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Cell Biochemistry and Biophysics

, Volume 65, Issue 2, pp 263–273 | Cite as

Circulating Tumor Cells (CTCs) Detected by Triple-Marker EpCAM, CK19, and hMAM RT-PCR and Their Relation to Clinical Outcome in Metastatic Breast Cancer Patients

  • Shu Zhao
  • Huike Yang
  • Minghui Zhang
  • Dekai Zhang
  • Yupeng Liu
  • Yan Liu
  • Ying Song
  • Xiaosan Zhang
  • Hongbin Li
  • Wenjie Ma
  • Qingyuan ZhangEmail author
Translational Biomedical Research

Abstract

In order to investigate the prognostic value of circulating tumor cells (CTCs) in patients with metastatic breast cancer (MBC), the blood cells from 98 MBC patients and 60 controls were evaluated by RT-PCR to detect the presence of markers EpCAM, CK19, and hMAM. Peripheral blood was obtained from all patients with MBC before any systemic therapy. Immunofluorescence staining experiment was conducted on CTCs samples from 10 patients to investigate the coexpression of EpCAM, CK19, and hMAM. In addition, analyses were carried out for their correlation with patients’ clinicopathologic features. EpCAM+, CK19+, and hMAM+ cells were detected in 50 (51.0 %), 43 (43.9 %), and 68 (69.4 %) of the 98 patients, respectively. Triple-marker-positive CTCs were detected in 86 of 98 (87.8 %) patients with a significantly higher rate than the control group. Among the 98 patients, 12 (12.2 %) patients were negative for three genes, 34 (34.7 %) positive for one gene, 29 (29.6 %) positive for any two genes, and 23 (23.5 %) positive for all three genes. Compared to single-marker detection, the triple combined marker detection exhibited significantly higher rate. Furthermore, the specificity of triple combined markers of serial test was 100 %. The expression of three genes was significantly correlated with lymph node metastasis, high histological grade, and high levels of serum CA153 and CEA. Double-immunofluorescence labeling confirmed the presence of following CTCs phenotypes: CK19+/hMAM+, CK19+/hMAM−, CK19−/hMAM+, CK19+/EpCAM+, CK19−/EpCAM+, CK19+/EpCAM−, hMAM+/EpCAM+, and hMAM+/EpCAM−. After 2 years of follow-up, the presence of CTCs with triple-marker positive in peripheral blood was an independent risk factor for reduced progression-free survival (PFS) and overall survival (OS), and the presence of CTCs before any chemotherapy predicts poor OS and PFS in patients with MBC.

Keywords

Breast cancer Circulating tumor cells Triple-marker EpCAM CK19 hMAM 

Notes

Acknowledgments

This experiment was performed in the Oncobiology Key Lab of the Heilongjiang Province Common Institution of Higher Learning. This work was supported by the National Natural Science Foundation of China [grant number 81071889] and Harbin science and technology innovation talents research special funds [grant number 2011RFXYS060].

Conflict of interest

All authors declared that there is no conflict of interest.

References

  1. 1.
    Pantel, K., & Brakenhoff, R. H. (2004). Dissecting the metastatic cascade. Nature Reviews Cancer, 4, 448–456.PubMedCrossRefGoogle Scholar
  2. 2.
    Slade, M. J., & Coombes, R. C. (2007). The clinical significance of disseminated tumor cells in breast cancer. Nature Clinical Practice Oncology, 4, 30–41.PubMedCrossRefGoogle Scholar
  3. 3.
    Lacroix, M. (2006). Significance, detection and markers of disseminated breast cancer cells. Endocrine-Related Cancer, 13, 1033–1067.PubMedCrossRefGoogle Scholar
  4. 4.
    Ignatiadis, M., Georgoulias, V., & Mavroudis, D. (2008). Circulating tumor cells in breast cancer. Current Opinion in Obstetrics and Gynecology, 20, 55–60.PubMedCrossRefGoogle Scholar
  5. 5.
    Stathopoulou, A., Vlachonikolis, I., Mavroudis, D., et al. (2002). Molecular detection of cytokeratin-19-positive cells in the peripheral blood of patients with operable breast cancer: evaluation of their prognostic significance. Journal of Clinical Oncology, 20, 3404–3412.PubMedCrossRefGoogle Scholar
  6. 6.
    Jotsuka, T., Okumura, Y., Nakano, S., et al. (2004). Persistent evidence of circulating tumor cells detected by means of RT-PCR for CEA mRNA predicts early relapse: a prospective study in node-negative breast cancer. Surgery, 135, 419–426.PubMedCrossRefGoogle Scholar
  7. 7.
    Pierga, J. Y., Bonneton, C., Vincent-Salomon, A., et al. (2004). Clinical significance of immunocytochemical detection of tumor cells using digital microscopy in peripheral blood and bone marrow of breast cancer patients. Clinical Cancer Research, 10, 1392–1400.PubMedCrossRefGoogle Scholar
  8. 8.
    Benoy, I. H., Elst, H., Philips, M., et al. (2006). Real-time RT-PCR detection of disseminated tumour cells in bone marrow has superior prognostic significance in comparison with circulating tumour cells in patients with breast cancer. British Journal of Cancer, 94, 672–680.PubMedGoogle Scholar
  9. 9.
    Ashworth, T. R. (1869). A case of cancer in which cells similar to those in the tumours were seen in the blood after death. Australian Medical Journal, 14, 146–149.Google Scholar
  10. 10.
    Van der Auwera, I., Peeters, D., Benoy, I. H., et al. (2010). Circulating tumour cell detection: a direct comparison between the cell- search system, the AdnaTest and CK-19/mammaglobin RT-PCR in patients with metastatic breast cancer. British Journal of Cancer, 102, 276–284.PubMedCrossRefGoogle Scholar
  11. 11.
    Bosma, A. J., Weigelt, B., Lambrechts, A. C., et al. (2002). Detection of circulating breast tumor cells by differential expression of marker genes. Clinical Cancer Research, 8, 1871–1877.PubMedGoogle Scholar
  12. 12.
    Ring, A. E., Zabaglo, L., Ormerod, M. G., et al. (2005). Detection of circulating epithelial cells in the blood of patients with breast cancer: comparison of three techniques. British Journal of Cancer, 92, 906–912.PubMedCrossRefGoogle Scholar
  13. 13.
    Cimino, A., Halushka, M., Illei, P., et al. (2010). Epithelial cell adhesion molecule (EpCAM) is overexpressed in breast cancer metastases. Breast Cancer Research and Treatment, 123, 701–708.PubMedCrossRefGoogle Scholar
  14. 14.
    Saloustros, E., & Mavroudis, D. (2010). Cytokeratin 19-positive circulating tumor cells in early breast cancer prognosis. Future Oncology, 6, 209–219.PubMedCrossRefGoogle Scholar
  15. 15.
    Roncella, S., Ferro, P., Bacigalupo, B., et al. (2006). Relationship between human mammaglobin mRNA expression in breast cancer tissue and clinico-pathologic features of the tumors. Journal of Experimental & Clinical Cancer Research, 25, 65–72.Google Scholar
  16. 16.
    Span, P. N., Waanders, E., Manders, P., et al. (2004). Mammaglobin is associated with low-grade, steroid receptor-positive breast tumors from postmenopausal patients, and has independent prognostic value for relapse-free survival time. Journal of Clinical Oncology, 22, 691–698.PubMedCrossRefGoogle Scholar
  17. 17.
    Zach, O., Kasparu, H., Krieger, O., et al. (1999). Detection of circulating mammary carcinoma cells in the peripheral blood of breast cancer patients via a nested reverse transcriptase polymerase chain reaction assay for mammaglobin mRNA. Journal of Clinical Oncology, 17, 2015–2019.PubMedGoogle Scholar
  18. 18.
    Ignatiadis, M., Kallergi, G., Ntoulia, M., et al. (2008). 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. Clinical Cancer Research, 14, 2593–2600.PubMedCrossRefGoogle Scholar
  19. 19.
    Mikhitarian, K., Martin, R. H., Ruppel, M. B., et al. (2008). Detection of mammaglobin mRNA in peripheral blood is associated with high grade breast cancer: interim results of a prospective cohort study. BMC Cancer, 8, 55–65.PubMedCrossRefGoogle Scholar
  20. 20.
    Osta, W. A., Chen, Y., Mikhitarian, K., et al. (2004). EpCAM is overexpressed in breast cancer and is a potential target for breast cancer gene therapy. Cancer Research, 16, 5818–5824.CrossRefGoogle Scholar
  21. 21.
    Stathopoulou, A., Gizi, A., Perraki, M., et al. (2003). Real-time quantification of CK-19 mRNA-positive cells in peripheral blood of breast cancer patients using the lightcycler system. Clinical Cancer Research, 9, 5145–5151.PubMedGoogle Scholar
  22. 22.
    Xenidis, N., Perraki, M., Kafousi, M., et al. (2006). Predictiveand prognostic value of peripheral blood cytokeratin-19 mRNA-positive cells detected by real-time polymer-ase chain reaction in node-negative breast cancer patients. Journal of Clinical Oncology, 24, 3756–3762.PubMedCrossRefGoogle Scholar
  23. 23.
    Xenidis, N., Vlachonikolis, I., Mavroudis, D., et al. (2003). Peripheral blood circulating cytokeratin-19 mRNA-positive cells after the completion of adjuvant chemotherapy in patients with operable breast cancer. Annals of Oncology, 14, 849–855.PubMedCrossRefGoogle Scholar
  24. 24.
    Hongbo, L., Xiaohui, L., Hong, K., et al. (2007). Assessing routine and serum markers of liver fibrosis in CHB patients using parallel and serial interpretation. Clinical Biochemistry, 40, 562–566.PubMedCrossRefGoogle Scholar
  25. 25.
    Paterlini-Brechot, P., & Benali, N. L. (2007). Circulating tumor cells (CTCs) detection: clinical impact and future directions. Cancer Letters, 253, 180–204.PubMedCrossRefGoogle Scholar
  26. 26.
    Cristofanilli, M., Budd, G. T., Ellis, M. J., et al. (2004). Circulating tumor cells, disease progression and survival in metastatic breast cancer. New England Journal of Medicine, 351, 781–791.PubMedCrossRefGoogle Scholar
  27. 27.
    Tewes, M., Aktas, B., Welt, A., et al. (2009). Molecular profiling and predictive value of circulating tumor cells in patients with metastatic breast cancer, an option for monitoring response to breast cancer related therapies. Breast Cancer Research and Treatment, 115, 581–590.PubMedCrossRefGoogle Scholar
  28. 28.
    Zhao, S., Liu, Y. P., Zhang, Q. Y., et al. (2011). The prognostic role of circulating tumor cells (CTCs) detected by RT-PCR in breast cancer: a meta-analysis of published literature. Breast Cancer Research and Treatment, 130, 809–816.PubMedCrossRefGoogle Scholar
  29. 29.
    Watson, M. A., & Fleming, T. P. (1996). Mammaglobin, a mammaryspecific member of the uteroglobin gene family, is overexpressed in human breast cancer. Cancer Research, 56, 860–865.PubMedGoogle Scholar
  30. 30.
    Chen, Y., Zou, T. N., Wu, Z. P., et al. (2010). Detection of cytokeratin 19, human mammaglobin, and carcinoembryonic antigen-positive circulating tumor cells by three-marker reverse transcription-PCR assay and its relation to clinical outcome in early breast cancer. International Journal of Biological Markers, 2, 59–68.Google Scholar
  31. 31.
    Androulakis, N. E. M., Perraki, M., Apostolaki, S., et al. (2011). Clinical relevance of circulating CK-19mRNA-positive tumor cells at first diagnosis in patients with metastatic breast cancer. Journal of Clinical Oncology, 29, abstr 10553.Google Scholar
  32. 32.
    Strati, A., Markou, A., & Parisi, C. (2011). Gene expression profile of circulating tumor cells in breast cancer by RT-qPCR. BMC Cancer, 11, 422–433.PubMedCrossRefGoogle Scholar
  33. 33.
    Shen, C. X., Hu, L. H., Xia, L., et al. (2009). The detection of circulating tumor cells of breast cancer patients by using multimarker (Survivin, hTERT and hMAM) quantitative real-time PCR. Clinical Biochemistry, 42, 194–200.PubMedCrossRefGoogle Scholar
  34. 34.
    Chen, C. C., Chang, T. W., Chen, F. M., et al. (2006). Combination of multiple mRNA Markers(PTTG1, Survivin, UbcH10 and TK1) in the diagnosis of Taiwanese patients with breast cancer by membrane array. Oncology, 70, 438–446.PubMedCrossRefGoogle Scholar
  35. 35.
    Ferro, P., Franceschini, M. C., Bacigalupo, B., et al. (2010). Detection of circulating tumour cells in breast cancer patients using human mammaglobin RT-PCR: association with clinical prognostic factors. Anticancer Research, 30, 2377–2382.PubMedGoogle Scholar
  36. 36.
    Marques, A. R., Teixeira, Elsa., Diamond, Joana., et al. (2009). Detection of human mammaglobin mRNA in serial peripheral blood samples from patients with non-metastatic breast cancer is not predictive of disease recurrence. Breast Cancer Research and Treatment, 114, 223–232.PubMedCrossRefGoogle Scholar
  37. 37.
    Grünewald, K., Haun, M., Urbanek, M., et al. (2000). Mammaglobin gene expression: a superior marker of breast cancer cells in peripheral blood in comparison to epidermal-growth-factor receptor and cytokeratin-19. Laboratory Investigation, 80, 1071–1077.PubMedCrossRefGoogle Scholar
  38. 38.
    Daskalaki, A., Agelaki, S., Perraki, M., et al. (2009). Detection of cytokeratin-19 mRNA-positive cells in the peripheral blood and bone marrow of patients with operable breast cancer. British Journal of Cancer, 101, 589–597.PubMedCrossRefGoogle Scholar
  39. 39.
    Ignatiadis, M., Xenidis, N., Perraki, M., et al. (2007). Different prognostic value of cytokeratin-19 mrna–positive circulating tumor cells according to estrogen receptor and HER2 status in early-stage breast cancer. Journal of Clinical Oncology, 25, 5194–5202.PubMedCrossRefGoogle Scholar
  40. 40.
    Brandt, B., Roetger, A., Heidl, S., et al. (1998). Isolation of bloodbored epithelium-derived c-erb-B2 oncoprotein-positive clustered cells from the peripheral blood of breast cancer patients. International Journal of Cancer, 76, 824–828.CrossRefGoogle Scholar
  41. 41.
    Wulfing, P., Brochard, J., Buerger, H., et al. (2006). HER2 positive circulating tumor cells indicate poor clinical outcome in stage I to III breast cancer patients. Clinical Cancer Research, 12, 1715–1720.PubMedCrossRefGoogle Scholar
  42. 42.
    McShane, L. M., Altman, D. G., Sauerbrei, W., et al. (2005). Reporting recommendations for tumor marker prognostic studies (REMARK). Journal of the National Cancer Institute, 97, 1180–1184.PubMedCrossRefGoogle Scholar
  43. 43.
    Harris, L., Fritsche, H., Mennel, R., et al. (2007). American Society of Clinical Oncology 2007 update of recommendations for the use of tumor markers in breast cancer. Journal of Clinical Oncology, 25, 5287–5312.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2012

Authors and Affiliations

  • Shu Zhao
    • 1
  • Huike Yang
    • 2
  • Minghui Zhang
    • 1
  • Dekai Zhang
    • 3
  • Yupeng Liu
    • 4
  • Yan Liu
    • 5
  • Ying Song
    • 1
  • Xiaosan Zhang
    • 1
  • Hongbin Li
    • 1
  • Wenjie Ma
    • 1
  • Qingyuan Zhang
    • 1
    Email author
  1. 1.Department of Medical OncologyThe Third Affiliated Hospital of Harbin Medical UniversityHarbinChina
  2. 2.Department of AnatomyHarbin Medical UniversityHarbinChina
  3. 3.Center for Infectious and Inflammatory Diseases at the Institute of Biosciences and TechnologyTexas A&M University Health Science CenterHoustonUSA
  4. 4.Department of EpidemiologyPublic Health College of Harbin Medical UniversityHarbinChina
  5. 5.Department of Medical OncologyThe Fourth Affiliated Hospital of Harbin Medical UniversityHarbinChina

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