Abstract
Background/Aim: The expression level of the voltage-dependent potassium channel Kv 11.1 was shown to be associated with the clinicopathological features, aggressiveness, and prognosis of human breast cancer. Canine mammary gland tumor (cMGT) is the most common tumor type in intact female dogs; however, the significance of Kv 11.1 in cMGT is unknown. The aim of this study was to identify Kv 11.1 expression in 57 benign and malignant cMGT tissues from dogs and to investigate the correlation of Kv 11.1 expression with the clinicopathological parameters and prognosis of cMGT. Materials and Methods: A total of 57 samples were collected from cMGTs surgically resected at the Veterinary Medical Teaching Hospital, Seoul National University and subjected to immunohistochemistry assay using rabbit anti-Kv 11.1 polyclonal antibody. Immunohistochemical staining results were evaluated as the sum of intensity and percentage scores. The correlation between immunohistochemistry scores and clinicopathological parameters was investigated. Results: Immunohistochemical analysis revealed that Kv 11.1 immunoreactivity was higher in benign cMGTs than in malignant cMGTs. Kv 11.1 expression was significantly associated with tumor malignancy (p<0.001), tumor size (p<0.001), histological grade (p<0.05), and age at the time of mastectomy (p<0.05). Conclusion: This study presents the first evidence of Kv 11.1 expression in cMGTs and indicates an inverse correlation between Kv 11.1 expression and tumor malignancy. Kv 11.1 expression can be used as a prognostic biomarker and a tool for the management of cMGTs.
Mammary gland tumors (MGTs) are the most prevalent type of tumor in intact female dogs (1). MGTs have different histological types and biological features, and their heterogeneity is reflected in the differences in their prognosis and treatment options (2, 3). Surgery is the treatment of choice for female dogs with MGTs, except for those with inflammatory carcinomas (4). Adjuvant postoperative chemotherapy is usually administered to women with breast cancer to increase their survival rate, and many advances have been made in the treatment of human breast cancer. In contrast, postoperative chemotherapy is not routinely administered to dogs (5).
There are only a few previous studies on the efficacy of adjuvant postoperative chemotherapy in reducing recurrence and prolonging survival in dogs with MGT. Due to the debate on the efficacy of chemotherapy as adjuvant therapy and its significant side effects, non-chemotherapeutic drugs such as desmopressin and cyclooxygenase inhibitors are actively studied in canine research (6-9), and an additional functional biomarker with novel target therapy is vital for dogs.
Previous studies have confirmed that ion channels and pumps regulate membrane potential, ion homeostasis, and electrical signaling in excitatory cells and play important roles in cell proliferation, migration, apoptosis, and differentiation (10-12). Furthermore, previous studies have shown that ion channels are novel biomarkers and targets for cancer therapy due to their easy druggability (13, 14).
The voltage-dependent potassium channel Kv 11.1 [human ether-a-go-go-related gene (hERG)] is over-expressed in neoplastic cell lines and human primary tumors (15-18). It is also over-expressed in human breast cancers of different histogenesis, and its expression level was reported to be associated with the clinicopathological features, aggressiveness, and prognosis of cancers (19, 20).
The expression of canine ether-a-go-go–related gene (cERG), a polypeptide that is 97% similar to hERG, has been detected in various tissues, including the heart, brain, and kidney (21). Numerous studies have been published on the expression of cERG in dogs with cardiac disease (22); however, there are no studies on Kv 11.1 expression in dogs with MGT.
Canine MGT (cMGT) and human breast cancer have similar features, including the histological appearance and biological behavior (23). Furthermore, due to the similarity between cERG and hERG, cERG can be detected using an anti-hERG primary antibody (24). Therefore, we believe that Kv 11.1 (hERG) is expressed in cMGTs and that investigating the expression level of Kv 11.1 will yield useful clinical data.
The aim of this study was to identify Kv 11.1 on cMGT from surgical cases and to investigate the correlation of Kv 11.1 expression with the clinicopathological parameters and prognosis of cMGT.
Materials and Methods
This study was approved by the Institutional Animal Care and Use Committee of Seoul National University (study approval number: SNU-200217-1). Written informed consent was obtained from dog owners before sampling.
Tissue samples and data collection. Fifty-seven cMGT samples were collected via surgical resection between May 2003 and February 2020 at Veterinary Medical Teaching Hospital, Seoul National University. The surgical specimens were fixed in 10% neutral buffered formalin, processed, and embedded in paraffin according to the standard protocol. Histological diagnosis was performed by a pathologist at the Veterinary Pathology Laboratory of Seoul National University or IDEXX Laboratories. Histological type was determined according to the criteria described by Goldschmidt et al. (25).
Clinical data, including dog breed, age, body weight, history of spaying, tumor size (diameter of largest tumor mass), presence of metastasis in the follow-up period, and prognostic outcome, were obtained from medical records or during telephone interviews with the dog owners. Metastasis and tumor recurrence were evaluated using palpation, thoracic radiography, abdominal ultrasonography, and fine-needle aspiration. If necessary, biopsy or computed tomography was performed. Disease-free survival (DFS) was calculated from the date of primary surgery to the date of detection of the first local recurrence or metastasis. Overall survival (OS) was defined as the time from surgery to death.
Antibody. Rabbit anti-Kv 11.1 polyclonal antibody with species reactivity to dogs was used for the detection of Kv 11.1 (1:180, MBS247167; Mybiosource, San Diego, CA, USA) according to the manufacturer’s instructions.
Immunohistochemistry (IHC). Serial 4-μm sections were mounted on amino-silane-coated slides. The sections were deparaffinized with xylene and rehydrated with graded ethanol Antigen retrieval was performed with citrate buffer (pH 6.0) for 20 min using a pressure cooker. Endogenous peroxidase activity was blocked with 0.3% H2O2, and nonspecific binding of antibodies was blocked by preincubation with normal goat serum.
To stain for Kv 11.1, the sections were incubated with primary antibody against Kv 11.1 overnight at 4°. After immunostaining steps were performed with a commercially available rabbit-specific horseradish peroxidase/diaminobenzidine detection IHC kit (AB64261; Abcam, Cambridge, UK) according to the manufacturer’s instructions, the sections were counterstained with Mayer’s hematoxylin. Negative controls were prepared by omitting the primary antibody.
Image analysis and scoring. Cells with brownish membrane and/or cytoplasm after immunostaining were considered positive for Kv 11.1. The immunohistochemical staining results were evaluated as the sum of the intensity and percentage scores. The percentage score was graded as 0 (0-10%), 1 (11-40%), 2 (41-80%), or 3 (81-100%) based on the proportion of positive-labeled cells among 1,000 randomly counted cells at 400× magnification.
Staining intensity was estimated by optical density (OD) using ImageJ (National Institutes of Health, Bethesda, MD, USA). OD was classified according to the criteria described by Koopman et al. (26). Staining intensity was rated on a scale of 0 to 3 (0: 0<OD≤0.085, 1+: 0.085<OD≤0.160, 2+: 0.160<OD≤0.259, and 3+: OD>0.259). Grading produced a final score (FS) of 0 to 6, and samples with FS>0 were classified as Kv 11.1 positive.
Statistical analysis. Statistical analyses were performed using SPSS (version 26.0 for Windows; IBM Inc., Armonk, NY, USA). Results of the image analysis, expressed as the mean±SD of the FS, were analyzed for their associations. Continuous variables were analyzed using Student’s t-test or Mann-Whitney U-test. Kv 11.1 expression was compared between the histopathological types using Kruskal-Wallis test. Survival analyses were performed using the log-rank test and Kaplan-Meier plots. Statistical significance was set at p<0.05.
Results
Clinical and histopathological features of dogs with cMGT. A total of 57 dogs with cMGT (41 intact female dogs and 16 female dogs spayed at 4-12 years of age) were assessed in this study. The mean age of the dogs at the time of surgery for MGT was 11 years (range=6-16 years). The breed distribution was as follows: 14 Maltese, 11 Yorkshire Terriers, 9 Shih-tzus, 6 Poodles, 4 mixed, 4 Dachshunds, 3 Schnauzers, 2 Jindos, 1 Cocker Spaniel, 1 Malinois, 1 Chihuahua, and 1 Japanese Spitz. The mean body weight was 5.1 kg (range=1.2-27.0 kg). Histological examination of 28 benign and 29 malignant samples was performed. The benign tumors included 12 complex adenomas, 8 mixed tumors, and 8 simple adenomas. The malignant tumors included 11 simple carcinomas, 10 complex carcinomas, 6 carcinosarcomas, 1 mixed carcinoma, and 1 adenosquamous carcinoma. Comparison of signalment data of benign and malignant MGTs in the 57 dogs is shown in Table I.
Comparison of signalment data between dogs with benign and those with malignant mammary gland tumors.
Kv 11.1 expression in MGTs. Immunohistochemistry staining of Kv 11.1 was mainly expressed in the cytoplasm of epithelial cells in the canine mammary glands. Representative images of the IHC assay performed with anti-Kv 11.1 antibody, relative to FS, are shown in Figure 1. Kv 11.1 expression (FS>0) was observed in 41 of the 57 (71.9%) dogs. All the benign tissues showed immunostaining; however, Kv 11.1 expression was observed in only 13 of the 29 (44.8%) malignant samples.
Representative images of immunohistochemical analysis performed using the anti-Kv 11.1 antibody. (A) No specific staining in the negative control. (B) Malignant mammary gland tumor (MGT) (complex carcinoma) showing no Kv 11.1 expression. (C) Malignant MGT (complex carcinoma) with final score (FS)=1 showing Kv 11.1 expression. (D) Benign MGT (simple adenoma) with FS=3 showing Kv 11.1 expression. (E) Malignant MGT (simple carcinoma) with FS=4 showing Kv 11.1 expression. (F) Benign MGT (complex adenoma) with FS=6 showing Kv 11.1 expression. Original magnification ×400, scale bar=50 micrometers.
Based on the scoring system used, the mean FS of all 57 samples was 2.58±2.05. The mean FS of the benign tumors was significantly higher than that of the malignant tumors (4.11±1.07 versus 1.10±1.66, p<0.001).
Associations between Kv 11.1 expression and other clinicopathological variables. Associations between Kv 11.1 expression and the clinicopathological features of cMGT are presented in Table II. There was no significant association between Kv 11.1 expression and factors such as metastasis during the follow-up period or spaying before/after mastectomy. However, with respect to the histological grade of malignancy, Kv 11.1 expression was significantly higher in grade I and II tumors than in grade III tumors (p<0.05). Tumor diameter >3 cm is known to be associated with MGT malignancy (3), and Kv 11.1 expression was found to be significantly lower in cMGTs of diameter >3 cm than in cMGTs of diameter ≤3 cm (p<0.001). Furthermore, Kv 11.1 expression was significantly lower in dogs that underwent surgery at an older age (≥10 years) than in dogs that underwent surgery before 10 years of age (p<0.05).
Clinicopathological variables associated with Kv 11.1 expression.
Kv 11.1 expression and survival. Kaplan-Meier estimates of the DFS and OS of the 29 dogs with malignant MGTs according to Kv 11.1 expression were analyzed (Figure 2). Of the 29 dogs with malignant cMGTs, 16 died, and the remaining 13 were lost to follow-up or were alive during the entire study period. The 13 dogs had positive Kv 11.1 expression, whereas the 16 dogs had negative Kv 11.1 expression. The median DFS and OS of the dogs with positive Kv 11.1 expression were 22 and 25 months, respectively. In contrast, the median DFS and OS of the dogs with negative Kv 11.1 expression were 14 and 24 months, respectively. These findings indicate that positive Kv 11.1 expression may be associated with longer DFS and OS than negative Kv 11.1 expression, even though statistical significance was not reached (p=0.581 and p=0.332, respectively).
Kaplan-Meier survival curve of (A) disease-free survival and (B) overall survival based on Kv 11.1 expression in 29 dogs with malignant mammary gland tumor.
Discussion
Human studies have suggested that Kv 11.1 can serve as a cancer biomarker for diagnostic and prognostic purposes (14, 16, 17). A recent study on human breast cancer found that high expression level of Kv 11.1 is associated with low cancer malignancy and other positive survival indicators (20). Another study detected Kv 11.1 expression in dog tissues, including heart, brain, and kidney tissues (21). However, Kv 11.1 expression in dogs with cancer, including cMGTs, has never been investigated. The histopathological, molecular, and clinical features of cMGTs and human breast cancers are quite similar (23). In this study, we identified similarities between cMGTs and human breast cancers.
The results of this study showed that Kv 11.1 is expressed in cMGTs. Furthermore, Kv 11.1 immunoreactivity was significantly higher in benign cMGTs than in malignant cMGTs. Grade III cMGTs had lower Kv 11.1 expression than grade I and II cMGTs. Moreover, Kv 11.1 expression was significantly lower in cMGTs >3 cm than in cMGTs ≤3 cm. The results showed that Kv 11.1 expression inversely correlates with cMGT malignancy and can be used as a cMGT malignancy biomarker. As benign cMGTs in dogs can gradually progress into malignant cMGTs (3), our findings suggest that loss of Kv 11.1 may be involved in the progression and malignant transformation of benign MGTs.
Kv 11.1 expression was positive in all the benign cMGT samples, but not in the malignant cMGT samples. Only 40% of the 10 grade I malignant cMGT samples in our study showed positive Kv 11.1 expression. Thus, Kv 11.1 expression may be used as a marker in histopathological examinations to differentiate benign and malignant MGTs with low malignancy. However, a greater number of grade I malignant MGT samples are needed in future studies to verify the effectiveness of Kv 11.1 expression as a marker.
Median DFS and OS were longer in dogs with positive Kv 11.1 expression than in dogs with negative Kv 11.1 expression. This finding suggests that Kv 11.1 expression may be associated with positive prognosis. However, as the statistical significance was low, it is necessary to conduct further studies using larger sample sizes over longer periods.
Our study also revealed that Kv 11.1 expression was low when surgical age was high. This finding could be attributed to the development of benign tumors at a younger age than malignant tumors (3).
We posit that hormonal effects on the mammary gland and the presence of metastasis correlate with Kv 11.1 expression level, but we did not observe any correlation between Kv 11.1 expression and spaying before/after mastectomy or the presence of metastasis in the follow-up period. In this study, most of the dogs that were spayed before mastectomy were neutered after 5 years of age. Spaying in old age may have little hormonal effect on the mammary gland compared to nonspayed dogs. Since the number of dogs with metastasis in this study was small for statistical analysis, further studies with larger sample sizes should be conducted.
A previous human in vitro study found that activating Kv 11.1 inhibited the proliferation of breast cancer cells by activating senescence. In that study, the Kv 11.1 activator NS 1643, a diphenylurea derivative, was found to irreversibly inhibit cell proliferation (27). Because the features of human breast cancer and cMGT are similar, a dog model can be used to identify human molecular markers (23). Based on the aforementioned similarities, future research may reveal that Kv 11.1 can prevent cMGT cell proliferation via a similar mechanism. Furthermore, future studies may show that Kv 11.1 activators can inhibit cMGT proliferation by acting on Kv 11.1 channels. Kv 11.1 activators could be used as antiproliferative drugs to delay the malignant transformation of benign MGTs or as adjuvant postoperative therapy.
In a previous canine heart-based study, the administration of the Kv 11.1 activator ICA-105574 resulted in drug-induced QT shortening due to Kv 11.1 activation (28). This finding suggests that Kv 11.1 activators can be used as canine medicine; however, they can also induce adverse cardiac effects. Future research on the proper use of Kv 11.1 activators to treat cMGT should focus on identifying methods to reduce the risk of cardiac adverse effects.
Due to the adverse effects and low efficacy of chemotherapy, efforts to find new medicines to treat cMGT are necessary. Future studies on Kv 11.1 activators may help increase the lifespan of dogs with cMGT and reduce the pain caused by various adverse effects of chemotherapy.
This is the first study to investigate Kv 11.1 expression in dogs with cMGT, and the study results showed that Kv 11.1 expression level can serve as a biomarker. We hope to support this research by conducting further studies with larger sample sizes and longer study periods in the future. Although this study focused on cMGTs, it may be helpful to include normal mammary gland tissues in future studies. We hope to supplement the results of this study by conducting further studies using real-time polymerase chain reaction or western blotting to improve our understanding of the role of Kv 11.1 in cMGTs.
Conclusion
This is the first study to demonstrate the presence of Kv 11.1 in cMGTs using IHC. We confirmed that Kv 11.1 expression is significantly higher in benign cMGTs compared to in malignant cMGTs. Regarding malignant cMGTs, Kv 11.1 expression was higher in grades I and II tumors than in grade III tumors, indicating that Kv 11.1 expression level may be used as a prognostic biomarker. In addition, detection of Kv 11.1 expression may have future applications, such as use of Kv 11.1 activators to treat cMGTs.
Acknowledgements
The Authors would like to thank Enago (https://www.enago.co.kr) for English language editing.
Footnotes
Authors’ Contributions
Conceptualization; Project Administration: NRL, Validation: SIL, WHK. Supervision: WHK.
Conflicts of Interest
The Authors declare no conflicts of interest in relation to this study.
Funding
The current work was funded by the Research Institute for Veterinary Science, Seoul National University, Seoul, Republic of Korea.
- Received September 11, 2023.
- Revision received October 10, 2023.
- Accepted October 11, 2023.
- Copyright © 2024 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
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