Nawaz Imran, Qiu Xiaoming, Wu Heng, Li Yang, Fan Yaguang, Hu Li-Fu, Zhou Qinghua, Ernberg Ingemar
Department of Microbiology; Tumor and Cell Biology; Karolinska Institute; Stockholm, Sweden; Department of Microbiology; Faculty of Life Sciences; University of Balochistan; Quetta, Pakistan.
Tianjin Key Laboratory of Lung Cancer Metastasis and Tumor Microenvironment; Tianjin Lung Cancer Institute; Tianjin Medical University General Hospital; Tianjin, PR China.
Epigenetics. 2014 Aug;9(8):1138-48. doi: 10.4161/epi.29499. Epub 2014 Jun 17.
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.
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。异常的启动子甲基化导致潜在的肿瘤抑制基因（TSGs）沉默是癌症发生和发展过程中的早期事件。因此，DNA中甲基化的癌症类型特异性TSGs可作为早期癌症检测的有用生物标志物。我们现已开发出一种“多重甲基化特异性PCR”（MMSP）检测方法，通过一次PCR反应分析多个潜在TSGs的甲基化状态。该方法将有助于非小细胞肺癌（NSCLC）的早期诊断和治疗效果研究。对来自中国的3例NSCLC患者的肺癌组织及配对远隔非癌组织进行了全基因组CpG甲基化和表达微阵列分析。选择38个潜在的TSGs，对亚硫酸氢盐处理后的DNA进行甲基化PCR分析。基于敏感性和特异性，选择HOXA9、TBX5、PITX2、CALCA、RASSF1A和DLEC1这6个标记基因建立MMSP检测方法。然后用该检测方法分析70例NSCLC患者的肺癌组织及配对远隔非癌组织，以及24例良性肺病变患者作为对照。该检测方法的敏感性为99%（69/70）。HOXA9和TBX5是最敏感的2个标记基因，分别为87%（61/70）和84%（59/70）。RASSF1A和DLEC1的特异性最高，为99%（69/70）。以至少鉴定出任意两个甲基化标记基因为标准，61/70的癌症样本呈阳性，敏感性为87%，特异性为94%。早期I期或II期NSCLC甚至可以100%的特异性和86%的敏感性被检测到。总之，MMSP有潜力发展成为一种基于人群的筛查工具，可用于NSCLC的早期诊断。它也可能适用于监测治疗效果和复发情况。
