Canine mammary carcinoma: An update Oscar Hern´an Rodríguez-Bejarano a,b,c,d, Lucía Botero e,f, Giovanni Vargas Hern´andez e,f, Carlos Parra-Lopez d,h,*, Manuel Alfonso Patarroyo g,h,** a Health Sciences Faculty, Universidad de Ciencias Aplicadas y Ambientales (U.D.C.A), Calle 222#55-37, Bogot´a 111166, Colombia b FIDIC Group, Fundaci´on para la Investigaci´on Traslacional Manuel Elkin Patarroyo (FITMEP), Calle 44#58-05, Bogot´a 111321, Colombia c PhD Programme in Biotechnology, Sciences Faculty, Universidad Nacional de Colombia, Carrera 45#26-85, Bogot´a 111321, Colombia d Immunology and Translational Medicine Group, Medicine Faculty, Universidad Nacional de Colombia, Carrera 45#26-85, Bogot´a 111321, Colombia e Veterinary Medicine and Zootechnics Faculty, Universidad Nacional de Colombia, Carrera 45#26-85, Bogot´a 111321, Colombia f Small Animal Clinic, Universidad Nacional de Colombia (CPA-UN), Carrera 45#26-85, Bogot´a 111321, Colombia g Grupo de Investigaci´on B´asica en Biología Molecular e Inmunología (GIBBMI), Fundaci´on para la Investigaci´on Traslacional Manuel Elkin Patarroyo (FITMEP), Calle 44#58-05, Bogot´a 111321, Colombia h Microbiology Department, Faculty of Medicine, Universidad Nacional de Colombia, Carrera 45#26-85, Bogot´a 111321, Colombia A R T I C L E I N F O Keywords:

Canine mammary cancer Canine biomodel Diagnosis Classification Carcinogenesis Treatment Biomarkers Translational application A B S T R A C T Canine mammary carcinoma (CMC) is the most frequently diagnosed tumour in unspayed canines and leads to one of the main causes of their deaths, thereby making it a major problem for veterinary medicine. Such neoplasia appears spontaneously and, due to its biological and clinical similarity with human breast cancer (HBC), it has been considered an excellent animal biomodel for studying this type of cancer. This review ad­ dresses CMC’s main characteristics, including clinical-diagnostic approach, histopathological and clinicopatho­ logical aspects, epidemiological features, carcinogenesis, metabolic reprogramming, therapeutic tools, related biomarkers and prognostic and predictive factors.

1. Introduction

Canines develop cancer spontaneously, whilst sharing several bio­ logical, clinical, pathological and molecular characteristics with humans (Zhang et al., 2018). Mammary tumours represent the commonest neoplasia in unspayed canines, accounting for 50–70% of all tumours diagnosed. Around half of these mammary tumours are canine mam­ mary carcinoma (CMC), highlighting such an important challenge for veterinary medicine (Razavirad et al., 2024). There is marked variability worldwide regarding its incidence; this is influenced by health practices such as early sterilisation which has dramatically decreased cases in developed regions. Concerning comparative oncology, CMC shares numerous clinical and molecular similarities with human breast cancer (HBC); it thus positions the canine as a useful biomodel for HBC research, thereby enabling advances benefitting both species based on being able to develop new drugs, therapies and carry out clinical trials (Nosalova et al., 2024). This review has been aimed at describing the current status of CMC and updating knowledge regarding its epidemi­ ological characteristics, carcinogenesis, metabolic reprogramming, clinical aspects (clinical signs, diagnostic approach, classification, grading, staging), therapeutic tools, biomarkers and prognostic and predictive factors.

2. Clinical signs and diagnostic approach

A complete history must be compiled and a physical examination made for suitable clinical evaluation of CMC. CMC might manifest as single or multiple nodules in the mammary glands and can be found during palpation of all 5 pairs of mammary glands and can be associated with glandular tissue or the teat. CMCs are usually firm, circumscribed nodules, ranging in size from millimetres to centimetres; however, they can be plaque-like lesions. Adherence to the skin and adjacent tissues,

* Corresponding author at: Immunology and Translational Medicine Group, Medicine Faculty, Universidad Nacional de Colombia, Carrera 45#26-85, Bogot´a

111321, Colombia.

** Corresponding author at: Grupo de Investigaci´on B´asica en Biología Molecular e Inmunología (GIBBMI), Fundaci´on para la Investigaci´on Traslacional Manuel Elkin Patarroyo (FITMEP), Calle 44#58-05, Bogot´a 111321, Colombia. E-mail addresses: caparral@unal.edu.co (C. Parra-Lopez), mapatarroyog@unal.edu.co (M.A. Patarroyo). Contents lists available at ScienceDirect The Veterinary Journal journal homepage: www.elsevier.com/locate/tvjl https://doi.org/10.1016/j.tvjl.2026.106668 Received 2 February 2026; Received in revised form 6 April 2026; Accepted 9 April 2026

317 (2026) 106668

Available online 15 April 2026 1090-0233/© 2026 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/bync-nd/4.0/ ).

local heat and oedema, inflammatory signs and ulceration are some of the clinical findings to consider regarding the appearance of CMC; however, the absence of one or more of these signs does not exclude aggressive behaviour.

In some cases involving some of these clinical signs (particularly ulceration of the epidermis overlying the tumour), canines may be misdiagnosed with severe, acute, erosive or ulcerative dermatitis. Regarding the affected mammary glands, the M4 and M5 glands are involved in 65%-70% of cases; multiple lesions usually develop in more than half canine cases. Each lesion must be evaluated individually when multiple lesions are present, since the prognosis depends on the most aggressive tumour’s clinical, histological and molecular characteristics. Furthermore, several types of tumour may coexist in the same mammary gland (Cassali et al., 2020).

Although most canines having CMCs are clinically asymptomatic at the time of diagnosis, symptoms such as weight loss, fatigue, lethargy, dyspnoea, cough, oedema or lameness may occur in advanced stages of the disease; such symptoms depend on metastasis extent and location. Previous studies have shown that around 50% of CMCs have metastasis in the regional lymph nodes; palpation of the regional lymph nodes is thus very important during a physical examination, since it has been seen that lymph node involvement can promote the development of distant metastases, most frequently occurring towards the lung and bone (Gray et al., 2020). The clinical course of CMC-related metastases is very similar to that seen for HBC, where ~7% of women will have metastatic involvement and 20% of those having local disease will eventually have metastatic lesions (Razavirad et al., 2024).

CMC is usually clinically diagnosed via veterinary consultation regarding the presence of one or multiple nodules in the mammary glands, or as an incidental finding during a physical examination for a reason unrelated to the mammary glands. The largest diameter of each primary tumour should be assessed during initial physical examination so as to be taken into account once clinical staging has been made (i.e. the largest primary tumour’s largest diameter should be taken), along with the period of tumour growth, clinical signs of invasion and in­ flammatory changes. The superficial axillary and inguinal lymph nodes should initially be evaluated to verify enlargement, adherence to deep planes and inflammatory changes (i.e. signs of tumour infiltration); however, the absence of such signs does not mean that there is no involvement of the regional lymph nodes). However, definitive diag­ nosis and tumour grading should be made through a histopathological study of the lesion (Abadie et al., 2018; Cassali et al., 2020). Fine-needle aspiration (FNA) can be performed before surgery dur­ ing the aforementioned clinical evaluation to differentiate other tumour types, inflammation and hyperplasia; nonetheless, it cannot be used for definitive CMC diagnosis as there is high heterogeneity in CMCs and an inherent variability in cellular morphology throughout tumour areas. An 88% sensitivity and 96% specificity have been demonstrated for CMC diagnosis by cytology (histopathology being the gold standard) (Dolka et al., 2018). Evaluating a lesion using cytology with FNA can aid in differential diagnosis, thereby helping identify mastitis, lipomas and mastocytomas. Although performing FNA of a tumour during clinical evaluation does not interfere with patients’ surgical planning, the type of surgery is determined by lesion size, the affected mammary glands and lymphatic drainage (Cassali et al., 2020). Histopathological evalu­ ation is usually made after surgery, evaluating both the primary tumour and all other mammary glands and regional lymph nodes, including the transition zone between a tumour and its adjacent tissues. Analysing surgical margins is significant for planning adjuvant therapy. When dealing with multiple tumours, each should have its own individual histopathological evaluation since different tumour types may be pre­ sent in the same canine (Sleeckx et al., 2011).

Studying distant metastases is essential for determining clinical stage and making treatment decisions. The lungs are the organs most frequently affected by distant metastasis in CMC, even though the lymph nodes (sublumbar, sternal, prescapular), liver, brain and bones may also be involved. A three-view radiograph and abdominal ultrasound are always recommended as extension studies in CMC. Clinical staging consists of evaluating a primary tumour, regional lymph node involve­ ment (axillary and superficial inguinal) and identifying distant metas­ tases (Gray et al., 2020).

3. Classification, grading and staging

3.1. Histological classification

Several attempts have been made to classify CMC based on its his­ topathological characteristics as it is morphologically and biologically heterogeneous, given such importance in predicting a tumour’s bio­ logical behaviour. Misdorp et al., (1974) made the first attempt to establish a histopathological classification of CMCs, this being approved by the World Health Organisation (WHO); Misdorp et al., updated their

1974 classification in 1999, such classification also being approved by

the WHO (Misdorp et al., 1999). Goldschmidt et al., (2011) proposed a new classification system which was revised and updated in 2019 by the WHO and the Davis-Thompson DVM Foundation (Burrai et al., 2023). The latter classification is based on assessing various tumour charac­ teristics, such as cellular differentiation, components and arrangement, basement membrane invasion and highlighted the fact that CMCs are morphologically highly variable (Gray et al., 2020). The “Consensus for the Diagnosis, Prognosis and Treatment of Canine Mammary Tumours” was published in 2020; it was based on proposals made at the IV Mammary Pathology Meeting: Diagnosis, Prognosis and Treatment of the Canine and Feline Mammary Neoplasia, held on Brazil in 2019, following Cassali et al.,’s histological classification as proposed in 2013 and updated in 2017 (Cassali et al., 2020).

3.2. Histological grading

The systems developed by Misdorp et al., and the Nottingham His­ tological Grade (NHG), adapted from Elston and Ellis’ numerical system for HBC, have traditionally been used for assessing CMC histological grade. Such grading systems differ slightly but include three histological parameters: mitotic count, nuclear pleomorphism and tubule formation. Each histological parameter is scored from 1 to 3; adding each score together yields a total score giving the degree of histological malignancy (Goldschmidt et al., 2011). CMC has three histological grades: Grade I (well-differentiated) having a total score of 3–5, Grade II (moderately differentiated) 6–7 and Grade III (poorly differentiated) a total score of 8–9 (Cassali et al., 2020). More recently, Pe˜na et al., presented their grading method for CMC which included all cell types for evaluating histological grade, modifying mitotic count and nuclear pleomorphism parameters; it is considered a canine adaptation of NHG (ca-NHG) (Pe˜na et al., 2013).

3.3. Clinical staging

Defining CMC clinical stage, or the anatomical extent of the disease, is essential for appropriate therapeutic decision-making. Simple assess­ ments are recommended for defining clinical stage before performing surgical resection since CMC is often associated with metastasis. A thorough examination for multiple mammary gland masses and FNA of enlarged regional lymph nodes is suggested (Goldschmidt et al., 2016). Chest X-ray is recommended for all but the smallest lesions; abdominal ultrasound enables deep inguinal lymph node and abdominal visceral parenchyma evaluation. Clinical stage also defines local invasiveness, as increasing tumour size is known to be associated with a higher likeli­ hood of significant local invasion (Fesseha, 2020a). CMC clinical staging is determined according to the TNM system approved by the WHO for canines in 1980; this defines CMC extent based on primary tumour evaluation, regional lymph node involvement (axillary and superficial inguinal) (N) and identification of distant metastases (M) (Owen, 1979). O.H. Rodríguez-Bejarano et al.

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Some studies have found significant differences regarding survival involving different tumour grades. Survival rates are worse concerning grade III CMC than grade I or II. Canines having simple carcinomas had a worse prognosis than those with other carcinomas. There were no sig­ nificant differences regarding survival between grades II and III, both having a very poor prognosis. Grade III CMCs have a higher risk of mortality than grades I and II (Karayannopoulou et al., 2005). Tumour size is one of the most important prognostic factors for CMC; it is classified in three groups: T1 (≤3 cm), T2 (3–5 cm) and T3 (≥5 cm). The larger tumours (T3) are associated with a higher proliferation rate and risk of malignancy (Ferreira et al., 2009). Regional lymph node status is classified according to metastasis presence or absence as N0 (no metastases) and N1 (presence of metastases); it is also important for CMC prognosis. The third parameter consists of detecting distant me­ tastases; these are most frequently located in the lungs and are classified based on metastases presence or absence as M0 (metastasis absence) and M1 (metastasis presence), this being an essential CMC prognostic factor (Sorenmo et al., 2011). Five clinical stages reflecting tumour progression in canines have been defined: regional lymph node involvement (stage IV) and distant metastasis (stage V) (Cassali et al., 2020).

3.4. Classification systems’ clinical and translational impact

CMC represents a significant translational model; consequently, histopathological classification and grading have undergone major up­ dates during recent years, particularly with the introduction of stand­ ardised systems incorporating more refined morphological criteria and grading schemes adapted from those used for HBC. Such modifications have enabled more precise characterisation of tumour subtypes, as well as better correlation between histological features and clinical outcomes.

Updating histological classification enabled more detailed subtype description and categorisation for improving prognostic stratification (clinical impact), thereby enabling a more precise, subtype-specific prognosis. The updated classification introduced histological subtypes having drastically different outcomes, such as a favourable prognosis for complex carcinoma and carcinoma arising from complex adenoma/ mixed tumour and poor prognosis for anaplastic carcinoma and carci­ nosarcoma. A histological description is therefore no longer merely descriptive, but directly guides CMC prognosis and clinical expectations (Rasotto et al., 2017a). Updated grading systems have enabled more robust risk stratification by correlating risk with disease-free survival (DFS), overall survival (OS) and the risk of metastasis and recurrence. Grade remains an independent predictor in multivariate models, alongside stage and lymph node status.

Veterinarians can therefore predict outcomes following surgery with greater accuracy, thereby enabling better planning of follow-up for ca­ nines suffering CMC (Chang et al., 2005). An integrated approach combining TNM staging and histological grade has improved the pre­ diction of metastasis-free survival and enabled the creation of composite prognostic scores (bio-scoring systems), thereby shifting the focus from single-parameter assessment to multidimensional risk models, similar to those used in human oncology (Sorenmo et al., 2019a). The Nottingham Prognostic Index (NPI) for invasive HBC combines lymph node status, tumour size and NHG; given that it has been validated in several studies for use in combination with predictive factors in HBC, the NPI has been adapted for CMC-related use. It is called the Veterinary Nottingham Prognostic Index (vet-NPI) which is calculated as follows: tumour size [cm] × 0.2 + NHG (1, 2 or 3, respectively, for grades I, II and III) + evidence of vascular invasion and/or regional lymph node metastasis (1/if absent or 2/present). NPI has been shown to have prognostic value for CMC (Santos et al., 2015). A similar vet-NPI has excluded informa­ tion on lymph node metastasis, called vascularisation-based vet-NPI (vet-NPI-V), one study finding that canines having a ≤4 vet-NPI-V had significantly longer overall survival compared to canines having > 4 vet-NPI-V (p < 0.001) (Canadas et al., 2019).

Risk-adapted treatment strategies can be used regarding their impact on clinical decision-making as updated classification enables the iden­ tification of specific histological subtypes having a particular grade and the absence/presence of lymph node involvement. Patients for whom more extensive surgery, closer monitoring and the consideration of adjuvant therapies (where available) are indicated can then be selected. Similarly, grading systems combined with classification can help predict the risk of local recurrence and orientate any decision concerning extended mastectomy and/or conservative surgery. Although margins can predict local recurrence, grade and histological subtype are better predictors of survival. Diagnostic standardisation for veterinary pa­ thology laboratories represents another key impact area as uniform diagnostic criteria can partially reduce inter-observer variability and ensure consistent reporting between institutions in different locations, thereby improving reproducibility and reliability concerning the conduct of multicentre studies and clinical trials (Rasotto et al., 2017a). Regarding comparative oncology’s translational relevance in sharing key characteristics with HBC, using similar classification systems for CMC enables considering parallel progression patterns, validating inte­ grated prognostic models for both species, supporting the identification of therapeutic targets and developing shared treatment strategies. Furthermore, it enables the stratification of canine patients in a manner similar to that for humans, with a view to precision medicine ap­ proaches, translational impact and comparative oncology research (Bergholtz et al., 2022).

4. Epidemiological characteristics

4.1. Distribution and frequency

CMCs are amongst the most frequently diagnosed tumours in canines worldwide (Garden et al., 2018). Although scarce epidemiological data related to CMC incidence rates has been reported in the literature, it should be born in mind that pertinent records are a fundamental tool for having data related to this canine neoplasia’s real occurrence and dis­ tribution and that such records should be progressively implemented in all countries. However, CMC incidence and prevalence vary consider­ ably between regions, mainly due to differences in population structure, reproductive management practices and the availability of cancer reg­ istries. The canine is by far the domestic species most frequently affected by mammary tumours, such prevalence being three times higher than in humans; approximately 50% of all tumours in canines are mammary tumours, about 45% of these being malignant (Fesseha, 2020b). CMC related evidence shows that their incidence varies in countries which have reported them, being higher in unspayed canines than in spayed or neutered canines (a canine population control measure) (Root Kustritz, 2018). CMC incidence has been significantly, due to the common practice of performing ovariohysterectomy on canines at an early age (Vazquez et al., 2023).

It should be kept in mind that direct comparison of studies’ epide­ miological data should be interpreted with caution due to substantial methodological heterogeneity. CMC-related information is still very limited globally, mainly due to the lack of censuses, registries and studies determining the actual canine population in different regions. Differences arise from study design (population-based registries vs. bi­ opsy submissions vs. insurance databases), diagnostic criteria and his­ topathological classification systems, inclusion criteria (all mammary lesions vs. only malignant tumours), population structure (insured vs. general population; breed-specific cohorts) and reproductive status distribution, strongly influencing incidence. Furthermore, the lack of standardised global cancer registries in veterinary medicine limits findings’ comparability and extrapolation. Such discrepancies may partially explain the extensive variation regarding reported incidence rates and subtype distributions in different countries. Structured regional analyses and the harmonisation of diagnostic criteria are thus essential for improving the reliability of international epidemiological O.H. Rodríguez-Bejarano et al.

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comparisons concerning CMC. Further studies in other regions and countries worldwide are thus needed to ensure the availability of a greater amount of data associated with CMCs’ global epidemiology, thereby enabling the results to be extrapolated to the general canine population, aimed at better identifying risk factors and establishing clearer prognosis (Brønden et al., 2007). The epidemiological evidence is thus presented by geographical region (Table 1 provides a summary) to facilitate interpretation regarding such complex context.

4.1.1. Europe

Europe has some of the most robust population-based datasets due to having veterinary cancer registries. Data from the Vet-OncoNet initia­ tive and the Portuguese Companion Animal Information System (CAIS) in Portugal has revealed that mammary tumours accounted for 21.6% of all canine neoplasms, 39.1% having been classified as CMCs. This system (which is aligned with the ‘One Health’ framework) integrates cancer surveillance with national animal databases, thereby improving epide­ miological resolution (Pinello et al., 2022). Long-term data from the Animal Tumour Registry (ATR) (1985–2002) in Italy has revealed that mammary tumours account for up to 70% of all canine tumours, inci­ dence rates reaching 191.8 cases per 100,000 canines per year (Merlo et al., 2008). Reported incidence rate in Sweden was 111 cases per 10,

000 canines per year from 1995 to 2002 (Egenvall et al., 2005). The

Norwegian Canine Cancer Register (NCCR) found incidence rates ranging from 16.3 to 83.6 cases per 1000 canines per year for three breeds (Moe, 2001). Danish Veterinary Cancer Registry (DVCR) data indicated that 27% of all canine neoplasms concerned mammary tu­ mours (44% CMC frequency) (Brønden et al., 2010). Longitudinal analysis in Spain (the Canary Islands) showed a decrease in CMCs’ relative frequency (from 62.7 to 48.9%), along with an increase in cases of multiple tumours (Rodríguez et al., 2022). Earlier studies in the United Kingdom estimated incidence rates regarding 205 cases per 100,

000 canines per year, whilst more recent analyses have suggested rates

of up to 1340.7 cases per 100,000 canines per year, likely reflecting differences in study design and population sampling (Dobson et al., 2002; Varney et al., 2023). The relatively lower incidence observed in some European countries has been associated with routine ovar­ iohysterectomy practices, significantly reducing CMC risk. Complex carcinoma and simple carcinoma are consistently reported amongst the most frequently occurring subtypes in European studies. Complex and simple carcinomas predominate in Italy, reflecting the widespread use of traditional histopathological classification systems (Merlo et al., 2008; Vascellari et al., 2016). The most frequently diag­ nosed subtypes in Spain include complex carcinoma, tubulopapillary carcinoma and carcinoma arising from benign mixed tumours, sug­ gesting a relatively high prevalence of mixed histogenetic origins (Rodríguez et al., 2022).

4.1.2. North America

A pioneering epidemiological study conducted by Schneider et al., (1969) in the United States, indicated an incidence of 145 cases per 100,

000 canines per year (Schneider et al., 1969). More recent data has

suggested that the widespread adoption of early neutering has contrib­ uted to a decline in incidence. Available data suggests a predominance of simple carcinomas, this being consistent with patterns observed concerning high-grade malignant tumours (Fesseha, 2020a; Root Kus­ tritz, 2018). Differences in classification criteria and reporting standards limit direct comparison with European datasets.

Table 1 Global epidemiology and histological subtype distribution of canine mammary carcinoma regarding geographical regions. CMC Epidemiological distribution Region Country Study type Period Key findings Most frequently occurring subtypes Reference Europe Portugal Registry (Vet-OncoNet and CAIS) Cross-sectional 2019–2020

• Mammary tumours 21.6% of all canine

neoplasms (39.1% CMCs)

• Not reported

(Pinello et al.,

2022)

Italy Registry (ATR) Retrospective case series 1985–2002

• Mammary tumours 70% of all canine tumours

• Incidence 191.8/ 100,000 per year

• Complex carcinoma

• Simple carcinoma

(Merlo et al.,

2008)

Sweden Retrospective case series 1995–2002

• Incidence 111/10,000 per year

• Age-dependent incidence

• Not reported

(Egenvall et al.,

2005)

Norway Registry (NCCR) Population-based case series 1992–1997

• Incidence 16.3–83.6/1000 per year for three

breeds

• Not reported

(Moe, 2001) Denmark Registry (DVCR) Case series 2005–2008

• Mammary tumours 27% of all canine

neoplasms (44% CMC frequency)

• Not reported

(Brønden et al.,

2010)

Spain Retrospective case series 2003–2020

• Decrease in CMC relative frequency (62.7% to

48.9%

• Increase in cases of multiple tumours

• Complex carcinoma

• Tubulopapillary carcinoma

• Mixed-derived carcinoma

(Rodríguez et al.,

2022)

UK Retrospective case series 1997–1998

• Incidence 205/100,000 per year

• Not reported

(Dobson et al.,

2002)

UK Nested case-control 2015–2016

• Incidence 1340.7/100,000 per year

• Not reported

(Varney et al.,

2023)

North America

USA

Epidemiological 1963–1966

• Incidence 145/100,000 per year

• Early baseline

• Simple carcinoma

(Schneider et al.,

1969)

Latin America Mexico Retrospective case series 2002–2012

• 47.5% of mammary lesions were malignant

• High prevalence in unspayed dogs

• Simple carcinoma

• Complex carcinoma

• Mixed carcinoma

(Salas et al., 2015) Colombia Retrospective case series 1986–2006

• Mammary tumours 17.81% of all canine

neoplasms

• Simple carcinoma

• Complex carcinoma

• Mixed carcinoma

(Caicedo, 2012) Asia India Retrospective case series 2008–2010

• Mammary tumours 46.79% of all canine

malignant neoplasms

• Papillary adenocarcinoma

• Mixed carcinoma

• Solid carcinoma

(Gupta, 2012) China Epidemiological 2017–2021

• Mammary tumours 46.71% of all canine

neoplasms (51.59% CMCs)

• Greater prevalence in unspayed canines

• Not reported

(Zheng et al.,

2022)

South Korea Retrospective case series 2008–2011

• Mammary tumours 52.6% of all canine

neoplasms (51.7% CMCs)

• Mixed carcinoma

• Complex carcinoma

• Tubulopapillary carcinoma

(Im et al., 2014) O.H. Rodríguez-Bejarano et al.

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4.1.3. Latin America

A largescale retrospective study in Mexico (2015) revealed that 47.5% of mammary lesions were malignant, a high prevalence occurring in unspayed canines, along with increased incidence during the last four years of this study; subtype distribution was characterised by a pre­ dominance of simple carcinoma, complex carcinoma and mixed carci­ noma, epithelial tumours being the most frequently recurring overall (Salas et al., 2015). Retrospective data from Colombia has indicated that mammary tumours were the second most common tumour in canines and simple carcinoma the most frequently occurring CMC subtype, fol­ lowed by complex and mixed carcinomas, although population-based incidence data is limited (Caicedo, 2012). Such findings may reflect similarities in reproductive management practices and diagnostic ap­ proaches in Latin America.

4.1.4. Asia

Studies in India have reported that CMCs accounted for around 46.79% of malignant canine tumours, the highest incidence being observed in dogs aged 10–12 years-old; the most prevalent subtypes included papillary adenocarcinoma, mixed carcinoma and solid carci­ noma, indicating a higher representation of aggressive epithelial phe­ notypes (Gupta, 2012). Epidemiological analyses in China have identified mammary tumours as being the most commonly occurring neoplasm (46.71%), having higher prevalence in unspayed canines and a predominance of malignant forms (51.59%) (Zheng et al., 2022). Mammary tumours in South Korea have accounted for 52.6% of neo­ plasms found in canines, 51.7% of these being CMCs; mixed carcinoma, complex carcinoma and tubulopapillary carcinoma were the most frequently reported, showing partial overlap with European patterns but maintaining regional variability (Im et al., 2014). These differences may have been influenced by biological and methodological factors, including tumour grading criteria.

4.2. Risk factors and aetiology

Although CMC aetiology is not yet fully understood, its occurrence is influenced by several factors such as age, race, hormones, genetic fac­ tors, nutritional status, cyclooxygenase-2 (COX-2) expression and envi­ ronmental factors. A recent retrospective, matched case-control study in Brazil was aimed at elucidating the relationship between epidemiolog­ ical and clinical risk factors and CMC histopathological diagnosis. Var­ iables such as canine size, breed, housing, diet type and body score were evaluated; canine size, breed, housing and being overweight were found to be CMC predictors. The highest risk of developing CMC was reported to be associated with large canines, Yorkshire or Poodle breeds, living outside and/or being overweight (da Silva et al., 2023). Such risk factors for CMC are discussed in detail below (Fig. 1).

4.2.1. Age

Regarding the age for being at risk for neoplasia, most CMCs develop in canines aged between 5- and 10-years-old, average presentation age ranging from 8- to 10-years-old (Pastor et al., 2018; Salas et al., 2015). A data trend has been observed; the highest presentation peak is reached at 10 years of age, followed by a marked decrease in CMC incidence (Egenvall et al., 2005; Salas et al., 2015) and between older age at diagnosis and CMC occurrence (Pastor et al., 2018). Canines having benign mammary tumours usually have a lower average presentation age (7–9 years) than those having CMCs (Burrai et al., 2020; Papparella Fig. 1. Potential risk factors for developing canine mammary cancer (created using BioRender.com, accessed on January 19th, 2026). O.H. Rodríguez-Bejarano et al.

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et al., 2022; Pastor et al., 2018). It is worth noting here that canine ageing is a complex, multifactorial biological phenomenon affecting biological systems at multiple organisational levels, beginning with biochemical changes at the molecular level and spreading through cells, tissues and organs. Physiological responses may become less efficient with ageing, resulting in a decline in metabolic, immune and hormonal functions, thereby leading to greater vulnerability to diseases, including cancer (Vaj´anyi et al., 2024). Other factors such as stress, exercise, nutrition and socialisation can also influence canine ageing; such factors appear to interact with biological pathways linked to inflammation and oxidative stress (McCune and Promislow, 2021). Furthermore, lifestyle factors such as exercise and nutrition influence the immune system which tends to become weakened with age (Larsen and Farcas, 2014). The hallmarks associated with canine ageing and cancer include epigenetic alterations, genomic instability, telomere shortening, loss of proteostasis, impaired metabolism, mitochondrial dysfunction, cellular senescence and immune system dysfunction (Guelfi et al., 2024). Epigenetics deals with modifications regulating gene expression without altering DNA sequences. Epigenetic changes directly contrib­ uting to ageing and age-related diseases such as cancer include DNA methylation and histone modifications, alterations in chromatin acces­ sibility, loss of histones and heterochromatin, aberrant histone modifi­ cations and dysregulated microRNA (miRNA) expression/activity. As in humans, canine ageing is characterised by distinctive epigenetic changes (Jin et al., 2024). It has been proposed that neoplastic diseases should be addressed by taking into account the close relationship be­ tween genetics and epigenetics, given that cancer-related gene expres­ sion regulation may be influenced by transcription factor mutations that can lead to changes in chromatin and gene expression. Mutations in genes encoding epigenetic effectors (chromatin remodellers, histone modifiers, DNA methyltransferases) may compromise their activity and alter gene expression, or mutations in regulatory sequences may directly affect gene expression programmes, as they could prevent the binding of transcription factors and epigenetic modifiers. Interactions between epigenetics and genetics play a key role in carcinogenesis and cancer progression through uncontrolled proliferation, tumour heterogeneity and metastasis (Recillas-Targa, 2022).

Canines have similar telomere biology to that of humans, having comparable telomere length, telomere shortening and a lack of telo­ merase activity in somatic cells, although canines lose telomeric DNA approximately 10 times faster than humans. Telomeres shorten as cells divide, leading to DNA damage, cell-cycle arrest and the expression of pro-inflammatory factors, all being associated with ageing (Fick et al., 2012). Cancer cells maintain telomere length to grow indefinitely by reactivating telomerase or through a recombination-based mechanism. The identification of mutations in the TERT promoter has improved our understanding of the mechanism of TERT transcriptional activation and other genetic mutations that regulate TERT expression and telomere length (Okamoto and Seimiya, 2019). Most cancers have shorter telo­ meres compared to that of normal tissues, despite the risk of crisis, apoptosis or senescence due to excessive telomere shortening. One possible explanation is that the maintenance of shortened telomeres activates specific genes which are necessary for cancer progression, i.e. interferon-stimulated genes (ISGs). Short telomeres correlate with greater cancer malignancy, and telomere length in cancer could be useful as a prognostic biomarker or risk predictor (Lou et al., 2009). After 50–60 cell-cycles, cells having shortened telomeres undergo replicative senescence due to chromosomal instability and p53 activa­ tion which is triggered by DNA damage response in response to telomere shortening. However, cancer cells can acquire replicative senescence through genetic mutations in p53 or other cell-cycle checkpoint proteins and continue to proliferate; telomeres therefore become critically shortened. However, a small population of these cells that activate telomerase or alternative telomere lengthening via recombination ac­ quire immortality at this point and proceed to carcinogenesis (Rossiello et al., 2022).

Genomic and chromosomal instability (hallmarks of cancer and ageing) determine cancer cells’ genomic composition, thereby influ­ encing their behaviour. Various genetic and epigenetic alterations in cancer and ageing have been linked to genomic instability, including DNA repair defects, oncogene-induced replicative stress and mitotic spindle assembly checkpoint dysfunction. One consequence of genomic and chromosomal instability is DNA leakage from the nucleus into the cytoplasm, thereby triggering signalling pathways such as those involving ISGs (Chen et al., 2022).

Cells maintain proteome homeostasis through regulated processes such as protein synthesis, folding, transport, post-translational modifi­ cation and degradation. Protein homeostasis loss (proteostasis) is a key feature of ageing. The proteostasis network preserves proteome stability by preventing the accumulation of misfolded or damaged proteins. This network includes heat shock proteins, chaperones, the ubiquitinproteasome system and the autophagy-lysosomal pathway. Cells accu­ mulate misfolded proteins with ageing due to a decline in proteostasis efficiency, leading to reduced viability and the onset of protein mis­ folding disorders (Tang and Xiao, 2023). Misfolded proteins, particu­ larly those with exposed hydrophobic regions, tend to form toxic aggregates that disrupt membranes and cellular components (Ajmal, 2023). Cellular stress induced by the abnormal accumulation of unfolded or misfolded proteins in the endoplasmic reticulum is emerging as a potential trigger for cancer. Oncogene overexpression stimulates protein synthesis and secretory demands, these being typical triggers of endoplasmic reticulum stress; misfolded protein response activation promotes oncogene transformation, whose signalling path­ ways contribute to tumour growth, along with angiogenesis (Hetz et al.,

2020).

Ageing leads to a loss of metabolic homeostasis, this being regulated by cellular pathways which monitor nutrient availability and energy status by interacting with hormones and growth factor signals; ageing leads to a gradual decline in these regulatory functions. IGF-1, mTOR, AMPK and sirtuin signalling have been the most extensively studied pathways. These pathways play an essential role in regulating cell pro­ cesses such as protein synthesis, autophagy, metabolism, oxidative stress and immunity, which are directly linked to carcinogenesis and tumour progression (Guelfi et al., 2024).

Mitochondrial dysfunction is a critical factor in ageing and the onset of age-related diseases. Mitochondrial function is essential for metabolic regulation and cellular homeostasis, contributing to bioenergetics, reactive oxygen species (ROS) production, catabolic and anabolic pro­ cesses, calcium and iron homeostasis, intrinsic apoptosis cascade and various signalling pathways. Mitochondria become larger and fewer in number with ageing, their oxidative phosphorylation decreases and sequential damage to mitochondrial DNA begins to occur. Mitochon­ drial ROS production increases with age which could trigger greater oxidative damage to lipids (lipid peroxidation) and DNA (mutation accumulation); the latter could lead to carcinogenic mutation acquisi­ tion (Xu et al., 2025).

Senescence is a cell process which leads to the permanent arrest of the cell-cycle and is associated with a distinctive phenotype charac­ terised by chromatin remodelling and alterations in gene expression, increased macromolecular damage, metabolic reprogramming, increased autophagy and the activation of a complex pro-inflammatory secretome (McHugh and Gil, 2018). Cellular senescence can be repli­ cative or stress-induced. In the first scenario, telomere shortening (which occurs with each cell division) induces senescence to prevent genomic instability, DNA damage and carcinogenesis, triggering a p53and p21-mediated response that halts the cell-cycle and prevents damaged cell proliferation. In the second scenario, senescence is induced by stress produced by the stimulation of extracellular inflam­ mation and fibrosis mediators (IL-1β, IL-6, IL-8, TGF-β and CCL2), thereby amplifying pro-inflammatory molecule and ROS production and leading to NF-kB pathway activation and dysfunction of Wnt signalling (Birch and Gil, 2020). Stress-induced senescence regarding ageing is O.H. Rodríguez-Bejarano et al.

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considered more relevant because the accumulation of cellular damage is more frequent. Senescence is an important tumour-suppressing mechanism that halts premalignant cell proliferation and facilitates affected cell elimination through immune surveillance. However, the inability to eliminate senescent cells due to deficient immune surveil­ lance can lead to a state of chronic inflammation that fosters a pro-tumour microenvironment conducive to malignant tumour initia­ tion, migration and metastasis (Ou et al., 2021). As senescent cells can secrete cytokines, chemokines and other proinflammatory factors, adopting the well-known senescence-associated secretory phenotype (SASP), SASP can have harmful effects on neigh­ bouring cells, promoting tissue dysfunction and inflammation, which may further exacerbate age-related diseases, including cancer. Such secretory profile contributes to local and systemic inflammation, thereby playing a role in the ‘age-associated inflammation’ phenomenon (“inflammageing”), chronic inflammation linked to elevated IL-1, IL-6, IL-8 and TNF-α levels. Inflammageing thus refers to immune changes in response to chronic stress and views the pro-inflammatory process as an adaptation which could lead to harmful effects on the immune system by reducing its ability to respond effectively to infection and cancer (impaired immunosurveillance). Chronic inflammation is common in older canines and increased inflammatory cytokine levels contribute to age-related diseases (Franceschi et al., 2018). One age-associated immunological change in canines is the decline in T- and B-cell pro­ duction, primarily due to thymic atrophy and haematopoietic stem cell inefficiency. The changes observed in haematopoietic stem cells with age play an important role in T- and B-cell repertory generation; this can alter antigen-specific adaptive immune responses. The decline in lymphocyte production and thymic function deterioration suggest that older canines have weakened immune responses (Alexander et al.,

2018).

Ageing affects both the innate and adaptive immune systems and involves a combination of changes in immune cells, lymphoid organs and circulating soluble factors that influence immune cells and their microenvironment. A low-grade inflammatory response is a hallmark of ageing and is responsible for the increased susceptibility to inflamma­ tory diseases observed with advancing age (Bottazzi et al., 2018). Immunosenescence is a term used to describe older people’s decline in immune response; this is associated with key characteristics such as a reduced ability to respond to new antigens, persistent low-grade inflammation, memory T-cell accumulation and a simultaneous decrease in naive T-cells. It is characterised by a greater propensity for autoimmune responses and a non-sustained memory response (Kaiser et al., 2021). T-cell generation during adulthood depends primarily on peripheral naive T-cell proliferation. For reasons that are not yet fully-understood, naive CD8+ T-cells are less successfully maintained with age than naive CD4+ T-cells. Fewer circulating CD8+ naive T-cells represents the most consistent and prominent marker of immune ageing in older adults (Goronzy and Weyand, 2017).

Genetic events causing cancer orchestrate the development of an inflammatory microenvironment and latent and unresolved inflamma­ tion drives malignant progression. Uncontrolled inflammatory media­ tors expression and production are key features of ageing at cellular and organ levels, thereby contributing to establishing the tumour microen­ vironment’s (TME) fundamental components that promote tumour progression. TME is now regarded as an essential component of malig­ nant tumours. Pro-tumour inflammatory cells are able to interact with tumour and stromal cells via various molecules, such as cytokines, chemokines and growth factors, thereby contributing to the creation of an immunosuppressive microenvironment promoting cancer cells’ pro­ liferation and aggressive behaviour (Rodríguez-Bejarano et al., 2024).

4.2.2. Race

Some studies have shown higher CMC incidence in pure breeds than in mixed breeds; however, there is no conclusive data regarding which breeds have a higher risk, since this factor varies greatly depending on geographical location, the type of study and associated biases (Vascellari et al., 2016). A Swedish study found that English Springer Spaniels, Dobermans and Boxers were the breeds having the highest risk of developing CMC (Egenvall et al., 2005). The highest CMC incidence in India was observed in cross-breed canines (19.61%), followed by German Shepherds (17.66%), Labradors (13.73%), Spitze and canines classified as belonging to undescribed breeds (11.76%) (Gupta, 2012). A Mexican study found that 80% of the canines having CMC belonged to mixed breeds and 20% were pure breeds (Poodles and Cocker Spaniels predominated in the small-breed group, whilst German Shepherds, Labrador Retrievers and Rottweilers predominated in the large-breed group) (Salas et al., 2015). Samoyed, Schnauzer, Poodle, German Pinscher and Cocker Spaniel were the breeds most associated with CMC in Spain, whilst Miniature Pinscher, American Staffordshire Terrier, English Pointer and some local breeds (such as the Canarian Hound / Bardino Majorero) were less associated (Rodríguez et al., 2022). Springer, Cocker Spaniel, Boxer, Staffordshire Bull Terrier and Lhasa Apsos were the most likely breeds for developing CMC in the UK (Varney et al., 2023) whilst the breeds having with the greatest CMC incidence in Italy were Samoyeds, Dobermans, Schnauzers and Yorkshire terriers (Vascellari et al., 2016). English Cocker Spaniels, Pekingese and German Shepherds were the breeds in Argentina having the highest CMC fre­ quency (Benavente et al., 2016). Another Spanish study identified Re­ trievers, flushing canines and water canines as being the breeds having the greatest CMC incidence (Pastor et al., 2018).

4.2.3. Hormonal factors

• Steroid hormones

Regarding CMC-related hormonal factors, sex steroids (oestrogens and progesterone, primarily synthesised by the ovaries) and prolactin are related to CMC carcinogenesis and HBC initiation, promotion and pro­ gression. Mammary gland oestrogen dependence is primarily driven by the activity of oestrogen receptor-α (ERα), a ligand-activated tran­ scription factor that mediates oestradiol’s proliferative effects on mammary epithelial cells. Following oestrogen binding, ERα dimerises and translocates to the nucleus where it regulates the transcription of genes involved in cell-cycle progression and survival, such as cyclin D1 and MYC, thereby promoting tumour growth (Canadas-Sousa et al., 2019). This pathway’s biological relevance is supported by epidemio­ logical evidence showing that cumulative exposure to ovarian hormones throughout life significantly increases unspayed canines’ risk of devel­ oping mammary tumours, whilst early ovariohysterectomy confers a markedly protective effect. The age at which canine ovariohysterectomy is performed influences the risk of developing CMC; the available evi­ dence shows that canines spayed before puberty have a 0.5% risk, whilst canines spayed after one oestrous cycle have an 8% risk. This rises to 26% after the second oestrous cycle and they have a risk similar to that of an unspayed canine after the third oestrous cycle (Benavente et al., 2016). The risk of developing CMC has been observed to increase with each additional cycle (Burrai et al., 2020). It has also been reported that unspayed canines have a four times greater risk of developing CMC than those spayed before the age of two (Schneider et al., 1969). It is worth noting that using exogenous progesterone as canine contraceptive treatment can induce benign canine mammary tumour development (Rao et al., 2009). Furthermore, oestrogen signalling in the CMC is reinforced by interaction with oncogenic pathways such as PI3K/AKT1/mTOR and MAPK/ERK which can enhance ERα transcrip­ tional activity and contribute to ligand-independent activation, a mechanism associated with tumour progression (Miricescu et al., 2020). Furthermore, high local oestrogen production in malignant mammary gland tissue, mediated by stromal aromatase expression, sustains a paracrine signalling circuit within the TME, thereby maintaining oestrogen-dependent growth even amidst systemic hormone level fluc­ tuations (Takagi et al., 2013). Progesterone receptor (PR) expression O.H. Rodríguez-Bejarano et al.

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reflects a functional oestrogen axis and contributes to epithelial prolif­ eration and hormone-sensitive progenitor cell expansion (de Andr´es et al., 2022a). Such mechanisms underpin ER+-related CMC, similar to the HBC luminal subtype.

It has been observed that benign canine mammary tumours and lowgrade CMCs are typically ER+ and are associated with a better prog­ nosis, whilst high-grade CMCs tend to have decreased ER and PR expression and are associated with a poorer prognosis (Brunetti et al., 2021). The loss of ER expression in high-grade CMCs is a phenomenon that reflects tumour dedifferentiation and progression towards a more aggressive, hormone-independent phenotype. Tumour cells undergo lineage plasticity, evolving towards a more basal or mesenchymal phenotype (i.e. epithelial-mesenchymal transition (EMT)) which is characterised by decreased hormone receptor expression (ER and PR) and increased basal/stem cell markers expression (CK5/6, SOX2 and GATA3), thereby losing their dependence on oestrogen-mediated sig­ nalling for proliferation and survival. Furthermore, EMT-associated transcription factors (SNAIL, TWIST, ZEB1) suppress ESR1 expression (Ye and Weinberg, 2015). ESR1 gene epigenetic repression (ESR1 pro­ moter hypermethylation, histone deacetylation and chromatin compaction, transcriptional repressor recruitment) is one of the best-established ER loss mechanisms, leading to stable ER expression silencing without the need for genetic mutations (Skrzypczak et al. 2024). Increased genomic instability is another mechanism underlying ER loss in high-grade CMC, leading to copy-number alterations affecting ESR1 or its regulators, mutations in transcription factors controlling ER expression and dysregulation of oncogenic signalling pathways such as PI3K/AKT/mTOR and MAPK/ERK. When hyperactive, these can sup­ press ER expression and promote ligand-independent growth, reducing a tumour’s dependence on oestrogen signalling (Osborne and Schiff, 2011). High-grade CMCs’ chronic inflammation and hypoxia in the TME can suppress ER expression and promote the emergence of tumour cell populations having a more aggressive, hormone-independent phenotype through the production of IL-6, TNF-α and hypoxia-inducible factor 1α (HIF-1α) (Hases et al., 2022). Taken together, such processes highlight high-grade CMC biological convergence towards an ER-negative state which is associated with a poor prognosis and limited therapeutic response.

• Prolactin

Prolactin (PRL) plays a role in normal mammary epithelium prolif­ eration and differentiation in most mammalian species and in post­ partum lactation stimulation. PRL in canines is an essential luteotropic factor during the second half of pregnancy (in addition to its role in mammary development). The PRL receptor (PRLR) belongs to the class I cytokine receptor superfamily, whose members are single-pass mem­ brane receptors lacking tyrosine kinase activity (Michel et al., 2014). Emerging evidence supports the hypothesis that CMC may act as a PRL-producing tumour via autocrine/paracrine mechanisms, thereby establishing a self-sustaining oncogenic signalling loop that is inde­ pendent of pituitary regulation. Tumour cells can thus synthesise PRL whilst expressing PRLR, thereby enabling continuous binding between ligand and receptor within the TME (Standing et al., 2023). PRL-mediated signalling constitutes a key pathway in CMC progression, acting via endocrine, autocrine and paracrine tumour growth-promoting mechanisms. PRL binding to PRLR (frequently overexpressed in malig­ nant mammary tumours) activates intracellular cascades such as JAK2/STAT5, PI3K/AKT/mTOR and RAS/RAF/MEK/ERK, promoting cell proliferation, survival and resistance to apoptosis (Clevenger and Rui, 2022). Sustained STAT5 activation induces the transcription of anti-apoptotic and cell-cycle regulatory genes, whilst the PI3K/AKT pathway contributes to metabolic adaptation and the evasion of cell death signals, essential characteristics in high-grade tumours. In addi­ tion to its mitogenic role, PRL participates in TME remodelling, indi­ rectly promoting processes such as angiogenesis and invasiveness. Furthermore, PRL-mediated SRC family kinase and Rho GTPase acti­ vation contributes to cytoskeletal remodelling and increased invasive potential. Evidence suggests that PRL may induce EMT-associated pro­ grammes, thereby increasing tumour cells’ migratory capability, although such mechanisms still require further characterisation in canine models. Taken together, PRL/PRLR axis activation emerges as a central modulator of tumour progression (Hathaway et al., 2023). It is worth noting that PRL signalling interacts significantly with ER, PR and growth factor pathways, amplifying mitogenic and survival signals and contributing to endocrine resistance. Such findings suggest that intra-tumour PRL production may play a central role in CMC pro­ gression by maintaining multiple converging signalling networks that promote tumour growth, plasticity and therapeutic resistance (de Andr´es et al., 2022a). Modulating the signalling of intra-tumour PRL could represent a promising therapeutic strategy in CMC, particularly regarding tumours that are hormone-independent or resistant to con­ ventional endocrine therapies.

A systematic review of various hormonal receptors highlighted a decrease in expression of ERα, PR, PRLR and growth hormone receptor (GHR) associated with CMC (Beauvais et al., 2012). Another study investigated the association between estradiol-17β, progesterone and PRL plasmatic concentrations and ERα, ERβ, PR, PRLR and GHR gene expression, regarding reproductive status (spayed vs. unspayed) and cycle phase (anestral vs. diestral). It found that the lowest gene expression for all receptors occurred in affected canines’ CMCs compared to that in their normal tissues. Steroid levels were not influ­ enced by the expression of their respective receptors in tumours, but increased PRL levels were negatively associated with low PRLR expression in CMCs (Spoerri et al., 2015). Other studies have shown that more than half CMCs are negative for ER and PR (Toniti et al., 2009). Regarding PRL, one study observed that canines having CMC had higher PRL plasma levels than healthy canines, and that PRL levels in mammary tissue were higher in CMC than in normal mammary tissue, leading to the hypothesis that CMC could be a source of PRL (Queiroga et al., 2005). It has also been hypothesised that malignant canine mammary tissue transformation is associated with decreased PRLR expression (Michel et al., 2012). A recent study has compared HBC and CMC samples from a hormonal perspective. Great similarity was observed between premenopausal HBC and CMC regarding hormone receptors. The amounts of hormones analysed in both species were seen to increase in tumours compared to amounts in normal mammary glands; an in­ crease was seen regarding all hormones analysed in HBC and CMC in tumours compared to control samples. Intra-tumour androgen levels were similar in both species, although progesterone and oestrogen levels were higher in HBC than in CMC. The study suggested that the major hormonal influence was oestrogenic in both species through the ER, with the α isoform being predominant in HBC (de Andr´es et al., 2022b).

4.2.4. Genetic factors

Several studies have found that genetic alterations do influence mammary tumours development and that certain breeds seem to show a greater predisposition to developing CMC.

A study has evaluated 10 HBC genes (BRCA1, BRCA2, CHEK2, ERBB2, FGFR2, LSP1, MAP3K1, RCAS1, TOX3 and TP53) seeking an association with CMC; it was found that BRCA1 and BRCA2 were significantly associated with CMC and that FGFR2 had a borderline as­ sociation. A stronger association was found for BRCA1 regarding cases of CMC. Both BRCA1 and BRCA2 had ~4 odds ratios (Rivera et al., 2009). A striking similarity of genomic features was found in another study involving whole exome and transcriptome analysis of 191 CMCs having archetypal HBC features, including frequent PIK3CA mutations (43.1%), aberrations of the PI3K-AKT pathway (61.7%) and key genes implicated in cancer onset and progression (KRAS, MKI67, TP53, NKX1–2, SETD1A,

PTEN, PIK3R1, MKI67, BRCA1/2, NBN, NSMCE1, POLD1, RECQL4,

RMI1, RTEL1, SLX4, SMC5, TOP3B and XRCC3). The relative absence of ERBB2 amplification and the HER-2-enriched subtype in CMC denoted O.H. Rodríguez-Bejarano et al.

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species-specific molecular mechanisms. Furthermore, 5.5% of canines having CMC analysed were shown to have germline BRCA1/2 variants predisposing to cancer. Mutations affecting the Ki-67 (MKI67) prolifer­ ation marker gene (encoding the Ki-67 protein) were also identified in CMC, but having a much lower case rate (6%) (Kim et al., 2020). A recent study used exome capture and next-generation sequencing to investigate somatic genetic aberrations occurring in CMC. The PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase subunit alpha) gene was identified as the most commonly occurring mutated gene in CMC, based on 55 tumour-normal tissue pairs; 25% of samples har­ boured mutations in this gene. The study identified a recurrent missense mutation, p.H1047R; this is homologous to the human PIK3CA point mutation found in different types of HBC. Mutations homologous to other known human point mutations were also identified, such as PIK3CA p.E545K and KRAS p.G12V/D. Copy number aberrations affecting significant oncogenic and tumour suppressor pathways were also identified, including deletions affecting the tumour suppressor gene PTEN (phosphatase and tensin homologue) (Arendt et al., 2023). A study of 40 CMC samples evaluated p53 expression; this high­ lighted significantly reduced expression in 8 samples, overexpression in

2 samples and normal expression in 30 samples; however, no correlation

was reported between TP53 gene expression and tumour aggressiveness. (Oliveira et al., 2017). A subsequent study using immunohistochemistry reported that 7 out of 35 CMC samples had immunostaining for p53, even though it could not be established that this biomarker significantly predicted prognosis (although this could have reflected the limited amount of CMC samples included in this study) (Munday et al., 2019). A more recent study evaluated 170 CMC samples, finding that only 0.5% (8/170) expressed p53, these being high-grade tumours having high proliferative activity which suggested that the p53 gene is involved in CMC progression (Brunetti et al., 2021). Cyclin D1 is a proto-oncogene which has been seen to have greater expression in CMCs; however, its usefulness as a prognostic factor is limited since its expression is also particularly high in canine mammary dysplasia (Sfacteria et al., 2003). CMC-related EGFR overexpression has been shown to be associated with tumour size, necrosis, mitotic grade, histological grade of malig­ nancy, tumour relapse, distant metastasis and clinical stage. Further­ more, it may contribute to increased CMC angiogenesis and aggressiveness (Queiroga et al., 2017). HER-2 mutations and over­ expression have been detected in CMC; it has been suggested that HER-2 participates in canine mammary gland carcinogenesis, since a positive correlation between HER-2 expression, malignancy and high histologi­ cal grade has been described (Matsuyama et al., 2001).

4.2.5. Nutritional status

Obesity conditions early high-grade CMC development in canines and has also been associated with lower survival rate; similarly, canines fed on a homemade diet have a higher body condition score (BCS) than those fed on a commercial diet (although no relationship has been observed between CMC-related diet and survival rates) (Tesi et al., 2020). It has been shown that young overweight female canines fed a red meat-rich diet have a higher risk of developing breast dysplasia and CMC, whilst the risk of CMC decreases in young (9–12 months old) spayed female canines having a lean body constitution (Alenza et al., 1998). Concerning HBC, a higher risk has been seen in obese women having a high-fat diet; obesity in such women leads to greater tumour proliferation, risk of recurrence and decreased survival (Kamineni et al., 2013). Overweight canines have higher aromatase expression (this being a fundamental enzyme in oestrogen synthesis), in addition to the early appearance of CMC and a higher histological grade (Lim et al.

2015).

4.2.6. COX-2 expression

The cyclooxygenases (COX) are enzymes which synthesise inflam­ matory mediators called prostaglandins from arachidonic acid. Isoforms COX-1 and COX-2 have specific biological functions. COX-2 is stimulated by low inflammatory responses, growth factors and/or on­ cogenes and their role in carcinogenesis; their association with adverse HBC prognostic factors has been established, i.e. angiogenesis, invasion, metastasis and apoptosis suppression (Solanki et al., 2018). COX-2 overexpression has been observed in various canine cancers, including CMC (Greg´orio et al., 2017; Millanta et al., 2016). COX-2 expression has been shown to occur more frequently and be higher in CMCs (56–100% of malignant cells) than in benign canine mammary tumours and is associated with lymph node metastasis during/following surgery and with the development of distant metastases. Reports have indicated that higher COX-2 expression in CMC is related to lower survival and a worse prognosis (Millanta et al., 2016).

5. Carcinogenesis

CMCs can arise from canine mammary gland epithelial or stromal cell components; these may undergo neoplastic transformation depen­ dent on genetic and metabolic modifications with oncogene expression and repression of tumour suppressor genes. Such tumour cells activate apoptosis evasion programmes, enabling increased cell proliferation accompanied by accumulated DNA damage, alterations regarding DNA replication, poor cell-cycle regulation, alterations in cell metabolism and misfolded protein accumulation (Abdelmegeed and Mohammed,

2018).

The TP53 gene becomes activated in cells having severely damaged DNA, thereby initiating cell-cycle arrest, inducing damage repair or triggering apoptosis if such damage is irreparable. Some CMC studies have shown that p53-positive tumours were high-grade and had high proliferative activity, suggesting that the TP53 gene is involved in CMC progression, even though its prognostic usefulness has not been estab­ lished (Brunetti et al., 2021). Cyclin D1 is cyclin-dependent kinases CDK4 and CDK6 regulatory subunit; it is involved in cell-cycle control and has been identified as having higher expression in CMCs (Sfacteria et al., 2003).

The EGFR proto-oncogene plays an important role in CMCs since the expression of its phosphorylated form is associated with increased angiogenesis and metastasis (Silva et al., 2014). The proto-oncogene c-erbB-2 encodes HER-2 (EGFR-2), a protein which normally regulates cell proliferation and plays a relevant role in the regulation of tumour cell growth and differentiation. One study found higher HER-2 and EGFR expression in low-grade CMCs compared to that for high-grade CMCs, having a high positive statistical correlation for the latter. Such results suggested that aggressive tumours tend to lose EGFR and HER-2 expression simultaneously; these markers’ loss of expression may thus be related to neoplastic progression in CMCs (Silva et al., 2014). HER-2 expression contributes to tumour progression by increasing angiogenesis in CMCs (Carvalho et al., 2013).

The PI3K/AKT/mTOR pathway participates in regulating prolifera­ tion, protein synthesis, apoptosis, cell motility and angiogenesis, and becomes deregulated in several CMCs [57,114]. Mutations have been identified in the PIK3CA, PTEN, PIK3R1 (phosphoinositol-3-kinase regulatory subunit 1) and AKT1 genes (serine/threonine kinase 1) regarding CMC (Kim et al., 2020). It has been suggested that oncogene KRAS or PIK3CA activation or loss of tumour suppressor gene PTEN could over-activate such signalling pathway and may be important for CMC development (Arendt et al., 2023).

Mutations in PTEN and PIK3R1 have been described in different histological subtypes of benign canine mammary tumour and CMC, whilst mutations in AKT1 have only been observed in complex CMC, thereby suggesting that they are tissue-specific (Kim et al., 2020). Phosphorylated AKT expression in CMC correlates with more aggressive subtypes, lymphatic invasion and worse survival rates (Asproni et al., 2021). PTEN expression is associated with less aggressive tumours, whilst its loss is associated with lower survival rates (Asproni et al., 2021; Ressel et al., 2009). PTEN overexpression in CMC inhibits cell proliferation by inducing apoptosis by upregulating caspase-3, caspase-9 O.H. Rodríguez-Bejarano et al.

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and Bax and downregulating AKT phosphorylation (Tong et al., 2016). KRAS oncogene mutations in CMC can activate the RAS signalling pathway and promote tumorigenesis (Kim et al., 2020). BRCA1 and BRCA2 are tumour suppressor genes which encode a nuclear phosphoprotein involved in cell-cycle regulation and response to DNA damage. When BCRA1 expression is lost, genomic instability occurs, leading to other alterations, such as tumour suppressor gene inactivation and oncogene activation, thereby inducing tumorigenesis (Montalban et al., 2021). BRCA1 and BRCA2 mutations may predispose certain canine breeds to developing CMC, BRCA1 being more strongly associated (Rivera et al., 2009). Furthermore, BRCA2 expression is lower in CMC compared to healthy mammary gland tissue and is a possible mechanism explaining tumorigenesis, although it remains unclear why such expression levels are low (Yoshikawa et al., 2015). Another char­ acteristic of CMCs is a shift in BRCA1 expression from exclusively nu­ clear to both nuclear and cytoplasmic (Nieto et al., 2003). Ovarian steroids (mainly oestrogens and progesterone) play a stim­ ulating role in canine mammary epithelium proliferation, thereby creating conditions for neoplastic proliferation since these steroids (particularly progesterone) could cause autocrine growth hormone (GH) production which could act on mammary epithelium stem cells. This is an important step in carcinogenesis which could stimulate local or sys­ temic secretion of insulin-like growth factor 1 (IGF-1) (Benavente et al., 2016). IGF-1 is a mammary gland cell proliferation factor, whose effect could be stimulated by local oestrogen levels, thereby promoting CMC development and maintenance (Queiroga et al., 2008). It has been suggested that these hormones could act as local growth factors stimulating autocrine/paracrine CMC development and/or pro­ gression (Queiroga et al., 2009). Oestrogens modulate gene expression and directly affect activation through phosphorylation of several protein kinases which can promote cell proliferation, in turn increasing the chance of acquiring new genetic errors (Torres et al., 2021). Oestrogens promote a procarcinogenic effect by inhibiting apoptosis and inducing genetic/epigenetic changes modulating the expression of genes involved in regulating cell proliferation and differentiation (Kumaraguruparan et al., 2006). Oestrogen-induced cell proliferation increases genetic alteration incidence.

Furthermore, oestradiol oxidative metabolism-derived metabolites can cause direct genotoxic effects [37, 38].

High steroid hormone levels have been identified in the serum and mammary tissue of canines having malignant tumours compared to that for those having benign tumours, thereby suggesting that steroid hor­ mones act as local growth factors, stimulating cell proliferation. Oes­ trogen and progesterone receptors are also present in both benign and malignant lesions. By inducing cell proliferation oestrogens increase the likelihood of genetic alterations and the products associated with oes­ trogen oxidative metabolism can cause direct genotoxic damage. Oes­ trogen mechanisms of action include classic genomic effects modulating gene transcription, and non-genomic effects, thereby triggering rapid effects upon oestrogen binding to specific receptors (ER), particularly ERα. Such responses modulate various intracellular signalling pathways thereby triggering the post-translational modification of several proteins (Torres et al., 2021).

As mentioned above, CMC-related COX-2 overexpression has been established; several studies have focused on this enzyme’s action in carcinogenesis. COX-2 enables prostaglandin production; these are arachidonic acid-derived mediators; particularly, prostaglandins E2 (PGE2) and H2 (PGH2) contribute to the malignant phenotype and have been implicated in tumorigenesis through their immunomodulatory ef­ fects and their effects on proliferative processes, apoptosis and angio­ genesis. COX-2’s antiapoptotic mechanism is related to a reduction in arachidonic acid content (Araújo et al., 2016).

6. Metabolic reprogramming

Tumour cells must undergo multiple modifications to achieve a higher proliferative rate with low cell death rate, thereby favouring tumour growth and even metastasis. Metabolic reprogramming is one of the most important alterations concerning malignant tumours; it is responsible for meeting bioenergetic demands and anabolic re­ quirements enabling tumour cell proliferation and growth. Tumour cells have modifications favouring an imbalance between proliferation rate and apoptosis; metabolic reprogramming is one such modification, this being a hallmark of carcinogenesis (Ohshima and Morii, 2021; Schiliro and Firestein, 2021) (Fig. 2). The Warburg effect has been described as a metabolic adaptation in which tumour cells capture greater amounts of glucose and use it as an energy source, but with decreased and unim­ paired mitochondrial activity (DeBerardinis and Chandel, 2020).

6.1. Metabolic alterations regarding canine mammary carcinoma

Regarding carbohydrate metabolism, hyperglycaemia has been described in canines carrying CMC, this apparently being related to tumour cell presence since blood glucose levels decrease considerably once mastectomy has been performed. It has been proposed that glycolysis dysregulation in CMC is associated with increased lactate release by tumour cells and greater glucose uptake and processing (Rodigheri et al., 2023). It has been shown that glycolytic enzymes and glucose transporters GLUT1 and GLUT3 have higher expression in CMC (Lee et al., 2020; Tamarindo et al., 2023). Macrophage polarisation in the M1/M2 intermediate state and M2 macrophage activation is one effect of increased lactate release in CMC, thereby promoting immuno­ suppression in the TME, correlating with tumour progression and aggressiveness (Bao et al., 2021). Carbohydrate catabolism and anabo­ lism are essential for meeting tumour growth demands; this becomes apparent when observing that enzymes related to the pentose phosphate pathway are deregulated in CMC; oestrogen context is also important as it seems to play an additional role because ER+ cells have low glucose-6-phosphate dehydrogenase (G6PDH) expression whilst ERcells have high expression (Tamarindo et al., 2023). As previously discussed regarding lipid metabolism, obesity leads to earlier CMC onset. Obesity is associated with more aggressive CMC, having a higher angiogenesis rate and greater tumour-associated macrophage (TAM) infiltration (Lim et al., 2022). It has been sug­ gested that increased adiponectin expression may prevent CMC devel­ opment and positively affect its prognosis, whilst a decrease in its expression in obese canines influences CMC aggressiveness (Lim et al., 2015). It remains a matter of debate whether high-fat diets or obesity correlate with CMC development and associated survival rates (Tamarindo et al., 2023). Key enzymes in fatty acid translocation from the cytoplasm to the mitochondria for their subsequent oxidation and ATP production modify their expression in high histological grade CMC, i.e. carnitine acylcarnitine translocase (CACT), carnitine palmitoyl transferase 1 and 2 (CPT1 and CPT2) and carnitine O-acetyltransferase (CRAT) (Cacciola et al., 2021). One study found CMC sphere-forming cells had higher palmitoleate, palmitate and dihomo-gamma-linolenic acid levels compared to those for adherent cells, indicating that certain free fatty acids having specific metabolic profiles are significant regarding CMC-related carcinogenesis. Higher omega-3 polyunsaturated fatty acid levels have been reported in healthy canines compared to those with CMC, whilst an opposite correlation has been observed for omega-6 fatty acids, suggesting a protective property for the former (Tuzlu et al., 2021).

Regarding amino acids (aa) metabolism, methionine, serine, aspar­ agine, glutamine, alanine, taurine and citrulline plasma levels are lower in healthy canines compared to those affected by CMC. Methionine, lysine, histidine, aspartate, serine, asparagine, glutamate, glutamine, alanine, taurine, citrulline and ornithine levels are higher in canines having metastatic CMC compared to those for healthy canines, sug­ gesting that different aa may play a relevant role in CMC origin and progression (Azuma et al., 2012). Tumour cells usually depend on glutamine metabolism; it has been seen that glutaminase A and O.H. Rodríguez-Bejarano et al.

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transglutaminase II usually have higher expression in CMC, especially in high-grade tumours (Ryu et al. 2018). Some aa transporters have altered expression in CMC, i.e. L-type aa transporter 1 (LAT1) associated with leucine, isoleucine, valine, phenylalanine, tyrosine, tryptophan, methi­ onine and histidine uptake; this has increased expression in tumour tissue compared to that in healthy breast tissue (Fukumoto et al., 2013). Peptidyl arginine deiminase 2 (PAD2), which converts arginine to citrulline, has decreased expression in CMC compared to healthy breast tissue, suggesting that this aa plays an important role in CMC (Cherrington et al., 2010).

Regarding mitochondrial alterations, high mitochondrial DNA (mtDNA) heterogeneity has been observed in different CMCs; this could be explained by a lack of histones and repair mechanisms, along with in mitochondria being the main source of ROS (Kowal et al., 2022). CMC-associated mtDNA mutations related to the electron transport chain function have been identified in genes such as ND2 (NADH de­ hydrogenase subunit 2), COXII (cytochrome C oxidase subunit II), ATP6 (ATP synthase F0 subunit 6) and COXIII (cytochrome C oxidase subunit III) (Surdyka and Slaska, 2017). Mitochondria are also closely related to cell death activation which has been explored as a therapeutic strategy for CMC in vitro with therapeutic alternatives, such as toosendanin and melatonin (Y. Yang et al. 2023).

6.2. The impact of canine mammary carcinoma-related metabolic

reprogramming on the tumour microenvironment CMC-related tumour progression is sustained by profound metabolic reprogramming which extends beyond intrinsic bioenergetic adaptation and actively shapes the TME. Such reprogramming enables tumour cells to meet anabolic and energetic demands whilst simultaneously orches­ trating immune evasion. Rather than representing isolated alterations in carbohydrate, lipid or amino acid metabolism, these changes constitute an interconnected network driven by nutrient availability, oncogenic signalling and microenvironmental pressures such as hypoxia (Halma et al., 2023). A critical yet under-explored aspect of CMC-related metabolic reprogramming is its direct impact on immune cell function within the TME. Metabolic intermediates are not mere by-products of tumour activity, but active signalling molecules that reshape immune responses. The Warburg effect (supporting rapid ATP generation and biosynthetic flow) leads to increased local lactate production and acid­ ifies the TME, thereby exerting profound immunomodulatory effects. Lactate acts as a metabolic by-product and signalling molecule stabil­ ising HIF-1α and activates pathways such as STAT3 in immune cells. These signals promote macrophage polarisation towards an M2 pheno­ type, inhibit cytotoxic T-cell and NK-cell activity and promote regula­ tory T-cell expansion, thereby establishing an immunosuppressive Fig. 2. Canine mammary carcinoma-related metabolic reprogramming. Metabolic reprogramming enables cancer cells to produce more energy and metabolic in­ termediates maintaining high bioenergetic and biosynthetic demands for maintaining rapid and efficient cell proliferation. An increase in glucose uptake with greater anaerobic glycolysis represents a significant metabolic modification in CMC (i.e. the Warburg effect) for satisfying anabolic demands, and an adjustment in energy metabolism with increased fatty acid synthesis and oxidation, having differentiated effects, depending on the amount of available lipids. Reprogramming amino acid (aa) metabolism includes alterations in glutamine, serine, glycine and arginine uptake and use, these being essential for energy production and biosynthesis and redox balance. Legend: amino acids (aa): acetyl-CoA: acetyl coenzyme A; TCA cycle: tricarboxylic acid cycle (created using BioRender.com, accessed on January 19th, 2026).

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niche. This shift boosts the production of anti-inflammatory cytokines (such as IL-10) and reduces pro-inflammatory mediators, thereby facil­ itating immune evasion (Aki et al., 2023).

Lipid metabolism represents another critical aspect of tumourimmune interaction in the CMC. Metabolic changes such as increased fatty acid availability and altered adipokine profiles contribute to tumour aggressiveness and immune modulation. TAMs adapt to this lipid-rich environment through a fatty acid oxidation-dependent meta­ bolism which promotes their immunosuppressive phenotype. Further­ more, lipid accumulation in dendritic cells impairs antigen presentation, thereby limiting T-cell activation. Such findings highlight lipid meta­ bolism as a source of energy and a determinant of immune cells’ fate within the TME (Arner and Rathmell, 2023). Tumour-induced metabolic reprogramming leads to competition for nutrients within the TME. Cancer cells outcompete immune cells for essential substrates such as glucose, glutamine and other aa, leading to effector T-cells’ metabolic exhaustion and a compromised anti-tumour response. This competitive advantage is reinforced by the overexpression of aa transporters, such as LAT1, which increases essential aa uptake and activates nutrient-sensing pathways, including mTORC1. Simultaneously, increased gluta­ minolysis promotes tumour growth whilst limiting glutamine avail­ ability for immune cells, further contributing to immune dysfunction. Moreover, arginine metabolism alterations (potentially linked to reduced PAD2 expression) may also influence nitric oxide (NO) pro­ duction and T-cell function, thereby reinforcing immunosuppression (Merlo Pich et al., 2025).

Such processes define a bidirectional, self-reinforcing metabolicimmune axis. Tumour-derived metabolites and nutrient depletion acti­ vate key signalling pathways in both tumour and immune cells (i.e. HIF- 1α, mTORC1, AMPK and STAT3), leading to coordinated cell function reprogramming. In turn, metabolically reprogrammed immune cells secrete cytokines such as IL-10 and TGF-β, further promoting tumour growth, immune evasion and metabolic adaptation. This positive feed­ back loop underscores metabolic reprogramming’s role as a central driver of CMC progression. From a translational perspective, metabolic pathway modulation represents a promising therapeutic strategy; inhibiting glycolysis, glutaminolysis or fatty acid oxidation may halt tumour growth and also restore anti-tumour immunity. Therapeutic approaches aimed at disrupting the metabolic-immune interaction could thus enhance existing treatments’ efficacy and offer new avenues for CMC interventions.

7. Therapeutic tools

The treatment of choice for almost all canines having CMC consists of surgical tumour resection; this is considered curative when less aggressive histological types are present or there is no lymphatic involvement or distant metastasis (Cassali et al., 2020; Horta et al., 2014). A physiological approach would state that surgery primarily re­ duces the tumour burden but does not eliminate disseminated tumour cells nor does it interfere with systemic processes underlying disease progression, i.e. EMT, intravasation and/or premetastatic niche forma­ tion. An inflammatory carcinoma provides the exception to initial sur­ gical management, palliative medical treatment and chemotherapy being preferred (Raposo et al., 2017).

Differing surgical approaches have been described, such as lumpec­ tomy (nodulectomy), simple mastectomy, regional mastectomy, unilat­ eral or bilateral mastectomy with or without lymphadenectomy and ovariohysterectomy, to be performed depending on clinical stage, ul­ ceration and inflammation. It should be stressed that surgical manage­ ment of CMC cases involving distant metastases which have been detected before an intervention does not prolong survival time, but could help improve the quality of life in canines having ulcerated and/or painful lesions. (Dias et al., 2016; Goldschmidt et al., 2016). A decision regarding the type of surgical procedure depends on clinical stage, whilst associated ulceration and inflammation are also taken into account. However, a surgical recommendation remains a matter of debate; some approaches prefer aggressive surgical procedures, considering the risk of new tumours developing in unresected mammary glands (Dias et al., 2016; Fossum, 2018).

There is no consensus regarding a surgical approach which offers the best local control and reduces the risk of recurrence and distant metastasis. Unilateral or bilateral mastectomy would thus seem to have a greater probability of local tumour control; however, the resection extension may be associated with increased postoperative complications and postoperative stress, this being an aggressive technique, and may not improve survival. These techniques may be considered over­ treatment for some canines, and caution should be exercised when choosing a surgical approach; although there is no professional consensus regarding the best surgical approach for breast tumours in canines, there is a unanimous need for further studies on the subject (Horta et al., 2015).

As metastasis in lymph nodes may preferentially drain mammary gland parenchyma, lymphadenectomy is recommended when perform­ ing mastectomy. Ovariohysterectomy has not been shown to be bene­ ficial regarding survival rate or CMC recurrence (Cassali et al., 2020). One study has reported that after a canine underwent regional mastec­ tomy to resect a single mammary gland tumour, a new tumour appeared in the same mammary chain in 58% of cases, leading to a second intervention in 77% of them. It was thus suggested that an initial radical mastectomy could prevent fresh neoplasia formation, thereby avoiding a second surgical intervention. Further studies are needed to reach a consensus regarding the best surgical approach to CMC. Some consen­ suses have proposed that the extent of mastectomy should be based on tumour lesion stage, size, number and location, along with lymphatic drainage. Prospective studies in this regard should attempt to unify practice and lead to large-scale data for CMC specialists’ discussion (Cassali et al., 2020; Sahai et al., 2020).

Surgical management cannot be considered curative regarding CMC involving vascular or lymphatic invasion since there is the possibility of regional or distant metastatic disease. An additional therapeutic option could be recommended for delaying metastatic involvement and improving survival. A standard adjuvant therapeutic regime has not yet been described for aggressive histological subtypes (solid carcinoma, micropapillary carcinoma, anaplastic carcinoma and/or carcinosar­ coma), locally advanced disease or metastatic disease. However, the probable benefit of adjuvant treatment involving hormonal therapy, chemotherapy, nonsteroidal anti-inflammatory drugs (NSAIDs), radio­ therapy and/or immunotherapy should be kept in mind, even when metastatic involvement is not evident in a lymph node or distant organ (Sahai et al., 2020).

Using hormone therapy directed at inhibiting hormone receptors (ER or PR) has been proposed when CMC are positive for such receptors, especially regarding well-differentiated carcinomas, including ER modulators, gonadotropin-releasing hormone (GnRH) agonists and/or progesterone antagonists. One study which evaluated goserelin, a GnRH agonist, found decreased tumour size and oestrogen and progesterone levels when used in hormone-dependent CMC (Lombardi et al. 1999). Another study evaluated the neoadjuvant effect of the PR antagonist, aglepristone (RU534), on CMC-related proliferation and apoptosis in relation to PR expression; an antiproliferative effect in PR(+) CMCs was found (Guil-Luna et al., 2011). However, the use of tamoxifen as hor­ monal treatment is not recommended due to the side effects observed in canines having CMC (pyometra, vulvar inflammation and signs of pseudopregnancy) (Tavares et al., 2010). Hormone therapy use on ca­ nines having CMC still requires further research. Some CMC-related chemotherapy protocols have been used/intro­ duced; however, additional prospective studies and randomised controlled trials are still needed to guide the use of such regimens and assess their impact on prognosis and survival rates. Chemotherapy has been proposed for CMC involving a high risk of metastasis or recurrence, i.e. defined as tumours larger than 3 cm, high histological grade, O.H. Rodríguez-Bejarano et al.

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regional lymph node involvement and lymphovascular invasion. Mechanistically, drugs such as doxorubicin induce tumour cell death by intercalating into DNA and inhibiting topoisomerase II, in turn leading to apoptosis. However, resistance mechanisms (including increased drug efflux, enhanced DNA repair capacity and metabolic reprogramming) can reduce therapeutic efficacy. Metabolic tumour adaptations, such as increased glycolysis and lactate production, may promote an immuno­ suppressive microenvironment and contribute to treatment resistance (Nosalova et al., 2024). Table 2 lists chemotherapy protocols which have been evaluated in small CMC trials.

COX-2 inhibitors have become an important therapeutic strategy concerning CMC. COX-2 overexpression leads to increased PGE2 production thereby promoting angiogenesis, tumour proliferation, im­ mune evasion and metastasis. COX-2 inhibition disrupts such processes by reducing downstream signalling pathways’ PGE2-mediated activa­ tion, i.e. PI3K/AKT/mTOR and MAPK/ERK (Sahu et al., 2023). Drugs inhibiting COX-2 are divided into selective and non-selective COX-2 inhibitors (Nosalova et al., 2024). Experiments have shown that that COX-2 inhibitors inhibit tumorigenesis by modulating apoptosis, sup­ pressing tumour cell growth, inhibiting cell proliferation and reducing tumour cells’ metastatic potential (Gurpinar et al., 2014). Piroxicam and meloxicam (selective COX-2 inhibitors) have been shown to induce cell-cycle arrest, apoptosis, cell migration suppression and cell prolif­ eration, or exhibit cytotoxic activity in in vitro assays using CMC cell lines. Furthermore, piroxicam has been shown to reduce tumour size in a murine CMC xenograft model (Hurst et al., 2019). Furthermore, COX-2 inhibition may modulate the TME by reducing immunosuppressive cell populations and enhancing antitumour immune responses (Sahu et al., 2023). One study has shown an improvement in clinical status and progression-free survival in canines having inflammatory subtype CMC which were treated with piroxicam compared to doxorubicin-based chemotherapy protocols, suggesting that piroxicam should be consid­ ered a single agent for treating inflammatory subtype CMC (de M Souza et al., 2009). Another study found that disease-free survival and overall survival rates were significantly higher in canines having high-grade CMC which had received adjuvant treatment with firocoxib; however, it suggested the need for further studies to compare the efficacy of chemotherapy drugs versus COX-2 inhibitors as adjuvant treatment. (Arenas et al., 2016). A more recent study evaluated firocoxib’s proap­ optotic effects in vitro in two CMC cell lines (one from primary tumour and one from bone metastasis) and in vivo with canines affected by CMC, suggesting firocoxib as potential neoadjuvant treatment for CMC (Brandi et al., 2022).

Radiotherapy has only been used in veterinary medicine with promising results for palliative treatment of some canine tumours, such as osteosarcoma, nasal carcinoma and lymphoma; however, it has not been thoroughly evaluated in CMC as a therapeutic modality. It has been suggested as adjuvant treatment to surgery regarding the inflammatory subtype or in metastatic cases, or when tumours have been partially resected (Hunley et al., 2010). Along with local tumour control, radio­ therapy can induce immunogenic cell death, thereby increasing antigen presentation and promoting immune cell infiltration, thus suggesting potential synergy with immunotherapeutic approaches (Robinson et al., 2025). More studies are needed to clarify/evaluate CMC-related radiotherapy.

Immunotherapy options for CMC are still limited but they do represent a promising area for research. The PD-1/PD-L1 immune checkpoint axis plays a key role in tumour immune evasion; targeting this pathway could restore T cell-mediated antitumour responses. A murine monoclonal antibody (mAb) against canine PD-L1 (cPD-L1) has been demonstrated to be able to recognise tissues from different canine cancers, including CMC, although its functional therapeutic potential has yet to be validated (Sirivisoot et al., 2023). Furthermore, the ability of recombinant canine IL-15-based (rcIL-15) adjuvant therapy to alle­ viate tumour-related inflammation and enhance anti-tumour immunity has been demonstrated in canines suffering CMC; it stimulates NK-cells and CD8 + T-cells, as evidenced by elevated IFN-γ levels and improved clinical parameters in treated canines (Kang et al., 2025). Neoadjuvant intratumoral immunotherapy using cowpea mosaic virus (CPMV) has also been applied in CMC, demonstrating rapid tumour reduction without sistemic toxicity, enabling surgical intervention in previously inoperable cases, promoting favourable immunomodulation within TME, and significantly improving overall survival compared with con­ trols (Alonso-Miguel et al., 2022; Valdivia et al., 2023). Neoadjuvant intratumoural administration of anti-canine PD-1 (acPD-1) has been evaluated, both as monotheraphy and in combination with CPMV, in CMC. It was found that both acPD-1 theraphy and CPMV/acPD-1 com­ bination achieved tumour control and induced regression of injected Table 2 Adjuvant chemotherapy protocols for canine mammary cancer. CMC adjuvant chemotherapy protocols Protocol Result Reference 5-fluorouracyl (150 mg/m2) + cyclophosphamide (100 mg/m2) − Surgery + adjuvant therapy: 100% survival after 2 years’ follow-up − Just surgery: 71.4% mortality due to metastasis − Significant difference regarding overall survival (24 months’ surgery + adjuvant chemotherapy vs just 6 months’ surgery) − Adverse effects: temporal leukopenia (Karayannopoulou et al., 2001) Doxorubicin (30 mg/m2) or docetaxel (30 mg/m2) − Surgery + adjuvant therapy: 231 days’ mean survival time − Just surgery: 390 days’ mean survival time − No significant differences regarding relapse-free in­ terval, time to metastasis and overall survival − Adverse effects: mild allergic skin reactions with docetaxel use (Simon et al., 2006) Carboplatin (300 mg/m2) + piroxicam (0.3 mg/kg/ día)/firocoxib (5/mg/kg/ day) − Just surgery: 63 days’ mean survival time − Surgery + chemotherapy:

did not reach mean survival rate − Surgery + chemotherapy + piroxicam: 390 days’ median survival rate − Surgery + chemotherapy + firocoxib: 570 days’ mean survival time − Adverse effects: death due to piroxicam’s adverse effects (Lavalle et al.,

2012)

Mitoxantrone (5.5 mg/m2) + firocoxib (5/mg/kg/ day) − Just surgery: 12.7 ± 0.8 months´survival rate − Surgery + chemotherapy:

16.5 ± 2.6 months

survival rate − Surgery + chemotherapy + firocoxib: 19.4 ± 2.1 months’ survival rate − Disease-free survival rate was significantly higher in surgery + mitoxantrone and surgery + firocoxib than just surgery − Adverse effects:

neutropenia, gastrointestinal toxicity, elevated urea and creatinine levels (Arenas et al.,

2016)

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tumours, elicited responses in non-injected tumours located in the ipsilateral and contralateral mammary chains, and promoted the stabi­ lisation and reduction of stablished pulmonary metastasis (Sergent et al.,

2024).

8. Biomarkers

Biomarkers are molecules which can be evaluated in body fluids or tissues and can provide specific disease-related information; biomarker evaluation is often used in veterinary oncology for diagnosis, prognosis or tumour staging (Henry, 2010; Mobasheri and Cassidy, 2010). Bio­ markers represent a crucial interface between tumour biology and clinical application, providing valuable information for diagnosis, prognosis, tumour staging and therapeutic decision-making. Biomarker use regarding HBC is highly useful for diagnosis and taking therapeutic decisions for patients, as this enables predicting response rates regarding certain treatments and provides prognostic information. Reliable biomarker identification and validation in veterinary oncology is a constantly evolving field, particularly regarding CMC. Although numerous molecular markers have been proposed based on their simi­ larity with HBC, their clinical implementation in canines remains limited due to methodological variability, a lack of standardisation and incomplete biological validation (Abadie et al., 2018; Gray et al., 2020). Immunohistochemistry (IHC) and serum testing have become widely used tools for the assessment of CMC biomarkers for diagnostic and research purposes, thereby enabling the analysis of protein expression in tumour tissues and the measurement of various molecules in serum. However, a major limitation concerning IHC evaluation lies in the absence of standardised protocols and the frequent use of antibodies developed for human, murine or rabbit antigens as this can lead to variability regarding specificity and reproducibility (Kaszak et al., 2018). Such technical heterogeneity contributes significantly to the in­ consistencies observed amongst studies and hinders biomarker finding application in routine clinical practice. It has been suggested that the IHC biomarker panel for CMCs should include ER, PR, Ki-67 and COX-2. HER-2, Top2α, PCNA, p53, VEGF, EGFR, E-cadherin, BRCA-1/2, CEA, CA 15–3, miRNA, LCAT and cancer stem cells (CSC) have also been proposed as biomarkers for evaluating CMC (De Campos et al., 2016).

8.1. Biomarkers and canine mammary carcinoma classification

HBC has traditionally been classified into four molecular subtypes (luminal A, luminal B, HER2-positive and triple-negative) based on ER, PR and human epidermal growth factor receptor 2 (HER-2) expression in tumour cells (Rakha et al., 2023). Unlike HBC, molecular classification in CMC has not yet been standardised. Several studies have attempted to categorise CMCs into HBC molecular subtypes based on ER, PR and HER-2 expression; however, reported distribution varies markedly be­ tween cohorts and, on occasion, contradictory results have been ob­ tained (Abadie et al., 2018; Varallo et al., 2019). CMC classification has thus not been based on these biomarkers’ expression. It has been observed that ERα expression can be found in 50% of benign canine mammary tumours (de las Mulas et al. 2005). It should be noted that the aforementioned subtypes’ biological and clinical relevance regarding CMC remains unclear. A major limitation concerns HER2 assessment; HER-2 status is rigorously defined whilst most CMC studies rely solely on non-standardised IHC approaches regarding HBC using validated IHC scoring systems, supplemented by fluorescence in situ hybridisation (FISH) to confirm gene amplification (Abadie et al., 2018; Varallo et al., 2019). Very few studies have used FISH to detect or assess HER-2 amplification in CMC and canine pulmonary adenocarcinomas (Brunetti et al., 2024; Muscatello et al., 2022). Consequently, HER2 amplification has not been consistently demonstrated in canine tumours and its prognostic and therapeutic value remains controversial. Such discrepancies highlight the limitations of directly extrapolating HBC molecular subtypes to CMCs which may differ concerning their genomic landscape, hormonal regulation and TME. There is thus an urgent need to develop canine-specific molecular classification systems, supported by genomic and transcriptomic data rather than relying solely on human-based frameworks.

8.2. Human epidermal growth factor receptors

Human epidermal growth factor receptor 1 (HER-1) promotes tumour cell migration and invasion, stimulating angiogenesis and metastasis (Sabbah et al., 2020). It has been shown that HER-1 over­ expression in CMC may contribute to increased angiogenesis and tumour aggressiveness, this being associated with disease progression (Carvalho et al., 2013). High HER-1 expression in CMC has been associated with large tumour size, age, tumour necrosis, high mitotic index, tumour malignancy histological grade, more advanced clinical stage and lower disease-free and overall survival (Carvalho et al., 2013; Guimar˜aes et al.,

2014).

HER-2 amplification is usually associated with tumour cell growth, proliferation, migration, invasion and the ease of developing metastasis affecting lymph nodes and blood vessels, along with poor prognosis in HBC (Wolff et al., 2023). One CMC study has observed a positive cor­ relation between HER-2 expression in serum and tumour tissue, whilst a positive correlation has been observed between HER-2 expression and tumour mitotic index, high histological grade and tumour size in another study (Campos et al., 2015). However, some studies have noted no differences between benign canine mammary tumours and CMC regarding HER-2 expression (Ressel et al., 2013). A paradoxical result was found in one study consisting of canines having CMC expressing HER-2 having a higher survival rate than those not expressing HER-2 (Hsu et al., 2009). It has been seen that homology regarding humans and canine HER-2-associated tumour antigens suggests a promising option for using immunotherapy based on antibodies directed against HER-2, in canines having HER-2 + CMC (Singer et al., 2012). Further research is thus needed for carefully evaluating HER2’s specific diag­ nostic and biological role in CMC (Burrai et al., 2015). HER-2 expression is correlated with Top2α expression; the combined detection of these two biomarkers has thus been suggested (N.-Y. Yang et al. 2023).

8.3. Biomarkers associated with proliferation

The wide range of biomarkers which has been proposed regarding CMC can be grouped into interconnected biological processes driving tumour progression. Biomarkers associated with proliferation (such as Ki-67), proliferating cell nuclear antigen (PCNA), topoisomerase II alpha (Top2α) and cyclin-dependent kinase inhibitors reflect dysregulated cell-cycle dynamics and are consistently associated with tumour grade, metastatic potential and a poor clinical prognosis. Ki-67 has been the most widely studied prognostic biomarker for proliferation regarding canine tumours and has been the most widely used regarding CMC (Carvalho et al., 2016). Ki-67 expression in CMC is affected by factors such as mastitis, tumour size, invasion of other tissues and/or lymph node metastasis and has significantly less expression in benign canine mammary tumours than CMC (N.-Y. Yang et al. 2023). Top2α is over­ expressed in malignant tumours and has been developed for prognostic purposes concerning a variety of tumours. High Top2α suggests tumour cells’ degree of malignancy in tumour cells and it has been shown that Top2α amplification in CMC makes it more sensitive to anthracycline chemotherapy (Yakkala et al., 2023; Yang et al. 2023). PCNA is considered a good proliferation biomarker for HBC, but only when evaluated together with other biomarkers such as ER, PR, Ki-67 and/or HER-2 (Kang et al., 2024). One CMC study of PCNA expression posi­ tively correlated it with tumour size, histological subtype, degree of differentiation, nuclear grade, mitotic index, histological grade of ma­ lignancy and lymph node metastasis (Aydogan et al., 2018). Cyclin-dependent kinase inhibitors p21 and p27 are regulated by p53; one CMC study suggested that loss of p21 overexpression is associated O.H. Rodríguez-Bejarano et al.

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with tumour metastasis, whereas reduced cell-cycle inhibition by p27 is associated with malignant progression and poor overall survival (Klopfleisch and Gruber, 2009).

8.4. Biomarkers associated with genomic instability

Biomarkers associated with genomic instability further contribute to our understanding of tumour heterogeneity and progression. Alterations in tumour suppressor genes (i.e. TP53) promote the accumulation of genetic damage and malignant transformation, along with defects in DNA repair pathways involving BRCA1 and BRCA2. TP53 produces the p53 protein which is extremely relevant regarding cell-cycle and apoptosis control (Valdivia et al., 2021). Mutated p53 accumulates and promotes tumorigenesis; it plays a role as an oncogene promoting ma­ lignant cell transformation, leading to tumour formation. p53 gene mutations have been considered the most common genetic alteration concerning HBC, particularly regarding the most aggressive types, positively correlating with a worse CMC-related prognosis and shorter survival time (Sorenmo et al., 2019b). p53 mutations and low expression have also been associated with CMC-related tumour progression (tumour size, histological grade, lymph node metastasis, clinical stage and postoperative recurrence) (Brunetti et al., 2021; Oliveira et al., 2017). BRCA1 and BRCA2 genes have been the most important tumour suppressor genes for CMC susceptibility reported to date (Varol et al., 2018). An initial study showed loss of BRCA1 expression was associated with high Ki-67 and ER-negative tumours (Nieto et al., 2003). Later studies found that BRCA1 and BRCA2 mutations were significantly associated with CMC, having a stronger association for BRCA1 (Enginler et al., 2014; Rivera et al., 2009). It has been postulated (not very convincingly) that BRCA2 is expressed in a reduced manner as a possible mechanism for explaining CMC-related tumorigenesis (Yoshikawa et al., 2015). The CMC-related BRCA1 and BRCA2 mutation and decreased expression mechanism has yet to be clearly established, so further studies are needed if they are to be proposed as clinical biomarkers.

8.5. Biomarkers associated with angiogenesis and inflammation

Angiogenesis and inflammation represent another critical axis of tumour progression. Vascular endothelial growth factor (VEGF) drives neovascularisation and facilitates metastatic dissemination, while cyclooxygenase-2 (COX-2) contributes to a pro-inflammatory TME which enhances tumour growth, immune modulation and angiogenesis. COX-2 expression is greater in CMC than in benign canine mammary tumours and has high expression, mainly when metastases occur in the lymph nodes, without being associated with prognosis (Araújo et al., 2016; Hugo et al., 2015). COX-2 expression level is also variable regarding different degrees of CMC (Millanta et al., 2016; Queiroga et al., 2011). High COX-2 expression in CMC has been associated with increased cell proliferation, angiogenesis, tumour infiltration by T-cells and TAMs and poor prognosis (Arenas et al., 2016; Carvalho et al., 2015). VEGF forms a protein family encoded by VEGF-A, VEGF-B, VEGF-C and VEGF-D genes. Whilst VEGF-A and VEGF-B are involved in angiogenesis, VEGF-C and VEGF-D are involved in lymphangiogenesis, promoting metastasis in CMCs (Ghalehbandi et al., 2023). Increased serum VEGF levels have been positively correlated with more advanced clinical stage, lymph node metastasis, poor prognosis and lower CMC survival rate (Qiu et al., 2008; Queiroga et al., 2011). The combined detection of VEGF and PTEN could form an important index for esti­ mating CMC biological behaviour and prognosis (Qiu et al., 2008).

8.6. Biomarkers associated with epithelial-mesenchymal transition

A loss of epithelial integrity and the acquisition of invasive properties are mediated by alterations in adhesion molecules such as epithelialcadherin (E-cadherin); this is a key EMT regulator and acts as a tumour suppressor gene whose expression becomes decreased in CMC (Ferreira et al., 2012). Reduced E-cadherin expression is consistently associated with increased tumour size, histological grade, disease stage, invasiveness, metastasis and worse overall prognosis in HBC and CMC, thereby highlighting its role as a mechanistic driver and potential prognostic biomarker (Alonso-Diez et al., 2022). It has been suggested that E-cadherin could be a marker for identifying benign canine mam­ mary tumours and CMCs, since a study found that E-cadherin expression rate in CMCs was 36.83%, lower than that for benign canine mammary tumours (Na et al., 2020). E-cadherin should be evaluated along with other biomarkers, such as Ki-67 (Kaszak et al., 2018).

8.7. Serum biomarkers

In addition to tissue markers, circulating biomarkers such as carci­ noembryonic antigen (CEA), CA 15–3, microRNAs (miRNAs), lecithincholesterol acyltransferase (LCAT) and mammary globin have been studied as minimally invasive tools for CMC detection and monitoring. CEA overexpression is positively associated with tumour size, grade and lymph node status; it is also useful for the early detection of recurrence and metastasis in HBC (Stieber et al., 2015). An initial study determined CA 15–3, CEA and lactic dehydrogenase (LDH) serum concentrations in canines suffering different stages of CMC, without finding significant CEA-related differences between groups (Campos et al., 2012). More recent studies have proposed that the combined evaluation of CA 15–3, CEA and serum ferritin (SF) may improve non-invasive CMC detection sensitivity, thereby providing a reference value for clinical application (Yu et al., 2022). CA 15–3 is often overexpressed during the malignant transformation of many human adenocarcinomas, thereby increasing tumour cells’ metastatic and invasive potential (Chen et al., 2021). CA 15–3 positively correlates with HBC-related tumour size, lymph node status and disease stage; however, it is recommended that it be evaluated in conjunction with CEA (Shao et al., 2015; Stieber et al., 2015). One study has demonstrated a high correlation between CA 15–3 serum levels and CMC-related tumour histological grade, indicating that this biomarker’s serum values increase as tumour stage progresses (Manuali et al., 2012). More recent studies have proposed that combined CA 15–3 and CEA assessment may provide a non-invasive technique for non-invasive detection of CMC (Jain et al., 2021). Further studies are required before CEA and CA 15–3 can be considered suitable CMC-related biomarkers.

miRNAs are small RNA molecules whose function is gene silencing and translational repression by binding to target mRNA, and have been linked to tumour-related processes, including proliferation, differentia­ tion, invasion, angiogenesis, metastasis, apoptosis and drug resistance (H. Yang et al., 2023). Similarities between HBC and CMC regarding miRNA expression pattern have been described, along with several onco-microRNAs (oncomiRs), being highly conserved in canines and humans, and have been shown to act as oncogenes or tumour-suppressor genes in CMC (Jeong et al., 2019).

One study has shown that miR-29b and miR-21 were significantly upregulated in HBC and CMC samples (Boggs et al., 2008). Other studies have shown that various CMC-associated miRNAs expression profiles had a more significant difference between the metastatic and non-metastatic groups, suggesting a more significant role for microRNAs in relation to metastasis (Bulkowska et al., 2017). A more recent study reported that serum miR-19b could be a candidate biomarker for diag­ nosis and miR-18a for prognosis (Fish et al., 2020). miRNAs are particularly promising due to their stability in biological fluids and their involvement in multiple cancer-related processes, such as proliferation, metastasis and drug resistance. However, their clinical applicability in CMC requires further validation in large and standardised cohorts (Petrouˇskov´a et al., 2022).

LCAT is a potential serum and plasma biomarker which can be used as an indicator of invasive HBC. A proteomic analysis study of CMC found that LCAT was selectively expressed in mixed CMC samples; LCAT was also found to be an indicator of aggressive and advanced-stage CMC. O.H. Rodríguez-Bejarano et al.

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It has been suggested that LCAT could be an HBC and aggressive CMC biomarker (Park et al., 2020). Mammary globin is a protein from the secretoglobin (SCGB) family which has recently been recognised as a HBC-associated protein (Milosevic et al., 2023). An initial study has suggested that mammary globin-B is secreted into the blood and can only be detected in the serum of canines having CMC and not in that from other diseases (Pandey et al., 2015). A recent study found a sig­ nificant increase in mammary globin-B expression in CMCs from the complex carcinoma histological subtype compared to those from healthy mammary gland tissue, thereby positively correlating with its tran­ scription and suggesting that mammary globin-B overexpression is associated with CMCs (Pandey et al., 2018).

8.8. Cancer stem cells and circulating tumour cells

Cancer stem cells (CSC) are a small subpopulation of self-renewing tumour cells which are thought to be produced from malignant trans­ formation with critical mutations of normal stem cells, from mature somatic cell dedifferentiation, from pluripotent cancer cell induction and from EMT and TME cellular plasticity remodelling (Zhang et al., 2023). Canine mammary CSCs have a marker profile and express surface markers similar to CSCs in HBC (Rybicka and Kr´ol, 2016). One CMC study found that the CD44+/CD24+ CSC phenotype was associated with less aggressive and low-grade histological types, whilst the CD44+/CD24- phenotype was associated with a higher grade, suggest­ ing that the CD44/CD24 phenotype was useful for evaluating tumour behaviour as well as CMC-related CSC properties (Im et al., 2015). As there is no universal method for detecting CSCs, and that high and dy­ namic heterogeneity in CSCs has been described, this makes its use as a diagnostic tool still not so clear (Akrap et al., 2016). Circulating tumour cells (CTC) in peripheral blood has also been considered an important biomarker of micro-metastatic potential regarding HBC (Liang et al., 2020). A few studies have identified po­ tential CMC-related CTC markers such as AGR2, ATP8B1, CRYAB, F3 IRX3 and SLC1A; however, further studies are needed to understand the true usefulness of such markers. It has been proposed that CRYAB could be a good marker for detecting CTCs in metastatic CMC (da Costa et al.,

2013).

8.9. Translational impact of biomarkers on canine mammary carcinoma

It is worth noting that biomarkers should not be interpreted in isolation, but rather as integrated molecular network components. The interaction between proliferative signalling, angiogenesis, inflamma­ tion, EMT and genomic instability collectively influences tumour behaviour, metastatic potential and therapeutic response. This integra­ tive perspective underscores the need to move beyond approaches based on individual markers and adopt multi-parameter panels or multi-omic strategies better capturing tumour complexity. Despite the growing amount of candidate biomarkers, only a limited subset has been demonstrated to have consistent prognostic or therapeutic value regarding HBC. This highlights the urgent need for standardised meth­ odologies, species-specific reagents and prospective clinical studies for validating biomarker performance. Whilst CMC shares several molecu­ lar characteristics with HBC, significant biological and methodological differences limit its direct translational application. Future research should prioritise the development of canine-specific biomarker panels and molecular classification systems, integrating genomic, tran­ scriptomic and proteomic data to improve prognostic stratification and guide therapeutic decision-making in veterinary oncology.

9. Prognostic and predictive factors

Tumour size, histological subtype, histological grade, regional lymph node metastasis and proliferative index are treatment prognostic factors which can help predict canines’ clinical course and survival after undergoing initial surgery. Regarding tumour size, survival is shorter regarding tumours larger than 5 cm (T3), compared to stages T1 and T2 (Nunes et al., 2018). Multivariate analysis was used for validating his­ tological subtype as an independent prognostic indicator in a study prospectively evaluating 229 2-year-old canine patients having CMC; subtype-specific median survival time and local recurrence/distant metastasis rates were identified. Carcinomas arising from benign mixed tumours, complex carcinoma and simple tubular carcinoma were found to have an excellent prognosis, along with prolonged survival. Simple tubulopapillary carcinoma, intraductal papillary carcinoma and malig­ nant carcinoma and myoepithelioma had a 10-fold increased risk of tumour-related death. Similarly, the prognosis was significantly worse for adenosquamous carcinoma, comedocarcinoma and solid carcinoma. Anaplastic carcinoma and carcinosarcoma had the worst prognosis, having the highest metastasis rates. Tumour diameter was recognised in the same study as a strong predictor of local recurrence/distant metas­ tasis and an independent prognostic factor for survival in multivariate analysis. Lymphatic invasion and histological grade were predictive of local recurrence/distant metastasis and survival in univariate analysis (Rasotto et al., 2017b). The number of lymph nodes involved and extracapsular tumour extension are also considered a poor prognostic factor (Cassali et al., 2020). Predictive markers enable CMC-related targeted and more specific therapy. COX-2, Ki-67, ER and PR expres­ sion are amongst predictive factors evaluated by immunohistochem­ istry, whilst there is still not sufficient evidence to include E-cadherin and HER-2 expression (Cassali et al., 2020; Varallo et al., 2019).

10. Conclusions and future perspectives

CMC is one of the most frequently diagnosed spontaneous malignant neoplasias in unspayed canines and great similarities with HBC can be found from different perspectives. This aspect highlights the canine as a relevant biomodel for studying breast cancer and paves a way where a better understanding of this neoplasia could enhance the development of new therapies, diagnostic tools and potential biomarkers benefitting both species. In this overview, we characterise CMC based on various topics, such as epidemiological characteristics, carcinogenesis, meta­ bolic reprogramming, clinical approach, pathological aspects, thera­ peutic tools, biomarkers, and prognostic and predictive factors. One aspect worth highlighting, which poses a challenge in the study of CMC, is the epidemiological context, which remains undetermined in many countries and regions around the world. Very few studies are available, and those that do exist vary greatly in terms of the time pe­ riods covered, making any comparison difficult. Frequency rates could be considered unreliable due to the lack of representative sampling and reliable measures of the size of the canine population in the places where these studies were conducted. However, the attempt to characterise the canine population at risk of CMC cannot be dismissed, as it allows for the assessment of this important animal health problem and provides a more appropriate focus for prevention, timely diagnosis and adequate treat­ ment. It should be noted that such epidemiological gaps reflect broader methodological limitations concerning current CMC studies, including the use of small and heterogeneous cohorts, the lack of longitudinal follow-up, sample processing variability and the absence of standardised experimental and clinical protocols. Such limitations hinder studies’ reproducibility and comparability, highlighting the need for multi­ centre, prospective research involving harmonised designs and unified reporting criteria.

Once CMC has been diagnosed, surgical resection remains the treatment of choice, but there is still no consensus regarding the selec­ tion of the type and extent of surgery for each case. The use of both neoadjuvant and adjuvant chemotherapy with drugs such as doxoru­ bicin, carboplatin, cyclophosphamide and 5-fluorouracil, NSAIDs, mitoxantrone and docetaxel, with variable results and toxicities, does not yet have well-defined criteria for the selection and implementation of standardised protocols in CMC, therefore, it is necessary to adopt O.H. Rodríguez-Bejarano et al.

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consensus and conduct a greater number of randomised controlled clinical trials to establish these protocols. The possibility of developing and evaluating new drugs for CMC in in vitro studies, in vivo studies, and randomised controlled clinical trials should be considered. Such situa­ tion further highlights the need for randomised controlled trials for establishing evidence-based guidelines for CMC treatment. Factors such as canine life expectancy, sample collection, surgical procedures, diagnostic imaging, amongst others, are easier to perform in canines than in mice, so canines could be positioned as a better animal model for studying carcinogenesis and HBC progression. However, despite such advantages, the obvious limitations of a canine biomodel must be recognised, i.e. breed-related genetic variability, differences regarding life expectancy and tumour progression times compared to that for humans, the limited availability of standardised clinical datasets and ethical and logistical constraints on controlled trials. Such aspects must be carefully considered when extrapolating the results to human oncology; recognising these limitations is essential for interpreting re­ sults correctly and avoiding over-generalisation to human disease. Although efforts have been made to further investigate the molecu­ lar, histopathological, and clinical characteristics of CMC, there are still not enough clinical studies on CMC that use standardised protocols, specific markers for canines (e.g. antibodies or serum markers), and large, homogeneous cohorts of canines to adequately validate the results obtained to date. In this regard, the standardisation and validation of canine-specific antibodies and molecular tools remains a fundamental unmet need, as many studies rely on cross-reactive human reagents which may compromise specificity and reproducibility. The develop­ ment, validation and widespread adoption of canine-specific antibodies, genomic panels and serum biomarkers are essential for ensuring robust and reproducible CMC molecular characterisation.

One field that has been attracting interest in both CMC and HBC is biomarkers, but there is still no ideal biomarker for either diagnosis or prognosis. The most widely studied biomarkers have been measured in blood because they are easy to obtain, but they are not always specific to breast tumours and do not provide clear information. Changes in these serum biomarkers can provide information even before the appearance of clinical signs, which is why there is growing interest in their use for the early detection of this type of neoplasm. However, although several studies have been conducted on the clinical application of HBC bio­ markers in CMC, the conclusions have not been the most promising, given that most of these studies were conducted on cohorts with few canines and/or very heterogeneous groups, using different molecular techniques, which casts doubt on the reliability of the results. Future efforts should focus on integrating multi-omic biomarkers and largescale validation studies for identifying reliable diagnostic and prog­ nostic signatures.

Then, available information regarding canines supporting a description of CMC’s multiple epidemiological, molecular, pathological and clinical characteristics is still limited. Further study and research are thus needed concerning different aspects related to this significant neoplasia. Collaboration between interdisciplinary veterinary oncology and human oncology groups should enable advances to be made towards a more suitable understanding of CMC. CMC and HBC comparative studies will promote a deeper understanding of the similarities and differences between both species, thereby strengthening comparative oncology and its clinical applicability.

Ultimately, progress regarding CMC research will depend on the creation of shared databases and biobanks involving interdisciplinary human and veterinary oncologist teams. Comparative studies between CMC and HBC will be essential for elucidating conserved and speciesspecific tumourigenesis mechanisms which will improve the findings’ clinical applicability and advance the field of comparative oncology. Future research should focus on integrating advanced technologies from human oncology, such as next-generation sequencing, spatial and singlecell transcriptomics and artificial intelligence-based analytical ap­ proaches. Applying these approaches to CMC could significantly improve our understanding of tumour heterogeneity, microenviron­ mental interactions and therapeutic response in CMC, thereby enhancing its translational value in comparative oncology. CRediT authorship contribution statement Carlos Parra-Lopez: Writing – review & editing, Supervision, Funding acquisition, Conceptualization. Manuel Alfonso Patarroyo: Writing – review & editing, Writing – original draft, Supervision, Conceptualization. Botero Lucia: Writing – review & editing. Vargas- Hernandez Giovanni: Writing – review & editing. Rodriguez-Bejar­ ano Oscar Hernan: Writing – review & editing, Writing – original draft, Visualization, Formal analysis, Data curation, Conceptualization. Ethical statement Ethical approval was not required for this review. All authors confirm that this work adheres to Elsevier’s Publishing Ethics Policy. Declaration of Generative AI and AI-assisted technologies in the writing process During the preparation of this work the authors did not use any AI or AI-assisted technologies.

Funding statement This review was supported by a grant from the Ministerio de Ciencia, Tecnología e Innovaci´on (MinCiencias) of Colombia, Program 92191, Project 92268-Contract No. 800-2023.

Declaration of Competing Interest The authors declare the following financial interests/personal re­ lationships which may be considered as potential competing interests: Carlos Parra-Lopez reports financial support was provided by Colombia Ministry of Science Technology and Innovation. If there are other au­ thors, they declare that they have no known competing financial in­ terests or personal relationships that could have appeared to influence the work reported in this paper.

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