Detection, clinical relevance and specific biological properties of disseminating tumour cells

Carcinomas, solid tumours derived from epithelial tissues, represent the majority of malignancies in the European Union with ~2 million newly diagnosed cases every year1. Carcinomas arise from glandular cells and their progenitors (such as breast and prostate) or epithe-lial cells lining the body compartments (such as lung and colon). Most deaths from this class of tumours are caused by haematogenous spread of cancer cells into distant organs and their subsequent growth to overt metastases2. The classical view is that metastatic spread is a late process in malignant progression, but recent work suggested that dissemination of primary cancer cells to distant sites might be an early event, particularly in breast cancer progression3.

Recent technical developments allow for detection and characterization of tumour cells, in particular the bone marrow (BM) and peripheral blood of cancer patients, at the single-cell level4. Evidence indicates that BM is the common organ to which tumour cells from many types of carcinoma home4,5. It can be speculated that the BM might also form an important reservoir of tumour cells, from which they might re-circulate into other distant organs where better growth conditions may exist, such as liver or lungs. The fact that tumour cells are detectable in the peripheral blood of patients with breast cancer months to years after complete removal of the primary tumour indicates that these cells might re-circulate between metastatic sites6,7. However, BM is accessible to aspiration compared with other organs like lung or liver, and an alternative hypothesis is therefore that the presence of tumour cells in BM could simply reflect the ability of these cells to survive in any distant organ.

The detection and characterization of tumour cells in BM and those circulating in the peripheral blood has there-fore gained considerable attention over recent years4,8,9. Research on the genotype and phenotype of dissemi-nating cancer cells provides new insights into the biology of tumour cell dissemination in cancer patients and will open new avenues for early detection of metastatic spread and its successful treatment.

A variety of nomenclature is used in the literature to describe metastatic cells in blood and BM. In general, minimal residual disease, or minimal residual cancer, is defined as the presence of tumour cells that are not detectable by the current routine diagnostic procedures used for tumour staging in cancer patients after surgical removal of the primary tumour. The tumour cells in the BM are named disseminated tumour cells (DTCs), and those in the peripheral blood, circulating tumour cells (CTCs)5.

The present Review will focus on the technical advance-ments in the detection and characterization of DTCs and CTCs, the use of DTCs and CTCs in cancer staging and real-time monitoring of systemic anticancer thera-pies, and the specific biological properties and molecular characteristics of these cells with a particular emphasis on the relevance of these findings for the development and use of new targeted therapies in oncology.

Technical advancementsThe two main approaches for the detection of DTCs and/or CTCs are immunological assays using monoclonal antibodies directed against histogenic proteins and PCR-based molecular assays exploiting tissue-specific

*Institute of Tumour Biology, Center of Experimental Medicine, University Medical Center Hamburg Eppendorf, Martinistrasse 52, Hamburg, Germany.‡Section of Tumour Biology, Department of Otolaryngology/Head-Neck Surgery, VU University Medical Center, De Boelelaan 1117, 1081 HV Amsterdam, the Netherlands.Correspondence to K.P. e-mail: [email protected]:10.1038/nrc2375Published online 11 April 2008

Detection, clinical relevance and specific biological properties of disseminating tumour cellsKlaus Pantel*, Ruud H. Brakenhoff‡ and Burkhard Brandt*

Abstract | Most cancer deaths are caused by haematogenous metastatic spread and subsequent growth of tumour cells at distant organs. Disseminating tumour cells present in the peripheral blood and bone marrow can now be detected and characterized at the single-cell level. These cells are highly relevant to the study of the biology of early metastatic spread and provide a diagnostic source in patients with overt metastases. Here we review the evidence that disseminating tumour cells have a variety of uses for understanding tumour biology and improving cancer treatment.

nATURE REvIEws | cancer volUME 8 | MAy 2008 | 329

REVIEWS

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mailto:[email protected]

Ferrofluid A suspension of 10 nm colloidal iron particles stabilized by polymers.

PharmacodynamicsThe effects on the biochemistry of the body resulting from treatment with a drug or combination of drugs.

ELISPOTAn antibody-capture-based method for enumerating specific T cells (CD4+ and CD8+) that secrete a particular cytokine (often interferon-γ).

transcripts. Although protocols for the direct analysis of unprocessed samples exist, most approaches require an enrichment of DTCs and/or CTCs before application of the detection technology. Enrichment is usually based on density-gradient centrifugation and immunomag-netic procedures10. These sensitive technologies are able to identify a DTC or CTC at frequencies of 1 per 106–107 nucleated blood or BM cells (FIG. 1).

Immunological approaches. Cytokeratins — cytoskel-etal proteins that are specifically expressed in epithelial cells — have become the standard markers for the detection of DTCs or CTCs in patients with epithelial tumours such as breast, prostate, colon or lung cancer. The workhorse for the field is density-gradient enrich-ment of viable nucleated cells and immunostaining of cytospins. Crucial steps in the procedure are the sampling techniques and the methods used, including the antibodies11–13. one problem with these methods is that the sensitivity might be suboptimal, particularly for detection of CTCs in the blood because these cells are usually present at lower levels. However, the devel-opment of an automated immunomagnetic enrichment and staining system for CTCs12,14,15 (Cellsearch™) has improved the situation. By this approach CTCs are enriched by ferrofluids coupled to antibodies against EpCAM (also known as tumour-associated calcium signal transducer 1 (TACsTD1)), a cell adhesion molecule commonly expressed on normal and malig-nant epithelial cells14,16. Tumour cells are identified by cytokeratin staining using fluorescent antibodies

and non-specific staining of haematopoietic cells is detected by counterstaining with CD45 (common leukocyte antigen, also known as PTPRC) antibodies. Cells detected and isolated by the system have been successfully analysed for mRnA expression and DnA mutations13,17. The system appears to provide clinically useful information on the prognosis of patients with metastatic breast, colon and prostate cancer15,18–20, and has the potential to evaluate CTCs in pharmacodynamic studies testing new targeted therapies21,22.

Most recently, a microfluidic platform (‘CTC chip’) mediating the interaction of target CTCs with antibody EpCAM-coated microposts under precisely controlled laminar flow conditions in whole blood has gained considerable attention9,23. Contrary to reports using other technologies, the CTC chip identified surprisingly high numbers of cytokeratin-positive CTCs in nearly all tested patients with lung, prostate, pancreatic, breast and colon cancer, including those without metastatic disease. surprisingly, patients with localized prostate cancer had more CTCs than patients with overt metastasis. Future studies are required to test whether these cells are viable CTCs with tumour-specific genomic characteristics9.

Although EpCAM-based enrichment methods are frequently used by many groups, they might not be optimal because the amount of EpCAM on tumour cells including DTCs varies widely and depends on the tumour type16,24,25. Therefore, alternative devices have been developed to circumvent this problem26. Ultra-speed automated digital microscopy in a system called fibre-optic array scanning technology (FAsT) applies laser-printing techniques to the rare-cell detection problem. By this method, laser-printing optics have been used to excite 300,000 cells per second, which have been decorated by fluorescent dye-conjugated antibodies27,28. A much simpler approach is based on separation by cell size (membrane microfilter devices)29,30. Considering that size and cell shape of DTCs and CTCs is rather heterogeneous, it is unclear whether this approach will have the potential to increase the sensitivity and reproducibility of DTC and CTC diagnostics.

A completely different antibody-based approach is the EPIsPoT assay, an adaptation of the ELISPOT technology, used to detect proteins released by CTCs and/or DTCs31,32. Using the EPIsPoT method only viable, protein-excreting cells are detected. nevertheless, the clinical utility of all of these new approaches needs to be validated in large-scale studies in cancer patients.

PCR-based assays. PCR methods targeting tissue-specific gene expression initiated a competitive race against immunocytochemistry for the detection of DTCs and CTCs at the beginning of the 1990s33. PCR-based assays are extremely sensitive and are able to detect a single cell in a sample of 2×107 or more white blood cells34. However, a few transcripts cause false-positive signals in non-cancer controls, and only since the introduction of the quantitative real-time PCR (qPCR) methods has this problem been addressed35. In view of the lack of true cancer-specific molecular targets, qPCR

At a glance

• Tumour cell dissemination is an early event in tumorigenesis and is relevant for metastatic progression (in particular for breast cancer). These data have led to the introduction of disseminating tumour cells (DTCs) in international tumour classification systems.

• Bone marrow (BM) is a common homing organ for tumour cells that are derived from various types of epithelial tumours including breast, prostate and colon cancer. Tumour cells may either establish overt metastases in the BM, as is seen for patients with breast or prostate cancer, or re-circulate to other organs, such as liver or lung, where they find better growth conditions, as is evident in patients with colon cancer.

• Significant technical advancements in immunological procedures and quantitative real-time PCR-based assays now allow DTCs to be identified and enumerated at frequencies of 1 per 106–107 nucleated blood or BM cells.

• Sophisticated molecular techniques such as whole-genome analysis or gene expression profiling have been applied to obtain initial information on the molecular characteristics of DTCs. The current data indicate that most DTCs are dormant (non-proliferative) in situ. However, these cells are viable and can proliferate in cell culture in response to appropriate growth factors, such as the stem cell growth factors epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF2).

• DTCs can express cancer stem cell profiles (such as CD44+/CD24– in breast cancer patients) and exhibit stem cell properties such as resistance to chemotherapy and long-term persistence in the BM.

• Identification of therapeutic targets on DTCs and circulating tumour cells (CTCs) and real-time monitoring of CTCs in cancer patients undergoing systemic therapy are the most important future clinical applications. In this context, the ERBB2 proto-oncogene has served as a proof-of-principle target for the monitoring and treatment of DTCs in human breast cancer.

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Nature Reviews | Cancer

Blood vessel

Intravital flow cytometry in miceIn vivo

In vitro

Venous puncture

FAST

Size Density Marker proteins

IMT

MEMS, ISET

EPISPOT

Low-density array

Protein secretion

CTC chip

DNA

FISHICC WGA qPCR

RNA

cDNA

became more or less the state-of-the-art quantitative method, allowing one to determine cut-off values of marker transcript numbers in samples of non-cancer controls, above which transcripts can be considered as tumour cell-derived (BOx 1).

Many reviews have addressed the technical problems associated with reliable qPCR detection and are beyond the scope of this article29,35–37. Moreover, the expression level of all known marker genes varies between tumours from different patients and even among cells of the same tumour. This cancer heterogeneity points to the use of multiple marker mRnAs38–40. Consequently, the discovery of sensitive mRnA markers has been approached using specific differential gene expression screening of primary tumours and normal tissue17,41,42.

In order to enhance the specificity and multiplicity of qPCR assays mRnA was also isolated from cells enriched by easy-to-perform immunomagnetic bead techniques17,43,44. As an alternative multi-marker assay omitting cell enrichment and qPCR, wu et al. developed a sensitive, high-throughput colorimetric membrane-array method using oligonucleotide probes and alkaline phosphatase detection for simultaneous detection of human telomerase reverse transcriptase (TERT), cytokeratin 19 (KRT19), carcinoembryonic antigen (CEA, here also known as CEA-related cell adhesion molecule 7 (CEACAM7)) and mucin 1 (MUC1) cDnA after reverse transcription of total blood RnA45. A small evaluation study on gastrointestinal cancer patients showed promising data45.

Figure 1 |Methodsforcirculatingtumourcell(cTc)enrichment,detectionandcharacterization.In vitro methods for processing CTCs after venous puncture have been established and approved in clinical trials. The enrichment methods are based on cell size (membrane microfilter devices (micro-electro-mechanical system (MEMS); isolation by size of epithelial tumour cells (ISET)), cell density, which is mainly used for disseminated tumour cell (DTC) enrichment from bone marrow (BM), marker protein expression or nucleic acid expression or mutation. Immunomagnetic bead techniques (IMT) using specific antibodies to surface proteins (such as EpCAM) are the most frequently applied and can be carried out in a semi-automated manner. Enriched cells are further characterized by additional immunocytochemistry (ICC) using antibodies for tumour-associated markers or on viable cells for protein secretion by EPISPOT. Nucleic acid analyses are carried out on enriched cells as well as directly from total RNA or mRNA from the blood. Fluorescence in situ hybridization (FISH) is used for gene aberrations and quantitative real-time PCR (qPCR) for mRNA detection of tumour-associated target genes. For exact quantification of gene dosage in a single cell a whole-genome amplification (WGA) can be introduced into the work flow to linearly increase the amount of target DNA. Furthermore, after reverse transcription of total blood RNA, a colorimetric membrane cDNA array method using oligonucleotide probes and alkaline phosphatase for simultaneous detection of the mRNA of a small number of genes have been applied, promising the realization of a future high-throughput analysis for CTC detection. The most recent CTC chip method is a microfluidic platform that targeted CTC by anti-EpCAM-antibodies coated on microposts. Omitting EpCAM-based separation, ultra-speed automated digital microscopy (fibre-optic array scanning technology (FAST)) and laser-printing techniques have been used to excite 300,000 cells per second to detect CTCs that have been decorated by fluorescence dye-conjugated antibodies directly on a slide. In vivo the CTC detection problem was already approached in mice by intravital flow cytometry.

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The search for the best mRnA marker transcripts is still ongoing (TABLE 1). KRT19 has been used by many groups, especially for breast cancer, despite its expression by immune cells. Moreover, the presence of processed pseudogenes in the genome hampers the design of primers that specifically detect only the transcripts in RnA preparations, which are often contaminated with genomic DnA38,39,46,47. Reverse transcriptase (RT)-PCR and qPCR assays targeting the epidermal growth factor receptor (EGFR) or CEA have been used successfully for CTC detection in cancer patients39,40,48,49. And, although new promising markers — such as MGB2 (also known as secretoglobin, family 2A, member 1 (SCGB2A1) for breast cancer; TM4SF3 (also known as tetraspanin 8 (TSPAN8)) and EpCAM for colon cancer; parathyroid hormone-like hormone (PTHLH) and SCCA (also known as SERPINB3 ) for head and neck cancer; and sCCA and surfactant, pulmonary-associated protein B (SFTPB) for lung cancer — have become available, these still need to be validated in large clinical studies. Interestingly, some markers such as EGFR could also provide important information for patients undergoing targeted therapies with new EGFR-blocking drugs.

Cancer staging revisitedThe efforts made in the detection of disseminating tumour cells over the past few decades culminated in the introduction of DTC and CTC detection in international tumour staging systems50,51. In 2007 CTCs and DTCs in BM were cited for the first time in the recommendations of the American society of Clinical oncology (AsCo) on tumour markers52.

Here we will review the current clinical studies on the use of DTC and/or CTC measurements for tumour staging and monitoring of minimal residual disease.

Prognostic implications of DTCs and CTCs. A large number of studies have documented DTCs in BM from patients with most types of epithelial cancers (for reviews, see REFS 5,31). Thus BM has emerged as a com-mon homing organ for disseminating carcinoma cells, independent of the primary tumour site and the pat-tern of overt metastases. various clinical studies have provided evidence for an association between the pres-ence of DTCs detected at the time of tumour resection and post-operative metastatic relapse in patients with cancers of the breast, prostate, lung and gastrointestinal tract (for reviews, see REFS 5,31). However, the clinical utility of DTC analyses as a prognostic indicator is still under debate.

At present, the most solid data exists for patients with primary breast cancer. several large studies have demonstrated that the detection of DTCs in BM is signifi-cantly associated with a poor prognosis (for review, see REF. 53). The pooled results from 12 different European centres and one Us centre, including 4,703 patients in total, revealed that approximately 30% of women with primary breast cancer harbour DTCs in their BM, and the 10 year follow-up of these patients revealed a signifi-cantly decreased overall and disease-free survival when compared with patients without DTCs in their BM54. The presence of DTCs in BM was significantly associated with a higher tumour stage, poorly differentiated tumour cells, the presence of lymph node metastasis and no or


40y = –3.3334x + 24.896R2 = 0.9998

30

CT

c[DNA]

20

0 0.001 0.001 0.01 1 10 100

Threshold line

Box 1 | Improvements using quantitative real-time PCR

Quantitative real-time PCR (qPCR) has improved detection of circulating tumour cells (CTCs) as it allows determination of cut-off values of marker transcript numbers, above which transcripts can be considered as tumour cell-derived. qPCR has further been improved by the development of new primer structures (for example, minor groove binders), and relies on internal probes that specifically hybridize to the amplified sequences. In addition, owing to the continuous measurement of the amplified signal, false-positive results, which could produce an abnormally shaped, non-linear amplification curve, can be easily identified and removed37,124. The figure shows a regression plot of the CT value (y-axis) versus the concentration of a standard sample (x-axis). A serial 10-fold dilution was performed in quintuplicates and the high precision is demonstrated by a low standard deviation. The graph illustrates the broad range of high sensitivity and accuracy of quantification by qPCR. Amplification efficiency calculated from the plot results in an underestimation of the gene dosage of ~7% for 30 PCR cycles (Econst = 10–1/slope = 1.9952). The figure insert shows corresponding real-time amplification curves, showing the change in fluorescence (∆Rn, y-axis) as a function of amplification cycles (x-axis). The horizontal red line indicates the threshold.

Beyond quantification, there remain unanswered biological problems, such as the discovery of the ‘ideal’ endogenous control gene that does not deviate between tumour and normal cells from different individuals125. This problem might be related to the fact that control genes can be upregulated in response to cytokine stimuli. But furthermore this is also the case for CTC marker genes (such as MUC1, EGFR, mammaglobin (also known as secretoglobin 2A2 (SCGB2A2)) or keratin 20 (KRT20)). For example, KRT20 mRNA levels are significantly higher in blood samples from patients with colorectal cancer than in those from healthy volunteers, whereas no difference could be detected between patients with colorectal cancer and chronic inflammatory disease26,127.

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Table 1 | Targets for DTC and/or CTC detection applied in recent studies

Markerandfunction Source enrichment Detectionmethod relevance refsSurvival markersM30, apoptosis-associated KRT18 fragment, generated by caspase

Bone marrow

Density gradient centrifugation

Immunocytochemistry Indicated therapy response in neoadjuvant therapy of advanced breast cancer

90

Survivin, apoptosis inhibitor

Peripheral blood

No enrichment qPCR Survivin-positive CTCs in patients with breast cancer; association with advanced pathological and clinical disease parameters; similar results for gastrointestinal cancers

67,86

Telomerase, telomere extension, inhibition of senescence

Peripheral blood

No enrichment Telomerase PCR enzyme-linked immunosorbent assay

Telomerase-positive CTCs in patients with advanced prostate cancer with undetectable serum PSA and patients with localized prostate cancer before radical prostatectomy

85

Stem cell-associated markersKRT19, potential stem cell marker

Peripheral blood

No enrichment qPCR, ELISPOT and RT-PCR Therapy monitoring of advanced NSCLC and breast cancer Early breast cancer

38,46

47BMI1, PcG of proto-oncogenes, gene regulation at chromatin level

Peripheral blood

No enrichment qPCR Patients with advanced breast cancer; correlation with positive p53 immunostaining and negative progesterone receptors as well as disease-free and overall survival in small subgroups (advanced stages)

105

EpCAM Peripheral blood

Immunomagnetic enrichment

Immunocytochemistry applied in semi-automated CTC detection system

Detection of CTCs for metastatic breast, colon, prostate cancer association with prognosis

13,14, 18,19,

23FGF2; KRT19+/MUC1–, stem cell marker profile

Peripheral blood; bone marrow

Immunomagnetic (EpCAM) enrichment; bone marrow, density gradient centrifugation enrichment

EPISPOT Detection of viable and stem cell protein-secreting DTCs in prostate and breast cancer

32

TWIST1 (basic helix–loop–helix transcription factor; implicated in cell lineage determination and differentiation)

Bone marrow

Immunomagnetic (EpCAM) enrichment

Expression microarray, qPCR TWIST1 expression associated with 1 year disease-free survival of patients with advanced breast cancer under neoadjuvant chemotherapy

129

PTEN, BRCA1, microsatellite instability

Peripheral blood

Density gradient, immunomagnetic cell enrichment

Immunocytochemistry, microsatellite PCR

PTEN deletions frequently observed in CTCs from patients with prostate cancer; BRCA1 associated with rapid biochemical recurrence

83

Therapeutic target markersERBB2 (oncogenic growth factor receptor)

Peripheral blood

Density gradient and immunomagnetic enrichment

Immunocytochemistry, PCR, FISH

Detection of ERBB2-positive CTCs is associated with early onset of metastasis in breast cancer; detection of ERBB2-positive CTCs for ERBB2-negative breast cancer; first evidence as a monitoring parameter under adjuvant therapy

90,93, 130

EGFR Peripheral blood; bone marrow

No enrichment or immunomagnetic (EpCAM) enrichment; qPCR, density gradient enrichment

Immunocytochemistry, FISH Prognostic relevant in patients with prostate cancer and castration-resistant prostate cancer, detected on DTCs; specific EGFR mRNA detection in breast, HNSCC and lung cancer

5,13, 39

IGFR1 Peripheral blood

Immunomagnetic (EpCAM) enrichment

Immunocytochemistry Monitoring of anti-IGFR therapy in hormone-refractory prostate cancer

21

Multimarker setsTERT, KRT19, KRT20, CEA

Peripheral blood

No enrichment RT-PCR Marker set positive in post-operative colorectal cancer patients with normal perioperative serum CEA levels Similar results for oesophageal cancer

40,48

102TERT, KRT19, CEA, MUC1

Peripheral blood

No enrichment Colorimetric membrane-array method using oligonucleotide probes and alkaline phosphatase detection

Gastric cancer patients and healthy individuals; four-marker set reached diagnostic accuracy of ~90%; independent predictor for post-operative recurrence or metastasis

45

CEA, carcinoembryonic antigen; CTC, circulating tumour cell; DTC, disseminated tumour cell; EGFR, epidermal growth factor receptor; EpCAM, epithelial cell adhesion molecule; FGF2, fibroblast growth factor 2; FISH, fluorescence in situ hybridization; HNSCC, head and neck squamous cell carcinoma; IGFR1, insulin-like growth factor 1; KRT, keratin; MUC1, mucin 1; NSCLC, non-small-cell lung cancer; PcG, polycomb group member; PSA, prostate-specific antigen; PTEN, phosphatase and tensin homologue; qPCR, quantitative real-time PCR; RT-PCR, reverse transcriptase PCR; TERT, telomerase reverse transcriptase.

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Iliac crestThe outer rim of the pelvic bone, accessed for needle aspiration of bone marrow.

low hormone receptor expression. Prognostic relevance was shown for all subgroups, even among those patients with small tumours and without lymph node metastases. Interestingly, the detection of DTCs in BM was not only correlated to the appearance of bone metastases but also to the occurrence of overt metastasis to other secondary organs, such as liver, lung or brain54. Although using different antibodies and detection methods, almost all investigators participating in this pooled analysis used anti-cytokeratin antibodies to screen for DTCs in the BM. As different antibodies and staining techniques can result in variations in the test parameters55,56, several interna-tional organizations have recognized the need for stand-ardization of the immunocytochemical assay11,57 (see also DIsMAl project URl in Further information).

Although aspiration of BM is a routine diagnostic proce-dure in the clinical management of patients with haema-tological malignancies, it is invasive, time-consuming, uncomfortable for the patients and difficult to stand-ardize with regard to the sample quality. Best practice is to draw bilateral samples of 3–4 ml from the iliac crest to prevent mixing with blood, and to monitor the presence of megakaryocytes in the cytospins as an intrinsic control for quality measurement. A major limitation is that BM aspiration is not easy to perform during control visits at outpatient centres, which hampers repeated analyses. Consequently, recent efforts have concentrated on the detection of CTCs in the peripheral blood of cancer patients. At present, there are only a limited number of studies comparing BM and peripheral blood examina-tions performed at the same time points7,58,59, and the clinical significance of CTCs in the peripheral blood is less clear than that for DTCs in BM. In all studies published thus far, there was a higher frequency of BM-positive than blood-positive samples from the same patients7,58,60, probably owing to the fact that BM might provide condi-tions for homing and survival of DTCs, thus contributing to their accumulation in this compartment, whereas blood analyses allow only a snapshot of tumour cell dissemina-tion. At present, the German sUCCEss trial (see URl in Further information) is the largest study (performed with the CellsearchTM system) to evaluate the prognostic relevance of CTCs in breast cancer patients without overt metastases61. when mature, the results of this study will contribute significantly to the guidelines on CTC detection in early-stage breast cancers.

The prognostic relevance of CTCs in the blood of patients with early-stage disease without overt metastasis is still under investigation, with encouraging results from smaller single-centre studies8,38,62,63. A recent study indicates that CTC detection predicts the prognosis in clinically relevant subgroups of early-stage breast cancer patients64. nevertheless, the few studies in which both compartments were assessed in the same breast cancer patients showed that the detection of DTCs in BM had superior prognostic significance over CTC measure-ments in the blood59,60. However, these comparisons were performed with suboptimal CTC detection methods and future studies using the improved detection technologies discussed above might help to clarify this important issue. Moreover, in other tumour entities such as gastrointestinal

cancer, a disease in which overt BM metastases are rare, CTC analyses have generated prognostic information and might therefore become helpful indicators of early systemic tumour cell spread to other distant organs such as lung or liver65,66.

Besides ‘natural’ dissemination, the role of surgical manipulation of the primary tumour and tumour cell dissemination and metastasis has been under debate for years. Using sensitive assays, evidence emerged that surgery might induce dissemination of CTCs and contribute to the development of metastasis in gastroin-testinal cancers45,49,67. Moreover, mathematical analyses of relapse patterns suggest that surgery might also inter-rupt dormancy of DTCs in breast cancer patients and might have a role in the spread of tumour cells68.

Monitoring of minimal residual disease. Besides tumour staging at the time of diagnosis, there is an urgent need for biomarkers for real-time monitoring of the efficacy of systemic adjuvant therapy in individual patients, analo-gous to the use of the blood glucose test for directing insulin in the treatment of diabetes. At present, the success or failure of anticancer therapies is only assessed retrospectively by the absence or presence of overt metastases during the post-operative follow-up period. However, overt metastatic disease is incurable by any of the current therapies. Monitoring of BM and periph-eral blood during and after systemic adjuvant therapy for DTCs and CTCs might provide unique information for the clinical management of the individual cancer patient, and allow an early change in therapy years before the appearance of overt metastasis signals incurability. The identification of patients at increased risk for recur-rence after completion of standard adjuvant chemothera-pies is therefore an application of high clinical relevance, as these patients might benefit from an additional second-line treatment with new drugs. Detection of these cells combined with biological characterization might be of tremendous value for the treatment choice of advanced-stage patients.

several studies have indicated that the presence of DTCs in BM after adjuvant therapy in breast cancer patients predicts a poor prognosis69–72. Braun et al. first reported that the presence of DTCs after taxane- or anthracycline-containing chemotherapy was associ-ated with an extremely poor prognosis and pointed to a heterogeneous response to treatment71. A recent European pooled analysis involving 696 patients from three large European academic breast cancer centres confirmed these initial findings73.

sequential peripheral blood analyses should be more acceptable than repeated BM aspirations and many research groups are currently assessing the clinical value of CTC analyses for therapy monitoring in clinical studies. In metastatic breast cancer patients, the detection of CTCs has provided significant prognostic information15,18, and seems to be superior to conventional imaging methods for response evaluation74. The clinical utility of these findings are now being prospectively addressed in a randomized trial, swoG s0500, led by the southwest oncology group (see URl in Further information). This

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AnoikisApoptosis induced in isolated cells leaving an epithelial tissue.

trial on patients with metastatic breast cancer is aimed at determining whether patients with increased levels of CTCs after 3 weeks of first-line chemotherapy show an improved overall survival and progression-free survival when changing to an alternative chemotherapy regimen at the next treatment course rather than waiting for clinical evidence of progressive disease.

The value of CTC measurements in patients with early-stage breast cancer (without overt metastases) is also under active investigation. In the GEPARQuattro trial (see URl in Further information), which investi-gates the efficacy of primary systemic chemotherapy (+/– trastuzumab), CTCs were detected in 22% of patients before primary systemic chemotherapy, and this rate decreased to 14% after chemotherapy75. In the sUCCEss trial (see URl in Further information) 1,767 patients have been recruited and CTCs were evident in 10% of the patients before adjuvant chemotherapy and in 7% after completion of therapy61. The ongoing clinical follow-ups of these trials will show whether the observed decreases in CTC rates will be associated with an improved survival rate of the cancer patients.

Together, the present data strongly support the view that DTCs and CTCs are relevant for metastatic progres-sion, can survive current chemotherapy, might indicate failure of therapeutic interventions potentially allowing a switch in treatment modality, and provide a diagnostic source of the lesions in metastatic cancer patients and the biological characteristics of micrometastatic cells in patients with early-stage cancer.

The biological characteristics of DTCs and CTCswhen this field of research began, and the first indica-tions of cytokeratin-positive cells in the BM were reported, a debate on the actual biological properties of these cells emerged. Crucial questions arose that were answered in subsequent years.

Haematopoietic or epithelial origin? The first question that arose was whether these cells were of haematopoi-etic or epithelial origin. Although haematopoietic cells can be a source of false-positive scores (as discussed above), most cytokeratin-positive cells in BM and blood samples are of epithelial origin. In general, cytokeratins are highly epithelial-specific histological markers and in previous reports using aspirates of 191 non-cancer controls that were stained for cytokeratin expression, only two samples of positive cells were seen76.

Are cytokeratin-positive cells tumour cells? This question was answered when the techniques to identify genetic changes of single cells became apparent. Using whole genome amplification and comparative genomic hybridi-zation the genomes of a single DTC and CTC could be explored77–80. All cytokeratin-positive cells seemed to show genetic changes, clearly indicating that the cells are genetically abnormal. However, in patients with early-stage breast cancer with no evidence of overt metastasis, the genetic patterns of different DTCs from single patients were heterogeneous81. This is in contrast to the cells isolated from late-stage patients with overt metastases,

these DTCs were genetically homogeneous. surprisingly, DTCs from patients with early-stage breast cancer did not usually contain the same genetic changes as the primary tumour80. Hence, the answer to this question is not so clear. without doubt we can state that these cells are genetically abnormal and invasive. whether they all have the (genetic) properties for extravasation and outgrowth to a new metastatic tumour is questionable.

on the basis of these genetic data new metastatic models emerged. It was hypothesized that DTCs in patients with breast cancer leave the primary tumour early during its development and that the subsequent genomic changes leading to overt metastasis might be independent from the changes important for primary tumour growth3,5,82 (FIG. 2). Recent data suggest that in other adenocarcinoma tumour types additional models might also exist. In multifocal and highly heterogeneous prostate cancers genetic aberrations of CTCs in early-stage patients are identical to those in distinct, even small areas of the primary tumour83, which suggests that a metastatic subclone already exists in the primary tumour84. Hence, the genetic information of DTCs and CTCs has led to new insight. In addition to molecular data obtained from DTCs and CTCs, profiling of primary tumours in terms of the presence or absence of DTCs and/or CTCs might also reveal relevant information for early tumour cell dissemination, and this is one of the main goals of the European DIsMAl consortium (see URl in Further information) (FIG. 3).

Are DTCs and CTCs viable? A third question dealt with whether these cells were dead or alive. Dispersed epithelial cells that are not encased in a tissue context will quickly undergo apoptosis owing to a process called anoikis. There is some indirect evidence that some CTCs may have an increased resistance to this physiological process through, for example, the expression of telom-erase85 and survivin67,86. Using EPIsPoT, it was shown that viable DTCs were detected in BM in more than 50% of patients with breast cancer 31,32, which is consistent with the fact that the BM is the predominant site of metastatic outgrowth in breast cancer. The viability of DTCs from patients with breast cancer or other epithelial tumours is also supported by the fact that these cells — isolated from a substantial fraction of patients — can be cultured in vitro87,88. By contrast, a substantial number of CTCs show apoptotic markers, indicating that CTCs are more prone to apoptosis and cell death than the DTCs found in the BM89–91. This view is supported by the notion that RnA from whole blood of cancer patients still contains the remnants of apoptotic cells, which can even be used as a biomarker.

Using various assays it has been shown that the blood of prostate cancer patients frequently harbours viable CTCs but whether such cells can survive long-term in culture is unknown32.

Proliferating or quiescent? Although there are many indications that at least a proportion of DTCs and CTCs are alive, a fourth question emerged regarding whether DTCs are proliferating or quiescent (dormant).

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http://www.cancer.gov/Templates/drugdictionary.aspx?CdrID=42265


Early dissemination

Early-stage cancer

Develop overt metastasis in parallel

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DTCs

Overt metastasis

No metastasis

DTCs

Genetic progression

DTCs

a b

The most important answers came from in vitro analyses. solakoglu et al. showed that, when cultured, these cells are able to proliferate with appropriate stimuli but have only limited proliferative capacity87. As in vitro culturing is complex and might cause bias and induce artefacts, many researchers also used co-immunostaining with

specific markers associated with proliferation5. These data confirmed the in vitro observations: most DTCs and also CTCs7,91 are Ki-67-negative, and seem to be non- or slow-proliferating cells.

The molecular nature of DTCs and CTCs. A fifth question addressed the molecular nature of these cells, and the relevance of their molecular make-up for prognosis. various studies revealed a striking inter-patient variability of DTCs with regard to the expres-sion of growth factor receptors, proteases, adhesion molecules and major histocompatibility complex antigens5,31. In particular, the ERBB2 (also known as HER2) proto-oncogene appears to define an aggressive subset of DTC and CTC that is associated with poor prognosis for patients with breast cancer92–94. Moreover, expression of the urokinase-type plasminogen activator receptor (uPAR, also known as PlAUR) on DTCs is correlated to metastatic relapse in gastric cancer95, and genes encoding ERBB2 and uPA (PlAU) are co-amplified in breast DTCs96. Thus, signalling mediated by ERBB2 and uPAR might be important for the transi-tion of DTCs from a dormant stage to an active growth phase, and future strategies aimed at inducing and/or maintaining tumour cell dormancy might include concomitant inhibition of these receptors97.

An intriguing issue related to the apparent dormant, non-proliferating nature of the DTCs is the trigger that might cause this peculiar state and, more importantly from the perspective of treatment, the trigger to push the tumour cells back into proliferation. As discussed above, particular signalling pathways might have a role, but recently an observation by Koebel et al. pointed to the immune system as a key factor98. In mouse models they convincingly showed that depletion of CD4+ and CD8+ cells and inhibition of interferon-γ initiated progres-sive growth in previously dormant tumours. Hence, the immune system might keep these tumours in a dormant state, although the relevance of immunosurveillance in controlling dormant metastatic cells in cancer patients is still unclear97. It should also be mentioned that in the mouse models dormancy was defined as a balance between cell proliferation and cell loss. This might not reflect the situation in cancer patients because most DTCs appear to be quiescent (that is, Ki67-negative) in situ, as stated above. Dormancy can be defined as either slow-growing tumours that appear after a long period of time; tumours that do not grow at all; or tumours that are in proliferative and apoptotic equilibrium. The mecha-nisms controlling tumour dormancy have been recently reviewed in detail97.

Cancer stem cells? Recent research has prompted a sixth question — that of the relationship of DTCs and CTCs to cancer stem cells and metastasis99,100,. The cancer stem cell concept hypothesizes that tumours arise from a small subpopulation of stem cells or progenitor cells. In the resulting tumour only a small fraction of the cells retain such stem cell-like properties and are capable of forming new tumours, whereas the large majority of cells in a tumour lose these characteristics after differentiation.

Figure 2 |ametastaticmodelderivedfromdisseminatedtumourcell(DTc)andcirculatingtumourcellstudiesinhumanpatients.In this model the concepts of the metastatic stem cell and the parallel metastatic progression have been integrated. a | Metastatic stem cell model. The cancer stem cell hypothesis indicates that the epithelial stem cells are the primary source of cancer formation. Tissues, such as mucosal linings128 or breast tissue, are hierarchically organized and consist of actively dividing and differentiating cells that form the large bulk of the tissue, as well as slowly cycling stem cells that have self-renewal capacity and form the primary source of the cells. This hierarchy is proposed to be retained on malignant progression, and results in a tumour with a small fraction of cancer stem cells (in orange), whereas the majority of cells have no stem cell phenotype (in green). Only the cancer stem cells have the capacity for self-renewal and form overt metastases on dissemination, whereas DTCs without stem cell properties have only a limited proliferative capacity. b | Parallel metastatic progression model. Besides the concept of the metastatic stem cells, the parallel metastatic progression model also needs to be considered, as genetic data from DTCs in early-stage cancers indicate that metastatic cells can exist in an early-stage tumour. Dissemination of early-stage tumours might lead to DTCs that progress independently from the primary tumour (in blue), forming overt metastases comprising tumour cells that are genetically different from the primary tumour cells (in green). These models seem to be in contradiction, but in fact are complementary.

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Breast, colon or head and neck cancerN0, M0, matched for age, T stage, grading

DTC- and/or CTC-positive

Microdissection

CGH or expression array

TMA

Bioinformatics

DNA

DNA copy number profile

Hierarchical cluster

RNA

Tissue section Tumour area

RNA

DNA

a

b

c

d

Immuno-histochemistry

Fluorescence in situhybridizationDNA

Protein

DTC- and/or CTC-negative

These cancer stem cell populations appear to be relevant for clinical outcome after treatment100. Consequently, it has been assumed that the founder cells of overt metastases might also be stem cells derived from the primary tumour (FIG. 2). Consistent with this hypothesis was the observation that DTCs in breast cancer patients frequently displayed a cancer stem cell marker phenotype (CD44+/CD24–/low)32,101. The link between metastasis and breast cancer stem cells

is further supported by the observation that stem cells enriched from the primary tumour using the CD44+/CD24–/low markers show an expression profile compared with primary breast cancer cells that is strongly associ-ated with metastatic relapse in breast cancer patients102. nevertheless, CD44 is a somewhat outdated stem cell marker103 and new ones such as aldehyde dehydroge-nase 1 (AlDH1, also known as AlDH1A1)104 or BMI1105 are now available. However, there are additional similari-ties between the properties of DTCs in BM and cancer stem cells, suggesting that the founder cells of overt metas-tases (‘metastatic stem cells’) might reside in the DTC population. For example, most CTCs or DTCs are non- proliferating and resistant to chemotherapy7,38,69,71, as has been shown for cancer stem cells. Moreover, many DTCs proliferate in vitro in response to epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF2)87,88 — two growth factors associated with stem cells. However, it is still unclear whether DTCs have self-renewal ability, the hallmark of cancer stem cells.

Stromal factors. Finally, in the context of metastatic progression the role of the stroma should be taken into consideration. An interesting observation was reported in mouse models, indicating that BM-derived haemato-poietic progenitor cells form a pre-metastatic niche at the sites of metastasis formation before the tumour cells arrive106. This suggests an intriguing interaction of tumour cells with the host and creation of their own micro-environment, and might explain the frequent homing of tumour cells to the BM that we observe in patients. Evidence that stromal elements have a crucial role in the formation of overt tumours is continuously increasing. There are various reports that the stromal cells frequently display genetic changes107,108. Although these data are mostly collected by one group from formalin-fixed paraffin-embedded material that might give rise to genetic artefacts, the number of reports in different tumour types is remarkable and deserves further attention. Genetic data on stromal cells in metastatic sites has not been published; however, it would be interesting to learn whether there is a link between the genetic changes in the stroma in primary tumours and corresponding metastasis.

Fighting minimal residual diseaseThe use of targeted therapies in addition to chemo-therapy and radiotherapy has started a new era in clinical oncology. The ERBB2 proto-oncogene is currently the most predominant biological target for systemic therapy with remarkable results of clinical trials using a humanized monoclonal antibody (trastuzumab) in breast cancer109. At present, the ERBB2 immunohistochemical score of breast carcinomas is used to guide therapy decisions for the application of humanized anti-ERBB2 monoclonal antibodies, leading to a significantly improved disease-free and overall survival110. However, to date, determina-tion of the ERBB2 score by tissue testing is a one-time event, and there are still difficulties with ERBB2 status determination on the primary tumour111. The detection of ERBB2-positive CTCs might serve to enable a real-time assessment of the ERBB2 status during the clinical

Figure 3 |Searchformoleculardeterminantsofearlytumourcelldissemination.Besides the direct analysis of disseminated tumour cells (DTCs) and/or circulating tumour cells (CTCs), the genetic profiling of primary tumours in relation to the presence or absence of DTCs or CTCs might provide unique information on putative molecular determinants of micrometastases in cancer patients. a | Early-stage cancer patients without lymph node metastasis (stage N0) and with no signs of overt metastasis (stage M0) are selected, and both groups (positive or negative for DTCs and/or CTCs) are matched for all other relevant parameters, such as age, tumour stage or differentiation grade. b | The best results are obtained from analysis of fresh frozen tumour tissue. To avoid contamination with normal tissue present in all tumours, areas containing tumour cells are laser-microdissected and the DNA and RNA from these areas are isolated. c | RNA is hybridized to microchips containing probes representing the entire pattern of expressed human genes. The extracted DNA is analysed by comparative genomic hybridization (CGH) using microarrays that cover the whole genome. The complex patterns obtained by these microchip experiments require a sophisticated bioinformatics analysis to reveal those signatures significantly associated with the presence of DTCs and/or CTCs. d | A further validation of the resulting candidate genes is required and can be performed rapidly on tissue microarrays (TMAs) containing hundreds of tumour samples from an independent cohort of cancer patients with known DTC and/or CTC status.

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course of disease. The clinical study AlTTo (see URl in Further information) will assess the question of whether anti-ERBB2 therapy can be improved by new agents and regimens with ancillary studies on CTC detection.

Furthermore, a striking discrepancy has been observed between the detection of ERRB2-positive DTCs and CTCs with the ERBB2 score of the corresponding primary tumour. ERBB2-positive DTCs and CTCs can also be detected in patients with ERBB2-negative primary tumours93,94,112, suggesting that additional patients could benefit from ERBB2-directed therapies. ongoing clinical studies will reveal whether the ERBB2 status of DTCs or CTCs could predict response to trastuzumab or other ERBB2-directed therapies.

Recent reports support the possibility that ERBB2 gene amplification can be acquired during cancer progression94. Moreover, it could be assumed that only a few ERBB2-overexpressing cancer cells have the potential to disseminate, subsequently leading to metastases and death. This is in line with previous results from experimental studies113–115. In addition, the upregulation of the chemokine receptor CXCR4 — recently shown to mediate cancer cell motility, particularly to BM — is essential for ERBB2-dependent cancer metastasis116. This connection between ERBB2 and CXCR4 signalling might explain the high detection rate of ERBB2-positive DTCs in BM92 and peripheral blood93,117.

several studies have examined ERBB2-positive CTCs in small numbers of patients treated with ERRB2-targeted therapies and showed that it is possible to monitor ERBB2 in this way. Thus, determination of the ERBB2 status on CTCs might become a relevant tool for both the risk assessment and stratification of patients to ERBB2-directed therapies, and also the identification of the actual therapeutic target, with important consequences for a more individualized therapy against minimal residual disease. This strategy, together with real-time

monitoring of CTCs (and/or DTCs), should also provide new insights into how the tumour cell population changes when subjected to specific therapies, and might be a new way of assessing other new targeted therapies69–71,118,119.

Conclusions and future directionsThe detection of DTCs and CTCs has been shown to be of clinical relevance in many tumour types, and partic-ularly in breast cancer. The significance of these data from meta-analysis led to the detection of DTCs being introduced into the TnM system. The workhorse for DTC detection is immunostaining of density-gradient-enriched BM aspirates, which is still of more prognostic value than the detection of CTCs in the blood of early-stage cancer patients. new platforms might open new avenues for CTC detection. Despite the proven clinical relevance of DTC detection in early-stage breast cancer, the clinical utility for the individual early-stage patient remains limited and improved platforms using additional detection methods or markers are still neces-sary. A most promising new diagnostic field has opened for advanced-stage patients: the sensitive CTC detection platforms allow monitoring of disease in advanced-stage patients when the tumour is removed, and the single cell technologies might allow profiling of these cells to adapt treatment regimens. The first clinical trials to determine the clinical utility of CTC detection for these purposes are currently in progress.

For the future, more data should be collected on tumour stem and/or progenitor cells, and the molecules and cellular processes involved in tumour cell dissemina-tion, especially in stromal interaction. Among the cellular processes, the role of metastasis-suppressor genes2,120 and metastasis-associated microRnAs121,122 in micrometas-tases might be interesting subjects of further investi-gation. Furthermore, the emerging role of the genetic background of the host in dissemination and homing of CTCs needs to be taken into consideration123.

1. Boyle, P. & Ferlay, J. Cancer incidence and mortality in Europe, 2004. Ann. Oncol. 16, 481–488 (2005).

2. Eccles, S. A. & Welch, D. R. Metastasis: recent discoveries and novel treatment strategies. Lancet 369, 1742–1757 (2007).

3. Husemann, Y. et al. Systemic spread is an early step in breast cancer. Cancer Cell 13, 58–68 (2008).Here it was reported for the first time that tumour cells can disseminate systemically from earliest epithelial alterations in ERBB2- and PyMT-transgenic mice and from ductal carcinoma in situ in women. A new hypothesis for cancer cell dissemination was proposed, which must be substantiated by further studies.

4. Pantel, K. & Alix-Panabieres, C. The clinical significance of circulating tumor cells. Nature Clin. Pract. Oncol. 4, 62–63 (2007).

5. Pantel, K. & Brakenhoff, R. H. Dissecting the metastatic cascade. Nature Rev. Cancer 4, 448–456 (2004).

6. Meng, S. et al. Circulating tumor cells in patients with breast cancer dormancy. Clin. Cancer Res. 10, 8152–8162 (2004).

7. Muller, V. 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. 11, 3678–3685 (2005).This report demonstrates that CTCs are in a quiescent state (that is, are non-proliferating and

Ki67-negative) and survive chemotherapy in a considerable fraction of treated patients with breast cancer.

8. Cristofanilli, M. & Mendelsohn, J. Circulating tumor cells in breast cancer: Advanced tools for “tailored” therapy? Proc. Natl Acad. Sci. USA 103, 17073–17074 (2006).

9. Uhr, J. W. Cancer diagnostics: one-stop shop. Nature 450, 1168–1169 (2007).

10. Paterlini-Brechot, P. & Benali, N. L. Circulating tumor cells (CTC) detection: clinical impact and future directions. Cancer Lett. 253, 180–204 (2007).

11. Fehm, T. et al. A concept for the standardized detection of disseminated tumor cells in bone marrow from patients with primary breast cancer and its clinical implementation. Cancer 107, 885–892 (2006).A recent concept for the standardization of DTC detection, describing in detail the confounding factors of immunocytochemical BM analyses and the recommended quality assurance procedures

12. Riethdorf, S. et al. Detection of circulating tumor cells in peripheral blood of patients with metastatic breast cancer: a validation study of the CellSearch system. Clin. Cancer Res. 13, 920–928 (2007).

13. Shaffer, D. R. et al. Circulating tumor cell analysis in patients with progressive castration-resistant prostate cancer. Clin. Cancer Res. 13, 2023–2029 (2007).

14. Kagan, M., Howard, D. & Bendele, T. in Tumor Markers: Physiology, Pathobiology, Technology and

Clinical Applications. (eds Diamandis, E., Fritsche, H., Lilja, H., Chan, D. & Schwarz, M.) 495–498 (AACC Press, Washington, DC, 2002).

15. Cristofanilli, M. et al. Circulating tumor cells, disease progression, and survival in metastatic breast cancer. N. Engl. J. Med. 351, 781–791 (2004).A pivotal clinical study that provided the first significant evidence for the clinical relevance of detecting CTCs in breast cancer patients with overt metastases.

16. Litvinov, S. V. et al. Expression of Ep-CAM in cervical squamous epithelia correlates with an increased proliferation and the disappearance of markers for terminal differentiation. Am. J. Pathol. 148, 865–875 (1996).

17. Smirnov, D. A. et al. Global gene expression profiling of circulating tumor cells. Cancer Res. 65, 4993–4997 (2005).

18. Hayes, D. F. et al. Circulating tumor cells at each follow-up time point during therapy of metastatic breast cancer patients predict progression-free and overall survival. Clin. Cancer Res. 12, 4218–4224 (2006).

19. Cohen, S. J. et al. Isolation and characterization of circulating tumor cells in patients with metastatic colorectal cancer. Clin. Colorectal Cancer 6, 125–132 (2006).

20. Moreno, J. G. et al. Circulating tumor cells predict survival in patients with metastatic prostate cancer. Urology 65, 713–718 (2005).

R E V I E W S




21. de Bono, J. S. et al. Potential applications for circulating tumor cells expressing the insulin-like growth factor-I receptor. Clin. Cancer Res. 13, 3611–3616 (2007).The reported data support the further evaluation of CTCs in pharmacodynamic studies and patient selection, particularly in advanced prostate cancer, exemplifying IGFR1 detection of CTCs under anti-IGFR1 therapy.

22. Danila, D. C. et al. Circulating tumor cell number and prognosis in progressive castration-resistant prostate cancer. Clin. Cancer Res. 13, 7053–7058 (2007).

23. Nagrath, S. et al. Isolation of rare circulating tumour cells in cancer patients by microchip technology. Nature 450, 1235–1239 (2007).First report on a microfluidic platform (‘CTC chip’) that identified CTCs in the peripheral blood from 99% of patients with lung, prostate, pancreatic, breast or colon cancer.

24. Braun, S., Hepp, F., Sommer, H. L. & Pantel, K. Tumor-antigen heterogeneity of disseminated breast cancer cells: implications for immunotherapy of minimal residual disease. Int. J. Cancer 84, 1–5 (1999).

25. Thurm, H. et al. Rare expression of epithelial cell adhesion molecule on residual micrometastatic breast cancer cells after adjuvant chemotherapy. Clin. Cancer Res. 9, 2598–2604 (2003).

26. He, W., Wang, H., Hartmann, L. C., Cheng, J. X. & Low, P. S. In vivo quantitation of rare circulating tumor cells by multiphoton intravital flow cytometry. Proc. Natl Acad. Sci. USA 104, 11760–11765 (2007).A method using intravital flow cytometry for non-invasive detection of rare CTCs in vivo as they flow through the peripheral vasculature after intravenous injection of a tumour-specific fluorescent ligand in mice.

27. Hsieh, H. B. et al. High speed detection of circulating tumor cells. Biosens. Bioelectron. 21, 1893–1899 (2006).

28. Krivacic, R. T. et al. A rare-cell detector for cancer. Proc. Natl Acad. Sci. USA 101, 10501–10504 (2004).

29. Zheng, S. et al. Membrane microfilter device for selective capture, electrolysis and genomic analysis of human circulating tumor cells. J. Chromatogr. A 1162, 154–161 (2007).

30. Pinzani, P. et al. Isolation by size of epithelial tumor cells in peripheral blood of patients with breast cancer: correlation with real-time reverse transcriptase-polymerase chain reaction results and feasibility of molecular analysis by laser microdissection. Hum. Pathol. 37, 711–718 (2006).

31. Alix-Panabieres, C., Muller, V. & Pantel, K. Current status in human breast cancer micrometastasis. Curr. Opin. Oncol. 19, 558–563 (2007).

32. Alix-Panabieres, C. et al. Detection and characterization of putative metastatic precursor cells in cancer patients. Clin. Chem 53, 537–539 (2007).Using a novel technology called EPISPOT this paper describes that some CTCs in patients with localized prostate cancer secrete FGF2, a stem cell growth factor.

33. Smith, B. et al. Detection of melanoma cells in peripheral blood by means of reverse transcriptase and polymerase chain reaction. Lancet 338, 1227–1229 (1991).

34. Brakenhoff, R. H. et al. Sensitive detection of squamous cells in bone marrow and blood of head and neck cancer patients by E48 reverse transcriptase-polymerase chain reaction. Clin. Cancer Res. 5, 725–732 (1999).

35. van Houten, V. M. et al. Molecular assays for the diagnosis of minimal residual head-and-neck cancer: methods, reliability, pitfalls, and solutions. Clin. Cancer Res. 6, 3803–3816 (2000).

36. Benoy, I. H. et al. Detection of circulating tumour cells in blood by quantitative real-time RT-PCR: effect of pre-analytical time. Clin. Chem. Lab. Med. 44, 1082–1087 (2006).

37. Nolan, T., Hands, R. E. & Bustin, S. A. Quantification of mRNA using real-time RT-PCR. Nature Protoc. 1, 1559–1582 (2006).

38. Quintela-Fandino, M. et al. Breast cancer-specific mRNA transcripts presence in peripheral blood after adjuvant chemotherapy predicts poor survival among high-risk breast cancer patients treated with high-dose chemotherapy with peripheral blood stem cell support. J. Clin. Oncol. 24, 3611–3618 (2006).

39. Xi, L. et al. Optimal markers for real-time quantitative reverse transcription PCR detection of circulating tumor cells from melanoma, breast, colon, esophageal, head and neck, and lung cancers. Clin. Chem 53, 1206–1215 (2007).

40. Wang, J. Y. et al. Molecular detection of circulating tumor cells in the peripheral blood of patients with colorectal cancer using RT-PCR: significance of the prediction of postoperative metastasis. World J. Surg. 30, 1007–1013 (2006).

41. Martin, K. J. et al. Linking gene expression patterns to therapeutic groups in breast cancer. Cancer Res. 60, 2232–2238 (2000).

42. Bai, V. U. et al. Identification of prostate cancer mRNA markers by averaged differential expression and their detection in biopsies, blood, and urine. Proc. Natl Acad. Sci. USA 104, 2343–2348 (2007).

43. de Cremoux, P. et al. Detection of MUC1-expressing mammary carcinoma cells in the peripheral blood of breast cancer patients by real-time polymerase chain reaction. Clin. Cancer Res. 6, 3117–3122 (2000).

44. Zieglschmid, V. et al. Combination of immunomagnetic enrichment with multiplex RT-PCR analysis for the detection of disseminated tumor cells. Anticancer Res. 25, 1803–1810 (2005).

45. Wu, C. H. et al. Development of a high-throughput membrane-array method for molecular diagnosis of circulating tumor cells in patients with gastric cancers. Int. J. Cancer 119, 373–379 (2006).A sensitive, high-throughput colorimetric membrane-array omitting cell enrichment and qPCR is reported using oligonucleotide probes and alkaline phosphatase detection for simultaneous detection of CTC target genes.

46. Chen, T. F. et al. CK19 mRNA expression measured by reverse-transcription polymerase chain reaction (RT-PCR) in the peripheral blood of patients with non-small cell lung cancer treated by chemo-radiation: an independent prognostic factor. Lung Cancer 56, 105–114 (2007).

47. Xenidis, N. et al. Clinical relevance of circulating CK-19 mRNA-positive cells detected during the adjuvant tamoxifen treatment in patients with early breast cancer. Ann. Oncol. 18, 1623–1631 (2007).

48. Wang, J. Y. et al. Multiple molecular markers as predictors of colorectal cancer in patients with normal perioperative serum carcinoembryonic antigen levels. Clin. Cancer Res. 13, 2406–2413 (2007).

49. Liu, Z., Jiang, M., Zhao, J. & Ju, H. Circulating tumor cells in perioperative esophageal cancer patients: quantitative assay system and potential clinical utility. Clin. Cancer Res. 13, 2992–2997 (2007).

50. Hermanek, P., Sobin, L. H. & Wittekind, C. How to improve the present TNM staging system. Cancer 86, 2189–2191 (1999).

51. Singletary, S. E., Greene, F. L. & Sobin, L. H. Classification of isolated tumor cells: clarification of the 6th edition of the American Joint Committee on Cancer Staging Manual. Cancer 98, 2740–2741 (2003).

52. Harris, L. et al. American Society of Clinical Oncology 2007 update of recommendations for the use of tumor markers in breast cancer. J. Clin. Oncol. 25, 5287–5312 (2007).

53. Slade, M. J. & Coombes, R. C. The clinical significance of disseminated tumor cells in breast cancer. Nature Clin. Pract. Oncol. 4, 30–41 (2007).

54. Braun, S. et al. A pooled analysis of bone marrow micrometastasis in breast cancer. N. Engl. J. Med. 353, 793–802 (2005).Pooled analysis of data from 12 European centres and one US centre, comprising 4,703 patients with primary breast cancer (that is, no signs of overt metastases), showing that the ~30% of women with DTCs in their BM have an unfavourable clinical outcome.

55. Borgen, E. et al. Immunocytochemical detection of isolated epithelial cells in bone marrow: non-specific staining and contribution by plasma cells directly reactive to alkaline phosphatase. J. Pathol. 185, 427–434 (1998).

56. Braun, S. & Pantel, K. Biological characteristics of micrometastatic cancer cells in bone marrow. Cancer Metastasis Rev. 18, 75–90 (1999).

57. Borgen, E., Naume, B. & Nesland, J. M. Standardization of the immunocytochemical detection of cancer cells in BM and blood: I. Establishment of objective criteria of the evaluation of immunostained cells. Cytotherapy 1, 377–388 (1999).

58. Pierga, J. Y. 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. 10, 1392–1400 (2004).

59. Wiedswang, G. et al. Comparison of the clinical significance of occult tumor cells in blood and bone

marrow in breast cancer. Int. J. Cancer 118, 2013–2019 (2006).

60. Benoy, I. H. et al. Real-time RT-PCR detection of disseminated tumour cells in bone marrow has superior prognostic significance in comparison with circulating tumour cells in patients with breast cancer. Br J. Cancer 94, 672–680 (2006).

61. Rack, B. K. et al. Circulating tumor cells (CTCs) in the peripheral blood of primary breast cancer patients. J. Clin. Oncol. 25, Abstract 10595 (2007).

62. Ntoulia, M. et al. Detection of Mammaglobin A- mRNA-positive circulating tumor cells in peripheral blood of patients with operable breast cancer with nested RT-PCR. Clin. Biochem 39, 879–887 (2006).

63. Xenidis, N. et al. Predictive and prognostic value of peripheral blood cytokeratin-19 mRNA-positive cells detected by real-time polymerase chain reaction in node-negative breast cancer patients. J. Clin. Oncol. 24, 3756–3762 (2006).

64. Ignatiadis, M. et al. Different prognostic value of cytokeratin-19 mRNA positive circulating tumor cells according to estrogen receptor and HER2 status in early-stage breast cancer. J. Clin. Oncol. 25, 5194–5202 (2007).This clinical study shows for the first time that CTCs detected by a sensitive qPCR assay are of prognostic value in particular subgroups of early-stage breast cancer patients who carry tumours with a high-risk molecular profile.

65. Wolfrum, F., Vogel, I., Fandrich, F. & Kalthoff, H. Detection and clinical implications of minimal residual disease in gastro-intestinal cancer. Langenbecks Arch. Surg. 390, 430–441 (2005).

66. Soeth, E. et al. Detection of tumor cell dissemination in pancreatic ductal carcinoma patients by CK 20 RT-PCR indicates poor survival. J. Cancer Res. Clin. Oncol. 131, 669–676 (2005).

67. Hoffmann, A. C. et al. Survivin mRNA in peripheral blood is frequently detected and significantly decreased following resection of gastrointestinal cancers. J. Surg Oncol. 95, 51–54 (2007).

68. Demicheli, R., Retsky, M. W., Hrushesky, W. J. & Baum, M. Tumor dormancy and surgery-driven interruption of dormancy in breast cancer: learning from failures. Nature Clin. Pract. Oncol. 4, 699–710 (2007).

69. Wiedswang, G. et al. Isolated tumor cells in bone marrow three years after diagnosis in disease-free breast cancer patients predict unfavorable clinical outcome. Clin. Cancer Res. 10, 5342–5348 (2004).This clinical study demonstrates that DTCs in BM survive chemotherapy and persist for at least 3 years after surgical removal of the primary tumour. The presence of these dormant DTCs was associated with an increased risk of metastatic relapse.

70. Janni, W. et al. The persistence of isolated tumor cells in bone marrow from patients with breast carcinoma predicts an increased risk for recurrence. Cancer 103, 884–891 (2005).

71. Braun, S. et al. Lack of effect of adjuvant chemotherapy on the elimination of single dormant tumor cells in bone marrow of high-risk breast cancer patients. J. Clin. Oncol. 18, 80–86 (2000).

72. Slade, M. J. et al. Persistence of bone marrow micrometastases in patients receiving adjuvant therapy for breast cancer: results at 4 years. Int. J. Cancer 114, 94–100 (2005).

73. Janni, W. J. et al. Persistence of disseminated tumor cells in bone marrow of breast cancer patients predicts increased risk for relapse- results of pooled European data. J. Clin. Oncol. 24, Abstract 10083 (2006).

74. Budd, G. T. et al. Circulating tumor cells versus imaging--predicting overall survival in metastatic breast cancer. Clin. Cancer Res. 12, 6403–6409 (2006).

75. Mueller, V. et al. Prospective monitoring of circulating tumor cells in breast cancer patients treated with primary systemic therapy—A translational project of the German Breast Group study GeparQuattro. J. Clin. Oncol. 25, Abstact 21085 (2007).

76. Braun, S. 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. 342, 525–533 (2000).

77. Klein, C. A. et al. Comparative genomic hybridization, loss of heterozygosity, and DNA sequence analysis of single cells. Proc. Natl Acad. Sci. USA 96, 4494–4499 (1999).

78. Schmidt-Kittler, O. et al. From latent disseminated cells to overt metastasis: genetic analysis of systemic breast cancer progression. Proc. Natl Acad. Sci. USA 100, 7737–7742 (2003).

R E V I E W S



79. Schardt, J. A. et al. Genomic analysis of single cytokeratin-positive cells from bone marrow reveals early mutational events in breast cancer. Cancer Cell 8, 227–239 (2005).

80. Gangnus, R., Langer, S., Breit, E., Pantel, K. & Speicher, M. R. Genomic profiling of viable and proliferative micrometastatic cells from early-stage breast cancer patients. Clin. Cancer Res. 10, 3457–3464 (2004).

81. Klein, C. A. et al. Genetic heterogeneity of single disseminated tumour cells in minimal residual cancer. Lancet 360, 683–689 (2002).

82. Gray, J. W. Evidence emerges for early metastasis and parallel evolution of primary and metastatic tumors. Cancer Cell 4, 4–6 (2003).

83. Schmidt, H. et al. Asynchronous growth of prostate cancer is reflected by circulating tumor cells delivered from distinct, even small foci, harboring loss of heterozygosity of the PTEN gene. Cancer Res. 66, 8959–8965 (2006).

84. Fidler, I. J. The pathogenesis of cancer metastasis: the ‘seed and soil’ hypothesis revisited. Nature Rev. Cancer 3, 453–458 (2003).

85. Fizazi, K. et al. High detection rate of circulating tumor cells in blood of patients with prostate cancer using telomerase activity. Ann. Oncol. 18, 518–521 (2007).

86. Yie, S. M., Luo, B., Ye, N. Y., Xie, K. & Ye, S. R. Detection of Survivin-expressing circulating cancer cells in the peripheral blood of breast cancer patients by a RT-PCR ELISA. Clin. Exp Metastasis 23, 279–289 (2006).

87. Solakoglu, O. et al. Heterogeneous proliferative potential of occult metastatic cells in bone marrow of patients with solid epithelial tumors. Proc. Natl Acad. Sci. USA 99, 2246–2251 (2002).

88. Pierga, J. Y. et al. Clinical significance of proliferative potential of occult metastatic cells in bone marrow of patients with breast cancer. Br. J. Cancer 89, 539–545 (2003).

89. Mehes, G., Witt, A., Kubista, E. & Ambros, P. F. Circulating breast cancer cells are frequently apoptotic. Am. J. Pathol. 159, 17–20 (2001).

90. Fehm, T. et al. Determination of HER2 status using both serum HER2 levels and circulating tumor cells in patients with recurrent breast cancer whose primary tumor was HER2 negative or of unknown HER2 status. Breast Cancer Res. 9, R74 (2007).

91. Schmidt, H. et al. Frequent detection and immunophenotyping of prostate-derived cell clusters in the peripheral blood of prostate cancer patients. Int. J. Biol. Markers 19, 93–99 (2004).

92. Braun, S. et al. ErbB2 overexpression on occult metastatic cells in bone marrow predicts poor clinical outcome of stage I–III breast cancer patients. Cancer Res. 61, 1890–1895 (2001).

93. Wulfing, P. et al. HER2-positive circulating tumor cells indicate poor clinical outcome in stage I to III breast cancer patients. Clin. Cancer Res. 12, 1715–1720 (2006).The first description of the prognostic value of ERBB2-positive CTCs in non-metastatic breast cancer. Furthermore, a discrepancy of the ERBB2-status between the primary tumour and the CTCs was reported.

94. Meng, S. et al. HER-2 gene amplification can be acquired as breast cancer progresses. Proc. Natl Acad. Sci. USA 101, 9393–9398 (2004).The acquisition of ERBB2 gene amplification in CTCs was reported for breast cancer patients whose primary tumor was ERBB2-negative. Herceptin-containing therapy achieved complete response and partial response in a few patients.

95. Heiss, M. M. et al. Minimal residual disease in gastric cancer: evidence of an independent prognostic relevance of urokinase receptor expression by disseminated tumor cells in the bone marrow. J. Clin. Oncol. 20, 2005–2016 (2002).

96. Meng, S. et al. uPAR and HER-2 gene status in individual breast cancer cells from blood and tissues. Proc. Natl Acad. Sci. USA 103, 17361–17365 (2006).

97. Aguirre-Ghiso, J. A. Models, mechanisms and clinical evidence for cancer dormancy. Nature Rev. Cancer 7, 834–846 (2007).

98. Koebel, C. M. et al. Adaptive immunity maintains occult cancer in an equilibrium state. Nature 450, 903–907 (2007).This experimental study provides evidence for an active role of the immune system in maintaining dormancy of tumour cells. In a carcinogen-induced

tumour model, the immune system of naive mice restrained cancer growth for extended time periods.

99. Wicha, M. S. Cancer stem cells and metastasis: lethal seeds. Clin. Cancer Res. 12, 5606–5607 (2006).

100. Bao, S. et al. Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature 444, 756–760 (2006).

101. Balic, M. et al. Most early disseminated cancer cells detected in bone marrow of breast cancer patients have a putative breast cancer stem cell phenotype. Clin. Cancer Res. 12, 5615–5621 (2006).Using multiple immunostaining of DTCs that are present in the BM of breast cancer patients, this is the first report indicating that many DTCs have a putative cancer stem cell phenotype (that is, CD44+/CD24–).

102. Liu, R. et al. The prognostic role of a gene signature from tumorigenic breast-cancer cells. N. Engl. J. Med. 356, 217–226 (2007).

103. Al-Hajj, M., Wicha, M. S., Benito-Hernandez, A., Morrison, S. J. & Clarke, M. F. Prospective identification of tumorigenic breast cancer cells. Proc. Natl Acad. Sci. USA 100, 3983–3988 (2003).

104. Ginestier, C., Hur, M. & Charafe-Jauffret, E. ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome. Cell Stem Cell 1, 555–567 (2007).

105. Silva, J. et al. Circulating Bmi-1 mRNA as a possible prognostic factor for advanced breast cancer patients. Breast Cancer Res. 9, R55 (2007).

106. Kaplan, R. N. et al. VEGFR1-positive haematopoietic bone marrow progenitors initiate the pre-metastatic niche. Nature 438, 820–827 (2005).

107. Patocs, A. et al. Breast-cancer stromal cells with TP53 mutations and nodal metastases. N. Engl. J. Med. 357, 2543–2551 (2007).

108. Weber, F. et al. Microenvironmental genomic alterations and clinicopathological behavior in head and neck squamous cell carcinoma. JAMA 297, 187–195 (2007).

109. Piccart-Gebhart, M. J. et al. Trastuzumab after adjuvant chemotherapy in HER2-positive breast cancer. N. Engl. J. Med. 353, 1659–1672 (2005).

110. Romond, E. H. et al. Trastuzumab plus adjuvant chemotherapy for operable HER2-positive breast cancer. N. Engl. J. Med. 353, 1673–1684 (2005).

111. Wolff, A. C. et al. American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. J. Clin. Oncol. 25, 118–145 (2007).

112. Fehm, T. et al. Presence of apoptotic and nonapoptotic disseminated tumor cells reflects the response to neoadjuvant systemic therapy in breast cancer. Breast Cancer Res. 8, R60 (2006).

113. Roetger, A. et al. Selection of potentially metastatic subpopulations expressing c-erbB-2 from breast cancer tissue by use of an extravasation model. Am. J. Pathol. 153, 1797–1806 (1998).

114. Brandt, B. H. et al. c-erbB-2/EGFR as dominant heterodimerization partners determine a motogenic phenotype in human breast cancer cells. FASEB J. 13, 1939–1949 (1999).

115. Thor, A. D., Edgerton, S. M., Liu, S., Moore, D. H. 2nd & Kwiatkowski, D. J. Gelsolin as a negative prognostic factor and effector of motility in erbB-2-positive epidermal growth factor receptor-positive breast cancers. Clin. Cancer Res. 7, 2415–2424 (2001).

116. Li, Y. M. et al. Upregulation of CXCR4 is essential for HER2-mediated tumor metastasis. Cancer Cell 6, 459–469 (2004).

117. Brandt, B. et al. Isolation of blood-borne epithelium-derived c-erbB-2 oncoprotein-positive clustered cells from the peripheral blood of breast cancer patients. Int. J. Cancer 76, 824–828 (1998).

118. Becker, S., Becker-Pergola, G., Wallwiener, D., Solomayer, E. F. & Fehm, T. Detection of cytokeratin-positive cells in the bone marrow of breast cancer patients undergoing adjuvant therapy. Breast Cancer Res. Treat 97, 91–96 (2006).

119. Becker, S., Solomayer, E., Becker-Pergola, G., Wallwiener, D. & Fehm, T. Primary systemic therapy does not eradicate disseminated tumor cells in breast cancer patients. Breast Cancer Res. Treat. 106, 239–243 (2007).

120. Steeg, P. S. Tumor metastasis: mechanistic insights and clinical challenges. Nature Med. 12, 895–904 (2006).

121. Ma, L., Teruya-Feldstein, J. & Weinberg, R. A. Tumour invasion and metastasis initiated by microRNA-10b in breast cancer. Nature 449, 682–688 (2007).

122. Tavazoie, S. F. et al. Endogenous human microRNAs that suppress breast cancer metastasis. Nature 451, 147–152 (2008).

123. Hunter, K. Host genetics influence tumour metastasis. Nature Rev. Cancer 6, 141–146 (2006).

124. Godfrey, T. E. & Kelly, L. A. Development of quantitative reverse transcriptase PCR assays for measuring gene expression. Methods Mol. Biol. 291, 423–445 (2005).

125. de Kok, J. et al. Normalization of gene expression measurements in tumor tissues: comparison of 13 endogenous control genes. Lab. Invest. 85, 154–159 (2005).

126. Kowalewska, M., Chechlinska, M., Markowicz, S., Kober, P. & Nowak, R. The relevance of RT-PCR detection of disseminated tumour cells is hampered by the expression of markers regarded as tumour-specific in activated lymphocytes. Eur J. Cancer 42, 2671–2674 (2006).

127. Dandachi, N. et al. Critical evaluation of real-time reverse transcriptase-polymerase chain reaction for the quantitative detection of cytokeratin 20 mRNA in colorectal cancer patients. J. Mol. Diagn. 7, 631–637 (2005).

128. Braakhuis, B. J., Tabor, M. P., Kummer, J. A., Leemans, C. R. & Brakenhoff, R. H. A genetic explanation of Slaughter’s concept of field cancerization: evidence and clinical implications. Cancer Res. 63, 1727–1730 (2003).Using genetic analyses of tumour and surrounding mucosa, a model for squamous cancers was established, strongly focused on the role of the epithelial stem cell.

129. Watson, M. A. et al. Isolation and molecular profiling of bone marrow micrometastases identifies TWIST1 as a marker of early tumor relapse in breast cancer patients. Clin. Cancer Res. 13, 5001–5009 (2007).

130. Apostolaki, S. et al. Circulating HER2 mRNA-positive cells in the peripheral blood of patients with stage I and II breast cancer after the administration of adjuvant chemotherapy: evaluation of their clinical relevance. Ann. Oncol. 18, 851–858 (2007).

AcknowledgementsWe thank I. Alpers for support in the manuscript preparation and D. Kemming for the technical assistance in art work. This work was supported by the European Commission (DISMAL project, contract no. LSHC-CT-2005-018911 and OVCAD project, contract no. LSHC-CT-2005-018698), Deutsche Forschungsgemeinschaft, Bonn, Germany and the Netherlands Organization for Scientific Research.

Competing interests statementThe authors declare competing financial interests: see web version for details.

DATABASESEntrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=geneALDH1A1 | BMI1 | CD24 | CD4 | CD44 | CEACAM7 | CXCR4 | EGF | EGFR | ERBB2 | FGF2 | KRT19 | KRT20 | MUC1 | PLAU | PLAUR | PTHLH | PTPRC | SCGB2A1 | SCGB2A2 | SERPINB3 | SFTPB | TACSTD1 | TERT | TSPAN8National Cancer Institute: http://www.cancer.gov/breast cancer | colon cancer | head and neck cancer | lung cancer | pancreatic cancer | prostate cancerNational Cancer Institute Drug Dictionary: http://www.cancer.gov/drugdictionary/trastuzumab

FURTHER INFORMATIONK. Pantel’s homepage: http://www.uke.uni-hamburg.de/institute/tumorbiologie/index_ENG.phpALTTO: http://www.alttotrials.comDISMAL: http://www.dismal-project.euGEPARQuattro trial: http://www.germanbreastgroup.de/geparquattro/Metastasis Research Society: http://www.metastasis-research.org/OVCAD: http://www.ovcad.eu/Southwest Oncology Group clinical trail: http://www.cancer.gov/clinicaltrials/SWOG-S0500/SUCCESS trial: http://www.success-studie.de/VU University Medical Center: http://www.vumc.nl/

alllinkSareacTiveinTheonlinepDf

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http://www.nature.com/nrc/journal/vaop/ncurrent/box/nrc2375_audecl.html

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene

























http://www.cancer.gov/

http://www.cancer.gov/cancertopics/types/breast

http://www.cancer.gov/cancertopics/types/colon-and-rectal

http://www.cancer.gov/cancertopics/types/head-and-neck

http://www.cancer.gov/cancertopics/types/lung

http://www.cancer.gov/cancertopics/types/pancreatic

http://www.cancer.gov/cancertopics/types/prostate

http://www.cancer.gov/drugdictionary/

http://www.cancer.gov/Templates/drugdictionary.aspx?CdrID=42265

http://www.uke.uni-hamburg.de/institute/tumorbiologie/index_ENG.php

http://www.uke.uni-hamburg.de/institute/tumorbiologie/index_ENG.php

http://www.alttotrials.com

http://www.dismal-project.eu

http://www.germanbreastgroup.de/geparquattro/

http://www.metastasis-research.org/

http://www.metastasis-research.org/

http://www.ovcad.eu/

http://www.cancer.gov/clinicaltrials/SWOG<2011>S0500/

http://www.success-studie.de/

http://www.vumc.nl/

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