LINE-1 Methylation Status in Canine Splenic Hemangiosarcoma Tissue and Cell-Free DNA

SIMPLE SUMMARY: In dogs, hemangiosarcoma is the most common cancer of the spleen. Although early diagnosis is effective for tumor control, benign and other malignant tumors also form splenic nodules. It is difficult to distinguish between these types of splenic tumors by imaging modalities, and splenectomy is required to confirm the diagnosis. Recently, the clinical application of cell-free DNA in plasma has been termed “liquid biopsy” and utilized as a non-invasive method for the detection of tumor-specific genetic and epigenetic alterations. LINE-1 hypomethylation correlates well with global DNA methylation status, and it has been evaluated as a potential non-invasive tumor biomarker. This study aimed to determine the diagnostic value of LINE-1 methylation in dogs with splenic masses using tissue and cfDNA samples. The results show that significant LINE-1 hypomethylation was observed in hemangiosarcoma samples compared with other malignant tumors and benign groups. In addition, non-significant but similar results were observed for the cfDNA samples, thereby indicating its potential role as an early diagnostic biomarker for canine splenic hemangiosarcoma. ABSTRACT: Splenic hemangiosarcoma is one of the most common malignant tumors in dogs, and early diagnosis is of great importance for achieving a good prognosis. DNA methylation plays an important role in cancer development. Long interspersed nuclear element 1 (LINE-1) is the most abundant repetitive element in the genome. LINE-1 hypomethylation has been shown to be related to carcinogenesis in humans, and it has been used as a novel cancer biomarker. This study aimed to evaluate the methylation status of LINE-1 in tumor tissue and circulating cell-free DNA and assess its clinical significance in canine splenic hemangiosarcoma. Genomic DNA was isolated from splenic masses of 13 dogs with hemangiosarcoma, 11 with other malignant tumors, and 15 with benign lesions. LINE-1 methylation was quantified using methylation-sensitive and -insensitive restriction enzyme digestion followed by real-time polymerase chain reaction. Additionally, blood samples were collected from eight patients to isolate cell-free DNA to determine LINE-1 methylation status changes during the treatment course. LINE-1 methylation in tumor samples was significantly lower in patients with hemangiosarcoma than in those with other malignant tumors and benign lesions. Non-significant but similar results were observed for the cell-free DNA samples. Our results demonstrate that LINE-1 methylation status is a potential biomarker for splenic hemangiosarcoma.