Development of a multiplex methylation specific PCR suitable for (early) detection of non-small cell lung cancer
Development of a multiplex methylation specific PCR suitable for (early) detection of non-small cell lung cancer
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DOI：
10.4161/epi.29499
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发表时间：
2014-08-01
期刊：
影响因子：
3.7
通讯作者：
Ernberg, Ingemar
中科院分区：
文献类型：
--
作者：
Nawaz, Imran;Qiu, Xiaoming;Ernberg, Ingemar
肺癌是一个全球性的健康问题，也是癌症相关死亡的主要原因。启动子异常甲基化导致的潜在肿瘤抑制基因（TSGs）沉默是癌症发生和发展的早期事件。因此，DNA中甲基化的癌症类型特异性TSG可以作为早期癌症检测的有用生物标志物。我们现在已经开发了一种“多重甲基化特异性PCR”（MMSP）测定法，用于通过单一PCR反应分析多种潜在TSG的甲基化状态。该方法可用于非小细胞肺癌（NSCLC）的早期诊断和疗效研究。对来自中国的三名NSCLC患者的肺癌组织和匹配的远处非癌组织进行全基因组CpG甲基化和表达微阵列。选择38个潜在的TSG，并通过亚硫酸氢盐处理的DNA上的甲基化PCR进行分析。在敏感性和特异性的基础上，选择HOXA 9、TBX 5、PITX 2、CALCA、RASSF 1A和DLEC 1六个标记基因来建立MMSP检测。然后使用该检测方法分析70名非小细胞肺癌患者的肺癌组织和匹配的远处非癌组织，以及24名良性肺部病变患者作为对照。灵敏度为99%（69/70）。HOXA 9和TBX 5是最敏感的2个标记基因，分别为87%（61/70）和84%（59/70）。RASSF 1A和DLEC 1的特异性最高，为99%（69/70）。使用鉴定至少任何两个甲基化标记基因的标准，61/70个癌症样品为阳性，对应于87%的灵敏度和94%的特异性。早期I或II期NSCLC甚至可以以100%的特异性和86%的灵敏度被检测到。总之，MMSP有可能发展成为一种基于人群的筛查工具，并可用于NSCLC的早期诊断。它也可能适用于监测治疗结果和复发。
Lung cancer is a worldwide health problem and a leading cause of cancer-related deaths. Silencing of potential tumor suppressor genes (TSGs) by aberrant promoter methylation is an early event in the initiation and development of cancer. Thus, methylated cancer type-specific TSGs in DNA can serve as useful biomarkers for early cancer detection. We have now developed a "Multiplex Methylation Specific PCR" (MMSP) assay for analysis of the methylation status of multiple potential TSGs by a single PCR reaction. This method will be useful for early diagnosis and treatment outcome studies of non-small cell lung cancer (NSCLC). Genome-wide CpG methylation and expression microarrays were performed on lung cancer tissues and matched distant non-cancerous tissues from three NSCLC patients from China. Thirty-eight potential TSGs were selected and analyzed by methylation PCR on bisulfite treated DNA. On the basis of sensitivity and specificity, six marker genes, HOXA9, TBX5, PITX2, CALCA, RASSF1A, and DLEC1, were selected to establish the MMSP assay. This assay was then used to analyze lung cancer tissues and matched distant non-cancerous tissues from 70 patients with NSCLC, as well as 24 patients with benign pulmonary lesion as controls. The sensitivity of the assay was 99% (69/70). HOXA9 and TBX5 were the 2 most sensitive marker genes: 87% (61/70) and 84% (59/70), respectively. RASSF1A and DLEC1 showed the highest specificity at 99% (69/70). Using the criterion of identifying at least any two methylated marker genes, 61/70 cancer samples were positive, corresponding to a sensitivity of 87% and a specificity of 94%. Early stage I or II NSCLC could even be detected with a 100% specificity and 86% sensitivity. In conclusion, MMSP has the potential to be developed into a population-based screening tool and can be useful for early diagnosis of NSCLC. It might also be suitable for monitoring treatment outcome and recurrence.