Canine Mammary Tumors as a Potential Model for Human Breast Cancer in Comparative Oncology

Clinical and molecular similarities between canine mammary tumors (CMTs) and human breast cancer (HBC) propel scientists to further study their application in comparative oncology as a model for human breast cancer. In total, 64 canine mammary tumors were selected to study the most common markers, which are applicable for human breast cancer treatment, including estrogen and progesterone receptors (ER and PR), human epidermal growth factor (HER2/neu), Ki67, and cyclooxygenase 2 (Cox2). Immunohistochemistry (IHC) was used to assess the protein expression. The Veterinary Nottingham Prognostic Index (Vet-NPI) was also computed. Moreover, univariate and multivariable Cox proportional hazard analyses were applied to estimate hazard ratios (HRs). The results demonstrated that Ki67 was strongly expressed in the triple-negative tumors, and Ki67 protein expression continuously increased over the increase of Cox2 protein expression (p < 0.001). Further analysis revealed a significant difference among canine mammary subtypes and Vet-NPI, in which triple-negative tumors displayed the highest mean score compared to other subtypes (p < 0.001). In addition, the multivariable analysis revealed that the regional mastectomy procedure (adjusted HR = 2.78 (1.14–6.8)), the triple-negative tumors (adjusted HR = 48.08 (7.74–298.8)), strong Ki67 protein expression group (adjusted HR = 7.88 (2.02–30.68)), and strong Cox2 protein expression group (adjusted HR = 29.35 (5.18–166.4)) demonstrated significantly lower disease-free survival rates compared to other corresponding groups. Overall, canine mammary tumors showed strong similarities to human breast cancer in terms of clinical and molecular aspects; therefore, they could be suggested as a model for human breast cancer in comparative oncology.


Background
Despite the progress in human breast cancer (HBC) research and treatment, their incidence is increasing by 0.5% annually; therefore, discovering new treatment strategies seems crucial [1].Although mouse laboratory models have signifcantly contributed to HBC research, traditional laboratory mouse models do not spontaneously develop tumors.Tus, genetically engineered or patient-derived xenograft (PDX) models are often required for more complex HBC studies [2].Given that canine mammary tumors (CMTs) arise spontaneously and share other characteristics with HBC, such as incidence rate, age at onset, hormonal infuence, subtype classifcation, and disease course [2,3], these models can therefore become an excellent alternative to human clinical studies [4].
In mammals, ovarian steroids, including estrogen and progesterone, afect the mammary epithelium, which can lead to malignant proliferation [5].Te steroid hormones exert their biological efects via specifc receptors, including estrogen and progesterone receptors (ER and PR) [6].Tere are two known isoforms of the ERs, ERα and ERß [7].Te role of ERα in human breast cancer and canine mammary tumors is well known; however, further research on the role of ERß is still required [8].Like HBC, the presence of ERα in CMT has been associated with pathological disease features and tumoral diferentiation [9].In addition, the progesterone receptor (PR) plays a vital role in the growth of mammary glands.It causes the expansion of the lobular units of the terminal ductal during puberty and pregnancy [10].
Over the past few decades, some molecular markers associated with tumor growth and development have been recognized in humans and canines.Tese include human epidermal growth factor receptor 2 (HER2/neu), Ki67, and cyclooxygenase 2 (Cox2) [11][12][13].HER2/neu is a protooncogene widely studied in human medicine and has shown extraordinary therapeutic results in response to anti-HER2 drugs, such as trastuzumab [14,15].Ki67 is a nuclear protein more expressed in the cell cycle and is reported as a marker for proliferation [16].Tere is credible evidence suggesting the role of Cox2 in tumor growth and development in both species [13,17].Researchers believe that the upregulation of Cox2 in human breast cancer is involved in prostaglandin production during tumorigenesis.Terefore, it could be a signifcant target for nonsteroid antiinfammatory medicines, such as celecoxib [18].
It is worth mentioning that there are still gaps in the research, including the need to determine the impact of surgical procedures on treatment outcomes and the ability to classify mammary tumors into distinct subtypes similar to HBC [2,[19][20][21].Te present study attempted to determine the molecular subtypes of canine mammary tumors according to ERα, PR, and HER2 statuses and measure the protein expression of Ki67 and Cox2 to compare the similarities and diferences between the two species.In addition, the disease-free survival (DFS, the time from surgical resection to tumor recurrence or metastasis) was computed by accurately recording clinical, surgical, and histopathological data.

Animals and Tumors.
Sixty-four spontaneous CMTs from veterinary clinics and hospitals in Tehran, Iran, were prospectively studied.Te histological classifcation of CMTs includes malignant epithelial neoplasms, malignant epithelial neoplasms of special types, malignant mesenchymal neoplasms-sarcoma, carcinosarcoma-malignant mixed mammary tumors, benign neoplasms, hyperplasia/dysplasia, neoplasms of the nipple, and hyperplasia/dysplasia of the nipple [22].All the specimens used in our study were diagnosed as (1) malignant epithelial neoplasms, such as (a) simple carcinoma, (b) micropapillary carcinoma, (c) complex carcinoma, (d) complex-mix carcinoma, (e) solid carcinoma, (f ) comedo carcinoma, (g) anaplastic carcinoma, (h) malignant myoepithelioma, and (i) intraductal papillary carcinoma, or (2) malignant epithelial neoplasms of special type, such as (a) squamous cell carcinoma, (b) adenosquamous carcinoma, and (c) spindle carcinoma.Informed consent was received from animal owners, and the study was approved by the Ethics Committee of Islamic Azad University of Tehran, Iran-Science & Research Branch (No. IR.IAU.SRB.REC.1398.180).Te median age of the animals was 10 years, ranging from 5 to 15 years.As presented in Table 1, clinicopathological features, such as age, tumor laterality, tumor size, type of surgery, lymph node status, metastasis, histological grade, and TNM staging, were available for the study.Tumor size was classifed according to the WHO criteria (≤3 cm, 3-5 cm, and ≥5 cm).Te surgical procedures were categorized into three types [19].Te frst type was simple mastectomy (36 cases), which included removing the afected gland only.Te second type was regional mastectomy (20 cases), which included removing the afected gland and glands that share lymphatic drainage along with associated lymph nodes.Te third type was radical mastectomy (8 cases), which included removing the entire mammary chain and associated lymph nodes either unilaterally or bilaterally.According to the TNM staging classifcation, 31 animals were in stage I, 20 were in stage II, and 13 were in stage III.In addition, 27 tumors were classifed as grade 1, 25 as grade 2, and 12 as grade 3. Veterinary Nottingham Prognostic Index (Vet-NPI) was taken from the study of Haybittle et al. [23] and computed as with specifc antibodies at room temperature for one hour, and rinsed with PBS thrice.Te sections were incubated in horseradish peroxidase (HRP), rinsed thrice with PBS, and counterstained by the Harris hematoxylin method.Primary antibodies against ERα (mouse monoclonal, orb389114, dilution 1 : 100), PR (rabbit polyclonal, orb106338, dilution 1 : 100), and HER2 (rabbit polyclonal, orb315778, dilution 1 : 100) were purchased from Biorbyt (Cambridge, UK).In addition, Ki67 (rabbit monoclonal, SKU: 325, ready-to-use) and Cox2 (rabbit monoclonal, SKU: 306, dilution 1 : 50) were purchased from Biocare Medical (CA, USA).

Molecular Subtypes of Canine Mammary Tumors.
Te expression of ERα and PR protein was quantifed based on the percentage of cells with nuclear positivity.HER2 protein expression was measured according to the percentage of cells with uniform intense complete membrane staining.We classifed canine mammary tumors into diferent subtypes as previously described [25].ER-positive, PR-positive, and HER2-negative were defned as luminal A tumors.ER-positive, PR-positive, and HER2-positive were defned as luminal B tumors.ER-negative, PR-negative, and HER2-negative were defned as triple-negative tumors.ER-negative, PR-negative, and HER2-positive were defned as HER2-enriched tumors.

Immunoreactivity Scoring Method. As shown in Figures 1 and 2, all formalin-fxed parafn-embedded (FFPE)
tissue sections were reviewed by an expert pathologist.Te expression of ER and PR was measured based on the percentage of positive nuclear cells, where a score <1 was considered negative, from 1 to 10 as weak, and >10 as positive protein expression [26].In addition, HER2 equal to 0 or +1 was considered negative, +2 as equivocal, and +3 as a positive expression [26].Ki67 ranged from 1 to 43 (in percentage) and was quantifed by scoring the nuclear staining intensity, where a score of <5 was considered weak, from 5 to 14 as moderate, and ≥15 as strong expression [25].Additionally, Cox2 status was determined according to the modifed Allred scoring system, which is a combination of two parameters, including the proportion and intensity scores, where scores 0 and 1 were considered as unexpressed, 2 and 3 as weak, from 4 to 5 as moderate, and from 6 to 8 as strong expression [27].3).Te same analysis was performed between canine mammary subtypes and Ki67 protein expression so that triple-negative tumors exhibited the highest expression among diferent subtypes (p < 0.001, Table 3).Additionally, further analysis revealed a signifcant diference between canine mammary subtypes and Vet-NPI, in which triple-negative tumors displayed the highest mean score compared to other subtypes (p < 0.001, Table 3).To provide a clearer picture of the diferences between diferent subtypes in relation to tumor size, Cox2 status, and Vet-NPI, a Tukey's post hoc test was performed (Figures 4(a)-4(c)).Tis analysis revealed that the diferences in tumor size were signifcant only between triple-negative tumors with luminal A, luminal B, and HER2-enriched subtypes (Figure 4(a)).However, for Cox2 and Vet-NPI statuses, the diferences were signifcant for all subtypes, except luminal B and  6

Canine Mammary Tumors Exhibited Various Clinical Features and Protein Expression
Veterinary Medicine International exhibited signifcantly the worst DFS compared to their corresponding subgroups (Table 2).Multivariable analysis adjusted with age, vascular invasion, perineural invasion, histological grade, and TNM stage for each marker individually revealed that the regional mastectomy procedure, the triple-negative tumors, strong Ki67 protein expression group, and strong Cox2 protein expression group demonstrated signifcantly lower disease-free survival rates compared with other corresponding subgroups (Table 2).Survival curves depicted by Kaplan-Meier plots and relevant log-rank p values supported the multivariable analysis, where the triple-negative tumors, strong Ki67 protein expression, and strong Cox2 protein expression exhibited the worst diseasefree survival rates compared to the relevant groups (log-rank p value <0.001, Figures 5(a)-5(c)).Tumors with the highest Vet-NPI score, classifed as a poor group, exhibited the lowest disease-free survival rate compared to others (Figure 5(d)).

Discussion
Mammary tumors are the most common type of tumor in intact female dogs, accounting for around 42% of all tumors [28,29].Recently, it has become increasingly clear that canine mammary tumors are clinically and molecularly similar to human breast cancer [30][31][32].Here, we discuss some clinical, pathological, and molecular similarities in both species, suggesting that CMT can be used as a reliable model for human breast cancer in comparative oncology.
Research indicates that despite having diferent lifespans, the average age of onset at which dogs are diagnosed with mammary tumors (after six years) is very similar to that of women (after 40 years) [33].In both species, tumor size is directly related to survival and has an independent prognostic value [31,34,35].Patients with non-necrotic tumors, without vascular and perineural invasions, with well-diferentiated tumor cells, and in the early stages experience signifcantly longer survival after primary mammary tumor surgery [36].Consistent with these fndings, our cohort indicated that the average age of onset is after six years, and tumors larger than 3 cm have the signifcantly lowest DFS compared to other relevant subgroups.We also found that the presence of vascular and perineural invasion, poorly diferentiated tumors, and advanced stages are of prognostic value for DFS.Tese variables are signifcantly associated with poor prognosis and low DFS.Hörnfeldt and Mortensen reviewed twelve studies on surgical dose and clinical outcome in the treatment of mammary gland tumors in female dogs.According to their analysis, no study shows a clear advantage in choosing one surgical method over the other [19].However, our study, with its potential to signifcantly infuence future research in this area, has uncovered signifcant fndings.We found that the dogs that underwent radical mastectomy had a signifcantly lower DFS rate than dogs that received other relevant surgical procedures.Moreover, our multivariable analysis indicated that the regional mastectomy method could be an independent prognostic factor.Tese fndings suggest that surgical procedures can be proposed as prognostic factors and help fll the gaps in this feld.Te choice of surgical procedure depends on the tumor size (pT), which can predict lymph node involvement.Terefore, a large sample size with various surgical procedure designs is necessary to compare the efcacy of treatment outcomes.
In recent years, extensive veterinary research has been conducted on tumor markers, such as ERα, PR, HER2, Ki67, and Cox [37][38][39][40][41]. Reports confrm the unique similarities between human breast cancer and canine mammary tumors regarding these markers' protein expression and prognostic values [42].ERα is upregulated in two-thirds of breast tumors and can infuence endocrine therapy in terms of  Veterinary Medicine International treatment selection or response to treatment [43].Terefore, ERα status can be considered a valuable prognostic indicator in CMT.We reported that the reduced expression of ERα is associated with the aggressive phenotype of the tumor and poor prognosis [42].In addition, decreased PR expression is associated with a poor prognosis and can be a reliable indicator of recurrence [44].Te combined status of ERα, PR, and HER2/neu indicated diferences in prognosis, whereas ERα-negative, PR-negative, and HER2/neu-negative tumors showed the worst prognosis [38,45].In line with these fndings, we reported that triple-negative tumors (ERα-, PR-, and HER2-) exhibited less disease-free survival and more recurrence compared with other relevant subtypes.Additionally, given that some studies classify canine mammary tumors into four subtypes, including luminal A, luminal B, triple negative, and HER2-enriched [2,20], others cannot classify HER2-enriched as an independent subtype [21].Terefore, more studies can be helpful to accurate classifcation of mammary tumor subtypes.Our classifcation in this study was consistent with previous studies that classifed canine mammary tumors into four subtypes.Furthermore, as shown in Figure 3, the frequency of subtypes in our study was almost similar to that in human breast cancer [46].Tese results could further prove that CMT could be a potential model for human breast cancer in comparative oncology.Te Ki67 is a nuclear protein highly expressed in proliferating cells [47].Although studies on Ki67 protein expression have used diferent cutof points, most have shown  Veterinary Medicine International a signifcant association between higher Ki67 protein expression and a worse prognosis [40,48].Similarly, we found comparable results indicating a correlation between higher Ki67 expression and a poor prognosis.In addition, human breast studies have revealed that Cox2 overexpression plays a crucial role in prostaglandin production over tumorigenesis and is involved in the early steps of mammary carcinogenesis [49,50].Studies on CMTs show a signifcant relationship between higher levels of Cox2 expression and decreased DFS [13,41].Interestingly, we observed similar fndings in our dataset, where higher Cox2 protein expression was signifcantly associated with higher Ki67 protein expression and reduced DFS.Most recently, our group in a case series investigated the expression of Cox2 in canine infammatory mammary carcinoma and concluded that this model could be suitable for comparative oncology [51].Tis study has potential limitations, such as limited sample size and lack of information on the breed, spaying status, diet, environmental factors, and comorbidities.Tese confounding factors may afect prognosis and survival.In addition, due to the limited sample size in each subgroup of histological classifcation, we could not correlate them with protein expression and clinicopathological parameters.Hence, a larger sample size with functional or genetic analysis of the tumor, including gene expression, mutation, or copy number variation profles, can provide more insights into the molecular mechanisms and pathways involved in the disease.Additionally, the study does not evaluate the response or resistance of the canine mammary tumors to any specifc treatments, such as chemotherapy, hormonal therapy, or targeted therapy, which could determine the suitability and translatability of the canine model for human breast cancer therapy.
Overall, the cumulative similarities presented here strengthen the hypothesis of considering CMTs as a source for increasing the understanding of HBC molecular pathogenesis.Despite the many similarities identifed between CMT and HBC that propose it as a reliable model in comparative oncology, limitations include high cost, being time-consuming, and reliance on dog owners in postoperative care.Nevertheless, this model can be used in human breast clinical trials to develop novel therapeutic strategies in comparative oncology.

Figure 1 :Figure 2 :
Figure 1: Representative images of CMTsubtypes by IHC.ERα and PR protein expression was measured according to the percentage of cells with nuclear positivity.HER2 protein expression was quantifed based on the percentage of cells with uniform intense complete membrane staining.Te labeled images indicate subtypes of canine mammary tumors, including luminal A, luminal B triple-negative, and HER2enriched.Scale bars are equal to 100 µm.

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Veterinary Medicine International subtypes and tumor size, in which triple-negative tumors possessed the highest mean tumor size compared to other subtypes (p < 0.001, Table

Figure 3 :Figure 4 :
Figure 3: Pie charts of subtype frequencies comparing canine mammary tumors and human breast cancer.Pie charts show the frequency of subtypes in canine mammary tumors and human breast cancer that are nearly equal.(a) Frequency (%) of CMT subtypes in our study.(b) Frequency (%) of human breast cancer subtypes.

Figure 5 :
Figure 5: Kaplan-Meier plots indicating disease-free survival probabilities among 64 available canine mammary tumors.(a) Dogs having triple-negative (TN) tumors demonstrate signifcantly poor disease-free survival compared with other subtypes of canine mammary tumors (log-rank p < 0.001).(b, c) Tumors expressing high Ki67 and Cox2 protein displayed signifcantly poor disease-free survival compared with other tumor subgroups (log-rank p < 0.001).(d) Dogs presenting poor Vet-NPI indicated signifcantly poor disease-free survival compared with other groups (log-rank p < 0.001).For each Kaplan-Meier plot, a corresponding log-rank p value was presented.

Table 1 :
Clinical and para-clinical data of 64 canine mammary tumors hospitalized in veterinary clinics and hospitals in Tehran during 2010-2020.
phosphate-bufered saline (PBS) and then blocked with 5% bovine serum albumin (BSA) at room temperature for one hour.Te sections were rinsed three times in PBS, incubated Analysis.One-way ANOVA was computed to compare a continuous variable with categorical explanatory variables.All values were expressed as mean and standard deviation (±SD).Tukey's post hoc test with a 95% confdence interval (CI) was performed to evaluate the association between diferent variables in pairs.Te Kaplan-Meier method was applied for survival analysis, and the log-rank test calculated the diferences.Te Cox proportional hazard model was employed for the univariate and multivariate survival analysis.In all statistical analyses, p < 0.05 was considered signifcant.Te RStudio statistical software version 1.2.5033 was used for statistical analysis.

Table 2 :
Univariate and multivariable analyses of prognostic factors for 64 available canine mammary tumors.
Veterinary Medicine International HER2-enriched pairwise subtypes (Figures4(b) and 4(c)).Likewise, one-way ANOVA was performed on tumor size, Ki67 protein expression, and Vet-NPI status, comparing them with tumors expressing Cox2 diferentially.Te results suggested a signifcant trend between these variables and Cox2 protein expression from negative to strong expression statuses (p < 0.001, Table4).3.3.Multivariable Analysis Indicated the Worst DFS for Triplenegative, Ki67-strong, and Cox2-Strong Tumors Comparedto Other Relevant Subgroups.Our univariate analysis showed that tumors greater than 5 cm, vascular invasion involvement, perineural invasion involvement, necrosis presence, histological grade 3, TNM stage III, radical mastectomy procedure, triple-negative subtype, strong Ki67, and strong Cox2

Table 3 :
Analyses of predictor variables in relation to subtypes of canine mammary tumors.

Table 4 :
Analyses of predictor variables in relation to canine mammary tumors expressing diferentially Cox2 protein.One-way ANOVA was applied to compute the p values among 64 canine mammary tumors that diferentially express Cox2 protein.All variables displayed signifcant diferences (p < 0.001), so that triple-negative tumors showed the highest mean compared with other subgroups.Veterinary Nottingham Prognostic Index (Vet-NPI) was computed as: Vet-NPI � [tumor size (cm) × 0.2] + histological grade (1, 2 or 3) + evidence of vascular invasion and/or regional lymph node metastases (1 or 2 if absent or present, respectively).SD, standard deviation.