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Tissue and Soluble Biomarkers in Breast Cancer and Their Applications: Ready to Use?

Tissue and Soluble Biomarkers in Breast Cancer and Their Applications: Ready to Use? Abstract Breast cancer therapies are in continuous evolution: From surgery to hormonal therapy, from classical and new combined chemotherapies to emerging targeted agents of recent introduction to the clinic. The attempt to personalize the best treatment for each patient is driven by efficacy and safety parameters and tumor biology investigations of markers for aggressiveness and response to treatment. The plethora of targeted therapies has provided momentum for the quest to better understand not only target mechanisms of action, but also tumor behavior. Moreover, how to monitor response to these agents is crucial today to achieve better resource-sharing and to find cheaper, less invasive, and standardized detection techniques for clinically validated biomarkers. In this report, we briefly summarize data on the major tissue and soluble biomarkers focusing on their actual use in daily practice, as well as their emerging role and possible future applications in breast cancer treatment. The therapeutic approach in breast cancer patients has changed over the last few years with the introduction of several biomarkers in daily practice. A biomarker is defined as a discriminating biological or biologically-driven substance (a metabolite) of a process, event, or condition (1). The first biomarkers routinely used for the selection of patients to receive a specific therapy were hormone receptors, where the presence of estrogen receptor (ER) and progesterone receptor predicted response to treatment both in the adjuvant and in the metastatic setting. In this era of emerging “targeted” agents, such as monoclonal antibodies, antiangiogenetics, tyrosine-kinase inhibitors and so on, the classical radiological scans and the clinical approach do not appear to be specific enough for the correct selection of patients who would benefit from these agents. Biomarkers, detectable in tissue samples during surgery or in peripheral blood of cancer patients, permit the selection of patients, thus resulting in better safety, efficacy profiles, and proper allocation of resources. They can also assume the function of a prognostic (to provide information on overall patient outcome, regardless of therapy) or a predictive (in advanced tumors, to estimate response or survival of a specific patient on a specific regimen, compared with another one) marker. Eventually, biomarkers could substitute a clinical endpoint, “surrogate biomarkers,” in research trials (2). Tissue Biomarkers Tissue biomarkers can be detected in histological samples. One example of their clinical application is the expression of c-Erb B2 in breast cancer, which led to the discovery of trastuzumab and its validation today supported by international clinical guidelines (3). Several other tissue biomarkers are now used with prognostic significance in daily practice: tumor grading, HER2 expression, Ki67, and vascular and lymphatic invasion (4). Their influence is evident in different treatment settings, in the early stages in particular, they define benefit from adjuvant therapies. An essential role includes the detection of estrogen receptor and progesterone receptor for hormonal therapy, whereas the eventual response to chemotherapy depends not only on hormonal sensitivity but moreover on Ki67 expression and tumor grading. In the adjuvant and metastatic settings, the need for addition of targeted therapies, such as trastuzumab, to classical chemotherapy is based on HER2 expression or FISH amplification (5). Recently, in the neoadjuvant setting, some articles reported that the evaluation of Ki67 levels before and after chemotherapy permits correct assessment and prediction of response (6). Other new emerging biomarkers such as p53, urokinase plasminogen activator/plasminogen activator inhibitor-1 could be considered for prognosis determination and prediction of response to treatment, but their real value is not yet well established (4). In the very near future, the routine choice of adjuvant treatment will be carried out by multiparameter gene expression analysis (ie, Oncotype DX, MammaPrint, and so on), especially for the prediction of recurrence risk in node-negative and ER-positive breast cancer patients (7,8). Despite current data suggesting that the introduction of these assays to daily practice could lead to great progress in determining the best treatment, we are still waiting for validation from two large, prospective, randomized ongoing clinical trials. Soluble Biomarkers Soluble biomarkers are easy to collect from blood for the evaluation of protein-based biomarkers (ie, serum tumor markers and vascular endothelial growth factor [VEGF]) and cellular biomarkers, such as circulating tumor cells (CTCs). Classical serum tumor markers include carcinoembryonic antigens, CA 15.3 and CA 27.59, which have a prognostic value in breast cancer and are routinely evaluated in conjunction with diagnostic imaging to monitor metastatic diseases during active therapy. They must be considered with caution: Early periodic peaks in CA 15.3 or CA 27.59 levels have been reported during the first 4–6 weeks of a new therapy regimen. Likewise, they are not recommended for screening, diagnosis, or routine surveillance after primary therapy (4). The trend in circulating VEGF level is a promising antiangiogenic biomarker: Baseline levels seem to correlate with outcome in some combined treatments in metastatic breast cancer. VEGF and other growth factor levels increase after bevacizumab treatment, but several different biomarkers are associated with poor outcome and show a role in resistance to anti-VEGF therapies (1). However, no prospective, large well-designed studies have validated this as well as other biomarkers for the selection of patients who will respond to antiangiogenic therapies and to monitor disease status during treatment. In the last few years, among the different circulating markers that have emerged in breast cancer (9), a new and promising assay is represented by the evaluation of CTCs. They are defined as tumor cells circulating in the peripheral blood of patients, shed from either the primary tumor or from its metastases. CTCs were first reported in 1869, now they are widely used as prognostic markers following the introduction of new detection techniques, which are easier to perform and interpret. In the “seed and soil” model, it has been proposed that the formation of metastatic colonies occurs through the dissemination of circulating cells derived from the primary tumor. It has been estimated that approximately 1 × 106 CTCs per gram per tumor are released into the circulation daily. CTCs can be found in blood samples, even after tumor eradication, probably deriving from dormant deposits (10,11). Nowadays, different advanced technologies permit the easy detection of CTCs using direct identification methods (ie, immunocytochemistry, immunofluorescence, and flow cytometry) or indirect techniques, nucleic acid based, with the detection of mRNA transcripts by reverse transcriptase-polymerase chain reaction (12). Considering that CTCs are a rare event, to increase assay sensitivity and specificity, a cell enrichment method based on immunomagnetic separation technology is required. The CellSearch System, the only system to have obtained US Food and Drug Administration approval, is based on the combination of an enrichment step and immunofluorescent analysis of cytokeratin expression. Cytokeratin is detected by antibodies directed to epithelial cell adhesion molecule (EpCAM)-1 expressed by breast cancer cells. A summary of the most widespread techniques used for CTC detection is reported in Table 1 (12). Table 1 Circulating tumor cell (CTC) enrichment and detection techniques. An enrichment step with different, specific, and validated approaches is often required. Several studies can be performed on these isolated cells. Recent techniques provide both steps* Assay  Description  Advantages  Disadvantages  Membrane microfilter–based approaches  Use of filters as enrichment step: filters retain tumor cells that are larger than leukocytes. Putative CTCs can be fixed and stained for different markers (CKs)  Easy and inexpensive; one-step process; preservation of cell viability for further studies  Loss of tumor cells that have smaller size or have undergone lysis; insensitive; lack of tumor specificity  Image-based immunologic approaches (eg MACS)  Isolation of cells for subsequent analysis using different tumor-specific antibodies with microbeads  Applicable for all tumor types; easy and inexpensive technique  Heterogeneity of tumor cells in size; relatively low purity of samples; final results depend on detection technique  AdnaTest  Combination of EpCAM and MUC1 (bound to magnetic beads) targeting epithelial CTCs; further analysis by isolation, direct lysis, mRNA extraction, and application of RT-PCR  Combined specific enrichment (EpCAM, MUC1); downstream analysis; possibility to characterize CTCs for stem cells and epithelial–mesenchymal transition  No flexibility, no automation; false-positive due to the expression of the same antigens on non-tumor cells; false-negative results due to loss of antigens on CTCs  Microchip technology (CTC chip)  Fluorescent nuclear and CK stain for positive selection and CD45 stain for negative selection; CTCs captured are directly recognized by cameras, based on morphology, viability, and expression of tumor markers  98% cell viability; high detection rate; further analysis possible  Only EpCAM+ CTCs detected; not commercially available; lack of validation studies in clinical settings  CellSearch  Semi-automated analyzer that enriches CTCs with ferrofluid nanoparticles coated with anti-EpCAM antibodies. CD45−, CK8+, CK18+, CK19+ cells are counted by a four-color semi-automated fluorescent microscope  Semi-automated; high sensitivity; CTC quantification; reproducibility; FDA approved  Only EpCAM+/CK+/CD45− CTCs detected; subjective image interpretation; no further analysis possible  MagSweeper  Immunomagnetic cell separator that enriches target cells and individual cells not bound to magnetic particles. Isolated cells can be extracted based on their physical characteristics  Cell preservation; preservation of cell viability for further studies  Lack of validation studies in clinical settings  EPISPOT technology  Immunological assay based on the enzyme-linked immunosorbent assay (identification and count of cells able to secrete proteins like MUC1 and CK19 in short-term culture), after immunomagnetic depletion of CD45+ cells  Analysis only on viable cells and high sensitivity  CTC isolation not possible, further analysis not available; requires active protein secretion; technically challenging  RT-PCR-based methods  Analysis of expression of candidate genes specific to epithelial tumor cells by mRNA evaluation, often combined with other enrichment techniques  High sensitivity  RNA degradation; false-positive results due to unspecific amplification, contamination, and pseudogenes; false-negative results due to low expression level  Flow cytometry  Simultaneous analysis of multiparameters such as size, viability, DNA content, and expression of different markers for CTC detection  High specificity and multiple parameters  Low sensitivity  Laser scanning cytometry  Cytofluorimetric analysis fluorescence performed after cells are counted using a forward scatter as a threshold parameter  No enrichment needed and visual confirmation of CTCs  Subjective CTC analysis and low specificity due to marker limitations  Assay  Description  Advantages  Disadvantages  Membrane microfilter–based approaches  Use of filters as enrichment step: filters retain tumor cells that are larger than leukocytes. Putative CTCs can be fixed and stained for different markers (CKs)  Easy and inexpensive; one-step process; preservation of cell viability for further studies  Loss of tumor cells that have smaller size or have undergone lysis; insensitive; lack of tumor specificity  Image-based immunologic approaches (eg MACS)  Isolation of cells for subsequent analysis using different tumor-specific antibodies with microbeads  Applicable for all tumor types; easy and inexpensive technique  Heterogeneity of tumor cells in size; relatively low purity of samples; final results depend on detection technique  AdnaTest  Combination of EpCAM and MUC1 (bound to magnetic beads) targeting epithelial CTCs; further analysis by isolation, direct lysis, mRNA extraction, and application of RT-PCR  Combined specific enrichment (EpCAM, MUC1); downstream analysis; possibility to characterize CTCs for stem cells and epithelial–mesenchymal transition  No flexibility, no automation; false-positive due to the expression of the same antigens on non-tumor cells; false-negative results due to loss of antigens on CTCs  Microchip technology (CTC chip)  Fluorescent nuclear and CK stain for positive selection and CD45 stain for negative selection; CTCs captured are directly recognized by cameras, based on morphology, viability, and expression of tumor markers  98% cell viability; high detection rate; further analysis possible  Only EpCAM+ CTCs detected; not commercially available; lack of validation studies in clinical settings  CellSearch  Semi-automated analyzer that enriches CTCs with ferrofluid nanoparticles coated with anti-EpCAM antibodies. CD45−, CK8+, CK18+, CK19+ cells are counted by a four-color semi-automated fluorescent microscope  Semi-automated; high sensitivity; CTC quantification; reproducibility; FDA approved  Only EpCAM+/CK+/CD45− CTCs detected; subjective image interpretation; no further analysis possible  MagSweeper  Immunomagnetic cell separator that enriches target cells and individual cells not bound to magnetic particles. Isolated cells can be extracted based on their physical characteristics  Cell preservation; preservation of cell viability for further studies  Lack of validation studies in clinical settings  EPISPOT technology  Immunological assay based on the enzyme-linked immunosorbent assay (identification and count of cells able to secrete proteins like MUC1 and CK19 in short-term culture), after immunomagnetic depletion of CD45+ cells  Analysis only on viable cells and high sensitivity  CTC isolation not possible, further analysis not available; requires active protein secretion; technically challenging  RT-PCR-based methods  Analysis of expression of candidate genes specific to epithelial tumor cells by mRNA evaluation, often combined with other enrichment techniques  High sensitivity  RNA degradation; false-positive results due to unspecific amplification, contamination, and pseudogenes; false-negative results due to low expression level  Flow cytometry  Simultaneous analysis of multiparameters such as size, viability, DNA content, and expression of different markers for CTC detection  High specificity and multiple parameters  Low sensitivity  Laser scanning cytometry  Cytofluorimetric analysis fluorescence performed after cells are counted using a forward scatter as a threshold parameter  No enrichment needed and visual confirmation of CTCs  Subjective CTC analysis and low specificity due to marker limitations  * CKs = cytokeratins; EpCAM = epithelial cell adhesion molecule; FDA = Food and Drug Administration; MACS = magnetic activated cell sorting system RT-PCR = reverse transcriptase-polymerase chain reaction. View Large In the last year, discordant opinion has stressed the possibility of a reduction in CTC detection from the EpCAM-based systems due to the lower expression of this adhesion molecule in normal-like breast cancers, a breast tumor subtype with aggressive features identified through a gene expression profile (13). Recently, the addition of CD146 (marker that detects breast cancer cells that lack EpCAM expression and can be recovered by anti-CD146 ferrofluids) as a detection marker for EpCAM-negative CTCs has been reported, with the same clinical relevance as EpCAM-positive CTCs alone in selecting poor prognosis patients (14). Ongoing studies are also in progress to identify new gene markers superior to EpCAM for the detection of CTCs (15). Clinical studies have recently investigated the correlation between CTC number, response to treatment, prognosis, also in comparison with computed tomography and positron emission tomography scans, first in metastatic breast cancer patients, and also in earlier settings (16). In early breast cancer, it has been demonstrated that as the presence of CTCs 18 months after neoadjuvant treatment is an independent prognostic factor predicting shorter relapse-free survival, as well as hormone receptor negativity and large tumor size (17). Serial evaluation of CTCs in early breast cancer could be useful in association with classical radiological evaluation to predict survival and response to treatment. Moreover, in the metastatic setting, CTCs can be detected in 50% of patients, and their presence correlates with a higher risk of recurrence and reduction in survival. Those particular patients, in whom CTCs are not detected and eventually progress, must be taken into account as the presence of other factors that contribute to relapse is still an open issue (18). CTC evaluation in daily practice could outline a “biological staging” of the disease, to be considered in addiction to TNM staging, as these rare events can predict prognosis during treatment in a real-time fashion (19). Moreover, in ongoing US trials, early modifications in CTC counts after 3 weeks of first-line chemotherapy is the milestone for continuing the same therapy or switching to other agents. Recently, in metastatic patients treated with first-line chemotherapy combined with bevacizumab, it has been reported that the antiangiogenic treatment modifies the predictive value of CTCs, possibly due to their impact on tumor vessel endothelium conditioning less intravasation ability of the cancer cells. In this setting, another biomarker, such as circulating endothelial cell count variations, could be useful as an early surrogate marker (20). On the other hand, indirect assays permit not only CTC count but also the ability to isolate them. Molecular and genetic studies may be performed on these cells allowing more in-depth study of the molecular alterations in breast cancer cells during the metastatic process as well as pharmacogenomic tests for the detection of better therapeutic agents to eradicate minimal disease. In this field, the debate is ongoing, due to the discovery of small amounts of free DNA (about 1 ng/mL) even in peripheral blood of healthy subjects that is increased in some diseases, including breast cancer. Point mutations, abnormal hypermethylation, and microsatellite aberrations have been identified in circulating DNA, with good correlation to the primary tumor. Many authors have investigated the possibility of performing such molecular studies to outline a correlation between circulating DNA levels and prognosis or the relationship of circulating mutations and the primary tumor, but conclusive results are still not available (21,22). Another current hot topic is the discordance of HER2 expression in breast cancer samples and CTCs, related to the relevance of CTCs as “noninvasive” and real-time biopsies. It has been demonstrated that in about 30% of patients, a shift in HER2 status between the tissue biopsy and CTCs occurence (23,24). Ongoing studies have been designed to explore the clinical value of this shift for HER2 therapies in HER2-negative tumors with HER2-positive CTCs and vice versa. Conclusions In the era of emerging molecularly driven agents, the need for specific biomarkers is critical for monitoring efficacy and safety and for cost considerations. Their application needs to be prospectively tested and validated in large clinical trials, and standardized detec clinical practice will be reflected in the proper use of antineoplastic agents, improving breast cancer treatment and patient survival. Funding This work has been partially supported by a research grant to Dr Marco Danova from the IRCCS Foundation San Matteo, Pavia. References 1. Jain RK,  Duda DG,  Willett CG, et al.  Biomarkers of response and resistance to antiangiogenic therapy,  Nat Rev Clin Oncol. ,  2009, vol.  6  6(pg.  327- 338) 2. Hodgson DR,  Wellings E,  Orr M, et al.  Circulating tumor-derived predictive biomarkers in oncology,  Drug Discov Today. ,  2010, vol.  15  3–4(pg.  98- 101) 3. Verma S,  Lavasani S,  Mackey J, et al.  Optimizing the management of HER2-positive early breast cancer: the clinical reality,  Curr Oncol. ,  2010, vol.  17  4(pg.  20- 33) 4. Harris L,  Fritsche H,  Mennel R, et al.  American Society of Clinical Oncology 2007 update of recommendations for the use of tumor markers in breast cancer,  J Clin Oncol. ,  2007, vol.  25  33(pg.  5287- 5312) 5. Weigel MT,  Dowsett M.  Current and emerging biomarkers in breast cancer: prognosis and prediction [published online ahead of print July 10, 2010],  Endocr Relat Cancer. ,  2010, vol.  17  4(pg.  245- 262) 6. Colleoni M,  Bagnardi V,  Rotmensz N, et al.  A nomogram based on the expression of Ki-67, steroid hormone receptors status and number of chemotherapy courses to predict pathological complete remission after preoperative chemotherapy for breast cancer,  Eur J Cancer. ,  2010, vol.  46  12(pg.  2216- 2224) 7. Buyse M,  Loi S,  van’t Veer L, et al.  Validation and clinical utility of a 70-gene prognostic signature for women with node-negative breast cancer,  J Natl Cancer Inst. ,  2006, vol.  98  17(pg.  1183- 1192) 8. Desmedt C,  Piette F,  Loi S, et al.  Strong time dependence of the 76-gene prognostic signature for node-negative breast cancer patients in the TRANSBIG multicenter independent validation series,  Clin Cancer Res. ,  2007, vol.  13  11(pg.  3207- 3214) 9. Nolen BM,  Marks JR,  Ta’san S, et al.  Serum biomarker profiles and response to neoadjuvant chemotherapy for locally advanced breast cancer,  Breast Cancer Res. ,  2008, vol.  10  3pg.  R45  10. Cristofanilli M,  Budd T,  Ellis MJ, et al.  Circulating tumor cells, disease progression and survival in metastatic breast cancer,  N Engl J Med. ,  2004, vol.  351  8(pg.  781- 791) 11. Andreopoulou E,  Cristofanilli M.  Circulating tumor cells as a prognostic marker in metastatic breast cancer,  Expert Rev Anticancer Ther. ,  2010, vol.  10  2(pg.  171- 177) 12. Alunni-Fabbroni M,  Sandri MT.  Circulating tumor cells in clinical practice: methods of detection and possible characterization,  Methods. ,  2010, vol.  50  4(pg.  289- 297) 13. Sieuwerts AM,  Kraan J,  Bolt J, et al.  Anti-epithelial cell adhesion molecule antibodies and the detection of circulating normal-like breast tumor cells,  J Natl Cancer Inst. ,  2009, vol.  101  1(pg.  61- 66) 14. Mostert B,  Kraan J,  Bolt-de Vries J, et al.  Detection of circulating tumor cells in breast cancer may improve through enrichment with anti-CD146 [published online ahead of print April 9, 2010],  Breast Cancer Res Treat. ,  2011, vol.  127  1(pg.  33- 41) 15. Obermayr E,  Sanchex-Cabo F,  Tea MK, 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, vol.  3  10pg.  666  16. De Giorgi U,  Valero V,  Rohren E, et al.  Circulating tumor cells and [18F]fluorodeoxyglucose positron emission tomography/computed tomography for outcome prediction in metastatic breast cancer,  J Clin Oncol. ,  2009, vol.  27  20(pg.  3303- 3311) 17. Pierga JY,  Bidard FC,  Mathior C, et al.  Circulating tumor cell detection predicts early metastatic relapse after neoadjuvant chemotherapy in large operable and locally advanced breast cancer in a phase II randomized trial,  Clin Cancer Res. ,  2008, vol.  14  21(pg.  7004- 7010) 18. Nagaiah G,  Abraham J.  Circulating tumor cells in the management of breast cancer,  Clin Breast Cancer. ,  2010, vol.  10  3(pg.  209- 216) 19. Dawood S,  Broglio K,  Valero V, et al.  Circulating tumor cells in metastatic breast cancer: from prognostic stratification to modification of the staging system?,  Cancer. ,  2008, vol.  113  9(pg.  2422- 2430) 20. Bidard FC,  Mathiot C,  Degeorges A, et al.  Clinical value of circulating endothelial cells and circulating tumor cells in metastatic breast cancer patients treated first line with bevacizumab and chemotherapy [published online ahead of print March 16, 2010],  Ann Oncol. ,  2010, vol.  21  9(pg.  1765- 1771) 21. Sorenson GD.  Detection of mutated KRAS2 sequences as tumor markers in plasma/serum of patients with gastrointestinal cancer,  Clin Cancer Res. ,  2000, vol.  6  6(pg.  2129- 2137) 22. Schwarzenbach H,  Pantel K,  Kemper B, et al.  Comparative evaluation of cell-free tumor DNA in blood and disseminated tumor cells in bone marrow of patients with primary breast cancer,  Breast Cancer Res. ,  2009, vol.  11  5pg.  R71  23. Punnoose EA,  Atwal SK,  Spoerke JM, et al.  Molecular biomarker analyses using circulating tumor cells,  PLoS One. ,  2010, vol.  5  9pg.  e12517  24. Flores LM,  Kindelberger DW,  Ligon AH, et al.  Improving the yield of circulating tumour cells facilitates molecular characterization and recognition of discordant HER2 amplification in breast cancer,  Br J Cancer. ,  2010, vol.  102  10(pg.  1495- 1502) 25. Criscitiello C,  Sotirou C,  Ignatiadis M.  Circulating tumor cells and emerging blood biomarkers in breast cancer [published online ahead of print August 11, 2010],  Curr Opin Oncol. ,  2010, vol.  22  6(pg.  552- 558) We gratefully thank Dr Martina Torchio (Medical Oncologist at the University of Pavia) for careful revision. © The Author 2011. Published by Oxford University Press. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png JNCI Monographs Oxford University Press

Tissue and Soluble Biomarkers in Breast Cancer and Their Applications: Ready to Use?

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Oxford University Press
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© The Author 2011. Published by Oxford University Press.
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Abstract

Abstract Breast cancer therapies are in continuous evolution: From surgery to hormonal therapy, from classical and new combined chemotherapies to emerging targeted agents of recent introduction to the clinic. The attempt to personalize the best treatment for each patient is driven by efficacy and safety parameters and tumor biology investigations of markers for aggressiveness and response to treatment. The plethora of targeted therapies has provided momentum for the quest to better understand not only target mechanisms of action, but also tumor behavior. Moreover, how to monitor response to these agents is crucial today to achieve better resource-sharing and to find cheaper, less invasive, and standardized detection techniques for clinically validated biomarkers. In this report, we briefly summarize data on the major tissue and soluble biomarkers focusing on their actual use in daily practice, as well as their emerging role and possible future applications in breast cancer treatment. The therapeutic approach in breast cancer patients has changed over the last few years with the introduction of several biomarkers in daily practice. A biomarker is defined as a discriminating biological or biologically-driven substance (a metabolite) of a process, event, or condition (1). The first biomarkers routinely used for the selection of patients to receive a specific therapy were hormone receptors, where the presence of estrogen receptor (ER) and progesterone receptor predicted response to treatment both in the adjuvant and in the metastatic setting. In this era of emerging “targeted” agents, such as monoclonal antibodies, antiangiogenetics, tyrosine-kinase inhibitors and so on, the classical radiological scans and the clinical approach do not appear to be specific enough for the correct selection of patients who would benefit from these agents. Biomarkers, detectable in tissue samples during surgery or in peripheral blood of cancer patients, permit the selection of patients, thus resulting in better safety, efficacy profiles, and proper allocation of resources. They can also assume the function of a prognostic (to provide information on overall patient outcome, regardless of therapy) or a predictive (in advanced tumors, to estimate response or survival of a specific patient on a specific regimen, compared with another one) marker. Eventually, biomarkers could substitute a clinical endpoint, “surrogate biomarkers,” in research trials (2). Tissue Biomarkers Tissue biomarkers can be detected in histological samples. One example of their clinical application is the expression of c-Erb B2 in breast cancer, which led to the discovery of trastuzumab and its validation today supported by international clinical guidelines (3). Several other tissue biomarkers are now used with prognostic significance in daily practice: tumor grading, HER2 expression, Ki67, and vascular and lymphatic invasion (4). Their influence is evident in different treatment settings, in the early stages in particular, they define benefit from adjuvant therapies. An essential role includes the detection of estrogen receptor and progesterone receptor for hormonal therapy, whereas the eventual response to chemotherapy depends not only on hormonal sensitivity but moreover on Ki67 expression and tumor grading. In the adjuvant and metastatic settings, the need for addition of targeted therapies, such as trastuzumab, to classical chemotherapy is based on HER2 expression or FISH amplification (5). Recently, in the neoadjuvant setting, some articles reported that the evaluation of Ki67 levels before and after chemotherapy permits correct assessment and prediction of response (6). Other new emerging biomarkers such as p53, urokinase plasminogen activator/plasminogen activator inhibitor-1 could be considered for prognosis determination and prediction of response to treatment, but their real value is not yet well established (4). In the very near future, the routine choice of adjuvant treatment will be carried out by multiparameter gene expression analysis (ie, Oncotype DX, MammaPrint, and so on), especially for the prediction of recurrence risk in node-negative and ER-positive breast cancer patients (7,8). Despite current data suggesting that the introduction of these assays to daily practice could lead to great progress in determining the best treatment, we are still waiting for validation from two large, prospective, randomized ongoing clinical trials. Soluble Biomarkers Soluble biomarkers are easy to collect from blood for the evaluation of protein-based biomarkers (ie, serum tumor markers and vascular endothelial growth factor [VEGF]) and cellular biomarkers, such as circulating tumor cells (CTCs). Classical serum tumor markers include carcinoembryonic antigens, CA 15.3 and CA 27.59, which have a prognostic value in breast cancer and are routinely evaluated in conjunction with diagnostic imaging to monitor metastatic diseases during active therapy. They must be considered with caution: Early periodic peaks in CA 15.3 or CA 27.59 levels have been reported during the first 4–6 weeks of a new therapy regimen. Likewise, they are not recommended for screening, diagnosis, or routine surveillance after primary therapy (4). The trend in circulating VEGF level is a promising antiangiogenic biomarker: Baseline levels seem to correlate with outcome in some combined treatments in metastatic breast cancer. VEGF and other growth factor levels increase after bevacizumab treatment, but several different biomarkers are associated with poor outcome and show a role in resistance to anti-VEGF therapies (1). However, no prospective, large well-designed studies have validated this as well as other biomarkers for the selection of patients who will respond to antiangiogenic therapies and to monitor disease status during treatment. In the last few years, among the different circulating markers that have emerged in breast cancer (9), a new and promising assay is represented by the evaluation of CTCs. They are defined as tumor cells circulating in the peripheral blood of patients, shed from either the primary tumor or from its metastases. CTCs were first reported in 1869, now they are widely used as prognostic markers following the introduction of new detection techniques, which are easier to perform and interpret. In the “seed and soil” model, it has been proposed that the formation of metastatic colonies occurs through the dissemination of circulating cells derived from the primary tumor. It has been estimated that approximately 1 × 106 CTCs per gram per tumor are released into the circulation daily. CTCs can be found in blood samples, even after tumor eradication, probably deriving from dormant deposits (10,11). Nowadays, different advanced technologies permit the easy detection of CTCs using direct identification methods (ie, immunocytochemistry, immunofluorescence, and flow cytometry) or indirect techniques, nucleic acid based, with the detection of mRNA transcripts by reverse transcriptase-polymerase chain reaction (12). Considering that CTCs are a rare event, to increase assay sensitivity and specificity, a cell enrichment method based on immunomagnetic separation technology is required. The CellSearch System, the only system to have obtained US Food and Drug Administration approval, is based on the combination of an enrichment step and immunofluorescent analysis of cytokeratin expression. Cytokeratin is detected by antibodies directed to epithelial cell adhesion molecule (EpCAM)-1 expressed by breast cancer cells. A summary of the most widespread techniques used for CTC detection is reported in Table 1 (12). Table 1 Circulating tumor cell (CTC) enrichment and detection techniques. An enrichment step with different, specific, and validated approaches is often required. Several studies can be performed on these isolated cells. Recent techniques provide both steps* Assay  Description  Advantages  Disadvantages  Membrane microfilter–based approaches  Use of filters as enrichment step: filters retain tumor cells that are larger than leukocytes. Putative CTCs can be fixed and stained for different markers (CKs)  Easy and inexpensive; one-step process; preservation of cell viability for further studies  Loss of tumor cells that have smaller size or have undergone lysis; insensitive; lack of tumor specificity  Image-based immunologic approaches (eg MACS)  Isolation of cells for subsequent analysis using different tumor-specific antibodies with microbeads  Applicable for all tumor types; easy and inexpensive technique  Heterogeneity of tumor cells in size; relatively low purity of samples; final results depend on detection technique  AdnaTest  Combination of EpCAM and MUC1 (bound to magnetic beads) targeting epithelial CTCs; further analysis by isolation, direct lysis, mRNA extraction, and application of RT-PCR  Combined specific enrichment (EpCAM, MUC1); downstream analysis; possibility to characterize CTCs for stem cells and epithelial–mesenchymal transition  No flexibility, no automation; false-positive due to the expression of the same antigens on non-tumor cells; false-negative results due to loss of antigens on CTCs  Microchip technology (CTC chip)  Fluorescent nuclear and CK stain for positive selection and CD45 stain for negative selection; CTCs captured are directly recognized by cameras, based on morphology, viability, and expression of tumor markers  98% cell viability; high detection rate; further analysis possible  Only EpCAM+ CTCs detected; not commercially available; lack of validation studies in clinical settings  CellSearch  Semi-automated analyzer that enriches CTCs with ferrofluid nanoparticles coated with anti-EpCAM antibodies. CD45−, CK8+, CK18+, CK19+ cells are counted by a four-color semi-automated fluorescent microscope  Semi-automated; high sensitivity; CTC quantification; reproducibility; FDA approved  Only EpCAM+/CK+/CD45− CTCs detected; subjective image interpretation; no further analysis possible  MagSweeper  Immunomagnetic cell separator that enriches target cells and individual cells not bound to magnetic particles. Isolated cells can be extracted based on their physical characteristics  Cell preservation; preservation of cell viability for further studies  Lack of validation studies in clinical settings  EPISPOT technology  Immunological assay based on the enzyme-linked immunosorbent assay (identification and count of cells able to secrete proteins like MUC1 and CK19 in short-term culture), after immunomagnetic depletion of CD45+ cells  Analysis only on viable cells and high sensitivity  CTC isolation not possible, further analysis not available; requires active protein secretion; technically challenging  RT-PCR-based methods  Analysis of expression of candidate genes specific to epithelial tumor cells by mRNA evaluation, often combined with other enrichment techniques  High sensitivity  RNA degradation; false-positive results due to unspecific amplification, contamination, and pseudogenes; false-negative results due to low expression level  Flow cytometry  Simultaneous analysis of multiparameters such as size, viability, DNA content, and expression of different markers for CTC detection  High specificity and multiple parameters  Low sensitivity  Laser scanning cytometry  Cytofluorimetric analysis fluorescence performed after cells are counted using a forward scatter as a threshold parameter  No enrichment needed and visual confirmation of CTCs  Subjective CTC analysis and low specificity due to marker limitations  Assay  Description  Advantages  Disadvantages  Membrane microfilter–based approaches  Use of filters as enrichment step: filters retain tumor cells that are larger than leukocytes. Putative CTCs can be fixed and stained for different markers (CKs)  Easy and inexpensive; one-step process; preservation of cell viability for further studies  Loss of tumor cells that have smaller size or have undergone lysis; insensitive; lack of tumor specificity  Image-based immunologic approaches (eg MACS)  Isolation of cells for subsequent analysis using different tumor-specific antibodies with microbeads  Applicable for all tumor types; easy and inexpensive technique  Heterogeneity of tumor cells in size; relatively low purity of samples; final results depend on detection technique  AdnaTest  Combination of EpCAM and MUC1 (bound to magnetic beads) targeting epithelial CTCs; further analysis by isolation, direct lysis, mRNA extraction, and application of RT-PCR  Combined specific enrichment (EpCAM, MUC1); downstream analysis; possibility to characterize CTCs for stem cells and epithelial–mesenchymal transition  No flexibility, no automation; false-positive due to the expression of the same antigens on non-tumor cells; false-negative results due to loss of antigens on CTCs  Microchip technology (CTC chip)  Fluorescent nuclear and CK stain for positive selection and CD45 stain for negative selection; CTCs captured are directly recognized by cameras, based on morphology, viability, and expression of tumor markers  98% cell viability; high detection rate; further analysis possible  Only EpCAM+ CTCs detected; not commercially available; lack of validation studies in clinical settings  CellSearch  Semi-automated analyzer that enriches CTCs with ferrofluid nanoparticles coated with anti-EpCAM antibodies. CD45−, CK8+, CK18+, CK19+ cells are counted by a four-color semi-automated fluorescent microscope  Semi-automated; high sensitivity; CTC quantification; reproducibility; FDA approved  Only EpCAM+/CK+/CD45− CTCs detected; subjective image interpretation; no further analysis possible  MagSweeper  Immunomagnetic cell separator that enriches target cells and individual cells not bound to magnetic particles. Isolated cells can be extracted based on their physical characteristics  Cell preservation; preservation of cell viability for further studies  Lack of validation studies in clinical settings  EPISPOT technology  Immunological assay based on the enzyme-linked immunosorbent assay (identification and count of cells able to secrete proteins like MUC1 and CK19 in short-term culture), after immunomagnetic depletion of CD45+ cells  Analysis only on viable cells and high sensitivity  CTC isolation not possible, further analysis not available; requires active protein secretion; technically challenging  RT-PCR-based methods  Analysis of expression of candidate genes specific to epithelial tumor cells by mRNA evaluation, often combined with other enrichment techniques  High sensitivity  RNA degradation; false-positive results due to unspecific amplification, contamination, and pseudogenes; false-negative results due to low expression level  Flow cytometry  Simultaneous analysis of multiparameters such as size, viability, DNA content, and expression of different markers for CTC detection  High specificity and multiple parameters  Low sensitivity  Laser scanning cytometry  Cytofluorimetric analysis fluorescence performed after cells are counted using a forward scatter as a threshold parameter  No enrichment needed and visual confirmation of CTCs  Subjective CTC analysis and low specificity due to marker limitations  * CKs = cytokeratins; EpCAM = epithelial cell adhesion molecule; FDA = Food and Drug Administration; MACS = magnetic activated cell sorting system RT-PCR = reverse transcriptase-polymerase chain reaction. View Large In the last year, discordant opinion has stressed the possibility of a reduction in CTC detection from the EpCAM-based systems due to the lower expression of this adhesion molecule in normal-like breast cancers, a breast tumor subtype with aggressive features identified through a gene expression profile (13). Recently, the addition of CD146 (marker that detects breast cancer cells that lack EpCAM expression and can be recovered by anti-CD146 ferrofluids) as a detection marker for EpCAM-negative CTCs has been reported, with the same clinical relevance as EpCAM-positive CTCs alone in selecting poor prognosis patients (14). Ongoing studies are also in progress to identify new gene markers superior to EpCAM for the detection of CTCs (15). Clinical studies have recently investigated the correlation between CTC number, response to treatment, prognosis, also in comparison with computed tomography and positron emission tomography scans, first in metastatic breast cancer patients, and also in earlier settings (16). In early breast cancer, it has been demonstrated that as the presence of CTCs 18 months after neoadjuvant treatment is an independent prognostic factor predicting shorter relapse-free survival, as well as hormone receptor negativity and large tumor size (17). Serial evaluation of CTCs in early breast cancer could be useful in association with classical radiological evaluation to predict survival and response to treatment. Moreover, in the metastatic setting, CTCs can be detected in 50% of patients, and their presence correlates with a higher risk of recurrence and reduction in survival. Those particular patients, in whom CTCs are not detected and eventually progress, must be taken into account as the presence of other factors that contribute to relapse is still an open issue (18). CTC evaluation in daily practice could outline a “biological staging” of the disease, to be considered in addiction to TNM staging, as these rare events can predict prognosis during treatment in a real-time fashion (19). Moreover, in ongoing US trials, early modifications in CTC counts after 3 weeks of first-line chemotherapy is the milestone for continuing the same therapy or switching to other agents. Recently, in metastatic patients treated with first-line chemotherapy combined with bevacizumab, it has been reported that the antiangiogenic treatment modifies the predictive value of CTCs, possibly due to their impact on tumor vessel endothelium conditioning less intravasation ability of the cancer cells. In this setting, another biomarker, such as circulating endothelial cell count variations, could be useful as an early surrogate marker (20). On the other hand, indirect assays permit not only CTC count but also the ability to isolate them. Molecular and genetic studies may be performed on these cells allowing more in-depth study of the molecular alterations in breast cancer cells during the metastatic process as well as pharmacogenomic tests for the detection of better therapeutic agents to eradicate minimal disease. In this field, the debate is ongoing, due to the discovery of small amounts of free DNA (about 1 ng/mL) even in peripheral blood of healthy subjects that is increased in some diseases, including breast cancer. Point mutations, abnormal hypermethylation, and microsatellite aberrations have been identified in circulating DNA, with good correlation to the primary tumor. Many authors have investigated the possibility of performing such molecular studies to outline a correlation between circulating DNA levels and prognosis or the relationship of circulating mutations and the primary tumor, but conclusive results are still not available (21,22). Another current hot topic is the discordance of HER2 expression in breast cancer samples and CTCs, related to the relevance of CTCs as “noninvasive” and real-time biopsies. It has been demonstrated that in about 30% of patients, a shift in HER2 status between the tissue biopsy and CTCs occurence (23,24). Ongoing studies have been designed to explore the clinical value of this shift for HER2 therapies in HER2-negative tumors with HER2-positive CTCs and vice versa. Conclusions In the era of emerging molecularly driven agents, the need for specific biomarkers is critical for monitoring efficacy and safety and for cost considerations. Their application needs to be prospectively tested and validated in large clinical trials, and standardized detec clinical practice will be reflected in the proper use of antineoplastic agents, improving breast cancer treatment and patient survival. Funding This work has been partially supported by a research grant to Dr Marco Danova from the IRCCS Foundation San Matteo, Pavia. References 1. Jain RK,  Duda DG,  Willett CG, et al.  Biomarkers of response and resistance to antiangiogenic therapy,  Nat Rev Clin Oncol. ,  2009, vol.  6  6(pg.  327- 338) 2. Hodgson DR,  Wellings E,  Orr M, et al.  Circulating tumor-derived predictive biomarkers in oncology,  Drug Discov Today. ,  2010, vol.  15  3–4(pg.  98- 101) 3. Verma S,  Lavasani S,  Mackey J, et al.  Optimizing the management of HER2-positive early breast cancer: the clinical reality,  Curr Oncol. ,  2010, vol.  17  4(pg.  20- 33) 4. 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JNCI Monographs – Oxford University Press

Published: Oct 1, 2011

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