Recibido: 19/06/2025 Aceptado: 15/10/2025 Publicado: 04/12/2025 UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico 1 of 5 UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico Hipometilación genómica global en tumores de mama canino Alicia Decuadro* 1 , María del Carmen Montenegro 2 , Silvia Llambí 2 , Nariné Balemian 2 , Mónica Cappetta* 3 ¹ Universidad de la República, Facultad de Medicina Veterinaria, Unidad Académica de Clínica de Pequeños Animales. Ruta 8, Km 18 Montevideo, Uruguay. ² Universidad de la República, Facultad de Medicina Veterinaria, Unidad Académica de Genética y Mejoramiento Animal. Ruta 8, Km 18 Montevideo, Uruguay. ³ Universidad de la República, Facultad de Medicina, Unidad Académica de Genética. Av. General Flores 2125, Montevideo, Uruguay. *Autor correspondencia: oncologiafvetudelar@gmail.com ABSTRACT Due to its inﬂuence in transcriponal potenal of genes, genec regulaon by means of epigenec mechanisms is essenal for normal growth and development. In mammary cancer, epigenec modiﬁcaons play a key role for its development and progression. In early carcinogenesis stages, due to genec alteraons or environmental factors, chroman structure alteraons due to DNA methylaon and post-translaonal modiﬁcaons of DNA-bound proteins may appear. As with other types of tumor, genome-wide hypomethylaon and hypermethylaon of speciﬁc genes, parcularly in CpG islands that normally are not methylated, are observed. In order to compare global DNA methylaon levels between tumor ssue and normal mammary ssue, we studied 11 intact female dogs with mammary tumors. Both types of ssue were collected during surgery, with subsequent clinical staging and histopathological classiﬁcaon of tumors. For each animal, DNA was extracted from paired samples of tumor ssue and normal mammary ssue. Global genomic methylaon levels were calculated by relave quantaon of 5-methyl-2’-deoxycydine (5mdC) with HPLC. Results showed that tumoral ssue had a global DNA hypomethylaon when compared with normal mammary ssue (P < 0.05). This diﬀerence was greater in high histopathological grade tumors, characterized by their aggressive clinical behavior and high metastac rate. These ﬁndings underscore the importance of addional studies in this line of research, with greater sample sizes. In the future, global DNA methylaon may be used as a prognosis biomarker for mammary cancer in dogs. Key words: Canine mammary tumors; epigenecs; global DNA methylaon RESUMEN La regulación génica mediante mecanismos epigenécos es esencial para el crecimiento y el desarrollo normal, ya que inﬂuye en el potencial transcripcional de los genes. En el cáncer de mama, las modiﬁcaciones epigenécas desempeñan un papel clave en su desarrollo y progresión. En etapas tempranas de la carcinogénesis, como consecuencia de lesiones genécas o factores ambientales, se producen alteraciones en la estructura de la cromana a través de la melación del ADN y de modiﬁcaciones postraduccionales de proteínas unidas al ADN. Al igual que varios tumores, se observa una hipomelación a nivel genómico y la hipermelación de ciertos genes, especialmente en islas CpG que normalmente no se encuentran meladas. Con el objevo de comparar el nivel de melación global del ADN entre tejido tumoral y tejido mamario sano, se estudiaron 11 perras enteras con tumores de mama. Ambos pos de tejido fueron extraídos durante la cirugía, y se realizó la estadiﬁcación clínica y clasiﬁcación histopatológica de los tumores. A parr de muestras pareadas de tejido tumoral y tejido mamario sano de cada animal, se extrajo ADN y se determinaron los niveles de melación genómica global mediante cuanﬁcación relava de 5-mel 2-desoxicidina (5mdC) ulizando HPLC. Los resultados revelaron una hipomelación global del ADN en el tejido tumoral en comparación con el tejido mamario sano (P < 0.05). Esta diferencia fue más pronunciada en tumores de alto grado histopatológico, caracterizados por un comportamiento clínico agresivo y una elevada tasa de metástasis. Estos hallazgos subrayan la importancia de connuar con esta línea de invesgación, ampliando el tamaño muestral. En el futuro, la melación global del ADN podría ulizarse como un biomarcador pronósco del cáncer de mama en perros. Palabras clave: Tumores de mama en caninos; epigenéca; melación global del ADN https://doi.org/10.52973/rcfcv-e361726 Revist a Científica, FCV-LUZ / Vol. XXXVI Global DNA hypomethylation in canine mammary tumors

Revista Cienﬁca, FCV-LUZ / Vol. XXXVI UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico INTRODUCTION Epigenecs refers to changes in genec expression impacng on phenotype and that involve no modiﬁcaons of the DNA sequence [1 ,[2]. Epigenec processes include DNA methylaon, histone post-translaonal modiﬁcaons, non-coding RNA funcons, and co-transcriponal modiﬁcaon of diﬀerent RNA molecules (mRNA, rRNA, tRNA, RNAmi or lncRNA) [3]. Genec regulaon by means of epigenec mechanisms is essenal for normal growth and development, condioning transcriponal potenal of genes. In normal cells, DNA methylaon in necessary for maintenance of cellular growth and metabolism, while abnormal DNA methylaon may cause diﬀerent diseases, like cancer [1]. In cancer, DNA methylaon is one of the most frequently altered epigenec processes [4 ,5]. DNA methylaon consists in a covalent chemical modiﬁcaon that adds a methyl group (CH3) to the ﬁﬅh carbon of the cytosine pyrimidine ring, leading to the formaon of 5-methylcytosine (5- me-C). This process is catalyzed by the DNA methyltransferases (DNMT), a family of enzymes that may carry out de novo DNA methylaon or establish methylaon of hemimethylated DNA [2 , 6]. In mammary cancer, epigenec modiﬁcaons are a key element in tumor development and progression. In early carcinogenesis stages, due to genec lesions or environmental impacts, alteraons in chroman structure take place through DNA methylaon and post-translaonal modiﬁcaons of DNA- bound proteins [7]. These alteraons impact cellular plascity and favor oncogenic reprogramming of tumor progenitor cells, promong the acquision of uncontrolled self-renewal properes. In later stages of cancer growth, further epigenec modiﬁcaons, combined with subclonal mutaons and microenvironmental signals, modulate tumor cell phenotype and inﬂuence its metastac propensity [8]. As for the role of DNA methylaon in mammary cancer, it has been demonstrated, similarly to cancer in general, both hypomethylaon at the genomic level and hypermethylaon of certain genes, parcularly in usually unmethylated CpG islands [2]. More than 100 genes with hypermethylated promoters have been idenﬁed; many of them are crical for tumor suppression, cell cycle regulaon, apoptosis, angiogenesis, ssue invasion, and metastasis [9]. On the other hand, DNA methylaon has been associated with clinical and pathological characteriscs of women with breast cancer, such as tumor clinical stage and grade. Addionally, speciﬁc methylaon proﬁles associated with diﬀerent breast cancer subtypes in women have been idenﬁed, suggesng they could play an important role in their development and progression [10]. Cancer is the best-characterized complex human disease associated with epigenec defects [11 ,[12], with epigenec modiﬁcaons observed in human tumors being shared with canines. This supports the possibility of using dogs (Canis lupus familiaris) as a model for studying such alteraons for diagnosis, prognosis and development of new therapies. Canines are an excellent study model, as they share the same environment with humans [13]. Humans and dogs share similar aspects of the disease, including spontaneous tumor development, hormonal components, age of incidence, and disease progression. Tumor size and lymph node invasion show no diﬀerences between both species. Likewise, humans and dogs share expression of certain factors such as steroid receptors, epidermal growth factors, proliferaon factors, and P53 mutaons [14]. DNA methylaon studies related to canine mammary tumor (CMT) development are scarce. Research includes the analysis of genome-wide methylaon proﬁles (methylome) [15] and quanﬁcaon of global DNA methylaon [16]. Brandão et al. [17] analyzed the DNA methylaon status of the ESR1 gene encoding estrogen receptor alpha in canine mammary tumors. Beetch et al. [18] analyzed samples from diﬀerent tumor stages, as well as from healthy donors, describing hypermethylaon paerns of cancer-related genes. In view of the above, this study aims to compare global DNA methylaon levels between tumor ssues and normal mammary ssue of female dogs with mammary tumors. MATERIALS AND METHODS The research was carried out at the Faculty of Veterinary Medicine Hospital, the Genecs and Animal Improvement Unit of the Faculty of Veterinary Medicine, and the Genecs Department of the Faculty of Medicine (University of the Republic). All procedures were approved under number 1383 by the Honorary Commission for Animal Experimentaon (CHEA). Eleven intact female dogs, with mammary tumors and ages ranging from 6 to 12 years of old that had been admied to the Hospital, were selected. Mammary tumor cases were selected according to the clinical characteriscs previously described in the species. The animals included in the study exhibited an acceptable general clinical condion and underwent pre-surgical studies (blood biochemistry and urinalysis) within reference ranges, which, combined with disease staging, allowed for the selecon of an appropriate surgical approach (anesthec risk classiﬁcaon: ASA I or II). Clinical staging of paents with mammary tumors was performed according to the TNM mammary tumor classiﬁcaon criteria of the World Health Organizaon (WHO) by means of triple view chest X-ray and abdomen ultrasound. Normal and tumoral ssue samples were collected the same day of the surgery, and were immediately stored in Eppendorf tubes with no soluon at -80°C in a freezer Thermo Fisher Scienﬁc, TSX Series (USA) unl their epigenec and histological and pathological analysis (TABLE I). Normal ssue samples were obtained from the mammary glands adjacent to those with tumors, using standardized sampling site criteria. This procedure was followed because regional excisions or radical mastectomies generally involve removal of normal mammary glands in the same surgical procedure, thus eliminang the need for addional biopsies for collecon of these types of samples. The DNA extracon from tumoral and normal mammary ssue samples was performed with the DNeasy Blood & Tissue kit (Qiagen). Extracted DNA concentraon and purity was measured with a DeNovix DS-11 spectrophotometer USA. Global genomic methylaon levels were measured by relave 2 of 5

Hypomethylaon in canine mammary tumors / Decuadro y cols. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico quanﬁcaon of 5-methyl-2’-deoxycydine (5mdC) with HPLC as described by Berdasco et al. [11] and Cappea et al. [12]. For enzymac hydrolysis, 1 μg of genomic DNA (in 10 μg of ultra- pure water) of each sample was denatured for 10 minutes (min) at 94 °C, followed by cooling on ice for 5 min. Subsequently, 1.5 μL of nuclease P1 (New England BioLabs), with its corresponding buﬀer was added, with subsequent incubaon at 37 °C for 15 hours (h) using a Mulgene II machine (Labnet Internaonal, Inc. USA Hydrolyzed DNA was incubated with alkaline phosphatase (Thermo Scienﬁc™ USA) for an addional hour at 37 °C and 10 min at 75 °C, according to the manufacturer›s instructions, in a reaction with a final volume of 50 μL. Nucleoside separaon following genomic hydrolysis was performed with an HPLC Agilent series 1100 equipment (USA) with UV detecon. 50 μL of hydrolyzed DNA were injected into a dC18 Atlans (Waters) reverse-phase column (2.1 × 20 mm; 5 mm parcle size), protected by a guard column (2.1 x 20 mm; 5 mm parcle size; Agilent), at a 0.150 mL/min constant ﬂow rate. The following buﬀers were used: 0.1 % formic acid in water (solvent A), and 0.1 % formic acid in water:methanol (40 %) (solvent B); in an isocrac gradient of 95 % solvent A and 5 % solvent B for 30 min. As standards, a 5 mM mixture of the following mononucleosides were used: deoxyadenosine (dA), deoxythymidine (dT), deoxyguanosine (dG), deoxycydine (dC), 5-methyl-2’-deoxycydine (5mdC), and deoxyuridine (dU) (Sigma-Aldrich). Quanﬁcaon of 5mdC and dC percentages was directly calculated from the area of their respecve peaks in the HPLC chromatogram. Samples were analyzed in duplicate, and those with a 5mdC content diﬀerences between duplicates greater than 3 % were excluded [12]. Global genomic DNA methylaon level is shown as the relave amount of 5mdC in total cydine residues; thereby, global genomic DNA methylaon percentage is: [mdC / (mdC + dC)] × 100. Stascal analysis The Shapiro-Wilk test was performed to assess normality of global methylaon percentage data distribuon. Upon conﬁrmaon of normality assumpons, Student’s t-test was performed. Cohen’s d eﬀect size was calculated to assess the magnitude of the diﬀerence between both groups [19]. RESULTS AND DISCUSSION Global genomic DNA methylaon was assessed in 11 female dogs of various breeds diagnosed with mammary cancer, comparing tumor ssue with normal mammary ssue from each animal studied. TABLE I summarizes age, breed, disease stage, histopathological classiﬁcaon and grade of tumors following surgery, as classiﬁed according to Goldshmidt et al. [20], as well as the DNA methylaon percentage of normal and tumor ssues. The histopathological analysis showed that 8 (72.7) and 3 (27.3 %) of the 11 female dogs analyzed had malignant and benign tumors respecvely. Malignant tumors comprised: 3 simple carcinomas, 2 mixed carcinomas, 1 papillary cysc carcinoma, 1 complex adenocarcinoma, and 1 solid carcinoma. Benign tumors corresponded to 2 mixed adenomas and 1 complex adenoma. This distribuon of tumor types is consistent with the most frequent tumors in CMT reported by Goldschmidt et al. [20]. TABLE I Descripon of paents by age, breed, disease stage, histopathological classiﬁcaon, and methylaon percentage in normal ssue and tumor ssue. Case Age Breed Stage Histopathological Grade Normal Tissue Methylaon Tumor Tissue Methylaon 1 10 Cimarrón III II Papillary cysc carcinoma 4.791892158 2.725887465 2 8 Mixed Breed III II Simple adenocarcinoma 2.144284287 2.336448598 3 12 Cocker III II Mixed carcinoma 2.413530977 2.285336856 4 8 Mixed Breed III III Simple adenocarcinoma 4.342321813 2.766662856 5 7 Mixed Breed III Benign Mixed adenoma 2.884515795 3.053574218 6 7 German Shepherd III II Complex adenocarcinoma 2.46986528 1.924168464 7 8 Pug I I Mixed carcinoma 3.143661972 2.307747267 8 8 German Shepherd I Benign Complex Adenoma 3.750999886 3.754702599 9 6 Poodle I Benign Mixed adenoma 3.879251734 3.617225363 10 8 Mixed Breed III II Simple Carcinoma 4.002230611 2.622711446 11 10 Poodle I II Solid Carcinoma 4.027690371 3.458671344 The breeds studied included 4 mixed breeds, 2 Poodles, 2 German Shepherds, 1 Cocker Spaniel, 1 Cimarrón, and 1 Pug. The prevalence of these breeds in the sample may reﬂect their overrepresentaon in the canine populaon of our country, which may vary according to geographical locaon [21]. Comparison of global genomic methylaon levels between normal ssue and tumor ssue from dogs histopathologically diagnosed with mammary tumors revealed a signiﬁcant diﬀerence (P< 0.05). Speciﬁcally, tumor ssues had lower methylaon levels than normal ssues animals (FIG. 1), indicang global hypomethylaon in tumor ssues of the analyzed This ﬁnding is consistent with exisng literature, where hypomethylaon is reported as a common epigenec alteraon in diﬀerent cancer types, including mammary cancer in women [12] and dogs [15 , 16], as well as other cancer types [15]. Cases 1, 4, and 10 showed the greatest diﬀerences in genomic methylaon percentages between tumor and normal ssue. These three female dogs (two mixed breeds and one 3 of 5

Revista Cienﬁca, FCV-LUZ / Vol. XXXVI UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico Cimarrón) were diagnosed with high histopathological grade tumors (grade III): one cysc papillary carcinoma and two simple adenocarcinomas. These tumors are characterized by their clinically aggressive behavior and high metastac rates. These results are consistent with those of Biondi et al. [16], who also observed signiﬁcant diﬀerences in global DNA methylaon paerns in CMT, where hypomethylaon was more frequent in malignant tumors. Although these authors used a diﬀerent detecon approach (5-methylcytosine immunostaining) and a larger sample size, the similarity of ﬁndings supports the existence of a relaonship between global DNA hypomethylaon and malignancy in canine mammary tumors, thus suggesng its potenal as a prognosc biomarker, parcularly in tumors with a more aggressive behavior and a higher relapse risk. FIGURE 1. Comparison of global DNA methylaon level percentages between normal mam- mary ssues and mammary tumor ssues. Boxes represent interquarle ranges, and lines across boxes the median values. Stascally signiﬁcant diﬀerences between normal mam- mary ssues and mammary tumors were calculated using Student’s t test (P < 0.05) The observed diﬀerence in methylaon between normal and tumor ssue in 11 of the samples, besides being stascally signiﬁcant, was also of considerable magnitude according to the calculated Cohen’s d eﬀect value of 0.84 (values above 0.8 indicate a large eﬀect). Although research on global genomic methylaon in female dogs with CMT is limited, there are studies focusing on methylaon in speciﬁc genes. For example, Brandão et al. [17] invesgated DNA methylaon status of the ESR1 gene encoding estrogen receptor alpha in CMT. Their ﬁndings showed no signiﬁcantly diﬀerences in ESR1 methylaon status between diﬀerent types of tumors, suggesng DNA methylaon would not be involved in ESR1 regulaon in canine mammary tumors. This contrasts with ﬁndings in human breast cancer, where DNA methylaon may have a signiﬁcant impact on ESR1 expression. Although this study focused in global genomic methylaon and not in speciﬁc genes such as ESR1, it is worth menoning the diﬀerenal eﬀect that methylaon may have at the global level, as well as the fact that modiﬁcaons in speciﬁc genes do not necessarily reﬂect global epigenec: modiﬁcaons. In addion, Jeong et al. [15] used bisulﬁte sequencing to invesgate whole genome methylaon proﬁles in both canine mammary tumors and peripheral blood mononuclear cells. Interesngly, they found unique methylaon signatures enriched in CMT, where genes associated with apoptoc pathways and transmembrane ion transport exhibited hypermethylaon, while genes involved in cellular proliferaon and oncogenes were hypermethylated in tumor ssues. The inclusion of peripheral blood samples by Jeong et al. [15] constutes a precedent for their use in CMT research. This poses a future line of research for our team, as these are less invasive samples that may be used as a suitable tool for diagnosis, prognosis and treatment monitoring. Beetch et al. [18] examined samples from various tumor stages alongside normal donor controls, characterizing hypermethylaon paerns in cancer-associated genes. Their ﬁndings demonstrated that genes involved in transcriponal regulaon, apoptoc processes, signal transducon pathways, and cellular migraon correlate with gene expression paerns and disease progression in canine paents. This suggests that methylaon proﬁling holds clinical potenal for disnguishing between diﬀerent stages of tumor progression. Although their approach diﬀers from ours, they agree that aggressive canine mammary tumors and benign tumors have diﬀerent methylaon paerns. Thus, DNA methylaon modiﬁcaons could guide disease progression monitoring, which is linked to prognosis. Human research focuses on achieving deeper understanding of epigenec mechanisms to obtain beer therapies, as well as leveraging therapies that promote global epigenec normalizaon to counteract epigenec aberraons. Such approaches promise to enhance the clinical ulity of epigenec therapeucs, opmizing research investment returns while improving paent outcomes [22]. Given the paucity of comparable studies in canine oncology, this ﬁndings contribute valuable data toward beer understanding the epigenec landscape of mammary tumor biology in dogs. CONCLUSIONS This study invesgated global DNA methylaon levels in female dogs with mammary tumors and normal mammary ssue (control samples). Lower DNA methylaon percentages were observed in tumor ssues, being this eﬀect more pronounced in high histopathological grade tumors when compared to normal ssue. Based on these results, we can conclude that tumor ssue DNA was hypomethylated when compared to normal ssue. This work highlights the importance of connuing this line of research with larger sample sizes, mainly so that in the future global DNA methylaon can be used as a biomarker to provide more accurate prognosis for this disease in dogs. ACKNOWLEDGMENTS This work was carried out in the Small Animal Clinic and Surgery Unit and the Academic Unit of Genecs and Animal Improvement at the Faculty of Veterinary Medicine, University of the Republic, and the Genecs Unit of the Faculty of Medicine. Funding was from the Sectoral Commission for Scienﬁc 4 of 5

Hypomethylaon in canine mammary tumors / Decuadro y cols. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico Research (SIC), the Research and Scienﬁc Development Commission of the Faculty of Veterinary Medicine (CIDER), and the Graduate Academic Programs of the Faculty of Veterinary Medicine, Uruguay. Conﬂict of interest The authors declare no conﬂicts of interest related to this report. BIBLIOGRAPHIC REFERENCES [1] Zhou WM, Liu B, Shavandi A, Li L, Song H, Zhang JY. Methylaon landscape: Targeng writer or eraser to discover an-cancer drug. Front Pharmacol. [Internet]. 2021; 12:690057. doi: hps://doi.org/qf7c [2] Karsli-Ceppioglu S, Dagdemir A, Judes G, Ngollo M, Penault-Llorca F, Pajon A, Bignon YJ, Bernard-Gallon D. Epigenec mechanisms of breast cancer: an update of the current knowledge. Epigenomics. [Internet]. 2014; 6(6):651-664. doi: hps://doi.org/gms37n [3] Bártová E. Epigenec and gene therapy in human and veterinary medicine. Environ. Epigenecs. [Internet]. 2024; 10(1):dvae006. doi: hps://doi.org/qf7f [4] Szczepanek J, Skorupa M, Jarkiewicz-Tretyn J, Cybulski C, Tretyn A. Harnessing Epigenecs for Breast Cancer Therapy: The Role of DNA Methylaon, Histone Modiﬁcaons, and MicroRNA. Int. J. Mol. Sci. [Internet]. 2023; 24(8):7235. doi:hps://doi.org/qf7h [5] Brookes E, Shi Y. Diverse epigenec mechanisms of human disease. Annu. Rev. Genet. [Internet]. 2014; 48:237-268. doi: hps://doi.org/qf7j [6] Nishiyama A, Nakanishi M. Navigang the DNA methylaon landscape of cancer. Trends Genet. [Internet]. 2021; 37(11):1012-1027. doi:hps://doi.org/ gm5thv [7] Menezo Y, Silvestris E, Dale B, Elder K. Oxidave stress and alteraons in DNA methylaon: two sides of the same coin in reproducon. Reprod. Biomed. Online. [Internet]. 2016; 33(6):668-683. doi: hps://doi.org/f9p3tc [8] Pasculli B, Barbano R, Parrella P. Epigenecs of breast cancer: Biology and clinical implicaon in the era of precision medicine. Semin. Cancer Biol. [Internet]. 2018; 51:22-35. doi: hps://doi.org/gd45tv [9] Wu Y, Sarkissyan M, Vadgama JV. Epigenecs in Breast and Prostate Cancer. In: Verma, M. (eds). Cancer Epigenecs. Methods Mol. Biol. [Internet]. 2015; 1238:425–466. doi: hps://doi.org/f6trhx [10] Győrﬀy B, Boai G, Fleischer T, Munkácsy G, Budczies J, Paladini L, Børresen-Dale A, Kristensen VN, Santarpia L. Aberrant DNA methylaon impacts gene expression and prognosis in breast cancer subtypes. Int. J. Cancer. [Internet]. 2016; 138(1):87-97. doi: hps://doi.org/ gjjd2s [11] Berdasco M, Fraga M, Esteller M. Quanﬁcaon of global DNA methylaon by capillary electrophoresis and mass spectrometry. In: Tost, J. (eds) DNA Methylaon. Methods Mol. Biol. [Internet]. 2009; 507:23–34. doi: hps://doi. org/cjz5p9 [12] Cappea M, Berdasco M, Hochmann J, Bonilla C, Sans M,Hidalgo PC, Artagaveya N, Kiles R, Marnez M, Esteller M, Bertoni B. Eﬀect of genec ancestry on leukocyte global DNA methylaon in cancer paents. BMC Cancer. [Internet]. 2015; 15:434. doi: hps://doi. org/f7dp9f [13] Xavier P, Müller S, Fukumasu H. Epigenec Mechanisms in Canine Cancer. Front. Oncol. [Internet]. 2020; 10:591843. doi: hps://doi.org/qf7k [14] Abdelmegeed S, Mohammed S. Canine mammary tumors as a model for human disease (Review). Oncol. Le. [Internet] 2018; 15(6):8195–8205. doi: hps://doi. org/mdsg [15] Jeong S, Lee K, Nam A, Cho J. Genome-wide methylaon proﬁling in canine mammary tumor reveals miRNA candidates associated with human breast cancer. Cancers. [Internet] 2019; 11(10):1466. doi: hps://doi. org/qf7m [16] Biondi L, Tedardi M, Genle L, Chamas P, Dagli M. Quanﬁcaon of global DNA methylaon in canine mammary gland tumors via immunostaining of 5-methylcytosine: Histopathological and clinical correlaons. Front. Vet. Sci. [Internet] 2021; 8:628241. doi: hps://doi.org/qf7n [17] Brandão Y, Toledo M, Chequin A, Cristo T, Sousa R, Ramos E, Klassen G. DNA methylaon status of the estrogen receptor α gene in canine mammary tumors. Vet Pathol. [Internet] 2018; 55(4):510–516. doi: hps://doi.org/qf7p [18] Beetch M, Harandi S, Yang T, Boyco C, Chen Y, Stefanskan B, Mohammed S. DNA methylaon landscape of triple- negave ductal carcinoma in situ (DCIS) progressing to the invasive stage in canine breast cancer. Sci. Rep. [Internet] 2020; 10:2415. hps://doi.org/qf7q [19] Cohen J. Stascal Power Analysis for the Behavioral Sciences. 2nd ed. New York University: Lawrence Erlbaum Associates. 1988. [20] Goldshmidt M, Peña L, Rasoo R, Zappulli, V. Classiﬁcaon and grading of canine mammary tumors. Vet. Pathol. [Internet] 2011; 48(1):117–131. doi: hps://doi.org/ cg36bk [21] Egenvall A, Bonne B, Ohagen P, Olson P, Hedhammar A, von Euler H. Incidence of and survival aﬅer mammary tumors in a populaon of over 80,000 insured female dogs in Sweden from 1995 to 2002. Prevent. Vet. Med. [Internet]. 2005; 69(1-2):109–127. doi: hps://doi.org/ fw7k7z [22] Hillyar C, Rallis K, Varghese J. Advances in epigenec cancer therapeucs. Cureus. [Internet] 2020; 12(11):e11725. doi: hps://doi.org/gqsrqz 5 of 5