Malignant Mammary Tumors Research Articles

Mammary carcinoma in a male cat following long-term medroxyprogesterone acetate treatment: case report and review of the literature.

In male cats, as in men, mammary carcinomas are rarely reported. However, like in females, hormonal therapy is a significant risk factor. This study reports the case of an 11-year-old male cat with multiple mammary tumours and a history of long-term medroxyprogesterone acetate therapy for the suppression of sexual behaviour, along with a brief review of the literature. Complete surgical removal of the right mammary chain and the ipsilateral inguinal lymph nodes was performed, and all tissues were submitted for histology. Histological examination revealed the presence of a tumour in the third and fourth mammary glands, consisting of neoplastic cells arranged in various structures, including tubulopapillary and tubular structures, sometimes cystically dilated, and solid areas. The inguinal lymph nodes were also involved. The morphology was consistent with a diagnosis of mammary carcinoma, tubulopapillary type, with nodal metastases. Immunohistochemistry revealed that tumour cells were positive for cytokeratin (clones AE1/AE3), while stromal cells were positive for vimentin (clone V9). The proliferation marker Ki-67, evaluated on both the primary tumour and the nodal metastases, was strongly expressed in the nuclei of neoplastic cells, with a Ki-67 proliferation index of 8.9% and 20% for the primary tumour and the metastases, respectively. This case highlights the importance of considering the possibility of malignant mammary tumours not only in female but also in male cats with a history of long-term hormonal treatment for suppression of sexual behaviour.

TGFβ in malignant canine mammary tumors: relation with angiogenesis, immunologic markers and prognostic role

Transforming growth factor-β (TGFβ) and FoxP3 regulatory T cells (Treg) are involved in human breast carcinogenesis. This topic is not well documented in canine mammary tumors (CMT). In this work, the tumoral TGFβ expression was assessed by immunohistochemistry in 67 malignant CMT and its correlation to previously determined FoxP3, VEGF, and CD31 markers and other clinicopathologic parameters was evaluated. The high levels of TGFβ were statistically significantly associated with skin ulceration, tumor necrosis, high histological grade of malignancy (HGM), presence of neoplastic intravascular emboli and presence of lymph node metastases. The observed levels of TGFβ were positively correlated with intratumoral FoxP3 (strong correlation), VEGF (weak correlation) and CD31 (moderate correlation). Tumors that presented a concurrent high expression of TGFβ/FoxP3, TGFβ/VEGF, and TGFβ/CD31 markers were statistically significantly associated with parameters of tumor malignancy (high HGM, presence of vascular emboli and nodal metastasis). Additionally, shorter overall survival (OS) time was statistically significantly associated with tumors with an abundant TGFβ expression and with concurrent high expression of TGFβ/FoxP3, TGFβ/VEGF, and TGFβ/CD31. The presence of lymph node metastasis increased 11 times the risk of disease-related death, arising as an independent predictor of poor prognosis in the multivariable analysis. In conclusion, TGFβ and Treg cells seem involved in tumor progression emerging as potential therapeutic targets for future immunotherapy studies.

Investigation of p27 tumor suppressor gene (CDKN1B) polymorphisms in dogs with malignant mammary tumors

Investigation of p27 tumor suppressor gene (CDKN1B) polymorphisms in dogs with malignant mammary tumors

Vitamin D status in female dogs with mammary gland tumors.

Little information exists about vitamin D status in bitches with mammary tumors. To determine whether low plasma vitamin D concentrations are found in bitches with mammary tumors. Eighty-five client-owned bitches with mammary tumors (n = 21 benign, n = 64 malignant) and 39 age-matched healthy bitches. Case-control study. Plasma ionized and total calcium, phosphorus, magnesium, urea, creatinine, albumin, total proteins, alanine aminotransferase, alkaline phosphatase, parathyroid hormone (PTH), calcitriol (1,25-dihydroxyvitamin D), and 25-hydroxyvitamin D concentrations were measured in all bitches at the time of clinical diagnosis and before any treatments. Statistical analysis was performed to compare variables among groups (control, benign, and malignant). No significant differences were found when plasma 25-hydroxyvitamin D concentrations in bitches with malignant (148.9 [59.9] ng/mL) and benign mammary tumors (150.1 [122.3] ng/mL) were compared with control group (129.9 [54.5] ng/mL). Parathyroid hormone was significantly higher in bitches with malignant (19.9 [20.5] pg/mL), and benign mammary tumors (14.6 [14.9] pg/mL) compared with control group (7.5 [7.5] pg/mL; P < .01). Only the presence of mammary tumors (P < .01) and age (P = .04; adjusted R2 = .22) was significant in predicting PTH. Bitches with mammary tumors do not have low 25-hydroxyvitamin D concentrations thus vitamin D supplementation is unlikely to be useful for prevention of mammary tumors in bitches.

The diagnostic value of selected immune parameters in peripheral blood of dogs with malignant mammary tumours - a preliminary study.

The main adaptive immune cells are T and B lymphocytes and they play key roles in the induction of immune responses against canine mammary tumours. Investigating these cell subpopulations may lead to more precise diagnosis of these malignancies. The percentages of CD3+, CD4+ and CD8+ T cells and of CD21+ B cells in the peripheral blood of bitches with malignant mammary tumours were compared with those in the blood of healthy animals. The phenotypic features of peripheral blood leukocytes were evaluated by flow cytometry. There was a significant difference in the mean percentages of CD3+ lymphocytes between healthy (66.7%) and metastatic dogs (46.1%), and between tumour-bearing non-metastatic (66.6%) and metastatic dogs. There was also a significant difference in CD4+ T helper cell percentages between healthy dogs (40.4%) and dogs with metastases (23.2%), and between the latter and dogs without them (35.5%). In the case of CD21+ lymphocyte subsets, a significant difference was noted between healthy animals (10.9%) and those with metastases (20.1%), and between the latter and patients without metastases (8.5%). There were also significant differences in CD3+/CD21+ ratios between the group with metastases (3.0), the healthy group (7.8), and the group without metastases (8.5). Similarly, a significant difference was noted in CD4+/CD8+ ratios between animals with metastases (1.4), bitches in the control group (2.2), and dogs without metastases (1.9). Peripheral blood leukocyte phenotypic characteristics are putative novel biomarkers. These findings may be useful in future studies improving mammary tumour diagnostic procedures, especially in metastasis detection.

Evaluation and Comparison of Different Surgico-chemotherapeutic Regimens for the Treatment of Malignant Canine Mammary Tumours

Background: Cancer has been regarded as the most dreaded disease for both human and animals because of its incurability. Canine mammary tumours are common and major problem encountered by academicians and practicing veterinarians. As a result, the current study was conducted to examine the effectiveness and overall impact of different surgico-chemotherapeutic approaches in treating malignant mammary tumors in dogs. Methods: The investigation involved 18 clinical instances of mammary tumors in different breeds, regardless of age or sex and were divided into three groups of six animals each. In Group S, only surgical excision of tumour was performed while animals of Group SD and Group SV were treated with surgical excision followed by administration of Doxorubicin (30 mg/m2) BSA and Vincristine sulphate (0.025 mg/kg) intravenously alongwith DNS at 7th and 14th post-operative days respectively. Different physiological and haemato-bichemical parameters i.e. Hb, PCV, TLC, TPC, DLC, Serum glucose, TSP, SUN, SC, ALT, AST and ALP were recorded preoperatively, postoperatively and following chemotherapy at 10th, 30th and 60th day intervals. Result: The present study showed transient change in the physiological, haematological and biochemical profiles following surgery and chemotherapy treatment. The histopathological analysis showed more cases of adenocarinoma which were followed by mixed carcinoma. Surgery combined with chemotherapy (doxorubicin and vincristine) resulted in minimal to no reoccurrence with few adverse reactions including inappetance, vomition, anaemia and alopecia. It was concluded that the most effective treatment for malignant mammary tumors in dogs is surgical excision followed by sequential vincristine therapy, which efficiently regresses the tumor without causing relapse.

Diagnosis of Canine Tumours and the Value of Combined Detection of VEGF, P53, SF and NLRP3 for the Early Diagnosis of Canine Mammary Carcinoma.

The average life of a dog is generally maintained at ten to fifteen years, and tumours are the predominant reason that leads to the death of dogs, especially canine mammary carcinoma. Therefore, early diagnosis of tumours is very important. In this study, tumor size, morphology, and texture could be seen through general clinical examination, tumor metastasis could be seen through imaging examination, inflammatory reactions could be seen through hematological examination, and abnormal cell morphology could be seen through cytological and histopathological examination. In the 269 malignant cases and 179 benign cases, we randomly selected 30 cases each, and an additional 30 healthy dogs were selected for the experiment (healthy dogs: dogs in good physical condition without any tumor or other diseases). We used RT-qPCR and ELISA to determine the relative expression of vascular endothelial growth factor (VEGF), tumor protein P53 (P53), serum ferritin (SF), and NOD-like receptor protein 3 (NLRP3) in 30 healthy dogs, 30 dogs with benign mammary tumours, and 30 dogs with malignant mammary tumours. In the results, the same expression trend was obtained both in serum and tissues, and the expression of the four markers was the highest in malignant mammary tumours, with highly significant differences compared with the benign and healthy/paracancerous groups. By plotting the ROC curves, it was found that the results of combined tests were better than a single test and the combination of the four markers was the best for the early diagnosis. In conclusion, this can assist the clinical early diagnosis to a certain extent, and also provides some references and assistance for the development of tumor detection kits in clinical practice.

Investigation of efficacy of two different chemotherapy protocols used in neoadjuvant chemotherapy in clinical stages II-IV canine malignant mammary tumours.

The first aim of this study is to demonstrate the clinical efficacy and reliability of two different neoadjuvant chemotherapy (NAC) protocols consisting of doxorubicin/cyclophosphamide (AC) and paclitaxel in dogs with clinical stages II-IV canine malignant mammary tumours (CMTs). Secondly, to determine the Luminal A, Luminal B, HER2-positive and triple-negative molecular subtypes and their value in predicting clinical response to NAC in biopsy samples, and thirdly, to reveal the changes in Ki-67, human epidermal growth factor receptor type 2 (HER2), oestrogen receptor (ER), and progesterone receptor (PgR) expression levels induced by NAC. Thirty dogs with clinical stages II-IV CMTs (T1-3N0-1M0) according to the modified TNM system were included in the study. Dogs in group-1 (n = 15) AC combination and dogs in group-2 (n = 15) were administered paclitaxel. Partial response (PR) was the most common clinical response in both treatment groups (66.66% and 86.66%, respectively). There was no difference between the groups regarding clinical response parameters (p = .001). The rate of treatment responders was higher than the rate of non-responders in both groups (p < .001). The adverse effects observed in both groups were mostly limited to grades 1 and 2 and all were easy to manage. The most frequently detected molecular subtype was Luminal A (59.25%). Complete response (CR) was achieved in 33.33% of dogs with triple-negative CMT in the AC group and 14.29% of the Luminal A subtype in the paclitaxel group. Alterations in Ki-67, HER2, ER, and PgR expressions after chemotherapy were not statistically significant (p > .05). As a result, we have shown that these neoadjuvant chemotherapy protocols are effective and safe alternative treatment options for CMTs.

Organopathology of the mammary gland in female carnivores: current state of research issues

Organopathology of the mammary gland in female carnivores: current state of research issues

Machine learning determines stemness associated with simple and basal-like canine mammary carcinomas

Simple and complex carcinomas are the most common type of malignant Canine Mammary Tumors (CMTs), with simple carcinomas exhibiting aggressive behavior and poorer prognostic. Stemness is an ability associated with cancer initiation, malignancy, and therapeutic resistance, but is still few elucidated in canine mammary tumor subtypes. Here, we first validated, using CMT samples, a previously published canine one-class logistic regression machine learning algorithm (OCLR) to predict stemness (mRNAsi) in canine cancer cells. Then, using the canine mRNAsi, we observed that simple carcinomas exhibit higher stemness than complex carcinomas and other histological subtypes. Also, we confirmed that stemness is higher and associated with basal-like CMTs and with NMF2 metagene signature, a tumor-specific DNA-repair metagene signature. Using correlation analysis, we selected the top 50 genes correlated with higher stemness, and the top 50 genes correlated with lower stemness and further performed a gene set enrichment analysis to observe the biological processes enriched for these genes. Finally, we suggested two promise stemness-associated targets in CMTs, POLA2 and APEX1, especially in simple carcinomas. Thus, our work elucidates stemness as a potential mechanism behind the aggressiveness and development of canine mammary tumors, especially in simple carcinomas, describing evidence of a promising strategy to target this disease.

Oral mirtazapine decreases the gastrointestinal adverse effects in cats on doxorubicin chemotherapy

Anorexia, depression, and vomiting are the common adverse effects of chemotherapy in humans and animals. Mirtazapine is primarily used as an appetite stimulant and antiemetic in dogs and cats. Therefore, we evaluated the efficacy of mirtazapine in reducing the gastrointestinal adverse effects in cats receiving doxorubicin chemotherapy. This single-masked, placebo-controlled crossover study enrolled 11 cats with malignant mammary gland tumors. The cats were randomly assigned to receive either mirtazapine (1.88 mg/cat) or placebo every 48 h for 2 weeks from the first initiation of doxorubicin chemotherapy. Each cat was then crossed over to the alternate group on the subsequent chemotherapy with a 1-week wash-out period. The owners were asked to record appetite score, activity score, episodes of vomiting and diarrhea for 2 weeks after each doxorubicin administration. Cats treated with mirtazapine showed significantly increased bodyweight compared with those on placebo (P = 0.010). The appetite and activity scores during mirtazapine treatment was significantly higher than those during placebo treatment (P = 0.005 and 0.018, respectively). Furthermore, the prevalence of episodes of vomiting during mirtazapine treatment was significantly lower than that during placebo treatment (P = 0.026). Our results demonstrate that mirtazapine can significantly increase bodyweight, appetite, and activity and reduce vomiting in cats after doxorubicin chemotherapy.

Immunohistochemical Study of MMP-9 Expression in Canine Malignant Mammary Tumors

A class of proteinases called matrix metalloproteinases (MMPs) is involved in the breakdown of the extracellular matrix (ECM) as well as other biological activities. Due to their capacity to degrade the matrix, they were considered in the invasion and metastasis of tumors. The primary goal of this study is to evaluate the association between MMP-9 expression in tumor-adjacent stromal cells and malignant mammary tumors in dogs, as well as the potential use of this protease as a predictive tumor marker. During surgery and necropsy, 32 canine malignant mammary gland tumors were discovered. For histopathological grading of tumors, the Pena et al. [1], approach and the Goldsmith et al. [2], method were employed. Utilizing immunohistochemical labeling, epithelial and stromal cell MMP-9 expression was identified. The level of tumor malignancy was associated with the degree of MMP-9 expression in stromal cells. In addition, stromal cells had much greater levels of MMP-9 expression. The expression of MMP-9 was well distributed and intense in all grade II and III carcinomas as well as poorly differentiated carcinosarcomas. High histologic grade tumors have greater levels of MMP-9 expression, according to semiquantitative examination of the gene’s expression. This finding lends credence to the concept that MMP-9 functions as an ECM component in tumor invasion and metastasis.

Advancing canine mammary tumor diagnostics: Unraveling the diagnostic potential of Cytokeratin 19 through droplet digital PCR analysis

Cytokeratin 19 (CK19) is a complex intracytoplasmic cytoskeletal protein primarily localized in the ducts of the mammary gland and skin epithelial cells. In humans, the expression of CK19 gene within circulating tumor cells (CTCs) extracted from blood samples of breast cancer patients reflects tumor cell activity, offering valuable insights for predicting early metastatic relapse or monitoring treatment effectiveness. However, knowledge of serum tumor markers is limited in veterinary oncology. Recently, droplet digital PCR (ddPCR), has been employed to explore rare target genes due to its heightened sensitivity and accuracy as a novel molecular diagnostic tool. The objectives of this study were to investigate the expression of the CK19 mRNA in CTCs, non-neoplastic mammary tissues, and both benign and malignant canine mammary tumors (CMTs) through ddPCR analysis. In Study I, we optimized the discard volume for blood samples to reduce CK19 contamination from skin epithelial cells post-venipuncture. The results revealed that discarding the initial 3 mL of blood was adequate and effective in eliminating CK19 mRNA contamination. In Study II, after the removal of the initial 3 mL of blood, we investigated CK19 mRNA-positive CTCs in the peripheral blood of normal healthy dogs, including those with benign and malignant CMTs. Intriguingly, CK19 mRNA was undetectable in all blood samples. The expression of CK19 mRNA in mammary tissues was investigated in Study III. The copy number (CN) ratios of the CK19 gene in non-neoplastic mammary tissues (14.77 ± 14.65) were significantly higher (P < 0.05) than those in benign (4.23 ± 3.35) and malignant groups (6.56 ± 5.64). Notably, no difference was observed between the benign and malignant groups. In conclusion, CK19 mRNA appeared unlikely to be a suitable candidate as a biomarker in the peripheral blood of CMTs, while the CN ratio in mammary tissues could serve as a potential discriminator between non-neoplastic and CMT groups, complementing the gold standard of histopathological examination.

Linfonodo ilíaco medial como alvo de metástase em cadelas com neoplasmas mamários malignos

ABSTRACT: The regional lymphatic connections of the mammary glands in canines are poorly investigated, both in healthy animals and in those with mammary neoplasms. The objective was to establish the occurrence of metastases in the medial iliac lymph nodes (MILN) in bitches with mammary neoplasm, in addition to evaluating the effectiveness of the technique of lymphatic labeling of the MILN, administering methylene blue (MB) dye intrauterinely or intradermally. Thirty-two bitches with tumors in different mammary glands were included in the study. In 13 bitches (GU group), the MILNs were stained with intrauterine MB, followed by unilateral lymphadenectomy, ovariohysterectomy (OVH) and unilateral mastectomy. Nineteen bitches (GI group) underwent OVH, followed by MILN staining with intradermal MB, lymphadenectomy, and unilateral mastectomy. In GU, 11 bitches (84.62%) exhibited MB-stained MILNs, but staining was not observed in two (15.38%). Metastasis to MILNs was observed in three bitches (9.38%) from the GI and two (6.25%) from the GU. Intrauterine and intradermal staining of MILNs in bitches with mammary tumors was efficient, but intradermal administration of MB produced better results. Given metastatic spread to MILNs in approximately 15.63% of patients, we recommend that screening for MILNs should be considered as part of the staging of animals with malignant mammary neoplasms.

Immunohistochemical Expression and Prognostic Value of COX-2 and Alpha-Smooth Muscle Actin-positive Cancer-associated Fibroblasts in Feline Mammary Cancer.

Cyclo-oxygenase-2 (COX-2) and cancer associated fibroblasts (CAFs) play an important role in the development and progression of tumor malignancy in humans and animals, showing that both can influence the tumor microenvironment. However, the impact of these two markers in feline mammary carcinogenesis has not yet been addressed. In the present study, the clinicopathological significance of COX-2 immunoexpression and alpha-smooth muscle actin (α-SMA)-positive cancer-associated fibroblasts (CAFs) was determined and correlated with disease-free and overall survival of 50 felines with malignant mammary tumors. COX-2 overexpression was positively associated with mitotic index (p=0.031), degree of malignancy (p≤0.001), lymph node metastasis (p≤0.001), vascular invasion (p=0.002), disease recurrence (p=0.019) and distant metastasis (p=0.036). α-SMA-positive CAFs were associated with mitotic index (p=0.004), lymph node metastasis (p=0.027), vascular invasion (p=0.05), disease recurrence (p≤0.001) and distant metastasis (p≤0.001). Additionally, both markers were correlated with disease-free and overall survival, emerging as predictors of poor prognosis. Our results indicate for the first time that the presence of two markers, COX-2 and α-SMA, is associated with carcinogenesis and worse prognosis in feline mammary cancer and that α-SMA-positive CAFs have a role in feline mammary tumorigenesis, cancer development, and clinical outcome.

Correlation between fine-needle aspiration biopsy and routine histopathology in the diagnosis of spontaneous feline mammary gland tumours

The present study evaluated the correlation between fine-needle aspiration biopsy and routine histopathological examination in the diagnosis of spontaneous mammary gland tumours in cats. From the studied 120 neoplastic growths, cytological diagnosis was correct in 111 cases (92.5%), and incorrect in 9 (7.5%) patients. Out of all studied tumours, 3 false positive (2.5%) and 6 false negative (5%) results were found out. Among benign tumours, cytological diagnosis was correct in 17 cases (94.44 %). Only one false negative diagnosis was demonstrated (5.56% of all benign tumours). As to malignant tumours, cytological diagnosis was correct in 94 (92.15%), and incorrect in 8 patients (7.85%). For diagnostics of feline mammary neoplasms, the sensitivity of cytology was 95.23%, the specificity - 75%, positive predictive value: 97.5% and negative predictive value: 60% compared to routine histopathology. The analysis with Cohen’s kappa test showed agreement of 99.17% (Cohen’s κ 0.96) between cytology and histopathology for benign and 94.17% (Cohen’s κ 0.75) for malignant mammary neoplasms.

Vascular Endothelial Growth Factor A (VEGF-A) concentrations in canine malignant mammary tumours with and without metastases to regional lymph nodes

Vascular Endothelial Growth Factor A (VEGF-A) concentrations in canine malignant mammary tumours with and without metastases to regional lymph nodes

YRNA and tRNA fragments can differentiate benign from malignant canine mammary gland tumors

Mammary gland tumors (MGT) are the most common tumors in sexually intact female dogs. The functional regulation of miRNAs, a type of noncoding RNAs (ncRNAs), in canine MGT has been extensively investigated. However, the expression of other ncRNAs, such as YRNAs and transfer RNA–derived fragments (tRFs) in canine MGT is unknown. We investigated ncRNAs other than miRNAs from our small RNA project (PRJNA716131) in different canine MGT histologic subtypes. This study included benign tumors (benign mixed tumor, complex adenoma) and malignant tumors (carcinoma in benign tumor and carcinoma with metastasis) samples. Aberrantly expressed ncRNAs were examined by comparisons among MGT subtypes. The relative expression trends were validated in canine MGT tissues, plasma, extracellular vesicles, and MGT cell lines using quantitative reverse transcription PCR. Three aberrantly expressed ncRNAs were identified by comparisons among MGT subtypes. YRNA and tRNA-Gly-GCC distinguished benign mixed tumor from other MGT histologic subtypes, while tRNA-Val differentiated complex adenoma, carcinoma in benign tumors, and carcinoma with metastasis. The ROC curve of the three ncRNAs showed they might be potential biomarkers to discriminate malignant from benign MGT. YRNA and tRFs expression levels were decreased in metastatic compared with primary canine MGT cell lines. To the best of our knowledge, this is the first investigation of YRNA and tRFs in canine MGT. The three identified ncRNAs may be biomarkers for differentiating MGT histologic subtypes. Suggested Reviewers: Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporatio

Identifying the Risk Factors for Malignant Mammary Tumors in Dogs: A Retrospective Study.

Mammary cancer is one of the main causes of death in female dogs worldwide, considering that many risk factors are involved in its development. This study aimed to elucidate the relationship between epidemiological and clinical risk factors with the histopathological diagnosis of malignant mammary tumors in dogs treated at the Veterinary Hospital of the Federal University of Uberlândia, which has one of the first veterinary oncology services in Brazil. A retrospective matched case-control study was conducted to identify risk factors for the development of malignant mammary tumors in dogs. The variables analyzed were size dog, breed, housing, type of diet, and body score. Potential risk factors were selected by univariate analysis (p < 0.25) before multivariate forward binary logistic regression. The most frequent benign tumor was the benign mixed tumor (35.2%), and the most frequent malignant tumor was the mixed carcinoma (27.4%). Size dog, breed, housing, and overweight are predictors of malignant mammary tumors in dogs. The highest risk of developing malignant mammary tumors is associated with large female dogs, Yorkshire or Poodle breeds, living outside the home, or being overweight.

Prognostic value of different forms of serum MMP-2 and MMP-9 in dogs with mammary tumors; a longitudinal study

Gelatinases (MMP-2 and MMP-9) are related to tumor invasion and metastasis. In humans, the diagnostic value of serum levels of gelatinases has been confirmed in breast cancer, but their diagnostic value in canine mammary tumors is still unknown. This study aimed to determine the serum level of gelatinases in dogs with mammary tumors in order to determine their value in the diagnosis of malignancy or benign tumors and also in predicting the possibility of metastasis and recurrence. Frequent measurement of MMP-2 and MMP-9 by gelatin zymography in serum before and after surgical treatment has not previously been studied for monitoring mammary tumors in dogs. Thus, the serum levels of MMP-2 and MMP-9 in 26 dogs with mammary tumors before surgical treatment and then 1, 4 and 12 months after surgery were evaluated by gelatin zymography. Serum samples of 26 healthy dogs with normal conditions were used as control.Dogs with benign and malignant mammary tumors showed bands of pro-MMP-2, pro-MMP-9 and active MMP-9. However, only pro-MMP-2 and pro-MMP-9 bands appeared in the serum of control group. Our results showed that the presence of active MMP-9, regardless of its level, was prognostically important for metastasis and or recurrence (M/R). However, the presence of active MMP-2 band was more important for M/R than active MMP-9, as its presence coincides with definitive M/R. It seems that serum gelatin zymography could possibly be used at regular intervals before and after surgery to evaluate the probability of M/R in dogs with mammary tumors. More research is needed in this regard.