中文摘要
研究背景及目的：肺癌目前在全世界屬癌症死亡率之首位，五年之存活率只有14%，高死亡率的原因主要是由於肺癌不易早期偵測及治療後的高復發率所致。對於早期(第一期)的非小細胞之肺癌，手術治療是對於此群病人的標準方式；僅管如此，第一期的非小細胞肺癌在手術後仍有30-40%的病人死於疾病的復發。因此，建立準確的預後(prognosis)分子指標一直是肺癌臨床研究的重要課題，也是對於此類病人輔於化學治療的新思維。目前傳統上預測肺癌預後之工具為腫瘤TNM分期，然而其準確性仍有其限制；因此，發展更準確預後工具實有其必要性。過去研究顯示，抑癌基因 (Tumor suppress genes) 啟動子過度甲基化 (promoter hypermethylation) 與癌症形成 (initiation) 及惡化 (progression) 有關，我們得到經由全基因組之啟動子過度甲基化的篩檢，可以找出預測早期非小細胞肺癌預後的過度甲基化基因。

研究方法及結果：此研究有兩個主要結果：（1）利用Illumina基因體甲基化分析法，以篩檢族群 (screening cohort) 之配對癌組織及血漿建立台灣肺癌基因體甲基化變異圖譜，綜合鑑別出高頻率甲基化區域，並配合病人病理資料分析，找出與肺癌形成與預後有關的甲基化基因體區域；（2）以驗證族群 (validation cohort) 研究肺癌病患及正常人之組織中基因啟動子甲基化之變異情形，計算出所篩檢之高頻率甲基化基因分子指標之敏感性及專一性。以新穎的Illumina基因體甲基化圖譜及Pyrosequencing定量甲基化分析法，挑選並鑑定與肺癌有關之抑癌基因過度甲基化指標，建立預測肺癌預後甲基化pyrosequencing平台。本研究完成了75位（51位screening cohort、24位validation cohort）具有預後資料（存活率、癌症復發及轉移）的早期肺癌病人 (stages I & II) 之基因體甲基化圖譜，確定了目標（1）與目標（2）肺癌預後較差有關的37個甲基化指標（肺腺癌18個，鱗狀上皮肺癌18個，兩種都有1個），28個基因於24位病人完成了pyrosequencing 甲基化定量分析，基因Probe 1、2、9、10、11、12、17、21、22、25 和28在肺腺癌與正常組織有顯著不同的統計意義，確定他們可能為肺腺癌患者分子指標。另一方面，基因Probe 25、26和27在鱗狀細胞肺癌與正常組織有顯著不同的統計意義，確定他們可能為鱗狀細胞肺癌患者分子指標。再從中挑出4個基因probe 25、26、27及28於120位病人以pyrosequencing 甲基化分析，確定基因Probe 25、26和27其肺癌組織甲基化程度顯著高於正常肺組織 (P<0.0001~P=0.018)，而且基因Probe 27和26的甲基化可預測肺癌的預後 (Cox regression, P=0.0011~0.0102)。

結論: 基因Probe 26和27的甲基化可預測肺癌的預後。

英文摘要
Background and Purpose: Lung cancer is the leading cause of cancer-related deaths worldwide. A more sensitive biomarker panel would allow for more accurate prognostic prediction of lung cancer than the current TNM stage methods. CpG island hypermethylation is known to be associated with tumor suppressor gene (TSG) silencing and can be detected in tissue. In addition, promoter hypermethylation of TSGs has been shown to correlate with cancer initiation and progression. Therefore, we performed epigenome-screening approach for analysis of methylation profiles of thousands of gene in parallel and aimed for the identification of methylation biomarkers critical to lung tumorigenesis. The potential methylation biomarkers were validated by pyrosequencing methylation assay. The goal of our study is to develop promotor gene methylation by pyrosequencing for survival prognosis of lung cancer.

Methods and Results: We performed epigenome-screening to select multiple methylation biomarkers in tumor tissue of a screening cohort of early staged (stages I and II) patients with known survival data. We confirmed the specificity and sensitivity of these multiple methylation biomarkers in a validation cohort. We evaluated if these methylated regions can be used as effective biomarkers for prognostic prediction. We have adapted the Illumina’s Infinium methylation assay (28,000 CpG dinucleotides for 14,000 genes) and pyrosequencing quantitative methylation assay to identify 37 hypermethylated regions (18 for lung adenocarcinoma; 18 for lung squamous carcinoma; 1 for lung adenocarcinoma and lung squamous carcinoma) with prognostic potential in 75 stages I and II lung cancer patients (51 in screening cohort; 24 in validation cohort) with survival data available. Among the 28 hypermethylated regions were validated for methylation status using pyrosequencing in DNA from 24 pair’s tumor and normal tissues from 12 ADC patients. Gene probes 1, 2, 9, 10, 11, 12, 17, 21, 22, 25 and 28 showed a significantly differential power between tumor and normal tissues, suggesting their association with lung cancer in 12 ADC patients. On the other hand, Gene probes 25, 26 and 27 showed a significantly differential power between tumor and normal tissues, confirming their association with lung cancer in 12 SCC patients. Gene probes 25, 26 and 27 with significantly differential power between tumor and normal tissues were further confirmed for their association with lung cancer in 120 patients (P<0.0001~P=0.018). The hypermethylation of gene probes 27 and 26 could predict the survival of the lung caner in 120 patients (Cox regression, P=0.0011~0.0102).

Conclusion: The hypermethylation of Gene probes 26 and 27 could predict the survival of the lung caner in 120 patients.

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