Targeted, Deep Sequencing Reveals Full Methylation Profiles of Multiple HPV Types and Potential Biomarkers for Cervical Cancer Progression
Targeted, Deep Sequencing Reveals Full Methylation Profiles of Multiple HPV Types and Potential Biomarkers for Cervical Cancer Progression
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靶向深度测序揭示了多种 HPV 类型的完整甲基化谱和宫颈癌进展的潜在生物标志物
DOI：
10.1158/1055-9965.epi-16-0368
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发表时间：
2017-04-01
影响因子：
3.8
通讯作者：
Rader, Janet S.
中科院分区：
文献类型：
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作者：
Liu, Pengyuan;Iden, Marissa;Rader, Janet S.
背景：浸润性宫颈癌（ICC）及其癌前阶段（宫颈上皮内瘤变；CIN1 - 3）通过妇科和病理学检查得以区分，但目前没有方法能够预测注定会进展为ICC的癌前病变。因此，非常需要开发可靠的检测方法来评估患者的预后。
方法：人乳头瘤病毒（HPV）DNA甲基化在宫颈疾病中发生显著改变。采用HPV富集方法和新一代DNA测序技术，在CIN（n = 2例CIN1；n = 2例CIN2；n = 20例CIN3）和ICC（n = 37）样本的病例 - 病例比较中对甲基化状态进行了表征。焦磷酸测序在更大的样本队列（n = 61例CIN3；50例ICC）中验证了感兴趣的CpG位点的甲基化变化。
结果：在所有HPV类型中，ICC的整体病毒甲基化水平显著高于CIN3。13种不同HPV类型的平均L1基因甲基化水平最能区分CIN3和ICC。单个CpG位点的甲基化水平作为一种定量分类器，在HPV16样本中检测ICC的敏感性和特异性>95%。焦磷酸测序证实，与CIN3相比，ICC中HPV16的E1基因的这些CpG位点的甲基化水平显著更高。
结论：ICC中的整体HPV甲基化水平显著高于CIN3，其中L1基因甲基化水平在区分CIN3和ICC方面表现最佳。HPV16的E1基因中CpG位点（972、978、1870和1958）的甲基化水平能够区分CIN3和ICC。
影响：在特定的E1 CpG位点较高的甲基化可能与HPV16阳性的CIN3病变进展为ICC的可能性增加有关。（C）2017美国癌症研究协会
Background: Invasive cervical cancer (ICC) and its premalignant phase (cervical intraepithelial neoplasia; CIN1-3) are distinguished by gynecologic and pathologic examination, yet no current methodologies can predict precancerous lesions that are destined to progress to ICC. Thus, development of reliable assays to assess patient prognosis is much needed. Methods: Human papillomavirus (HPV) DNA methylation is significantly altered in cervical disease. Using an HPV enrichment approach and next-generation DNA sequencing, methylation status was characterized in a case–case comparison of CIN (n = 2 CIN1; n = 2 CIN2; n = 20 CIN3) and ICC (n = 37) samples. Pyrosequencing validated methylation changes at CpGs of interest in a larger sample cohort (n = 61 CIN3; 50 ICC). Results: Global viral methylation, across HPV types, was significantly higher in ICC than CIN3. Average L1 gene methylation in 13 different HPV types best distinguished CIN3 from ICC. Methylation levels at individual CpG sites as a quantitative classifier achieved a sensitivity and specificity of >95% for detecting ICC in HPV 16 samples. Pyrosequencing confirmed significantly higher methylation of these CpGs in E1 of HPV 16 in ICC compared with CIN3. Conclusions: Global HPV methylation is significantly higher in ICC than CIN3, with L1 gene methylation levels performing best for distinguishing CIN3 from ICC. Methylation levels at CpGs in the E1 gene of HPV 16 (972, 978, 1870, and 1958) can distinguish between CIN3 and ICC. Impact: Higher methylation at specific E1 CpGs may associate with increased likelihood of progression to ICC in HPV 16–positive CIN3 lesions. Cancer Epidemiol Biomarkers Prev; 26(4); 642–50. ©2017 AACR.