表皮生長因子接受器（EGFR）突變的偵測，在肺腺癌中可作為預測患者對表皮生長因子接受器標靶治療效果的依據，當腫瘤細胞存在表皮生長因子接受器基因突變時，使用酪胺酸激酶抑制劑 (TKIs)可提供良好的治療反應。然而診斷肺癌以及分析表皮生長因子接受器突變所需的檢體，通常來自侵入性方式，檢體經常很小且有限，甚至難以獲得適當之檢體以供檢測。
為了增加表皮生長因子接受器突變檢測的可行性和敏感度，我們建立一個高敏感度的鐵克曼突變偵測系統（TMDA）方法，來分析肺癌常見之惡性肋膜積液之表皮生長因子接受器突變。本試驗一共收集了70例患者的肋膜積液，採取其中之游離DNA進行檢測，其中有50例的患者，先前已使用組織蠟塊或肋膜積液的細胞蠟塊，採用直接定序 (Sanger sequencing) 或Scorpions ARMS的方法測得表皮生長因子接受器突變結果。我們以鐵克曼突變偵測系統檢測相同患者的惡性肋膜積液游離DNA，以比較其與組織或細胞蠟塊檢體檢測表皮生長因子接受器突變狀況的一致性。
由於表皮生長因子接受器突變主要發生在酪胺酸激酶所在之Exon 18-21，尤其以Exon 19缺失 (deletion) 和Exon 21 (L858R) 突變占最多，約90%，故我們主要檢測Exon 19缺失及L858R，檢測後得到的結果在70例的患者，其中有3例沒有訊號、可能因DNA的量不足，扣除這3例患者，而其餘的67例患者中，有L858R突變或Exon 19缺失共有40例，所佔比率為59.7%。在所有突變的40例患者中，L858R突變有26例、佔所有突變的65%，Exon 19缺失有20例、佔所有突變的比率為50%，其中包括6例同時有L858R突變與Exon 19缺失，佔所有檢測病例之15%。
由我們的研究顯示，以鐵克曼突變偵測系統檢測肺癌惡性肋膜積液中之表皮生長因子接受器突變狀態是可行的方法，且可考慮在切片之前即以惡性肋膜積液進行表皮生長因子接受器突變分析，以縮短檢測時間，使患者可以更快接受治療。

Epidermal growth factor receptor (EGFR) mutation status is important for the selection of the candidates for target therapy with tyrosine kinase inhibitor in lung adenocarcinomas. However, the specimens for analysis of EGFR mutation are usually derived from invasive procedures and not always available.
To increase the availability and sensitivity of EGFR mutation detection, we used a highly sensitive Taqman Mutation Detection Assay (TMDA) for EGFR mutation analysis in 70 patients of malignant pleural effusion, including 50 patients previously analyzed for EGFR mutation by Sanger sequencing or real-time PCR method on either biopsy/surgical specimen or cell block of pleural effusion. Cell-free DNA in pleural effusion was isolated to perform EGFR analysis by this assay. A comparison of the EGFR mutation status between the cell-free DNA of pleural effusion and the previous specimen from the same patient was also made.
EGFR mutations mainly occur in the tyrosine kinase domain, exons 18–21, in which deletions in exon 19 and point mutation in exon 21 (L858R) account for approximately 90%, therefore the main EGFR mutation testing in the present study has focused on exon 19 deletions and L858R mutation. The result showed no signal in 3 out of the 70 patients, which may be due to insufficient amount of DNA. In the other 67 patients, 40 (59.7%) of them showed L858R mutation or exon 19 deletions. There are 26 patients (65%) with L858R mutation and 20 patients (50%) with exon 19 deletions. Both L858R mutation and exon 19 deletions are found in 6 patients, accounting for 15% in all EGFR mutation cases.
Our study indicates that TMDA is a feasible technique to detect EGFR mutation status in malignant pleural effusion, and potentially useful for the discovery of false-negative cases analyzed by Sanger sequencing. The malignant pleural effusion could be firstly used for EGFR mutation analysis to shorten the turnaround time.

論文審定書…………………………………………………………… . i
中文摘要………………………………………………………….……ii- iii
英文摘要………………………………………..…………………….iv-v
第一章 背景資料…………………………………………………………1
1.1 肺癌的病因………………………………………………………………1
1.2 肺癌的種類………………………………………………………………2
1.3 非小細胞肺癌依據細胞型態可分成主要三種…………………….2
1.4 肺癌臨床診斷之分期……………………………………………………2
1.5表皮生長因子…………………………………………………………3
1.6 肋膜積液…………………………………………………………………4
1.7肋膜積液之分子檢測…………………………………………………5
1.8 實驗目的………………………………………………………………6
第二章 材料與方法………………………………………………………7
2.1 檢體來源………………………………………………………………7
2.2 肋膜積液取樣方法……………………………………………………7
2.3 抽取肋膜積液中的游離DNA…………………………………………7
2.4 測試即時聚合酶鏈反應效率最佳之游離DNA濃度………………8
2.5利用內部陽性對照組 (Internal positive control) 的實驗設計排除聚 合酶鏈反應偽陰性的結果 (false negative) ……………………………9
2.6 評估以鐵克曼突變偵測系統檢測肋膜積液之表皮生長因子接受器突變之可行性…………………………………………………………………9
2.7 以鐵克曼突變偵測系統分析肋膜積液游離 DNA之表皮生長因子接受器突變………………………………………………………………………10
2.8 對照組之臨床檢體的表皮生長因子接受器突變分析檢測……10-11
第三章 結果…………………………………………………………………12-13
第四章 討論………………………………………………………………14-16
第五章 結論………………………………………………………………17
參考文獻……………………………………………………………27-30
附錄……………………………………………………………………31-38

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