The quest for the ‘bony Grail’ of detecting circulating tumour cells in patients with prostate cancer
Authors: C. Massard, A. Chauchereau, K. Fizazi
Journal: Annals of Oncology (2009)

Prostate cancer is the most common cancer and the second leading cause of death from cancer in males in most Western countries. Most prostate cancer deaths are caused by haematogenous metastatic spread and subsequent tumour cell growth in distant organs, especially the bones [1.Attard G. Sarker D. Reid A. et al.Improving the outcome of patients with castration-resistant prostate cancer through rational drug development.Br J Cancer. 2006; 95: 767-774Crossref PubMed Scopus (69) Google Scholar]. Currently, treatment of patients with disseminated prostate cancer is based on direct cytotoxicity against tumour cells, via androgen deprivation therapy or docetaxel-based chemotherapy [2.Tannock I.F. de Wit R. Berry W.R. et al.TAX 327 Investigators. Docetaxel plus prednisone or mitoxantrone plus prednisone for advanced prostate cancer.N Engl J Med. 2004; 351: 1502-1512Crossref PubMed Scopus (4835) Google Scholar].Cells shed by carcinoma circulate in the blood of patients with solid tumours and circulating tumour cells (CTCs) are present in patients with various carcinomas with a wide range of incidences and frequencies [3.Pantel K. Brakenhoff R.H. Brandt B. Detection, clinical relevance and specific biological properties of disseminating tumour cells.Nat Rev Cancer. 2008; 8: 329-340Crossref PubMed Scopus (953) Google Scholar]. The detection of CTCs in cancer patients yields prognostic information and might help tailor systemic therapies to the individual needs of cancer patients [4.Alix-Panabières C. Riethdorf S. Pantel K. Circulating tumor cells and bone marrow micrometastasis.Clin Cancer Res. 2008; 14: 5013-5021Crossref PubMed Scopus (196) Google Scholar].A major problem in the evaluation of the treatment of advanced prostate cancer is the lack of strong surrogate markers for disease outcome and clinical benefit. Prostate cancer does not usually lead to radiologically measurable metastatic disease because the bone is typically the site of metastatic spread [5.Berthold D.R. Tannock I.F. Are prostate cancer clinical trial enrollment criteria and endpoints appropriate?.Nat Clin Pract Oncol. 2006; 3: 74-75Crossref PubMed Scopus (1) Google Scholar]. The use of prostate-specific antigen (PSA) is therefore currently widely used as a marker of treatment activity although PSA does not always correlate with decreased tumour growth, overall survival (OS) or quality of life [6.Fitzpatrick J.M. Anderson J. Sternberg C.N. et al.Optimizing treatment for men with advanced prostate cancer: expert recommendations and the multidisciplinary approach.Crit Rev Oncol Hematol. 2008; 68: S9-S22Crossref PubMed Scopus (27) Google Scholar]. Detecting CTCs has a number of potential applications in patients with prostate cancer including the assessment of prognosis, recurrence after definitive local treatment, response to therapy and obtaining a tumour sample to assess target modulation by drugs without invasive procedure in patients with metastases. Different CTC isolation methods are available with their advantages and drawbacks. CTC detection can rely on slide-based methods (immunochemistry or FISH), molecular analysis (RT-PCR) or flow-based cell sorting methods after immunofluorescent identification and immunomagnetic labelling of cells (anti-Ep-CAM and CK against CTC and anti-CD45 against leukocytes) [3.Pantel K. Brakenhoff R.H. Brandt B. Detection, clinical relevance and specific biological properties of disseminating tumour cells.Nat Rev Cancer. 2008; 8: 329-340Crossref PubMed Scopus (953) Google Scholar]. CTCs can also be detected using telomerase activity in the great majority of patients with prostate cancer [7.Fizazi K. Morat L. Chauveinc L. et al.High detection rate of circulating tumor cells in blood of patients with prostate cancer using telomerase activity.Ann Oncol. 2007; 18: 518-521Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar].CTC enrichment can be achieved with cell positive/negative selection. The CellSearch™ System (Veridex®, Warren, NJ) allows the analysis of CTCs in a standardized, objective manner. CTC enrichment, specific for epithelial cells, is carried out automatically with antibody-coated magnetic beads and fluorescent labelling. The specific antibodies for epithelial CTC enrichment and detection are, respectively, Ep-CAM and Cytokeratin 8/18/19. The leukocyte component is detected with CD45 antibodies. CTCs are defined as nucleated cells lacking CD45 expression and exhibiting Ep-CAM and cytokeratin expressions. Using this system, CTCs are detectable in metastatic carcinomas with a wide range of incidences and frequencies and not in benign diseases [8.Allard W.J. Matera J. Miller M.C. et al.Tumor cells circulate in the peripheral blood of all major carcinomas but not in healthy subjects or patients with nonmalignant diseases.Clin Cancer Res. 2004; 10: 6897-6904Crossref PubMed Scopus (1980) Google Scholar]. An alternative method for CTC enrichment uses filters with pores that retain the large tumour cells but not the smaller blood cells (The Metagenex® system, Paris, France). The iSET (isolation by size of epithelial tumour cell) allows CTC counting and CTC isolation for cytomorphological and molecular characterisation (FISH). This method is independent of CTC epithelial marker expression and allows the enrichment of epithelial and nonepithelial CTCs [9.Vona G. Estepa L. Béroud C. et al.Paterlini-Bréchot P impact of cytomorphological detection of circulating tumor cells in patients with liver cancer.Hepatology. 2004; 39: 792-797Crossref PubMed Scopus (160) Google Scholar]. At the present time, the CellSearch System (Veridex®) is the only method that has obtained approval for monitoring metastatic breast, colorectal and prostate cancers [10.de Bono J.S. Scher H.I. Montgomery R.B. et al.Circulating tumor cells predict survival benefit from treatment in metastatic castration-resistant prostate cancer.Clin Cancer Res. 2008; 14 (1): 6302-6309Crossref PubMed Scopus (1739) Google Scholar].Recent studies indicated that CTC counting is a promising biomarker predictive for clinical outcome in patients with disseminated [11.Cristofanilli M. Budd G.T. Ellis M.J. et al.Circulating tumor cells, disease progression, and survival in metastatic breast cancer.N Engl J Med. 2004; 351 (Oncol Rep 2008; 10(2): 137–146): 781-791Crossref PubMed Scopus (3567) Google Scholar] and localised [12.Pierga J.Y. Bidard F.C. Mathiot 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; 14: 7004-7010Crossref PubMed Scopus (314) Google Scholar] breast cancer. In the January issue of Annals of Oncology, Olmos et al. [13.Olmos D. Arkenau H.T. Ang J.E. et al.Circulating tumour cell (CTC) counts as intermediate end points in castration-resistant prostate cancer (CRPC): a single-centre experience.Ann Oncol. 2009; 20: 27-33Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar] demonstrate using the CellSearch® System that the CTC count is a strong prognostic factor for OS in patients with advanced castration-resistant prostate cancer (CRPC) at baseline, but also during the clinical course of the disease. Patients with advanced CRPC and a low CTC count enjoy better OS compared to patients with a high CTC count. Moreover, patients with a high CTC count present with aggressive disease and other poor prognostic factors. The CTC count could also be a reliable predictor of treatment efficacy in advanced metastatic cancer. Olmos et al. also showed that the monitoring of the CTC count during systemic therapies could identify patients with a favourable pattern of response, so that CTC counting may be a more specific, more sensitive and earlier predictive factor of response than radiologic evaluation assessed by the RECIST criteria.Besides the quantitative use of the CTC count to predict the prognosis or response to treatment, the collection of CTCs may be used as an elegant technique to sample malignant tissue from an individual for molecular study purposes, provided that the biological features of CTCs are similar to those of metastatic cells. Detecting molecular abnormalities in a subpopulation of CTCs will complete information on tumour biology or tumour sensitivity to treatment. Thus, characterisation of CTCs may lead to a breakthrough in the understanding of the metastatic phenotype and may constitute a surrogate marker of primary or metastatic tumours that ultimately permit a more personalised therapeutic approach. However, to our knowledge, there are still no studies that compare molecular profiles between CTCs, the primary tumour and metastases, and available CTC purification techniques do not yield reliable overall gene expression patterns from CTCs in a large cohort of patients. Although high-throughput gene expression from CTCs is technically challenging, the potential payoff in yielding a more profound understanding of the molecular features of metastatic cancer justifies its implementation.It is commonly admitted that CTCs, at least a subset of them, correspond to one of the mandatory steps of the metastasis spread from the primary tumour [3.Pantel K. Brakenhoff R.H. Brandt B. Detection, clinical relevance and specific biological properties of disseminating tumour cells.Nat Rev Cancer. 2008; 8: 329-340Crossref PubMed Scopus (953) Google Scholar]. This has prompted the search for markers previously used to characterise the primary tumour. However, some molecular discrepancies have been reported between the primary tumour and circulating cells. For example, HER2-positive CTCs from breast cancer correlated significantly with disease-free survival and OS although HER-2 was not expressed in the corresponding primary tumours [14.Wülfing P. Borchard J. Buerger H. et al.HER2-positive circulating tumor cells indicate poor clinical outcome in stage I to III breast cancer patients.Clin Cancer Res. 2006; 12: 1715-1720Crossref PubMed Scopus (228) Google Scholar]. In systems that allow automated detection and enumeration of CTCs like the CellSearch™ System, CTC detection relies on the expression of the epithelial cell adhesion molecule and on the cytokeratin profile of these cells. However, it should be borne in mind that the selection of CTCs by cytokeratin immunostaining may exclude some cells that have undergone intrinsic modifications of their phenotype during the shedding process. For example, the down-regulation of cytokeratin 18 was reported in prostate disseminating tumour cells [15.Weckermann D. Muller P. Wawroschek F. et al.Disseminated cytokeratin positive tumor cells in the bone marrow of patients with prostate cancer: detection and prognostic value.J Urol. 2001; 166: 699-703Crossref PubMed Scopus (70) Google Scholar] and down-regulation of cytokeratin expression compensated by increased vimentin expression has been reported in micrometastatic breast cancer cells [16.Willipinski-Stapelfeldt B. Riethdorf S. Assmann V. et al.Changes in cytoskeletal protein composition indicative of an epithelial-mesenchymal transition in human micrometastatic and primary breast carcinoma cells.Clin Cancer Res. 2005; 11: 8006-8014Crossref PubMed Scopus (251) Google Scholar]. Until now, changes in gene expression associated with the epithelial–mesenchymal transition that can affect CTCs were not taken into account in isolation procedures. Moreover, the molecular bases of the dormancy of CTCs are poorly understood and the relationship between enumerated CTCs and metastasis is still an open question. Comprehensive molecular characterisation of CTCs may certainly provide important insights into the biology of prostate cancer metastasis and may contribute to better identification of patients who need additional targeted therapy.Greater knowledge of prostate cancer biology has led to the isolation of many new and promising targets, and agents targeting these molecules are currently under development in large randomised phase III trials, to improve OS and the quality of life of patients with metastatic CRPC. Evidence that the androgen receptor axis is still active in the setting of CRPC has also been provided [1.Attard G. Sarker D. Reid A. et al.Improving the outcome of patients with castration-resistant prostate cancer through rational drug development.Br J Cancer. 2006; 95: 767-774Crossref PubMed Scopus (69) Google Scholar], and drugs directly or indirectly targeting the androgen receptor such as abiraterone [18.Scher H.I. Beer T.M. Higano C.S. et al.Phase I/II study of MDV3100 in patients (pts) with progressive castration-resistant prostate cancer (CRPC)ASCO.J Clin Oncol. 2008; 26 (Suppl); (Abstr 5006)Crossref Google Scholar], new specific peripheral anti-androgens [19.Fizazi K. Le Maitre A. Hudes G. et al.Meta-analysis of Estramustine in Prostate Cancer (MECaP) Trialists' Collaborative Group. Addition of estramustine to chemotherapy and survival of patients with castration-refractory prostate cancer: a meta-analysis of individual patient data.Lancet Oncol. 2007; 8: 994-1000Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar], and others [20.K Miller K Fizazi BJU Int, in pressGoogle Scholar], are among the most promising new agents in CRPC. In this list and in addition to drugs targeting angiogenesis (bevacizumab, VEGF Trap), are agents that target molecules involved in the onset of bone metastases such as ZD 4054, which targets the endothelin1 receptor A [21.Fizazi K. et al.J Clin Oncol. 2009; Google Scholar], denosumab which targets the Rank ligand [22.K Fizazi. J Clin Oncol in pressGoogle Scholar], and radiopharmaceutical agents [23.de Bono J.S. Attard G. Adjei A. et al.Potential applications for circulating tumor cells expressing the insulin-like growth factor-I receptor.Clin Cancer Res. 2007; 13: 3611-3616Crossref PubMed Scopus (173) Google Scholar]. Analysis of some of these molecular targets in CTCs from patients participating to ongoing phase I, II or III trials is underway or planned and shall help to better identify those patients likely to benefit [23.de Bono J.S. Attard G. Adjei A. et al.Potential applications for circulating tumor cells expressing the insulin-like growth factor-I receptor.Clin Cancer Res. 2007; 13: 3611-3616Crossref PubMed Scopus (173) Google Scholar]. For example, the discovery of recurrent gene fusions in prostate cancers has important clinical and biological implications [24.Kumar-Sinha C. Tomlins S.A. Chinnaiyan A.M. Recurrent gene fusions in prostate cancer.Nat Rev Cancer. 2008; 8: 497-511Crossref PubMed Scopus (561) Google Scholar]. The detection of the translocation of TMPRSS2 to the ERG gene in CTCs could be used as a biomarker in clinical drug development.CTC counting but also molecular analysis of harvested CTCs are likely to accelerate clinical research in CRPC during the next decade by providing researchers and physicians with the tools they were lacking to access bone metastases. This is why the Royal Marsden Hospital team should be congratulated and their report [13.Olmos D. Arkenau H.T. Ang J.E. et al.Circulating tumour cell (CTC) counts as intermediate end points in castration-resistant prostate cancer (CRPC): a single-centre experience.Ann Oncol. 2009; 20: 27-33Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar] will certainly remain a seminal paper in the difficult quest for the ‘bony Grail’ in CRPC. Prostate cancer is the most common cancer and the second leading cause of death from cancer in males in most Western countries. Most prostate cancer deaths are caused by haematogenous metastatic spread and subsequent tumour cell growth in distant organs, especially the bones [1.Attard G. Sarker D. Reid A. et al.Improving the outcome of patients with castration-resistant prostate cancer through rational drug development.Br J Cancer. 2006; 95: 767-774Crossref PubMed Scopus (69) Google Scholar]. Currently, treatment of patients with disseminated prostate cancer is based on direct cytotoxicity against tumour cells, via androgen deprivation therapy or docetaxel-based chemotherapy [2.Tannock I.F. de Wit R. Berry W.R. et al.TAX 327 Investigators. Docetaxel plus prednisone or mitoxantrone plus prednisone for advanced prostate cancer.N Engl J Med. 2004; 351: 1502-1512Crossref PubMed Scopus (4835) Google Scholar]. Cells shed by carcinoma circulate in the blood of patients with solid tumours and circulating tumour cells (CTCs) are present in patients with various carcinomas with a wide range of incidences and frequencies [3.Pantel K. Brakenhoff R.H. Brandt B. Detection, clinical relevance and specific biological properties of disseminating tumour cells.Nat Rev Cancer. 2008; 8: 329-340Crossref PubMed Scopus (953) Google Scholar]. The detection of CTCs in cancer patients yields prognostic information and might help tailor systemic therapies to the individual needs of cancer patients [4.Alix-Panabières C. Riethdorf S. Pantel K. Circulating tumor cells and bone marrow micrometastasis.Clin Cancer Res. 2008; 14: 5013-5021Crossref PubMed Scopus (196) Google Scholar]. A major problem in the evaluation of the treatment of advanced prostate cancer is the lack of strong surrogate markers for disease outcome and clinical benefit. Prostate cancer does not usually lead to radiologically measurable metastatic disease because the bone is typically the site of metastatic spread [5.Berthold D.R. Tannock I.F. Are prostate cancer clinical trial enrollment criteria and endpoints appropriate?.Nat Clin Pract Oncol. 2006; 3: 74-75Crossref PubMed Scopus (1) Google Scholar]. The use of prostate-specific antigen (PSA) is therefore currently widely used as a marker of treatment activity although PSA does not always correlate with decreased tumour growth, overall survival (OS) or quality of life [6.Fitzpatrick J.M. Anderson J. Sternberg C.N. et al.Optimizing treatment for men with advanced prostate cancer: expert recommendations and the multidisciplinary approach.Crit Rev Oncol Hematol. 2008; 68: S9-S22Crossref PubMed Scopus (27) Google Scholar]. Detecting CTCs has a number of potential applications in patients with prostate cancer including the assessment of prognosis, recurrence after definitive local treatment, response to therapy and obtaining a tumour sample to assess target modulation by drugs without invasive procedure in patients with metastases. Different CTC isolation methods are available with their advantages and drawbacks. CTC detection can rely on slide-based methods (immunochemistry or FISH), molecular analysis (RT-PCR) or flow-based cell sorting methods after immunofluorescent identification and immunomagnetic labelling of cells (anti-Ep-CAM and CK against CTC and anti-CD45 against leukocytes) [3.Pantel K. Brakenhoff R.H. Brandt B. Detection, clinical relevance and specific biological properties of disseminating tumour cells.Nat Rev Cancer. 2008; 8: 329-340Crossref PubMed Scopus (953) Google Scholar]. CTCs can also be detected using telomerase activity in the great majority of patients with prostate cancer [7.Fizazi K. Morat L. Chauveinc L. et al.High detection rate of circulating tumor cells in blood of patients with prostate cancer using telomerase activity.Ann Oncol. 2007; 18: 518-521Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar]. CTC enrichment can be achieved with cell positive/negative selection. The CellSearch™ System (Veridex®, Warren, NJ) allows the analysis of CTCs in a standardized, objective manner. CTC enrichment, specific for epithelial cells, is carried out automatically with antibody-coated magnetic beads and fluorescent labelling. The specific antibodies for epithelial CTC enrichment and detection are, respectively, Ep-CAM and Cytokeratin 8/18/19. The leukocyte component is detected with CD45 antibodies. CTCs are defined as nucleated cells lacking CD45 expression and exhibiting Ep-CAM and cytokeratin expressions. Using this system, CTCs are detectable in metastatic carcinomas with a wide range of incidences and frequencies and not in benign diseases [8.Allard W.J. Matera J. Miller M.C. et al.Tumor cells circulate in the peripheral blood of all major carcinomas but not in healthy subjects or patients with nonmalignant diseases.Clin Cancer Res. 2004; 10: 6897-6904Crossref PubMed Scopus (1980) Google Scholar]. An alternative method for CTC enrichment uses filters with pores that retain the large tumour cells but not the smaller blood cells (The Metagenex® system, Paris, France). The iSET (isolation by size of epithelial tumour cell) allows CTC counting and CTC isolation for cytomorphological and molecular characterisation (FISH). This method is independent of CTC epithelial marker expression and allows the enrichment of epithelial and nonepithelial CTCs [9.Vona G. Estepa L. Béroud C. et al.Paterlini-Bréchot P impact of cytomorphological detection of circulating tumor cells in patients with liver cancer.Hepatology. 2004; 39: 792-797Crossref PubMed Scopus (160) Google Scholar]. At the present time, the CellSearch System (Veridex®) is the only method that has obtained approval for monitoring metastatic breast, colorectal and prostate cancers [10.de Bono J.S. Scher H.I. Montgomery R.B. et al.Circulating tumor cells predict survival benefit from treatment in metastatic castration-resistant prostate cancer.Clin Cancer Res. 2008; 14 (1): 6302-6309Crossref PubMed Scopus (1739) Google Scholar]. Recent studies indicated that CTC counting is a promising biomarker predictive for clinical outcome in patients with disseminated [11.Cristofanilli M. Budd G.T. Ellis M.J. et al.Circulating tumor cells, disease progression, and survival in metastatic breast cancer.N Engl J Med. 2004; 351 (Oncol Rep 2008; 10(2): 137–146): 781-791Crossref PubMed Scopus (3567) Google Scholar] and localised [12.Pierga J.Y. Bidard F.C. Mathiot 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; 14: 7004-7010Crossref PubMed Scopus (314) Google Scholar] breast cancer. In the January issue of Annals of Oncology, Olmos et al. [13.Olmos D. Arkenau H.T. Ang J.E. et al.Circulating tumour cell (CTC) counts as intermediate end points in castration-resistant prostate cancer (CRPC): a single-centre experience.Ann Oncol. 2009; 20: 27-33Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar] demonstrate using the CellSearch® System that the CTC count is a strong prognostic factor for OS in patients with advanced castration-resistant prostate cancer (CRPC) at baseline, but also during the clinical course of the disease. Patients with advanced CRPC and a low CTC count enjoy better OS compared to patients with a high CTC count. Moreover, patients with a high CTC count present with aggressive disease and other poor prognostic factors. The CTC count could also be a reliable predictor of treatment efficacy in advanced metastatic cancer. Olmos et al. also showed that the monitoring of the CTC count during systemic therapies could identify patients with a favourable pattern of response, so that CTC counting may be a more specific, more sensitive and earlier predictive factor of response than radiologic evaluation assessed by the RECIST criteria. Besides the quantitative use of the CTC count to predict the prognosis or response to treatment, the collection of CTCs may be used as an elegant technique to sample malignant tissue from an individual for molecular study purposes, provided that the biological features of CTCs are similar to those of metastatic cells. Detecting molecular abnormalities in a subpopulation of CTCs will complete information on tumour biology or tumour sensitivity to treatment. Thus, characterisation of CTCs may lead to a breakthrough in the understanding of the metastatic phenotype and may constitute a surrogate marker of primary or metastatic tumours that ultimately permit a more personalised therapeutic approach. However, to our knowledge, there are still no studies that compare molecular profiles between CTCs, the primary tumour and metastases, and available CTC purification techniques do not yield reliable overall gene expression patterns from CTCs in a large cohort of patients. Although high-throughput gene expression from CTCs is technically challenging, the potential payoff in yielding a more profound understanding of the molecular features of metastatic cancer justifies its implementation. It is commonly admitted that CTCs, at least a subset of them, correspond to one of the mandatory steps of the metastasis spread from the primary tumour [3.Pantel K. Brakenhoff R.H. Brandt B. Detection, clinical relevance and specific biological properties of disseminating tumour cells.Nat Rev Cancer. 2008; 8: 329-340Crossref PubMed Scopus (953) Google Scholar]. This has prompted the search for markers previously used to characterise the primary tumour. However, some molecular discrepancies have been reported between the primary tumour and circulating cells. For example, HER2-positive CTCs from breast cancer correlated significantly with disease-free survival and OS although HER-2 was not expressed in the corresponding primary tumours [14.Wülfing P. Borchard J. Buerger H. et al.HER2-positive circulating tumor cells indicate poor clinical outcome in stage I to III breast cancer patients.Clin Cancer Res. 2006; 12: 1715-1720Crossref PubMed Scopus (228) Google Scholar]. In systems that allow automated detection and enumeration of CTCs like the CellSearch™ System, CTC detection relies on the expression of the epithelial cell adhesion molecule and on the cytokeratin profile of these cells. However, it should be borne in mind that the selection of CTCs by cytokeratin immunostaining may exclude some cells that have undergone intrinsic modifications of their phenotype during the shedding process. For example, the down-regulation of cytokeratin 18 was reported in prostate disseminating tumour cells [15.Weckermann D. Muller P. Wawroschek F. et al.Disseminated cytokeratin positive tumor cells in the bone marrow of patients with prostate cancer: detection and prognostic value.J Urol. 2001; 166: 699-703Crossref PubMed Scopus (70) Google Scholar] and down-regulation of cytokeratin expression compensated by increased vimentin expression has been reported in micrometastatic breast cancer cells [16.Willipinski-Stapelfeldt B. Riethdorf S. Assmann V. et al.Changes in cytoskeletal protein composition indicative of an epithelial-mesenchymal transition in human micrometastatic and primary breast carcinoma cells.Clin Cancer Res. 2005; 11: 8006-8014Crossref PubMed Scopus (251) Google Scholar]. Until now, changes in gene expression associated with the epithelial–mesenchymal transition that can affect CTCs were not taken into account in isolation procedures. Moreover, the molecular bases of the dormancy of CTCs are poorly understood and the relationship between enumerated CTCs and metastasis is still an open question. Comprehensive molecular characterisation of CTCs may certainly provide important insights into the biology of prostate cancer metastasis and may contribute to better identification of patients who need additional targeted therapy. Greater knowledge of prostate cancer biology has led to the isolation of many new and promising targets, and agents targeting these molecules are currently under development in large randomised phase III trials, to improve OS and the quality of life of patients with metastatic CRPC. Evidence that the androgen receptor axis is still active in the setting of CRPC has also been provided [1.Attard G. Sarker D. Reid A. et al.Improving the outcome of patients with castration-resistant prostate cancer through rational drug development.Br J Cancer. 2006; 95: 767-774Crossref PubMed Scopus (69) Google Scholar], and drugs directly or indirectly targeting the androgen receptor such as abiraterone [18.Scher H.I. Beer T.M. Higano C.S. et al.Phase I/II study of MDV3100 in patients (pts) with progressive castration-resistant prostate cancer (CRPC)ASCO.J Clin Oncol. 2008; 26 (Suppl); (Abstr 5006)Crossref Google Scholar], new specific peripheral anti-androgens [19.Fizazi K. Le Maitre A. Hudes G. et al.Meta-analysis of Estramustine in Prostate Cancer (MECaP) Trialists' Collaborative Group. Addition of estramustine to chemotherapy and survival of patients with castration-refractory prostate cancer: a meta-analysis of individual patient data.Lancet Oncol. 2007; 8: 994-1000Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar], and others [20.K Miller K Fizazi BJU Int, in pressGoogle Scholar], are among the most promising new agents in CRPC. In this list and in addition to drugs targeting angiogenesis (bevacizumab, VEGF Trap), are agents that target molecules involved in the onset of bone metastases such as ZD 4054, which targets the endothelin1 receptor A [21.Fizazi K. et al.J Clin Oncol. 2009; Google Scholar], denosumab which targets the Rank ligand [22.K Fizazi. J Clin Oncol in pressGoogle Scholar], and radiopharmaceutical agents [23.de Bono J.S. Attard G. Adjei A. et al.Potential applications for circulating tumor cells expressing the insulin-like growth factor-I receptor.Clin Cancer Res. 2007; 13: 3611-3616Crossref PubMed Scopus (173) Google Scholar]. Analysis of some of these molecular targets in CTCs from patients participating to ongoing phase I, II or III trials is underway or planned and shall help to better identify those patients likely to benefit [23.de Bono J.S. Attard G. Adjei A. et al.Potential applications for circulating tumor cells expressing the insulin-like growth factor-I receptor.Clin Cancer Res. 2007; 13: 3611-3616Crossref PubMed Scopus (173) Google Scholar]. For example, the discovery of recurrent gene fusions in prostate cancers has important clinical and biological implications [24.Kumar-Sinha C. Tomlins S.A. Chinnaiyan A.M. Recurrent gene fusions in prostate cancer.Nat Rev Cancer. 2008; 8: 497-511Crossref PubMed Scopus (561) Google Scholar]. The detection of the translocation of TMPRSS2 to the ERG gene in CTCs could be used as a biomarker in clinical drug development. CTC counting but also molecular analysis of harvested CTCs are likely to accelerate clinical research in CRPC during the next decade by providing researchers and physicians with the tools they were lacking to access bone metastases. This is why the Royal Marsden Hospital team should be congratulated and their report [13.Olmos D. Arkenau H.T. Ang J.E. et al.Circulating tumour cell (CTC) counts as intermediate end points in castration-resistant prostate cancer (CRPC): a single-centre experience.Ann Oncol. 2009; 20: 27-33Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar] will certainly remain a seminal paper in the difficult quest for the ‘bony Grail’ in CRPC. Authors would like to thank Dr Ludovic Lacroix (Translational Research Unit, Institut Gustave Roussy) for helpful discussions and Lorna Saint Ange for editing.