LINE-1 and Alu Methylation in hrHPV-Associated Precancerous Cervical Samples

OBJECTIVE: This study was conducted to determine global DNA methylation patterns in cervical cells cytologically identified as atypical squamous cells of unknown significance (ASCUS) with a normal, LSIL, or HSIL histopathological result. METHODS: Methylation patterns of long interspersed nuclear elements (LINE-1) and short interspersed element (Alu) sequences were assessed using the combined bisulfite restriction analysis (COBRA) method in cervical samples with cytology-diagnosed cervical lesions. RESULTS: In cervical precancerous lesions with hrHPV positive, the percentage of overall ((m)C) and (m)C(m)C LINE-1 methylation levels showed a stepwise increase from hrHPV positive normal to HSIL with significant differences (p<0.001). However, both methylation levels were significantly higher in hrHPV negative normal than in hrHPV positive normal (p<0.001). The overall ((m)C) Alu methylation in hrHPV positive LSIL and HSIL was lower than in hrHPV positive normal, with a significant difference (p<0.05). Remarkably, the percentage of (u)C(m)C and (m)C(u)C of LINE-1 and Alu in three different hrHPV positive cervical lesions showed a stepwise decrease from hrHPV positive normal, LSIL and HSIL, respectively. Furthermore, receiver operating characteristic (ROC) curve analyses revealed that the LINE-1 (m)C and (m)C(m)C patterns have high sensitivity and specificity for distinguishing HSIL from normal/LSIL in hrHPV positive cases at the appropriate cutoff levels. CONCLUSION: We have demonstrated the LINE-1 and Alu methylation data in normal and premalignant cervical epithelia. LINE-1 hypomethylation was found in hrHPV positive normal cells, with lower methylation levels associated with cancer features. In cytologically diagnosed Atypical Squamous Cells of Unknown Significance (ASCUS), the levels of (m)C and the (m)C(m)C pattern could be utilized in concert with hrHPV detection to classify the ASCUS sample prior to colposcopy.