Department of Molecular & Clinical Cancer Medicine, University of Liverpool, Liverpool, United Kingdom.
Cancer Res. 2012 Nov 15;72(22):5692-701. doi: 10.1158/0008-5472.CAN-12-2309. Epub 2012 Sep 7.
The exceptional high mortality of lung cancer can be instigated to a high degree by late diagnosis. Despite the plethora of studies on potential molecular biomarkers for lung cancer diagnosis, very few have reached clinical implementation. In this study, we developed a panel of DNA methylation biomarkers and validated their diagnostic efficiency in bronchial washings from a large retrospective cohort. Candidate targets from previous high-throughput approaches were examined by pyrosequencing in an independent set of 48 lung tumor/normal paired. Ten promoters were selected and quantitative methylation-specific PCR (qMSP) assays were developed and used to screen 655 bronchial washings from the Liverpool Lung Project (LLP) subjects divided into training (194 cases and 214 controls) and validation (139 cases and 109 controls) sets. Three statistical models were used to select the optimal panel of markers and to evaluate the performance of the discriminatory algorithms. The final logit regression model incorporated hypermethylation at p16, TERT, WT1, and RASSF1. The performance of this 4-gene methylation signature in the validation set showed 82% sensitivity and 91% specificity. In comparison, cytology alone in this set provided 43% sensitivity at 100% specificity. The diagnostic efficiency of the panel did not show any biases with age, gender, smoking, and the presence of a nonlung neoplasm. However, sensitivity was predictably higher in central (squamous and small cell) than peripheral (adenocarcinomas) tumors, as well as in stage 2 or greater tumors. These findings clearly show the impact of DNA methylation-based assays in the diagnosis of cytologically occult lung neoplasms. A prospective trial is currently imminent in the LLP study to provide data on the enhancement of diagnostic accuracy in a clinical setting, including by additional markers.
肺癌极高的死亡率在很大程度上可以归因于晚期诊断。尽管有大量研究探讨了肺癌诊断的潜在分子生物标志物，但很少有研究能够转化为临床应用。在本研究中，我们开发了一组 DNA 甲基化生物标志物，并在大型回顾性队列的支气管灌洗液中验证了其诊断效率。通过焦磷酸测序对先前高通量方法中的候选靶点进行了独立的 48 对肺肿瘤/正常样本检测。选择了 10 个启动子，并开发了定量甲基化特异性 PCR（qMSP）检测方法，用于筛选来自利物浦肺癌项目（LLP）的 655 例支气管灌洗液。这些样本根据培训（194 例病例和 214 例对照）和验证（139 例病例和 109 例对照）集分为两组。使用三种统计模型选择最佳的标志物组合，并评估鉴别算法的性能。最终的对数回归模型纳入了 p16、TERT、WT1 和 RASSF1 的高甲基化。该 4 基因甲基化特征在验证集中的性能表现为 82%的敏感性和 91%的特异性。相比之下，在该组中单独使用细胞学检查的敏感性为 100%特异性的 43%。该标志物组合的诊断效率在年龄、性别、吸烟状况和非肺部肿瘤存在方面没有任何偏差。然而，在中央（鳞癌和小细胞癌）肿瘤中，敏感性明显高于周围（腺癌）肿瘤，以及在 2 期或更晚期肿瘤中。这些发现清楚地表明了基于 DNA 甲基化的检测方法在诊断细胞学隐匿性肺肿瘤中的重要作用。目前 LLP 研究中即将进行前瞻性试验，以提供关于在临床环境中提高诊断准确性的数据，包括通过额外的标志物。
