Research in Veterinary Science 105 (2016) 124133

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Research in Veterinary Science

journal homepage: www.elsevier.com/locate/rvsc

Evaluation of apoptosis-associated protein (Bcl-2, Bax, cleaved caspase-3

and p53) expression in canine mammary tumors:
An immunohistochemical and prognostic study
Izabella Dolka a,, Magdalena Krl b, Rafa Sapierzyski a
a
Department of Pathology and Veterinary Diagnostics, Faculty of Veterinary Medicine, Warsaw University of Life Sciences-WULS, Nowoursynowska 159c, 02-776 Warsaw, Poland
b
Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw University of Life Sciences-WULS, Nowoursynowska 159c, 02-776 Warsaw, Poland

a r t i c l e i n f o a b s t r a c t

Article history: Apoptosis is an important process involved in pathogenesis and progression of neoplasia. However, it has been
Received 29 June 2015 not so far extensively investigated in canine mammary tumors (CMTs). Therefore the aim of our study was to de-
Received in revised form 26 January 2016 termine Bcl-2, Bax, cleaved caspase-3 (CC3) and p53 expression in CMTs and evaluate their correlation with host/
Accepted 5 February 2016
tumor factors, and overall survival (OS). Bcl-2 expression was often found in benign lesions and in patients with
Available online xxxx
low TNM stage. Expression of Bax, CC3 and p53 was observed in malignant CMTs. The expression of apoptosis-
Keywords:
associated proteins was not signicantly associated with OS. A positive-p53 status was signicantly related
Dog with poorer tumor differentiation, higher mitotic index (MI), more invasive growth, necrosis, and occurred
Mammary tumor often in CMTs from large breed dogs. In the shorter-survival group of dogs (18 months), a positive correlation
Apoptosis was found between CC3 and Bcl-2 expression; CC3 and MI, ER and p53 expression, while in the longer-survival
Immunohistochemistry group (N 18 months) CC3 expression was negatively correlated with ER, whereas p53 expression was positively
Overall survival correlated with MI. We conrmed the usefulness of such parameters as: tumor size, MI, type of growth, tumor
metastasis and TNM stage in predicting OS in a univariate analysis. In multivariate analysis we identied age
as an independent prognostic factor for OS. Expression of single apoptosis-associated protein should not be
used as a prognostic marker. However, we showed signicant correlation patterns of expression of proteins in-
volved in apoptotic-signaling pathways in shorter- and longer survival groups. So far, there have been only a
few similar reports published.
2016 Elsevier Ltd. All rights reserved.

1. Introduction
A variety of factors are involved in the pathogenesis and progression
of canine mammary tumors (CMTs), including activation of steroid hor-
mone receptors (especially estrogens and progesterones), several mo-
lecular pathways, e.g. dysregulation of cellular proliferation or
apoptosis-associated molecules (Klopeisch et al., 2011). Better under-
standing of CMTs biology is very important for clinicians and patholo-
gists. Although numerous studies on CMTs have been published in the
past, expression of apoptosis-associated proteins and its potential to
predict outcome has not yet been fully described.
Apoptosis, a programmed cell death, has an essential inuence on
development but also carcinogenesis of mammary gland cells. It is reg-
ulated by a wide variety of factors, especially these belonging to the Bcl-
2 family: inhibitors (i.e. Bcl-2 protein), promoters (i.e. Bax protein) and
by activation of the apoptotic cascade via caspase-3. The cleaved (acti-
vated) caspase-3 (CC3) is considered as an executioner of apoptotic
Corresponding author.
E-mail address: izabella_dolka@sggw.pl (I. Dolka).
pathway and its activation is required for induction of apoptosis. During
the past few decades the apoptosis-associated proteins have been indi-
cated as potential tumor markers for human breast cancer (HBC)
(Kymionis et al., 2001; Yildirim et al., 2014). For example several HBC
studies showed that Bcl-2 and estrogen receptor alpha ER (NR3A1
nuclear receptor subfamily 3, group A, member 1) is an indicator of a fa-
vorable outcome of endocrine treatment in breast cancer patients with
early disease stage. It was also indicated as a predictor of treatment re-
sponse in patients with advanced disease (Ciocca and Elledge, 2000).
Other studies showed that increased Bcl-2 expression overall correlated
to better survival rates, while over-expression of Bax played a role in as-
sessment of sensitivity for chemotherapy. Correlation of CC3 expression
with overall survival (OS) is unclear (Nakopoulou et al., 2001). How-
ever, expression of Bcl-2, Bax and CC3 has not been widely investigated
in CMTs. Only a few scientic papers reported Bcl-2 expression in CMTs
(Yang et al., 2006; Dolka et al., 2011; Yildirim et al., 2014). In contrary,
there are many published papers about a role of p53 tumor suppressor
in CMTs. The p53 gene and its protein play an important role in DNA re-
pair, regulation of cell cycle and in apoptosis. Over-expression of p53
protein was correlated to higher malignancy grade of CMTs (Lee et al.,
2004).

http://dx.doi.org/10.1016/j.rvsc.2016.02.004
0034-5288/ 2016 Elsevier Ltd. All rights reserved.
 I. Dolka et al. / Research in Veterinary Science 105 (2016) 124133
The aim of this study was to evaluate the expression levels of Bcl-2,
Bax, CC3, p53 in CMTs and to determine their correlation with prolifer-
ative activity, expression of steroid hormone receptors, clinicopatholog-
ical features and OS of these patients. In above analyses we considered
survival time after mastectomy of these dogs. Patients were divided
into two groups: 1) shorter-survival group ( 18 months), and
2) longer-survival group (N 18 months). As far as we realize, this is the
rst wide-scale analysis of apoptosis-associated proteins expression in
CMTs considering its correlation with many other factors.
2. Materials and methods
2.1. Tumor samples and histopathological examination
Canine mammary tumor tissue sections (n = 40) were derived from
the archives of the Department of Pathology and Veterinary Diagnostics,
Faculty of Veterinary Medicine, Warsaw University of Life Sciences.
Samples were surgically collected during routine mastectomies from
40 female dogs with single mammary tumor. Single mass tumor was
sent by referring veterinarian and submitted to the Department of Pa-
thology and Veterinary Diagnostics. The dogs' owners gave written or
verbal permission for the use of their animals' tissue samples. The
study was approved by the 3rd Local Ethical Committee (approval no.
8/2012, 17.01.2012) of the Warsaw University of Life Sciences. Samples
were xed in 10% buffered formalin and then dehydrated and embed-
ded in parafn. Parafn blocks were cut into 4 m tissue sections,
stained with hematoxylin and eosin (HE) and subjected to immunohis-
tochemical (IHC) procedure. Histopathological diagnosis was per-
formed according to Goldschmidt et al. (2011) and histologic tumor
grade (I, II and III) was assessed with respect to tubule formation, degree
of differentiation and mitotic index (Clemente et al., 2010). The mitotic
index (MI) was determined as the mean number of cells in mitosis eval-
uated in 10 high-power elds (HPF) under 40 magnication with an
objective lens (eld area: 0.239 mm2) (Dutra et al., 2008). Histological
evaluation was conducted by three independent pathologists. Question-
able samples were reviewed jointly using a multi-headed microscope.
The following data of patient history was collected and considered in
further analysis: age at diagnosis, spay status, breed, weight, tumor
size (the largest diameter of tumor in centimeter), local recurrence (de-
ned as recurrent tumor at the surgery site with histopathologic conr-
mation), type of growth (expansive tumor mass that pushes normal
tissues aside, well-circumscribed or encapsulated; invasive inltrative
growth or blood/lymphatic vessels invasion), and clinical stage. The
clinical stage was classied according to the modied TNM staging sys-
tem (for tumors, nodes and metastases), which was performed as fol-
low: stage I (T1N0M0), stage II (T2N0M0), stage III (T3N0M0), stage
IV (TanyN1M0), and stage V (TanyNanyM1) (Lana et al., 2007). Regional
lymph node metastases were determined by histopathology at the time
of diagnosis. Thoracic radiographs were performed at the time of diag-
nosis (before mastectomy) and during the follow-up period, if clinically
indicated. Overall 2-year survival was dened as time from surgery to
death or euthanasia due to any cause or end of the follow-up. The
follow-up could be ended because of cancer-related mortality, death
by other causes or a nal examination of dogs that were alive.
2.2. Immunohistochemical examination
For the purposes of the immunohistochemical examination the fol-
lowing polyclonal antibodies diluted in 1% BSA (Sigma-Aldrich,
Germany) were used: anti-Bcl-2, anti-Bax, anti-p53 protein (all ob-
tained from Santa Cruz Biotechnology, USA) diluted 1:50, anti-CC3
(Sigma-Aldrich) diluted in 1:500. In addition the following monoclonal
antibodies were used: anti-Ki67 (Dako, Denmark) diluted in 1:75, anti-
estrogen receptor alpha (ER, Dako, Denmark), anti-progesterone re-
ceptor (PR, Immunotech, Beckman Coulter Co., USA), in ready-to-use
kit. Dewaxed and rehydrated sections were subjected to antigen
125
retrieval with 0.02 M citrate buffer (pH 6.0) by heating in microwave
oven. Sections with anti-ER were incubated in Dako Target Retrieval
Solution (pH 9.0). After cooling in distilled water (20 min), endogenous
peroxidase activity was blocked with hydrogen peroxide (3% H2O2) for
10 min at room temperature (RT). Next, the tissue sections were rinsed
with TRIS buffer (pH 7.4) and incubated with the primary antibody for
1 h at RT. Then EnVision + System-HRP (Dako, Denmark) was
employed. The brown color precipitate (reecting stained antigen)
was obtained with a freshly prepared solution of chromogen 3,3-diami-
nobenzidine (DAB) (Dako, Denmark). After washing in distilled water
for 5 min, tissue sections were counterstained with Mayer's hematoxy-
lin for 10 min, washed in distilled water, dehydrated in graded baths of
alcohol, cleared in xylene, and mounted with DPX medium (Gurr,
Sigma-Aldrich). Parafn-embedded neoplastic canine tissues and non-
neoplastic mammary gland tissue with known positive reactivity were
used as positive controls. Some inammatory cells exhibited strong
Bcl-2, Bax or CC3 immunoreactivity and were used as an internal posi-
tive control. Negative controls were obtained by omitting incubation
with primary antibodies. All immunohistochemical slides were exam-
ined using a standard light microscope Olympus BX41 (Olympus,
Tokyo, Japan) at high-power elds (40 objective and 10 eyepiece).
Computer image analysis and CellSens software (Olympus, Tokyo,
Japan) were used for the interpretation of the IHC staining results. The
number of positively stained neoplastic cells was assessed from an ex-
amination of at least 10 images in high-power elds. Areas with necrosis
were omitted. Positive immunostaining for Bcl-2, Bax and CC3 proteins
were dened as cytoplasmic pattern (brown color representing the pre-
cipitate). Cases of CMTs with cytoplasmic and nuclear Bax and CC3 ex-
pression were considered to be positive. Furthermore, Bcl-2, Bax and
CC3 expression were evaluated by the following scale: 0 negative
staining, 1 b25% positive cells (mild); 2 25 to 75% positive cells
(moderate); 3 over 75% positive cells (strong) (Vakkala et al., 1999;
Koda et al., 2005). Positive immunostaining for Ki67, ER, PR and p53
was dened as nuclear expression pattern (presence of brown precipi-
tate in nucleus). The number of immunoreactive cells for anti-ER,
-PR and -p53 antibodies was classied as: 0 () = negative (included
negative immunostaining pattern or immunopositivity in less/or equal
than 10% of tumor cells) and 1 (+) = positive (positive cells constituted
more than 10%) (Lee et al., 2004; Kumaraguruparan et al., 2006). CMTs
samples were classied into two categories: low (b20%) Ki67 score and
high (20%) Ki67 score (Romero et al., 2014).
2.3. Statistical analysis
The statistical analyses were conducted with the Statistica 10 soft-
ware (StatSoft Inc. USA) and p-value b 0.05 was assumed as signicant
(*). Data were grouped as follows: age at diagnosis ( 8 years and
N8 years), breed (pure/mixed breeds), weight of pure-breed dogs
(small/large), spay status at the time of diagnosis (intact/spayed before
diagnosis/spayed at the time of mastectomy), tumor size (b3 cm and
3 cm), tumor type based on clinical behavior (benign/malignant), car-
cinoma type (simple/complex and arising in a benign tumor), histologic
grade (I and II/III), mitotic index MI (00.9, 1.01.9 and 2.0 in HPF),
necrosis (yes/no), type of growth (expansive/invasive), tumor metasta-
sis (yes/no), cause of death (cancer-related mortality/non-cancer-
related mortality), TNM (I, II, III/IV, V), Ki67 (low/high), ER (nega-
tive/positive), PR (negative/positive), Bcl-2 (negative/positive), Bax
(negative/positive), CC3 (negative/positive), and p53 (negative/posi-
tive). Univariate and multivariate statistical analyses were applied.
The patients' age, tumor size and mitotic index were categorized
based on previous studies of CMTs (Martin De Las Mulas et al., 2005;
Goldschmidt et al., 2011; Santos et al., 2013). Survival analysis was con-
ducted using the Kaplan-Meier method. The effect of selected factors on
probability of overall survival was evaluated by multivariate survival
analysis using the Cox proportional hazards model of regression. The
hazard ratios were estimated with 95% condence interval. Associations
126 I. Dolka et al. / Research in Veterinary Science 105 (2016) 124133

Fig. 1. The Kaplan-Meier curves of 40 female dogs with CMTs presenting the signicant relationship between tumor factors and overall survival. (A) Tumor size 3 cm (n = 17, blue line)
and tumor size b 3 cm (n = 23, red dotted line). p = 0.006. (B) Mitotic index: 0.00.9/HPF (n = 27, blue line), 1.01.9/HPF (n = 7, red dotted line), and 2.0/HPF (n = 6, green dotted
line), p = 0.016. (C) Expansive type of tumor growth (n = 30, blue line) and invasive type of tumor growth (n = 10, red dotted line), p = 0.005. (D) Presence of metastases (n = 7, blue
line) and no metastases (n = 24, red dotted line), p = 0.021. (E) TNM I + II + III stage (n = 36, red dotted line) and TNM IV + V stage (n = 4, blue line), p = 0.030. (For interpretation of
the references to color in this gure legend, the reader is referred to the web version of this article.)

Fig. 2. The Kaplan-Meier curves of 40 female dogs with CMTs presenting the relation between apoptosis-associated protein expression and overall survival. There was not signicant
association (NS) with OS. (A) Bcl-2-positive tumors (n = 30, blue line) and Bcl-2-negative tumors (n = 10, red line), p = 0.135. (B) Bax-positive tumors (n = 24, blue line) and Bax-
negative tumors (n = 16, red line), p = 0.518. (C) CC3-positive tumors (n = 24, blue line) and CC3-negative tumors (n = 16, red dotted line), p = 0.732. (D) p53-positive tumors
(n = 12, blue line) and p53-negative tumors (n = 28, red dotted line), p = 0.382. (For interpretation of the references to color in this gure legend, the reader is referred to the web
version of this article.)
 I. Dolka et al. / Research in Veterinary Science 105 (2016) 124133 127

Table 1
Incidence of host and tumor factors, and association between studied variables with survival times.

Host and tumor factors Total Shorter-survival group 18 monthsa Longer-survival group N 18 monthsa p value Mean OS time in
months SEM
N Mean time in months SEM N Mean time in months SEM
(median survival
(median survival in months) (median survival in months)
in months)

Age at diagnosis (years)

8 years 14 1 16.0 (16.0) 13 23.3 1.0 (23.0) 0.021 22.8 1.5 (22.5)
N8 years 26 11 14.9 1.1 (16.0) 15 23.5 1.0 (23.0) 29.8 0.8 (19.0)
Breed NS
Pure 29 7 15.7 0.8 (16.0) 22 23.4 0.7 (23.0) 21.6 0.9 (22.0)
Mixed 11 5 14.0 2.3 (16.0) 6 23.3 1.9 (21.0) 19.1 2.0 (20.0)
Weight pure-breed dogs NS
Small (b20 kg) 20 3 15.7 1.9 (17.0) 17 24.1 0.8 (24.0) 22.9 1.0 (23.0)
Large (20 kg) 9 4 15.8 0.6 (16.0) 5 21.0 1.3 (19.0) 28.7 1.2 (19.0)
Spay status at the time of diagnosisb 0.007
Intact 4 0 4 23.3 2.4 (21.5) 23.3 2.4 (21.5)
Spayed before diagnosisc 9 3 15.0 0.6 (15.0) 6 21.5 1.1 (21.0) 19.3 2.3 (19.0)
Spayed concomitantly at the time of 26 9 15.0 1.4 (16.0) 17 23.8 0.9 (23.0) 20.7 1.1 (20.5)
mastectomy
Tumor size 0.007
b3 cm 23 3 17.0 0.6 (17.0) 20 23.4 0.8 (23.0) 22.6 0.9 (22.0)
3 cm 17 9 14.3 1.3 (16.0) 8 23.4 1.4 (23.0) 18.6 1.5 (18.0)
Tumor type NS
Benign 9 1 16.0 (16.0) 8 24.5 1.3 (23.5) 23.6 1.5 (23.0)
Malignant 31 11 14.9 1.1 (16.0) 20 23.0 0.8 (22.0) 20.1 1.0 (20.0)
Benign lesions ND
Benign mixed tumor 1 0 1 25.0 (25.0) 25.0 (25.0)
Simple adenoma 7 0 7 24.4 1.5 (23.0) 24.4 1.5 (23.0)
Complex adenoma 1 1 16.0 (16.0) 0 16.0 (16.0)
Carcinoma type NS
Simple type 18 6 13.5 1.9 (15.0) 12 22.5 1.1 (21.0) 19.5 1.4 (19.5)
Complex and arising in benign tumor 13 5 16.6 0.5 (17.0) 8 23.6 1.1 (24.0) 20.9 1.2 (22.5)
Individual carcinoma types
Tubulopapillary 16 6 13.5 1.9 (15.0) 10 21.2 0.9 (20.0) 0.001 18.3 1.3 (19.0)
Solid 1 0 1 30.0 (30.0) ND 30.0 (30.0)
Tubular 1 0 0 28.0 (28.0) 28.0 (28.0)
Carcinoma arising in benign tumor 10 4 16.8 0.6 (17.0) 6 22.3 1.0 (23.0) 0.014 20.1 1.1 (19.5)
Complex carcinoma 3 1 16.0 (16.0) 2 27.5 0.5 (27.5) 1.00 23.7 3.8 (27.0)
Histologic graded NS
I and II 24 8 14.5 1.5 (16.0) 16 22.9 0.8 (22.0) 20.1 1.1 (20.0)
III 7 3 16.0 0.0 (16.0) 4 23.3 2.7 (22.0) 20.1 2.0 (19.0)
Mitotic index 0.001
00.9/HPF 27 6 15.2 0.8 (15.5) 21 23.5 0.7 (23.0) 21.7 0.9 (22.0)
1.01.9/HPF 7 6 14.8 2.0 (16.5) 1 28.0 (28.0) 16.7 2.5 (17.0)
2.0/HPF 6 0 6 22.2 1.8 (20.0) 22.2 1.8 (20.0)
Necrosis NS
Yes 11 4 16.3 0.3 (16.0) 7 23.0 1.7 (22.0) 20.5 1.5 (19.0)
No 29 8 14.4 1.5 (15.5) 21 23.5 0.8 (23.0) 21.0 1.0 (21.0)
Type of growth 0.005
Expansive 30 8 14.1 1.5 (15.5) 22 23.1 0.7 (22.5) 20.7 1.0 (20.5)
Invasive 10 4 16.8 0.5 (16.5) 6 24.5 1.9 (25.5) 21.4 1.7 (19.0)
Tumor metastasisd 0.001
Yes 7 4 12.8 2.9 (14.0) 3 25.7 3.0 (27.0) 18.3 3.2 (18.0)
No 24 7 16.1 0.5 (16.0) 17 22.5 0.8 (22.0) 20.6 0.8 (20.0)
Cause of deathd NS
Cancer-related mortality 6 4 12.8 2.9 (14.0) 2 19.0 0.0 (19.0) 14.8 2.2 (17.0)
Non-cancer-related mortality 4 2 16.5 0.5 (16.5) 2 25.0 3.0 (25.0) 20.8 2.8 (19.5)
TNM NS
I + II + III 36 11 14.7 1.1 (16.0) 25 23.0 0.7 (22.0) 20.5 0.9 (20.0)
IV + V 4 1 18.0 (18.0) 3 26.7 2.0 (27.0) 24.5 2.6 (25.0)
Ki67 index NS
Low 27 7 14.9 1.7 (16.0) 20 23.7 0.8 (23.5) 21.4 1.0 (22.0)
High 13 5 15.2 1.0 (16.0) 8 22.6 1.5 (21.0) 19.8 1.4 (19.0)
ER NS
Negative 20 8 14.8 1.5 (16.0) 12 24.7 1.1 (24.5) 20.7 1.4 (20.5)
Positive 20 4 15.5 1.3 (16.0) 16 22.4 0.8 (22.0) 21.1 0.9 (20.0)
PR NS
Negative 13 6 15.2 0.7 (16.0) 7 23.1 1.3 (24.0) 19.5 1.4 (19.0)
Positive 27 6 14.8 2.0 (16.5) 21 23.5 0.8 (23.0) 21.6 1.0 (21.0)
Bcl-2 NS
Negative 10 5 15.2 1.0 (16.0) 5 23.6 1.7 (23.0) 19.4 1.7 (19.0)
Positive 30 7 14.9 1.7 (16.0) 23 23.3 0.8 (23.0) 21.4 1.0 (21.5)
Bax NS
Negative 16 4 16.0 0.8 (16.0) 12 22.7 1.1 (21.5) 21.0 1.1 (20.0)
Positive 24 8 14.5 1.5 (16.0) 16 23.9 0.9 (23.0) 20.8 1.2 (21.0)
CC3 NS

(continued on next page)

128 I. Dolka et al. / Research in Veterinary Science 105 (2016) 124133

Table 1 (continued)

Host and tumor factors Total Shorter-survival group 18 monthsa Longer-survival group N 18 monthsa p value Mean OS time in
months SEM
N Mean time in months SEM N Mean time in months SEM
(median survival
(median survival in months) (median survival in months)
in months)

Negative 16 6 15.2 0.8 (15.5) 10 22.9 1.0 (23.0) 20.0 1.2 (19.5)
Positive 24 6 14.8 2.0 (16.5) 18 23.7 0.9 (22.0) 21.5 1.2 (20.5)
p53 NS
Negative 28 8 14.3 1.5 (15.5) 20 23.8 0.8 (23.0) 21.1 1.1 (22.0)
Positive 12 4 16.5 0.5 (16.0) 8 22.4 1.5 (20.0) 20.4 1.3 (19.0)

N - number of cases; NS - not signicant; ND - not done, because of small number of cases.
a
Chi-square test.
b
Spay status at the time of diagnosis was unknown only for one dog.
c
Spayed at least one year prior diagnosis.
d
Malignant tumors only.
p-value b 0.05 was assumed as signicant.
p-value b 0.01.

between the factors analyzed and category of survival (two subgroups:
18 months and N18 months) were performed using the chi-square
test. Relationships between selected variables were evaluated on the
basis of Spearman's rank correlations.
3. Results
3.1. Animals and tumor samples characteristics
Forty female dogs with a single mammary tumor were included in
this study. The clinical and histopathological data are summarized in
(Table A.1). The mean age of dogs at the time of surgery was 9.9
2.5 years (range: 514 years). Twenty nine dogs were purebred (72%),
predominated small breeds (n = 20, 69%), especially Dachshunds
(n = 10) and 11 of the patients (28%) were mixed breeds. Most of the
bitches (n = 26, 67%) were spayed at the time of mastectomy. The
mean tumor diameter was 2.6 1.5 cm (range: 0.55 cm). Mammary
tumors were most often found in the 4th and 5th pair of glands (n =
27, 79%). In most cases the left mammary chain was affected (56%,
n = 20). Seventy seven percent of CMTs (n = 31) were diagnosed as
malignant, while 23% (n = 9) as benign lesion (simple or complex ade-
nomas and benign mixed tumor). Among malignant tumors the most
frequent were: simple carcinomas (n = 18, 58%), especially
tubulopapillary carcinomas (n = 16, 88%), carcinomas arising in a be-
nign tumor (n = 10, 32%), and complex carcinomas (n = 3, 10%). His-
tological grading of the malignant CMTs revealed that 4 simple
carcinomas and 1 complex carcinoma were classied as a grade I of ma-
lignancy, 9 simple carcinomas, 5 carcinomas arising in a benign tumor
and 1 complex carcinoma as grade II and 5 simple carcinomas, 1 car-
cinoma arising in a benign tumor and 1 complex carcinoma were classi-
ed as grade III tumors of malignancy. Most of the CMTs had a low
mitotic index in the range of 00.9/HPF (n = 27, 67%). The presence
of necrosis was observed in 27% (n = 11) cases, all of them were malig-
nant. Expansive growth was observed in all benign tumors and in 21
malignant tumors. Overall, 23% (n = 7) of dogs with malignant CMTs
developed metastases and 57% (n = 4) of them died during the fol-
low-up period. Most of the metastatic CMTs were diagnosed as simple
carcinomas (n = 4, 57%), tubulopapillary type (n = 3) and classied
as grade II in terms of malignancy (n = 4, 57% of these cases). Three pa-
tients were diagnosed with lymph node metastasis. Three dogs were

Fig. 3. Immunohistochemical images of canine mammary tumors. (A) Simple, tubular carcinoma: neoplastic cells with moderate cytoplasmic immunolabeling of Bcl-2 expression. IHC,
bar = 20 m. (B) Solid carcinoma: intense cytoplasmic immunostaining for Bax with focal nuclear immunoreactivity. IHC, bar = 50 m. Inset: higher magnication shows the nuclear
(arrow) and cytoplasmic positivity (arrowhead). IHC, bar = 10 m. (C) Complex carcinoma: intense cytoplasmic immunostaining for CC3. IHC, bar = 20 m. (D) Solid carcinoma: p53
expression shows intense nuclear immunoreactivity. IHC, bar = 50 m.
 I. Dolka et al. / Research in Veterinary Science 105 (2016) 124133 129

Table 2
Incidence and relationship between apoptosis-associated proteins expression and clinicopathological variables.

Host and tumor factors Bcl-2 Bax CC3 p53

Pos. Neg. Pos. Neg. Pos. Neg. Pos. Neg.

Age at diagnosis (years)

8 years 11 3 7 7 7 7 5 9
N8 years 19 7 17 9 17 9 7 19
p value NS NS NS NS
Breed
Pure 24 5 17 12 16 13 8 21
Mixed 6 5 7 4 8 3 4 7
p value NS NS NS NS
Weight pure-breed dog
Small (b20 kg) 17 3 12 8 11 9 2 18
Large (20 kg) 7 2 5 4 5 4 6 3
p value NS NS NS 0.002
Spay status at the time of diagnosisa
Intact 2 2 4 0 3 1 2 2
Spayed before diagnosisb 8 1 5 4 4 5 4 5
Spayed concomitantly at the time of mastectomy 19 7 14 12 17 9 6 20
p value NS NS NS NS
Tumor type
Benign 7 2 3 6 3 6 0 9
Malignant 23 8 21 10 21 10 12 19
p value NS NS NS NS
Benign lesions
Benign mixed tumor 1 0 0 1 0 1 0 1
Simple adenoma 6 1 3 4 3 4 0 7
Complex adenoma 0 1 0 1 0 1 0 1
p value NS NS NS NS
Carcinoma type
Simple type 12 6 12 6 12 6 9 9
Complex and arising in benign tumor 11 2 9 4 9 4 3 10
p value NS NS NS NS
Individual carcinoma types
Tubulopapillary 10 6 11 5 10 6 8 8
Solid 1 0 1 0 1 0 1 0
Tubular 1 0 0 1 1 0 0 1
Carcinoma arising in benign tumor 9 1 7 3 6 4 1 9
Complex carcinoma 2 1 2 1 3 0 2 1
p value NS NS NS NS
Histologic grade
I and II 17 7 16 8 16 8 5 19
III 6 1 5 2 5 2 7 0
p value NS NS NS 0.001
Mitotic index
00.9/HPF 20 7 14 13 13 14 3 24
1.01.9/HPF 5 2 6 1 7 0 3 4
2.0/HPF 5 1 4 2 4 2 6 0
p value NS NS 0.042 0.000
Necrosis
Yes 9 2 8 3 9 2 9 2
No 21 8 16 13 15 14 3 26
p value NS NS NS 0.000
Type of growth
Expansive 24 6 17 13 16 14 6 24
Invasive 6 4 7 3 8 2 6 4
p value NS NS NS 0.017
Tumor metastasisc
Yes 3 4 5 2 5 2 3 4
No 20 4 16 8 16 8 8 16
p value 0.031 NS NS NS
Survival
Alive 24 6 17 13 17 13 8 22
Died 6 4 7 3 7 3 4 6
p value NS NS NS NS
Cause of deathc
Cancer-related mortality 4 2 3 3 3 3 3 3
Non-cancer-related mortality 2 2 4 0 4 0 1 3
p value NS NS NS NS
Overall survival
18 months 7 5 8 4 6 6 4 8
N18 months 23 5 16 12 18 10 8 20
p value NS NS NS NS
TNM
I + II + III 29 7 21 15 22 14 10 26
IV + V 1 3 3 1 2 2 2 2
p value 0.014 NS NS NS
130 I. Dolka et al. / Research in Veterinary Science 105 (2016) 124133
developed lung metastases during the follow-up period. One animal
had evidence of both local and distant metastases. Recurrence was ob-
served in 2 patients. Follow-up data regarding OS was available in all
40 cases. The majority of dogs (n = 30, 75%) were alive at the end of
the follow-up period. Ten dogs (25%) died or were humanely eutha-
nized (n = 6, 60%) due to existence of cancer-related issues (metastasis,
local recurrence of invasive carcinoma). Cancer-related mortality was
signicantly associated with presence of metastasis (p b 0.05). In all
dogs with cancer-related issues the primary tumor was diagnosed as
malignant. The cause of death in 40% (n = 4) of dogs was not associated
with CMTs, but it was caused by: car accident, urinary bladder carci-
noma, brain tumor and one dogs died due to old age. Data was analyzed
for associations between animal and tumor factors, apoptosis-
associated proteins expression and OS (Figs. 1 and 2) and separately
for survival groups: 18 months and N 18 months (Table 1). Overall
Kaplan-Meier survival curves revealed that tumor size, MI, type of
growth, tumor metastasis and TNM stage, inuenced patient OS
(Fig. 1). CMTs over 3 cm (p b 0.01) with MI in the range of 1.01.9/
HPF (p b 0.05), invasive type of growth (p b 0.01), presence of metasta-
ses (p b 0.05) and advanced clinical stages (TNM IV + V) (p b 0.05) were
signicantly associated with shorter overall survival. Between the two
groups of shorter and longer survivors, signicant differences were
noted in their: age (p b 0.05), spay status (p b 0.01), tumor size
(p b 0.01), tumor MI (p b 0.001), type of growth (p b 0.01) and presence
of metastases (p b 0.001) (Table 1). Signicantly shorter survival (
18 months, Table 1) was observed in 11 (42%) older dogs, three (33%)
dogs spayed before diagnosis, nine dogs (53%) with tumors larger
than 3 cm, six dogs (86%) with tumors having a MI 1.01.9/HPF, four
dogs (40%) with tumors showing an invasive growth pattern and four
(57%) dogs with metastatic tumor. In a detailed analysis of histologic
type of these tumors, the only signicant difference was observed be-
tween tubulopapillary carcinoma and carcinoma arising in benign
tumor. Signicantly shorter survival (p b 0.001) was observed in six
(38%) dogs with tubulopapillary carcinoma, whereas six (60%) dogs
with carcinoma arising in benign tumor had signicantly longer survival
(p b 0.05, Table 1). Due to small number of deaths application of Cox re-
gression model was difcult to apply in some cases and results did not
often. However, multivariate analysis indicated age of dogs as only sig-
nicantly independent prognostic factor (HR: 0.004; 95% CI: 0.000
0.398; p = 0.019) for OS.
Nuclear ER and PR expression patterns were found in 20/40 (50%)
and 27/40 (68%) of CMTs, respectively. Among benign tumors, expres-
sion of steroid-hormone receptors was found the most often in simple
adenomas (ER: n = 5, 56%; PR: n = 6, 67%) whereas among malignant
tumors it was found mostly in tubulopapillary carcinomas (ER and PR:
n = 9, 29%). The ER expression was signicantly associated with ex-
pansive type of growth (p b 0.05). The PR-positive CMTs were fre-
quently noted in small pure-breed dogs (p b 0.01), among dogs
spayed at the time of mastectomy (p b 0.05) and among grade I and II
carcinomas (p b 0.01). Most patients with CMTs expressing steroid-
hormone receptors were alive at the follow-up completion (ER: n =
16, 40% and PR: n = 22, 55%), had OS over 18 months (ER: n = 16,
40% and PR: n = 21, 53%), and did not show any metastases (ER:
n = 12, 39% and PR: n = 14, 45%). However, these results were not sig-
nicant. In the group of dogs with a survival period N 18 months the cor-
relation between ER and PR expression was signicantly positive (r =
0.457, p b 0.01), and the correlation between PR expression and MI was
signicantly negative (r = 0.0410, p b 0.05).
Notes to Table 2:
NS - not signicant; Pos. - positive; Neg. - negative.
a
Spay status at the time of diagnosis was unknown only for one dog.
b
Spayed at least one year prior diagnosis.
c
Malignant tumors only.
p-value b 0.05 was assumed as signicant.
p-value b 0.01.
p b 0.001.
3.2. Expression of Bcl-2 in canine mammary tumors
Cytoplasmic expression pattern for Bcl-2 was found in 75% (30/40)
of all the examined CMTs (Fig. 3A). Seventy eight percent (7/9) of
them were benign and 74% (23/31) were malignant (Table 2). The
Bcl-2 expression was evaluated as mild in 27% (n = 11), moderate
and strong in 48% (n = 19) of the examined CMTs. The Bcl-2 expression
was more often in malignant tumors of grade I and II (55%, 17/31) com-
pared to malignant tumors of grade III (19%, 6/31). Moderate and strong
expressions were detected mostly in benign CMTs (67%, 6/9) and in the
group of malignant tumors of grade I and II (32%, 10/31). Of the 46% (6/
31) complex carcinomas and carcinomas arising in benign tumor, as
well as 52% (15/29) CMTs without necrosis were scored moderate and
strong for Bcl-2 expression. Among the benign tumors, simple adeno-
mas most commonly (56%, 5/9) showed moderate and strong Bcl-2 ex-
pression. There were no signicant differences between histological
types of CMTs and Bcl-2 expression. Moderate and strong Bcl-2 expres-
sion in CMTs (18/19, 95%) were more frequent in dogs with longer sur-
vival after surgery (p b 0.01). Positive Bcl-2 status was signicantly
associated with metastatic status and TNM stage (both p b 0.05)
(Table 2). Most long-survivors without metastases and with TNM
stage I, II, III had Bcl-2-positive CMTs (83%, 20/24 and, 81%, 29/36 re-
spectively), and 50% of them (50%, 12/24, and 50%, 18/36, respectively)
showed moderate or strong Bcl-2 expression. Interestingly, only cases
with cancer-related mortality (17%, 1/6) revealed moderate or strong
Bcl-2 expression. Six dogs (60%, 6/10) with Bcl-2 positive tumors died,
and 4 of them (4/6) died due to cancer-related issues. However, there
was no signicant correlation between Bcl-2 expression and OS
(Fig. 2A). In the group of dogs with a follow-up period 18 months,
Bcl-2 expression was correlated with CC3 expression (r = 0.602,
p b 0.001).
3.3. Expression of Bax in canine mammary tumors
Expression of Bax protein was detected in the cytoplasm of 60% (24/
40) of the CMTs (Table 1), however in three cases there was both cyto-
plasmic and nuclear reactivity of Bax (Fig. 3B). Positive reaction for Bax
was more common in malignant (53%, 21/40) than benign tumors (8%,
3/40) (Table 2). Forty two percent (13/31) of the carcinomas whereas
none of the benign tumor were dened as moderate and strong for
Bax expression. Tubulopapillary carcinoma (23%, 7/31) was the most
frequent type of carcinoma within the CMTs with moderate and strong
Bax expression. Analysis of Bax expression did not reveal any signicant
association with clinicopathological features (age, tumor size, histolog-
ical type, grade, etc.) and OS (Table 2, Fig. 2B). Positive expression of Bax
was common in dogs with metastatic diseases (71%, 5/7), however a
signicant correlation was not observed. Seventy percent (7/10) of the
dogs with Bax-positive CMTs died during the follow-up period. In 50%
(3/6) of the dogs that died due to cancer-related issues Bax-positive tu-
mors were diagnosed. However, these results, are not signicant. There
were no signicant correlations between individual variables like prolif-
erative status, expression of steroid-hormone receptors, p53, Bcl-2 and
CC3 expression and survival.
3.4. Expression of CC3 in canine mammary tumors
The cleaved caspase-3 (CC3) was detected in 60% (24/40) of all CMTs
(Table 1). Its expression was detected in the cytoplasm (Fig. 3C) but in
 I. Dolka et al. / Research in Veterinary Science 105 (2016) 124133
three cases also nuclear staining pattern was noted. Among the CC3-
positive 53% (21/40) were malignant tumors and 8% (3/40) were be-
nign cases (Table 2). Carcinomas arising in benign tumor and complex
carcinomas (19%, 6/31) were most often diagnosed as having moderate
or strong CC3 expression. However, CC3 expression neither its degree
were not signicantly correlated with host/tumor factors, or OS. How-
ever, they were signicantly associated to the MI (p b 0.05) (Table 2).
Most of the CMTs diagnosed with 1.01.9 or 2.0 MI were CC3-
positive (100%, 7/7 and 67%, 4/6; respectively).
Seventy percent (7/10) of the dogs with a positive-CC3 mammary
tumor died during the follow-up period of this study, and 50% (3/6) of
dogs that died due to cancer-related issues had mammary tumor posi-
tive for CC3 (Table 1, Fig. 2C). However, these results were not signi-
cant. In the group of shorter survivals the CC3 expression was
correlated with the MI and Bcl-2 expression (r = 0.937, p b 0.001, and
r = 0.602, p b 0.05, respectively). In the group of longer survivals, the
correlation between CC3 and ER expression was signicantly negative
(r = 0.390, p b 0.05).
3.5. Expression of p53 in canine mammary tumors
Expression of p53 was observed in 30% of CMTs (12/40), all of them
were malignant (Table 2, Fig. 3D). The presence of p53 antigen signi-
cantly correlated with III malignancy grade in examined CMTs (100%,
7/7, p b 0.01). Moreover, p53 expression was signicantly associated
with the MI (p b 0.001), presence of necrosis (p b 0.001) and type of
growth (p b 0.05). All CMTs with a mitotic index 2.0/HPF (100%, 6/
6), 90% (9/10) tumors with necrosis, and 60% (6/10) with invasive
growth showed positive immunostaining for p53 protein. One-half
(n = 8) of tubulopapillary carcinomas and only one carcinoma arising
in benign tumor were p-53 positive. We found signicant association
of p53 expression and weight of pure-breed dogs (p b 0.01) (Table 2).
Expression of p53 was detected in 67% (6/9) of large breed dogs with
CMTs (German Shepherd predominated), compared to 10% (2/20) of
small breed dogs. Most patients without metastatic diseases which
stayed alive during the study were negative for p53 expression in
CMTs (67%, 16/24 and 73% 22/30; respectively). Forty percent (4/10)
of dogs with p53-positive CMTs died and 50% (3/6) of these death
cases were cancer-related, however these values were not signicant
(Table 2). Although, patients with the p53-positive CMTs had shorter
survival times than those with the p53-negative CMTs, these results
were not signicant (Fig. 2D). In the group of dogs with shorter survival
time there was signicant correlation found between p53 and ER ex-
pression (r = 0.577, p b 0.05), whereas in the group of dogs with longer
survival, signicant correlation was observed between p53 and MI (r =
0.765, p b 0.001).
4. Discussion
In this study we present results of an immunohistochemical analysis
of apoptosis-associated protein expression in CMTs and its correlation
with host and tumor factors. We analyze and describe their possible
prognostic signicance in dogs after mastectomy. The present study
demonstrated that the most of the Bcl-2-positive tumors were benign.
In addition, Bcl-2 was strongly expressed in benign lesions, in the
grade I and II differentiated carcinomas, and in dogs with lower clinical
stages of tumor according to the TNM classication. These results were
in accordance with the nding of one study conducted on CMTs (Yang
et al., 2006) and several studies conducted on HBC indicating that Bcl-
2 expression is noted in early stages of mammary tumorigenesis (Zaha
and Lazr, 2012). The Bcl-2 protein could allow cells to survive after mu-
tation and to accumulate genetic alterations. Decreasing expression of
Bcl-2 during malignant transformation may be accompanied by addi-
tional genetic changes and constitute a feature of tumor progression
(Koda et al., 2004; Martnez-Arribas et al., 2007). In contrast, Yildirim
et al. (2014) reported that Bcl-2 was over-expressed in highly
131
malignant CMTs such as solid and tubulopapillary adenocarcinomas.
The role of Bcl-2 as a prognostic marker has been extensively evalu-
ated in HBC studies, and most of them reported that patients with
the Bcl-2-positive tumors had better survival than those with
lower Bcl-2 expression (Kymionis et al., 2001; Hwang et al., 2012;
Yildirim et al., 2014). Although in our study Bcl-2 expression seemed
to correlate with favorable outcome, we did not show any signicant
correlation. To the authors' knowledge there is only one analysis of
correlation between OS and Bcl-2 expression in 27 cases of CMTs
(Yildirim et al., 2014). Being the anti-apoptotic protein, Bcl-2 is a tar-
get of therapy in breast cancer patients. Additionally, Bcl-2 correla-
tion to steroid-hormone receptors may be used to enhance tumor
response in combination therapy with tamoxifen (Deng and Letai,
2013). To date, correlation of Bcl-2 and ER expression in CMTs
has been examined only in one study and conclusions were similar
to ours (Yang et al., 2006). However, as far as we realize, this study
is the rst to evaluate correlation between PR and Bcl-2 in CMTs. Re-
garding follow-up data, we demonstrated that PR and Bcl-2 expres-
sions do not correlate. This is a relative new observation, and to
date, an inuence of progesterone on anti-apoptotic Bcl-2 protein
has been demonstrated mainly in HBC (Ciocca and Elledge, 2000;
Kymionis et al., 2001; Martnez-Arribas et al., 2007; Zaha and Lazr,
2012). However, the present study is the rst to report a positive cor-
relation between Bcl-2 and CC3 expression in CMTs in short survivals
group. It is well known that Bcl-2 inhibits caspase cascade activation,
however Bcl-2 apoptotic response can also be modulated by CC3
which is related to short survival (Hu et al., 2014). The status when
both proteins are overexpressed has been called paradoxical effects
of Bcl-2 (Grigoriev et al., 2002). We also showed that similarly to
CC3, a high level of Bax expression was frequently noted in malig-
nant CMTs and their expressions were equally distributed among
the tumor sample. However, we examined Bax and CC3 expression
only in tumor tissue and not in adjacent tissues. Kumaraguruparan
et al. (2006) reported lower CC3 and Bax expressions in CMTs com-
pared to adjacent normal tissues examined using Western blot.
Interestingly, in a few cases of CMTs we detected nuclear CC3 and
Bax staining patterns. The possible explanation for this is that Bax
forms complexes with wild type p53 as a response to factors stimulating
apoptosis (Raffo et al., 2000; Vargas-Roig et al., 2008). Other authors
suggested that nuclear Bax translocation may temporarily attenuate
the cells' ability to trigger apoptosis through an intrinsic pathway. How-
ever, the mechanism and biological signicance of this phenomenon
still remain undened (Vargas-Roig et al., 2008). Regarding the nuclear
CC3 expression, some authors have suggested that the CC3 protein can
be actively translocated from the cytoplasm into the nucleus during ap-
optosis (Vakkala et al., 1999; Grigoriev et al., 2002; Kamada et al., 2005).
Because of the small number of samples with nuclear Bax and CC3 ex-
pression we did not perform any survival analysis.
We observed a high level of Bax expression in metastatic tumors. We
did not nd signicant associations between Bax or CC3 expression and
clinicopathological features. This is in accordance with previous results
showing that Bax and CC3 expression had no prognostic inuence on
OS (Kymionis et al., 2001; Yildirim et al., 2014; Bongiovanni et al.,
2015). In contrary, in metastatic HBC reduced Bax expression was asso-
ciated with shorter survival (Krajewski et al., 1995).
We noted signicant association between CC3 expression and MI in
all CMTs as well as in the group of dogs with shorter survival time. This
nding conrms impaired balance between proliferation and apoptosis
in CMTs (Vakkala et al., 1999; Huang et al., 2015).
Our results revealed a signicant negative correlation between CC3
and ER expression in CMTs. This nding supports a dual mode of ac-
tion for estrogen, which besides stimulation of growth and survival pro-
tects cells against apoptosis (Lewis-Wambie and Jordan, 2009).
Interestingly, the estrogen-mediated protection against apoptosis was
observed in a group of dogs with longer survival times. Still very little
is known about the underlying mechanisms by which estrogen inhibits
132 I. Dolka et al. / Research in Veterinary Science 105 (2016) 124133

apoptosis. However, the CC3 and ER status may be clinically important size, mitotic index, type of growth, tumor metastasis and TNM stage
determinants of treatment response (Lewis-Wambie and Jordan, 2009). are still the most reliable factors for survival prediction.
The expression of p53 in CMTs has been previously investigated and
was found to be associated with aggressive tumor behavior Conict of interest
(opuszyski et al., 2010). The p53 expression was more frequently
demonstrated in CMTs of dogs with shorter survival, however it was The authors declare they have no competing interests.
not signicant even if some authors have previously claimed that p53
protein expression in CMTs was correlated with poor patient survival Acknowledgements
(Lee et al., 2004; opuszyski et al., 2010). Thus, its prognostic signi-
cance still remains questionable (Klopeisch et al., 2011). Interestingly, Part of this paper was presented during a poster session at the Meet-
p53-positive CMTs were mostly observed in large breed dogs. Veldhoen ing of the European Society of Veterinary Pathology and European Col-
et al. (1999) found high expression of p53 in Boxers with mammary lege of Veterinary Pathologists (ESVP/ECVP) Belgrade, 8-11
cancer. This suggests a link between heritable p53 gene mutations and September 2010 and published as an abstract in the Proceedings of
breed predisposition to cancer. If this nding is conrmed in further the ESVP/ECVP 2010, pp. 168 and in Journal of Comparative Pathology
studies, it may be especially important for breeders because of a poten- 2010, vol. 143, issue 4, p. 315. This paper was supported by the funds
tially expected inherited predisposition to cancer in particular breeds or of Department of Pathology and Veterinary Diagnostics, Faculty of
families of dogs. In our study p53 status was not a predictive factor for Veterinary Medicine, WULS. The authors would like to express their
overall survival, however a statistically signicant interaction between gratitude to Professor Elbieta Malicka for the assistance with histo-
p53 and MI was identied in the group of dogs with longer survival pathological examination of canine mammary gland tumors.
time. A plausible explanation for this surprising result could be rela-
tively small number of events such as death in total population. We
Appendix A
demonstrated that p53 over-expression was associated with the pres-
ence of necrosis. Similar results were reported in a breast cancer study
(Kato et al., 2002). Some of the studies have also indicated that mutant Table A.1
p53 promotes the nuclear factor kappa B (NFB) activity by the tumor The clinical and histopathological data of 40 female dogs with CMTs.
necrosis factor (TNF) and sustains secretion of pro-inammatory cyto-
Host and tumor factors N %
kines as well as leads to apoptosis inhibition (Estrela-Lima et al., 2010;
Walerych et al., 2012). The present study revealed a positive correlation Age at diagnosis (years)
8 years 14 35
between ER and p53 in the group of dogs with shorter survival. This is
N8 years 26 65
a relatively new nding that has been not demonstrated previously. Breed
Some references indicated that ER status of CMTs was attributed to fa- Pure 29 72
vorable prognosis and a good response to endocrine treatment, similar Mixed 11 28
Weight pure-breed dogs
to HBC reports (Nieto et al., 2000; Zaha and Lazr, 2012). Berger et al.
Small (b20 kg) 20 69
(2012) showed that over-expression of p53 increased the expression Large (20 kg) 9 31
of ER in the MCF-7 cell line. It is not surprising because p53 is a direct Spay status at the time of diagnosisa
transcriptional target of ER. This cross-talk may be important for clini- Intact 4 10
cians to make a decision about the hormone therapy. Spayed before diagnosisb 9 23
Spayed concomitantly at the time of mastectomy 26 67
According to previously published papers the univariate analysis of
Tumor size
mammary tumor-bearing dogs demonstrated several factors signi- b3 cm 23 57
cantly associated with OS after surgery, including tumor size, mitotic 3 cm 17 43
index, type of growth, metastasis and TNM stage (Philibert et al., Affected chainc
Left 20 56
2003; Dutra et al., 2008; Pea et al., 2013). Our study revealed signi-
Right 16 44
cant differences between longer- and shorter-survivals within the fol- Affected glandd
lowing factors: age, spay status, tumor size, carcinoma type (only I 5 15
tubulopapillary carcinoma and carcinoma arising in benign tumor), mi- II, III 2 6
totic index and type of tumor growth. In the multivariate analysis, youn- IV, V 27 79
Tumor type
ger dogs with CMTs had signicantly longer OS compared to older dogs.
Benign 9 23
This nding was in line with previous studies in dogs, which conrmed Benign mixed tumor 1 11
that age was the best prognostic clinical feature (Hellmn et al., 1993). Adenoma simple 7 78
Adenoma complex 1 11
Malignant 31 77
Simple type 18 58
5. Conclusions Tubular 1 6
Tubulopapillary 16 88
The evidence of changes in expression of apoptosis-associated pro- Solid 1 6
teins in CMTs at different developmental/malignant transformation Carcinoma arising in benign tumor 10 32
Complex carcinoma 3 10
stages conrmed that these signaling pathways are important during
Histologic grade
tumorigenesis. In this study, the expression of apoptosis-associated pro- I 9 29
teins had no prognostic correlation with OS of dogs with mammary tu- II 15 48
mors. We showed that the Bcl-2 expression is related to early stage of III 7 23
CMTs. The malignant progression of CMTs was associated with Bax, Mitotic index
00.9/HPF 27 67
CC3 and p53 protein expression. The higher expression of p53 was asso- 1.01.9/HPF 7 18
ciated with unfavorable clinicopathological parameters. Our study dem- 2.0/HPF 6 15
onstrated possible cross-talk between various proteins, for example Bcl- Necrosis
2 and CC3; p53, CC3 and ER in CMTs in short- or long-survival groups. Yes 11 27
No 29 73
However, more studies involving higher numbers of patients are re-
Type of growth
quired. Based on our results, we conclude that age of a dog, tumor
 I. Dolka et al. / Research in Veterinary Science 105 (2016) 124133 133

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Host and tumor factors N % breast epithelium, benign mammary dysplasia and in breast cancer. Folia Morphol.
(Warsz) 63, 337341.
Expansive 30 75 Koda, M., Przystupa, W., Jarzabek, K., Wincewicz, A., Kanczuga-Koda, L., Tomaszewski, J.,
Invasive 10 25 Sulkowska, M., Wolczyski, S., Sulkowski, S., 2005. Expression of insulin-like growth
Tumor metastasis factor-I receptor, estrogen receptor , Bcl-2 and Bax proteins in human breast cancer.
Yes 7 23 Oncol. Rep. 14, 9398.
No 24 77 Krajewski, S., Blomqvist, C., Franssila, K., Krajewska, M., Wasenius, V.M., Niskanen, E.,
Survival Nordling, S., Reed, J.C., 1995. Reduced expression of proapoptotic gene Bax is associ-
Alive 30 75 ated with poor response rates to combination chemotherapy and shorter survival in
Died 10 25 women with metastatic breast adenocarcinoma. Cancer Res. 55, 44714478.
Cause of death Kumaraguruparan, R., Karunagaran, D., Balachandran, C., Manohar, B.M., Nagini, S., 2006.
Cancer-related mortality 6 60 Of humans and canines: a comparative evaluation of heat shock and apoptosis-
associated proteins in mammary tumors. Clin. Chim. Acta 365, 168176.
Non-cancer-related mortality 4 40
Kymionis, G.D., Dimitrakakis, C.E., Konstadoulakis, M.M., Arzimanoglou, I., Leandros, E.,
TNM staging
Chalkiadakis, G., Keramopoulos, A., Michalas, S., 2001. Can expression of apoptosis
I 22 55
genes, bcl-2 and bax, predict survival and responsiveness to chemotherapy in
II 13 32 node-negative breast cancer patients? J. Surg. Res. 99, 161168.
III 1 3 Lana, S.E., Rutteman, G.R., Withrow, S.J., 2007. Tumors of the mammary gland. In:
IV 4 10 Withrow, S.J., Vail, D.M. (Eds.), Withrow & MacEwen's Small Animal Clinical Oncol-
V 0 0 ogy, fourth ed. Saunders Elsevier. St. Louis, Missouri, pp. 619636.
Lee, Ch., Kim, W., Lim, J., Kang, M., Kim, D., Kweon, O., 2004. Mutation and overexpression
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d
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