Solid malignant adenomyoepithelioma in the mammary gland of a male dog – first description

doi:10.21203/rs.3.rs-4907241/v1

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Case Report

Solid malignant adenomyoepithelioma in the mammary gland of a male dog – first description

https://doi.org/10.21203/rs.3.rs-4907241/v1

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Canine mammary tumors are well known and described in a high frequency in female dogs, and most of them are malignant. However, in male dogs, a very low rate, and benign occurrence seems to be more common. This is the first case report to describe the histologic and immunophenotypic features of a solid malignant adenomyoepithelioma in the mammary gland of a male dog. A sample was received from an intact male dog, Shih Tzu, 12-years-old, for histopathological evaluation. According to clinical history, the mass was located near the prepuce, non-adherent, evolution time unknown. Histologically, the mammary nodule consisted of a neoplastic proliferation of cells in a solid pattern, supported by a scant fibrovascular stroma. Cells had epithelioid form, moderated cytoplasm, lightly eosinophilic, and variably vacuolization. Nuclei are round to ovoid, with occasional karyomegaly and bizarre nuclei. Moderated pleomorphism; high mitotic count. Interspersed with the proliferation, a moderate amount of myxoid matrix production. Immunohistochemical analysis was performed and revealed neoplastic cells positive for p63 (90%), Pan-CK (10%), and PR (75%); HER-2 less than 10%; COX-2 and ER-negative. The results confirmed the diagnosis of solid malignant adenomyoepithelioma. Despite the low occurrence of MGT in male dogs, further studies with a high number of cases are needed. The role of age, breed, sexual status, and hormonal influences is unknown. Understanding the correlation between histological types, their behavior, and immunohistochemistry markers is crucial.

canine tumors

immunohistochemistry

gland neoplasia

Canine mammary tumors (CMTs) have been intensively studied by several areas of Veterinary Medicine over decades (Peña et al. 2014). It’s already known and well-described that these tumors have a high frequency in female dogs, and most of them are malignant (Oliveira Filho et al. 2010; Sleeckx et al. 2011; Cassali et al. 2014; Nunes et al. 2018). Nevertheless, when it comes to male dogs, the literature presents a different perspective.

The exact cause of mammary cancer in male dogs remains unclear (Gopal et al. 2022). The association between hormonal disorders, testicular and mammary tumors is controversial. Further research is needed to uncover the impact of obesity, diabetes, castration, and sex hormonal therapies in canine mammary tumors in male dogs (Bearss et al. 2012; Maiti et al. 2014; Han et al. 2016; Kwon et al. 2017; Gopal et al. 2022; Tkaczyk-Wlizło et al. 2024).

Studies have concluded that cases of CMT in male dogs are rare, compared to female dogs, (Saba et al. 2007; Bearss et al. 2012; Han et al. 2016; Kwon et al. 2017; Zuchi et al. 2018; Nunes et al. 2018; Silva et al. 2019; Zheng et al. 2022) with a ratio of 1:62 male vs. female (Saba et al. 2007). The frequency of malignant tumors in male dogs is markedly lower than in female dogs (Tkaczyk-Wlizło et al. 2024). However, aggressive tumor types such as carcinosarcoma, high-grade invasive carcinoma, inflammatory carcinoma, grade II tubulopapillary carcinoma, and grade II carcinoma in mixed tumor, have been described (Lather et al. 2017; Kwon et al. 2017; Silva et al. 2019; Machado et al. 2020; Gopal et al. 2022).

The present report is the first to describe the histologic and immunophenotypic features of a solid malignant adenomyoepithelioma (MAM) in the mammary gland of a male dog.

A sample was received from an intact male dog, Shih Tzu, 12-years-old, for histopathological evaluation. According to clinical history, the mass was located near the prepuce, non-adherent, evolution time unknown. The dog had undergone castration without histopathological analysis because the testicles had no macroscopic alteration.

Grossly, the sample consisted of a soft to firm mass measuring 9.0 x 2.0 x 1.0 cm, recovered by skin with ulceration focus measuring 2.0 x 1.0 cm and one nipple present. On the cut surface, a solid, homogenous, and pale-brown pattern was observed. Fragments were collected and submitted for routine histopathology. Lymph node absence.

Histologically, the mammary nodule consisted of a neoplastic proliferation of cells in a solid pattern, supported by a scant fibrovascular stroma (Fig. 1a,b). Cells had epithelioid form, moderated cytoplasm, lightly eosinophilic, and variably vacuolization. Nuclei are round to ovoid, dispersed chromatin, and there are multiple or single prominent and large nucleoli, occasional karyomegaly, and bizarre nuclei. Moderated pleomorphism (Fig. 1c). The total mitotic count was 40 mitoses in 10 high-power fields (i.e., 2.37mm² of area) (Fig. 1d). Foci of isolated metaplastic sebaceous cells (Fig. 1e). Interspersed with the proliferation, a moderate amount of amorphous and slightly basophilic material was observed, indicating myxoid matrix production (Fig. 1f). Also, multiple and extended areas of necrosis and hemorrhage were noted with moderated mononuclear inflammation. To characterize the neoplastic formation such areas were avoided. Based on histologic morphology, the mammary tumor was diagnosed as a solid pattern Tumor compatible with malignant adenomyoepithelioma.

The neoplasm was subjected to immunohistochemical evaluation for antibodies against p63, pan-cytokeratin (Pan-CK), cyclooxygenase-2 (COX-2), estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER-2) (Table 1) as previously proposed (Cassali et al. 2014; Nakagaki et al. 2022).

Table 1

Antibodies and immunohistochemical protocols applied
Antibody	Clone	Dilution	Antigen retrieval
p63	DAK-P63	1:100	Water bath heating, 95ºC, 20 min; citrate buffer pH 6.0
Pan-CK	AE1/AE3	1:500	Pressurized heat, 125ºC, 2 min; citrate buffer pH 6.0
COX-2	SP1	1:50	Pressurized heat, 125ºC, 2 min; citrate buffer pH 6.0
ER	EP-1	ready-to-use	Pressurized heat, 125ºC, 2 min; citrate buffer pH 6.0
PR	hPRa2	1:50	Pressurized heat, 125ºC, 2 min; citrate buffer pH 6.0
HER-2	polyclonal	1:200	Pressurized heat, 125ºC, 2 min; citrate buffer pH 6.0

Immunohistochemical analysis revealed neoplastic cells positive for p63 (90%), Pan-CK (10%), and PR (75%) (Fig. 2a-c); HER-2 less than 10% (Fig. 2d); COX-2 and ER-negative (Fig. 2e-f). The results confirmed the diagnosis of malignant adenomyoepithelioma, according to recent literature features (Nakagaki et al. 2022).

As a follow-up, we contacted the Hospital, and the dog was euthanized a month after surgery. He presented multiple episodes of seizures, walking in circles, and salivation. Due to the poor health condition, the tutor has chosen the euthanasia. No neurological exams or necropsy were authorized or performed.

Mammary tumors must be recognized as potential differentials for ventral abdominal nodules in male dogs, affecting mainly caudal abdominal and inguinal mammary glands and around prepuce (Han et al. 2016; Silva et al. 2019). Analyzing individual animal characteristics is essential to identify factors related to mammary tumor development, including age, breed, hormonal exposure, and sexual status (Tkaczyk-Wlizło et al. 2024).

The present patient was sexually intact at the moment of mastectomy and had no other known health issues. Although no macroscopic alterations were found in the testicles, the possibility of a microscopic neoplasm cannot be entirely dismissed. Similar conditions have been reported in past studies (Bearss et al. 2012; Maiti et al. 2014; Han et al. 2016; Gopal et al. 2022). However, in other cases testicular neoplastic such as Leydig Cell Adenoma (Kwon et al. 2017), a diffuse seminoma and a solid interstitial cell tumor (Zuchi et al. 2018), Seminoma, Leyding cell tumor and sertolioma (Machado et al. 2020) have been also reported. During a literature analysis, Tkaczyk-Wlizło et al. (2024) observed the absence of testis tumors in 48 of 74 male dogs. Just in seven cases (12.7%), testicular neoplastic was diagnosed concurrently with mammary gland tumors (MGTs), or the disease was diagnosed in the past or after MGTs.

Metastasis to regional lymph node and lungs was reported in cases of triple-negative tumors, confirming the aggressiveness of such tumor type (Gopal et al., 2022; Machado et al., 2020). In this case, as the dog had neurological symptoms and no further exams were performed, we cannot completely rule out the occurrence of metastasis to the central nervous system.

Large-scale studies are needed to determine the impact of sex hormones on mammary tumor development in male dogs. (Gopal et al. 2022). As well as the importance of other factors such as age and breed (Tkaczyk-Wlizło et al. 2024), obesity, diabetes, and sex hormone therapy (Bearss et al. 2012). Limited data on male MGTs makes it difficult to assess the potential protective effect of castration or the impact of excess weight. (Tkaczyk-Wlizło et al. 2024).

When it comes to immunohistochemical analysis, the present case revealed neoplastic cells diffusely positive for p63 (90%) and multiple foci of Pan-CK (10%). In 2022, Nakagaki et al. subclassified tumors described as solid carcinoma in the mammary gland of female dogs into six subtypes based on morphological and immunohistochemical findings. One of them, and also the most frequent (50,4%; 68/135) was the malignant adenomyoepithelioma. The criteria used by the authors to define this subtype was 10–90% of neoplastic cells immunopositive to p63 and cells with moderately sized vacuolated cytoplasm. The present case fulfills these criteria. This tumor type has also been described in female cats (Nunes et al. 2021).

In our case, 75% of neoplastic cells were positive for the progesterone receptor, while the estrogen receptor was negative. Previous studies show considerable variation in hormonal expression (Mamom et al. 2012; Machado et al. 2020; Gopal et al. 2022; Tkaczyk-Wlizło et al. 2024).

A study with eight male dogs had strong and diffuse ER expression within one malignant and seven benign tumors. All dogs revealed PR expression, less intense and less diffuse than for ER but still present (Saba et al. 2007). The ER was negative in one malignant case (Lather et al. 2017), ER and PR negative in one malignant case (Kwon et al. 2017) and three triple-negative malignant cases also occurred (Machado et al. 2020; Gopal et al. 2022). Similar to our findings, a study described two cases with intense PR expression and ER-negative (Mamom et al. 2012). However, these two particular cases were diagnosed as benign tumors, simple adenoma and fibroadenoma.

The HER-2 expression, in our case, was low, less than 10% in neoplastic cells. This result corroborates with previous findings of low e/or negative HER-2 expression (Mamom et al. 2012; Machado et al. 2020; Gopal et al. 2022). In humans, the overexpression of this marker is associated with aggressive tumors. In female dogs, the HER2 (over)expression remains unclear (Peña et al. 2014). In male dogs, no robust data are available.

In female dogs, COX-2 overexpression in mammary glands is well documented and linked to a poor prognosis and shorter overall survival (Lavalle et al. 2009). Albeit this case is about a male dog, COX-2 expression was negative. This suggests that it might be a less aggressive tumor but Cox-2 inhibitors could not be an alternative for treatment (Lavalle et al. 2009).

To the authors' knowledge, this is the first morphological and immunophenotypic description of a solid malignant adenomyoepithelioma in the mammary gland of a male dog. Despite the low occurrence of MGT in male dogs, further studies with a high number of cases are needed to understand this complex neoplasm. The role of age, breed, sexual status, and hormonal influences is unknown. Understanding the correlation between histological types, their behavior, and immunohistochemistry markers is crucial.

Competing interests - The authors declare no competing interests.

Author Contributions Statement - All authors contributed to the study's conception. Material preparation, data collection and analysis were performed by EAV, JGW, MMN, MLMSR, GDC and KYRN. The first draft of the manuscript was written by EAV and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Funding - This work was financially supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa de Minas Gerais –FAPEMIG [Rede Mineira de Pesquisa Translacional em Imunobiológicos e Biofármacos no Câncer (REMITRIBIC, RED-00031-21)] and Coordenação de Aperfeiçoamento Pessoal de Nível Superior (CAPES), Brazil.

Data availability - No datasets were generated or analyzed during the current study.

Ethical approval - Not applicable.

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Solid malignant adenomyoepithelioma in the mammary gland of a male dog – first description

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