Are CD44⁺/CD24⁻ cells the assumed cancer stem cells in breast cancer?
Identification and characterization of the population of cancer stem cells (CSC) depends on several cellular markers, which combination is specific for the phenotype of CSC in the corresponding tumor. Several markers of CSC have already been identified in breast cancer (BC), but there are no univers...
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
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| Цитувати: | Are CD44⁺/CD24⁻ cells the assumed cancer stem cells in breast cancer? / D.E. Ryspayeva, I.I. Smolanka, A.S. Dudnichenko, A.A. Lyashenko, Yu.A. Grinevich, V.G. Gurianov, M.V. Koshubarova, A.A. Seleznev // Experimental Oncology. — 2017 — Т. 39, № 3. — С. 224–228. — Бібліогр.: 20 назв. — англ. |
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nasplib_isofts_kiev_ua-123456789-1385422025-02-09T16:47:41Z Are CD44⁺/CD24⁻ cells the assumed cancer stem cells in breast cancer? Ryspayeva, D.E. Smolanka, I.I. Dudnichenko, A.S. Lyashenko, A.A. Grinevich, Yu.A. Gurianov, V.G. Koshubarova, M.V. Seleznev, A.A. Original contributions Identification and characterization of the population of cancer stem cells (CSC) depends on several cellular markers, which combination is specific for the phenotype of CSC in the corresponding tumor. Several markers of CSC have already been identified in breast cancer (BC), but there are no universal indicators that could specifically identify the CSC in BC. Aims: To determine the validation of the CSC model for cell surface markers such as CD44 and CD24 and their clinical significance. Materials and Methods: Primary tumor samples of 45 patients with invasive BC without chemotherapy prior to surgery exposure were examined in paraffin blocks. CD44 and CD24 antigens expression was evaluated by the percentage of positive cells using different chromogens and the MultiVision detection system by immunohistochemical method. In this research the evaluation was determined by the following criteria: (-), negative — expression in < 10% of tumor cells; (+), positive — expression in ≥10% of cells. The same scoring system was applied for the expression of CD44⁺/CD24⁻. Results: 62.2% of investigated patients are patients older than 50 years and most of them with stage II of disease (71.0%) and luminal tumor subtypes (68.9%). We analysed the expression of CD44, CD24 and CD44⁺/CD24⁻ for different patients with dividing them into two groups. The group A consists of patients with unfavorable prognosis (relapses and metastases have occurred in the first three years after diagnosis), and the group B — with a favourable prognosis (the development of metastases after three years). Median disease-free survival in the group A is 19 months, in the group B — 46 months. The difference between the overall survival (OS) curves in the groups A and B is statistically significant (p < 0.001), the risk of death was higher in the group A (hazard ratio (HR) 5.9; confidence interval (CI) 2.3–15.2). The content of CD44 cells did not differ statistically between groups A and B (p = 0.18), but there was a tendency for increasing in OS with the existence of CD44+ cells (p = 0.056). The distribution of the expression of CD24 marker did not differ between the groups (p = 0.36) as well as the OS curves (p = 0.59). Analysis of the expression of CD44+/CD24⁻ which were considered as possible CSC, revealed a paradoxical increase (p = 0.03) of the frequency in patients of the group B (40.9%) compared to the group A (8.7%). Nevertheless, the comparison of the clinical outcomes did not reveal a statistically significant difference in the survival curves in the groups with existence and absence of CD44⁺/CD24⁻ expression (p = 0.08). The analysis showed the increasing of the risk of worse clinical outcomes in the cases of expression absence of CD44⁺/CD24⁻ (HR 2.8; CI 1.1–6.8). Conclusions: As a result of our research, the analysis of the quantity of assumed stem cells of the BC, which were identified by immunohistochemistry as CD44 and CD24 cells, failed to detect a statistically significant relation between groups of patients with different prognosis, and the identification of their expression is not enough for the characteristics of CSC. The obtained data demonstrating the worst clinical outcome in the cases of absence of CD44⁺/CD24⁻ expression apparently require further investigations and the validation of the immunohistochemical method with the determination of the cut-off line in defining of CD44 and CD24 status. 2017 Article Are CD44⁺/CD24⁻ cells the assumed cancer stem cells in breast cancer? / D.E. Ryspayeva, I.I. Smolanka, A.S. Dudnichenko, A.A. Lyashenko, Yu.A. Grinevich, V.G. Gurianov, M.V. Koshubarova, A.A. Seleznev // Experimental Oncology. — 2017 — Т. 39, № 3. — С. 224–228. — Бібліогр.: 20 назв. — англ. 1812-9269 https://nasplib.isofts.kiev.ua/handle/123456789/138542 en Experimental Oncology application/pdf Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| institution |
Digital Library of Periodicals of National Academy of Sciences of Ukraine |
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DSpace DC |
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English |
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Original contributions Original contributions |
| spellingShingle |
Original contributions Original contributions Ryspayeva, D.E. Smolanka, I.I. Dudnichenko, A.S. Lyashenko, A.A. Grinevich, Yu.A. Gurianov, V.G. Koshubarova, M.V. Seleznev, A.A. Are CD44⁺/CD24⁻ cells the assumed cancer stem cells in breast cancer? Experimental Oncology |
| description |
Identification and characterization of the population of cancer stem cells (CSC) depends on several cellular markers, which combination is specific for the phenotype of CSC in the corresponding tumor. Several markers of CSC have already been identified in breast cancer (BC), but there are no universal indicators that could specifically identify the CSC in BC. Aims: To determine the validation of the CSC model for cell surface markers such as CD44 and CD24 and their clinical significance. Materials and Methods: Primary tumor samples of 45 patients with invasive BC without chemotherapy prior to surgery exposure were examined in paraffin blocks. CD44 and CD24 antigens expression was evaluated by the percentage of positive cells using different chromogens and the MultiVision detection system by immunohistochemical method. In this research the evaluation was determined by the following criteria: (-), negative — expression in < 10% of tumor cells; (+), positive — expression in ≥10% of cells. The same scoring system was applied for the expression of CD44⁺/CD24⁻. Results: 62.2% of investigated patients are patients older than 50 years and most of them with stage II of disease (71.0%) and luminal tumor subtypes (68.9%). We analysed the expression of CD44, CD24 and CD44⁺/CD24⁻ for different patients with dividing them into two groups. The group A consists of patients with unfavorable prognosis (relapses and metastases have occurred in the first three years after diagnosis), and the group B — with a favourable prognosis (the development of metastases after three years). Median disease-free survival in the group A is 19 months, in the group B — 46 months. The difference between the overall survival (OS) curves in the groups A and B is statistically significant (p < 0.001), the risk of death was higher in the group A (hazard ratio (HR) 5.9; confidence interval (CI) 2.3–15.2). The content of CD44 cells did not differ statistically between groups A and B (p = 0.18), but there was a tendency for increasing in OS with the existence of CD44+ cells (p = 0.056). The distribution of the expression of CD24 marker did not differ between the groups (p = 0.36) as well as the OS curves (p = 0.59). Analysis of the expression of CD44+/CD24⁻ which were considered as possible CSC, revealed a paradoxical increase (p = 0.03) of the frequency in patients of the group B (40.9%) compared to the group A (8.7%). Nevertheless, the comparison of the clinical outcomes did not reveal a statistically significant difference in the survival curves in the groups with existence and absence of CD44⁺/CD24⁻ expression (p = 0.08). The analysis showed the increasing of the risk of worse clinical outcomes in the cases of expression absence of CD44⁺/CD24⁻ (HR 2.8; CI 1.1–6.8). Conclusions: As a result of our research, the analysis of the quantity of assumed stem cells of the BC, which were identified by immunohistochemistry as CD44 and CD24 cells, failed to detect a statistically significant relation between groups of patients with different prognosis, and the identification of their expression is not enough for the characteristics of CSC. The obtained data demonstrating the worst clinical outcome in the cases of absence of CD44⁺/CD24⁻ expression apparently require further investigations and the validation of the immunohistochemical method with the determination of the cut-off line in defining of CD44 and CD24 status. |
| format |
Article |
| author |
Ryspayeva, D.E. Smolanka, I.I. Dudnichenko, A.S. Lyashenko, A.A. Grinevich, Yu.A. Gurianov, V.G. Koshubarova, M.V. Seleznev, A.A. |
| author_facet |
Ryspayeva, D.E. Smolanka, I.I. Dudnichenko, A.S. Lyashenko, A.A. Grinevich, Yu.A. Gurianov, V.G. Koshubarova, M.V. Seleznev, A.A. |
| author_sort |
Ryspayeva, D.E. |
| title |
Are CD44⁺/CD24⁻ cells the assumed cancer stem cells in breast cancer? |
| title_short |
Are CD44⁺/CD24⁻ cells the assumed cancer stem cells in breast cancer? |
| title_full |
Are CD44⁺/CD24⁻ cells the assumed cancer stem cells in breast cancer? |
| title_fullStr |
Are CD44⁺/CD24⁻ cells the assumed cancer stem cells in breast cancer? |
| title_full_unstemmed |
Are CD44⁺/CD24⁻ cells the assumed cancer stem cells in breast cancer? |
| title_sort |
are cd44⁺/cd24⁻ cells the assumed cancer stem cells in breast cancer? |
| publisher |
Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| publishDate |
2017 |
| topic_facet |
Original contributions |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/138542 |
| citation_txt |
Are CD44⁺/CD24⁻ cells the assumed cancer stem cells in breast cancer? / D.E. Ryspayeva, I.I. Smolanka, A.S. Dudnichenko, A.A. Lyashenko, Yu.A. Grinevich, V.G. Gurianov, M.V. Koshubarova, A.A. Seleznev // Experimental Oncology. — 2017 — Т. 39, № 3. — С. 224–228. — Бібліогр.: 20 назв. — англ. |
| series |
Experimental Oncology |
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224 Experimental Oncology 39, 224–228, 2017 (September)
ARE CD44+/CD24– CELLS THE ASSUMED CANCER STEM CELLS
IN BREAST CANCER?
D.E. Ryspayeva1, *, I.I. Smolanka1, A.S. Dudnichenko2, A.A. Lyashenko1, Yu.A. Grinevich1,
V.G. Gurianov3, M.V. Koshubarova1, A.A. Seleznev4
1National Cancer Institute, Kyiv 03022, Ukraine
2Medical Academy of Postgraduate Education, Kharkiv 61176, Ukraine
3O.O. Bogomolets National Medical University, Kyiv 03115, Ukraine
4CSD Health Care, private laboratory, Kyiv 03022, Ukraine
Identification and characterization of the population of cancer stem cells (CSC) depends on several cellular markers, which combina-
tion is specific for the phenotype of CSC in the corresponding tumor. Several markers of CSC have already been identified in breast
cancer (BC), but there are no universal indicators that could specifically identify the CSC in BC. Aims: To determine the validation
of the CSC model for cell surface markers such as CD44 and CD24 and their clinical significance. Materials and Methods: Primary
tumor samples of 45 patients with invasive BC without chemotherapy prior to surgery exposure were examined in paraffin blocks.
CD44 and CD24 antigens expression was evaluated by the percentage of positive cells using different chromogens and the MultiVi-
sion detection system by immunohistochemical method. In this research the evaluation was determined by the following criteria: (-),
negative — expression in < 10% of tumor cells; (+), positive — expression in ≥10% of cells. The same scoring system was applied for
the expression of CD44+/CD24−. Results: 62.2% of investigated patients are patients older than 50 years and most of them with stage
II of disease (71.0%) and luminal tumor subtypes (68.9%). We analysed the expression of CD44, CD24 and CD44+/CD24− for diffe-
rent patients with dividing them into two groups. The group A consists of patients with unfavorable prognosis (relapses and metastases
have occurred in the first three years after diagnosis), and the group B — with a favourable prognosis (the development of metastases
after three years). Median disease-free survival in the group A is 19 months, in the group B — 46 months. The difference between
the overall survival (OS) curves in the groups A and B is statistically significant (p < 0.001), the risk of death was higher in the group
A (hazard ratio (HR) 5.9; confidence interval (CI) 2.3–15.2). The content of CD44 cells did not differ statistically between groups
A and B (p = 0.18), but there was a tendency for increasing in OS with the existence of CD44+ cells (p = 0.056). The distribution of the
expression of CD24 marker did not differ between the groups (p = 0.36) as well as the OS curves (p = 0.59). Analysis of the expression
of CD44+/CD24− which were considered as possible CSC, revealed a paradoxical increase (p = 0.03) of the frequency in patients
of the group B (40.9%) compared to the group A (8.7%). Nevertheless, the comparison of the clinical outcomes did not reveal a sta-
tistically significant difference in the survival curves in the groups with existence and absence of CD44+/CD24– expression (p = 0.08).
The analysis showed the increasing of the risk of worse clinical outcomes in the cases of expression absence of CD44+/CD24− (HR 2.8;
CI 1.1–6.8). Conclusions: As a result of our research, the analysis of the quantity of assumed stem cells of the BC, which were identi-
fied by immunohistochemistry as CD44 and CD24 cells, failed to detect a statistically significant relation between groups of patients
with different prognosis, and the identification of their expression is not enough for the characteristics of CSC. The obtained data
demonstrating the worst clinical outcome in the cases of absence of CD44+/CD24− expression apparently require further investigations
and the validation of the immunohistochemical method with the determination of the cut-off line in defining of CD44 and CD24 status.
Key Words: breast cancer, cancer stem cells, CD44, CD24, CD44+/CD24− cells.
According to modern concepts tumors originate
from normal cells because of accumulated genetic and
epigenetic changes, but to identify these tumor-forming
cells is rather difficult. Stem cells were suggested as at-
tractive models of “tumor-initiating” or “tumorigenic”
cells [1, 2] as they possess a lot of characteristics similar
to tumor cells, including the self-renewal capacity and
differentiation into various clones and an ability to in-
duce cellular heterogeneity, to migrate and penetrate
into surrounding tissues. Cancer stem cells (CSC) have
been identified as a subgroup of tumor cells with stem
properties. They are responsible for the growth, progres-
sion and relapse of the tumor [1, 3].
The hypothesis of the key role of CSC in predic-
tion the biological aggressiveness of cancer was
proposed many years ago and has been revived with
new experimental approaches recently [4]. However,
the high heterogeneity of CSC within a tumor as well
as the absence of certain criteria for “stem” (belonging
to CSC) require further investigations in this direction.
The identification and the description of the popula-
tion of CSC depend mainly on several cellular markers,
the combination of which is specific for the phenotype
of CSC in the corresponding tumor. Several supposing
CSC markers have already been characterized in breast
cancer (BC) but so far there is no universal combination
of markers which could specifically identify CSC of BC.
Thus, expression of the cell surface marker
CD44 was identified in experiments on cell lines as-
sociated with proliferation and invasion of BC cells [5].
The dominant tumorogenic effect of CD44 with
Submitted: July 31, 2017.
*Correspondence: E-mail: ryspayeva1@gmail.com
Abbreviations used: AJCC — American Joint Committee on Can-
cer; ASCO/CAP — American Society of Clinical Oncology/College
of American Pathologists; BC — breast cancer; CD — cluster of dif-
ferentiation; CI — confidence interval; CSC — cancer stem cells;
ER — estrogen receptor; HER2 — human epidermal growth factor
receptor 2; HR — hazard ratio; IHC — immunohistochemistry;
LN — lymph node; PFS — progression-free survival; OS — overall
survival; PR — progesterone receptor.
Exp Oncol 2017
39, 3, 224–228
Experimental Oncology 39, 224–228, 2017 (September) 225
evidence of worse clinical behavior and a shorter
progression-free survival (PFS) [6, 7] has provided the
rationale using of the CD44 marker alone or in combi-
nation with other surface markers in order to determine
cells with “stem” properties.
The other marker, the membrane protein CD24,
also plays the certain role in tumorigenesis and its
expression may be sufficient in promotion tumor me-
tastasis and increasing proliferation of tumor cells [8].
At the same time, the effect of CD24 expression
on tumorigenicity and invasiveness is inconsistent and
varies from positive to negative [9].
The combination of these markers in BC was in-
vestigated by Al-Hajj et al. [10]. They demonstrated
for the first time that CD44+/CD24−/low cells from the
tumor pleural effusion of patients with BC were more
tumorigenic in mice and received xenografts have
reproduced the heterogeneity of the original tumor.
This specific immunophenotype CD44+/CD24−/low has
been presented as CSC of BC and as a powerful pre-
dictor of short PFS, overall survival (OS) and as a risk
of distant metastases [10, 11].
But, unfortunately, the further researches did not
provide the convincing evidence that CD44+ tumor
cells are stem cells in reality [12]. This is confirmed
by the research [13] in which the analysis of the number
of supposed stem cells in BC (identified immunohisto-
chemically as CD44+/CD24− cells) was not related with
clinical outcomes and survival, although the tumors
which have developed distant metastases (mainly
in the bone) had a higher share of CD44+/CD24− cells.
The other study [14] points out that the signature
CD44+/CD24− has identified a more basal cell phe-
notype than tumorigenicity and CD44−/CD24+ cells
showed signs of luminal epithelial cells.
Hence, these investigations have demonstrated
the ability to form breast tumors by tumor cells with
CD44+/CD24−/low characteristics and simultaneously the
amount of CD44+/CD24− in BC cell lines did not predict
a tumorigenic potential. Our task was to determine the
validation of the CSC model by the surface markers
CD44 and CD24, CD44+/CD24− and indicate their clini-
cal significance and connection with patient outcomes.
MATERIALS AND METHODS
In the present research, primary tumor samples
of 45 patients with invasive BC were studied in paraffin
blocks. The patients were operated at the National Can-
cer Institute (Kyiv) for the period from 2008 to 2012 and
they did not receive chemotherapy before surgery. All the
patients gave their written informed consent. The study
was approved by the Local Medical Ethics Committee.
The evaluation variables were age, axillary lymph
node (LN) metastasis status, tumor stage, estrogen re-
ceptor (ER) status, progesterone receptor (PR), and hu-
man epidermal growth factor receptor 2 (HER2) status.
We also analyzed PFS and censored OS. The corre-
lations between marker expression and patient survival
time from the date of surgery were studied as well. The
average follow-up time was 53.9 ± 20.4 months (M ± SD).
The disease stage was collected from the pa-
tient’s medical history and was coded according
to the American Joint Committee on Cancer (AJCC),
the tumor phenotype was classified according to the
St. Gallen consensus [15]. ER and PR status were de-
termined by immunohistochemistry (IHC) using Ameri-
can Society of Clinical Oncology/College of American
Pathologists (ASCO/CAP) guidelines [16]. HER2 status
was also determined by IHC. HER2-positive tumors
were defined as 3+ and HER2-negative tumors were
defined as 0 or 1+ using ASCO/CAP guidelines [17].
The baseline characteristics of the patients are sum-
marized in Table 1.
Тable 1. Patient characteristics
Patient characteristics Number of patients, n (%)
All cases 45
Age, years
< 50 17 (37.8)
≥ 50 28 (62.2)
LN metastasis
N− 20 (44.4)
N+ 25 (55.6)
Stage (AJCC)
I 4 (9.0)
IIA 16 (35.5)
IIB 16 (35.5)
IIIА 9 (20.0)
Intrinsic subtypesa
Luminal А 14 (31.1)
Luminal В (HER2-negative) 17 (37.8)
Triple negative BC 11 (24.4)
HER2-positive 3 (6.7)
Note: aSt. Gallen’s intrinsic subtype’s classification.
Sections of tumor tissue 3 μm thick were made
on SuperFrost Plus adhesive glasses (Menzel, Ger-
many) from formalin fixed paraffin blocks, which then
were deparaffinized in xylene and rehydrated in a 96%
ethanol solution. For antigen retrieval, paraffin tissue
sections were immersed into citrate buffer (pH 6.0 for
Ki-67 and HER2) and into TRIS-EDTA buffer (pH 9.0 for
the rest) heated to 98 °C for the period of 40 min. Then
the sections were rinsed out with TRIS-HL buffer.
The cells were incubated with antibodies at room
temperature within 30 min. CD44 and CD24 were
detected by the MV Detection System (MultiVision
anti-rabbit/HRP + anti-mouse/AP polymers — Thermo
Scientific, USA) with anti-mouse alkaline phospha-
tase + anti-rabbit peroxidase polymers using various
chromogenes — blue chromogen CD24 and red
chromogen CD44 (Table 2). Then the sections were
stained with Light Green for a short period of time, de-
hydrated and mounted. Staining was performed with
EnVision™ FLEX+ System (Dako, Denmark) to evaluate
the expression of ER, PR, HER2, Ki-67.
The expression of cell surface markers CD44 and
CD24 was graded in terms of the percentage of posi-
tive cells with membrane staining in each block as well
as the intensity of staining. Pathologist who was blind-
ed to clinical diagnosis scored all cases. Staining for
each of CD44, CD24 and CD44+/CD24− was evaluated
separately using our scoring system.
There is no common cut-off value for evaluating
of the expression of CD44 and CD24 nowadays and
in the research the evaluation was determined accord-
ing to the following criteria: (−), negative — expression
226 Experimental Oncology 39, 224–228, 2017 (September)
in < 10% of tumor cells; (+), positive — expression of
≥ 10% of tumor cells. The same scoring system de-
scribed for the antigens CD44 and CD24 was applied for
the expression of the proportion of CD44+/CD24− [18].
Table 2. Antibodies and immunohistochemical techniques
Anti-
body Clone Source Dilution
Incubation
temperature/
time
ER EP1 Rabbit mono-
clonal
DAKO, Denmark RTU 4 °C/overnight
PR PgR 636
Mouse monoclonal
DAKO, Denmark RTU 4 °C/overnight
HER2 Rabbit polyclonal DAKO, Denmark 1:750 Room tempera-
ture, 30 min
Ki-67 MIB1
Mouse monoclonal
DAKO, Denmark RTU Room tempera-
ture, 30 min
CD24 SN3
Mouse monoclonal
Thermo
Scienti fic, USA
1:200 Room tempera-
ture, 30 min
CD44 Rabbit polyclonal Thermo
Scientific, USA
1:100 Room tempera-
ture, 30 min
Note: RTU — ready–to–use.
Statistical analysis. The data were analysed
using MedCalc v.17.6 (MedCalc Software Inc, Broek-
straat, Belgium). In order to analyze and report the
data descriptive statistics were used. The χ2 test was
used for comparison of qualitative data. For survival
time estimation the Kaplan — Meier survival analysis
was used. The survival proportions (with standard
error) were calculated. The median survival times are
reported with their 95% confidence interval (CI). For
the comparison between the survival curves logrank
test was used, hazard ratio (HR) with CI was calculated.
The significance threshold was set at p < 0.05.
RESULTS AND DISCUSSION
We have analyzed the patients’ clinicopathological
data (Table 1). 62.2% of patients were the patients over
50 years old and the majority of them (71.0%) had the
stage II of disease and luminal tumor subtypes (68.9%).
The patients were divided into two groups:
group A — with an unfavorable prognosis (the occur-
rence of relapses and metastases in the first three
years after staging the diagnosis), and group B — with
a favorable prognosis (development of metastases
after three years of the disease). Median PFS in group
A was 19 months (CI 17–22 months), in group B —
46 months (CI 43–52 months). In addition, the 1-year
old PFS in group A was 82.6 ± 7.9% vs 100% in group B.
The difference between OS curves in groups A and
B is statistically significant (p < 0.001) (Fig. 1). Median
OS in group A was 53.3 months (CI 39–58 months),
in group B — 80 months (CI 75–95 months). The
5-year OS of patients in group A was 23.4 ± 10.2%
and in group B — 92.3 ± 7.4%, respectively, the risk
of death was higher in group A (HR 5.9; CI 2.3–15.2).
The analysis of clinicopathological data has not
correlated with the existence of metastases in regional
LN (p = 0.10) and the tumor phenotype (p = 0.12) of the
patients in group A with an unfavorable clinical out-
come given above (Table 3). Although there were more
patients with non-luminal subtypes (43.5%) in group
A associated with a worse prognosis than group B
(18.2%), the distribution between the groups did not
differ statistically significant (p = 0.12). The statistically
significant difference was in the distribution by stages
(p = 0.009); there were more patients with stages I and
IIA in group B.
Su
rv
iva
l p
ro
ba
bi
lit
y,
%
100
80
60
40
20
0
0 20 40 60 80 100 120
Time, months
A
B
Fig. 1. Survival analysis according group A (patients with an un-
favorable prognosis) and B (patients with a favorable prognosis)
Using the alleged data an unfavorable clinical out-
come may be associated with the existence of CSC,
we performed an analysis of the expression of cell sur-
face markers of identified CD44 and CD24, their combi-
nations of CD44+/CD24− in different groups of patients.
Table 3. Clinicopathological characteristics of patients’ groups with dif-
ferent outcomes and expression of CD44, CD24, CD44+/CD24−
Clinicopathological characteristics Group A,
n (%)
Group B,
n (%) p-value
All cases 23 (51.1) 22 (48.9)
Age, years
< 50 6 (26.1) 11 (50) 0.18
≥ 50 17 (73.9) 11 (50)
LN metastasis
N− 7 (30.4) 13 (59.1) 0.10
N+ 16 (69.6) 9 (40.9)
Stage (AJCC)
I 0 (0.0) 4 (18.2) 0.009
IIA 7 (30.4) 9 (40.9)
IIB 13 (56.5) 3 (13.6)
IIIА 3 (13.1) 6 (27.3)
Intrinsic subtypes
Luminal А 5 (21.7) 9 (40.9) 0.12
Luminal В (HER2-negative) 8 (34.8) 9 (40.9)
Triple negative BC 7 (30.4) 4 (18.2)
HER2-positive 3 (13.1) 0 (0.0)
CD44
Positive (%) 6 (26.1) 11 (50.0) 0.18
Negative (%) 17 (73.9) 11 (50.0)
CD24
Positive (%) 8 (34.8) 4 (18.2) 0.36
Negative (%) 15 (65.2) 18 (81.8)
CD44+/CD24−
Positive (%) 2 (8.7) 9 (40.9) 0.03
Negative (%) 21 (91.3) 13 (59.1)
We had been observing a different expression
of CD44, CD24, and a different distribution in groups
A and B (Fig. 2). Some tumors contained only CD44+
(red) or only CD24+ (blue) cells, while the others had
a combination of two cell types.
Expression of cell surface markers CD44 and
CD24 and their combinations CD44+/CD24− were
analyzed.
Expression of CD44 cell did not differ statistically be-
tween groups A and B (p = 0.18). Nevertheless, the lack
of expression was observed in 73.9% of cases in group A.
The expression of the CD44+ phenotype was observed
Experimental Oncology 39, 224–228, 2017 (September) 227
in half of the group B. Some studies showed that the
tumors consisting mainly of CD44+ cells may have worse
clinical behavior [7]. Probably a certain role in the expres-
sion of CD44+ is represented by the TGF-β signaling path-
way which is known to play a dual role in the progression
of tumors. On the one hand, it is one of the most powerful
inhibitors of cell proliferation; on the other, it promotes
the invasion, angiogenesis, epithelial-mesenchymal
transition and metastasis [6, 9, 19].
Our results show that cells with different phenotypes,
even within the same type of tumor and tissue, respond
differently to the activation of TGF-β. Median OS in the
absence of CD44+ expression was 63 months (CI 54–
78 months) and with high expression — 88 months
(CI 56–95 months) (p = 0.056). There is a tendency for
an increasing in OS with the existence of CD44+ cells.
The distribution of the expression of another surface
marker of CD24 did not differ either between the groups
(p = 0.36) or the OS curves (p = 0.59). Median OS with
low expression or absence of CD24+ was 71 months
(CI 57–88 months) and in the presence of expression —
56 months (CI 39–78 months). In this case, low expression
or lack of expression were noted in 81.8% of cases in group
B, whereas in group A there were more cells with positive
expression of the phenotype CD24+. This observation
contradicts the hypothesis that CD24+ cells are more dif-
ferentiated and less tumor-bearing cells. But it conforms
the concept of linking the expression of CD24 with tumor
progression and metastatic behavior [8].
Since the detection of CD24+ and CD44+ cells in-
volves several steps and it cannot be ruled out the pos-
sibility that the procedure has changed the expression
of some antigens. We analyzed the simultaneous de-
tection of CD44+/CD24− as presumed CSC. The state
of expression of cells with immunophenotype CD44+/
CD24− revealed a paradoxical increase (p = 0.03) of its
frequency in patients of group B (40.9%) compared
with group A (8.7%). The existing data conflicts with
earlier reports indicating that phenotype CD44+/CD24−
correlated with an unfavorable prognosis [18, 20].
Nevertheless, the comparison of clinical outcomes
did not reveal a statistically significant difference in the
survival curves in the groups with the presence and
absence of CD44+/CD24− expression (p = 0.08). Median
OS in the presence of CD44+/CD24− expression was
88 months (CI 88–95 months), whereas the absence
of it was 63 months (CI 54–78 months) (Fig. 3). The
same trend of higher survival in the pre sence of CD44+/
CD24− expression was observed for 3 years of survival
(100% vs 84.4 ± 6.2%, respectively) and 5-year of sur-
vival (85.7 ± 13.2% vs 50.2 ± 9.7%, respectively). This
analysis allows to make a conclusion that the risk of un-
favourable clinical outcomes increases in the absence
of CD44+/CD24− expression (HR 2.8; CI 1.1–6.8).
Fig. 2. Immunohistochemical profiles of expression CD44 (red) and CD24 (blue): a — CD44+ cells; b — CD24+ cells; c — CD44+/
CD24− cells (group A); d — CD44+/CD24− cells (group B). Immunohistochemical staining using various chromogenes patterns
of CD44, CD24 on paraffin-embedded tissue sections of breast carcinoma, × 100
228 Experimental Oncology 39, 224–228, 2017 (September)
Su
rv
iva
l p
ro
ba
bi
lit
y,
%
100
80
60
40
20
0
0 20 40 60 80 100 120
Time, months
-
+
Fig. 3. Survival analysis according to the presence (+) and
absence (−) of expression CD44+/CD24−
According to the results of our investigation,
the analysis of the number of supposed stem cells
of BC identified by IHC as CD44+ and CD24– cells did
not reveal a statistically significant relation between
groups of patients having different prognosis. Immuno-
histochemical analysis of tumors among the samples
of the BC showed high heterogeneity in the expression
of the selected immunophenotypes. The expression
of a single cell surface marker may not be sufficient
to identify it without an alternative and to determine its
tumorigenic potential and, hence, the clinical outcome.
The data obtained which demonstrate the worst clinical
outcome in the absence of CD44+/CD24− expression are
discordant to the data of other researchers [9, 18]. This
result is also confirmed by the observed trend of higher
survival in the presence of CD44+/CD24− and CD44+ phe-
notypes. But, the obtained data also indicate that studies
in experimental systems may not reflect the behavior of cells
in the tumor in patients. In addition, the usage of differ-
ent immunohistochemical scoring method in defining
CD44 and CD24 status and the classification of the status
of CD44 and CD24 expression may be the cause of the dis-
crepancy between our investigation and previous studies.
We acknowledge the limitations of this study. Firstly,
this is strictly a population-based cancer registry in-
vestigation. Secondly, it includes an insufficiently ad-
equate number of BC cases. Thirdly, there is no definite
technique for evaluating stem cells and the search for
markers identifying CSC still continues.
To sum it up, based on our data, the markers CD44 and
CD24 do not reflect the features of CSC and unfavor-
able prognosis and do not clarify the role and clinical
significance of the immunophenotype CD44+/CD24−.
These markers require further studies, especially clinical
trials and validation of the immunohistochemical method.
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