Abstract
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.
Keywords: dog, target therapy, veterinary oncology
Mammary gland tumors are a common diagnosis in small animal practice, with many similarities in etiology, histopathologic classification, risk factors, and treatment between mammary tumors in humans and dogs. Therefore, the study of mammary gland tumors in bitches may be a useful animal model for comparative oncology research and evaluation of human clinical trials [6]. Dogs may present different grades of mammary tumors, and histopathological classification is essential for establishing the prognosis and survival in these patients [3]. This classification includes histopathological grading based on the cell type identified in the histopathological examination [5].
Mammary tumors in dogs are a heterogeneous group of naturally occurring neoplasms, possibly with a hormonal factor involved. Early spaying up to one year of age may be a preventive method, as it blocks the hormonal influence on the mammary gland cells. In some studies, early-spayed bitches appear to have a lower incidence of mammary tumors than those intact [3, 6]. In general, treatment for mammary tumors consists of removing the tumor and adjacent mammary glands, associated with lymphadenectomy, when necessary [3, 6]. One study found that 41 to 53% of mammary gland tumors are malignant, and most are classified as carcinomas [19]. Adjuvant chemotherapy may be necessary in the postoperative period, depending on the histological grade [19, 23].
Complementary treatments to surgery and chemotherapy can help improve mammary tumor therapy in dogs. Targeted therapy promotes a cytotoxic effect directed specifically at tumor cells, enabling treatment with a less systemic cytotoxic effect than chemotherapy. Sodium dichloroacetate (DCA) has been described as a targeted antitumor therapy, promoting a metabolic switch from cytoplasmic glycolysis to mitochondrial oxidative phosphorylation, resulting in stimulation of autophagy, reduction in metastatic potential, and tumor proliferation [10, 22].
DCA is an analog of acetic acid that may promote a metabolic switch from cytoplasmic glycolysis to mitochondrial oxidative phosphorylation, inhibiting pyruvate dehydrogenase kinase (PDHK) and stimulating the activation of pyruvate dehydrogenase (PHD) [20]. The effect of DCA in neoplastic cells induces oxidative glycolysis, decreasing the depolarization of the mitochondrial membrane and sensitizing tumor cells to apoptosis [17]. This metabolic switch made by DCA promotes autophagy, reduction in the rate of metastasis, and reduction in the multiplication of tumor cells [10, 22].
Normal cells utilize the mitochondrial tricarboxylic acid cycle to generate energy through glucose oxidation. However, tumor cells use large amounts of glucose to generate ATP (adenosine triphosphate), even in the presence of oxygen, through aerobic fermentative glycolysis as an energy source [17, 21]. Although there are no in vivo studies with DCA for mammary gland tumors in dogs, an in vitro study demonstrated the cytotoxic effect of DCA in four canine mammary tumor cell lines [7]. This study aimed to analyze the effect and safety of DCA as a preoperative neoadjuvant therapy for mammary gland tumors in bitches.
MATERIALS AND METHODS
Study subjects
Patient selection and data collection took place from January to December 2019 at the Veterinary Oncology Service at CVE-PUCPR. The inclusion criteria were bitches females with a clinical and cytopathological diagnosis of mammary gland tumor, the presence of measurable tumors at diagnosis, and acceptance of the dog owner for participation in the study. Animals with pre-existing chronic liver or kidney disease, previous chemotherapy treatment, cytopenias (anemia, thrombocytopenia, or neutropenia), and those in estrus, pregnancy, or lactation were excluded. Nineteen animals were included in the study, all of whom underwent a unilateral total mastectomy after treatment with DCA. Consent forms were signed by all selected dog owners. This study was approved by the Institutional Ethics Committee on Animal Use of the Pontifical Catholic University of Parana (PUCPR), Curitiba, Brazil (Ethical approval number: 01179/2017- second version).
Diagnosis, staging, treatment, and surgical excision
Medical history was collected, including age, breed, weight, reproductive status, disease onset time, comorbidities, previous surgical excision history, number of lesions, tumor location, presence of skin ulceration, and metastasis. Metastasis diagnosis involved abdominal ultrasound and thorax radiographic evaluation before DCA treatment, and all affected mammary glands were recorded for tumor location evaluation. The 25 animals participating in the study received DCA at a dose of 10 mg/kg orally every 12 hr, for 15 days, in the preoperative period. Drogavet Veterinary Pharmacy in Curitiba, Brazil produced the drug, formulated at a concentration of 10 mg/mL in liquid form. Throughout the study period, all dogs received only DCA to eliminate potential interference from other medications. After treatment with DCA in the preoperative period, the standard technique of unilateral total mastectomy with inguinal and axillary lymphadenectomy was applied, when possible, in all selected dogs in the study. The surgical approach included reconstructive surgery techniques when necessary.
Hematological and adverse effects evaluation
Blood samples for blood count and serum for serum biochemical analysis (alanine aminotransferase, alkaline phosphatase, urea, creatinine, total protein, and albumin) were collected from all selected dogs. Hematological analysis was performed on days 0, 7, and 15 of treatment. Dogs returned weekly for clinical examination and blood collection, and owners were asked about any gastrointestinal adverse effects during the last seven days of DCA treatment. Toxicity was evaluated and classified according to the Consensus for Adverse Effects [23], considering hematological and gastrointestinal adverse effects. Other changes in behavior or general condition were also recorded.
Tumor volume analysis
Tumors were measured using a caliper on day 0, day 7, and day 15 of treatment. The final tumor volume was calculated by multiplying the width, length, and height in centimeters. The final tumor volume was the result of multiplying the three dimensions in centimeters (cm3). When multiple tumors were present, volumes from up to three lesions were added to calculate the total tumor volume, according to the previously described methodology [16].
The response to treatment was evaluated by comparing the initial tumor volume on day 0 with the final volume on day 15. Response categories included reduction (30% or more reduction in tumor volume), stable disease (less than 30% reduction or up to 20% increase in tumor volume), or progression (an increase of more than 20% of the tumor volume) [16].
Histopathology
Samples of neoplastic tissue were collected from all 19 dogs after mastectomy. The tumor samples were fixed in formalin buffered with 10% phosphate saline for 72 hr, followed by processing using a routine histological protocol. Slides with 4 µm thick sections were prepared using a rotating microtome and stained with hematoxylin-eosin. Regional lymph node evaluation was conducted for eight cases, and histologic classification was done following the methodology described by Cassali et al. (2020) [3].
Immunohistochemistry
The Tissue Microarray (TMA) technique described by Mattioli et al. (2011) was applied to embedded samples [12]. Using a 5 mm diameter metal drill, fragments were collected from each tumor, and new paraffin blocks were made by grouping the collected fragments.
Immunoreactivity for the Ki67 proliferation marker was performed on all samples. The TMA blocks were sectioned in a rotary microtome to obtain sections with a thickness of 4 µm. The samples were then deparaffinized with xylene, rehydrated with absolute ethyl alcohol, and treated with hydrogen peroxide (5% in methanol) for 15 min to block endogenous peroxidase. Target Retrieval Solution TM (Dako, Glostrup, Denmark) was used for heat-induced antigen retrieval, as per the manufacturer’s instructions. The samples were then incubated with the mouse anti-human Ki-67 primary antibody (clone MIB-1; Dako, Santa Clara, CA, USA), followed by secondary antibodies AdvanceTM (Dako). After incubation, samples were visualized using DAB 1,1 chromogen (OriGene, Rockville, MD, USA) before counterstaining with Harris Hematoxylin. The same routine was followed for antigen retrieval of COX-2 in all tumor samples. AdvanceTM (Dako) anti-COX-2 polyclonal antibodies were incubated in all samples, and the immunoreactions were visualized using DAB 1,1 chromogen (OriGene), followed by counterstaining using Harris Hematoxylin.
For Ki67 marker evaluation, immunoreactivity was considered when staining occurred in the cell nucleus, regardless of the degree of intensity. The evaluation was carried out through the observation of positive nuclei in 1,000 cells, with the determination of the percentage of positive nuclei. The cut-off point used for classification was ≥20% of positive cores, following the methodology described by Carvalho et al. (2016) [2]. The evaluation of the immunohistochemical samples for the COX-2 marker was performed using a semi-quantitative score that takes into account the cytoplasmic COX-2 distribution and staining intensity [9]. In this scoring system, the assessment of cytoplasmic COX-2 distribution and staining intensity are both taken into account. The scoring scale is as follows: score 0 indicates labeling in 0% of cells, score 1 indicates labeling in less than 10% of cells, score 2 indicates labeling in 10% to 30% of cells, score 3 indicates labeling in 31% to 60% of cells, and score 4 indicates labeling in more than 60% of neoplastic cells. Additionally, the staining intensity is evaluated, with score 0 indicating no staining, score 1 indicating weak staining, score 2 indicating moderate staining, and score 3 indicating strong staining. The values of the two scores are multiplied to obtain a total score that ranges from 0 to 12. Tumors with a total score of 6 or higher are considered positive for COX-2 [9].
Statistical analysis
Descriptive statistical measures and absolute and relative frequencies were used to analyze clinical and treatment variables. Means (or medians) before and after treatment were compared using Student’s t-test and Wilcoxon test for paired samples. The data distribution was verified using the Kolmogorov-Smirnov test. Statistical analysis was performed using the Statistical Package for Social Sciences (SPSS) program, version 15.0. The significance level used was 5%.
RESULTS
Clinical data and staging
The study involved bitches with mammary gland tumors, and the results were presented in a comprehensive manner. The mean age of the animals was 10.0 years with a standard deviation of ± 2.5, ranging from 5 to 14 years. Most of the animals were classified as a mixed breed (n=10, 52.6%), followed by Boxer (n=3, 15.8%), Poodle (n=2, 10.5%), and one (5.3%) representing each of the following breeds: Schnauzer, Rottweiler, Shih-Tzu, and Pinscher. The mean weight was 17.0 kg (SD= ± 9.2), ranging from 3.7 to 31.7 kg. Regarding reproductive status, four (21.1%) bitches were spayed, and 15 (78.9%) were intact.
The average time of disease onset was 3.0 months (SD= ± 6.8), ranging from 1 to 15.5 months. Four (21.1%) cases had comorbidities. Most bitches had two or more mammary nodules (n=15, 78.9%), while four had a single lesion (21.1%). The inguinal mammary gland was the most affected (n=10, 52.6%), followed by the caudal abdominal gland (n=8, 42.1%). None of the cases showed abdominal metastasis on ultrasonography, while two dogs had possible lung metastasis identified in thorax radiographs (10.5%).
Hematological analysis
The analysis of hematological parameters revealed a significant reduction in hematocrit (P=0.02) and leukocyte (P=0.04) means after DCA treatment. Table 1 summarizes the hematological parameters assessed during the study period. Results regarding the occurrence of grade 1 anemia was observed in five (26.3%) bitches, and the hemoglobin value decreased in two (10.5%) cases. Despite the variations for these two hematological parameters, the means remained within the reference range for the species (Table 1).
Table 1. Comparison of complete blood count and serum biochemical markers in bitches with mammary gland tumors, before (day 0) and at the end of treatment with sodium dichloroacetate (DCA) (day 15) (n=19).
| Parameter | Values before DCA treatment (day 0) n=19 | Values after DCA treatment (day 0) n=19 | P |
|---|---|---|---|
| Mean (SD) | Mean (SD) | ||
| Erythrocytes (106/µL) | 6.9 (± 0.9) | 6.4 (± 0.9) | 0.08 |
| Hemoglobin (g/dL) | 16.7 (± 3.0) | 16.3 (± 2.7) | 0.61 |
| Hematocrit (%) | 47.8 (± 6.3) | 43.6 (± 7.6) | 0.02 |
| Mean corpuscular volume (MCV) (fL) | 70.0 (± 4.1) | 69.8 (± 4.1) | 0.86 |
| Mean corpuscular hemoglobin (MCH) (pg) | 24.4 (± 2.6) | 23.9 (± 2.4) | 0.50 |
| Mean corpuscular hemoglobin concentration (%) | 34.8 (± 2.8) | 34.3 (± 2.9) | 0.53 |
| Total plasma protein (g/dL) | 7.5 (± 0.9) | 7.4 (± 1.0) | 1.00* |
| Platelets (103/µL) | 293.9 (± 97.8) | 310.9 (± 84.5) | 0.50 |
| Leukocytes (103/µL) | 11.7 (± 4.9) | 9.6 (± 4.6) | 0.04* |
| Eosinophils (/µL3) | 676.8 (± 565.3) | 513.6 (± 372.6) | 0.21* |
| Lymphocytes (/µL) | 1,994.3 (± 914.7) | 1,587.9 (± 636.1) | 0.06 |
| Monocytes (/µL) | 658.2 (± 336.4) | 512.5 (± 324.6) | 0.20* |
| Neutrophils (/µL) | 8,369.3 (± 4,439.0) | 6,993.9 (± 4,155.8) | 0.08* |
| Albumin (g/dL) | 3.0 (± 0.3) | 3.0 (± 0.3) | 0.94 |
| Alanine aminotransferase (UI) | 36.9 (± 17.7) | 34.4 (± 12.5) | 0.49 |
| Creatinine (mg/dL) | 1.0 (± 0.2) | 1.0 (± 0.3) | 0.49 |
| Alkaline phosphatase (UI) | 103.1 (± 166.3) | 106.5 (± 161.1) | 0.86* |
| Urea (mg/dL) | 34.4 (± 14.2) | 30.2 (± 11.3) | 0.28 |
*Wilcoxon test for paired samples. Other values were calculated using Student’s t-test for paired samples.
Isolated episodes of cytopenia were observed, such as the occurrence of grade 1 anemia in five (26.3%) bitches after the start of DCA administration, with a reduction in total hematocrit values (ranging from 32% to 35%) (Table 1). The hemoglobin value decreased in two (10.5%) cases, from 16.9 to 11.0 g/dL and 15.4 to 11.1 g/dL (reference value: 12.0 to 18.0 g/dL). There were no cases of neutropenia or thrombocytopenia during DCA treatment.
Gastrointestinal adverse effects
Two dogs experienced grade 1 vomiting after seven days of DCA treatment, and one episode of grade 1 diarrhea was observed. There were no cases of anorexia or inappetence. The vomiting and diarrhea had a spontaneous resolution, and it was not necessary to discontinue the medication.
Tumor volume
At the time of the first evaluation, the mean volume of the nodules was 33.3 cm3 (SD= ± 41.9), ranging from 1.7 cm3 to 107.8 cm3. The tumor volume response to DCA treatment compared initial and final tumor volumes. Nine cases showed a reduction in tumor volume (47.4%), while nine bitches (47.4%) maintained stable disease (Fig. 1). Only one bitch had progressive disease with a 267% increase in initial tumor volume (Table 2).
Fig. 1.
A female dog with grade I tubular carcinoma at various stages of dichloroacetic acid (DCA) treatment: day 0 (A), day 7 (B), and day 15 (C) (case 9 detailed in Table 2). The dog exclusively received DCA during the treatment, resulting in a 30.8% reduction in the total tumor volume. Observable changes include a decrease in size, ulceration area, and inflammation throughout the treatment. Following the completion of DCA treatment, the patient underwent ovariohysterectomy before mastectomy; hence, the suture is visible in Fig. 1C.
Table 2. Distribution of tumors according to histopathological classification, immunohistochemical evaluation, preoperative tumor volume before (day 0) and after (day 15) treatment with sodium dichloroacetate (DCA) (n=19), and response to treatment.
| Case | Histopathological classification | Immunohistochemistry | Preoperatory tumor volume | Percentage of reduction (−) or increase (+) in tumor volume | Response classification | ||
|---|---|---|---|---|---|---|---|
| Ki67 (%) | Score COX-2 | Before DCA treatment (day 0) | After DCA treatment (day 15) | ||||
| 1 | Solid carcinoma | >20 | 2 | 13.8 | 1.3 | −90.5 | Reduction |
| 2 | Complex adenoma | <20 | 0 | 34.2 | 8.2 | −76.0 | Reduction |
| 3 | Solid carcinoma grade I | >20 | 2 | 95.6 | 25.7 | −73.1 | Reduction |
| 4 | Fibroadenoma | <20 | 2 | 1.4 | 0.5 | −64.2 | Reduction |
| 5 | Benign mixed tumor | <20 | 1 | 52.5 | 28.9 | −44.9 | Reduction |
| 6 | Carcinoma in mixed tumor grade II | <20 | 0 | 33.2 | 18.8 | −43.4 | Reduction |
| 7 | Carcinoma in mixed tumor grade I | >20 | 1 | 1.0 | 0.6 | −40.0 | Reduction |
| 8 | Carcinoma in mixed tumor | <20 | 1 | 13.9 | 9.2 | −33.8 | Reduction |
| 9 | Tubular carcinoma grade I | <20 | 1 | 3.9 | 2.7 | −30.8 | Reduction |
| 10 | Solid carcinoma | >20 | 4 | 16.7 | 11.7 | −29.9 | Stable |
| 11 | Complex carcinoma grade II | <20 | 3 | 85.7 | 68.2 | −20.4 | Stable |
| 12 | Carcinoma in mixed tumor grade I | <20 | 3 | 1.0 | 0.8 | −20.0 | Stable |
| 13 | in situ carcinoma | >20 | 4 | 2.1 | 1.7 | −19.0 | Stable |
| 14 | Carcinoma in mixed tumor | <20 | 1 | 3.2 | 2.7 | −15.6 | Stable |
| 15 | Solid carcinoma | <20 | 2 | 63.5 | 57.2 | −9.9 | Stable |
| 16 | Carcinoma in mixed tumor | >20 | 3 | 107.8 | 98.1 | −9.0 | Stable |
| 17 | Solid carcinoma | <20 | 0 | 124.9 | 121.2 | −3.0 | Stable |
| 18 | Solid carcinoma grade II | <20 | 0 | 8.0 | 7.8 | −2.5 | Stable |
| 19 | Carcinoma in mixed tumor | <20 | 0 | 107.7 | 366.0 | +239.0 | Progression |
Histopathological diagnosis and immunohistochemical evaluation
The most frequently observed histopathological diagnosis was carcinoma in mixed tumor (n=7, 36.8%), followed by solid carcinoma (n=6, 31.6%). Most selected cases (n=16, 84.2%) were malignant mammary gland tumors. Evaluation of axillary and inguinal lymph nodes was performed in eight cases (42.1%), revealing metastasis in one case (grade II carcinoma in mixed tumor) (5.2%) (Table 2).
Table 3 displays the immunohistochemical classification based on the evaluation of the Ki67 nuclear marker and COX-2 score. Results indicate that stability or increase in tumor volume after DCA treatment was significantly associated with a COX-2 score of 3 and 4 when compared to a score of 0 to 2. Furthermore, patients with a COX-2 score of 3 and 4 had a significantly lower mean percentage reduction in tumor volume after DCA treatment when compared to those with a score of 0 to 2.
Table 3. Comparison of immunohistochemical patterns (Ki67 and COX-2 score), in bitches with mammary gland tumor, according to clinical characteristics and preoperative tumor volume after sodium dichloroacetate (DCA) treatment.
| Variables | Ki67 (%) | COX-2 score | ||||||
|---|---|---|---|---|---|---|---|---|
| ≥20 | <20 | P | 3 and 4 | 0 to 2 | P | |||
| Mean (SD) | Mean (SD) | Mean (SD) | Mean (SD) | |||||
| Age (years) | 9.7 (2.7) | 9.4 (2.3) | 0.83* | 10.6 (1.8) | 9.1 (2.6) | 0.25* | ||
| Weight (Kg) | 11.2 (10.8) | 16.6 (8.1) | 0.22** | 14.7 (10.5) | 15.0 (9.0) | 0.95* | ||
| Percentage of tumor reduction after treatment | 43.6 (31.9) | 30.4 (23.3) | 0.33 | 19.7 (7.4) | 40.6 (28.8) | 0.03 | ||
| N (%) | N (%) | P# | N (%) | N (%) | P# | |||
| Neutered | ||||||||
| No | 5 (83.3) | 8 (61.5) | 0.61 | 3 (60.0) | 10 (71.4) | 1.00 | ||
| Yes | 1 (16.7) | 5 (38.5) | 2 (40.0) | 4 (28.6) | ||||
| Number of lesions | ||||||||
| 2 or more | 4 (66.7) | 11 (84.6) | 5 (100.0) | 10 (71.4) | 0.53 | |||
| 1 | 2 (33.3) | 2 (15.4) | 0.56 | 0 (0.0) | 4 (28.6) | |||
| Skin ulceration | ||||||||
| Yes | 1 (16.7) | 6 (46.2) | 0.33 | 1 (20.0) | 6 (42.9) | 0.60 | ||
| No | 5 (83.3) | 7 (53.8) | 4 (80.0) | 8 (57.1) | ||||
| Metastasis | ||||||||
| Yes | 1 (16.7) | 2 (15.4) | 1.00 | 1 (20.0) | 2 (14.3) | 1.00 | ||
| No | 5 (83.3) | 11 (84.6) | 4 (80.0) | 12 (85.7) | ||||
| Tumor volume after DCA treatment | ||||||||
| Stable disease or progression | 3 (50) | 7 (53.8) | 1.00 | 5 (100) | 5 (35.7) | 0.03 | ||
| Reduction | 3 (50) | 6 (46.2) | 0 (0.0) | 9 (64.3) | ||||
*Student’s t-test. **Mann-Whitney U test. #Fisher’s exact test.
DISCUSSION
This study showed that DCA promoted a cytoreductive effect in approximately half of the cases. Similar results were obtained with cell lines from canine mammary tumors, in which treatment with DCA led to a reduction in proliferation and metastatic potential for all cell lines [7]. In vitro and in vivo experiments demonstrated that DCA can inhibit tumor growth and reduce metastasis in mammary tumor cell lines in mice. This effect is dose and time-dependent, with higher doses and longer administration times resulting in better outcomes [21].
The selected dogs had an average age of 10 years. Adult and elderly bitches are more predisposed to mammary gland tumors, possibly due to the higher likelihood of mutations throughout life, leading to the development of solid tumors [6]. Although there is no consensus, the reproductive status may influence the incidence of mammary tumors in bitches, with intact animals being more predisposed to mammary tumors [3, 6]. In this study, most of the bitches treated were intact or underwent late spaying; which is consistent with previous studies [19, 24].
The caudal glands (caudal abdominal and inguinal) showed a higher occurrence of multiple mammary tumors in this study. The higher concentration of mammary tissue in the caudal glands justifies the increase in lesions in this region and has been widely described [18, 19].
The malignancy grade of a tumor is determined by several characteristics such as differentiation, mitosis figures, and cell count. Mammary tumors can affect multiple glands with different histopathology classifications [3, 6]. The histopathological evaluation of patients in this study revealed a higher incidence of mixed mammary tumors, with most diagnoses classified as malignant. Table 3 shows seven dogs with carcinoma in mixed tumors varying from grade I to II (cases 6, 7, 8, 12, 14, 16, and 19). The development of a mixed mammary tumor involves two cell types (epithelial and mesenchymal) and the classification in grade is important for prognosis [3, 18]. A reduction in tumor size was observed in five dogs with benign tumors (Table 2). This observation may be linked to the inherently lower malignancy of these tumors, suggesting a more favorable overall response following chemotherapy treatment or surgery [2, 3, 5].
The expression of the inducible enzyme COX-2 has been correlated with increased angiogenesis, proliferation, and metastatic potential in canine mammary tumors [1, 14]. COX-2 staining was evaluated using a semiquantitative system proposed by Lavalle et al. (2009), which assigns scores based on the intensity of COX-2 staining and the percentage of COX-2 positive cells. Although none of the samples in this study showed high immunoreactivity to COX-2 (score >6) due to reduced staining intensity in the evaluated samples, samples with scores of 3 or 4 were significantly associated with stable disease or progression [9]. The low COX-2 scores identified in the samples of this study could serve as a favorable prognostic factor for these animals. Given that, this marker is associated with reduced survival and a higher occurrence of metastasis in dogs [2, 9, 14], it may also influence the treatment response. It was observed that dogs with higher COX-2 scores (ranging from 3 to 4) exhibited stable disease or progression after DCA treatment (Table 3).
Solid carcinoma and carcinoma in mixed tumors were the most likely to reveal positive cells for Ki67 in previous studies [3, 18]. In this study, there was no statistically significant association between Ki67 immunohistochemical staining and tumor characteristics or clinical response after DCA treatment, possibly to the small sample size. Ki67 is a nuclear marker used as a proliferation index of canine mammary tumors and is related to the prognosis [8, 15].
DCA has been found to target cells that use glycolysis for energy, making it a potential targeted therapy [13]. Its targeted action on tumor cells may reduce the rate of side effects compared to traditional chemotherapy [21]. While adverse effects associated with DCA, such as vomiting, diarrhea, and apathy, have been reported, they were transient and of low intensity in this study [4, 11]. No adverse gastrointestinal effects were reported that prevented the continuation of treatment.
In mice, an effective antineoplastic dose of DCA was found to be 200 mg/kg, which reduced the rate of metastasis and tumor volume in mice with implanted mammary tumors [21]. However, due to the limited clinical studies in canine species, a higher dose of DCA for dogs may carry an elevated risk of adverse effects. The DCA dose described as safe for dogs ranges from 6.25 to 12.5 mg/kg [11]. The dose used in this study was within the safety range for dogs (10 mg/kg).
Hematologic toxicity is an important factor in the evaluation of targeted therapy. This study observed a significant reduction in mean hematocrit and total leukocyte concentration. Five dogs presented grade 1 anemia after DCA treatment, while no animal had individual leukopenia. The occurrence of anemia have been reported in dogs treated with DCA, and should be considered when assessing toxicity [4, 11]. Although the precise mechanism of anemia related to DCA administration is not entirely clear, one potential explanation is alterations in the multiplication and maturation of erythrocytes due to DCA treatment [4]. The use of doses above 90 mg/kg may cause leukopenia, thrombocytopenia, and hepatotoxicity in dogs treated with DCA [4].
Variation in tumor volume should be evaluated with caution, as several factors contribute to the final volume of a solid tumor, such as the estrous cycle, connective tissue edema, inflammation, or associated mastitis [6, 24]. Despite external factors, there was a promising effect of DCA treatment in this study. The evaluation of solid mammary tumors demonstrated a cytoreductive potential of DCA in nine cases that showed a reduction in tumor volume. In one case, the reduction was more than 90% in tumor volume after treatment. Reductions in erythema and swelling of the tumors were also observed, which may indicate an anti-inflammatory side effect of DCA on mammary tumors in bitches. It is important to emphasize that only one case had a disease classified as progressive, with an increase in tumor volume.
The mechanism responsible for the reduction in tumor volume with DCA treatment still requires further clarification. One in vitro study suggested that DCA’s cytotoxic potential for mammary tumor cells might be due to a decrease in cell proliferation by mechanisms not associated with apoptosis [7]. Furthermore, the effectiveness of DCA appears to be dependent on both the dose and duration of treatment, with high doses decreasing the likelihood of metastases in 58% of breast cancer cell lines [17]. The duration of treatment utilized in this study was derived from a previous investigation that assessed the toxicity of dogs receiving DCA [11]. The 15-day period proved to be safe for the treated dogs, and the dosage was deemed appropriate. It is conceivable that longer treatment durations with higher doses may potentially result in more favorable outcomes in future studies.
This study had limitations, such as sample size and follow-up time. Additionally, the complexity of the etiology and multiple histopathological diagnoses may compromised the assessment of response to antineoplastic treatments for mammary gland tumors [24]. The concomitant treatment of benign and malignant tumors may also have influenced the final cytoreduction result. Furthermore, due to the study’s duration, it was not possible to evaluate the occurrence of long-term distant metastases in bitches treated with DCA.
The results of this study demonstrate a potential beneficial cytoreductive effect with the use of DCA as preoperative neoadjuvant therapy in the treatment of mammary gland tumors in bitches. Adverse effects associated with DCA were transient and of low intensity, and did not interfere with treatment continuation. Additionally, reductions in tumor erythema and edema were observed after DCA treatment.
CONFLICT OF INTEREST
The authors declare no conflicts of interest.
Acknowledgments
This study was funded by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior–Brasil (CAPES)–Finance Code 001.
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