2015, Volume 31, Number 1, Page(s) 030-035
CXCR-4 and COX-2 Expression in Basal Cell Carcinomas and Well-Differentiated Squamous Cell Carcinomas of the Skin; Their Relationship with Tumor Invasiveness and Histological Subtype
Oya Nermin SİVRİKOZ1, Belkız UYAR2, Filiz DAĞ3, Funda TAŞLI1, Salahattin M. SANAL4
1Department of Pathology, Şifa University, Faculty of Medicine, İZMİR, TURKEY
Department of Dermatology, Şifa University, Faculty of Medicine, İZMİR, TURKEY
3Department of Pathology, Tınaztepe Private Hospital, İZMİR, TURKEY
4Department of Oncology, Tınaztepe Private Hospital, İZMİR, TURKEY
Keywords: CXCR-4, COX-2, Basal cell carcinoma, Squamous cell carcinoma, Skin neoplasms
Both CXCR-4 and COX-2 are biological markers that play
a significant role in several neoplastic processes. We explored the
differences in expression of these markers in certain subtypes of basal
cell carcinoma, and squamous cell carcinomas in general.
Material and Method: In this study, we investigated the differences
between 38 patients with basal cell carcinoma (nodular, infiltrative
and micro-nodular subtypes) and 24 patients with well-differentiated
squamous cell carcinomas with respect to their depth of invasion,
tumor location, age, and CXCR-4 and COX-2 expression.
Results: Statistically, we found no significant difference between
squamous cell carcinomas and basal cell carcinoma in terms of
CXCR-4 and COX-2 expression; however, the degree of marker
expression became stronger with increasing depth of invasion in both
tumors. The expression of both markers was also higher in infiltrative
type basal cell carcinoma compared to all the other subtypes.
The results were statistically significant (p<0.05). Additionally, a
significantly positive correlation also existed between COX2 and
CXCR4 expression (p < 0.05).
Conclusion: The degree of expression of CXCR-4 and COX-2 is
related to invasiveness in both malignancies; thus, infiltrative type
of basal cell carcinoma displays the highest degree of CXCR-4 and
COX-2 expression among all the subtypes. Furthermore, our results
indicate that these two biological markers may both be involved in
the process of carcinogenesis and require investigation with further
molecular and genetic studies in larger series.
The most common types of non-melanoma skin carcinomas
are basal cell carcinoma (BCC) and squamous cell carcinoma
. UV light exposure plays an important role in
the etiology of both tumors3
. While SCC has significant
metastatic potential, BCC rarely metastasizes.
BCCs are histologically classified as superficial, nodular,
micronodular, infiltrative, nodulocystic, fibroepithelioma
like, metatypical, and mixed types. The micronodular
and infiltrative types tend to show local recurrence more
frequently than the others and have a higher potential to
metastasize4. Superficial and metatypical BCCs also have
increased rate of recurrence.
In general, SCC has a more aggressive behavior than BCC.
SCC often metastasizes to neighboring lymph nodes and
distant organs. Currently, convincing scientific information
demonstrates that chemo-attractants, like CXCR-4, secreted
by stromal immune cells, enable cancer cells invade tissues
where they have secondary growth potential5.
Chemokines are structurally 8-14 kDa sized polypeptides
that have the properties of a signaling molecule. CXCR-4
is a member of chemokine receptor family. The interaction
between CXCR-4 and stromal cell-derived factor (SDF-
1α), its ligand, is known to play an important role in
tumorigenesis, metastasis, and angiogenesis6-8. This
biological role is demonstrated frequently in lung, breast,
melanoma, glioblastoma, pancreatic and cholangiocellular
Lately, UV-induced abnormal prostaglandin synthesis is
reported to be an important factor in the development of
cancer. COX is an enzyme that catalyses the conversion of
free arachidonic acid to prostaglandins. It has two isoforms
known as COX-1 and COX-2. COX-1 is found in most
normal tissues and is required for normal physiological
function14. COX-2 is not detected under normal
conditions and is expressed in circumstances such as growth
factors, cytokines, oncogene activation and hypoxia. COX-2
is also known to be expressed in many neoplastic processes.
It has been found to stimulate cell division, angiogenesis,
and inhibit apoptosis15,16.
We investigated the importance of CXCR-4 and COX-
2 expression in both BCC and SCC. Furthermore, we
examined if these biological markers showed any difference
with respect to degree of invasiveness in each BCC subtype,
which recently has been found to play a significant role in
the development of metastatic process.
A total of 62 patients consisting of 24 SCC and 38 BCC were
evaluated in our study. Clinical information of all cases
(age, gender and tumor site) was obtained from the hospital
records. Paraffin blocks of each patient were obtained from
the archival tissues. Sections (4 micron thick) were taken
from the paraffin blocks, one for H&E staining and two
for immunohistochemical staining on two (+) charged
slides. H&E-stained profiles were evaluated under the light
microscope. Basal cell carcinomas were divided into two
groups as aggressive (infiltrative, micronodular) and nonaggressive
(nodular). All SCCs were well differentiated and
all were invasive. The other types of basal cell carcinoma
and poorly- and moderately-differentiated squamous cell
carcinomas were excluded from the study because of their
limited number for analysis. All cases of SCCs were the
well-differentiated, keratinized type. None had any lymph
node or distant organ metastasis.
The positive charged slides were stained with DAKO
Autostainer 48 Link (Dako, Denmark) and COX-2 (DAKO,
Clone CX- 294, 1/100 dilution) and CXCR-4 (Abcam, Clone
ab 2074, 1/50 dilution) antibodies. The slides were then
evaluated under the light microscope by two pathologists.
Cytoplasmic staining in both was accepted as positive and
was scored as follows: 0= no staining, 1+: 1-25% cells (1+:
Poor staining), 2+: 26-50% cells, 3+: 51-100% cells (2+, 3+:
Statistical method: The Statistical Package for the Social
Sciences (SPSS) 21 and Medcalc 9 (Acacialaan 22, B-8400
Ostend, Belgium) programs were utilized for data analysis.
For analysis of quantitative data, compliance with the
normal distribution was evaluated with the Kolmogorov-
Smirnov test, Shapiro-Wilk test and Variation coefficients.
Parametric methods were used for the analysis of variables
with a normal distribution and nonparametric methods
were used for the analysis of variables without a normal
distribution. The Independent Samples t- test was used in
the comparison of two independent groups and the One-
Way Anova test in the comparison of multiple groups with
each other. The Pearson Chi-Square and Linear-by-Linear
Association tests were used for the comparison of categorical
data. Data were examined at the 95% confidence level and p
values smaller than 0.05 were accepted as significant.
There were 62 patients consisting of 38 (61.3%) BCC and
24 (38.7%) SCC cases. The ages of the cases varied between
41 and 91 the mean age was 72 years. Among BCC patients,
19 were male and 19 female. According to histological subtype, they were distributed as 27 nodular, 7 infiltrative
and 4 micronodular. Among SCC patients, there were 17
male and 7 female patients.
The anatomic distribution of both malignancies were as
follows: Among SCC patients, the malignancy arose from
facial structures in 12 (50.0%), from the ear in 2 (8.3%),
and from the trunk in 5 (20.8%) whereas among BCC
patients the malignancy arose from the facial structures
in 26 (68.4%), from the scalp in 3 (7. 9%), from the ear in
4 (10.5%), and from the trunk in 5 (13.2%). Statistically
no significant relationship was found between tumor
type, location, patient age and CXCR-4 as well as COX-2
Among BCC patients, COX-2 staining was absent in 57.9%,
poor (+1) in 15.8%, and strong (+2, +3) (Figure 1) in
36.3%. All patients with infiltrative subtype stained strongly
positive, while none showed absent or weak staining. The
multinodular subtypes showed absent or poor staining
in 75% and strong staining in only 25%. The staining was
absent or poor in 92.6% of the nodular subtype cases. Strong
staining was observed in only 7.4%. These differences were
statistically significant (p<0.05). When cases were distributed
between aggressive and non-aggressive groups, 27.2% of
cases in the aggressive group showed either absence of
staining or at the most poor staining properties while 72.8%
showed strong positivity. In the non-aggressive group 92.6%
showed absence or poor staining characteristics. Only 7.4%
of cases in the non-aggressive group showed strong staining
COX-2 staining was absent or poor (+1) (Figure 2) in 50%
of the SCC cases and strong in 50%. Among the BCC cases,
36.6% stained strongly positive. However, when staining characteristics were compared between the histological
types, 42.1% of SCCs stained positively whereas, when
the presence (1+ to 3+) or complete absence (0) of any
staining was taken into consideration, 84.6% of the BCC
cases stained positively. The difference between the staining
characteristics of these two histologies was not statistically
When COX-2 staining was evaluated according to depth of
invasion in BCC cases, there was no staining in cases with
papillary dermis invasion and 4 cases with reticular dermis
invasion. The staining was absent in 77.8%, poor in 16.7%,
and strong in only 5.6%. Strong staining was present in 75%
of the cases with fat and deeper tissue invasion (Table I).
The results were statistically significant (p<0.05).
When COX-2 staining properties were investigated in SCC
cases, there was absence of staining or at the most poor
staining in all of the cases with papillary dermis invasion.
In cases with reticular dermis invasion, 40% showed
absence or poor staining properties; whereas, in cases with
subcutaneous fat or deep dermis invasion, 87.5% showed
strong staining properties.
Among SCC patients, CXCR-4 staining was absent or poor
(0, +1) (Figure 3) in 45.8% and strong (+2, +3) (Figure 4)
in 54.1%. There was no statistically significant difference
(p>0.05). Furthermore, again among BCC patients, CXCR-
4 staining was absent or poor (0, +1) in 68.4% and strong
(Figure 5) in 31.6%. With respect to the relationship between
BCC tumor subtypes and CXCR-4 staining characteristics,
14.3% of infiltrative types were poorly stained or none at
all, and 85.7% were strongly stained. Micronodular types
showed 0 and +1 staining in 75.0% and strong positivity in
25.0%. The nodular types showed absent or poor staining in 81.5% and strong positivity in only 20.0% (Table II). These
differences were statistically significant (p<0.05).
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|Table II: CXCR4 distribution in basal cell carcinoma subtypes and squamous cell carcinomas
When the relationship between depth of invasion and
CXCR-4 staining characteristics were evaluated in both
BCC and SCC, cases with papillary dermis invasion showed
absent or poor staining (0, 1+) in 85.7%, and strong (2+, 3+) staining in 14.3%. Among cases with reticular dermis
invasion, 71.4% either did not show any staining or poorly
stained; whereas, 28.6% were strongly stained. In those with
fat and deeper tissue invasion, the rate of poor staining was
25.0%, and strong staining 75% (Table III). The results were
statistically significant (p<0.05).
Furthermore, there was no correlation between the age and
CXCR-4 as well as COX-2 staining properties according
to partial correlation test when the tumor type and BCC
subtypes were sanctioned. A strongly positive correlation
was observed between COX-2 staining properties and
invasion. Similarly, a moderately positive correlation was
observed between CXCR-4 staining and degree of invasion.
A strongly positive correlation was also found between
CXCR-4 and COX-2 (r: 0,725).
BCC and SCC are the two most common types of nonmelanoma
skin carcinomas. UV light exposure plays an
important role in the pathogenesis of both tumors. BCC is
a localized disease and rarely metastasizes while SCC has
the potential to metastasize2
Chemokine receptor expression in a malignant cell is
associated with increased proliferation and cell motility.
This results in tumor growth and metastasis7,11. In a
study of 19 malignant melanoma cases, Toyozav et al.
found CXCR-4 expression to be associated with tumor
aggressiveness and poor prognosis11.
Katayama et al. discovered that CXCR-4 expressing cells
have high proliferation and migration capability in a study
they conducted on head and neck SCCs. Furthermore,
they also discovered that CXCR-4 expression decreased
together with cellular proliferation and migration following
treatment with IFN-alpha. Additionally, when multivariate
analysis was conducted, CXCR-4 positivity emerged as
an independent factor in tumor-specific mortality17.
Previous studies conducted on BCC showed CXCR-
4 expression to have a role in BCC tumorigenesis and
angiogenesis6. Chu et al. reported SDF-1α/ CXCR4 to be
an important factor for BCC invasiveness4.
We found CXCR-4 expression to be significantly increased
in basal cell carcinoma, especially in the infiltrative subtype.
In addition, a high rate of CXCR-4 positivity was found
in squamous cell carcinomas. The increased CXCR-4
positivity with increasing invasiveness for both tumor types
is important in identifying tumor aggressiveness.
We found the CXCR-4 expression rate to be higher (2+, 3+)
in SCC than BCC. Our finding suggests that SCC may have
a higher metastatic potential when compared to BCC.
Another molecule playing a role in skin carcinogenesis
is COX-2. Studies have shown that COX-2 expression
stimulates cell division, angiogenesis, and inhibits apoptosis
in neoplastic process7,8.
In 1999, Petland et al. found increased COX-2 expression
in UV-induced skin tumors; conversely, decreased new
tumor formation was demonstrated when a selective COX-
2 inhibitor was administered18. Another study on oral
SCC cases showed that the majority of COX-2-positive
tumors showed cervical lymph node metastasis and
tumors with low COX-2 expression responded better to
Yalcin et al. examined COX-2 and p53 expression in
BCC, actinic keratosis and SCC. They discovered that
no significant difference existed between aggressive and
non-aggressive BBC cases in terms of COX-2 expression.
However, the positivity rate was higher in SCC than BCC
cases3. Conversely, in our study, COX-2 expression was
significantly higher in the aggressive subtypes of BCC than
non-aggressive ones and the findings were statistically
significant. Furthermore, COX-2 expression became more
pronounced as invasion depth increased. Our higher degree
of (2+, 3+) COX-2 positivity in squamous may be cell
carcinomas, compared to basal cell carcinomas indicates
that this molecule may be important in terms of tumor
aggressiveness and poor prognosis.
In conclusion, we found that aggressiveness in UV-induced
skin tumors, namely BCC and SCC increased as COX-2
and CXCR-4 expression increased. We did not come across
any study in the literature where these two markers, which
can easily be applied immunohistochemically on a routine
basis, were evaluated together in both BCC and SCC. Our
study indicates that targeted treatments to inhibit CXCR-
4 and COX-2 can be useful for both tumors. We also
demonstrated significantly positive correlation between
these two molecular markers. Henceforth, we feel that there
may also be a corresponding relationship in the process of
carcinogenesis. Our findings may be instrumental for a
genetically based study on a larger series of patients.
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