Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Rockville, MD, USA.
Int J Cancer. 2013 Mar 15;132(6):1412-22. doi: 10.1002/ijc.27750. Epub 2012 Aug 20.
We explored the association of human papillomavirus type 16 (HPV16) DNA methylation with age, viral load, viral persistence and risk of incident and prevalent high grade CIN (CIN2+) in serially collected specimens from the Guanacaste, Costa Rica cohort. 273 exfoliated cervical cell specimens (diagnostic and pre-diagnostic) were selected: (1) 92 with HPV16 DNA clearance (controls), (2) 72 with HPV16 DNA persistence (without CIN2+) and (3) 109 with CIN2+. DNA was extracted, bisulfite converted and methylation was quantified using pyrosequencing assays at 66 CpGs across the HPV genome. The Kruskal-Wallis test was used to determine significant differences among groups, and receiver operating characteristic curve analyses were used to evaluate how well methylation identified women with CIN2+. In diagnostic specimens, 88% of CpG sites had significantly higher methylation levels in CIN2+ after correction for multiple tests compared with controls. The highest area under the ROC curve (AUC) was 0.82 for CpG site 6457 in L1, and a diagnostic sensitivity of 91% corresponded to a specificity of 60% for CIN2+. Prospectively, 17% of CpG sites had significantly higher methylation in pre-diagnostic CIN2+ specimens (median time of 3 years before diagnosis) versus controls. The strongest pre-diagnostic CpG site was 6367 in L1 with an AUC of 0.76. Age-stratified analyses suggested that women older than the median age of 28 years have an increased risk of precancer associated with high methylation. Higher methylation in CIN2+ cases was not explained by higher viral load. We conclude that elevated levels of HPV16 DNA methylation may be useful to predict concurrently diagnosed as well as future CIN2+.
我们探索了人乳头瘤病毒 16 型（HPV16）DNA 甲基化与年龄、病毒载量、病毒持续存在以及在哥斯达黎加瓜纳卡斯特队列的连续采集标本中发生和现患高级别宫颈上皮内瘤变（CIN2+）的风险之间的关联。选择了 273 个宫颈脱落细胞标本（诊断和预诊断）：（1）92 个 HPV16 DNA 清除（对照组），（2）72 个 HPV16 DNA 持续存在（无 CIN2+）和（3）109 个 CIN2+。提取 DNA，用亚硫酸氢盐转化，使用焦磷酸测序法在 HPV 基因组的 66 个 CpG 位点上定量甲基化。Kruskal-Wallis 检验用于确定组间的显著差异，受试者工作特征曲线分析用于评估甲基化对 CIN2+女性的识别能力。在诊断标本中，与对照组相比，在调整了多个检验后，88%的 CpG 位点在 CIN2+中的甲基化水平显著升高。L1 中 CpG 位点 6457 的曲线下面积（AUC）最高为 0.82，CIN2+的诊断灵敏度为 91%，特异性为 60%。前瞻性地，在预诊断 CIN2+标本（诊断前 3 年的中位时间）中，有 17%的 CpG 位点的甲基化水平显著高于对照组。最强的预诊断 CpG 位点是 L1 中的 6367，AUC 为 0.76。年龄分层分析表明，年龄大于 28 岁中位年龄的女性发生与高甲基化相关的癌前病变的风险增加。CIN2+病例中较高的甲基化水平不能用较高的病毒载量来解释。我们得出结论，HPV16 DNA 甲基化水平升高可能有助于预测同时诊断和未来的 CIN2+。
