Evaluation of p53 and Ki67 Expression Profiles in Basal Cell Carcinomas in a Usual and an Unusual Location
Nuran SÜNGÜ1, Merve Meryem KIRAN1, Hayriye TATLI DOĞAN1, Aydan KILIÇARSLAN1, Emre KARAKÖK1, Mesut AKYOL2
1Department of Pathology, Ankara Yıldırım Beyazit University, Ankara Ataturk Research and Training Hospital, ANKARA, TURKEY
2Department of Biostatistics, Ankara Yıldırım Beyazit University, Ankara Ataturk Research and Training Hospital, ANKARA, TURKEY
Keywords: Basal cell carcinoma, p53, Ki67, Location
Owing to their importance in cell proliferation in cutaneous malignancies, we aimed to immunohistochemically compare the
expression profiles of p53 and Ki67 in basal cell carcinoma (BCC) cases in both a usual and an unusual locations in this study.
Material and Method: In this study we included 12 in an unusual location of BCC cases and 21 BCC cases in a usual location. Immunohistochemical
expression of p53 and Ki67 antibodies were studied in 33 paraffin-embedded tissue specimens of basal cell carcinoma. We compared the p53 and
Ki67 staining scores with clinicopathologic features.
Results: The tumor size was found to be greater in BCC cases in an unusual location than those in a usual location. The relationship between
age and tumor size was also evaluated in both groups and it was found that tumor size increased with age. A comparative study between the two
groups showed no difference p53 and Ki67 expression percentages. There was a linear correlation between the Ki67 and p53 marker staining
rates (ρ=0.420; p=0.015). In the samples taken from cases in a usual region, there was a linear and moderate relationship between the markers
(ρ=0.513; p=0.017). Median tumor diameter results were similar to the marker staining score (p>0.05).
Conclusion: This is the first study comparing the expression profiles of p53 and Ki67 of BCC cases in an unusual and a usual location. No
significant difference was found concerning Ki67 and p53 expression levels between the two groups.
Basal cell carcinomas (BCC) are the most frequent type
of non-melanoma malignancies and their frequency
increases with aging (1,2). BCC cases have a slow clinical
course and are seen with local recurrences (2). However,
regional lymph node, lung and bone metastases have only
been reported in rare cases. The main etiological reasons
are exposure to sunlight and genetic factors. Additionally,
several etiological factors such as immunosuppression,
male gender, scar tissue, arsenic intake and nevus syndrome
may be responsible for the etiology (3).
BCC is characterised by basaloid cells showing peripheral
palisading and also peritumoral spaces associated with a
surrounding loose fibromucinous stroma (Figure 1) (4).
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|Figure 1: The nodular type of BCC is characterised by basaloid
cells showing peripheral palisading (short arrow) and also
peritumoral cleavages associated with a surrounding loose
fibromucinous stroma (long arrow) (H&E; x100).
Although BCC is seen quite often, it occurs rarely in some
parts of the body. Those in an unusual location constitute
1.2% of all cases (4).
P53 gene mutation is also responsible for BCC progression,
as in several other tumor types. P53 is a tumor suppressor
gene which is defective in malignancies and p53 mutation
has been reported in more than 50% of human malignancies (5-7). The p53 gene mutation is important in BCC cases
that have defined UV light exposure. Various expressions
have been demonstrated by p53 immunohistochemical
testing of BCC cases. Additionally, the relation between p53
expression and aggressive progression has been shown (7).
DNA damage is caused by endocellular factors such as
oxygen radicals or extracellular factors such as UV light.
Under normal conditions, the p53 tumor suppressor
gene arrests the cell cycle or induces apoptosis after DNA
damage. In fact, if there is a mutation in the p53 gene, these
functions are lost. Then the apoptosis capacity is decreased
and a tumor can develop (5). Ki67 is a high molecular
weight non-histone protein that has a controlling effect
at several points in the cell cycle. It is usually associated
with proliferation and particularly with cell proliferation.
Increased Ki67 expression is seen in several tumor types
and in skin damaged due to exposure to sunlight (7).
Owing to their importance in cell proliferation in
cutaneous malignancies, we aimed in this study to
immunohistochemically compare the expression profiles of
p53 and Ki67 in BCC cases located in both common and
We included 1208 patients with a BCC diagnosis who were
consulted in our hospital between 2006 and 2017 in this
study. Their results were then analysed retrospectively. BCC
cases involving the head, neck, body, scalp and extremities
were accepted as common areas. BCC cases in the vulva,
inguinal, axilla, nipple-areola complex, umbilicus, thigh,
and the periungual, scrotal and anal regions were accepted
as rare areas (8). H&E sections of 12 BCC cases in an
unusual location were re-examined. During the same time
period, 21 BCC cases in a usual location were selected and
the nodular type was the most frequent histopathological
Anti-p53(p53-DO7-L-CE) and Ki67(MIB-1) antibodies
with a 1/100 dilution rate were performed in all cases.
Immunohistochemical staining was performed by preparing
3-4 μ thickness surface sections from formalin-fixed
paraffin blocks of the cases to poly-l-lysine coated slides
using a Leica Bond-Max automatic immunohistochemical
staining machine. Subsequently immunohistochemical
stained slides were examined in two groups. The expression
levels of p53 and Ki67 antibodies were evaluated in the peak
staining density area by a percentage value (%). Additionally,
scoring was also carried out and a 5% cut-off rate for p53
and Ki-67 antibody were performed. Ki-67 and p53 positive
cells were scored as follows: 0-5% positive cells=negative,
6-25%=1+, 26-50%=2+, 51-75%:3+, >75%=4+.The scores
were reclassified as low for score 12+ and high for score
In the representation of descriptive statistics, median
(InterQuartile Range) for age and tumor size, and n (%) for
categorical variables were used. Mann Whitney U test was
used to compare p53 and Ki67 staining scores according to
age, tumor diameter, sex, and tumor location. Relationships
between marker staining rates according to age, tumor
diameter, sex, and sample location were investigated by
Spearman rank correlation coefficient (ρ). Sample locations
according to sex were compared with Chi-Square test. Chi-
Square (Likelihood ratio) test was used to compare Ki67
proliferation index according to tumor location and sex.
The Kruskal-Wallis nonparametric variance analysis was
used to compare tumor size according to p53 marker
staining score. The Mann Whitney U test was used to
compare tumor diameters according to the Ki67 marker
staining score, since Ki67 marker was a single high intensity
The MS-Excel 2010 and IBM SPSS Statistics 22.0 (IBM
Corp. Release 2013. IBM SPSS Statistics for Windows,
Version 22.0, Armonk, NY, USA) programs were used for
statistical analysis and calculations. Statistical significance
was accepted as p <0.05.
The total number of BCC cases who were sent to our clinic
between 2006 and 2017 was 1208. 622 (51.6%) of the cases
were male and 586 (48.6%) of the cases were female. The
mean age of the cases was 65.19 years (min 16- max 94
In this study, nineteen of the total 33 cases (57.6%) were
female, 14 (42.4%) were male. Twenty-one (63.6%) of
the samples were in the usual location and 12 (36.4%)
were in unusual locations. These unusual locations were
2 cases involving the vulva, 2 in the inguinal region, 2 in
the umbilicus and one each in the acral (periungal), anal,
nipple-areola complex, sacral and axilla regions. The
usual location rate of samples taken from female patients
was 63.2% (n=12) and the rate of tumors that have usual
locations from male patients was 64.3% (n=9). The usual
location of the samples taken from male and female
patients was similar (x2=0.004; p=0.947). The median age
of the patients with a sample from a usual and an unusual
location was statistically insignificant (Z=1.124, p=0.274).
The diameters of the tumors ranged from 0.3 to 8.0 mm,
while the median was 0.8 (IQR=1.1) mm. There was only a
weak correlation between patient age and tumor diameters
in the linear direction (r=0.365, p=0.037). According to sex,
there was no significant correlation between age and tumor
diameter in male patients (r=0.280; p=0333) and there was a strong correlation between age and tumor diameter in
female patients (r=0.567; p=0.011). Tumor diameters in the
samples taken from male and female patients were similar
(p=0.602). Tumors which arise from unusual locations were
larger than the ones that have usual locations. This result
was statistically significant (p=0.001) (Table I).
Tumor cells were stained with p53 at a rate of at least 1.0%
and at most 90.0%. The staining median for p53 was 20%
(IQR=30.0%). With Ki67, these values were determined as
3.0%-75.0% respectively and the median was 10.0% (10.0%).
The staining rates of both markers did not differ according
to the location of the specimen (p>0.05) (Table II).
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|Table II: p53 and Ki67 marker staining percentages according to sample location
There was a linear correlation between the Ki67 and p53
marker staining rates but it was only moderate (ρ=0.420;
p=0.015). In the samples taken from female patients, there was a linear and moderate relationship between the
markers (ρ=0.501; p=0.029), but no significant relationship
was observed between the markers in the male patients
(ρ=0.269; p=0.353). In the samples taken from the usual
region, there was a linear and moderate relationship
between the markers (ρ=0.513; p=0.017), whereas there
was no significant relationship between the markers in the
samples taken from the unusual region (ρ=0.504; p=0.095).
When the p53 and Ki67 staining rates were re-scored as 0,
low and high, 22 (66.6%) of the p53 staining scores was
low and 5 (15.2%) were high (Figure 2) while 6 were
(18.2%) 0. 19 (57.6%) of the Ki67 staining scores were low
(Figure 3), 1 (3.0%) was high and 13 (39.4%) were 0. The
Ki67 staining score was found to be independent of the
sample location (χ 2=2.240; p=0.326). For the other marker, p53, the staining score classification was not comparable
according to the sample location (Table III).
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|Figure 2: Immunohistochemical p53 high score expression
of a BCC case in the head and neck region (IHC; x400).
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|Table III: p53 and Ki67 marker staining scores according to sample location
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|Figure 3: Ki67 staining had a low score in a BCC case
in the anal region (IHC; x400).
Gender and p53 staining score were not compared. Ki67
staining score was found to be independent of sex, and
there was no difference in staining score according to sex
When the tumor diameter was compared according to the
p53 and Ki67 staining scores, the median tumor diameter
was 0.9 (IQR=1.8) mm in the high staining score cases
while the median tumor diameter was 1.0 (IQR=1.7) mm
in the low staining score cases for p53. Tumor diameter
median was similar to the markers staining score (p>0.05).
BCC cases are most commonly seen in sun-exposed areas
(1-13). Cases in the vulva, inguinal region, axilla, nippleareola
complex, umbilical, periungal, scrotal and anal
regions are accepted to be unrelated to sunlight (8).
A previous study reported that the rate of BCC cases in
an unusual location was 1.12% among all cases (14). We
found similar results in our study with BCC cases that were
consulted to our clinic between 2006 and 2016 (15). The
rate was found to be 0.99%. in our study
The risk of developing BCC increases over 60 years(16).
Metastatic tumors are also usually larger in size. In our
study, the tumor size increased as the age increased. This
result was statistically significant (p=0.037).
Between 1 and 10% of UV light reaches earth as UV-B
and 90% of it as UV-A. UV-B light causes p53 mutation
because of genotoxicity and absorption by keratinocytes.
Wild-type (wt) p53 protects the genome and it ends the
cell cycle during the G1 phase after DNA damage. Wt p53
is increased after DNA damage. Increase of p53 in the
epidermis after sunlight damage causes an arrest of the cell
cycle or recovery of the damage. Mutant p53 is more stable
than wt p53 and it accumulates in the cell (9).
Immunohistochemical p53 expression is accepted as an
indirect marker of mutation. It is thought that P53 mutation
occurs in the earlier period of cutaneous neoplasms.
Additionally, p53 mutation is also seen in actinic keratoses
and normal cutaneous epidermis (6,9).
Previous studies have found p53 expression in cutaneous
neoplasms to vary between 42% and 84% (17,18). In our
study, the expression was found to be 81.8%. The reason for
this variability seems to be the different staining techniques
and different evaluation systems for p53 immunoreactivity.
Previous studies did not find any difference related to p53
expression levels among aggressive and non-aggressive
BCC variants. De Rosa et al. compared p53 expression
levels between aggressive and non-aggressive BCC variants.
p53 expression levels among aggressive BCC cases were
discovered to be higher (19). We could not evaluate the
relation between BCC types and p53 expression because of
our limited case number. In fact, p53 expression levels of
subtypes should be evaluated with larger study groups.
It is emphasized that masses that metastasize in BCC are
generally larger in size (16,21). It has been reported that p53
expression is common in malignant epithelial lesions, but
not in association with malignant phenotype or metastasis
(20). We also found no correlation between tumor size and
p53 expression (p=0.235).
P53 gene mutation is significant in skin tumors following
UV light exposure. The p53 staining score would be expected
to be different between cases in an unusual location that
were not exposed to the sun compared to those from in a
usual location with exposure to the sun but there was no
difference in some studies (17,19,21). Ansarin et al. reported
no correlation regarding the p53 immunoreactivity pattern
and the site of the lesion.
Ki67 is associated with condensed chromatin surface and
chromosomes. It is localized in the nucleocytoplasm before
its nucleus location after cell division. Ki67 is expressed as
late G1-M and G2-M phase (9). Ki67 proliferation index
among BCC cases was found to be between 1% and 61%
in previous studies (22). In our study, Ki67 expression was
seen in all cases. The Ki67 proliferation indexes of our cases
were determined to be between 3% and 75%. No correlation
between tumor size and Ki67 expression levels was found in
the literature (22). Moreover, we did not find any correlation
between tumor size and Ki67 expression levels and this was
compatible with the literature (p=0.500).
In our study, a correlation was found between Ki67 and p53
expression percentage levels (p=0.015).This is an expected
result which determines that Ki67 is a marker showing
proliferation and p53 is related with an aggressive progress
(19,22). Furthermore, in our study, there was a linear and
moderate correlation (p=0.029) between the markers in the
samples taken from the female patients, but no significant
correlation was observed between the markers in the
male patients (p=0.353). In the samples taken from the
usual region, there was a linear and moderate relationship
(p=0.017) between the markers, whereas there was no
significant relationship between the markers in the samples
taken from the unusual region (p=0.095). The correlation
between both markers in both women and the usual areas
may be due to increased sun exposure with the use of a
tanning bed for women.
The patient number enrolled in our study group with BCC
in an unusual location was quite limited. In our study, p53
antibody staining, which was positive in all BCC cases in an
unusual location, was studied immunohistochemically. In
fact it should be studied with an increased number of cases
and further genetic techniques.
In conclusion, this is the first study comparing the expression
profiles of p53 and Ki67 in BCC cases in an unusual
location and BCC cases in an usual location. No significant
difference was found concerning Ki67 and p53 expression
levels between the two groups. This result demonstrates
that several factors such as advanced age, trauma, arsenic
intake, male gender and immunosuppression also had
effects on p53 damage besides exposure to sunlight.
Indeed, further studies with larger groups are necessary to
research p53 and Ki67 expression levels in more aggressively
progressing BCC cases and those in an unusual location.
CONFLICT of INTEREST
The authors declare no conflict of interest.
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