臺灣博碩士論文加值系統

第一章
研究目的：先前報告指出表皮生長因子受器突變常發生在女性、不曾抽菸者、腺性非小細胞肺癌的病人。本研究是探討性別是否為決定非小細胞肺癌表皮生長因子受器突變相關因素中的混擾因子。
實驗材料與方法：回溯性收集病人人口學、 抽菸史、腫瘤分期、病理組織次分類及表皮生長因子受器突變狀況資料，採用Pearson’s 卡方檢定、多重邏輯迴歸分析及依抽菸史分層策略來檢驗性別是否為決定表皮生長因子受器突變相關因素中的混擾因子。
結果：在426名非小細胞肺癌病人中，百分之四十七為女性、百分之五十七為不曾吸菸者、百分之八十四為肺腺癌。多變項迴歸分析顯示不曾抽菸者(勝算比, odds ratio,為3.49，95%信賴區間值為1.99-6.13; P值小於0.001)、肺腺癌(勝算比為9.43，95%信賴區間值為3.62-24.56; P值小於0.001)和表皮生長因子受器突變顯著相關。而性別則和表皮生長因子受器突變無顯著相關(勝算比為1.25，95%信賴區間值為0.73-1.25; P值為0.416)；進一步分析顯示性別也和表皮生長因子受器突變亞型第19外子缺失 (exon 19 deletion)及L858R點突變(L858R point mutation)無顯著相關(勝算比為1.19 ， 95%信賴區間值為0.56-2.50; P值為0.650)。在把病人進一步依抽菸史進行分層分析顯示:女性和男性的表皮生長因子受器突變的發生率不管是在不曾吸菸(64.8% vs. 55.8%, P值為0.204)或曾吸菸(27.8% vs. 24.2%, P值為0.775)的族群中並無顯著不同。因此，若只限定不曾吸菸、女性且為肺腺癌的非小細胞肺癌病患接受表皮生長因子受器突變檢測，將有高達百分之四十的表皮生長因子受器突變個案會被預先排除接受表皮生長因子受器抑制劑治療。
結論：我們的研究結果顯示性別是決定非小細胞肺癌表皮生長因子受器突變相關因素中的混擾因子，同時建議性別可能和表皮生長因子受器突變非小細胞肺癌的腫瘤發生無關。

第二章
研究目的:肺癌的病人很容易有的情形，表皮生長因子受器突變第19外子缺失及L858點突變為最常見非小細胞肺癌致癌基因之一，本論文為探討表皮生長素受器突變和非小細胞肺癌腦轉移間的相關性。
實驗材料與方法:回溯性分析病人臨床病理及表皮生長因子受器突變因子等和腦部轉移累計發生率的相關性及預測性，同時使用肺癌細胞株來研究表皮生長素受器突變和肺癌細胞散佈的相關性。
結果:多變相回歸分析顯示在384個案中，表皮生長因子受器突變和整體腦部轉移累計發生率相關(勝算比, odds ratio=2.24, P值=0.001)。在第三及第四期非小細胞肺癌個案中，進一步採用死亡為競爭風險因子的事件發生時間(time-to-event)分析模式顯示除整體存活長短外，表皮生長因子受器突變可以預測後續腦部轉移的發生 (37.1% vs. 10.6%, 相對危險比, hazard ratio =2.98, P=0.002)，且此能力獨立於個案年齡(相對危險比=2.00, P=0.012)，性別，病理組織類型和抽菸史。比起年紀大且為原野型表皮生長因子受器的個案，起年紀輕且為突變型表皮生長因子受器的個案，期發生後續腦部轉移的風險顯著升高 (58.1% vs.10.9%,相對危險比=6.57, P<0.001)。雖然第19外子缺失個案會比L858點突變個案有些微較長的整體存活期 (20.6 vs. 14.2 月, P=0.368)，但兩者在預測後續腦部轉移的發生的能力上則不分軒輊 (39.5% vs. 34.5%,相對危險比=0.91, P=0.770)。在體外細胞實驗中，表皮生長因子受器突變過度表現會導致肺癌細胞朝向類似間質細胞形狀的改變，同時也會提升肺癌細胞散佈能力。臨床上，比起原野型表皮生長因子受器的個案，表皮生長素受器突變個案的腫瘤有較高比率表現出波型蛋白 (75.3% vs. 51.2%; P=0.007)，且個案發生後續腦部轉移的中位數存活時間較短(23.5 月vs. not reached, P=0.017)。
結論:本結果建議比起原野型表皮生長因子受器的非小細胞肺癌個案，在追蹤期間，表皮生長素受器突變個案應接受較為密集之腦部影像學評估，以利早期發現腦轉移。

Chapter 1
Background: Mutations in epidermal growth factor receptor (EGFR) commonly occur in non-small-cell lung cancer (NSCLC) patients characterized by female gender, never-smoker status and adenocarcinoma histology. The aim of this study was to determine whether gender is a confounding factor for EGFR mutations in NSCLC.
Materials and methods: Demographic data and smoking history of patients, tumor stage and subtype of NSCLC, and EGFR mutation status were retrospectively collected. To elucidate the confounding effect, Pearson's χ2 test, and a multivariable logistic regression model were used to correlate these characteristics with EGFR mutations.
Results: Of those 426 NSCLC patients, 47% were females, 57% were non-smokers and 84% had adenocarcinomas. The multivariate logistic regression analysis demonstrated that never-smoker status [odds ratio (OR) = 3.49, 95% confidence interval (CI): 1.99-6.13; P <0.001)] and adenocarcinoma (OR = 9.43, 95% CI 3.62-24.56; P<0.001) were associated with EGFR muta¬tions; however, gender was not (OR = 1.25, 95% CI: 0.73-2.15; P = 0.416). Furthermore, gender was not associated with EGFR mutation subtypes (OR = 1.19, 95% CI: 0.56-2.50; P = 0.650). The frequency of EGFR mutations between females and males was not different in non-smokers (64.8% vs. 55.8%, P = 0.204) or ever smokers (27.8% vs. 24.2%, P = 0.775). Therefore, if the assessment for EGFR mutation status was limited to non smoking females with adenocarcinoma, up to 40% of the patients harboring EGFR mutations would be precluded from the benefit of EGFR inhibitor therapy.
Conclusions: Our results indicated that gender is a confounding factor for EGFR mutations in NSCLC and suggested that gender may not be associated with tumorigenesis in NSCLC harboring EGFR mutations.

Chapter 2
Background: Non-small cell lung cancer (NSCLC) patients tend to develop brain metastases (BM), but the link between BM occurrence and driver mutations in NSCLC is not very clear. We explored whether activating mutations of epidermal growth factor receptors (EGFRs) in exon 19 deletion or L858R predict BM in NSCLC. Materials and methods: Retrospective analyses were used to determine the association of clinic-pathological, EGFR mutation and BM. In vitro experiments were conducted to explore the link between EGFR mutation and lung cancer cell dissemination.
Results: A multivariable logistic regression analysis of 384 patients demonstrated that the presence of mutated-EGFRs was associated with overall BM (OR=2.24, P=0.001) compared to that of wild-type EGFR (WT-EGFR). Moreover, the time-to-event analysis model considering death as a competing risk revealed that, irrespective of survival, mutated-EGFRs predicted subsequent BM (SBM) in stage IIIB-IV patients (37.1% vs. 10.6%, HR=2.98, P=0.002) after adjusting for age (HR=2.00, P=0.012), gender, histological subtype, and smoking history. Notably, the younger mutated-EGFR subgroup was at a higher risk for SBM compared to the older WT-EGFR one (58.1% vs.10.9%, HR=6.57, P<0.001). Additionally, EGFR exon 19 deletion, despite having a slightly longer overall survival (20.6 vs. 14.2 months, P=0.368), was comparable to L858R mutation in predicting SBM (39.5% vs. 34.5%, HR=0.91, P=0.770). In vitro, the overexpression of mutated-EGFRs induced morphological changes towards a mesenchymal-like phenotype and promoted mobility in lung cancer cells. Clinically, mutated-EGFR NSCLC displayed a higher proportion of vimentin-positive expression (75.3% vs. 51.2%; P=0.007) and a shorter median time to SBM (23.5 months vs. not reached, P=0.017) than WT-EGFR NSCLC. Conclusion: These results suggest that NSCLC patients carrying mutated-EGFRs may require a higher frequency of brain imaging assessments than those with WT-EGFR to facilitate earlier SBM detection during follow-up.

Content 1
Chapter 1 Histological subtype and smoking status, but not gender, are associated with epidermal growth factor receptor mutations in non-small cell lung cancer 2
中文摘要 3
Abstract 5
Introduction 7
Patients and methods 9
Results 12
Discussion 15
Conclusion 20
Tables 21
References 29
Chapter 2 Brain Metastases in Patients with Non-Small Cell Lung Cancer: the Role of Mutated-EGFRs with an Exon 19 Deletion or L858R Point Mutation in Cancer Cell Dissemination 35
中文摘要 36
Abstract 38
Introduction 40
Materials and methods 42
Results 52
Discussion 60
Conclusion 66
Figures 67
Tables 78
References 85

1. Mujoomdar A, Austin JH, Malhotra R, Powell CA, Pearson GD, Shiau MC, Raftopoulos H Clinical predictors of metastatic disease to the brain from non-small cell lung carcinoma: primary tumor size, cell type, and lymph node metastases. Radiology. 2007;242: 882-888.
2. Li T, Kung HJ, Mack PC, Gandara DR Genotyping and genomic profiling of non-small-cell lung cancer: implications for current and future therapies. J Clin Oncol. 2013;31: 1039-1049.
3. Doebele RC, Lu X, Sumey C, Maxson DA, Weickhardt AJ, Oton AB, Bunn PA Jr, Barón AE, Franklin WA, Aisner DL, Varella-Garcia M, Camidge DR. Oncogene status predicts patterns of metastatic spread in treatment-naive nonsmall cell lung cancer. Cancer. 2012;118: 4502-4511.
4. Shin DY, Na II, Kim CH, Park S, Baek H, Yang SH EGFR mutation and brain metastasis in pulmonary adenocarcinomas. J Thorac Oncol. 2014;9: 195-199.
5. Li B, Sun SZ, Yang M, Shi JL, Xu W, Wang XF, Song MM, Chen HM The correlation between EGFR mutation status and the risk of brain metastasis in patients with lung adenocarcinoma. J Neurooncol. 2015;124: 79-85.
6. Iuchi T, Shingyoji M, Itakura M, Yokoi S, Moriya Y, Tamura H, Yoshida Y, Ashinuma H, Kawasaki K, Hasegawa Y, Sakaida T, Iizasa T. Frequency of brain metastases in non-small-cell lung cancer, and their association with epidermal growth factor receptor mutations. Int J Clin Oncol. 2015;20: 674-679.
7. Riely GJ, Pao W, Pham D, Li AR, Rizvi N, Venkatraman ES, Zakowski MF, Kris MG, Ladanyi M, Miller VA Clinical course of patients with non-small cell lung cancer and epidermal growth factor receptor exon 19 and exon 21 mutations treated with gefitinib or erlotinib. Clin Cancer Res. 2006;12: 839-844.
8. Yang JC, Wu YL, Schuler M, Sebastian M, Popat S, Yamamoto N, Zhou C, Hu CP, O'Byrne K, Feng J, Lu S, Huang Y, Geater SL, et al. Afatinib versus cisplatin-based chemotherapy for EGFR mutation-positive lung adenocarcinoma (LUX-Lung 3 and LUX-Lung 6): analysis of overall survival data from two randomised, phase 3 trials. Lancet Oncol. 2015;16: 141-151.
9. Lee CK, Wu YL, Ding PN, Lord SJ, Inoue A, Zhou C, Mitsudomi T, Rosell R, Pavlakis N, Links M, Gebski V, Gralla RJ, Yang JC Impact of Specific Epidermal Growth Factor Receptor (EGFR) Mutations and Clinical Characteristics on Outcomes After Treatment With EGFR Tyrosine Kinase Inhibitors Versus Chemotherapy in EGFR-Mutant Lung Cancer: A Meta-Analysis. J Clin Oncol. 2015;33: 1958-1965.
10. Karachaliou N, Mayo-de las Casas C, Queralt C, de Aguirre I, Melloni B, Cardenal F, Garcia-Gomez R, Massuti B, Sánchez JM, Porta R, Ponce-Aix S, Moran T, Carcereny E, et al. Association of EGFR L858R Mutation in Circulating Free DNA With Survival in the EURTAC Trial. JAMA Oncol. 2015;1: 149-157.
11. Verbeek BS, Adriaansen-Slot SS, Vroom TM, Beckers T, Rijksen G Overexpression of EGFR and c-erbB2 causes enhanced cell migration in human breast cancer cells and NIH3T3 fibroblasts. FEBS Lett. 1998;425: 145-150.
12. Thomas SM, Coppelli FM, Wells A, Gooding WE, Song J, Kassis J, Drenning SD, Grandis JREpidermal growth factor receptor-stimulated activation of phospholipase Cgamma-1 promotes invasion of head and neck squamous cell carcinoma. Cancer Res. 2003;63: 5629-5635.
13. Lo HW, Hsu SC, Xia W, Cao X, Shih JY, Wei Y, Abbruzzese JL, Hortobagyi GN, Hung MC. Epidermal growth factor receptor cooperates with signal transducer and activator of transcription 3 to induce epithelial-mesenchymal transition in cancer cells via up-regulation of TWIST gene expression. Cancer Res. 2007;67: 9066-9076.
14. Hsiao SH, Chung CL, Lee CM, Chen WY, Chou YT, Wu ZH, Chen YC, Lin SE. Suitability of computed tomography-guided biopsy specimens for subtyping and genotyping of non-small-cell lung cancer. Clin Lung Cancer. 2013;14: 719-725.
15. Hsiao SH, Chung CL, Chou YT, Lee HL, Lin SE, Liu HE Identification of subgroup patients with stage IIIB/IV non-small cell lung cancer at higher risk for brain metastases. Lung Cancer. 2013;82: 319-323.
16. Chou YT, Lin HH, Lien YC, Wang YH, Hong CF, Kao YR, Lin SC, Chang YC, Lin SY, Chen SJ, Chen HC, Yeh SD, Wu CW EGFR promotes lung tumorigenesis by activating miR-7 through a Ras/ERK/Myc pathway that targets the Ets2 transcriptional repressor ERF. Cancer Res. 2010;70: 8822-8831.
17. Tiruppathi C, Malik AB, Del Vecchio PJ, Keese CR, Giaever I. Electrical method for detection of endothelial cell shape change in real time: assessment of endothelial barrier function. Proc Natl Acad Sci U S A. 1992;89: 7919-7923.
18. Keese CR, Bhawe K, Wegener J, Giaever I.Real-time impedance assay to follow the invasive activities of metastatic cells in culture. Biotechniques. 2002;33: 842-844, 846, 848-850.
19. Chitale et.al: Memorial Sloan-Kettering Cancer Center (http://cbio.mskcc.org/public/lung_array_data/)
20. TCGA (https://tcga-data.nci.nih.gov/docs/publications/luad_2014/).
21. Hendriks LE, Smit EF, Vosse BA, Mellema WW, Heideman DA, Bootsma GP, Westenend M, Pitz C, de Vries GJ, Houben R, Grünberg K, Bendek M, Speel EJ,et al. EGFR mutated non-small cell lung cancer patients: more prone to development of bone and brain metastases ? Lung Cancer. 2014;84:86-91
22. Rangachari D, Yamaguchi N, VanderLaan PA, Folch E, Mahadevan A, Floyd SR, Uhlmann EJ, Wong ET, Dahlberg SE, Huberman MS, Costa DB. Brain metastases in patients with EGFR-mutated or ALK-rearranged non-small-cell lung cancers Lung Cancer. 2015;88:108-11.
23. Baek MY, Ahn HK, Park KR, Park HS, Kang SM, Park I, Kim YS, Hong J, Sym SJ, Park J, Lee JH, Shin DB, Cho EK. Epidermal growth factor receptor mutation and pattern of brain metastasis in patients with non-small cell lung cancer. Korean J Intern Med. 2016 Apr 20. doi: 10.3904/kjim.2015.158.
24. Mok TS, Wu YL, Thongprasert S, Yang CH, Chu DT, Saijo N, Sunpaweravong P, Han B, Margono B, Ichinose Y, Nishiwaki Y, Ohe Y, Yang JJ, et al. Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma. N Engl J Med. 2009;361: 947-957.
25. Hsu F, De Caluwe A, Anderson D, Nichol A, Toriumi T, Ho C EGFR mutation status on brain metastases from non-small cell lung cancer. Lung Cancer 2016 ;96:101-7.
26. Han G, Bi J, Tan W, Wei X, Wang X, Ying X, Guo X, Zhou X, Hu D, Zhen W. Oncotarget. 2016 Jul 29. doi: 10.18632/oncotarget.10933. A retrospective analysis in patients with EGFR-mutant lung adenocarcinoma: is EGFR mutation associated with a higher incidence of brain metastasis?
27. Lal A, Glazer CA, Martinson HM, Friedman HS, Archer GE, Sampson JH, Riggins GJ. Cancer Res. 2000;62:3335-9. Mutant epidermal growth factor receptor up-regulates molecular effectors of tumor invasion
28. De Craene B, Berx G. Regulatory networks defining EMT during cancer initiation and progression. Nat Rev Cancer. 2013 Feb;13(2):97-110. doi: 10.1038/nrc3447
29. Omuro AM, Kris MG, Miller VA, Franceschi E, Shah N, Milton DT, Abrey LE. High incidence of disease recurrence in the brain and leptomeninges in patients with nonsmall cell lung carcinoma after response to gefitinib. Cancer. 2005;103: 2344-2348.
30. Lee YJ, Choi HJ, Kim SK, Chang J, Moon JW, Park IK, Kim JH, Cho BC. Frequent central nervous system failure after clinical benefit with epidermal growth factor receptor tyrosine kinase inhibitors in Korean patients with nonsmall-cell lung cancer. Cancer. 2010;116: 1336-1343.
31. Heon S, Yeap BY, Britt GJ, Costa DB, Rabin MS, Jackman DM, Johnson BE. Development of central nervous system metastases in patients with advanced non-small cell lung cancer and somatic EGFR mutations treated with gefitinib or erlotinib. Clin Cancer Res. 2010;16: 5873-5882
32. Pitz MW, Desai A, Grossman SA, Blakeley JO. Tissue concentration of systemically administered antineoplastic agents in human brain tumors. J Neurooncol. 2011;104: 629-638.
33. Togashi Y, Masago K, Masuda S, Mizuno T, Fukudo M, Ikemi Y, Sakamori Y, Nagai H, Kim YH, Katsura T, Mishima M Cerebrospinal fluid concentration of gefitinib and erlotinib in patients with non-small cell lung cancer. Cancer Chemother Pharmacol. 2012;70: 399-405.
34. Ballard P, Yates JW, Yang Z Kim DW, Yang JC, Cantarini M, Pickup K, Jordan A, Hickey M, Grist M, Box M, Johnström P, Varnäs K, Malmquist J, Thress KS, Jänne PA, Cross D. Preclinical Comparison of Osimertinib with Other EGFR-TKIs in EGFR-Mutant NSCLC BrainMetastases Models, and Early Evidence of Clinical Brain Metastases Activity. Clin Cancer Res. 2016;22:5130-5140.
35. Zeng Q, Wang J, Cheng Z, Chen K, Johnström P, Varnäs K, Li DY, Yang ZF, Zhang X. Discovery and Evaluation of Clinical Candidate AZD3759, a Potent, Oral Active, Central Nervous System-Penetrant, Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor. J Med Chem. 2015 Oct 22;58(20):8200-15
36. Wu WS, Chen YM, Tsai CM, Shih JF, Lee YC, Perng RP, Whang-Peng J The epidermal growth factor receptor-tyrosine kinase inhibitor era has changed the causes of death of patients with advanced non-small-cell lung cancer. J Chin Med Assoc. 2013;76: 682-685.
37. Mamon HJ, Yeap BY, Jänne PA, Reblando J, Shrager S, Jaklitsch MT, Mentzer S, Lukanich JM, Sugarbaker DJ, Baldini EH, Berman S, Skarin A, Bueno R. High risk of brain metastases in surgically staged IIIA non-small-cell lung cancer patients treated with surgery, chemotherapy, and radiation. J Clin Oncol. 2005;23:1530-7
38. Yatabe Y, Matsuo K, Mitsudomi T. Heterogeneous distribution of EGFR mutations is extremely rare in lung adenocarcinoma. J Clin Oncol. 2011;29: 2972-2977.
39. Wu CC, Hsu HY, Liu HP, et al. Reversed mutation rates of KRAS and EGFR genes in adenocarcinoma of the lung in Taiwan and their implications. Cancer. 2008;113: 3199-3208.