- Email: [email protected]
BM micrometastases and circulating tumor cells in breast cancer patients: where have we been, where are we now and where does the future lie?
Cytotherapy (2005) Vol. 7, No. 6, 478 /482
BM micrometastases and circulating tumor cells in breast cancer patients: where have we been, where are we now and where does the future lie? V Mu¨ller1 and K Pantel2 1
Department of Gynecology and 2Institute of Tumor Biology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
Over the past 15 years early tumor cell dissemination has been detected in patients with breast cancer using sensitive immunocytochemical and molecular assays based on the use of MAb and PCR, respectively. Clinical studies involving more than 4000 breast cancer patients have now demonstrated that the presence of disseminated tumor cells in BM identified with immuncytochemical assays at primary diagnosis is a strong and independent prognostic factor. The published studies for the detection of disseminated tumor cells in BM fulfill the highest level of evidence as prognostic markers in primary breast cancer. In addition, various assays for the detection of circulating tumor cells in the peripheral blood have been developed recently and some studies suggest a potential clinical relevance of this parameter as a prognostic and predictive factor. Comparative analyzes indicate that the prognostic information derived from BM and blood screening
seems to be complementary and not redundant. Advanced methods for molecular characterization of single tumor cells and the surrounding environment have been developed lately, and this approach allows new insights into the metastatic cascade and characterization of targets for therapeutic approaches. Taken together, these findings provide the basis for the implementation of disseminated tumor cells in BM or blood as markers for stratification and assessment of therapies in prospective clinical trials. The valuable information derived from these trials should help to improve future treatment of breast cancer patients.
Introduction
have shown that the presence of disseminated tumor cells (DTC) in BM represents an additional clinical marker that should be useful for clinical decision making in order to establish risk-adapted adjuvant treatment strategies. First reports indicate that, with recently developed detection techniques, blood could be a more convenient source for DTC analysis. Another important and unique application of the detection of DTC in blood and BM could be the monitoring of therapeutic efficacy in the adjuvant setting with no measurable disease, also in the context of new therapeutic approaches. Here, we will briefly sum up the history of DTC detection, highlight important current aspects in the field of DTC detection in BM and blood of breast cancer patients, and discuss perspectives for clinical and research applications.
With the use of traditional prognostic factors in breast cancer, it is still not possible to identify reliably those patients who will relapse with metastatic disease [1]. Therefore, much research effort has been undertaken to identify additional factors enabling individual risk assessment. In addition, new therapeutic approaches directed against molecular mechanisms of malignant cell growth (e.g. HER-2/neu) allow individualized treatment [2]. However, identification of patients suitable for the different approaches is difficult. Immunocytochemical and molecular assays now enable specific detection of metastatic tumor cells even at the single cell stage, and allow detection of systemic tumor cell dissemination in the blood and BM, as one of the first crucial steps in the metastatic cascade. Several current studies in breast cancer
Keywords blood, BM, breast cancer, disseminated tumor cells.
Correspondence to: Professor Klaus Pantel, Institute of Tumor Biology, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany. – 2005 ISCT
DOI: 10.1080/14653240500360980
BM micrometastases and circulating tumor cells in breast cancer patients
Detection of disseminated tumor cells in BM The medullary space of the iliac crest is a site of intensive cellular exchange between blood and the mesenchymal interstitium. Occult tumor cells are even detected in the BM of patients who have cancers that do not preferably metastasize to the bone [3/5], indicating that the BM is a particularly good site for the detection of occult tumor cells. Early studies used histologic BM samples and revealed only low detection rates and no prognostic impact of DTC detection in BM [6 /9]. The low frequency of DTC leads to the need for more sensitive detection techniques. To date, most experience with BM screening for occult metastatic breast cancer cells exists for immunocytochemical analyzes using Ficoll density gradient centrifugation for tumor cell enrichment. For the identification of DTC, some marker Ag, such as epithelial membrane Ag or mucin-1, may not be suited for routine use because they also appear to be expressed on a subset of hematopoetic cells. Many studies have used cytokeratins (CK) as marker Ag; these proteins are stable and expressed in a majority of epithelial tumors [10,11]. A combination of several Ab to various CK Ag or broad-spectrum anti-CK Ab has been used because of the antigenic heterogeneity of tumor cells [12 /14]. The most recent studies [15 /20] consistently reported that the presence of DTC in BM has a strong prognostic impact on patient survival. This was also confirmed in a pooled analysis of Braun et al. [21] including 4703 patients with a 10-year follow-up. However, even in these studies, different Ab were used for the identification of epithelial cells in BM and the number of cells analyzed per patient varied or was not stated, illustrating a substantial variation in methodology. In the context of evaluation of methodology, two recent reports of the group of Naume et al. are also of considerable importance. One report [19] confirmed the independent prognostic value of DTC detection in BM in a cohort of 817 patients. In addition, the prognostic information derived from the established enrichment of mononuclear cells by Ficoll gradient was compared with an immunomagnetic enrichment technique that used Ab to deplete white blood cells and that was able to increase the detection rate for DTC. Surprisingly, the higher rate of positive patients with the more effective enrichment technique did not improve the prognostic value of DTC detection. In a second report [22], the same group showed that morphologic evaluation of the cells
479
detected by positive staining with an anti-cytokeratin Ab is important for their prognostic impact. Also, it was demonstrated that an increasing number of DTC in BM per patient reflects an increasingly bad prognosis. This again highlights the need for standardized methodology in DTC detection. The most recent TNM classification for breast cancer [23] does not qualify the presence of circulating tumor cells (CTC) in peripheral blood or DTC in BM as metastasis, but it optionally reports the presence of such cells together with their detection method, e.g. M0(i/) for the immunocytochemical detection or M0(mol/) for detection by molecular methods. Another important application of DTC detection is the monitoring of therapeutic efficacy in the adjuvant setting. Several studies have found DTC in BM even several years after surgery and adjuvant therapy, and it seems that the presence of DTC after adjuvant treatment could perhaps be used to identify patients with an increased risk for recurrence [24 /27]. In addition to simply identifying DTC, recent technical developments allow examination of the genome of even single cells. Using a combination of immunocytochemistry and FISH, several groups have reported numerical chromosomal aberrations in cytokeratin-positive cancer cells in BM and blood, indicating the malignant origin of these disseminated cells [28 /30]. The availability of protocols for the amplification of the whole genome has enabled a more detailed analysis of DTC and demonstrated that DTC are genetically heterogeneous [31,32]. Surprisingly, these cells have shown little resemblance to their respective primary tumor [32]. This suggests that the cells may have separated from their primary tumor at an early stage and evolved independently, influenced by the specific selective pressures of the BM environment. This is a new model of the events leading to manifest metastases. However, it is unclear whether the cells analyzed are capable of developing into metastases and which of the genomic alterations observed in DTC are clinically relevant. In this context, it also seems important to understand the environment in BM that enables tumor cell dormancy and eventually regrowth. One study, by Solomayer et al. [33] suggested profound and long-lasting negative effects on the BM immune system by adjuvant chemotherapy, and a another publication by Campbell et al . [34] suggested an increased incidence of DTC in patients with immune dysfunction.
480
V Mu¨ller and K Pantel
In addition, several recent publications have addressed the question regarding which properties of the primary tumor enable tumor cell dissemination. Woelfle et al . [35] have found that early dissemination of tumor cells is associated with a specific gene expression signature and the molecular pathways associated with hematogeneous spread are unrelated to those leading to lymphatic dissemination. Fehm et al . [36] and Gerber et al . [37] have described certain biologic factors of the primary tumors (e.g. expression of p53 or high proliferation) as correlated with the presence of DTC in BM.
Circulating tumor cells in the peripheral blood Peripheral blood would be an ideal source for the detection of DTC because of an easy sampling procedure. However, the prognostic significance of CTC is much less clear than for DTC in BM. For tumor cells, blood is only a temporary compartment, and it is not known whether a significant part of CTC survives and is subsequently capable to form detectable metastases. Also, the frequency of CTC in the blood seems to be lower and therefore Ficoll gradient centrifugation might not provide sufficient enrichment of malignant cells. An important progress seems to be the development of new enrichment systems for CTC [38,39]. Using new detection systems, it has been shown that the presence of CTC in breast cancer patients detected by immunocytochemical or molecular methods is correlated with the stage and course of the disease [38,40]. Cristofanilli et al. [39] showed in a prospective study that CTC detection provided significant prognostic information in patients with metastatic breast cancer using an automated enrichment and detection system. These findings demonstrate that CTC detection could have clinical relevance Pierga et al . [20] and also our group [38]. Our group [20] and also Mu¨ller et al. [38] have recently shown a correlation between the detection of CTC in the blood and DTC in BM of patients with primary breast cancer. However, the prognostic role of CTC detection in primary breast cancer is unclear and it seems possible that the information derived from BM and blood screening could to be complementary and not redundant.
Where does the future lie? Various immunocytochemical and molecular methods have been applied to detect occult hematogeneous tumor cell spread in breast cancer patients. For patients with primary breast cancer, prospective international studies
have demonstrated the highest level of evidence that the presence of DTC in BM is an independent prognostic factor. Final consensus is now needed regarding quality control issues and criteria for acceptable technical assay performance in order to permit multicenter clinical studies. For immunocytochemistry, such a standardization will be achieved by use of identical Ab, staining procedures and criteria for cells judged to be positive. Such criteria have been published recently by the Tumor Evaluation Committee of the International Society for Hematotherapy and Graft Engineering (now called the International Society for Cytotherapy) [41]. Interlaboratory quality control programs will also be established to ensure consistency of tests performed. These issues are part of a international project supported by the European Union called DISMAL (Disseminated Malignancies). With these developments, the final step towards implementation into the clinical setting will be taken. In addition to standardization of technical issues, more detailed marker implementation into current risk classification systems, such as the Tumor-Node-Metastasis (TNM) Classification System, is needed. Beyond adding another prognostic factor in breast cancer, the potential of occult hematogeneous tumor cell spread as a tool for prediction or monitoring the efficacy of systemic therapy is of great importance. In contrast to lymph nodes, which are generally removed at primary surgery, BM and blood can be obtained repeatedly in the post-operative course of treatment. Therapeutic efficacy of adjuvant systemic therapy can be assessed currently only retrospectively in large-scale clinical trials following an observation period of at least 5 years. Consequently, progress in this form of therapy is slow and it is not possible to tailor therapy to an individual patient. The potential of a surrogate marker assay that permits immediate assessment of therapy-induced effects on occult metastatic cells is therefore evident. It could be possible to identify patients who need additional adjuvant therapy, e.g. bisphosphonate treatment, which might eliminate tumor cells in BM persisting after adjuvant treatment [42]. Prospective clinical studies are now required to evaluate whether eradication of DTC in BM and blood after systemic therapy translates into a longer disease-free period and overall survival. Several studies are already ongoing or planned to address these issues. An additional important goal is the possibility of identifying tumorspecific targets to improve therapy regimens. Studies have
BM micrometastases and circulating tumor cells in breast cancer patients
shown that it is possible to identify therapeutic targets on DTC and some evidence suggests that single DTC show different properties than cells of the primary tumor [43 /45]. This is of importance, for example, in the context of new therapeutic approaches, such as Ab treatment directed against HER-2/neu, which has been demonstrated to reduce relapse rates in the adjuvant setting. In addition, the research in the field of tumor cell dissemination should lead to an increased understanding of the metastatic cascade. This could allow the development of new therapeutic approaches suppressing the development of metastatic disease when applied in the early stage of micrometastases before the development of manifest metastatic disease. In summary, these developments will, in our opinion, lead to progress in the treatment of breast cancer patients.
References 1 2 3
4
5
6
7
8
9
10
11
Pantel K, Brakenhoff RH. Dissecting the metastatic cascade. Nat Rev Cancer 2004;4:448 /56. Johnson ML, Seidman AD. Emerging targeted therapies for breast cancer. Oncology (Williston Park) 2005;19:611 /8. Lindemann F, Schlimok G, Dirschedl P et al . Prognostic significance of micrometastatic tumour cells in bone marrow of colorectal cancer patients. Lancet 1992;340:685 /9. Braun S, Schindlbeck C, Hepp F et al . Occult tumor cells in bone marrow of patients with locoregionally restricted ovarian cancer predict early distant metastatic relapse. J Clin Oncol 2001;19:368 /75. Roder JD, Thorban S, Pantel K et al . Micrometastases in bone marrow: prognostic indicators for pancreatic cancer. World J Surg 1999;23:888 /91. Funke I, Schraut W. Meta-analyses of studies on bone marrow micrometastases: an independent prognostic impact remains to be substantiated. J Clin Oncol 1998;16:557 /66. Porro G, Menard S, Tagliabue E et al . Monoclonal antibody detection of carcinoma cells in bone marrow biopsy specimens from breast cancer patients. Cancer 1988;61:2407 /11. Mathieu MC, Friedman S, Bosq J et al . Immunohistochemical staining of bone marrow biopsies for detection of occult metastasis in breast cancer. Breast Cancer Res Treat 1990;15:21 /6. Courtemanche DJ, Worth AJ, Coupland RW et al . Detection of micrometastases from primary breast cancer. Can J Surg 1991;34:15 /9. Moll R, Franke WW, Schiller DL et al . The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells. Cell 1982;31:11 /24. Fuchs E, Cleveland DW. A structural scaffolding of intermediate filaments in health and disease. Science 1998;279:514 /9.
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
481
Osborne MP, Wong GY, Asina S et al . Sensitivity of immunocytochemical detection of breast cancer cells in human bone marrow. Cancer Res 1991;51:2706 /9. Pantel K, Schlimok G, Angstwurm M et al . Methodological analysis of immunocytochemical screening for disseminated epithelial tumor cells in bone marrow. J Hematother 1994;3:165 /73. Braun S, Muller M, Hepp F et al . Re. Micrometastatic breast cancer cells in bone marrow at primary surgery: prognostic value in comparison with nodal status. J Natl Cancer Inst 1998;90:1099 /101. Braun S, Pantel K, Muller P et al . Cytokeratin-positive cells in the bone marrow and survival of patients with stage I, II, or III breast cancer. N Engl J Med 2000;342:525 /33. Diel IJ, Kaufmann M, Costa SD et al . Micrometastatic breast cancer cells in bone marrow at primary surgery: prognostic value in comparison with nodal status. J Natl Cancer Inst 1996;88:1652 /8. Gebauer G, Fehm T, Merkle E et al . Epithelial cells in bone marrow of breast cancer patients at time of primary surgery: clinical outcome during long-term follow-up. J Clin Oncol 2001;19:3669 /74. Mansi JL, Gogas H, Bliss JM et al . Outcome of primary-breastcancer patients with micrometastases: a long-term follow-up study. Lancet 1999;354:197 /202. Wiedswang G, Borgen E, Karesen R et al . Detection of isolated tumor cells in bone marrow is an independent prognostic factor in breast cancer. J Clin Oncol 2003;21:3469 /78. Pierga JY, Bonneton C, Vincent-Salomon A et al . Clinical significance of immunocytochemical detection of tumor cells using digital microscopy in peripheral blood and bone marrow of breast cancer patients. Clin Cancer Res 2004;10:1392 /400. Braun S, Vogl FD, Naume B et al . International pooled analysis of prognostic significance of bone marrow micrometastasis in patients with stage I, II, or III breast cancer. N Engl J Med 2005;353:793 /802. Naume B, Wiedswang G, Borgen E et al . The prognostic value of isolated tumor cells in bone marrow in breast cancer patients: evaluation of morphological categories and the number of clinically significant cells. Clin Cancer Res 2004;10:3091 /7. Singletary SE, Allred C, Ashley P et al . Revision of the American Joint Committee on Cancer staging system for breast cancer. J Clin Oncol 2002;20:3628 /36. Slade MJ, Singh A, Smith BM et al . Persistence of bone marrow micrometastases in patients receiving adjuvant therapy for breast cancer: results at 4 years. Int J Cancer 2005;114:94 /100. Janni W, Rack B, Schindlbeck C et al . The persistence of isolated tumor cells in bone marrow from patients with breast carcinoma predicts an increased risk for recurrence. Cancer 2005;103:884 / 91. Wiedswang G, Borgen E, Karensen R et al . Isolated tumor cells in bone marrow three years after diagnosis in disease-free breast cancer patients predicts unfavorable clinical outcome. Clin Cancer Res 2004;10:5342 /8.
482
V Mu¨ller and K Pantel
27 Janni W, Hepp F, Rjosk D et al . The fate and prognostic value of occult metastatic cells in the bone marrow of patients with breast carcinoma between primary treatment and recurrence. Cancer 2001;92:46 /53. 28 Mueller P, Carroll P, Bowers E et al . Low frequency epithelial cells in bone marrow aspirates from prostate carcinoma patients are cytogenetically aberrant. Cancer 1998;83:538 /46. 29 Fehm T, Sagalowsky A, Clifford E et al . Cytogenetic evidence that circulating epithelial cells in patients with carcinoma are malignant. Clin Cancer Res 2002;8:2073 /84. 30 Solakoglu O, Maierhofer C, Lahr G et al . Heterogeneous proliferative potential of occult metastatic cells in bone marrow of patients with solid epithelial tumors. Proc Natl Acad Sci USA 2002;99:2246 /51. 31 Klein CA, Blankenstein TJ, Schmidt-Kittler O et al . Genetic heterogeneity of single disseminated tumour cells in minimal residual cancer. Lancet 2002;360:683 /9. 32 Schmidt-Kittler O, Ragg T, Daskalakis A et al . From latent disseminated cells to overt metastasis: genetic analysis of systemic breast cancer progression. Proc Natl Acad Sci USA 2003;100:7737 /42. 33 Solomayer EF, Feuerer M, Bai L et al . Influence of adjuvant hormone therapy and chemotherapy on the immune system analysed in the bone marrow of patients with breast cancer. Clin Cancer Res 2003;9:174 /80. 34 Campbell MJ, Scott J, Maecker HT et al . Immune dysfunction and micrometastases in women with breast cancer. Breast Cancer Res Treat 2005;91:163 /71. 35 Woelfle U, Cloos J, Sauter G et al . Molecular signature associated with bone marrow micrometastasis in human breast cancer. Cancer Res 2003;63:5679 /84. 36 Fehm T, Becker S, Pergola-Becker G et al . Influence of tumor biological factors on tumor cell dissemination in primary breast cancer. Anticancer Res 2004;24:4211 /6.
37
38
39
40
41
42
43
44
45
Gerber B, Krause A, Muller H et al . Simultaneous immunohistochemical detection of tumor cells in lymph nodes and bone marrow aspirates in breast cancer and its correlation with other prognostic factors. J Clin Oncol 2001;19:960 /71. Mu¨ller V, Stahmann N, Riethdorf S et al . Circulating tumor cells in breast cancer: correlation to bone marrow micrometastases, heterogeneous response to systemic therapy and low proliferative activity. Clin Cancer Res 2005;11:3678 /85. Cristofanilli M, Budd GT, Ellis MJ et al . Circulating tumor cells, disease progression, and survival in metastatic breast cancer. N Engl J Med 2004;351:781 /91. Terstappen LW, Rao C, Gross S et al . Peripheral blood tumor cell load reflects the clinical activity of the disease in patients with carcinoma of the breast. Int J Oncol 2000;17:573 /8. Borgen E, Naume B, Nesland JM et al . Standardization of the immunocytochemical detection of cancer cells in BM and blood. I. Establishment of objective criteria for the evaluation of immunostained cells. Cytometry 1999;1:377 /88. Rack B, Janni W, Schoberth A et al . Secondary adjuvant therapy with zoledronate in patients with early breast cancer: is there an effect on persisting isolated tumor cells (ITC) in the bone marrow (BM)? Breast Cancer Res Treat 2004;88(Suppl 1):abstract 6019. Meng S, Tripathy D, Shete S et al . HER-2 gene amplification can be acquired as breast cancer progresses. Proc Natl Acad Sci USA 2004;101:9393 /8. Pantel K, Schlimok G, Braun S et al . Differential expression of proliferation-associated molecules in individual micrometastatic carcinoma cells. J Natl Cancer Inst 1993;85:1419 /24. Braun S, Schlimok G, Heumos I et al . ErbB2 overexpression on occult metastatic cells in bone marrow predicts poor clinical outcome of stage I /III breast cancer patients. Cancer Res 2001;61:1890 /5.
BM micrometastases and circulating tumor cells in breast cancer patients: where have we been, where are we now and where does the future lie? V Mu¨ller1 and K Pantel2 1
Department of Gynecology and 2Institute of Tumor Biology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
Over the past 15 years early tumor cell dissemination has been detected in patients with breast cancer using sensitive immunocytochemical and molecular assays based on the use of MAb and PCR, respectively. Clinical studies involving more than 4000 breast cancer patients have now demonstrated that the presence of disseminated tumor cells in BM identified with immuncytochemical assays at primary diagnosis is a strong and independent prognostic factor. The published studies for the detection of disseminated tumor cells in BM fulfill the highest level of evidence as prognostic markers in primary breast cancer. In addition, various assays for the detection of circulating tumor cells in the peripheral blood have been developed recently and some studies suggest a potential clinical relevance of this parameter as a prognostic and predictive factor. Comparative analyzes indicate that the prognostic information derived from BM and blood screening
seems to be complementary and not redundant. Advanced methods for molecular characterization of single tumor cells and the surrounding environment have been developed lately, and this approach allows new insights into the metastatic cascade and characterization of targets for therapeutic approaches. Taken together, these findings provide the basis for the implementation of disseminated tumor cells in BM or blood as markers for stratification and assessment of therapies in prospective clinical trials. The valuable information derived from these trials should help to improve future treatment of breast cancer patients.
Introduction
have shown that the presence of disseminated tumor cells (DTC) in BM represents an additional clinical marker that should be useful for clinical decision making in order to establish risk-adapted adjuvant treatment strategies. First reports indicate that, with recently developed detection techniques, blood could be a more convenient source for DTC analysis. Another important and unique application of the detection of DTC in blood and BM could be the monitoring of therapeutic efficacy in the adjuvant setting with no measurable disease, also in the context of new therapeutic approaches. Here, we will briefly sum up the history of DTC detection, highlight important current aspects in the field of DTC detection in BM and blood of breast cancer patients, and discuss perspectives for clinical and research applications.
With the use of traditional prognostic factors in breast cancer, it is still not possible to identify reliably those patients who will relapse with metastatic disease [1]. Therefore, much research effort has been undertaken to identify additional factors enabling individual risk assessment. In addition, new therapeutic approaches directed against molecular mechanisms of malignant cell growth (e.g. HER-2/neu) allow individualized treatment [2]. However, identification of patients suitable for the different approaches is difficult. Immunocytochemical and molecular assays now enable specific detection of metastatic tumor cells even at the single cell stage, and allow detection of systemic tumor cell dissemination in the blood and BM, as one of the first crucial steps in the metastatic cascade. Several current studies in breast cancer
Keywords blood, BM, breast cancer, disseminated tumor cells.
Correspondence to: Professor Klaus Pantel, Institute of Tumor Biology, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany. – 2005 ISCT
DOI: 10.1080/14653240500360980
BM micrometastases and circulating tumor cells in breast cancer patients
Detection of disseminated tumor cells in BM The medullary space of the iliac crest is a site of intensive cellular exchange between blood and the mesenchymal interstitium. Occult tumor cells are even detected in the BM of patients who have cancers that do not preferably metastasize to the bone [3/5], indicating that the BM is a particularly good site for the detection of occult tumor cells. Early studies used histologic BM samples and revealed only low detection rates and no prognostic impact of DTC detection in BM [6 /9]. The low frequency of DTC leads to the need for more sensitive detection techniques. To date, most experience with BM screening for occult metastatic breast cancer cells exists for immunocytochemical analyzes using Ficoll density gradient centrifugation for tumor cell enrichment. For the identification of DTC, some marker Ag, such as epithelial membrane Ag or mucin-1, may not be suited for routine use because they also appear to be expressed on a subset of hematopoetic cells. Many studies have used cytokeratins (CK) as marker Ag; these proteins are stable and expressed in a majority of epithelial tumors [10,11]. A combination of several Ab to various CK Ag or broad-spectrum anti-CK Ab has been used because of the antigenic heterogeneity of tumor cells [12 /14]. The most recent studies [15 /20] consistently reported that the presence of DTC in BM has a strong prognostic impact on patient survival. This was also confirmed in a pooled analysis of Braun et al. [21] including 4703 patients with a 10-year follow-up. However, even in these studies, different Ab were used for the identification of epithelial cells in BM and the number of cells analyzed per patient varied or was not stated, illustrating a substantial variation in methodology. In the context of evaluation of methodology, two recent reports of the group of Naume et al. are also of considerable importance. One report [19] confirmed the independent prognostic value of DTC detection in BM in a cohort of 817 patients. In addition, the prognostic information derived from the established enrichment of mononuclear cells by Ficoll gradient was compared with an immunomagnetic enrichment technique that used Ab to deplete white blood cells and that was able to increase the detection rate for DTC. Surprisingly, the higher rate of positive patients with the more effective enrichment technique did not improve the prognostic value of DTC detection. In a second report [22], the same group showed that morphologic evaluation of the cells
479
detected by positive staining with an anti-cytokeratin Ab is important for their prognostic impact. Also, it was demonstrated that an increasing number of DTC in BM per patient reflects an increasingly bad prognosis. This again highlights the need for standardized methodology in DTC detection. The most recent TNM classification for breast cancer [23] does not qualify the presence of circulating tumor cells (CTC) in peripheral blood or DTC in BM as metastasis, but it optionally reports the presence of such cells together with their detection method, e.g. M0(i/) for the immunocytochemical detection or M0(mol/) for detection by molecular methods. Another important application of DTC detection is the monitoring of therapeutic efficacy in the adjuvant setting. Several studies have found DTC in BM even several years after surgery and adjuvant therapy, and it seems that the presence of DTC after adjuvant treatment could perhaps be used to identify patients with an increased risk for recurrence [24 /27]. In addition to simply identifying DTC, recent technical developments allow examination of the genome of even single cells. Using a combination of immunocytochemistry and FISH, several groups have reported numerical chromosomal aberrations in cytokeratin-positive cancer cells in BM and blood, indicating the malignant origin of these disseminated cells [28 /30]. The availability of protocols for the amplification of the whole genome has enabled a more detailed analysis of DTC and demonstrated that DTC are genetically heterogeneous [31,32]. Surprisingly, these cells have shown little resemblance to their respective primary tumor [32]. This suggests that the cells may have separated from their primary tumor at an early stage and evolved independently, influenced by the specific selective pressures of the BM environment. This is a new model of the events leading to manifest metastases. However, it is unclear whether the cells analyzed are capable of developing into metastases and which of the genomic alterations observed in DTC are clinically relevant. In this context, it also seems important to understand the environment in BM that enables tumor cell dormancy and eventually regrowth. One study, by Solomayer et al. [33] suggested profound and long-lasting negative effects on the BM immune system by adjuvant chemotherapy, and a another publication by Campbell et al . [34] suggested an increased incidence of DTC in patients with immune dysfunction.
480
V Mu¨ller and K Pantel
In addition, several recent publications have addressed the question regarding which properties of the primary tumor enable tumor cell dissemination. Woelfle et al . [35] have found that early dissemination of tumor cells is associated with a specific gene expression signature and the molecular pathways associated with hematogeneous spread are unrelated to those leading to lymphatic dissemination. Fehm et al . [36] and Gerber et al . [37] have described certain biologic factors of the primary tumors (e.g. expression of p53 or high proliferation) as correlated with the presence of DTC in BM.
Circulating tumor cells in the peripheral blood Peripheral blood would be an ideal source for the detection of DTC because of an easy sampling procedure. However, the prognostic significance of CTC is much less clear than for DTC in BM. For tumor cells, blood is only a temporary compartment, and it is not known whether a significant part of CTC survives and is subsequently capable to form detectable metastases. Also, the frequency of CTC in the blood seems to be lower and therefore Ficoll gradient centrifugation might not provide sufficient enrichment of malignant cells. An important progress seems to be the development of new enrichment systems for CTC [38,39]. Using new detection systems, it has been shown that the presence of CTC in breast cancer patients detected by immunocytochemical or molecular methods is correlated with the stage and course of the disease [38,40]. Cristofanilli et al. [39] showed in a prospective study that CTC detection provided significant prognostic information in patients with metastatic breast cancer using an automated enrichment and detection system. These findings demonstrate that CTC detection could have clinical relevance Pierga et al . [20] and also our group [38]. Our group [20] and also Mu¨ller et al. [38] have recently shown a correlation between the detection of CTC in the blood and DTC in BM of patients with primary breast cancer. However, the prognostic role of CTC detection in primary breast cancer is unclear and it seems possible that the information derived from BM and blood screening could to be complementary and not redundant.
Where does the future lie? Various immunocytochemical and molecular methods have been applied to detect occult hematogeneous tumor cell spread in breast cancer patients. For patients with primary breast cancer, prospective international studies
have demonstrated the highest level of evidence that the presence of DTC in BM is an independent prognostic factor. Final consensus is now needed regarding quality control issues and criteria for acceptable technical assay performance in order to permit multicenter clinical studies. For immunocytochemistry, such a standardization will be achieved by use of identical Ab, staining procedures and criteria for cells judged to be positive. Such criteria have been published recently by the Tumor Evaluation Committee of the International Society for Hematotherapy and Graft Engineering (now called the International Society for Cytotherapy) [41]. Interlaboratory quality control programs will also be established to ensure consistency of tests performed. These issues are part of a international project supported by the European Union called DISMAL (Disseminated Malignancies). With these developments, the final step towards implementation into the clinical setting will be taken. In addition to standardization of technical issues, more detailed marker implementation into current risk classification systems, such as the Tumor-Node-Metastasis (TNM) Classification System, is needed. Beyond adding another prognostic factor in breast cancer, the potential of occult hematogeneous tumor cell spread as a tool for prediction or monitoring the efficacy of systemic therapy is of great importance. In contrast to lymph nodes, which are generally removed at primary surgery, BM and blood can be obtained repeatedly in the post-operative course of treatment. Therapeutic efficacy of adjuvant systemic therapy can be assessed currently only retrospectively in large-scale clinical trials following an observation period of at least 5 years. Consequently, progress in this form of therapy is slow and it is not possible to tailor therapy to an individual patient. The potential of a surrogate marker assay that permits immediate assessment of therapy-induced effects on occult metastatic cells is therefore evident. It could be possible to identify patients who need additional adjuvant therapy, e.g. bisphosphonate treatment, which might eliminate tumor cells in BM persisting after adjuvant treatment [42]. Prospective clinical studies are now required to evaluate whether eradication of DTC in BM and blood after systemic therapy translates into a longer disease-free period and overall survival. Several studies are already ongoing or planned to address these issues. An additional important goal is the possibility of identifying tumorspecific targets to improve therapy regimens. Studies have
BM micrometastases and circulating tumor cells in breast cancer patients
shown that it is possible to identify therapeutic targets on DTC and some evidence suggests that single DTC show different properties than cells of the primary tumor [43 /45]. This is of importance, for example, in the context of new therapeutic approaches, such as Ab treatment directed against HER-2/neu, which has been demonstrated to reduce relapse rates in the adjuvant setting. In addition, the research in the field of tumor cell dissemination should lead to an increased understanding of the metastatic cascade. This could allow the development of new therapeutic approaches suppressing the development of metastatic disease when applied in the early stage of micrometastases before the development of manifest metastatic disease. In summary, these developments will, in our opinion, lead to progress in the treatment of breast cancer patients.
References 1 2 3
4
5
6
7
8
9
10
11
Pantel K, Brakenhoff RH. Dissecting the metastatic cascade. Nat Rev Cancer 2004;4:448 /56. Johnson ML, Seidman AD. Emerging targeted therapies for breast cancer. Oncology (Williston Park) 2005;19:611 /8. Lindemann F, Schlimok G, Dirschedl P et al . Prognostic significance of micrometastatic tumour cells in bone marrow of colorectal cancer patients. Lancet 1992;340:685 /9. Braun S, Schindlbeck C, Hepp F et al . Occult tumor cells in bone marrow of patients with locoregionally restricted ovarian cancer predict early distant metastatic relapse. J Clin Oncol 2001;19:368 /75. Roder JD, Thorban S, Pantel K et al . Micrometastases in bone marrow: prognostic indicators for pancreatic cancer. World J Surg 1999;23:888 /91. Funke I, Schraut W. Meta-analyses of studies on bone marrow micrometastases: an independent prognostic impact remains to be substantiated. J Clin Oncol 1998;16:557 /66. Porro G, Menard S, Tagliabue E et al . Monoclonal antibody detection of carcinoma cells in bone marrow biopsy specimens from breast cancer patients. Cancer 1988;61:2407 /11. Mathieu MC, Friedman S, Bosq J et al . Immunohistochemical staining of bone marrow biopsies for detection of occult metastasis in breast cancer. Breast Cancer Res Treat 1990;15:21 /6. Courtemanche DJ, Worth AJ, Coupland RW et al . Detection of micrometastases from primary breast cancer. Can J Surg 1991;34:15 /9. Moll R, Franke WW, Schiller DL et al . The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells. Cell 1982;31:11 /24. Fuchs E, Cleveland DW. A structural scaffolding of intermediate filaments in health and disease. Science 1998;279:514 /9.
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
481
Osborne MP, Wong GY, Asina S et al . Sensitivity of immunocytochemical detection of breast cancer cells in human bone marrow. Cancer Res 1991;51:2706 /9. Pantel K, Schlimok G, Angstwurm M et al . Methodological analysis of immunocytochemical screening for disseminated epithelial tumor cells in bone marrow. J Hematother 1994;3:165 /73. Braun S, Muller M, Hepp F et al . Re. Micrometastatic breast cancer cells in bone marrow at primary surgery: prognostic value in comparison with nodal status. J Natl Cancer Inst 1998;90:1099 /101. Braun S, Pantel K, Muller P et al . Cytokeratin-positive cells in the bone marrow and survival of patients with stage I, II, or III breast cancer. N Engl J Med 2000;342:525 /33. Diel IJ, Kaufmann M, Costa SD et al . Micrometastatic breast cancer cells in bone marrow at primary surgery: prognostic value in comparison with nodal status. J Natl Cancer Inst 1996;88:1652 /8. Gebauer G, Fehm T, Merkle E et al . Epithelial cells in bone marrow of breast cancer patients at time of primary surgery: clinical outcome during long-term follow-up. J Clin Oncol 2001;19:3669 /74. Mansi JL, Gogas H, Bliss JM et al . Outcome of primary-breastcancer patients with micrometastases: a long-term follow-up study. Lancet 1999;354:197 /202. Wiedswang G, Borgen E, Karesen R et al . Detection of isolated tumor cells in bone marrow is an independent prognostic factor in breast cancer. J Clin Oncol 2003;21:3469 /78. Pierga JY, Bonneton C, Vincent-Salomon A et al . Clinical significance of immunocytochemical detection of tumor cells using digital microscopy in peripheral blood and bone marrow of breast cancer patients. Clin Cancer Res 2004;10:1392 /400. Braun S, Vogl FD, Naume B et al . International pooled analysis of prognostic significance of bone marrow micrometastasis in patients with stage I, II, or III breast cancer. N Engl J Med 2005;353:793 /802. Naume B, Wiedswang G, Borgen E et al . The prognostic value of isolated tumor cells in bone marrow in breast cancer patients: evaluation of morphological categories and the number of clinically significant cells. Clin Cancer Res 2004;10:3091 /7. Singletary SE, Allred C, Ashley P et al . Revision of the American Joint Committee on Cancer staging system for breast cancer. J Clin Oncol 2002;20:3628 /36. Slade MJ, Singh A, Smith BM et al . Persistence of bone marrow micrometastases in patients receiving adjuvant therapy for breast cancer: results at 4 years. Int J Cancer 2005;114:94 /100. Janni W, Rack B, Schindlbeck C et al . The persistence of isolated tumor cells in bone marrow from patients with breast carcinoma predicts an increased risk for recurrence. Cancer 2005;103:884 / 91. Wiedswang G, Borgen E, Karensen R et al . Isolated tumor cells in bone marrow three years after diagnosis in disease-free breast cancer patients predicts unfavorable clinical outcome. Clin Cancer Res 2004;10:5342 /8.
482
V Mu¨ller and K Pantel
27 Janni W, Hepp F, Rjosk D et al . The fate and prognostic value of occult metastatic cells in the bone marrow of patients with breast carcinoma between primary treatment and recurrence. Cancer 2001;92:46 /53. 28 Mueller P, Carroll P, Bowers E et al . Low frequency epithelial cells in bone marrow aspirates from prostate carcinoma patients are cytogenetically aberrant. Cancer 1998;83:538 /46. 29 Fehm T, Sagalowsky A, Clifford E et al . Cytogenetic evidence that circulating epithelial cells in patients with carcinoma are malignant. Clin Cancer Res 2002;8:2073 /84. 30 Solakoglu O, Maierhofer C, Lahr G et al . Heterogeneous proliferative potential of occult metastatic cells in bone marrow of patients with solid epithelial tumors. Proc Natl Acad Sci USA 2002;99:2246 /51. 31 Klein CA, Blankenstein TJ, Schmidt-Kittler O et al . Genetic heterogeneity of single disseminated tumour cells in minimal residual cancer. Lancet 2002;360:683 /9. 32 Schmidt-Kittler O, Ragg T, Daskalakis A et al . From latent disseminated cells to overt metastasis: genetic analysis of systemic breast cancer progression. Proc Natl Acad Sci USA 2003;100:7737 /42. 33 Solomayer EF, Feuerer M, Bai L et al . Influence of adjuvant hormone therapy and chemotherapy on the immune system analysed in the bone marrow of patients with breast cancer. Clin Cancer Res 2003;9:174 /80. 34 Campbell MJ, Scott J, Maecker HT et al . Immune dysfunction and micrometastases in women with breast cancer. Breast Cancer Res Treat 2005;91:163 /71. 35 Woelfle U, Cloos J, Sauter G et al . Molecular signature associated with bone marrow micrometastasis in human breast cancer. Cancer Res 2003;63:5679 /84. 36 Fehm T, Becker S, Pergola-Becker G et al . Influence of tumor biological factors on tumor cell dissemination in primary breast cancer. Anticancer Res 2004;24:4211 /6.
37
38
39
40
41
42
43
44
45
Gerber B, Krause A, Muller H et al . Simultaneous immunohistochemical detection of tumor cells in lymph nodes and bone marrow aspirates in breast cancer and its correlation with other prognostic factors. J Clin Oncol 2001;19:960 /71. Mu¨ller V, Stahmann N, Riethdorf S et al . Circulating tumor cells in breast cancer: correlation to bone marrow micrometastases, heterogeneous response to systemic therapy and low proliferative activity. Clin Cancer Res 2005;11:3678 /85. Cristofanilli M, Budd GT, Ellis MJ et al . Circulating tumor cells, disease progression, and survival in metastatic breast cancer. N Engl J Med 2004;351:781 /91. Terstappen LW, Rao C, Gross S et al . Peripheral blood tumor cell load reflects the clinical activity of the disease in patients with carcinoma of the breast. Int J Oncol 2000;17:573 /8. Borgen E, Naume B, Nesland JM et al . Standardization of the immunocytochemical detection of cancer cells in BM and blood. I. Establishment of objective criteria for the evaluation of immunostained cells. Cytometry 1999;1:377 /88. Rack B, Janni W, Schoberth A et al . Secondary adjuvant therapy with zoledronate in patients with early breast cancer: is there an effect on persisting isolated tumor cells (ITC) in the bone marrow (BM)? Breast Cancer Res Treat 2004;88(Suppl 1):abstract 6019. Meng S, Tripathy D, Shete S et al . HER-2 gene amplification can be acquired as breast cancer progresses. Proc Natl Acad Sci USA 2004;101:9393 /8. Pantel K, Schlimok G, Braun S et al . Differential expression of proliferation-associated molecules in individual micrometastatic carcinoma cells. J Natl Cancer Inst 1993;85:1419 /24. Braun S, Schlimok G, Heumos I et al . ErbB2 overexpression on occult metastatic cells in bone marrow predicts poor clinical outcome of stage I /III breast cancer patients. Cancer Res 2001;61:1890 /5.