Mixed Tumor Carcinoma Research Articles

Treatment of mammary gland tumors in bitches: effects of sodium dichloroacetate as neoadjuvant therapy.

Mastectomy is the standard treatment for mammary gland tumors in dogs. In addition to traditional therapy, sodium dichloroacetate (DCA) can act as target therapy, as it may promote autophagy, reduce metastatic potential, and tumor proliferation in mammary tumor cell lines. This study aimed to analyze the effects of DCA as preoperative therapy for mammary tumors in bitches. Nineteen animals were selected, and they received DCA at a dose of 10 mg/kg orally every 12 hr, for 15 days. The periodic evaluation included hematological analysis (complete blood count and biochemical markers), evaluation of gastrointestinal adverse effects, evaluation of tumor volume, histopathological analysis, and immunohistochemical evaluation (Ki67 and cyclooxygenase-2/COX-2 markers). After treatment, there was a significant reduction in hematocrit (P=0.02) and leukocyte (P=0.04) means. Despite the variations for these two hematological parameters, the means remained within the reference range for the species. There were two cases of vomiting and one case of diarrhea. Most cases were classified as carcinoma in mixed tumor (n=7, 36.8%), followed by solid carcinoma (n=6, 31.6%). Nine cases (47.4%) showed reduced tumor volume, nine (47.4%) had stable disease, and one showed progressive disease. While there was no sample with a COX-2 score higher than 6, tumor samples with COX-2 scores 3 and 4 were significantly associated with stable disease or progression. DCA preoperative treatment for bitches with mammary gland tumors showed safety and potential cytoreduction in some cases.

GATA-3 expression and its correlation with prognostic factors and survival in canine mammary tumors.

The transcription factor GATA-3 plays a significant role in mammary gland development and differentiation. Recent studies on human oncology have demonstrated its association with favorable pathologic factors in breast cancer. Canine mammary tumours, proposed as comparative and translational study models, have epidemiological, clinical, biological, and genetic characteristics similar to those of human breast cancers. Here, we evaluated the frequency of GATA-3 expression in mammary tumors of dogs and its relationship with prognostic factors and survival. Tumor samples were obtained from 40 female dogs and grouped according to histological type into benign tumors (n = 10), carcinoma in mixed tumors (CMTs) (n = 20), and aggressive tumors (n = 10). CMTs were further separated according to histological grade, and data on clinical staging and diagnosis, histopathological grading, and survival rate were collected. GATA-3 and estrogen receptor (ER) expression were higher in benign and well-differentiated carcinomas than in aggressive tumors, which showed greater Ki-67 expression. The expression rate of ER in the studied groups was equivalent to that of GATA-3. We identified a strong positive correlation between GATA-3 and ER expression frequencies and a negative correlation between those of GATA-3 and Ki-67. There were associations between GATA-3 (p < 0.001), Ki-67 (p = 0.003), tumor size (p < 0.001), clinical stage (p = 0.002), lymph node metastasis (p < 0.001), and histological grade (p < 0.001) by univariate survival analysis. The parameters ER (p = 0.015) and GATA-3 (p = 0.005) also influenced survival in a multifactorial manner. Kaplan-Meier analysis of survival curves validated our previous findings that dogs with GATA-3 expression in ≥79.4% of cells had significantly higher survival rates (p < 0.001). The performance analysis showed that the expression of GATA-3 in ≥79.4% of cells effectively predicted survival or death in dogs with mammary tumors. Collectively, these results suggest that GATA-3 can be a relevant marker in the study of mammary tumor progression and has potential as a prognosis marker for predicting outcomes in canine mammary tumors.

Canine Mammary Neoplasms - Evaluation of Tumor Microenvironment

Background: The tumor microenvironment is an important target of studies in different types of neoplasms. Understanding the role of general components such as immune, vascular and fibroblastic cells has the objective of contributing to prognosis and treatment. The aim of this study was to evaluate the relationship between mast cells and angiogenesis in benign and malignant mammary neoplasms by investigating the role of degranulation and microlocation of mast cells and neoformed vessels in canine mammary neoplasms.Materials, Methods &amp; Results: Mammary glands (n = 122) from 50 female dogs submitted to mastectomy without chemotherapy were evaluated and categorized into 3 groups: control group (n = 46); malignant group (n = 57) and benign group (n = 19). Lymph nodes without changes (n = 59) and with metastases (n = 6) were also evaluated. To evaluate the MCD (mast cell density) and angiogenesis, Toluidine Blue (0.1%) and Gomori’s Trichrome techniques were performed and adapted from previous studies. Photomicrographs of 10 hotspot areas on a 40x objective lens of the mammary glands and lymph nodes were captured to assess MCD and angiogenesis. In the absence of these areas, random fields were captured. For the mammary glands of the malignant and benign groups, 20 fields were analyzed, as the analysis considered the microlocation (peritumoral and intratumoral). Counting was performed manually using ImageJ software version 1.42q by 2 observers. The statistical analysis were performed using SPSS software version 19.0. The most frequent histological type in the malignant group was carcinoma in mixed tumor (68.42%; 39/57) and in the benign group was benign mixed tumor (57.89%; 11/19). Female dogs without breed pattern were more frequently affected represented 70% of the animals and the mean age was 9 years and 8 months ± 3 years and 1 month. The granulated density of mast cells and peritumor vessels was higher in the malignant group (P = 0.03; P = 0.02). There was also a positive correlation between intratumor and total vessel density and mast cell density. There was no significance between the malignant and benign groups in regard with fibrosis density.Discussion: In this study were observed a greater density of blood vessels in malignant group, suggesting the participation of blood vessels for neoplastic proliferation. Furthermore, these vessels were located in the peritumoral region as in previous studies. The positive correlation between MCD and blood vessels was similar to a previous study performed in canine breast carcinomas and breast cancer in women. Regarding microlocation, another study also found higher MCD in the peritumoral region than in the intratumoral region of canine carcinomas. Although there are already studies for this purpose in cases of oral squamous cell carcinoma in humans, we believe this is the first study to investigate the role of mast cell degranulation in mammary neoplasm of bitches. The MCD was not significant among the malignant and benign groups and in the mammary glands of the control group the MCD was higher, as observed by other studies. Future studies should be associated the survival time and the presence of metastases in order to confirm the findings. In view of these findings, we may conclude that a higher density of mast cells is related to a higher density of blood vessels and that these are more abundant in malignant neoplasms, which reinforces the crucial role of angiogenesis in the neoplastic development.Keywords: tumor microenvironment, mammary tumor, mast cells, angiogenesis, bitches.

Expression of VCAN and its receptors in canine mammary carcinomas with or without myoepithelial proliferation

The proteoglycan versican (VCAN) plays a complex role in cancer. The expression of this molecule has been related to invasion and progression in malignant mixed tumors, such as carcinoma in mixed tumors (CMT) of the canine mammary gland. In addition, its interaction with surface cell receptors EGFR, HER-2 and CD44 in malignant epithelial cells may be responsible for proliferation and cellular motility in early stages of cancer. We comparatively evaluated the expression of this proteoglycan and its receptors in in situ and invasive areas of simple carcinomas (SC) and CMT to investigate similarities and differences between these histological types. Immunohistochemistry was performed with anti-VCAN, anti-CD44, anti-EGFR and anti-HER-2 antibodies in 32 cases of SC or CMT. VCAN was highly expressed in stroma adjacent to invasive areas in SC and CMT. CMTs presented comparatively higher expression of VCAN in stroma adjacent to in situ and in invasive areas than in corresponding areas in SCs. In CMT, EGFR and HER-2 expressions were higher in situ compared to invasive areas. In contrast, increased CD44 and EGFR expression was found in invasive areas in SC compared to CMT. These results indicate that versican expression is similarly associated with invasiveness in SC and CMT, however higher levels were seen in CMT suggesting that the presence of myoepithelial proliferation in this tumor type participates in stromal composition and promoting an increase in the expression of versican.

Hypercalcemia in bitches with malignant mammary neoplasms

Malignant hypercalcemia is a paraneoplastic syndrome. It has been detected in several types of malignant tumours in dogs, such as lymphomas, and also in mammary neoplasms, without the exact determination of the mechanisms involved in its occurrence as well as its incidence. In this study, 100 bitches with mammary malignant neoplasia, diagnosed by histopathological analysis, were submitted to clinical evaluation for disease staging, haematological evaluation, serum biochemistry, including renal function tests, hepatic and total calcium serum levels and ionized fraction. These parameters were analyzed at the time of the initial attendance and 30 days after the treatment. The most frequent histological pattern was carcinoma in mixed tumors (26%). In 52% of the diseased dogs, hypercalcemia was observed by means of ionized calcium dosing, which was not verified by means of total calcium dosing, where only 4% presented hypercalcemia. No correlation was found between hypercalcemia and the histological pattern of the neoplasms. However, the correlation was verified between hypercalcemia and more advanced stages of the disease, mainly from stage III, suggesting that the detection of hypercalcemia may be correlated with worse prognosis, showing the importance of the research concerning the presence of paraneoplastic syndromes in bitches with mammary neoplasms.

Electrocardiography in Bitches with Mammary Tumors

Background: The incidence of cancer has increased due to greater longevity of the animals as a consequence of better control of other diseases, improvement of nutrition and good practices in preventive medicine. Malignant tumors can cause paraneoplastic syndrome as hypercalcemia, anemia, cachexia, among others and consequent cardiovascular disorders. The electrocardiography (ECG) is a complementary exam that can reveal with its traces these rhythms disorders. Based on that, the objective of this study was to evaluate the ECG in bitches with mammary tumors in order to detect in which type of tumor would be found significant changes as in cardiac rhythm, ECG waves, segments or cardiac axis.Material, Methods &amp; Results: Twenty-nine female dogs were used in this study, being 19 bitches with mammary tumor, and they were allocated in three groups: G1: control group (n=10), G2: benign neoplasia group (n=6) and G3: malignant neoplasia group (n=13). The most prevalent type of neoplasia in G2 was the benign mixed tumor (83%), followed by adenoma (17%) whereas in G3: carcinoma in mixed tumor (61%); papillary carcinoma (23%). Regarding cardiac rhythm, it was found sinus arrhythmia (SA) and normal sinus rhythm (NS): G1: 50% SA and 50% NS; G2: 67% SA and 33% NS; G3: 54% SA and 46% NS. No ventricular or atrial arrhythmias were detected. For other parameters in G1, G2 and G3, respectively (mean±Std error): FC (bpm): 110±9.2, 120±8.5, 124±7,5; P (ms): 48±1.6, 51±1.8, 50±1.2; P (mV) 0.19±0.02, 0,2±0.02, 0,19±0.02; PR (ms): 94±4.3, 93±5.5, 89±3.9; QRS (ms): 56±1.54, 60±4, 62±1.2; R (mV): 1.1±0.06, 1.2±0.24, 0.9±0.13; QT (ms): 203±9.4; 204±7.9; 182±15.6; and cardiac axis (°): 66±6.2, 61±7.9, 70±7.5. There were no significant differences for all cardiac parameters and also for ST interval and T wave morphology.Discussion: All electrocardiographic parameters found are in accordance with other studies carried out in the canine species. The results regarding tumor types differ from what was found in another study, in which in 18 bitches with mammary tumors, 55% were benign, with prevalence of adenomas (38%) followed by benign mixed tumors, and 45% malignant, with adenocarcinoma prevailing (22%). In another study, it was verified in 63 bitches the predominance of tubular carcinoma (26.56%) and carcinoma in mixed tumor (23.44%). Diverging from the electrocardiographic parameters of this study, a significant difference was found in the R wave amplitude value in the research by Barros et al., (2015) who performed computerized electrocardiography in 50 dogs, not only with mammary neoplasms (55% mammary carcinomas), but also in mastocytomas, lymphomas, benign tumors and other sarcomas. This author found out that the R wave amplitude values of the neoplasia group were lower when compared to the control group. In addition, we suspect that other types of tumors could result in more paraneoplastic syndrome than the mammary neoplasms found in this research. Neoplasms as lymphomas (T cells), apocrine gland anal sac adenocarcinomas, multiple myelomas and thymomas are known as a cause of hypercalcemia of malignancy and histiocytic sarcomas, myelomas, leukemia and lymphomas causing anemia. In conclusion, ECG has no changes in benign or malignant mammary tumors in dogs. However, this fact does not exclude the importance of its performance in pre-anesthetic evaluations.

Presence of BRCA1 Gene Mutation in Bitches with Malignant Mammary Tumors

Background: Mammary tumors (MTs) in bitches are similar to breast cancers in women. Thus, they can be used as a model for human breast cancer and findings can be extrapolated for use in human medicine. BRCA1 is a tumor suppressor gene. When the gene has a mutation, it cannot repair damaged DNA, which causes genetic instability and tumorigenesis. Therefore, we aimed to study the frequency of single nucleotide polymorphisms (SNPs) in the BRCA1 gene that are associated with distinct histological types of malignant MT in bitches.Materials, Methods &amp; Results: The study population consisted of 91 bitches, including a control group of 6 animals with healthy mammary glands and 85 animals with MTs. All animals underwent a presurgery evaluation consisting of a questionnaire administered to the person responsible for the animal, a physical examination, collection of peripheral blood for hematological and serum biochemistry evaluations, an electrocardiogram, and a preanesthesia evaluation. In addition, distant metastasis was studied via chest radiography and abdominal ultrasound. After evaluations were complete, the animals that could undergo surgery were administered general anesthesia and underwent a mastectomy or mammary gland sample collection. Histopathological examination and molecular analysis were performed to identify mutations in the BRCA1 gene. Histopathological examinations found 10 different types of malignant tumors in 36 sick animals. Tumor samples plus samples from the 6 control animals were subjected to DNA extraction, polymerase chain reaction (PCR) analysis, and genetic sequencing. The tumor with the highest incidence (33.33%) was a complex carcinoma, followed by carcinoma in mixed tumor (13.88), tubular carcinoma (13.88) and carcinosarcoma (13.88). Molecular analysis revealed 3 different SNP points in 5 samples (4006G&gt;A, 3619A&gt;G, and 3761C&gt;T). The allelic variant 4006G&gt;A (1/36) resulted in the alteration of the amino acid valine by isoleucine (V1336 I). The mutation 3619A&gt;G (2/36) inserted the amino acid alanine instead of threonine (T1207 A). The mutation 3761C&gt;T (2/36) led to the alteration of the amino acid serine by phenylalanine (S1254 F), a mutation for which there are no published reports. The histological types that showed BRCA1mutations were complex carcinoma (1/5), carcinoma in mixed tumor (1/5), papillary carcinoma (1/5) and tubular carcinoma (2/5). Software analysis identified the new SNP (nucleotide 3761) in BRCA1 and 2 points mutations in nucleotides 4006 and 3619 and responsible for genetic instability.Discussion: The development of breast cancer is caused by many endogenous and exogenous factors. The results of our study show that these factors have a greater presence in female, mixed breed, uncastrated, and older dogs, confirming the data in the veterinary literature. In the present study, we found different histological types of malignant breast tumors with mutations in the BRCA1 gene, as other authors have reported. However, we also found the mutation 3761C&gt;T, which, to the best of our knowledge, has not been reported in the literature. This shows the need for studies in veterinary medicine that assess mutations in the BRCA1 gene and the most common histological types. In conclusion, SNPs in the BRCA1 gene cause genetic instability, resulting in additional mutations that lead to the development of breast tumors. They are point mutations that affect transcription, resulting in truncated proteins. These proteins may have a loss of function, leading to carcinogenesis.Keywords: polymorphism, BRCA1 gene, cancer, dogs.Descritores:polimorfismo, gene BRCA1, câncer, cães. Título: Presença da mutação do gene BRCA1 em cadelas com tumores mamários malignos.

Hyperresistinemia in Obese Female Dogs With Mammary Carcinoma in Benign-Mixed Tumors and Its Correlation With Tumor Aggressiveness and Survival.

Resistin is associated with metabolic, inflammatory, and neoplastic disorders, and is also considered a prognostic marker in human oncology. Canine mammary tumors have epidemiological, clinical, biological, and genetic characteristics similar to those of women and are proposed as a comparative study model. Here, we evaluate the serum levels of resistin in female dogs with or without mammary carcinoma in mixed tumors (CBMT) and its correlation with the proliferative potential of the tumor, obesity, and survival. Eighty dogs grouped according to the presence (50) or absence (30) of CBMT, reproductive status and body condition were assessed for weight, fat percentage, and canine body mass index. The characteristic of the proliferative potential of the tumor (Ki-67) was evaluated. Ki-67 levels (p = 0.024), staging (p = 0.004), and grade (p = 0.016) influenced the survival of the female dogs. Through a multifactorial analysis, it could be seen that the parameters proliferation index (Ki-67) (p = 0.044) and staging (p = 0.036) influenced the survival of the animals. Neutered and overweight dogs from the control and CBMT groups showed hyperresistinemia. Ki-67 expression and resistin levels in dogs with CBMT were higher in overweight dogs than in dogs with normal weight (p = 0.0001). The survival rate of dogs with CBMT, obese and with high levels of resistin (8,400 μg L−1) was lower when compared to those with lower levels of resistin. These results showed an important relationship between hyperresistinemia, tumor proliferative potential and excessive body fat, suggesting that resistin levels may act as an interesting prognostic marker in patients with CBMT.

Investigation of Neoplastic Cells in the Bone Marrow of Female Dogs with Mammary Gland Tumors

Background The mammary glands are the second most common tumor development site in female dogs. One of the ways of staging such tumors is to evaluate the presence or absence of distant metastasis, including in bone marrow. Such findings in human medicine are associated with poor survival of women with breast tumors. However, in veterinary medicine, this clinical staging is used more for patients with lymphomas and mastocytomas. Studies using bone marrow biopsies as a staging method for mammary tumors are scarce. Objectives The present study was to evaluate mammary lesions and bone marrow in 23 female dogs, searching for disseminated tumor cells or metastatic foci. Results: Grade I carcinoma in mixed tumors was the type most observed (22.4%), and there was no statistical difference in relation to tumor size or presence of metastasis in lymph nodes. In the bone marrow of one female dog with carcinosarcoma (4.35%), there was cytoplasmic marking of a probable disseminated tumor cell of epithelial origin, and immunohistochemical evaluation showed presence of cytokeratin-19 antibodies. None of the female dogs presenting reduced cellularity or medullary fibrosis, confirmed through Masson’s trichrome technique, had cell marking in immunohistochemical analyses. Conclusions Bone marrow evaluation can be used as a staging method for mammary gland tumors in female dogs, since disseminated tumor cells present the potential to become secondary lesions and to disseminate to distant foci, thereby causing tertiary metastases over an indeterminate period of time.

Relationship of obesity with clinical and laboratory changes in female dogs with mammary neoplasia

ABSTRACT: Obesity may be associated with the onset of mammary tumors in women. In companion animals, these data are still scarce, mainly associating the clinic of the patient with laboratory and histological findings. This study aimed to evaluate the clinical and laboratory aspects of female dogs with mammary neoplasia, investigating whether they are related to obesity. Four groups of spayed or non-spayed female dogs were studied, divided into (G1) female dogs without mammary tumor and normal body condition score (BCS), (G2) female dogs without mammary tumor and obese, (G3) female dogs with mammary tumor and normal BCS and (G4) female dogs with mammary tumor and obese. BCS, canine body mass index (CBMI), blood count, glucose, total cholesterol, triglycerides and cytopathological and histopathological examinations were evaluated. Mixed-breed animals were the most prevalent in this study (67.5%). There was a limitation regarding the use of CBMI related to body proportionality. Considering the 28 tumors evaluated, carcinoma in mixed tumor was the most prevalent malignant histological type, while adenomyoepithelioma was the most prevalent benign histological type. It was possible to conclude that obesity was not related to clinical and laboratory changes in the female dogs affected with mammary neoplasias. In addition, no relationship was noted between BCS and CBMI with the histological type or malignancy of mammary neoplasia, since the malignant histological types were the most prevalent tumors in both the ideal score group and the overweight to obese group.

Clinical and Ultrasonographic Aspects of Benign and Malignant Mammary Tumors in Female Dogs

Background: Mammary neoplasms in dogs are commonly observed in veterinary clinical routine, most of which being malignant. Hormonal stimulation, endogenous or exogenous, may possibly influence its development. In addition to clinical evaluation, ultrasound analysis can provide information about the characteristics of breast lumps. The association between clinical-epidemiological and pathological data is important for diagnosis. Therefore, given the importance of this pathology for the health of affected dogs, we aimed to evaluate the clinical and ultrasound alterations, along with the factors associated with the development of benign and malignant mammary neoplasms in female dogs. Materials, Methods &amp; Results: We examined 47 samples from the mammary tumors of 35 female dogs at the Small Animal Clinic of the Veterinary Hospital (HV) of the Santa Cruz State University (UESC). The dogs underwent a complete clinical examination, with clinical staging, via TNM classification, followed by hematological, biochemical, radiological and ultrasound, abdominal, and breast exams. Breast ultrasound examination was used to evaluate the shape parameters such as, limits, margins or contour, ecotexture, echogenicity, hyperechoic halo, posterior acoustic shading, surrounding changes, and nodule components. These criteria were associated with the histopathological classification of neoplasms. Epidemiological data was studied through an adapted questionnaire containing information on risk factors associated with breast cancer. The same questionnaire was applied to tutors of 19, age-matched, female dogs with no history of breast cancer. The results revealed that most female dogs with neoplasia were over eight years of age, with no specific breed and were not castrated, and 31.4% of them had already been administered with contraceptives during the reproductive period. Ovariohysterectomy acted as a protective factor (OR 0.06) to the development of breast tumors, while contraceptive use was considered as a risk factor (OR 6.99). The average time reported between tumor perception and clinical care was 13.2 months. The caudal and inguinal abdominal breasts were the most affected. Among the samples evaluated, 76.6% were malignant, with mixed tumor carcinoma being the most frequent type and 69.4% graded in grade I. Nodules classified as malignant showed the largest diameter (P &lt; 0.05). Breast ultrasound results revealed that tumors with heterogeneous echotextures and mixed components were associated with malignancy (P &lt; 0.05). Discussion: The fact that the average age of female dogs with breast cancer was over eight years of age corroborates the literature. Considering that a greater age would mean a longer exposure to the carcinogenic initiators responsible for mutations, and to promoters, such as hormonal changes. Contraceptives increase the risk of breast lumps, while reduce that of ovariohysterectomy, in female dogs, even when performed after the second heat. The size of the nodules and ultrasound criteria related to echotexture and the type of component of the neoplasia may be used as prognostic parameters of female breast nodules. Additionally, most nodules evaluated in this study were malignant (mixed tumor carcinoma was the most common subtype), possibly due to the owners' delay in seeking veterinary care after tumor observation. Although malignant, most nodules presented with a low histopathological grading.Background: Mammary neoplasms in dogs are commonly observed in veterinary clinicalroutine, most of which being malignant. Hormonal stimulation, endogenous or exogenous,may possibly influence its development. In addition to clinical evaluation, ultrasound analysiscan provide information about the characteristics of breast lumps. The association betweenclinical-epidemiological and pathological data is important for diagnosis. Therefore, given theimportance of this pathology for the health of affected dogs, we aimed to evaluate the clinicaland ultrasound alterations, along with the factors associated with the development of benignand malignant mammary neoplasms in female dogs.Materials, Methods &amp;amp; Results: We examined 47

Epidemiological, clinical and pathological evaluation of overall survival in canines with mammary neoplasms

ABSTRACT Canine mammary neoplasms (CMNs) are the most frequent lesions and in female dogs. However, studies correlating pathological criteria with clinical evolution in female dogs with mammary neoplasms are scarce. The present study aims to present epidemiological, clinical-pathological and overall survival data to help establish the prognosis and understand the biological behavior of CMNs. A total of 1539 cases were included (85% malignant and 13% benign). Tumor size was an important prognostic factor and was associated with overall patient survival (P&lt; 0.0001). Most dogs diagnosed with malignant neoplasms (83%) had initial clinical staging, although 17% had regional or distant metastases at the time of diagnosis and lower overall survival (P&lt; 0.0001). Carcinoma in mixed tumor was the most frequent histological type and had a better prognosis. Solid carcinomas, micropapillary carcinomas and carcinosarcomas were considered histological types with aggressive biological behavior and were associated with a worse prognosis and lower overall survival (P&lt; 0.0001).

BI-RADS classification and gray level histogram of malignant mammary tumors in bitches

ABSTRACT: Mammary tumor is the most frequent among the tumors that affect canine females, with relevant importance in veterinary medicine. The objective of this study was to determine the image characteristics of mammary tumors in female dogs, and compare different ultrasonographic techniques for neoplastic evaluation. During the experiment, 30 bitches with presence of nodular lesion in the mammary gland were used. Initially females were submitted to clinical and laboratory evaluations, and subsequent to the ultrasound examination of the tumor mass, as well as abdominal ultrasound and thoracic x-ray for the metastasis investigation. Quantitative analysis by histogram of the gray levels and categorization of the tumor masses by the BI-RADS system were performed. Later, the bitches were submitted to surgical resection of the tumors, where samples of the neoplastic tissue were collected for histopathological analysis. Carcinoma in mixed tumor showed a higher rate (33.3%), and the malignancy degree of epidermal tumors were classified in grade 1 (n=9), grade 2 (n=12) and grade 3 (n=3). Malignancy degree showed positive correlation with BI-RADS (r=0.55; P&lt;0.05) and with the parameter echotexture - histogram base width (r=0.42, P&lt;0.05). BI-RADS graduation also showed a positive correlation with the echotexture parameters (standard deviation of average echogenicity r=0.66, P&lt;0.05 and base width r=0.55, P&lt;0.05). It was concluded that the BI-RADS method in combination with the echotexture of tumors, can be used to evaluate mammary tumors in dogs and establish the planning of treatment.

Mixed tumors of the canine mammary glands: Evaluation of prognostic factors, treatment, and overall survival

Mixed tumors are the most frequent mammary gland neoplasms in bitches; however, studies that thoroughly describe their clinicopathological data, treatment approaches, and the survival of bitches with mixed tumors are scarce. This study evaluated the epidemiological and clinicopathological data, prognostic factors, and therapeutic approaches for bitches with mixed tumors. In all, 162 benign mixed tumors, 682 carcinomas in mixed tumors, and 60 carcinosarcomas were included. Regarding tumor size, T3 lesions were predominantly associated with carcinosarcomas, while T1 and T2 lesions occurred more frequently in benign mixed tumors and in carcinomas in mixed tumors. Based on clinical staging, most bitches with benign mixed tumors presented with stage I tumors; 92% of bitches with carcinomas in mixed tumors presented with stage I–III tumors, while 8% presented with stage IV–V tumors; and 70% of bitches with carcinosarcomas presented with stage I–III tumors, while 30% presented with stage IV–V tumors. Surgery was curative for bitches with benign mixed tumors and for those with stage I–III carcinomas in mixed tumors. Combination therapy in bitches with carcinomas in mixed tumors (IV–V) and carcinosarcomas resulted in a higher overall survival compared with bitches who underwent surgery only. Carcinosarcomas presented higher relapse rates and distant metastases than carcinomas in mixed tumors did.

Relationship between the inflammatory tumor microenvironment and different histologic types of canine mammary tumors

Mammary neoplasms are the tumors with higher incidence in female dogs. Among the factors that contribute for the development of this and other neoplasms, the inflammatory tumor microenvironment plays a crucial role. Several studies reported important roles for lymphocytes, macrophages, plasma cells, neutrophils, eosinophils and mast cells in this context. In the present study, our aim was to evaluate the number of profile cells of inflammatory cells and area of tumor fibrosis and the relation of these features with canine mammary tumors of different histologic and clinical presentation (benign mixed tumor, carcinoma in mixed tumor, solid carcinoma and tubular carcinoma) Counting and staining of inflammatory cells and tumor fibrosis were performed through histochemistry, while counting and staining of CD4+, TCD8+ and FOXP3+ lymphocytes were performed through immunohistochemistry. Statistical analysis of the association between densities of inflammatory cells, tumor fibrosis and histologic types revealed significant difference for plasma cells (p = .035), neutrophils (p = .0113), macrophages (p = .0047), and tumor fibrosis (p = .05). The found data suggest associations between high number of neutrophils and aggressive mammary tumors, between high densities of plasma cells, macrophages and CD8+ cells and between low number of profile cells of CD4+ cells and less aggressive tumors. Larger areas of tumor fibrosis showed relation to more aggressive canine mammary tumors.

CD4+, CD8+, FoxP3+ and HSP60+ Expressions in Cellular Infiltrate of Canine Mammary Carcinoma in Mixed Tumor

Background: Cancer is a complex process that receive many influences of the tumor microenvironment. The participation of immune system cells and proteins in tumor microenvironment is not yet completely understood. Thus, the aim of this study was to evaluate the infiltrate cellular, subpopulations of T-lymphocytes and HSP60 of canine mammary carcinoma in mixed tumor (CMCMT).Materials, Methods &amp; Results: Female dogs (n = 20) were selected after Canine mammary tumor (CMT) diagnosis and data were achieved throughout clinical-pathological information. Clinical staging was evaluated and tumor biopsies were processed by histology and cellular infiltrate was performed according criteria and grade. Survival curve were generated by Kaplan-Meier and the lymphocytic infiltrate were compared by Log-Rank followed Chi-Square χ². For immunolabeling it was used anti-CD4, anti-CD8, anti-FoxP3 and HSP60 monoclonal antibodies and were attributed scores from 0 to 3. Clinical-pathological relationship was analyzed using Spearman correlation. This study was approved by the Committee for Ethics in Research using Animals (CEUA-UECE), protocol 12247080-2. Our data showed a mean age of 9.3 years-old, the size of tumors presented more than 5 cm (50%), which were located in inguinal mammary glands (70%), and CMTs shows I (70%) and II (30%) grade. The cellular infiltrate was distributed both in peri and intratumoral regions, dispersed multifocally with moderate intensity and lymphocytes were the major populations found into tumors (n = 826 ± 220). In relationship to cellular infiltrate with CMT grade it was observed that lymphocytes (ρ = 0.28) and plasma cells (ρ = 0.22) showed a slight positive correlation, and an opposed negative correlation of neutrophils (ρ = -0.1) and macrophages (ρ = -0.38). CMT presents moderate lymphocytic infiltrate (&lt; 800 lymphocytes), shows higher (P = 0.01) survival rates as compared to intense lymphocytic infiltrate (≥ 800 lymphocytes). FoxP3+ showed lower intensity while CD4+ and CD8+ expression were concentrated surrounding of lymphocytic infiltrate tumor region. HSP60+ was observed in the inflammatory and tumor cells.Discussion: Our data are according to a greater risk to the development of breast tumor in old bitches, not castrated and before or after puberty, as well as the use of contraceptives based on progesterone and estrogen. In relation to size of tumor, these findings reinforce that there is a relationship of tumor size with a higher malignancy grade and with a worse prognosis. The predominant tumor location was in the inguinal breasts that is attributed to the high activity of the mammary glands to hormonal stimuli. CMT with low clinical staging are associated with greater overall survival of affected bitches. In relation to tumor microenvironment, it has been reported that heterogeneous populations of the immune system cells often infiltrate the mammary tumors, whose lymphocytes are the main cells. It is suggested that tumor lymphocytosis may be necessary for malignant behavior of the tumor microenvironment. On the other hand, macrophages and neutrophils play an important role that may favor or inhibit the tumor cells development in the tumor microenvironment. In our work, CD4, CD8 and FoxP3 labeling were distributed in peri and intratumoral regions, and consequently, these markers can be used as prognostic for CMT, as well as being a potential target for anticancer therapies. This is the first work that presents results about the participation of HSP60 in CMT, however this data needs further investigation. HSP60 participates as a potent activator of the immune system through its peptides and other HSP types were studied in mammary carcinomas in bitches and presenting results that indicate the association of these proteins with the carcinogenesis process.

Prognostic Factors in Canine Mammary Carcinomas and HER2 Expression Relationship

Background: The human epidermal growth factor type 2 (HER2) receptor is a membrane glycoprotein tyrosine kinase. In woman, HER2 expression is diagnosed in 30% of breast carcinomas and it is associated with a worse prognosis, higher rate of recurrence and mortality. In the bitch, the HER2 overexpression in canine mammary tumors is still controversial and the prognostic value remains uncertain. Thus, we aimed to verify the HER2 expression in canine mammary carcinomas and relate it to the type and histological grade, lymph node metastasis and clinical staging.Materials, Methods &amp; Results: Ninety bitches diagnosed with mammary carcinoma were included in this study. The inclusion criteria were bitches with complete clinical examination, thoracic radiographic examination and submitted unilateral or bilateral mastectomy. Ninety-nine samples of mammary carcinoma were used and the fragments of tumor and regional lymph nodes were fixed in 10% neutral formalin for histopathological and immunohistochemistry analysis. The lesions were evaluated by two pathologists and classified according to the type and histological grade. HER2 expression was performed by semi-quantitative analysis of the slides according to the HerceptTestTM (Dako) recommended score. Simple carcinomas were the most frequent (51.51%) followed by complex carcinomas (46.47%) and in situ carcinoma (2.02%). The histological grade of 97 carcinoma samples was attributed, except in situ carcinoma, 37 (38.14%) of the neoplasms were grade I, 50 (51.55%) grade II and only 10 (10.31%) tumors were classified as grade III. Forty bitches were submitted to clinical staging (TNM) and 42.50% of the bitches received staging in grade I and, 25% of the bitches staged in grade IV and V, with metastases. The HER2 expression, 13/99 samples (13.13%) received score +2, 19/99 (19.19%) score +1 and absence of marking (score 0) was identified in 67 samples (67.80 %). Immunostaining in hyperplastic or normal epithelial cells was evidenced, often in association with weak or moderate cytoplasmic labeling. Of the samples expressing +2 score for HER2 (n = 13), eight samples (17.39%) were complex carcinoma and five (9.80%) simple carcinomas. There was no relationship between HER2 immunostaining with age, tumor size, TNM, histological type, histological gradation, lymph node metastasis and distance. Animals with lymph node metastasis, as well as those diagnosed with distant metastasis, did not present HER2 expression in the tumors.Discussion: The simple carcinoma seems to be the most frequent type histological diagnosed in canine mammary carcinomas, followed by carcinoma in mixed tumor and complex carcinoma. Tubulopapillary carcinomas are more invasive in the female dogs as well as in the woman. Carcinomas grade I and II are more frequent and present a better prognosis for the dog. However, bitches with grade III carcinoma survived for a shorter time when compared to dogs with grade I or II tumors. A factor that may have contributed to the lower number of bitches at worst prognostic stage (EC IV and V) is the current owners’ awareness that they have sought veterinary help earlier, as soon as they detect small nodules in mammary gland. Overexpression of HER2 in women breast cancer is diagnosed in 20-30% of cases, whereas in bitches, this expression is variable. Also the different percentages of canine HER2 immunostaining are due to the lack of standardization for the analysis of the immunostaining, the immunohistochemical techniques employed and the non-specificity of the HER2 antibody. In canine mammary carcinomas the HER2 expression in low and this immunostaining is not related to other established prognostic factors. This study reinforces the hypothesis put forward by other authors that in the bitch the expression of HER2 may not be related to malignancy and tumor progression.

Consensus regarding the diagnosis, prognosis and treatment of canine mammary tumors: benign mixed tumors, carcinomas in mixed tumors and carcinosarcomas

Consensus regarding the diagnosis, prognosis and treatment of canine mammary tumors: benign mixed tumors, carcinomas in mixed tumors and carcinosarcomas

Validation of a low-cost modified technique for constructing tissue microarrays for canine mammary tumor analysis

Compared with conventional histological paraffin blocks, tissue microarray (TMA) represents a “high-throughput tool” that provides rapid results, a time- and cost-effective approach and simultaneous investigation of several tissue samples under the same conditions. Given the large number of cases of dogs affected with mammary tumors, the complexity of these tumors and their similarity with breast cancer in women, this study aimed to validate a low-cost modified method to construct TMAs for canine mammary tumor analysis using immunomarkers. Carcinoma cases were selected from canine mammary carcinomas in mixed tumors (CMT) because this tumor type is the most heterogeneous among the histopathological types of mammary tumors observed in female dogs. Through a histopathological examination, tumor representativity was compared between conventional sections and histological sections obtained from the TMA block; both were stained with hematoxylin and eosin. An immunohistochemistry analysis was performed to compare the percentages of immunoreactive cells obtained in whole tissue sections versus those obtained from sections from the TMA block. Streptavidin-biotin peroxidase complex and anti-PCNA, anti-vimentin and anti-pancytokeratin antibodies were used. Statistical analysis consisted of the nonparametric Friedman’s test (p≤0.05) and descriptive statistical analysis. Histopathological analysis showed tumor representativity in all TMA cores selected for the study. There was no difference between the immunohistochemical analysis of mammary tumors using conventional histological sections or sections obtained from a single 1-mm-diameter TMA core, regardless of the marker used: PCNA (p=0.279), pancytokeratin (p=0.243) and vimentin (p=0.967). The results did not change even when the means of any number of cores were compared among each other and with the conventional histological section: PCNA (p=0.413), pancytokeratin (p=0.177) and vimentin (p=1.0). Therefore, this study validates the use of a low-cost and modified TMA construction technique proposed by Pires et al. for canine mammary tumor analysis.

Paraneoplastic Hypercalcemia Secondary to Canine Mammary Tumors

Background: Paraneoplastic syndromes are complexes symptom that occur at a distinct site from the primary tumor or its metastasis by the production of hormone by the tissue in which the tumor appears. Paraneoplastic hypercalcemia is associated with an abnormal elevation of serum calcium levels and the mainly tumor related to this syndrome in canine is lymphoma, anal sac apocrine gland adenocarcinoma and multiple myeloma. In mammary tumors, the most frequent tumor that affect female dogs, this syndrome was also observed. The aims of this study were to evaluate serum calcium levels in female dogs with malignant mammary tumors and correlate calcium levels with clinicopathological parameters.Materials, Methods &amp; Results: It was evaluated fifty-one female dogs with mammary carcinomas (simple carcinomas and carcinoma in mixed tumors) for serum calcium levels using colorimetric test. Clinical-histopathological data as spray status, pseudopregnancy, tumor size, ulceration, clinical staging, histopathological type and tumor grade were also evaluated in association with serum calcium levels. All dogs were treated with unilateral mastectomy. It was observed that 18 animals (35%) had calcium serum levels increased (&gt;11.5 mg/dL) and 56% (10/18 cases) of these animals had serum calcium levels higher than 12 mg/dL. All dogs with hypercalcemia were asymptomatic, including two female dogs that presented the highest levels (13.43 mg/dL and 14.28 mg/dL). Hypercalcemia of malignancy was related to mammary carcinomas after the exclusion of other causes of hypercalcemia through laboratory tests (complete blood count and serum biochemistry) and abdominal ultrasound. No correlation was verified between the corrected serum calcium values with clinical and histopathological parameters evaluated.Discussion: In this study, it was observed a high incidence of paraneoplastic hypercalcemia associated with canine mammary tumors (35%). In humans, this syndrome is related in up to 10% of all patients with advanced cancer and with worse prognosis. The most frequent clinical signs of hypercalcemia are nonspecific and can be confused with other diseases, such as polyuria, polydipsia, anorexia, constipation, lethargy and weakness. The treatment of this syndrome is based on tumor resection and when necessary other treatments can be performed with fluid containing 0.9% sodium chloride, furosemide, prednisolone and calcitonin. Patients with asymptomatic or mildly symptomatic hypercalcemia (calcium levels &lt;12 mg/ dL) do not require immediate treatment. Clinical signs occur more frequently with serum calcium levels higher than 15 mg/dL. Calcium levels higher than 18 mg/dL are considered a medical emergency and the clinical signs observed are trigger seizures, cardiac arrhythmia, acute renal failure and death. Most animals of this study presented mild hypercalcemia, that could justify the absence of clinical signs related to this syndrome, and the treatment for this syndrome was the tumor removal. The high serum calcium levels did not show correlation with more aggressive tumors and poorer prognosis, conditions evaluated by histological type, tumor grade and clinical stage. The evaluation of serum calcium levels is an important clinical test to be done in female dogs with mammary tumors, besides to be an affordable and technically simple test. The clinical signs related to this syndrome are nonspecific and may be confused with other diseases commonly observed in older dogs. The data suggest that there are no correlation between serum calcium levels with aggressiveness of canine mammary tumors and with other clinical features.