ABSTRACT
Background
Mammary tumours are the most common neoplasm in female dogs. Canine mammary tumours (CMTs) are appropriate models for human studies.
Objectives
The present study aimed to diagnose CMTs pathologically and compare different tumour types by immunohistochemistry (IHC).
Methods
Histological features of various tumours were recorded. Moreover, haematological, biochemical and immunohistochemical changes were evaluated in affected animals.
Results
Among 130 mastectomized dogs in veterinary hospitals in Tehran, Iran, 74 dogs were affected by various types of mammary tumours. The highest frequency of malignant tumours was related to carcinoma types (38/74; 51.4%) and the highest frequency of benign tumours was related to fibroadenoma (6/74; 8.1%). In malignant tumours, anaemia and monocytosis were observed compared to benign tumours. Serum ALT and ALP levels were also significantly increased in malignant tumours (p < 0.05) compared to benign tumours. The IHC showed the profile of adenoma (ductal and intraductal papillary) ER+/PR−/HER2−, benign mixed tumour ER−/PR−/HER2−, carcinoma (mucinous, anaplastic) ER−/PR−/HER2− and metastatic mast cell tumour ER−/PR+/HER2−. The highest expression of αSMA was recorded in benign tumours and the highest expression of Ki67 was recorded in metastatic mast cell tumour, benign mixed tumour and adenoma (p < 0.05).
Conclusion
Together, although the dog is a good model for studying mammary tumours, the variability of results in CMTs requires further studies in this field. An interesting finding of this study was the triple‐negative profile in the benign mixed tumour, which had previously been reported only for malignant mammary tumours. In addition, the significant positivity of Ki67 expression (34%–66%) in benign CMTs is noted for the first time.
Keywords: dogs, immunohistochemistry, mammary tumours, triple‐negative profile
The study examined 130 dogs that had mastectomies in Tehran, Iran. Malignant tumours were most commonly carcinomas, while the most frequent benign tumours were fibroadenomas. Blood tests indicated that dogs with malignant tumours had lower red blood cell and haemoglobin levels, signifying anaemia and higher monocyte counts compared to those with benign tumours. Significant increases in serum ALP and AST enzymes were also noted in malignant tumours. The interesting IHC finding was a triple‐negative profile (ER−/PR−/HER2−) in the mixed benign tumour, with significant positivity of Ki‐67 expression in benign canine mammary tumours.
1. Introduction
Mammary tumours are the second most common neoplasm in dogs and the most common neoplasm in female dogs (Goldschmidt et al. 2011; Kaszak et al. 2018). Mammary tumours are most commonly found in dogs 5 years of age or older. Dachshunds, Cocker Spaniels, Toy Poodles, German Shepherds and Mixed‐breed dogs have been reported to have the highest incidence of mammary neoplasms (Tavasoly et al. 2013). Several immunohistochemical markers have been used as a panel for clinical prognosis in women with breast tumours. Analysis of this panel of markers can be important in determining the specific phenotype of the tumour associated with a specific biological behaviour (Varallo et al. 2019). Early, accurate and minimally invasive diagnosis is essential, as some CMTs are malignant and have a poor prognosis. Fine‐needle aspiration cytology (FNAC) and blood testing are both minimally invasive diagnostic methods that have been used in veterinary medicine. However, appropriate biomarkers need to be identified to diagnose and assess the prognosis of CMTs. To date, the most studied biomarkers are FNAC‐related, such as Ki67, human epidermal growth factor receptor 2 (HER2), oestrogen receptor (ER), progesterone receptor (PR), p53, E‐cadherin and cyclooxygenase 2 (COX2) (Kaszak et al. 2018; Varallo et al. 2019). However, in veterinary medicine, there are currently few studies focused on this topic, and the published results are also contradictory (Varallo et al. 2019; Xinyi et al. 2025).
Approximately 50% of CMTs are malignant. The most common malignant types in dogs are adenocarcinoma (tubular carcinoma), followed by papillary carcinoma, solid carcinoma, complex carcinoma and carcinosarcoma. The most common types of canine benign mammary tumours are fibroadenoma, ductal papilloma, mixed benign tumours and simple adenoma (Kaszak et al. 2018). There have been controversies and challenges regarding grading, prognosis and survival after resection of CMTs. As noted in many studies, lymphatic/vascular invasion and lymph node metastasis have been associated with a poor prognosis (Goldschmidt et al. 2011). The present study aimed to provide a clinico‐pathological and immunohistochemical characterization of canine mammary tumours (CMTs), with particular emphasis on accurate histological classification (including mixed tumours), evaluation of hormonal receptor status (ER, PR and HER2), assessment of myoepithelial integrity (αSMA) and determination of proliferative index (Ki67). An additional objective was to integrate these pathological findings with pertinent clinical data, such as haematological, ultrasonographic and therapeutic history, to establish a practical diagnostic approach that could inform appropriate clinical decisions and improve prognostic accuracy in affected bitches.
2. Material and Methods
This study was conducted on mammary masses (paraffinized samples, samples obtained after surgery and necropsy) of 130 dogs referred to small animal treatment centres in Tehran city in different age groups over a period of 3 years from 1 October 2020 to 1 October 2023. Given that the formation of tumour tissues in the mammary glands of dogs takes time and the chance of encountering dogs with mammary cancer at an older age is higher, the dogs studied were generally older than 3 years of age. This study included dogs with a mean age of 10.16 ± 2.92 years referred to small animal clinics in Tehran, along with tissue samples obtained from dogs undergoing surgery or collected from private small animal clinics and veterinary laboratories in Tehran (Labra Laboratory, Tehran Azma Laboratory, Adonis Veterinary Hospital and Tehran Veterinary Hospital). Given that non‐spayed dogs are more likely to develop mammary cancer, all of the referred cases in this study were non‐spayed females; spayed dogs were excluded from the analysis.
2.1. Haematology and Biochemistry
Blood samples containing EDTA, which were taken from the jugular vein of dogs with mammary tumours using a syringe needle (21 gauge), were analysed using a CELL‐DYN 3500 automated haematology analyser (Abbott Diagnostics Division, Santa Clara, CA, USA). On the other hand, biochemical tests were performed to measure ALT, AST and ALP levels using commercial kits (Pars Azmoon, Tehran, Iran). Because haematological and biochemical data were unavailable for some dogs, the analyses were performed on the confirmed 38 cases for which complete blood and serum samples were obtained.
2.2. Pathology
Mammary tissue samples obtained from surgery were first placed in a 10% formalin buffer solution at a ratio of at least 1/10 (tissue volume/fixative volume) for at least 48 h for tissue fixation. After fixation, the samples were cut with a maximum thickness of 5 mm, and after placing them in special containers, the tissue processing operation (dehydration, clarification and staining) was performed using an Autotechnicon device (Leica TP1020, Germany). Then, tissue sections with a thickness of 4–6 µm were prepared using a rotary microtome (Leica RM2125 RTS), and after floating in a hot water bath and mounting them on glass slides, routine haematoxylin and eosin (H&E) staining steps were performed on them. Also, if necessary, in addition to H&E staining, tissue sections were stained with Masson's trichrome, Alcian blue and Toluidine blue (Amniattalab and Rezazadeh 2022; Amoorahim and Amniattalab 2025). Evaluation and classification of benign and malignant tumours were performed based on the guidelines proposed by Goldschmidt et al. (2011, 2017).
2.3. IHC
After confirming the mammary tumours by light microscopy and determining their type and pathological pattern, tissue sections were stained with the immunohistochemical markers ER, PR, HER2, αSMA and Ki67 using the EnVision + Dual Link System‐HRP method to compare the tumours with normal mammary tissues. Briefly, deparaffinized sections were rehydrated with ethanol, and the slides were incubated in 3% hydrogen peroxide to inhibit endogenous peroxidase activity for 30 min. Antigen retrieval was performed using 0.01 M citrate buffer at pH 6.0 for 25 min. Slides were incubated in 5% bovine serum albumin in Tris‐buffered saline (TBS) to prevent nonspecific staining for 30 min. The slides were then washed in TBS and water and incubated at room temperature. Subsequently, the slides were incubated overnight at 4°C with the following primary antibodies αSMA (Dako, USA; 1:200), Ki67 (Dako, USA; 1:100), ER (Dako, USA; 1:35), PR (Novocastra, UK; 1:40) and HER2 (Dako, USA; 1:250). Finally, the slides were washed with phosphate‐buffered saline (PBS) and treated with streptavidin‐horseradish peroxidase for 20 min. The slides were washed and treated with diaminobenzidine for 10 min, and the sections were counterstained with Harris haematoxylin. Healthy mammary tissue from the same animal (obtained from the contralateral normal gland, distant from the tumour) was used as the positive control. For negative controls, non‐immune IgG was used instead of the primary antibodies (Amoorahim and Amniattalab 2025).
2.4. IHC Scoring
The percentage of positive cells was scored according to Varallo et al. (2019): 0 = no labelling, 1 ≤ 1%, 2 = 1%–10%, 3 = 11%–33%, 4 = 34%–66% and 5 = 67%–100% (Varallo et al. 2019). A threshold of 10% was used to define positivity (Peña et al. 2014). The intensity of immunoreaction was scored as 0 = absent, 1 = weak, 2 = moderate and 3 = strong (Varallo et al. 2019). Cell counting followed Nunes et al. (2022), counting ≥ 500 cells at 400× magnification (Nunes et al. 2022).
In addition, the intensity of the immunoreaction for the markers was scored as follows: 0 = absent, 1 = weak, 2 = moderate and 3 = strong (Varallo et al. 2019). It is important to note that two types of benign neoplastic lesions, including benign mixed tumour of the mammary and adenoma (ductal and intraductal papillary) (three samples of each type), and two types of malignant neoplastic lesions, including carcinoma (mucinous and anaplastic) and metastatic mast cell tumour (three samples of each type), were considered and prepared for the comparative scoring of immunohistochemical slides.
2.5. Statistical Analysis
The obtained data were analysed using SPSS 27 software. Descriptive findings of the studied variables, including indicators such as absolute and relative frequency, mean and standard deviation, were calculated and reported. First, the normal distribution of serum levels of enzymes and blood parameters, as well as the data on the number of cells positive for αSMA, ER, PR, HER2 and Ki67 in mammary neoplastic masses, was examined using the Shapiro–Wilk test. In the case of normal distribution of the residuals, one‐way analysis of variance and Tukey's post hoc test were used to compare between treatments, and for data with a non‐normal distribution, the non‐parametric Kruskal–Wallis test was used.
3. Results
Out of 130 dogs examined with mammary lesions suspected of being tumours, 74 were definitely found to be involved in benign or malignant mammary tumours after conducting tests and paraclinical examinations. The aforementioned cases underwent mastectomy surgery after additional tests were conducted and then tissue sampling was performed.
3.1. Haematology and Biochemistry
Comparative results of haematology and blood enzymatic changes are presented in Table 1. According to this table, there was a significant decrease (p < 0.05) in the number of red blood cells (RBCs) and also in the amount of haemoglobin (Hb) in the malignant tumour groups compared to benign tumours. However, in other parameters, including HCT and PLT, no significant difference was observed between benign and malignant tumours (p > 0.05). In terms of white blood cell parameters, only the number of monocytes (Mon) in the malignant tumour groups showed a significant increase (p < 0.05) compared to benign tumours. The remaining parameters, including the total number of WBCs, Neut, Eos and Lym, despite the changes, did not differ significantly between the benign and malignant groups (p > 0.05). Analysis of the results of blood enzymes showed that the levels of ALT and ALP in malignant tumours increased significantly (p < 0.05) compared to benign tumours. However, the level of AST did not differ significantly between the different groups (p > 0.05).
TABLE 1.
Haematological and biochemical parameters in dogs with different mammary tumour types. Data presented as mean ± SD.
| Mammary tumour (n = 38) | ||||||||
|---|---|---|---|---|---|---|---|---|
| Parameter | Benign mixed tumour (n = 4) | Fibroadenoma (n = 5) | adenoma (ductal and intraductal papillary) (n = 4) | Miscellaneous tumour (benign) (n = 4) | Carcinoma (mucinous, anaplastic) (n = 10) | Metastatic tumour (n = 3) | Malignant mixed tumour (MMG) (n = 8) | p‐value |
| RBC (1012/L) | 7.02 ± 0.56 | 6.33 ± 0.77 | 6.79 ± 0.30 | 7.12 ± 0.48 | 5.52 ± 0.13 | 5.12 ± 0.66 | 5.88 ± 0.70 | 0.035 |
| HCT (%) | 41.4 ± 1.54 | 38.91 ± 1.11 | 39.20 ± 2.23 | 40.75 ± 1.68 | 35.33 ± 1.18 | 34.66 ± 1.49 | 35.81 ± 0.99 | 0.651 |
| Hb (g/L) | 166.44 ± 8.71 | 159.12 ± 5.20 | 158.38 ± 4.88 | 161.30 ± 6.39 | 134.70 ± 6.80 | 130.61 ± 5.70 | 134.16 ± 4.44 | 0.048 |
| PLT (109/L) | 276.33 ± 67.55 | 223.59 ± 41.59 | 289.72 ± 58.50 | 228.49 ± 69.45 | 243.11 ± 55.65 | 189.56 ± 38.60 | 207.28 ± 67.78 | 0.733 |
| WBC (109/L) | 11.67 ± 3.33 | 8.05 ± 2.40 | 12.02 ± 2.20 | 10.42 ± 3.33 | 12.49 ± 7.54 | 12.85 ± 6.03 | 10.15 ± 3.81 | 0.479 |
| Neut (109/L) | 5.24 ± 0.89 | 6.73 ± 1.06 | 6.33 ± 1.44 | 5.90 ± 1.20 | 7.39 ± 1.61 | 9.66 ± 1.75 | 7.28 ± 1.88 | 0.580 |
| Lym (109/L) | 3.16 ± 0.30 | 2.89 ± 0.71 | 3.07 ± 0.22 | 3.45 ± 0.18 | 3.36 ± 0.69 | 3.19 ± 0.38 | 2.85 ± 0.25 | 0.614 |
| Mon (109/L) | 0.61 ± 0.26 | 0.55 ± 0.57 | 0.66 ± 0.16 | 0.47 ± .036 | 1.36 ± 0.58 | 1.69 ± 0.37 | 1.17 ± .081 | 0.035 |
| Eos (109/L) | 0.50 ± 0.24 | 0.41 ± 0.18 | 0.24 ± 0.10 | 0.57 ± 0.23 | 0.83 ± 0.24 | 1.37 ± 0.45 | 0.98 ± 0.24 | 0.668 |
| ALT (U/L) | 31.04 ± 10.48 | 21.32 ± 12.70 | 25.88 ± 6.88 | 31.45 ± 5.78 | 83.41 ± 86.73 | 48.1 ± 23.5 | 112.5 ± 96.78 | 0.032 |
| AST (U/L) | 27.17 ± 8.83 | 61.49 ± 14.94 | 44.5 ± 31.82 | 23.12 ± 12.57 | 34.32 ± 19.04 | 56.47 ± 10.60 | 32.13 ± 18.75 | 0.628 |
| ALP (U/L) | 111.34 ± 40.23 | 81.55 ± 26.99 | 105.11 ± 64.21 | 90.13 ± 35.72 | 277.74 ± 75.68 | 119.73 ± 37.88 | 179.71 ± 175.62 | 0.004 |
Note: The reference ranges for different parameters are RBC (5.8–8.50); HCT (37–55); Hb (140–190); PLT (150–400); WBC (6–13); Neut (3–10.50); Lym (1–4); Mon (0.15–1.2); Eos (0.0–1.3); ALT (17–95); AST (18–56); ALP (7–115). p < 0.05 is significant.
3.2. Pathology and IHC
Pathological sections of mammary masses from dogs that had undergone mastectomy or autopsy (for metastatic tumours) were prepared at a thickness of 6 µm and stained with H&E. Of the 130 dogs initially presenting with mammary lesions suspected to be tumours, histopathological examination confirmed benign or malignant mammary tumours in 74 cases. The remaining 56 cases were diagnosed as non‐neoplastic conditions (e.g., mammary hyperplasia, mastitis, cysts) and were excluded from further analysis. Among 74 tissue samples of mammary tumours, the most common benign tumour type was fibroadenoma (6 cases) and the most common malignant tumour type was carcinoma (mucinous and anaplastic) (38 cases). Of 74 cases of malignant mammary tumours, definitive metastasis to the liver, lungs and mammary tissues was documented in 24 cases (16 carcinomas, 5 malignant mixed tumours and 3 mast cell tumours). In a single case of benign mixed mammary tumour, concurrent liver metastasis was noted; nevertheless, the presence of an additional neoplasm precluded a conclusive determination as to whether the metastases derived from the primary mammary tumour or the coexisting lesion. The appearance of some CMT lesions is presented in Figure 1. Also, the pathological pattern of some of the tumours recorded is presented in Figure 2. Toluidine blue staining was used to confirm tumour mast cells in metastatic samples, and Alcian blue staining was used to confirm cartilage or bone cells in mixed benign tumours, the results of which are presented in Figure 2. The mean ± standard error of the number of cells with positive immunoreactivity against αSMA, ER, PR, HER2 and Ki67 in mammary neoplastic masses is presented in Table 2. Given the normal distribution of residuals in all groups in the Shapiro–Wilk test, one‐way analysis of variance and Tukey's post hoc test were used to compare the number of cells with positive immunoreactivity against αSMA, ER, PR, HER2 and Ki67 antibodies in mammary neoplastic masses. The counting of positive cells (performed on at least 500 mammary tissue cells at 400× magnification in randomly selected opposite fields; Table 2) showed that the average number of cells positive for ER, PR and Ki67 differed significantly among all benign and malignant tumour groups (p < 0.001) (3 cases of each benign or malignant tumour type were examined). In the case of αSMA marker, there was a significant difference (p < 0.001) in the remaining groups, except for the carcinoma (mucinous and anaplastic) and adenoma (ductal and intraductal papillary) groups, which were not significantly different. Also, in the case of HER2 marker, there was a significant difference (p < 0.001) in the remaining groups, except for the carcinoma and metastatic mast cell tumour groups, which were not significantly different. Regarding the results of scoring the expression intensity of immunohistochemical markers, according to Table 3, except for the expression intensity of αSMA, which was not significant in different tumour groups, the expression intensity of other markers showed significant differences (p < 0.05) in different tumour groups. The results of staining with immunohistochemical markers ER, PR, HER2, αSMA and Ki67 are presented in Figure 3. Finally, the characteristics of benign mammary tumours diagnosed in the current study are presented in Table 4.
FIGURE 1.
Macroscopic appearance of some canine mammary tumours. (A) Intraductal papillary adenoma. (B) Carcinoma. (C) Malignant mixed tumour.
FIGURE 2.
(A) Anaplastic carcinoma tumour with moderately pleomorphic cell masses (arrow in main image), low mitotic count and different duct shapes (small and large) and inflammation in some areas of the tissue. Anaplastic cells with cytoplasm and a few nuclei (arrow in small image) are also visible. (B) Ductal adenoma. Uniform benign tumour cells with a villous proliferation pattern and with many layers (arrow) without mitotic figures, inflammation or haemorrhage. Higher magnification of tumour cells of the duct wall with many layers and villous shape (asterisk). (C) Metastatic mast cell tumour (in mammary tissue). Uniform hyperplastic cells of the mammary ducts are visible, with some of them falling into the duct (arrowhead), and the mammary parenchyma is occupied by many tumour mast cells (asterisk). These mast cells have many mitotic figures and are giant cells with cytoplasm and large and hyperchromatic nuclei (arrow in small image). (D) Benign mixed tumour. Presence of masses of cartilage and sometimes bone cells (asterisk) as well as desmoplasia in the mammary parenchyma. In the thumbnail, a uniform, multilayered epithelial tissue of some mammary glands (arrow) can be observed. (E) Fibroadenoma tumour. Increased number of glandular cells (arrow) and connective stroma (arrowhead) in the mammary parenchyma without mitotic figures and with slight inflammation in some areas of the tumour. In the thumbnail, glandular cells (arrow) and connective tissue (arrowhead) can be seen. (F) Intraductal papillary adenoma. Proliferation of glandular cells and mammary ducts in a villous manner (arrowhead) within cystic spaces and the presence of single cysts (asterisk) of inflammatory cells in some areas of the mammary parenchyma. In the thumbnail, mammary glands with a single row of cuboidal epithelial cells (arrow) without pleomorphism or mitotic figures are seen. (A–F; H&E). (G) Metastatic mast cell tumour; tumour mast cells (arrowhead) with purple metachromatic granules occupying the mammary parenchyma in large numbers (Toluidine blue staining). (H) Benign mixed tumour; bone cells with pinkish‐orange nuclei (arrow) and blue‐stained cartilage material (arrowhead) (Alcian blue staining).
TABLE 2.
Comparison of immunohistochemical marker expression (mean ± SD) in mammary tumour groups.
| Group | Antibody | ||||
|---|---|---|---|---|---|
| α‐SMA | ER | PR | HER‐2 | Ki67 | |
| Carcinoma (mucinous, anaplastic) | 228.66 ± 8.57b | 9.93 ± 0.20b | 8.66 ± 0.60b | 29.00 ± 2.59c | 64.33 ± 4.22a |
| Metastatic mast cell tumour | 318.00 ± 8.89c | 42.50 ± 2.08c | 131.66 ± 3.11c | 27.1 ± 1.36c | 205.00 ± 5.19c |
| Benign mixed tumour | 169.33 ± 5.78a | 3.66 ± 0.33a | 2.50 ± 0.28ab | 0.00 ± 0.00a | 125.83 ± 1.64b |
| adenoma (ductal and intraductal papillary) | 254.16 ± 2.68b | 89.00 ± 1.75d | 1.33 ± 0.60a | 18.33 ± 1.74b | 270.16 ± 5.08d |
| p‐value | < 0.001 | < 0.001 | < 0.001 | < 0.001 | < 0.001 |
Note: Different superscript letters (a,b,c,d) within columns indicate significant differences at p < 0.05.
TABLE 3.
Immunoreactivity intensity scores for αSMA, ER, PR, HER2 and Ki67 in mammary tumour groups. Data presented as median (25th–75th percentile).
| Group | Antibody | ||||
|---|---|---|---|---|---|
| α‐SMA | ER | PR | HER‐2 | Ki67 | |
| Carcinoma (mucinous, anaplastic) | 3 (3–3) | 1 (1–1)a | 1 (1–1)a | 1 (1–1)ab | 2 (1–2)a |
| Metastatic mast cell tumour | 3 (3–3) | 1 (1–1)a | 2 (2–2)b | 1 (1–1)ab | 3 (3–3)b |
| Benign mixed tumour | 3 (2–3) | 1 (1–1)a | 1 (1–1)a | 0 (0–0)a | 3 (3–3)b |
| adenoma (ductal and intraductal papillary) | 3 (3–3) | 2 (2–2)b | 1 (0–1)a | 2 (1–2)b | 2 (2–2)a |
| p‐value | < 0.392 | < 0.023 | < 0.023 | < 0.021 | < 0.015 |
Note: Different letters within columns indicate significant differences at p < 0.05.
FIGURE 3.
Immunohistochemical findings of four types of benign and malignant canine mammary tumours for markers ER, PR, HER2, αSMA and Ki67. (1) Carcinoma; in this malignant tumour, as can be observed, the expression of the three markers ER, PR and HER2 is triple‐negative (ER−/PR−/HER2−), but the expression of αSMA and Ki67 is positive. It should be noted that in the case of HER2, weak expression (1) was observed in a small number of cuboidal epithelial cells of the mammary glands (less than 5% of the cells), which was recorded as negative because an expression threshold of at least 10% is considered for the expression of ER, PR and HER2 markers. αSMA expression was 34%–66% for myoepithelial cells of the gland wall and muscular cells of the duct wall, and its expression intensity was also recorded as strong (3). Ki67 expression was also recorded focally in 11%–33% of the gland epithelial cells with an expression intensity as moderate (2). (2) Metastatic mast cell tumour; in this tumour, ER−/PR+/HER2−, PR expression was in the range of 11%–33% and the intensity of expression was moderate (2). This was explained by the explanation that the positive expression of progesterone was related to a lineage of tumour mast cells and not the cells of the mammary tissue itself. In addition, the nuclear expression of Ki67 was also in the range of 34%–66% and the intensity of expression was strong (3), which was also related to tumour mast cells present in the mammary parenchyma. The expression of αSMA was also in the range of 66%–34% and its intensity of expression was strong (3). (3) Benign mixed tumour; despite its benignity, ER−/PR−/HER2− was recorded in this tumour. The expression of SMAα was also in the range of 34%–66% and its intensity of expression was strong (3). The nuclear expression of Ki67 was also in the range of 11%–33% and the intensity of expression was strong (3). (4) intraductal papillary adenoma; in this benign tumour, they were ER+/PR−/HER2−. ER expression was recorded in the range of 11%–33% and its intensity of expression was moderate (2). SMAα expression was also in the range of 34%–66%, and its intensity of expression was strong (3). Nuclear expression of Ki67 was also in the range of 34%–66% in the glandular epithelial cells as well as in the myoepithelial cells, and in the range of 34%–66% and its intensity of expression was moderate (2). IHC.
TABLE 4.
Characteristics of the canine benign mammary tumours.
| Tumour type | Number | Immunophenotype | Size (cm) | Haemorrhage | Ki67 nuclear expression (%) | Existence of concurrent tumour in mammary tissue |
|---|---|---|---|---|---|---|
| Benign mixed tumour | 3 | ER−/PR−/HER2− | 1–4 |
− (n = 2) + (n = 1) |
11–33 | 1/3 * |
| Adenoma (ductal and intraductal papillary) | 3 | ER+/PR−/HER2− | 2–7 | — |
34–66 (n = 2) 11–33 (n = 1) |
— |
Concurrent tumour type not diagnosed.
4. Discussion
The results of the present study showed that among 130 dogs of different breeds that were referred to veterinary hospitals in Tehran for mastectomy (and sometimes necropsy) over 3 years, 74 were definitely diagnosed with benign and malignant tumours based on pathological observations, with the highest frequency of malignant tumours being carcinomas (mucinous and anaplastic) (38 cases; 51.4%) and the highest frequency of benign tumours being fibroadenomas (6 cases; 8.1%) (Table 5). Also, the highest frequency of mammary tumours was in the Terrier Mix breed (40.5%), followed by the Shih Tzu breed (18.9%) (Table 6), which may be due to the greater popularity of these breeds for keeping. However, in the study by Fattahian et al. (2010), it was stated that Terrier and Dachshund breeds had the highest frequency of referrals for mammary tumours in Tehran. In addition, about 43% of the mammary tumours identified in this study were malignant and the remaining 57% were benign and the age range of dogs with tumours was 5.7–6.3 years (Fattahian et al. 2010). While in the present study, the age range of dogs with tumours was 10–12 years; 25.7% of the tumours were benign and 74.3% were malignant. Another study by Razavirad et al. (2024) on 64 CMTs in Tehran, Iran, reported that 35.94% of the tumours occurred at the age of ≥ 8 years, 32.81% at the age of 9–11 years and 31.25% at the age of ≤ 12 years (Razavirad et al. 2024). According to the sources, most benign tumours of the canine mammary gland are of the ductal adenoma, fibroadenoma or mixed tumour type. In the current study, fibroadenoma and adenoma (ductal and intraductal papillary) tumours were more frequent. In addition, it was mentioned that the most common malignant tumours of the canine mammary gland are carcinomas (solid carcinoma, tubular and papillary adenocarcinoma and anaplastic carcinoma) (Goldschmidt et al. 2011; Nosalova et al. 2024). In the present research, all the aforementioned malignant tumours were observed, and in addition to them, metastatic tumours and fibrosarcoma were also recorded. In addition to previously established risk factors for canine malignant mammary tumours such as age, breed, body size, housing conditions, body condition score and reproductive status (Gonçalves da Silva et al. 2023), the findings of the present study suggest that several additional variables may have substantially influenced the progression to malignancy in the majority of cases examined. These include: (a) psychological non‐acceptance of the disease by owners, stemming from strong emotional attachment to their pets, which often results in the neglect of therapeutic follow‐up despite the presence of early clinical signs; (b) the considerable financial burden associated with treatment regimens and chemotherapeutic protocols; (c) inconsistent owner compliance in referring animals for routine periodic veterinary examinations, thereby diminishing the likelihood of early neoplastic detection; and (d) failure to perform timely ovariohysterectomy at the appropriate developmental stage. It is important to note that the aforementioned factors may collectively introduce a potential source of selection bias within the study population.
TABLE 5.
Distribution of canine mammary tumours by age and tumour type.
| Variable | Absolute abundance of mammary tumour (number) | Relative abundance (%) |
|---|---|---|
| Age (year) | ||
| ≤ 3 | 0 | 0.0 |
| 4–6 | 9 | 12.2 |
| 7–9 | 22 | 29.7 |
| 10–12 | 24 | 32.4 |
| ≥ 12 | 19 | 25.7 |
| Tumour type | ||
| Carcinoma (mucinous and anaplastic) | 38 | 51.4 |
| Malignant mixed tumour (MMG) | 13 | 17.6 |
| Fibroadenoma | 6 | 8.1 |
| adenoma (ductal and intraductal papillary) | 5 | 6.8 |
| Miscellaneous tumours | 5 | 6.8 |
| Metastatic tumour | 4 | 5.4 |
| Benign mixed tumour | 3 | 4.1 |
TABLE 6.
Distribution of mammary tumours by breed, age and fertility status in 74 affected dogs (n = 74).
| Breed | Absolute abundance of mammary tumour (number) | Age range (years) | Fertility | Relative abundance (%) |
|---|---|---|---|---|
| Terrier mix | 30 | 5–14 | + | 40.5 |
| Shih Tzu | 14 | 6–13 | + | 18.9 |
| Shih Tzu Terrier mix | 7 | 7–14 | + | 9.5 |
| Mixed | 4 | 5–13 | + | 5.4 |
| Spitz | 3 | 9 | + | 4.1 |
| Golden Retriever | 3 | 8–9 | + | 4.1 |
| Bichon Frisé | 2 | 10 | + | 2.7 |
| German Shepherd | 2 | 10–12 | + | 2.7 |
| Dachshund | 2 | 9 | + | 2.7 |
| Miniature Pinscher | 1 | 11 | + | 1.4 |
| Terrier | 1 | 12 | + | 1.4 |
| Yorkshire Terrier | 1 | 9 | + | 1.4 |
| Husky | 1 | 5 | + | 1.4 |
| Pointer | 1 | 5 | + | 1.4 |
| Pomeranian | 1 | 5 | + | 1.4 |
| Great Dane | 1 | 5 | + | 1.4 |
| Total | 74/130 | 5–14 | + | 56.9 |
In terms of haematology, the current research found that malignant tumours had significantly fewer RBCs and Hb, indicating anaemia and more Mon compared to benign tumours. Also, serum ALT and ALP levels were higher in malignant tumours. In the immunohistochemical study on CMTs conducted by Sasani et al. (2003), out of 141 dogs, 6 female dogs with tumours (5 carcinoma samples and 1 mixed mammary tumour sample), none of the samples showed a positive reaction to oestrogen and PRs, which seems to be due to the malignancy of the tumours or lack of antigen protection (Sasani et al. 2003). The findings of another study suggested that in CMTs, benign tumours express both oestrogen and PRs. In contrast, low ER expression is linked to more aggressive cancer. Tumours with an ER+/PR+/HER2− profile have low malignancy, unlike triple‐negative tumours, which are more aggressive and have a poor prognosis (Pastor et al. 2020). Also, it has been reported that in CMTs showed that Ki67 was highly expressed in triple‐negative tumours (p < 0.001) (Razavirad et al. 2024). In the present research, according to the results and Figure 3, in carcinoma and mixed benign tumour (despite being benign), we had ER−/PR−/HER2− (triple‐negative). In cases of metastatic mast cell tumour, it was ER−/PR+/HER2− and in cases of adenomas (ductal and intraductal papillary), it was ER+/PR−/HER2−. Considering a 10% expression threshold for assuming ER, PR and HER2 markers as positive, it should be noted that the carcinomas in the present study are consistent with the results of the study as mentioned earlier in terms of triple negativity of ER−/PR−/HER2− and since PR is usually considered a marker for tumour recurrence (Abdelmegeed and Mohammed 2018) and HER2 is also considered a marker for tumour prognosis, therefore, in carcinomas, according to the histopathological and immunohistochemical picture, recurrence and poor prognosis are expected. On the other hand, positive ER expression in adenomas (ductal and intraductal papillary) is also consistent with the benign nature of this tumour. Also, positive expression of PR (in mast cell tumour) can indicate the metastatic and recurrent nature of this tumour, although the mammary cells themselves were negative for PR. However, an interesting and unexpected result of the present study was ER−/PR−/HER2− (triple‐negative) for the benign mixed tumour, which is in contrast to the results of previous studies. Some authors have suggested a myoepithelial origin for myofibroblasts of mammary tissue (Peña et al. 2014), but the absence of CK and p63 immunoreactivity, together with strong Vim expression in a study, suggests a mesenchymal nature of canine mammary sarcomas (CMSs) (Dolka et al. 2013). On the other hand, some IHC studies refer to the origin of myoepithelial cells based on their immunophenotype. These cells seem capable of a metaplastic transformation into mesenchymal cells, although the exact mechanisms remain unclear (Peña et al. 2014). In the present investigation, IHC findings were mostly based on carcinomas and benign tumours. However, the results showed that αSMA expression was higher in benign tumours with higher fibroplasia and was lower in carcinomas, most of which did not have much fibroplasia in their stroma, except in cases where the carcinoma was complex and included tumorigenesis of both mammary tissue cells and myofibroblasts. The results of the αSMA marker for carcinomas included all three types of simple, complex and tubular carcinomas (adenocarcinoma), which were naturally considered as averages for them. However, in the case of benign tumours, in most cases where fibroplasia was also high, αSMA expression and its intensity were also high (34%–66%; Strong), which indicated the involvement of myofibroblast cells in the formation of these tumours. A study in India involving 42 CMTs found that most malignant tumours were triple‐negative for ER, PR and HER2, showing a poor prognosis. Benign tumours had higher ER and PR expression, while malignant tumours often had lower ER levels and variable PR expression. Positive receptor expression indicated better outcomes compared to triple‐negative tumours (Kavisha et al. 2023). In the present study, IHC results for HER2 showed a lack of expression in all benign and malignant tumours studied. However, in carcinomas, expression was observed below 5% in cuboidal epithelial cells of the mammary gland. However, due to the consideration of a 10% threshold for positivity, these cases were considered negative for HER2. Our results align with Kavisha et al. for HER2. In dogs, the significance of HER2 overexpression in mammary tumours is unclear. Furthermore, the variability of increased expression of this marker in malignant tumours (17.6%–48%) has led to a lack of consensus regarding its prognostic value. However, the most important reason for the nonstandard interpretation of findings for HER2 is the lack of a biological threshold for positive labelling in CMTs. On the other hand, in human mammary cancer, the threshold for positivity is defined by the percentage of ER and PR that ensures the effectiveness of hormone therapy, and has recently been standardized at 1%; in CMTs, this threshold has not been established, and the few reports on the prognostic value of ER and PR detection are still conflicting (Peña et al. 2014). In the present research, although HER2 was negative in both benign and malignant tumours studied, the good or bad prognosis in relation to this marker should be considered together with the ER and PR markers. With this description, it can be said that in the present study, carcinomas with ER−/PR−/HER2− profile had the worst prognosis compared to the other cases studied. Ki67 antigen is a nuclear protein that is highly expressed in proliferating cells before mitosis and has been identified in various types of human and canine tumours. IHC indicates that high Ki67 expression in mammary cancer is associated with a poor prognosis (Abdelmegeed and Mohammed 2018). Further, nuclear Ki67 expression is more accurate than mitotic index (MI) for measuring cell division, as it shows activity in all cell cycle phases (Kaszak et al. 2018). However, Ki67 is a marker of cell proliferation in CMTs but should not be used alone to define malignancy; interpretation requires histopathological context (e.g., architecture, invasion, atypia, MI, metastasis) (Carvalho et al. 2016; Vazquez et al. 2023). It has also been noted that Ki67 levels are lower in older female dogs than in younger dogs, which is probably because tumour proliferation processes are slower in older dogs (Kaszak et al. 2018). However, in the current study, according to the immunohistochemical results and considering the age of the studied dogs (10–14 years), the range of Ki67 expression in malignant tumours was recorded as 11%–66% and was variable. But the most interesting point of the present study is that in benign mixed and adenoma (ductal and intraductal papillary) tumours, Ki67 expression was in the range of 11%–33% and 34%–66%, and the age range was 4–5 years and 12–14 years, respectively. According to our findings, tumour growth and proliferation do not seem to be strongly related to the age of the dog. It should be noted, however, that most of the malignant tumours found were in the age range of 10–14 years, which may have been the reason. But the important point in the present study is why, in benign tumours, although it was very rare to find mitotic states in H&E staining, there was significant nuclear Ki67 expression in these tumours in IHC. This issue may be explained by the fact that interphase and other stages of the cell cycle that cannot be detected by H&E are more abundant in benign tumours and therefore can be stained with Ki67. Of course, proving this hypothesis requires further studies. It is worth noting that the Ki67+ immunophenotype has been reported in previous studies, such as the research conducted by Kurilj et al. (2011) on the IHC of CMTs. They conducted this study between 2005 and 2007 and reported an expression of less than 20% for Ki67 in benign tumours (Kurilj et al. 2011). Also, in another study on the IHC of benign mammary lesions in cats by Soares et al. (2022), they reported 29% Ki67 expression in simple adenoma tumours, while in the same study, the expression of this marker was only 1% in benign fibroadenomatous changes (Soares et al. 2022). In dogs, Ki67 expression may be driven by shared growth‐promoting mutations or microenvironmental factors across synchronous tumours in the mammary glands, allowing genetically similar CMTs to show divergent phenotypes (Hansen et al. 2026). A study found Ki67 expression in benign areolar duct cells from women. It suggests Ki67 may indicate risk and response in benign mammary tissue related to hyperplasia and atypia (Khan et al. 2005). In the present study, in benign mixed lesions and adenomas (ductal and intraductal papillary), cellular atypia was not observed and ER, PR and HER2 expression were also negative. However, Ki67 expression was in the range of 11%–33% and 34%–66%, which is higher than the values reported so far in benign mammary tumours. It is worth noting that follow‐up data for all studied cases were unavailable, but review of benign cases showed no malignancy, recurrence, or concurrent tumours, with normal survival except for one case of benign mixed tumour in which a tumour was present concurrently, although the type of concurrent tumour was not determined. Although proliferation has prognostic value in CMTs, findings are inconsistent: histological malignancy does not always predict clinical behaviour, and despite known prognostic factors, tumour behaviour remains incompletely understood (Carvalho et al. 2016). RNA‐based molecular assessment of Ki67 may complement immunohistochemistry to better reflect proliferative activity (Hansen et al. 2026). The present research reports, for the first time in an animal model, the presence of a triple‐negative (ER−/PR−/HER2−) immunoprofile accompanied by Ki67 positivity in benign mixed mammary tumours of canines. This finding is unexpected, as the triple‐negative phenotype has previously been associated exclusively with malignant mammary tumours, predicting aggressive behaviour and poor prognosis (Pastor et al. 2020; Razavirad et al. 2024), and is virtually synonymous with malignancy in human mammary cancer. The observation raises important questions regarding the relationship between immunohistochemical phenotype and biological behaviour in CMTs, suggesting that in canines this immunophenotype may not invariably predict malignant behaviour. Nevertheless, these findings should be considered preliminary evidence, requiring validation through larger prospective studies with long‐term follow‐up to determine whether such benign triple‐negative lesions represent indolent variants or precursor lesions with potential for malignant transformation. The study supports the value of the canine model for mammary tumour research while highlighting the need for standardized immunohistochemical thresholds and further investigation of benign lesion biology. In this study, given the lack of owner cooperation in post‐mastectomy follow‐up, inattention to tumour signs, and late clinical referral, prophylactic strategies including early neutering, hormonal management, regular clinical examinations, nutritional and weight counselling and breed management are recommended to reduce the risk of occurrence, enable early detection, or prevent CMT malignancy (Jing et al. 2024). One limitation of this study is the relatively small number of benign tumours compared to malignant cases. This distribution reflects the clinical population observed in the veterinary clinics under study, where malignant CMTs predominate among the animals sampled and evaluated histopathologically. Therefore, it is recommended that future studies be conducted with a larger number of benign tumours.
5. Conclusion
The study examined 130 dogs that had mastectomies in Tehran, Iran, over 3 years from 1 October 2020 to 1 October 2023, finding that 74 were diagnosed with either benign or malignant mammary tumours. The affected dogs were mostly aged 10–12 years, with terrier mixes and Shih Tzus showing the highest tumour rates. Malignant tumours were most commonly carcinomas (mucinous and anaplastic), while the most frequent benign tumours were fibroadenomas. Blood tests indicated that dogs with malignant tumours had lower RBC and Hb levels, signifying anaemia and higher monocyte counts compared to those with benign tumours. Significant increases in serum ALP and ALT enzymes (p < 0.05) were also noted in cases of malignant tumours compared to those of benign tumours. Immunohistochemical tests revealed various tumour profiles, including unexpected triple‐negative (ER−/PR−/HER2−) tumours, with significant positivity of Ki67 expression (34%–66%) observed in some benign tumours. The study suggests that while dogs are good models for studying mammary tumours, varied results indicate the need for further research. It recommends standardizing positivity thresholds for immunohistochemical markers and considering multiple assessment parameters for better tumour prognosis in canine cases.
Author Contributions
Hossein Lak: project administration, investigation, formal analysis, software, writing – original draft. Amir Amniattalab: conceptualization, formal analysis, investigation, software, writing – original draft, writing – review and editing.
Funding
This research did not receive any specific grant from funding agencies in the public, commercial, or not‐for‐profit sectors. All costs associated with this study were covered by the authors.
Ethics Statement
The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to. This research has been evaluated and approved by the Research Ethics Committee of the Islamic Azad University‐Urmia Branch (Approval ID: IR.IAU.URMIA.REC.1401.113, dated 18 December 2022). All procedures involving animals were conducted in accordance with international, national, and institutional guidelines for humane animal treatment, and the appropriate ethical review committee approval was obtained prior to the commencement of the study.
Conflicts of Interest
The authors declare no conflicts of interest.
Acknowledgements
The authors would like to thank the TUMS Cancer Institute for preparing the immunohistochemical sections.
Data Availability Statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.