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HER-2 expression in canine morphologically normal, hyperplastic and neoplastic mammary tissues and its correlation with the clinical outcome
Research in Veterinary Science 94 (2013) 299–305
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HER-2 expression in canine morphologically normal, hyperplastic and neoplastic mammary tissues and its correlation with the clinical outcome Lorenzo Ressel a,d, Roberto Puleio c, Guido Ruggero Loria c, Iacopo Vannozzi b, Francesca Millanta a, Santo Caracappa c, Alessandro Poli a,⇑ a
Dipartmento di Patologia Animale, Facoltà di Medicina Veterinaria, Università di Pisa, Viale delle Piagge 2, I-56124 Pisa, Italy Dipartmento di Clinica Veterinaria, Facoltà di Medicina Veterinaria, Università di Pisa, Viale delle Piagge 2, I-56124 Pisa, Italy Istituto Zooprofilattico Sperimentale della Sicilia, Via Marinuzzi 3, I-90129 Palermo, Italy d Department of Veterinary Pathology, School of Veterinary Sciences, University of Liverpool, Crown Street, Liverpool L69 7ZJ, United Kingdom b c
a r t i c l e
i n f o
Article history: Received 8 February 2012 Accepted 21 September 2012
Keywords: Dog HER-2 Immunohistochemistry Mammary tumours Overall-survival Prognosis
a b s t r a c t The proto-oncogene HER-2/neu (c-erbB-2) encodes a transmembrane receptor protein with tyrosinekinase activity. Previous studies have shown that HER-2 protein over-expression is present in canine mammary tumours, however, possible prognostic and predictive analogies between protein over-expression patterns in canine and human species are still controversial. Thirty-five canine mammary carcinomas, 11 mammary adenomas, and normal, hyperplastic or dysplastic tissues taken at the marginal area of the tumours were evaluated by immunohistochemistry (IHC) for HER-2 expression, using the Hercept TestÒ system scoring guidelines. HER-2 over-expression was detected in 3/11 adenomas and 10/35 carcinomas. Normal, hyperplastic and dysplastic mammary tissues were also found to be positive. The correlations between HER-2 expression and tumour histological grading, mitotic index, the presence of lymphatic invasion, and overall survival (OS) were evaluated. In carcinomas, HER-2 positive status only correlated with the mitotic index. A positive correlation was also found between HER-2 positive status and the presence of HER-2 over-expression in normal, hyperplastic or/and dysplastic mammary tissues surrounding the tumours. The percentage of HER-2 over-expressing tumours was similar to the percentage previously observed in canine benign and malignant mammary tumours. However an investigation regarding morphologically normal and hyperplastic or dysplastic tissues surrounding neoplastic lesions also showed HER-2 over-expression. In contrast with human mammary tumours, this study confirmed that in canine species, HER-2 over-expression does not identify a subgroup of tumours with a poor prognosis. In fact, we found HER-2 over-expression in morphologically non-neoplastic mammary tissues, surrounding hyperplastic and neoplastic lesions. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction The proto-oncogene HER/neu (also known as c-erbB-2) is located on human chromosome 17 and encodes a 185-kDa transmembrane glycoprotein that belongs to the human epidermal growth factor receptor (tyrosine kinase receptor family) (Lian and Tan, 2002). HER-2 plays an important role in the regulation of essential cellular processes such as cell growth, survival and differentiation (Gutierrez and Schiff, 2011). In approximately 20–30% of patients with breast cancer, tumour cells show a gene amplification and/or protein over expression of c-erbB-2 (Hanna et al., 1999), which is routinely used as a prognostic marker on its own and in correlation with other proteins (p53, Ki67, ER, PR) (Selvarajan et al., 2004). In addition to its prognostic value, HER-2 repre⇑ Corresponding author. Tel.: +39 050 2216982; fax: +39 050 2216914. E-mail address: [email protected] (A. Poli). 0034-5288/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.rvsc.2012.09.016
sents a predictive marker: analysis of HER-2 status is necessary for the selection of patients with mammary carcinomas who could benefit from treatment with trastuzumab (Herceptin, Genetech, San Francisco, CA, USA), a humanized anti HER-2 monoclonal antibody. In veterinary oncology, the role of HER-2 has been investigated in feline and canine mammary tumours by in situ hybridization (Cish and Fish) and immunohistochemistry. In feline species, immunohistochemical HER-2 protein over-expression has been found in 56% (36.0–59.6%) of mammary carcinomas, a higher percentage than observed in human breast carcinomas. However, as in women, HER-2 over-expression appears to be correlated with morphological parameters suggestive of malignancy and poor prognosis (De Maria et al., 2005; Millanta et al., 2005; Ordás et al., 2007). In recent studies, HER-2 over-expression in feline mammary carcinomas appeared to be markedly lower (5.5%) than in the previous published human and canine reports and HER-2 expression was
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not correlated to poor prognosis in a 1 year post-surgical clinical follow-up (Rasotto et al., 2011). Moreover, amplification of cerbB-2 was detected only in 2/9 (20%) feline carcinomas with protein over-expression, a lower percentage than observed in humans (Ordás et al., 2007). In canine mammary tumours, previous studies have shown that both c-erbB-2 amplification (Ahern et al., 1996) and HER-2 over-expression (Rungsipipat et al., 1999; Martin de las Mulas et al., 2003; Hsu et al., 2009) are detectable, however no gene amplification has been observed by in situ hybridization (Martin de las Mulas et al., 2003). Different percentages of HER-2 over-expressing carcinomas have been reported (ranging from 19.1% to 35.4%); and the relevance of HER-2 over-expression, in canine mammary tumours, as a prognostic factor has not yet been clearly determined (Rungsipipat et al., 1999; Dutra et al., 2004; Hsu et al., 2009). In order to clarify the role of HER-2 over-expression in canine mammary tumours, we evaluated the receptor expression status in neoplastic mammary tissues and in normal and hyperplastic tissues surrounding tumours. This was done by immunohistochemical study, by correlating its expression in cancer cells with clinical–pathological features and overall survival. 2. Materials and methods 2.1. Animals Tissues were supplied by the Animal Tumours Registry at the University of Pisa. The tissue samples were from 46 female dogs of different breeds, bearing benign or malignant mammary neoplasms. The cases belonged to the Veterinary Training Hospital (Clinical Department of Veterinary Medicine of the University of Pisa) and came from operations carried out between January 2004 and December 2007 or from referring practitioners in the surrounding areas. In addition to individual data (age, breed, body size and history of ovariohysterectomy), data on tumour size (maximum length), adherence to underlying tissues, and skin ulceration were also recorded. Ipsilateral regional lymph node sampling was performed in 29/35 dogs with grossly infiltrating carcinomas. All the animals with invasive carcinomas were submitted to a 2-year follow-up after surgery, to evaluate the post-surgical course of the disease. Overall survival was defined as the time from the day of diagnosis until the day of death or last follow-up. Death due to tumour associated causes was recorded as data and confirmed by necropsy.
according to the Hercept test (DakoÒ), thus the sections were dewaxed and rehydrated in distilled water through a series of graded alcohols. Epitope retrieval was carried out in the microwave oven using an epitope retrieval solution (citrate buffer, pH 6.0) at 98 °C for 40 min. Endogenous peroxidase activity was blocked by incubating the sections in 12 ml of methanol containing 200 ml of 30% hydrogen peroxide for 30 min, followed by washing in TBS/Tween. The prediluted HER2/neu antibody was applied for 30 min, followed by rinsing in TBS/Tween. A visualization reagent was applied for 30 min and rinsed with TBS/Tween, followed by a DAB solution (3, 30 -diaminobenzidine tertahydrochloride, Sigma D5637, dissolved in 11 ml Tris/hydrochloric acid buffer at pH 7.6, to which 100 ml of 1% hydrogen peroxide was added just before use) for 10 min. DAB was removed by rinsing with distilled water and the slides were then counterstained with haematoxylin, dehydrated in increasing grades of ethanol, than cleared in xylene, and mounted (DepexÒ). A human breast tumour known to react with HER-2 antibody was used as a positive control, while negative controls were performed by omitting the primary antibody and replacing the primary monoclonal antibody with a subtype matched unrelated primary antibody. The Hercept test scoring system was used to establish the degree of membrane staining. No staining or membrane staining observed in <10% of tumour cells was given a score of 0; faint/barely perceptible membrane staining detected in >10% of tumour cells was scored as 1+; a weak to moderate and strong complete membrane staining observed in >10% of tumour cells were graded as 2+ and 3+, respectively. A score of 1+ was considered negative and 2+ and 3+ were considered positive. A human breast tumour known to exhibit a score of 2+ was considered as a reference for 2+ scores. Slides supplied with the Hercept test system were used as a reference for scores of 1+ and 3+. 2.4. Statistical analysis Statistical analysis was performed using SPSS Advanced Statistics 13.0 (SPSS Inc., Chicago, IL, USA). The Chi square test was used to investigate the significance of the relationship between HER-2 over-expression and individual variables. Statistical significance was based on a 5% (0.05) significance level. Pearson’s correlation test was used to correlate the HER-2 expression in neoplastic mammary tissues and in tissues surrounding tumours. Overall survival analysis was performed using the Kaplan–Meier method, and the Tarone–Ware test was used to investigate the relationship between HER-2 over-expression and overall survival.
2.2. Histology
3. Results
Representative portions of each mammary surgical sample and regional lymph nodes when present were fixed in 10% buffered formalin, embedded in paraffin wax, and routinely processed. Five-micrometer-thick sections were obtained and stained with haematoxylin and eosin (HE) for histological examination. Lesions were classified according to the World Health Organization (WHO) classification (Misdorp et al., 1999), and tumours displaying multiple features were classified according to the most pronounced histologic differentiation. Tumours were graded following Elton and Ellis’s criteria (Elston and Ellis, 1991), as previously described (Millanta et al., 2005). Mitotic index and lymphatic invasion or necrosis were also recorded for each tumour.
We studied 46 mixed breed dogs (Crossbreed: n = 11; Boxer: n = 7; English setter: n = 4; Toy poodle, German shepherd and Doberman: n = 3, respectively; Kurzhaar, Rottweiler and Yorkshire terrier: n = 2, respectively; Beagle, Breton, Cirneco, Cocker spaniel, Drahathar, Labrador retriever, Samoiedo, Pitbull and Maremman shepherd: n = 1, respectively). In eleven bitches (23.9%) mammary adenomas were diagnosed, while in 35 subjects (76.1%) a mammary carcinoma was diagnosed. The mean age of the 11 bitches with mammary adenomas was 9.0 years ± 2.0 population SD (range: 6–12 years), while of the 35 subjects with mammary carcinomas, the mean age was 9.7 years ± 2.1 population SD (range 5– 14 years; median 10 years), of all these subjects 14 had an age < the median value, while 21 had an age P this value. The mean maximum diameter of the adenomas was 2.5 ± 1.8 cm (range: 0.3–3 cm), while the mean maximum diameter of the carcinomas was 3.7 ± 2.8 cm (range: 0.2–8 cm). Thirty-one of the 35 carcinomas had a maximum tumour diameter >5 cm, while only 4 were 65 cm. The mitotic index in the mammary adenomas was
2.3. Immunohistochemical study Tissue sections (5 lm thick) were cut from each block, mounted on coated slides (Superfrost plus, Menzel-Glaser, Germany) and dried at 37 °C for 24 h. Immunohistochemistry was performed
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or normal epithelial cells, often in association with a weak or moderate cytoplasmic staining. Positive and negative controls were as expected in all of the tests. HER-2 over-expression was detected in three of the 11 adenomas (27.3%; Fig. 1A), two adenomas scored +2 and one tumour scored +3. In 10 of the 35 carcinomas (28.6%; Fig. 1B), seven tumours scored +2 and three tumour scored +3. Table 2 summarizes the immunohistochemical data, listing the incidence of HER-2 over-expression for each category considered in the 35 bitches with mammary carcinomas. Of the 11 complex carcinomas, two were HER-2 over-expressing and 9 HER-non overexpressing, while of the simple carcinomas, 8/24 were HER-2 over-expressing. Of the ten HER-2 over-expressing carcinomas, six were detected in bitches of an age below the median value (6/21) and four in subjects of an age higher than the median value (4/14). Six of the 24 WDCs-MDCs carcinomas were HER-2 overexpressing, as well as four of the 11 PDCs tumours. Only one of the 17 neoplasms with a mitotic index below the media value was HER-2 over-expressing, while of the 18 carcinomas with a mitotic index above the median value, nine had a HER-2 overexpressing neoplasm. Four HER-2 over-expressing carcinomas were detected in tumours with lymphatic invasion (4/10), and six in tumours where lymphatic invasion was absent. Six HER-2 over-expressing tumours were detected in the 21 subjects alive at the end of the follow-up period and four in the 14 dead bitches. Presence of the HER-2 over-expressing tumours did not correlate with older age or tumour type (simple vs complex carcinomas), tumour grading or lymphatic invasion, while higher expression was observed in tumours with a mitotic index above the median (P < 0.005). Poor prognosis was not correlated with HER-2 status. Kaplan–Meier survival curves showed that, when HER-2 was over-expressed, bitches with malignant tumours tended to have the same survival time as subjects with HER-2 non over-expressing carcinomas (Fig. 2). The staining pattern revealed in the mammary tissues surrounding the examined tumours and the correlation between HER-2 expression in these neoplastic tissues and HER-2 expression in normal, hyperplastic and dysplastic mammary tissues surrounding these tumours are reported in Table 3. Of the 29 normal mammary tissue samples, 11 were HER-2 over-expressing (4/9 at the periphery of benign neoplasms and 7/20 surrounding malignant tumours). Seven of the 26 lobular hyperplasias were HER-2 overexpressing, one surrounding benign neoplasms (1/6), and six in tis-
0.7 ± 1.0 mitosis/10 hpf (range: 0–3) and in the mammary carcinomas, it was 11.6 ± 9.7 mitosis/10 hpf (range: 1–42 mitosis/hpf; median 9 mitosis/hpf). Eighteen of the mammary carcinomas had a mitotic index that was above the median value and 17 below the median value. At the end of the clinical follow-up, 21 out of 35 bitches with mammary carcinomas (60%) were still alive and 14 (40%) had died from mammary tumours (no deaths by non-tumour related causes). Three adenomas were of simple adenomas, while eight were complex. Simple carcinomas were the most frequent type of malignant tumors (24/35, 68.6%), whereas complex carcinomas was less frequently observed (11/35, 31.4%). Histologic grading of the 35 mammary carcinomas revealed that 24 (68.6%) were well-differentiated (WDCs; grade I) or moderately differentiated carcinomas (MDCs; grade II), and 11 (31.4%) were poorly differentiated carcinomas (PDCs; grade III). Only one of the 11 complex carcinomas was a PDC, while all the others were WDCs or MDCs. Ten of the 24 simple carcinomas were PDCs and 14 were WDCs or MDCs. The presence of lymphatic invasion was observed in 10 of the 35 carcinomas (28.6%), whereas in the other 25 carcinomas (71.4%), only a local stromal invasion was detected. In the follow-up study, poor prognosis (2 years after surgery) was significantly associated with simple carcinomas (P < 0.025), PDCs (P < 0,001), mitotic index above the median value (P < 0.01) and lymphatic invasion presence (P < 0.001), whereas poor prognosis was not related with tumor size and age of subject, although all the subjects with carcinomas with a diameter 65 cm were alive at the end of the follow-up period (Table 1). This finding is in agreement with data reported on morphologic prognostic markers in canine mammary carcinomas (Lana et al., 2007), which demonstrates that our population is a representative sample of the canine mammary tumour population. Thirty-seven hyperplastic lesions were diagnosed in tissues surrounding neoplastic alterations: 26 lobular hyperplasia (70.3%), six cases surrounding benign and 20 malignant tumours, and 11 ductal hyperplasia (29.8%), two cases surrounding benign and 9 malignant tumours. In addition, in 29/46 samples, a residual normal mammary gland was also detected in nine cases at the periphery of benign neoplasms and in 20 cases, in tissues surrounding the 35 carcinomas. The immunohistochemical analysis of HER-2 revealed membrane labeling of tumour cells as well as neighboring hyperplastic
Table 1 Correlation among clinical–pathological features of the 35 examined canine malignant mammary tumours and overall survival of bitches bearing these cancer. Clinico-pathological features
No. of cases
Overall survival
P
Alive (No. of cases)
Dead (No. of cases)
Tumour type Complex Simple
11 24
10 11
1 13
0.025
Tumour size 65 cm >5 cm
4 31
4 17
0 14
NS
Age
14 21
8 13
6 8
NS
Tumour grading WDC–MDC PDC
24 11
19 2
5 9
0.001
Mitotic index
17 18
14 7
3 11
0.01
Lymphatic invasion Absent Present
25 10
21 0
4 10
0.001
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Fig. 1. HER-2 expression in canine mammary gland lesions. (A) Simple adenoma. Presence of a complete membranous staining of a strong intensity (score 3 +) (Immunohistochemistry; Bar = 50 lm). (B) Solid carcinoma. Complete membranous staining of weak to moderate intensity (score 2 +) (Immunohistochemistry; Bar = 50 lm). (C and D) HER-2 over-expressing hyperplastic mammary tissues were observed at the periphery of a HER-2 over-expressing adenoma (score 2+) (C) and HER-2 non overexpressing (score 1+) (D) carcinomas (Immunohistochemistry; Bar = 200 lm).
Table 2 Correlation among HER-2 expression, clinical–pathological features of the 35 mammary tumours examined and overall survival of the bitches bearing these carcinomas. Clinico-pathological features
No. of cases
HER-2 expression
P
Non over-expressing (0 - + 1)
Over-expressing (+2 - + 3)
Tumour type Complex Simple
11 24
9 16
2 8
NS
Age
14 21
10 15
4 6
NS
Tumour grading WDC–MDC PDC
24 11
18 7
6 4
NS
Mitotic index
17 18
16 9
1 9
0.005
Lymphatic invasion Absent Present
25 10
19 6
6 4
NS
Tumour related death Alive Dead
21 14
15 10
6 4
NS
sues surrounding carcinomas (6/20), while three ductal hyperplasias were HER-2 over-expressing, one surrounding a benign neoplasm (1/2) and two (2/9) a malignant neoplasm. The presence of normal or hyperplastic HER-2 over-expressing mammary tissues was observed at the periphery of both HER-2 over-expressing benign (Fig. 1C) and malignant tumours, and HER-2 non overexpressing benign and malignant (Fig. 1D) tumours. Out of the nine normal mammary tissue samples surrounding adenomas, seven (five HER-2 non over-expressing and two HER-2 over-expressing) were detected at the periphery of HER-2 non over-expressing tumours, and two, both HER-2 over-expressing, in tissues surrounding HER-2 over-expressing tumours, with a significant correlation (r = 0.706; P < 0.03). Eight hyperplastic alterations were detected in tissues surrounding these benign alterations: six lobular and two ductal hyperplasias. HER-2 non
over-expressing hyperplastic alterations were always detected at the periphery of HER-2 non over-expressing adenomas. On the other hand, the two HER-2 over-expressing hyperplastic lesions were detected at the periphery of HER-2 over-expressing adenomas. The correlation between HER-2 expression in lobular hyperplasias surrounding adenomas and HER-2 expression in these tumours was significant for lobular hyperplasias (r = 800; P < 0.05), but was not explored for ductal hyperplasias due to the limited number of samples. Out of 20 normal mammary samples detected in tissues surrounding malignant lesions, 12 HER-2 non over-expressing samples were detected at the periphery of HER2-non-over-expressing carcinomas. On the other hand, of the eight samples at the periphery of the HER-2–over-expressing tumours, one was HER-2 non-over-expressing, and seven were HER-2 over-expressing with a significant correlation (r = 0.826;
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In breast cancer, the Hercept TestÒ, approved by the US Food and Drug Administration (FDA), guarantees the standardized and reproducible immunohistochemical detection of HER-2 protein in analytical conditions (Thomson et al., 2001). Previous studies have confirmed the cross reactivity between anti-human protein antibodies and canine tissues (Rungsipipat et al., 1999; Martin de las Mulas et al., 2003; Hsu et al., 2009). In this investigation, we also used the Hercept test grading system in order to compare the results with previous investigations on canine mammary carcinomas (Martin de las Mulas et al., 2003; Hsu et al., 2009). HER-2 protein over-expression was observed in 10/35 (28.6%) canine malignant mammary tumours, but also in 3/11 (27.3%) benign mammary tumours. The percentage of HER-2 over-expressing malignant mammary neoplasms was similar to published reports. Other authors have demonstrated HER-2 over-expression with the same methodology in 17.6% and 29.7% canine mammary carcinomas (Martin de las Mulas et al., 2003; Hsu et al., 2009). Rungsipipat et al. (1999) determined a value of 19.1% of malignant mammary tumours over-expressing HER-2 protein using anti-human HER-2 polyclonal antibodies. On the basis of our and all these previous studies it is possible to argue that HER-2 over-expression in canine mammary malignant tumours ranges between 16.7% and 29.7%, a percentage similar to that reported in human breast cancer (20– 30%) (Révillion et al., 1998; Almasri and Al Hamad, 2005). In our study, a higher percentage of benign mammary tumours were classified as HER-2 over-expressing. This result is in contrast with data reported by Hsu et al. (2009), who analyzed six benign mammary tumours and two normal mammary glands that scored negative for HER-2 over-expression. On the other hand, Rungsipipat et al. (1999) reported that 50% of 32 benign mammary tumours over-expressed HER-2. These different results may be related to the larger number of benign lesions included in our study and in the latter study than those carried out by Hsu et al. (2009). Unfortunately an accurate comparison between the results observed by Rungsipipat and those of our study cannot be performed due to the different scoring systems adopted. In addition, we detected HER-2 over-expression in hyperplastic or dysplastic lesions surrounding the neoplastic tissues and in a similar percentage in morphologically normal mammary gland tissues surrounding these neoplastic tissues. HER-2 over-expression in hyperplastic lesions has been previously demonstrated in canine and feline mammary tissues (Rungsipipat et al., 1999; De Maria et al., 2005, Ordás et al., 2007; Rasotto et al., 2011). Our data confirmed that in canine mammary glands, as described in the feline species (Rasotto et al., 2011), abnormal levels of HER-2 protein could be involved in cellular growth in hyperplastic or benign neoplastic alterations. Interestingly, in our study, a positive correlation
Fig. 2. Kaplan–Meier survival curves for the group of 35 bitches with malignant mammary tumours (10 HER-2 over-expressing and 24 HER-2 non over-expressing neoplasms) showing the relationship between HER-2 over-expression and two-year follow-up after surgical removal.
P < 0.0001). In tissues surrounding the malignant tumours, 20 lobular hyperplasias were detected: 13 at the periphery of HER-2 non over-expressing tumours that were always HER-2 non overexpressing, and seven at the periphery of HER-2 over-expressing carcinomas (in one case HER-2-non-over-expressing and in six cases HER-2 over-expressing). Also in this case, the correlation between HER-2 expression in neoplastic tissues and lobular hyperplasias was significant (r = 0.907; P < 0.0001). Finally, nine ductal hyperplasias were detected in tissues surrounding these neoplasms, seven HER-2 non over-expressing at the periphery of HER-2 non over-expressing carcinomas, and two HER-2-overexpressing at the periphery of HER-2-over-expressing tumours. Again, a significant correlation was detected (r = 0.839; P < 0.005) between HER-2 status in the ductal hyperplasias surrounding neoplastic tissues. 4. Discussion In this study we investigated immunohistochemical HER-2 expression in morphologically normal, hyperplastic and neoplastic canine mammary gland tissues along with its prognostic potential.
Table 3 HER-2 expression in the different canine mammary tissues surrounding neoplastic lesions and correlation among HER-2 expression in neoplastic tissues and in these normal and hyperplastic tissues. Tissues surrounding tumours
Benign tumour tissues Normal mammary tissues Lobular hyperplasia Ductal hyperplasia Malignant tumour tissues Normal mammary tissues Lobular hyperplasia Ductal hyperplasia
c-erbB-2 expression
c-erbB-2 expression Normal
Over
Normal Over Normal Over Normal Over
5 2 5 0 1 0
0 2 0 1 0 1
Normal Over Normal Over Normal Over
12 0 13 0 7 0
1 7 1 6 0 2
Pearson
P
0.706
0.03
0.800
0.05
ND
0.826
0.0001
0.907
0.0001
0.839
0.005
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was observed between the HER-2 status of hyperplastic or dysplastic lesions and that of the respective tumours. As expected, in our study several factors were significantly associated with a poor survival prognosis 2 years after surgery, including tumour size >5 cm, histological classification of simple carcinoma, tumour grading of PDCs, mitotic index above the median value, and the presence of lymphatic invasion. These results confirm the findings of previous studies regarding the importance of these prognostic factors for canine mammary carcinomas (Chang et al., 2005; Hsu et al., 2009) and highlight that the sample we studied is representative of the population of mammary tumors in dogs. In human oncology, many studies have demonstrated that gene amplification of c-erbB-2 and/or overexpression of its receptors are linked to shorter disease-free intervals, increased risk of metastasis, and resistance to many types of therapy. Receptor activation, in fact, improves tumor cell motility, protease secretion and invasion. It also modulates the cell cycle checkpoint function, DNA repairing, and the apoptotic response (Eccles, 2001). The HER status of breast tumors is of clinical predictive importance, since anticancer therapies targeted at specific tumor-associated gene products are expected to be effective in identifying therapeutic regimens. A humanized anti-HER-2 monoclonal antibody was developed to bind HER-2 protein and inhibit tumor cell growth (Cobleigh et al., 1999) and has been widely introduced for the treatment of HER-2 over-expressing carcinomas (Hortobagyi, 2001; Vogel et al., 2002). It is therefore important to accurately evaluate the status of HER-2. Several studies have thus indicated that analysis of DNA and protein levels are equally useful (Van de Vijver, 2001). Immunohistochemical demonstration of HER-2 over-expression could represent a simple and reliable method for examining protein expression status. In addition, it provides similar results to other standard methods such as fluorescent in situ hybridization (Jacobs et al., 1999; Kaya et al., 2001). A number of studies have attempted to identify a similar prognostic role for HER-2 expression in canine mammary carcinomas. In a previous investigation (Dutra et al., 2004), HER-2 expression was found to be associated with some well known morphological indicators of poor prognosis. In particular a correlation was found between HER-2 expression and tumour histological grading. A similar correlation has been previously reported in several cases of human breast cancer, where HER-2 amplification (Tsuda et al., 1992; Gramlich et al., 1994) or expression (Leal et al., 1995, 2001) was correlated with histological grade. In our study, the correlation between HER-2 over-expression and tumour histological grading was not detected, while HER-2 over-expression was correlated with tumour mitotic activity. Tsuda et al. (1990) obtained similar data regarding the correlation between mitotic count and HER-2 over-expression, in breast cancer. Despite this, in our Kaplan–Meier analysis, HER-2 over-expression was not correlated with overall survival, and bitches with HER-2over-expressing carcinomas did not present a poorer prognosis than those with HER-2-non-over-expressing tumours. These data confirm previous studies on canine mammary carcinomas, where HER-2 over-expression was associated with higher survival rate (Dutra et al., 2004; Hsu et al., 2009). On the other hand the data are in contrast with another study where three bitches with HER-2-over-expressing mammary carcinomas had metastasis or died <6 months after surgery (Martin de las Mulas et al., 2003). The results obtained in our study together with the majority previous investigations carried out on dogs to date contrast findings in human breast cancers, where c-erbB-2 gene amplification and protein over-expression predict shorter disease-free intervals and overall survival times (Révillion et al., 1998; Almasri and Al Hamad, 2005), suggesting that c-erbB-2 should be considered as an independent adverse prognostic factor. However the reason for
the difference in HER-2 over-expression between human breast cancer and canine mammary tumours remains unknown (Hsu et al., 2009). In conclusion, our findings further suggest that c-erbB-2 seems to play a role in the proliferation of mammary gland tissues in bitches, but that it is probably not involved in malignant transformation and is not associated with a shorter overall survival time. Although additional large scale studies are warranted to further explore the significance of HER-2 protein over-expression in canine mammary lesions, the lack of correlations between HER-2 overexpression and poor prognosis must be taken into account in assessing target therapies for dogs.
Conflict of interest None of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper. Acknowledgment We would like to thank Adrian John Wallwork for his help during the process of reviewing the manuscript. References Ahern, T.E., Bird, R.C., Bird, A.E., Wolfe, L.G., 1996. Expression of the oncogene cerbB-2 in canine mammary cancers and tumor-derived cell lines. American Journal of Veterinary Research 57, 693–696. Almasri, N.M., Al, Hamad.M., 2005. Immunohistochemical evaluation of human epidermal growth factor receptor 2 and estrogen and progesterone receptors in breast carcinoma in Jordan. Breast Cancer Research 7, R598–R604. Chang, S.-C., Chang, C.-C., Chang, T.-J., Wong, M.-L., 2005. Prognostic factors associated with survival 2 years after surgery in dogs with malignant mammary tumors: 79 cases (1998–2002). Journal of the American Veterinary Medical Association 227, 1625–1629. Cobleigh, M.A., Vogel, C.L., Tripathy, D., Robert, N.J., Scholl, S., Fehrenbacher, L., Wolter, J.M., Paton, V., Shak, S., Lieberman, G., Simon, D.J., 1999. Multinational study of the efficacy and safety of humanized anti HER-2 monoclonal antibody in women who have HER-2 overexpressing metastatic breast cancer that has progressed after chemotherapy for metastatic disease. Journal of Clinical Oncology 17, 2639–2648. De Maria, R., Olivero, M., Iussich, S., Nakaichi, M., Murata, T., Biolatti, B., Di Renzo, M.F., 2005. Spontaneous feline mammary carcinoma is a model of HER2 overexpressing poor prognosis human breast cancer. Cancer Research 65, 907– 912. Dutra, A.P., Granja, N.V., Schmitt, F.C., Cassali, G.D., 2004. C-erbB-2 expression and nuclear pleomorphism in canine mammary tumors. Brazilian Journal of Medical and Biological Research 37, 1673–1681. Eccles, S.A., 2001. The role of c-erbB-2/HER2/neu in breast cancer progression and metastasis. Journal of Mammary Gland Biology and Neoplasia 6, 393–406. Elston, C.W., Ellis, I.O., 1991. Pathological prognostica factors in breast cancer. I. Value of histological grade breast cancer: experience from a large study with long-term follow-up. Histopathology 19, 403–410. Gramlich, T.L., Cohen, C., Fritsch, C., DeRose, P.B., Gansler, T., 1994. Evaluation of cerbB-2 amplification in breast carcinoma by differential polymerase chain reaction. Anatomic Pathology 101, 493–499. Gutierrez, C., Schiff, R., 2011. HER2: biology, detection, and clinical implications. Archives of Pathology & Laboratory Medicine 135, 55–62. Hanna, W., Kahn, H.J., Trudeau, M., 1999. Evaluation of HER-2/neu (erbB-2) status in breast cancer: from bench to bedside. Modern Pathology 12, 827–834. Hortobagyi, G.N., 2001. Overview of treatment results with trastuzumab (Herceptin) in metastatic breast cancer. Seminars in Oncology 28 (Suppl. 18), 43–47. Hsu, W.-L., Huang, H.-M., Liao, J.-W., Wong, M.-L., Chang, S.-C., 2009. Increased survival in dogs with malignant mammary tumours overexpressing HERprotein and detection of a silent single nucleotide polymorphism in the canine HER-2 gene. The Veterinary Journal 180, 116–123. Jacobs, T.W., Gown, A.M., Yazjii, H., Barnes, M.J., Schnitt, S.J., 1999. Comparison of fluorescence in situ hybridisation and immunohistochemistry for the evaluation of HER-2/neu in breast cancer. Journal of Clinical Oncology 17, 1974–1982. Kaya, H., Ragazzini, T., Aribal, E., Güney, I., Kotilo, L.E., 2001. Her-2/neu Gene amplification compared with HER-2/neu protein overexpression on ultrasound guided core-needle biopsy specimens of breast carcinoma. Pathology and Oncology Research 7, 279–283.
L. Ressel et al. / Research in Veterinary Science 94 (2013) 299–305 Lana, S.E., Rutteman, G.R., Withrow, S.J., 2007. Tumours of the mammary gland. In: Withrow, S.J., Vail, D.M. (Eds.), Small Animal Clinical Oncology, fourth ed. Saunders, Elsevier, St. Louis, MI, USA, pp. 619–936. Leal, C.B., Schimtt, F.C., Bento, M.J., Maia, N.C., Lopes, C., 1995. Ductal carcinoma in situ of the breast. Histologic categorization and its relationship to ploidy and immunohistochemical expression of hormone receptors, p53, and c-erbB-2 protein. Cancer 75, 2123–2131. Leal, C.B., Henrique, R., Monteiro, P., Lopes, C., Bento, M.J., De Suosa, C.P., Lopes, P., Olson, S., Silva, M.A., Page, D.L., 2001. Apocrine ductal carcinoma in situ of the breast: histologic classification and expression of biologic markers. Human Pathology 32, 487–493. Lian, S.C., Tan, P.H., 2002. CerbB2 status in breast cancer: pathologic issues. Annals Academy Science of Singapore 31, 793–798. Martin de las Mulas, J., Orda´, S., Milla´n, Y., Ferna´ndez-Soria, V., Ramo´n y Cajal, S., 2003. Oncogene HER-2 in canine mammary gland carcinomas: an immunohistochemical and chromogenic in situ hybridization study. Breast Cancer Research and Treatment 80, 363–367. Millanta, F., Calandrella, M., Citi, S., Della Santa, D., Poli, A., 2005. Overexpression of HER-2 in feline invasive mammary carcinomas: an immunohistochemical survey and evaluation of its prognostic potential. Veterinary Pathology 42, 30– 34. Misdorp, W., Else, R.W., Hellme´n, E., Lipscomb, T.P., 1999. Histological Classification of Mammary Tumors of the Dog and the Cat, second series, vol. VII, Armed Forces Institute of Pathology, American Registry of Pathology, Washington, DC and the World Health Organization Collaborating Center for Worldwide Reference on Comparative Oncology, pp. 1–59. Ordás, J., Millán, Y., Dios, R., Reymundo, C., de Las Mulas, J.M., 2007. Proto-oncogene HER-2 in normal, dysplastic and tumorous feline mammary glands: an immunohistochemical and chromogenic in situ hybridization study. BMC Cancer 7, 179. Rasotto, R., Caliari, D., Castagnaro, M., Zanetti, R., Zappulli, V., 2011. An immunohistochemical study of HER-2 expression in feline mammary tumours. Journal of Comparative Pathology 144, 170–179.
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Contents lists available at SciVerse ScienceDirect
Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
HER-2 expression in canine morphologically normal, hyperplastic and neoplastic mammary tissues and its correlation with the clinical outcome Lorenzo Ressel a,d, Roberto Puleio c, Guido Ruggero Loria c, Iacopo Vannozzi b, Francesca Millanta a, Santo Caracappa c, Alessandro Poli a,⇑ a
Dipartmento di Patologia Animale, Facoltà di Medicina Veterinaria, Università di Pisa, Viale delle Piagge 2, I-56124 Pisa, Italy Dipartmento di Clinica Veterinaria, Facoltà di Medicina Veterinaria, Università di Pisa, Viale delle Piagge 2, I-56124 Pisa, Italy Istituto Zooprofilattico Sperimentale della Sicilia, Via Marinuzzi 3, I-90129 Palermo, Italy d Department of Veterinary Pathology, School of Veterinary Sciences, University of Liverpool, Crown Street, Liverpool L69 7ZJ, United Kingdom b c
a r t i c l e
i n f o
Article history: Received 8 February 2012 Accepted 21 September 2012
Keywords: Dog HER-2 Immunohistochemistry Mammary tumours Overall-survival Prognosis
a b s t r a c t The proto-oncogene HER-2/neu (c-erbB-2) encodes a transmembrane receptor protein with tyrosinekinase activity. Previous studies have shown that HER-2 protein over-expression is present in canine mammary tumours, however, possible prognostic and predictive analogies between protein over-expression patterns in canine and human species are still controversial. Thirty-five canine mammary carcinomas, 11 mammary adenomas, and normal, hyperplastic or dysplastic tissues taken at the marginal area of the tumours were evaluated by immunohistochemistry (IHC) for HER-2 expression, using the Hercept TestÒ system scoring guidelines. HER-2 over-expression was detected in 3/11 adenomas and 10/35 carcinomas. Normal, hyperplastic and dysplastic mammary tissues were also found to be positive. The correlations between HER-2 expression and tumour histological grading, mitotic index, the presence of lymphatic invasion, and overall survival (OS) were evaluated. In carcinomas, HER-2 positive status only correlated with the mitotic index. A positive correlation was also found between HER-2 positive status and the presence of HER-2 over-expression in normal, hyperplastic or/and dysplastic mammary tissues surrounding the tumours. The percentage of HER-2 over-expressing tumours was similar to the percentage previously observed in canine benign and malignant mammary tumours. However an investigation regarding morphologically normal and hyperplastic or dysplastic tissues surrounding neoplastic lesions also showed HER-2 over-expression. In contrast with human mammary tumours, this study confirmed that in canine species, HER-2 over-expression does not identify a subgroup of tumours with a poor prognosis. In fact, we found HER-2 over-expression in morphologically non-neoplastic mammary tissues, surrounding hyperplastic and neoplastic lesions. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction The proto-oncogene HER/neu (also known as c-erbB-2) is located on human chromosome 17 and encodes a 185-kDa transmembrane glycoprotein that belongs to the human epidermal growth factor receptor (tyrosine kinase receptor family) (Lian and Tan, 2002). HER-2 plays an important role in the regulation of essential cellular processes such as cell growth, survival and differentiation (Gutierrez and Schiff, 2011). In approximately 20–30% of patients with breast cancer, tumour cells show a gene amplification and/or protein over expression of c-erbB-2 (Hanna et al., 1999), which is routinely used as a prognostic marker on its own and in correlation with other proteins (p53, Ki67, ER, PR) (Selvarajan et al., 2004). In addition to its prognostic value, HER-2 repre⇑ Corresponding author. Tel.: +39 050 2216982; fax: +39 050 2216914. E-mail address: [email protected] (A. Poli). 0034-5288/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.rvsc.2012.09.016
sents a predictive marker: analysis of HER-2 status is necessary for the selection of patients with mammary carcinomas who could benefit from treatment with trastuzumab (Herceptin, Genetech, San Francisco, CA, USA), a humanized anti HER-2 monoclonal antibody. In veterinary oncology, the role of HER-2 has been investigated in feline and canine mammary tumours by in situ hybridization (Cish and Fish) and immunohistochemistry. In feline species, immunohistochemical HER-2 protein over-expression has been found in 56% (36.0–59.6%) of mammary carcinomas, a higher percentage than observed in human breast carcinomas. However, as in women, HER-2 over-expression appears to be correlated with morphological parameters suggestive of malignancy and poor prognosis (De Maria et al., 2005; Millanta et al., 2005; Ordás et al., 2007). In recent studies, HER-2 over-expression in feline mammary carcinomas appeared to be markedly lower (5.5%) than in the previous published human and canine reports and HER-2 expression was
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not correlated to poor prognosis in a 1 year post-surgical clinical follow-up (Rasotto et al., 2011). Moreover, amplification of cerbB-2 was detected only in 2/9 (20%) feline carcinomas with protein over-expression, a lower percentage than observed in humans (Ordás et al., 2007). In canine mammary tumours, previous studies have shown that both c-erbB-2 amplification (Ahern et al., 1996) and HER-2 over-expression (Rungsipipat et al., 1999; Martin de las Mulas et al., 2003; Hsu et al., 2009) are detectable, however no gene amplification has been observed by in situ hybridization (Martin de las Mulas et al., 2003). Different percentages of HER-2 over-expressing carcinomas have been reported (ranging from 19.1% to 35.4%); and the relevance of HER-2 over-expression, in canine mammary tumours, as a prognostic factor has not yet been clearly determined (Rungsipipat et al., 1999; Dutra et al., 2004; Hsu et al., 2009). In order to clarify the role of HER-2 over-expression in canine mammary tumours, we evaluated the receptor expression status in neoplastic mammary tissues and in normal and hyperplastic tissues surrounding tumours. This was done by immunohistochemical study, by correlating its expression in cancer cells with clinical–pathological features and overall survival. 2. Materials and methods 2.1. Animals Tissues were supplied by the Animal Tumours Registry at the University of Pisa. The tissue samples were from 46 female dogs of different breeds, bearing benign or malignant mammary neoplasms. The cases belonged to the Veterinary Training Hospital (Clinical Department of Veterinary Medicine of the University of Pisa) and came from operations carried out between January 2004 and December 2007 or from referring practitioners in the surrounding areas. In addition to individual data (age, breed, body size and history of ovariohysterectomy), data on tumour size (maximum length), adherence to underlying tissues, and skin ulceration were also recorded. Ipsilateral regional lymph node sampling was performed in 29/35 dogs with grossly infiltrating carcinomas. All the animals with invasive carcinomas were submitted to a 2-year follow-up after surgery, to evaluate the post-surgical course of the disease. Overall survival was defined as the time from the day of diagnosis until the day of death or last follow-up. Death due to tumour associated causes was recorded as data and confirmed by necropsy.
according to the Hercept test (DakoÒ), thus the sections were dewaxed and rehydrated in distilled water through a series of graded alcohols. Epitope retrieval was carried out in the microwave oven using an epitope retrieval solution (citrate buffer, pH 6.0) at 98 °C for 40 min. Endogenous peroxidase activity was blocked by incubating the sections in 12 ml of methanol containing 200 ml of 30% hydrogen peroxide for 30 min, followed by washing in TBS/Tween. The prediluted HER2/neu antibody was applied for 30 min, followed by rinsing in TBS/Tween. A visualization reagent was applied for 30 min and rinsed with TBS/Tween, followed by a DAB solution (3, 30 -diaminobenzidine tertahydrochloride, Sigma D5637, dissolved in 11 ml Tris/hydrochloric acid buffer at pH 7.6, to which 100 ml of 1% hydrogen peroxide was added just before use) for 10 min. DAB was removed by rinsing with distilled water and the slides were then counterstained with haematoxylin, dehydrated in increasing grades of ethanol, than cleared in xylene, and mounted (DepexÒ). A human breast tumour known to react with HER-2 antibody was used as a positive control, while negative controls were performed by omitting the primary antibody and replacing the primary monoclonal antibody with a subtype matched unrelated primary antibody. The Hercept test scoring system was used to establish the degree of membrane staining. No staining or membrane staining observed in <10% of tumour cells was given a score of 0; faint/barely perceptible membrane staining detected in >10% of tumour cells was scored as 1+; a weak to moderate and strong complete membrane staining observed in >10% of tumour cells were graded as 2+ and 3+, respectively. A score of 1+ was considered negative and 2+ and 3+ were considered positive. A human breast tumour known to exhibit a score of 2+ was considered as a reference for 2+ scores. Slides supplied with the Hercept test system were used as a reference for scores of 1+ and 3+. 2.4. Statistical analysis Statistical analysis was performed using SPSS Advanced Statistics 13.0 (SPSS Inc., Chicago, IL, USA). The Chi square test was used to investigate the significance of the relationship between HER-2 over-expression and individual variables. Statistical significance was based on a 5% (0.05) significance level. Pearson’s correlation test was used to correlate the HER-2 expression in neoplastic mammary tissues and in tissues surrounding tumours. Overall survival analysis was performed using the Kaplan–Meier method, and the Tarone–Ware test was used to investigate the relationship between HER-2 over-expression and overall survival.
2.2. Histology
3. Results
Representative portions of each mammary surgical sample and regional lymph nodes when present were fixed in 10% buffered formalin, embedded in paraffin wax, and routinely processed. Five-micrometer-thick sections were obtained and stained with haematoxylin and eosin (HE) for histological examination. Lesions were classified according to the World Health Organization (WHO) classification (Misdorp et al., 1999), and tumours displaying multiple features were classified according to the most pronounced histologic differentiation. Tumours were graded following Elton and Ellis’s criteria (Elston and Ellis, 1991), as previously described (Millanta et al., 2005). Mitotic index and lymphatic invasion or necrosis were also recorded for each tumour.
We studied 46 mixed breed dogs (Crossbreed: n = 11; Boxer: n = 7; English setter: n = 4; Toy poodle, German shepherd and Doberman: n = 3, respectively; Kurzhaar, Rottweiler and Yorkshire terrier: n = 2, respectively; Beagle, Breton, Cirneco, Cocker spaniel, Drahathar, Labrador retriever, Samoiedo, Pitbull and Maremman shepherd: n = 1, respectively). In eleven bitches (23.9%) mammary adenomas were diagnosed, while in 35 subjects (76.1%) a mammary carcinoma was diagnosed. The mean age of the 11 bitches with mammary adenomas was 9.0 years ± 2.0 population SD (range: 6–12 years), while of the 35 subjects with mammary carcinomas, the mean age was 9.7 years ± 2.1 population SD (range 5– 14 years; median 10 years), of all these subjects 14 had an age < the median value, while 21 had an age P this value. The mean maximum diameter of the adenomas was 2.5 ± 1.8 cm (range: 0.3–3 cm), while the mean maximum diameter of the carcinomas was 3.7 ± 2.8 cm (range: 0.2–8 cm). Thirty-one of the 35 carcinomas had a maximum tumour diameter >5 cm, while only 4 were 65 cm. The mitotic index in the mammary adenomas was
2.3. Immunohistochemical study Tissue sections (5 lm thick) were cut from each block, mounted on coated slides (Superfrost plus, Menzel-Glaser, Germany) and dried at 37 °C for 24 h. Immunohistochemistry was performed
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or normal epithelial cells, often in association with a weak or moderate cytoplasmic staining. Positive and negative controls were as expected in all of the tests. HER-2 over-expression was detected in three of the 11 adenomas (27.3%; Fig. 1A), two adenomas scored +2 and one tumour scored +3. In 10 of the 35 carcinomas (28.6%; Fig. 1B), seven tumours scored +2 and three tumour scored +3. Table 2 summarizes the immunohistochemical data, listing the incidence of HER-2 over-expression for each category considered in the 35 bitches with mammary carcinomas. Of the 11 complex carcinomas, two were HER-2 over-expressing and 9 HER-non overexpressing, while of the simple carcinomas, 8/24 were HER-2 over-expressing. Of the ten HER-2 over-expressing carcinomas, six were detected in bitches of an age below the median value (6/21) and four in subjects of an age higher than the median value (4/14). Six of the 24 WDCs-MDCs carcinomas were HER-2 overexpressing, as well as four of the 11 PDCs tumours. Only one of the 17 neoplasms with a mitotic index below the media value was HER-2 over-expressing, while of the 18 carcinomas with a mitotic index above the median value, nine had a HER-2 overexpressing neoplasm. Four HER-2 over-expressing carcinomas were detected in tumours with lymphatic invasion (4/10), and six in tumours where lymphatic invasion was absent. Six HER-2 over-expressing tumours were detected in the 21 subjects alive at the end of the follow-up period and four in the 14 dead bitches. Presence of the HER-2 over-expressing tumours did not correlate with older age or tumour type (simple vs complex carcinomas), tumour grading or lymphatic invasion, while higher expression was observed in tumours with a mitotic index above the median (P < 0.005). Poor prognosis was not correlated with HER-2 status. Kaplan–Meier survival curves showed that, when HER-2 was over-expressed, bitches with malignant tumours tended to have the same survival time as subjects with HER-2 non over-expressing carcinomas (Fig. 2). The staining pattern revealed in the mammary tissues surrounding the examined tumours and the correlation between HER-2 expression in these neoplastic tissues and HER-2 expression in normal, hyperplastic and dysplastic mammary tissues surrounding these tumours are reported in Table 3. Of the 29 normal mammary tissue samples, 11 were HER-2 over-expressing (4/9 at the periphery of benign neoplasms and 7/20 surrounding malignant tumours). Seven of the 26 lobular hyperplasias were HER-2 overexpressing, one surrounding benign neoplasms (1/6), and six in tis-
0.7 ± 1.0 mitosis/10 hpf (range: 0–3) and in the mammary carcinomas, it was 11.6 ± 9.7 mitosis/10 hpf (range: 1–42 mitosis/hpf; median 9 mitosis/hpf). Eighteen of the mammary carcinomas had a mitotic index that was above the median value and 17 below the median value. At the end of the clinical follow-up, 21 out of 35 bitches with mammary carcinomas (60%) were still alive and 14 (40%) had died from mammary tumours (no deaths by non-tumour related causes). Three adenomas were of simple adenomas, while eight were complex. Simple carcinomas were the most frequent type of malignant tumors (24/35, 68.6%), whereas complex carcinomas was less frequently observed (11/35, 31.4%). Histologic grading of the 35 mammary carcinomas revealed that 24 (68.6%) were well-differentiated (WDCs; grade I) or moderately differentiated carcinomas (MDCs; grade II), and 11 (31.4%) were poorly differentiated carcinomas (PDCs; grade III). Only one of the 11 complex carcinomas was a PDC, while all the others were WDCs or MDCs. Ten of the 24 simple carcinomas were PDCs and 14 were WDCs or MDCs. The presence of lymphatic invasion was observed in 10 of the 35 carcinomas (28.6%), whereas in the other 25 carcinomas (71.4%), only a local stromal invasion was detected. In the follow-up study, poor prognosis (2 years after surgery) was significantly associated with simple carcinomas (P < 0.025), PDCs (P < 0,001), mitotic index above the median value (P < 0.01) and lymphatic invasion presence (P < 0.001), whereas poor prognosis was not related with tumor size and age of subject, although all the subjects with carcinomas with a diameter 65 cm were alive at the end of the follow-up period (Table 1). This finding is in agreement with data reported on morphologic prognostic markers in canine mammary carcinomas (Lana et al., 2007), which demonstrates that our population is a representative sample of the canine mammary tumour population. Thirty-seven hyperplastic lesions were diagnosed in tissues surrounding neoplastic alterations: 26 lobular hyperplasia (70.3%), six cases surrounding benign and 20 malignant tumours, and 11 ductal hyperplasia (29.8%), two cases surrounding benign and 9 malignant tumours. In addition, in 29/46 samples, a residual normal mammary gland was also detected in nine cases at the periphery of benign neoplasms and in 20 cases, in tissues surrounding the 35 carcinomas. The immunohistochemical analysis of HER-2 revealed membrane labeling of tumour cells as well as neighboring hyperplastic
Table 1 Correlation among clinical–pathological features of the 35 examined canine malignant mammary tumours and overall survival of bitches bearing these cancer. Clinico-pathological features
No. of cases
Overall survival
P
Alive (No. of cases)
Dead (No. of cases)
Tumour type Complex Simple
11 24
10 11
1 13
0.025
Tumour size 65 cm >5 cm
4 31
4 17
0 14
NS
Age
14 21
8 13
6 8
NS
Tumour grading WDC–MDC PDC
24 11
19 2
5 9
0.001
Mitotic index
17 18
14 7
3 11
0.01
Lymphatic invasion Absent Present
25 10
21 0
4 10
0.001
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Fig. 1. HER-2 expression in canine mammary gland lesions. (A) Simple adenoma. Presence of a complete membranous staining of a strong intensity (score 3 +) (Immunohistochemistry; Bar = 50 lm). (B) Solid carcinoma. Complete membranous staining of weak to moderate intensity (score 2 +) (Immunohistochemistry; Bar = 50 lm). (C and D) HER-2 over-expressing hyperplastic mammary tissues were observed at the periphery of a HER-2 over-expressing adenoma (score 2+) (C) and HER-2 non overexpressing (score 1+) (D) carcinomas (Immunohistochemistry; Bar = 200 lm).
Table 2 Correlation among HER-2 expression, clinical–pathological features of the 35 mammary tumours examined and overall survival of the bitches bearing these carcinomas. Clinico-pathological features
No. of cases
HER-2 expression
P
Non over-expressing (0 - + 1)
Over-expressing (+2 - + 3)
Tumour type Complex Simple
11 24
9 16
2 8
NS
Age
14 21
10 15
4 6
NS
Tumour grading WDC–MDC PDC
24 11
18 7
6 4
NS
Mitotic index
17 18
16 9
1 9
0.005
Lymphatic invasion Absent Present
25 10
19 6
6 4
NS
Tumour related death Alive Dead
21 14
15 10
6 4
NS
sues surrounding carcinomas (6/20), while three ductal hyperplasias were HER-2 over-expressing, one surrounding a benign neoplasm (1/2) and two (2/9) a malignant neoplasm. The presence of normal or hyperplastic HER-2 over-expressing mammary tissues was observed at the periphery of both HER-2 over-expressing benign (Fig. 1C) and malignant tumours, and HER-2 non overexpressing benign and malignant (Fig. 1D) tumours. Out of the nine normal mammary tissue samples surrounding adenomas, seven (five HER-2 non over-expressing and two HER-2 over-expressing) were detected at the periphery of HER-2 non over-expressing tumours, and two, both HER-2 over-expressing, in tissues surrounding HER-2 over-expressing tumours, with a significant correlation (r = 0.706; P < 0.03). Eight hyperplastic alterations were detected in tissues surrounding these benign alterations: six lobular and two ductal hyperplasias. HER-2 non
over-expressing hyperplastic alterations were always detected at the periphery of HER-2 non over-expressing adenomas. On the other hand, the two HER-2 over-expressing hyperplastic lesions were detected at the periphery of HER-2 over-expressing adenomas. The correlation between HER-2 expression in lobular hyperplasias surrounding adenomas and HER-2 expression in these tumours was significant for lobular hyperplasias (r = 800; P < 0.05), but was not explored for ductal hyperplasias due to the limited number of samples. Out of 20 normal mammary samples detected in tissues surrounding malignant lesions, 12 HER-2 non over-expressing samples were detected at the periphery of HER2-non-over-expressing carcinomas. On the other hand, of the eight samples at the periphery of the HER-2–over-expressing tumours, one was HER-2 non-over-expressing, and seven were HER-2 over-expressing with a significant correlation (r = 0.826;
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In breast cancer, the Hercept TestÒ, approved by the US Food and Drug Administration (FDA), guarantees the standardized and reproducible immunohistochemical detection of HER-2 protein in analytical conditions (Thomson et al., 2001). Previous studies have confirmed the cross reactivity between anti-human protein antibodies and canine tissues (Rungsipipat et al., 1999; Martin de las Mulas et al., 2003; Hsu et al., 2009). In this investigation, we also used the Hercept test grading system in order to compare the results with previous investigations on canine mammary carcinomas (Martin de las Mulas et al., 2003; Hsu et al., 2009). HER-2 protein over-expression was observed in 10/35 (28.6%) canine malignant mammary tumours, but also in 3/11 (27.3%) benign mammary tumours. The percentage of HER-2 over-expressing malignant mammary neoplasms was similar to published reports. Other authors have demonstrated HER-2 over-expression with the same methodology in 17.6% and 29.7% canine mammary carcinomas (Martin de las Mulas et al., 2003; Hsu et al., 2009). Rungsipipat et al. (1999) determined a value of 19.1% of malignant mammary tumours over-expressing HER-2 protein using anti-human HER-2 polyclonal antibodies. On the basis of our and all these previous studies it is possible to argue that HER-2 over-expression in canine mammary malignant tumours ranges between 16.7% and 29.7%, a percentage similar to that reported in human breast cancer (20– 30%) (Révillion et al., 1998; Almasri and Al Hamad, 2005). In our study, a higher percentage of benign mammary tumours were classified as HER-2 over-expressing. This result is in contrast with data reported by Hsu et al. (2009), who analyzed six benign mammary tumours and two normal mammary glands that scored negative for HER-2 over-expression. On the other hand, Rungsipipat et al. (1999) reported that 50% of 32 benign mammary tumours over-expressed HER-2. These different results may be related to the larger number of benign lesions included in our study and in the latter study than those carried out by Hsu et al. (2009). Unfortunately an accurate comparison between the results observed by Rungsipipat and those of our study cannot be performed due to the different scoring systems adopted. In addition, we detected HER-2 over-expression in hyperplastic or dysplastic lesions surrounding the neoplastic tissues and in a similar percentage in morphologically normal mammary gland tissues surrounding these neoplastic tissues. HER-2 over-expression in hyperplastic lesions has been previously demonstrated in canine and feline mammary tissues (Rungsipipat et al., 1999; De Maria et al., 2005, Ordás et al., 2007; Rasotto et al., 2011). Our data confirmed that in canine mammary glands, as described in the feline species (Rasotto et al., 2011), abnormal levels of HER-2 protein could be involved in cellular growth in hyperplastic or benign neoplastic alterations. Interestingly, in our study, a positive correlation
Fig. 2. Kaplan–Meier survival curves for the group of 35 bitches with malignant mammary tumours (10 HER-2 over-expressing and 24 HER-2 non over-expressing neoplasms) showing the relationship between HER-2 over-expression and two-year follow-up after surgical removal.
P < 0.0001). In tissues surrounding the malignant tumours, 20 lobular hyperplasias were detected: 13 at the periphery of HER-2 non over-expressing tumours that were always HER-2 non overexpressing, and seven at the periphery of HER-2 over-expressing carcinomas (in one case HER-2-non-over-expressing and in six cases HER-2 over-expressing). Also in this case, the correlation between HER-2 expression in neoplastic tissues and lobular hyperplasias was significant (r = 0.907; P < 0.0001). Finally, nine ductal hyperplasias were detected in tissues surrounding these neoplasms, seven HER-2 non over-expressing at the periphery of HER-2 non over-expressing carcinomas, and two HER-2-overexpressing at the periphery of HER-2-over-expressing tumours. Again, a significant correlation was detected (r = 0.839; P < 0.005) between HER-2 status in the ductal hyperplasias surrounding neoplastic tissues. 4. Discussion In this study we investigated immunohistochemical HER-2 expression in morphologically normal, hyperplastic and neoplastic canine mammary gland tissues along with its prognostic potential.
Table 3 HER-2 expression in the different canine mammary tissues surrounding neoplastic lesions and correlation among HER-2 expression in neoplastic tissues and in these normal and hyperplastic tissues. Tissues surrounding tumours
Benign tumour tissues Normal mammary tissues Lobular hyperplasia Ductal hyperplasia Malignant tumour tissues Normal mammary tissues Lobular hyperplasia Ductal hyperplasia
c-erbB-2 expression
c-erbB-2 expression Normal
Over
Normal Over Normal Over Normal Over
5 2 5 0 1 0
0 2 0 1 0 1
Normal Over Normal Over Normal Over
12 0 13 0 7 0
1 7 1 6 0 2
Pearson
P
0.706
0.03
0.800
0.05
ND
0.826
0.0001
0.907
0.0001
0.839
0.005
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was observed between the HER-2 status of hyperplastic or dysplastic lesions and that of the respective tumours. As expected, in our study several factors were significantly associated with a poor survival prognosis 2 years after surgery, including tumour size >5 cm, histological classification of simple carcinoma, tumour grading of PDCs, mitotic index above the median value, and the presence of lymphatic invasion. These results confirm the findings of previous studies regarding the importance of these prognostic factors for canine mammary carcinomas (Chang et al., 2005; Hsu et al., 2009) and highlight that the sample we studied is representative of the population of mammary tumors in dogs. In human oncology, many studies have demonstrated that gene amplification of c-erbB-2 and/or overexpression of its receptors are linked to shorter disease-free intervals, increased risk of metastasis, and resistance to many types of therapy. Receptor activation, in fact, improves tumor cell motility, protease secretion and invasion. It also modulates the cell cycle checkpoint function, DNA repairing, and the apoptotic response (Eccles, 2001). The HER status of breast tumors is of clinical predictive importance, since anticancer therapies targeted at specific tumor-associated gene products are expected to be effective in identifying therapeutic regimens. A humanized anti-HER-2 monoclonal antibody was developed to bind HER-2 protein and inhibit tumor cell growth (Cobleigh et al., 1999) and has been widely introduced for the treatment of HER-2 over-expressing carcinomas (Hortobagyi, 2001; Vogel et al., 2002). It is therefore important to accurately evaluate the status of HER-2. Several studies have thus indicated that analysis of DNA and protein levels are equally useful (Van de Vijver, 2001). Immunohistochemical demonstration of HER-2 over-expression could represent a simple and reliable method for examining protein expression status. In addition, it provides similar results to other standard methods such as fluorescent in situ hybridization (Jacobs et al., 1999; Kaya et al., 2001). A number of studies have attempted to identify a similar prognostic role for HER-2 expression in canine mammary carcinomas. In a previous investigation (Dutra et al., 2004), HER-2 expression was found to be associated with some well known morphological indicators of poor prognosis. In particular a correlation was found between HER-2 expression and tumour histological grading. A similar correlation has been previously reported in several cases of human breast cancer, where HER-2 amplification (Tsuda et al., 1992; Gramlich et al., 1994) or expression (Leal et al., 1995, 2001) was correlated with histological grade. In our study, the correlation between HER-2 over-expression and tumour histological grading was not detected, while HER-2 over-expression was correlated with tumour mitotic activity. Tsuda et al. (1990) obtained similar data regarding the correlation between mitotic count and HER-2 over-expression, in breast cancer. Despite this, in our Kaplan–Meier analysis, HER-2 over-expression was not correlated with overall survival, and bitches with HER-2over-expressing carcinomas did not present a poorer prognosis than those with HER-2-non-over-expressing tumours. These data confirm previous studies on canine mammary carcinomas, where HER-2 over-expression was associated with higher survival rate (Dutra et al., 2004; Hsu et al., 2009). On the other hand the data are in contrast with another study where three bitches with HER-2-over-expressing mammary carcinomas had metastasis or died <6 months after surgery (Martin de las Mulas et al., 2003). The results obtained in our study together with the majority previous investigations carried out on dogs to date contrast findings in human breast cancers, where c-erbB-2 gene amplification and protein over-expression predict shorter disease-free intervals and overall survival times (Révillion et al., 1998; Almasri and Al Hamad, 2005), suggesting that c-erbB-2 should be considered as an independent adverse prognostic factor. However the reason for
the difference in HER-2 over-expression between human breast cancer and canine mammary tumours remains unknown (Hsu et al., 2009). In conclusion, our findings further suggest that c-erbB-2 seems to play a role in the proliferation of mammary gland tissues in bitches, but that it is probably not involved in malignant transformation and is not associated with a shorter overall survival time. Although additional large scale studies are warranted to further explore the significance of HER-2 protein over-expression in canine mammary lesions, the lack of correlations between HER-2 overexpression and poor prognosis must be taken into account in assessing target therapies for dogs.
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