]
In this review, we focus on CTCs and ctDNAs detection
methods and practical utilization of these materials
as diagnosis, prognosis and therapeutic response
monitoring biomarkers in breast oncology.
Table 1: The advantages of liquid biopsy collection over standard biopsy
Fig 1: Breast cancer related circulating tumor cells
(CTCs) and circulating tumor DNA (ctDNA)
fragments are released into blood circulation by
tumor cells and they can be isolated from the
blood of breast cancer patients.
Roles of CTCs in BC
Tumor cells that detach from their primary site and enter
to blood stream or lymphatic vessels to metastasize
are called CTCs.[] It is estimated that, nearly 1 million
tumor cells enter circulation everyday, but 85% of them
disappear within 5 minutes. Therefore, only one CTC
can be found in 1 ml of blood sample or 1 CTC per
billion of nucleated hematopoietic cells in blood.[]
So, even though CTCs were first described in 1869,[]
recent advancements in single cell analysis techniques
in the last decade rendered CTC research area as one
of the hotspot research topics of cancer research, especially
for BC.
CTC detection methods
Since CTCs are in less number than the other cell types
in blood samples, several two-step approaches are used
as breast CTC detection methods. At the first step,
CTCs are selected in LB samples either by positive selection
(enrichment) or negative selection (depletion)
methods.[] Immunomagnetic-/microfluidic-based methods and filtration (isolation by size) method are
used as positive selection methods, while densitygradient
centrifugation method is used as negative
selection method at the first step. Epithelial cell adhesion
molecule (EpCAM) is the most frequently used
biomarker targeted by immunomagnetic enrichment
methods.[] ISET (isolation by size of epithelial tumor
cells) filtration of CTCs method is used based
on the fact that CTCs are slightly bigger than red and
white blood cells.[] Cells, other than CTCs in the
blood are cross-linked to erythrocytes and removed
for negative selection with density gradient centrifugation.[] At the second step, the isolated cells are either
subsequently immunostained to detect CTCs by fluorescence
microscopy, flow cytometry or tumor-related
messenger ribonucleic acid (mRNA) transcripts are
detected by reverse transcription-polymerase chain reaction
(RT-PCR) method.[]
CellSearch system is currently the gold standard
method for breast CTC detection by being the only
Food and Drug Administration (FDA)-approved
method.[,] This system targets EpCAM molecule
on cell surface of CTCs for positive selection at the first
step of CTC detection. EpCAM-positive CTCs are isolated
from blood by immunomagnetic selection and
then fluorescently labeled for DAPI, CD45 and CK19 to
be sure about breast CTCs at the second step of the system.[,] Currently, CTCs isolated with CellSearch
system are in utilization for prognosis evaluation of
BC patients. Downstream high-throughput genomic,
transcriptomic or proteomic characterization methods
can also be used as alternatives of validation step of the
system.[,] However, mRNA expression detection
based RT-PCR or probe-based methods are currently not alternatives because these methods have less specificity
compared to the other methods. Thus utilization
of mRNA expression detection methods have been discontinued
as part of the detection systems, but studies
are ongoing.[,]
The other frequently used methods for CTC detection
from LB of BC patients are AdnaTest, which is
another immunomagnetic enrichment based method,
EPISPOT-assay that detects CTCs via specific proteins
such as CK19, fluorescence in situ hybridization
(FISH) technique that detects chromosome aberrations
in the CTCs, micro RNA (miRNA)-profiling that
detects CTCs by profiling altered miRNA expression,
"CTC-chips" that detect EpCAM-expressing cells and
mRNA-based PCR detection methods can be used.[]
Clinical utilization of CTCs in breast oncology
Breast tumor cells encountered at secondary homing
sites, such as bone marrow (BM) and PB, are currently
seen as surrogate markers and precursors of distant
metastasis.[] At the late 90"s, several studies investigated
the role of BM disseminated tumor cells (DTC)
in the micrometastatic process of BC. After that, several
groups developed different techniques to detect
DTCs in BM of early BC patients, mostly based on
epithelial cell staining and cytological visual screening.
[,] However, BM DTC detection methods have
not been implemented in the routine clinical workup
of early BC patients and only limited attempts were initiated
to demonstrate clinical utility of these methods
because these techniques were labor-intensive.[] On
the other hand, technological advancements on single
cell research techniques and minimally invasive nature
of LB, attracted attention on roles of CTCs in breast oncology
and numerous clinical trials were performed in
relative easy mostly using recently developed methods
such as CellSearch and AdnaTest.
The SUCCESS trial, which is the largest clinical
trial on the prognostic relevance of CTCs in early BC,
observed CTC status of prechemotherapy and postchemotherapy
BC patients and determined that CTCnegative
patients have higher overall survival (OS) and
disease-free survival (DFS) values both before and after
chemotherapy compared to CTC-positive patients.
Thus, the SUCCESS trial demonstrated that CTC persistence
correlates with shorter DFS and OS in early
BC.[] In addition, this trial determined that, women
with at least five CTCs in 30 ml of blood samples had
highest risk for relapse at early BC, and this cut off
value is still in clinical use. Another study determined
that at least one CTC presence in 7.5 ml blood of early
BC patients is an independent predictor of shorter DFS
and OS.[] Although evidence from CTC-based clinical
trials showed that persistence of CTCs in blood is
an important predictor of worse survival in large trials,
data supporting prognostic relevance of specific
CTC subtypes in early BC is limited; because expression
profiles of CTCs may not correlate with their
corresponding primary tumor subtype.[] However,
clinical trials on possible use of CTC monitoring to decide
therapy choices for early BC are still ongoing. The
ongoing TREAT CTC trial is the first LB-based large
study evaluating the concept of targeting chemoresistant
early BC.[] In this clinical study, HER2 status of
the CTCs are assessed and effects of trastuzumab treatments
are evaluated based on CTC counts.
Possible utilizations of CTCs as prognosis, therapy
monitoring and therapy selection tool in metastatic BC
are also evaluated with several clinical trials. In general,
results of these trials indicate that, 40?80% of patients
with metastatic BC have CTCs in PB. In addition, the
study of Cristofanilli et al. demonstrated that, patients
who have CTC counts above the cutoff value of at least
5 CTCs in 7.5 ml blood when they were diagnosed are
associated with impaired clinical outcome.[,] The
prognostic value of this cutoff value has been further
verified by several studies and still in utilization.[,]
Besides the prognostic role of CTC status, alteration
in CTC levels during treatment has also been shown to
reflect therapy response in metastatic BC. The study of
Hayes et al. indicated that, a decrease in CTC levels under
the threshold of five-cells/7.5 ml PB predicted better
PFS and OS in metastatic BC.[] In addition, treatment
efficacy assessment with CTC counting provided
better prediction results compared to the standard radiological
imaging in metastatic BC patients.[]
Although prognostic significance of CTC counting
has been proven for metastatic BC, studies on effects of specific CTC types on patient survival have contradictory
results. For example, while one study determined
that patients with HER2-positive CTCs had significantly
longer PFS,[] another study showed that
patients with HER2-positive CTCs had significantly
worse survival;[] and another study determined that
HER2 status of CTCs in metastatic BC had no correlation
with clinical outcome.[] An ongoing clinical
trial called DETECT may provide results to resolve this
enigma. Because in this study women who have HER2-
negative metastatic BC with at least one HER2-positive
CTC; and women who have HER2-negative BC (hormone
receptor-positive or triple-negative) and exclusively
HER2-negative CTCs are in study groups.
Roles of ctDNAs in BC
cfDNAs are short (160-180 bp), non-cellbound nucleic
acid fragments in blood circulation. The discovery of
cfDNA dates back to 1940s; Mandel and Metais reported
presence of cfDNA in cell-free blood compartment
in 1948.[] They detected cfDNA in bloodstream of
healthy individuals and patients.[] In 1965, Bendich
and colleagues became the first researchers hypothesized
that cancer-induced cfDNA could be associated
with metastasis.[] Two different groups observed
presence of same K-RAS and N-RAS mutations in tumor
tissues and isolated cfDNA in blood samples of
cancer patients in 1994.[,] At the following years,
in addition to RAS mutations, other known cancer tissue
specific mutations (such as TP53 mutations) were
detected in ctDNA as part of the total cfDNA pool,
which specifically derived from tumors in plasma
isolated cfDNAs of the patients with several different
cancers including breast, colon, lung, melanoma and
hepatocellular carcinoma.[] In addition to genetic
alterations, cancer tissue specific epigenetic alterations
such as hyper-methylation in promoter of suppressor
genes were also identified in blood ctDNAs of cancer
patients.[] Recent advancements in genomics and
bioinformatics research techniques cause increase in
cfDNA detection method development and clinical
utility investigation research studies.
cfDNA detection methods
cfDNAs can be detected in both from plasma and serum.
Because the lower background concentration of
cfDNAs, researchers mostly prefer to isolate them from
plasma rather than serum.[] The analysis of tumor
specific cfDNA requires sensitive detection techniques
to separate small fraction of tumor specific circulating
DNA from others. There are mainly two approaches to analyze plasma-collected cfDNA; quantification of the
presence of ctDNA from whole cfDNA in plasma, and
identification of tumor specific genomic alterations
including point mutations, chromosomal and microsatellite
alterations and methylation changes. The sensitivity
of traditional approaches to DNA analysis is insufficient
for detection of somatic mutations in plasma
ctDNA from patients with cancer. To overcome these
limitations a variety of digital PCR (dPCR) methods
have been developed with a high level of analytical sensitivity
and specificity as alternative techniques to classical
quantitative-RT-PCR (q-RT-PCR) for absolute
quantification and detection of genetic alterations in
ctDNA isolated from LB cancer patients.[] dPCR has
permitted to detection of fragmented and low abundant
cell free nucleic acid targets from body liquids in
a short time period.[] It has been shown that dPCR
identifies copy number variations that differ by only 1
copy and identifies allele frequencies lower than 0.1%.
[] Furthermore, dPCR detects point mutations, genetic
alterations (loss of heterozygosity, aneuploidy),
and copy number alterations in ctDNA.[] dPCR
technology improves ctDNA recovery and decreases
the lower limit of detection to 0.01%.[,]
One of the successful dPCR molecular techniques
is called BEAMing (Beads, Emulsion, Amplification
and Magnetics) that consist of emulsion PCR with
magnetic beads and flow cytometry for highly sensitive
detection and quantification of ctDNA fragments.[]
The more recently developed technology of dPCR is
droplet digital PCR (ddPCR).[38] In ddPCR method, a
DNA sample is partitioned into 10.000 to 20.000 droplets
to provide a digital counting of nucleic acid targets
in the chip-based platform.[]
Due to high efficiency and low-cost, high-throughput
NGS technologies have started to be used frequently
to identify genetic alterations in plasma ctDNA.[]
Many different targeted deep sequencing approaches
(Tamseq, Safeseq, Ion-Ampliseq CAPP-seq) are in use
to analyze known cancer-related mutations such as
EGFR, BRAF, KRAS.[] In addition, non-targeted genome-
wide analyses enable the identification of tumor
specific changes without prior knowledge about the aberrations
present in the tumor. Furthermore, such approaches
can be used to discover genetic changes underlying
therapy resistance and to identify new feasible
targets for cancer patients.
In future, dPCR and NGS methods will likely to be
used as complementary methods for LB analyses fluid
biopsy assays. The dynamic individual mutations can
be detected by using former approach, but it requires prior knowledge about the mutant allele. New generation
methods make it possible to discover novel mutated
variants, but have higher costs and cannot be easily
applied to long-term patient follow up.
Use of cfDNAs in breast oncology
There are a number of studies that try to evaluate utility
blood cfDNA level determination approach for distinguishment
of benign and malignant breast tumors.
These studies identified positive correlation with cfDNA
counts and BC compared to healthy people.[]
However, more studies are required for clinical utility
of this approach, because the defined ranges are wide
and overlapping. Thus, currently cfDNA quantification
approach for BC diagnosis and screening is not eligible
to use in breast oncology.
The size and integrity of isolated ctDNAs of different
stage BC patients and healthy donors were also
compared in several studies to identify utility of this
approach for early diagnosis and stage determination
for BC. Umetani et al. identified that, mean serum
DNA integrity was significantly higher in stage II-IV
BC patients compared to healthy donors, but not statistically
significantly different between normal and
stage 0 or stage I individuals.[] Iqbal et al. also found
higher DNA integrity in BC patients, especially in stage
IV patients, compared to healthy controls.[] However,
clinical utility of this approach is low because of its
low ability on distinguishment of early BC patients and
healthy women.
The utility of identification and quantification of
BC specific alterations in ctDNAs of BC patients were
also studied. Chimonidou et al. identified promoter
methylation in CST6 gene from cfDNAs in 13?40% of
BC patients but in none of healthy controls.[] Dulaimi
et al. determined hypermethylation of RASSF1A,
APS, and DAP kinase gene promoters in the ctDNAs of
70% BC patients and none in serum DNA from healthy
women.[] Oshiro et al. found PIK3CA mutations in
cfDNAs in 22% of BC patients who have PIK3CA mutations
in their tumors, but none in healthy women or
patients with non-PIK3CA mutated BC.[] Investigation
of BC specific genetic alterations in ctDNAs of BC
patients seem promising for its clinical utility due to
high specificity for BC, but sensitivity of this approach
need to be increased with additional studies.
Several other studies have also conducted to determine
prognosis and therapeutic response monitoring
functions of breast ctDNAs. Studies in BC patients have
determined that cfDNA concentrations decrease after
surgery and chemotherapy,[,] and post-operative detection of ctDNAs was predictive of early relapse for
BC.[] Thus, breast ctDNAs can be potentially used for
therapy response observation for BC patients. However,
current studies indicate that ctDNA levels do not reflect
BC prognosis, and further studies are required.[]
Conclusion and Future Perspectives
Since CTCs and ctDNA fragments may be originated
from a number of metastatic sites, these LB derived
materials are potentially better representatives of the
whole disease compared to single site biopsy. However,
most of the current CTC detection methods are able to
isolate and detect only epithelial type CTCs by targeting
EpCAM-positive epithelial CTCs; yet it is known
that mesenchymal-type of CTCs have also been observed
as a result of epithelial-to-mesenchymal transition
(EMT) in human BC patients.[] For example,
EpCAM-negative breast CTCs, which metastasize to
brain have recently been identified.[] Identification
of these CTCs suggests that, EpCAM-negative CTC
sub-populations may be present in LB samples of BC
patients that need to be identified. In order to detect
all heterogeneous types of breast CTCs novel antigenindependent
CTC enrichment techniques need to be
developed in the future.
As presented, CTCs are invaluable tools, which have
clinical utilization during disease stage evaluation, disease
progression monitoring and targeted personalized
therapy development applications for breast oncology.
The real-time monitoring and characterization of
CTCs can provide administration of suitable and personalized
targeted therapy for BC patients compared to
other methods. For example, human epidermal growth
factor receptor 2 (HER2), estrogen receptor (ER) and
progesterone receptor (PgR) status of breast cancer
patients can be monitored with CTCs in real-time to
evaluate stage and progression of the disease as well as
to decide suitable targeted and personalized therapies
for BC patients. However, more studies are needed.
The quantitative and qualitative analysis of ctDNA
in BC demonstrate tumor-associated genetic and/or
epigenetic alterations and treatment response in BC patients.[,,,] Before utilization of ctDNA analysis
methods in clinical practice, more data should be
obtained by using different methods, and more clinical
studies are needed. Since the patient-derived ctDNAs
only inform us about the dying tumor cell genomes,
the obtained data may be misleading about the genetic
alterations in resistant tumor cell populations, and this
possibility should also be further investigated.
In conclusion, CTCs and ctDNAs have potential
to be major biomarkers of diagnosis, prognosis monitoring
and therapy monitoring tools for personalized
therapy in breast oncology, in the near future.
Disclosure Statement
The authors declare no conflicts of interest.
