Epstein–Barr virus (EBV) is a ubiquitous γ-herpesvirus (human herpesvirus – type 4), with double-stranded genomic DNA of about 170 kb in length. The virus is strongly related to the development of several malignancies in humans (32, 35), particularly lymphoproliferative disorders like Hodgkin´s disease, diffuse large B-cell lymphoma, endemic Burkitt´s lymphoma, and EBV-associated NK/T-cell lymphoma (10, 12). Chronic infections with EBV have also been associated with other types of epithelial cell tumours that may include undifferentiated nasopharyngeal carcinoma and EBV-associated gastric carcinoma (29, 34). EBV induces the production of inflammatory cytokines and activates significant signalling pathways, including NF-κB, mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK), and phosphatidylinositol 3-kinase (PI3K)/Akt, favouring a highly inflammatory microenvironment and initiation and promotion of tumoral cells (24, 31). EBV has been suggested as a probable aetiological agent of breast cancer in women (11); however, the results of several studies have been inconsistent and in some cases contradictory. No expression of various EBV viral gene products including Epstein–Barr virus-encoded small RNAs (EBERs), Epstein–Barr nuclear antigen 1 (EBNA1), latent membrane protein 1 (LMP1), and latent membrane protein 2A (LMP2A) was detected in breast cancer cells (7), while others suggested that EBV was not associated with breast cancer despite the virus being present in some breast carcinoma cells (usually in <1% of the cells) (23). On the other hand, EBV gene sequences were detected in 68% of invasive breast cancer specimens that were also positive for human papilloma virus (HPV) and mouse mammary tumour virus (MMTV) gene sequences, demonstrating that individual or multiple viruses could be present in breast cancer (8). Most recently EBV was shown to infect mammary epithelial cells
Canine mammary tumours have epidemiological, histopathological, and molecular resemblance to human breast cancer and dogs have been proposed as a useful animal model to study breast cancer biology. Similarities have previously been reported between canine and human mammary tumours in terms of increased proliferation, altered cell differentiation, and decreased cell adhesion assessed by gene expression profiles of metastatic and non-metastatic tumours (17). From the perspective of the pathogenesis of neoplastic diseases, it is interesting to assess the presence of potential aetiological agents of human mammary tumours in the dog (27). In this regard, the EBV-specific
Histological classification and EBV-positivity of canine mammary tumours
Code | Breed | Age (years) | Sex | Histological diagnosis | Grade | PCR EBNA-1 | PCR |
---|---|---|---|---|---|---|---|
01 | French Poodle | 8 | F | Mixed carcinoma | I | − | − |
02 | Schnauzer | ND | F | Mixed carcinoma | I | − | − |
03 | Golden | 3 | F | Mixed carcinoma | I | − | − |
04 | Creole | 9 | F | Complex carcinoma | II | − | − |
05 | Hound | 2 | F | Solid carcinoma | III | − | − |
06 | Chow Chow | 12 | F | Benign mixed tumour | - | − | − |
07 | French Poodle | 2 | F | Mixed carcinoma | II | − | − |
08 | French Poodle | 8 | F | Simple carcinoma | II | + | − |
09 | French Poodle | 10 | F | Mixed carcinoma | II | − | − |
10 | Poodle | 13 | F | Mixed carcinoma | II | − | − |
11 | Pointer | 9 | F | Simple carcinoma | III | − | − |
12 | ND | 8 | F | Mixed carcinoma | III | − | − |
13 | Pinscher | ND | F | Simple adenoma | - | − | − |
14 | ND | 14 | M | Mixed carcinoma | II | − | − |
15 | French Poodle | 10 | F | Mixed carcinoma | III | − | − |
16 | ND | 12 | F | Mixed carcinoma | I | − | − |
17 | French Poodle | 8 | F | Complex carcinoma | II | − | − |
18 | Schnauzer | ND | F | Complex carcinoma | I | − | − |
19 | Shih-tzu | 14 | F | Simple carcinoma | III | − | − |
20 | French Poodle | 8 | F | Complex carcinoma | III | − | − |
21 | Pastor Malinois | 12 | F | Mixed carcinoma | I | − | − |
22 | Rhodesian Ridgeback | 8 | F | Mixed carcinoma | I | − | − |
23 | ND | 17 | F | Solid carcinoma | III | − | − |
24 | Rottweiler | 13 | F | Mixed carcinoma | III | − | − |
25 | French Poodle | 8 | F | Carcinoma–in situ | - | − | − |
26 | Akita | 8 | F | Mixed carcinoma | III | − | − |
27 | Maltese | 13 | F | Mixed carcinoma | I | − | − |
28 | Chihuahua | 15 | F | Mixed carcinoma | II | − | − |
29 | Labrador | 10 | F | Mixed carcinoma | II | − | − |
30 | Labrador | 9 | F | Simple carcinoma | I | − | − |
31 | French Poodle | 10 | F | Mixed carcinoma | II | − | − |
32 | Creole | 8 | F | Solid carcinoma | I | − | − |
33 | German Shepherd | 5 | F | Simple carcinoma | I | − | − |
34 | Pinscher | 11 | F | Complex carcinoma | I | − | − |
35 | French Poodle | 3 | F | Mixed carcinoma | I | − | − |
36 | Chihuahua | 9 | F | Simple carcinoma | I | − | − |
37 | Siberian Husky | 12 | F | Carcinoma–in situ | II | − | − |
38 | French Poodle | 16 | F | Simple carcinoma | I | − | − |
39 | Schnauzer | 11 | F | Mixed carcinoma | I | − | − |
40 | Pug | 7 | F | Simple carcinoma | I | − | − |
41 | French Poodle | 13 | F | Mixed carcinoma | I | − | − |
42 | Labrador | 11 | F | Complex carcinoma | I | − | − |
43 | French Poodle | 10 | F | Complex carcinoma | I | − | − |
44 | Crossbreed | 7 | F | Complex carcinoma | II | − | − |
45 | French Poodle | 12 | F | Simple carcinoma | II | − | − |
46 | Schnauzer | 9 | F | Complex carcinoma | I | − | − |
47 | Golden | 12 | F | Complex carcinoma | II | − | − |
F – female; M – male; “+” – positive; “−” – negative; ND – not determined
Histological features of canine mammary tumours are shown in Fig. 1. The single tissue positive for the EBNA-1 gene was the sample 8, coming from an 8-year-old French Poodle and showing a simple carcinoma of the tubulopapillary subtype, grade II. The mitotic index, calculated in 10 fields of 400×, was low, with an average of 0.5 mitosis/field. An infiltrative growth pattern was noted, with tubular architecture and multiple sizes, some of them with small papilla in the lumen of the tubule and covered by several layers of dysplastic epithelial cells (Fig. 1A). Mild cellular pleomorphism and anisocytosis were also observed. The nuclei varied from rounded to ovoid with finely granular chromatin and displacement toward the periphery. Deposition of cholesterol crystal foci and necrosis (Fig. 1B) were also present.
Sample 32 corresponded to a solid carcinoma which was characterised by an expansive and infiltrative growth pattern. The cells were arranged in solid cords and formed dense lobules supported by a fine fibro-vascular stroma (Fig. 1C). In a more enhanced view, the nuclei were prominent and had lost polarity, causing an obvious irregularity (Fig. 1D). Sample 11 was a tubular carcinoma, with tissue differentiation and cells organised in tubules without full definition (Fig. 1E). A simple carcinoma of the tubulopapillary type in sample 33 was associated with a moderate lymphoplasmacytic inflammatory reaction (Fig. 1F). In the following image another tubulopapillary carcinoma (sample 41) was observed, with irregular tubules, cellular anisocytosis, and nuclear pleomorphism (Fig. 1G). In the case of complex carcinoma (sample 34), different cell populations were easily observed and the marked epithelial proliferation contrasted with the myoepithelial growth pattern, which still conserved areas with glandular architecture (Fig. 1H). The cystic-papillary carcinoma (sample 36) showed pleomorphism and anisocytosis with malignant papillary proliferation, which was inserted into a dilated ductal segment with cystic morphology (Fig. 1I). Papillae extended into cystic tubular lumina and were supported by a fine fibrovascular connective tissue stroma (Fig. 1J). Sample 43 was another case of complex carcinoma. The epithelial cells were pleomorphic and arranged in irregular tubules, and the myoepithelial cells were spindle shaped within the interstitium (Fig. 1K). Finally, a simple adenoma was found in sample 13, where displastic glandular cells acquired a tubular organisation with no clear findings of malignancy (Fig. 1L).
EBV is estimated to infect more than 90% of the world´s adult population (6); however, only some people are susceptible to developing neoplasm induced by the virus. The exact role that EBV plays in the promotion of benign tumours, and the progression of breast cancer in women is still uncertain, despite several studies showing an association between the virus and this type of malignancy (11). A meta-epidemiological study concluded that EBV infection may increase the risk of breast cancer (1). EBV is known by its capacity to induce cellular growth and alter the tumoural environment (24). In this study, we found a high frequency of the mixed type of mammary tumours in dogs. Because only one sample was positive for EBV, it was not possible to establish a relationship between a tumour type and the presence of the virus. A recent study assessed the prognostic significance of the histologic subtypes and evidenced that survival was higher in dogs with benign mixed tumours, complex carcinoma, or simple tubular carcinoma than with simple tubulopapillary carcinoma or other subtypes (25). Similarly, another study reported that dogs with simple carcinomas had a less favourable prognosis than dogs with other types of carcinoma (16). Domestic dogs can be naturally infected with the EBV-like γ-herpesvirus (14). The reported findings of viral DNA sequences and viral oncogenic proteins in serum (3, 20) and in multiple canine tumours (4, 14) led some researchers to propose that dogs may represent an exceptional model in the study of EBV-related disease (14, 15). Lymphoid malignancies in both human and canine tumours retain morphologic, biological, and molecular similarities (15).
To our knowledge, this study for the first time reports the detection of EBV in canine mammary tumours, showing the amplification of the EBNA-1 gene by PCR. A previous study that assessed the presence of EBV and other viruses in tumoural and normal mammary tissues of dogs did not detect any virus in these tissues (30). In the same way, the EBV EBNA-1 and EBNA-2 proteins could not be detected by immunohistochemistry in lymphoid neoplasms (14). The EBV
In this study, the detection of EBV gene sequence was dependent on the volume of amplification product from the first round of PCR that was used in the nested PCR. Only one sample was positive for the presence of EBV genes, and the possible reason for this result may be that EBV detection in canine and human tumours, and other tissues is largely affected by technical factors. The quantity and quality of viral DNA available for the identification, including the type of tissue chosen (fixed in formalin and embedded in paraffin), DNA concentration, primer sequences, and particular conditions of the PCR technique used to amplify the viral gene (simple or nested PCR and volume of reaction) may all exert influence. These factors may also partially explain the fact that although antibodies to EBV are sometimes detected, gene sequences are not always amplified. In a previous study, in spite of having detected antibodies to EBV viral capsid antigens in 41% of canine lymphomas, the authors failed to detect EBV-specific DNA and RNA sequences including those of the
We conclude that in the development of mammary gland tumours of dogs there may be a large number of aetiological elements besides the potential presence of EBV. This preliminary study reveals a lack of association between EBV and these tumours in dogs. Tumour origin and progression is a multifactorial process and therefore it is necessary to establish more accurately, in addition to the presence of viral sequences the mechanisms used by this virus to interact with animal tissues and the progression to neoplastic cells if such is the case. Additional studies are required to identify a causal relationship between EBV and mammary gland tumours in dogs, which could eventually have an impact on public health prevention and control practice.