Methylation of LINE-1 in cell-free DNA serves as a liquid biopsy biomarker for human breast cancers and dog mammary tumors

Abstract

Breast cancer (BC) is one of the most common cancers in both women and female dogs. Methylation changes of LINE-1 have been reported in human cancers. The aim of this study was to determine the hypomethylation of canine LINE-1 in liquid biopsies for canine mammary tumors (CMT) and to assess its diagnostic performance in human plasma. BC associated LINE-1 methylation was measured by methylation sensitive (HpaII) and insensitive (MspI) restriction enzyme digestion followed by real-time PCR using the cfDNA isolated from 300 µl of plasma. The relative level of methylated canine LINE-1 was less than 0.4 in the benign and malignant CMTs (0.29 ± 0.061 and 0.39 ± 0.066, respectively) when it was 0.92 ± 0.067 in the healthy controls. The area under the ROC curve (AUC) was significantly high in both benign and malignant tumors (0.97 and 0.93). Furthermore, this approach was also successfully implemented in a set of 26 human BCs with 10 healthy controls (AUC = 0.78). Altogether, our data suggest that the comparative approach using a dog model might be helpful to rapidly develop a new diagnostic biomarker and that the methylation of LINE-1 in cfDNA may be a good target as a diagnostic marker of both human BC and CMT.

Tumor microenvironment complexity and therapeutic implications at a glance

“…In the context of cancer, they are involved in horizontal gene transfer and oncogenic transformation of normal cells as well as in the metastasis development [197]. Although there is no clear mechanistic evidence for their oncogenic potential, several proposals include (ii) overexpression of several pro-metastatic genes through the tolllike receptor 9 (TLR9)/ myeloid differentiation primary response 88 (MYD88) independent pathway [198]; (ii) transposable elements [199] and finally the cellular uptake of exosomes [200,201]. Another consequence of cell-free contacts involving cfDNA is the increased chemo-, radioresistance of cancer cells, as radiotherapy produces oxidized DNA which triggers reactive oxygen species and induces DNA damage response pathways [202].…”

Evaluation of nucleosome concentrations in healthy dogs and dogs with cancer

“…Though there are no published studies specifically describing cancer detection using nucleosomes in dogs, several publications have described the utility of cfDNA (13)(14)(15)(16)(17).…”

DNA methylation landscape of triple-negative ductal carcinoma in situ (DCIS) progressing to the invasive stage in canine breast cancer

“…DNA methylation patterns in CGIs of ESR1, encoding for ERα, and BRCA1, an important tumor suppressor gene, have recently been examined 32,33 , reporting changes in DNA methylation of these genes in malignant canine tumors. Moreover, LINE-1 methylation in cell free DNA (cfDNA) from liquid biopsies was used in a comparative approach of canine and human breast tumors 34 . LINE-1 is a transposable element whose methylation has classically been used as in indicator of changing DNA methylation patterns in human cancer models.…”

“…Hypomethylation of these elements robustly differentiated canine breast tumors from normal breast tissue. As such, the cut-off level of LINE-1 methylation based on canine data for distinguishing normal breast tissue from breast tumors was implemented in human cfDNA 34 . This approach successfully predicted the presence of human breast tumors.…”