Pathology – Research and Practice 212 (2016) 10–16

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Pathology – Research and Practice

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Original Article

Immunohistochemical expression of cancer stem cell related markers

CD44 and CD133 in endometrial cancer
Samah S. Elbasateeny a,∗ , Amira A. Salem a , Walid A. Abdelsalam b , Reham A. Salem c
a
Pathology Department, Faculty of Medicine, Zagazig University, Egypt
b
Gynecology and Obstetrics Department, Faculty of Medicine, Zagazig University, Egypt
c
Clinical Oncology and Nuclear Medicine Department, Faculty of Medicine, Zagazig University, Egypt

a r t i c l e i n f o a b s t r a c t

Article history: The goal of this study was to detect the presence of cancer stem cell markers CD44 and CD133 in
Received 5 June 2015 immunohistochemically stained samples of endometrial cancer and correlate their expression with clin-
Received in revised form icopathological variables to identify the impact of CD44 or CD133 expression on tumor behavior and
29 September 2015
endometrial carcinogenesis. Marker expression was analyzed in 62 endometrial cancer samples (57
Accepted 21 October 2015
endometrioid carcinoma and 5 carcinosarcoma) and 15 proliferative endometrium samples. We detected
CD133 and CD44 expression in 87.09% and 79.03% respectively of the studied endometrial cancers, and the
Keywords:
expression was significantly different from the normal group. CD44 expression decreased with myome-
Immunohistochemical
Cancer stem cell
trial invasive depth and lymph-vascular space invasion (LVSI), and these inverse relationships were
CD44 significant (p = 0.034, p = 0.019, respectively). CD133 was more expressed by early stage tumor (FIGO
CD133 I-II) compared with those having FIGO III to IV stage disease (p = 0.021). The most notable conclusion of
Endometrial cancer the present study is that CD44 and CD133 might participate in early-stage endometrial cancer carcino-
genesis, and their overexpression may facilitate the early diagnosis of endometrial cancers. Analysis of our
results supports the hypothesis that CD44 expression tends to decrease as the disease becomes invasive
and progressive. So, we concluded that CD44 down-regulation might warn of a more aggressive course
and may have a link with poorly prognosis carcinosarcomas. Further examination of the expression and
function of CD44 and CD133 with a greater number of carcinosarcomas is warranted.
© 2015 Elsevier GmbH. All rights reserved.

1. Introduction carcinomas rather than uterine sarcomas [2]. Prognosis of endome-

Endometrial carcinoma is the most common invasive neoplasm
in the female genital tract. Based on clinicopathological and molec-
ular genetic features, it can be divided into two major groups
referred to as type I and type II. The endometrioid subtype, which
is the prototype of type I carcinoma, is associated with unopposed
estrogenic stimulation, as well as endometrial hyperplasia [1].
Carcinosarcoma (malignant-mixed Müllerian tumor) is a biphasic
neoplasm composed of distinctive and separate, but admixed,
carcinomatous and sarcomatous elements. Recent immunohisto-
chemical and molecular genetic studies support the hypothesis
that both tumor components (epithelial- and mesenchymal-like
elements) have a common clonal origin. Accordingly carcinosarco-
mas are currently thought to be “undifferentiated” or “metaplastic”
∗ Corresponding author at: Pathology Department, Faculty of Medicine, Zagazig
University, Egypt.
E-mail address: samah elbasateeny@yahoo.com (S.S. Elbasateeny).
trial carcinoma is dependent on some well accepted clinical and
pathological parameters, including the histological type and grade
of the tumor, the depth and pattern of myometrial invasion, the
degree of disease extension beyond the uterine corpus, adnexal
involvement and pelvic and para-aortic lymph node metastasis [1].
The risk of endometrial cancer recurrence has been well character-
ized and ranges from 7.7% to 63.3%, depending on the presence
or absence of specific prognostic factors [3]. For these reasons,
novel targeted therapies are currently being developed with the
aim to achieve greater specificity for a selected population of cancer
cells [4]. It has recently been reported that a small subpopulation
of malignant tumors has a high proliferative potential and con-
tributes to tumorigenesis, proposing the concept of cancer stem
cells (CSCs) [5]. Cancer stem cells can be defined as a population of
undifferentiated tumorigenic cells responsible for tumor initiation,
maintenance, and spreading [6]. In accordance with the paradigm
already established for hematopoietic stem cells CSC display unlim-
ited proliferation potential, ability to self-renew, and capacity to
generate a progeny of differentiated cells that constitute the major

http://dx.doi.org/10.1016/j.prp.2015.10.008
0344-0338/© 2015 Elsevier GmbH. All rights reserved.
 S.S. Elbasateeny et al. / Pathology – Research and Practice 212 (2016) 10–16 11

tumor population [7]. The existence of CSCs was first reported in
acute myeloid leukemia (AML) [8]. Thereafter, emerging evidence
of the existence of CSCs has been demonstrated in a variety of solid
tumors, including brain [9], breast [10], colon, prostate, ovary [11]
and endometrium [12]. Many of these studies have used the surface
expression of CD133, a 5-transmembrane glycoprotein, as a marker
for the isolation of CSCs [13]. CD133 (human prominin-1) is a mem-
brane glycoprotein with a putative function in plasma membrane
organization. Current evidence suggests that CD133 is a reliable
marker for the isolation of the endometrial CSC population [11].
In endometrial cancer CD133+ cells showed a higher proliferative
capacity in vitro than the CD133− subset. Moreover, this population
exhibited clonogenic growth activity in soft agar studies, forming
colonies from a single cell, whereas the CD133− failed to do it. Inter-
estingly, CD133 could be useful as a prognostic factor in endome-
trial carcinoma [14]. Besides CD133, there are other interesting pro-
teins that may be expressed in CSC of solid tumors, such as endome-
trial carcinoma. Among these candidate markers is CD44 [14]. CD44
is a separate family of cell adhesion molecules that includes the
standard (CD44s) and variant isoforms, which are products of alter-
native splicing [15,16]. CD44 has multiple biological functions,
initially implicated in the process of invasion and metastasis, but
now recognized as a marker for tumor initiating cells (CSCs) in dif-
ferent tissues. Interestingly, CD44 is expressed in the spheres that
are generated from endometrial cancer cell lines [17].
The goal of this study was to detect the presence of cancer
stem cell markers CD44 and CD133 in immunohistochemically
stained samples of endometrial cancers and correlate their expres-
sion with clinicopathological variables to identify the impact of
CD44 or CD133 expression on tumor behavior and endometrial
carcinogenesis.
alcohols, and placed in 0.5% hydrogen peroxide in methanol for
10 min to block endogeneous peroxidase activity. Antigen retrieval
was carried out by incubation in 0.01 M citrate buffer (pH 6.0)
for 5 min in a pressure cooker. The sections were exposed to the
primary antibody for 60 min at room temperature. The standard
strept avidin-biotin-peroxidase complex method was used for
CD44 (mouse monoclonal antibody, Clone 156-3C11, catalog no.
MS-668-P0Lab Vision, California, USA) and CD133 (mouse mono-
clonal antibody, clone 171-191, catalog no. 144305, Lab Vision,
California, USA) by employing diaminobenzidine (DAB) as the chro-
mogen. Human tonsil and renal tubular epithelial cells were used as
a positive control for CD44 and CD133 respectively, while negative
controls for both markers were obtained by omitting the primary
antibody.
2.3. Evaluation of immunohistochemical staining
We calculated the “immunohistochemical score” (IHS) of CD44
and CD133 for each case. The scoring system used for both CD44
and CD133 was similar to previously published methods [18–20].
The extent of positively stained epithelial cells was estimated
and classified on a four-point scale as follows: no staining = 0%,
1 = 1–10%, 2 = 11–25%, 3 = 26–50%, and 4 = 51–100%. The intensity
of the immunoexpression was categorized into three groups: weak
Table 1
Clinicopathological characteristics of the studied 62 endometrial cancer patients.
Variable n
Age at surgery (y)
Mean ± SD 57.35 ± 7.799
Range 37–68

2. Material and methods Mass size

≤4 38
>4 24
2.1. Patients and clinical data
Histopathology
A cross sectional study was conducted at the Departments Endometrioid adenocarcinoma 57 (91.93%)
Carcinosarcoma 5 (8.06%)
of Pathology, Gynecology, and Oncology, Zagazig University Hos-
pital, Egypt in the period from 2011 to 2014. Tissues were Grading
G1 27 (43.5%)
obtained from surgically resected specimens of 62 patients with
G2 21 (33.9%)
endometrial cancer who underwent surgical staging. Pelvic lym- G3 14 (22.6%)
phadenectomies were done only in 55 patients; the remaining
Depth of myometrial invasion
7 patients were disqualified from lymph node dissection due to
No 6 (9.7%)
poor status performance, advanced age, obesity, or other comor- Superficial (<50%) 26 (41.9%)
bidities. We obtained written informed consent from all patients Deep (≥50%) 30 (48.4%)
for tumor tissue collection. Tumors were staged according to Involvement of the cervix
the 2010 FIGO (the International Federation of Gynecology and No 36 (58.1%)
Obstetrics staging system). All tissue samples were formalin-fixed Yes 26 (41.9)
and paraffin-embedded. The corresponding hematoxylin–eosin Adnexal/serosa involvement
No 47 (75.8%)
slides were reviewed to determine the grade, the histotype, the
Yes 15 (24.2%)
depth of myometrial invasion (if present), and the presence or
absence of lymphovascular invasion. 57 cases showed endometri- Distant metastases (lymph nodes not included)
No 57 (91.9%)
oid histology, and only 5 cases were carcinosarcoma (metaplastic Yes 5 (8.06%)
carcinoma). Clinicopathological parameters including the age, the
Lymph node metastases (in 55 lymphadenectomies)
macroscopic sizes, and the FIGO stage of all tumors were obtained
Negative 42 (76.4%)
from the patient’s medical records. The control group consisted Positive 13 (23.6%)
of 15 normal Proliferative phase endometrium tissue samples
LVSI
were obtained from patients who underwent hysterectomy for
Negative 48 (77.4%)
myoma uteri. Patients with endometrial hyperplasia, adenomyosis, Positive 14 (22.6%)
or endometriosis were excluded.
FIGO stage
I 36 (58.1%)
II 6 (9.8%)
2.2. Immunohistochemical staining
III 15 (24.2%)
IV 5 (8.1%)
The sections (4–5 ␮m) obtained from representative tissue sam-
Total cases 62 (100%)
ple blocks were deparaffinised with xylene, rehydrated in graded
12 S.S. Elbasateeny et al. / Pathology – Research and Practice 212 (2016) 10–16

Fig. 1. Examples of immunohistochemical expression of CD44. (A) Normal proliferative endometrium show negative staining (×200). (B) Strong membranous staining in
endometroid carcinoma G I that does not invade the myometrium (×400). (C) Moderate diffuse membranous staining in endometroid carcinoma GII (×400). (D) A case of
carcinosarcoma shows weak or no expression of CD44 in either epithelial or mesenchymal component (×400).

(+1), moderate (+2), and strong (+3). A final IHS score was obtained
by multiplying the score for extent and the score for intensity.
Therefore, the combined immunoreactivity score ranged from 0 to
12. We used IHS cut-off point 10 to separate cases with weak to
moderate expression from cases with strong expression. According
to this, we categorized cases into 3 groups as follows: 0 (absent),
IHS ≤ 10 (weak to moderate) and IHS > 10 (strong).
2.4. Statistics
The results from the analysis of the continuous variables are
expressed as a means ± standard deviation (SD). Analysis of cat-
egorical data was performed using the 2 or Fisher’s exact test,
spearman correlation was performed to assess the correlation
between the two cancers stem cell markers CD44 and CD133. All
statistical analyses were performed using SPSS software (version
19.0; SPSS, Chicago, IL, USA) and p ≤ 0.05was considered to indicate
a statistically significant difference.
3. Results
3.1. Clinicopathologic features
Among the studied 62 endometrial cancer patients 57 (91.9%)
had endometrioid adenocarcinoma, and only five cases (8.06%) had
carcinosarcoma (metaplastic carcinoma). All cancer patients clini-
copathologic characteristics are summarized in Table 1.
in endometrial cancer with a statistically significant difference
(p < 0.0005) (Table 2). CD44 tends to be less expressed in carcinosar-
coma than in endometroid carcinoma, with borderline significance
relationship (p = 0.077). CD44 expression was found to be signif-
icantly correlated with LVSI and depth of myometrial invasion
(p = 0.019 and p = 0.034, respectively). In our analysis, the CD44
tends to be expressed less in node-positive patients, with border-
line significance relationship (p = 0.061). However, no significant
relationship was detected between CD44 expression and other clin-
icopathologic features (p > 0.05) (Table 3).
3.3. Immunohistochemical expression of CD133
CD133 expression was detected in 87.09% (54 out 62) of
the studied endometrial carcinoma cases. The neoplastic glands
showed predominantly membranous CD133 immunoreactivity
(Fig. 2). CD133 was less expressed in normal (proliferative)
endometriuma than in endometrial cancer with a statistically sig-
nificant difference (p < 0.0005) (Table 2). In our analysis CD133
tends to be more expressed in patients with early-stage tumors
(FIGO I-II) compared with those having FIGO III to IV stage disease,
Table 2
Comparison of cases with endometrial carcinoma and normal proliferative
endometrium.
IHS score Proliferative Endometrial
endometrium (n = 15) cancer (n = 62)

0 13 13
3.2. Immunohistochemical expression of CD44 CD44 IHS ≤ 10 2 33
IHS > 10 0 16
p value <0.0005
CD44 expression was detected in 79.03% (49 out 62) of the 0 10 8
studied endometrial cancer cases. The neoplastic glands showed CD133 IHS ≤ 10 5 35
predominantly membranous CD44 immunoreactivity (Fig. 1). CD44 IHS > 10 0 19
p value <0.0005
was less expressed in normal (proliferative) endometriuma than
 S.S. Elbasateeny et al. / Pathology – Research and Practice 212 (2016) 10–16 13

Table 3
Correlation of clinicopathological parameters with CD133 and CD44 expressions in 62 endometrial cancer cases.

Variable CD44 CD133

Absent (13) IHS ≤ 10 (33) IHS > 10 (16) Absent (8) IHS ≤ 10 (35) IHS > 10 (19)

Age at surgery (y)

<55 4 12 4 5 10 5
≥55 9 21 12 3 25 14
p value 0.820 0.170

Mass size
≤4 6 23 9 7 20 11
>4 7 10 7 1 15 8
p value 0.299 0.284

Histopathology
Endometrioid carcinoma 10 31 16 8 31 18
Carcinosarcoma 3 2 0 0 4 1
p value 0.077 0.872

Grading
G1 4 13 10 2 17 8
G2 3 14 4 2 11 8
G3 6 6 2 4 7 3
p value 0.147 0.401

Depth of myometrial invasion

No 0 2 4 2 4 0
Superficial (<50%) 3 15 8 4 13 9
Deep (≥50%) 10 16 4 2 18 10
p value 0.034 0.215

Involvement of the cervix

No 7 16 13 3 23 10
Yes 6 17 3 5 12 9
p value 0.087 0.307

Adnexal/serosa involvement
No 9 26 12 6 26 15
Yes 4 7 4 2 9 4
p value 0.796 0.917

Distant metastases (lymph nodes not included)

No 12 31 14 6 33 18
Yes 1 2 2 2 2 1
p value 0.824 0.189

Lymph node metastases (in 55 lymphadenectomies)

Negative 6 26 10 5 26 11
Positive 6 6 1 3 5 5
p value 0.061 0.302

LVSI
Negative 6 28 14 4 28 16
Positive 7 5 2 4 7 3
p value 0.019 0.145

FIGO stage
I-II 9 21 12 2 26 14
III-IV 4 12 4 6 9 5
p value 0.820 0.028

with a statistically significant difference (p = 0.021). No significant
relationship was detected between CD133 expression and other
clinico-pathologic features (p > 0.05) (Table 3).
3.4. The correlation analysis between CD44 and CD133
expressions among the studied 62 endometrial carcinoma
Based on a correlation analysis between the results of expres-
sion of the two stem cell related marker among the studied 62
endometrial carcinoma, a significant positive correlation was found
between the expression of CD44 and CD133 proteins (Spearman
correlation (r) = 0.525, p < 0.0005, Table 4).
4. Discussion
In CSC theory, a tumor shows a hierarchy analogous to the nor-
mal tissue hierarchy, with a restricted pool of cells within the tumor
having the ability to initiate and sustain tumor growth, showing
self-renewal, and differentiation potential [21,22]. These cells have
been termed cancer stem cells to reflect their ‘stem-like proper-
ties and ability to continually sustain tumorigenesis [13]. Cancer
stem cells express a variety of markers on their cell surface. The
expression or absence of these markers has been used to isolate
subpopulations of cancer cells in the examination of CSC properties.
Some cell surface markers, including CD133, CD44, CD24, and THY1
are common to several solid cancers, including breast cancer, brain
tumors, colorectal, and endometrial cancers [13]. In the present
study, we examined the expression of CSC markers CD133 and CD44
in 62 endometrial cancer (57 endometrioid carcinoma and 5 car-
cinosarcomas) and 15 normal proliferative endometrium samples.
We analyzed the marker expressions to identify the impact of CD44
or CD133 on tumor behavior and endometrial carcinogenesis.
In the present study, we detected CD133 and CD44 expression
in 87.09% and 79.03% respectively of the studied endometrial
14 S.S. Elbasateeny et al. / Pathology – Research and Practice 212 (2016) 10–16

Fig. 2. CD133 immunoreactivity in representative cases of endometrial cancer. (A) Strong diffuse immunoreactivity in G II endometrial carcinoma (×200). (B) Weak staining
in G II endometrial carcinoma (×400). (C) Strong immunoreactivity in G III endometroid carcinoma (×400). (D) Carcinosarcoma shows strong expression of CD133 in both
epithelial and mesenchymal components (×400).

cancers, and the expression was significantly different from the
normal group. This was supported by other studies that detected
a higher level of CD133 in primary human endometrial tumors
compared to expression in benign proliferative and secretory
endometrium [14,23]. Also, in most reports the CD44 and its
variants are expressed more in endometrial cancer tissue than in
normal endometrium [24–27]. Moreover, they reported that the
average CD44 scores in either simple/complex or atypical hyper-
plastic lesions were statistically lower than those of endometrial
carcinomas [24–26,28,29]. Hofmann et al. [28] and Inoue [29]
reported that the CD44 gene promoter is activated by the K-ras
oncogene product, which also affects splicing of CD44 mRNA.
In 10–37% of the endometrial cancers, an activating mutation is
reportedly found in codons 12 or 13 of the K-ras proto-oncogene,
and also in 6–16% of atypical hyperplasia, but not in normal
endometrium; this implies that CD44 affects early-stage EC
oncogenesis—more in well-differentiated cancers of endometrial
morphology. Therefore, we could infer that CSCs related proteins
CD133 and CD44 might participate in early-stage endometrial
cancer carcinogenesis and their overexpression may facilitate the
early diagnosis of endometrial carcinoma.
In our study CD44 expression decreased with myometrial inva-
sive depth, and this inverse relationship was significant. Such a
result is consistent with a hypothesis that highly invasive cancers
tend to lose CD44 expression [26,30–32]. A similar but insignificant
relationship of CD44 expression was found by Wojciechowski et al.
[27]. Stokes et al. [33] also found that CD44v6 expression strongly
correlated with lack of myometrial invasion. He suggests this could
be a marker for cancers with myometrial involvement, which
would thus facilitate preoperative qualification for lymphadenec-
tomy. On the other hand, Leblanc et al. [34] reported CD44 expres-
sion to increase with depth of myometrial invasion. Such inconsis-
tent results may be due to different methodology and small patient
cohorts (Leblanc: 33 patients; Stokes: 40; Wojciechowski et al.: 51).
In our group of patients, we reported a significant relation-
ship between decreased CD44 expression and LVSI. In the relevant
literature, the correlation between CD44 and LVSI also varies.
Many reported that loss of CD44, particularly CD44v6, is of inter-
est in advanced-stage disease, which is similar to our findings
[26,31–33]. Leblanc et al. [34] and Yorishima et al. [35] found pos-
itive relationships between LVSI and CD44v6 expression, which
implies that molecules affect local invasion. Other reports showed
no relationship between CD44 and LVSI [27]. In our analysis the
CD44 tended to be expressed less in node-positive patients with
borderline significance relationship, this observation could also
support the hypothesis that CD44 is lost in more invasive cancers.

Table 4
The correlation analysis between CD44 and CD133 expression among the studied 62 cases of endometrial cancer.

CD133 Total

Absent IHS ≤ 10 IHS > 10

Absent count (% of total) 7 (11.3%) 2 (3.2%) 4 (6.5%) 13 (21%)

CD44 IHS ≤ 10 count (% of total) 1 (1.6%) 29 (46.8%) 3 (4.8%) 33 (53.2%)
IHS > 10 count (% of total) 0 (0%) 4 (6.5%) 12 (19.4%) 16 (25.8%)

Total count (% of total) 8 (12.9%) 35 (56.5%) 19 (30.6%) 62% (100%)

Spearman correlation (r) = 0.525, p value < 0.0005.

 S.S. Elbasateeny et al. / Pathology – Research and Practice 212 (2016) 10–16
A similar relationship with also borderline significance was found
by Wojciechowski et al. [27], although other authors’ observa-
tions do not concur [20,32–34]. Only Yorishima et al. [35] and
Hoshimoto et al. [36] found positive, significant correlations of
CD44v6 and CDv3 respectively, with node involvement, which sug-
gests dominating roles for variant forms of CD44 in highly invasive
tumors.
In our study, we reported that CD44 tends to be less expressed
in carcinosarcoma than in endometroid carcinoma, with borderline
significance relationship. As carcinosarcoma is a highly aggressive
tumor with a worse prognosis [37,38], our results could indicate
a link between CD44 down-regulation and poor prognosis. Also,
this finding is consistent with a hypothesis that highly invasive
cancers tend to lose CD44 expression [26,27,30–32]. However, we
had too few patients (5 women) with carcinosarcoma to confirm
this hypothesis. As with other authors [20,27,32–34], we found no
correlation between CD44 expression and age, tumor size, grade,
involvement of the cervix or adnexa, distant metastasis, or FIGO
stage.
In the present study, the CD133 was more expressed by early
stage tumors (FIGO I-II) compared with those having a FIGO III to
IV stage disease. A similar relationship between CD133 expression
and FIGO staging was found by Rutella et al. [14] who found that
CD133 was preferentially expressed by early stage tumors. He sug-
gests that selective targeting of CD133-expressing cancer cells has
a greater potential to eradicate the neoplastic disease and to limit
disease recurrence in this subgroup of patients, although in other
relevant literature the CD133 expression does not correlate with
FIGO staging [39,40].
In our analysis, CD133 expression did not vary significantly by
tumor histological type as endometrioid and carcinosarcoma did
not differ significantly in the percentage of CD133-expressing cells.
This is similar to the observation obtained by Rutella et al. [14] who
found that endometrioid and non-endometrioid tumors did not dif-
fer significantly in CD133 expression. This may be attributable to
the low number of non-endometrioid tumors in our study (only 5
patients) and also in the Rutella study (21 patients). Also, in our
analysis no correlation was detected between CD133 expression
and age, tumor size, depth of myometrial invasion, involvement of
the cervix or adnexa, distant metastasis, lymph node metastasis, or
LVSI. This is consistent with the results obtained by previous related
studies [39,40].
In the present study, a significant positive correlation was found
between the expression of the two stem cell related markers (CD44
and CD133) among the studied 62 endometrial cancer. This finding
is consistent with the observations in colorectal cancers obtained
by Haraguchi et al. [41] who concluded that the CD133 (+) CD44
(+) population may be the best to identify tumor initiating cells of
human colon cancer. Also in cancer colon, Wang et al. [42] found
that the combination of CD44 and CD133 correlated with more fea-
tures of CSCs, but they cannot be generalized and applied to all colon
cancer cells.
5. Conclusion
The most notable conclusion of the present study is that CD44
and CD133 might participate in early-stage endometrial cancer
carcinogenesis, and their overexpression may facilitate the early
diagnosis of endometrial cancers. Analysis of our results supports
the hypothesis that CD44 expression tends to decrease as the dis-
ease becomes invasive and progressive. So, we concluded that CD44
down-regulation might warn of a more aggressive course and may
have a link with poorly prognosis carcinosarcomas. Further exam-
ination of the expression and function of CD44 and CD133 with a
greater number of carcinosarcomas is warranted.
15
Conflicts of interest
There are no conflicts of interest.
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