The clinicopathologic and prognostic significance of CD44+/CD24(-/low) and CD44-/CD24+ tumor cells in invasive breast carcinomas.

Abstract

Cells with distinct phenotypes and stem cell-like properties have been reported to exist in breast cancer. The aim of the present study was to investigate the clinicopathologic and prognostic significance of the CD44(+)/CD24(-/low) and CD44(-)/CD24(+) tumor phenotypes' prevalence. Double immunohistochemistry was applied on a series of 155 paraffin-embedded breast tissue specimens to detect CD44 and CD24. Evaluation of the phenotypes was performed by image analysis. The prevalence of CD44(+)/CD24(-/low) and CD44(-)/CD24(+) tumor cells was 58.7% and 82.6%, respectively. The dominance of the CD44(+)/CD24(-/low) tumor cells was inversely associated with lymph node metastasis (P = .019) and tended to inversely associate with the stage of the disease (P = .068). Moreover, the prevalence of CD44(+)/CD24(-/low) was found to exert no significant impact on patients' prognosis although it displayed a tendency toward an increase in disease-free survival (P = .074). On the other hand, the prevalence of CD44(-)/CD24(+) tumor cells was found to have no clinicopathologic significance. However, it was found to exert an unfavorable impact on both relapse-free (P = .009) and overall survival (P = .046) of the patients with breast carcinomas of intermediate differentiation (grade 2). In breast tissue, CD44(+)/CD24(-/low) tumor cells seem to be associated with lack of lymph node metastasis and a tendency toward an increase of the relapse-free survival of the patients. On the contrary, tumor cells with the phenotype CD44(-)/CD24(+) seem to identify patients with worse disease-free and overall survival within the group of intermediate-grade differentiation patients whose prognosis is difficult to assess.

Full text

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Original contribution
The clinicopathologic and prognostic significance of
CD44+/CD24−/lowand CD44−/CD24+tumor cells in invasive
breast carcinomas
Eleni Mylona MD, Ioanna Giannopoulou BSc, Emmanouil Fasomytakis BSc,
Alexandros Nomikos MD, Christina Magkou MD, Panagiotis Bakarakos MD,
Lydia Nakopoulou PhD⁎
Department of Pathology, School of Medicine, National and Kapodistrian University of Athens, GR-11527 Athens, Greece
Received 5 October 2007; revised 1 December 2007; accepted 6 December 2007
Keywords:
Stem cells;
Breast cancer;
CD44+/CD24−/low;
CD44−/CD24+;
Metastasis
Summary Cells with distinct phenotypes and stem cell–like properties have been reported to exist in
breast cancer. The aim of the present study was to investigate the clinicopathologic and prognostic
significance of the CD44+/CD24−/lowand CD44−/CD24+tumor phenotypes' prevalence. Double
immunohistochemistry was applied on a series of 155 paraffin-embedded breast tissue specimens to
detect CD44 and CD24. Evaluation of the phenotypes was performed by image analysis. The prevalence
of CD44+/CD24−/lowand CD44−/CD24+tumor cells was 58.7% and 82.6%, respectively. The
dominance of the CD44+/CD24−/lowtumor cells was inversely associated with lymph node metastasis
(P = .019) and tended to inversely associate with the stage of the disease (P = .068). Moreover, the
prevalence of CD44+/CD24−/lowwas found to exert no significant impact on patients' prognosis
although it displayed a tendency toward an increase in disease-free survival (P = .074). On the other
hand, the prevalence of CD44−/CD24+tumor cells was found to have no clinicopathologic significance.
However, it was found to exert an unfavorable impact on both relapse-free (P = .009) and overall
survival (P = .046) of the patients with breast carcinomas of intermediate differentiation (grade 2). In
breast tissue, CD44+/CD24−/lowtumor cells seem to be associated with lack of lymph node metastasis
and a tendency toward an increase of the relapse-free survival of the patients. On the contrary, tumor
cells with the phenotype CD44−/CD24+seem to identify patients with worse disease-free and overall
survival within the group of intermediate-grade differentiation patients whose prognosis is difficult
to assess.
© 2008 Published by Elsevier Inc.
1. Introduction
Currently, a growing body of evidence has been reported
supporting the notion that tumors are organized in a
hierarchy of heterogeneous cell populations with different
biological properties and that the capability to sustain tumor
⁎Corresponding author.
E-mail address: lnakopou@cc.uoa.gr (L. Nakopoulou).
www.elsevier.com/locate/humpath
0046-8177/$ – see front matter © 2008 Published by Elsevier Inc.
doi:10.1016/j.humpath.2007.12.003
Human Pathology (2008) 39, 1096–1102

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formation and growth exclusively resides in a small
proportion of tumor cells termed cancer stem cells. The
latter are believed to share the feature of self-renewal with
stem cells as well as the differentiation into diverse cell types
[1,2]. More specifically, there is substantial evidence in favor
of the stem cell theory of breast carcinogenesis based on the
observation of phenotypes common to breast stem cells and
tumor cells and on the demonstration that breast epithelial
cells with stem cell features were more susceptible to
neoplastic transformation [3]. Cancer stem cells were
reported to have been identified in breast cancer as CD44+/
CD24−/lowcells, capable of driving tumor formation when
injected into the mammary fat pad of NOD/SCID mice [4].
However, much concern has been expressed with regard to
whether the cells that have been separated from solid tumors
are truly cancer stem cells [5]. Moreover, CD44+/CD24−/low
cells have not been cultured as adherent cells yet and their
clinicopathologic or prognostic significance was never
proved or remains controversial, respectively [6,7].
In addition, molecular profiling studies have classified
breast cancers and breast cancer lines into 5 types with
distinct prognostic significance [8,9]. Of these, luminal and
basal are the most important types corresponding with the
2 types of normal breast epithelial cells (luminal and basal/
myoepithelial) [10]. Interestingly, in vitro studies have
shown that all cell lines that contained CD44+/CD24−/low
population belonged to the basal group, whereas luminal cell
types contained higher levels of CD44−/CD24+, suggesting
that the latter population may contain cancer stem cells, thus
corresponding to the luminal type of tumors, which represent
the majority of breast cancers [3,11].
Given the aforementioned data, we identified CD44+/
CD24−/lowand CD44−/CD24+tumor cells in paraffin-
embedded tissue sections of patients with breast cancer, by a
double-stainingimmunohistochemicaltechniqueandsearched
for their potential pathologic and prognostic significance.
2. Material and methods
2.1. Patients and samples studied
One hundred and sixty paraffin blocks with tumor
samples were available from patients with resectable breast
cancer who had undergone surgery. We only selected women
with histologically proven, clearly invasive breast carcino-
mas, regardless of their initial stage, in whom axillary lymph
node dissection had been performed and who had all their
resected materials studied histologically. The patients were
aged from 25 to 86 years (mean age, 56.74 years). None of
them had received radiation or chemotherapy preoperatively.
Lastly, an informed consent was obtained from patients for
the material derived from them to be used in research.
A routine histologic examination was performed with
hematoxylin-eosin staining. All carcinomas were classified
Table 1
CD44+/CD24−/lowand CD44−/CD24+phenotypes as related to clinicopathologic parameters and immunohistochemical markers
CD44+/CD24−/lowtumor cellsCD44−/CD24+tumor cells
n Median Range
P MedianRangeP
Menopausal status Before
After
Ductal
Lobular
1
2
3
1
2
3
b2
2-5
N5
Not infiltrated
Infiltrated
1
2
3
Negative
Positive
Negative
Positive
46
99
115
29
18
88
35
52
50
42
31
93
20
58
85
24
97
22
59
85
66
78
2
2
2
3
0
2
2
2
2
2
3
2
1
4
1
4
2
1
3
2
3
2
0-59
0-70
0-70
0-30
0-33
0-70
0-59
0-47
0-70
0-70
0-70
0-70
0-47
0-42
0-70
0-70
0-70
0-30
0-70
0-70
0-70
0-70
NS70
70
70
72
65
70
72
66
70
70
64
71
71
70
69
64
71
69
70
70
70
69
5-90
3-91
5-90
3-91
5-87
3-91
6-90
5-91
3-90
9-87
8-90
3-91
6-87
8-91
3-89
8-90
3-91
6-87
3-89
5-91
3-87
5-91
NS
Histologic type NS NS
Histological grade NSNS
Nuclear grade NSNS
Tumor sizeNS NS
LN 0.019NS
Stage 0.068NS
ERNS NS
PR NS NS
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according to the criteria of the World Health Organization
[12] and were recorded as invasive ductal or invasive lobular.
The combined histologic grade (1, 2, and 3) of infiltrating
ductal carcinoma was obtained according to a modified
Scarff-Bloom-Richardson histologic grading system with
guidelines as suggested by Nottingham City Hospital
pathologists [13]. Nuclear grading was based on nuclear
polymorphism. Staging at the time of diagnosis was based on
the TNM system [14]. Tumor size (b2, 2-5, N5 cm) and
lymph node status were evaluated separately. The clinico-
pathologic characteristics of the series are shown in Table 1.
During the immunohistochemical procedure, some speci-
mens were destroyed and were considered to be too small to
be evaluated for both immunomarkers. Therefore, the
number of cases finally included in the statistical analysis
was 155.
Follow-up was available for all 147 patients. Mean
survival time was 111 months, whereas mean disease-free
survival time was 106 months (range, 2-135 months). Patient
outcome was defined as disease-free and overall survival. All
patients received conventional postoperative treatment
depending on the extent of the disease, including radiation
therapy and medical antiestrogen therapy, when indicated.
Premenopausal patients with axillary involvement were
treated with 6 courses of adjuvant chemotherapy.
2.2. Immunohistochemistry
A double immunohistochemical staining was applied on
4-μm paraffin-embedded tissue sections using the following
monoclonal antibodies: (a) anti-CD24 (clone SN3b, Neo-
markers, Fremont, CA) at a dilution of 1:60 and (b) anti-
CD44 (clone 156-3C11, Neomarkers) at a dilution of 1:200.
Antigen retrieval was achieved by treating the slides in a
microwave for 15 minutes in sodium citrate 10 mmol/L, pH
6.0. After deparaffinization, rehydration, and quenching of
the endogenous peroxidase activity, CD24 was applied for
1 hour at room temperature and immunodetected, using the
3-step immunohistochemical technique of streptavidin-
biotin-alkaline phosphatase (SuperSensitive Link-Label ISH
Detection System, Biogenex, San Ramon, CA). Fast red was
used as a chromogen. Subsequently, CD44 was added for
1 hour at room temperature and immunodetected with a
polymer-linked peroxidase molecule, conjugated to second-
ary antibody (Impress Re, 2-step polymer-based technique,
Vector Lab, Burlingame, CA). Diaminobenzidine/nickel
(SK-400, Vector Lab) was used as a chromogen. Finally,
sections were counterstained with hematoxylin and mounted.
Paraffin sections of normal tonsilar tissue were used as a
positive control for CD44 and CD24 antibodies staining.
2.3. Image analysis
CD44 was identified as black and CD24 as red membrane
staining. Cells with black color staining without much
interference from red color were identified as CD44+/
CD24−/lowtumor cells, whereas cells with intense red
staining in the absence of black color were characterized as
CD44−/CD24+tumor cells. To avoid the evaluation bias
inherent to subjective pathologic inspection, automatic
image analysis was performed. Images were acquired using
a Zeiss Axiolab microscope (Carl Zeiss Jena GmbH, Jena,
Germany) with a mechanical stage and fitted with a Sony-iris
CCD video camera (Sony Corp, Tokyo, Japan). The video
camera was connected to a Pentium IIÉ personal computer
loaded with Image Scan software (Jandel Scientific, Erkrath,
Germany). Image analysis was done for 15 randomly
selected areas (on 400× high-power field) of each case.
Fig. 1
benzidine/nickel stained only) in invasive breast cancer. CD44 is
also present in the stromal cells (double-staining immunohisto-
chemistry, ×200).
Detection of the CD44+/CD24−/lowtumor cells (diameno-
Fig. 2
stained only) in invasive breast cancer. CD24 expression is obvious
in the cytoplasm of the malignant cells, as well (double-staining
immunohistochemistry, ×200).
Detection of the CD44−/CD24+tumor cells (fast red
1098E. Mylona et al.

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Each area was photographed and stored as a JPEG file (1600
× 1200 pixels, 16.7 million colors). The total number of
CD44+/CD24−/lowand CD44−/CD24+was calculated.
2.4. Statistical analysis
Correlations were assessed using the Spearman rank
correlation test for 2 continuous variables and the Mann-
Whitney U test and Kruskall-Wallis test when one variable
was continuous and the other categorical. To investigate the
effect of CD44+/CD24−/lowand CD44−/CD24+prevalence
on clinical outcome, we used the median values as cutoff
values and especially for CD44+/CD24−/lowas well as the
value 10%, as previously described [6]. Overall and disease-
free survival distribution curves were assessed by the
Kaplan-Meier test and log-rank statistics followed by
Cox's proportional hazards regression model. For all tests,
a 2-tailed P of less than .05 was considered significant. All
statistical tests were performed using SPSS software (release
13.0, SPSS Inc, Chicago, IL).
3. Results
CD44+/CD24−/lowand CD44−/CD24+phenotypes were
detected in the tumor cells (Figs. 1 and 2) as well as in the
normal epithelium and in in situ carcinomas, when the latter
were observable in the examined specimens. Moreover,
Fig. 3
on the overall (A) and disease-free (B) survival of the patients
(Kaplan-Meier curves).
Effect of the prevalence of CD44+/CD24−/lowtumor cells
Fig. 4
on the overall (A) and disease-free (B) survival of the patients
(Kaplan-Meier curves).
Effect of the prevalence of CD44−/CD24+tumor cells
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CD44 protein was immunodetected in the tumor stroma,
whereas CD24 was also localized in the cytoplasm of the
malignant cells.
In breast cancer tissues, CD44+/CD24−/lowtumor cells
ranged between 0% and 70% (median value, 2). Of the 155
evaluable specimens, 91 (58.7%) depicted more than 1%,
whereas 23 (12.4%) displayed more than 10% CD44+/
CD24−/lowtumor cells. As far as the CD44−/CD24+
phenotype is concerned, CD44−/CD24+tumor cells ranged
from a low of 3% to a high of 91% (median value, 70). The
majority of tumors (82.6%) displayed more than 50%
CD44−/CD24+tumor cells.
The prevalence of the CD44+/CD24−/lowtumor cells was
inversely associated with lymph node metastasis (P = .019,
Mann-Whitney test) and tended to inversely associate with
the stage of the disease (P = .068, Kruskal-Wallis test),
although the relation was not statistically significant
(Table 1). No relation was found with any of the rest of
the clinicopathologic parameters (age, menopausal status,
histologic and nuclear grade, histologic type, estrogen and
progesterone receptors' expression) (Table 1). As far as
the survival is concerned, the prevalence of the CD44+/
CD24−/lowcells was found to exert no significant impact on
patients' disease-free and overall survival, although it
displayed a tendency toward an increase in disease-free
survival (P = .074) (Fig. 3).
The prevalence of CD44−/CD24+tumor cells was found
to have no clinicopathologic significance (Table 1). Contrary
to the CD44+/CD24−/lowphenotype, the prevalence of the
CD44−/CD24+tumor cells was found to exert an unfavor-
able impact on patients' both relapse-free (P = .016) and
overall survival (P = .078), although in the latter a borderline
one (Fig. 4). However, the unfavorable prognostic impact
was found to be more powerful in the subgroup of patients
with grade 2 (intermediate differentiation) invasive breast
cancer (P = .009 for relapse-free and P = .046 for the overall
survival) (Fig. 5). Finally, an inverse correlation was found
between the 2 phenotypes under investigation (P = .005, r =
−0.226 Spearman test).
4. Discussion
The potential existence of a stem-like cell in tumors is an
old concept [15]. The recent study of Al-Hajj et al [4] in
breast cancer is believed to have provided evidence
supporting the existence of stem cells in a solid tumor.
However, much concern has been expressed about both
theoretical and technical issues. Specifically, the likelihood
of variations of stemness with time as well as whether these
cells sorted from tumors are truly cancer stem cells [5,16].
Moreover, data from in vitro studies suggest that the CD44−/
CD24+population may contain cancer stem cells corre-
sponding to the luminal type of tumors [10]. In the present
study, we immunodetected cancer cells with the phenotypes
CD44+/CD24−/lowand CD44−/CD24+and searched for their
clinicopathologic value.
According to our findings, the prevalence of CD44+/
CD24−/lowand CD44−/CD24+cells was high (ranged
between 0% and 70% and between 3% and 91% respec-
tively) which is considered to be against their capacity
as cancer stem cells, as the latter would have been expected
to be very sparse (≪1% of the tumor cells) [5]. As far as
the CD44+/CD24−/lowphenotype is concerned, of the
155 evaluable specimens studied, 58.7% depicted 1% or
more, whereas 12.4% displayed more than 10% CD44+/
CD24−/lowtumor cells, with the latter percentage to be in
Fig. 5
the overall (A) and disease-free (B) survival of the patients with
grade 2 invasive breast carcinomas (Kaplan-Meier curves).
Effect of the prevalence of CD44−/CD24+tumor cells on
1100E. Mylona et al.

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accordance with the prevalence reported by Abraham
et al [6].
The present study showed CD44+/CD24−/lowtumor
cells to be more often detected in tumors with better
phenotype (without lymph node metastasis and of a lower
stage), tending to increase the relapse-free survival of the
patients. These results are in conflict with the expected
ones according to the reported capacity of CD44+/
CD24−/lowcells as cancer stem cells [4] as well as the
reported invasiveness gene signature these cells have been
reported to have [17], and unfavorably influencing patients'
disease-free survival [7,17]. However, our approach differed
from that of the aforementioned authors in that we identified
CD44+/CD24−/lowtumor cells in solid tumor specimens and
not in cell suspensions, which means that we did not disturb
the relation between the potential cancer stem cells and
their environment which is well known to affect the ability
of the cells to mature and differentiate [5]. Furthermore,
our findings disagree with those of Abraham et al [6].
However, the latter have considered the prevalence of
CD44+/CD24−/lowtumor cells as a categorical value having
arbitrarily used 10% as cut point.
The unexpected relation of the prevalence of CD44+/
CD24−/lowtumor cells with a favorable tumor phenotype
cannot justify their characterization as cancer stem cells
unless we suggest that tumor cells may not originate
from normal adult stem cells but from a transit-cell
population in the normal breast. Thus, tumor stem cells
and the tumor they generate might have very different
characteristics depending on which of these normal
populations the tumor stem cells arise from—for
instance, it could mean the difference between tumors
that are poorly differentiated and highly aggressive or
relatively well differentiated and noninvasive [16]. More-
over, the finding of the CD24-negative cell population
being inversely associated with lymph node metastasis is
in accordance with that of Baumann et al [18] who
showed that upon CD24 expression, breast cancer cells
acquired enhanced spreading, motility, and invasive
properties that facilitated metastasis.
In addition, the present study is the first to associate
the prevalence of CD44−/CD24+tumor cells with a worse
clinical outcome, especially those patients with carcino-
mas of intermediate-grade differentiation (grade 2), a
group whose prognosis is difficult to assess. The finding
of the CD44-negative cell population being associated
with unfavorable clinical impact is in accordance with that
of Lopez et al [19] who in a mouse model of
spontaneously metastasizing breast cancer provided in
vivo evidence that loss of CD44 promoted remote
metastasis. Moreover, the same authors showed in vitro
that invasion of CD44-positive tumor cells was inhibited
in hyaluronan-containing matrices [19], thus providing
further data that during breast cancer progression,
hyaluronan-CD44 interaction occurring through epithe-
lial-stromal interactions is protective against metastasis, a
finding which further supports the aforementioned inverse
relation between the CD44+/CD24−/lowphenotype and
lymph node metastasis.
In conclusion, although CD44+/CD24−/lowbreast tumor
cells may be highly efficient in initiating tumors in
experimental settings, according to the present study's
findings in solid tumor specimens, CD44+/CD24−/low
tumor cells seem to be associated with lack of lymph
node metastasis and a tendency toward an increase of the
relapse-free survival of the patients. On the contrary,
tumor cells with the phenotype CD44−/CD24+seem to
help in identifying patients with worse disease-free and
overall survival, especially those pertaining to the group
of intermediate-grade differentiation patients whose prog-
nosis is difficult to assess.
Acknowledgment
We are grateful to S Koulyras for editing the manuscript.
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