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Detection, clinical relevance and
specific biological properties of
disseminating tumour cells
Klaus 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 singlecell 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.
*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]
doi:10.1038/nrc2375
Published online 11 April 2008
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 epithelial 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.
nature reviews | cancer
The detection and characterization of tumour cells in BM
and those circulating in the peripheral blood has therefore gained considerable attention over recent years4,8,9.
Research on the genotype and phenotype of disseminating 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 advancements 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 therapies, 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 advancements
The 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
volume 8 | may 2008 | 329
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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 (nonproliferative) 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.
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 immunomagnetic 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).
Ferrofluid
A suspension of 10 nm
colloidal iron particles
stabilized by polymers.
Pharmacodynamics
The effects on the biochemistry
of the body resulting from
treatment with a drug or
combination of drugs.
ELISPOT
An antibody-capture-based
method for enumerating
specific T cells (CD4+ and
CD8+) that secrete a particular
cytokine (often interferon-γ).
Immunological approaches. Cytokeratins — cytoskeletal 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 enrichment 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 development 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 malignant epithelial cells14,16. Tumour cells are identified
by cytokeratin staining using fluorescent antibodies
330 | may 2008 | volume 8
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 fibreoptic array scanning technology (FAST) applies laserprinting 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 tissuespecific gene expression initiated a competitive race
against immunocytochemistry for the detection of
DTCs and CTCs at the beginning of the 1990s33. PCRbased 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
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Intravital flow cytometry in mice
In vivo
Blood vessel
Venous puncture
In vitro
FAST
Size
Density
Marker proteins
IMT
RNA
cDNA
MEMS, ISET
Low-density array
CTC chip
Protein secretion
DNA
ICC
FISH
WGA
qPCR
EPISPOT
Figure 1 | Methods for circulating tumour cell (CTC) enrichment, detection and characterization. In vitro methods
for processing CTCs after venous puncture have been established and approved in clinical trials. TheNature
enrichment
Reviews | Cancer
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 tumourassociated 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.
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.
nature reviews | cancer
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 membranearray 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.
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Box 1 | Improvements using quantitative real-time PCR
CT
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
40
y = –3.3334x + 24.896
indicates the threshold.
Threshold
R2 = 0.9998
Beyond quantification, there remain unanswered
line
biological problems, such as the discovery of the ‘ideal’
endogenous control gene that does not deviate
30
between tumour and normal cells from different
125
individuals . 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,
20
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
0
healthy volunteers, whereas no difference could be
0.001
0.001
0.01
1
10
100
detected between patients with colorectal cancer and
c[DNA]
chronic inflammatory disease26,127.
Nature Reviews | Cancer
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 revisited
The 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.
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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 common homing organ for disseminating carcinoma cells,
independent of the primary tumour site and the pattern of overt metastases. Various clinical studies have
provided evidence for an association between the presence 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 significantly 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 significantly 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
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Table 1 | Targets for DTC and/or CTC detection applied in recent studies
Marker and function
Survival markers
M30, apoptosisassociated KRT18
fragment, generated by
caspase
Survivin, apoptosis
inhibitor
Source
Enrichment
Detection method
Relevance
Refs
Bone
marrow
Density gradient
centrifugation
Immunocytochemistry
Indicated therapy response in neoadjuvant
therapy of advanced breast cancer
Peripheral
blood
No enrichment
qPCR
Peripheral
blood
No enrichment
90
Survivin-positive CTCs in patients with breast
cancer; association with advanced pathological
and clinical disease parameters; similar results
for gastrointestinal cancers
Telomerase PCR enzymeTelomerase-positive CTCs in patients with
linked immunosorbent assay advanced prostate cancer with undetectable
serum PSA and patients with localized prostate
cancer before radical prostatectomy
67,86
No enrichment
qPCR, ELISPOT and RT-PCR
38,46
BMI1, PcG of protoPeripheral
oncogenes, gene
blood
regulation at chromatin
level
No enrichment
qPCR
EpCAM
Immunomagnetic
enrichment
Immunocytochemistry
applied in semi-automated
CTC detection system
EPISPOT
Telomerase, telomere
extension, inhibition of
senescence
Stem cell-associated markers
KRT19, potential stem Peripheral
cell marker
blood
Peripheral
blood
FGF2; KRT19+/MUC1–, Peripheral
stem cell marker profile blood;
bone
marrow
TWIST1 (basic helix–
loop–helix transcription
factor; implicated in cell
lineage determination
and differentiation)
PTEN, BRCA1,
microsatellite instability
Bone
marrow
Peripheral
blood
Therapeutic target markers
ERBB2 (oncogenic
Peripheral
growth factor receptor) blood
EGFR
Peripheral
blood;
bone
marrow
IGFR1
Peripheral
blood
Therapy monitoring of advanced NSCLC and
breast cancer
Early breast cancer
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)
Detection of CTCs for metastatic breast, colon,
prostate cancer association with prognosis
Immunomagnetic
(EpCAM) enrichment;
bone marrow,
density gradient
centrifugation
enrichment
Immunomagnetic
Expression microarray, qPCR
(EpCAM) enrichment
Detection of viable and stem cell proteinsecreting DTCs in prostate and breast cancer
Density gradient,
immunomagnetic
cell enrichment
PTEN deletions frequently observed in CTCs
from patients with prostate cancer; BRCA1
associated with rapid biochemical recurrence
Immunocytochemistry,
microsatellite PCR
Density gradient and Immunocytochemistry,
immunomagnetic
PCR, FISH
enrichment
TWIST1 expression associated with 1 year
disease-free survival of patients with advanced
breast cancer under neoadjuvant chemotherapy
Detection of ERBB2-positive CTCs is associated
with early onset of metastasis in breast cancer;
detection of ERBB2-positive CTCs for ERBB2negative breast cancer; first evidence as a
monitoring parameter under adjuvant therapy
No enrichment or
Immunocytochemistry, FISH Prognostic relevant in patients with prostate
immunomagnetic
cancer and castration-resistant prostate
(EpCAM) enrichment;
cancer, detected on DTCs; specific EGFR mRNA
qPCR, density
detection in breast, HNSCC and lung cancer
gradient enrichment
Immunomagnetic
Immunocytochemistry
Monitoring of anti-IGFR therapy in hormone(EpCAM) enrichment
refractory prostate cancer
Multimarker sets
TERT, KRT19, KRT20,
CEA
Peripheral
blood
No enrichment
RT-PCR
TERT, KRT19, CEA,
MUC1
Peripheral
blood
No enrichment
Colorimetric membranearray method using
oligonucleotide probes
and alkaline phosphatase
detection
85
47
105
13,14,
18,19,
23
32
129
83
90,93,
130
5,13,
39
21
Marker set positive in post-operative colorectal 40,48
cancer patients with normal perioperative serum
CEA levels
Similar results for oesophageal cancer
102
Gastric cancer patients and healthy individuals;
45
four-marker set reached diagnostic accuracy of
~90%; independent predictor for post-operative
recurrence or metastasis
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, insulinlike 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 crest
The 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 international organizations have recognized the need for standardization of the immunocytochemical assay11,57 (see also
DISMAL project URL in Further information).
Although aspiration of BM is a routine diagnostic procedure in the clinical management of patients with haematological malignancies, it is invasive, time-consuming,
uncomfortable for the patients and difficult to standardize 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 examinations 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 conditions for homing and survival of DTCs, thus contributing
to their accumulation in this compartment, whereas blood
analyses allow only a snapshot of tumour cell dissemination. 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 measurements 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
334 | may 2008 | volume 8
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 gastrointestinal cancers45,49,67. Moreover, mathematical analyses
of relapse patterns suggest that surgery might also interrupt 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, analogous 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 peripheral 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 recurrence after completion of standard adjuvant chemotherapies 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 associated 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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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 investigates 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 progression, 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 CTCs
When this field of research began, and the first indications 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 haematopoietic 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.
Anoikis
Apoptosis induced in isolated
cells leaving an epithelial
tissue.
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 hybridization 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 earlystage 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,
nature reviews | cancer
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 earlystage 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 telomerase85 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).
volume 8 | may 2008 | 335
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REVIEWS
a
b
Early dissemination
Early-stage cancer
DTCs
Genetic progression
DTCs
Advanced-stage cancer
Develop overt
metastasis in parallel
DTCs
No metastasis
Overt metastasis
Nature
Reviews | Cancer
Figure 2 | A metastatic model derived
from
disseminated tumour cell (DTC) and circulating
tumour cell studies in human patients. 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 selfrenewal 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 earlystage 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.
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
336 | may 2008 | volume 8
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 interpatient variability of DTCs with regard to the expression 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 cancer 95,
and genes encoding ERBB2 and uPA (PLAU) are coamplified in breast DTCs96. Thus, signalling mediated
by ERBB2 and uPAR might be important for the transition 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 progressive 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 slowgrowing 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 mechanisms 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.
www.nature.com/reviews/cancer
© 2008 Nature Publishing Group
REVIEWS
a
DTC- and/or CTC-positive
Breast, colon or head and neck cancer
N0, M0, matched for age, T stage, grading
DTC- and/or CTC-negative
b
RNA
Microdissection
Tissue section
Tumour area
DNA
c
DNA
DNA copy number profile
RNA
CGH or expression array
Bioinformatics
Hierarchical cluster
d
DNA
Protein
TMA
Fluorescence
in situ
hybridization
Immunohistochemistry
Figure 3 | Search for molecular determinants of early tumourNature
cell dissemination.
Reviews | Cancer
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.
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
nature reviews | cancer
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 associated with metastatic relapse in breast cancer patients102.
Nevertheless, CD44 is a somewhat outdated stem cell
marker103 and new ones such as aldehyde dehydrogenase 1 (ALDH1, also known as ALDH1A1)104 or BMI1105
are now available. However, there are additional similarities between the properties of DTCs in BM and cancer
stem cells, suggesting that the founder cells of overt metastases (‘metastatic stem cells’) might reside in the DTC
population. For example, most CTCs or DTCs are nonproliferating 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 disease
The use of targeted therapies in addition to chemotherapy 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 diseasefree and overall survival110. However, to date, determination 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 realtime assessment of the ERBB2 status during the clinical
volume 8 | may 2008 | 337
© 2008 Nature Publishing Group
REVIEWS
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
progression 94. 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 ERBB2directed 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
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Acknowledgements
We 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 statement
The authors declare competing financial interests: see web
version for details.
DATABASES
Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.
fcgi?db=gene
ALDH1A1 | BMI1 | CD24 | CD4 | CD44 | CEACAM7 | CXCR4 |
EGF | EGFR | ERBB2 | FGF2 | KRT19 | KRT20 | MUC1 | PLAU |
PLAUR | PTHLH | PTPRC | SCGB2A1 | SCGB2A2 | SERPINB3 |
SFTPB | TACSTD1 | TERT | TSPAN8
National Cancer Institute: http://www.cancer.gov/
breast cancer | colon cancer | head and neck cancer |
lung cancer | pancreatic cancer | prostate cancer
National Cancer Institute Drug Dictionary:
http://www.cancer.gov/drugdictionary/
trastuzumab
FURTHER INFORMATION
K. Pantel’s homepage: http://www.uke.uni-hamburg.de/
institute/tumorbiologie/index_ENG.php
ALTTO: http://www.alttotrials.com
DISMAL: http://www.dismal-project.eu
GEPARQuattro trial:
http://www.germanbreastgroup.de/geparquattro/
Metastasis Research Society: http://www.metastasisresearch.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/
All links are active in the online pdf
www.nature.com/reviews/cancer
© 2008 Nature Publishing Group