Quantification of Global DNA Methylation in Canine Melanotic and Amelanotic Oral Mucosal Melanomas and Peripheral Blood Leukocytes From the Same Patients With OMM: First Study.

Oral mucosal melanomas (OMMs) are aggressive and resistant cancers of high importance in veterinary oncology. Amelanotic OMM produces comparatively less melanin and is considered to be more aggressive than melanotic OMM. Global DNA methylation profiles with hypomethylated or hypermethylated patterns have both been associated with aggressive neoplasms; however, global DNA hypomethylation seems to correlate to higher aggressiveness. Accordingly, global DNA methylation in peripheral blood leukocytes has been investigated to understand the role of systemic or environmental factors in cancer development. This study aimed to quantify global DNA methylation in canine melanotic and amelanotic OMM samples and in the peripheral blood leukocytes of the same dogs. Tumor tissue samples were collected from 38 dogs, of which 19 were melanotic and 19 were amelanotic OMM. These were submitted to immunohistochemistry (IHC) with anti-5-methylcytosine (5mC) and anti-Ki67 primary antibodies. Ki67- and 5mC-positive nuclei were manually scored with the help of an image analysis system. Peripheral blood samples were collected from 18 among the 38 OMM-bearing dogs and from 7 additional healthy control dogs. Peripheral blood leukocytes were isolated from the 25 dogs, and DNA was extracted and analyzed by high-performance liquid chromatography (HPLC) for global DNA methylation. The pattern of global DNA methylation in both canine melanotic and amelanotic OMM indicated higher percentages of weakly or negatively stained nuclei in most of the OMM cells, presuming predominant global DNA hypomethylation. In addition, Ki67 counts in amelanotic OMM were significantly higher than those in melanotic OMM (p < 0.001). Global DNA methylation different immunostaining patterns (strong, weak or negative) correlated with Ki67 scores. Global DNA methylation in circulating leukocytes did not differ between the 9 melanotic and 9 amelanotic OMM or between the 18 OMM-bearing dogs and the 7 healthy dogs. This study provides new information on canine melanotic and amelanotic OMM based on global DNA methylation and cell proliferation.

Inhibitory Effects of a Reengineered Anthrax Toxin on Canine Oral Mucosal Melanomas.

Canine oral mucosal melanomas (OMM) are the most common oral malignancy in dogs and few treatments are available. Thus, new treatment modalities are needed for this disease. Bacillus anthracis (anthrax) toxin has been reengineered to target tumor cells that express urokinase plasminogen activator (uPA) and metalloproteinases (MMP-2), and has shown antineoplastic effects both, in vitro and in vivo. This study aimed to evaluate the effects of a reengineered anthrax toxin on canine OMM. Five dogs bearing OMM without lung metastasis were included in the clinical study. Tumor tissue was analyzed by immunohistochemistry for expression of uPA, uPA receptor, MMP-2, MT1-MMP and TIMP-2. Animals received either three or six intratumoral injections of the reengineered anthrax toxin prior to surgical tumor excision. OMM samples from the five dogs were positive for all antibodies. After intratumoral treatment, all dogs showed stable disease according to the canine Response Evaluation Criteria in Solid Tumors (cRECIST), and tumors had decreased bleeding. Histopathology has shown necrosis of tumor cells and blood vessel walls after treatment. No significant systemic side effects were noted. In conclusion, the reengineered anthrax toxin exerted inhibitory effects when administered intratumorally, and systemic administration of this toxin is a promising therapy for canine OMM.

Global DNA methylation of peripheral blood leukocytes from dogs bearing multicentric non-Hodgkin lymphomas and healthy dogs: A comparative study.

Non-Hodgkin lymphomas are among the most common types of tumors in dogs, and they are currently accepted as comparative models of the disease in humans. Aberrant patterns of DNA methylation seem to play a key role in the development of hematopoietic neoplasms in humans, constitute a special mechanism of transcriptional control, and may be influenced by genetic and environmental factors. Blood leukocyte DNA global methylation has been poorly investigated in dogs. The aim of this study is to examine whether peripheral blood global DNA methylation is associated with canine multicentric lymphomas. Peripheral venous blood samples from ten healthy dogs and nine dogs bearing multicentric lymphomas were collected, and the buffy coat was separated. Global DNA methylation was analyzed by High Performance Liquid Chromatography (HPLC) and immunocytochemistry (ICC). In both analyses, leukocytes from dogs with lymphoma presented lower global DNA methylation than in healthy dogs (HPLC: p = 0.027/ 5MeCyt immunoreactivity scores: p = 0.015). Moderate correlation was observed between the results obtained by HPLC and ICC (correlation coefficient = 0.50). For the identification of differently methylated genes between both groups, the Infinium Human Methylation (HM) EPIC BeadChip (850K) was used. Of the 853,307 CpGs investigated in the microarray, there were 34,574 probes hybridized in the canine samples. From this total, significant difference was observed in the methylation level of 8433 regions, and through the homologous and orthologous similarities 525 differently methylated genes were identified between the two groups. This study is pioneer in suggesting that dogs bearing non-Hodgkin lymphoma presented DNA global hypomethylation of circulating leukocytes compared with healthy dogs. Although canine samples were used in an assay developed specifically for human DNA, it was possible to identify differently methylated genes and our results reiterate the importance of the use of peripheral blood leukocytes in cancer research and possible new biomarkers targets.