Therapeutic Advances in Medical Oncology Review

Ther Adv Med Oncol

Circulating tumour cells: their utility in cancer (2010) 2(6) 351—365
DOI: 10.1177/
management and predicting outcomes 1758834010378414
! The Author(s), 2010.
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Matthew G. Krebs, Jian-Mei Hou, Tim H. Ward, Fiona H. Blackhall and Caroline Dive http://www.sagepub.co.uk/
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Abstract: Recent advances in technology now permit robust and reproducible detection of
circulating tumour cells (CTCs) from a simple blood test. Standardization in methodology has
been instrumental in facilitating multicentre trials with the purpose of evaluating the clinical
utility of CTCs. We review the current body of evidence supporting the prognostic value of CTC
enumeration in breast, prostate and colorectal cancer, using standardized approaches, and
studies evaluating the correlation of CTC number with radiological outcome. The exploitation of
CTCs in cancer management, however, is now extending beyond prognostication. As technol-
ogies emerge to characterize CTCs at the molecular level, biological information can be
obtained in real time, with the promise of serving as a ‘surrogate tumour biopsy’. Current
studies illuminate the potential of CTCs as pharmacodynamic and predictive biomarkers and
potentially their use in revealing drug resistance in real time. Approaches for CTC charac-
terization are summarized and the potential of CTCs in cancer patient management exempli-
fied via the detection of epidermal growth factor receptor mutations from CTCs in patients with
non-small cell lung cancer. The opportunity to learn more about the biology of metastasis
through CTC analysis is now being realized with the horizon of CTC-guided development of
novel anticancer therapies.

Keywords: biomarker, circulating tumour cells, circulating tumour microemboli, clinical

trials, personalized medicine, pharmacodynamic biomarker, predictive biomarker, prognosis

Introduction biological significance is being revealed. This Correspondence to:

Professor Caroline Dive,
Metastatic disease is responsible for over 90% of review focuses on some of the more developed PhD
cancer deaths [Wittekind and Neid, 2005; Weiss, methods for CTC detection and the current Paterson Institute for
Cancer Research,
2000]. The hypothesis that circulating tumour body of evidence supporting the clinical utility Wilmslow Road,
cells (CTCs) are a fundamental prerequisite to of detecting and characterizing CTCs. Manchester, M20 4BX, UK
cdive@picr.man.ac.uk
metastasis was first proposed in the mid 19th
Matthew G. Krebs,
Century by Thomas Ashworth, an Australian Potential therapeutic uses of circulating MBChB
pathologist [Ashworth, 1869]. Today, the identi- tumour cells Jian-Mei Hou, PhD
Tim H. Ward, PhD
fication and molecular characterization of CTCs The potential applications of CTC enumeration Fiona H. Blackhall, PhD,
in cancer patients remains key to unprecedented and characterization are far-reaching and could FRCP
Clinical and Experimental
insights into the metastatic process and is antic- facilitate several key areas of cancer therapeutics Pharmacology Group,
ipated to unmask novel therapeutic targets for the (Table 1). Most clinical studies, so far, have Paterson Institute for
Cancer Research and
treatment of cancer. Progress, however, has been focused on CTC enumeration in guiding progno- School of Cancer and
hindered until quite recently by the technical sis in metastatic cancer patients and current Enabling Sciences,
University of Manchester,
challenges posed by CTC detection, analogous research is exploring the pharmacodynamic and Manchester Cancer
to looking for the proverbial ‘needle in a hay- predictive biomarker utility of CTCs. A much- Research Centre and
Manchester Academic
stack’. However, there have been major techno- anticipated application relates to the detection Health Sciences Centre,
logical advances, in recent years, leading to of CTCs in patients with early stage, resectable Manchester, UK

the genuine prospect of comprehensive interroga- cancers with the potential to discriminate selec-
tion of CTCs. Tumour cells can now be reliably tively for patients most likely to benefit from
identified in the peripheral bloodstream of can- adjuvant treatment and indeed to monitor the
cer patients with metastatic disease and their efficacy of therapy during the adjuvant course.

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Therapeutic Advances in Medical Oncology 2 (6)
Table 1. Potential applications of circulating tumour cell (CTC) analysis.
Enumeration of CTCs
 Guide prognosis
 Assist in measuring response to anticancer
therapy — predictive and/or pharmacodynamic
biomarker
 May lead to more accurate prognosis when
added to existing staging classifications
 Select patients for adjuvant chemotherapy
 Detect recurrent disease
 Aid diagnostic process
However, most methods in current use are not
sensitive enough for reliable enumeration of
CTCs in early stage cancer patients. In addition,
CTC number surveillance, upon completion of
therapy, may detect relapse at a much earlier
stage than traditional clinical or radiological
parameters and this is under investigation.
The molecular characterization of CTCs offers a
unique ability to assess genotypic and phenotypic
features of a cancer without the need for invasive
biopsy. As our understanding of tumour biology,
the signalling pathways on which tumour forma-
tion and maintenance depends and treatment
resistance mechanisms improves, it becomes
increasingly clear that mechanism-based antican-
cer therapeutics must be tailored according to
individuals’ tumour characteristics: the concept
of personalized medicine. Traditionally, treat-
ment decisions have been empiric based on the
histology of tumour biopsies, normally taken for
diagnosis, that in some cases were procured sev-
eral months or even years prior to the patient
treatment decisions at hand. The characteriza-
tion of CTCs, derived from a ‘simple’ blood
test, have the potential to serve as an invaluable
‘real-time tumour biopsy’ permitting an up-to-
date snapshot of tumour biology, ideally without
the need for invasive tests. Furthermore, CTCs
can be analysed on a serial basis, permitting the
identification of emergent treatment resistance
profiles in real time. If successful, this application
could herald a reduction in the incidence of
unnecessary toxicity experienced by a patient
taking futile treatment regimens. However, well-
designed, prospective, randomized, multicentre
clinical trials coupled with robust CTC method-
ologies will be needed to confirm that changes in
therapy based on CTC evaluation, imposed ear-
lier in a treatment course than possible with
352
Molecular characterisation of CTCs
 Surrogate for biological activity of underlying
tumour — ‘real-time biopsy’
 Elucidate prognostic and predictive molecular
features
 Detection of treatment-resistant profiles —ease
of serial sampling
 Improve understanding of mechanisms of
biological processes
 Discover and identify new targets for therapeu-
tic manipulation
traditional radiological methods, make a signifi-
cant difference to patient outcomes. Early data to
support these potential clinical utilities of CTCs
are discussed herein, with specific focus on prog-
nostication, correlation of CTC number with
radiological outcomes and a survey of approaches
used to characterize CTCs at the molecular level.
The metastatic process and difficulties
presented for CTC detection
In order to appreciate the challenges of CTC
detection, it is important to consider the con-
cepts and theories relating to the metastatic pro-
cess itself. Paget first described the ‘seed and soil’
theory of tumour invasion and dissemination in
1889 to explain the nonrandom formation of
metastasis [Paget, 1889]. The intrinsic properties
of the tumour cells (seeds) and host microenvi-
ronment (soil) are vital determinants of sites of
formation. The anatomical structure of the vas-
culature is also thought to play a (more mechan-
ical) role in the determination of the destination
of tumour cells that enter the circulation [Fidler
and Poste, 2008; Coman et al. 1951; Ewing,
1928]. The entire process of metastasis appears
to be inefficient in that only the ‘decathlon cham-
pions’ of CTCs (as coined by Isaiah Fidler
[Fidler, 2003]) are successful in establishing dis-
tant metastatic disease. In support of this hypoth-
esis, preclinical models demonstrate that within
24 hours of intravenous administration of tumour
cells, less than 0.1% cells remain viable and that
less than 0.01% of these surviving CTCs can pro-
duce metastasis [Fidler, 1970]. This low ‘success
rate’ could be explained by a stochastic/random
survival and growth of a few neoplastic cells or,
more likely, by the selection of a unique subpop-
ulation of cells with greater metastatic potential.
Indeed, the hypothesis that some CTCs express
‘tumour-initiating’ properties has been purported
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and perhaps it is these cells that are CTC
‘decathlon champions’ capable of seeding distant
metastatic disease [Theodoropoulos et al. 2010;
Aktas et al. 2009]. The identification of clusters
of contiguous tumour cells in the circulation of
cancer patients, termed tumour microemboli
[Friedl and Gilmour, 2009; Ilina and Friedl,
2009; Liotta et al. 1976], has given rise to an as
yet untested hypothesis that these cells, via
cell—cell contact survival signaling, have a sur-
vival advantage over single CTCs and thus
might be more likely to contain the minority of
culprit tumour cells that form metastasis.
Contemporary reviews of the metastatic process
include the suggestion that a reversible epithelial-
to-mesenchymal transition (EMT), describing a
major phenotypic change in a subset of cells
within the primary tumour, is essential for metas-
tasis to proceed [Thiery and Sleeman, 2006;
Yang et al. 2006; Thiery, 2003]. During EMT,
epithelial tumour cells lose cell-to-cell to contacts
and develop a more motile and invasive mesen-
chymal phenotype, facilitating their entry into the
bloodstream, and revert back to an epithelial
phenotype upon extravasation in host tissue
(the so-called mesenchymal-to-epithelial transi-
tion (MET)) [Christiansen and Rajasekaran,
2006; Thiery and Sleeman, 2006]. Many CTC
detection techniques depend on capture of CTCs
based on defined epithelial protein expression
(e.g. EpCam and cytokeratins); thus, the very
process of CTC detection may be inherently
flawed if EMT has occurred. However, it is rec-
ognized that EMT is not a homogenous ‘black
and white’ cellular scenario and it seems likely
that CTCs can express both epithelial and
mesenchymal properties, to varying degrees,
giving rise to heterogeneous CTC populations
[Christiansen and Rajasekaran, 2006]. A recent
in vivo study postulated a model of cell coopera-
tivity where mesenchymal expressing tumour
cells were responsible for breakdown of the extra-
cellular matrix and vascular wall invasion, which
facilitated entry of both mesenchymal and
‘passenger’ epithelial tumour cells into the circu-
lation [Tsuji et al. 2009]. Only the epithelial
expressing tumour cells had the capabilities to
subsequently form distant metastases in this
model. The relationships between EMT and
collective cell migration are not yet clear.
With this framework of metastatic tumour cell
behaviour in mind, the challenge for CTC
research is thus not only to discriminate tumour
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MG Krebs, J-M Hou et al.
cells circulating in the bloodstream from the vast
majority of red and white blood cells (in 10 ml of
blood there are an estimated 100 million leuco-
cytes and 50 billion erythrocytes) but additionally
to identify that subpopulation of CTCs with
the elite, lethal metastatic potential that are ulti-
mately responsible for mortality.
Methods of CTC detection
A battery of novel CTC detection technologies
has emerged over the last few years, elegantly
summarized in recent reviews [Alunni-Fabbroni
and Sandri, 2010; Pantel et al. 2009; Paterlini-
Brechot and Benali, 2007; Stebbing and Jiao,
2009]. In principle, methods can be divided
into nucleic-acid-based and cytometric
approaches.
Nucleic-acid-based methods were predominantly
adopted throughout the 1990s following the
development of the polymerase chain reaction
(PCR) and indeed can be very sensitive tech-
niques, relying on the detection of specific
DNA or RNA sequences differentially expressed
by tumour cells [Alunni-Fabbroni and Sandri,
2010; Paterlini-Brechot and Benali, 2007].
However, in recent years there has been a prefer-
ential shift toward cytometric assays where cells
remain intact, hence morphology can be visual-
ized, cells can be enumerated and further analysis
by techniques such as fluorescent in situ hybridi-
zation (FISH) or even DNA/RNA extraction are
practically or theoretically possible, contrasting
with the more limited capabilities using nucleic-
acid-based methods (Table 2).
Cytometric approaches use immunostaining pro-
files to identify and characterize CTCs. These
assays need to be highly sensitive, highly specific
and highly reproducible if they are to be useful in
the clinical setting and used to make patient
treatment decisions. Most methods employ an
initial enrichment step to optimize the probability
of rare cell detection, achievable through immu-
nomagnetic separation, centrifugation or filtra-
tion (Table 2). Cytometric-based techniques
subsequently interrogate cells by fluorescence
microscopy or immunohistochemistry.
TM
CellSearch system
The most widely used cytometric CTC tech-
nology currently in clinical testing is the
TM
CellSearch platform (Veridex LLC,
Huntingdon Valley, PA, USA) and is the only
technology to have received FDA approval for
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Therapeutic Advances in Medical Oncology 2 (6)
Table 2. Main advantages and disadvantages of techniques used for circulating tumour cell (CTC) enrichment and analysis
[Alunni-Fabbroni and Sandri, 2010; Paterlini-Brechot and Benali, 2007].
Advantages
Enrichment
Immunomagnetic Cells selected on basis of antigen expression
separation (manual) using antibody coated magnetic beads.
Versatility for positive or negative
cell selection.
Immunomagnetic Semi-automated magnetic separation — easy
separation to use. Blood samples stable for up to
(CellSearch) 96 hours. Automation permits reproducible
results between users, laboratories and
samples thus robust for multisite trials.
Centrifugation Simple method. Isolates mononuclear cells
on the basis of differences in density gradient.
Nonexpensive.
Filtration (ISET) Isolates cells on basis of size differences (CTC
large; WBC small). Effective method. Tumour
cells amenable for molecular characterization.
Analysis
Cytometric Cells directly visualized by IHC or IF - permits
evaluation of morphology and CTC
enumeration. Cells can be extracted for
molecular characterization, e.g. FISH, RT-PCR.
In combination this allows greater specificity
than nucleic acid methods.
Nucleic Acid Based Highly sensitive in particular with multimarker
(RT-PCR) assay. Can be performed without enrichment
step but at expense of sensitivity. Quantitative
RT-PCR permits relative quantification.
FISH, fluorescent in situ hybridization; IF, immunofluorescence; IHC, immunohistochemistry; ISET, isolation by size of epithelial tumour cells;
RT-PCR, reverse transcription polymerase chain reaction; WBC, white blood cell.
the enumeration of CTC in whole blood in spe-
cific cohorts of cancer patients [Miller et al.
2010]. The major advantage of this system is
its semi-automation and proven reproducibility,
reliability, sensitivity, linearity and accuracy
[Riethdorf et al. 2007; Allard et al. 2004].
These are features crucial to any biomarker tech-
nology to ensure validity of results in clinical test-
ing across multiple sites and have so far been
lacking with previous techniques. CellSearch
employs immunomagnetic bead-based separation
to enrich for CTCs, and the platform character-
istics have been described in detail previously
[Miller et al. 2010; Allard et al. 2004].
Application of CTC technologies to
clinical testing
Prior to the introduction of the CellSearch plat-
form, a variety of combinations of enrichment
techniques and detection assays had been applied
in an attempt to draw clinically relevant conclu-
sions regarding the prevalence of CTCs in cancer
354
Disadvantages
Dependent on epithelial marker expression
which tumour cells may or may not express;
cells may be lost in sample preparation
Dependent on epithelial marker (EpCam)
expression. Challenging to extract pure CTC
for further characterisation but enrichment
good compared with other techniques.
Expensive.
Poor enrichment for CTCs - high contamination
with WBCs. Large number CTCs potentially
lost in processing
Small tumour cells may not be detected by this
method. Lower sensitivity compared with
other techniques.
Detection depends on epithelial or tumour
marker expression. No pure CTC marker
exists. False-positive cells may be enriched.
Current techniques probably less sensitive
than nucleic acid methods.
Cells cannot be visualized directly for
morphology or enumeration. Clinical
correlations reported but difficult to
standardize techniques across laboratories.
No transcripts purely specific for tumour cells
thus high false-positive rate.
patients. Unfortunately, many of these studies
accrued small numbers of patients with insuffi-
cient statistical power for translation to routine
clinical practice. The inclusion within a single
study of patients with different disease stage
and treatments also hindered the ability to draw
robust conclusions. Other variables between
reported studies included: the timing of blood
samples in relation to treatment interventions;
whether or not the first few millilitres of blood
drawn should be discarded on the basis that
this will avoid contamination with skin epithelial
cells; and, importantly, few studies reported con-
ditions for sample handling, sample transport
and the conditions and duration of sample stor-
age prior to analysis. All of these factors, if not
standardized, are likely sources of variability.
Other biological factors have yet to be examined
in sufficient depth, such as the relationship
between the rate of cancer cell entry into the cir-
culation and circadian rhythms although studies
in 8 prostate and 51 breast cancer patients
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suggested that multiple sampling over a 24-hour
period, within a given individual, did not fluctu-
ate [Martin et al. 2009; Moreno et al. 2001].
Once the patients’ blood samples are presented
for evaluation, the choice of antigen for CTC
capture in cytometric assays is critical and even
if the same capture antigen is exploited across
different studies and sites, if different antibodies
are used they may have different sensitivity and
specificities. Similarly, different reverse transcrip-
tion (RT)-PCR primers for target sequences in
nucleic-acid-based approaches, coupled with
subtle differences in assays, might amplify
cDNA with different efficiency with wide ranging
variation in PCR product. Criteria for the defini-
tion of CTCs have also varied. The requirements
for exploratory CTC research and the application
of CTCs as biomarkers in clinical trials have dif-
ferent requirements, where standardization is
essential for the latter application.
CellSearch technology has addressed many of
these variables with its semi-automation, stan-
dardized kits and user-proficiency assessments
and is consequently the predominate technology
for clinical testing. Its robust validation permits
for reliable comparisons of studies and, as such,
will remain a focus for this review. Many of the
next-generation CTC technologies hold great
promise for improving sensitivity and specificity
for CTC detection but validation studies are out-
standing prior to their integration into large-scale
clinical studies. In this regard, the ‘CTC-chip’ is
a very promising approach based on microfluidics
and immunomagnetic cell capture and its capa-
bility in lung cancer will be discussed [Nagrath
et al. 2007].
Clinical studies using CellSearch technology
The first reported study using CellSearch
assessed CTC numbers in 145 healthy women
volunteers, 199 women with nonmalignant dis-
ease and 964 patients with a range of metastatic
epithelial cancer types [Allard et al. 2004]. Of all
healthy women donors and women with nonma-
lignant disease, only one sample contained 2
CTCs (CTC number (#) is expressed per stan-
dard 7.5 ml blood). In contrast, out of 2183 sam-
ples taken from 964 metastatic carcinoma
patients CTC# ranged from 0 to 23,618 and
36% of specimens had 2 CTCs. The presence
varied widely in samples from different carcino-
mas with frequency being highest in metastatic
prostate, unknown primary, ovary and breast car-
cinomas [Allard et al. 2004]. Results were highly
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MG Krebs, J-M Hou et al.
reproducible between duplicate measurements
and between measurements performed in differ-
ent laboratories, paving the way for further
studies aimed at determining the prognostic,
pharmacodynamic and predictive value of CTC
enumeration in patients with advanced cancers.
Three seminal studies in breast, prostate and
colorectal cancer [Cohen et al. 2008; de Bono
et al. 2008; Cristofanilli et al. 2004] have subse-
quently shown that CTC#, over a specified cut-off
value, predicts for worse prognosis, and a change
in CTC# following initiation of therapy demon-
strates predictive value for survival outcome
(Tables 3 and 4). These studies have led to FDA
approval of the CellSearch system as an aid to
prognosis and aid to monitoring patients on ther-
apy in these three clinical situations. Several smal-
ler studies have supported these findings and our
own group has begun to investigate the role of
CTCs in the management of small cell lung
cancer (SCLC) and non-small cell lung cancer
(NSCLC) [Krebs et al. 2010; Hou et al. 2009].
CTC number as a prognostic biomarker
Breast cancer
The first prospective, multicentre prognostic
study using CellSearch was performed in patients
with breast cancer. Out of 177 metastatic breast
cancer patients starting a new line of therapy, a
high CTC# at baseline defined as 5 CTCs
compared with patients with <5 CTCs, was an
independent predictor for poor outcome in terms
of progression-free survival (PFS; 2.7 versus 7.0
months p < 0.0001) and overall survival (OS;
10.9 versus 21.9 months p < 0.0001) [Hayes
et al. 2006; Cristofanilli et al. 2005, 2004].
Assessment of CTC levels at any subsequent
time point (3 weeks onwards) reproducibly pre-
dicted clinical outcome with the worst PFS and
OS occurring in those patients with CTC counts
5 at any time point tested. Further analysis
determined that a dynamic change in CTC#
from baseline, up to 15—20 weeks after initiation
of therapy, predicted more discriminately for sur-
vival outcome. For example, patients with persis-
tently elevated CTC counts (5 at baseline and
follow up) exhibited significantly worse OS com-
pared with patients with persistently low CTC
counts (<5 at baseline and follow up).
Furthermore, patients who converted from an
elevated CTC count (5) at baseline to a low
CTC count (<5) at follow up survived signifi-
cantly longer than those patients who converted
from a low baseline CTC count to elevated CTC
355
356
Table 3. Summary of selected prognostic studies published to date using CellSearch technology.
Study and cancer Breast [Hayes Breast Castrate Castrate Castrate Castrate Colorectal Colorectal Small cell
type et al. 2006; [Nole et al. resistant resistant resistant resistant [Cohen [Tol et al. lung cancer
Cristofanilli 2008] prostate prostate prostate prostate et al. 2009, 2010] [Hou et al.
et al. 2005, cancer [de cancer cancer cancer 2008] 2009]
2004] Bono et al. [Scher et al. [Olmos [Danila
2008] 2009] et al. 2009] et al. 2007]
No of patients 177 80 219 156 119 112 413 451 50
% Positive for 49% (5) 61% (5) 58% (5) 55% (5) 50% (5) 62% (5) 26% (3) 29% (3) 86% (1)
CTCs at BL
(above cut-off)
Line of therapy Any 1st, 2nd or Any 1st Any Any 1st, 2nd or 1st 1st
Therapeutic Advances in Medical Oncology 2 (6)

3rd 3rd
CTC prognostic 5 5 5 no cut-off 5 no cut-off 3 3 no cut-off
cut-off

Median OS — — — — — — — — —
according to BL
CTC count:
<cut-off 18 m — 21.7 m — >30 m — 20.6 m 22.0 m —
cut-off 10.6 m — 11.5 m — 19.5 m — 9.4 m 13.7 m —
P value <0.001 — <0.0001 — <0.0001 — <0.0001 <0.0001 —

Median OS — — — — — — — — —
according to BL
and FU CTC
counts:
<cut-off at BL 22.6 m — >26 m — >30 m — 17.7 m 21.9 m —
<cut-off at FU
 cut-off at BL 19.8 m — 21.3 m — 21.4 m — 11.0 m 14.5 m —
<cut-off at FU
<cut-off at 10.6 m — 9.3 m — 11.2 m — 10.9 m 6.3 m —
BLcut-off at FU
cut-off at BL 4.1 m — 6.8 m — 9.2 m — 3.7 m 6.3 m —
cut-off at FU
Timing of FU Up to 15-20 — Up to 13-20 — After 1-2 — 3—5 weeks 1—2 weeks —
blood sampling: weeks weeks cycles of
novel agent
in phase 1
setting

(continued)

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 Table 3. Continued.
Study and cancer Breast [Hayes Breast Castrate Castrate Castrate Castrate Colorectal Colorectal Small cell
type et al. 2006; [Nole et al. resistant resistant resistant resistant [Cohen [Tol et al. lung cancer
Cristofanilli 2008] prostate prostate prostate prostate et al. 2009, 2010] [Hou et al.
et al. 2005, cancer [de cancer cancer cancer 2008] 2009]

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2004] Bono et al. [Scher et al. [Olmos [Danila
2008] 2009] et al. 2009] et al. 2007]
Additional CTC values Patients CTC counts Higher Patients Higher CTC values CTC values Higher
comments assessed for with 5 predicted baseline with >50 baseline assessed assessed baseline
correlation CTC at OS better CTCs (as a CTC at CTCs (as a for correla- for correla- CTCs (as a
with radiologi- baseline or than PSA continuous baseline continuous tion with tion with continuous
cal response follow up decrement variable) had poorer variable) radiological radiological variable)
(see Table 4) compared algorithms predicted OS com- predicted response response predicted
to <5 CTC at all time for worse pared with for worse (see Table (see Table for worse
predicted points OS (HR patients OS. 4) 4) OS (HR 1.1,
worse PFS; 1.58, with 5—50 Predictive p ¼ 0.015)
S not p < 0.0001) or <5 CTCs model
reached (6.3 vs 21.1 improved
vs 30 with inclu-
months, sion of PSA
p < 0.001). and albu-
min levels

CTC, circulating tumour cell; HR, hazard ratio; m, months; OS, overall survival; PFS, progression free survival; FU, follow up; BL, baseline.
MG Krebs, J-M Hou et al.

357
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Table 4. Summary of selected CellSearch-based studies comparing circulating tumour cell numbers with radiological outcomes and overall survival.

Study and cancer Type Breast [Budd et al. Breast CRC [Cohen et al. CRC [Tol et al. 2010] Breast [Nakamura Breast [Liu et al.
2006] [De Giorgi et al. 2008] et al. 2009] 2009]
2009]

Number of patients 138 102 364 307 107 68

Imaging technique CT PET-CT CT CT CT CT
Timing of imaging from 10 weeks 9—12 weeks 6—12 weeks 9 weeks 12 weeks 9—12 weeks
baseline
Timing of CTC from baseline 4 weeks 9—12 weeks Within 1 month 1—2 weeks 3—4 weeks 3—12 weeks
imaging/death
CTC cut off 5 5 3 3 5 5
Concordance (% patients with 76% 75% 78% 90% Not reported 70%
<cut off CTC and NPD by
imaging and patients
with  cut off CTC and PD
by imaging)
Median OS according to
Imaging and CTC
parameters:
1. NPD by Imaging and CTC 26.9 m Not reached 18.8 m 21.6 m Not reported Not reported
<cut off
Therapeutic Advances in Medical Oncology 2 (6)

2. PD by Imaging and CTC 19.9 m 14.7 m 8.3 m 7.1 m

<cut off
3. NPD by Imaging and CTC 15.3 m 4.9 m 7.1 m 9.4 m
cut off
4. PD by Imaging and CTC 6.4 m 9.8 m 3.1 m 3.4 m
cut off
Statistical significance
between groups:
1v2 0.0785 0.0018 <0.0001 <0.0001 Not reported Not reported
1v3 0.0389 <0.0001 <0.0001 <0.0001
1v4 <0.0001 <0.0001 <0.0001 <0.0001
2v3 0.7549 0.1073 0.4662 Not reported
2v4 0.0039 0.2244 0.0001 Not reported
3v4 0.0777 0.6441 0.0330 Not reported
Additional comments CTCs strongest CTCs strongest CTCs strong predic- CTCs at baseline In cases where If 5 CTCs detected
predictor for OS predictor for OS tor for OS in and at 1—2 CTCs increased at time of imag-
in multivariate in multivariate multivariate weeks, strongest at 3—4 weeks, ing, 3—5 weeks
analysis. analysis. In this analysis. predictor for PFS 63.6% patients prior to imaging
However, both series imaging However, both and OS in multi- showed PD at or 7—9 weeks
CTC values and helpful in deter- CTC values and variate analysis. time of CT scan. prior to imaging,
imaging were mining prognosis imaging were However, both In cases where Hazard Ratio for
useful in combi- in patients with useful in combi- CTC values and CTCs decreased PD at scan ¼ 6.3,
nation in predict- <5 CTCs. In nation in predict- imaging were by >90% at 3—4 3.1, 4.9 respec-
ing survival patients with  5 ing survival useful in combi- weeks, 85.7% tively (p < 0.001).
outcome. CTCs, difference outcome nation in predict- patients showed Study included
in survival was ing survival complete or par- patients on hor-
not significant outcome tial response at monal therapy as
between those imaging well as
with PD or NPD chemotherapy
by imaging

FU, follow up; OS, overall survival; PD, progressive disease; NPD, nonprogressive disease (defined as combination of complete response, partial response and stable disease); CTC,
circulating tumour cell; CRC, colorectal cancer.

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count at follow up (Tables 3 and 4). These were
the first data to support CTCs as a potentially
useful marker for determining response to
therapy.
Metastatic castrate-resistant prostate cancer
Studies in metastatic castrate-resistant prostate
cancer (mCRPC) have shown CTC# determined
by CellSearch to be prognostic. In 219 patients
with mCRPC commencing a new line of chemo-
therapy, baseline CTC# 5 predicted for signif-
icantly worse OS compared with patients with <5
CTCs (median OS 11.5 months versus 21.7
months respectively, p < 0.0001) [de Bono et al.
2008]. Blood samples were taken at monthly
intervals following commencement of chemo-
therapy and CTCs 5 at any subsequent time
point consistently predicted for worse OS, in a
pattern similar to that seen in the breast cancer
study (Table 3).
Not only were CTCs a strong predictor for OS in
multivariate analysis in this study but their pre-
dictive value for survival was superior to that of
monitoring a reduction in prostate-specific anti-
gen (PSA) of >30% or >50%, a routinely tested
tumour maker with potential predictive value for
survival in mCRPC [de Bono et al. 2008].
The use of a low cut-off value (5 CTCs) for
separating patients into ‘favourable’ and ‘unfa-
vourable’ prognostic groups has been questioned
by statistical considerations that challenge the
ability of CTC assays to accurately enumerate
small numbers of cells, with a risk of stratifying
patients into the wrong prognostic group [Allan
and Keeney, 2010; Tibbe et al. 2007]. An alter-
native approach is to consider CTC# as a con-
tinuous variable where one would hypothesize
the higher the number of CTCs, the worse the
prognosis. To this end, data from the above-
detailed prostate cancer study were reanalysed
considering baseline and post-treatment CTC
values as continuous variables [Scher et al.
2009]. Results confirmed that the higher the
CTC#, the worse the OS for patients, irrespec-
tive of cut off (Table 3). Two smaller prostate
cancer studies exhibited similar results. [Olmos
et al. 2009; Danila et al. 2007] (Table 3).
Colorectal cancer
Studies in colorectal cancer (CRC) have also
shown CTC# determined by CellSearch to be
prognostic. In a study of 413 patients with
metastatic CRC commencing first-, second- or
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MG Krebs, J-M Hou et al.
third-line therapy, patients with baseline CTC#
3 had significantly worse median PFS (4.4
versus 7.8 months, p ¼ 0.004) and OS (9.4
versus 20.6 months, p < 0.0001) compared with
patients with <3 CTCs [Cohen et al. 2009,
2008]. CTC# at any subsequent blood draw,
up to 13—20 weeks after initiation of treatment,
was consistently able to predict for worse PFS
and OS on the basis of 3 CTCs. A dynamic
change in CTC number from baseline to follow
up predicted more discriminately for survival
outcome as seen for patients with breast and
prostate cancer, e.g. patients with persistently
elevated CTC counts (3 at baseline and
follow up) exhibited significantly worse OS com-
pared with patients with persistently low CTC
counts (<3 at baseline and follow up) (Table 3).
A second study in metastatic CRC assessed the
prognostic value of CTCs in patients starting
first-line treatment with chemotherapy and tar-
geted agents [Tol et al. 2010]. The same patterns
of results were identified (Table 3).
Lung cancer
Our group has shown that CTCs are detectable
in much higher numbers in patients with SCLC
compared with other disease types. In a cohort of
50 patients, the median number of CTCs
detected was 28, ranging from 0 to 44,896 per
7.5 ml blood. Higher numbers of CTCs were
associated with worse survival in univariate anal-
ysis with >300 CTCs associated with signifi-
cantly worse survival compared with patients
with <2 CTCs (4.5 months versus 14.8 months
respectively, p < 0.005) [Hou et al. 2009]. A per-
sistently elevated CTC count after one cycle of
chemotherapy was a stronger predictor for OS.
Studies are ongoing regarding the prognostic sig-
nificance of CTC number in both SCLC and
NSCLC where in the latter the range of CTC#
is smaller than the former.
Summary of prognostic studies
Common to all of these studies is that CTC#
evaluated by CellSearch, prior to commencing a
new line of chemotherapy, has prognostic value
in breast, prostate and colorectal cancer, leading
to FDA approval in these disease types.
Furthermore, changes in CTC number during
treatment predict survival outcome, highlighting
the potential to use CTCs as a surrogate end-
point biomarker. In the clinical setting this may
influence changes in treatment strategy, within
just a few weeks of starting therapy, in cases
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Therapeutic Advances in Medical Oncology 2 (6)
where CTCs remain above prognostic cut off or
demonstrate an increasing trend. Indeed, at least
one randomized study is currently underway to
test this hypothesis. The SWOG 0500 study ran-
domizes women with metastatic breast cancer to
either continuation of the same chemotherapy or
change in therapy on the basis of a persistently
elevated CTC count at 3 weeks (5 CTCs), in
those with an initially elevated count (5 CTCs)
at baseline. Target accrual is 500 patients and
primary outcome measures are PFS and OS.
Results are anticipated early 2012.
CTCs as a surrogate response biomarker
Hand-in-hand with changes in CTC number
predicting survival outcome is the question of
whether changes in CTC number correlate with
traditional radiological outcomes, the ‘gold stan-
dard’ for monitoring disease status. Several stud-
ies have investigated this correlation, some of
which were reported in parallel to the aforemen-
tioned prognostic studies (Table 4).
The approach has been to take CTC samples at a
given time point after commencing a new line of
therapy and to compare CTC values with
response to treatment according to standard-of-
care imaging techniques. Studies correlated CTC
number (above and below a specified cut off)
with response to imaging according to Response
Evaluation Criteria in Solid Tumours (RECIST)
and OS was calculated according to both param-
eters independently or as combined modalities
(Table 4).
Although studies were slightly heterogeneous in
their methodological approaches with regard to
timing of CTC samples and imaging after com-
mencing treatment, the results favoured CTCs
over conventional imaging to be the most signifi-
cant factor in determining prognosis in multivari-
ate analysis. Concordance between CTCs and
imaging was reported in the range 70—90%
although this should be interpreted with caution
as patients with stable disease were grouped
together with patients with partial response (PR)
and complete response (CR), collectively termed
nonprogressive disease (NPD). This means that
in cases where CTC# was reduced but stable dis-
ease was seen by imaging, a positive correlation was
concluded, as opposed to drawing positive correla-
tion only when reduction in CTC# was seen with a
true radiological response (i.e. PR or CR).
Analysing CTCs and imaging, however, as com-
bined modalities enhanced the ability to predict
360
survival. For example, in a study of breast cancer,
both CTC values at 4 weeks and imaging results at
10 weeks were independently predictive for sur-
vival [Budd et al. 2006]. In combination, however,
greater discrimination for survival was identified,
e.g. patients who exhibited PD by CT scan at 10
weeks, could be categorized into those with a very
poor prognosis (5 CTCs; median OS 6.4
months) and those with a better prognosis (<5
CTCs; median OS 19.9 months, p ¼ 0.0039).
Further details are summarized in Table 4.
Studies in CRC showed similar results. In multi-
variate analysis, CTCs were the most significant
factor for determining prognosis but the combi-
nation of CTCs and imaging provided additive
data (Table 4) [Tol et al. 2010; Cohen et al.
2008]. Such studies have not been reported in
prostate cancer as it is notoriously difficult to
assess tumour response by standard imaging in
this disease.
For future clinical use, the biological significance
of CTC# in predicting patient survival soon after
commencing chemotherapy is encouraging and
may, in certain situations, be more informative
than response by imaging, e.g. with novel tar-
geted therapies which are often cytostatic in
their effect. There is also a body of evidence to
support response by standard CT imaging as an
unreliable surrogate for survival [Mandrekar et al.
2010; Desar et al. 2009]. On the basis of data
reported so far, a particularly pertinent clinical
setting to which CTCs could be applied is that
of phase 1 trials. CTC# may be used to predict
the activity of a drug early in its development,
potentially facilitating go/no-go decision-making
and thus expediting the drug development pro-
cess and reducing expenditure by avoiding the
all-too-frequently seen ‘drug fails late and expen-
sive’ scenario. The ability to ascertain drug—tar-
get expression and downstream measures of
drug—target hitting in CTCs are other exciting
prospects beginning to be realized (as discussed
in the following).
CTCs as a real-time biopsy
The characterization of CTCs at the molecular
level will facilitate personalized medicine via
analysis of pharmacodynamic and/or predictive
biomarkers, and biomarkers of drug sensitiv-
ity or inherent/acquired resistance. This is
already being realized, exemplified by studies of
human epidermal growth factor receptor-2
(HER2) status in patients with breast cancer.
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Expression of HER2 has been detected in CTCs
in metastatic breast cancer patients in cases
where primary tumours were negative at original
diagnosis [Pestrin et al. 2009; Fehm et al. 2007;
Hayes et al. 2002]. This suggests change in bio-
logical activity over time and may justify treat-
ment with HER2-receptor antagonists, such as
trastuzumab, in patients who previously would
not have been eligible. Indeed one study
showed 9 of 24 breast cancer patients whose pri-
mary tumour was HER2-negative each acquired
HER2 gene amplification in their CTCs during
cancer progression [Meng et al. 2004]. Four of
the nine patients were treated with trastuzumab-
containing therapy, three of whom showed
evidence of complete or partial response.
An exemplary model for personalized medicine
lies in the detection of epidermal growth factor
receptor (EGFR) mutations in patients with
NSCLC, predicting for response to EGFR tyro-
sine kinase inhibitors such as gefitinib and erloti-
nib [Sequist et al. 2007]. In a large phase 3 study
of East Asian patients with NSCLC, randomized
to gefitinib (250 mg/day) or carboplatin (area
under the curve 5 or 6 mg/ml/minute) with pac-
litaxel (200 mg/m2) (CP), patients with EGFR
mutations treated with gefitinib exhibited signif-
icantly longer PFS compared with patients trea-
ted with CP (hazard ratio [HR] 0.48, 95%
confidence interval [CI], 0.36—0.64, p < 0.001).
Conversely, patients negative for the EGFR
mutation exhibited significantly longer PFS if
they received CP chemotherapy versus gefitinib
(HR with gefitinib 2.85, 95% CI 2.05—3.98,
p < 0.001) [Mok et al. 2009]. Mutation analyses
were performed using tumour specimens; how-
ever, EGFR-mutation analysis is now possible
in CTCs.
CTC chip technology and EGFR
mutation analysis
Pre-eminent studies using a novel CTC technol-
ogy have demonstrated that EGFR mutations
can be detected from CTCs [Sequist et al.
2009; Maheswaran et al. 2008; Nagrath et al.
2007]. The ‘CTC chip’ isolates CTCs by passing
whole blood through a microchip containing
78,000 geometrically arranged microposts
coated in EpCam. Sensitivity and specificity is
reported as 99.1% and 100%, respectively,
and CTCs were detected in 115/116 samples
taken from 68 patients with a range of tumours
including NSCLC (n ¼ 55), prostate (n ¼ 26),
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MG Krebs, J-M Hou et al.
pancreatic (n ¼ 15), breast (n ¼ 10) and colon
(n ¼ 10) [Nagrath et al. 2007].
Using this technology, DNA was extracted from
CTCs isolated from patients with NSCLC and
EGFR-mutation analysis performed using the
scorpion amplification refractory mutation
system (SARMS) [Maheswaran et al. 2008].
CTCs were detected in 27/27 patients with a
median number of 74 cells/ml. Twenty patients
with known EGFR-mutant primary tumours had
CTCs available for molecular analysis, from
whom EGFR mutations were detected in CTCs
from 19 patients (95%). In addition to the pri-
mary activating mutation, the resistance muta-
tion T790M, predictive for EGFR tyrosine
kinase inhibitor resistance [Kobayashi et al.
2005], was detected in 11/20 patients. Serial
molecular analysis of CTCs in four patients dem-
onstrated evolving genotypes with the emergence
of T790M drug-resistance mutation in all four
patients, consistent with development of clinical
drug resistance.
The potential to use this test in the clinical setting
is extremely promising for predicting both
response to therapy and emergence of resistance
genotypes. It exemplifies perfectly the crucial role
CTCs may play in tandem with development of
novel molecular agents and routine clinical
management.
CTC characterization after enrichment using the
CellSearch platform
Several other methods exist to characterize CTCs
with the common goal of facilitating personalized
medicine and to learn more about the biology of
metastasis. Our own group has characterized the
malignant origin of CTCs isolated by CellSearch
in patients with SCLC. The CellSearch system
has a channel, not required for CTC identifica-
tion, that can be used to detect an appropriately
fluorochrome labelled antibody for further CTC
characterization. This was exploited in our stud-
ies to examine a neuroendocrine marker, CD56
expression in SCLC CTCs. The heterogeneity in
CTC expressions of CD56 broadly mirrored that
found in matched tumour biopsy [Hou et al.
2009].
In a series of phase I studies, the CellSearch spare
channel was used for the detection of insulin-
like growth factor-1 receptor (IGF-1R) in
patients receiving a monoclonal antibody against
IGF-1R either alone or in combination with
361
Therapeutic Advances in Medical Oncology 2 (6)
chemotherapy [de Bono et al. 2007]. Out of 26
patients with detectable CTCs, 23 had evidence
of IGF-1R-positive CTCs. In patients with pros-
tate cancer receiving anti-IGF-1R treatment and
docetaxel, there was some evidence to support a
greater and more rapid decline in PSA in those
patients with positive IGF-1R CTCs than
in those patients with no CTCs, suggesting a
potential predictive marker.
Revealing the genotype/phenotype of CTCs will
further our understanding of the heterogeneous
populations of CTCs that are beginning to be
observed and potentially reveal the CTC ‘decath-
lon champions’ with the highest malignant poten-
tial. To the best of the authors’ knowledge, only
one study, so far, has reported global gene
expression profiling from CTCs isolated by
CellSearch [Smirnov et al. 2005]. This was per-
formed using samples from three patients, one
with breast, one with prostate and one with colo-
rectal cancer, each having >100 CTCs/7.5 ml
sample. Differences in genetic profiles were
seen between each of the patients and candidate
tumour-specific genes were selected and prospec-
tively validated by qPCR in a cohort of 74 met-
astatic cancer patients and 50 healthy donors.
The CTC gene signatures were successfully
able to discriminate cancer patients from healthy
donors, and discriminate between the three dif-
ferent cancer types. This is promising initial data
but methods will need to advance further to reli-
ably isolate RNA from smaller numbers of CTCs
and to improve purification from contaminating
white blood cells which overwhelm and dilute the
tumour gene signatures. The move toward single
cell gene expression profiling from purified
CTCs is greatly anticipated.
FISH has also been performed in CTCs follow-
ing enrichment by CellSearch to confirm malig-
nant origin or identify predictive aberrations.
Three studies in prostate cancer have assessed
for FISH abnormalities in CTCs including
amplification of androgen receptor (AR) and
MYC [Leversha et al. 2009]; gene copy number
for chromosomes 1, 7, 8 and 17 [Swennenhuis
et al. 2009]; and fusions of TMPRSS2-ERG
together with AR and phosphatase and tensin
homologue (PTEN) amplifications [Attard et al.
2009]. All have shown genetic aberrations of
CTCs confirming their malignant origin.
Progress has been made in terms of ability to
characterize CTCs but there is much still to be
362
learned of their biology. The genetic and epige-
netic determinants of tumour cell invasion, cell
survival within the circulation and ability to col-
onize distant organs is an area of intense research
[Nguyen et al. 2009]. CTC characterization of
single cells, once achievable, will offer unprece-
dented opportunity to explore this further.
Conclusion
Since their first description in 1869, a multitude
of studies have provided evidence for CTCs in
the blood of cancer patients. Until recently
there has been substantial variability in CTC
data using several different methods of detection
and analysis. CellSearch technology provides
robust, reproducible CTC enumeration and by
unifying methodology across the scientific com-
munity meaningful interpretations are being
drawn. The limitations of this approach, how-
ever, are founded on the bias towards high
enough levels of EpCam expression on CTCs,
and evidence is emerging to suggest that CTCs
are heterogeneous in this regard. Nevertheless,
the prognostic utility of CTCs, enumerated
using CellSearch, has been demonstrated for
breast, prostate and colorectal cancer and our
own studies have shown prognostic significance
in SCLC. The potential for serial CTC enumer-
ation to predict response to therapy has also been
demonstrated in breast, prostate and colorectal
cancer and a prospective evaluation of changing
therapy based on CTC enumeration alone, early
in the treatment course, is underway in breast
cancer.
Whilst data are emerging on several cancer types
(breast, prostate, CRC and lung), there remains
little information regarding CTCs in many others
(e.g. melanoma, pancreatic cancer, head and
neck cancer, biliary cancer) and much more
research across the cancer spectrum is required.
The promise of CTC research in early stages of
cancer is largely unmet, requiring more sensitive
technologies. However, the ‘real-time biopsy’
potential based on CTC molecular characteriza-
tion is intuitive and promising and remains a key
area for further intensive research. The envi-
sioned future is of a model synonymous to anti-
microbial therapy whereby a simple blood test
will permit molecular tumour characterization,
identification of treatment targets and aid in
selection of the most appropriate targeted ther-
apy from an armamentarium of targeted agents.
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Funding
Supported by Cancer Research UK grant C147/
A12328. M.K. was supported by a Cancer
Research UK/AstraZeneca Clinical Pharmacology
Fellowship.
Conflict of interest statement
The authors declare no conflict of interest in the
preparation of this manuscript.
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