Prognostic impact of CD44-positive cancer stem-like cells at the invasive front of gastric cancer
British Journal of Cancer
Prognostic impact of CD44-positive cancer stem-like cells at the invasive front of gastric cancer
Hirokazu Kodama 0 3 4
Satoshi Murata 0 3 4
Mitsuaki Ishida 1 2 3 4
Hiroshi Yamamoto 0 3 4
Tsuyoshi Yamaguchi 0 3 4
Sachiko Kaida 0 3 4
Tohru Miyake 0 3 4
Katsushi Takebayashi 0 3 4
Ryoji Kushima 1 3 4
Masaji Tani 0 3 4
0 Department of Surgery, Shiga University of Medical Science , Otsu , Japan
1 Division of Diagnostic Pathology, Department of
2 Division of Diagnostic Pathology , Kansai
3 Clinical Laboratory Medicine, Shiga University of Medical Science , Otsu , Japan
4 Medical University , Osaka , Japan
Background: The invasive tumour front may provide prognostic information. We examined the relationship between the presence of cancer stem cells (CSCs) at the invasive tumour front and prognosis in gastric cancer (GC). Methods: CD44 is a CSC marker; accordingly, CD44 standard (CD44s), CD44 variant-6 (CD44v6), and CD44 variant-9 (CD44v9) expression were examined in 123 resected primary GCs and the clinical significance of CSCs at the invasive tumour front was analysed. Results: Thirteen (10.6%), 79 (64.2%), and 47 (38.2%) GCs were CD44s-, CD44v6-, and CD44v9-positive, respectively. Patients with CD44-positive expression at the invasive tumour front had significantly poorer disease-specific survival than those with negative expression (CD44s: Po0.00001, CD44v6: P ? 0.013, CD44v9: P ? 0.0002). CD44s expression at the invasive tumour front was an independent prognostic factor in resectable GC patients (hazard ratio ? 3.13; 95% confidence interval, 1.09-9.01; P ? 0.035) and was significantly associated with peritoneal (Po0.001), lymphatic (Po0.001), and haematogenous recurrences (P ? 0.008). In addition, the number of CD44 isoforms expressed in cancer cells at the invasive tumour front was associated with patient prognosis. No conventional clinicopathological factors were independently associated with CD44 expression at the invasive tumour front. Conclusions: CD44-positive cancer stem-like cells at the invasive tumour front indicate poor survival and can be a unique
CD44; cancer stem cells; prognosis; gastrectomy; recurrence; survival
biological prognostic factor for GC.
Cancer stem cells (CSCs) are a subpopulation of tumour cells with
the ability to initiate tumours as well as reconstitute the cellular
heterogeneity typical of their tumours of origin
CD44-positive fractions of gastric cancers (GCs) can generate
spheroid colonies under non-adherent conditions, and small
numbers of CD44-positive cells can generate tumours in severe
combined immunodeficiency (SCID) mice
(Takaishi et al, 2009)
indicating that CD44-expressing cancer cells include CSCs. Several
reports have suggested that CSCs can initiate and facilitate cancer
progression by inducing cancer metastasis and therapeutic resistance
in GC, resulting in poor survival
(Klonisch et al, 2008; Ishigami et al,
2010; Wang et al, 2011)
CD44 is a family of transmembrane glycoprotein receptors that
bind to hyaluronic acid, and the resultant intracellular signalling is
linked to diverse cellular functions, including cell adhesion,
migration, and invasion
(Jackson et al, 1992; Sneath and
. The gene encoding the CD44 standard isoform
(CD44s) consists of 20 exons, wherein the distal extracellular
domain is responsible for binding to hyaluronic acid and the
proximal extracellular domain is the site of alternative mRNA
splicing, which results in multiple variant isoforms (CD44v2?v10)
(Sneath and Mangham, 1998). Among the variant isoforms, the
roles of CD44 variant-6 (CD44v6) and CD44 variant-9 (CD44v9)
are well-studied in GC. CD44v6-positive advanced GCs are
associated with haematogenous metastasis, and the survival of
CD44v6-positive patients is significantly poorer than that of
(Yamaguchi et al, 2002)
interacts with and stabilises xCT, a glutamate-cystine transporter,
resulting in increased intracellular levels of reduced glutathione.
CD44v9-positive cells have an enhanced ability to suppress reactive
oxygen species (ROS) production, resulting in the therapeutic
resistance, recurrence, and metastasis of tumours
(Ishimoto et al,
2011; Tsugawa et al, 2012; Yae et al, 2012)
The invasive front of tumours has been proposed to contain
important prognostic information owing to the lack of
cohesiveness, secretion of proteolytic enzymes, reorganisation of the
extracellular matrix, and increased cell proliferation
Weiss, 2009; Sharma et al, 2013)
However, no studies have evaluated CD44 expression at the
invasive tumour front (ITF). Here, we examined the expression
and localisation of CD44 isoforms in GC tissues to determine the
association between the presence of cancer stem-like cells at the
ITF and the prognosis of patients with resectable GC. In order to
evaluate the prognostic significance of CD44 isoforms based on
immunohistochemical analyses of whole-tumour sections and the
ITF, the primary end points were disease-specific survival and
patterns of relapse in resectable GC patients, and the secondary
end point was to stratify the survival risk of GC cases according to
the prognostic analysis.
MATERIALS AND METHODS
Patients and study materials. One hundred and twenty-three
primary gastric adenocarcinoma specimens were obtained from
patients who underwent gastrectomy between January 2007 and
December 2009 at the Shiga University of Medical Science
Hospital, Japan. Specimens from patients with distant metastasis,
peritoneal dissemination, or mucosal infiltration of the tumour
(pT1(M)) were excluded. The histological type was determined as
differentiated or undifferentiated; specifically, highly and
moderately differentiated tubular or papillary adenocarcinomas were
classified as the differentiated type, and poorly differentiated
adenocarcinomas and signet-ring cell carcinomas were classified as
the undifferentiated type. Mucinous adenocarcinomas were
classified depending on other predominant elements. Tumour
stage and pathological classification were defined according to the
Japanese Gastric Cancer Association, 2011
. Postoperative adjuvant
chemotherapy was given to 51 patients with pathological T2?T4
tumours or lymph node metastasis. Among these 51 patients, 25
were treated with tegafur, 5-chloro-2,4-dihydroxypyridine, and
oxonic acid (S1), 11 were treated with tegafur and uracil (UFT), 8
were treated with S1 ? docetaxel, 6 were treated with S1 ?
irinotecan, and 1 was treated with S1 ? paclitaxel. The median
follow-up time was 68 months (range: 1?97). A total of 22 patients
died of GC-related causes during the study period. Peritoneal,
lymphatic, and haematogenous recurrences occurred in 17, 8, and
9 cases, respectively. Written informed consent was obtained from
all patients in accordance with the local ethical guidelines. The
study was approved by the Institutional Review Board of the Shiga
University of Medical Science.
Pathological examination. Formalin-fixed, paraffin-embedded
tissue blocks of the resected GC specimens were cut into
3-mmthick sections, deparaffinised, and rehydrated. Each section was
stained with haematoxylin and eosin and then used for
immunostaining. Immunohistochemical analyses were performed
using an autostainer (Benchmark XT System; Ventana Medical
Systems, Tucson, AZ, USA) according to the manufacturer?s
instructions. The following primary antibodies were used: primary
antibodies against CD44s (H-CAM, 1 : 200 dilution; Novocastra
Laboratories Ltd, Newcastle upon Tyne, UK), CD44v6 (VFF-18,
1 : 500 dilution; Abcam, Cambridge, UK), and CD44v9 (RV3,
1 : 5000 dilution; Cosmo Bio, Tokyo, Japan). Lymph nodes were
used as the positive control for CD44s expression, and skin tissue
was used as the positive control for CD44v6 and CD44v9
(Sneath and Mangham, 1998)
. The negative control
was evaluated by substituting the primary antibodies with similarly
diluted non-immunised mouse serum. Immunostaining
examinations were assessed independently by two pathologists blinded to
the clinical or pathological features. When there were
discrepancies, a final decision was established by reassessment using a
The ITF was evaluated at the deepest invasive section of the
(Sentani et al, 2014)
. CD44s, CD44v6, and CD44v9
expression was semiquantitatively analysed in whole-tumour tissue
sections or at the ITF as the percentage of positive tumour cells.
The percentage of positive tumour cells was evaluated in
lowpower fields ( 40) in the whole-tumour section; subsequently,
positive membrane staining of the tumour cells was confirmed at
100 magnification. A result was defined as positive when 45%
of cancer cells were positively stained and negative when o5% of
the cancer cells were positively stained. This cut-off value of 5%
was consistent with that used in the previous reports
(Yasui et al,
1998; Okayama et al, 2009; Ryu et al, 2012; Wu et al, 2015)
Tumour and stromal cells were identified based on the
morphological features of the positive cells. When it was not clear whether
positive cells were tumour cells or stromal cells using morphological
methods, especially for poorly differentiated adenocarcinomas,
immunostaining for cytokeratin (AE1/AE3, 1:10 dilution; Abcam,
Cambridge, UK) was combined with morphological methods.
CD44 expression, initial recurrence pattern, and survival. Initial
recurrence patterns, based on the first recurrent lesion detected
during postoperative follow-up, were determined using computed
tomography at intervals of at least 6 months. Recurrence patterns
were classified into three types; peritoneal, lymphatic, and
haematogenous (i.e., liver, lung, brain, and bone metastasis).
Diseasespecific survival was calculated from the date of gastric surgery to
either the date of death from GC or the date of the last follow-up.
Statistical analysis. Univariate and multivariate analyses were
performed using the Cox proportional hazards model to evaluate
the associations between clinical covariates and disease-specific
survival. Variables associated with survival with Po0.05 in a
univariate analysis were used for a multivariate analysis. Kaplan?
Meier survival curves were constructed to compare patients with
positive and negative CD44 expression. Significant differences were
determined using the log-rank test. Associations of
clinicopathological variables with CD44s, CD44v6, or CD44v9 expression were
evaluated using the w2-test or Fisher?s exact test, as appropriate.
Variables that showed differences at Po0.10 were included in the
multivariate logistic regression analyses. Cumulative incidence
rates were calculated to evaluate the associations between initial
recurrence patterns and CD44s, CD44v6, or CD44v9 expression.
The cumulative incidence of each recurrence type was estimated
using the competing-risk method, in which death from other
causes was considered a competing risk. Comparisons between
patients with positive and negative CD44 expression were
performed using the Gray test. All P-values were two-sided, and
the significance level was set at Po0.05. Statistical analyses, except
the Gray test, were performed using SPSS 22.0 (SPSS Inc., Chicago,
IL, USA). Gray tests were performed using EZR (Saitama Medical
Center, Jichi Medical University, Saitama, Japan), which is a
graphical user interface for R (The R Foundation for Statistical
Computing, Vienna, Austria).
Expression of CD44 isoforms in GC. Patient and tumour
characteristics are summarised in Table 1. Positive staining was
observed for CD44s in 27 (22.0%) or 13 (10.6%) of the GC patients,
positive staining for CD44v6 in 108 (87.8%) or 79 (64.2%) GC
patients, and positive for CD44v9 in 72 (58.5%) or 47 (38.2%) GC
patients in whole-tumour tissue sections or at the ITF, respectively.
The immunohistochemical staining patterns of CD44s, CD44v6,
and CD44v9 in whole-tumour sections and at the ITF of GC
patients are shown in Figure 1. Membranous immunoreactivity
was observed for CD44s, CD44v6, and CD44v9.
CD44 isoform expression and patient outcome. To evaluate the
prognostic potential of the presence of cancer stem-like cells at the
ITF, we examined the relationship between CD44 isoform expression
in whole-tissue sections or at the ITF and disease-specific survival
(Table 2). A univariate Cox analysis showed that stage (hazard ratio
(HR) ? 6.84; 95% confidence interval (CI), 2.67?17.54; Po0.0001),
blood vessel invasion (HR ? 12.5; 95% CI, 1.67?90.90; P ? 0.014),
tumour size 45 cm (HR ? 4.18; 95% CI, 1.63?10.75; P ? 0.003),
CD44v9 expression in whole-tissue sections (HR ? 2.87; 95% CI,
1.06?7.75; P ? 0.039), CD44s expression at the ITF (HR ? 6.80; 95%
CI, 2.73?16.95; Po0.0001), CD44v6 expression at the ITF
HR (95% CI)
CD44v9 expression at invasive tumour front
Negative 1 (reference)
Positive 4.65 (1.89?11.49)
(HR ? 4.15; 95% CI, 1.23?14.08; P ? 0.022), and CD44v9 expression
at the ITF (HR ? 4.65; 95% CI, 1.89?11.49; P ? 0.001) were
associated with disease-specific survival. These results suggest that
CD44 isoform expression at the ITF was a strong prognostic factor
for GC. Furthermore, a multivariate Cox analysis revealed that
CD44s expression at the ITF was a poor independent prognostic
factor (HR ? 3.13; 95% CI, 1.09?9.01; P ? 0.035).
Kaplan?Meier survival curves according to the expression of each
CD44 isoform at the ITF are shown in Figure 2. Patients with
CD44s-, CD44v6-, or CD44v9-positive expression at the ITF showed
significantly poorer survival than those with negative expression
(Po0.0001, P ? 0.013, and P ? 0.0002, respectively) (Figure 2A?C).
patients: patients with negative expression for all CD44 isoforms
(all-negative) (n ? 41), patients with positive expression for one
CD44 isoform (single-positive) (n ? 38), patients with positive
expression for two CD44 isoforms (double-positive) (n ? 31),
and patients with positive expression for all CD44 isoforms
(all-positive) (n ? 13) (Po0.0001). The all-positive group showed
the worst survival, that is, a 51.9% 5-year survival rate; the
doublepositive and single-positive groups showed 73.7% and 90.8% 5-year
survival rates, respectively, and the all-negative group showed the
best 5-year survival rate, that is, 97.4%. This stratified model based
on the number of the CD44 isoforms detected predicts GC patient
Predictive model for survival. To generate a detailed predictive
model for patients with GC surgery, we stratified patients based on
the expression of each CD44 isoform at the ITF (Figure 2D). There
were significant differences in survival among the four groups of
CD44 expression at the ITF and clinicopathological features. To
clarify the factors affecting the presence of cancer stem-like cells at
the ITF, we examined the correlation between conventional
clinicopathological variables and CD44 isoform expression at
P < 0.0001
the ITF. Univariate analyses revealed that CD44s expression at the
ITF was not correlated with any clinicopathological variables
(Table 3a). However, CD44v6 expression at the ITF was
significantly correlated with the depth of tumour invasion
(P ? 0.004), lymphatic invasion (P ? 0.005), and blood vessel
invasion (P ? 0.011) (Table 3b). CD44v9 expression at the ITF was
significantly correlated with tumour size (P ? 0.036) (Table 3c).
However, according to a subsequent multivariate logistic regression
analysis, no independent clinicopathological factors were
associated with the expression of any CD44 isoform at the ITF.
CD44 expression at the ITF and recurrence. We examined
whether the expression of each CD44 isoform at the ITF was
associated with recurrence after surgery for resectable GC. As
summarised in Table 4, the cumulative recurrence rate at 5 years
for each recurrence type was significantly higher in patients with
CD44s-positive expression at the ITF than in patients with
CD44snegative expression at the ITF (peritoneal recurrence: 43.4%, 95% CI,
14.1?70.0% vs 5.8%; 95% CI, 2.4?11.5%, Po0.001; lymphatic
recurrence: 51.3%, 95% CI, 12.1?80.9% vs 4.3%, 95% CI,
1.4?10.0%, Po0.001; haematogenous recurrence: 29.4%, 95% CI,
5.4?59.7% vs 6.2%; 95% CI, 2.5?12.2%, P ? 0.008). The cumulative
peritoneal recurrence rate at 5 years was significantly higher in
patients with CD44v6- or CD44v9-positive expression at the ITF than
in those with negative expression (CD44v6 expression: 13.8%, 95% CI,
7.0?22.8% vs 2.4%, 95% CI, 0.2?11.2%, P ? 0.007; CD44v9 expression:
21.2%, 95% CI, 10.3?34.6% vs 2.9%; 95% CI, 0.5?9.0%, P ? 0.041).
The results of this study showed that the presence of CD44-positive
cancer stem-like cells at the ITF, independent of any conventional
clinicopathological factors, is strongly correlated with poor survival
of patients with resectable GC.
Cancer stem cells are a malignant subset found in hierarchically
organised tumours and are capable of tumour initiation,
selfrenewal, and differentiation to the bulk of non-tumorigenic cancer
cells. CD44 is a CSC surface marker in several cancers, including
(Al-Hajj et al, 2003; Collins et al, 2005; Dalerba et al, 2007;
Takaishi et al, 2009)
. Furthermore, in GC, CD44 variant isoforms
have recently been reported to affect tumour initiation and cancer
cell maintenance (Ishimoto et al, 2010), and may play a functional
role in the protection of CSCs from high levels of ROS in the
(Ishimoto et al, 2011)
. Several studies
have reported that CD44 isoform expression in GC suggests a poor
(Wakamatsu et al, 2012; Cao et al, 2014; Xie et al, 2015)
however, most studies of CSC marker expression in tumours assess
the intensity of expression or proportion of cells expressing CSC
markers using whole-tumour sections or tissue microarrays. The
ITF, the deepest and most invasive area of the tumour, has been
reported to contain useful prognostic information. In addition,
(c) CD44v9 expression at invasive tumour front
n ? 47 (38.2%)
Undifferentiated 21(42.0%) 29(58.0%) 0.474
Abbreviations: CD44s ? CD44 standard isoform; CD44v6 ? CD44 variant isoform 6; CD44v9 ? CD44 variant isoform 9; CI ? confidence interval; LN ? lymph node.
various critical molecular events that regulate tumour proliferation,
adhesion/migration, and angiogenesis occur in the ITF
and Piffko, 2000; Rowe and Weiss, 2009; Sharma et al, 2013)
Accordingly, the ITF is mainly responsible for the clinical behaviour
of the tumour
(Khanh do et al, 2011; Tanaka et al, 2014)
. However, it
is not clear whether the presence of CSCs at the ITF has clinical
significance. In our study, CD44v9 expression in the whole-tumour
sections was associated with survival, and the expression of all tested
CD44 isoforms at the ITF was strongly associated with survival
(CD44s: Po0.00001, CD44v6: P ? 0.013, CD44v9: P ? 0.0002).
These findings show that CD44-positive cancer stem-like cells at
the ITF are more important prognostic factors than those in
wholetumour sections. Moreover, a multivariate logistic regression analysis
showed that the expression of each CD44 isoform at the ITF was
independent of conventional clinicopathological factors, suggesting
that the expression of CD44 isoforms at the ITF is a distinctive
prognostic factor. Additional studies are needed to reveal the
association between the expression of CD44 isoforms and molecular
or morphological changes in tumours at the ITF.
Epithelial-mesenchymal transition (EMT) is an important step
in cancer invasion and metastasis
(Wu and Zhou, 2008; Voulgari
and Pintzas, 2009)
. A switch from CD44v to CD44s is essential for
EMT and tumour progression in breast cancer (Brown et al, 2011).
Furthermore, the ratio of vimentin/E-cadherin mRNA expression
is significantly correlated with the CD44s/CD44v9 ratio in
colorectal cancer, indicating that CD44 switching might induce
(Mashita et al, 2014)
. These findings suggest that CD44s
confers mesenchymal properties, unlike other CD44-variants, and
is associated with metastasis. Our data also showed that CD44s
expression at the ITF is associated with peritoneal, lymphatic, and
haematogenous recurrences, suggesting that CD44s-expressing
cancer stem-like cells at the ITF are likely to metastasise in various
directions. Moreover, CD44s expression was also recognised an
independent prognostic factor. These findings support the
hypothesis that CD44s switching affects EMT and metastasis in
GC; however, experimental verification is still needed to
conclusively evaluate the hypothesis.
We recently observed cancer cells that disseminated into the
peritoneal cavity during curative gastrectomy in GC; moreover,
these cancer cells have proliferative and tumorigenic capacity,
which could result in peritoneal metastasis
(Takebayashi et al,
. We also recently confirmed the presence of disseminated
CD44s-expressing cancer stem-like cells during gastrectomy
(Murata et al, unpublished data). In addition, we detected viable,
proliferative, and clustered cancer cells, including
CD44s-expressing cancer stem-like cells, in remnant gastric lumens immediately
before gastrointestinal reconstruction
(Murata et al, 2016)
. In this
study, the presence of CD44s-expressing tumour cells at the
invasive front of the gastric wall was significantly correlated with
peritoneal recurrences and poor survival. Cancer stem-like cells
may spill out easily during resection when they are present at the
ITF; CD44s-positive cancer cells that have mesenchymal properties
may easily leave the ITF of the gastric wall and disseminate into the
When patients were classified based on the number of CD44
isoforms expressed at the ITF, we found that the prognosis was
worse in patients expressing more CD44 isoforms. This result
suggests that each CD44 isoform in GC may have a distinct
function. GC frequently exhibits marked histological heterogeneity
with various differentiated cancer cells, and these might be
produced by different undifferentiated cells with the properties of
each CSC. Therefore, the functional role of each CD44 isoform in
lue a 14 26 65
va-P .00 .0 0
the tumour differentiation pathway, heterogeneity, and therapeutic
resistance should be elucidated in the future. All patients with
CD44s-positive cells in this study also had CD44v6- and
CD44v9positive cells at the ITF, and GC patients with CD44s-positive cells
at the ITF had the worst prognosis in the predictive model for
survival. The number of CD44 isoforms expressed was an indicator
of patient outcome. In particular, the all-negative group showed
the best survival among all predictive model groups. These findings
suggest that both the expression of CD44s itself and the number of
CD44 isoforms expressed have prognostic value in GC patients.
From a clinical perspective, patients with positive expression for all
CD44 isoforms at the ITF have a very poor prognosis, suggesting
the need for more intensive adjuvant chemotherapy. Patients with
negative expression of all CD44 isoforms at the ITF have extremely
good outcomes, suggesting that adjuvant chemotherapy is not
necessary after gastrectomy. To verify the utility of these
biomarkers, that is, CD44 isoforms, at the ITF for making
treatment decisions, clinical studies are needed.
In conclusion, the presence of CD44-expressing cancer
stemlike cells at the ITF was significantly associated with poor survival
in GC patients, indicating that CD44 isoforms are promising
biomarkers for GC.
We thank Mrs Ikuko Arikawa and Mrs Miho Yamamoto for their
excellent technical assistance. We also thank Mrs Ryoko Tanaka
and Mrs Masami Yamato for their help with clinical data
preparation. We are also grateful to Mr Yoshimitsu Miyahira at
the Department of Clinical Laboratory Medicine for his valuable
assistance with cell staining. We would like to thank Editage
(www.editage.jp) for English language editing. This work was
supported by JSPS KAKENHI Grant Number JP15K10096.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
This work is published under the standard license to publish
agreement. After 12 months the work will become freely available and
the license terms will switch to a Creative Commons
AttributionNonCommercial-Share Alike 4.0 Unported License.
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