Báo cáo khoa học: "Mutation and overexpression of p53 as a prognostic factor in canine mammary tumors" pptx

-2851$/ 2)
9H W H U L Q D U \ 
6FLHQFH
J. Vet. Sci.
(2004),
/
5
(1), 63–69
Mutation and overexpression of
p53
as a prognostic factor in canine
mammary tumors
Chung-Ho Lee, Wan-Hee Kim, Ji-Hey Lim, Min-Soo Kang
1
, Dae-Yong Kim
1
and Oh-Kyeong Kweon*
Department of Veterinary Surgery, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Korea
1
Department of Veterinary Pathology, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Korea
We concentrated ourselves to evaluate the prognostic
significance of the
p53
gene mutations, its protein
expression and MIB-1 index as a proliferative marker in
canine mammary tumors. In the present study, a total of 20
cases were examined, among which there were 5 malignant
mixed tumors, 4 mammary gland adenocarcinomas, 1
papillary adenocarcinoma, 8 benign mixed tumors and 2
mammary gland adenomas. Positive immunostaining for
p53 with PAb240 antibody was found in 2 benign (20%)

and 3 malignant (30%) tumors. However, PAb421 antibody
did not give positive result at all. In Western blot analysis,
the p53 expression in benign and malignant tumors was
detected in 4 and 3 cases, respectively.
p53
mutations were
found in 6 cases out of the cases with detected p53 protein
expression. The MIB-1 index in benign and malignant
tumors were 17.6
±
20.8% and 29.0
±
27.2%, respectively
and there was no significant difference between tumor
types. There was a significant correlation between
p53
mutations and p53 overexpression (correlation coefficient =
0.5,
p
< 0.05). In Kaplan-Meier survival analysis, the p53
index was associated with significantly shortened survival
time (
p
< 0.01). In multivariate analysis, p53 overexpression
was only an independent factor for indicator of worse
prognosis in canine mammary tumors (
p
= 0.01). These
results demonstrated that
p53

gene mutations and protein
overexpression using the PAb240 anti-p53 antibody were
useful predictors of increased malignant potential and poor
prognosis in canine mammary tumors.
Key words:
canine, mutation, overexpression,
p53
, prognosis
Introduction
Canine mammary tumors account for half of all tumors in
bitches and approximately 40-50% of them are considered
malignant [2,3,24]. Effective treatment method with prompt
accurate diagnostic procedure is the prime importance for
this life threatening neoplasm. In surgical intervention,
about 48% of dogs died or euthanized even within 1 year
after their surgery due to recurrence or metastasis [10].
Despite of the intensive clinico-pathological investigation, a
very little is known about the prognosis and causes of canine
mammary tumor [2]. Precise clinical and pathologic
stratagies are subjected to numerous errors, and imaging
methods are not very sensitive to initial tumor spread [21].
Therefore, accurate and additional prognostic aids are
required to identify patients at high risk.
Recent advances in tumor biology have identified a
number of markers that may form a basis for tumor
stratification [7,10,26]. Numerous studies have been focused
on the investigation of the significant role of the
p53
tumor
suppressor gene in the tumorigenesis of human and canine

cancers. Mutations of the
p53
gene are believed to be the
most common genetic alteration in canine mammary tumors
like other human and dog malignancies and many studies
also indicated that
p53
mutation is associated with tumor
progression [11,16,17,30,33]. Mammary carcinomas in
dogs have similarities of prevalence, metastasis and disease
pattern compared with the breast cancer in human [27]. In
humans,
p53
gene mutations have been documented in
breast cancer by numerous intensive studies [3,6]. These
mutations have been detected in 15-34% of cases and have
been considered an important indicator of poor prognosis
and shortened survival rate [3,8]. Some abnormalities of the
p53
gene have been documented in spontaneous thyroid
carcinoma, oral papiloma, circumanal gland adenoma,
osteosarcoma and lymphoma in dogs [5,14,18,19,32]. Our
previous report with the data in the present study
demonstrated that
p53
mutations were in 7 out of 20 cases
studied and 3 out of 4 dogs died of mammary carcinoma had
a
p53
mutation [15].

In the present study, the relationship among the clinical
and histological parameters, the
p53
gene mutations, its
protein expression and MIB-1 index as a proliferative
marker in canine mammary tumors was evaluated to get the
prognostic markers.
*Corresponding author
Phone: +82-2-880-1248; Fax: +82-2-888-2866
E-mail:
64 Chung-Ho Lee
et al.
Materials and Methods
Tumor specimens
Twenty female dogs were selected which were referred to
the Veterinary Medical Teaching Hospital (VMTH), Seoul
National University, for diagnosis and treatment. The
individual basic data were described in our previous report
[15]. Metastasis suspicions were solved by thoracic
radiographs and ultrasonographs of liver, kidney and spleen
before surgery. Each case was classified according to the
clinical TNM staging of canine mammary tumors modified
from the World Health Organization [24]. All patients
underwent either by lumpectomy or mastectomy and none
of the patients had experienced preoperative systemic
chemotherapy or radiotherapy.
Mean follow-up period was 16 months (range, 2-38
months) and the last clinical assessment was used to
determine final status. Survival time was defined as the time
from tumor biopsy or excision to the time of death due to

progression of disease or the last clinical assessment.
Recurrence was defined as the occurrence of mammary
tumor again after surgery at any stage or grade. Progression
of the disease was considered at the death of the animal from
cancer or remote lymph node or organs metastasis.
Tissue blocks of each tumor were frozen in liquid nitrogen
immediately after surgical removal and stored at
−
70
o
C for
DNA and protein extraction. Some adjacent sections were
immediately fixed in 10% neutral buffered formalin and
routinely processed for embedding in paraffin. Serial
sections were cut 3
µ
m from each specimen block and
prepared for immunohistochemistry and histopathology.
Mutational analysis
The mutational analysis of
p53
was performed as
described in our previous report [15].
Western blot analysis of anti-P53 antibody
Protein samples were prepared by homogenizing tumor
specimens in buffer solution containing 50 mM Tris-HCl
(pH 8.0), 150 mM NaCl, 0.02% sodium azide, 1% TritonX-
100, 1
µ
g/ml aprotinin and 100

µ
g/ml phenylmethylsulfonyl
fluoride (PMSF) using a Teflon pestle. They were then
boiled at 100
o
C for 5 minutes. The lysates were sonicated
and centrifuged at 12,000 rpm for 10 minutes. Supernatant
protein concentrations of the lysates were measured using
the BioRad protein assay kit (BioRad, Hercules, USA).
Equal amounts of protein (20
µ
g) from each tissue sample
were then boiled for 5 minutes and electrophoresed on a
10% SDS/polyacrylamide gel with prestained size markers
(Color markers, Sigma, Saint Louis, USA). Following
electrophoresis, proteins in the gels were transferred onto
nitrocellulose membrane using Mini Trans-Blot
®
apparatus
(BioRad, Hercules, USA). Relative protein concentration
per lane and transfer efficiency were checked by staining
nitrocellulose membranes with Ponceau S (Amresco Inc.,
Solon, USA). Membranes were blocked non-specific
binding by incubating in blocking solution containing Tris-
buffered saline (TBS)/0.05% Tween-20 (TBST) with 5%
(w/v) skimmed milk overnight at 4
o
C. The blotted
membrane was incubated in monoclonal mouse anti-human
p53 protein antibody (PAb421, Oncogene

TM
research
products, San Diego, CA, USA) diluted at 1 : 100 with
blocking solution for one hour at room temperature and then
rinsed three times for 5 minutes each with TBST, followed
by anti-immunoglobulin G horseradish peroxidase
conjugate secondary antibody (horseradish peroxidase
conjugated goat anti-mouse IgG, Zymed Lab. Inc., So. San
Francisco, CA, USA) diluted at 1 : 2000 with blocking
solution. The membrane was washed three times for 5
minutes each with TBST and once for 5 minutes with TBS.
Membranes were processed using enhanced
chemiluminescence (ECL) Western blotting detection
reagents (Amersham Pharmacia biotech, Buckinghamshire,
England) and autoradiography according to the
manufacturers instructions.
Immunohistochemistry
The immunohistochemical study was performed using the
antibodies against the p53 protein and MIB-1 on formalin-
fixed, paraffin-embedded tissue specimens from initial
tumors. PAb240 and PAb 421 (monoclonal antibody to p53
protein of mouse origin, 1:50 dilution, Oncogene
TM
research
products), which recognize different epitopes of the p53
product, were used for the detection of overexpression of
mutant p53 protein, and MIB-1 (monoclonal antibody to Ki-
67 antigen of mouse origin, 1 : 50 dilution, Immunotech,
Marseille, France) for the detection of Ki-67 antigen.
Formalin-fixed sections were deparaffinized in two

changes of xylene for five minutes each and rehydrated
through sequential immersions in four changes of graded
concentrations of ethanol. Sections were then rinsed in
distilled water. For unmasking of nuclear antigen, tissue
sections were boiled for six minutes using a microwavable
pressure cooker on a citrate buffer (10 mM, pH 6.0), and
were allowed to cool down gradually to the room
temperature and then rinsed in PBS. In p53 staining, slides
were digested in 0.1% porcine trypsin for 20 minutes at
37
o
C and rinsed three times with PBS. Endogenous
peroxidase present within the tissue was inactivated by
immersion of the slides in 3% hydrogen peroxide in
methanol and the sections blocked with a protein blocker
(Histostain SP kit, Zymed Lab. Inc., So. San Francisco, CA,
USA). Each tissue section was incubated overnight at 4 with
the appropriate primary antibody to p53 protein and MIB-1.
Slides were rinsed three times in PBS, and then incubated
for 30 minutes with biotinylated secondary antibody
(Histostain SP kit, Zymed Lab. Inc.). PBS-washed sections
were then incubated for 20 minutes in the streptavidin-
p53
as a prognostic factor in cmt 65
peroxidase conjugate solution (Histostain SP kit, Zymed
Lab. Inc.) for detection of bound primary antibody. After
washing in PBS three times, slides were incubated in 3, 3-
diaminobenzidine solution. Color change was monitored on
positive-control slides and was stopped by immersion in
distilled water, and then briefly counterstained with

hematoxylin only in MIB-1 immunostaining. Slides were
dehydrated through ascending alcohol and xylene and then
coverslip applied. All steps were carried out at room
temperature in a humidified chamber unless otherwise
indicated.
Formalin-fixed, paraffin-embedded human gastric cancer
and oral squamous cell carcinoma tissue block were used as
positive controls. Negative controls were provided by
treating with non-immune serum, instead of the primary
antibody. Histologically normal mammary gland tissue
block served as negative tissue controls, and nonneoplastic
tissue on each slide provided internal negative controls.
Microscopic evaluation
Light microscopic evaluation of immunohistochemically
treated sections for positive nuclear staining was performed.
The quality of each immunohistochemically stain was
assessed by comparing the sections with an accompanying
positive control slide.
A tumor sample was regarded as p53 positive if nuclear
staining was clearly detected, but cytoplasmic staining alone
was not recorded as positive. Positively staining was
evaluated semi-quantitatively using a previously described
system where 0 = no staining; 1 = <10%; 2 = 10-50%; and 3
= >50% of cells. Based upon previous reports [25,29], we
considered tumors to be p53 positive by receiving 2 or 3
score.
Proliferative activity was examined by staining with an
anti-Ki-67 specific antibody, MIB-1, and was evaluated
separately in each case after counting at least 500 nuclei in
3-5 randomly selected high-power fields of the section

(
×
400). Proliferation indexes were calculated as the
percentage of cells with positive nuclear staining compared
with the total nuclear area.
Statistical analysis
MIB-1 index was analysed with Mann-Whitney U test to
determine whether differences per tumor type were
significant. Correlation was estimated among
clinicopathological parameters,
p53
mutations, p53 index
and MIB-1 index. Survival curves on each prognostic
variables were computed using the Kaplan-Meier survival
analysis and compared curves by log rank test. Multivariate
Coxs regression analysis was performed to determine the
prognostic value of several parameters.
All statistical analyses were performed with software
package SPSS (Release 8.0, SPSS inc.) and a
P
-value of
<0.05 was considered as statistically significant.
Results
Clinical features of the canine patients
Histopathologic study revealed that there were 5
malignant mixed tumors (2 stage V, 1 stage IV, 2 stage III), 4
mammary gland adenocarcinomas (1 stage V, 3 stage IV), 1
papillary adenocarcinoma (1 stage II), 8 benign mixed
tumors ( 2 stage IV, 3 stage II, 3 stage I) and 2 mammary
gland adenoma (1 stage II, 1 stage I). 4 dogs with malignant

tumors and 2 with benign tumors had palpably enlarged
lymph nodes in axillary and inguinal region. It was found
that 16 dogs were alive and 4 died. Local recurrence
occurred in 4 dogs within 2, 6, 12 and 13 months after the
first operation respectively, and further recurrence was found
in a dog even after 1 month of re-excision.
Identification of tumor-associated
p53
gene alterations

p53
gene alteration was found in 7 cases (35%) and their
different mutational characteristics also identified. four mis-
sense and 1 non-sense mutations were detected in 10
malignant lesions (40%), and 2 mis-sense and 1 silent
mutations were found in 10 benign mammary tumors
(30%). Among the 6 mis-sense mutations, 5 mutations were
located in highly conserved domains II, III, IV and V. In a
case, the codon change CGA
→
TGA results in the
introduction of a stop codon at position 213 and another one
showed the presence of a silent mutation. G:C
→
A:T
transitions were detected in 5 mutations and transversions
were shown in 3 dogs.
Overexpression of p53 protein and MIB-1
Various positive nuclear immunostainig was detected in
each of the control sections of human gastric cancer and oral

squamous cell carcinoma. Staining was not observed in
negative controls treated with non-immune serum in place
of the primary antibody.
Positive immunostaining for p53 protein with PAb240
antibody was found in 5 case (25%). The proportion of
benign and malignant lesions stained for p53 are 20%
and 30% respectively (Fig. 1b, 2b). However, PAb421
antibody did not give positive result at all. There was a
significant correlation between
p53
mutations and p53
overexpression (correlation coefficient = 0.50,
p
<0.05,
Table 1).
In Western blot analysis, the p53 protein expression in
benign and malignant tumors was detected in 4 and 3 cases,
respectively (Fig. 2).
p53
gene mutations were found in 6
cases out of the cases with detected p53 protein expression.
The MIB-1 positive range was from 2% to 75% (23.3
±
24.3%). The MIB-1 index in benign and malignant tumors
were 17.6
±
20.8% and 29.0
±
27.2% (Fig. 1c, and 2c).
There was no significant difference in the MIB-1 index

between tumor types.
66 Chung-Ho Lee
et al.
p53 nuclear overexpression, survival time and prognostic
value
In Kaplan-Meier survival analysis, the p53 index was
associated with significantly shortened survival time (Fig. 3,
p
< 0.01). The results of multivariate analysis for
determining the prognostic value of several parameters are
shown in Table 2. P53 overexpression was only an
independent factor for indicator of worse prognosis in
canine mammary tumors (
p
= 0.01).
Discussion
In the present study, p53 immunohistochemical
expression by using PAb240 anti-human p53 antibody is
found in 25% of the canine mammary tumors. Similar
expression rate was reported by other investigators [9,28,
F
ig. 1.
Photomicrographs of a section of the case with stage II mammary gland adenoma (1a, 1b, 1c) and of a section of the case with sta
ge
V
malignant mixed tumor (2a, 2b, 2c) stained with hematoxylin and eosin (a), immunohistochemically for p53 with an anti-p53 antibo
dy
(
PAb240, Oncogene) (b) and MIB-1 with an anti-Ki-67 antibody (MIB-1, Immunotech) (c). (1a) Note well-differentiated and we
ll-

c
apsulated neoplastic cells. H&E stain,
×
200. (1b) Note weak p53 nuclear positive immunostaining of several tumor cells. No countersta
in,
×
200. (1c) Note moderate proliferative activity of several neoplastic cells expressed as diffuse MIB-1 immunostaining. Hematoxy
lin
c
ounterstain,
×
200; (2a) Note pleomorphic tumor cells with a moderate amount of cytoplasm and hyperchromatic 2 to 3 nuclei . H&
E
s
tain,
×
200. (2b) Note diffuse strong p53 nuclear positive immunostaining of several tumor cells. No counterstain,
×
200. (2c) Note hi
gh
p
roliferative activity of several neoplastic cells expressed as diffuse MIB-1 immunostaining. Hematoxylin counterstain,
×
200.
p53
as a prognostic factor in cmt 67
34]. The PAb240 antibody used in this study has an epitope
within amino acid residues 371-380 of human p53 and is
able to stain tumor cells with
p53

mis-sense mutations. In
many other studies, immunoreactivity of the canine p53
protein towards CM-1 (rabbit anti-human p53 polyclonal
antibody), PAb240 (mouse anti-human p53 monoclonal
antibody), BP53-12 and PAb122 (mouse anti-human p53
monoclonal antibody), which recognize different epitopes of
the p53 product, has been found in various canine
neoplasms by immunohistochemical analysis [1,9,12,25,31,
35]. Veldhoen and Milner [31] suggested that canine p53
protein had a strong reactivity in an immunoprecipitation
assay towards monoclonal anti-human antibody, PAb421. In
order to define the immunoreactivity of canine p53 further,
PAb421 antibody was used in this study by
immunohistochemistry. However, PAb421 antibody did not
give positive result at all. Albaric
et al
. [1] and Haga
et al
.
[12] suggested that p53 positive result was able to alter
according to different p53 antibodies and especially Ab-7
and DO-7 anti-human p53 antibodies did not react in canine
tumors. This demonstrated that there might be local
differences in the nature and organization of amino acid
residues on the surface of the canine p53 molecule when
compared to human p53 proteins.
Multivariate regression analysis and Kaplan-Meier
survival analysis in the present study revealed that the p53
overexpression index is an independent risk factor for
increased recurrence and death from these tumors and

significantly shortened the survival time. Similarly it has
been suggested that alterations in p53 expression correlated
with highly aggressive tumor behavior as a promising new
parameter to evaluate the cellular biology and prognosis of
human mammary ductal carcinoma [22,25]. P53 expression
tends to be more frequent in phyllodes tumors with higher
malignant potential [29]. However, reported elsewhere
immunohistochemistry for p53 expression is not a suitable
prognostic markers in canine mammary carcinoma and
female breast cancer [20,34].
Positive staining of p53 protein was detected in two
benign mammary tumors accompanied by increased index
of MIB-1 in this study. A recent study by Rohan
et al
. [23]
concluded that p53 staining in benign breast biopsies was
associated with an increased risk of future breast cancer.
Thus, p53 protein levels of wild type or mutant protein may
be associated with the subsequent development of canine
mammary and human breast cancer Many investigations
have been focused on the role of immunohistochemical
overexpression in predicting
p53
mutation [11]. Done
et al
.
[6] concluded that p53 inactivation occurred prior to
invasion in breast carcinogenesis, with mutations being
uniformly identified in ductal carcinoma in situ associated
with

p53
-mutated invasive carcinomas.
Immunohistochemical analysis of MIB-1, as a
proliferative marker is a good approach for evaluation of the
growth fraction [4,13]. MIB-1 is a monoclonal antibody
against recombinant parts of the Ki-67 antigen and true Ki-
67 equivalents [4]. Sarli
et al
. [26] suggested that MIB-1
index revealed a significant association with prognosis in
canine malignant mammary tumors. The MIB-1
immunostaining found in this study tended to be more
frequent in malignant mammary tumors, but it was not
Table 1.
Correlation coefficient rates between clinicopathological parameters,
p53
mutations, p53 index and MIB-1 index
Stage Tumor type
P53
mutations P53 index MIB-1 index
Stage
Tumor type
P53
mutations
P53 index
MIB-1 index
1
a)
0.677
a)

0.196
0.155
-0.048
a)
1
0.105
0.153
0.226
1
a)
0.501
b)
0.429
1
0.397 1
a)
p
<0.01,
b)
p
<0.05
F
ig. 2.
P53 protein expression in benign (a) and malignant (
b)
m
ammary tumors by Western blot.
Table 2.
Multivariate analysis of clinicopathological factors,
p53

mutations, p53 overexpression and MIB-1 index.
Prognosis
Age
Stage
Tumor type
P53
mutations
P53 overexpression
MIB-1 index
N.S.
N.S.
N.S.
N.S.
P
= 0.0122
N.S.
N.S.: not significant
68 Chung-Ho Lee
et al.
significant.
The present study suggested that
p53
gene mutations and
protein overexpression using the PAb240 anti-p53 antibody
were useful predictors of increased malignant potential and
worse prognosis in canine mammary tumors.
References
1. Albaric O, Bret L, Amardeihl M, Delverdier M.
Immunohistochemical expression of p53 in animal tumors: a
methodological study using four anti-human p53 antibodies.

Histol Histopathol 2001, 16, 113-121.
2. Benjamin SA, Lee AC, Saunders WJ. Classification and
behavior of canine mammary epitherial neoplasms based on
life-span observations in beagles. Vet Pathol 1999, 36, 423-
436.
3. Bergh J, Norberg T, Sjogren S, Lindgren A, Holmberg L,
Coplete J. Sequencing of the
p53
gene provides prognostic
information in breast cancer patients, particularly in relation
to adjuvant systemic therapy and radiotherapy. Nature Med
1995, 1, 1029-1034.
4. Cattoretti G, Becker MHG, Key G, Duchrow M, Schluter
C, Galle J, Gerdes J. Monoclonal antibodies against
recombinant parts of the Ki-67 antigen (MIB 1 and MIB 3)
detect proliferating cells in microwave-processed formalin-
fixed paraffin sections. J Pathol 1992, 168, 357-363.
5. Devilee P, Van Leeuwen IS, Voesten A, Rutteman GR, Vos
JH, Cornelisse CJ. The canine
p53
gene is subject to
somatic mutations in thyroid carcinoma. Anticancer Res
1994, 14, 2039-2046.
6. Done SJ, Arneson NCR, Özçelik H, Redston M, Andrulis
IL. p53 protein accumulation in non-invasive lesions
surrounding
p53
mutation positive invasive breast cancers.
Breast Cancer Res Treat 2001, 65, 111-118.
7. Donnay I, Rauis J, Devleeschouwer N, Wouters-Ballman

p, Leclercq G,Verstegen J. Comparison of estrogen and
progesterone receptor expression in normal and tumor
mammary tissues from dogs. Am J Vet Res 1995, 56, 1188-
1194.
8. Elledge RM, Allred DC. Prognostic and predictive value of
p53 and p21 in breast cancer. Breast Cancer Res Treat 1998,
52, 79-98.
9. Gamblin RM, Sagartz JE, Couto CG. Overexpression of
p53 tumor suppressor protein in spontaneously arising
neoplasms of dogs. Am J Vet Res 1997, 58, 857-863.
10. Graham JC, Myers RK. The prognostic significance of
angiogenesis in canine mammary tumors. J Vet Intern Med
1999, 13, 416-418.
11. Greenblatt MS, Bennett WP, Hollstein M, Harris CC.
Mutation in the
p53
tumor suppressor gene: clues to cancer
etiology and molecular pathogenesis. Cancer Res 1994, 54,
4855-4878.
12. Haga S, Nakayama M, Tatsumi K, Maeda M, Imai S,
Umesako S, Yamamoto H, Hilgers J, Sarkar NH.
Overexpression of the
p53
gene product in canine mammary
tumors. Oncol Rep 2001, 8, 1215-1219.
13. Ikegami S, Yoshimura I, Tsuji A, Deta K, Kimura F,
Odajima K, Asano T, Hayakawa M. Immunohistochemical
study of p53 and Ki-67 overexpression in grade 3 superficial
bladder tumor in relationship to tumor recurrence and
prognosis. Nippon

Hinyokika Gakkai Zasshi 2001, 92, 656-
665.
14. Johnson AS, Couto CG, Weghorst CM. Mutation of the
p53
tumor suppressor gene in spontaneously occurring
osteosarcomas of the dog. Carcinogenesis 1998, 19, 213-217.
15. Lee CH, Kweon OK. Mutations of
p53
tumour suppressor
gene in spontaneous canine mammary tumors. J Vet Sci
2002, 3, 321-325.
16. Levine AJ, Momand J, Finlay CA. The
p53
tumour
suppressor gene. Nature 1991, 351, 453-456.
17. Mayr B, Dressler A, Reifinger M, Feil C. Cytogenetic
alterations in eight mammary tumors and tumor-suppressor
gene
p53
mutation in one mammary tumor from dogs. Am J
Vet Res 1998, 59, 69-78.
18. Mayr B, Schaffner W, Botto I, Reifinger M, Loupal G.
Canine tumour suppressor gene
p53
-mutation in a case of
adenoma of circumanal glands. Vet Res Commun 1997, 21,
369-373.
19. Mayr B, Schellander K, Schleger W, Reifinger M.
Sequence of an exon of the canine

p53
gene-mutation in a
papilloma. Br Vet J 1994, 150, 81-84.
20. Pietilainen T, Lipponen P, Aaltomaa S, Eskelinen M.
Kosma VM, Syrjanen K. Expression of p53 protein has no
independent prognostic value in breast cancer. J Pathol 1995,
177, 225-232.
21. Popov Z, Hoznek A, Colombel M, Bastuji-Garin S,
Lefrere-Belda MA, Bellot J, Abbou CC, Mazerolles C,
Chopin DK. The prognostic value of p53 nuclear
overexpression and MIB-1 as a proliferative marker in
transitional cell carcinoma of the bladder. Cancer 1997, 80,
1472-1481.
22. Rajan PB, Scott DJ, Perry RH, Griffith CDM. p53 protein
expression in ductal carcinoma in situ (DCIS) of the breast.
Breast Cancer Res Treat 1997, 42, 283-290.
F
ig. 3. Kaplan-Meier survival curves for dogs with p53 positi
ve
(
score
≥
2) and negative (score < 2) (
P
= 0.0089).
p53
as a prognostic factor in cmt 69
23.
Rohan TE, Hartwick W, Miller AB, Kandel RA.
Immunohistochemical detection of c-erbB-2 and p53 in

benign breast disease and breast cancer risk. J Natl Cancer
Inst 1998,
90
, 1262-1269.
24.
Rutteman GR, Withrow SJ, MacEwen EG.
Tumors of the
mammary gland. In: Small animal clinical oncology. 3rd ed.
pp. 455-467, Withrow, S. J. and MacEwen, E. G. (Ed.), W. B.
Saunders, Philadelphia, 2001.
25.
Sagartz JE, Bodley WL, Gamblin RM, Couto CG,
Tierney LA, Capen CC.
p53 tumor suppressor protein
overexpression in osteogenic tumors of dogs. Vet Pathol
1996,
33
, 213-221.
26.
Sarli G, Preziosi R, Benazzi C, Castellani G, Marcato PS.
Prognostic value of histologic stage and proliferative activity
in canine malignant mammary tumors. J Vet Diagn Invest
2002,
14
, 25-34.
27.
Sartin EA, Barnes S, Kwapien RP, Wolfe LG.
Estrogen
and progesterone receptor status of mammary carcinomas
and correlation with clinical outcome in dogs. Am J Vet Res

1992,
53
, 2196-2200.
28.
Schafer KA, Kelly G, Schrader R, Griffith WC,
Muggenburg BA, Tierney LA, Lechner JF, Janovitz EB,
Hahn FF.
A canine model of familial mammary gland
neoplasia. Vet Pathol 1998,
35
, 168-177.
29.
Shpitz B, Bomstein Y, Sternberg A, Klein E, Tiomkin V,
Kaufman A, Groisman G, Bernheim J.
Immunoreactivity
of p53, Ki-67, and c-erbB-2 in phyllodes tumors of the breast
in correlation with clinical and morphologic features. J Surg
Oncology 2002,
79
, 86-92.
30.
van Leeuwen IS, Hellmen E, Cornelisse CJ, van den
Burgh B, Rutteman GR.
P53
mutations in mammary tumor
cell lines and corresponding tumor tissues in the dog.
Anticancer Res 1996,
16
, 3737-3744.
31.

Veldhoen N, Milner J.
Isolation of canine p53 cDNA and
detailed characterization of the full length canine p53 protein.
Oncogene 1998,
16
, 1077-1084.
32.
Veldhoen N, Stewart J, Brown R, Milner J.
Mutations of
the
p53
gene in canine lymphoma and evidence for germ line
p53 mutations in the dog. Oncogene 1998,
16
, 249-255.
33.
Veldhoen N, Watterson J, Brash M, Milner J.
Identification of tumour-associated and germ line
p53
mutations in canine mammary cancer. Br J Cancer 1999,
81
,
409-415.
34.
Wakui S, Muto T, Yokoo K, Yokoo R, Takahashi H,
Masaoka T, Hano H, Furusato M.
Prognostic status of
p53
gene mutation in canine mammary carcinoma. Anticancer
Res 2001,

21
, 611-616.
35.
Wolf JC, Ginn PE, Homer B, Fox LE, Kurzman ID.
Immunohistochemical detection of
p53
tumor suppressor
gene protein in canine epithelial colorectal tumors. Vet Pathol
1997,
34
, 394-404.