Shafiee et al. Cancer Cell International 2013, 13:79
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PRIMARY RESEARCH
Open Access
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Diagnosis, classification and grading of canine
mammary tumours as a model to study human
breast cancer: an Clinico-Cytohistopathological
study with environmental factors influencing
public health and medicine
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Radmehr Shafiee1, Javad Javanbakht2*, Nahid Atyabi2, Pegah Kheradmand3, Danial Kheradmand4,
Alimohammad Bahrami5, Hasti Daraei6 and Farshid Khadivar1
Abstract
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Background: The human “Elston and Ellis grading method” was utilized in dogs with mammary tumor to examine
its relation to prognosis in this species, based on a 2-year follow-up period. Although cytopathology is widely used
for early diagnosis of human neoplasms, it is not commonly performed in veterinary medicine. Our objectives in
this study were to identify cytopathology criteria of malignancy for canine mammary tumors and the frequency of
different types of mammary lesions and their relationship with histologic grade was investigated. Another aim of
this study was to differentiate the simple and adenocarcinoma tumors from the complex or mixed tumor described
by Elston and Ellis grading method.
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Methods: The study was performed in 15 pure or mixed-breed female dogs submitted to surgical resections of
mammary tumours. The mammary tumours were excised by simple mastectomy or regional mastectomy, with or
without the superficial inguinal lymph nodes. Female dogs were mainly terriers (9 dogs) or mixed (3 dogs), the 3
other animals were a German shepherd, Dachshund and Pekingese. Before surgical excision of the tumour, FNAC
was performed using a 0.6 mm diameter needle attached to a 10 ml syringe held in a standard metal syringe
holder. The cytological sample was smeared onto a glass slide and either air-dried for May-Grünwald-stain, or
ethanol-fixed for Papanicolaou stain and masses were surgically removed, the tumours were grossly examined and
tissue samples were fixed in 10%-buffered-formalin and embedded in paraffin. Sections 4 μm thick were obtained
from each sample and H&E stained.
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* Correspondence: [email protected]
2
Department of Pathology, Faculty of Veterinary Medicine, Tehran University,
Tehran, Iran
Full list of author information is available at the end of the article
© 2013 shafiee et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
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Results: We obtained a correct cytohistological correlation in 14/15 cases (93.3%) when all cytopathological
examinations were considered. Of the 15 cases examined, 2(13.3%) had well-differentiated (grade I), 6(40%) had
moderately differentiated (grade II) and 7(46.7%) had poorly differentiated (grade III) tumours. Classification of all
canine mammary gland lesions revealed 13(86.7%) malignant and 2(13.3%) benign tumors. The histological
examination showed that the most common tumor types of mammary glands in bitches were: complex carcinoma,
adenocarcinoma, malignant mixed tumour, benign mixed tumour, simple carcinoma– (5/15; 33.3%), (3/15; 20%),
(3/15; 20%) and (2/15;13.3%), respectively. Simple carcinoma and cystic hyperplasia were less common - (1/15; 6.7%),
and (1/15; 6.7%), respectively. Moreover, the most often tumors occur in inguinal mammary (60%) and abdominal
(27%) glands.
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Conclusions: Our results demonstrate that, because of the similarity of the cytohistopathological findings in the
human and canine mammary gland tumours, it is possible to use the same cytopathological criteria applied in
human pathology for the diagnosis of canine mammary gland tumours. Furthemoer, routine use of this human
grading method would help the clinician to make a more accurate prognosis in the interests of post-surgical
management in dogs with mammary carcinomas. Furthermore, this research will allow a more discriminating
classification of mammary tumors and probably has a bearing on cytohistopathology, epidemiology, pathogenesis
and prognosis. The most often tumors occur in inguinal mammary (60%) and abdominal (27%) glands. This
interesting regional difference may be due to a) the duration of the growth before the diagnosis; b) the age of the
dogs; and c) high prevelance of unspayed animals. Moreover, the most common type of tumor was complex
carcinoma – 33.3% (5 cases).
Keywords: Grading of tumours, Cytohistopathology, Dog, Mammary glands, Tumour
diagnosis and prognosis. Radiographic screening, surgical biopsy or aspiration biopsy are beneficial tools for
early diagnosis of tumours. Although the first attempt is
to make a differential diagnosis with inflammation or
hyperplasia these tools should be used to understand the
biological behavior of the mass that also contribute to
the prognosis [12].
Cytological examination has important benefits in clarifying some aspects in early diagnosis of mammary lesions.
This procedure is commonly used in palpable lesions such
as mammary glands, thyroid, lymph nodes and salivary
glands. It also prevents the need for a surgical attempt
and complications that might occur during surgery [13].
Fine needle aspiration cytology (FNAC) is a method
largely used to obtain samples for cytological diagnosis in
several organs. When applied to mammary gland lesions,
the method shows good diagnostic accuracy. Moreover, it
is a simple, rapid and low cost method, with minimal risk
for the patient [11-13]. Paget (1854) [14] was the first to
use samples of aspirated mammary tumours for microscopic examination. In veterinary medicine, the use of cytology as a diagnostic method is very recent and has been
growing in the last 20 years [7].
Our objectives in this study were to identify cytopathology criteria of malignancy for canine mammary tumors and the frequency of different types of mammary
lesions and their relationship with histologic grade was
investigated. Another aim was to differentiate the simple
tumor from the complex or mixed tumor described by
Elston and Ellis grading method.
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Background
Mammary gland tumours are one of the most common
neoplasms in female dogs [1]. These spontaneous tumors are very similar to those in humans, but the incidence rate among bitches is 3 times larger than in
women [2], and there are some molecular and biological
similarities between canine and human mammary tumours [1-4]. Malignant cases occur in approximately
half of canine mammary tumours (CMTs) [5,6], with a
3-fold higher incidence when compared with breast tumours in human females.2 Similar to their human counterparts, these tumours occur almost exclusively in
females, and only rarely in male dogs [2]. Generally, in
canine malignant mammary tumours (CMMT), metastatic spread occurs via lymphatic vessels to the regional
lymph nodes or haematogenously and the lungs are the
most frequent site of distant metastases [7].
A number of new methods are therefore tested within
tumor pathology to improve the diagnostic and prognostic
accuracy. To make a diagnosis, a number of clinical and
paraclinical tests are used because it is essential to find the
most aggressive malignant tumors that need radical surgery
and supplementary treatment as soon as possible. After excision, a hematoxylin-eosin stain is often used to provide a
histopathological diagnosis, but even though this is
performed by experienced pathologists, the disorderly structure of some mammary tumors may cause confusion and
may lead to an erroneous diagnosis of malignancy [8-11].
Anamnesis and physical examination coupled with
epidemiological findings have an important role in the
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Results
In the other malignant tumours, some nuclear anomalies were identified such as double nucleus in 8 samples
(53%) of malignant tumours (Figure 3B, 3C, 3E and 3F),
giant nucleus in 8 samples (53%), mitotic figures in 7
samples (46%) and abnormal chromatin structures in 4
samples (26%). In 5 cases spindle shaped cells were associated with tumour cells (33%) (Figure 3E and 3G). In
the 2 benign tumours (dog n°3 and 9), the mammary
gland structure remained uniform (Figure 3H).
Histopathology findings
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All the tumor samples were divided into six histopathologic groups: hyperplasia (one cases), adenocarcinoma (3
cases), complex carcinoma (5), simple carcinoma (1), benign mixed tumor (2 cases), and malignant mixed tumor
(3cases) (Figure 4A), adenocarcinomas was further divided into papillary (one adenocarcinomas), and solid
(two adenocarcinomas) types. According to their maximum diameter, the tumours were classified as T1 in 8/
15 (53.3%), T2 in 3/15 (20%) and T3 in 4/15 (26.7%)
dogs. The most frequently represented tumour type was
complex carcinoma (5/15; 33.3%), followed by adenocarcinoma (3/15; 20%), malignant mixed tumor (3/15; 20%),
benign mixed tumor (2/15;13.3% cases), simple carcinoma (1/15; 6.7%)and cystic hyperplasia (1/15; 6.7%), as
presented in Table 1 (Figure 2).
The histological grades of the 15 cases were as follows:
grade I, 2(13.3%); grade II, 6(40%); grade III, 7(46.7%)
with high mitotic index. The relationship between
tumour grading and histological type is presented in
Table 2. Of the 15 dogs in which mammary examination
was performed, 2 had well-differentiated grade I
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The macroscopic specifications of the multifarious mammary masses were summarized in the Figure 1 and Table 1.
The preferential localisation of mammary neoplasms were
the inguinal lobes (60% of cases), abdominal lobes (%27)
and thoracic lobes (%13). Furthermore, 45% of the inguinal
masses affected the cranio-inguinal lobe, %33 caudoabdominal lobe and %22 cranio and caudo abdominal
lobes, whereas thoracic masses were found in two cases
and abdominal masses in four of cases. Eventually, % 67 of
tumour masses were found in the left mammary lobes and
%33 in the right mammary lobes. Eighty % of mammary
tumours exhibited a small size, with weight inferior to 50 g
but a relatively high proportion of these masses (20%)
weighted more than 100 g, even reaching 110 g and 180 g
(cases n° 13 and 17 respectively). The majority of the tumours showed a hard or an elastic consistency but some of
them appeared fluctuant (cases n°3, 6 and 8). In the great
majority of cases (93%), the aspect of the tumour on the
cut surface was grayish-white and lobed. Some cystic structures or blood spot districts areas were also often found (in
40% and 20% of cases, respectively) (Figure 1).
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Macroscopic details and findings
Cytology findings
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All the tumor masses were divided into four cytologic
groups: hyperplasia (one case), adenocarcinoma (2 cases),
carcinoma (9cases), benign secretory (3 cases) (Figure 2).
In all cases with malign characteristics (malign (dogs n
°2,4,5,6,8,10,11,12,13 and 15), and malign suspected
masses(dogs n°1,7 and 14)), clusters of cells with
anisocytosis, anisokaryosis and hyperchromasia were
observed (Figure 3A).
Figure 1 The relative risk of neoplasms of the mammary gland in female dogs in individual age and tumor size categories.
Shafiee et al. Cancer Cell International 2013, 13:79
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Table 1 Cytological and histopathological analysis of pre-operative and sampled during surgery from the mammary
neoplasms of the 15 females dogs together with signalment of bitches included in the this study
The number of cases of mammary Tumour localisation
tumours in 15 female dogs
Cytological
type
Cytological
classification
Histopathological
type
Histopathological
classification
Right cranio and caudo inguinal carcinoma
lobes / left caudo-inguinal lobe
Malignant
Complex
carcinoma
Malignant
2
Left thoracic lobe
carcinoma
Malignant
Malignant mixed
tumour
Malignant
3
Left cranio abdominal lobe
Benign
secretory
Benign
Benign mixed
tumour
Benign
4
Left cranio and caudo inguinal
lobes
carcinoma
Malignant
Complex
carcinoma
Malignant
5
Right cranio inguinal lobe
carcinoma
Malignant
Complex
carcinoma
Malignant
6
Left caudo inguinal lobe
carcinoma
Malignant
7
Left cranio inguinal lobe
adenocarcinoma Malignant
8
Left cranio and caudo inguinal
lobe and right caudo-inguinal
lobe
carcinoma
9
Right caudo inguinal lobe
Benign
secretory
10
Left cranio inguinal lobe
11
Left cranio and caudo
abdominal lobes
12
Right cranio inguinal lobe
13
Simple carcinoma
Malignant
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1
Malignant mixed
tumour
Malignant
Complex
carcinoma
Malignant
Benign mixed
tumour
Benign
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Malignant
A
Benign
Malignant
Complex
carcinoma
Malignant
carcinoma
Malignant
Solid
adenocarcinoma
Malignant
hyperplasia
Benign
Cystic hyperplasia
Malignant
Left cranio inguinal lobe
carcinoma
Malignant
Malignant mixed
tumour
Malignant
14
Right cranio-abdominal lobe
adenocarcinoma Malignant
Papillary
adenocarcinoma
Malignant
15
Left thoracic lobe
adenocarcinoma Malignant
Solid
adenocarcinoma
Malignant
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carcinoma
Figure 2 Cytological analysis of pre-operative fine needle aspirates and histopathological analysis of mammary tumour masses
sampled during surgery in the 15 females dogs.
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Figure 3 Evaluation of accuracy of fine needle aspiration cytology for diagnosis of canine mammary tumours. A: Fine-needle aspirate
from a mammary carcinoma in adog. Variation in cell (anisocytosis) and nuclear. (anisokaryosis) size are present, May-Grunwald-Giemsa staining
method, 1000X. B: Adenocarcinoma: Malignant mammary epithelial cells: Fine needle aspirate with hypercellular pleomorphic, large
hyperchromatic naked cells with coarse and abundant chromatin granules and vacuolar changes, May-Grunwald-Giemsa staining method, 1000X.
C: Malignant multinucleated mammary epithelial cell; nuclei exhibit nuclear criteria of malignancy; nuclei superimposed and in different focal
planes. May-Grunwald-Giemsa staining method, 1000X. D: Cytological appearance of spindle shape cells, May-Grunwald-Giemsa staining method,
400X. E: Myoepithelial cells (spindle shape) with abundant chromatin granules (red arrows) in adenocarcinoma, May-Grunwald-Giemsa staining
method, 1000X. F and G: This cluster of cells shows multinucleated cells containing several irregularly sized nucleiare found in some cases, MayGrunwald-Giemsa staining method, 1000X. H: Fine-needle aspiration biopsy. Benign mammary tumor epithelial cell cluster. Cytological
appearance of uniform epithelial cells, May-Grunwald-Giemsa staining method, 400X.
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tumours (Cystic hyperplasia and benign mixed tumour),
6 had moderately differentiated grade II (1 papillary
adenocarcinoma, 3 complex carcinoma, 1 benign mixed
tumour and 1 malignant mixed tumor) and 7 had poorly
differentiated grade III (1 solid adenocarcinoma, 1 papillary adenocarcinoma, 1 simple carcinoma, 2 complex
carcinoma and 2 malignant mixed tumor) (Table 2).
Of the 15 canine mammary cancers (CMTs) included,
13 of the 15 cases exhibited a range of morphologies, a
highly pleomorphic cell population and polygonal were a
prominent feature of all neoplasms, accounting for
greater than 86.7%of the tumour cell population in most
cases. Also, in the group of CMTs, 46.7% (7/15) of cases
were composed of highly cellular areas with a homogeneous population of spindle cells (Figure 4B, 4C and
4D). On the other hand, a high mitotic rate (More than
three mitotic figures per high-power field (400×) was
identified in 73.37% (11/15cases), with atypical mitoses
conspicuous in all tumours (Figure 3E and 4E). In
addition, 11 of the 15 (73.37%) CMTs cases showed necrotic foci and10 of the 15 (66. 7%) CMTs cases showed
infiltrates of various numbers inflammatory foci primarily consisting of lymphocytes, plasma cells, and neutrophils cells (Figure 4G).
Furthermore, in the group of CMTs, 66.7% (10/15) of
cases exhibited haemorrhage localized in the different
regions of the tumor tissue (Figure 4L). Most CMTs this
study increased mitotic activity, cellularity, nuclear pleomorphism and the presence of lesional necrosis are ominous features and suggest an increased risk of local
recurrence (Figure 4I). According to local invasiveness,
33.3% of the tumours (5 out of 15) were found (Figure 3K
and 4J). In addition, the neoplastic cells within the blood
vessels were observed as well (Figure 4I). Moreover, 7
of the 15(46.7%) CMTs cases revealed that these tissue
sections were comprised of cancerous epithelial cells
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Figure 4 Histopathological evaluation of mammary gland tumours in dogs. A: Solid adenocarcinoma; The presence of neoplastic, mitotic
and inflammatory cells (H&E, X 200.), B: Cartilage tissue cells due to metaplasia in malignant mixed tumour H&E, X 400. C: Myoepithelial reaction
included with spindle shape cells in carcinoma, H&E, X 400. D and E: A cystic tumour of adenocarcinoma showing local invasion of interlobular
connective tissue and note the high mitotic index and also different mitotic figures (H&E, (H&E, × 400 and 200). F: Papillary adenocarcinoma
proliferating neoplastic epithelial cells in the form of papillary projections and presence of mitotic figures. H&E, 200×, G and H: Beinign mixed
mammay tumour. Tumour tissue showing epithelial and myoepithelial components, 400×, I: The presence of neoplastics emboli within the
dermal lympathic vessels, which was occasionally observed with some of the most aggressive adenocarcinoma, leads to blockage of the
superficial dermal lymphatic drainage (H&E, X 200), J: Cholesterol cleft in malignant mixed tumour, (H&E, X 400). K: Cells exhibit variable numbers
of mitoses are found; or, the second population of cells may have oval to fusiform vesicular nuclei with an extensive amount of eosinophilic
cytoplasm and distinct cells margins (H&E, X 400) L: Haemorrhage foci and the central necrotic areas are interpreted as an indication that the
neoplastic cells are growing faster and that there is therefore a higher risk of progression to invasive carcinoma (H&E, X 400).
characterized by hyperchromasia, enlarged nuclei, prominent nucleoli of mammary gland. Also, 4 of the 15(26.7%)
CMTs cases exhibited cholesterol clefts in the lumina of
the ducts (Figure 4J).
Discussion
Clinical and cytopathological similarities between canine
mammary tumours and human breast cancer have been
described in recent decades [7].
Considering the breed distribution, cross breeds, terrier, mixed, German shepherd, Dachshund and Pekingese were predominant, which is similar as in other
studies [15-17]. The age at diagnosis ranged from 6 to
14 years, with a median of 10 years. This interval of risk
age is in agreement with other studies [17-20].
Mammary tumors are the most common neoplasms in
female dogs1. Malignant tumors may carry a poor prognosis and necessitate surgery. Few data are available on
the value of cytologic examination as a diagnostic or
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Grade III
Total
Cystic hyperplasia
1(6.7%)
_
_
1(6.7%)
Solid adenocarcinoma
_
_
1(6.7%)
1(6.7%)
Papillary adenocarcinoma
_
1(6.7%)
1(6.7%)
2(13.3%)
Simple carcinoma
_
_
1(6.7%)
1(6.7%)
Complex carcinoma
_
3(20%)
2(13.3%)
5(33.3%)
Benign mixed tumour
1(6.7%)
1(6.7%)
_
2(13.3%)
Malignant mixed tumor
_
1(6.7%)
2(13.3%)
3(20%)
Total
2(13.3%)
6(40%)
7(46.7%)
15(100%)
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prognostic tool for mammary tumors in dogs. FNAC is
considered a fast, accurate and cost-effective method for
the diagnosis of human mammary tumours [8-11]. However, the evaluation of its accuracy is poorly reported in
veterinary medicine. There is a difference concerning
the frequency of lesions diagnosed in human versus the
canine mammary gland. We performed a validity study
to further characterize sensitivity and specificity values,
as well as the accuracy of FNAC in the diagnosis of
CMTs.
In our study, we found 93.3% cytological and histological diagnostic agreement. In previously reported
studies of the canine mammary lesions, the agreement
between the cytological and histological diagnosis ranged
from 25% to 47% [5,21-23]. These results are low when
compared with results of studies of human breast lesions
published by Choi et al. [24] and Ciatto et al. [25]. They
described high levels of agreement between cytological
and histological diagnoses, ranging from 64.8% to 74.1%.
In addition, when the authors excluded the inconclusive
cases, their level of agreement increased to 93.1 and
96.7%, respectively. The results were at variance with
findings of Simeonov and Stoikov [26], who reported
84.6% of correlation between cytological and histological
diagnoses of mammary tumours.
In some studies, the fine needle aspiration cytology specimens contained many individual bizarre, multi-nucleated,
and/or giant cells having hyperchromatic pleomorphic nuclei, prominent nucleoli, and relatively abundant cytoplasm, admixed with numerous mitotic figures in a
hemorrhagic or inflammatory background in human. A
small amount of sheet-like or three-dimensional clusters
of malignant cells coexisted [20,22].
Histopathologic examination is considered the gold
standard for the diagnosis of CMTs. The histological
analysis of CMTs usually includes a spindle cell component. However, according to Allen et al. 1986, the presence of spindle cells in cytological samples of breast
neoplasms is not restricted to mixed tumours, as these
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Histological type
cells may be observed in other breast lesions, including
myofibroblastomas, fibromatoses and even spindle cell
carcinomas [27]. Despite the similar cytological and
histological features between canine and human mixed
tumours of mammary gland, in canines, these tumours
are very common, while in humans they are very rare.
Most of the canine mammary tumours are benign or
malign mixed tumours that are composed with epithelial
and myoepithelial proliferations with generally cartilage,
bone and squamous metaplasia [28]. Allen et al. 1986
reported that the existence of spindle shaped cells within
cytological aspirates should not be limited to mixed tumours as these cells might also exist in other mammary
lesions such as myofibroblastoma . Haziroglu et al., 2010,
present spindle shaped cells reported in one case of
malignant mixed tumour and in one case of complex carcinoma [29]. In the present study, spindle shaped cells
were encountered in two cases of malignant mixed
tumour, in two cases of complex carcinoma and in one
cases of solid adenocarcinoma, agreeing in this way with
the previous reports.
Histopathological examination of the biopsy specimens
was established as the most reliable diagnostic approach
and revealed the characteristics of the tumour in many
terms, which included pleomorphism, mitotic index, differentiation level, presence of necrosis, and the stromal
invasion (the infiltration with neoplastic cells of the
blood and lymph vessels and the cutaneous and soft tissue and the sur-gical margins). This data have been accepted as a golden standard in diagnosis due to its great
importance in terms of the biological behaviour and the
prognostic outcome of the neoplasia [30].
According to some authors, [31-34] tumors might have
the potential to feed themselves via alternative pathways
by vascular channels covered by deregulated neoplastic
cells.
The presence of neoplastic emboli within the dermal
lymphatic vessels, which was occasionally observed with
some of the most aggressive CMTs, leads to blockage of
the superficial dermal lymphatic drainage. The outcome
is a clinical presentation that resembles an inflammatory
process (inflammatory mammary cancer), which has a
poor prognosis and a rapid, fatal clinical course, since all
the available treatments are usually palliative [35-37].
The central necrotic areas are interpreted as an indication that the neoplastic cells are growing faster and that
there is therefore a higher risk of progression to invasive
carcinoma [38,39].
Histopathological diagnosis of CMTs is crucial in prediction of tumour behaviour after surgical excision.
Moreover, histopathologic typing of the tumour is also
important in establishing a post-operative chemotherapy
plan to increase the survival time following the surgery
since several protocols have been used with success in
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Table 2 Relationship between histological grading and
tumour type together with number and percentage of
cases in 15 dogs with mammary tumour
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In the present study, a correlation between histological
type and grade was evident. Carcinomas with a comparatively favourable prognosis, such as Complex carcinomas [30,53], were usually of grade II or III. On the
other hand, simple carcinoma (the most malignant type)
was usually grade III. Similar observations were reported
in human patients by Elston and Ellis [51].
Because of the diversity of histological typing criteria,
grading methods and endpoints used in different studies
on the prognostic value of histological grading in canine
mammary cancer, the results of such studies are difficult
to compare [5,42,53]. In the only study 58 similar to
ours(Due to the high percentage of tumor growth in the
grade III), 50% of dogs with grade I mammary tumours,
64% with grade II, and 79% with grade III died within 2
years of surgical treatment. These results differed from
our findings, possibly due to the use of a less refined
grading method and the inclusion of sarcomas, which
have the least favorable prognosis of all mammary tumours [50,54].
Conclusions
Our results demonstrate that, because of the similarity of
the cytological findings in human and canine mammary
gland tumours, it is possible to use the same cytological
criteria applied in human pathology for the diagnosis of
canine mammary gland tumours. This study is hoped to
open the way up for further cytopathology studies.
This study demonstrated that the Elston and Ellis
method of histological grading in canine mammary
tumor is a reliable prognostic factor. That is correlated
with histopathological classification. Histological grading
of canine mammary carcinomas by the Elston and Ellis
method was significantly related to prognosis, especially
in cases of simple carcinoma. Its routine use should be
helpful in indicating appropriate post-surgical treatment.
The estimation of the proliferative activity of tumours
by well standardized mitotic counting techniques should
have a central position in histopathology research and
practice.
Tumors of the mammary glands were most common
in 6 – 14 year old bitches.
The most often tumors occur in inguinal mammary
(60%) and abdominal (27%) glands. This interesting regional difference may be due to a) the duration of the
growth before the diagnosis; b) the age of the dogs; and
c) high prevelance of unspayed animals.
The most common type of tumor was complex carcinoma – 33.3% (5 cases).
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dogs [40]. Various classification systems [41-43] have
been developed to estimate the prognosis of the disease.
Several studies revealed that half (42.0-55.0%) of the surgically removed mammary tumors in bitches were malignant 7. Meuten reported that about 20.0-40.0% of bitches
with mammary tumors developed malignant kinds. 49 Although Simeonov and Stoikov reported that only 19.0%
benign and 81.0% mammary tumors were malignant [26].
Moreover, Tavasoly et al., 2013 reported that all samples
(n = 37) were malignant. In the present study only 13.3%
benign and 86.7% mammary tumors were malignant [44].
Tavasoly et al., 2013 reported, 86.5% (n = 32), and 13.5%
(n = 5) of mammary tumors were carcinomas and sarcomas, respectively. The most frequently represented tumor
type was simple carcinoma 56.8% (n = 21), followed by
complex carcinoma 13.5% (n = 5), sarcoma 13.5% (n = 5),
carcinoma arising from benign tumor 10.8% (n = 4) and
special type of carcinoma 5.4% (n = 2).
Rezaie et al. found that 70.6% of bitches had
tubulopapillary carcinoma, 23.5%- solid carcinoma, and
5.9% - cribriform carcinoma [45] . Ežerskytė et al. indicated that the most common tumor types of mammary
glands in bitches were simple carcinoma, complex carcinoma and carcinosarcoma 46.0%, 27.0% and 13.0%, respectively [46].
In the present study, the most frequently represented
neoplasm type was complex carcinoma (5/15; 33.3%),
followed by adenocarcinoma (3/15; 20%), malignant
mixed tumor (3/15; 20%), simple carcinoma (1/15; 6.7%)
and cystic hyperplasia (1/15; 6.7%).
However, in spite of this high percentage of malignant
mammary tumors, according to WHO classification, the
vast majority of malignant tumors were well differentiated
adenocarcinomas, mostly complex and tubulopapillary,
whereas special types of carcinomas and sarcomas were
rare, which is similar as in other studies [16,17,47-49].
The measurement of only one of the parameters (variations of nucleus dimensions, giant nucleus formation,
nucleus / cytoplasm distortion and rate, nuclear pleomorphism, changes in chromatin structures (altered dimensions, irregular chromatin shapes in nucleus,
clearing of the parachromatin areas), variation in nucleolus number, abnormal nucleolus shape and presence of
macronucleolus, mitotic count) associated with histological grade is unlikely to provide powerful prognostic
information [50-52]. Complete histological grading is
therefore preferable to nuclear grading for accurate
prognosis. In the present study the main criteria retained
to determine malignancy were tubule formation, the nucleus pleomorphism and dimension and a significant
variation in the mitotic rates.
Most grading systems of mammary carcinomas in dogs
are a modification of the numeric method of Elston and
Ellis.
Materials and methods
Animals characteristics
The study was performed in 15 pure or mixed-breed female dogs submitted to surgical resections of ‘mammary
Shafiee et al. Cancer Cell International 2013, 13:79
http://www.cancerci.com/content/13/1/79
Page 9 of 11
an assessment of (1) tubule formation, (2) nuclear pleomorphism, and (3) mitotic counts, each feature being
scored 1 to 3 points. The scores were then added to obtain the tumour grade, as follows: 3–5 points, welldifferentiated (grade I); 6–7 points, moderately differentiated (grade II); 8–9 points, poorly differentiated (grade
III). Grading was carried out by one veterinary pathologist and, without prior knowledge of the results, confirmed by a second pathologist.
C
LE
tumours’ in the Veterinary School Hospital of Tehran
University Faculty with the complaints of mass existence
in different mammary lobes. The animals, aged 6–
14 years (mean ± SD = 10.5 ± 1.8), showed with or without clinical or radiological evidence of distant metastasis.
Female dogs were mainly terriers (9 dogs) or mixed (3
dogs), the 3 other animals were a German shepherd,
Dachshund and Pekingese. They were selected from
cases treated surgically between July 2011 and February
2013. The mammary tumours were excised by simple
mastectomy or regional mastectomy [50], with or without the superficial inguinal lymph nodes.
Classification of tumours
The final diagnosis was classified in the protocol
according to the following five categories: (1) benign, (2)
suspicious-probably benign, (3) suspicious-probably malignant, (4) malignant and (5) insufficient/inadequate
material for the diagnosis. However, to establish a comprehensive histological correlation, the two categories of
suspicious cases were classified in a generic group entitled ‘suspicious-not otherwise specified.
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Abbreviations
CMTs: Canine mammary tumours; CMMT: Canine malignant mammary
tumours; FNAC: Fine needle aspiration cytology; HE: Haematoxylin and eosin.
Competing interests
The authors declare that they have no competing interests.
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Before surgical excision of the tumour, Fine needle aspiration cytology (FNAC) was performed using a 0.6 mm
diameter needle attached to a 10 ml syringe held in a
standard metal syringe holder. The cytological sample
was smeared onto a glass slide and either air-dried for
May-Grünwald-stain, or ethanol-fixed for Papanicolaou
stain. Subsequently, dogs were induced with propofol
(4 mg/kg, IV, Propofol, Abbott) and anaesthetized with
isoflurane (2-3%, Isoflurane, Adeka) and masses were
surgically removed, the tumours were grossly examined
and tissue samples were fixed in 10%-buffered-formalin
and embedded in paraffin. Sections 4 μm thick were
obtained from each sample and H&E stained. The
cytopathological criteria adopted were those proposed
by Bibbo [55] and for histopathological analysis of tumors it was used the Veterinary [30] and Human [56]
classification. We considered the histopathological diagnosis as the ‘gold standard.
TI
Cytological evaluation
A
Clinical and histopathological evaluation
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R
Tumour size: Mammary neoplasms were classified by
size according to the World Health Organization Clinical Staging System TNM 19, as T1 (<3 cm maximum
diameter), T2 (3–5 cm maximum diameter) and T3
(>5 cm maximum diameter). In cases of multiple tumours, the largest one was used as the basis for
classification.
Tumour type: Representative sections of each tumour
(from the central core to periphery) and the excised
lymph nodes were fixed in 10% buffered formalin,
processed by routine methods, embedded in paraffin
wax, sectioned at 5 μm and stained with haematoxylin
and eosin (HE). Histopathological findings were
recorded and used to classify the tumours according to
the criteria of a recently validated system [30]. In cases
with multiple tumours, the most malignant one as defined by Misdorp [50] was recorded.
Tumour grade: Histological grading was performed on
HE-stained sections. According to the Elston and Ellis
method [51], the grade for each case was derived from
Authors’ contributions
NA and RSH participated in the histopathological evaluation, performed the
literature review, acquired photomicrographs and drafted the manuscript
and gave the final histopathological diagnosis and designed and carried out
all the experiments. JJ is the principal investigator of the laboratory in which
the research was performed and contributed to the interpretation of the
data and writing of the manuscript. PKH, DKH, AMB, HD and FKH edited the
manuscript and made required changes and wrote the manuscript. All
authors have read and approved the final manuscript.
Acknowledgements
The authors thank staff of the Department of pathology, Faculty of
Veterinary Medicine, Tehran University for their valuable technical assistance.
Author details
1
Faculty of Veterinary Medicine, Tehran University, Tehran, Iran. 2Department
of Pathology, Faculty of Veterinary Medicine, Tehran University, Tehran, Iran.
3
Semnan University of Medical Science, Faculty of Medicine, Semnan, Iran.
4
MD, Graduate Islamic Azad University of Mashhad, Faculty of Medicine,
Mashhad, Iran. 5MD, Graduate Paraveterinary Faculty of Ilam, University of
Ilam, Ilam, Iran. 6Department of Environmental Health Engineering, Alborz
University of Medical Sciences, Karaj, Iran.
Received: 15 July 2013 Accepted: 5 August 2013
Published: 9 August 2013
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doi:10.1186/1475-2867-13-79
Cite this article as: Shafiee et al.: Diagnosis, classification and grading of
canine mammary tumours as a model to study human breast cancer:
an Clinico-Cytohistopathological study with environmental factors
influencing public health and medicine. Cancer Cell International
2013 13:79.
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