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The quest for the ‘bony Grail’ of detecting circulating tumour cells in patients with prostate cancer
1 Department of Medicine, Institut Gustave Roussy, University Paris XI, Villejuif
2 Prostate Cancer Translational Research Unit, Institut Gustave Roussy, Villejuif, France
* (E-mail: fizazi{at}igr.fr)
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]. 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].
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]. The detection of CTCs in cancer patients yields prognostic information and might help tailor systemic therapies to the individual needs of cancer patients [4].
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]. 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]. 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]. CTCs can also be detected using telomerase activity in the great majority of patients with prostate cancer [7].
CTC enrichment can be achieved with cell positive/negative selection. The CellSearchTM 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]. 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]. 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].
Recent studies indicated that CTC counting is a promising biomarker predictive for clinical outcome in patients with disseminated [11] and localised [12] breast cancer. In the January issue of Annals of Oncology, Olmos et al. [13] 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]. 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]. In systems that allow automated detection and enumeration of CTCs like the CellSearchTM 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] and down-regulation of cytokeratin expression compensated by increased vimentin expression has been reported in micrometastatic breast cancer cells [16]. 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], and drugs directly or indirectly targeting the androgen receptor such as abiraterone [18], new specific peripheral anti-androgens [19], and others [20], 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], denosumab which targets the Rank ligand [22], and radiopharmaceutical agents [23]. 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]. For example, the discovery of recurrent gene fusions in prostate cancers has important clinical and biological implications [24]. 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] will certainly remain a seminal paper in the difficult quest for the ‘bony Grail’ in CRPC.
acknowledgements
Authors would like to thank Dr Ludovic Lacroix (Translational Research Unit, Institut Gustave Roussy) for helpful discussions and Lorna Saint Ange for editing.
references
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