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

9HWHULQDU\ 6FLHQFH 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-Kyeo

9HWHULQDU\ 6FLHQFH

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: ohkweon@snu.ac.kr

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 −70o

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

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, 1µg/ml aprotinin and 100 µg/ml phenylmethylsulfonyl

fluoride (PMSF) using a Teflon pestle They were then

boiled at 100o

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 (20µg) 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%

C The blotted membrane was incubated in monoclonal mouse anti-human

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, OncogeneTM

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

37o

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-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

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-mis-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

introduction of a stop codon at position 213 and another one

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

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,

Fig 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 stage

V malignant mixed tumor (2a, 2b, 2c) stained with hematoxylin and eosin (a), immunohistochemically for p53 with an anti-p53 antibody (PAb240, Oncogene) (b) and MIB-1 with an anti-Ki-67 antibody (MIB-1, Immunotech) (c) (1a) Note differentiated and well-capsulated neoplastic cells H&E stain, × 200 (1b) Note weak p53 nuclear positive immunostaining of several tumor cells No counterstain,

× 200 (1c) Note moderate proliferative activity of several neoplastic cells expressed as diffuse MIB-1 immunostaining Hematoxylin counterstain, × 200; (2a) Note pleomorphic tumor cells with a moderate amount of cytoplasm and hyperchromatic 2 to 3 nuclei H&E stain, × 200 (2b) Note diffuse strong p53 nuclear positive immunostaining of several tumor cells No counterstain, × 200 (2c) Note high proliferative activity of several neoplastic cells expressed as diffuse MIB-1 immunostaining Hematoxylin counterstain, × 200

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 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

1

a)

0.677a)

0.196 0.155 -0.048a)

1 0.105 0.153 0.226

1

a)

0.501b)

0.429

1

a)p < 0.01, b)p < 0.05

Fig 2 P53 protein expression in benign (a) and malignant (b)

mammary 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

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.

Fig 3 Kaplan-Meier survival curves for dogs with p53 positive

(score≥ 2) and negative (score < 2) (P = 0.0089).

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.