2016, Volume 32, Number 3, Page(s) 148-157
Circulating Tumor Cells in Breast Cancer: Correlation with Clinicopathological Parameters, Hormone Profile and MicroRNA Polymorphisms
Cherry BANSAL1,4, Mukta PUJANI2, Sanjeev MISRA3, AN SRIVASTAVA1, US SINGH4
1Department of Pathology, Era's Medical College and Hospital, Lucknow, UTTAR PRADESH, INDIA
2ESIC Medical College, FARIDABAD, INDIA
3Department of Surgical Oncology, AIIMS, Jodhpur, RAJASTHAN, INDIA
4Department of Pathology, King George Medical University, Lucknow, UTTAR PRADESH, INDIA
Keywords: Breast cancer, Circulating tumor cells, Hormone receptors, microRNA , Flow cytometry
Circulating tumor cells are isolated tumor cells in the peripheral blood that serve as important prognostic indicators for many kind
of tumors. The study was conducted to know the rate of detection of circulating tumor cells among breast cancer patients in comparison with
benign breast diseases and control subjects and to know the association between CTC positivity and various clinicopathological parameters,
hormonal profile and microRNA polymorphisms.
Material and Method: In the present case control study, we included 182 healthy controls, 108 cases of benign breast disease and 114 breast
carcinoma cases. Various clinicopathological details of cases were recorded. Immunohistochemistry was performed for estrogen (ER) and
progesterone receptors (PR) and Her-2 neu. Circulating tumor cells were analyzed using flow cytometry (EpCAM, CK, CD45). Genotypic
frequency of micro RNA polymorphisms was determined by PCR-RFLP assay.
Results: Circulating tumor cell positivity was observed in 11/114 (9.64%) breast cancer cases but absent in benign and control groups, and was
significantly associated with tumor size, histologic type, tumor grade, metastasis and skin infiltration (p<0.05). Circulating tumor cell positivity
did not show any correlation with the immunohistochemical profile. No significant associations between pre-miRNA genetic variations
miR-196a2 C/T (rs11614913), miR-146a G/C (rs2910164) and miR-499 T>C (rs3746444) polymorphisms and circulating tumor cell positivity
Conclusion: The flow cytometry protocol for detection and molecular characterization of circulating tumor cells is a time and cost-effective
technique, suitable for routine clinical use. However, more elaborate studies are needed to establish the findings as our study was limited by small
Breast cancer is by far the most common cancer among
women worldwide. According to the incidence of cancers,
breast cancer ranks second in the world1
. According to
the National Cancer Registry Program's (NCRP) recent
report for 2008, the load of breast and cervical cancers
together was 23.6-38.7% of total cancers in the Northeastern
states of India, while in all the other states these two cancers
contributed 35.2-57.7% of the total cancers2
published reports of cancer registries in India indicate
rising trends in breast cancer incidence3
The tumor cells shed into blood circulation from primary
or metastatic cancers are referred to as circulating tumor
cells (CTC). Although rare, CTC serves as a biomarker to
evaluate the tumor genotypes during the course of treatment
and progression of the disease. A proportion of CTC are capable of initiating a metastatic clone. CTC have been
identified in a variety of epithelial cancers, predominantly
breast, prostate, lung and colon. CTC are more likely to be
detected in patients with metastatic disease, and they have
also been reported in localized cancers4,5.
For detection of CTC, a number of techniques are currently
available, but none of these approaches constitute a
desired optimal level to serve as a gold standard. Available
techniques for CTC isolation and detection include either
nucleic acid based detection (free DNA or RNA) (cell free
circulating DNA, cfDNA) or intact CTC detection based
on their physical properties (large cell size, differences
in density, charge, migratory properties, granules etc.)
or detection of CTC by directing antibodies against cell
surface antigens (Cell Search System- FDA approved
method, Isoflux and Flow cytometry). Among the cell surface antigens used with these technologies, the most
widely used antibody is directed against epithelial cell
adhesion molecule (EpCAM)4-7.
CTC serve as important prognostic indicators. Various
studies have concluded that CTC serve as independent
prognostic markers in cancers of breast, prostate, lung and
colorectum. The potential applications for CTC include
isolation and identification of CTC (early diagnosis
and prognosis), alteration in CTC levels to evaluate the
response to new therapies (prognosis and prediction) and
CTC phenotype and genotype (diagnosis, prognosis and
In the present case control study, we included 182 healthy
controls, 108 cases of benign breast disease and 114
carcinoma breast cases. Healthy controls and diseased
studied in the present work were of North Indian ethnicity
and unrelated to each other. Patients were recruited (Dec
2010- Nov 2012) from the surgical oncology department;
King George Medical University, Lucknow, India. Breast
carcinoma patients included were those who had not
received neoadjuvant chemotherapy yet. Controls were
from healthy population and unrelated to diseased subjects.
Informed consent in written was taken from all the study
subjects. Approval from the institutional ethical committee
was taken for the study protocols and the work done. The
World Medical Association Declaration of Helsinki's norms
were followed by the authors. Controls included fulfilled
the criteria: no chronic disease, no history of present/ past
malignancy or premalignant lesion. Cancer cases were
frequency-matched to all the controls for characteristics
like age, gender, and ethnicity.
Immunohistochemistry for estrogen receptor (ER),
progesterone receptor (PR) and her-2 neu were performed
on representative blocks of paraffin embedded tumor
tissue. 4μm thick sections were taken on poly-L-lysine
coated slides and submitted for immunohistochemistry.
Antigen retrieval was done using citrate buffer at pH 2.5
for hormone receptors and pH 6 for her-2 neu. The normal
breast ducts served as internal positive control for ER/PR.
Breast carcinoma with known her-2 neu overexpression
served as an external positive control for her-2 neu staining.
ER or PR were considered positive when more than 1% of
tumor cell nuclei were immunoreactive.
For interpretation of Her-2 neu staining the following
method was used8:
Score 0 (Negative): No staining is observed or membrane
staining is observed in less than 10% of the tumor cells Score 1+ (Negative): A faint/barely perceptible membrane
staining is detected in more than 10% of the tumor cells.
The cells are only stained in part of their membrane
Score 2+ (Weakly positive): A weak to moderate complete
membrane staining is observed in more than 10% of the
Score 3+ (Strongly positive): A strong complete membrane
staining is observed in more than 30% (formerly 10%) of
the tumor cells
Score 3+ was considered as positive immunostaining for
This was performed on Beckton-Dickinson Fluorescence
Activated Cell Sorter (FACS). The samples were
immunostained with EpCAM peridinin chlorophyll
protein complex, CD45 fluorescein isothiocyanate, and
pan cytokeratin (CK – 8/18/19-phycoerythrin (PE) (all
from BD Biosciences, San Jose, CA) for 30 minutes at 4°C.
BD FACS lyse buffer (BD Biosciences) was added for 15
minutes after staining to lyse RBCs. A total of 500,000
events were collected for analysis on a 2-laser, 6-color BD
FACS Canto device using BD FACS Diva software (both
from BD Biosciences). The data were exported as FCS 3.0
files and analyzed using Flowjo (Tree Star, Ashland, OR)
Genotypic frequency of miRNA polymorphisms was
determined by PCR-RFLP assay. Details of genotyping
and statistical analysis for miRNA's have been given in our
The Statistical analysis was done by SPSS Software version
15.0 and graph pad prism version 5.01. We applied Chisquare
and Fisher's exact test wherever required.
|Characteristic Profile of Controls, Benign and
The present study included 404 study subjects, out of which
114 were breast carcinoma cases, 108 benign breast disease
and 182 controls. Benign or malignant cases were biopsy/
cytology-proven. Mean ages were 36, 33 and 64 years for
controls, benign and malignant disease respectively. Most
of the patients in control group (69%), benign breast disease
group (62.28%) and breast carcinoma group (69.29%) were
Hindus followed by Muslims. Premenopausal patients
formed the majority in all study groups i.e. controls (88.46%), benign (100%) and carcinoma cases (62.28%).
Details are shown in Table I
Clinico-Pathological Profile of Breast Cancer and CTC
Eleven out of 114 breast cancer cases were positive for
CTC (9.64%), with no CTC positive case in either control
or benign group. In the <40 years age group, 3/36 (8.33%)
patients were found to be CTC positive while 8/78 (10.25%)
were CTC positive in > 40 years age group. Out of all CTC
positive cases (11), 72.72% (8/11) were above 40 years
of age (Figure 1). CTC positivity in premenopausal vs
postmenopausal group was found to be 5.63% vs 16.27%
respectively. Most of the CTC positive cases (63.69%; 7/11)
were postmenopausal (Figure 2). Neither age of patients
nor menopausal status was found to have any association
with CTC positivity (Table II).
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|Figure 1: Comparison of age distribution in CTC positive and
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|Figure 2: Comparison of menopausal status in CTC positive and
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|Table II: Clinicopathological profile of breast carcinoma cases and circulating tumor cells (CTC) positive cases
None of the CTC positive cases belonged to T1 group
(tumor size <2 cm). In the T2 group (tumor size 2- 5 cm),
there were 2/61 (3.27%) cases while CTC positivity was very high (19.56%) in the tumors >5cm in size (T3). The
difference was found to be statistically significant (p=
0.0049). 9/11 (81.82%) CTC positive cases belonged to T3
group while 2/11 (18.18%) belonged to T2 group (Figure
Regarding histologic type, the number of cases of invasive
ductal carcinoma (IDC) with CTC positivity was 9/111
(8.1%) compared to 2/3 (66.67%) for invasive lobular
carcinoma, which was statistically significant (p= 0.0242).
Out of all CTC positive cases, 81.82% belonged to IDC while
18.18% belonged to ILC (Figure 4, 5). CTC positivity when
seen in relation to grade of tumor was highest for grade
3 (31.25%) followed by grade 2 (1.31%) and grade 1 (0)
and the difference was found to be statistically significant
(p<0.0001) (Figure 6).
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|Figure 4: Distribution of tumor type among CTC positive and
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|Figure 5: Comparison of in situ component among CTC positive
and negative cases.
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|Figure 6: Distribution of MRB grade among CTC positive and
Although CTC positivity was higher in node positive group
(11.39%), we found 2 CTC positive cases (2/35; 5.71%) in
node negative group as well but no association was found
between CTC positivity and lymph node status. 81.82% of all CTC positive cases had lymph node metastasis (Figure
7). Positive CTC cases were 50% in patients with skin
infiltration by the tumor compared to only 3.06% in those without skin infiltration, the difference being statistically
significant (p<0.0001). Among all CTC positive cases,
72.73% had skin infiltration by tumor (Figure 8).
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|Figure 7: Comparison of lymph node status among CTC positive
and negative cases.
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|Figure 8: Comparison of skin infiltration among CTC positive
and negative cases.
75% of CTC positive cases were observed in the metastatic
breast cancer group while CTC positivity was 1.96% in the
non metastatic group, which was statistically significant
(p< 0.0001). 81.82% of all CTC positive cases had distant metastasis (Figure 9). There was not much difference
in CTC positivity between breast cancer cases with and
without intratumoral and peritumoral lymphocytes (9.43
vs 9.83) (Figure 10). Details are shown in Table III.
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|Figure 9: Comparison of metastatic status among CTC positive
and negative cases.
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|Figure 10: Distribution of tumoral lymphocytes among CTC
positive and negative cases.
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|Table III: Distribution of breast cancer cases and CTC positive/negative cases
Hormone Receptor Status of Breast Cancer and CTC
Out of all the CTC positive cases, 72.72% were ER negative,
54.54% were PR negative, 63.64% were her-2 neu negative
while 72.72% were triple negative as depicted in Table IV.
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|Table IV: Hormone receptor status of breast carcinoma cases and circulating tumor cells (CTC) positive cases
Most of the CTC positive breast cancer cases were estrogen
receptor (ER) negative (16.32% vs. 4.61% in CTC positivity
in ER negative vs ER positive groups). However, the difference was not statistically significant (p= 0.053). CTC
positivity did not show much difference in progesterone
receptor (PR) negative vs PR positive groups (9.83% vs.
9.43%) (Table V).
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|Table V: Distribution of breast cancer cases and CTC positive/negative cases in relation to hormone receptor status
In the Her-2 neu positive group, CTC were detected in 4/55
patients (7.27%) compared to 7/59 (11.86%) in the her-2
neu negative group. In the triple negative tumors (ER, PR,
Her-2 neu negative), CTC positivity was observed in 8 out
of 32 cases (25%). The details are depicted in Table V.
In nutshell, CTC positivity was observed to be significantly
associated with tumor size, histologic type, tumor grade,
metastasis and skin infiltration.
Pre-miRNA Genetic Variations (miR-196a2 C/T
(rs11614913), miR-146a G/C (rs2910164) and miR-
499 T>C (rs3746444) Polymorphisms and Circulating
Tumor Cell (CTC) Status
In the present study, we did not find any significant
associations between pre-miRNA genetic variations miR-
196a2 C/T (rs11614913), miR-146a G/C (rs2910164) and
miR-499 T>C (rs3746444) polymorphisms and Circulating
tumor cells (CTC) positivity in susceptibility to breast
cancer (data not shown). Due to very low sample size, there
were not significant cases in each group, so we were not
able to analyze the association between the pre-miRNA
genetic variations (miR-196a2 C/T (rs11614913), miR-146a G/C (rs2910164) and miR-499 T>C (rs3746444)
polymorphisms and Circulating tumor cells (CTCs) unlike
our previous work in which we could find associations
between miR and breast cancer risk9.
According to GLOBOCAN 2012 (WHO), breast cancer is
the second most common cancer in the world and, by far,
the most frequent cancer among women with an estimated
1.67 million new cancer cases diagnosed in 2012 (25% of
all cancers). Breast cancer ranks as the fifth cause of death
from cancer overall (522,000 deaths) and is the most
frequent cause of cancer death in women in less developed
regions (324,000 deaths, 14.3% of total). An estimated
70218 women died in India due to breast cancer, which
is highest than any other country in the world10
early diagnosis by adequate screening of the breast lump is of prime importance to safeguard the health of women
globally and particularly for our country.
Many patients continue to die of the disease especially in
developing countries like India, including those diagnosed
at an early stage despite advances in early detection
and treatment. It is believed that after the completion of
primary therapy, minimal residual disease ultimately leads
relapse and distant metastases. Circulating tumor
cells are isolated epithelial cells with similar characteristics
to the tumor cells of the primary site that have been
identified in the peripheral blood of many solid cancers
like breast, prostate and colon. The greatest challenge lies
in the detection of these rare cells (1 in 106 to 1 in 107 of all
nucleated cells) from among numerous hematopoietic cells5,6.
A variety of methods have been developed for detection of
CTC. To increase the chances of detecting these cells, we
require techniques that utilize different methods to increase
the concentration of CTC in blood, namely, differential
centrifugation, Ficoll enrichment and cell separation by
immunomagnetic technique. Another limitation is the loss
of malignant cells on account of their fragility. The positive
detection of CTC has been used in a number of techniques
like immunohistochemistry, immunofluorescence,
Fluorescent in situ hybridization (FISH), flow cytometry,
southern blot, Northern Blot, Polymerase chain reaction
(PCR), Real time PCR, etc4-7.
Out of this exhaustive list, the Cell Search System is the
most commonly utilized, commercially available technique,
which is FDA approved. It is a semi quantitative device
for detection of CTC based on expression of epithelial
cell adhesion molecule (EpCAM) with antibody coated
magnetic beads as an enrichment media. CTC are defined
as cytokeratin +/CD 45 – nucleated cells4-7. Cristofanilli
et al.11 used cell search system to detect CTC and theirs'
was the first study to establish a threshold of 5 CTC per
7.5ml blood for differentiating between patients with
favourable and unfavourable prognosis.
Flow cytometry has also been applied for detection of
CTC in patients with metastatic cancers by Riethdorf et al.12, however, they found it to be less sensitive. Cruz et al.13 comparatively evaluated different cytokeratin types
(CK 7, CK 20, pan CK, CK8/CK18, CK 8 and CK 18) by
flowcytometry for identification of best combination of
DNA/ CK staining for detecting scarce circulating breast
cancer cells. They observed that CK 18 was the brightest
and more sensitive staining for breast cancer cells by flow
cytometry. The advantage of this method is that a special machine is not required, so it can be of great utility in
resource poor settings especially in developing countries
Hristozova et al.14 described a sensitive and reliable
multicolor flow cytometry protocol for CTC detection by
using an electronic threshold during data acquisition.
There is an ongoing debate as to which is better:
morphologic or molecular detection of CTC. Slide based
counting has the advantage of being highly specific, but
many authors believe that this method has low sensitivity
compared to quantitative mRNA techniques.
The importance of detecting CTC is more if it can be done
in early stage cancers when metastasis has not taken place
so that appropriate therapeutic remedy can be provided
to patient. Lucci et al.15 studied the prognostic value
of CTC in early stage breast cancer: 73 patients had >
1 CTC, 29 patients had > 2 CTC while 16 had > 3 CTC
per 7.5 ml blood. They did not observe any correlation
between primary tumor features and CTC detection.
However, presence of CTC was associated with significant
short progression free survival. On the contrary, in the
present study, CTC positivity significantly correlated with
tumor size, histologic type, tumor grade, metastasis and
skin infiltration. 66.66% (2/3) of ILC cases as compared
to 8.1% (9/111) cases of IDC were positive for CTC, this
could be due to absence of E-Cadherin which leads to early
dissemination of cancer cells into the blood stream.
Molecular methods in which mRNA of tumor cells is
amplified can also be used to detect CTC and have greater
sensitivity. Multimarker assay can be used instead of single
probe assay to further improve sensitivity. Disadvantages
of amplification-based tests are the false positivity,
heterogeneity in expression levels of particular target
transcripts as well as false negative6.
Pukazhendhi and Glück4 reviewed 81 manuscripts on
CTC in breast cancer and categorized them into those in
discovery datasets, prognostic factor in metastatic breast
cancer, predicting clinical utility in early breast cancer.
Based on this, they commented that the current diagnostic
modalities for CTC mainly focus on epithelial markers,
however measurement of circulating DNA is the best
Giordano et al.16 studied the clinical impact of CTC in
various molecular subtypes of breast cancer. Baseline CTC
detection had good prognostic value in all breast cancer
subtypes except Her 2 neu positive cancer. Guiliano et
al.17 observed the effect of different first line systemic treatment on the prognostic value of CTC in 492 advanced
breast cancer patients. A pre treatment level more than
or equal to 5 CTC/7.5 ml blood was associated with an
increased baseline number of metastatic sites compared
to those with less than 5 CTC/7.5 ml (p=0.0077). They
had 4 different treatment groups, out of which groups
with endocrine treatment and CT alone, high CTC was
associated with worse prognosis while the groups receiving
either her 2 neu targeted treatment or biological agent, did
not maintain the negative prognostic value of high CTC at
Krishnamurthy et al.18 evaluated the presence of CTC
in peripheral blood and its correlation between various
clinicopathological characteristics and hormone receptor
profile. CTC were found in 13 out of 43 T1 tumors
while in T2 tumors 12/38 were CTC positive. There was
no correlation between detection of CTC and standard
prognostic factors contrary to our findings of significant
association of CTC with tumor size, histologic type, tumor
grade, metastasis and skin infiltration. In our study, 31.25%
of grade 3 tumors were positive for CTC, followed by 1.31%
of grade 2 tumors, signifying that higher the grade, more
the positivity for CTC.
Turker et al.19 determined the effectiveness of CTC in
22 metastatic and 12 Early stage breast cancer cases for
prediction of progression free survival (PFS) and overall
survival (OS) as an adjunct to standard treatment care in
breast cancer management. CTC was positive in 3 (13.6%)
patients before chemotherapy (CT) and 6 (27.3%) patients
during CT in the metastatic subgroup whereas positive in
only one patient in early stage subgroup before and during
CT. CTC positivity was confirmed as a prospective marker
in this study even with small patient group.
Franken et al.20 undertook a study to explore whether
the presence of CTC at the time of diagnosis was associated
with recurrence free survival (RFS) and breast cancer
related death (BRD) in 404 breast cancer patients. Patients
were stratified into unfavorable (CTC > 1) and favorable
(CTC =0 in 30 ml peripheral blood). They concluded that
CTC in breast cancer patients before undergoing surgery
with curative intent is associated with an increased risk of
Peeters et al.21 explored potential differences in the
detection and prognostic significance of CTCs in MBC
according to immunohistochemical subtypes of breast
cancer. They did not observe any significant differences in
the absolute CTC counts (P=0.120) or in CTC positivity
rates according to ≥1 and ≥5 CTCs per 7.5 ml blood detection thresholds (P=0.165 and P=0.651, respectively)
between immunohistochemical subtypes. Very high CTC
counts, defined as ≥80 CTCs per 7.5 ml, were observed
more frequently in patients with Luminal A and triple
negative (TN) breast cancer (P=0.024). In the total study
population, the presence of ≥5 CTCs was the single most
significant prognostic factor for both PFS and OS in
multivariate analysis (P<0.001).
Rack et al.22 analyzed CTC in 2026 patients with early
breast cancer before adjuvant chemotherapy and in 1492
patients after CT using Cell Search System. Before CT,
CTC were detected in 21.5% of patients (435/2036), out
of which node negative versus node positive patients
with CTC were 19.6% vs. 22.4% (p<0.001), similar to the
current study where the node status significantly correlated
with CTC positivity. However, no association was found
with tumor size, tumor grade or hormone receptor status
which is contrary to our results as we found a significant
association with tumor size and tumor grade.
This study had many limitations. The sample size is too low
and confidence intervals are very wide, and the power of
the study is too low to reach to any significant conclusion.
The study strongly needs to be validated and replicated in
a bigger sample size.
In conclusion, the flow cytometry protocol for detection
and molecular characterization of CTCs is a time and
cost-effective technique, suitable for routine clinical use.
However, more elaborate studies are needed to establish
the role of flowcytometry in detection of circulating tumor
cells as a prognostic marker. One added advantage of
flow cytometric immunophenotyping is that panels can
be expanded to get additional information. Estrogen and
Progesterone receptors and Her2neu status in metastatic
breast carcinomas or BRAF mutation status (using of
mutation-specific antibodies) can be very useful in the
current approach towards personalized treatment.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
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