Department of Cytopathology, The Third Affiliated Hospital of Zhengzhou University, No. 7 Front Kangfu Street, Er'qi District, Zhengzhou 450052, China.
The Cancer Hospital Affiliated to Zhengzhou University, No. 127 Dongming Street, Jinshui District, Zhengzhou 450008, China.
Clin Biochem. 2015 Sep;48(13-14):855-9. doi: 10.1016/j.clinbiochem.2015.05.006. Epub 2015 May 14.
Methylation-sensitive high-resolution melting (MS-HRM) is a new technique for DNA methylation analysis, but it is rarely used for the detection of viral DNA methylation. In this study, we investigated the HPV-16L1 gene methylation that is detected by MS-HRM as a potential biomarker for prognosing cervical dysplasia and cancer.
A total of 114 HPV-16 infected patients (normal (17), CIN1 (25), CIN2 (29), CIN3 (32), SCC (11)) who underwent liquid-based cytology test and biopsy were included in this study. 17 cases with HPV-16 infection and negative cytologic and histologic results served as the control group. The HPV-16L1 gene methylation statuses of these samples were investigated using a methylation-sensitive high-resolution melting (MS-HRM) assay after bisulfite modification.
The HPV-16L1 gene methylation statuses of all the 114 specimens were successfully detected by MS-HRM, and we observed increasing methylation levels in severe lesions, as determined using histologic assays. In addition, the methylation levels of CIN2+ (CIN2, CIN3 and SCC) were significantly higher than that of CIN2- (normal and CIN1, P<0.001). When taking CIN2+ as the reference, our HPV-16L1 DNA methylation assay achieved 91.7% sensitivity and 59.5% specificity, respectively.
The results of the present work demonstrated that HPV-16L1 gene methylation was closely associated with cervical precancerosis and cancer. Moreover, using MS-HRM to detect HPV-16L1 gene methylation may be a powerful assay for the triage of HPV-16-positive females, which could identify patients with high risk of invasive cancer.
甲基化敏感高分辨率熔解（MS-HRM）是一种新的 DNA 甲基化分析技术，但很少用于检测病毒 DNA 甲基化。本研究旨在探讨 HPV-16L1 基因甲基化作为预测宫颈上皮内瘤变和宫颈癌的潜在生物标志物。
共纳入 114 例 HPV-16 感染患者（正常 17 例、CIN1 25 例、CIN2 29 例、CIN3 32 例、SCC 11 例），均行液基细胞学检查和活检。17 例 HPV-16 感染且细胞学和组织学结果均为阴性的患者作为对照组。采用亚硫酸氢盐修饰后甲基化敏感高分辨率熔解（MS-HRM）检测这些样本中 HPV-16L1 基因的甲基化状态。
通过 MS-HRM 成功检测到 114 例标本中 HPV-16L1 基因的甲基化状态，且我们观察到严重病变的甲基化水平逐渐升高，这与组织学检查结果一致。此外，CIN2+（CIN2、CIN3 和 SCC）的甲基化水平明显高于 CIN2-（正常和 CIN1，P<0.001）。以 CIN2+为参考，我们的 HPV-16L1 DNA 甲基化检测的灵敏度和特异性分别为 91.7%和 59.5%。
本研究结果表明 HPV-16L1 基因甲基化与宫颈癌前病变和癌症密切相关。此外，采用 MS-HRM 检测 HPV-16L1 基因甲基化可能是一种有效的 HPV-16 阳性女性分流检测方法，可以识别具有侵袭性癌症高风险的患者。
