Lab Med Online.  2012 Apr;2(2):59-73.

Circulating Tumor Cells in Breast Cancer: Detection Systems, Molecular Characterization, and Future Challenges

Affiliations
  • 1Laboratory of Analytical Chemistry, Department of Chemistry, University of Athens, Athens, Greece.

Abstract

BACKGROUND
Circulating tumor cell (CTC) analysis is a promising new diagnostic field for estimating the risk for metastatic relapse and metastatic progression in patients with cancer. CONTENT: Different analytical systems for CTC isolation and detection have been developed as immunocytochemical and molecular assays, most including separation steps by size or biological characteristics, such as expression of epithelial- or cancer-specific markers. Recent technical advancements in CTC detection and characterization include methods based on multiplex reverse-transcription quantitative PCR and approaches based on imaging and microfilter and microchip devices. New areas of research are directed toward developing novel assays for CTC molecular characterization. QC is an important issue for CTC analysis, and standardization of micrometastatic cell detection and characterization methodologies is important for the incorporation of CTCs into prospective clinical trials to test their clinical utility. The molecular characterization of CTCs can provide important information on the molecular and biological nature of these cells, such as the status of hormone receptors and epidermal and other growth factor receptor family members, and indications of stem-cell characteristics. This information is important for the identification of therapeutic targets and resistance mechanisms in CTCs as well as for the stratification of patients and real-time monitoring of systemic therapies. SUMMARY: CTC analysis can be used as a liquid biopsy approach for prognostic and predictive purposes in breast and other cancers. In this review we focus on state-of-the-art technology platforms for CTC isolation, imaging, and detection; QC of CTC analysis; and ongoing challenges for the molecular characterization of CTCs.


MeSH Terms

Biopsy
Breast
Humans
Neoplastic Cells, Circulating
Polymerase Chain Reaction
Population Characteristics
Recurrence

Figure

  • Fig. 1 Main approaches for CTC isolation-enrichment. (A) Enrichment by density-gradient centrifugation in the presence of ficol. (B) Immunomagnetic separation [Fehm et al. (18), Königsberg et al. (19), Sieuwerts et al. (20), Mostert et al. (21), Schindlbeck et al. (22), Deng et al. (23)]. (B1) Negative selection through removal of leukocytes by anti-CD45; (B2) positive selection through an antibody against a pan-epithelial differentiation antigen, EpCAM; (B3) combined use of antibodies against CTC surface markers (anti-CD146, anti-CD176, anti-CK-19, and others). (C) ISET system [Vona et al. (24)]. (D) Microfluidic device: the CTC chip captures EpCAM-expressing cells in peripheral blood by use of anti-EpCAM-coated microposts [Nagrath et al. (27)]. (E) A portable filter-based microdevice filtration based on the size difference between CTCs and human blood cells [Lin et al. (25), Zheng et al. (26)]. Abbreviations: PBMCs, peripheral blood mononuclear cells; PDMS, polydimethylsiloxane.

  • Fig. 2 Main approaches for CTC detection and molecular characterization. (A) Image-based approaches: (A1) classic ICC; (A2) CellSearch system (FDA cleared); (A3) Ariol system; (A4) laser-scanning cytometry; (A5) EPISPOT assay (detects tumor-specific proteins released by CTCs). (B) Molecular assays, based on nucleic acid analysis in CTCs: (B1) classic RT-PCR; (B2) multiplex RT-PCR, AdnaTest BreastCancer; (B3) RT-qPCR; (B4) liquid bead array.


Reference

1. Ashworth TR. A case of cancer in which cells similar to those in the tumours were seen in the blood after death. Med J Australia. 1869. 14:146–147.
2. Fidler IJ. The pathogenesis of cancer metastasis: the 'seed and soil' hypothesis revisited. Nat Rev Cancer. 2003. 3:453–458.
Article
3. Kaiser J. Cancer's circulation problem. Science. 2010. 327:1072–1074.
Article
4. Pantel K, Alix-Panabieres C, Riethdorf S. Cancer micrometastases. Nat Rev Clin Oncol. 2009. 6:339–351.
Article
5. Hayes DF, Smerage JR. Is there a role for circulating tumor cells in the management of breast cancer? Clin Cancer Res. 2008. 14:3646–3650.
Article
6. Kasimir-Bauer S. Circulating tumor cells as markers for cancer risk assessment and treatment monitoring. Mol Diagn Ther. 2009. 13:209–215.
Article
7. Braun S, Vogl FD, Naume B, Janni W, Osborne MP, Coombes RC, et al. A pooled analysis of bone marrow micrometastasis in breast cancer. N Engl J Med. 2005. 353:793–802.
Article
8. Stathopoulou A, Mavroudis D, Perraki M, Apostolaki S, Vlachonikolis I, Lianidou E, et al. Molecular detection of cytokeratin-19-positive cells in the peripheral blood of patients with operable breast cancer: evaluation of their prognostic significance. J Clin Oncol. 2002. 20:3404–3412.
Article
9. Xenidis N, Vlachonikolis I, Mavroudis D, Perraki M, Stathopoulou A, Lianidou E, et al. Peripheral blood circulating cytokeratin-19 mRNA-positive cells after the completion of adjuvant chemotherapy in patients with operable breast cancer. Ann Oncol. 2003. 14:849–855.
Article
10. Xenidis N, Perraki M, Kafousi M, Apostolaki S, Lianidou ES, Georgoulias V, et al. Predictive and prognostic value of peripheral blood cytokeratin-19 mRNA-positive cells detected by real-time polymerase chain reaction in nodenegative breast cancer patients. J Clin Oncol. 2006. 24:3756–3762.
Article
11. Ignatiadis M, Kallergi G, Ntoulia M, Lianidou E, Georgoulias V, Mavroudis D, 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. 2008. 14:2593–2600.
Article
12. Ignatiadis M, Xenidis N, Perraki M, Lianidou E, Sotiriou C, Georgoulias V, et al. Different prognostic value of cytokeratin-19 mRNA positive circulating tumor cells according to estrogen receptor and HER2 status in early-stage breast cancer. J Clin Oncol. 2007. 25:5194–5202.
Article
13. Xenidis N, Ignatiadis M, Apostolaki S, Perraki M, Georgoulias V, Mavroudis D, et al. Cytokeratin-19 mRNA-positive circulating tumor cells after adjuvant chemotherapy in patients with early breast cancer. J Clin Oncol. 2009. 27:2177–2184.
Article
14. Cristofanilli M, Budd GT, Ellis MJ, Allard WJ, Terstappen LW, Hayes DF, et al. Circulating tumor cells, disease progression, and survival in metastatic breast cancer. N Engl J Med. 2004. 351:781–791.
Article
15. Mostert B, Sleijfer S, Foekens JA, Gratama JW. Circulating tumor cells (CTCs): detection methodsand their clinical relevance in breast cancer. Cancer Treat Rev. 2009. 35:463–474.
Article
16. Lianidou ES, Mavroudis D, Sotiropoulou G, Agelaki S, Pantel K. What's new on circulating tumor cells? A meeting report. Breast Cancer Res. 2010. 12:307.
Article
17. Tibbe AG, Miller MC, Terstappen LW. Statistical considerations for enumeration of circulating tumor cells. Cytometry A. 2007. 71:154–162.
Article
18. Fehm T, Solomayer EF, Meng S, Tucker T, Lane N, Wang J, et al. Methods for isolating circulating epithelial cells and criteria for their classification as carcinoma cells. Cytotherapy. 2005. 7:171–185.
Article
19. Königsberg R, Obermayr E, Bises G, Pfeiler G, Gneist M, Wrba F, et al. Detection of EpCAM positive and negative circulating tumor cells in metastatic breast cancer patients. Acta Oncol. 2011. 50:700–710.
Article
20. Sieuwerts AM, Kraan J, Bolt J, van der Spoel P, Elstrodt F, Schutte M, et al. Anti-epithelial cell adhesion molecule antibodies and the detection of circulating normal-like breast tumor cells. J Natl Cancer Inst. 2009. 101:61–66.
Article
21. Mostert B, Kraan J, Bolt-de Vries J, van der Spoel P, Sieuwerts AM, Schutte M, et al. Detection of circulating tumor cells in breast cancer may improve through enrichment with anti-CD146. Breast Cancer Res Treat. 2011. 127:33–41.
Article
22. Schindlbeck C, Stellwagen J, Jeschke U, Karsten U, Rack B, Janni W, et al. Immunomagnetic enrichment of disseminated tumor cells in bone marrow and blood of breast cancer patients by the Thomsen-Friedenreich-Antigen. Clin Exp Metastasis. 2008. 25:233–240.
Article
23. Deng G, Herrler M, Burgess D, Manna E, Krag D, Burke JF. Enrichment with anti-cytokeratin alone or combined with anti-EpCAM antibodies significantly increases the sensitivity for circulating tumor cell detection in metastatic breast cancer patients. Breast Cancer Res. 2008. 10:R69.
Article
24. Vona G, Sabile A, Louha M, Sitruk V, Romana S, Schütze K, et al. Isolation by size of epithelial tumor cells: a new method for the immunomorphological and molecular characterization of circulating tumor cells. Am J Pathol. 2000. 156:57–63.
25. Lin HK, Zheng S, Williams AJ, Balic M, Groshen S, Scher HI, et al. Portable filter-based microdevice for detection and characterization of circulating tumor cells. Clin Cancer Res. 2010. 16:5011–5018.
Article
26. Zheng S, Lin HK, Lu B, Williams A, Datar R, Cote RJ, et al. 3D microfilter device for viable circulating tumor cell (CTC) enrichment from blood. Biomed Microdevices. 2011. 13:203–213.
Article
27. Nagrath S, Sequist LV, Maheswaran S, Bell DW, Irimia D, Ulkus L, et al. Isolation of rare circulating tumour cells in cancer patients by microchip technology. Nature. 2007. 12. 20. 450:1235–1239.
Article
28. Stott SL, Hsu CH, Tsukrov DI, Yu M, Miyamoto DT, Waltman BA, et al. Isolation of circulating tumor cells using a microvortex-generating herringbone-chip. Proc Natl Acad Sci U S A. 2010. 107:18392–18397.
Article
29. Adams AA, Okagbare PI, Feng J, Hupert ML, Patterson D, Göttert J, et al. Highly efficient circulating tumor cell isolation from whole blood and label-free enumeration using polymer-based microfluidics with an integrated conductivity sensor. J Am Chem Soc. 2008. 130:8633–8641.
Article
30. Wang X, Qian X, Beitler JJ, Chen ZG, Khuri FR, Lewis MM, et al. Detection of circulating tumor cells in human peripheral blood using surfaceenhanced raman scattering nanoparticles. Cancer Res. 2011. 71:1526–1532.
Article
31. Pachmann K, Clement JH, Schneider CP, Willen B, Camara O, Pachmann U, et al. Standardized quantification of circulating peripheral tumor cells from lung and breast cancer. Clin Chem Lab Med. 2005. 43:617–627.
Article
32. Riethdorf S, Fritsche H, Müller V, Rau T, Schindlbeck C, Rack B, et al. Detection of circulating tumor cells in peripheral blood of patients with metastatic breast cancer: a validation study of the CellSearch system. Clin Cancer Res. 2007. 13:920–928.
Article
33. Balic M, Rapp N, Stanzer S, Lin H, Strutz J, Szkandera J, et al. Novel immunofluorescence protocol for multimarker assessment of putative disseminating breast cancer stem cells. Appl Immunohistochem Mol Morphol. 2011. 19:33–40.
Article
34. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 2009. 55:611–622.
Article
35. Slade MJ, Smith BM, Sinnett HD, Cross NC, Coombes RC. Quantitative polymerase chain reaction for the detection of micrometastases in patients with breast cancer. J Clin Oncol. 1999. 17:870–879.
Article
36. Stathopoulou A, Gizi A, Perraki M, Apostolaki S, Malamos N, Mavroudis D, et al. Real-time quantification of CK-19 mRNA-positive cells in peripheral blood of breast cancer patients using the lightcycler system. Clin Cancer Res. 2003. 9:5145–5151.
37. Stathopoulou A, Ntoulia M, Perraki M, Apostolaki S, Mavroudis D, Malamos N, et al. A highly specific real-time RT-PCR method for the quantitative determination of CK-19 mRNA positive cells in peripheral blood of patients with operable breast cancer. Int J Cancer. 2006. 119:1654–1659.
Article
38. Ring AE, Zabaglo L, Ormerod MG, Smith IE, Dowsett M. Detection of circulating epithelial cells in the blood of patients with breast cancer. Comparison of three techniques. Br J Cancer. 2005. 92:906–912.
Article
39. Fehm T, Braun S, Muller V, Janni W, Naume B, Pantel K, et al. A concept for the standardized detection of disseminated tumor cells in bone marrow from patients with primary breast cancer and its clinical implementation. Cancer. 2006. 107:885–892.
Article
40. Obermayr E, Sanchez-Cabo F, Tea MK, Singer CF, Krainer M, Fischer MB, et al. Assessment of a six gene panel for the molecular detection of circulating tumor cells in the blood of female cancer patients. BMC Cancer. 2010. 10:666.
Article
41. Reinholz MM, Nibbe A, Jonart LM, Houghton R, Zehentner B, Roche PC, et al. Evaluation of a panel of tumor markers for molecular detection of circulating cancer cells in women with suspected breast cancer. Clin Cancer Res. 2005. 11:3722–3732.
Article
42. Aktas B, Tewes M, Fehm T, Hauch S, Kimmig R, Kasimir-Bauer S. Stem cell and epithelialmesenchymal transition markers are frequently overexpressed in circulating tumor cells of metastatic breast cancer patients. Breast Cancer Res. 2009. 11:R46.
Article
43. Fehm T, Hoffmann O, Aktas B, Becker S, Solomayer EF, Wallwiener D, et al. Detection and characterization of circulating tumor cells in blood of primary breast cancer patients by RTPCR and comparison to status of bone marrow disseminated cells. Breast Cancer Res. 2009. 11:R59.
Article
44. Sieuwerts AM, Kraan J, Bolt-de Vries J, van der Spoel P, Mostert B, Martens JW, et al. Molecular characterization of circulating tumor cells in large quantities of contaminating leukocytes by a multiplex real-time PCR. Breast Cancer Res Treat. 2009. 118:455–468.
Article
45. Markou A, Strati A, Malamos N, Georgoulias V, Lianidou ES. Molecular characterization of circulating tumor cells in breast cancer by a liquid bead array hybridization assay. Clin Chem. 2011. 57:421–430.
Article
46. Alix-Panabières C, Vendrell JP, Slijper M, Pellé O, Barbotte E, Mercier G, et al. Full-length cytokeratin-19 is released by human tumor cells: a potential role in metastatic progression of breast cancer. Breast Cancer Res. 2009. 11:R39.
Article
47. Khleif SN, Doroshow JH, Hait WN. AACR-FDA-NCI Cancer Biomarkers Collaborative. AACR-FDA-NCI Cancer Biomarkers Collaborative consensus report: advancing the use of biomarkers in cancer drug development. Clin Cancer Res. 2010. 16:3299–3318.
Article
48. Fehm T, Müller V, Aktas B, Janni W, Schneeweiss A, Stickeler E, et al. HER2 status of circulating tumor cells in patients with metastatic breast cancer: a prospective, multicenter trial. Breast Cancer Res Treat. 2010. 124:403–412.
Article
49. Kraan J, Sleijfer S, Strijbos MH, Ignatiadis M, Peeters D, Pierga JY, et al. External quality assurance of circulating tumor cell enumeration using the CellSearch® system: a feasibility study. Cytometry B Clin Cytom. 2011. 80:112–118.
Article
50. Balic M, Dandachi N, Hofmann G, Samonigg H, Loibner H, Obwaller A, et al. Comparison of two methods for enumerating circulating tumor cells in carcinoma patients. Cytometry B Clin Cytom. 2005. 68:25–30.
Article
51. Punnoose EA, Atwal SK, Spoerke JM, Savage H, Pandita A, Yeh RF, et al. Molecular biomarker analyses using circulating tumor cells. PLoS One. 2010. 5:e12517.
Article
52. Becker S, Becker-Pergola G, Banys M, Krawczyk N, Wallwiener D, Solomayer E, et al. Evaluation of a RT-PCR based routine screening tool for the detection of disseminated epithelial cells in the bone marrow of breast cancer patients. Breast Cancer Res Treat. 2009. 117:227–233.
Article
53. Van der Auwera I, Peeters D, Benoy IH, Elst HJ, Van Laere SJ, Prové A, et al. Circulating tumour cell detection: a direct comparison between the CellSearch System, the AdnaTest and CK-19/mammaglobin RT-PCR in patients with metastatic breast cancer. Br J Cancer. 2010. 102:276–284.
Article
54. Mego M, Mani SA, Cristofanilli M. Molecular mechanisms of metastasis in breast cancerclinical applications. Nat Rev Clin Oncol. 2010. 7:693–701.
Article
55. Kallergi G, Markomanolaki H, Giannoukaraki V, Papadaki MA, Strati A, Lianidou ES, et al. Hypoxia-inducible factor-1alpha and vascular endothelial growth factor expression in circulating tumor cells of breast cancer patients. Breast Cancer Res. 2009. 11:R84.
Article
56. Tewes M, Aktas B, Welt A, Mueller S, Hauch S, Kimmig R, et al. 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 Res Treat. 2009. 115:581–590.
Article
57. Meng S, Tripathy D, Shete S, Ashfaq R, Haley B, Uhr J, et al. HER-2 gene amplification can be acquired as breast cancer progresses. Proc Natl Acad Sci U S A. 2004. 101:9393–9398.
58. Bozionellou V, Mavroudis D, Perraki M, Stathopoulou A, Lianidou E, Georgoulias V, et al. Trastuzumab administration can effectively target chemotherapy-resistant cytokeratin-19 messenger RNA-positive tumor cells in the peripheral blood and bone marrow of patients with breast cancer. Clin Cancer Res. 2004. 10:8185–8194.
Article
59. Riethdorf S, Müller V, Zhang L, Rau T, Loibl S, Komor M, et al. Detection and HER2 expression of circulating tumor cells: prospective monitoring in breast cancer patients treated in the neoadjuvant GeparQuattro trial. Clin Cancer Res. 2010. 16:2634–2645.
Article
60. Cao S, Li Y, Li J, Li CF, Zhang W, Yang ZQ, et al. Quantitative determination of HER2 expression by confocal microscopy assay in CTCs of breast cancer. Oncol Rep. 2010. 23:423–428.
Article
61. Ignatiadis M, Rothé F, Chaboteaux C, Durbecq V, Rouas G, Criscitiello C, et al. HER2-positive circulating tumor cells in breast cancer. PLoS One. 2011. 6:e15624.
Article
62. Apostolaki S, Perraki M, Pallis A, Bozionelou V, Agelaki S, Kanellou P, et al. Circulating HER2 mRNA-positive cells in the peripheral blood of patients with stage I and II breast cancer after the administration of adjuvant chemotherapy: evaluation of their clinical relevance. Ann Oncol. 2007. 18:851–858.
Article
63. Flores LM, Kindelberger DW, Ligon AH, Capelletti M, Fiorentino M, Loda M, et al. Improving the yield of circulating tumour cells facilitates molecular characterisation and recognition of discordant HER2 amplification in breast cancer. Br J Cancer. 2010. 102:1495–1502.
Article
64. Munzone E, Nolé F, Goldhirsch A, Botteri E, Esposito A, Zorzino L, et al. Changes of HER2 status in circulating tumor cells compared with the primary tumor during treatment for advanced breast cancer. Clin Breast Cancer. 2010. 10:392–397.
Article
65. Payne RE, Yagüe E, Slade MJ, Apostolopoulos C, Jiao LR, Ward B, et al. Measurements of EGFR expression on circulating tumor cells are reproducible over time in metastatic breast cancer patients. Pharmacogenomics. 2009. 10:51–57.
Article
66. Liu Z, Fusi A, Schmittel A, Tinhofer I, Schneider A, Keilholz U. Eradication of EGFR-positive circulating tumor cells and objective tumor response with lapatinib and capecitabine. Cancer Biol Ther. 2010. 10:860–864.
Article
67. Ntoulia M, Stathopoulou A, Ignatiadis M, Malamos N, Mavroudis D, Georgoulias V, et al. Detection of Mammaglobin A-mRNA-positive circulating tumor cells in peripheral blood of patients with operable breast cancer with nested RT-PCR. Clin Biochem. 2006. 39:879–887.
Article
68. Diehn M, Cho RW, Clarke MF. Therapeutic implications of the cancer stem cell hypothesis. Semin Radiat Oncol. 2009. 19:78–86.
Article
69. Meng S, Tripathy D, Frenkel EP, Shete S, Naftalis EZ, Huth JF, et al. Circulating tumor cells in patients with breast cancer dormancy. Clin Cancer Res. 2004. 10:8152–8162.
Article
70. Korkaya H, Paulson A, Iovino F, Wicha MS. HER2 regulates the mammary stem/progenitor cell population driving tumorigenesis and invasion. Oncogene. 2008. 27:6120–6130.
Article
71. Bonnomet A, Brysse A, Tachsidis A, Waltham M, Thompson EW, Polette M, et al. Epithelial-tomesenchymal transitions and circulating tumor cells. J Mammary Gland Biol Neoplasia. 2010. 15:261–273.
Article
72. Balic M, Lin H, Young L, Hawes D, Giuliano A, McNamara G, et al. Most early disseminated cancer cells detected in bone marrow of breast cancer patients have a putative breast cancer stem cell phenotype. Clin Cancer Res. 2006. 12:5615–5621.
Article
73. Theodoropoulos PA, Polioudaki H, Agelaki S, Kallergi G, Saridaki Z, Mavroudis D, et al. Circulating tumor cells with a putative stem cell phenotype in peripheral blood of patients with breast cancer. Cancer Lett. 2010. 288:99–106.
Article
74. Magnifico A, Albano L, Campaner S, Delia D, Castiglioni F, Gasparini P, et al. Tumor-initiating cells of HER2-positive carcinoma cell lines express the highest oncoprotein levels and are sensitive to trastuzumab. Clin Cancer Res. 2009. 15:2010–2021.
Article
75. Klein CA. Parallel progression of primary tumours and metastases. Nat Rev Cancer. 2009. 9:302–312.
Article
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