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323
Absence of p53 Protein Overexpression in
Precancerous Lesions of the Vulva
Petra D. Kohlberger, M.D.1
Reinhard Kirnbauer, M.D.2
Dagmar Bancher, M.D.1
Gerald Gitsch, M.D.1
Alexander Reinthaller, M.D.1
Sepp Leodolter, M.D.3
Erwin Tschachler, M.D.2
Christian Kainz, M.D.1
Gerhard Breitenecker, M.D.4
BACKGROUND. Recently the authors reported the prognostic value of p53 protein
overexpression in invasive squamous cell carcinoma of the vulva. The aim of this
study was to evaluate the status of p53 overexpression and human papillomavirus
(HPV) infection in patients with precancerous lesions of the vulva.
METHODS. Biopsy specimens of 28 women (mean age, 44.2 years; range, 19-71
years) with warty and/or basaloid type vulvar intraepithelial neoplasia (VIN) of
Grade 1 to 3 were examined retrospectively for p53 protein overexpression by
immunohistochemistry. The presence of the HPV genome was assessed using a
nested polymerase chain reaction (PCR) method with consensus primers directed
against the L1 coding region.
1
Department of Gynecology and Obstetrics,
University of Vienna Medical School, Vienna,
Austria.
2
Division of Immunology, Allergy and Infectious Diseases, Department of Dermatology,
University of Vienna Medical School, Vienna,
Austria.
3
Ludwig Boltzmann Association, Austrian Society for the Promotion of Scientific Research,
Vienna, Austria.
4
Department of Pathology, Gynecopathological
Unit, University of Vienna Medical School, Vienna, Austria.
RESULTS. Neither the preoperative punch biopsy specimen nor the subsequent
surgical specimen contained immunohistochemically detectable levels of p53 in
this study of a group of younger women with preinvasive vulvar lesions. These
results are in contrast to those obtained previously in older women with keratinizing squamous cell carcinoma demonstrating p53 protein overexpression in approximately 50% of patients. HPV DNA was detected in the vast majority of VIN cases
(92.8%) using a highly sensitive nested PCR method. The current data indicate
that p53 protein is not overexpressed in basaloid/warty VIN when evaluated by
immunohistochemistry. In addition, this study confirms previous reports demonstrating the presence of HPV DNA in the majority of these lesions.
CONCLUSIONS. These data suggest that p53 protein overexpression is not an early
event in the pathogenesis of basaloid/warty type vulvar dysplasia and that HPV
infection may contribute to the development of VIN. Cancer 1998;82:323–7.
q 1998 American Cancer Society.
KEYWORDS: p53, human papillomavirus (HPV), vulvar intraepithelial neoplasia, immunohistochemistry, polymerase chain reaction.
B
The authors thank G. Dudek, A. Armstrong, and
P. Haderer for technical assistance.
Address for reprints: Petra D. Kohlberger, M.D.,
Department of Gynecology and Obstetrics, University of Vienna Medical School, Waehringer
Guertel 18-20, A-1090 Vienna, Austria.
Received February 20, 1997; revision received
July 9, 1997; accepted July 9, 1997.
ased on epidemiologic and virologic evidence, vulvar carcinoma
recently has been divided into two etiologically distinct groups.1
The first group comprises the vast majority of vulvar malignancies
that occur mostly in older women. These neoplasias histologically are
keratinizing squamous cell carcinomas, and human papillomavirus
(HPV) DNA is detected less often.1,2 The pathogenesis and nature of
the precursor lesions of these carcinomas still are unclear. A recent
study indicated that squamous hyperplasia is not the predominant
precursor for HPV negative carcinoma3 because only 33% of keratinizing squamous carcinomas are associated with atypical squamous hyperplasia.
The second group comprises a minority of vulvar malignancies
occurring in younger women. These tumors usually show a basaloid
or warty histology, often are associated with adjacent vulvar intraepithelial neoplasia (VIN), and frequently are positive for HPV DNA.
These patients are at increased risk for developing multifocal malig-
q 1998 American Cancer Society
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CANCER January 15, 1998 / Volume 82 / Number 2
nancies of the lower genital tract.4,5 A study examining
risk factors for intraepithelial precursor lesions and
vulvar carcinomas found a similar risk profile for VIN
and basaloid/warty carcinomas, indicating that a common transmissible agent (HPV) is responsible for both
diseases.2 Therefore basaloid and warty-type VIN are
regarded as the precursor lesions of basaloid and warty
vulvar carcinomas.1
Degradation of p53 by E6, as well as the functional
inactivation of the retinoblastoma protein Rb by E7,
is regarded as the main mechanism by which the expression of HPV E6 and E7 oncoproteins subverts the
function of negative regulators of the cell cycle, which
are vital mechanisms to maintain the stability of the
cellular genome.6 p53 mutations appear to be the most
common genetic alterations in human cancers. In contrast, somatic p53 mutation is rare in cervical carcinoma and results in the expression of an altered p53
protein.7 – 9 Many studies demonstrate an inverse relationship between the presence of HPV DNA and p53
protein overexpression.8,10 – 12 In HPV negative tumors
a somatic mutation of the p53 gene may result in functional inactivation of p53 protein.6,7 This model, based
on the presence of mutant p53 in HPV negative tumors
and wild-type p53 inactivation in HPV positive tumors,
still is subject to some controversy, because several
authors have not observed such an inverse relationship.13 – 15
Recently we reported that p53 protein overexpression was associated with a poor survival rate in
patients with invasive squamous cell carcinomas of
the vulva.16 The objective of the current study was to
examine whether basaloid/warty type VIN overexpresses p53 protein, and if so, to examine a possible
association with recurrent disease after surgical treatment. In addition, the presence of HPV DNA was evaluated in the same lesions to determine the relationship between HPV DNA positivity and p53 overexpression.
MATERIAL AND METHODS
Paraffin embedded tissue specimens of 28 patients
with VIN were identified retrospectively and examined
for p53 protein overexpression using immunohistochemical methods, and for the presence of HPV DNA
using a nested consensus primer-based polymerase
chain reaction (PCR).
The clinical diagnosis had been confirmed histologically with a punch biopsy and was followed by
either local excision or partial vulvectomy. Tissues
were fixed in formaldehyde and processed for histologic assessment. The patients were seen in regular
intervals for a median of 22.3 months (range, 1 – 81
months) at the General Hospital, Department of Gynecology and Obstetrics, University of Vienna Medical
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School. The mean patient age at the time of diagnosis
was 44.2 years (range, 19 – 71 years).
Histologic typing was performed according to the
recommendation of the International Society of Gynecological Pathologists.1,17 Histologic diagnosis was VIN
I in 2 patients (7.1%), VIN II in 10 patients (35.7%),
and VIN III in 16 patients (57.1%). Twenty patients
(71.4%) exhibited basaloid-type VIN, which is characterized clinically by a smooth erythematous surface.
Histologically, the epithelium is comprised of cells
with typical immature parabasal cell-like morphology.
In contrast, warty-type VIN was present in 8 patients
(28.6%) with a clinically typical condylomatous appearance and, histologically, parakeratosis and hyperkeratosis with a spiked surface. However, basaloid and
warty histologic features were not mutually exclusive
for one tissue specimen, because in nearly all cases
both histologic types were observed within one lesion.
Therefore, specimens were classified according to the
dominant histologic pattern and the highest grade of
dysplasia. Immunohistochemical staining was performed as described previously.16
DNA Extraction and HPV Detection
Extraction of DNA from paraffin embedded tissue was
performed as described by Wright et al.18 Five 15-mm
sections from blocks of formalin fixed tissue specimens were deparaffinized twice in xylene followed by
2 washes with ethanol to remove the solvent. After
overnight proteinase K digestion and phenol/chloroform extraction, the DNA was ethanol precipitated and
resuspended in 50 mL of TE buffer.
For the detection of HPV 3 mL of DNA solution
were subjected to GP5//GP6/ general primer PCR for
detection of the HPV L1 gene (primers: GP5/ 5* ttt gtt
act gtg gta gat act ac; GP6/ 5* gaa aaa taa act gta aat
cat att c) as described previously.19 PCRs were performed in 50 mL reaction using standard conditions as
recommended by the manufacturer. For HPV amplification, a 4-minute denaturation step at 94 7C was
followed by 40 cycles of amplification using 1U thermostable DNA polymerase (Amplitaq; Perkin Elmer,
Oak Brook, IL) with a PCR processor (480; Perkin Elmer). Each cycle included a denaturation step at 94
7C for 1 minute, a primer annealing step at 40 7C for
2 minutes, and a chain elongation step at 72 7C for 1.5
minutes. The final elongation step was prolonged by 4
minutes. To control for cross-contamination between
samples, thin sections of breast carcinoma were processed similarly and amplified in between every fifth
VIN specimen. To ensure integrity of the DNA, samples were amplified in parallel with primers specific
for b-actin (primers: BS 5* ccc ccg tag cag cgg gcg ctt;
BAs 5* gtg ggg cac gac gac tgg ctc)20 using extremely
hot polymerase (Advanced Biotechnologies). Each cy-
W: Cancer
FIGURE 1. Basaloid vulvar intraepithelial neoplasia type III without p53 protein overexpression (original magnification 132).
cle included a denaturation step at 94 7C for 1 minute,
a primer annealing step at 50 7C for 30 seconds, and
a chain elongation step at 72 7C for 2 minutes. The
final elongation step was prolonged by 4 minutes. Only
samples positive in the b-actin PCR were included in
the analysis.
RESULTS
Overexpression of p53 protein was not detectable in
any of the VIN specimens by immunohistochemistry
(Fig. 1). Positive control slides for each staining charge
were prepared from endometrial or vulvar carcinoma
specimens and consistently showed strong nuclear reactivity (Fig. 2).
HPV DNA was detected in 26 cases (92.9%) using
a consensus primer PCR for detection of the L1 gene
encoding the viral major capsid protein. Because the
vast majority of genital HPV positive dysplastic intraepithelial lesions previously have been shown to contain high risk HPV types (mostly types 16 or 18), and
determination of these types was not the major thrust
of this study, HPV typing was not performed. After
treatment of VIN lesions with either local excision or
partial vulvectomy, two women (7.1%) developed recurrent dysplastic lesions of the vulva.
DISCUSSION
The immunohistochemical detection of mutated p53
protein is based on protein accumulation as a consequence of stabilization, whereas the expression level
of wild-type p53 protein is much lower and cannot
be detected by immunohistochemistry. p53 mutations
often are comprised of missense mutations in exons
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FIGURE 2. Overexpression of p53 protein in a squamous cell carcinoma
of the vulva (original magnification 164).
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326
CANCER January 15, 1998 / Volume 82 / Number 2
5 – 8 that lead to the synthesis of an ineffective truncated protein with a longer half-life.21 In addition, nonsense or frameshift mutations can result in the production of an unstable truncated protein, which is
negative by immunostaining. These mutations only
are etected at the DNA level using single-strand conformation polymorphism (SSCP) and/or DNA sequencing.22 In this study we chose the immunohistochemical method for detection of p53 protein overexpression due to the limited amount of available tumor
tissue because VIN lesions usually are very small. Although immunohistochemical studies have a lower
sensitivity and the evaluation is more subjective, they
provide the closest examination of the protein in situ
in a given epithelium. In tumor samples only a minority of the cells may contain a mutated p53 gene and the
wild-type sequences may dilute the mutated signal,
which then may be missed by SSCP experiments.
To our knowledge only two reports have examined
HPV infection and p53 protein overexpression in patients with VIN. Tervahauta et al reported one case of
VIN and found p53 protein overexpression concomitant with the detection of HPV DNA.23 A study of 12
cases of vulvar carcinomas with adjacent VIN lesions
found p53 protein overexpression in those VIN lesions
with adjacent p53 positive vulvar carcinoma.24 These
different results compared with the current study may
be explained by a difference in the study populations.
VIN examined by Milde-Langosch et al.24 was in close
proximity to invasive carcinoma, whereas VIN examined in the current study was not associated with invasive disease.
In the current study HPV DNA was detected in
92.8% of cases using a sensitive PCR method. Our results are in accordance with those of Hording et al,
who reported HPV infection in 90% of VIN lesions.5
This high percentage of HPV positivity may be a relative overestimate, because HPV infection can cause
multifocal genital lesions and subclinical or latent disease and thus it is likely that a small percentage of
biopsies taken from normal tissue of VIN patients or
even healthy controls would harbor HPV DNA detectable by PCR. However, these biopsies were not available for testing and therefore we can only speculate
which percentage of biopsies from normal-appearing
tissue would test positve for HPV by PCR. Nevertheless, it appears very unlikely that the percentage of
HPV DNA-positivity in normal tissue would exceed a
relatively small number compared with the 92% positivity rate found in VIN lesions.
A review of the literature concerning p53 protein
overexpression in cervical intraepithelial neoplasia
(CIN) reveals a higher percentage of p53 positivity in
invasive cervical carcinoma compared with intraepithelial lesions.14 Sparse nuclear staining for p53 was
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observed in 3 of 70 CIN lesions.25 Akasofu et al found
p53 accumulation in basal and suprabasal atypical
cells of CIN type III and in invasive carcinoma.26 Harmsel et al reported a low level of p53 protein in CIN
and high levels in carcinoma of the cervix.27 In contrast
to these studies, Jeffers et al also reported a positive
immunohistochemical staining reaction for p53 in
nonneoplastic cervical squamous epithelium and in
21 – 59% of CIN types I – III.28 We suspect that these
results indicate a positive immunohistochemical
staining for wild-type p53. Other possible causes for
the detection of wild-type p53 with immunohistochemical methods are inflammation prolonging p53
half-life time or DNA damage-induced up-regulation
of p53.29,30
We previously have shown that p53 protein is
overexpressed in keratinizing squamous cell vulvar
carcinoma and that detection of p53 is associated with
worse prognosis.16 This study demonstrates that p53
is not detected in preinvasive basaloid/warty lesions
of the vulva, supporting the concept that p53 protein
overexpression is not an early event in the pathogenesis of warty/basaloid type VIN. The high percentage
of HPV DNA positivity in VIN lesions suggests that
persistent HPV infection may contribute to the pathogenesis of vulvar intraepithelial dysplasia.
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