Circulating tumor cells in breast cancer: functional heterogeneity, pathogenetic and clinical aspects
Each patient has a unique history of cancer ecosystem development, resulting in intratumor heterogeneity. In order to effectively kill the tumor cells by chemotherapy, dynamic monitoring of driver molecular alterations is necessary to detect the markers for acquired drug resistance and find the new...
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irk-123456789-1376042018-06-18T03:05:58Z Circulating tumor cells in breast cancer: functional heterogeneity, pathogenetic and clinical aspects Cherdyntseva, N.V. Litviakov, N.V. Denisov, E.V. Gervas, P.A. Cherdyntsev, E.S. Reviews Each patient has a unique history of cancer ecosystem development, resulting in intratumor heterogeneity. In order to effectively kill the tumor cells by chemotherapy, dynamic monitoring of driver molecular alterations is necessary to detect the markers for acquired drug resistance and find the new therapeutic targets. To perform the therapeutic monitoring, frequent tumor biopsy is needed, but it is not always possible due to small tumor size or its regression during the therapy or tumor inaccessibility in advanced cancer patients. Liquid biopsy appears to be a promising approach to overcome this problem, providing the testing of circulating tumor cells (CTC) and/or tumor-specific circulating nucleic acids. Their genomic characteristics make it possible to assess the clonal dynamics of tumors, comparing it with the clinical course and identification of driver mutation that confer resistance to therapy. The main attention in this review is paid to CTC. The biological behavior of the tumor is determined by specific cancerpromoting molecular and genetic alterations of tumor cells, and by the peculiarities of their interactions with the microenvironment that can result in the presence of wide spectrum of circulating tumor clones with various properties and potentialities to contribute to tumor progression and response to chemotherapy and prognostic value. Indeed, data on prognostic or predictive value of CTC are rather contradictory, because there is still no standard method of CTC identification, represented by different populations manifesting various biological behavior as well as different potency to metastasis. Circulating clasters of CTC appear to have essentially greater ability to metastasize in comparison with single CTC, as well as strong association with worse prognosis and chemoresistance in breast cancer patients. The Food and Drug Administration (USA) has approved the CTC-based prognostic test for clinical application in patients with advanced breast cancer. Prospective clinical trials have demonstrated that measuring changes in CTC numbers during treatment is useful for monitoring therapy response in breast cancer patients. Molecular and genetic analysis of CTC gives the opportunity to have timely information on emergence of resistant tumor clones and may shed light on the new targets for pathogenetic antitumor therapy. 2017 Article Circulating tumor cells in breast cancer: functional heterogeneity, pathogenetic and clinical aspects / N.V. Cherdyntseva, N.V. Litviakov, E.V. Denisov, P.A. Gervas, E.S. Cherdyntsev // Experimental Oncology. — 2017 — Т. 39, № 1. — С. 2-11. — Бібліогр.: 84 назв. — англ. 1812-9269 http://dspace.nbuv.gov.ua/handle/123456789/137604 en Experimental Oncology Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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Reviews Reviews Cherdyntseva, N.V. Litviakov, N.V. Denisov, E.V. Gervas, P.A. Cherdyntsev, E.S. Circulating tumor cells in breast cancer: functional heterogeneity, pathogenetic and clinical aspects Experimental Oncology |
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Each patient has a unique history of cancer ecosystem development, resulting in intratumor heterogeneity. In order to effectively kill the tumor cells by chemotherapy, dynamic monitoring of driver molecular alterations is necessary to detect the markers for acquired drug resistance and find the new therapeutic targets. To perform the therapeutic monitoring, frequent tumor biopsy is needed, but it is not always possible due to small tumor size or its regression during the therapy or tumor inaccessibility in advanced cancer patients. Liquid biopsy appears to be a promising approach to overcome this problem, providing the testing of circulating tumor cells (CTC) and/or tumor-specific circulating nucleic acids. Their genomic characteristics make it possible to assess the clonal dynamics of tumors, comparing it with the clinical course and identification of driver mutation that confer resistance to therapy. The main attention in this review is paid to CTC. The biological behavior of the tumor is determined by specific cancerpromoting molecular and genetic alterations of tumor cells, and by the peculiarities of their interactions with the microenvironment that can result in the presence of wide spectrum of circulating tumor clones with various properties and potentialities to contribute to tumor progression and response to chemotherapy and prognostic value. Indeed, data on prognostic or predictive value of CTC are rather contradictory, because there is still no standard method of CTC identification, represented by different populations manifesting various biological behavior as well as different potency to metastasis. Circulating clasters of CTC appear to have essentially greater ability to metastasize in comparison with single CTC, as well as strong association with worse prognosis and chemoresistance in breast cancer patients. The Food and Drug Administration (USA) has approved the CTC-based prognostic test for clinical application in patients with advanced breast cancer. Prospective clinical trials have demonstrated that measuring changes in CTC numbers during treatment is useful for monitoring therapy response in breast cancer patients. Molecular and genetic analysis of CTC gives the opportunity to have timely information on emergence of resistant tumor clones and may shed light on the new targets for pathogenetic antitumor therapy. |
| format |
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| author |
Cherdyntseva, N.V. Litviakov, N.V. Denisov, E.V. Gervas, P.A. Cherdyntsev, E.S. |
| author_facet |
Cherdyntseva, N.V. Litviakov, N.V. Denisov, E.V. Gervas, P.A. Cherdyntsev, E.S. |
| author_sort |
Cherdyntseva, N.V. |
| title |
Circulating tumor cells in breast cancer: functional heterogeneity, pathogenetic and clinical aspects |
| title_short |
Circulating tumor cells in breast cancer: functional heterogeneity, pathogenetic and clinical aspects |
| title_full |
Circulating tumor cells in breast cancer: functional heterogeneity, pathogenetic and clinical aspects |
| title_fullStr |
Circulating tumor cells in breast cancer: functional heterogeneity, pathogenetic and clinical aspects |
| title_full_unstemmed |
Circulating tumor cells in breast cancer: functional heterogeneity, pathogenetic and clinical aspects |
| title_sort |
circulating tumor cells in breast cancer: functional heterogeneity, pathogenetic and clinical aspects |
| publisher |
Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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2017 |
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Reviews |
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http://dspace.nbuv.gov.ua/handle/123456789/137604 |
| citation_txt |
Circulating tumor cells in breast cancer: functional heterogeneity, pathogenetic and clinical aspects / N.V. Cherdyntseva, N.V. Litviakov, E.V. Denisov, P.A. Gervas, E.S. Cherdyntsev // Experimental Oncology. — 2017 — Т. 39, № 1. — С. 2-11. — Бібліогр.: 84 назв. — англ. |
| series |
Experimental Oncology |
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2025-07-10T04:06:27Z |
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2025-07-10T04:06:27Z |
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| fulltext |
2 Experimental Oncology 39, 2–11, 2017 (March)
CIRCULATING TUMOR CELLS IN BREAST CANCER: FUNCTIONAL
HETEROGENEITY, PATHOGENETIC AND CLINICAL ASPECTS
N.V. Cherdyntseva1, 2, *, N.V. Litviakov1, 2, E.V. Denisov1, 2, P.A. Gervas2, E.S. Cherdyntsev3
1Tomsk Cancer Research Institute, Tomsk National Research Medical Centre of Russian
Academy of Science, Tomsk 634050, Russia
2Tomsk State University, Tomsk 634050, Russia
3Tomsk Polytechnic University, Tomsk 634050, Russia
Each patient has a unique history of cancer ecosystem development, resulting in intratumor heterogeneity. In order to effectively
kill the tumor cells by chemotherapy, dynamic monitoring of driver molecular alterations is necessary to detect the markers for
acquired drug resistance and find the new therapeutic targets. To perform the therapeutic monitoring, frequent tumor biopsy
is needed, but it is not always possible due to small tumor size or its regression during the therapy or tumor inaccessibility in advanced
cancer patients. Liquid biopsy appears to be a promising approach to overcome this problem, providing the testing of circulating
tumor cells (CTC) and/or tumor-specific circulating nucleic acids. Their genomic characteristics make it possible to assess the
clonal dynamics of tumors, comparing it with the clinical course and identification of driver mutation that confer resistance
to therapy. The main attention in this review is paid to CTC. The biological behavior of the tumor is determined by specific cancer-
promoting molecular and genetic alterations of tumor cells, and by the peculiarities of their interactions with the microenvironment
that can result in the presence of wide spectrum of circulating tumor clones with various properties and potentialities to contribute
to tumor progression and response to chemotherapy and prognostic value. Indeed, data on prognostic or predictive value of CTC
are rather contradictory, because there is still no standard method of CTC identification, represented by different populations
manifesting various biological behavior as well as different potency to metastasis. Circulating clasters of CTC appear to have es-
sentially greater ability to metastasize in comparison with single CTC, as well as strong association with worse prognosis and
chemoresistance in breast cancer patients. The Food and Drug Administration (USA) has approved the CTC-based prognostic test
for clinical application in patients with advanced breast cancer. Prospective clinical trials have demonstrated that measuring
changes in CTC numbers during treatment is useful for monitoring therapy response in breast cancer patients. Molecular and
genetic analysis of CTC gives the opportunity to have timely information on emergence of resistant tumor clones and may shed light
on the new targets for pathogenetic antitumor therapy.
Key Words: breast cancer, circulating tumor cells, intratumor heterogeneity, chemotherapy, liquid biopsy, prognosis of the disease course.
CLONAL EVOLUTION OF TUMORS
PROVIDES INTRATUMOR HETEROGENEITY
The modern oncology paradigm was declared
by Peter Nowell, who was the first to describe cancer
as complex and branching evolutionary trajectories,
in parallel with Darwin’s iconic evolutionary speciation
tree. His concept was described in 1976 in his paper
entitled “Clonal evolution of tumor cell population”,
which was published in “Science” journal [1]. A classi-
cal or Darwinian evolutionary system embodies a basic
principle: purposeless genetic variation of reproduc-
tive individuals, united by common descent, coupled
with natural selection of the fittest variants [2].
Cancer is a process of a clonal evolution where
sequential acquisition of mutations with concomi-
tant, successive subclonal dominance or selective
sweeps results in the tumors with various molecular
aberrations, thus requiring personalized approach
to treatment. Intratumor clonal heterogeneity, mani-
festing as a coexistence of tumor cells with different
genotypes and phenotypes within the same tumor,
is considered to be an essential driving force providing
tumor clonal evolution, progression, and resistance
to chemo- and radiotherapy. The clonal heterogeneity
of different primary tumor sites and differences in tu-
mor clones between primary tumors and metastases
fail to provide a proper or accurate diagnosis as well
as successful prognosis and treatment of cancer.
Breast cancer (BC) is the most common malignancy
and is the leading cause of cancer-related mortality
of women in developing countries [3]. The high BC mor-
tality is directly endowed by the failure in early detection
of the disease and the lack of effective markers to esti-
mate the risk of cancer progression and to predict tumor
response to chemotherapy and radiotherapy. Largely
unsuccessful attempts to develop new approaches
to prediction of response to therapy, and effective
evaluation of the clinical course of BC can be mainly
attributed to high intratumoral variety of this disease.
Invasive carcinoma of no special type characterized
by a significant intratumor morphological heterogeneity
is the most common histological type of BC, accounting
for approximately 80% of all cases. We determined the
tubular structures as well as trabecular, solid, alveolar,
Submitted: December 06, 2016.
*Correspondence: E-mail: nvch@tnimc.ru
Abbreviations used: ALDH — aldehydedehydrogenase; BC —
breast cancer; СK — cytokeratin; CNV — copy number variation;
CTC — circulating tumor cells; DTC — disseminated tumor cells;
EGFR — epidermal growth factor receptor; EMT — epithelial-me-
senchymal transition; EpCAM — epithelial cell adhesion molecule;
HER2/neu — receptor to epidermal growth factor; PCR — poly-
merase chain reaction; TIMP1 — tissue inhibitor of matrix metal-
loprotease-1; TKIs — tyrosine kinase inhibitors.
Exp Oncol 2017
39, 1, 2–11
REVIEW
Experimental Oncology 39, 2–11, 2017 (March)39, 2–11, 2017 (March) (March) 3
and discrete groups of tumor cells displaying archi-
tectural arrangement within the primary tumors [4, 5].
The morphological structure of the primary tumor was
reported to be evidently related to clinical outcome,
resulting from cancer progression clinically manifested
as invasion, lymph node involvement, and distant
metastases consistent with results obtained from
analyzing the large cohorts of BC patients, the mor-
phological structure of the primary tumor was reported
to be evidently related to clinical outcome, resulting
from cancer progression clinically manifested as inva-
sion, lymph node involvement, and distant metastases.
A contribution of intratumor morphological heteroge-
neity to chemotherapy efficiency was also shown [4,
5]. High risk of lymphogenous metastasis was shown
to be related with the presence of alveolar structures
in tumors of postmenopausal women and with the
increased number of different types of morphological
structures in premenopausal women. The detection
of alveolar structures and the greatest morphological
diversity of breast tumors appeared to be associated
with resistance to neoadjuvant chemotherapy [4–6].
In our study, the microarray transcriptional analysis
of different morphological structures of breast tumors
showed specific gene expression sets and non-coding
sequences for each type of morphological structures
as compared to normal breast epithelial cells. Con-
sistent with our data, different types of morphological
structures in breast tumors belong to the functionally-
separated populations of tumor cells displaying ex-
pression of various specific genes. Essential variations
in the expression pattern of signaling pathways endow-
ing tumor chemoresistance and contributing to tumor
cell invasion of different structures were also shown
(data are being prepared for publication).
Significant differences in the spectrum and func-
tional activity of the major cancer-related signaling
pathways in different types of the above mentioned
parenchymal structures corroborate their different
contribution to tumor progression and serve as the
basis for the identification of new prognosis markers
and targets for therapeutic intervention.
Chemotherapy may destroy certain sensitive can-
cer clones and erode their habitats, but it can also
provide a potent selective pressure for the expansion
of resistant variants. Chemotherapy and radiotherapy
induce the clonal evolution of tumor cell population,
leading to the expansion of dominating drug resistant
tumor clones, which are considered to be the major
cause of tumor progression and anticancer treatment
failures. Identification of the molecular mechanisms
responsible for multidrug resistance could provide the
opportunity for more effective control of tumor growth
and progression [7].
Tumor clone evolution is forwarded through the
interaction between tumor cells and microenviron-
ment as well as under the influence of genotoxic
factors resulting in the occurrence of new mutations
under conditions of high genetic instability. Tumors are
composed of cells with driver changes and cell clones
carrying neutral mutations, which are not selected,
as well as a plurality of cells with random changes,
which often may promote tumor growth. On the other
hand, tumor cells can also dramatically influence to the
microenvironment during evolution process, suppor-
ting tumor growth.
The primary driver mutations result in the activation
of oncogenic signalling pathways and/or inactivation
of tumor suppressors. They are important for tumor
(carcinogenesis) and actually provide its biological
behavior, determining the clinical course and outcome
of the malignant process. The increased genetic insta-
bility in the primary malignant clone leads to the gene-
ration of new mutations (so-called secondary driver
mutations) giving rise to new subclones, which may
become dominant in the tumor over time. Passenger
mutations are random single mutations that can both
contribute to cancer progression and do not have any
effect on the tumor behavior [8–11] (Fig. 1).
b ca
Fig. 1. Different types of tumor clonal architecture. Tumor
progression is presented as a growing tree. The distribution
of primary (1) and secondary (2) driver mutations as well as pas-
senger mutations (3) within the tumor tree. Three types of tumor
clonal architecture varying in the subclone amount were identi-
fied. The poor response likelihood depends on the type of tumor
tree. Type “a” tumors contain more primary driver mutations
and show good response to treatment. Other tumors (“c”) have
a high genetic diversity, manifested in a greater representation
of the secondary driver mutations and mutations “passengers”,
and as a result, display poor therapeutic effect. Type “b” tumor
response to therapy has intermediate value [10–12]
The dynamics of the somatic cell evolution is de-
pendent on the rate of mutation processes, genetic
diversity and clonal expansion, and may be modified
by such events as clonal interference (competition
of clones having an advantage in the same adaptive
environment) or parallel expansion with subsequent
appearance of dominant clones [12–14]. The epige-
netic alterations, which are acquired more rapidly than
genetic changes, contribute significantly to the clonal
evolution. They can be inherited during cell division
and may determine the tumor phenotype [15, 16].
Cancer tissue ecosystems provide the architectonic
space and driving determinants for fitness selection,
i.e. the so-called adaptive landscape [7, 17]. The tumor
microenvironment is composed of multiple dynamically
interacting components that can influence cancer clone
evolution. For example, TGF-β, promoting dissemina-
tion of cancer cells through the induction of epithelial-
mesenchymal transition (EMT), is one of the critically
important molecules regulating the tumor ecosystem,
along with other inflammatory cytokines [12, 18].
The reciprocal interactions between tumor cells
and the tumor microenvironment are regulated
by both systemic factors (nutrients, hormones), and
4 Experimental Oncology 39, 2–11, 2017 (March)
mediators, which are produced by tumor-associated
fibroblasts, endothelial cells, mesenchimal progeni-
tor cells or different types of infiltrating inflammatory
cells and cancer cells itself. In each case, the exter-
nal environment, lifestyle, exposures to genotoxic
agents, constitutive genetics of the host cells, sys-
temic regulators, local regulators (microenvironment)
and architectural constraints provide the evolution
of somatic cells, tumor biological behavior and out-
come [7].
Tumor cells can re-modulate the microenviron-
ment and create niches to endow their competitive
advantage in growth and dissemination. The interac-
tion between tumor cells and the microenvironment
can be dramatically modulated by chemotherapy
or radiation therapy. Although the majority of cancer
cells can be killed by cytotoxic agents, the landscape
remodeling creates conditions for the selection and
expansion of minor variants of tumor cells insensitive
to treatment [19].
Thus, each patient has a unique history of the
tumor development, and to have benefit from che-
motherapy, it is necessary to monitor molecular
changes that can serve as both markers of acquired
resistance and targets for the effects of other drugs.
Chemotherapy, including targeted therapy, can
be considered as the guiding factor in the cancer cell
clone evolution, inducing the expansion of resistant
clones instead of dead sensitive cells. For success-
ful treatment, it is necessary to know what a new
driver clone has appeared during the chemotherapy
to choose a proper drug for further therapy. For ex-
ample, patients with mutations in the tyrosine kinase
domain of epidermal growth factor receptor (EGFR),
whose tumors initially responded to chemotherapy
with tyrosine kinase inhibitors (TKIs), would develop
resistance to these TKIs. The acquired resistance
to TKIs is provided by the appearance of additional
mutations in the EGFR gene (particularly, T790M) [20]
or in the chimeric gene ALK [21], altering the spatial
structure of the coding protein, thereby abrogating
its interaction with the inhibitor.
The identification of new driver mutations promotes
the development of new drugs. For example, afatinib,
a recently introduced new TKI, can be used for lung
cancer patients. This inhibitor manifests its activity
against T790M mutation occurring in 50% of patients
who showed resistance during the treatment with
EGFR-targeted TKIs gefitinib or erlotinib [22].
The development of drug resistance phenotype,
contributing to insensitivity of tumor to chemotherapy
in 80–90% of cases is one of the crucial mechanisms
of cancer progression [23, 24].
To estimate chemoresistance of intratumor
morphological heterogeneity of BC, we studied the
expression levels of different genes, coding the ABC-
transporter family, in various parenchymal structures,
such as alveolar, tubular, trabecular, solid structures
and individual cells. We showed the different gene
expression profiles in various structures and revealed
that the activation of ABC gene expression occurred
most frequently in the trabecular structures [25].
In our study we also directly showed the partial
destruction of tumor clones during neoadjuvant
chemotherapy for BC patients, using the detec-
tion of copy number variation (CNV) as a numerical
chromosome aberration, namely deletions or ampli-
fications, in various loci. To study the CNV, microar-
ray analysis was performed using the high density
microarray platform Affymetrix (USA), CytoScanTM
HD Array [26, 27]. We tested CNV in breast tumor
biopsy before treatment and in surgical specimens
after preoperative chemotherapy. We observed par-
tial elimination of tumor clones carrying deletions
and amplifications in a patient who had achieved
clinical partial regression of the primary tumor after
preoperative chemotherapy (Fig. 2). In the patient
E, whose tumor progressed while recei ving chemo-
therapy, along with the disappearance of the clones,
the emergence of new clones was observed, which
appeared to provide drug resistance and tumor pro-
gression (metastasis to bones, soft tissues, lungs
and cervical lymph nodes).
ba
Fig. 2. Change of tumor clones during the preoperative che-
motherapy in patients with BC. CNV of DNA (deletion or ampli-
fication of chromosomal regions and individual chromosomes)
in breast tumor cells were determined. High density microarray
(Affimetrix Cytoscan HD Array), which is able to detect the entire
spectrum of mutant tumor clones was used. Red shows dele-
tions, blue — amplification. The genetic landscape of a tumor
prior (a) to treatment and after (b) treatment [27]
The biological behavior of the tumor is determined
by specific cancer-promoting molecular and genetic
alterations of tumor cells, and by the peculiarities
of their interactions with the microenvironment. There-
fore, the study of the properties of tumor cells is ne-
cessary to provide successful cancer care of patients,
including diagnosis, choice of therapy, monitoring
of the treatment efficacy, prognosis of disease course
and prediction of therapy response.
Moreover, the molecular profiling of tumors and the
development of the so-called precision oncology are
becoming increasingly important in the management
and therapy of cancer patients. A precision approach
requires monitoring of the natural molecular evolution
of individual tumors to develop the appropriate tar-
geted therapies for each patient [28]. The personalized
medicine assumes the systemic use of patient-specific
genetic information (both germline and somatic) and
molecular or/and cellular tumor characteristics to se-
lect the optimal treatments with the goal of improved
therapeutic efficacy and reduced toxicity [29–31]. The
precision medicine is considered as a general trend
in the development of targeted therapy.
Experimental Oncology 39, 2–11, 2017 (March)39, 2–11, 2017 (March) (March) 5
CIRCULATING TUMOR CELLS: DEFINITION
AND DETECTION
In clinical practice, the objective obstacles exist
to obtain a sufficient amount of tumor material that can
be tested at different steps of examination and treat-
ment of cancer patients. The restrictions are caused
by impossibility to perform multiple biopsies, lack
of sufficient amount of tumor tissue or its complete
disappearance on pathological examination in patients
with complete pathological response. It is also impor-
tant that the material derived from a limited number
of tumor sites, may not accurately reflect the tumor
heterogeneity, because the different tumor sites may
contain different cell clones with specific molecular
characteristics and biological behavior. This can lead
to diagnostic errors and misinterpretation of molecular
testing results to prescribe specific targeted therapies.
The testing of tumor properties using circulating
tumor cells (CTC) and/or circulating tumor-specific
nucleic acids derived from blood-sample (“liquid bio-
psy”) is considered a promising alternative to analysis
of tumor biopsy. Molecular characteristics of circulat-
ing tumor-specific DNA/RNA or CTC will enable the
assessment of tumor clonal dynamics related to clini-
cal course and detection of driver genetic changes
conferring resistance to therapy. In this review we fo-
cused on CTC that gave rise to the term “liquid biopsy”,
proposed by the National Institute of Health (USA),
as opposed to a standard tissue biopsy and later
nucleic acids included in notion “liquid biopsy” [32].
However, it should be noted that the phenomenon
of intratumoral heterogeneity resulted from the pro-
cess of tumor clonal evolution through the interaction
between tumor cells and microenvironment as well
as under the influence of chemotherapeutic agents
may provide the failure of “liquid biopsy” for disease
prognosis and prediction of chemotherapy response.
This is due to the presence of cell clones within
a tumor with different functional properties providing
diversity in their biological behavior responsible for
clinical course of disease (metastasis) and sensitivity
(resistance) to therapy. Therefore, one can expect
the coexistence of wide spectrum of circulating tu-
mor clones with various properties and potentialities
to contribute to tumor progression and response
to chemotherapy. Considering the behavioral varia-
bility of distinct subpopulations of CTC in the blood
we can assumed that they have different prognostic
and predictive value.
In this review numerous experimental and clinical
results were analyzed to show the diversity and vari-
ability in phenotype and genotype of CTC and their
prognostic significance. Data on prognostic or predic-
tive value of CTC are rather contradictory, because
there are still no standard appropriate methods of CTC
identification, represented by different populations
manifesting various biological behavior endowing
different potency to metastasis.
Tumor cells in the bloodstream are detected after
the tumor removal in 30% of patients with different
malignancies, including BC. They are also detected
in the bone marrow of 20–60% of BC patients (so-
called disseminated tumor cells — DTC). It is known
that BC metastases may occur even 20 years after the
treatment, and more than 30% of the patients without
clinical evidence of the disease appeared to have CTC
in the blood stream, indicating that tumor cells can
survive in a state of dormancy for long periods [33].
Since it is known that the intravasation of tumor
cells from primary tumor into the circulation is a obliga-
tory prerequisite for metastasis [34], it is quite obvi-
ous that the presence of CTC may reflect the risk
of metastatic disease in a particular patient. There
are many published studies evaluating the prognostic
significance of CTC as a factor of high risk for meta-
stasis [35, 36]. However, there are also other studies
that have not shown that CTCs are able to predict the
risk of cancer dissemination [37, 38].
There are the following reasons for these contro-
versial results: lack of a standard method of identifi-
cation of CTC, lack of fundamental knowledge of the
phenotypic and genetic characteristics of different
CTC populations, mechanisms of metastasis, and the
absence of validated information about their relation
with the clinical course of the disease.
Along with single CTC, the circulating clusters
or micrometastases (2–50 united cell) have been
found in the blood of patients with various malignan-
cies [39]. CTC clasters, which are responsible for the
development of future metastases, are a result of the
collective invasion and subsequent intravasation of tu-
mor cells into lymphatic or blood vessels. It is assumed
that their penetration into the blood vessels occurs
in the areas of destruction of the endothelium [40],
and is promoted by tumor cells undergoing EMT [41]
as well as by cooperation with tumor-associated fi-
broblasts [42], eventually contributing to proteolysis
of the vascular walls. Vascular endothelial growth
factor has been shown to contribute to the collective
cell intravasation into blood stream and accumulation
of tumor clusters [43].
A recent study using a mouse model of BC has
shown that CTC clusters have oligoclonal origin from
primary tumor cell groups, thus confirming the fact
of intravasation and denying the opportunity of a ran-
dom aggregation of clusters from single CTC [44].
Along with the mechanism of cluster origin by invasion
of cell groups to the blood vessels, there are published
data on circulating clusters organized from single
proliferated cancer stem cell adhered to the endothe-
lium [45], or by the aggregation of single cancer stem
cell with blood cells, in particular with lymphocytes
and platelets. A recent study confirmed that CTC are
composed of tumor cells, although some cases (< 5%)
demonstrated the presence of immune system cells
expressing CD45, CD68, CD14 and other markers
in their structure. All CTC showed a low expression
of transcripts encoding epithelial CTC markers, such
as keratin, mucin 1 (MUC1), epithelial cell adhesion
molecule (EpCAM) and/or CDH1 [46, 47].
6 Experimental Oncology 39, 2–11, 2017 (March)
Numerous CTC detection techniques have been
developed so far. The cytological CTC identification
method was first proposed by Nabar in 1959. However,
this method was found to be low in specificity. The most
commonly used CTC detection approach is based
on the use of monoclonal antibodies against epithe-
lial markers, such as EpCAM and cytokeratin (CK),
provided that CTC do not express hematopoietic cell
markers. The Cellsearch System™ ® (Veridex, Warren,
NJ, USA), the first Food and Drug Administration-
approved commercial automated system developed
for the identification of EpCAM+, CKs 8, 18, 19+, CD45−
and nuclear cells, is considered as an effective tool for
determining prognosis in patients with metastatic BC,
prostate cancer and colorectal cancer [48].
Considering the fact that the CTC detection using
different markers can identify cell subpopulations
with different biological behavior, it is obvious that
the Cellsearch system has significant limitations be-
cause it detects the epithelial markers only. However,
it is known that EpCAM expression is downregulated
during EMT, and gain of mesenchymal markers such
as vimentin and fibronectin was found to correlate with
a worse prognosis more effectively than CK-positive
cells [49]. Recent studies show that CTC markers may
change over the course of therapy [50].
AdnaTestBreast™ test is based on the CTC detec-
tion by assessing the EpCAM gene expression [37].
However, a prospective German multicenter trial
(DETECT) showed no correlation between CTC and
disease-free or overall survival [37].
The microfluidic platform (the “CTC-chip”) deals
with small blood volumes and uses antibodies against
the common epithelial cell surface marker EpCAM [51].
Recently, a refined methodology called “herringbone-
chip”, or “HB-Chip”, has been developed to provide
an enhanced platform for CTC isolation. The CTC chip
was created for isolation of rare CTC in patients with
breast, lung and prostate cancers [52].
The immunospot assay (Elispot) method is also used
for detection of CTC or bone marrow DTC. Using this
method, only viable tumor cells are detected and appro-
priate cytokine secretion is studied at the individual cell
level. The reverse transcription quantitative polymerase
chain reaction (PCR), is used for detecting of CTC
marker gene expression, such as CK-19, mucin 1, mam-
moglobin, EGFR and CK-20. In the metastatic BC set-
ting, plasma levels of the miR-200 family, as well as a few
other circulating microRNAs, are highly correlated with
CTC in the blood and show great potential in predicting
the survival of these patients [53].
CTC isolated from blood by different methods can
be assessed by immunohistochemistry for specific
markers. Fluorescence in situ hybridization is used
to detect gene amplification or translocations. DNA
or RNA extracted from the CTC can be used for the
reverse transcription quantitative PCR to assay gene
expression profiling. However, the isolation of CTC
with high purity as well as the getting alive cells
to be cultivated in vitro is a technical problem. All
these approaches focus on the detection of already
known molecular changes, whereas the full genome
sequencing can be used for the detection of new aber-
rations, and the expression profiling for the detection
of previously unknown activated signaling pathways
providing CTC function.
Thus, it is obvious that the efficiency of using CTC
as markers depends on the detection method, as dif-
ferent types of CTC with different metastatic potential
can be identified. Different technologies may detect
different CTC subpopulation with different sensitivity
and purity.
PROPERTIES AND PHENOTYPE
OF CIRCULATING TUMOR CELLS
The common definition of CTC designate them
as epithelial cells with characteristics of the primary
tumor cells, which are able to initiate the metastasis,
and therefore, they can be regarded as a marker
for prediction of distant metastases [53]. However,
it is only partially true, because in order to leave the
primary tumor, cancer cells must acquire certain
properties, namely: locomotor phenotype due to EMT,
ability to survive in the bloodstream, avoiding anoikis
(cell death induced by the loss of attachment to the
epithelial cell layer), resistance to chemotherapy and
immune-mediated death in the bloodstream. For this,
the clonogenic potential and ability to return to the
epithelial phenotype (mesenchymal-epithelial transi-
tion) are important conditions for providing tumor cell
homing to secondary organs and formation of mac-
rometastasis [53].
CTC are extremely rare, estimated as one CTC per
billion normal blood cells in the circulation of patients
with advanced cancer. They can passively enter the
blood circulation during vessel damage or surgery,
as well as via an active migration process, mov-
ing away from the primary tumor and entering the
blood circulation due to their conversion to the EMT
phenotype. The level of epithelial E-cadherin expres-
sion decreases, while the level of neural N-cadherin
expression increases, thus resulting in the violation
of cell adhesion. At the same time, the extracellular
matrix is destroyed by matrix metalloproteinases and
components of the urokinase plasminogen activator
system. Increased expression of EMT markers has
been found in the CTC and bone marrow disseminated
cells. Depending on the origin of the primary tumor,
CTC have different properties and different surface
markers: epithelial cell markers, such as EpCAM, CK+,
EMT-related genes such as vimentin, fibronectin, etc.,
and stem cell markers, such as CD44+ CD24− ALDH+
(aldehydedehydrogenase). CTC can also express
different molecules of major cell signaling pathways,
such as EGFR, PI3K, Akt, etc. [54]. It is important
to note that CTC can also colonize their primary tu-
mors, accelerating cancer progression [55].
The activation of WNT signaling pathway involved
in the regulation of cell adhesion was found in 30%
of CTC in contrast to only 1% of WNT activated cells
Experimental Oncology 39, 2–11, 2017 (March)39, 2–11, 2017 (March) (March) 7
found in the primary tumor [44]. The WNT pathway
is known to provide inhibition of tumor cell death via
anoikis, that results in their survival in bloodstream.
The co-expression of EMT markers TWIST and stem
cell markers ALDH1 on circulating pan-cytokeratin
positive cells was found in 30% of early BC patients
and in 80% of metastatic BC patients. In a recent study,
Schindlbeck et al. compared CTC enumeration with
DTC detection using CellSearch technology in patients
with primary or metastatic BC [56]. The authors found
a significant concordance (69.4%) between DTC and
CTC, which increased in patients with metastases.
Other authors identified EMT markers, such as PI3Kα,
Akt-2, and TWIST1, and stem cell markers, such
as ALDH1, Bmi1 and CD44 in CTCs of patients with
early BC [57].
As for circulating clusters, they were characterized
by the increased levels of tissue inhibitor of matrix
metalloprotease-1 (TIMP1) and platelet transcripts,
and were in a hybrid state of EMT when the expres-
sion of both epithelial and mesenchymal transition
took place [58, 59]. This state of EMT was reported
to be chara cteristic of collective invasion and was as-
sociated with more aggressive cancer [59, 60].
Another study identified the adhesion molecule
plakoglobin, as being higher expression in circula-
ting cell cluster than in single CTC [44]. Cheung et al.
reported that CTC clusters and lung metastases fre-
quently expressed epithelial cytoskeletal protein, kera-
tin 14 (K14). The RNA sequencing analysis revealed
that K14 positive cells were enriched for desmosome
and hemidesmosome adhesion complex gene, and
were depleted for MHC class II genes. Suppression
of K14 expression resulted in the inhibition of distant
metastases, likely, through a violation of the acti vity
of numerous molecular players, including tenas-
cin C (Tnc), Jagged 1 protein (Jag 1) and epiregulin
(Ereg) [39].
CIRCULATING TUMOR CELLS: RELATION
TO METASTASIS AND THERAPY
RESPONSE
Evaluation of the prognostic significance of CTC
in patients with early BC is intensively carried out.
There are many reports indicating that the pre-
sence of CTC is associated with lower overall and
progression-free survival rates [61, 62]. The authors
argue that EMT-associated markers in CTC predict
unfavorable prognosis more effectively than epithelial
markers [49]. It indicates more aggressive metastatic
potential of cells carrying EMT markers due to the high
potential for extravasation and subsequent adaptation
in the microenvironment at secondary sites, namely
premetastatic niches.
The properties of tumor cells, providing their meta-
static potential, are significantly modified under the
influence of microenvironment, including endothelial
cells, fibroblasts, inflammatory cells (macrophages,
neutrophils), and mesenchymal stem cells [63]. Tu-
mor-associated macrophages enhance the invasive-
ness of the primary tumor cells, producing epidermal
growth factors. Blood platelets aggregate with tumor
cells, protecting them from damage in the blood-
stream. This likely explains the association between
thrombocytosis and lower survival rates, and the ef-
fective use of anticoagulants to reduce the incidence
of metastasis [63].
Cristofanilli et al. [64] in their study with a large co-
hort of BC patients concluded that the number of CTC
was an independent predictor of progression-free sur-
vival as well as overall survival. The level of CTC of less
than 5 tumor cells per 7.5 ml of blood was associated
with high rates of disease-free and overall survival, but
not with tumor response to chemotherapy [64].
As it was mentioned above, a validated test for CTC
detection has been approved for clinical use in patients
with metastatic BC. Numerous studies report that the
detection rate of CTC is significantly higher in patients
with disseminated BC than in patients with early BC
(50–80% vs 5–13%) [56]. Thus, it can be concluded
that the count of CTC with metastatic potential is higher
in patients with advanced stages than in patients with
early stages. The sensitivity of CTC detection tech-
niques can be reduced due to the low frequency of CTC
in patients with early BC [65].
Krishnamurthy et al. [36] found no relationship
between the frequency of CTC detection in patients
with locally advancer BC and the standard prognostic
factors, including hormone receptors and HER2/neu
status. Circular tumor cells are believed can indepen-
dently predict dissemination to different secondary
sites. The detection of CTC and DTC is considered
as useful in selecting patients for adjuvant chemo-
therapy [36].
Rack et al. [66] evaluated 1767 BC patients for the
presence of CTC and found the correlation between
the CTC level and positive lymph nodes. Lang et al. [67]
showed the correlation between CTC detection and
bone marrow micrometastases and HER2 status of the
primary tumor.
The tumors belonging to distinct molecular
subtypes are known to have different responses
to treatment and different clinical courses. Igna-
tiadis et al. [68] showed a prognostic significance
of CK19 mRNA-positive CTC in patients with estrogen
receptor-negative, triple-negative and HER2-positive
BC. The authors reported elimination of CK 19 mRNA+
CTC during the treatment with trastuzumab, a hu-
manized anti-HER2 monoclonal antibody. The iden-
tification of CTC after adjuvant chemotherapy could
serve as an independent predictor of tumor progres-
sion [68]. It was shown that in patients with metastatic
BC, the treatment with lapatinib significantly reduced
the number of HER2-positive CTC regardless of the
HER2 status of the primary tumor. This finding gives
the opportunity to monitor molecular changes during
the target therapy to control its efficacy [69].
The c-erbB2 (HER2) gene amplification was shown
to play a critical role in the pathogenesis of human
BC. The activation of c-erbB2 gene was observed
8 Experimental Oncology 39, 2–11, 2017 (March)
in 20–30% of early BC. It was also demonstrated
that HER2-positive CTC had a high metastatic po-
tential [33]. The heterogeneity of HER2 status was
revealed, some tumor cells had HER2/neu gene
amplification and others show normal copy numbers.
It is interesting to note that HER2-positive CTC were
found in 89% of HER2 negative patients, and these pa-
tients had a survival benefit after trastuzumab-based
therapy [70]. The CTC test may provide the additional
information for determining HER2 status of the tumor
and administering trastuzumab [71].
The CTC monitoring during trastuzumab treat-
ment allows prediction of resistance to this drug and
gives the opportunity of using appropriate inhibitors
affecting STAT3 or PI3K/mTOR signaling pathways
contributing to cancer progression. A series of studies
were conducted to evaluate the response to therapy
in BC patients with heterogeneity of HER2/neu ex-
pression in cells of the primary tumor and in CTC [33,
70–73].
By studying the relationship between CTC with
HER2 amplification and efficiency of targeted therapy,
it can be assumed that it is crucial to determine the
grade of heterogeneity of the targets for specific
therapy, which influence the treatment outcome. For
this, large-scale clinical trials are required.
Prospective studies provided data on the efficacy
of CTC detection for monitoring chemotherapy [61,
62]. Molecular studies of CTC might discover new
molecular targets for treatment and predictors of poor
response due to the emergence of resistant clones
giving the opportunity to choose the drug. Molecular
characterization of CTC may contribute to the develop-
ment of novel anticancer drugs.
In the SUCCESS (Simultaneous Study of Gem-
citabine-Docetaxel Combination Adjuvant Treatment,
as well as Extended Bisphosphonate and Surveillance)
trial, including 2026 patients with stage I–III BC, the
CTC count detected before treatment using the Cell-
Search technology was an independent predictor
of disease-free, overall and BC-specific survivals.
CTC were tested during chemotherapy in 1492 pa-
tients and were detected in 22% of these patients.
The median follow-up was 36 months. Recurrence
was found in 28% of patients who had at least 5 CTC
in the blood before the start of systemic treatment and
in 7% of patients who had no CTC. A total of 14% of the
CTC-positive patients died of BC compared with 3%
of the CTC-negative patients. The presence of CTC
both before the start of systemic adjuvant treatment
and after completion of chemotherapy was associated
with deteriorated survival [66].
It is known that CTC-clusters have a stronger abi-
lity to induce metastasis than the equivalent number
of single CTC. This fact was reported in the early 70-ies
of the last century, when CTC-clusters and single
CTC were injected to the experimental animals and
the number of metastatic foci were compared [74].
A recent study confirmed that CTC clusters indicated
a metastatic potential 23 to 50 times greater than
single CTC, and metastases developing from clusters
led to dramatically reduced survival [48].
In mouse models of BC, experimentally aggre-
gating tumor cells into clusters displayed a >15-fold
increase in colony formation ex vivo and a >100-fold
increase in metastasis formation in vivo [39]. In this
study, the authors observed CTC-clusters at different
stages of metastasis, including collective invasion,
local dissemination, intravasation, circulation and
formation of micrometastases, and they demonstrated
that the polyclonal dissemination of CTC-clusters
is a frequent mechanism in a common mouse model
of BC, accounting for more than 90% of all metas-
tases [39]. The recent studies demonstrate that the
monitoring of CTC in combination with CTC-clusters
provides a higher prognostic value in assessing the
risk of metastatic spread of advanced BC compared
to CTC detection alone [75].
There are also reports that CTC-clusters show
higher resistance to chemotherapy than single
CTC [47, 76]. The increased metastatic potential
of CTC-clusters and their resistance to chemotherapy
are likely to relate with the lack of proliferating cells and
the ability of cells to avoid anoikis [76, 77]. In addition,
an enhanced survival advantage of CTC clusters also
might be afforded by continued production of auto-
crine pro-migratory factors, matrix proteases and
protection of cells from immunological surveillance
by lymphocytes and natural-killer cells.
Several studies have shown a significant portion
of tumor cells involving in EMT within the cluster
cell population, thus also explaining their resistance
to chemotherapy [46, 77]. It is related to the increased
activity of ABC transporters in EMT-cells, which are
responsible for drug resistance. There are also reports
that CTC clusters contain cells with a cancer stem cell
phenotype, characterized by high metastatic poten-
tial [78].
The potential of CTC to predict relapse and overall
survival in early BC patients may depend on timing
of blood sampling, duration of follow-up and more
importantly on the method of CTC detection. The dif-
ferent results obtained by different authors may be due
to a number of objective reasons: the nature and pro-
perties of CTC in each individual patient are not known;
CTC may shift from an active state to a dormant state,
being resistant to chemotherapy and immune attack;
there is no clear evidence whether chemotherapy
results in the increase or decrease of CTC number.
It is particularly important that the conditions for the
development of macrometastases are objectively va-
ried in different individuals. According to fundamental
knowledge about the biology of tumor dissemination,
the metastasis development is determined not only
by the tumor cell behavior, but also the environment
influencing their mobility, migration from the primary
tumor and survival in the bloodstream and distant
sites. Indeed, the Paget’s “seed and soil” hypothesis
(1889) was proposed to explain that metastasis is a re-
sult of cross-talk between selected cancer cells (the
Experimental Oncology 39, 2–11, 2017 (March)39, 2–11, 2017 (March) (March) 9
“seeds”) and specific organ microenvironments (the
“soil”) [79]. This idea was later developed by Lyden
into the concept of “metastatic niches” [80]. Primary
tumor cells provide tumor invasion and regulate cre-
ation of pre-metastatic niches by secreting various
cytokines and growth factors, which promote the re-
lease of cells from the tumor and mobilization of bone
marrow cells into metastatic niches [81].
It is known that in patients with certain types of tu-
mors, for example, with luminal subtype of BC, meta-
stases can occur many years after initial diagnosis
[38]. This is due to the phenomenon of “dormant
tumor”, when tumor cells extravasated in the secon-
dary sites (pre-metastatic niches) do not proliferate
and do not form macrometastases.
Initiation of secondary tumor growth is induced
by the specific conditions related to various types
of injury (trauma, surgery, radiotherapy, chemo-
therapy), other stress, and inflammatory processes
that are associated with the activation of regeneration
processes [82, 83].
The presence of tumor cells in the circulation and
metastatic sites is assumed not to be a sufficient con-
dition for macrometastasis, because even if CTC are
present, the conditions of “soil” may not be fully taken
into account. However, it should be noted that the un-
derestimation of the effect of “soil” does not detract
from the value of CTC detection, as “side effect” in this
case will always be a benefit for the patient’s survival,
if there are no objective conditions for the development
of macrometastases.
CONCLUSION
The numerous reports may justify the prospect
of using the CTC detection not only in metastatic
cancer, but also in the early stages of BC, thus im-
proving efficiency of the treatment for early BC. The
fundamental knowledge about the nature of CTC,
their properties, and the correlation between CTC and
clinical course of disease indicate their high potential
value as markers of tumor progression and targets for
therapeutic intervention. High phenotypic and func-
tional heterogeneity of CTC open the opportunity for
determining the molecular profile of different CTC sub-
populations to identify metastasis-related prognostic
phenotype and genotype, providing the evaluation
of metastatic potential.
Detection of molecular changes in CTCs during
chemotherapy, resulted in chemoresistance, is the
promising way to shift the course of chemotherapy
right time as well as to find new therapeutic targets.
The identification of CTC activated signaling pathways,
contributing to clinical outcome, can provide an ef-
fective search for new drug targets. For example, the
activation of WNT signaling pathway observed in 30%
of CTC vs 1% of primary tumor cells provides inhibition
of anoikis, showing the potential to identify the targets
for pathogenetic therapy.
CTC are increasingly recognized as the main
source for recurrence and metastasis. The develop-
ment of novel therapeutic techniques that target CTC
includes:
• dialysis after surgery to remove CTCs from the
bloodstrem [84];
• drugs that target cancer stem cells;
• targeted drugs for various subclones of CTC;
• identification and inactivation of signaling pathways
that allow CTC to survive in the bloodstream and
home to secondary sites.
Thus, we can conclude that further large-scale
evidence-based clinical trials are needed to determine
the prognostic and predictive value of CTC in BC, ta-
king into consideration the “price/benefit” ratio.
ACKNOWLEDGMENTS
This work was supported by the Russian Science
Foundation, Grant 14–15–00318, and Tomsk State
University Competitiveness Improvement Program.
CONFLICT OF INTEREST
None declared.
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