Quantification of Global DNA Methylation in Canine Mammary Gland Tumors via Immunostaining of 5-Methylcytosine: Histopathological and Clinical Correlations.

Luiz Roberto Biondi,Maria Lucia Zaidan Dagli,Marcello Vannucci Tedardi,Luciana Boffoni Gentile,Patricia Pereira Costa Chamas

doi:10.3389/fvets.2021.628241

Luiz Roberto Biondi, Maria Lucia Zaidan Dagli + Show 3 more

Open Access

https://doi.org/10.3389/fvets.2021.628241

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Abstract

Mammary tumors are the most prevalent neoplasms in non-neutered female dogs, with genetic and epigenetic alterations contributing to canine mammary carcinogenesis. This study quantified global DNA methylation in 5-methylcytosine (5mC)-immunostained canine mammary tumor samples and established histopathological and clinical correlations. A total of 91 formalin-fixed paraffin-embedded mammary tumor samples from female dogs were retrospectively selected and subjected to immunohistochemistry using an anti-5mC mouse monoclonal antibody. We evaluated 5mC+ stained nuclei of neoplastic epithelial cells in canine mammary glands to obtain semiquantitative histoscores based on staining intensity. Survival rates were estimated based on owners' or veterinary records. Histological samples comprised 28 and 63 benign and malignant canine mammary gland tumors, respectively. Results revealed significant differences between global DNA methylation patterns when mammary samples were categorized as benign or malignant (p = 0.024), with hypomethylated patterns more prevalent in malignant tumors and those with higher relapse behavior (p = 0.011). Of note, large diameter (>5 cm) tumors revealed a lower methylation pattern (p = 0.028). Additionally, we found non-statistically significant differences when tumors were grouped by histopathological characteristics, clinical parameters, or survival. These findings propose global DNA methylation assessment as a promising tool for detecting canine mammary tumors with relapse propensity.

Highlights

Mammary tumors are the most prevalent neoplasm in non-neutered female dogs [1] representing ∼50% of tumor diagnoses [2]
We retrospectively evaluated global DNA methylation status in canine mammary tumors and correlated these results with clinical parameters, survival, and tumor relapse
Methylation in canine neoplastic disease, and none have focused on global DNA methylation in mammary neoplasms

Summary

Introduction

Mammary tumors are the most prevalent neoplasm in non-neutered female dogs [1] representing ∼50% of tumor diagnoses [2]. Methylation occurs at cytosine bases located at the 5′ end of guanine bases, promoting the formation of CpG dinucleotide islands. These CpG dinucleotide islands play a central role in transcriptional repression (when most CpG dinucleotides in an island are methylated). Methylation, which occurs at cytosine bases (C), located 5′ to guanine (G) bases to form CpG dinucleotide islands, can be affected by loss or gain of DNA methyltransferases or demethylases. Aberrant hypermethylation, can silence tumor suppressor gene promoter regions These are both wellestablished mechanisms through which cancer cells may acquire critical features on their pathway to transformation [9,10,11,12]. Like genetic mutations, epigenetic alterations can be mitotically inherited, resulting in a rapidly growing cancer cell population by conferring advantages to tumor cells, resulting in uncontrolled growth [13, 14]

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