Mammary gland tumors are seen in female dogs of all ages and often lead to death due to their extreme aggression and high recurrence rate. Therefore, diagnosis is important for the prevention of progression and for CMT treatment. In this study, dogs with CMTs presented to the veterinarian with one or more nodules within the mammary gland. When tumors were present, the dogs showed non-specific symptoms such as fatigue, lethargy, weight loss, dyspnea, cough, lymphedema or lameness. The results of our study were in agreement with the results of a previous study [
22]. Routine blood analysis revealed that monocyte and lymphocyte levels in dogs with CMTs were lower than in healthy dogs, but leukocyte levels were higher in dogs with CMTs. This observation suggests that the reduction in monocyte and lymphocyte levels and the increase in leukocytes may be related to the occurrence or development of CMTs. In blood biochemical examination, ALP and TBIL in dogs with CMTs were higher than in healthy dogs, and this difference may be caused by the tumors. Moreover, ALP was associated with prognosis, which further provides a basis for subsequent diagnosis and treatment. [
23]. As the tumor grows, the level of CRP continues to rise; this is in line with previous studies [
24]. This “acute-phase response”, in which CRP rapidly changes in inflammation and tissue injury in response to a variety of stimuli, was observed with the progression of some malignancies and in the activity of various diseases [
25]. Changes in these indicators indicate multiple direct or paraneoplastic changes associated with tumors that can be detected/suspected by hematology examination. To our knowledge, these changes have not been mentioned in previous studies. Regarding serological molecular biological detection, the feasibility of using human antigen kits to determine HER-2 serum concentrations in dogs was demonstrated. The HER-2 serum concentrations were significantly different between healthy dogs and dogs with malignant tumors. However, these results are in disagreement with the results of a previous study [
26]. ELISA for serum HER-2 is a dynamic test, however there is randomness in clinical samples that can lead to variation in the results. Previous studies have shown that HER-2 over-expression was detected in CMTs. Several studies have attempted to identify a similar prognostic role of HER-2 expression in canine mammary carcinomas. In previous investigations, HER-2 expression was found to be associated with some well-known morphological indicators of poor prognosis [
11,
27,
28]. These were also demonstrated in our experiment. Moreover, ELISA for serum HER-2 can be performed at any time and can be used when primary tumor samples are unavailable, eliminating the need for a biopsy [
29]. In this study, mRNA expression of HER-2, E-cadherin, N-cadherin, Vimentin, CEA, CA15-3 and SF was measured in CMT tissues by qPCR. The expression of these tumor markers (except for E-cadherin) in the malignant tumor group was significantly higher than that in the benign group and the healthy control group (
p < 0.05), however, there was no significant difference between the benign mammary tumor disease group and the healthy control (
p > 0.05). The mRNA expression of E-cadherin in tumor tissues was significantly lower than that in tumor-adjacent healthy tissues. This result was in line with some in-vitro studies, and a previous study demonstrated reduced expression of E-cadherin in malignant CMTs compared with normal mammary glands, suggesting that down-regulation of E-cadherin was a common event in canine mammary tumors [
30]. Deregulation of E-cadherin is related to the infiltrative and metastatic ability of the tumor, due to of disruption of the E-cadherin, with consequent loss of cell adhesion and a concomitant increase in cell motility. Moreover, a reduction in E-cadherin expression was significantly associated with infiltrative growth and vessel invasion [
31]. In conclusion, there is a significant relationship between loss of E-cadherin expression and other known factors for poor prognosis in CMTs, such as tumor size, ulceration, histological type, type of growth, lymph node metastasis and necrosis [
31,
32,
33]. This indicates that a loss of E-cadherin expression may have prognostic value in malignant CMTs. The expression level of E-cadherin in tumor tissue is correlated with disease progression, and it can be used as a diagnostic marker for mammary tumors [
34]. Further evidence should be sought in future studies in which the clinical course of the disease is carefully monitored.
From a clinical point of view, imaging was used to further assess for the presence of tumors that are not visible to the naked eye, and to provide complete imaging information [
22]. Pathology examinations are mainly used to diagnose mammary tumors. The evaluation of the morphological appearance and malignant growth behavior of the tumor help to identify the nature of the tumor (benign or malignant) [
4]. However, pathological examinations have limited predictive value for the prognostic status of patients, and X-ray examinations also have limitations. For example, overlapping images cannot always be distinguished. Hematological examinations cannot directly determine the presence of tumors, but they can be used as auxiliary tests to guide further diagnosis. Therefore, we discussed the importance of serological tests and molecular biological tests in the diagnosis of CMT. To date, the most studied and reliable biomarkers of CMT are HER-2, E-cadherin, N-cadherin, Vimentin, CEA, CA15-3 and SF, which can be detected in both serum and tissue samples using different molecular methods. Notably, these biomarkers can be used for the early detection and prognosis of CMT. Early detection, diagnosis and treatment can improve survival and quality of life in both humans and dogs, which is significant for mammary tumor research [
35,
36]. In both human and veterinary medicine, many studies on the biomarkers of mammary gland tumors have been conducted. In recent years, the use of serological molecular biological detection and molecular biological detection to assess the status and prognosis of tumor patients has shown several prospective clinical applications [
4], and these methods are important in assessing whether a mammary tumor will recur after surgery [
37]. In addition, these methods can be used to monitor tumors through changes in blood composition and to detect the presence of tumors much earlier than conventional diagnostic methods. Serological tests and molecular biological tests have advantages over histopathology, as the procedure of detection is non-invasive, and they can reveal dynamic changes of physiological and pathological states before clinical signs appear. Furthermore, serological and molecular biological testing is more cost-effective than traditional diagnostic methods [
38,
39]. It is not only suitable for early diagnosis of CMTs, but also for occasional physical examination of healthy dogs. However, the early detection of CMTs, the causative factors and the clinical evaluation of tumors will continue to be a challenge. For this reason, the early detection of CMTs is crucial for the dog’s clinical outcomes. Unfortunately, serological canine tumor biomarkers are rarely applied in veterinary clinics. To obtain more reliable results, we always recommend the evaluation of more than one biomarker [
40]. Measurement of biomarkers in dogs by serological and molecular biological assays is a milestone in the early diagnosis of tumors and assessment of disease progression.