去氧核糖核酸甲基化與台灣非小細胞肺癌之關聯

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

吳則樂

肺癌 ； 甲基化 ； lung cancer ； methylation

臺灣大學臨床醫學研究所學位論文

2007年

碩士

余忠仁

繁體中文

研究背景與目的 肺癌目前在台灣與世界各地均為癌症的主要死亡原因之一，台灣衛生署統計2006年台灣十大死因，癌症高居十大死因第一位，其中尤以肺癌居首，占所有癌症死亡率百分之十九點七；非小細胞肺癌約占原發性支氣管肺癌百分之八十至八十五，五成以上肺癌病人在診斷時已為第三、第四期，未經治療的第四期非小細胞肺癌，平均存活四至五個月，少於百分之十的病人可存活超過一年 [Blackhall et al.,2006]；若能早期發現肺癌，早期治療，將有助於降低肺癌死亡率。 甲基化 (hypermethylation) 是抑制腫瘤抑制基因或其他癌症相關基因的主要機轉，在細胞過度增生或變性時便有可能產生，已被視為人類癌症的早期指標，目前已發現會因甲基化而抑制的基因，遍布整個細胞週期；此外，甲基化在人類癌症通常具組織特異性，所以可以當做腫瘤標記來進行檢查。而甲基化基因常隨種族、抽菸狀況、病理種類與癌症分期而有所不同，此研究主要是針對台灣非小細胞肺癌病患，希望能找出台灣肺癌病人常見的高甲基化基因，幫助我們早期診斷、早期治療病患，並進一步了解肺癌與甲基化變異的關聯。 研究方法與材料 我們取得西元1995年9月至西元2002年11月，共一百零六位經細胞學或病理組織切片確診為非小細胞肺癌，且年滿二十歲病患的開刀後檢體，利用甲基化聚合酶鏈鎖反應與甲基化實序聚合酶鏈鎖反應進行研究，比較甲基化基因與肺癌分期、細胞種類、抽菸、性別、存活率之關聯；並自第一至第四期共取十位肺癌病患檢體，進行甲基化微陣列--差異性甲基化雜合 (Differential Methylation Hybridization, DMH)，篩選在台灣肺癌有出現甲基化變異的基因。 用SPSS 13 (SPSS Inc., Chicago, IL, USA)與StatsDirect軟體分析甲基化聚合酶鏈鎖反應結果；肺癌病患存活率與甲基化基因分析利用Kaplan-Meier method與two-sided log-rank test；病人特性與甲基化基因分析，若為類別變數用Fisher’s exact test分析，連續變數則用Mann-Whitney U test分析；利用cox regression進行多變數分析；p<0.05代表有顯著意義。 甲基化微陣列利用Gene Pix Pro 4.1掃描，晶片上的螢光比值經過校正、對數轉換(log transformation)與標準化(standardization)處理，畫成散佈圖(scatter plots)；並分早期或晚期肺癌，以Cy5/Cy3 ratio≧1.5選取高甲基化基因。 結果與討論 106位病人中共62位肺癌病患出現甲基化RASSF1A (58.5%)，相對於肺鱗狀細胞癌(11/28, 39.3%)，甲基化RASSF1A主要出現在肺腺癌(49/72, 68.1%)(p=0.0087)。在腫瘤出現甲基化RASSF1A的病患有明顯較差的無復發存活率 (p=0.002)，但總存活時間兩者並無明顯差異。在多變數分析可發現甲基化RASSF1A與肺癌晚期均為肺癌病患的獨立預後因子，危險性分別為2.08倍(95%信賴區間為1.194~3.625，p=0.01))及2.939倍(95%信賴區間為1.749~4.939，p<0.0001)。甲基化RASSF1A出現的頻率不受分期、性別、抽菸與否影響，故可以做為臺灣肺癌病患早期診斷或早期偵測開刀後復發的標記，及早進行肺癌治療，同時也可當做評估預後的指標。 此外，EGFR突變主要出現在女性肺腺癌，而甲基化RASSF1A在肺腺癌也有明顯升高的趨勢(p=0.0087)，比較甲基化RASSF1A與EGFR突變兩者出現的頻率，顯示並無相關，p值=0.1655，故推測兩者造成癌化的途徑應沒有關聯。 甲基化微陣列篩選出的高度甲基化的基因，在早期與晚期肺癌均出現轉錄、轉譯相關基因，結構蛋白、細胞傳輸相關的基因，酵素相關基因；此外，訊息傳遞、細胞生長相關基因的甲基化主要出現在早期肺癌，而晚期肺癌則出現包括細胞黏著、細胞發展分化相關基因，細胞調節相關基因的高甲基化，顯示癌症的產生可能有階段性的甲基化基因變化。

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.

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