LINE-1 and Alu Methylation Patterns in Lymph Node Metastases of Head and Neck Cancers

Background: The potential use of hypomethylation of Long INterspersed Element 1 (LINE-1) and Alu elements (Alu) as a biomarker has been comprehensively assessed in several cancers, including head and neck squamous cell carcinoma (HNSCC). Failure to detect occult metastatic head and neck tumors on radical neck lymph node dissection can affect the therapeutic measures taken. Objective: The aim of this study was to investigate the LINE-1 and Alu methylation status and determine whether it can be applied for detection of occult metastatic tumors in HNSCC cases. Methods: We used the Combine Bisulfite Restriction Analysis (COBRA) technique to analyse LINE-1 and Alu methylation status. In addition to the methylation level, LINE-1 and Alu loci were classified based on the methylation statuses of two CpG dinucleotides in each allele as follows: hypermethylation ((CC)-C-m-C-m), hypomethylation ((CC)-C-u-C-u), and 2 forms of partial methylation ((CC)-C-m-C-u and (CC)-C-u-C-m). Sixty-one lymph nodes were divided into 3 groups: 1) non-metastatic head and neck cancer (NM), 2) histologically negative for tumor cells of cases with metastatic head and neck cancer (LN), and 3) histologically positive for tumor cells (LP). Results: Alu methylation change was not significant. However, LINE-1 methylation of both LN and LP was altered, as demonstrated by the lower LINE-1 methylation levels (p<0.001), higher percentage of (CC)-C-m-C-u (p<0.01), lower percentage of (CC)-C-u-C-m (p<0.001) and higher percentage of (CC)-C-u-C-u (p<0.001). Using receiver operating characteristic (ROC) curve analysis, %(CC)-C-u-C-m and %(CC)-C-m-C-u values revealed a high level of AUC at 0.806 and 0.716, respectively, in distinguishing LN from NM. Conclusion: The LINE-1 methylation changes in LN have the same pattern as that in LP. This epigenomic change may be due to the presence of occult metastatic tumor in LN cases.