ABSTRACT
Mammary hyperplasia is characterized by an increase in the whole
mammary gland, considered benign and with a good prognosis. A
two-year-old dog showed a volume increase in the entire mammary chain
after hormone stimulation. To characterize the process prognosis, a
immunohistochemical procedure was used to evaluate the Maspin staining,
considered good prognosis marker, and the Ki-67, a proliferation
marker, whose high expression indicates malignancy. As a result, we
obtained a low expression of Ki-67 and a high expression of Maspin,
suggesting an excellent prognosis for the animal.
Keywords: Female dog, imunohistochemistry, neoplasia, prognosis.
RESUMO
IMUNOEXPRESSÃO DE MASPIN E KI-67 COMO
MARCADORES PROGNÓSTICOS DA CARACTERIZAÇÃO BENIGNA DA HIPERPLASIA
FIBROEPITELIAL MAMÁRIA CANINA
A hiperplasia mamária é caracterizada por um aumento de toda a glândula
mamária, considerada benigna e de bom prognóstico. Uma cadela de
dois anos apresentou aumento de volume de toda a cadeia mamária após
estímulo hormonal. Para caracterizar o prognóstico do processo foi
utilizada a técnica de imunohistoquímica com avaliação da marcação do
Maspin, considerado um marcador de bom prognóstico e o Ki-67, marcador
de proliferação celular e que, portanto, mostraria alta expressão na
malignidade. Como resultado obteve-se uma baixa expressão do Ki-67 e
uma alta expressão do Maspin, sugerindo um excelente prognóstico para o
animal.
Palavras-chave: Cadela, imunohistoquímica, neoplasia, prognóstico.
INTRODUCTION
The mammary gland hyperplasia (fibroepithelial hyperplasia) is
characterized by fast and abnormal growth of the mammary gland, and it
is considered a benign non-neoplastic proliferation (NELSON &
COUTO, 1998).
This alteration usually occurs in female cats, under 2 years old, 1 or
2 weeks after the oestrus or at the beginning of the pregnancy. It
occurs when they are exposed to (endogenous or exogenous) progesterone
for a long time. Therefore, old female cats also develop this condition
when exposed to megestrol acetate (WITHROW & MacEWEN, 1989) or in
recurrence of pseudo pregnancy cases (LORETTI
et al.,
2005). It has also been described in spayed male cats or in those that
are not exposed to progesterone. There is sufficient evidence to
conclude that this is a hormone-dependent condition. Nevertheless, it
has already been described in ovariohysterectomized female cats (NELSON
& COUTO, 1998; SOUZA
et al., 2002; VASCONCELLOS, 2003). This disorder is very uncommon in dogs (SOUZA
et al., 2002).
Immunohistochemistry has been used as a diagnostic method for
prognostic markers of mammary gland cancer in dogs (THOMAS &
BERNER, 2000; KANDIOLER-ECKERSBERGER
et al., 2000; KIM
et al.,
2010). However, there is no report relative to the use of Ki-67 and
Maspin as immunohistochemical markers to characterize fibroepithelial
hyperplasia of the mammary gland of dogs, renal cell carcinoma (TURUNC
et al., 2010), nasopharyngeal carcinoma (FENG
et al., 2010), lung cancer (NAM & PARK, 2010), and cell proliferation (CHANO
et al.,
2010). The aim of the present study is to use the immunohistochemical
markers – Maspin and Ki-67 – as possible molecular markers in the
prognosis of fibroepithelial hyperplasia of mammary glands in female
dogs.
MATERIALS AND METHODS
A 2-year-old female Dachshund dog was evaluated. Two months prior to
examination, the bitch was reported to have developed an enlargement of
all mammary glands and had a diagnosis of pseudo pregnancy (
Figure 1A).
The dog was then medicated with progesterone (unknown dosage), which
resulted in enlargement of the mammary glands. The skin at these areas
was very irritated, there was edema at the lower extremities, and she
had gained over 4 kg of weight. The bitch was brought to the veterinary
care service and submitted to ovariohysterectomy with regression of
more than a half of the glands after 60 days. The biopsy exam confirmed
mammary hyperplasia (
Figure 2A)
and the dog was also prescribed metergolina, at doses of 0.1 mg / kg
orally BID for 7 days, returning to normal within 3 months (
Figure 1B).
Anti-Maspin and anti-Ki67 antibodies were used to predict the clinical
outcome and were considered prognostic markers in mammary neoplasia.
The immunohistochemical procedure had the blockage of the endogenous
peroxidase carried out with hydrogen peroxide (10%), and its antigenic
retrieval was performed in a steam pan with a citrate buffer solution
(pH = 7.0). The incubation was performed with primary antibody
(diluited in BSA+PBS) in an overnight process at – 4ºC with anti-Maspin
(Novocastra™) 1:50; the antibody anti-Ki-67 (MIB-11 Dako, 1:50). After
the incubation with a secondary antibody (Solution A-Kit Dako LSAB+,
Universal- Peroxidase), the slides were incubated with
Streptavidina/Peroxidase complex (Solution B-Kit Dako LSAB+,
Universal-Peroxidase) for 30 minutes. The development was performed
with a DAB chromogenic substrate (chromogen 20 mG + 1 mL Buffered,
Dako™) and counterstained with Harris Hematoxylin (
Figure 2B and 2C). A negative control was utilized deprived of primary antibody (
Figure 2)
RESULTS AND DISCUSSION
The present study highlights the perspective of new molecular markers
that can be used for the differential diagnosis of neoplasia and benign
diseases, such as this unusual hyperplasia case of the mammary gland in
dog. The study warns for the aggravation of hyperplasia with the use of
progesterone and shows that the ovariohisterectomy was effective in
controlling the size of the mammary gland.
The use of molecular markers by the immunohistochemistry has been
described in the literature as predictive of the diagnosis and
prognosis of neoplasias (KANDIOLER-ECKERSBERGER
et al.,
2000). The present study used these markers in a benign disease and
showed that their expressions were consistent with their proposal. The
outcomes showed a strong expression of the anti-Maspin antibody
(Erviegas) and a weak staining of the anti-Ki-67 antibody (Dako), which
was normally expected in the benign processes corroborating a good
prognostic for the female dog. According to the literature (ZUCCARI
et al.,
2004), Ki-67 is a good cell proliferation marker; thus, the prognosis
is considered good when its expression is low. On the other hand, it
has been considered that Maspin, as a cell adhesion molecule, has a
strong expression when the adhesion is preserved, a characteristic of
benign tumors. Experiments have demonstrated an inverse correlation
between loss of Maspin expression and increased malignancy of mammary
gland tumor and the worst disease outcome (MAASS
et al., 2001; STARK
et al., 2010; PRASAD
et al.,
2010). Studies have indicated that Ki-67 is a marker to characterize
malignant tumors due to intensive cell proliferation in mammary gland
tumors (ZUCCARI
et al.,
2004). In this report we can see an agreement with the literature in
relation to these markers in accordance with its expression by
immunohistochemistry.
CONCLUSION
During the present study, the low expression of Ki-67 and the strong
expression of Maspin suggest a benign process, indicating their
possible use to confirm it as a benign mammary disease. Because it was
favorable to this case, a greater number of real tests are necessary
for the confirmation of the potential markers ki-67 and Maspin.
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Submetido
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