The present study investigated the expression of miR-21, alterations in CA 15 − 3, and CEA as serum mammary tumor markers and utilization of immunohistochemistry in the diagnosis of CMT. Elevated levels of thrombocytes, although within the normal reference range, was the most common abnormality recorded via hematology.
CMT is frequently diagnosed, especially in female dogs [19]. In the present study, intact female dogs, with a median age of 9 years, were affected. Hormonal factors such as estrogen could be implicated [20]. The onset of CMT is believed to be influenced by age, hormones, and genetic predisposition and is rarely seen in young dogs (< 3 years of age) [21].
The hormonal effect was also studied in human breast cancer pathology [22]. An incidence rate of 73% was previously reported in intact female dogs with an approximate age range of 8–13 years [23], and another study reported a mean age of 9.5 years [24]. In the present study, the German Shepherd was the most affected breed. Cocker Spaniels and German Shepherds were postulated to be at higher risk compared with other breeds [25]; however, Labrador Retrievers and Poodles were overrepresented in another study [20].
CMT diagnosis is relatively feasible using clinical data, which is collected and analyzed, and a combination of visual and palpation provides the practitioner with the necessary information to narrow differential diagnosis lists [26]. In the present study, two females showed multiple masses, and tumor size was taken as the size of the largest tumor, as described previously [27]. Radiological examination was conducted to confirm the absence of distant metastasis. It is recommended to conduct the left and right lateral view and ventrodorsal view to confirm or rule out the presence of metastasis [26].
Histological examination showed malignant mammary gland tumors that were either carcinoma or sarcoma. The most frequently recorded CMT was mixed-type, which is in line with the findings from previous studies [28, 29]. Tumor type according to histological classification may be correlated with grade and prognosis [30]. Further immunohistochemical studies are useful to identify the origin of neoplastic cells [31]. Keratin immunoreactivity was limited to epithelial cells. Myofibroblasts express both α-actin and vimentin, whereas connective tissue cells express vimentin. Myoepithelial cells are a major component of carcinomas, which usually express CK14, CK5, SMA, calponin, and p63 [32]. However, proliferating myoepithelial cells show a decreased expression of these markers with an increased expression of vimentin, and eventually become a fibroblast-like cell expressing only vimentin [10]. Identification of the type of neoplastic cells involved in the tumor was suggested to be important for tumor prognosis. Tumors with or without myoepithelial cells should be identified since it was proposed that myoepithelial cells presence in malignant tumors exhibit an inhibitory effect on neoplastic cells with subsequent favorable prognosis [28, 33]. In the present study, it was difficult to diagnose myoepithelial cells using hematoxylin and eosin-stained tissue sections; therefore, they were identified by P63 immunoreactivity but were negative for SMA. P63 is more specific for myoepithelial cells and shows no cross-reactivity with stromal myofibroblasts compared with SMA and calponin [10].
Hematological alterations showed no significant reduction in erythrogram and MCV, and MCHC were within the normal range. PCV and Hb were reported to be reduced, along with nearly normal values for other hematological parameters in CMTs [7, 34, 35]. Thrombocytes were elevated, but were within the normal reference range. Thrombocytosis and leukopenia were previously reported to be the most prominent change in malignant mammary tumors [36]. However, leukocytosis and anemia were associated with chronicity and extent of advanced stage [37]. Thrombocyte elevation was associated with a systematic inflammatory response [38]. Immune-mediated involvements and non-treated solid neoplasm were postulated as a cause [36].
The present study showed that CA 15 − 3 levels were elevated in association with CMT. Elevation of CA 15 − 3 is correlated with disease stage, neoplastic cells activities, and its associated products [2, 12]. The most widely used serum markers in breast cancer are CA 15 − 3 and CEA. They are relatively inexpensive and require a less invasive method of sample collection compared with immunohistochemical tests, which have been used in veterinary medicine [12]. In human medicine, CA 15 − 3 is considered a specific diagnostic and monitoring tool for mammary tumors [39]. It is mainly a monitoring tool for women breast cancer [40, 41], and is elevated in around 70% of advanced cases. Benign breast tumors also show elevated CA15-3 levels [42]. Higher levels of CA 15 − 3 are positively correlated with tumor stage; for instance, stage III is associated with higher serum concentration compared with stage I, and this high concentration may be associated with poor outcomes [2, 43]. The present study did not detect changes in CEA between the control and disease groups, in line with a previous study [12]. However, other studies showed that a change is expected [27, 44]. Liver metabolism, kidney clearance rate, and method of assay could be implicated [12, 45]. Moreover, the lower positivity rate of CEA compared with CA 15 − 3 in breast cancer makes it a controversial marker for the diagnosis of this type of tumor [46]. Previous studies [47, 48] reported that CEA lacks predictive ability in metastatic breast cancer, although other studies correlated it with poor prognosis [49, 50]. Furthermore, a strong relationship between elevated serum CA 15 − 3 and advanced CMT has been observed, indicating CA 15 − 3 has better diagnostic value than CEA [12].
The present study found that miRNA-21 expression was upregulated 12.84-fold in female dogs with CMT, which is in agreement with the findings of a previous study [51] that reported increased miRNA-21 expression in mammary gland neoplasms and a fivefold increase in carcinoma samples. Furthermore, miR-21 was significantly upregulated in CMTs [52]. The use of microRNAs as indicators is becoming more common in human medicine as they are stable, noninvasive, easy to assess markers [53]. miR-21, miR-155, and miR-10b are established as oncogenic and metastasis-promoting miRNAs in breast cancer [54, 55]. The oncogenes, miR-21 and miR-29b, are associated with several cancers, including human mammary cancer, and have consistently found to be upregulated in mammary cancers using microarray analysis [56–59]. Moreover, miR-21 showed an increase in copy number in human mammary tumor tissues [60]. miR-21 targets both PTEN and TPM1, and loss of this miRNA results in increased caspase activity and subsequent apoptosis [59, 61]. Comparisons between normal mammary gland and neoplastic tissues showed that miR-21, miR-143, miR-194, and miR-203 were significantly increased and therefore matched the definition of an oncogenic miRNA [62]. miR-21 is a substantial biomarker for the diagnosis of breast cancer in human patients [63]. Despite the broad use of miR-21and its misregulation in association with different carcinomas, it remains a promising marker for the prognosis, diagnosis, and prediction of cancer [53].
The tumors presented in the present study showed positive vimentin immunoreactivity, either partially or completely, with high expression of miR-21, indicating a mesenchymal origin of the tumor cells. Similarly, in a previous study on esophageal squamous cell carcinoma, miR-21 expression was predominantly present in fibroblasts associated with cancer [64]. Therefore, elevation of miR-21 may be directly attributed to mesenchymal cells expressing vimentin.
Limitations of this study, small number enrolled while strains of this study comparison between miR-21 and serum tumor marker. The attribution between high expression of miR-21 and mesenchymal origin of tumor cell required further investigation.