ARTICLE
Auteur(s) : Habib
Ansarin, Mina Daliri, Razieh Soltani-Arabshahi
Department of Dermatology, Hazrat-e Rasool University Hospital,
Iran University of Medical Sciences, Niyayesh Street, Sattarkhan
Avenue, Tehran 14455, Iran
accepté le 11 Mai 2006
Basal cell carcinoma (BCC) is the most common cutaneous cancer
throughout the world and the most prevalent malignancy overall in
Caucasian individuals [1]. Up to 70% of primary BCCs, 85% of
metastatic cases and 90% of recurrent ones occur on the head or
neck [2]. BCC is generally characterized by slow growth and minimal
tissue invasion; however, a small number of tumors behave
aggressively with relentless deep tissue invasion, recurrence and
even local or distant metastases, causing significant morbidity or
mortality. Such tumors present a therapeutic challenge for both the
physician and the patient [3]. Early diagnosis is essential to
improve the management of patients with aggressive types of BCC.
There is no single specific marker available for distinguishing
aggressive BCCs from non-aggressive ones. It has been shown that
growth pattern and the histological subtype of the tumor are
related to aggressive behavior [3-5]. The associations of tumor
aggressiveness with proliferative indices and cell-cycle and
apoptosis-related proteins [6, 7], markers of keratinocyte
differentiation [8], decreased number of nerve fibers within the
tumor [9], and increased tumor vascularity have been studied. Yet,
the results are not consistent. Identifying specific markers
associated with aggressive behavior in BCC may result in earlier
diagnosis and better opportunities for therapeutic
interventions.Mutations in the tumor suppressor gene p53 have been
found in several malignant neoplasms, including melanoma and
non-melanoma skin cancers [10]. Various studies have reported
over-expression of p53 protein in 40-90% of basal cell carcinomas
[11-14]. With regard to the pivotal role of p53 mutations in the
pathogenesis of BCC [15], and its association with tumor
invasiveness in several other epithelial tumors like head and neck
squamous cell carcinoma [16], this gene might be a possible
candidate to predict the aggressive behavior in BCC. Several
investigators have addressed this issue with different results
[17-21]. In this study, we examined the relationship between p53
protein expression and histopathological type of basal cell
carcinoma, site of the tumor, age, and history of previous
radiotherapy.
Materials and methods
Cases studied
From March 2003 to April 2004, 33 tissue samples, each from a
different patient, with the diagnosis of primary basal cell
carcinoma were examined in the Department of Pathology, Hazrat-e
Rasool University Hospital, Tehran. Second excisions of residual
tumors were excluded. All formalin-fixed hematoxylin and eosin
stained sections were reviewed by a pathologist and
histopathological subtypes were defined as follows [22].
Nodular: Circumscribed nests of tumor cells embedded in the
dermis;
Superficial: Buds and irregular proliferations of tumor tissue
attached to the undersurface of the epidermis with little
penetration into the dermis;
Infiltrative: Irregular spiky elongated strands of basaloid
cells only a few layers thick which invade the dermis;
Morpheic: Irregular groups of elongated narrow tumor strands
embedded in a dense fibrous stroma. In cases with hybrid
morphology, we categorized the neoplasm according to the element
representing more than 50% of the tumor. With regard to previous
studies which indicated a relationship between certain pathologic
growth patterns and aggressive behavior in BCCs [3-5, 22-29]
circumscribed and superficial tumors were categorized as
non-aggressive, while infiltrative and morpheic ones were
identified as aggressive tumors.
We reviewed the medical records of all of the patients to
collect clinical, demographic, and epidemiological data. The
medical ethics committee of the university approved the study.
Immunohistochemistry
Sections of paraffin-embedded formalin-fixed tissue (4 micron) were
tested for the presence of p53 by means of monoclonal mouse
anti-p53 IgG1 (PAb240, DakoCytomation, Denmark; dilution 1:75) with
steam heat-induced antigen retrieval method. The recognized epitope
of this antibody is exposed only on mutant forms of p53 protein and
is inaccessible in its wild types [30]. Endogenous peroxidase
activity was blocked with incubation in 3%
H2O2 and the non-specific antibody binding
was inhibited by incubation with a protein block system (Protein
Block Serum-Free, DakoCytomation, Denmark). Immunoreactivity was
illustrated by an avidin-biotinylated enzyme complex kit (LSAB+
System-HRP, DakoCytomation, Denmark) in combination with
3,3′-diaminobenzidine (DAB) and the slides were counterstained with
hematoxylin. As negative control samples, serial sections were
stained as described above, but incubated with non-human reactive
mouse IgG1 alone instead of primary antibody. A section of colon
cancer was included as a positive control. The percentage of
positively stained cells was recorded by counting p53 positive
cells per 1000 tumor cells and p53 immunoreactivity was scored as:
1+, < 25% of tumor cells show nuclear reactivity; 2+, 25-50%;
3+, 50-75%; and 4+, > 75% of the whole mass of tumor stains
[17].
The Chi-Square test was used for analysis of the difference in
p53 immunoreactivity between groups, using a significance level of
p < 0.05 and 2-tailed tests. The analysis was done using SPSS
v.10.0 software.
Results
Of the 33 tissue samples, 26 were from males and 7 from females.
The patients’ mean age at the time of excision was 63.9 ± 8.03
years (range, 48-78 years). Four patients gave a history of
childhood scalp radiotherapy for tinea capitis. In 31 cases, the
tumor was located on the head or neck region, while the trunk was
involved in 2 cases. According to the pathological criteria
mentioned, 22/33 of the tumors were non-aggressive and 11/33 were
aggressive. The clinical characteristics of the patients and the
pathological subtypes are shown in table 1( Table 1 ).
All of the tumor specimens studied showed some reactivity for
p53. Staining was diffusely distributed in the tumor but more
intense in the peripheral palisading zone and in the deeper
advancing parts of the tumor (( figure 1 )). The percentage
of p53 positive tumor cells in different specimens varied from 12%
to 95%. ( figure
2 ) compares p53 immunoreactivity in pathologically
aggressive and non-aggressive subtypes of basal cell carcinoma. All
of the 11 specimens of aggressive tumors exhibited intense nuclear
staining in more than 50% of the tumor cells; while in the 22
non-aggressive tumors less than 50% of cells showed positive
nuclear staining (figures 3 and 4). p53 immunoreactivity was
significantly higher in aggressive BCCs than non-aggressive ones
(x2 test; p < 0.01).
Of the 4 specimens from patients with a history of previous
radiotherapy, three showed nodular and one had infiltrative
histology; all of these 4 tumors were positive for p53 with strong
nuclear staining. p53 immunoreactivity was significantly higher in
patients with past history of radiotherapy than patients without
this risk factor (x2 test; p < 0.01). There was no
significant association between p53 immunoreactivity and sex, age
of the patient, or sun-exposure (site of the tumor).
Table 1 The clinical and pathological characteristics
of the patients
|
Variable
|
Cases (Percent)
|
|
Sites
|
|
|
Sun-exposed
|
28 (84.85)
|
|
Nose
|
7 (21.21)
|
|
Cheek
|
5 (15.15)
|
|
Scalp
|
5 (15.15)
|
|
Frontal
|
3 (9.09)
|
|
Ear
|
3 (9.09)
|
|
Nasolabial fold
|
3 (9.09)
|
|
Temporal
|
2 (6.06)
|
|
Non sun-exposed
|
5 (15.15)
|
|
Trunk
|
2 (6.06)
|
|
Inner canthus
|
3 (9.09)
|
|
Pathological subtype
|
|
|
Non-aggressive
|
22 (66.67)
|
|
Circumscribed nodular
|
17 (51.52)
|
|
Superficial
|
5 (15.15)
|
|
Aggressive
|
11 (33.33)
|
|
Infiltrative
|
9 (27.27)
|
|
Morpheic
|
2 (6.06)
|
Discussion
Mutations in p53 are the most common gene alterations in human
cancers [10]. Up to 90% of human BCCs harbor mutations in the p53
gene. About 65% of these mutations involve C to T and CC to TT
transitions at dipyrimidine sequences, termed UV signature
mutations [15]. Expression of the p53 protein has also been
detected in normal sun-exposed epidermis and in the normal
epidermis adjacent to BCCs [31]. These observations provide strong
evidence that ultraviolet (UV) exposure is the major cause of p53
mutations; however, we did not find any difference in the rate of
p53 mutations between sun-exposed and sun-protected sites. This was
similar to the results of De Rosa et al. [17] and Auepemkiate et
al. [18] who found no correlation between p53 immunoreactivity and
site of the tumor or age of the patient, and Bolshakov et al. who
found no association between p53 mutation frequency and higher
lifetime sun exposure [20]. This controversy might be explained in
two ways. First, other UV induced p53-independent mechanisms (e.g.
PTCH and PTCH2 mutations) might be involved in the development of
basal cell carcinoma [24]. Furthermore, UV radiation is not the
only causative factor in p53 gene alteration. Tobacco smoke,
arsenic, free radicals and several other mutagens could also induce
mutations in this tumor suppressor gene [15]. It must be noted that
in our analysis of the relationship between p53 expression and the
site of the tumor, we categorized inner canthus as a non-exposed
site, as it appears to be well protected from large direct doses of
ultraviolet radiation (UVR) by the protective effect of facial
structures such as eyelids, eye brow ridge, nasal bridge and cheek
[32]. However, some investigators believe that this small region
receives a high dose of solar radiation indirectly through
reflection from the eyes [33].
Our finding of strong immunoreactivity for p53 in all patients
with previous X-ray radiation confirms the role of p53 mutation in
X-ray induced tumorigenesis. It is also in concordance with the
more aggressive behavior of BCCs in these patients despite the
predominance of nodular histology which usually favors an indolent
course [24, 34].
Our study was performed on a Caucasian population with a
moderate and steady level of sun exposure, which differs from
intense episodes of sun bathing in Australia, Europe and United
States. Yet, the findings support the results of previous studies
about correlation of p53 expression with aggressive behavior in BCC
[13, 14, 18-20]. p53 expression correlates positively with
proliferative activity in BCC and melanoma [21]. De Rosa et al.
have demonstrated that BCCs with aggressive morphological features
or relapse express a higher level of p53 immunopositivity in
comparison to more indolent tumors [17]. Their later studies
revealed that expression of p53 correlates inversely with cellular
differentiation; thus, the appearance of p53 positive clones in a
BCC indicates transition from a low to a high grade malignancy with
worse clinical behavior [6, 35].
On the other hand, Healy et al. failed to confirm any
association between p53 expression and tumor aggressiveness in
BCCs; however, they demonstrated higher expression of another
proliferation marker Ki-67 in aggressive BCCs [7]. In another
histochemical study, no correlation was found between the
immunoreactivity of p53 protein and the rate of recurrence, pattern
and diameter of BCC [19]. Bolshakov et al. found a higher rate of
p53 expression in aggressive BCCs than non-aggressive ones, but the
difference was not significant [20]. These conflicting results
might be due to differences in the sensitivity of various methods
used for detection of p53 mutations. In addition, differences in
the genetic characteristics of the populations studied, and the
level of background sun exposure might influence the results.
Our study has several limitations. First, directly assessing
mutations in p53 through genetic methods would be more accurate
than immunohistochemical staining. In addition, we did not use
clinical criteria of aggressiveness; neither did we follow the
clinical course of the tumors to detect cases with recurrence or
distant metastasis. Yet, our findings support the value of p53
overexpression to predict aggressive behavior in BCCs. This may
serve as a useful tool for prediction of tumor progression and help
to design a more rational treatment protocol.
Acknowledgments
The authors would like to thank Dr. Pirooz Salehian for his great
assistance in reading the pathology slides. The study was supported
by a research grant from Deputy of Research, Iran University of
Medical Sciences.
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