Clinical Study Of The Methylation Status Of RASSF1A Promoter And Its Clinical Significance In Primary Lung Cancer

Background: Aberrant methylation of CpG islands in the promoter region of tumor suppressor genes (TSGs) and tumor-related genes has become established as the major mechanism for gene silencing. Inactivation of TSGs by DNA methylation is regarded as one of the fundamental processes for the development of human malignant tumors, including lung cancers. We studied the methylation status of RASSF1A and clinical features in primary lung cancer. We also examined weather hypermethylation of RASSF1A promoter is an independent prognostic factor. Methods: We assessed the methylation status of their respective promoters in 212 resected non-small-cell lung cancers (NSCLCs), 31 resected small-cell lung cancers (SCLCs), 34 corresponding normal tissues, and 25 nonmalignant tissues by using a methylation-specific polymerase chain reaction (MSP). The PCR products were purified using gel extraction kit. PCR products were ligated into the pTA2 vector and several clones were sequenced for confirmation. Results: RASSF1A promoter hypermethylation was detected in 38.2%(81/212) of primary NSCLCs, 74%(23/31) of rescted SCLCs, but none of the corresponding normal tissues and nonmalignant tissues. There was no relationship between the hypermethylation of RASSF1A and clinicopathologic features, such as age, gender, smoking duration time, TNM stage, tumor size and histological type in NSCLCs. However, hypermethylation of RASSF1A promoter was found to be significantly associated with the age at starting smoking (P=0.015); the association between the differentiation of tumor and methylation of RASSF1A was statistically significant (P=0.000); the association between lymphatic permeation and methylation of RASSF1A was statistically significant (P=0.001). Analyzing the effect of methylation of RASSF1A on survival of 150 NSCLC patients, the Kaplan-Meier survival curves show that the patients with hypermethylation of RASSF1A have a poor survival rate, and the relationship between the survival rate and hypermethylation of RASSF1A was statistically significant (P=0.004). Then by using stepwise Cox proportional hazard regression testing, methylation status of RASSF1A was an independently factor affecting the patients'survival (RR=1.584, 95%CI=1.040-2.411, P=0.032). Conclusion: The promoter region of the RASSF1A gene has a hypermethylation tendency. The methylation-specific polymerase chain reaction (MSP) analysis of RASSF1A hypermethylation enabled distinction between patients diagnosed with lung cancer and those with nonneoplastic lung disease. These results suggested that detection of hypermethylation of the RASSF1A promoter is a promising molecular tool for diagnosis of primary lung cancer and it may be an independent prognostic factor in primary non-small cell lung cancer.