Abstract
The biological and clinical significance of circulating tumor cells (CTC) in the peripheral blood of breast cancer patients is not known. To study this question, we used a direct visualization assay to correlate the number of CTC with disease stage and progression. The CTC were enriched from the nucleated cell fraction by filtration and enumerated visually following immunostaining with anti-cytokeratin 8 (CK8) antibody CAM 5.2. In mixing experiments, we achieved a limit of detection of 5 MCF7 cells per 5 ml of blood or 5 × 107 peripheral blood leukocytes (PBL). We did not detect CTC in any control subjects (0/20). In 131 breast cancer patients, we found a higher incidence of CTC in patients with distant metastatic 36/51 (71%) than those with node-positive 17/36 (47%)(p= 0.026), or node-negative 17/44 (39%)(p= 0.001) disease. The distribution of the highest numbers of CTC observed in individual patients by repeated sampling over time ranged from 1 to 700 per 5 ml of blood with a trend toward higher numbers in those with distant metastases. In comparison with previous studies of equal specificity, based on a similar absence of CTC in controls, we report a higher incidence of CTC in node-negative and node-positive patients, suggesting a more frequent detection of CTC by our approach. This higher incidence was achieved, in part, by repeated sampling of our study population over time. Our results support the concept that CTC can be detected and enumerated in peripheral blood and that this minimally invasive assay merits further evaluation as a potential prognostic indicator and marker of disease progression.
References
Lalle M, De Rosa L, Marzetti L, Montuoro A: Detection of breast cancer cells in the bone marrow or peripheral blood: methods and prognostic significance. Tumori 86: 183–190, 2000
Schoenfeld A, Kruger KH, Gomm J, Sinnett HD, Gazet JC, Sacks N, Bender HG, Luqmani Y, Coombes RC: The detection of micrometastases in the peripheral blood and bone marrow of patients with breast cancer using immunohistochemistry and reverse transcriptase polymerase chain reaction for keratin 19. Eur J Cancer 33: 854–861, 1997
Slade MJ, Smith BM, Sinnett HD, Cross NCP, Coombes RC: Quantitative polymerase chain reaction for the detection of micrometastases in patients with breast cancer. J Clin Oncol 17: 870–879, 1999
Choy A, McCulloch P: Induction of tumour cell shedding into effluent venous blood breast cancer surgery. Brit J Cancer 73: 79–82, 1996
Brandt B, Roetger A, Heidl S, Jackisch C, Lelle RJ, Assmann G, Zänker KS: Isolation of blood–borne epithelium–derived c–erbB–2 oncoprotein–positive clustered cells from the peripheral blood of breast cancer patients. Int J Cancer 76: 824–828, 1998
Racila E, Euhus D, Weiss AJ, Rao C, McConnell J, Terstappen LWMM, Uhr JW: Detection and characterization of carcinoma cells in the blood. Proc Natl Acad Sci USA 95: 4589–4594, 1998
Martin VM, Siewert C, Scharl A, Harms T, Heinze R, Ohl S, Radbruch A, Miltenyi S, Schmitz J: Immunomagnetic enrichment of disseminated epithelial tumor cells from peripheral blood by MACS. Exp Hematol 26: 252–264, 1998
Krag DN, Ashikaga T, Moss TJ, Kusminsky RE, Feldman S, Carp NZ, Moffat Jr FL, Beitsch PD, Frazier TG, Gaskin TA, Shook JW, Harlow SP, Weaver DL: Breast cancer cells in the blood: a pilot study. Breast J 5: 354–358, 1999
Smith BM, Slade MJ, English J, Graham H, Lüchtenborg M, Sinnett HD, Cross NCP, Coombes C: Response of circulating tumor cells to systemic therapy in patients with metastatic breast cancer: comparison of quantitative polymerase chain reaction and immunocytochemical techniques. J Clin Oncol 18: 1432–1439, 2000
Terstappen LWMM, Rao C, Gross S, Weiss AJ: Peripheral blood tumor cell load reflects the clinical activity of the disease in patients with carcinoma of the breast. Int J Oncol 17: 573–578, 2000
Kraeft S–K, Sutherland R, Gravelin L, Hu G–H, Ferland LH, Richardson P, Elias A, Chen LB: Detection and analysis of cancer cells in blood and bone marrow using a rare event imaging system. Clin Cancer Res 6: 434–442, 2000
Beitsch PD, Clifford E: Detection of carcinoma cells in the blood of breast cancer patients. Am J Surg 180: 446–449, 2001
Mé hes G, Witt A, Kubista E, Ambros PF: Circulating breast cancer cells are frequently apoptotic. Am J Pathol 159: 17–20, 2001
Witzig TE, Bossy B, Kimlinger T, Roche PC, Ingle JN, Grant C, Donohue J, Suman VJ, Harrington D, Torre–Bueno J, Bauer KD: Detection of circulating cytokeratinpositive cells in the blood of breast cancer patients using immunomagnetic enrichment and digital microscopy. Clin Cancer Res 8: 1085–1091, 2002
Kahn HJ, Yang L–Y, Blondal J, Lickley L, Holloway C, Hanna W, Narod S, McCready DR, Seth A, Marks A: RTPCR amplification of CK19 mRNA in the blood of breast cancer patients: correlation with established prognostic parameters. Breast Can Res Treat 60: 143–151, 2000
Vona G, Sabile A, Louha M, Sitruk V, Romana S, Schü tze M, Capron F, Franco D, Pazzagli M, Vekemans M, Lacour B, Bréchot C, Paterlini–Bréchot P: Isolation by size of epithelial tumor cells. Am J Pathol 156: 57–63, 2000
Oud PS, Zahniser DJ, Garcia GL, van Boekel MCG, Haag DJ, Hermkens HG, Pahlplatz MMM, Vooijs PG, Herman CJ: Pressure–fixation method of transferring cells from polycarbonate filters to glass slides. Anal Quant Cytol 6: 131–138, 1984
Kahn HJ, Bailey D, Marks A: Monoclonal antibody D2–40, a new marker of lymphatic endothelium, reacts with Kaposi's sarcoma and a subset of angiosarcomas. Mod Pathol 15: 434–440, 2002
Braun S, Pantel K, Muller P, Janni W, Hepp F, Kentenich CRM, Gastroph S, Wischnik A, Dimpfl T, Kindermann G, Riethmuller G, Schlimok G: Cytokeratinpositive cells in the bone marrow and survival of patients with stage I, II, or III breast cancer. New Engl J Med 342: 525–533, 2000
Braun S, Hepp F, Kentenich CRM, Janni W, Pantel K, Riethmüller G, Willgeroth F, Sommer HL: Monoclonal antibody therapy with edrecolomab in breast cancer patients: monitoring of elimination of disseminated cytokeratin–positive tumor cells in bone marrow. Clin Cancer Res 5: 3999–4004, 1999 HJ Kahn et al. 246
Ménard S, Squicciarini P, Luini A, Sacchini V, Rovini D, Tagliabue E, Veronesi P, Salvadori B, Veronesi U, Colnaghi MI: Immunodetection of bone marrow micrometastases in breast carcinoma patients and its correlation with primary tumour prognostic features. Brit J Cancer 69: 1126–1129, 1994
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Kahn, H.J., Presta, A., Yang, LY. et al. Enumeration of Circulating Tumor Cells in the Blood of Breast Cancer Patients After Filtration Enrichment: Correlation with Disease Stage. Breast Cancer Res Treat 86, 237–247 (2004). https://doi.org/10.1023/B:BREA.0000036897.92513.72
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DOI: https://doi.org/10.1023/B:BREA.0000036897.92513.72