Cancer Diagnostics and Molecular Pathology

The
Oncologist®
Cancer Diagnostics and Molecular Pathology
Circulating Tumor Cells: Evolving Evidence and Future Challenges
E
b
FRAT DOTAN,a STEVEN J. COHEN,a KATHERINE R. ALPAUGH,a NEAL J. MEROPOL
aDepartment of Medical Oncology, Fox Chase Cancer Center, Philadelphia, Pennsylvania, USA; bDivision of
Hematology-Oncology, University Hospitals Case Medical Center, Case Comprehensive Cancer Center, Case
Western Reserve University, Cleveland, Ohio, USA
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Key Words. Circulating tumor cells • Breast cancer • Colon cancer • Prostate cancer
Disclosures: Efrat Dotan: None; Steven J. Cohen: Honoraria: Veridex; Katherine R. Alpaugh: None; Neal J. Meropol: None.
The content of this article has been reviewed by independent peer reviewers to ensure that it is balanced, objective, and free from
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commercial bias. No financial relationships relevant to the content of this article have been disclosed by the independent peer
reviewers.
ABSTRACT
Circulating tumor cells (CTCs) are rare malignant cells
itor therapy response, and a method to understand ba-
found in the peripheral blood that originate from the
sic tumor characteristics. This review covers the
primary tumor or metastatic sites. New techniques have
different techniques available for isolation of CTCs, the
by on February 16, 2011
been developed to isolate and characterize these cells.
clinical utility of CTCs in breast, prostate, and colon
CTC enumeration has been incorporated into different
cancer, and future directions in this field. The Oncologist
fields of oncology as a prognostic marker, a tool to mon-
2009;14:1070 –1082
INTRODUCTION
mation into mobile and invasive cells (Fig. 1) [2–5].
Solid tumors diagnosed in an early stage are typically
Through protein modifications and transcriptional changes,
treated by local resection, with or without additional che-
the cells lose their intercellular adhesion and epithelial cell
motherapy aimed at eliminating microscopic metastatic
polarity, and gain a new shape that promotes movement.
disease. These metastases are initiated by the invasion of
Loss or decreased expression of E-cadherin and other epi-
tumor cells into the systemic circulation. Detection and
thelial markers is believed to be an important step in the
characterization of metastatic or circulating tumor cells
process [2, 5]. This invasive potential is enhanced by the
(CTCs) may provide important prognostic and predictive
expression of integrins and activation of various metallo-
information to guide monitoring and treatment. Tumor cells
proteinases, and through constant interaction with the en-
may gain access to the circulation through normal neigh-
dothelial surface. Tumor-derived cytokines/growth factors
boring vessels, or via newly formed capillaries by tumor-
can stimulate expression of adhesion molecules on endo-
induced angiogenesis [1]. In a process called epithelial–
thelial cells that facilitate intravasation [6]. The acquired
mesenchymal transition (EMT), cancer cells undergo
mesenchymal traits enable these tumor cells to enter a
changes in phenotype that facilitate their escape from the
new tissue environment through extravasation [2, 5]. It is
structural constraints of the tissue architecture and transfor-
hypothesized that cells undergo the reverse process of
Correspondence: Neal J. Meropol, M.D., University Hospitals Case Medical Center, Case Western Reserve University, 11100 Euclid
Avenue, Lakeside 1200, Cleveland, Ohio 44106-5065, USA. Telephone: 216-844-5220; Fax: 216-844-5234; e-mail: neal.
meropol@case.edu
Received May 15, 2009; accepted for publication October 13, 2009; first published online in The Oncologist Ex-
press on November 6, 2009. ©AlphaMed Press 1083-7159/2009/$30.00/0 doi: 10.1634/theoncologist.2009-0094
The Oncologist 2009;14:1070 –1082 www.TheOncologist.com

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Dotan, Cohen, Alpaugh et al.
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Figure 1. Schema of the complex metastatic process. Epithelial–mesenchymal transition (EMT) alters the cell phenotype to
allow intravasation into the systemic circulation. Mesenchymal– epithelial transition (MET) occurs at the site of distant
metastases.
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mesenchymal– epithelial transition enabling them to ad-
METHODS FOR CTC DETECTION
here to the new environment and develop into a distant
metastasis [2, 5].
Enrichment Techniques
Cells in the peripheral blood that possess the phenotype
CTCs can be distinguished from normal cells in the periph-
of cancer are referred to as CTCs. These cells may represent
eral blood on the basis of their physical and biologic prop-
by on February 16, 2011
the tumor population that is most likely to develop overt
erties.
Significant
technical
challenges
exist
in
metastases. The presence of these cells was described as
identification of these rare cells in the circulation and in dis-
early as 1869 by Ashworth through identification of tumor-
tinguishing them from more prevalent hematologic cells
like cells in the peripheral blood of cancer patients at au-
and normal epithelial cells. Sensitive enrichment methods
topsy [7]. Sensitive molecular techniques developed in
for the tumor cell population can significantly increase the
recent years are able to enumerate CTCs in the peripheral
yield. Capture and enrichment are performed using specific
blood easily and accurately. CTCs are an attractive tumor
morphologic and phenotypic characteristics of tumor cells
surrogate that may represent the genetic and phenotypic
such as size, density, and specific protein expression. These
composition of the primary tumor. The detection, quantifi-
methods were found to be specific to cancer patients, with
cation, and characterization of CTCs in peripheral blood
zero or only rare cells found in healthy individuals [8, 9].
have the potential for application to various scenarios in
The isolation by size of epithelial tumor cells (ISET) as-
oncology. In early-stage disease, CTCs can potentially
say is an enrichment method using CTC size to separate
help in early detection of malignancy, prediction of the
cells that are
8 m [4, 10]. To date, studies have not val-
risk for metastatic disease, and prognosis. In more ad-
idated the assumption that all CTCs are
8
m [11]. Be-
vanced disease, CTCs may give prognostic information
cause tumor cells are thought to be of lower density than the
and aid the physician in monitoring response to treat-
other cells found in the circulation, some commercially
ment. Moreover, CTCs can potentially represent tumor
available assays (Ficoll-Hypaque, OncoQuick ; Greiner
characteristics, inform treatment decision making, and
Bio-One, Monroe, NC) use a density gradient for cell en-
serve as a surrogate for the prediction of activity of mo-
richment [4]. These methods still lack the required sensitiv-
lecularly targeted agents. This paper reviews the meth-
ity and result in low purity samples secondary to loss of
ods for CTC detection, clinical trials evaluating CTCs as
tumor cells during the isolation process [12].
a prognostic marker and surrogate of treatment effect,
The most commonly used methods for enrichment are
the clinical applicability of current technology, and fu-
based on immunomagnetic techniques—MACS Systems
ture research directions.
(Milteni Biotech, Bergisch Gladbach, Germany), RARE™
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1072
Circulating Tumor Cells
(StemCell Technologies, Vancouver, BC, Canada), Ad-
of this method, which can be improved by the use of mul-
naTest (AdnaGen AG, Langenhagen, Germany), macro-
tiple markers. Multiple automated assays have been devel-
iron beads, magnetic beads, CellSearch (Veridex, Raritan,
oped using this method for CTC characterization and these
NJ) [4, 11, 13, 14]. These are able to separate tumor cells
are summarized in Table 2.
from other cells in the circulation based on specific surface
Nucleic acid– based techniques assess DNA/RNA
markers. Some of these methods use negative selection by
changes specific to tumor cells. RT-PCR is the most com-
isolating mononuclear cells with anti-CD45 antibody, a
monly used method in the category. It is known to be a sen-
panleukocyte antibody. Others use positive selection by ap-
sitive technique able to amplify and identify minimal
plying monoclonal antibody targeting epithelial markers lo-
amounts of tumor-associated RNA, which serves as indi-
cated on tumor cells [15]. These systems have been found to
rect evidence for the existence of tumor cells in the circu-
be more sensitive than some of the density gradient assays
lation. Most other available techniques do not count
[12]. Yet because of a lack of highly specific tumor anti-
degrading cells that lack a nucleus even if they express the
gens, some CTCs may be lost during the process. In addi-
phenotype of CTCs, whereas RT-PCR identifies all the
tion, false-negative results are possible secondary to loss of
mRNA related to tumor cells. Because of its high sensitiv-
tumor-specific antigens by the CTCs. Table 1 compares the
ity, RT-PCR can produce false-positive results by recogni-
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different enrichment methods.
tion of nonspecific gene expression from normal cells [22].
In addition, the question is raised as to the clinical signifi-
CTC Identification
cance of mRNA identification that is not proven to be an
The isolated cells can be further characterized using direct
actual CTC. To date, an automated assay using RT-PCR for
techniques based on immunocytochemistry (ICC) for iden-
CTC identification has not been approved by the U.S. Food
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tifying tumor-specific proteins or by nucleic acid evalua-
and Drug Administration (FDA).
tion using reverse-transcriptase polymerase chain reaction
(RT-PCR) in an attempt to detect tumor-specific mRNA
Automated Assays Combining Enumeration and
transcripts [11, 16]. The advantage of ICC methods over
Identification Techniques
nucleic acid methods is the preservation of the cell during
The CellSearch assay for enumeration of CTCs in periph-
the process, which enables further morphologic and molec-
eral blood is the only method cleared by the U.S. FDA for
ular analysis. ICC and RT-PCR differ significantly, making
clinical use. In this method, 7.5 ml of blood is centrifuged,
by on February 16, 2011
comparison between them difficult. Ring et al. [17] com-
resulting in separation of the cells from the plasma and
pared three different techniques (two using ICC and one us-
buffer layer. Following removal of the plasma and buffer
ing RT-PCR) in patients with metastatic breast cancer
layer, the CTCs are captured using ferrofluid covalently
(MBC) and reported significant differences in the preva-
linked to an antibody against the surface epithelial cell ad-
lence of CTCs, with RT-PCR being the most sensitive
hesion molecule (EpCAM). The cells are captured using
method. Similarly, Smith and colleagues showed higher
EpCAM-labeled ferrofluid and phenotypically differenti-
sensitivity of RT-PCR compared with ICC in samples from
ated from leukocytes by labeling the product with a panel of
patients with MBC [18]. However, despite being more sen-
monoclonal antibodies—pan-CK antibody (anti-CK8, anti-
sitive, most of the large clinical trials reported to date have
CK18, and anti-CK19 antibody), anti-CD45 antibody to
used ICC-based methods because of their ability to identify
identify WBCs, and nucleic acid dye (4 ,6-diamidino-2-
intact cells.
phenylindole) to detect intact cells. The labeled sample is
ICC uses monoclonal antibodies targeting different ep-
loaded into a cassette and analyzed (CellTracks Analyzer
ithelial antigens for different tumors, for example, cytoker-
II; Immunicon, Huntington Valley, PA) by an automated
atin (CK) and mammaglobin for breast cancer, and
fluorescence detection system that identifies CTCs as nu-
carcinoembryonic antigen (CEA) and CK20 for colon can-
cleated cells expressing CK but lacking CD45 [23, 24]. The
cer [1, 16, 19, 20]. False-positive rates in the range of 22%–
system was initially validated by Allard et al. [8] through
61% were found with these methods, varying among
the analysis of
900 patients with metastatic carcinomas.
different antibodies and staining techniques [20]. Cells ex-
The technique was found to be accurate and highly repro-
pressing epithelial markers can easily be identified, yet
ducible, with high correlation between local and reference
false-positive results can occur because of the presence of
laboratories [8, 25]. In addition, there was no significant
nonmalignant cells carrying the same marker [4, 21]. In ad-
difference in CTC number when samples were processed
dition, tumor cells that have lost expression of epithelial
immediately after collection versus after 72 hours of stor-
surface markers during EMT are not captured [4]. The lack
age in CellSave (Veridex) preservative tubes [25]. Two or
of highly tumor-specific marker deters from the sensitivity
more CTCs were found in 57% of patients with prostate

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Dotan, Cohen, Alpaugh et al.
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Table 1. Comparison of enrichment techniques
Assay
Description
Advantages
Disadvantages
Size-based methods
ISET™ [10, 11]
Use of filters with pores of 8
● Inexpensive
● Loss of tumor cells that are of
m retains the tumor cells that
● One-step process
smaller size or have
are larger than leukocytes
● Preserve cell viability
undergone lysis
● Cells available for
● Insensitive
additional studies
● Lack of tumor specificity
Density-based methods
OncoQuick [4, 11, 12,
Separation of CTCs from other
● Inexpensive
● Poor sensitivity because
73]
cells based on lower density
● Single step of
mononuclear cells have a
Ficoll-Hypaque [4, 11, 73]
of CTCs,
1.077 g/ml
centrifugation
similar density
● Preserve cell viability
● Possible loss of tumor cells
● Cells available for
● Lack of tumor specificity
additional studies
Immunomagnetic methods
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MACS [4, 11, 13]
Isolation of cells using different
● Maintain cell structure for
● False positives result from
tumor-specific antibodies
additional evaluation
identification of normal cells
studies
expressing the same antigens
● Multiple antibodies
● False negatives result from
available
CTCs depleted of specific
antigens
● Not automated
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RARE™ [4, 74]
Combination of density
● Maintain cell structure for
● False positives result from
gradient and negative
additional evaluation
identification of normal cells
selection of CD45 cells
studies
expressing the same antigens
● Combination of density
● False negatives result from
gradient for higher
CTCs depleted of specific
sensitivity
antigens
● Not automated
AdnaTest [75, 76],
Combination of EpCAM and
● Maintain cell structure for
● False positives result from
MUC1 targeting epithelial
additional evaluation
identification of normal cells
CTCs; further analysis by
studies
expressing the same antigens
by on February 16, 2011
RT-PCR
● Linked to RT-PCR for
● False negatives result from
additional cell evaluation
CTCs depleted of specific
antigens
● Not automated
CellSearch [8, 13, 15, 25,
Semiautomated system
● Maintain cell structure for
● False positives result from
74, 77]
combining negative selection
additional evaluation
identification of normal cells
with anti-CD45 antibodies
studies
expressing the same antigens
and positive selection with
● Available commercially
● False negatives result from
antiepithelial cells antibodies
(semiautomated, FDA
CTCs depleted of specific
(EpCAM)
approved)
antigens
● Multiple large validation
● Semiautomated systems are
studies
expensive and require
training
CTC-Chip [9, 27, 68]
Array of 78,000- m sized posts
● Highly sensitive
● Limited data
in a specific nongeometric
● Detection of rare CTCs
● Not available outside research
pattern and coated with
from whole blood
setting
antibodies to EpCAM
● Gentle method—maintains
viability of cells
● Cells available for genetic
analysis
Abbreviations: CTC, circulating tumor cell; EpCAM, epithelial cell adhesion molecule; FDA, Food and Drug
Administration; MUC1, mucin 1; RT-PCR, reverse-transcriptase polymerase chain reaction.
cancer, 37% of patients with breast cancer, and 30% of pa-
three or five cells per 7.5 ml, a recent study by Goodman et
tients with colorectal cancer (CRC) . The finding of two or
al. [26] in advanced prostate cancer patients raised the ques-
more CTCs among samples from healthy volunteers and
tion as to the optimal cutoff and found a high predictive
patients with nonmalignant disorders was extremely rare
value for a cutoff of more than four cells per 7.5 ml. Thus,
(zero of 145 and one of 199, respectively) [8]. While most
despite large-scale validation studies and FDA clearance,
CellSearch -based clinical trials used a cutoff of more than
the optimal cutoff may still be debated.
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Circulating Tumor Cells
Table 2. Comparison of different CTC identification techniques
Method
Description
Advantages
Disadvantages
ICC
Use of monoclonal antibodies
● Inexpensive
● False positives result from
targeting antigens on tumor
● Can be coupled with many
identification of normal
cells
enrichment techniques
cells expressing similar
antigens
● False negatives result
from CTCs depleted of
specific antigens
● Operator dependent
● Labor intensive
Commercial methods for enumeration and ICC identification of CTCs
CellSearch [8, 13,
Identification of cells that are
● Maintain cell structure for
● False positives result from
15, 25, 74, 77]
CD45 and CK8 , CK18 ,
additional evaluation
identification of normal
and CK19
studies
cells expressing the same
● Available commercially
antigens
(semiautomated, FDA
● False negatives result
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approved)
from CTCs depleted of
● Multiple large validation
specific antigens
studies
● Semiautomated systems
are expensive and require
training
CTC-Chip [9, 27, 68]
Fluorescent nuclear and CK
● Highly sensitive
● Limited data
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stain for positive selection
● Detection of rare CTCs
● Not available outside
and CD45 stain for negative
from whole blood
research setting
selection
● Gentle method—maintains
viability of cells
● Cells available for genetic
analysis
FAST [78]
Identification of fluorescently
● Able to scan larger amount
● Limited data
labeled cells on glass slide
of cells than automated
● Early stage of
digital microscopy
development
● Able to detect rare cells
● Not available outside
by on February 16, 2011
without the need for an
research setting
enrichment process
RT-PCR–based methods
Analysis of expression of
● Highly sensitive
● Measures RNA, not intact
[16, 17]
candidate genes specific to
● Reproducible
cells
epithelial tumor cells by
● Samples can be stored for
● Analysis does not
mRNA evaluation
future analysis
maintain cell structure
● Inexpensive
● Risk of RNA degradation
● Need for tumor-specific
primers
● False-positive results
secondary to nonspecific
gene recognition
● Does not allow for
additional phenotypic or
molecular characterization
● Not automated
Abbreviations: CK, cytokeratin; CTC, circulating tumor cell; FAST, fiberoptic array scanning technique; FDA, Food and
Drug Administration; ICC, immunocytochemistry; RT-PCR, reverse-transcriptase polymerase chain reaction.
A recently described microfluidic platform (CTC-
cancers, and in none of the samples obtained from
Chip) can efficiently isolate CTCs from the peripheral
healthy volunteers. This highly sensitive method isolated
blood of patients with common epithelial tumors [9, 27].
even small numbers of CTCs, which may make it suitable
This device flows peripheral blood through an array of
for real-time treatment monitoring. This method is also
microposts coated with anti-EpCAM known as TAC-
unique in its ability to sort cells directly from whole
STD1. Using this method, Nagrath et al. [9] were able to
blood in a single step, without the need for preparatory
isolate CTCs in 99% of blood samples from patients with
procedures such as centrifugation, washing, or incuba-
metastatic lung, prostate, breast, pancreatic, or colorectal
tion. The microfluidic approach reportedly maintains the

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Dotan, Cohen, Alpaugh et al.
1075
viability of the cells, which can be used for performing
observed to have a shorter median OS duration (15 months,
additional molecular and genetic analyses.
versus 28.3 months for patients with fewer than five CTCs;
Currently, a gold standard test is not available for mea-
p
.0001). This association was independent of their hor-
suring CTCs. Many of these methods—that is, MACS ,
monal or human epidermal growth factor receptor (HER)-
ISET™ (Metagenex, Paris, France), RARE™, FAST—
2/neu status and the site of metastatic disease.
have not been validated in large clinical trials, and data are
To evaluate whether CTC enumeration adds clinical in-
lacking regarding their exact sensitivity and specificity.
formation to imaging results, Budd et al. [34] compared
Even for the FDA-cleared CellSearch , debate still exists
CTC counts with imaging studies for the prediction of dis-
regarding the optimal threshold for CTC identification [26].
ease progression and OS. The patient population was com-
Limited studies have been performed comparing the sensi-
prised of 138 patients with MBC from the original study
tivity and specificity of these assays. Comparison of the
who had measurable disease and imaging studies per-
features of these methods is summarized in Tables 1 and 2.
formed before and 10 weeks after initiation of therapy along
with CTC measurements 4 weeks after initiation of therapy.
CLINICAL EVALUATION OF CTCS
Significant inter-reader variability was noted in the assess-
Evaluation of CTCs has been attempted in many malignant
ment of imaging studies, as compared with minimal vari-
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diseases, including lung cancer, ovarian cancer, pancreatic
ability in CTC testing. Among all patients with
cancer, melanoma, and others [28 –31]. Presently, the most
nonprogressive disease on imaging, there was a significant
extensive and compelling evidence for their clinical utility
difference in OS between the patients with five or more
is in metastatic breast, prostate, and colorectal cancers. We
CTCs and the patients with fewer than five CTCs (15.3
review below the disease-specific studies for these clinical
months versus 26.9 months; p
.038). Similarly, patients
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indications.
with evidence of progression on imaging scans and low
CTC counts had a longer OS time than those with high CTC
Metastatic Breast Cancer
counts (19.9 months versus 6.4 months; p
.0039). These
The prevalence of detecting CTCs in peripheral blood ob-
data suggest that CTC measurement adds additional prog-
tained from patients with MBC varies in the range of 26%–
nostic information to that of imaging.
49% among different studies using various detection
Currently available data support the role of CTCs as a
methods [8, 25, 32–35]. The prognostic significance of
prognostic marker in patients with MBC. In selected pa-
by on February 16, 2011
CTCs in MBC was evaluated prospectively by Cristofanilli
tients, such as patients with nonmeasurable disease, CTCs
et al. [32] in 177 patients with measurable metastatic dis-
may be particularly useful for monitoring response to ther-
ease using the CellSearch system. That study evaluated
apy. However, there are no available data to address
the number of CTCs in patients before starting a new line of
whether altering treatment based on a change in CTC count
therapy and at the first follow-up visit. The number of CTCs
improves outcome. This question is currently being ad-
before initiation of therapy was found to be an independent
dressed in a National Cancer Institute (NCI)-sponsored
predictor of progression-free survival (PFS) and overall
study conducted by the Southwest Oncology Group
survival (OS). Patients with five or more CTCs per 7.5 ml
(SWOG 0500, NCI registration number, 00382018). This
blood at baseline had a shorter median PFS time than pa-
clinical trial will enroll 500 women with MBC who will un-
tients with fewer than five CTCs per 7.5 ml blood (2.7
dergo evaluation of CTCs at baseline and after one cycle of
months versus 7.0 months; p
.001). The OS time of pa-
first-line chemotherapy. Those with persistently elevated
tients with five or more compared with fewer than five
CTC counts after initiation of therapy will be randomized to
CTCs was also significantly shorter (10.1 months versus
either continue initial therapy until clinical/radiographic
18 months; p
.001). Inferior PFS and OS times were
evidence of disease progression or switch to a different che-
also noted for patients with five or more CTCs compared
motherapeutic agent (Fig. 2).
with those with fewer than five CTCs at follow-up time
Recently, a few groups have published reports of bio-
points [35]. In addition, Cristofanilli and colleagues
characterization of CTCs in patients with advanced breast
showed that a reduction in the number of CTCs 4 weeks af-
cancer. These mainly concentrate on evaluation of the
ter initiation of therapy for the subgroup of patients begin-
HER-2/neu status of CTCs [37– 40]. Pestrin and colleagues
ning first-line therapy for MBC was associated with longer
assessed HER-2 status by immunofluorescence and fluo-
PFS and OS times [36]. Dawood et al. [33] confirmed the
rescence in situ hybridization (FISH) on CTCs of patients
prognostic significance of CTCs in a large retrospective
with MBC [39]. Twenty-nine percent of patient with
study of newly diagnosed MBC patients. Five or more
HER-2
primary tumors were found to have HER-2
CTCs were found in 38.4% of patients at baseline who were
CTCs. Furthermore, 42% of patients with HER-2 primary
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1076
Circulating Tumor Cells
using RT-PCR for risk stratification in the setting of early-
stage breast cancer.
Breast Cancer Summary
The clinical utility of CTCs in patients with breast cancer
remains unclear. Although CTCs are clearly a prognostic
factor for newly diagnosed MBC patients, it remains uncer-
tain whether outcome can be improved with CTC-guided
treatment decisions. Although radiographic studies remain
the gold standard for making treatment decisions, CTCs
may have an important role in the management of patients
with nonmeasurable MBC, such as bone-only involvement.
The American Society of Clinical Oncology 2007 guide-
lines for the use of tumor markers in breast cancer stated
Figure 2.
Study design of Southwest Oncology Group
that measurement of CTCs should not be used for diagnosis
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(SWOG) Trial S0500, a randomized phase III trial that is test-
or treatment modifications in patients with breast cancer
ing the strategy of changing therapy versus maintaining ther-
[19]. The SWOG S0500 study continues to accrue patients,
apy for metastatic breast cancer patients who have elevated
and its results may guide the further use of CTCs for treat-
circulating tumor cell (CTC) levels after the first cycle of
treatment.
ment decision making in patients with breast cancer.
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Metastatic/Castration-Resistant Prostate Cancer
tumors were found to have HER-2 CTCs. Meng et al. [37,
The percentage of castration-resistant prostate cancer
38] reported response to HER-2–targeted therapy in pa-
(CRPC) patients with CTCs is in the range of 35%– 65% in
tients with HER-2 CTCs despite a primary tumor that was
studies using different methods for CTC identification [44 –
HER-2 . Currently, ongoing phase II trials are studying the
49]. Using immunomagnetic separation techniques,
critical clinical question of whether these HER-2
CTCs
Moreno et al. [44] documented that 23 of 37 patients with
can be used as a reliable marker for drug targeting.
metastatic disease had five or more CTCs per 7.5 ml blood
by on February 16, 2011
and that the median survival duration of this group was
1
Early-Stage Breast Cancer
year, compared with
4 years for patients with fewer than
The role of CTCs in the management of patients with early-
five CTCs. A follow-up study by Shaffer and colleagues
stage breast cancer remains uncertain. Ignatiadis and col-
confirmed this frequency of CTCs, with 65% of patients
leagues found CK19 mRNA positivity in the peripheral
having more than five CTCs using CellSearch [46]. Ol-
blood of 40.8% of 444 patients with stage I–II breast cancer
mos et al. [48] used CellSearch to evaluate the association
by RT-PCR [41]. After a median follow-up of 56.4 months,
between CTCs before and after treatment and OS in 191 pa-
patients with CK19 mRNA positivity experienced shorter
tients with CRPC. A high CTC count at baseline was asso-
disease-free survival (DFS) (p
.001) and OS (p
.001)
ciated with high-risk features such as elevated alkaline
times than those who were negative by PCR at diagnosis.
phosphatase, lower hemoglobin, elevated prostate-specific
These differences were more pronounced among patients
antigen (PSA), and the presence of bone involvement. Pa-
with estrogen receptor–negative breast cancer. Four hun-
tients with five or more CTCs had an OS time of 19.5
dred thirty-seven patients from this cohort were further
months, compared with an OS time
30 months for pa-
evaluated for CK19 mRNA positivity following adjuvant
tients with fewer than five CTCs (p
.012). The authors
chemotherapy [42]. Detection of CK19 mRNA following
also demonstrated that a drop of
30% in the number of
chemotherapy was associated with more extensive nodal
CTCs during the first two cycles of chemotherapy in pa-
disease, higher rates of clinical relapse, and disease-related
tients with five or more CTCs at baseline was associated
deaths. DFS and OS times were also significantly shorter in
with longer OS.
the group with CK19 mRNA positivity upon treatment
Similar results were documented by the group led by de
completion. The same group evaluated expression of
Bono, who prospectively studied 231 patients with CRPC
CK19, mammaglobin-A, and HER-2 mRNA in a similar
and PSA
5 ng/ml beginning a new therapy using the
group of patients [43]. Patients found to have expression of
CellSearch system. CTCs were assessed before starting
all three markers had a shorter DFS time than patients lack-
treatment and monthly thereafter [47]. Patients were strati-
ing this expression. These reports suggest the potential for
fied to either a favorable (CTCs
5/7.5 ml) or unfavorable

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Dotan, Cohen, Alpaugh et al.
1077
(CTCs
5/7.5 ml) group. Patients with unfavorable CTC
CRPC [52]. That study found a strong association between
counts at baseline had a significantly shorter OS time than
change in CTC count at 4, 8, and 12 weeks and survival,
those with a favorable baseline CTC count (11.5 months
whereas the change in PSA titer had only a weak associa-
versus 21.7 months; p
.0001). Patients exhibiting an im-
tion. Given the frequency of bone-only metastatic disease,
provement in their CTC count after therapy had a longer OS
CTCs may have a role in monitoring treatment effects in
time than patients who remained in the unfavorable group
these patients. For early-stage prostate cancer, CTCs appear
throughout therapy (21.3 months versus 6.8 months; p
to have limited clinical utility in management.
.0001). In addition, CTC enumeration during therapy was
found to be more predictive of clinical outcome than
Metastatic Colorectal Cancer
post-therapy changes in PSA at different time points dur-
Cohen et al. [53] demonstrated, in a pilot study, that CTCs
ing therapy.
could be isolated from patients with metastatic CRC
(mCRC) using immunomagnetic separation, and that a
Early Prostate Cancer
change in CTC number during therapy appeared to predict
Davis et al. [50] evaluated the correlation between CTCs
clinical outcome. A subsequent prospective study of 430
and tumor volume, pathological stage, and Gleason score in
patients beginning a new line of therapy was conducted to
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patients with localized prostate cancer. Peripheral blood
test the hypothesis that the CTC count at baseline and on
was analyzed by the CellSearch technique from 97 pa-
treatment using the CellSearch system is prognostic [54,
tients before and after radical prostatectomy and compared
55]. Patients were stratified to unfavorable (CTCs
3/7.5
with that of 25 patients with an elevated PSA level yet no
ml blood) and favorable (CTCs
3/7.5 ml blood) groups.
evidence of tumor in a prostate biopsy. CTCs were detected
Shorter PFS (4.5 versus 7.9 months; p
.0002) and OS (9.4
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in 21% of patients with cancer and 20% of controls. There
versus 18.5 months; p
.0001) times were seen in the un-
was no correlation found between tumor volume, patholog-
favorable, compared with the favorable, CTC group. A
ical stage, or Gleason score and CTC number. Similarly,
change from unfavorable to favorable CTC status during
detection of circulating prostate cancer cells using RT-PCR
therapy was associated with longer survival than in patients
with primers specific to the PSA gene in this population
who continued to exhibit an unfavorable CTC count during
showed no correlation with clinical stage, serum PSA level,
therapy. CTC counts before and during treatment were
or Gleason score [51].
found to be independent predictors of PFS and OS in pa-
by on February 16, 2011
tients with mCRC. The groups were further analyzed based
Prostate Cancer Summary
on line of therapy, liver involvement, treatment with oxali-
In contrast to breast cancer, prostate cancer has a widely
platin, irinotecan, and bevacizumab, age, and performance
used biomarker for clinical monitoring. PSA has tradition-
status. In all subgroups, an unfavorable CTC status was as-
ally been used to assess disease status in prostate cancer,
sociated with a worse prognosis [55].
both to follow the course of metastatic disease and as a
In a second large trial, CTCs were prospectively col-
marker of recurrence after definitive local therapy. Thus,
lected from 467 patients enrolled in the CAIRO 2 study,
the threshold to incorporate CTC evaluation into clinical
which randomized patients with mCRC to receive treat-
practice may be higher in prostate cancer. CTCs have been
ment with capecitabine, oxaliplatin, and bevacizumab with
evaluated in the metastatic/refractory setting and in early-
or without cetuximab [56]. Results of the CTC analysis
stage prostate cancer. Identification of CTCs has been per-
were very similar to those found by Cohen et al. [54]. At
formed using different techniques, including the recently
baseline, 29% of the patients exhibited three or more CTCs
FDA-cleared CellSearch
system, which has been vali-
per 7.5 ml blood, with this percentage decreasing during
dated in metastatic prostate cancer. CTCs are a prognostic
therapy. Significant differences were seen in PFS and OS
factor for patients with CRPC and may be more sensitive
between patients with three or more CTCs and those with
than PSA in assessing treatment response. Developing a
fewer than three CTCs (PFS, 8.2 months versus 10.5
study with a primary endpoint of prospectively comparing
months; p
.0005; OS, 13.7 months versus 22.2 months;
CTCs and PSA for treatment decision making is likely not
p
.0001). Similar trends were noted at follow-up time
feasible, given the resource requirements and debate re-
points. The CTC value at any point during therapy was a
garding what specific PSA decrease is a reasonable surro-
better predictor of PFS and OS than other factors such as
gate for clinical benefit of a therapy. Such an attempt was
disease site, lactate dehydrogenase level, and treatment reg-
outlined in a recent publication by Scher and colleagues
imen. Both prospective evaluations of CTCs in patients
comparing the predictive value of changes in CTC count
with mCRC provide strong support for CTCs as an inde-
with PSA level following front-line metastatic therapy for
pendent prognostic marker.
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1078
Circulating Tumor Cells
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Figure 3. Suggested study designs for the use of circulating tumor cell (CTC) enumeration in metastatic colorectal cancer.
Early-Stage Colorectal Cancer
Colorectal Cancer Summary
Studies have evaluated the role of CTCs in early-stage
CTCs have been evaluated in both the metastatic and earlier
CRC. Using quantitative PCR for detection, CEA and
disease settings. The use of CTC counts in early disease is
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CK20 transcripts in blood and peritoneal lavage were eval-
still under investigation as a potential tool for stratifying pa-
uated in samples from 39 patients undergoing curative re-
tients. CTC enumeration provides prognostic information
section of CRC [57]. Eleven patients (28%) had a positive
in patients with mCRC. However, prospective studies are
sample, of whom eight had stage III disease. Patients with
needed to assess the benefit of treatment modification based
positive quantitative PCR had shorter DFS and OS times
on CTC level in CRC patients. Evaluation of CTCs during
than patients who were negative by PCR. Patel et al. [58]
therapy and modification of therapy based on these results
evaluated the prevalence of positive RT-PCR using primers
may spare patients unnecessary toxicity, and provide them
by on February 16, 2011
for CEA and CK20 before and after curative surgery. RT-
with more effective therapy. In addition, CTC enumeration
PCR was positive in 81 of 116 patients (69%) before sur-
could theoretically guide the selection of “low-risk” pa-
tients who might be safely offered treatment breaks or dein-
gery, and significantly decreased 24 hours after surgery. No
tensification of therapy (e.g., discontinuing oxaliplatin).
correlation was found between the prevalence of preopera-
More definitive testing of CTCs as a clinical tool is some-
tive RT-PCR positivity and tumor stage. Sixty-one percent
what limited by cell yield. For example, only 12% of pa-
of the patients who were positive before surgery had nega-
tients with metastatic disease have an unfavorable CTC
tive PCR results 24 hours after the procedure. However, pa-
status on treatment using the CellSearch system [53]. Pos-
tients with node-positive disease were less likely to have
sible clinical trial designs to address the use of CTCs in
reversal of PCR positivity after surgery. Additional studies
treatment assignment are shown in Figure 3.
found RT-PCR positivity or detection of CTCs by ICC fol-
lowing curative surgery to be of prognostic value [59 – 63].
FUTURE DIRECTIONS
Alternatively, Bessa et al. [64] evaluated the prognostic
Detection of CTCs in the peripheral blood has been shown
value of CTCs 24 hours after surgery detected by RT-PCR
to be a reliable surrogate for prognosis in breast, prostate,
for CEA mRNA in 66 patients with CRC and found no
and colorectal cancers (Table 3). Although clinical trial
prognostic value. Inconsistent results were found in other
data reported to date suggest that patients with persistently
trials evaluating the frequency and prognostic significance
elevated CTC counts are on an ineffective therapy, they do
of CTCs isolated from the portal vein during curative sur-
not prove that an early change is beneficial. It is likely that
gery for CRC [65, 66]. Because conflicting results were
their greatest utility is in disease settings in which radio-
seen in these studies, larger studies are needed to establish
graphic imaging is unclear, such as nonmeasurable breast
the role of CTCs in early-stage CRC. Potentially, the iden-
or prostate cancer bone metastases. Prospective clinical tri-
tification of CTCs may assist clinicians with decisions re-
als are required to address this issue. The SWOG S0500
garding adjuvant therapy, or early initiation of systemic
clinical study will begin to address this question in breast
treatment at the time of subclinical relapse.
cancer. Similar large prospective trials are needed in CRC

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Dotan, Cohen, Alpaugh et al.
1079
Table 3. Summary of studies evaluating CTCs as a prognostic marker in metastatic cancers
Study
n of patients
Results (methods)
p-value
Metastatic breast cancer
Cristofanilli et al (2004) [32]
177
Baseline CTC count, >5 versus <5
(CellSearch )
PFS, 2.7 mos versus 7.0 mos
.001
OS, 10.1 mos versus
18 mos
.001
Cristofanilli et al. (2005) [36]
83
Baseline CTC count, >5 versus <5
(CellSearch )
Undergoing first-line therapy
PFS, 4.9 mos versus 9.5 mos
.0014
OS, 14.2 mos versus
18 mos
.0048
At first follow-up, CTC count >5
CTC versus <5:
PFS, 2.1 mos versus 8.9 mos
.007
OS, 11.1 mos versus
18 mos
.0029
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Budd et al. (2006) [34]
138 patients undergoing imaging
Patients without radiologic
.0389
(CellSearch )
and CTC evaluation during
progression: CTC count, >5 versus
therapy for MBC
<5
OS, 15.3 mos versus 26.9 mos
Patients with radiologic progression:
CTC count, >5 versus <5
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OS, 6.4 mos versus 19.9 mos
.0039
Dawood et al. (2008) [33]
185 patients with newly
Baseline CTC count, >5 versus <5
(CellSearch )
diagnosed MBC
OS, 15 mos versus 28.3 mos
.0001
HR for death with
5 CTCs, 3.64
(95% CI, 2.11–6.30)
Metastatic prostate cancer
Olmos et al. (2008) [48]
119 patients
Baseline CTC count, >5 versus <5
(CellSearch )
OS, 19.5 mos versus
30 mos
.012
by on February 16, 2011
Baseline CTC count, >5 versus <5
OS, 6.3 mos versus 21.1 mos
.001
de Bono et al. (2008) [47]
231 patients
Baseline CTC count, >5 versus <5
(CellSearch )
OS, 11.5 mos versus 21.7 mos
.0001
Metastatic colorectal cancer
Cohen et al. (2008) [54]
430 patients
Baseline CTC count, >3 versus <3
(CellSearch )
PFS, 4.5 mos versus 7.9 mos
.0002
OS, 9.4 mos versus 18.5 mos
.0001
Koopman et al. (2008) [56]
467 patients enrolled in the
Baseline CTC count, >3 versus <3
(CellSearch )
CAIRO 2 study
PFS, 8.2 mos versus 10.5 mos
.0005
OS, 13.7 mos versus 22.2 mos
.0001
Abbreviations: CAIRO 2, Phase III study of first-line therapy for metastatic colorectal cancer with capecitabine, oxaliplatin
and bevacizumab with or without cetuximab; CI, confidence interval; CTC, circulating tumor cell; HR, hazard ratio; MBC,
metastatic breast cancer; OS, overall survival; PFS, progression-free survival.
and prostate cancer. Perhaps the use of more sensitive tech-
expressed by normal cells, or alternatively not expressed by
niques, such as CTC-Chip, will improve detection rates in
cancer cells. In addition, EMT may cause cells to lose some
these diseases [9].
of their epithelial markers when entering the circulation.
CTCs appear to clearly be prognostic across several ad-
The malignant potential of an individual CTC and its rela-
vanced malignancies, with persistent debate as to what
tionship to either the primary tumor or metastatic tumor de-
these CTCs truly represent. Despite the use of sensitive
posits is understudied, and more research is needed to
techniques and tumor-specific markers, identification of
identify a specific CTC phenotype with the ability to form
these cells is still challenging. Some of these markers can be
metastases. Assuming that CTCs are indeed of malignant
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