宫颈发育不良：评估 CCNA1、DAPK1、HS3ST2、PAX1 和 TFPI2 的甲基化状态（Methylight），以提高诊断准确性。

Cervical dysplasia: assessing methylation status (Methylight) of CCNA1, DAPK1, HS3ST2, PAX1 and TFPI2 to improve diagnostic accuracy.

机构信息

Oncology Research Institute, 28 Medical Drive, 117456, Singapore.

出版信息

Gynecol Oncol. 2010 Nov;119(2):225-31. doi: 10.1016/j.ygyno.2010.07.028. Epub 2010 Aug 13.

DOI:10.1016/j.ygyno.2010.07.028

PMID:20708786

Abstract

PURPOSE

Diagnosis of cervical neoplasia hinges upon microscopic inspection of cervical samples. This has inherent operator-dependent variability. Testing for high-risk human papilloma virus (HPV) may help to triage patients with pre-invasive disease in determining clinical intervention and follow-up. However, HPV presence/absence does not reflect the cervical epithelial cell's molecular status. Epigenetic modifications, e.g. DNA methylation, have been observed in the early stages of neoplastic change, preceding gene mutations. Here, we assess the correlation between cytologic/histologic results and combined DNA methylation data of 5 genes in different grades of cervical dysplasia.

EXPERIMENTAL DESIGN

Cervical specimens collected via the liquid-based cytology system were each microscopically examined. Residual cells were subjected to DNA methylation analysis (Methylight) of gene loci CCNA1, PAX1, HS3ST2, DAPK1 and TFPI2. Methylation data were compared with cytologic/histologic reports. Statistical methods were applied to assess the ability of DNA methylation status to subtype the cervical neoplastic lesions according to their corresponding cytologic/histologic reports.

RESULTS

A total of 165 subjects provided cytologically proven 63 HSIL, 49 LSIL and 53 normal samples. All patients with HSIL and LSIL underwent colposcopic examination. Patients with LSIL were all found to be CIN1; patients with HSIL were subsequently subdivided into 10 squamous cell carcinoma (SCC), 31 CIN3, 10 CIN2 and 12 CIN1. For each gene, there was increasing frequency of methylation from normal and LSIL (CIN1), through HSIL (CIN2 and CIN3), to SCC. Methylation of ≥1 of genes investigated was observed in 88% of combined HSIL (CIN2 and CIN3) and SCC cases. All genes showed significant increase in methylation level (PMR value) with increasing disease grade (p<0.005). CCNA1 was the only gene that was able to distinguish CIN2 from CIN3 specimens (p=0.016). Based on receiver operating characteristic (ROC) analysis, HS3ST2 was the most significant candidate in segregating HSIL/SCC from normal/LSIL cases (p<0.0001); at an optimal cutoff value, sensitivity and specificity between 70% and 80% were obtained.

CONCLUSIONS

Development of DNA methylation status of a gene panel to improve diagnostic accuracy in cervical neoplasia is warranted.

摘要

目的

宫颈癌的诊断取决于对宫颈样本的显微镜检查。这存在固有操作者依赖性的变异性。检测高危型人乳头瘤病毒（HPV）可以帮助对有潜在侵袭性疾病的患者进行分类，以确定临床干预和随访。然而，HPV 的存在/缺失并不能反映宫颈上皮细胞的分子状态。在肿瘤发生的早期阶段，已经观察到表观遗传修饰，例如 DNA 甲基化。这里，我们评估了细胞学/组织学结果与不同等级宫颈发育不良中 5 个基因的联合 DNA 甲基化数据之间的相关性。

实验设计

通过液体细胞学系统收集宫颈标本，每个标本均进行显微镜检查。残留细胞进行基因座 CCNA1、PAX1、HS3ST2、DAPK1 和 TFPI2 的 DNA 甲基化分析（Methylight）。将甲基化数据与细胞学/组织学报告进行比较。应用统计方法评估 DNA 甲基化状态根据相应的细胞学/组织学报告对宫颈肿瘤病变进行亚分类的能力。

结果

总共 165 名患者提供了经细胞学证实的 63 例 HSIL、49 例 LSIL 和 53 例正常样本。所有 HSIL 和 LSIL 患者均接受了阴道镜检查。LSIL 患者均为 CIN1；HSIL 患者随后分为 10 例鳞状细胞癌（SCC）、31 例 CIN3、10 例 CIN2 和 12 例 CIN1。对于每个基因，从正常和 LSIL（CIN1），到 HSIL（CIN2 和 CIN3），再到 SCC，甲基化的频率逐渐增加。在所研究的基因中，≥1 个基因的甲基化在 88%的合并 HSIL（CIN2 和 CIN3）和 SCC 病例中观察到。随着疾病程度的增加（p<0.005），所有基因的甲基化水平（PMR 值）均显著增加。CCNA1 是唯一能够区分 CIN2 和 CIN3 标本的基因（p=0.016）。基于受试者工作特征（ROC）分析，HS3ST2 是区分 HSIL/SCC 与正常/LSIL 病例的最显著候选基因（p<0.0001）；在最佳截断值时，获得了 70%至 80%之间的敏感性和特异性。