摘要
中文總摘要 目的 本研究目的是藉由非侵襲性的方法，分析膀胱癌病人尿液檢體中腫瘤細胞之抑癌基因不正常甲基化的表現，以提早偵測出膀胱癌存在情形。並進一步探討經尿道膀胱腫瘤刮除術(TURBT)後，其病理學正常組織中抑癌基因不正常甲基化的結果與膀胱癌復發的相關性。 材料與方法 利用Methylation-specific PCR實驗方法，分析膀胱癌病人其術前尿液檢體及術後腫瘤組織檢體其抑癌基因啟動子的甲基化情形。並同時分析健康成人尿液檢體當作實驗對照組。其次，收集膀胱癌組織及病理學正常的膀胱組織檢體。利用Methylation-specific PCR方法分析組織檢體中抑癌基因甲基化的表現，分析病人的臨床病理資料，進一步評估抑癌基因不正常甲基化與膀胱癌復發的相關性。 結果 57位膀胱癌病人中有47位(83%)尿液檢體至少一個抑癌基因有不正常的甲基化表現。而實驗對照組的健康成人尿液，並沒發現不正常的DNA甲基化情形。從尿液檢體中分析RASSF1A、 p14 或E-cadherin 任一抑癌基因甲基化來評估膀胱癌發生則敏感度可高達83%。本研究發現表淺性(superficial)膀胱癌病人中，傳統尿液細胞學檢查為23% (6/26)。相較之下，利用Methylation-specific PCR分析E-cadherin、 p14或 RASSF1A 不正常基因甲基化偵測膀胱癌敏感度，在表淺性(superficial) 膀胱癌為 85% (22/26)、low grade膀胱癌為85% (11/13)。重要的是，在膀胱癌病人細胞學檢查為陰性或非典型細胞atypia的表淺性(superficial) 膀胱癌結果中發現若以Methylation-specific PCR偵測膀胱癌其敏感度可達到90% (18/20) 。 在膀胱癌病人中，病理學正常組織的膀胱檢體，發現抑癌基因具有不正常甲基化的現象，且隨著癌症進展程度愈高，其基因不正常甲基化的程度也隨著愈高E-cadherin (p=0.067)、p16(p＜0.001)、p14 (p=0.01)、RASSF1A(p=0.01)。統計分析結果也發現病理學正常組織的膀胱檢體若有p14抑癌基因不正常甲基化，則可能與膀胱癌的復發具有相關性(p=0.019)。 結論 利用Methylation-specific PCR方法偵測尿液檢體中E-cadherin、 p14 或 RASSF1A 抑癌基因不正常甲基化是個可信賴的非侵襲性診斷方法，可藉此提早偵測superficial、low grade膀胱癌的存在。 且在病理學正常組織的膀胱檢體，發現其p14抑癌基因不正常甲基化的結果可能是一個預測膀胱癌復發的重要因子。
並列摘要
bstract Objectives: To identify a better set of DNA methylation markers to detect superficial, low grade cancer cell in urine sediment. To investigate the hypermethylation of E-cadherin, p16, p14 and RASSF1A in pathologically normal urothelium to predict recurrence of bladder cancer after transurethral resection. Materials and Methods: Methylation-specific PCR (MSP) assay was used to detect promoter hypermethylation in four genes in primary tumor DNA and urine sediment DNA from 57 bladder cancer patients. Urine DNA from twenty healthy individuals were used as normal controls. Furthermore, the status of promoter hypermethylation was investigated by methylation-specific polymerase chain reaction in 50 bladder cancer samples and paired normal urothelia. The clinicopathological data were also analyzed in order to evaluate the clinical implication of aberrant methylation in bladder cancer recurrence. Results: Fifty-one (90%) tumor DNA and 47 urine DNA (83%) samples from bladder cancer patients revealed hypermethylation in at least one of the four analyzed genes whereas all urine samples from normal controls were negative. MSP detected hypermethylation in the urine from 80% (37/46) bladder cancer patients. Moreover, hypermethylation analysis of E-cadherin, p14 or RASSF1A genes in urine sediment DNA detected in 85% (22/26) of superficial, 85% (11/13) of low grade bladder cancers. More Importantly, hypermethylation was detected in the urine DNA of 90% (18/20) superficial tumors with negative or atypia cytology. Promoter hypermethylation in paired normal urothelia increased with progression of bladder cancer at E-cadherin (p=0.067), p16 (p＜0.001), p14 (p=0.01), and RASSF1A (p=0.01). Besides, p14 hypermethylation in pathologically normal urothelia was associated with shorter recurrence-free interval (p=0.019). Conclusions: Hypermethylation of E-cadherin, p14 or RASSF1A in urine sediment DNA is a reliable marker for detecting superficial, low grade cancer. P14 hypermethylation in pathologically normal urothelium samples should be considered a predictor of bladder cancer recurrence.