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Circulating Tumor Cells in Gastrointestinal Malignancies: Current Techniques and Clinical Implications

Authors:
  • KMG Klinik Silbermühle GmbH
  • Hirslanden Medical Center, Zurich and Stadspital Triemli, Zurich, Switzerland

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Since their introduction more than 50 years by Engell, circulating tumor cells (CTCs) have been evaluated in cancer patients and their detection has been correlated with clinical outcome, in esophageal, gastric, and colorectal cancer. With the availability of refined technologies, the identification of CTCs from peripheral blood is emerging as a useful tool for the detection of malignancy, monitoring disease progression, and measuring response to therapy. However, increasing evidence suggests a variety of factors to be responsible for disease progression. The analysis of a single CTC marker is therefore unlikely to accurately predict progression of disease with sufficient resolution and reproducibility. Here we discuss the current concept of CTCs, summarize the available techniques for their detection and characterization, and aim to provide a comprehensive update on the clinical implications of CTCs in gastrointestinal (GI) malignancies.
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Hindawi Publishing Corporation
Journal of Oncology
Volume 2010, Article ID 392652, 9pages
doi:10.1155/2010/392652
Review Article
Circulating Tumor Cells in Gastrointestinal Malignancies:
Current Techniques and Clinical Implications
Georg Lurje,1Marc Schiesser,1Andreas Claudius Hoffmann,2and Paul Magnus Schneider1
1Department of Visceral- and Transplantation Surgery, Department of Surgery, University Hospital Zurich, 8091 Zurich, Switzerland
2Department of Medicine (Cancer Research), West German Cancer Center, University Hospital Essen, 45122 Essen, Germany
Correspondence should be addressed to Paul Magnus Schneider, paul.schneider@usz.ch
Received 12 April 2009; Accepted 28 September 2009
Academic Editor: Vassilis Georgoulias
Copyright © 2010 Georg Lurje et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Since their introduction more than 50 years by Engell, circulating tumor cells (CTCs) have been evaluated in cancer patients and
their detection has been correlated with clinical outcome, in esophageal, gastric, and colorectal cancer. With the availability of
refined technologies, the identification of CTCs from peripheral blood is emerging as a useful tool for the detection of malignancy,
monitoring disease progression, and measuring response to therapy. However, increasing evidence suggests a variety of factors to
be responsible for disease progression. The analysis of a single CTC marker is therefore unlikely to accurately predict progression
of disease with sucient resolution and reproducibility. Here we discuss the current concept of CTCs, summarize the available
techniques for their detection and characterization, and aim to provide a comprehensive update on the clinical implications of
CTCs in gastrointestinal (GI) malignancies.
1. Introduction
Circulating tumor cells (CTCs), also known as the “leukemic
phase” of solid tumors, comprise the hematogenous route of
metastasis and have been associated with clinical outcome in
various malignancies, including breast, esophageal, gastric,
and colorectal cancer.
Tumor cells, which detach from the primary tumor
into the bloodstream and travel via circulation to distant
sites, where they potentially develop into secondary tumors,
can cause metastatic lesions, which are a major source of
mortality in patients with cancer. The detection of malignant
cells in blood has been established since years [1], and recent
studies have demonstrated the malignant nature of CTCs
[2,3]. This includes the following steps; tumor growth and
neovascularization (tumor-associated angiogenesis), tumor
invasion and epithelial to mesenchymal transition (EMT),
intravasation, dissemination, arrest in organs, extravasation,
proliferation, and formation of distant metastases. Tumors of
epithelial or hematopoietic origin show a dierent tendency
to metastasize; where some tumors show no or delayed
metastatic spread, others show metastases at the time of
diagnosis.
With the availability of refined technologies, the iden-
tification of CTCs from peripheral blood is emerging as
a useful tool in the detection of particular malignancies,
monitoring disease progression, and measuring response
to therapy. This review will discuss the current value of
CTCs, summarize the currently available techniques for
their detection and characterization, and should provide
a comprehensive update on their clinical implications in
gastrointestinal (GI) malignancies.
2. The Concept of Circulating Tumor Cells
Even though most solid tumor patients undergo multi-
modality therapies (e.g., various combinations of surgery,
chemotherapy, and radiation therapy), dissemination of
malignant tumor cells, and metastatic disease continues
to be the major cause of cancer related death. As a
primary tumor grows, neovascularization and sustained
tumor angiogenesis, the formation of new blood vessels
from endothelial precursors, are prerequisites for the growth
and progression of solid malignancies [4]. Initially, tumors
grow as avascular masses, which can proliferate on pre-
existent vasculature within the microenvironment. When a
2Journal of Oncology
tumor grows beyond a certain size of approximately 2-3 mm,
the tumor requires its own new and dedicated vasculature
[5]. The so-called “angiogenic switch,” the induction of
tumor vasculature or switch to an angiogenic phenotype,
is considered a hallmark of the malignant process and
is required for tumor propagation and progression [68].
Tumor cells may invade the neighbouring normal blood
vessels that were already in place at the primary tumor site, or
they may use the new-formed capillaries that grow intrinsic
to the tumor as a result of tumor-associated angiogenesis.
In either case, there is a tissue und tumor specific induction
of epithelial to mesenchymal transition (EMT), a change in
the expression of cell adhesion molecules (e.g., integrins,
laminins) and activation of proteases pathways (e.g., matrix
metalloproeteinases) that eventually allow tumor cells to
enter the host circulation [9].
Since Stephen Paget’s historic discovery of the “seed and
soil” concept, namely, CTCs tend to metastasize to certain
organ sites, which are selective for cells derived from specific
tumors [10], research on a global scale has attempted to
unravel the underlying molecular mechanisms that bring
“seed and soil” together to promote metastases [11,12].
A current definition of the “seed and soil” hypothesis
comprises three principles; at first, neoplasms contain genet-
ically diverse tumor cell subpopulations, each with dierent
metastatic potential. Secondly, metastases will be formed by
those cells, which will succeed in completing all steps in the
metastatic process and thirdly, the specific choice of “soil”
is mostly attributed to interactions between the tumor cell
and the organ microenvironment. These interactions include
tumor cell specific recognition of endothelial cell antigens
and response to local growth factors [11].
2.1. Immune System and CTC Interaction. Since CTCs and
their primary tumor are generally considered as foreign
tissue for the patients immune system, they have the ability
to form clusters of microtumors, thus escaping the hosts’
immune surveillance and gain a selective growth advantage
over local cells at the distant site [9]. Indeed, aggregation
of 5–10 occult CTCs has been shown to escape the hosts’
immune system, potentially promoting the recruitment of
proangiogenic (growth) factors from the local microenvi-
ronment and expression of new cell surface markers [13].
In fact, some CTCs show heterogenic expression patterns of
cell surface markers in comparison to their primary tumors.
As such, CTCs may aect the interaction with the patients’
immune defense, making it somewhat dicult to predict the
“biologic fate” of CTC-derived microtumors [13].
2.2. Local Invasion, Tumor-Dissemination, Extravasation/
Intravasation, and Implantation. Many aspects of dissem-
inated tumor cells remain to be determined. It is well
recognized though that metastatic disease is predominantly
driven by the tumors’ molecular characteristics (seed) and
hosts’ microenvironment (soil) [10,11]. The unregulated
growth of tumors is attributed to the serial acquisition
of genetic events, which are characterized at least in part
by enhanced cell proliferation, silencing of genes involved
in the inhibition of cell dierentiation and circumventing
genes involved in apoptosis [14]. Dierentiated cells in
adult bowel epithelium demonstrate only a short life-
span with a complete self-renewal time of approximately
2–7 days [15]. Due to their longevity and self-renewing
properties “cancer stem cells,” a small subgroup of cells in
malignant tumors, have a greater propensity to accumulate
these carcinogenic mutations [16,17]. They are therefore
considered responsible for tumor growth and progression
[1820]. This enables a complex crosstalk between cells
from the adjacent structures, including the stroma, and
cancer cells. Even before cancer cells penetrate the basal
membrane and become invasive, they succeed to stimulate
neovascularization on the stromal side of the membrane,
apparently through dispatching angiogenic factors, such as
VEGF and EGF [5]. In addition, tumor necrosis leads to
the recruitment of inflammatory cells that may enhance the
local production of chemokines (IL-8, IL1B) and growth
factors by adjacent stroma cells [7,21]. As a result, matrix
metalloproteinases (MMPs) are produced by inflammatory
cells within the adjacent stroma and are capable of degrading
the extracellular matrix (ECM), further facilitating local
invasion and phenotypic alterations of the tumor, ultimately
leading to tumor cell dissemination and metastatic disease
[22].
Tumor cells that have passed the basal membrane
are highly mobile and quickly penetrate the vascular
endotheliums or lymphatic vessels (intravasation) where
they circulate as CTCs and may extravasate at distant
organ sites. These circulating tumor cells show a geno-
and phenotypic variability. In fact, there is accumulating
evidence that some of them are apoptotic, while others
express markers of “pluripotent progenitor cells” [23,24].
Once the target organ is reached, CTCs often change to
their more epithelial phenotype suppressed during the EMT
[25]. In this way the mesenchymal-like CTCs can undergo
mesenchymal-epithelial transition (MET) and regain the
ability to proliferate [26]. In some cases, tumor cells can also
invade as multicellular clusters or microtumors in a process
termed “collective migration” [27]. These microtumors are
like circulating tumor microemboli (CTM) and are thought
to have a high metastatic potential. As such, they may give
rise to metastases without extravasation by attaching to the
walls of vessels and proliferating within the vasculature and
ultimatively induce the rupture of these vessel walls. As
such micro- and macrometastasis may derive either from
extravasated CTCs or from nonextravasated CTMs after
limited proliferation and rupture of vessel walls [28,29].
3. Methodological Considerations for
the Detection of CTCs
A variety of methods are currently available for enriching
and detecting CTCs from peripheral blood [3,29]. However,
their detection and characterization has been hindered by
the usage of nonstandardized, often nonspecific, and\or
nonsensitive detection methods leading to discordant study
results. In fact, no ideal method is currently available and
Journal of Oncology 3
Tab le 1: Common techniques for CTC-enrichment and detection. MACS (Magnetic Activated Cell Separation), ISET (Isolation by Size of
Epithelial Tumor Cells), FISH (Fluorescent in Situ Hybridization), RT-PCR (Reverse Transcription Polymerase Chain Reaction).
System Blood volume
per test (mL)
Time for
enrichment
(minute)
Principle of CTC enrichment and
detection Sensitivity Reference
OncoQuick 15–35 mL 45 minutes Cellular density gradient,
immunolabeling 1cell/4.5uL [31,34]
MACS 5–15 mL 120 minutes Capture by immunobeads,
immunolabeling and FISH or RT PCR 1cell/0.3uL [35]
CellSearch 7,5 mL 40 minutes
Capture by immunobeads (negative
selection by CD-45; positive selection
by CK-8, -18, -19)
1cell/0.5uL [33,36]
ISET 10 mL 15 minutes Direct filtration and immunolabeling 1 cell/mL [37]
several limitations apply to each of the following methods
described. Some of them however may be overcome if they
are used in combination [25,3033]. The most common
techniques for CTC enrichment and detection are discussed
as follows (Tabl e 1).
3.1. Density Gradient Separation of Mononucleated Cells.
Density gradient separation of mononucleated cells and
CTCs from other cells in the blood is performed using
commercially available kits such as Lymphoprep (Nycomed,
Oslo, Norway), OncoQuick (Greiner, Frickenhausen, Ger-
many), or other similar density gradient liquids. This process
generates a layered separation of cell types based on the
cellular density. After centrifugation, from bottom to top,
the following layers are found: erythrocytes, neutrophils,
density gradient, mononuclear cells, and plasma, which is
the top layer. Although the addition of a porous barrier
(OncoQuick) above the density gradient has prevented its
mix with whole blood, there are still limitations with this
method. These particularly include migration of CTCs into
the plasma fraction and the formation of aggregates at the
bottom of the gradient.
3.2. Direct Enrichment of Circulating Epithelial Cells by
Filtration. Direct enrichment of epithelial cells by filtration
is based on the observation, that the vast majority of
peripheral blood cells belong to the smallest cells in the
human body (8 to 11 um). They can be eliminated by blood
filtration through polycarbonate membrane calibrated pores
of 8 um [29]. Even though this method involves only one
single step, it is not highly eective in the process of enriching
CTCs.
3.3. Immunomagnetic Cell Enrichment. Immunomagnetic
cell enrichment is the most commonly used technique
for CTC enrichment. This approach can be performed by
itself or integrated with other physical (density gradient,
direct filtration) or nonphysical downstream molecular
methods such as quantitative real-time reverse transcrip-
tion polymerase chain reaction (RT-PCR) or fluorescence
in situ hybridization (FISH). Several protocols have been
developed and are now commercially available. Their yield
ranges from 1 ×104-to2×105-fold CTC enrichment
and in contrast to RT-PCR usually avoid cell lysis, thus
allowing a direct count of CTCs [29]. These systems rely
on positive selection of CTCs from samples through their
binding of antibodies coupled to magnetic beads that target
epithelial- or tumor-specific cell surface antigens, such as
cytokeratins (CKs), human epithelial antigens, epithelial
cell adhesion molecules, as well as prostate specific antigen
(PSA), carcinoembryonic antigen (CEA), human epithelial
growth factor receptor-2 (HER-2). Since no available anti-
body is 100% tumor or tissue specific [45], false-positive
(specifity) and false-negative (sensitivity) results continue
to impose a significant problem with immuno-magnetic
detection techniques. Recently, negative selection of white
blood cells by CD45 and CD61 specific antibodies in addition
to aforementioned positive selection methods was applied
in an attempt to further increase specific enrichment of
CTCs from peripheral blood (CellSearch, Veridex LLC,
Raritan, NJ, USA) [33,36,46]. The use of semiautomated
devices for the microscopic screening of large amounts of
immunostained slides has already helped to increase speed
and reproducibility of immuochemical analyses [47]. Among
the commercially available semiautomated approaches, the
CellSearch system (Veridex LLC, Raritan, NJ, USA), which
has been federal drug and food administration- (FDA-)
approved for the use in metastatic breast and colon cancer
patients, has gained considerable attention lately, because
it allows both automated immunomagnetic epithelial cell
adhesion molecule-based (EpCAM) enrichment, as well as
cyotokeratin staining of CTC in blood samples. Even though
this technique eliminates the majority of leukocytes, the
presence of nonepithelial cells and CTCs that do not express
epithelial antigens (by EMT) is still considered a major pitfall
of this approach. The major advantages of immunomagnetic
separation are the direct visualization and quantification of
CTCs and the detection of living cells without the need of
cell lysis, as it is the case for PCR-based methods.
3.4. Reverse-Transcriptase Polymerase Chain Reaction (RT-
PCR). Detection of CTCs from peripheral blood of cancer
patients by conventional reverse-transcriptase PCR (RT-
PCR) has been reported for several genes, for example,
4Journal of Oncology
CEA, CK-19, or -20 [48,49]. Since its introduction almost
20 years ago, RT-PCR has emerged as the most common
used technique for the detection of CTCs [25]. The main
advantage of this approach is its sensitivity that is considered
higher than other currently available protocols. This is
particularly true for protocols where mRNA is isolated after
enrichment of CTCs from whole blood [31]. As such, RT-
PCR is often considered the most sensitive assay to detect
tumor-specific molecular markers. In addition, RT-PCR does
also oer high specifity, as primers are designed for the
particular gene of interest and the whole genomic DNA or
RNA can be analyzed in one single reaction. Furthermore,
RT-PCR of tumor-specific mRNA is characterized by a
higher sensitivity in comparison to protein-based methods
and usually ranges within a detection range of 1 to 10
tumor cells among 106-107blood mononuclear cells [50,
51]. Nevertheless, the high sensitivity of RT-PCR, although
important for its clinical use, is problematic when false
positive results are encountered, as it is the case with sample
contamination (genomic DNA versus cDNA), illegitimate
transcription (low-level nonspecific transcription of certain
genes), or amplification of pseudogenes [52,53]. Another
limitation is the selection of mRNA markers used, since
the ideal marker would likely be highly overexpressed in
all tumor cells from a given tumor, but not expressed at
all in white blood cells and nonepithelial circulating cells.
Indeed there are several studies, which report detection of
CK19, CK20, CEA, and PSA in circulating nontumerous
(epithelial) cells [38,5155]. Therefore, it is extremely
important to carefully design primer and probes, choose
the “right candidate genes” and ideally combine some of
the aforementioned physical enrichment techniques with
molecular based methods such as RT-PCR or FISH [33].
3.5. Microchip Technology (CTC-Chip). Newer methods for
enrichment have recently been reported that utilize microflu-
idic platforms (the “CTC-chip”) capable of ecient and
selective separation of viable CTCs from peripheral whole
blood samples [56]. Nagrath et al. described the “CTC-
chip” that separates CTCs in whole blood using EpCAM
coated microposts and controlled laminar flow conditions
to ensure optimal interaction between the cells and the
microposts. Since the CTC-chip can process whole blood
directly, the authors claim that a level of purity, that is
up to 100-fold higher than what other technologies oer
can be achieved [56]. Nevertheless, further validation within
prospective clinical trials is warranted.
4. Clinical Implications of CTCs in
Gastrointestinal Malignancies
The presence of CTCs in patients with metastatic carcinoma
is generally associated with poor clinical outcome [9].
Initially, this has been shown for malignancies outside
the GI tract, such as breast cancer. Cristofanilli et al.
demonstrated that patients with metastatic breast cancer
could be segregated into favorable and nonfavorable groups
(with reference to PFS—progression free survival and OS—
overall survival) based on their CTC count. This was assessed
by an immuno-magnetic separation technique (CellSearch,
Veridex LLC, Raritan, NJ) that led to FDA approval for the
use in metastatic breast cancer in 2005 [36].
In a prospective multicenter study by Cristofanilli et al.
177 patients with measurable metastatic breast cancer (as
assessed according to the criteria of the World Health
Organization) were tested for blood levels of CTCs, both
before the patients were to start a new line of treatment
and at the first follow-up visit. Outcomes were assessed
according to levels of circulating tumor cells at baseline,
that is, before the patients started a new treatment for
metastatic disease. Patients with levels of CTCs equal to
or higher than 5 per 7.5 mL of whole blood, as compared
with the group with fewer than 5 circulating tumor cells per
7.5 mL, had a shorter progression-free survival (2.7 months
versus 7.0 months, P<.001) and shorter overall survival
(10.1 months versus >18 months, P<.001). This dierence
between the groups persisted (progression-free survival, 2.1
months versus 7.0 months; P<.001; overall survival, 8.2
months versus >18 months; P<.001) at the time of the first
follow-up visit, suggesting that the reduced proportion of
patients (from 49 percent to 30 percent) in the group with
an unfavorable prognosis showed a benefit from therapy.
The authors concluded that the number of CTC’s before
treatment is an independent predictor of PFS and OS in
patients with metastatic breast cancer [36].
Compared to breast cancer, there are fewer studies avail-
able in GI-cancer and the results are less consistent (Tab le 2).
However, given the recent focus on how k-ras mutations
aect clinical outcome in metastatic colorectal cancer and
anti-EGFR therapy with cetuximab [3941,57], evaluation
of patients for mutations in k-ras is rapidly becoming part
of routine practice in clinical oncology and had so far
mostly relied on formalin-fixed paran-embedded (FFPE)
tumor tissue. Testing of circulating tumor DNA in peripheral
blood to screen for mutations resident in the parent tumor
(blood biopsy) is unencumbered by many of the factors that
limit testing of FFPE-derived specimens. As such, blood is
easily accessible, not prone to selection bias, and provides a
continuous source of DNA. Accordingly, tests for circulating
tumor DNA are able to screen for mutations, such as for
mutations of the k-ras oncogene and are present at the time
of treatment unlike tests that rely on archived tissue samples
that were acquired previously [42,58].
4.1. CTCs in Esophageal Cancer. Even though not many
studies have been conducted and results have been incon-
sistent, the majority of studies that have explored CTCs as
a molecular marker for clinical outcome have demonstrated
their clinical utility [31,43,59,60]. In fact, a recent
study by Liu et al. aimed at establishing a quantitative
system for evaluating the role of CTCs in peripheral blood
from patients who underwent surgical resection for the
treatment of esophageal cancer [43]. One hundred fifty-
five peripheral blood samples from 53 esophageal cancer
patients were collected before surgery (B 1), immediately
Journal of Oncology 5
Tab le 2: CTCs studies and clinical outcome in gastrointestinal malignancies.
Patients analyzed Type of
cancer Method of CTC detection Results Reference
53 esophageal cancer patients
22 benign patients who under-
went thoracotomy
30 healthy volunteers
Esophageal
cancer
(1) Density gradient separation
(OncoQuick)
(2) Real-time RT-PCR (CEA-based)
Patients with high levels of
CTCs (CEA) were more
likely to show recurrent
disease
Liu et al. [43]
62 esophageal cancer patients (25
SCC/37 EA)
Esophageal
cancer
(1) Density gradient separation
(OncoQuick)
(2) Real-time RT-PCR (survivin-based)
Survivn mRNA levels fall
after surgical resection
Homann et al.
[60]
44 patients with GI malignancy
-24 esophageal cancer
-5 gastric cancer
-8 colorectal cancer
-3 pancreatic
GI
malignancies
(1) Density gradient separation
(OncoQuick)
(2) Real-time RT-PCR (survivin-based)
Survivn mRNA levels fall
after surgical resection
Homann et al.
[31]
29 locally advanced esophageal
cancer patients (11 SCC/18 EA)
Esophageal
cancer
(1) Density gradient separation
(OncoQuick)
(2) Real-time RT-PCR (ERCC1-based)
ERCC1 mRNA expression
associated with response to
neoadjuvant
radiochemotherapy
Brabender et al.
[71]
59 locally advanced esophageal
cancer patients (24 SCC/35 EA)
Esophageal
cancer
(1) Density gradient separation
(2) Methylation specific real-time
RT-PCR (DAPK and APC-based)
DAPK and APC
methylation associated with
unfavourable prognosis
Homann et al.
[44]
57 gastric cancer patients
15 patients with benign diseae
15 healthy volunteers
Gastric
cancer
(1) Density gradient separation
(2) Real-time RT-PCR (CEA-based)
Patients with high levels of
CTCs (CEA) were more
likely to show recurrent
disease
Miyazono et al.
[63]
59 gastric cancer patients
15 patients with benign diseae
Gastric
cancer
(1) CTC-enrichment was not performed
(2) Real-time RT-PCR (CEA-based)
High levels of CEA were
associated with lower risk
of tumor recurrence
Ikeguchi et al.
[64]
430 mCRC patients beginning a
new first-, second-, or third-line
systemic therapy
Colorectal
cancer
Immunomagnetic CTC enumeration
(CellSearch, Veridex)
Patients with high CTCs
hadpoorPFSandOS
Cohen et al.
[66]
430 mCRC patients beginning
a new first-, second-, or third-
line systemic therapy (follow-up
study)
Colorectal
cancer
Immunomagnetic CTC enumeration
(CellSearch, Veridex)
Patients with high CTCs
hadpoorPFSandOS(also
within subgroups with
longer follow up)
Cohen et al.
[67]
after surgery (B0), and on the 3rd day postoperatively
(B + 3). A direct quantitative real time RT-PCR method
based on carcinoembryonic antigen (CEA) mRNA gene
expression was designed for the detection of CTCs. The
authors showed a significant dierence between B 1and
B0 (P=.0001) and between B 1andB+3(P=.0209).
Fifty percent of patients with R>0.4(R=CTC ratio
of B + 3 over B0) showed tumor recurrence within 1 year
after surgery, whereas the probability was only 14.3% for
patients with R<0.4(P=.043) [43]. These findings are
consistent with a recent report for members of our group
exploring CTCs from peripheral blood as assessed by a direct
quantitative RT-PCR method, using CTC survivin-mRNA
expression as a surrogate for CTC count in peripheral blood
[31,60]. In their preliminary findings, the authors could
show that direct quantitative real-time RT-PCR analysis of
survivin mRNA expression in peripheral blood of patients
with gastrointestinal cancers is technically feasible and that
survivin mRNA levels fall signicantly, following complete
surgical resection (R0-resection according to UICC) and may
be a promising molecular marker for the completeness of
surgical resection (molecular R0 marker) [31]. A follow-up
study by the same group analyzed peripheral blood samples
from 62 esophageal cancer patients, who were scheduled for
surgical resection. 25 (40.3%) patients had squamous cell
carcinomas and 37 (59.7%) adenocarcinomas. In 24 (38.7%)
patients neoadjuvant chemoradiation was performed for
locally advanced disease. Whole blood was drawn one day
before the operation and 10 days post R0-resection in
all patients. Postoperative survivin levels were significantly
lower than preoperative levels in 41.2% of resected patients.
In patients receiving neoadjuvant chemoradiation a decrease
of postoperative survivin mRNA expression was detected
in 83.3% of patients with adenocarcinomas compared to
50% with squamous cell carcinoma (Mann-Whitney test:
P<.04), suggesting that survivin levels were particu-
larly decreased in adenocarcinomas following neoadjuvant
chemoradiation and surgical resection. Other studies by the
same group revealed that esophageal cancer patients who
showed minor histopathological response to neoadjuvant
6Journal of Oncology
radiochemotherapy demonstrated significant higher CTC
levels as assessed by ERCC1 mRNA-expression levels in
their blood [44]. Furthermore, similar results could be
shown for death-associated protein kinase (DAPK) and
adenomatous polyposis coli gene (APC) methylation status
as assessed by methylation-specific real-time RT-PCR. In
fact, 36 out of 59 patients (61.0%) with esophageal cancer
had detectable levels of methylated DAPK or APC promoter
DNA and preoperative detection was significantly associated
with an unfavorable prognosis in the multivariable model
[44].
4.2. CTCs in Gastric Cancer. Gastric adenocarcinoma is
the forth most common type of cancer and the second
leading cause of cancer-related death worldwide [61]. Even
though combined chemoradiation following complete sur-
gical resection has oered modest improvements in OS and
PFS (MAGIC-trial) [62], tumor recurrence after curative
resection continues to be a significant problem in the man-
agement of patients with localized gastric adenocarcinoma.
In an attempt to identify markers for tumor recurrence
Miyazono et al. investigated blood samples from 57 patients
with gastric cancer for the presence of CTCs [63]. After
density gradient separation of CTCs, CEA-specific real-
time RT-PCR was performed and correlated with the time
course during a surgical procedure and the advent of hepatic
tumor recurrence. Interestingly, the authors could show
that CEA-mRNA could not be detected in a control group
of healthy volunteers and 15 patients with benign disease.
In contrast, a total of 21 gastric cancer patients (36.8%)
were positive for CTC as detected by CEA-specific RT-PCR
and positive rates correlated with depth of tumor invasion.
Furthermore, the authors found that gastric cancer patients
with high levels of CEA were more likely to develop systemic
disease. More recently, Ikeguchi and Kaibara confirmed these
findings; even though the authors noted that CTCs may
be eliminated very quickly from the hosts circulation and
as such may be present only for a very short period of
time [64]. Other groups have reported similar findings for
relatively heterogenic study populations of patients with
gastrointestinal malignancies [65].
4.3. CTCs in Colorectal Cancer. CTCs have been studied
in colorectal cancer (CRC) [30,66] and recent studies
have shown their clinical utility in patients with metastatic
CRC [33,67]. In a prospective multicenter clinical trial,
430 patients beginning a new first-, second-, or third-
line systemic therapy had peripheral blood obtained for
enumeration of CTCs (CellSearch, Veridex LLC, Raritan, NJ,
USA) at baseline (pretreatment) and subsequent time points.
As demonstrated by the authors, CTC count at baseline, and
during therapy, was the strongest independent prognostic
marker compared with other clinical factors for PFS and
OS [33]. As the patient population was heterogeneous in
this study, the authors reported on the same study cohort
with extended follow-up time to evaluate the prognostic
significance of baseline CTC count by line of therapy, type
of therapy, and other important clinical characteristics [67].
Interestingly, the results remained remarkably consistent in
comparison to the original trial, with a near doubling in PFS
and OS for patients with favorable compared to unfavorable
baseline CTCs. In addition, elevated baseline CTC count
within patient and clinical subgroups was associated with
adverse clinical outcome in all subgroups. PFS was also
generally inferior in patients with elevated baseline CTCs,
even though this finding did not reach statistical significance
at the P<.05 level. Therefore, the authors state that
while an elevated CEA may be a poor prognostic factor for
resectable CRC [68], no present data support its prognostic
value for metastatic CRC. Thus, the authors suggest that the
next large metastatic CRC trial evaluating a new systemic
therapy should utilize CTCs as a stratification factor for OS
[67].
5. Summary and Future Perspectives
Progress in cancer biology has led to an expansion of our
understanding of the molecular and cellular mechanisms
of cancer development, cancer metastases, and resistance
of cancer cells to (radio-) chemotherapy. Despite recent
advancements in the detection, surgical resection, and (neo-)
adjuvant (radio-) chemotherapy, selection of the most ben-
eficial treatment strategies in gastrointestinal malignancies
remains a challenge and is also hindered by the lack of
validated predictive and prognostic molecular markers [68].
A multidisciplinary approach, including surgery, tailored
(radio-) chemotherapy, alone or in combination will be
necessary to improve the outcome for patients with GI
cancer. In addition, the high incidence of tumor drug
resistance remains a major stumbling block for eective
cancer treatment. For the clinician, it is important to
distinguish between prognostic and predictive molecular
markers. Predictive markers are associated with treatment
specific therapy and are mostly evaluated through clinical
response, time to progression, or toxicity. In contrast,
prognostic markers reflect the nature/aggressiveness of the
disease, independently of a specific treatment, and are
usually evaluated in terms of OS. In some cases, predictive
markerscanalsocarryprognosticweight[69], and both play
important roles in the prospective evaluation for a given
treatment regimen.
Over the last decades several candidate molecular mark-
ers have been discovered. Nevertheless, for almost all of these
markers it is necessary to obtain tumor tissue for molecular
analysis by at least a biopsy. In contrast, a noninvasive
molecular marker, such as CTCs [67], germline polymor-
phisms [70], or DNA-methylation [71]mayoer a quick
and noninvasive approach. For instance, monitoring of pre-
and postoperative CTC survivin-mRNA levels may provide
important clinical utility in predicting the completeness of
a surgical resection (molecular R0 marker) [31]. Although
tumor markers such as CEA are widely used for the follow-
up of patients with GI malignancies, their lack of sensitivity
remains unsolved. As such, detection and characterization
of CTCs may oer a novel approach, that can be used for
the estimation of risk for tumor recurrence and metastatic
Journal of Oncology 7
disease, stratification of patients to chemotherapy, iden-
tification of novel therapeutic targets, and monitoring of
systemic anticancer therapies. Nevertheless, it is becoming
increasingly apparent that disease progression depends on
numerous complex pathways, and that the analysis of one
single CTC-marker is unlikely to precisely predict progres-
sion of disease with sucient accuracy and reproducibility.
In addition, currently available studies are hampered by
small sample sizes, heterogeneous patient populations, and
most importantly the lack of standardized methodologies for
the detection and characterization of CTCs. Ongoing and
future clinical trials hold promise for further improvements
in optimizing and specifying (radio-) chemotherapy individ-
ually, not only prolonging lives but also improving quality of
life.
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... They have been found to be of clinical utility in predicting response to treatment and prognosis in several malignancies like breast and prostate cancer [1][2][3][4]. There are, however, limited reports about the significance of CTC in patients with gastrointestinal malignancy [5]. In a study about the significance of CTC in patients with diverse gastrointestinal cancers including 23 patients with metastatic esophageal cancer, about 21% (5/23) of these patients were observed to be CTC-positive and had a significantly shorter overall survival than CTC-negative patients [6]. ...
... In addition, multivariate analysis identified the presence of CTC as a strong, independent prognostic marker of tumor recurrence and overall survival [7]. Technically-different methods for the detection of CTC have been developed, including immunocytochemistry, reverse transcription polymerase chain reaction, flow-cytometry, and isolation by size (filtration) [5,[8][9][10]. ...
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Background: Isolation of circulating tumor cells (CTC) holds the promise to improve response-prediction and personalization of cancer treatment. In this study, we test a filtration device for CTC isolation in patients with non-metastatic esophageal adenocarcinoma (EAC) within recent multimodal treatment protocols. Methods: Peripheral blood specimens were drawn from EAC patients before and after neoadjuvant chemotherapy (FLOT)/chemoradiation (CROSS) as well as after surgery. Filtration using ScreenCell® devices captured CTC for cytologic analysis. Giemsa-stained specimens were evaluated by a cytopathologist; the cut-off was 1 CTC/specimen (6 mL). Immunohistochemistry with epithelial (pan-CK) and mesenchymal markers (vimentin) was performed. Results: Morphologically diverse malignant CTCs were found in 12/20 patients in at least one blood specimen. CTCs were positive for both vimentin and pan-CK. More patients were CTC positive after neoadjuvant therapy (6/20 vs. 9/15) and CTCs per/ml increased in most of the CTC-positive patients. After surgery, 8/13 patients with available blood specimens were still CTC positive. In clinical follow-up, 5/9 patients who died were CTC-positive. Conclusions: Detection of CTC by filtration within multimodal treatment protocols of non-metastatic EAC is feasible. The rate of CTC positive findings and the quantity of CTCs changes in the course of multimodal neoadjuvant chemoradiation/chemotherapy and surgery.
... Circulating tumor cells (CTC), various metabolites found in bile, blood, or urine, as well as extracellular vesicles represent some of other biomarkers currently evaluated for diagnosis and outcome prediction in patients with CCA [69]. While metabolites and extracellular vesicles have been investigated mostly for diagnostic purpose and differentiation between CCA and other hepatic malignancies or non-malignant biliary diseases, CTC have shown an association with DFS and OS in numerous malignancies. ...
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Cholangiocarcinoma (CCA) is the second most common primary liver cancer and subsumes a heterogeneous group of malignant tumors arising from the intra- or extrahepatic biliary tract epithelium. A rising mortality from CCA has been reported worldwide during the last decade, despite significant improvement of surgical and palliative treatment. Over 50% of CCAs originate from proximal extrahepatic bile ducts and constitute the most common CCA entity in the Western world. Clinicopathological characteristics such as lymph node status and poor differentiation remain the best-studied, but imperfect prognostic factors. The identification of prognostic molecular markers as an adjunct to traditional staging systems may not only facilitate the selection of patients who would benefit the most from surgical, adjuvant or palliative treatment strategies, but may also be helpful in defining the aggressiveness of the disease and identifying patients at high-risk for tumor recurrence. The purpose of this review is to provide an overview of currently known molecular prognostic and predictive markers and their role in CCA.
... Other methods, such as immunocytochemical characterization, which separate CTCs by their distinct morphological features, are also available [14]. As already mentioned, the detection of CTCs in an individual patient's blood as well as the number of CTCs has been correlated with metastasis, disease status [15], and clinical outcome, for example, in HER-2-positive breast cancer [16] or thyroid cancer [17]. However, before becoming CTCs, the epithelial tumor cells have to undergo epithelial-to-mesenchymal transition (EMT) to be able to migrate, invade their surroundings, and finally enter the circulation [18]. ...
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The perioperative use of regional anesthesia and local anesthetics is part of almost every anesthesiologist’s daily clinical practice. Retrospective analyses and results from experimental studies pointed towards a potential beneficial effect of the local anesthetics regarding outcome—i.e., overall and/or recurrence-free survival—in patients undergoing cancer surgery. The perioperative period, where the anesthesiologist is responsible for the patients, might be crucial for the further course of the disease, as circulating tumor cells (shed from the primary tumor into the patient’s bloodstream) might form new micro-metastases independent of complete tumor removal. Due to their strong anti-inflammatory properties, local anesthetics might have a certain impact on these circulating tumor cells, either via direct or indirect measures, for example via blunting the inflammatory stress response as induced by the surgical stimulus. This narrative review highlights the foundation of these principles, features recent experimental and clinical data and provides an outlook regarding current and potential future research activities.
... GC is responsible for the highest number of cancer-related mortalities (74), and the egregious mortality of GC is immeasurably more acute than ever before, primarily since the majority of patients have a terminal disease at stage III or IV at the time of diagnosis (75). In addition, there are many investigated biomarkers, such as CEA and CA724, which lack sufficient sensitivity and specificity for early GC diagnosis (76), and universal screening tools, such as endoscopic examinations and biopsies, are invasive, unpleasant, and inconvenient, leading to potential errors in GC detection. ...
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Recently, cancer research microRNA studies have drawn great attention. However, the results of these studies have been inconsistent and variable regarding the availability of circulating miRNAs in gastric cancer (GC) diagnosis. Thus, results should be interpreted cautiously. The purpose of the present study was to assess the diagnostic performance of circulating miRNAs in GC diagnosis. We conducted a systematic and comprehensive approach for the inclusion of studies. The sensitivity, specificity, and diagnostic odds ratio were pooled with random effects models, and a summary of receiver operator characteristic (SROC) curves were plotted. The potential heterogeneity was assessed with Q test and I2 statistics. Subgroup analyses and meta‑regressions further investigated the sources of heterogeneity. A total of 77 studies from 48 articles were eligible for the meta‑analysis. The results revealed a sensitivity of 0.76, a specificity of 0.81, and an AUC of 0.86 for gastric cancer diagnosis with circulating miRNAs. In addition, subgroup analyses indicated that multiple miRNAs assays, non‑microarray screening approaches, and serum‑based miRNA assays exhibited good diagnostic performance in contrast to a single miRNA assay, microarray expression profiling screening, and plasma‑based miRNA group analysis. The diagnostic ability of miRNAs in early stage I‑II groups and the high expression group were approximately similar to that in the stage I‑IV groups and the low expression group. For the circulating miRNAs, our meta‑analysis identified a combination of multiple miRNAs, non‑microarray chip screening, and serum‑based miRNA assays were associated with the most effective GC diagnostic performance. However, many unclear molecular mechanisms limited the accuracy of the diagnostic results, and should be interpreted with caution. Further large‑scale prospective studies are required for validating the diagnostic applicability of circulating miRNAs in gastric cancer patients.
... [21] Conforming to the historical concept of "seed and soil", by Stephen Paget the CTCs are believed to metastasize to certain organs depending on the origin, type and site of the primary tumor. [22] An average number of 11 million leukocytes and 5-7 billion erythrocytes are present in one ml of blood. Detection of single CTC per ml of blood is expected to have clinical importance. ...
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Objectives: To evaluate the incidence of CTCs in peripheral blood of OSCC patients. To correlate the incidence of CTCs with the lymph node status of OSCC patients. To correlate the incidence of CTCs with istopathological grading of OSCC and to correlate the incidence of CTCs pre-operatively and 6 weeks post-operatively. Design: This study was carried out in the Dept. of Oral Pathology and Microbiology, Sharad Pawar Dental College. The study group consisted of 60 cases of histopathologically diagnosed OSCC and 10 Controls from normal healthy individuals. 2ml peripheral blood was collected pre-operatively and 6 weeks post-operatively from histopathologically diagnosed OSCC patients. Smears were prepared from the collected blood samples and were stained using haematoxylin-eosin(H&E) stain. The prepared slides were examined for circulating tumour cells under light microscope under 10x, 40x & 100x. Results: We detected CTCs in 40% of the cases of OSCC included in our study. The incidence of CTCs increases progressively from well to moderate to poorly differentiated squamous cell carcinoma. Though CTC could be frequently detected in N2b cases, they were also found in N0 cases. There was no correlation between size of tumour and number of CTCs. In most cases post-op analysis reveal lack of CTCs but in few cases they were found significantly.Conclusion: The counting of CTCs at diagnosis, after surgical resection and during chemotherapy can anticipate clinical outcome and ultimately change the direction of treatment.
... The detection of CTCs in peripheral blood has received growing enthusiasm in the clinical utility of various cancers. Since 1990s many studies have been performed to indicate the correlation between CTCs and the clinicopathological characteristics of gastric tumor [33][34][35][36][37][38][39][40][41][42][69][70][71]. These studies suggest that CTCs may play a role in predicting tumor prognosis and response to treatment. ...
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Circulating tumor cells (CTCs) are rare cancer cells released from tumors into the blood stream that are thought to have a key role in cancer metastasis. Investigation of CTCs is an exciting area of research but remains in its infancy, and the presence of CTCs has been associated with worse prognosis in several major cancer types. Gastric cancer (GC) is a highly lethal malignancy and a serious public health concern in East Asia especially in China. There is an urgent need for identifying new, better prognostic markers to enhance diagnosis and prognosis, facilitate drug development, and to improve the treatment of gastric cancer patients. There are considerable interests in gastric CTCs given their potential use as gastric cancer biomarkers. This review highlights recent advances in studies of gastric CTCs, including the isolation and biological molecular characteristics of gastric CTCs, and their clinical significance.
... CTCs can play a crucial role in the development of metastasis. Several groups have explored techniques to detect CTCs (Hong & Zu, 2013;Lurje, Schiesser, Claudius, & Schneider, 2010). However, CTCs are present at extremely low concentrations-as low as one CTC in 10 6 -10 7 leukocytes in peripheral blood of cancer patients (Hong & Zu, 2013). ...
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Metastasis is the complex process by which primary tumor cells migrate and establish secondary tumors in an adjacent or distant location in the body. Early detection of metastatic disease and effective therapeutic options for targeting these detected metastases remain impediments to effectively treating patients with advanced cancers. If metastatic lesions are identified early, patients might maximally benefit from effective early therapeutic interventions. Further, monitoring patients whose primary tumors are effectively treated for potential metastatic disease onset is also highly valuable. Finally, patients with metastatic disease can be monitored for efficacy of specific therapeutic interventions through effective metastatic detection techniques. Thus, being able to detect and visualize metastatic lesions is key and provides potential to greatly improve overall patient outcomes. In order to achieve these objectives, researchers have endeavored to mechanistically define the steps involved in the metastatic process as well as ways to effectively detect metastatic progression. We presently overview various preclinical and clinical in vitro and in vivo assays developed to more efficiently detect tumor metastases, which provides the foundation for developing more effective therapies for this invariably fatal component of the cancerous process.
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