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Letters to the Editor |
1 Virology Department, Lapeyronie Hospital, University Medical Center Montpellier, Montpellier, France
2 INSERM U475, Immunopathology of Tumor Disease and Autoimmunity, Montpellier, France
3 Urology Department, Beau Soleil Clinic, Montpellier, France
4 Institute of Tumor Biology, University Medical Center, Hamburg-Eppendorf, Hamburg, Germany
5 Department of Gynecology, University Medical Center, Hamburg-Eppendorf, Hamburg, Germany
6 Centre Régional de Lutte contre le Cancer (CRLC), Val d’Aurelle Clinic, Montpellier, France
aAddress correspondence to this author at: Institute of Tumor Biology, Center for Experimental Medicine, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, D-20246 Hamburg, Germany. Fax 49-40-42803-5374; e-mail pantel{at}uke.uni-hamburg.de.
To the Editor:
Metastasis is the major cause of cancer-related death. Single disseminated tumor cells (DTC) can be detected in the bone marrow and peripheral blood years before the occurrence of clinically detectable metastases. Recent clinical studies, however, have clearly indicated that a significant fraction of breast cancer patients with DTC never develop distant metastases (1). Thus, DTC detected by sensitive immunocytochemical and molecular assays may be apoptotic or may lack stem cell properties and never give rise to an overt metastasis. The ability to detect and characterize viable DTC is therefore of utmost importance.
We applied a novel technique for the detection and ex vivo characterization of single viable DTC derived from epithelial tumors. Our technique for detection of specific secreted proteins, epithelial immunospot (EPISPOT), is an adaptation of the enzyme-linked immunospot assay. EPISPOT detects only viable tumor cells and can detect protein secretion at an individual cell level, allowing the direct determination of protein-secreting cell (SC) frequencies. Immunospots are the protein fingerprint left only by the viable SCs. Cell culture is needed for a sufficient amount of secreted marker proteins to accumulate to form immunospots; dying tumor cells do not secrete adequate amounts of protein and thus are not detected.
We first studied blood samples from breast and prostate cancer patients with gross metastases. SCs for the circulating tumor antigens mucin 1 (MUC1) or prostate-specific antigen (PSA) were detected in the majority of patients with metastatic breast (100%) and prostate (83.3%) cancer, respectively, whereas such SCs were not observed in healthy controls or in patients with benign prostatic hyperplasia (2). Consistent with our previous findings, the EPISPOT assay revealed viable DTC in the peripheral blood of 65% of prostate cancer patients, even in the absence of overt metastases (stage M0) (2), but the number of PSA-SCs in localized prostate cancer patients (median, 9; range, 2–197) was significantly lower (P = 0.01) than in patients with metastatic cancer (median, 29; range, 1–684), a finding that is in accordance with the different disease stages and total tumor burdens. Our findings, that MUC1- or PSA-SCs were present in patients with breast or prostate cancer but not in controls, suggest that these cells could be DTC released from the primary tumor.
To characterize DTC, we focused on fibroblast growth factor 2 (FGF2), a known stem cell growth factor also relevant for the in vitro growth of micrometastatic cells (3). We developed a dual fluorescent PSA/FGF2-EPISPOT to characterize PSA-SCs for the secretion of FGF2 and applied it to blood samples from 19 patients with localized prostate cancer. This study received ethics review board approval, and sample donors gave written informed consent. PSA-SCs were detected in 15 patients, and a subset of these SCs also secreted FGF2, suggesting that a significant fraction of DTC may secrete a factor potentially relevant to their outgrowth.
In addition, we tested whether the EPISPOT assay could also be applied to the analysis of bone marrow. In addition to MUC1, we screened for secretion of cytokeratin-19 (CK19), an intermediate filament of epithelial cells. Interestingly, our data provide the first evidence that CK19 can be secreted (Fig. 1A
). The enumeration of both MUC1- and CK19-SCs allowed the detection of viable DTC in 90% and 54% of breast cancer patients with (Fig. 1B
) and without (Fig. 1C
) overt distant metastasis, respectively. These incidences are in the range of those obtained with sensitive reverse transcription PCR–based techniques (4). The number of DTC per sample was considerably lower in M0-patients than M1-patients, but most of the DTC detected in M0 patients showed the CK19+/MUC1– phenotype (Fig. 1C
). DTC with this phenotype might have a particular biological potential, because recent findings of Gudjonsson et al. (5) suggested that MUC1–/CK19+ cells in the human breast may have stem cell–like properties. In addition to being structural proteins, cytokeratins play an unexpected role in influencing cell growth and size by regulating protein synthesis. A further characterization of the discovered MUC1–/CK19+ subset of DTC may be an important step toward the identification of the putative metastatic stem cells.
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Taken together, our results demonstrate that DTC in patients with cancer of the prostate or breast are viable and heterogeneous with regard to the secretion of relevant proteins. Many secreted proteins influence metastatic progression (e.g., growth factors and proteases). The EPISPOT assay, a multiparameter technology that reveals a unique fingerprint of proteins secreted by single viable DTC, opens a new avenue in the understanding of the biology of the metastatic cascade. This information may be used in the future for improved molecular staging and treatment monitoring of cancer patients.
Acknowledgments
This work was supported by grants from the Ministère de l’Economie des Finances et de l’Industrie; the University Medical Center of Montpellier, France; and the European Commission (DISMAL project, contract no. LSHC-CT-2005-018911).
References
The following articles in journals at HighWire Press have cited this article:
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  C. Alix-Panabieres, S. Riethdorf, and K. Pantel Circulating Tumor Cells and Bone Marrow Micrometastasis Clin. Cancer Res., August 15, 2008; 14(16): 5013 - 5021. [Abstract] [Full Text] [PDF]
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 G. J. Strewler Cancer Stem Cells and Bone, and Cancer Cell Dormancy and Bone Metastasis: Meeting Report from Skeletal Complications of Malignancy V: October 25-27, 2007 in Philadelphia, Pennsylvania, USA IBMS BoneKEy, August 1, 2008; 5(8): 285 - 288. [Full Text] [PDF]
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D. F. Hayes and J. Smerage Is There a Role for Circulating Tumor Cells in the Management of Breast Cancer? Clin. Cancer Res., June 15, 2008; 14(12): 3646 - 3650. [Abstract] [Full Text] [PDF]
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M. Ignatiadis, G. Kallergi, M. Ntoulia, M. Perraki, S. Apostolaki, M. Kafousi, G. Chlouverakis, E. Stathopoulos, E. Lianidou, V. Georgoulias, et al. 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., May 1, 2008; 14(9): 2593 - 2600. [Abstract] [Full Text] [PDF]
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 K. Pantel, C. Alix-Panabieres, and S. Riethdorf Circulating Tumor Cells: Detection and Clinical Relevance Am. Assoc. Cancer Res. Educ. Book, April 12, 2008; 2008(1): 603 - 610. [Abstract] [Full Text] [PDF]
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C. Alix-Panabieres and K. Pantel EPISPOT Assay: Detection of Proteins Secreted by Viable Disseminating Tumor Cells in Solid Tumor Patients Am. Assoc. Cancer Res. Educ. Book, April 12, 2008; 2008(1): 611 - 615. [Abstract] [Full Text] [PDF]
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