Canine mammary tumours: A quantitative DNA study using static cytometry

Rev Esp Patol. 2011;44(4):195---201

Patología R E V I S TA

E S PA Ñ O L A

D E

www.elsevier.es/patologia

ORIGINAL

Canine mammary tumours: A quantitative DNA study using static cytometry G.D. Cassali a,∗ , A.C. Bertagnolli b , Fatima Gärtner c , Fernando Schmitt c a

Instituto de Ciências Biológicas --- Universidade Federal de Minas Gerais, Belo Horizonte, Brazil Faculdade de Ciências da Saúde, Universidade Vale do Rio Doce, Governador Valadares, Brazil c Instituto de Patologia e Imunologia Molecular da Universidade do Porto --- Porto, Portugal b

Received 8 December 2010; accepted 8 May 2011 Available online 30 July 2011

KEYWORDS Dog; Mammary; Cancer; Neoplasm; Immunohistochemistry

PALABRAS CLAVE Perro; Cáncer; Mama; Neoplasia; Inmunohistoquímica

∗

Abstract Static cytometric analysis of DNA content was performed on formalin-fixed paraffin wax-embedded samples of 56 canine mammary neoplasms (23 benign and 33 malignant) and histology, patient age and immunohistochemical markers were compared between diploid and aneuploid tumours. 10 benign tumours (43.47%) and 16 malignant (48.48%) were aneuploid. No association was found between age and ploidy (P > 0.05). Aneuploidy was not related to age or tumour malignancy (P < 0.05). The expression of the following markers: progesterone receptor, MIB-1, CD-31, p53, c-erbB2, and cyclin D1 did not show significant differences between diploid and aneuploid tumours (P < 0.05). Epithelial and mesenchymal components of benign mixed tumours, complex adenomas and carcinomas in mixed tumours had an identical DNA content in 74.0% of cases suggesting a common cell origin of both components. © 2010 SEAP y SEC. Published by Elsevier España, S.L. All rights reserved.

Tumores mamarios caninos: estudio de ADN mediante análisis de citometría estática Resumen Un análisis del contenido de ADN por citometría estática fue realizado en muestras fijadas en formalina e incluidas en parafina de 56 neoplasias mamarias caninas (23 benignas y 33 malignas). El contenido de ADN se ha correlacionado con el aspecto histológico, la edad y con marcadores inmunohistoquímicos de las neoplasias. Diez tumores benignos (43,47%) y dieciséis malignos (48,48%) fueron aneuploides. La aneuploidía no estuvo relacionada con la edad o con el carácter maligno de los tumores (P < 0,05). La expresión de los marcadores receptor de progesterona, MIB-1, CD-31, p53, c-erbB2, y ciclina D1 no mostró diferencias significativas entre los tumores diploides y aneuploides (P < 0,05). Los componentes epiteliales y mesenquimales de los tumores mixtos benignos, adenomas complejos y carcinomas en tumores mixtos tienen un contenido de ADN idéntico en 74,0% de los casos lo que sugiere un origen celular común de ambos componentes. © 2010 SEAP y SEC. Publicado por Elsevier España, S.L. Todos los derechos reservados.

Corresponding author. E-mail address: [email protected] (G.D. Cassali).

1699-8855/$ – see front matter © 2010 SEAP y SEC. Published by Elsevier España, S.L. All rights reserved. doi:10.1016/j.patol.2011.05.005

196

Introduction Genetic aberrations have a profound impact on the prognosis of many neoplasms. Therefore, DNA content is one of the prognostic markers evaluated in breast cancer.1 In female breast cancer, neoplastic cells with irregularly increased DNA content (aneuploidy) have been associated with a poor prognosis and a lower rate of survival when compared to tumour cells with regular DNA content (euploidy).2,3 Aneuploidy has also been significantly correlated to steroid receptor. . . negativity, age. . . and percentage of S-phase fraction.4,5 Nuclear DNA content may be determined by means of image cytometry analysis (also denominated static cytometry) or flow cytometry. Static cytometry is performed by means of image analysis of Feulgen-stained paraffin sections. Image analysis of thin sections allows direct visualization of the cells analyzed and simultaneously measures other morphological information, when flow cytometry is not possible.6 This technique has the advantage of allowing the selection of individual nuclei, facilitating the rejection of artefacts and the detection of cells which comprise a small proportion of the total population.7 Furthermore, the technique eliminates the need for mechanical or chemical treatments for cell dissociation, which is required for flow cytometry. Mammary tumours are the most frequent neoplasms in female dogs and show histomorphological heterogeneity.8 A small number of studies have determined DNA ploidy in canine mammary neoplasms with flow cytometry and have verified that aneuploid lesions are associated with factors known to indicate a poor prognosis, such as lymphatic infiltration by neoplastic cells and metastasis.9,10 A further study measured DNA by image cytometry in order to clarify the histogenesis of mixed tumours.11 The use of image cytometry to analyze canine mammary neoplasms seems to be the most recommended procedure, since it distinguishes both cell populations of malignant and benign mixed tumours, which are common in this species. The aim of the present study was to analyze DNA content of canine mammary tumours using static cytometry and to verify its relationship with clinical and immunohistochemical prognostic factors. In addition, DNA content from epithelial and mesenchymal components of mixed mammary tumours was also studied.

Materials and methods Samples A total of 56 mammary tumours were surgically removed from female dogs at the ‘‘Hospital Veterinário’’ (Veterinary Teaching Hospital), ‘‘Universidade Federal de Minas Gerais’’ (UFMG) (Federal University of Minas Gerais), Brazil. The age of the bitches ranged from 3 to 15 years old, with a mean of 8.73 years. Tissue samples were fixed in 10% buffered formalin and embedded in paraffin following routine procedures. Ten consecutive sections (4 ␮m thick) were cut from the paraffin block. One section was stained with hematoxylin and eosin (H&E), another by the Feulgen procedure to detect DNA,

G.D. Cassali et al. and six sections were used for immunohistochemistry. Histological examination was conducted by three pathologists on H&E-stained sections, using the WHO diagnostic criteria for the classification of tumours in domestic animals.8

Static DNA cytometry In order to analyze DNA by static cytometry, a section of each sample was stained by the Feulgen technique. This technique is based on Bohm post-fixation (50 min) prior to the rehydration phase. After being hydrated, slides are submitted to acid hydrolysis in HCl 5N at 27 ◦ C for 60 min. They are stained (in the absence of light) by Schiff reagent for 90 min and then dehydrated and mounted. DNA content was quantified with diploid value (AC) of 30 lymphocyte nuclei as baseline. In each case, 200 nuclei of neoplastic cells were analyzed with the Leica Q550 IW system. In complex adenomas, mixed tumours and carcinomas in mixed tumours, this analysis was performed separately for epithelial and myoepithelial components, including areas of osseous and chondroid metaplasia. The images obtained were processed by the Qwin software and analyzed by Qploidy (Leica). Analysis of DNA content was performed by quantifying integrated optical density for neoplastic cells and compared to that of lymphocytes with the DNA index (DI). Cases with DI = 1.0 ± 0.15 have been considered as diploid DNA and those located out of this interval as aneuploids. DI =

mean integrated optical density of the sample population mean integrated optical density of the control population

Immunohistochemistry For immunohistochemical staining by the streptavidin---biotin-peroxidase (Ultra vision large volume detection system anti-polyvalent) technique, 5 ␮m thick sections were prepared and the antigenic retrieval technique was performed when necessary, according to the recommendation of the manufacturer of the primary antibody (Table 1). The primary antibodies and other reagents were applied using an automatic system of immunohistochemistry (Lab Vision Autostainer Model LV-1). Slides were incubated with the primary antibody for 30 min and 10 min for each one of the other stages: blocking endogenous peroxidase, blocking serum secondary antibody (Biotin Goat, Lab Vision), streptavidin biotin peroxidase (Streptavidin Peroxidase, Lab Vison) and developing chromogen 3.3-diaminobenzidine tetrahydrochloride (DAB Substrate System, Lab Vision). For positive controls, previously tested samples of human breast neoplasms were used and negative controls were obtained by substituting primary antibody for normal serum. The immunohistochemically stained sections were examined at ×400 magnification and evaluated according to the antibody used. In order to evaluate progesterone receptor (PR) immunostaining, a system based on the proportion and intensity of nuclear staining was used. The percentage of tumour cells with positive staining (proportion score, PS) was graded by eye from 0 to 3. Zero meant less than 5% staining, 1 represented 5---19%, 2 represented 20---60% and 3 indicated that more than 60% of the tumour nuclei were

Canine mammary tumours: A quantitative DNA study using static cytometry Table 1 study.

197

Primary antibodies, characteristics, sources, previous treatment and dilutions tested in the immunohistochemical

Antibody

Characteristics

Source

Previous Treatment

Dilution

PR Ki-67 c-erbB2 p53 CD31 Cyclin D1

Monoclonal, 10 A 9 Monoclonal, MIB-1 Polyclonal Polyclonal, CM1 Monoclonal Monoclonal

Immunotech, France Immunotech, France Dako, Denmark Novocastra, United Kingdom Dako, Denmark NeoMarkers, USA

TCBa TCBa TCBa TCBa TEb TCBa

1:10 1:30 1:80 1:150 1:5 1:20

a b

TCB --- treatment with heat/double-boiler. ET --- enzymatic treatment.

stained positively. The average staining intensity (intensity score, IS) of positive tumour cells was scored as 0 = negative, 1 = slight staining, 2 = moderate staining and 3 = strong staining. If the staining intensity within the tumour was heterogeneous, the predominant intensity was scored. The PS and IS scores were added to obtain a total score (TS) (range 0---6). PR was considered positive when PS was >1 and TS >2.12 The intensity of staining for human epidermal growth factor receptor 2(c-erbB-2) was scored by two observers according to the HercepTest scoring system.13 Slides were scored on a 0---3 scale as follows: 0, staining in <10% of tumour cells or no staining; 1+, faint and partial membrane staining in >10% of tumour cells; 2+, weak to moderate complete membrane staining in >10% of tumour cells; or 3+, moderate to strong complete membrane staining in >10% of tumour cells. Scores of either 2+ or 3+ were defined as positive. P53 staining was regarded as positive for tumours with more than 5% of positive stained nuclei.14 Cyclin D1 immunohistochemical intensity and distribution were semiquantitatively scored using the Allred score method.15 With this method, the intensity of the immunohistochemical reaction as viewed under the light microscope was recorded as 0, negative (no staining of any nuclei even at high magnification); 1, weak (only visible at high magnification); 2, moderate (readily visible at low magnification); or 3, strong (strikingly positive even at low power magnification). The proportion of tumour nuclei showing positive staining was also recorded as either: 1, <1%; 2, 1---10%; 3, 10---33%; 4, 34---67% and 5, ≥67%. The proportion and intensity scores were then added to obtain a total score, which ranged from 2 to 8. Specimens scored as 3 were considered or more were considered positive for cyclin D1. The proliferative index was expressed as the percentage of nuclei marked with MIB-1 per 1000 tumour cells. CD31 positivity was indicated by the presence of membrane staining. For the analysis of microvessel density (immunohistochemistry with CD31), any isolated marked cell or cell group, clearly apart from adjacent groups, with or without lumen, was considered as an individual vessel, according to Weidner et al.16 Areas of fibrosis, necrosis and inflammation, as well as vessels with muscular walls, were excluded from the counting. Microvessels were counted in the three most vascularized areas, known as hot spots, in 200 magnification fields (correspond to 0.76 mm2 , approximately), from which the media was obtained.

Statistical analysis Fisher’s test was used to compare DNA content between benign and malignant tumours. The Mann---Whitney test was used to verify differences in the expression of PR, c-erbB-2, p53, and cyclin D1 MIB-1 and CD31 markers between diploid and aneuploid tumours. Spearman analysis was used to evaluate the correlation between DNA content and the epithelial and mesenchymal components from mixed tumours, complex adenomas and carcinomas in mixed tumours.

Results From the 56 analyzed tumours, 23 were benign and 33 were malignant. The benign mixed tumour was the most frequent benign neoplasm, whilst the most frequent malignant tumour was the invasive ductal carcinoma (Table 2). The mean age of animals with benign tumours was 8.86 years (range 5---13 years). For animals with malignant tumours the mean age was 8.64 years (range 3---15 years). The mean age of animals with benign and malignant tumours did not differ significantly (P = 0.0287). The analysis of DNA content by static cytometry revealed that 43.47% (10/23) of benign and 48.48% (16/33) of malignant canine mammary gland tumours were aneuploid (P > 0.05) (Table 2). The mean age of animals with diploid and aneuploid tumours was 9.04 and 8.56 years old, respectively. No association has been verified between age and ploidy (P > 0.05). PR expression was observed in 100% of the benign (23/23) and 33.33% (11/33) of the malignant tumours and the difference between the groups was statistically significant (P < 0.05). Positivity for c-erbB-2, p53 and cyclin D1 in benign tumours was found in 0.00% (23/23), 47.82% (11/23) and 26.08% (6/23), respectively. In malignant tumours positivity for c-erbB-2, p53 and cyclin D1 was found, respectively, in 3.33% (1/33), 57.57% (19/33) and 48.48% (16/33). Positivity for c-erbB-2, p53 and cyclin D1 did not differ between benign and malignant tumours. All tumours had Ki67 positive cells and the proliferative index ranged from 3 to 34.0% (mean 8.73) in benign tumours and from 2 to 76.0% (mean 24.78) in malignant tumours. The mean microvessel density of benign tumours was 35.86 (range 7.70---107.00 vessels). For malignant tumours the mean microvessel density was 55.78 (range 10.67---199.67 vessels). The malignant tumour

198 Table 2

G.D. Cassali et al. Analysis of DNA content in canine mammary tumours using static cytometry.

Histological diagnosis

n

Benign Adenoma Benign mixed tumour Complex adenoma Papiloma Malignant Carcinoma in benign mixed tumour Invasive ductal carcinoma Invasive papillary carcinoma Squamous cell carcinoma Invasive micropapillary Ca Osteosarcoma Secretory carcinoma Apocrine carcinoma Fibrosarcoma

Diploids (n/%)

23 6 12 3 2 33 8 11 3 3 2 2 2 1 1

Aneuploids (n/%)

4 (66.67) 7 (58.33) 2 (66.67) ---

2 (33.33) 5 (41.67) 1 (33.33) 2 (100)

5 (62.5) 7 (63.64) 3 (100.0) --1 (50.0) --1 (50.0) -----

3 (37.5) 4 (36.36) --3 (100.0) 1 (50.0) 2 (100.0) 1 (50.0) 1 (100.0) 1 (100.0)

BMT --- benign mixed tumour.

Table 3 Expression of the markers PR, p53, c-erbB2, and cyclin D1 in diploid and aneuploid canine mammary tumours. Marker

Diploid positive/negative

Aneuploid positive/negative

n PR p53 c-erbB2 Cyclin D1

36 25/11a 13/23a 20/16a 1/35a

26 13/13a 10/16a 13/13a 0/26a

Equal letters in the same row indicate no significant difference (P > 0.05).

had a higher proportion of Ki-67 expression, and high mean microvessel density than benign tumours (P < 0.05). There was no significant difference in the expression of PR, c-erbB2, p53, cyclin D1, MIB-1 and CD31 markers among other diploid tumour and aneuploidy tumours (P > 0.05) (Tables 3 and 4). DNA content of epithelial and mesenchymal (spindle and stellate-shaped myoepithelial cells proliferating in the interstitial areas, chondroid metaplasia and osseous metaplasia) component was compared in 23 tumours: 12 benign mixed tumours, 8 carcinomas in benign mixed tumours and 3 complex adenomas (Figs. 1 and 2). The pattern of DNA content was identical in epithelial and mesenchymal components of 75.0% (9/12) of benign

mixed tumours, 66.67% (2/3) of complex adenomas and 75.0% (6/8) of carcinomas in mixed tumours evaluated (r = 0.4843, P > 0.05). 3 benign mixed tumours revealed aneuploidy only in the epithelial component and one complex adenoma revealed aneuploidy in the mesenchymal component. In 2 carcinomas in benign mixed tumours the aneuploid was observed only in the epithelial malignant component (Table 5).

Discussion The percentage of aneuploid benign tumours found in this study (43.47%) was higher than that reported in other studies using static cytometry or flow cytometry, whose values varied from 6.0% to 17%.10,17---21

Table 4 Expression of the markers MIB-1 and CD31 in diploid and aneuploid canine mammary tumours. Marker

Diploid mean

Aneuploid mean

MIB-1 CD31

13.710 ± 13.3 45.476 ± 24.27

19.81 ± 17.23 50.230 ± 43.35

Equal letters in the same row indicate no significant difference (P > 0.05).

Figure 1 Canine mammary gland. Benign mixed tumour. Mixed proliferation of tubular epithelial cells. Note mesenchymal component containing myxoid and chondroid matrix (hematoxylin and eosin ×200).

Canine mammary tumours: A quantitative DNA study using static cytometry

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Table 5 Distribution of DNA content in mixed tumours, complex adenomas and carcinomas in benign mixed tumours in epithelial and myoepithelial areas and areas of chondroid and/or osseous metaplasia. Histological diagnosis

n (%)

Benign mixed tumour Benign mixed tumour Benign mixed tumour Benign mixed tumour Complex adenoma Complex adenoma Complex adenoma Carcinoma in BMT Carcinoma in BMT Carcinoma in BMT Carcinoma in BMT Total

12 7 (58.33) 3 (25.0) 2 (16.67) 3 2 (66.67) 1 (33.33) 8 5 (62.5) 2 (25.0) 1 (12.5) 23

The high frequency of aneuploid benign tumours may be due to intra-tumour heterogeneity of the types of tumours studied, since 52.17% of the benign tumours studied were benign mixed tumours. Benign mixed tumours are histologically characterized by a mixture of epithelial and stromal components and can develop carcinomatous foci resulting in carcinomas.8 Perhaps the highest percentage of aneuploidy cases in our sample might reflect the emergence of clones with potential biological aggressivity, which may become manifest as a relapse or malignant transformation. This hypothesis is also supported by the finding that 25% (3/12) of benign mixed tumours were aneuploids only in their epithelial component, which are known to undergo malignant transformation into benign mixed tumours. A study of DNA content by flow cytometry in 32 pleomorphic adenomas of human salivary glands, a histological subtype similar to benign mixed tumours, found that 21.88% of cases were aneuploid and 31.25% were peridiploid.22

Figure 2 Canine mammary gland. Carcinoma in benign mixed tumour. Proliferation of typical epithelial cells, which show infiltrative growth characterized by presence of clusters of tumour cells penetrating the stroma without myoepithelial cells. Note the mesenchymal component containing myxoid and chondroid matrix (hematoxylin and eosin ×200).

Epithelial component

Mesenchymal component

Diploid Aneuploid Aneuploid

Diploid Diploid Aneuploid

Diploid Diploid

Diploid Aneuploid

Diploid Aneuploid Aneuploid

Diploid Diploid Aneuploid

To date, only one case of DNA content analysis in mammary adenomyoepithelioma in women, a histological subtype characterized by epithelial and myoepithelial proliferation, has been reported in the literature; the tumour was aneuploid and the epithelial component presented atypias with an increased number of mitotic figures.23 However, the frequency of aneuploid malignant tumours found in our series of cases was similar to that seen by other authors who reported values from 38.0% to 62.0%.10,17---19 In human mammary neoplasms, aneuploidy has been seen in 44.0---66.0% of malignant tumours.2 The average age of animals in the sample studied was 8.73 years, which corresponds to about 50 years of age in a woman.24 No significant association has been made between the animal’s age and ploidy. This result agrees with Hellmén et al.17 who did not find a correlation between age and ploidy in canine mammary tumours. Reports of human data are controversial; some studies found an association between aneuploidy in neoplasms of younger women, whereas others did not.4,25 In this study, no difference has been found in immunostaining reactivity for PR, MIB-1, cyclin D1, p53 and c-erbB2 or microvessel density by immunoreactivity with CD31 between diploid and aneuploid tumours. Despite not having verified a significant difference of expression of these markers between diploid and aneuploid tumours, the highest MIB-1, CD31 and cyclin D1 labelling was seen in aneuploid tumours. This trend may be confirmed when larger and more homogeneous sampling is used. In a previous study using flow cytometry analysis in 24 canine mammary tumours, a close relationship was found among progesterone receptor (PR), negative immunostaining and higher microvessel density.21 When DNA is analyzed by flow cytometry, a larger number of neoplastic cells are evaluated, which may account for this finding. In humans, aneuploid tumours are negative for oestrogen receptors4,5 and present a higher expression of cyclin D1,26 MIB-1,27 c-erbB2,28 and p53.29 However, this association has not been verified regarding angiogenesis.30 Our observations showed that the different cellular components of the tumours had identical DNA contents in 74.0% of the 23 cases with mixed cell population

200 (benign mixed tumours, complex adenomas and carcinomas in mixed tumours). This similarity in DNA content has already been shown by Gärtner et al.11 In this study we confirm the role myoepithelial cells play in mesenchymal metaplasia in mixed tumours and suggest the possibility of a common origin for both components from a totipotent cell with a capacity for divergent differentiation. Evidence for this hypothesis can be found in several studies. Nieto et al.31 verified that BRCA, a protein involved in the regulation of mammary stem cells, was expressed both in epithelial and in mesenchymal components of canine mixed tumours. Later, Erdélyi et al.32 confirmed the expression of filaments typical of epithelial and mesenchymal cells in epithelial formation of mixed tumours indicating the presence of a partial programme of myoepithelial differentiation in these cells. Moreover, Ramalho et al.33 identified the presence of cells with a myoepithelial phenotype (CK5, p63, vimentin and ASMA+) in areas of myxoid matrix, indicating that these cells play a role in the genesis of the mesenchymal component and of cells with basal phenotype (CK5+), which would probably give rise to the epithelial component. Considering that positive CK5 cells may represent progenitor cells or adult stem cells from which luminal epithelium and myoepithelial cells originate, we suggest that both components may originate from the same cell. In another study, both epithelial and mesenchymal components of benign mixed tumours and carcinomas in benign mixed tumours presented a similar D-loop mtDNA haplotype in their constituent cellular components, suggesting these cell types share a common origin.34 Nevertheless, these studies do not rule out the possibility that collision tumours may originate separately from epithelial and mesenchymal cells. From 6 (27.27%) of our cases of biphasic tumours which did not present the same pattern of DNA content, a higher probability of finding tumours with different origins could be expected. There are few reports that analyze the DNA content in carcinomas composed of a mixed population in humans. Pitts et al.35 analyzed samples of the epithelial component of eight metaplastic carcinomas with flow cytometry and observed 62.5% aneuploidy. Flint et al.36 studied DNA content with static cytometry in 11 metaplastic carcinomas and verified aneuploidy in the epithelial component of 100% of neoplasms. Differences among authors are likely to be smaller when the evaluation of metaplastic carcinomas is standardized adopting criteria proposed by Wargotz and Norris37 and when the same method is employed for the analyses of DNA content. The evaluation of DNA content with static cytometry would seem to be the most recommended procedure for tumours with a double cell population. Ottense et al.38 found 79% of reproducibility when comparing flow cytometry and image analysis of imprint material, in human breast carcinoma samples. The imprint material is the best method of preserving nuclei integrity, although in this case, tissue architecture is lost and distinguishing cells in mixed tumours and metaplastic carcinomas is difficult. Good results in the correlation of flow cytometry and static cytometry are described for nuclei extracted from material included in paraffin and cytocentrifuged, albeit with the same morphological restrictions of the imprint material.39

G.D. Cassali et al.

Conclusions In this study, using static cytometry analysis and immunohistochemistry, no relationship was observed between aneuploidy and malignant neoplastic behaviour or tumour markers PR, MIB-1, CD-31, p53, c-erbB2 and cyclin D1. The finding that DNA content was similar in epithelial and mesenchymal components of mixed tumours, complex adenomas and carcinomas in benign mixed tumours supports the hypothesis that these components have a common histogenesis from a stem cell with the capacity for divergent differentiation.

Conflict of interest The authors have no conflict of interest to declare.

Responsabilidades éticas Protección de personas y animales. Los autores declaran que los procedimientos seguidos se conformaron a las normas éticas del comité de experimentación humana responsable y de acuerdo con la Asociación Médica Mundial y la Declaración de Helsinki. Confidencialidad de los datos. Los autores declaran que en este artículo no aparecen datos de pacientes. Derecho a la privacidad y consentimiento informado. Los autores declaran que en este artículo no aparecen datos de pacientes.

References 1. Kuliffay P, Sanislo L, Galbavy S. Chromatin texture, DNA index, and S-phase fraction in primary breast carcinoma cells analysed by laserscanning cytometry. Bratisl Lek Listy. 2010;111: 4---8. 2. Machado-Santelli GM, Mori L, Pereira CA. Prediction of relapse in patients with breast cancer by DNA cytometry. Anal Cell Pathol. 1994;7:321---34. 3. Yildirim-Assaf S, Coumbos A, Hopfenmuller W, Foss HD, Stein H, Kühn W. The prognostic significance of determining DNA content in breast cancer by DNA image cytometry: the role of high grade aneuploidy in node negative breast cancer. J Clin Pathol. 2007;60:649---55. 4. Wenger CR, Bearsdale S, Owens MA, Pounds G, Oldaker T, Vendeley P, et al. DNA ploidy, S-phase, and steroid receptors in more than 127000 breast cancer patients. Breast Cancer Res Treat. 1993;28:0---20. 5. Pinto AE, André S, Pereira T, Silva G, Soares J. Anal Cell Pathol. DNA flow cytometry but not telomerase activity as predictor of disease-free survival in pT1-2/N0/G2 breast cancer. Pathobiology. 2006;73:63---70. 6. Gilchrist KW, Gray R, Van Driel-Kulker AMJ, Kulker AMJ, Mesker WE, Ploem-Zaaijer JJ, et al. High DNA content and prognosis in lymph node positive breast cancer. A case control study by the university of Leiden and ECOG. Breast Cancer Res Treat. 1993;28:1---8. 7. Siitonen SM, Olli-Peka K, Helin HJ, Isola JJ. Prognostic value of cells with more than 5c DNA content in node-negative breast

Canine mammary tumours: A quantitative DNA study using static cytometry

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

cancer as determined by image cytometry from tissue sections. Human Pathol. 1993;24:1348---53. Misdorp W, Else RW, Hellmen E, et al. Histological classification of the mammary tumors of the dog and the cat. In: SCHULMAN FY, editor. World Health Organization International Histological Classification tumors of Domestic animals, Second Series, vol. 7. Washington, DC: AFIP; 1999. p. 23. Möllermark G, Kängström LE, Eliasson I, Azawedo E, Barrios C, Larsson O, et al. Distribution of nuclear DNA-content in canine mammary tumours. J Small Anim Pract. 1988;29:309---14. Destexhe E, Bicker E, Coignoul F. Image analysis evaluation of ploidy, S-phase fraction and nuclear area in canine mammary tumours. J Comp Pathol. 1995;113:205---16. Gärtner F, Geraldes M, Cassali GD, Rema A, Schmitt F. DNA measurement and immunohistochemical characterization of epithelial and mesenchymal cells in canine mixed mammary tumours: putative evidence for a common histogenesis. Vet J. 1999;158:39---47. Sannino P, Shousha S. Demonstration of oestrogen receptors in paraffin wax sections of breast carcinoma using the monoclonal antibody 1D5 and microwave oven processing. J Clin Pathol. 1994;47:90---2. Dutra AP, Granja NV, Schmitt FC, Cassali GD. c-erbB-2 expression and nuclear pleomorphism in canine mammary tumors. Braz J Med Biol Res. 2004;37:1673---81. Castiglioni T, Elstner B, Curutchet HP, Montesinos M, Debonis D. Immunohistochemical analysis of p53 and c-erbB-2 in breast cancer. Medicina. 1995;55:415---20. Allred DC, Harvey JM, Berardo M, Clark GM. Prognostic and predictive factors in breast cancer by immunohistochemical analysis. Mod Pathol. 1998;11:155---68. Weidner N, Moore DH, Vartanian R. Correlation of Ki-67 antigen expression with mitotic figure index and tumor grade in breast carcinomas using the novel ‘‘parafin’’-reactive MIB-1 antibody. Hum Pathol. 1994;25:337---42. Hellmén E, Lindgren A, Linell F, Matsson P, Nilsson A. Comparison of histology and clinical variables to DNA ploidy in canine mammary tumors. Vet Pathol. 1988;25:219---26. Rutteman GR, Cornelisse CJ, Dijkshoorn NJ, Poortman J, Misdorp W. Flow cytometric analysis of DNA ploidy in canine mammary tumors. Cancer Res. 1988;48:3411---7. Scanziani E, Caniatti M, Sen S, Erba E, Cairoli F, Battocchio M. Flow cytometric analysis of cellular DNA content in paraffin wax-embedded specimens of canine mammary tumours. J Comp Path. 1991;105:p.75---82. Pérez Alenza MD, Rutteman GR, Kuipers-Dijkshoorn NJ, Pe˜ na L, Montoya A, Misdorp W, et al. DNA flow cytometry of canine mammary tumours: the relationship of DNA ploidy and S-phase fraction to clinical and histological features. Res Vet Sci. 1995;58:238---43. Cassali GD, Salvador AC, Freitas C, Dutra A, Schmitt FC. DNA flow cytometry of canine mammary tumors: comparative aspects with human breast tumors. Arq Bras Med Vet Zootec. 2007;59:1163---8. Desinan L, Scott CA, Pizzolitto, Avellini C, Bardus P, Rimondi G, et al. DNA flow cytometry and glial fibrillary acidic protein reactivity in pleomorphic adenomas of the salivary glands. Anal Quant Cytol Histol. 1996;18:355---60. Nomura K, Fukunaga M, Uchida K, Aizawa S. Adenomyoepithelioma of the breast with exaggerated proliferation of epithelial cells: report of case. Pathol Int. 1996;4612:1011---4.

201

24. Lebeau A. L’âge du chien et celui de l’homme essai de statistique sur la mortalité canine. Bul Acad Vét. 1953;26:229---32. 25. Lewis WE. Prognostic significance of flow cytometry DNA analysis in node-negative breast cancer patients. Cancer. 1990;65:2315---20. 26. Collechi P, Passoni A, Rocchetta M, Gnesi E, Baldini E, Bevilacqua G. Cyclin-D1 expression in node positive (N+) and node-negative (N−) infiltrating human mammary carcinomas. Inst J Cancer. 1999;84:139---44. 27. Masood S, Bui MM, Lu L. Comparison of proliferation activity in breast carcinoma by flow cytometry analysis of S-phase and quantitative analysis of MIB-1. Ann Clin Lab Sci. 1998;28:315---23. 28. Baak JP, Chin D, Van Diest PJ, Ortiz R, Matze-Cok P, Bacus SS. Comparative long-term prognostic value of quantitative HER2/neu protein expression, DNA ploidy, and morphometric and clinical features in paraffin-embedded invasive breast cancer. Lab Invest. 1991;64:215---23. 29. Midulla C, De Iorio P, Nagar C, Pisani T, Cenci M, Valli C, et al. Immunohistochemical expression of p53, nm23-HI, Ki67 and DNA ploidy: correlation with lymph node status and other clinical pathologic parameters in breast cancer. Anticancer Res. 1999;19:4033---7. 30. Marinho A, Soares R, Ferro J. Angiogenesis in breast cancer is related to age but not other prognostic parameters. Pathol Res Pract. 1997;193:267---73. 31. Nieto A, Perez-Alenza MD, Del Castillo N, Tabanera E, Casta˜ no M, Pe˜ na L. BRCA1 expression in canine mammary dysplasias and tumors: relationship with prognostic variables. J Comp Pathol. 2003;128:260---8. 32. Erdélyi I, Van Asten AJAM, Van Dijk JE, Nederbragt H. Expression of versican in relation to chondrogenesis-related extracellular matrix components in canine mammary tumors. Histochem Cell Biol. 2005;124:139---49. 33. Ramalho LNZ, Ribeiro-Silva A, Cassali GD, Zucoloto S. The expression of p63 and cytokeratin 5 in mixed tumors of the canine mammary gland provides new insights into the histogenesis of these neoplasms. Vet Pathol. 2006;43: 424---9. 34. Bertagnolli AC, Soares P, Van Asch B, Amorim A, Cirnes L, Máximo V, et al. An assessment of the clonality of the components of canine mixed mammary tumours by mitochondrial DNA analysis. Vet J. 2009;182:269---74. 35. Pitts WC, Rojas VA, Gaffey MJ, Rouse RV, Esteban J, Frierson HF, et al. Carcinomas with metaplasia and sarcomas of the breast. Am J Clin Pathol. 1991;95:623---32. 36. Flint A, Oberman HA, Davenport RD. Cytophotometric measurements of metaplastic carcinoma of the breast: correlation with pathologic features and clinical behavior. Mod Pathol. 1988;1:193---7. 37. Wargotz ES, Norris HJ. Metaplastic carcinoma of the breast: III. Carcinosarcoma. Cancer. 1989;64:1490---9. 38. Ottesen GL, Christensen IJ, Larsen JK, Larsen J, Christiansen J, Baldetorp B, et al. DNA ploidy analysis in breast carcinoma. Comparison of unfixed and fixed tissue analyzed by image and flow cytometry. Anal Quant Cytol Histol. 1997;19: 413---22. 39. Williams RA, Baak JP, Meijer GA, Charlton IG. Testing the value of a measurement protocol for DNA image cytometry and comparing different cytologic preparations. Anal Quant Cytol Histol. 1996;18:429---37.