EGFR and microvessel density in canine malignant mammary tumours

Research in Veterinary Science 95 (2013) 1094–1099

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EGFR and microvessel density in canine malignant mammary tumours Maria Isabel Carvalho a, Maria João Guimarães b, Isabel Pires c, Justina Prada c, Ricardo Silva-Carvalho d,e, Carlos Lopes b, Felisbina L. Queiroga a,f,⇑ a

Department of Veterinary Sciences, University of Trás-os-Montes and Alto Douro, 5001-801 Vila Real, Portugal Instituto de Ciências Biomédicas de Abel Salazar, University of Porto, 4099-003 Porto, Portugal c CECAV, Department of Veterinary Sciences, University of Trás-os-Montes and Alto Douro, 5001-801 Vila Real, Portugal d Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal e ICVS/3B’s – PT Government Associate Laboratory, Braga/Guimarães, Portugal f CECA, University of Porto, Porto, Portugal b

a r t i c l e

i n f o

Article history: Received 13 May 2013 Accepted 2 September 2013

Keywords: EGFR Angiogenesis MVD CD31 Canine mammary tumours

a b s t r a c t The epidermal growth factor receptor (EGFR) is a transmembrane tyrosine kinase receptor which has been shown to have an important role in human breast cancer. Its role appears to be associated with increased angiogenesis and metastasis. In order to clarify its role in canine mammary tumours (CMT), 61 malignant neoplasms were studied by using immunohistochemistry, comparing expression of EGFR, microvessel density (MVD) by CD31 immunolabelling and characteristics of tumour aggressiveness. High EGFR immunoexpression was statistically significantly associated with tumour size, tumour necrosis, mitotic grade, histological grade of malignancy and clinical stage. High CD31 immunoreactivity was statistically significantly associated with tubule formation, histological grade of malignancy and clinical stage. A positive correlation between EGFR and CD31 immunoexpression (r = 0.843; P < 0.001) was also observed. Results suggest that an over-expression of EGFR may contribute to increased angiogenesis and aggression in malignant CMT, presenting the possibility of using EGFR inhibitors in the context of metastatic disease treatment. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction The epidermal growth factor receptor (EGFR, HER-1 or c-erbB1), a tyrosine kinase receptor, is a member of the human epidermal growth factor receptor (HER) family and has been receiving great attention in the literature concerning humans tumours (Walker and Dearing, 1999; Kim et al., 2001; Tsutsui et al., 2002; Herbst, 2004). EGFR pathways contribute to several processes involved in tumour survival, including cell proliferation/differentiation, metastasis and angiogenesis (Herbst, 2004; Dannenberg et al., 2005; De Luca et al., 2008). Therefore, within the tumour microenvironment, the EGFR system is an important mediator, of autocrine and paracrine circuits which results in enhanced tumour growth (Herbst, 2004). In human breast cancer, the role of EGFR appears to be associated with a poor clinical outcome (Klijn et al., 1992; Walker and Dearing, 1999; Tsutsui et al., 2002). In canine mammary tumours (CMT), studies regarding EGFR immunoexpression and/or content are scarce. There is a limited number of old reports based on differ⇑ Corresponding author at: Department of Veterinary Sciences, University of Trás-os-Montes and Alto Douro, 5001-801 Vila Real, Portugal. Tel.: +351 91 782 69 82. E-mail address: [email protected] (F.L. Queiroga). 0034-5288/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.rvsc.2013.09.003

ent methodologies, demonstrating the presence of EGFR in mammary gland tissues and neoplasias (Nerurkar et al., 1987; Donnay et al., 1993, 1996; Rutteman et al., 1994) and more recent works that indicate the link between EGFR immunoexpression and high tumoural aggressiveness (Gama et al., 2009; Kim et al., 2013). A study, which was recently carried out by our team, in a small series of cases (n = 43), showed a link between EGFR immunoexpression and more aggressive tumour phenotypes (Guimarães et al., 2013). Angiogenesis, regarded as a hallmark of cancer progression, is a multi-step process which consists of the development of new blood vessels from preexisting ones and is essential for tumour growth and metastatic spread of tumour cells (Hanahan and Folkman, 1996; Zetter, 1998; Queiroga et al., 2011; Sleeckx et al., 2013). The transition from a quiescent to an invasive tumour reflects a change in the balance between cell death and proliferation and is accompanied by the acquisition of angiogenic properties (Hanahan and Folkman, 1996). High levels of angiogenic factors and histologic evidence of increased tumour neovascularization are considered to be associated with malignancy and prognosis in human breast cancer (Hanahan and Folkman, 1996; Locopo et al., 1998; Gasparini, 2000, 2001; Leek, 2001) and different studies have indicated similar results in CMT (Restucci et al., 2000; Lavalle et al., 2009; Queiroga et al., 2011).

M.I. Carvalho et al. / Research in Veterinary Science 95 (2013) 1094–1099

EGFR signaling and angiogenesis have been independently evaluated as targets for therapy in human studies (Fox et al., 1994; Weidner and Gasparini, 1994; Nieto et al., 2007; Nickerson et al., 2012) and several factors support the potential interaction between them: tumour cells can be stimulated in a paracrine manner by growth factors; endothelial cells and tumour cells can stimulate each other’s growth in the tumour microenvironment (Rowe et al., 2004); both EGF and TGF-a (ligands for EGFR) induce angiogenesis (Perrotte et al., 1999); EGFR expression and function in tumour-associated endothelial cells have also been described (Rak et al., 1995). The interest of doing studies in the scope of EGFR expression is further enhanced by the availability and by the US Food and Drug Administration approval of specific EGFR tyrosine kinase inhibitors, which are currently being tested in several tumours of humans and dogs by in vivo and in vitro studies (Baselga and Arteaga, 2005; Mitry et al., 2010; Demetri, 2011; Lyles et al., 2012; Takeuchi et al., 2012; Fahey et al., 2013). The reports of the role of EGFR in the processes of cell proliferation, metastasis and angiogenesis described in human breast cancer (Shien et al., 2005) combined with the availability of a new drug (the tyrosine kinase inhibitors) raise the possibility of a new therapeutic approach to be researched in CMT. Therefore, the aims of our study are to determine the relationship between EGFR expression and microvessel density in CMT in order to clarify the viability of using tyrosine kinase inhibitors as anti-angiogenic therapy in these tumours. 2. Material and methods 2.1. Animals and clinical procedures This study included 61 female dogs with spontaneous malignant CMT, received at the Veterinary Teaching Hospital of the University of Trás-os-Montes and Alto Douro and several other veterinary private practices for diagnosis and treatment. The animals ranged in age from 5 to 16 years and were of different breeds. During their first visit and physical examination, all the mammary glands and regional lymph nodes (axillary and inguinal) were evaluated. In cases with regional lymph node enlargement, the involvement was investigated by fine needle aspiration and confirmed by histological analysis after surgical removal. Each mammary tumour was evaluated according to the following clinical characteristics: size (T1 < 3 cm; T2 > 3 and <5 cm; T3 > 5 cm), and skin ulceration. Surgical excision of the tumours was carried out in all animals. Clinical staging was performed using a modified TNM system (Owens, 1980) and animals were classified in two stages: local stage (without lymph node involvement) and regional stage (lymph node involvement). 2.2. Histopathologic evaluation The samples for histopathology were fixed in a10% neutral buffered formalin for an average time of ten days (minimum 6 and maximum 14 days) before being embedded in paraffin and cut in 3 lm sections, following routine methods. The tumours were classified according to the World Health Organization (WHO) criteria for canine mammary neoplasms (Misdorp et al., 1999) and graded in accordance with the method proposed by Goldschmidt and colleagues (Goldschmidt et al., 2011). The histological parameters evaluated were the presence of tumour necrosis, mitotic grade, nuclear pleomorphism, tubule formation and histological grade of malignancy. 2.3. Immunohistochemical analysis For the immunohistochemical detection of EGFR and CD31, 3 lm consecutive sections of each tumour were cut and mounted

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on silane-coated slides. The detection of EGFR was carried out by a two-step methodology using a polymer detection system (Novolink Polymer Detection System, Novocastra, Newcastle, UK). For CD31, the streptavidin–biotin–peroxidase methodology with a commercial detection system (Ultra Vision Detection System, Lab Vision Corporation, Fremont, CA, USA) was used, following the manufacturer’s instructions. All the washes and dilutions were made in Phosphate buffered saline (PBS) solution (pH = 7.4). The sections were deparaffinized in xylene and rehydrated in a graded alcohol series, ending in tap water. For EGFR, antigen retrieval was carried out by enzyme digestion: the sections were incubated with 0.4% pepsin (Dako) in HCl 0.01 N solution (pH = 2) for 30 min at 37 °C. For CD31, antigen retrieval was performed by microwave treatment for 3  5 min at 750 W in 0.01 M citrate buffer, pH = 6.0, followed by cooling for 20 min at room temperature. Endogenous peroxidase was blocked, through incubation with 3% hydrogen peroxidase for 30 min. The slides were then dried and the sections outlined with a hydrophobic pen (Liquid Blocker, Daido Sangyo Co., Tokyo, Japan), washed in PBS for 5 min and the blocking serum was applied for 15 min (Ultra V Block, Labvision Corporation, Fremont, CA, USA). Subsequently, all sections were incubated with the specific primary antibody: EGFR (clone 31G7, Invitrogen, Paisley, Scotland, UK, at 1:100 dilution), for 45 min at room temperature; CD31 (Clone JC70A, Dako, Glostrup, Denmark, at 1:20 dilution), for 24 h at 4 °C. After the incubation time, the sections were washed in PBS for 5 min at room temperature and then post primary block and novolink polymer were applied for 30 min each (both included in the Novolink Polymer Detection System, Novocastra, Newcastle, UK) for EGFR. For CD31, biotinylated serum and streptavidin peroxidase were applied for 10 min each (both included in the Ultravision Detection System kit, Labvision Corporation, Fremont, CA, USA). The antibody reaction products were observed with the cromagen 3,30 -diaminobenzidine tetrachloride (DAB) at 0.05% with 0.01% H2O2 (30%). After being washed in distilled water, the sections were counterstained with Gill haematoxylin, dehydrated, cleared and mounted. The primary antibody was replaced with phosphate buffered saline (PBS) for negative controls. The positive controls used were canine epidermis for EGFR and dog angiosarcoma for CD31. 2.4. Quantification of immunoreactivity The quantification of immunoreactivity was performed simultaneously by two observers (F.L.Q and M.I.C.). To evaluate the EGFR expression a method adapted from Ceccarelli and colleagues was used (Ceccarelli et al., 2005), based on the estimates of the percentage of positive cells and the staining intensity. The percentage of positive cells was scored as extension 1 (1–10% positive cells), 2 (11–50% positive cells), 3 (51–80% positive cells) or 4 (>80%). The staining intensity was scored as 0 (negative), 1 (weak), 2 (moderate) or 3 (strong). Subsequently, the two scores were combined (sum) and a final score was obtained: Low immunoreactivity (score values 6 5) and High immunoreactivity (score values 6–7). The assessment of microvessel density (MVD) by CD31 immunolabelling was based on a method applied to the study of angiogenesis in CMT (Queiroga et al., 2011). Any positive cells or cell clusters were considered as a single countable microvessel. Under low-power magnification (40), two hot spots were identified. Subsequently, under a 200 magnification the stained microvessels were counted. Three different fields were counted in each of these areas (Schor et al., 1998). The somatory of the number of microvessels (MVD) was subsequently calculated from the two vascular hot spots. The cut-off point applied to split CD31 into two groups (low and high counting) was defined by the mean value of CD31 in malignant CMT, since the sample studied follows a

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normal distribution, according to the Smirnov–Kolmogorov test, as previously described for other molecular markers (Carvalho et al., 2011). 2.5. Statistical analysis The statistical software SPSS (Statistical Package for the Social Sciences) version 19.0 was used for statistical analysis. The Chisquare test and the Fisher’s exact test were used to study the categorical variables. ANOVA test was used for analyzing continuous variables. The Pearson’s correlation test was performed in order to verify the presence of association between values of EGFR and CD31. All values are expressed as Means ± S.D. In all statistical comparisons, P < 0.05 was accepted as denoting significant difference. 3. Results 3.1. Tumours

Fig. 2. Immunoreactivity for EGFR in tubulopapillary carcinoma, note the intense cytoplasmatic staining in luminal cells, bar = 30 lm.

The tumours were classified as tubulopapillary carcinomas (n = 31; 53.6%), complex carcinomas (n = 9; 16.1%), solid carcinomas (n = 11; 12.5%), anaplastic carcinomas (n = 2; 3.6%), In situ carcinomas (n = 2; 3.6%) and carcinosarcomas (n = 6; 10.7%). 3.2. Expression of EGFR and CD31 in canine mammary tumours The immunoreactivity for EGFR was observed in the cytoplasmatic membrane and within the cytoplasm of the neoplastic cells, in a diffuse pattern. In some tumours evident basal cell labelling was observed, while in others, labelling was noted in luminal cells (Figs. 1 and 2). In this study we also examined the possible association of the type of staining and the different tumour types, however, there was no statistical difference observed. The expression of EGFR was low in 27/61 (44.3%) tumours and high in 34/61 (55.7%) cases. The CD31 expression was evaluated in 52/61 of the mammary tumour samples analysed and the staining appeared in endothelial cells as a subtle outline of microvessels and occasionally in macrophages (Fig. 3). In 9 cases, tissue destruction during the technical methodology prevented accurate immunohistochemical quantification. The values of MVD (n = 52) showed a range between 8.0– 106.0 microvessels with a mean value of 42 and a standard deviation of 23.

Fig. 3. Immunoreactivity for CD31 in tubulopapillary carcinoma, bar = 30 lm.

3.3. Association between immunoreactivity of EGFR, MVD and clinicopathological variables The EGFR immunoexpression was statistically significantly associated with tumour size (P = 0.006), tumour necrosis (P = 0.035), mitotic grade (P = 0.006), histological grade of malignancy (P = 0.022) and clinical stage (P = 0.012). For MVD, statistically significant associations were observed for tubule formation (P = 0.026), histological grade of malignancy (P = 0.016) and clinical stage (P = 0.039). MVD was higher in cases with regional clinical stage (with lymph node metastasis). Tumours undifferentiated, with less tubular formation and tumours with high HGM also showed high MVD. Detailed information is provided in Table 1.

3.4. Association of EGFR and MVD

Fig. 1. EGFR immunostaining in anaplastic carcinoma, with predominantly membrane localization, bar = 30 lm.

The MVD in tumours with high EGFR expression (58.1 ± 17.2 microvessels) was higher than in tumours with low EGFR immunoreactivity (21.4 ± 10.1 microvessels) (Fig. 4).

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M.I. Carvalho et al. / Research in Veterinary Science 95 (2013) 1094–1099 Table 1 Association of clinicopathological variables with EGFR and microvessel density (MVD). Clinicopathological parameters

EGFR

P

High

Low

Tumour size 1 (<3 cm) 2 (3–5 cm) 3 (>5 cm)

5 8 19

13 9 4

Skin ulceration Present Absent

9 22

Histological type Solid carcinoma Tubulopapillary carcinoma Complex carcinoma Carcinosarcoma Anaplastic carcinoma In situ carcinoma

MVD

P

High

Low

0.006 (n = 58)

6 6 12

11 8 8

0.179 (n = 51)

4 20

0.171 (n = 55)

8 16

6 19

0.472 (n = 49)

7 18 3 3 2 1

4 13 6 3 0 1

0.518 (n = 61)

5 13 4 2 2 0

2 13 6 4 0 1

0.278 (n = 52)

Tumour necrosis Present Absent

18 14

6 19

0.035 (n = 57)

12 12

10 16

0.465 (n = 50)

Mitotic grade 1 2 3

9 10 13

19 3 4

0.006 (n = 58)

9 7 10

16 5 4

0.082 (n = 51)

Nuclear pleomorphism 1 2 3

1 14 18

3 13 9

0.488 (n = 58)

12

13

0.228 (n = 51)

16

7

Tubule formation 1 2 3

6 10 17

6 13 6

0.127 (n = 58)

4 7 15

7 13 5

0.026 (n = 51)

Histological grade of malignancy I II III

10 9 14

16 6 3

0.022 (n = 58)

7 6 13

14 8 3

0.016 (n = 51)

Clinical stage Regional (with lymph node involvement) Local (without lymph node involvement)

17 15

5 18

0.012 (n = 55)

13 11

6 18

0.039 (n = 48)

n – number of samples; P – statistical significance.

4. Discussion

Fig. 4. Association of EGFR expression and MVD in malignant canine mammary tumours.

3.5. Correlation between the EGFR expression and microvessel density In this study a positive and statistically significant correlation was observed between a tumoural EGFR expression and a higher MVD (n = 52; r = 0.843, P < 0.001).

The link between EGFR and MVD was studied in several human tumours (Perrotte et al., 1999; Baumann and Krause, 2004; Iivanainen et al., 2009; Diaz et al., 2010; Jin et al., 2011), including breast cancer (Fox et al., 1994; Weidner and Gasparini, 1994; Nieto et al., 2007; Nickerson et al., 2012). In CMT, MVD accessed by CD31 immunoexpression was studied by some authors (Graham and Myers, 1999; Restucci et al., 2000; Lavalle et al., 2009; Queiroga et al., 2011), and EGFR immunoexpression was also described (Gama et al., 2009; Guimarães et al., 2013; Kim et al., 2013). However, to the best of the authors’ knowledge, there are no studies that investigated the relationship between EGFR and MVD in these tumours. Our findings revealed a possible role for EGFR in malignant mammary tumour aggressiveness and progression. Similar results were observed by our team in a small set of tumours (Guimarães et al., 2013). In one study of Gama and collaborators the EGFR expression showed a statistically significant association with respect to the animal’s age and larger tumour size and a tendency for a worse prognosis (Gama et al., 2009). A recent study demonstrated that the EGFR expression was higher in grade III tumours and in tumours with central necrosis and lymphatic infiltration (Kim et al., 2013). The EGFR becomes activated by overexpression of the EGFR ligands, which is frequent in cancer (Hanahan and

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Weinberg, 2011). Activation of this receptor leads to the initiation of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase/Akt pathways which are important in cancer aggressiveness and progression, since it triggers cell growth, proliferation, survival, migration and invasion (Scaltriti and Baselga, 2006). In relation to the microvessel density a statistically significant association was verified with regards to tubule formation, histological grade of malignancy and clinical stage. The present results are in agreement with previous reports on human breast cancer (Rowe et al., 2004; Muhammadnejad et al., 2013) and on CMT (Graham and Myers, 1999; Restucci et al., 2000; Queiroga et al., 2011). During tumour progression, angiogenesis is almost always activated and remains in that state to support the expansion of the tumour (Pouyssegur et al., 2006; Hanahan and Weinberg, 2011). This complex process is regulated through the production of several proangiogenic and anti-angiogenic factors (Cristofanilli et al., 2002). Present results support the idea that in CMT an angiogenic phenotype is also a fundamental step for tumour progression and invasion as observed in human breast cancer. The link between EGFR and MVD was a subject of study in human breast cancer, with some researchers describing correlations with increased risk of metastasis and shorter overall survival, although with no consistent findings among different authors (Fox et al., 1994; Weidner and Gasparini, 1994; Nieto et al., 2007; Nickerson et al., 2012). The higher expression of EGFR in the current study was significantly associated with a high microvessel density. Furthermore a positive and statistically significant correlation was also observed. Angiogenesis is required for multistage carcinogenesis and EGFR might be an important mediator of the tumour angiogenic switch acquisition in malignant CMT, similarly to that described for breast cancer in humans (Weidner and Gasparini, 1994). In tumour cells, EGFR signaling regulates the synthesis and secretion of several different angiogenic factors (VEGF, IL-8, bFGF) by activation of phosphatidylinositol3-kinase (PI3K)/AKT, mitogen-activated protein kinase (MAPK) and hypoxia inducible factor- 1a (HIF-1) pathways (Normanno and Gullick, 2006; De Luca et al., 2008, 2011). A switch of sensitivity to EGFR ligands in endothelial cells might also promote angiogenesis (Normanno et al., 2006; De Luca et al., 2011).Studies carried out on induced tumours in mice (Perrotte et al., 1999; Iivanainen et al., 2009; Diaz et al., 2010) demonstrated that the tyrosine kinase inhibitors dramatically reduced MVD. A recent study performed by Katanasaka and collaborators in malignant gliomas demonstrated that the mRNA and protein expressions of angiopoietin-like 4 (Angptl4), a secreted protein involved in angiogenesis and metabolism regulation via inducing c-myc, were significantly triggered by EGFRvIII overexpression, both in vitro and in vivo (Katanasaka et al., 2013). The results of our work suggest that in CMT similar signaling pathways may possibly be present and the inhibition of EGFR downstream signaling might reduce the expression of angiogenic factors and consequently have an anticancer therapeutic action. These findings emphasize the need for additional studies with selective inhibitors of EGFR in order to verify their effect in CMT angiogenesis and their true clinical effectiveness in advanced oncological disease.

5. Conclusion The present results demonstrate that increased EGFR expression and increased microvessel density demonstrated a significant positive correlation in malignant CMT. This suggests that EGFR inhibitors may block angiogenesis and could be an alternative for the treatment and control of advanced neoplastic mammary disease in female dogs.

Conflict of interest The authors declare they have no competing interests.

Acknowledgements The authors thank Mrs Lígia Bento for expert technical assistance. This work was supported by the Centro de Ciência Animal e Veterinária (CECAV) – University of Trás-os-Montes and Alto Douro (UTAD), Vila Real, Portugal and by the strategic research project Pest-OE/AGR/UI0772/2011 financed by the Foundation for Science and Technology (FCT).

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