic separation (immunomagnetic separation) after Ficoll on peripheral blood of breast cancer patients, the percentage of detection of CK+ cells by ICC in blood was 76% in metastatic stages versus 40% in our series without enrichment. In a series of operable breast cancers, reported by Krag et al. (36) , tumoral cells were detected using high gradient magnetic cell sorting in blood samples preceding surgical removal of the primary tumor in 18 of 19 patients. Prognostic factors for disease-free survival in our series were classical: tumor size; clinical nodal status; tumor emboli; and BM micrometastatic disease. Pathological nodal status was not a prognostic factor in this series but was assessed after neoadjuvant chemotherapy in the majority of nonmetastatic patients. ERs are crucial factors because all receptor-positive patients received tamoxifen. Hormonal receptors have been described as an early prognostic factor, which loses its significance with long follow-up. This could explain the high value of this parameter in this series with short follow-up (37) .
The clinical value of circulating epithelial cells has yet to be established. BM cells appear to be more predictive of relapse than peripheral blood cell detection. BM may indeed act as a filter to concentrate breast cancer cells, as reflected by the propensity of breast cancer cells to form metastases in bone. A similar observation has been reported in Ewing’s sarcoma, where RT-PCR was performed to look for EWS-FLI-1 fusion transcripts, resulting from the specific t(11;22) translocations. Among patients with localized tumors, BM micrometastasis predicted significantly poorer disease-free survival rates, but the presence of CTCs was not predictive of relapse (38) . With a larger number of patients and longer follow-up, BM micrometastasis remained a major prognostic factor, and CTC became associated with a poor outcome among patients with clinically localized disease (39) .
Because of the simplicity of repeated sampling, CTCs would constitute a possible alternative or complement to BM surveillance. It could be used to monitor the efficacy of treatment in advanced or metastatic breast cancer. According to Smith et al. (40) , the number of cancer cells evaluated by RT-PCR or ICC reflects the outcome of systemic treatment in the majority of patients. This assay could screen for high-risk patients, determine the need for and monitor response to adjuvant or neoadjuvant therapy, and detect early recurrence of breast cancer.
In conclusion, the presence of tumor cells in blood is strongly correlated with the presence of these cells in BM. The number of cells detectable in blood by ICC is lower than in BM. Using an automated detection device, ICC is feasible for CTC detection in blood, but without any immunoselection technique, the level of detection is low even in metastatic patients with a high tumor burden. The prognostic value of CK+ cell detection in blood is lower than that in BM, but this may because of a lower detection sensitivity of the present method (low sampling volume). The prognostic value of BM involvement is clearly demonstrated by a number of large studies, which is not the case for blood. Blood could be more effectively used for sequential monitoring of treatment in adjuvant or metastatic situations.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: Jean-Yves Pierga, Département d’Oncologie Médicale, Institut Curie, 26 rue d’Ulm, 75231 Paris Cedex 05, France. Phone: 33-1-44-32-46-70; Fax: 33-1-44-32-46-71; E-mail: jean-yves.pierga{at}curie.net
Received 1/22/03; revised 7/ 8/03; accepted 11/ 6/03.
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REFERENCES |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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