台灣肺腺癌與表皮生長因子受體基因突變流行病學研究

Abstract

本論文以三個子計劃來探討肺腺癌病人其EGFR基因突變在台灣地區的流行病學特性以及其可能發生的致突變機轉。第一部分評估EGFR基因突變盛行率在性別分布的差異，並探討其與主動抽煙及二手煙暴露之相關性。 第二部分評估EGFR基因突變與雌激素生合成及其代謝之基因多型性的相關。第三部分除了探討EGFR基因突變與EGFR 基因本身之基因多型性相關性外，並進而評估DNA 修補基因、異物代謝基因是否與EGFR基因突變有相關。 研究一、肺腺癌病人EGFR基因突變盛行率在性別分布的差異、與主動抽煙及二手煙暴露之相關性研究 目的: 肺癌是全球高發生率及高死亡率的癌症之一，每年約有百萬人被診斷並死於肺癌。在台灣所有惡性腫瘤死因中，肺癌也居首位。在台灣，過去30 年腺癌佔所有肺癌的比例始終高居第一，其中女性、非吸菸者有較高的比率會罹患肺腺癌。根據流行病學的特徵顯示主動抽煙、二手煙以及基因易感受性都是肺腺癌的可能危險因子。過去研究指出癌症組織的EGFR 基因會在特定的地方產生突變(如在exon19 產生in-frame deletion 、exon21產生 L858R點突變) ，而此些突變會與腫瘤發生的有關。因此本研究將探討性別、香煙暴露(含主動抽煙及二手煙) 差異是否會影響EGFR 基因突變。 方法: 研究收集台中榮總肺腺癌病人共617 名。此些個案完成EGFR 基因突變之定序分析。其中132 位個案同時參與臺灣女性肺腺癌之遺傳流行病學研究 (GEFLAC)，因此由其中取得二手煙暴露之問卷資料。結果：相較於不抽煙者，主動抽煙與EGFR 基因突變成反比，同時29 歲前開始抽煙、抽煙的暴露達21 包年、或者戒煙小於21 年都與EGFR 基因突變的頻率成反比。至於二手煙的暴露沒有呈現劑量效應相關，然而當暴露的時間小於16 年時，相較於未暴露者，EGFR 基因有較低的突變比率。由此可見，EGFR 基因有較高的突變比率出現在不抽煙族群。進一步分析發現，不抽煙族群中，男性相較於女性有較高的exon19 in-frame deletion 突變比率(36.1% vs. 23.5%)，相反地，女性相較於男性有較高的L858R突變比率 (28.8% vs. 20.4%)。結論：香煙暴露與EGFR 基因突變比率成反比。在不抽煙族群中，特定型別的EGFR 基因突變呈現性別差異，此現象推測可能有不同比重的分子機轉參與EGFR特定型別的致突變性。 研究二、進行女性不抽煙的肺腺癌病人EGFR基因L858R點突變與及荷爾蒙生合成代謝之基因多型性的相關性研究 目的: EGFR基因突變會與肺癌標靶治療的反應性有關，也會與腫瘤的發生有關。因此本研究檢視雌激素生合成、代謝基因的多型性是否會與EGFR 基因突變有相關。方法: 617 名肺腺癌病人組織檢體中有410 位是不抽煙者。據此評估CYP17、CYP19A1、ERα 及COMT 之易感受基因型與EGFR 基因突變發生的相關性。結果: 在女性不抽煙者，EGFR L858R突變與 CYP19A1(TTTA)n、ERα rs2234693、COMT rs4680 基因型有統計上的顯著相關，其校正後的風險對比值分別為：2.6 (95 % 信賴區間：1.2-5.7)、2.1 (95 % 信賴區間：1.1-4.0)、1.8 (95 % 信賴區間：1.0-3.2)，CYP17 rs743572基因型其校正後的風險對比值為 1.5 (95 % 信賴區間：0.8-2.7)，其相關性未達統計顯著水準。進一步地合併CYP17、CYP19A1、ERα 及COMT 等易感受等位基因型 (allele)，經趨勢檢定之後，與EGFR L858R突變呈現劑量效應相關。此外，ERα 基因型也與EGFR exon19 in-frame deletion 及其他突變型(非L858R及exon19 in-frame deletion) 有統計上的顯著相關，其校正後的風險對比值分別為：2.9 (95 % 信賴區間：1.1-7.6)、4.3 (95 % 信賴區間：1.3-14.0)。再者，COMT rs4680在男性不抽煙者也呈現統計上的顯著相關，其校正後的風險對比值分別為3.6 (95 % 信賴區間：1.1-11.3)。結論：在不抽煙的肺腺癌病人，尤其是女性，EGFR L858R突變與雌激素生合成、代謝基因的多型性有相關。 研究三、EGFR exon 19 in-frame deletion與EGFR 基因、DNA 修補基因、及異物代謝基因之易感受基因型的相關性研究 目的: 檢視參與DNA 修補、解毒、異物代謝之易感受基因和EGFR 基因的基因多型性與EGFR基因exon 19 in-frame deletion 發生是否有相關。方法: 以410名不抽煙之肺腺癌病人組織，透過非條件羅輯迴歸模式，檢視上述易感受基因型與EGFR 基因突變的相關性。結果: 在女性不抽煙者，EGFR in-frame deletion與ERCC4 rs744154、NQO1 rs1800566 基因型有統計上的顯著相關，其校正後的風險對比值分別為：1.9 倍 (95 % 信賴區間：1.0-3.6)、2.2倍 (95 % 信賴區間：1.0-4.8)，EXO1 rs1047840基因型其校正後的風險對比值為 7.6倍 但未達統計顯著水準。進一步地合併ERCC4、NQO1、及EXO1等易感受等位基因型 (allele)，經趨勢檢定之後，與EGFR in-frame deletion呈現劑量效應相關。除此之外，在不抽煙者及女性不抽煙者EGFR (CA)n 基因型與EGFR in-frame deletion的發生也有統計上的顯著相關，其校正後的風險對比值分別為：1.7 倍 (95 % 信賴區間：1.0-2.9) 及1.9 倍 (95 % 信賴區間：1.0-3.5)。結論：在不抽煙的肺腺癌病人，尤其是女性，EGFR in-frame deletion的發生與DNA 修補、解毒及EGFR 本身的基因型有相關。 綜合本論文之研究顯示，EGFR 特定突變型別頻率在性別上呈現差異，此外，在女性不抽煙之肺腺癌病人EGFR基因突變，特別是L858R 及 exon19 in-frame deletion 分別與雌激素生合成、代謝機轉以及DNA 修補、解毒機轉的易感受基因型有關。同時，EGFR exon19 in-frame deletion也與EGFR 本身的易感受基因型有關。 此些似乎暗喻這兩種突變型的發生可能部分來自不同的分子機轉，部分共享相同的分子機制。有關基因與環境交互作用方面，環境暴露特別是環境荷爾蒙以及與肺腺癌相關的危險因子如二手煙，是否能修飾EGFR基因突變的發生有待進一步研究。雌激素受體之易感受基因型與各類型的EGFR突變皆有關，此現象暗示著抗雌激素藥物在EGFR突變的肺腺癌病人的預後應用值得進一步探討。

This dissertation included 3 studies to investigate the associations between EGFR mutation status and the genetic background and environmental carcinogens in never-smoking lung adenocarcinoma. Therefore, there will be several analyses and assays to be carried out: (1) Using the questionnaires, the relationships between exposures to the cigarette smoke, environmental tobacco smoke (ETS) and EGFR hotspot mutations will be delineated. (2) The genetic polymorphisms of the biosynthesis and catabolism of estrogen and DNA repair pathways will be studied in terms of the mechanisms of EGFR hotspot mutations. Study1: The Associations between Sex and Smoking Status on EGFR Mutations in Adenocarcinoma Background and Aims: To assess the impact of cigarette smoke and environmental tobacco smoke (ETS) are associated with the EGFR mutations status in adenocarcinoma patients. Methods: We enrolled 617 patients with lung adenocarcinoma. The history of cigarette smoking-related characteristics was obtained from a questionnaire; Among 132 patients who were also participated in the Genetic Epidemiological Study of Lung Adenocarcinoma (GELAC) study, the history of ETS exposure were obtained. Results: Patients with more than 21 pack-years of smoking histories or smokers with the fist use cigarette before the age of 29, or those who quit smoking less than 21 years had a significant lower frequency of EGFR mutations than never-smokers. Furthermore, there was a significantly inverse trend between smoking dose and EGFR mutation (p<0.0001). With regard to ETS, there was not a significant dose-response relationship of patients exposed to ETS with EGFR mutations. But a significant association was found for patients exposed to ETS for ≦ 16 years compared to patients not exposed to ETS (p=0.05). Additionally, Sex was significantly associated with EGFR mutation status in never-smokers (p=0.03). The percentage of in-frame deletion was higher in males than females (36.1% vs. 23.5%), and the percentage of L858R mutation was higher in females than males (28.8% vs. 20.4%). Conclusion: The amount and duration of cigarette smoking history were inversely associated with the EGFR mutations status. However, the impact of ETS remains inconclusive. In never-smokers, a significant difference was found between EGFR mutation subtypes and sex, this sex difference implies different mechanisms for the EGFR mutagenesis in EGFR mutation subtypes. Study2: EGFR L858R Mutation and Polymorphisms of Genes related to Estrogen Biosynthesis and Metabolism in Never-smoking Female Lung Adenocarcinoma Patients Background and Aims: To assess whether polymorphisms of genes related to estrogen biosynthesis and metabolism are associated with EGFR mutations. Methods: We studied 617 patients with lung adenocarcinoma, including 410 never-smoking patients. On the basis of multiple candidate genes approach, the effects of polymorphisms of CYP17, CYP19A1, ERα and COMT in association with the EGFR mutations status were evaluated using logistic regression analysis. Results: In female never-smokers, significant associations with EGFR L858R mutation were found for the (TTTA)n repeats in CYP19A1 (odds ratio [OR], 2.6; 95% confidence interval [CI], 1.2-5.7 for one or two alleles with (TTTA)n repeats >7 compared to both alleles with (TTTA)n repeats ≦7), and the rs2234693 in ERα (OR,2.1; 95% CI, 1.1-4.0 for C/T and C/C genotypes compared to T/T genotype). The C/C genotype (vs. T/T genotype) of ERα was significantly associated with EGFR L858R mutation (OR, 3.0; 95% CI, 1.1-8.1), in-frame deletion (OR, 2.9; 95% CI, 1.1-7.6) and other mutations (OR, 4.3; 95% CI, 1.3-14.0). The genotype of COMT rs4680 was significantly associated with EGFR L858R mutation in female and male never-smokers showing OR’s (95% CI) of 1.8 (1.0-3.2) and 3.6 (1.1-11.3), respectively, for genotypes G/A and G/G compared to genotype A/A. The number of risk alleles of CYP17, CYP19A1, ERα and COMT was associated with an increasing OR of EGFR L858R mutation in female never-smokers (P=.0002 for trend). Conclusions: The L858R mutation of EGFR is associated with polymorphisms of genes related to estrogen biosynthesis and metabolism in never-smoking female lung adenocarcinoma patients. The findings provide a clue for the genesis of EGFR mutations.

Study3: EGFR Exon 19 In-frame Deletion and Polymorphisms of EGFR and DNA Repair Genes in Never-smoking Female Lung Adenocarcinoma Patients Background and Aims: To explore whether the genetic polymorphisms in genes related to DNA repair and detoxification metabolism and in epidermal growth factor receptor (EGFR) are associated with EGFR mutations. Methods: We studied 410 never-smoking patients with lung adenocarcinoma. Multivariate-adjusted OR [aOR] with the corresponding 95% confidence intervals [CI] of EGFR mutation status in association with genotypes were evaluated using logistic regression analysis. Results: The rs744154C/G in ERCC4 was significantly associated with EGFR exon19 in-frame deletion both in never-smokers (aOR, 1.7 with 95% CI, 1.0-3.0) and female never-smokers (aOR, 1.9 with 95% CI, 1.0-3.6), respectively. A significant association with EGFR exon19 in-frame deletion was found for the rs1800566C/T in NQO1 (aOR, 2.2 with 95% CI, 1.0-4.8) in female never-smokers. The rs1047840A/A genotype in EXO1 showed a 7.6-fold increase in the exon19 in-frame deletion in female never-smokers, but the association was marginally significant. Furthermore, the number of risk alleles in NQO1, ERCC4 and EXO1 was associated with an increasing aOR of EGFR exon19 in-frame deletion both in never-smokers and female never-smokers (P= 0.007 and 0.002 for trend, respectively). Additionally, the CA-repeat polymorphism in EGFR was significantly associated with exon19 in-frame deletion both in never-smokers (aOR, 1.7 with 95% CI, 1.0-2.9) and female never-smokers (aOR, 1.9 with 95% CI, 1.0-3.5), respectively, for both alleles with (CA)n repeats > 18 vs. one or two alleles ≦18). Conclusions: The exon19 in-frame deletion of EGFR is associated with polymorphisms in EGFR and DNA repair and detoxification metabolism genes in never-smoking lung adenocarcinoma patients, especially in females. The findings provide a clue about the genesis of EGFR mutations.

In summary, EGFR hotspot mutations were more frequently found in never-smoking lung adenocarcinoma. Moreover, the sex difference in EGFR mutation subtypes was found in never-smoking lung adenocarcinoma, the exon 19 in-frame deletion was more frequently associated with male gender, while exon 21 mutations were more frequent in females. Additionally, from the viewpoints of molecular mechanisms of mutagenesis, the exon19 in-frame deletion of EGFR is associated with polymorphisms in EGFR and DNA repair and detoxification metabolism genes, while the L858R mutation of EGFR is associated with polymorphisms of genes related to estrogen biosynthesis and metabolism in never-smoking lung adenocarcinoma patients.