Scattone Nayra Villar, Epiphanio Tatiane Moreno Ferrarias, Caddrobi Karine Germano, Ferrão Juliana Shimara Pires, Hernandez-Blazquez Francisco Javier, Loureiro Ana Paula de Melo, Massoco Cristina de Oliveira, Dagli Maria Lucia Zaidan
Laboratory of Experimental and Comparative Oncology, School of Veterinary Medicine and Animal Science, University of São Paulo, São Paulo, Brazil.
PROVET Veterinary Hospital, São Paulo, Brazil.
Front Vet Sci. 2021 Aug 24;8:680181. doi: 10.3389/fvets.2021.680181. eCollection 2021.
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 ( < 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.
口腔黏膜黑色素瘤（OMM）是兽医肿瘤学中极具侵袭性且难治的重要癌症。无黑色素性OMM产生的黑色素相对较少，被认为比有黑色素性OMM更具侵袭性。具有低甲基化或高甲基化模式的全基因组DNA甲基化谱均与侵袭性肿瘤有关；然而，全基因组DNA低甲基化似乎与更高的侵袭性相关。因此，人们对外周血白细胞中的全基因组DNA甲基化进行了研究，以了解全身或环境因素在癌症发生发展中的作用。本研究旨在量化犬有黑色素性和无黑色素性OMM样本以及同一犬只外周血白细胞中的全基因组DNA甲基化情况。从38只犬收集肿瘤组织样本，其中19只为有黑色素性OMM，19只为无黑色素性OMM。这些样本用抗5-甲基胞嘧啶（5mC）和抗Ki67一抗进行免疫组织化学（IHC）检测。借助图像分析系统对Ki67和5mC阳性细胞核进行手动计数。从38只患有OMM的犬中的18只以及另外7只健康对照犬采集外周血样本。从这25只犬中分离外周血白细胞，提取DNA并通过高效液相色谱（HPLC）分析全基因组DNA甲基化情况。犬有黑色素性和无黑色素性OMM中的全基因组DNA甲基化模式表明，在大多数OMM细胞中，弱阳性或阴性染色细胞核的比例较高，推测主要存在全基因组DNA低甲基化。此外，无黑色素性OMM中的Ki67计数显著高于有黑色素性OMM（<0.001）。全基因组DNA甲基化的不同免疫染色模式（强、弱或阴性）与Ki67评分相关。9只患有有黑色素性OMM的犬和9只患有无黑色素性OMM的犬之间，以及18只患有OMM的犬和7只健康犬之间，循环白细胞中的全基因组DNA甲基化没有差异。本研究基于全基因组DNA甲基化和细胞增殖为犬有黑色素性和无黑色素性OMM提供了新信息。
