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Canines, as companion and duty/service animals, are highly liked domesticated animals. Therefore, numerous conditions which affect their health needs to be taken care of, for their well-being. Among various fatal conditions which diminish their work potential and shorten their lifespan are tumors and malignancies. Canine mammary tumor is one such important concern among both owners and Veterinarians. Though frequency of mammary tumors varies in different species, canines are the most frequently affected ones, with prevalence about 3 times higher than humans. Therefore pen side tests and point-of-care diagnostics are to be developed. Treatment regimens and therapeutics are kept up-to-date for efficient management of such conditions.

Canine domestication is an age-old practice which started with guard for humans and other animals, dogs being the favorite companion animals. They have many positive social effects on lives of their owners including enhanced emotional sense and companionship and even reduction of anxiety and mental disorders. Though dogs have proven good for human health, it should be kept in mind that are prone to many disorders and infectious diseases. Healthy life of dogs is attributed to healthy lifestyle, good feeding practices and regular medical care. In addition, an equally important practice is vaccination. Since dogs live in close proximity to human beings and their environment, regular check on their immunity becomes essential, especially when kept as indoor pets. Their illnesses vary from minor issues like dental tartar to fatal diseases like tumors and rabies. Depending upon lifestyle patterns and subsequent body parameters, many predisposing factors increases the susceptibility of dogs to various diseases.

Mammary tumors are either of benign or malignant types. Benign tumors are defined as mass or lump of cells unable to invade the neighboring tissue/ organ and less likely to re-grow after removal. However, malignant tumors are the tumors which have the ability to invade other tissues/ organs and tend to re-grow even after surgical removal. Malignant tumors grow at faster rate as compared to benign tumors. Nearly, 40-50% of canine mammary tumors (CMTs) are malignant.

CMTs can be classified on the basis of morphologic features, tissue of origin (epithelial, myoepithelial or mesenchymal tissue) or prognosis. The World Health Organization (WHO) classification of canine and feline mammary tumors has categorized CMT into three major categories i) malignant, ii) benign and iii) unclassified tumors. On the basis of histological characteristics, the mammary tumors have been classified into fibroadenomas, adenomas, solid carcinomas (with transformed epithelial cells), tubular adenocarcinomas (with tubule like arrangements), papillary adenocarcinomas (having papillary like projections), anaplastic carcinomas (with highly pleomorphic cells), sarcomas (involvement of connective tissue) and malignant tumors (Fig. 2). Fibroadenomas and adenomas are the types of benign mammary tumors the former contains some connective tissues component in between, and carcinomas, adenocarcinomas and sarcomas come under malignant tumors.

Human breast cancer (HBC) is a common malady concomitant with a massive death rate throughout the world. It has been reported that around 1.7 million women are globally identified with breast neoplasia, and out of this, half a million female dies from this terrible disease (Torre et al., 2015). Due to the shocking impacts of breast cancer, prevention, and therapeutic interventions have become a priority-based objective for many researchers to understand the biology of breast cancer disease during its progression and development.

The incidence of mammary tumor accounts for 25% of all the recorded tumors in dogs worldwide. Overall, about 198 cases of CMT are recorded per one lakh female dogs annually throughout the world which is quite an alarming percentage (Gray et al., 2020). Malignancy has also been recorded up to 45% which increases the fatality among morbid cases. Epidemiology of CMT is poorly documented particularly in India. In an epidemiological study conducted on canine tumors in Jammu in 2018, the overall prevalence was only 2% over a period of one year. The malignant tumors accounted 41.66% of the mammary tumors (Sharma et al., 2018). The rate of benign and malignant tumors incidence was reported as 46% and 54%, respectively. In another study conducted on the incidence of CMT in Ludhiana (Punjab) in 2020, it was found that 33.96% tumors were benign and 66.03% were malignant (Dash et al., 2020).

6.1 Age Age is one of the most important risk factors for developing mammary tumors in dogs. A comprehensive study carried on dogs has found that the risk of mammary tumors become significant by 8 years of age and increases linearly thereafter. The age of onset of tumors, however, may vary depending on the life span of the dog breed. An average age of CMT onset is 8-11 years, indicating as a condition of the middle-aged or older dogs. Mammary tumors are generally rare in dogs under 3 years of age, unless the dogs have been given exogenous hormones such as progestins 6.2 Breed Most consistently listed breeds in which mammary tumor occur include, English springer spaniel, Brittany, Cocker spaniel, English setter, Pointer, Afghan hound and German shepherd, in addition to the smaller breeds such as Poodle, Maltese, Chihuahua, Beagle, Dachshund and Terriers. These studies clearly show that the risk for mammary tumors has some breed association and, therefore, having a genetic component. Pure breeds of dogs are generally at a greater risk i.e., known to account for 80% of the submitted cases. Some studies have further shown that certain lines within specific breeds are at more risk than others.

There are countless biomarkers present in cells related to and determining the progress of a disease. Identifying these markers could aid in detecting cells with metastatic potential (Ramaswamy et al., 2003). The elevation of cyclo-oxygenase (Cox-2) expression was observed in CMT cases through enzyme immunoassay (Queiroga et al., 2005). In further study, enhanced expression of BRAC1and BRAC2 genes, certain enzymes such as matrix metalloproteinases (MMP-2 and MMP-9) by immunohistochemical examinations, and Bcl-2 & Bcl-XL elevated level in dogs with mammary neoplasm have been reported by Queiroga et al. (2011b).

A thorough history with complete physical examination including careful palpation of the mammary glands should be performed. As there are chances that dogs may be suffering from any other systemic ailments during middle to old age. In dogs with multiple tumors, all tumors should be excised and their location and size should be noted. Histopathological examination of the excised tumors should be carried out to classify mammary gland tumors. Mammary tumors are classified based on some of the parameters i.e., tumor size, lymph nodes, metastasis. Therefore, to stage a dog with mammary tumors information of these parameters needs to be collected and recorded. If lymph nodes are palpable and/or clinically enlarged, fine-needle aspiration should be performed for cytological evaluation. The technique is less invasive but with higher accuracy. Thoracic radiographs remain the mainstay for diagnosing the metastatic condition. The mammary tumors most commonly metastasize into lungs in dogs. The staging of CMTs becomes necessary as it is likely to confer some prognostic significance and provide guidance to veterinarians to make treatment decisions.

Surgical removal is the routine treatment method for mammary tumors, however, there is no consensus on the optimal procedure. There are supporting factors for every surgical procedure conducted for mammary tumors including lumpectomy (removal of tumor only), mastectomy (affected gland removal only), radical mastectomy (complete mammary gland and associated lymphatic tissue removal) and modified radical mastectomy (removal of complete mammary gland, lymph nodes and associated chest muscles).The more extensive procedures do not increase survival in dogs when compared to the other procedures, and the benefits of the simpler procedures are clear. Further, the radical mastectomy increases the disease-free interval but not the survival duration in cats.

In addition to the conventional treatment methods i.e., surgery, chemotherapy and radiotherapy, there are some adjuvant targeted therapies which block the growth of cancer by altering the individually expressed specific molecules responsible for tumor cell proliferation, survival, metastasis or microenvironment. The majorly studied adjuvant therapies include use of hormones, tyrosine kinase receptors, anti-cyclooxygenases, anti-tumor suppressor gene p53 and epithelial-mesenchymal transition inhibition. The main aim of hormonal therapy (Table 2) is to inhibit the hormone induced stimulation of carcinoma cells, which could be achieved either by ovariohysterectomy or different treatments like use of specific estrogen receptor modulators (SERMS), aromatase inhibitors for suppression of estrogen synthesis etc. A commonly used estrogen receptor (ER) antagonist drug tamoxifen for treatment of ER positive breast cancer in women, has also been tested against CMTs. However, it is not a viable treatment option in dogs due to associated side effects. Ovariohysterectomy is considered as a better and practical solution. The use of hormonal therapy in CMTs is based on the evidence of hormone dependency of these tumors as well as lessons learnt from hormonal therapy outcomes from human breast cancer. This therapy significantly ameliorates the recurrence of breast cancers and prolongs the survival rates in women having breast cancers positive for hormone receptors. However, the results of the canine investigations, which are all retrospective and non-randomized, are contradictory. Detailed research and prospective randomized trials, where effect of OHE is studied in context of hormone receptor status, are required to determine the role of hormonal therapy in CMTs.

One of the most promising areas of veterinary research these days is cancer immunotherapy and the development of CMT vaccines. DNA vaccines, also known as gene vaccines, are designed to transfer functional genes to target cells, allowing them to express functional proteins. Because endonucleases may easily access bare DNA, it requires a reliable and safe delivery route, which might be biological or non-biological. Different delivery strategies have been tried in human breast tumors to transport tumor antigens such Mucin-1 (MUC-1), Twist, HER-2, mammaglobin-1 and p53, as well as immunostimulatory molecules like IL-12 and IL-6. There have been various studies/ trials in canine animals using different molecules like IL-12, HER-2 and IFN-γ as vaccine candidates, with no clinically relevant outcomes or longlasting cell mediated and humoral immune response outcomes

Although there have been multiple biomarkers for mammary tumors both in human and canines reported over the time (Table 3), but no absolute biomarker for diagnosis and prognosis of CMT has been found yet. Out of several studies on clinical applications of different mammary tumor biomarkers, some showed a particular promising biomarker, on the other hand controversial results also existed for the same. For example, HER-2, which is one of the most prominent bio-marker both in HBC and CMT. HER-2 overexpression is associated with poor prognosis and reduced overall survival of the subject, but there are studies available which suggest no difference in HER-2 expression between benign and malignant tumors and studies showing HER-2 expression suggesting prolonged survival (Kaszak et al., 2018). Various biomarkers of mammary tumors are not mammary tumor specific such as p53, COX-2, PCNA etc. These markers undergo alterations in their expression level in all tumors. By keeping this in mind we need to device a panel of different markers to ensure the correct prognosis and diagnosis of canine mammary tumors. There are limited studies and only few commercial kits (for human) available using such combinations (Table 4).

In GADVASU, under the DBT sponsored project on “Development and evaluation of serodiagnostic assay for timely diagnosis and prognosis of mammary tumors”, we have generated and used hyper-immune sera raised against certain canine recombinant mammary tumor associated proteins for the standardization of sandwich and indirect ELISA for serodiagnosis of canine mammary tumor. The sensitivity and specificities of the assay’s ranges between 85-88% and 80-85%, respectively. However, an antibody against a single marker when used for different histotypes of CMT, its specificity reduced significantly. Multiplex assays are capable to detect many specific antigens in a single run whereas a traditional ELISA is used only to detect a single antigen. It has high-throughput potential, providing more results per sample, with lower sample volumes and with utmost sensitivity and specificity. Attempts are being made to increase the sensitivity and specificities of the assays by multiplexing various markers together (Fig.4).

Mammary tumors are the most common type of neoplasia found in female dogs with high percentage of malignancy and rate of mortality. The rate of incidence of these mammary neoplasia and dysplasia is continuously advancing nationally and internationally over the last 45 years. There are different risk factors such as age, diet, hormonal exposure and breeds, which are required to be monitored in order to control the incidence of CMTs. In addition to conventional therapies such as surgical excision, radiotherapy and chemotherapy, additional precision therapeutic agents targeting TKIs, p53 tumor suppressor gene, cyclooxygenases, epithelial-mesenchymal transitions and various hormones/ hormonal receptors could be beneficial for the treatment of CMTs. Scientific efforts are also being made to develop different biomarker panels and tests for prognosis/diagnosis of the diseases as well as to develop vaccines against CMTs.

Agostini M., Enzo M. V., Bedin C., Belardinelli V., Goldin E., Del Bianco P., and Nitti D. (2012). Circulating cell-free DNA: a promising marker of regional lymphnode metastasis in breast cancer patients. Cancer Biomarkers 11 (2-3), 89-98. Badowska-Kozakiewicz A. M., and Malicka E. (2012). Immunohistochemical evaluation of expression of heat shock proteins HSP70 and HSP90 in mammary gland neoplasms in bitches. Polish Journal of Veterinary Sciences 15 (2), 115-18. Beffagna G., Sammarco A., Bedin C., Romualdi C., Mainenti M., MolloA. and Zappulli V. (2017). Circulating cell-free DNA in dogs with mammary tumors: short and long fragments and integrity index. PLoS One 12 (1), e0169454. Birdi R., Kumar B. S., Gupta K., Kashyap N., & KumarA. (2019). Circulating level of heat shock protein 27 is elevated in dogs with mammary tumors. 3 Biotech 9 (6), 1-9. Campos L. C., Lavalle G. E., Estrela-Lima A., Melgaço de Faria J. C., Guimaraes J. E., DutraA. P. and Cassali G. D. (2012). CA 15.3, CEA and LDH in dogs with malignant mammary tumors. Journal of Veterinary Internal Medicine, 26 (6), 1383-88.

A Adenocarcinomas 3, 7 Adenomas 7 ALDH1 17, 18 Anaplastic carcinomas 7 B Babesiosis 1 Bcl 17, 21, 23 Benign tumors 3, 5 Biomarkers 17, , 29, 30, 31 BRCA 31 C Canine 1, 2, 5, 7, 8, 9, 10, 11, 12, 13, 17, 19, 19, 22, 23, 24, 27, 29, 30, 32, 33, 34 Canine distemper 1 Canine Mammary Tumors 1, , 5, 7, 8, 13, 17, 19, 29, 32, 33 Canine parvoviral diarrhoea 1 Carcinogenesis 10, 25 CHEK2 15 Classification and Grading 7 Companion animals 1, 9 COX inhibitors 21, 26 Cyclooxygenases 23, 26, 35 D Deracoxib 21, 26 Diagnosis 19, 20 Doxorubicin 21 E EMT 17, 25 Epidemiology 11 Estrogen receptors 20 FGFR2 15