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

Evi S. Lianidou; Athina Markou

doi:10.3343/lmo.2012.2.2.59

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

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Diagnostic Genetics

Laboratory Medicine Online

Laboratory Medicine Online 2012; 2(2): 59-73.

Published online: 1 April 2012

DOI: https://doi.org/10.3343/lmo.2012.2.2.59

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

Evi S. Lianidou, Athina Markou

Laboratory of Analytical Chemistry, Department of Chemistry, University of Athens, Athens, Greece.

Translators: Jong-Rak Choi. Department of Laboratory Medicine, Severance Hospital, Yonsei University College of Medicine, Seoul, Korea. (cjr0606@yuhs.ac). This article is translated from an English language article, originally published in Clinical Chemistry under agreement between the publishers of Laboratory Medicine Online and Clinical Chemistry. Acknowledgement and other information that is not directly related to the content of the article has not been translated, and thus omitted in this article. References are listed as they were in the original work. Original copyright © 2011 American Association for Clinical Chemistry, Inc.

Received 9 January 2012 Revised 9 January 2012 Accepted 27 January 2012

(open-access):

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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.

Figures and Tables

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.

Table 1

Overview of analytical methodologies for the detection and molecular characterization of CTCs

Abbreviations: hMAM, human MAM; CEA, carcinoembryonic antigen; GABA, γ-aminobutyric acid; PTPRC, protein tyrosine phosphatase, receptor type, C; CCNE2, cyclin E2; EMP2, epithelial membrane protein 2; MAL2, myelin and lymphocyte 2; PPIC, peptidylprolyl isomerase C; SLC6A8, solute carrier family 6 (neurotransmitter transporter, creatine), member 8; BST, bone marrow stromal cell antigen; MAGE, melanoma-associated antigen; PBGD, porphobilinogen deaminase.

Table 2

Comparison studies between different laboratories and different methodologies for the same clinical samples

Table 3

Molecular characterization of CTCs

Abbreviations: hMAM, human MAM; MAGE, melanoma antigen; HIF, hypoxia-inducible factor; pFAK, phosphorylated-focal adhesion kinase.

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