ARTICLE
Auteur(s) : Isabelle Conscience1, Nicolas
Jovenin2, Christelle Coissard3, Marianne
Lorenzato3, Anne Durlach3, Florent
Grange1, Philippe Birembaut3, Christine
Clavel3, Philippe
Bernard1
1CHU Reims, Hôpital Robert Debré, Service de
Dermatologie, Avenue du Général Koenig, Reims F-51092, France
2CHU Reims, Hôpital Robert Debré, Department
d’Informatique Médicale, Reims F-51092
3CHU Reims, Hôpital Maison Blanche, Laboratoire Pol
Bouin, Reims F-51092
accepté le 1 Mai 2006
P16 is a tumor suppressor protein encoded by the INK4a/CDKN2A gene
located on chromosome 9p21 [1]. It is a cell cycle regulatory tumor
suppressor protein that negatively regulates D-type cyclins in the
G1 cell cycle phase via intimate interplay with the retinoblastoma
gene (Rb). Besides cell cycle control, P16 has been implicated in
other processes such as senescence, apoptosis, cell migration and
angiogenesis. Ki67 antigen is a non-histone protein associated with
ribosomes. It is expressed in all phases of the cell cycle, except
G0 and is a marker of proliferation expressed in many cancers [2].
Studies on Ki67 expression of non-melanoma skin cancers led to
contradictory results. A relationship was not constantly
demonstrated between its expression and occurrence of premalignant
and malignant skin lesions [3-6].Recently, several studies have
shown a P16 overexpression in precancerous and cancerous cervical
lesions, with a correlation between P16 expression and the grade of
dysplasia [7]. Cervical squamous carcinomas are induced by
persistent infection with oncogenic and mucosal human
papillomaviruses (HPV), especially types 16 and 18. HPV E6 and E7
oncoproteins interact with cell cycle-regulating proteins p53 and
pRb, respectively. E7 oncoprotein inactivates and degrades pRb and
inhibits transcription of the cyclin-dependent kinase inhibitor
gene p16INK4a. An increased expression of E7 oncogene in
dysplastic cervical cells might thus be reflected by an increased
expression of p16INK4a.In the skin, the oncogenic role
of HPV was first demonstrated in epidermodysplasia verruciformis,
which is a very rare inherited disease with susceptibility to
specific HPV types and subsequent skin carcinomas. The relationship
between HPV infection and skin carcinomas (SCC) was then
demonstrated in organ transplant recipients. Although HPV-DNA
sequences have been identified in a proportion of common SCC [8],
the role of HPV in cutaneous carcinogenesis is still unclear in
non-immunocompromised patients and UV radiation remains the key
environmental risk factor of skin carcinomas in the general
population. Besides, some studies have shown an overexpression of
P16 correlated with the level of malignancy, suggesting that P16
could represent a biomarker of tumoral progression [9-13], while
others reported a loss of P16 expression correlated with higher
malignancy [14, 15]. A number of mutations of P16 have been
identified in premalignant actinic keratoses and SCC such as
homozygous gene deletions, hypermethylation of the p16 promoter
and, more rarely, mutations in the INK4a locus. In SCC, the p16
gene may be mutated in up to 24%. Those mutations can be hereditary
or acquired by UV-induction.The aim of the present study was first
to evaluate P16 expression in different types of non-melanoma skin
cancers, in comparison with normal human skin and begnin tumors in
order to determinate if P16 could be considerered as a marker of
malignancy and/or tumor progression. Second, to further delineate
the possible pathophysiological mechanism, we also characterized
the expression of Ki67 and the putative role of UV radiation.
Material and methods
Study design
For this retrospective, comparative study, we used formalin-fixed
and paraffin-embedded skin biopsy specimens from 76 non-selected
patients with either SCC (n = 30), Bowen’s disease (BD; n = 17),
basal cell carcinoma (BCC; n = 10), seborrheic keratosis (SK; n =
10) or normal human skin (NHS; n = 9).
Those biopsies were obtained from 76 patients, with mean age of
71.9 years (range 39-47), seen between 2001 and 2003 in our
department of Dermatology. Normal skin biopsies (n = 9) were issued
from mammoplasty pieces. Histological analysis was made by two
pathologists. Selection criteria included unequivocal histologic
features of either SCC, BD, BCC, SK and NHS.
Clinical and demographic data were determined from the patients’
files. The location of skin carcinomas (on sun exposed versus non
exposed areas) was recorded for all cases. Cases of
immunosuppression and their causes were specified.
Serial sections (4 μm thick) of formalin-fixed and
paraffin-embedded biopsy specimens were cut. The first and the last
sections were stained with hematoxylin and eosin to confirm
histopathologic diagnosis, serial sections were used for
immunohistochemistry and for detection and typing of HPV-DNA (4
sections, 5 μm).
Immunohistochemistry
Protein expression of P16 and Ki67 were studied in all patients.
Serial sections (4 μm thick) of formalin-fixed and
paraffin-embedded biopsies were cut. To remove paraffin, sections
were immersed in xylene followed by rehydration through graded
alcohol and rinsed in phosphate-buffered saline. Antigen retrieval
was performed by incubation in 10 mM citrate buffer (pH 6.0)
at 98 °C for 40 m in an autoclave. The following steps
were performed at room temperature. The endogenous peroxydase
activity was blocked by incubation in 1% hydrogen peroxide (H2O2)
for 10 m. Sections were preincubated with 1.5% normal serum
for 20 m to block non-specific binding. After removing the
serum, the primary P16 monoclonal antibody and the primary Ki67
monoclonal antibody (DAKO, France) diluted respectively 1:25 and
1:50 was added for 30 m. The sections were stained with
diaminobenzidine chromogen and counterstained with
aminoethylcarbazole for image analysis. A negative control was
prepared for each staining series. Immunostained sections were
submitted to an image cytometric measurement using a ZEISS image
analyser (Le Pecq, France), and KS 300 SOFTWARE, especially adapted
for the need of the laboratory. In the normal skin, we have taken
in account for the quantification of Ki-67 the positivities of the
epidermis. In tumor specimens, only tumoral tissue was assessed.
Using image analysis, cut-offs for defining overexpression of P16
and Ki67 were defined by the median of the percentage of
immunostainings on tumoral tissue of skin malignant tumors assessed
on a mean field number of five. P16 and Ki67 were considered as
overexpressed when immunoreactivity was above 10% and 5% of the
tumoral cells, respectively (figures 1 and 2).
Statistical analysis
Comparisons of characteristics between groups (with or without P16
and Ki67 expression) were made using the chi-square test and
Fisher’exact test for qualitative variables and student’s t test
for quantitative variables. A p-value of less than 0·05 was
considered significant.
Statistical analysis was performed with the SAS software,
version 8.2 for Windows (SAS institute, Cary, NC).
Results
Clinical characteristics are detailed in table 1( Table 1 ). Immunosuppression consisted in renal
transplantation in 17 patients, chronic lymphoid leukaemia in 5 and
chemotherapy in 8. Seventy-seven percent of all skin carcinomas
were located on sun-exposed areas. Results of p16 and Ki67
overexpression according to histology are summarized in table 2(
Table 2 ). P16 overexpression was
significantly more frequent in the group of cutaneous carcinomas
(58%) than in SK or NHS (0%) (p = 0.006). Within the group of
carcinomas, there was no difference according to the histologic
subtype.
The two groups of patients with P16-positive and P16-negative
tumors did not differ significantly by sex and age (mean: 75 years
versus 70 years, respectively, p = 0.2). However, overexpression of
P16 was significantly associated with skin carcinomas located on
sun-exposed areas. Indeed, 68% of tumors located on sun-exposed
areas versus 23% of those located on non-exposed areas
overexpressed P16 (p = 0.02).
Ki67 over-expression was significantly more frequent in the
group of cutaneous carcinomas (70%) than in the group including SK
(30%) and NHS (0%) (p = 0.04). Within the group of carcinomas, no
difference was observed for Ki67 expression according to the
histologic subtype. The two groups of patients with Ki67-positive
and Ki67-negative tumors did not differ significantly by sex and
age (mean: 73 years versus 69, respectively; p = 0.3).
P16 and Ki67 co-expression is detailed in table 3( Table 3 ). Twenty-three of 57 (40%) tumors
co-expressed P16 and Ki67. However, the concordance between P16 and
Ki67 expression remained below significancy (p = 0.07).
Table 1 Clinical characteristics of patients according
to the different skin lesions
|
SCC
|
BD
|
BCC
|
SK
|
NHS
|
|
No of patients
|
30
|
17
|
10
|
10
|
9
|
|
Mean age (years)
|
71.6
|
79.2
|
69.8
|
54.7
|
59.7
|
|
Sex ratio H/F
|
1.3
|
0.54
|
2.33
|
0.66
|
0
|
|
Immunosuppression
|
23%
|
35%
|
30%
|
0%
|
0%
|
|
Location on sun-exposed area
|
76%
|
65%
|
70%
|
30%
|
0%
|
Table 2 P16 and Ki67 overexpression in skin carcinomas,
seborrehic keratosis (SK) and normal human skin (NHS)
|
SCC
|
BD
|
BCC
|
SK
|
NHS
|
|
P16
|
18/30
|
10/17
|
5/10
|
0/10
|
0/10
|
|
Ki67
|
16/30
|
13/17
|
8/10
|
3/10
|
0/9
|
Table 3 Co-overexpression of P16 and KI 67 in skin
carcinomas
|
Ki 67
|
Total
|
|
P16
|
|
+
|
–
|
|
|
+
|
23
|
10
|
33
|
|
–
|
17
|
7
|
24
|
|
Total
|
40
|
17
|
57
|
Discussion
In our study, P16 was overexpressed in approximatively half of the
cases of BCC, BD and SCC, without significant difference between
the types of tumors. In contrast, P16 was not expressed in any NHS
or SK. When used as a diagnositic test for malignant tumors, P16
showed a sensitivity of 48% and a specificity of 100% in our study.
So far, in the literature, only a few studies have evaluated P16
immunohistologically in cutaneous SCC and its precursors, with
contradictory results. Some studies found an overexpression of P16
correlated with the level of malignancy, suggesting that P16 could
be a biomarker of tumoral progression [11-14]. Hodges and Smoller
[11] described P16 positivity in all of 10 actinic keratoses (AK),
and 10 invasive SCC, with a level of positivity which correlated
with the progression from normal human skin to SCC. Salama et al.
[12] found a positivity of P16 in 84.1% of BD cases, 6.8% of AK
cases and none of 29 controls. Willeke et al. [13] found that P16
was more frequently positive in high-grade keratinocytic
intraepithelial neoplasia (KIN) than in low-grade KIN, suggesting
that P16 may be a sensitive and specific marker for distinguishing
premalignant from malignant squamous cutaneous lesions. Mortier et
al. [14] found P16 expression in 66% of AK but only 10% of SCC. In
contrast, other authors reported a loss of P16 expression,
correlated with the level of malignancy. Chang et al. [15] have
found a correlation between the loss of p16 expression and the
occurrence of metastases in SCC, suggesting that loss of expression
of the p16 gene may play a critical role in tumor progression. Many
others studies showed mutations in the p16 gene and suggested that
a deficiency of this tumor suppressor gene may be important in the
development of SCC. Our results demonstrate that P16 overexpression
is associated with nonmelanoma skin malignancies. However, we
failed to show differences between different subtypes of carcinomas
(i.e. SCC, BD, and BCC), which suggests that P16 overexpression
does not correlate with the degree of proliferation and malignancy.
In addition, P16 labelling cannot represent a discriminating test
between histological types of cutaneous tumors.
In our study, Ki67 was significantly more frequently expressed
in the group of malignant tumors than in the group of normal skin
and SK, whatever the type of tumors. Only a few studies have
previously evaluated Ki67 expression in cutaneous carcinomas.
Kanitakis et al. [5] showed that an aggressive course of SCC was
not correlated with the expression of Ki67. Scurry et al. [6] did
not find any difference in Ki67 expression in lichen sclerosus of
the vulva, between patients with or without associated squamous
cell carcinoma. In contrast, three studies observed an
overexpression of Ki67 in recurrences of BCC [2], in SCC [3], and
in BD [4], respectively. Our study confirms and extends these
results, suggesting that Ki67 is strongly associated with skin
carcinomas, whatever the histological subtype. The design of
present study did not allow us to evaluate a possible prognostic
value of Ki67 expression.
The possible pathophysiological role of P16 overexpression in
skin carcinogenesis remains unclear. A first hypothesis is derived
from the model of cervix carcinomas in which a P16 overexpression
was observed in high-risk HPV-infected patients [16, 17]. However,
to date, except in specific clinical contexts (immunosuppression,
epidermodysplasia verruciformis, unusual localizations such as
extremities), the oncogenic role of HPV in non-melanoma skin cancer
remains very speculative [17-20]. In skin carcinomas, only two
studies have suggested a possible correlation between P16
expression and HPV [21, 22]. In our present series, HPV-DNA types
18 and 52, respectively, were found in only two SCC, both
associated with P16 overexpression. However, we only performed PCR
using primers for mucosal oncogenic HPV and not for skin HPV types.
Therefore, we cannot exclude in this study the presence of
cutaneous-type HPV with a possible role in inducing P16
overexpression.
Our study points out another possible mechanism for P16
overexpression and associated carcinogenesis. We found that P16 was
overexpressed in 68% of skin carcinomas located on sun-exposed
areas, versus 23% of those on others sites. This result is in
accordance with previous studies using cultured human melanocytes
or keratinocytes, which strongly suggested the role of UVB
radiation in inducing P16 overexpression [23, 24]. Subsequent
mechanisms of carcinogenesis remain unclear. However, the potential
role of a mutated P16 protein with a longer half-life, but impaired
regulatory functions after UV exposure, can be suspected [25, 26].
In this hypothesis, DNA mutations induced by UV radiations could
not be repaired by the P16-dependent arrest of the cell cycle,
therefore leading to the development of skin carcinomas. Finally,
the recently reported efficacy of imiquimod, a topical
immunomodulator, in the treatment of superficial BCC [27], which
are believed to be induced by acute and repeated exposure to UV
radiation [28], is in accordance with that hypothesis.
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