This study allowed us to comprehensively explore the histological variations of sarcomas and carcinosarcomas, as well as possible relations between these variations, clinical presentations, and morphological criteria of aggressiveness.
Few reports have addressed the frequency of mammary sarcomas in female dogs. In the female dogs we assessed, the frequency of mammary neoplasms with malignant mesenchymal components was similar to data from a previous report (13%) (Misdorp et al. 1971). However, in a recent survey with a fuller description, other authors reported that neoplasms with malignant mesenchymal components accounted for 5% of the total number of tumours (Nunes et al. 2018).
As previously published (Nunes et al. 2018), carcinosarcomas were the most frequent type of malignant mesenchymal neoplasm. The low frequency of pure sarcomas and sarcomas in mixed tumors corroborated the understanding that these histologic types are extremely rare in the mammary glands of female dogs (Nunes et al. 2018).
Among the assessed cases, the prevalence of carcinosarcomas and sarcomas larger than 5.0 cm is consistent with the unfavourable prognoses that are usually attributed to these types of tumours. Since survival rates are lower among female dogs with larger-diameter tumours, tumour size is an important prognostic factor for female dogs with mammary neoplasms (Ferreira et al. 2009). Besides this, large tumours have been associated with other prognostic factors, such as the histologic subtype, losses in hormone receptor expression, and the proliferative index (Langenbach et al. 1998; Nieto et al. 2000; Sarli et al. 2002; Ferreira et al. 2009).
The first challenge in performing a histologic diagnosis of mammary neoplasms with mesenchymal components is determining the benign or malignant character of the mesenchymal proliferation. Carcinosarcomas must be histologically distinguished from carcinomas in mixed tumors that are characterized by carcinomatous and benign mesenchymal components. To do so, the key is to identify malignant characteristics in the mesenchymal cells, such as the level of differentiation in the matrix, high cellularity, elevated nuclear and cellular pleomorphism, karyomegaly, irregular nuclear membrane, prominent nucleoli, and multinucleation (Cassali et al. 2017; Goldschmidt et al. 2017). The same aspects should be considered when distinguishing between sarcomas and benign mesenchymal tumours.
For all the cases in our study, we performed an immunohistochemical analysis of the expression of intermediate vimentin and cytokeratin AE1/AE3 filaments. This made it possible to discriminate between epithelial and mesenchymal proliferation. All of the mesenchymal components of the sarcomas and carcinosarcomas presented vimentin markers (cell markers of mesenchymal origin), and all the epithelial components of the carcinosarcomas presented cytokeratin AE1/AE3 markers (cellular markers of epithelial origin) (Boos et al. 2011; Dolka et al. 2013). Besides this, in some cases, the use of immunohistochemistry made it possible to detect small foci of sarcomatous and carcinomatous proliferation in lymph nodes. These were not detected with H&E staining.
The correct classification of sarcomas by the type of proliferation is another challenge in morphological diagnosis. This is due to the varying nature of the matrix in the same tumour. Different combinations of mesenchymal proliferation (osteosarcomatous, chondrosarcomatous, fibrosarcomatous, and liposarcomatous) and epithelial proliferation were observed. Similarly, this has previously been described in human (Yakan et al. 2014) and canine mammary glands (Cassali et al. 2017). The histologic variety of the mesenchymal components of neoplasms has been attributed to myoepithelial cell transformations (Cassali et al. 2012) epithelial-mesenchymal transition (EMT) (Kokkinos et al. 2007), or totipotent cells with a high differentiation capacity (Hellmen and Lindgren 1989).
We classified all tumours that presented a malignant osteoid matrix as osteosarcomas because, among the sarcomas found in different anatomical locations, this type exhibits the most aggressive type of proliferation (Goldschmidt et al. 2017).
Although the most frequent mesenchymal subtype in our study was osteosarcoma, another report on sarcomas in the mammary glands of female dogs demonstrated a higher frequency of fibrosarcomas (Dolka et al. 2013). On the other hand, another investigation revealed that, among extraskeletal osteosarcomas in dogs, the mammary gland is the most frequent location (Kokkinos et al. 2007). There were no pure chondrosarcomas in our findings. However, this histologic type has already been found in the mammary glands of female dogs (Serin and Aydogan 2009; Tavasoly et al. 2013).
Whether they are located in the mammary glands or other anatomical locations, osteosarcomas can be classified into different types, due to the heterogeneity of their cellular populations and diverse extracellular matrix formation. At times, the same neoplasm can simultaneously present different types and this can make them difficult to classify. Moreover, it corroborates the highly pleomorphic behaviour of this component. Any correlations between histologic types and prognoses are still uncertain, but skeletal fibroblastic osteosarcomas have been associated with better prognoses while the opposite has been reported for the telangiectatic type (Thompson and Dittmer 2017). Other researchers have found no correlation between the histologic subtypes of skeletal osteosarcomas and histologic grades (Nagamine et al. 2015). Similarly, we found no correlation between histologic subtypes and other clinical or pathological variables. However, other studies with larger numbers can give answers about the importance of this correlation.
Our findings concerning the highest frequency of the mesenchymal lymph node metastasis corroborate those of previous studies that related mammary gland sarcomas in female dogs with greater metastatic potential and a worse prognosis (Hellmen et al. 1993). However, the similarity between the frequency of lymph node metastases in sarcomas and carcinosarcomas (30% and 21,5% respectively), substantiated the malignancy of both histologic types (Goldschmidt et al. 2017). The prevalence of mesenchymal lymph node metastases with osteosarcoma components confirmed its malignancy and capacity to disseminate along the lymphatic pathway (Hellmen 2014) and reinforces the importance of assessing regional lymph nodes.
Our finding of a low frequency of cases with radiographic evidence of distant metastasis was different from other results in the literature. These studies examining the mammary glands of humans and female dogs related mammary gland sarcomas to the frequent occurrence of metastasis to other organs (Benjamin and Lee 1999; Yakan et al. 2014). The lack of radiographic exam results weakens knowledge regarding the behaviour of the tumours, from the time of the first diagnosis onwards, and this divergence was probably due to issues of access to the results of pre-surgical radiographic exams, as well as the ones carried out during the clinical history.
Determining the histologic grade is important for issuing a prognosis and deciding on a therapeutic plan for invasive mammary carcinomas (Karayannoupoulou et al. 2005). However, there is no established system for grading the mesenchymal components of mammary sarcomas in female dogs.
A previous study used a grading system to assess mammary sarcomas in female dogs, and most of the cases demonstrated a high histologic grade (Dolka et al. 2013). By using a grading system, the authors also observed that sarcomas with a higher histologic grade were associated with more proliferative activity (Ki67 index). This suggests there may be implications regarding the grade and prognosis of these neoplasms. We used the same grading system and also observed a prevalence of high-grade sarcoma components among the sarcomas and carcinosarcomas we assessed. However, further prognostic studies with larger sample sizes are needed to validate this grading system.
The interest in evaluating COX-2 expression in canine malignant mesenchymal tumours stems from research findings regarding the association between COX-2 overexpression (6-12) and lymph node metastasis by the time of surgery, the development of distant metastasis, and the decline in both overall survival and disease-free survival times (Queiroga et al. 2010; Lavalle et al. 2012; Carvalho et al. 2016). Besides this, the enzyme is a therapeutic target for non-steroidal anti-inflammatory drugs that have already been included in therapy protocols for the treatment of other histological types of mammary neoplasms in female dogs (Souza et al. 2009; Lavalle et al. 2012).
There are few published reports regarding COX-2 expression in the mesenchymal components of the mammary glands in female dogs. One study found intense expression in mammary fibrosarcoma (Arenas et al. 2016). Another investigation discovered significant COX-2 expression in appendicular osteosarcomas in dogs (Millanta et al. 2012).
The percentage of carcinosarcomas with a high COX-2 score was lower than the number previously described by Queiroga et al. (2010) and Carvalho et al. (2016). However, it was greater than the percentage reported by Lavalle et al. (2012). Probable justifications for these differences in results include the use of dissimilar antibodies, inherent distinctions in components and antibodies, or inherent variations in immunohistochemical exams.
Recently, COX-2 overexpression was associated with more aggressive histological types, such as carcinosarcomas (Pastor et al. 2020). This overexpression in carcinosarcomas and other mammary carcinomas was also linked to immunohistochemical markers for angiogenesis, proliferation and inflammation, shorter disease-free survival, and shorter global survival (Queiroga et al. 2010; Carvalho et al. 2016).
In conclusion, sarcomas and carcinosarcomas were found to be clinically similar and developed as large tumours, mainly in the abdominal and inguinal mammary glands, with frequent intratumoral necrosis but a low frequency of nodal metastasis. Sarcomatous histological patterns varied and, when compared with the other types of mesenchymal proliferation, the osteosarcomatous subtype was the only one that resulted in vascular invasion, metastasis in regional lymph nodes, and a higher histologic grade. Besides this, the findings of carcinosarcomas and sarcomas with high COX-2 expression suggest that, in some cases, these types of neoplasms may respond to therapy with COX-2 inhibitors. However, the usefulness of COX-2 inhibitors in treating these neoplasms remains to be further investigated.