Gynecologic Oncology 95 (2004) 336 – 340

www.elsevier.com/locate/ygyno

HER-2/neu expression in Paget disease of the vulva and the female breast
O. Brummera,*, H.-E. Stegnerb, G. Bfhmera, H. Kqhnlea, K.-U. Petrya
a
Department of Gynecologic Oncology, Medizinische Hochschule Hannover, 22763, Germany
b
Department of Gynecologic Histopathology, Universitätsklinik Hamburg Eppendorf, 22763 Hamburg, Germany
Received 30 January 2004

Abstract

Objectives. Paget disease of the vulva is a rare lesion that accounts for b1% of vulva neoplasms. A 12% prevalence of invasive Paget
carcinoma and a 4% prevalence of associated adenocarcinomas are described. Furthermore, a high recurrence rate of 30% after surgical
therapy is observed. This study aims to search for therapeutic strategies for recurrent Paget disease, which are less mutilating and less
aggressive than reexcision, x-ray therapy, or chemotherapy. Trastuzumab (Herceptink) is a recombinant monoclonal antibody against HER-
2/neu, approved by the U.S. FDA for the treatment of patients with HER-2/neu-positive metastatic breast carcinomas. The results of recent
studies indicate that HER-2/neu oncoprotein may play a role in the pathogenesis of extramammary Paget disease.
Methods. Using HercepTestk, we analyzed HER-2/neu overexpression in seven noninvasive Paget lesions, two invasive lesions, and one
Paget disease of the vulva with underlying adenocarcinoma. In addition, we investigated five mammary Paget diseases.
Results. Overexpression of HER-2/neu oncoprotein labeling exclusively the membranes of Paget cells was demonstrated in 8 out of 10
cases. One noninvasive and one with underlying adenocarcinoma stained negatively. Overexpression of HER-2/neu was demonstrated in all
five cases of mammary Paget disease.
Conclusion. Using HercepTestk as a standardized detection system, overexpression of HER-2/neu can be demonstrated in a majority of
both noninvasive and invasive Paget disease of the vulva. The use of Trastuzumab should be considered for the treatment of patients with
recurrent Paget disease of the vulva with overexpression of HER-2/neu.
D 2004 Elsevier Inc. All rights reserved.

Introduction development, the cells of the stratum germinativum give rise

Paget disease of the vulva is a rare lesion that accounts for
b1% of vulva neoplasms [1]. It is a manifestation of
extramammary Paget disease. Mammary Paget disease was
originally described as a breast lesion with associated
underlying invasive ductal adenocarcinoma. According to
the location, it is classified as mammary or extramammary
disease [2]. Although controversy still exists as to the true
nature of vulvar Paget disease, the majority of lesions appear
to be an intraepithelial form of malignancy of apocrine origin
involving the skin and its appendages. Paget disease of the
vulva is an epidermal neoplasm. During embryological
* Corresponding author. Allgemeines Krankenhaus Hamburg Altona,
Gyn7kologie und Geburtshilfe, Paul-Ehrlich-Str. 1, 22763 Hamburg,
Germany. Fax: +49 40 8822 4707.
E-mail address: oliver.brummer@gmx.de (O. Brummer).
0090-8258/$ - see front matter D 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.ygyno.2004.07.043
to the skin appendages including the apocrine glands and the
overlying mature squamous epithelium [3]. Also, the
mammary gland is an apocrine differentiated skin append-
age. The appearance of apocrine differentiated Paget cells as
a neoplasm of the nipple as well as a disease of the vulva
evidences this embryological kinship. A 12% prevalence of
invasive vulva Paget disease and a 4% prevalence of
associated vulva adenocarcinoma are described [1]. There
is also an association with other malignancy in 20% of the
cases [2]. After surgical treatment, which appears to be the
established primary therapy, 30% of the patients experience
local recurrences within 8 years [1,4]. In excising the lesion,
it is important to recognize that the lesion frequently extends
beyond the clinically apparent borders. Due to the high
recurrence rate, different therapeutic options like reexcision,
chemotherapy (5-FU), and x-ray therapy have been dis-
cussed. Since these are burdening therapies for a noninvasive
 O. Brummer et al. / Gynecologic Oncology 95 (2004) 336–340 337

disease, it is crucial to search for therapeutic strategies with
higher tolerability.
HER-2/neu, also known as c-erbB-2, is a transmembrane
receptor with overexpression in 25–30% of primary breast
carcinomas [5,6]. HER-2/neu receptor protein has tyrosine
kinase activity [7,8]. Prior studies demonstrate a 95%
positivity for HER-2/neu in Paget disease of the nipple with
underlying intraductal and invasive carcinomas [9]. It has
been demonstrated that HER-2/neu overexpression in breast
carcinomas is associated with poor patient prognosis and
aggressive disease potential [6,10–13]. Similar results could
be found in the majority of extramammary vulva Paget
disease [14–17].
Trastuzumab (Herceptink, Gentech, South San Fran-
cisco L.A.) is a recombinant monoclonal antibody against
HER-2/neu for the treatment of patients with metastatic
breast carcinoma, with overexpression of HER-2/neu. Initial
clinical trials with Trastuzumab suggest a prolongation of
survival for patients with advanced metastatic breast cancer
[18]. The U.S. FDA approved the usefulness of a stand-
ardized diagnostic kit for detection of HER-2/neu over-
expression. This test is an immunohistochemical staining
system for semiquantitative evaluation of HER-2/neu
expression. The standardized results of the HercepTestk
correspond with the therapeutic effect of Trastuzumab
against breast cancer [19,20].
It is the objective of this study to evaluate the HER-2/neu
expression status in Paget disease of the vulva using the
HercepTestk as a standardized detection system. For a
possible new indication for Herceptink treatment, it is
crucial to demonstrate a HER-2/neu overexpression in Paget
disease of the vulva.
protein expression. The sections were deparaffinized in
xylene and rehydrated through graded ethanols to distilled
water. The sections were immersed in 958C preheated Dako
Epitope Retrieval Solution (10 mM citrate buffer, pH 6).
Following this procedure, the slides were heated at 958C for
40 min. After a 20-min cool-down period at room temper-
ature, the sections were washed with Dako Wash Buffer, a
procedure that followed every subsequent incubation.
Endogenous peroxidase was blocked with Dako Blocking
Buffer for 5 min at room temperature. The slides were
incubated with primary polyclonal rabbit antihuman HER-2/
neu antibody, supplied in the kit, for 30 min at room
temperature. Bound primary antibody was labeled by
incubating the slides with Dako Visualization Reagent
(horseradish peroxidase-labeled dextran polymer conjugated
to affinity-purified goat antirabbit immunoglobulins in Tris-
HCl) for 30 min. Color development was achieved with 3,3-
diaminobenzidine (DAB) for 10 min. The sections were
counterstained with hematoxylin. To confirm validation of
the staining, control cell slides, provided in the kit and
consisting of three pelleted, formalin-fixed, paraffin-embed-
ded human breast cell lines with known HER-2/neu positivity
(MDA-231: 0; MDA-175: 1+; SK-BR-3: 3+), were stained
simultaneously. The negative controls underwent a similar
staining procedure with the exclusion of primary antibody.

Materials and methods

Paraffin blocks from 15 cases (n = 15) with Paget disease

were chosen from the archive of the Department of
Pathology/University of Hanover.
Seven patients were diagnosed by the histopathologist
with noninvasive and two patients with invasive Paget
disease of the vulva. One patient showed noninvasive Paget
disease of the vulva with underlying adenocarcinoma. It is a
small group but a rare disease. These 10 cases represent all
patients with this diagnosis in our Colposcopy Clinic from
1996 to 2002.
Five patients were chosen with Paget disease of the nipple,
three of them with ductal carcinoma in situ (DCIS) and two of
them with invasive carcinoma. Sections of 5-Am thickness
were cut and transferred onto slides, previously coated with
aminopropyltriethoxysilane (APES) (Sigma, Gillingham,
UK). The 15 patients were diagnosed, treated, and observed
by the Department of Gynecologic Oncology, University of
Hanover.
HercepTestk was performed in accordance with the Fig. 1. Noninvasive Paget disease of the vulva, HE staining. Paget cells in
manufacturer’s guidelines for evaluation of HER-2/neu the basal layers. Magnification 240.
338 O. Brummer et al. / Gynecologic Oncology 95 (2004) 336–340

Fig. 3. Paget disease of the vulva with underlying adenocarcinoma with

negative HercepTest immunostaining in the area of adenocarcinoma.
Magnification 240.

Following the U.S. FDA-approved scoring guidelines for

Fig. 2. HercepTest immunostaining of noninvasive Paget disease of the
vulva. 3+ Staining exclusively in the membranes of Paget cells, whereas
normal epithelial is negative. Same case and same area as Fig. 1.
Magnification 240.
breast carcinomas, only membrane staining intensity and
pattern were evaluated using the 0 to 3+ scale as illustrated
in the HercepTestk kit scoring guidelines (0 for no staining
at all or membrane staining in less than 10% of the tumor
cells; 1+ for only partial, weak staining of the cell
membrane of more than 10% of the tumor cells; 2+ for
moderate staining of the complete cell membrane in more
than 10% of the tumor cells; 3+ for intense staining of the
complete membrane in more than 10% of the tumor cells).
In accordance with the HercepTestk kit guide, HER-2/neu
overexpression was assessed as negative for scores of 0 and
1+ and positive for scores of 2+ and 3+.

This antibody was replaced by a normal rabbit serum (Dako Results

Negative Control Reagent). The antibody used in Hercep-
Testk did not exhibit cross-reactivity to HER-3 and HER-4 Noninvasive Paget disease of the vulva
in Western blot analysis (manufacturer’s instructions).
Using HercepTestk and the U.S. FDA-approved
Table 1
scoring system, overexpression of HER-2/neu was dem-
HER-2/neu expression in Paget disease of the vulva and the female breast onstrated in six out of seven cases: five cases with 3+, one
Patient Diagnosis HercepTestk score
1 IEP 3+
2 IEP 3+
3 IEP 3+
4 IEP 3+
5 IEP 3+
6 IEP 2+
7 IEP 1+
8 IP 3+
9 IP 3+
10 PA 1+/
11 PDN 3+
12 PDN 3+
13 PDN + DCIS 3+
14 PDN + DCIS + IC 3+
15 PDN + DCIS + IC 2+
Abbreviations: IEP, intraepithelial Paget disease; IP, invasive Paget disease;
PA, Paget disease and adenocarcinoma; PDN, Paget disease of the nipple; Fig. 4. Paget disease of the nipple with underlying DCIS, HE staining.
DCIS, ductal carcinoma in situ; IC, invasive breast carcinoma. Magnification 120.
 O. Brummer et al. / Gynecologic Oncology 95 (2004) 336–340
Fig. 5. HercepTest, Paget disease of the nipple with underlying DCIS,
strong immunostaining (3+) in DCIS. Same case and same area as Fig. 4.
Magnification 120.
case with 2+. Staining was visible exclusively in the
membranes of Paget cells, whereas normal epithelium was
negative (Figs. 1 and 2). Case number 7 showed a 1+
score (Table 1).
Invasive Paget carcinoma
Two cases of invasive Paget carcinoma showed clear
membrane signals, scoring 3+ in all Paget cells (invasive as
well as intraepithelial tumor areas). Normal epithelium
stained negatively.
Paget disease of the vulva and underlying adenocarcinoma
The adenocarcinoma stained negatively for HER-2/neu
(Fig. 3). The tumor areas with intraepithelial Paget cells
were rare in the investigated sections and stained 1+.
Paget disease of the nipple
Three cases with underlying DCIS showed intensive
staining with 3+ in Paget disease as well as in DCIS (Figs. 4
and 5). One case, with underlying invasive carcinoma,
stained 3+, the other one stained 2+ in Paget disease and
invasive carcinoma.
Discussion
At the time of initial presentation, most patients with
Paget disease of the vulva are misdiagnosed, because
biopsies are usually not performed [21,22]. Application of
steroid creams and antifungal or antibiotic agents are usually
the first therapies. The average interval from symptom onset
to histological diagnosis is 3 years [21–23]. Because of the
intraepithelial nature of the disease, tumor markers such as
squamous cell carcinoma (SCC) antigene, Ca 19-9, or
carcinoembryogenic antigene (CEA), are not helpful [24].
339
The extent of histologically demonstrated disease is far
greater than the visible lesion because the lesion tends to be
multicentric and diffuse in origin [23,25]. Several authors
have shown that positive margins after surgery do not
correlate with recurrent disease [4,23,25,26]. The consensus
regarding treatment is that wide local excision is adequate for
lesions without invasive growth. Woodruff [27] recommen-
ded that excision of the underlying tissue should be deep
enough to include the skin appendages to rule out invasive
growth in the subepithelial glands. The high recurrence rate
of about 30%, even after extensive local excision, represents
a therapeutic dilemma. The adequate management of patients
with extensive lesions or recurrent disease is still far from
being fully resolved. Morbidity from repeated surgery,
especially in elderly women, can be high. Only a few reports
discuss the use of radiotherapy [28,29]. A dose higher than
50 Gy is recommended, with the well-known side effects of
vulvar radiotherapy [30]. There are also some studies
reporting on chemoradiotherapy for invasive and noninva-
sive Paget disease [31–33]. HER-2/neu overexpression has
been demonstrated in mammary Paget disease in several
studies [9,34], but rarely investigated in Paget disease of the
vulva. HER-2/neu oncoprotein expression may play a role in
promoting the intraepithelial spread of adenocarcinoma cells
[17].
Trastuzumab-based combination therapy has been pro-
ven effective of the treatment for patients with metastatic
breast carcinomas with overexpression of HER-2/neu. The
U.S. FDA has approved HercepTest as a standardized
diagnostic kit to detect HER-2/neu overexpression. HER-2/
neu targeted immunotherapy in our department requires
either HER-2/neu protein overexpression in case of 3+
staining or HER-2/neu gene amplification by FISH analysis
in case of 2+ staining. The therapeutic effect of Trastuzumab
against breast carcinoma is reflected by the results obtained
with HercepTest.
The present study evaluates HER-2/neu overexpression
status in Paget disease of the vulva. Overexpression in eight
out of ten cases (80%) could be demonstrated. Two of them
were invasive Paget carcinomas. One case of noninvasive
Paget disease and one case of underlying adenocarcinoma
stained negatively. Five investigated cases of mammary
Paget disease showed strong expression. Staining, exclu-
sively in Paget cells, was observed in all cases. In
conclusion, based on the experience in HER-2/neu-positive
metastatic breast carcinoma, Trastuzumab therapy should be
considered as a possible new therapeutic strategy in selected
cases of extensive or recurrent Paget disease of the vulva
with overexpression of HER-2/neu.
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