DNA Hypermethylation in Taiwanese Patients with Non-small Cell Lung Cancer

• Main Authors: Tzer-yueh Wu, 吳則樂
• Other Authors: Chong-Jen Yu
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/55437430084723976221

碩士 === 臺灣大學 === 臨床醫學研究所 === 95 === Background Lung cancer is the major cause of cancer-related death in Taiwan and worldwide. Lung cancer accounted for 19.7% of all cancer deaths in 2006 in Taiwan, and 20% to 30% worldwide. About 80% to 85% of all lung cancers are non-small-cell cancer (NSCLC), with over 50% of patients presenting with locally advanced and/or unresectable disease. Detection of lung cancer at earlier stages could allow early treatment and potentially improve survival. During carcinogenesis, molecular aberrations including genetic and epigenetic changes precede morphological changes. Aberrant promoter hypermethylation is a major mechanism for silencing tumor suppressor or other cancer-associated genes. The purpose of our study is to determine the prevalent methylated genes in Taiwanese lung cancer patients, and to identify whether there is a relationship existed between methylation and age, gender, smoke exposure, histology type and tumor stage in resected NSCLC. The relationship between aberrant methylation and survival in surgically treated NSCLC patients was further explored. Materials and Methods This study was approved by National Taiwan University Hospital (NTUH) Institutional Review Board. Genomic DNA was extracted from 106 Taiwanese NSCLC patients who underwent curative surgery between 1995 and 2002 at NTUH. The inclusion criteria include male or female with histologically- and/or cytologically-diagnosed non-small cell lung cancer, and 20 years of age or older. Patients diagnosed with malignancies other than NSCLC within 5 years were excluded. Patients who received neoadjuvant chemotherapy were also excluded. Demographic data of the patients were shown in table 4. The commonest cell type was adenocarcinoma (72, 67.9%) as contrasted to squamous cell carcinoma (28, 26.4%). Other cell types consisted of poorly differentiated carcinoma and adenosquamous cell carcinoma. 58 patients had stage I disease, 14 stage II, 20 stage IIIA, 8 stage IIIB with T4 but not N3 disease, and 6 stage IV. Among the 6 stage IV patients, 2 had solitary distant metastasis at brain, 2 at different pulmonary lobes, and the other two patients were found to have bone metastasis shortly after the surgery. DNA from whole blood of healthy volunteers was treated with SssI methyltransferase, and then subjected to bisulfite treatment as a positive control for methylated alleles. Methylation-specific PCR was performed to detect methylation in both non-tumor and tumor parts from surgically-treated lung cancer patients. Bisulfite-sequencing PCR was performed to ensure complete methylation of cytosines in C in 5''-CG-3'' sequence. Microarray-based techinique (differential methylation hybridization, DMH) was utilized in 10 patients to detected novel hypermethylated genes of lung cancer. Taiwanese NSCLC patients with or without gene methylation were compared using Fisher’s exact test for categorized variables, using Mann-Whitney U test for continuous variables. Kaplan-Meier estimation was used to plot survival curves, and log-rank tests were used to test the difference between groups. Two-sided p values <0.05 were considered statistically significant. All data were analyzed with SPSS 13 (SPSS Inc., Chicago, IL, USA). DMH chips were scanned and analyzed with Gene Pix Pro 4.0. Results & conclusion RASSF1A methylation is significantly higher in Taiwanese neoplastic lung tissues (p<0.0001), especially in adenocarcinoma when comparing with squamous cell carcinoma (p=0.0087). NSCLC patients with RASSF1A hypermethylation also have shortened relapse free survival (p=0.002) but no differences was noted in overall survival (p=0.827). The frequency of RASSF1A methylation was statistically not associated with stage (p=0.292), patients’ age (p=0.557), gender (p=0.421), or smoking status (p=0.221). In multivariate analysis, RASSF1A methylation (RR=2.080, p=0.010) and advanced stage (RR=2.939, p<0.0001) both appeared to be independent prognostic factors of relapse free survival in surgically treated Taiwanese NSCLC patients. Methylated RASSF1A can be utilized as a potential marker in for early diagnosis of lung cancer or early detection of lung cancer recurrences in Taiwanese patients regardless of age, gender and smoking status. Early medical intervention can therefore be instituted for better prognosis. The epidermal growth factor receptor (EGFR) was found to be mutated in 37 (45.7%) of the 81 patients analyzed. The result showed that 33 of 55 (60%) adenocarcinomas and only 2 of 22 (9.1%) squamous cell carcinomas contained EGFR mutation (p<0.0001). RASSF1A hypermethylation and EGFR mutation both predominantly appear in Taiwanese lung adenocarcinomas. However, the association between RASSF1A hypermethylation and EGFR mutation was not statistically significant (p=0.165). DMH analysis of 10 Taiwanese NSCLC patients showed that hypermethylation mainly occurred in genes associated signal transduction, transcription and translation, cell cycle regulation, structural proteins, intercellular adhesion and developmental pathways. Genes controlling cell growth tend to be methylated in early stages, whereas genes involving intercellular adhesion and development are methylated in late stages. Further research is required to establish the methylation profiles in Taiwanese NSCLC patients, and to clarify the methylation mechanism in lung cancer.